JNEUROSCI.1256-23.2024.full-html.html

TITLE:

Impairment of the glial phagolysosomal system drives prion-like propagation in a

Drosophila

model of Huntington’s disease

ABBREVIATED TITLE:

Phagocytic glial defects drive huntingtin proteotoxicity

AUTHORS and AFFILIATIONS:

Graham H. Davis

1,2,3

, Aprem Zaya

, and Margaret M. Panning

Pearce

1,2,3,

Rowan University, Department of Biological and Biomedical Sciences, Glassboro, NJ 08028,

Saint

Joseph’s University, Department of Biology, Philadelphia, PA 19131, and

University of the Sciences,

Department of Biological Sciences, Philadelphia, PA 19104

*Correspondence should be addressed to Maggie Panning Pearce at pearcem@rowan.edu.

Number of pages: 48

Number of Figures and Tables: 18 Figures and 4 Tables

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ABSTRACT

Protein misfolding, aggregation, and spread through the brain are primary drivers of

neurodegenerative diseases pathogenesis. Phagocytic glia are responsible for regulating the load of

pathogenic protein aggregates in the brain, but emerging evidence suggests that glia may also act as

vectors for aggregate spread. Accumulation of protein aggregates could compromise the ability of glia

to eliminate toxic materials from the brain by disrupting efficient degradation in the phagolysosomal

system. A better understanding of phagocytic glial cell deficiencies in the disease state could help to

identify novel therapeutic targets for multiple neurological disorders. Here, we report that mutant

huntingtin (mHTT) aggregates impair glial responsiveness to injury and capacity to degrade neuronal

debris in male and female adult

Drosophila

expressing the gene that causes Huntington’s disease

(HD). mHTT aggregate formation in neurons impairs engulfment and clearance of injured axons and

causes accumulation of phagolysosomes in glia. Neuronal mHTT expression induces upregulation of

key innate immunity and phagocytic genes, some of which were found to regulate mHTT aggregate

burden in the brain. Finally, a forward genetic screen revealed Rab10 as a novel component of Draper-

dependent phagocytosis that regulates mHTT aggregate transmission from neurons to glia. These data

suggest that glial phagocytic defects enable engulfed mHTT aggregates to evade lysosomal

degradation and acquire prion-like characteristics. Together, our findings reveal new mechanisms that

enhance our understanding of the beneficial and potentially harmful effects of phagocytic glia in HD and

potentially other neurodegenerative diseases.

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INTRODUCTION

Neuron-glia crosstalk is critical for maintaining homeostasis in the central nervous system

(CNS), and disruption of these intercellular interactions is increasingly recognized as a central

component of many neurological disorders, including neurodegenerative diseases. Glia perform

immune surveillance functions in the CNS and respond to neuronal injury by altering gene expression

(Magaki et al., 2018) and clearing damaged cells (Raiders et al., 2021; Zheng and Tuszynski, 2023).

These glial responses may initially be neuroprotective, but prolonged glial reactivity propels the

neurodegenerative disease state, for example, by driving premature loss of living neurons or functional

synapses (Neniskyte et al., 2011; Hong et al., 2016; Dejanovic et al., 2022) and inducing

neuroinflammation (Liddelow et al., 2017). Expanding our understanding of how glia influence neuron

function and survival could reveal promising new therapeutic strategies for neurodegenerative

diseases.

A pathological hallmark of most neurodegenerative diseases is the accumulation of misfolded

proteins into intra- or extracellular amyloid aggregates in vulnerable regions of the CNS (Knowles et al.,

2014). Protein aggregates form due to age-associated decline in cellular protein folding capacity

(Santra et al., 2019; Stein et al., 2022) and overwhelming of degradative pathways, including the

ubiquitin-proteasome system, autophagy, and phagocytosis (Aman et al., 2021; Duong et al., 2021;

Wodrich et al., 2022). As professional phagocytes of the brain, microglia and astrocytes clear damaged

and dysfunctional cells (Paolicelli et al., 2011; Wakida et al., 2018; Herzog et al., 2019; Lee et al., 2021)

and other pathogenic material, such as protein aggregates (Liu et al., 2017; Choi et al., 2020).

Defective glial clearance of debris may be a driving force underlying neurodegenerative disease,

highlighted by a growing list of genetic risk variants associated with phagocytic and endolysosomal

pathways

(Podleśny-Drabiniok et al., 2020). Endolysosomal impairment promotes protein aggregate

accumulation; in turn, aggregates can drive further endolysosomal dysfunction, including deacidification

and membrane permeabilization of intracellular vesicles (Krasemann et al., 2017; Heckmann et al.,

2019; Burbidge et al., 2022; Lee et al., 2022). Aggregates that escape degradation may gain the ability

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to spread and seed soluble proteins in a prion-like manner (Jucker and Walker, 2018; Monaco and

Fraldi, 2020).

Mechanisms by which pathogenic aggregates dysregulate endolysosomal processing remain

largely unknown. Intracellular membrane fusion events that regulate endo/phagosome maturation are

catalyzed by Rab GTPases (Ng and Tang, 2008; Chan et al., 2011; Langemeyer et al., 2018), enzymes

that cycle between active (GTP-bound) and inactive (GDP-bound) states to organize endomembranes

into distinct functional domains (Hall, 1990; Chan et al., 2011). Rab dysfunction is implicated in several

neurodegenerative diseases

—e.g., upregulation of Rab4, Rab5, Rab7, and Rab27 has been observed

in Alzheimer's disease (AD) (Ginsberg et al., 2011), and several Rabs are known substrates of leucine

rich repeat kinase 2 (LRRK2), a genetic risk factor in familial Parkinson’s disease (PD) (Jeong et al.,

2018). Intriguingly, spread of

ɑ-synuclein between cultured neuronal and enteroendocrine cells is

mediated by the LRRK2 substrate Rab35 (Bae et al., 2018; Rodrigues et al., 2022), suggesting that

Rab-dependent processes may contribute to formation and propagation of pathogenic aggregate

seeds.

Here, we investigated the impact of protein aggregates generated in neurons on phagocytic glial

cell functions in a

Drosophila

model of

Huntington’s disease (HD). We report that neuronal expression

of aggregation-prone mutant huntingtin (mHTT) protein reduces the ability of glia to clear axonal debris

100

and to mount phagocytic and innate immunity transcriptional responses following acute nerve injury.

101

We also observed mHTT-induced changes to numbers of glial lysosomes and Rab+ vesicles in

102

uninjured brains, and identify Rab10 as a novel modifier of prion-like spreading of mHTT aggregates in

103

adult fly brains. Together, these studies shed light on mechanisms by which phagocytic glia respond to

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and are impaired by accumulation of pathogenic aggregates in neurons.

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MATERIALS AND METHODS

106

Fly husbandry

107

Fly stocks and crosses were raised on standard cornmeal/molasses media on a 12 hr light/12 hr

108

dark cycle at 25°C, unless otherwise noted. No sex-specific differences were observed in any

109

experiments, so both males and females were utilized in this study. Transgenic or mutant flies were

110

either generated for this study (described below), purchased from Bloomington Drosophila Stock

111

Center, or kindly provided by collaborators. Genotypes and sources of all fly stocks used in this study

112

are listed in Table 1, and complete genotypes of flies used in each figure are listed in Table 2.

113

Acute antennal nerve injury was performed by bilateral removal of the second and third antennal

114

segments from anesthetized adult flies (MacDonald et al., 2006). For quantitative PCR analyses,

115

maxillary palps were removed in addition to third antennal segments to sever all olfactory receptor

116

neuron (ORN) axons. Axotomized flies were incubated for the indicated times on standard media prior

117

to dissection and processing for imaging.

118

119

Cloning and Drosophila transgenesis

120

pUASTattB(HTT

ex1

Q25-V5) and pUASTattB(HTT

ex1

Q91-V5) plasmids were cloned by PCR

121

amplification of HTT exon 1 (HTT

ex1

) cDNA using a reverse primer that inserted an in-frame, C-terminal

122

V5 epitope tag (GKPIPNPLLGLDST) and ligation into the pQUASTattB vector backbone via XhoI and

123

XbaI restriction sites. pQUASTattB(HTT

ex1

Q25-GFP) and pQUASTattB(HTT

ex1

Q91-GFP) were

124

generated by replacing the mCherry sequence in pQUASTattB(HTT

ex1

Q25-mCherry) and

125

pQUASTattB(HTT

ex1

Q91-mCherry) plasmids previously generated by our lab (Pearce et al., 2015) with

126

an in-frame, C-terminal GFP sequence via Gibson Assembly (New England Biolabs, Inc. Ipswich, MA).

127

pUASTattB(pHluorin-HTT

ex1

Q25-tdTomato) and pUASTattB(pHluorin-HTT

ex1

Q91-tdTomato) plasmids

128

were generated by PCR amplification of HTT

ex1

cDNAs and subcloning to replace the CD4 sequence in

129

the pUASTattB(MApHS) plasmid (Han et al., 2014) via In-Fusion cloning (Takara Bio USA, Inc.,

130

Mountain View, CA). pUASTattB(mCherry-Galectin-3) and pUASTattB(mCherry-Galectin-8) were

131

cloned by PCR amplification of human LGals3 and LGals8 cDNAs from the pHAGE-mKeima-LGALS3

132

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and pHAGE-FLAG-APEX2-LGALS8 plasmids (Addgene plasmids #175780 and #175758, Watertown,

133

MA) (Eapen et al., 2021) and insertion downstream of an in-frame mCherry sequence in the pUASTattB

134

vector backbone via Gibson Assembly.

135

Plasmids were microinjected into

embryos with the su(Hw)attP8, attP24, VK19, or VK27

136

φC31 attP integration sites at BestGene, Inc (Chino Hills, CA). Table 1 lists detailed genotype

137

information for all transgenic flies generated in this study.

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139

Drosophila brain dissection and sample preparation

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Adult fly brains were dissected in ice-cold phosphate-buffered saline (PBS) containing either no

141

detergent or 0.03% (PBS/0.03T), 0.1% (PBS/0.1T), or 0.3% (PBS/0.3T) Triton X-100. Dissected brains

142

were fixed in 4% paraformaldehyde (PFA) in the dark at room temperature (RT) for 20 minutes. For

143

imaging of GFP or mCherry fluorescence signals, brains were washed 7X in PBS/0.03T buffer before

144

incubation in Slowfade Gold Antifade Mountant (Invitrogen, Carlsbad, CA). When imaging pHluorin-

145

tagged and other pH-sensitive constructs, such as GFP-LAMP1, brains were washed 7X in PBS for at

146

least 50 minutes at RT before incubation in Slowfade. For immunostaining, brains were washed 7X in

147

PBS/0.3T after fixation, blocked in PBS/0.3T containing 5% normal goat serum (Lampire Biological

148

Laboratories, Pipersville, PA) for 30 min at RT, incubated in primary antibodies diluted in blocking

149

solution for 24-48 hours at 4°C, washed 7X in PBS/0.3T, and then incubated in secondary antibodies

150

diluted in blocking solution for 16-20 hours at 4°C. After a final set of 7X PBS/0.3T washes, dissected

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brains were incubated in Slowfade. For Magic Red and LysoTracker staining, brains were dissected in

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PBS and incubated in 1:1,000 LysoTracker Red DND-99 (Invitrogen, Carlsbad, CA) or 1:1,250 Magic

153

Red (ImmunoChemistry, Davis, CA) diluted in PBS for 20 minutes at RT. Brains were washed 5X in

154

PBS for 15 min total, fixed in 4% PFA in PBS/0.1T for 20 minutes at RT, washed 6X in PBS/0.1T for 30

155

min total, and incubated in Slowfade. Following incubation in Slowfade for 1-24 hours at 4°C in the

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dark, all brains were bridge-mounted in Slowfade on glass microscopy slides under #1.5 cover glass,

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and edges were sealed using clear nail polish.

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Primary antibodies used in this study include: chicken anti-GFP (RRID: AB_300798; 1:1,000;

159

Abcam, Cambridge, UK), chicken anti-GFP (RRID: AB_2534023; 1:500; Thermo Fisher Scientific Inc.,

160

Waltham, MA), rat anti-N-cadherin (clone DN-Ex #8; RRID: AB_528121; 1:75; Developmental Studies

161

Hybridoma Bank, Iowa City, IA), mouse anti-Repo (clone 8D12; RRID: AB_528448; 1:25;

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Developmental Studies Hybridoma Bank, Iowa City, IA), mouse anti-V5 (RRID: AB_2556564; 1:125;

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Thermo Fisher Scientific Inc., Waltham, MA), and rabbit anti-Draper (1:500; a kind gift from Marc

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Freeman, Vollum Institute, Portland, OR). Secondary antibodies include: AlexaFluor 405 goat anti-

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rabbit (RRID: AB_221605; 1:250), FITC-conjugated donkey anti-chicken (RRID: AB_2340356; 1:250;

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Jackson Immuno Research Labs, West Grove, PA), AlexaFluor 568 goat anti-mouse (RRID:

167

AB_2534072; 1:250), AlexaFluor 647 goat anti-rabbit (RRID: AB_2535812; 1:250), and AlexaFluor 647

168

goat anti-rat IgGs (RRID: AB_141778; 1:250) (Invitrogen, Carlsbad, CA).

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Image acquisition

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All microscopy data were collected on a Leica SP8 laser-scanning confocal system equipped

172

with 405 nm, 488 nm, 561 nm, and 633 nm lasers and 40X 1.3 NA or 63X 1.4 NA oil objective lenses.

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Leica LAS-X software was used to establish optimal settings during each microscopy session and to

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collect optical z-slices of whole-mounted brain samples with Nyquist-optimized sampling criteria.

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Optical zoom was used to magnify and establish a region of interest (ROI) in each sample. For images

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showing a single glomerulus, confocal slices were collected to generate ~73 x 73

x 20 μm (

xyz

) stacks,

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with z-axis boundaries established using fluorescent signal in DA1 or VA1lm ORN axons. For images of

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a single antennal lobe, confocal slices were collected to generate ~117 x 117 x 26 μm (

xyz

) stacks,

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which were located using HTT

ex1

fluorescence in ORN axons.

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Post-imaging analysis

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Raw confocal data were analyzed in 2D using ImageJ/FIJI (RRID:SCR_002285; NIH, Bethesda,

183

MD) or in 3D using Imaris image analysis software (RRID:SCR_007370; Bitplane, Zürich, Switzerland).

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Methods used for image segmentation and semi-automated quantification of fluorescent signals were

185

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previously described (Donnelly et al., 2020). Briefly, raw confocal data were cropped to establish an

186

ROI for further analysis and displayed as a 2D sum intensity projection (ImageJ) or a 3D volume

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(Imaris). Background fluorescence was subtracted from raw confocal images. mCD8-GFP and HTT

ex1

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mCherry fluorescent signals

was segmented using the ‘Surfaces’ function in Imaris (surface detail =

189

0.25

μm, background subtraction = 0.75 μm). Using the ‘split touching objects’ option, seed point

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diameter was set to 0.85

μm. pHluorin- and tdTomato-labeled VA1lm ORN axons were quantified in

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central 30 x 30 pixels, 50-slice ROIs from sum intensity projections of each VA1lm glomerulus.

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pHluorin- and tdTomato-labeled Or83b+ ORN axons or MBN soma were quantified from central 100 x

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100 pixels, 75-slice ROIs of each antennal lobe or mushroom body calyx, and background intensity was

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subtracted from sum intensity projections. Quantification of Toll-6

MIMICGFP

fluorescence was performed

195

using the ‘Spots’ function in Imaris (

diameter = 0.5

μm,

-diameter = 1.0

μm), and glial nuclei-

196

associated GFP-Jra signal was quantified

using the “Surfaces” function to identify Repo+/GFP+ nuclei

197

(surface detail = 0.2

μm, background subtraction = 1.6 μm, number of voxels >10).

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mCherry-tagged mHTT

ex1

and GFP-tagged wtHTT

ex1

aggregates were identified and quantified

199

as previously reported (Donnelly et al., 2020). Briefly, mCherry+ surfaces were segmented and

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measured in Imaris (surface detail = 0.2

μm, background subtraction = 0.45 μm, seed point diameter =

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0.85

μm). Seeded wtHTT

ex1

aggregates were identified as mHTT

ex1

-mCherry+ objects that overlapped

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with wtHTT

ex1

-GFP signal.

Intracellular vesicles were identified using the ‘Surfaces’ algorithm in Imaris

203

(surface detail = 0.2 μm, background subtraction = 0.4 μm, volume >0.001 μm

) to segment fluorescent

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signals associated with lysosomes (MR+, LTR+, LAMP1-GFP+, GFP-LAMP1+, mCherry-Galectin-3+,

205

or mCherry-Galectin-8+) or phagosomes (YRab+ or YFP-Rab+). Intracellular vesicles associated with

206

mHTT

ex1

aggregates were identified by filtering for lysosomal or phagosomal surfaces within

0.2 μm of

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mHTT

ex1

objects

using the “Shortest Distance” calculation in Imaris.

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209

qPCR

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Transgenic

Drosophila

were flash frozen in liquid nitrogen, and heads were collected using a

211

microsieve with a 230 nm filter to separate bodies from heads and 170 nm filter to separate heads from

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appendages. Total RNA was extracted from isolated fly heads using the Zymo Direct-Zol RNA miniprep

213

kit (Zymo Research, Irvine, CA). Extracted RNA was quantified on a Nanodrop 2000 (Thermo Fisher

214

Scientific), and equal quantities of each sample were subjected to cDNA synthesis using the High-

215

Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA). qPCR was performed

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on a T100 Thermal Cycler with a CFX384 Real-Time System (Bio-Rad Laboratories, Hercules, CA)

217

using 10 ng of input RNA per replicate and TB Green Premix Ex Taq II (Takara Bio, Kusatsu, Japan).

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Sequences of all qPCR primers used in this study are listed in Table 3.

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Experimental design and statistical analyses

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All quantified data were organized and analyzed in Prism 9 software (Graphpad, San Diego,

222

CA). Power analyses to determine appropriate sample sizes for each experiment were calculated using

223

a Sample Size calculator available at ClinCalc.com (

ɑ = 0.05, β = 0.2). All quantifications are graphed

224

as mean ± standard error of the mean (s.e.m). A single glomerulus or antennal lobe represented one

225

biological replicate. Statistical comparisons were performed using the following tests where appropriate:

226

unpaired, two-tailed

-test when comparing two samples and ANOVA followed by

post hoc

multiple

227

comparison tests

when comparing ≥3 samples. Detailed statistical information for each experiment,

228

including sample sizes (

), means, and test results are summarized in Table 4.

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RESULTS

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Expression of mHTT exon 1 fragments in neurons inhibits phagocytic clearance of axonal debris

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HD is a monogenic neurodegenerative disease caused by expansion of a CAG repeat region in

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exon 1 of the

huntingtin

(

HTT/IT15)

gene beyond a pathogenic threshold of 37 repeats, leading to

233

production of mHTT proteins containing expanded N-terminal polyglutamine (polyQ

≥37) tracts

234

(Scherzinger et al., 1999). mHTT proteins are prone to misfolding and accumulate into insoluble

235

aggregates (Wanker, 2000), whereas wild-

type HTT (wtHTT) proteins containing polyQ≤36 tracts

236

remain soluble unless seeded by a pre-formed HTT aggregate (Preisinger et al., 1999; Chen et al.,

237

2001). To recapitulate these molecular features of HD, we generated transgenic flies that express N-

238

terminal exon 1 fragments of human mHTT (mHTT

ex1

) containing a polyQ91 tract or wtHTT (wtHTT

ex1

)

239

with a polyQ25 tract via the GAL4-UAS (Brand and Perrimon, N., 1993) or QF-QUAS binary expression

240

systems (Potter and Luo, 2011). As we have previously reported (Pearce et al., 2015; Donnelly et al.,

241

2020), fluorescent protein fusions of mHTT

ex1

and wtHTT

ex1

appeared punctate (i.e., aggregated or

242

insoluble) or diffuse (i.e., soluble), respectively, in axon termini of the DA1 type of olfactory receptor

243

neurons (ORNs), which synapse in the DA1 glomerulus of the antennal lobe in the central brain of adult

244

flies (Couto et al., 2005) (Fig. 1A and B).

245

To monitor the capacity of glia to maintain CNS homeostasis in the presence of mHTT

ex1

246

aggregates, we performed a series of experiments that quantified glial responses to acute injury in the

247

adult fly CNS. Surgical removal of the second and third antennal segments initiates Wallerian

248

degeneration of ORN axons, inducing a robust phagocytic glial response that involves upregulation of

249

the scavenger receptor, Draper, and clearance of axonal debris within 7 days (MacDonald et al., 2006).

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To determine if neuronal mHTT

ex1

expression affects the ability of glial cells to efficiently clear axonal

251

debris, we co-expressed mCherry-tagged HTT

ex1

transgenes with membrane-targeted GFP (mCD8-

252

GFP) in DA1 ORNs and measured GFP and mCherry fluorescence intensities following antennal nerve

253

axotomy (Fig. 1A-D). Interestingly, mHTT

ex1

expression was associated with reduced steady-state

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mCD8-GFP levels in DA1 ORN axons in 2 and 4 week-old flies (Fig. 1E), likely due to neurotoxicity

255

caused by accumulation of mHTT

ex1

aggregates over time. Quantification of DA1 ORN axons remaining

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after antennal nerve injury revealed that clearance of axonal debris was reduced in flies co-expressing

257

neuronal mHTT

ex1

compared with controls expressing wtHTT

ex1

(Fig. 1C). This effect could be observed

258

as early as 1 day post-injury, suggesting that mHTT

ex1

causes defects in both clearance and

259

engulfment of axonal debris. Delayed clearance of axonal debris was exacerbated in older (14 and 28

260

day-old) mHTT

ex1

-expressing flies (Fig. 1C), possibly related to a decline in Draper activity during

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normal aging (Purice et al., 2016) and/or enhanced glial dysfunction compounded by mHTT

ex1

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aggregate accumulation. Quantification of mCherry fluorescence indicated that clearance of axonal

263

mHTT

ex1

was also slowed compared to wtHTT

ex1

(Fig. 1D), suggesting that glia are deficient in

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degrading both axonal debris and neuronal mHTT

ex1

aggregates.

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We also tested whether formation of mHTT

ex1

aggregates in glial cells impacts ORN axonal

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debris clearance following acute injury. Restricting expression of mHTT

ex1

to glia resulted in

267

appearance of heterogeneously-sized mHTT

ex1

aggregates throughout the brain (Fig. 2A). Glial

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mHTT

ex1

aggregates also slowed injury-induced clearance of mCD8-GFP-labeled axons compared with

269

wtHTT

ex1

-expressing controls (Fig. 2B-D), though glial mHTT

ex1

expression did not affect DA1 ORN

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axon abundance in 1 day-old flies (Fig. 2E). Together, these findings indicate that mHTT

ex1

aggregates

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originating in either neurons or glia slow efficient clearance of injured ORN axons by phagocytic glia.

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Neuronal mHTT aggregates impair nascent phagosome formation

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Phagocytosis occurs via 4 major steps: (1) extension of phagocyte membranes toward

275

extracellular “find me” cues, (2) recognition of “eat me” signals by scavenger receptors, (3) cytoskeletal

276

and plasma membrane reorganization to surround and internalize extracellular material, and (4)

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maturation of nascent phagosomes through sequential endomembrane fusions that culminate at the

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lysosome (Vieira et al., 2002). We have previously reported that the conserved scavenger receptor,

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Draper/Ced-1/MEGF10 (MacDonald et al., 2006; Evans et al., 2015; Iram et al., 2016), regulates

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engulfment, clearance, and intercellular spreading of mHTT

ex1

aggregates originating in ORN axons

281

(Pearce et al., 2015; Donnelly et al., 2020). To determine whether delayed clearance of injured axons

282

containing mHTT

ex1

aggregates (Fig. 1C and 2D) could result from defective Draper-dependent

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engulfment, we co-expressed V5-tagged mHTT

ex1

or wtHTT

ex1

with the ratiometric membrane-

284

associated pH sensor (MApHS), consisting of the transmembrane domain of CD4 flanked by N-terminal

285

ecliptic pHluorin and C-terminal tdTomato (Han et al., 2014), in the VA1lm type of ORNs. Ecliptic

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pHluorin is brightest at pH 7.5 and dims as pH drops, quenching at pH <6.0 (Miesenböck et al., 1998),

287

whereas tdTomato fluorescence is pH resistant. Thus, internalization of MApHS-labeled ORN axonal

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debris into a rapidly-acidified nascent phagosome can be monitored by calculating pHluorin:tdTomato

289

fluorescence intensity ratios (Han et al., 2014). Indeed, pHluorin:tdTomato ratios in VA1lm axons were

290

decreased at 14 and 25 hours post-axotomy (Fig. 3A-C), and this effect was lost when

draper

was

291

deleted from glia using the tissue-specific CRISPR/Cas9-TriM method (Fig. 3C and E-G) (Poe et al.,

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2019), demonstrating that this construct accurately reports nascent phagosome acidification following

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engulfment. Notably, the decrease in pHluorin:tdTomato ratio following injury was less pronounced in

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VA1lm ORN axons co-expressing mHTT

ex1

compared with wtHTT

ex1

-expressing controls (Fig. 3A-C),

295

suggesting that mHTT

ex1

aggregates impair nascent phagosome formation following engulfment.

296

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Neuronal mHTT accumulates in low pH intracellular compartments

298

mHTT

ex1

expression was associated with a slight but significant decrease in steady-state

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pHluorin:tdTomato ratios in MApHS-labeled axons compared with wtHTT

ex1

-expressing controls (Fig.

300

3D), suggesting that even in the absence of acute injury, mHTT

ex1

signals for ORN axon engulfment. To

301

test this, we generated transgenic flies that express mHTT

ex1

or wtHTT

ex1

fused to N-terminal pHluorin

302

and C-terminal tdTomato fluorescent proteins, herein referred to as mHTT-associated pH sensor

303

(mHApHS) or wtHTT-associated pH sensor (wtHApHS). Accumulation of HTT

ex1

protein in low pH

304

cellular compartments would cause a decrease in the ratio of pHluorin:tdTomato fluorescence for these

305

constructs. wtHApHS and mHApHS transgenes were expressed in either Or83b+ ORNs, which

306

encompass 70-80% of all adult ORNs (Fig. 4A-B) (Larsson et al., 2004), or mushroom body neurons

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(MBNs; Fig. 4D-E), which are downstream in the fly olfactory circuit and innervate the learning and

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memory center of the fly CNS (McGuire et al., 2001). In both ORN axons and MBN soma,

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pHluorin:tdTomato ratios associated with mHApHS were significantly decreased compared with

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wtHApHS controls (Fig. 4C and F), suggesting that mHTT

ex1

proteins accumulate in acidified cellular

311

compartments. To discriminate mHTT

ex1

proteins engulfed by glia from mHTT

ex1

internalized into

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neuronal autophagolysosomes, we measured VA1lm ORN axonal mHApHS-associated fluorescence in

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animals with glial

draper

loss-of-function (Fig. 4G-H). Glial

draper

knockout increased

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pHluorin:tdTomato ratios for axonal mHApHS (Fig. 4I), suggesting that at least some portion of

315

mHTT

ex1

aggregates accumulate in acidic portions of the glial phagolysosomal system.

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Neuronal mHTT impairs injury-responsive gene upregulation

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Glia alter their transcriptional profile to elicit cellular responses to insult or injury in the brain. For

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example, acute CNS injury in

Drosophila

increases transcription of many genes involved in

320

phagocytosis and innate immunity, including the cell surface receptors, Draper and Toll-6, and

321

components of their downstream signaling pathways (Fig. 5A) (Purice et al., 2017; Byrns et al., 2021;

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Alphen et al., 2022). To test whether the reduced ability of glia to clear mHTT

ex1

-containing axonal

323

debris correlates with reduced injury responsiveness at a transcriptional level, we used qPCR and

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GFP-tagged reporters to quantify changes in gene expression of key components of these phagocytic

325

and innate immunity pathways following mHTT

ex1

accumulation in neurons. mHTT

ex1

expression in

326

uninjured Or83b+ ORNs increased relative expression of

toll-6,

relish

, and

drpr-I

transcripts between

327

1.2- and 1.5-fold (Fig. 5B) and levels of GFP-tagged Toll-6 and Jra proteins in the CNS (Fig. 5C-H),

328

suggesting that neuronal mHTT

ex1

aggregates activate a mild injury response in the brain. Jra-GFP

329

expression increased throughout the CNS and in Repo+ nuclei (Fig. 5F-H), suggesting that mHTT

ex1

330

induced upregulation of this subunit of the AP-1 transcription factor can be at least partially attributed to

331

glia. To test whether mHTT

ex1

impairs the ability of glia to respond to acute neural injury, we monitored

332

gene expression changes after bilateral antennal and maxillary palp nerve ablation. Similar to previous

333

reports (Purice et al., 2017), expression of

toll-6, dorsal, relish, drpr-I

mmp1,

and

ets21c

genes was

334

significantly increased 3 hrs after injury to ORN axons expressing either mCD8-GFP or wtHTT

ex1

(Fig.

335

5B). However, injury-induced upregulation of each of these genes was significantly reduced in animals

336

expressing mHTT

ex1

in Or83b+ ORNs (Fig. 5B), suggesting that mHTT

ex1

aggregation attenuates glial

337

JNeurosci Accepted Manuscript

transcriptional responses to injury. We further analyzed the impact of mHTT

ex1

on downstream immune

338

responses in the brain by measuring induction of antimicrobial peptide (AMP) genes, well-established

339

transcriptional targets of activated Relish/NF



B following Toll-6 or immune-deficiency pathway

340

activation (Swanson et al., 2020b, 2020a; Alphen et al., 2022). Levels of five AMP genes, including

341

drosomycin

attacinA

attacinD

diptericinA

, and

metchnikowin

, were significantly increased 3 hours

342

after ORN axotomy (Fig. 6A-E), similar to previous reports (Katzenberger et al., 2013; Swanson et al.,

343

2020b; Marischuk et al., 2021). Interestingly, mHTT

ex1

expression in ORNs alone was sufficient to

344

induce upregulation of

drosomycin

and

attacinD

(Fig. 6A & C), albeit to a lesser extent than following

345

acute injury. Further, injury-induced upregulation of

drosomycin

and

attacinA

was significantly reduced

346

by mHTT

ex1

expression in ORNs, suggesting that the activity of Relish-dependent signaling is altered by

347

accumulation of mHTT

ex1

aggregates. Together, these data indicate that neuronal mHTT

ex1

aggregates

348

trigger a mild immune response in the brain, but also inhibit the ability of glia to mount robust

349

transcriptional responses to neural injury.

350

Our findings are in agreement with previously published work demonstrating that expression of

351

mHTT

ex1

and Aβ in neurons activates Draper-dependent phagocytosis, likely in an effort to reduce

352

levels of these pathogenic proteins in the brain (Pearce et al., 2015; Ray et al., 2017). We further

353

investigated this by immunostaining adult fly brains expressing mHTT

ex1

in ORNs to monitor expression

354

levels and localization of endogenous Draper protein. In 1 week-old adult flies expressing neuronal

355

mHTT

ex1

, Draper immunolabeling increased ~1.2-fold in the vicinity of ORN axons compared with age-

356

matched controls expressing wtHTT

ex1

(Fig. 7A-C). Closer analysis revealed that in some cases,

357

Draper immunofluorescence was directly adjacent to or surrounding mHTT

ex1

-mCherry fluorescence

358

(Fig. 7D). To further examine these interactions, we used image segmentation and three-dimensional

359

reconstruction of confocal stacks to represent mHTT

ex1

-mCherry+ aggregates and Draper+ glial

360

membranes as individual

“surfaces” (Fig. 7E-F), as previously described (Donnelly et al., 2020). Close

361

physical association of mHTT

ex1

with Draper and other glial proteins was defined as surfaces located

362

≤0.2 μm from a mHTT

ex1

object. Interestingly, whereas almost no Draper signal was detected near

363

wtHTT

ex1

(Fig. 7E), ~14% of mHTT

ex1

aggregates were closely associated with Draper surfaces (Fig.

364

JNeurosci Accepted Manuscript

7F1-3). These findings are consistent with Draper+ glial membranes being recruited to neuronal

365

mHTT

ex1

aggregates to facilitate engulfment.

366

Synaptic neuropil in the

Drosophila

brain is primarily inhabited by two glial subtypes,

367

ensheathing glia and astrocytic glia (Doherty et al., 2009). In adult flies, ensheathing glia

368

compartmentalize synaptic regions and respond to CNS injury by upregulating Draper and clearing

369

debris via phagocytosis (Doherty et al., 2009). Consistent with our previous findings in all repo+ glia

370

(Pearce et al., 2015), mz0709+ ensheathing glia were vulnerable to prion-like conversion of glial

371

wtHTT

ex1

proteins by mHTT

ex1

aggregates generated in DA1 ORN axons (Fig. 8A, C-D). Seeded

372

wtHTT

ex1

aggregates were defined as wtHTT

ex1

signal that colocalized with mHTT

ex1

objects identified

373

by image segmentation of confocal stacks (Donnelly et al., 2020). Conversely, seeding of wtHTT

ex1

374

expressed in alrm+ astrocytic glia, which lack detectable Draper expression in the adult fly brain

375

(Doherty et al., 2009), was not observed (Fig. 8B-D). Interestingly, ensheathing glial-specific RNAi

376

knockdown of Toll-6, Relish, and NijA increased mHTT

ex1

aggregate numbers in DA1 ORN axons,

377

similar to the effects of Draper-I knockdown (Fig. 9A-B). Further, adult-specific, pan-glial knockdown of

378

Ets21c, which was found to be required for normal development, also increased numbers of mHTT

ex1

379

aggregates in DA1 ORN axons (Fig. 9C-D). Mean mHTT

ex1

aggregate volume was not affected by

380

ensheathing glial knockdown of these genes except for a ~20% increase following NijA depletion (Fig.

381

9E), suggesting that aggregate or vesicle size was unaffected by these genetic manipulations. Thus,

382

several glial genes with established roles in phagocytic and innate immune signaling regulate basal

383

turnover of mHTT

ex1

aggregates in ORN axons.

384

385

Neuronal mHTT aggregates are associated with defects in multiple endolysosomal compartments

386

Our findings thus far indicate that neuronal mHTT

ex1

aggregates elicit mild injury responses in

387

the brain and reduce the ability of phagocytic glia to respond transcriptionally and functionally to acute

388

nerve injury. We have previously postulated that prion-like spreading of mHTT

ex1

in the fly CNS could

389

be facilitated by escape of engulfed aggregates from the endolysosomal compartment of phagocytic

390

glia (Pearce et al., 2015; Donnelly et al., 2020). Therefore, we sought to determine whether neuronal

391

JNeurosci Accepted Manuscript

mHTT

ex1

aggregates are associated with defects in endolysosomal processing in non-injured brains.

392

We first measured effects of neuronal mHTT

ex1

expression on the quantity, size, and function of

393

lysosomes using dyes that label active cathepsins and low pH cellular compartments. Expression of

394

mHTT

ex1

in Or83b+ ORNs increased numbers of lysosomes labeled by the active cathepsin dye, Magic

395

Red (MR) (Fig. 10A-C), and the low pH sensor, LysoTracker Red (LTR) (Fig. 10D-F), compared with

396

control brains expressing wtHTT

ex1

. High resolution analysis of confocal stacks and filtering for

397

segmented MR+ and LTR+ surfaces within

0.2 μm of a mHTT

ex1

object revealed close association of

398

MR+ and LTR+ signals with mHTT

ex1

aggregates (Fig. 10C and F-H).

399

We next employed

Drosophila

genetic tools to assess the impact of neuronal mHTT

ex1

400

specifically on glial lysosomes by driving expression of lysosomal-associated membrane protein 1

401

(LAMP1) tagged at its cytosolic C-terminus with GFP (LAMP1-GFP) in all glia. Neuronal mHTT

ex1

402

expression increased the overall number of glial LAMP1-GFP+ vesicles and the number of LAMP1-

403

GFP+ vesicles in close proximity to HTT

ex1

signal (Fig. 11A-C and G-H). We typically observed only

404

partial overlap of LAMP1-GFP signal with mHTT

ex1

aggregate surfaces identified by this method,

405

possibly due to incomplete labeling or rupture of lysosomal membranes as a result of aggregate size or

406

structural features. Interestingly, this subpopulation of LAMP1-GFP+ lysosomes closely associated with

407

mHTT

ex1

were enlarged compared with all lysosomes (Fig. 11I). To examine whether neuronal mHTT

ex1

408

aggregates affect lysosome integrity, we expressed a transgene encoding LAMP1 fused at its N-

409

terminus to GFP in glia. This construct integrates into the lysosomal membrane such that GFP is

410

exposed to the lumen, and loss of GFP signal can thus be used to monitor LAMP1+ lysosome

411

degradative activity (Pulipparacharuvil et al., 2005). Interestingly, the quantity and mean volume of

412

GFP-LAMP1+ vesicles were significantly increased in brains expressing mHTT

ex1

in Or83b+ ORNs

413

(Fig. 11D-G), suggesting lysosomal enlargement and dysfunction due to mHTT

ex1

aggregates. GFP-

414

LAMP1+ surfaces were also more associated with mHTT

ex1

aggregates compared to wtHTT

ex1

controls

415

(Fig. 11F and I).

416

To directly test whether glial endolysosomes experience increased membrane damage due to

417

neuronal mHTT

ex1

expression, we generated transgenic flies expressing mCherry-tagged Galectin-3 or

418

JNeurosci Accepted Manuscript

Galectin-8, lectins that translocate from the cytoplasm to the lumen of ruptured lysosomes and

419

endosomes, respectively (Aits et al., 2015; Daussy and Wodrich, 2020; Jia et al., 2020). Neuronal

420

mHTT

ex1

expression increased overall numbers of glial Galectin-3/8+ surfaces (Fig. 12A-E), Galectin-

421

3/8+ surfaces closely associated with mHTT

ex1

aggregates (Fig. 12F), and mean volume of mHTT

ex1

422

associated Galectin-3/8+ vesicles (Fig. 12G). Together, these data suggest that neuronal mHTT

ex1

423

aggregates induce non cell-autonomous accumulation, enlargement, and membrane damage of

424

endolysosome vesicles in glial cells.

425

“Seeding-competent” mHTT aggregates are defined by their ability to nucleate or “seed” the

426

aggregation of normally-soluble wtHTT proteins, a defining characteristic of infectious prion and other

427

prion-like proteins (Jucker and Walker, 2018; Donnelly et al., 2022). Many studies have pointed to a

428

role for defective clearance by endolysosomal pathways in promoting the propagation of pathogenic

429

aggregates (Freeman et al., 2013; Jiang et al., 2017; Chen et al., 2019; Jiang and Bhaskar, 2020;

430

Polanco and Götz, 2022). To test whether altered glial lysosome function affects seeding competency

431

of mHTT

ex1

, we used RNAi to individually knockdown proteins with known roles in lysosome

432

degradation in glia and examined the ability of neuronal mCherry-tagged mHTT

ex1

to seed aggregation

433

of glial, GFP-tagged wtHTT

ex1

. Depletion of two subunits of the vacuolar ATPase (V-ATPase), Vha68-3

434

(Portela et al., 2018) and rabconnectin-3A (Yan et al., 2009), and Spinster, a late-endosomal and

435

lysosomal efflux permease (Rong et al., 2011), increased numbers of glial wtHTT

ex1

aggregates

436

detected as GFP+ surfaces that colocalized with mHTT

ex1

aggregates (Fig. 13A-C) (Donnelly et al.,

437

2020). Knockdown of Vha16-1, a V-ATPase subunit that regulates endolysosome membrane fusion

438

(Finbow et al., 1994; Dunlop et al., 1995), did not affect wtHTT

ex1

seeding; however, it did cause

439

accumulation of mHTT

ex1

aggregates in DA1 ORN axons (Fig. 13A and B). Together, these findings

440

suggest that disruption of normal glial lysosome acidification and/or degradative capacity promotes

441

formation of seeding-competent mHTT

ex1

aggregates.

442

443

The GTPase Rab10 mediates prion-like transmission of mHTT aggregates

444

JNeurosci Accepted Manuscript

Lysosome dysfunction could occur secondary to upstream defects in endo/phagosome

445

maturation. Our prior work supports a model in which a portion of mHTT

ex1

aggregates engulfed by glia

446

evade degradation during phagosome maturation and/or phagolysosome formation (Pearce et al.,

447

2015; Donnelly et al., 2020). To test this model, we used forward genetic screening to interrogate roles

448

for glial Rab GTPases in prion-like conversion of cytoplasmic wtHTT

ex1

proteins by engulfed neuronal

449

mHTT

ex1

aggregates. The

Drosophila

genome encodes 31 Rab and Rab-like proteins, all of which have

450

mammalian orthologs, and most of these GTPases are implicated in vesicle and target membrane

451

fusion in cells (Zhang et al., 2007). To determine whether any

Drosophila

Rabs mediate escape of

452

phagocytosed mHTT

ex1

aggregates and seeding of wtHTT

ex1

in the glial cytoplasm, we individually

453

knocked down each

Rab in repo+ glia using RNAi. Glial-restricted silencing of 23 of the 31 Rabs

454

produced viable adults, and these flies were used to monitor effects of Rab knockdown on mHTT

ex1

455

induced aggregation of wtHTT

ex1

in glia. Only two Rab RNAi lines,

Rab10

RNAi

and

Rab23

RNAi#2

456

significantly altered numbers of induced wtHTT

ex1

aggregates (Fig. 14A-C). Of note, 3 additional Rab23

457

RNAi lines had no significant effects on numbers of wtHTT

ex1

aggregates, suggesting that

Rab23

RNAi#2

458

may cause off-target effects. Strikingly, Rab10 depletion reduced numbers of seeded wtHTT

ex1

459

aggregates, phenocopying effects of Draper knockdown (Fig. 14A and C) and suggesting that Draper

460

and Rab10 function in the same pathway. To test whether this effect of Rab10 loss-of-function was

461

mediated via a reduction in Draper expression, we measured endogenous Draper immunofluorescence

462

rab10

null flies. Draper protein levels were ~18% lower in

rab10

-/-

animals compared to wild-type

463

controls (Fig. 14D). This reduction is unlikely to fully account for decreased seeding of wtHTT

ex1

464

following Rab10 knockdown, as numbers of wtHTT

ex1

aggregates are similar between wild-type and

465

draper

heterozygotes (Fig. 14G) (Pearce et al., 2015). To further explore this, we attempted to restore

466

Draper function via overexpression of Draper-I, which rescues loss of Draper function in aged flies

467

(Purice et al., 2016). However, transgenic expression of Draper-I in glia failed to rescue the effects of

468

Rab10 knockdown on wtHTT

ex1

aggregate seeding (Fig. 14E). These findings suggest that Rab10 acts

469

downstream of Draper rather than by regulating Draper activity. We further tested for interactions

470

between

drpr

and

rab10

using loss-of-function alleles to examine effects on mHTT

ex1

aggregate

471

JNeurosci Accepted Manuscript

transmission from presynaptic DA1 ORNs to postsynaptic projection neurons (PNs), a process

472

previously reported to require transport though Draper+ glia (Donnelly et al., 2020). Because reduced

473

survival of

rab10

null flies (Kohrs et al., 2021) was exacerbated by transgenic HTT

ex1

expression, we

474

tested for genetic interaction between

drpr

and

rab10

in heterozygous and trans-heterozygous animals.

475

While we detected no change in mHTT

ex1

aggregate numbers (Fig. 14F), significantly fewer wtHTT

ex1

476

aggregates formed in PNs of

rab10

+/-

drpr

+/-

transheterozygotes than in individual heterozygotes (Fig.

477

14G). This same effect was not observed in

rab14

+/-

drpr

+/-

transheterozygous animals (Fig. 14G),

478

indicating that the genetic interaction is specific to

drpr

and

rab10

. Thus, Draper and Rab10 appear to

479

function in the same phagocytic pathway that regulates prion-like transmission of phagocytosed

480

mHTT

ex1

aggregates in the fly brain.

481

482

Neuronal mHTT aggregates alter numbers of early and late glial phagosomes

483

Our forward genetic screen indicates that at least one glial Rab GTPase, Rab10, regulates the

484

seeding capacity of neuronal mHTT

ex1

aggregates. Interestingly, Rab10 has been reported to regulate

485

phagosome maturation in mammalian cells (Cardoso et al., 2010; Seto et al., 2011; Lee et al., 2020;

486

Wang et al., 2023), and Rab10 expression and activity are altered in neurodegenerative diseases,

487

including AD and PD (Eguchi et al., 2018; Tavana et al., 2018; Yan et al., 2018). Because little is known

488

about Rab10’s role in glia, we sought to characterize this GTPase alongside two additional Rabs with

489

well-established roles in endocytosis, Rab5 and Rab7, markers of early and late endo/phagosomes,

490

respectively (Hutagalung and Novick, 2011). Interestingly, we found that

rab10

rab5

, and

rab7

were

491

upregulated between ~1.4 and 2.4-fold following acute injury to ORN axons, and mHTT

ex1

expression in

492

Or83b+ ORNs alone caused upregulation of

rab10

and

rab7

genes by ~1.2-fold (Fig. 15A).

493

Interestingly, injury-induced upregulation of

rab5

and

rab7

was inhibited by ~50% and ~100%,

494

respectively, in flies expressing mHTT

ex1

compared to controls (Fig. 15A). Altogether, these results

495

identify

rab5

rab7

, and

rab10

as novel injury-response genes in the fly CNS and suggest that neuronal

496

mHTT

ex1

aggregates impair injury-induced responses of

rab5

and

rab7

497

JNeurosci Accepted Manuscript

We next examined effects of neuronal mHTT

ex1

expression on the localization of Rab10, Rab5,

498

and Rab7 proteins endogenously-tagged with YFP-myc at their N-termini, herein referred to as

499

YRab10, YRab5, and YRab7 (Dunst et al., 2015). YRab+ vesicles were identified in confocal stacks as

500

segmented YFP+ surfaces with a mean diameter of 0.3-8

μm (Fig. 15B-G), consistent with vesicle sizes

501

reported in other fly tissues (Prince et al., 2019). Expression of mHTT

ex1

in Or83b+ ORN axons caused

502

an overall increase in numbers of YRab10+ and YRab7+ vesicles, but a decrease in the number of

503

YRab5+ vesicles compared with wtHTT

ex1

controls (Fig. 15H). Further, each of these YRab+ vesicles

504

subpopulations were closely associated with mHTT

ex1

aggregates more frequently than with wtHTT

ex1

505

(Fig. 15I), suggesting that mHTT

ex1

protein interacts with each of these intracellular vesicle

506

subpopulations in the brain.

507

To assess effects of neuronal mHTT

ex1

aggregates specifically on glial Rab+ compartments, we

508

expressed YFP-tagged Rab5, 7, and 10 transgenes in all glia. Similar to our findings with endogenous

509

YRabs, expression of mHTT

ex1

in Or83b+ ORN axons was associated with increased numbers of glial

510

YFP-Rab10+ and -Rab7+ vesicles (Fig. 16A-B, E-F, and G); however, we observed a significant

511

decrease in glial YFP-Rab5+ vesicle abundance (Fig. 16C-D and G). YFP-Rab10+, -5+, and -7+

512

vesicles increased their association with axonal mHTT

ex1

aggregates compared with wtHTT

ex1

(Fig.

513

16H), in many cases with closely associated YFP-Rab+ signal partially surrounding a mHTT

ex1

514

aggregate (Fig. 17). Of note, only a small fraction of YFP-Rab+ vesicles were identified as associated

515

with mHTT

ex1

aggregates, possibly due to transient interactions with vesicle compartments as

516

aggregates transit the glial phagolysosomal system, heterogeneous labeling of phagosomes by these

517

markers in intact brain tissue, or because our selection filter excluded YFP-Rab+ surfaces located >0.2

518

μm away from an aggregate. The mean volume of YFP-Rab7+ vesicles that interacted with mHTT

ex1

519

aggregates was significantly increased compared with wtHTT

ex1

controls (Fig. 16I), suggesting that

520

mHTT

ex1

leads to enlargement of Rab7+ late phagolysosomes. Together, these data suggest that

521

accumulation of phagocytosed mHTT

ex1

aggregates in Rab7+ or Rab10+ late phagosomes and

522

decreased association with early Rab5+ phagosomes could be a key mechanism underlying protein

523

aggregate-induced toxicity and spreading in HD.

524

JNeurosci Accepted Manuscript

DISCUSSION

525

Toxic amyloid aggregates have been a primary target in neurodegenerative disease drug

526

development for decades, with some recent promise using immunotherapy to reduce aggregate loads

527

in the brain (Karran and De Strooper, 2022). Microglia and astrocytes have also emerged as attractive

528

therapeutic targets in efforts to boost neuroprotective glial functions or reduce neuroinflammation.

529

However, approaches that target glial cells must effectively strike a balance between amplifying

530

beneficial and reducing harmful effects of these cells in the brain. Here, we tested for interactions

531

between phagocytic glia and pathogenic protein aggregates in a

Drosophila

model of HD. We report

532

that aggregates formed by mHTT

ex1

protein fragments impair glial transcriptional and functional

533

responses to CNS injury, induce upregulation of stress response and innate immunity genes, and alter

534

numbers of endolysosomal vesicles detected in uninjured brains. A targeted forward genetic screen

535

revealed that Rab10, a GTPase previously reported to regulate phagosome maturation, mediates prion-

536

like conversion of cytoplasmic wtHTT

ex1

proteins by phagocytosed mHTT

ex1

aggregates. Together,

537

these findings suggest that neuronal mHTT

ex1

aggregates compromise intracellular membrane integrity

538

as they transit endolysosomal systems, generating toxic, seeding-competent aggregates that propagate

539

disease phenotypes.

540

Glia respond to neural injury by altering their transcriptional, morphological, and metabolic

541

profiles to promote neuronal survival and clear debris from the brain; however, failure of glia to return to

542

a resting state elicits harmful neuroinflammatory consequences (Liddelow et al., 2020). We have

543

previously reported that activated phagocytic glia can have both beneficial (i.e., elimination of toxic

544

aggregates) and harmful (i.e., as vectors for aggregate spread) effects in the brain (Pearce et al., 2015;

545

Donnelly et al., 2020). We report here that key glial injury-responsive pathways, i.e., Draper-mediated

546

phagocytosis and Toll-6-mediated innate immune signaling, are induced in the presence mHTT

ex1

547

aggregation in the adult fly brain. These findings are in line with studies from other labs demonstrating

548

that

Drosophila

Toll-6 and mammalian Toll-Like Receptor signaling pathways are upregulated in

549

response to dying neurons during development (McLaughlin et al., 2019) and in patient and mammalian

550

models of neurodegenerative disease (Casula et al., 2011; Miron et al., 2018; Kouli et al., 2020).

551

JNeurosci Accepted Manuscript

Interestingly, increased microglial NF-



B signaling mediates tau spread and toxicity in mice, further

552

linking innate immunity to prion-like mechanisms of disease progression (Wang et al., 2022). Thus,

553

activation of glial immune pathways may contribute to feed-forward mechanisms involving aggregate

554

formation, pathology propagation, and neuroinflammatory signaling.

555

Genome-wide association studies have revealed numerous genes associated with increased

556

risk of AD and other neurodegenerative diseases, and many of these risk variants are enriched in

557

pathways that control key glial cell functions. For example, rare risk-associated variants of the

558

microglial

TREM2

gene alter amyloid aggregate accumulation and seeding in cells and animal models

559

(Leyns et al., 2019; Parhizkar et al., 2019; Jain et al., 2023). A number of additional genes involved in

560

endolysosomal processing are associated with increased risk of AD, PD, FTD, and/or ALS, such as the

561

phagocytic receptor

CD33

, endosomal genes

BIN1

and

RIN3

, and

GRN

, which encodes the lysosomal

562

progranulin protein (Podleśny-Drabiniok et al., 2020; Welikovitch et al., 2023). Although

563

Draper/MEGF10 variants are not known risk factors in neurodegenerative disease, MEGF10 is highly

564

expressed in phagocytic astrocytes (Chung et al., 2013), mediates A



aggregate engulfment (Singh et

565

al., 2010; Fujita et al., 2020), and acts as a receptor for C1q, a mediator of early synapse loss in AD

566

mouse models (Hong et al., 2016; Iram et al., 2016). Our finding that upregulation of

draper

and other

567

phagocytic genes is inhibited by mHTT

ex1

expression suggests that glial responsiveness and

568

phagocytic capacity is attenuated in the presence of protein aggregate pathology in neurons. Genetic or

569

environmental risk factors that impact glial health could accelerate these defects and exacerbate

570

aggregate-induced neurotoxicity in the brain.

571

Lysosomes are essential for cell survival, not only to clear damaged or toxic materials from

572

cells, but also as intracellular centers for macromolecule recycling, energy metabolism, and cell-cell

573

communication. Lysosomal abnormalities, including vesicle enlargement, deacidification, and

574

membrane leakiness, have been reported in patient brains and animal and cell models of multiple

575

neurodegenerative diseases, suggesting that these defects play a central role in disease progression

576

(Bonam et al., 2019; Polanco and Götz, 2022; Udayar et al., 2022). We report here that Rab7+,

577

Rab10+, and Lamp1+ phagolysosomes accumulate in glia as a result of mHTT

ex1

expression in

578

JNeurosci Accepted Manuscript

neurons and that lysosome dysfunction and membrane permeabilization increase in the presence of

579

mHTT

ex1

aggregates. We also observed that neuronal aggregates were associated with decreased

580

nascent phagosome formation and reduced numbers of early (Rab5+) phagosomes in glia. These

581

findings are consistent with a “traffic jam” model (Small et al., 2017) in which mHTT

ex1

aggregates

582

accumulate over time in glial endolysosomal compartments, preventing proper flow of materials into

583

(i.e., engulfment) and out of (i.e., degradation) the pathway (Fig. 18). We postulate that persistence of

584

aggregates in faulty endolysosomes promotes formation and/or release of degradation-resistant

585

aggregates with enhanced toxicity and seeding capacity. This hypothesis is in agreement with recent

586

studies in tauopathy models showing that A



aggregation occurs secondary to lysosome deacidification

587

and membrane permeabilization (Lee et al., 2022), and hypophagocytic glia contribute to tau aggregate

588

propagation (Hopp et al., 2018; Brelstaff et al., 2021). Release of seeding-competent aggregates from

589

dysfunctional lysosomes could occur via active exocytosis, perhaps in an effort to alleviate cell toxicity,

590

or passively due to vesicle rupture (Flavin et al., 2017; Falcon et al., 2018; Yuste-Checa et al., 2021).

591

Among the growing list of genes linked to neurodegenerative disease pathogenesis are Rab

592

GTPases and genes that modify Rab functions (Kiral et al., 2018). Rab proteins are essential for vesicle

593

sorting and trafficking in all cells and use GTP hydrolysis to differentially associate with and organize

594

intracellular membranes (Homma et al., 2021). In a forward genetic screen aimed at identifying Rabs

595

that regulate phagocytic processing of neuronal mHTT

ex1

aggregates, we uncovered glial Rab10 as a

596

modifier of prion-like spreading of mHTT

ex1

from neurons to glia. Interestingly, several Rab proteins

597

have been previously reported to alter secretion or cell-to-cell propagation of tau or



-synuclein

598

aggregates (Rodriguez et al., 2017; Bae et al., 2018; Ugbode et al., 2019; Rodrigues et al., 2022),

599

suggesting that Rab-dependent endomembrane fusion could be exploited by prion-like aggregates. Our

600

data suggest that Rab10 acts downstream of Draper to enable engulfed mHTT

ex1

aggregates to evade

601

lysosomal degradation, perhaps escaping to the cytoplasm during Rab10-dependent vesicle fusion.

602

While relatively understudied compared with other Rabs, Rab10 has been reported to localize to and

603

regulate maturing endo/phagosomes and lysosomes in human macrophages and microglia (Cardoso et

604

al., 2010; Lee et al., 2020). Interestingly, Rab10 has already been implicated in multiple

605

JNeurosci Accepted Manuscript

neurodegenerative diseases: (1)

RAB10

gene expression is altered in human AD brains (Ridge et al.,

606

2017), (2) rare

RAB10

polymorphisms are linked to protection against AD (Ridge et al., 2017; Tavana

607

et al., 2018), (3) Rab10 depletion is associated with reduced A



levels (Udayar et al., 2013; Ridge et

608

al., 2017), and (4) Rab10 is a substrate of the PD risk factor gene leucine-rich repeat kinase 2 (LRRK2)

609

(Steger et al., 2016; Seol et al., 2019). Mutant LRRK2-mediated Rab phosphorylation leads to

610

endocytic defects (Liu et al., 2020; Rivero-Ríos et al., 2020; Streubel-Gallasch et al., 2021), and

611

LRRK2-modified Rab10 localizes to and promotes secretion from stressed lysosomes (Eguchi et al.,

612

2018; Kluss et al., 2022). Interestingly, phosphorylated Rab10 (pRab10) levels are elevated in the CNS

613

of AD and PD patients, and pRab10 has been reported to colocalize with pathological tau (Yan et al.,

614

2018; Tezuka et al., 2022) suggesting that post-translational modification of Rab10 modifies the

615

disease state. Whether Rab10’s phosphorylation status affects its ability to regulate prion-like activity of

616

mHTT

ex1

or other amyloid aggregates warrants further investigation.

617

In conclusion, our findings demonstrate that axonal mHTT

ex1

aggregates activate glial

618

phagocytosis but also impair normal glial responses to acute neural injury. Aggregate engulfment-

619

induced defects in endolysosomal processing, such as Rab-mediated vesicle membrane fusion, could

620

facilitate the formation and spread of prion-like aggregate seeds in the brain. These findings point to

621

central roles for phagocytic glia in regulating pathological aggregate burden in HD and highlight the

622

importance of exploring therapeutic interventions that target non-neuronal cells.

623

JNeurosci Accepted Manuscript

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989

990

JNeurosci Accepted Manuscript

FIGURE LEGENDS

991

Figure 1. mHTT

ex1

expression in ORNs impairs clearance of injured axons

. (

A-B

) Maximum

992

intensity projections of mCD8-GFP-labeled DA1 ORN axons expressing (

) HTT

ex1

Q25- (wtHTT

ex1

) or

993

(

) HTT

ex1

Q91-mCherry (mHTT

ex1

) in 7 day-old uninjured flies (

left

) or 14 day-old flies subjected to

994

bilateral antennal nerve axotomy 7 days earlier (

right

). Scale bars = 5

μm. (

C-D

) Quantification of (

)

995

mCD8-GFP+ and (

) HTT

ex1

-mCherry+ DA1 ORN axons remaining in 7, 14, and 28 day-old uninjured

996

flies or flies at 1, 3, and 5 days post-injury. (

) Quantification of mCD8-GFP+ DA1 ORN axons in 7, 14,

997

and 28 day-old flies expressing HTT

ex1

Q25- or HTT

ex1

Q91-mCherry in DA1 ORNs. All quantified data

998

were normalized to uninjured 1 day-old adults and graphed as mean ± s.e.m.; *

<0.05, **

<0.01,

999

***

<0.001, ****

<0.0001 by unpaired two-tailed

-test.

1000

1001

Figure 2. mHTT

ex1

expression in glia is associated with reduced ORN axon clearance post-injury.

1002

(

A-B

) Maximum intensity projections of antennal lobes from 5-6 day-old flies expressing HTT

ex1

Q25-

1003

(

top

) or HTT

ex1

Q91-V5 (

bottom

) in glia and immunostained with anti-

V5. Scale bars = 10 μm. (

B-C

)

1004

Maximum intensity projections of mCD8-GFP-labeled DA1 ORN axons in 1 day-old flies expressing (

)

1005

HTT

ex1

Q25- or (

) HTT

ex1

Q91-V5 in repo+ glia. Scale bars = 5

μm. (

) Quantification of mCD8-GFP+

1006

DA1 ORN axons in flies expressing HTT

ex1

Q25- or HTT

ex1

Q91-V5 in repo+ glia, either uninjured or at 1

1007

and 3 days post-injury. (

) Quantification of mCD8-GFP+ DA1 ORN axons in 1 day-old flies expressing

1008

HTT

ex1

Q25- or HTT

ex1

Q91-mCherry in repo+ glia. All data were normalized to uninjured 1 day-old adult

1009

flies and graphed as mean ± s.e.m.; ***

<0.001 by unpaired two-tailed

-test.

1010

1011

Figure 3. mHTT

ex1

expression inhibits engulfment of injured ORN axons.

(

A-B

) Maximum intensity

1012

projections of VA1lm ORN axons co-expressing the ratiometric phagocytic indicator, MApHS, and (

)

1013

HTT

ex1

Q25- or (

) HTT

ex1

Q91-V5 from 7 day-old uninjured flies (

left

) and flies 25 hours post-injury

1014

(

right

). Scale bars = 10μm.

(

) pHluorin:tdTomato fluorescence intensity ratios calculated in VA1lm

1015

glomeruli from 7-day-old uninjured flies and flies at 14 or 25 hours post-injury. Data were normalized to

1016

the uninjured condition for each genotype. (

) pHluorin:tdTomato fluorescence intensity ratios in 7 day-

1017

JNeurosci Accepted Manuscript

old flies co-expressing MApHS with HTT

ex1

Q25- or HTT

ex1

Q91-V5 in VA1lm ORNs, normalized to

1018

wtHTT

ex1

controls.

Data are shown as mean ± s.e.m.; **

<0.01, ***

<0.001, ****

<0.0001 by unpaired

1019

two-tailed

-test. (

E-F

) Maximum intensity projections of the central brain from 6-7 day-old flies

1020

expressing (

) repo-Cas9 and (

) repo-Cas9 plus gRNAs targeting

draper

(“Draper KO”). Brains were

1021

immunostained with anti-Draper. Scale b

ars = 10 μm. (

) Quantification of Draper immunofluorescence

1022

in brains from flies shown in (

E-F

), normalized to control.

1023

1024

Figure 4. Neuronal mHTT

ex1

accumulates in acidic cellular compartments. (A-B, D-E)

Maximum

1025

intensity projections of (

A-B

) Or83b+ ORN axons or (

D-E

) OK107+ MBN soma expressing (

A,D

)

1026

HTT

ex1

Q25- or (

B,E

) HTT

ex1

Q91-associated pH sensor (HApHS) from 13-14 day-old flies. Scale bars =

1027

μm. (

C,F

) pHluorin:tdTomato fluorescence intensity ratios of data shown in (

A-B

and

D-E

1028

normalized to wtHTT

ex1

controls. (

G-H

) Maximum intensity projections of VA1lm ORN axons co-

1029

expressing mHApHS and (

) repo-Cas9 or (

) repo-Cas9 and gRNAs targeting

draper

. Scale bars = 5

1030

μm. (

) pHluorin:tdTomato fluorescence intensity ratios calculated in VA1lm glomeruli from 9-10 day-old

1031

flies as shown in (

G-H

). Data were normalized to flies not expressing gRNAs. All quantified data are

1032

shown as mean ± s.e.m.; **

<0.01, ***

<0.001, ****p<0.0001 by unpaired two-tailed

-test.

1033

1034

Figure 5. mHTT

ex1

expression in ORNs upregulates phagocytic and innate immunity genes and

1035

impairs injury-induced transcriptional responses.

(

) Diagrams of Toll-6 (purple) and Draper (blue)

1036

signaling pathways. (

)

qPCR analysis of the indicated genes in 8-11 day-old flies expressing GFP,

1037

HTT

ex1

Q25-, or HTT

ex1

Q91-GFP in Or83b+ ORNs. RNA was isolated from heads of uninjured flies or

1038

flies 3 hours after bilateral antennal and maxillary palp nerve injury. Data are shown as mean ± s.e.m

1039

and normalized to the housekeeping gene

rpl32

. *

<0.05, **

<0.01, ***

<0.001 by one-way ANOVA;

1040

asterisks and hashtags indicate statistical significance comparing -/+ injury or genotypes, respectively.

1041

(

C-D and F-G

) Maximum intensity projections of Or83b+ ORN axons from 14-15 day-old flies

1042

expressing (

C,F

) HTT

ex1

Q25- or (

D,G

) HTT

ex1

Q91-V5 in (

C-D

) Toll-6

MIMICGFP

(Toll-6-GFP) or (

F-G

) Jra-

1043

GFP genetic backgrounds. Brains were immunostained with GFP, V5, and N-Cadherin (

C-D

) or GFP,

1044

JNeurosci Accepted Manuscript

Repo, and N-Cadherin (

F-G

) antibodies. In panel (

), diffuse wtHTT

ex1

signal was adjusted post-

1045

acquisition for increased visibility. Scale bars = 10

μm. (

E,H

) Quantification of (

) Toll-6-GFP or (

) Jra-

1046

GFP expression from flies show in (

C-D

and

F-G

). Data are graphed as mean ± s.e.m.; *

<0.05,

1047

****p<0.0001 by unpaired two-tailed t-test.

1048

1049

Figure 6. mHTT

ex1

expression in ORNs increases expression and impairs injury-induced

1050

upregulation of some AMP genes.

(

A-E

) qPCR analysis of the indicated AMP genes in 8-11 day-old

1051

flies expressing HTT

ex1

Q25- or HTT

ex1

Q91-GFP in Or83b+ ORNs. RNA was isolated from heads of

1052

uninjured flies or flies 3 hours after bilateral antennal and maxillary palp nerve injury. Data are shown

1053

as mean ± s.e.m and normalized to the housekeeping gene

rpl32

. **

<0.01, ***

<0.001 by unpaired

1054

two-tailed t-test; asterisks and hashtags indicate statistical significance comparing -/+ injury or

1055

genotypes, respectively.

1056

1057

Figure 7. A subset of mHTT

ex1

aggregates are closely associated with Draper+ glial membranes

1058

(

A-B

) Maximum intensity projections of Or83b+ ORN axons from 7-8 day-old flies expressing (

)

1059

HTT

ex1

Q25- or (

) HTT

ex1

Q91-mCherry and immunostained for Draper. Scale bars = 10

μm. (

)

1060

Quantification of Draper immunofluorescence from flies show in (

A-B

). Data are normalized to control

1061

and graphed as mean ± s.e.m.; *

<0.05 by unpaired two-tailed

-test; n=12. (

) Single

0.35 μm

1062

confocal slice showing magnified HTT

ex1

Q91-mCherry aggregate and associated Draper signal. Scale

1063

bar = 0.5

μm. (

E-F

) High-magnification c

onfocal stacks of Draper signal within ≤0.2 μm of either (

)

1064

HTT

ex1

Q25- or (

F1-3

) HTT

ex1

Q91-mCherry surfaces. Raw data are shown to the left of segmented

1065

surfaces generated from each fluorescence signal. In (

), diffuse wtHTT

ex1

signal was adjusted post-

1066

acquisition for increased visibility. Scale bars = 1

μm.

1067

1068

Figure 8. Seeded aggregation of wtHTT

ex1

protein expressed in ensheathing glia by neuronal

1069

mHTT

ex1

aggregates.

(

A-B

) Maximum intensity projections of DA1 glomeruli from 4-5 day-old flies

1070

expressing HTT

ex1

Q91-mCherry in DA1 ORNs and HTT

ex1

Q25-GFP

in (

) mz0709+ ensheathing glia or

1071

JNeurosci Accepted Manuscript

(

) Alrm+ astrocytic glia. Scale bars = 5

μm. (

C-D

) Quantification of (

) HTT

ex1

Q91-mCherry

(“mHTT”)

1072

and (

) seeded HTT

ex1

Q25-GFP

(“mHTT+wtHTT”) aggregates from flies shown in (

A-B

). Data are

1073

shown as mean ± s.e.m.; ***

<0.001 by unpaired two-tailed

-test.

1074

1075

Figure 9. Glial phagocytic and innate immunity genes regulate numbers of mHTT

ex1

aggregates

1076

in ORN axons. (A

) Maximum intensity projections of DA1 glomeruli from 7 day-old flies expressing

1077

HTT

ex1

Q91-mCherry in DA1 ORNs and siRNAs targeting the indicated genes in ensheathing glia. Scale

1078

bars = 5

μm. (

Quantification of HTT

ex1

Q91-mCherry aggregates detected in DA1 glomeruli from flies

1079

shown in (

). (

) Maximum intensity projections of DA1 glomeruli from 7 day-old flies expressing

1080

HTT

ex1

Q91-mCherry in DA1 ORNs and siRNAs targeting mCherry or Ets21c in repo+ glia in the

1081

presence of tubP-Gal80

. Adult flies were raised at the permissive (18°C,

top

) or restrictive (29°C,

1082

bottom

) temperatures to regulate siRNA expression in adults. Scale bars = 5

μm. (

) Quantification of

1083

HTT

ex1

Q91-mCherry aggregates detected in DA1 glomeruli from flies shown in (

). (

) Mean volumes

1084

for HTT

ex1

Q91-mCherry aggregates detected in DA1 glomeruli from flies shown in (

A and C

). All

1085

graphed data are shown as mean ± s.e.m.; *

<0.05, **

<0.01, ***

<0.001 by one-way ANOVA or

1086

unpaired two-tail

-test compared to no RNAi or mCherry

RNAi

controls.

1087

1088

Figure 10. Neuronal mHTT

ex1

expression increases numbers of acidified and active lysosomes in

1089

adult brains.

(A-B

) Maximum intensity projections of antennal lobes from 9-10 day-old adult flies

1090

expressing (

) HTT

ex1

Q25- or (

) HTT

ex1

Q91-GFP in Or83b+ ORNs and stained with Magic Red (MR)

1091

to label active cathepsins. Scale bars = 10

μm. (

) Quantification of MR+ surfaces (

left

) and HTT

ex1

1092

associated MR+ surfaces (

right

) from flies shown in (

and

); HTT

ex1

-associated vesicles were defined

1093

by filtering for MR+ surfaces

≤0.2 μm from HTT

ex1

fluorescent signal in confocal stacks.

(D-E

) Maximum

1094

intensity projections of antennal lobes from 15 day-old flies expressing (

) HTT

ex1

Q25- or (

)

1095

HTT

ex1

Q91-GFP in Or83b+ ORNs and stained with Lysotracker Red (LTR) to label low pH

1096

compartments. Scale bars = 10

μm. Diffuse wtHTT

ex1

signal was adjusted post-acquisition for increased

1097

visibility in panels (

and

). (

) Quantification of LTR+ surfaces (

left

) and HTT

ex1

-associated LTR+

1098

JNeurosci Accepted Manuscript

surfaces (

right

) from flies shown in (

and

); HTT

ex1

-associated vesicles were defined by filtering for

1099

LTR+ surfaces ≤0.2 μm from HTT

ex1

fluorescent signal in confocal stacks. Data are shown as mean ±

1100

s.e.m.; ****

<0.0001 by unpaired two-tailed

-test. (

G-H

) High-magnification images of boxes indicated

1101

in (

B and E

) showing co-localization of MR (

) or LTR (

) with mHTT

ex1

fluorescent signals. Scale bars

1102

= 1

μm.

1103

1104

Figure 11.

LAMP1+ vesicle accumulation in fly brains expressing mHTT

ex1

in ORNs. (A-B

)

1105

Confocal stacks showing antennal lobes from 19-22 day-old flies expressing (

) HTT

ex1

Q25- or (

)

1106

HTT

ex1

Q91-mCherry in Or83b+ ORNs and LAMP1 tagged at its cytoplasmic C-terminus with GFP

1107

(LAMP1-GFP) in glia. Brains were immunostained with anti-GFP to amplify LAMP1-GFP signal.

1108

LAMP1-GFP+ or HTT

ex1

+ segmented surfaces are shown to the right of each set of raw fluorescence

1109

images. Insets show magnified regions of interest from each image. Scale bars = 10

μm. (

) High-

1110

magnification confocal stack showing a LAMP1-

GFP+ surface within 0.2 μm of two HTT

ex1

Q91-

1111

mCherry+ aggregates. Scale bar = 1

μm.

(D-E

) Confocal stacks showing antennal lobes from 21-22

1112

day-old flies expressing (

) HTT

ex1

Q25- or (

) HTT

ex1

Q91-mCherry in Or83b+ ORNs and LAMP1

1113

tagged at its luminal N-terminus with GFP (GFP-LAMP1) in glia. GFP-LAMP1+ or HTT

ex1

+ segmented

1114

surfaces are shown to the right of each set of raw fluorescence images. Insets show a single GFP-

1115

LAMP1+ surface of interest from each image. Scale bars = 10

μm. Diffuse wtHTT

ex1

signal was

1116

adjusted post-acquisition for increased visibility in panels (

and

). (

) High-magnification confocal

1117

stack showing a GFP-

LAMP1+ surface within 0.2 μm of a HTT

ex1

Q91-mCherry+ aggregate. Scale bar =

1118

1μm. (

G-I

) Quantification of total LAMP1-GFP+ or GFP-LAMP1+ surfaces (

), LAMP1-GFP+ or GFP-

1119

LAMP1+ surfaces

≤0.2 μm from HTT

ex1

surfaces (

), and mean LAMP1-GFP+ or GFP-LAMP1+

1120

surface volume (

) in brains expressing HTT

ex1

Q25- or HTT

ex1

Q91-mCherry. The dark red bars in (

)

1121

represent LAMP1+ surfaces that co-localized with mHTT

ex1

. All graphed data are shown as mean ±

1122

s.e.m.; *

<0.05, **

<0.01, ***

<0.001, ****

<0.0001 by unpaired two-tailed

-test.

1123

1124

JNeurosci Accepted Manuscript

Figure 12. Increased association of Galectins-3 and -8 expressed in glia with neuronal mHTT

ex1

1125

aggregates.

(A-D

) Confocal stacks showing antennal lobes from 16-18 day-old flies expressing

1126

HTT

ex1

Q25- (

A and C

) or HTT

ex1

Q91-GFP (

B and D

) in Or83b+ ORNs together with Galectin-3 (

A-B

) or

1127

Galectin-8 (

C-D

) tagged with mCherry in glia. Segmented Galectin+ or HTT

ex1

+ surfaces are shown to

1128

the right of each set of raw fluorescence images. Insets show Galectin+ surfaces of interest from each

1129

image. Scale bars = 10

μm. (

E-G

) Quantification of total Galectin+ surfaces (

), Galectin+ surfaces

1130

≤0.2 μm from HTT

ex1

surfaces (

), and mean Galectin+ surface volume (

) in brains expressing

1131

HTT

ex1

Q25- or HTT

ex1

Q91-mCherry. The dark red bars in (

) represent Galectin+ surfaces that co-

1132

localized with mHTT

ex1

. All graphed data are shown as mean ± s.e.m.; *

<0.05, **

<0.01, ***

<0.001,

1133

****

<0.0001 by unpaired two-tailed

-test.

1134

1135

Figure 13.

Knockdown of genes regulating lysosome acidification alters seeded aggregation of

1136

glial wtHTT

ex1

protein by neuronal mHTT

ex1

aggregates.

(

) Confocal stacks showing DA1 glomeruli

1137

from 8-9 day-old flies expressing HTT

ex1

Q91-mCherry in DA1 ORNs and HTT

ex1

Q25-GFP plus siRNAs

1138

targeting the indicated genes in repo+ glia. Negative controls expressed siRNAs targeting mCherry.

1139

Scale bars = 5

μm. (

B-C

) Quantification of (

) HTT

ex1

Q91-mCherry or (

) seeded HTT

ex1

Q25-GFP

1140

aggregates from brains shown in (

). Data are shown as mean ± s.e.m.; *

<0.05, ***

<0.005 by

1141

unpaired two-tailed

-test.

1142

1143

Figure 14. Rab10 is required for seeded aggregation of glial wtHTT

ex1

by neuronal mHTT

ex1

1144

aggregates.

(

) Confocal stacks of DA1 glomeruli from 7 day-old flies expressing HTT

ex1

Q91-mCherry

1145

in DA1 ORNs and HTT

ex1

Q25-GFP plus siRNAs targeting firefly luciferase (FFLuc), Draper, or Rab10 in

1146

repo+ glia. Surfaces representing mHTT

ex1

aggregates (

red

) and seeded wtHTT

ex1

aggregates are

1147

superimposed on the raw data.

Scale bars = 5μm. (

B-C

) mHTT

ex1

(

) or mHTT

ex1

+wtHTT

ex1

(

)

1148

aggregates quantified from flies expressing HTT

ex1

Q91-mCherry in DA1 ORNs and HTT

ex1

Q25-GFP

1149

plus siRNAs targeting Draper-I (Drpr) or 23 different Rab GTPases in repo+ glia. Negative controls

1150

expressed no siRNAs or siRNAs targeting FFLuc or mCherry (

black bars

). (

) Normalized Draper

1151

JNeurosci Accepted Manuscript

immunofluorescence in the central brain of 4-5 day-old wild-type (

rab10

+/+

) and

rab10

null (

rab10

-/-

)

1152

flies. (

) Quantification of seeded wtHTT

ex1

aggregates in flies expressing HTT

ex1

Q91-mCherry in DA1

1153

ORNs and HTT

ex1

Q25-GFP plus siRNAs targeting Rab10, without or with Draper-I cDNAs in repo+glia.

1154

(

F-G

) Quantification of mHTT

ex1

(

) or mHTT

ex1

+wtHTT

ex1

(

) aggregates from 10 day-old flies

1155

expressing HTT

ex1

Q91-mCherry in DA1 ORNs and HTT

ex1

Q25-GFP in PNs, either heterozygous or

1156

trans-heterozygous for

draper

(

drpr/+

rab10

(

rab10/+

), or

rab14

(

rab14/+

) mutant alleles. All graphed

1157

data are shown as mean ± s.e.m.; *

<0.05, **

<0.01, ***

<0.005 by one-way ANOVA or unpaired two-

1158

tailed

test comparing to controls.

1159

1160

Figure 15. Association of neuronal mHTT

ex1

aggregates with Rab GTPases that label early,

1161

maturing, and late phagosomes. (A)

qPCR analysis of

rab10

rab5

and

rab7

expression in 8-11 day-

1162

old flies expressing GFP, HTT

ex1

Q25-, or HTT

ex1

Q91-GFP in Or83b+ ORNs. RNA was isolated from

1163

heads of uninjured flies or flies 3 hours after bilateral antennal and maxillary palp nerve injury. Data are

1164

shown as mean ± s.e.m and normalized to the housekeeping gene

rpl32

. *

<0.05, **

<0.01, ***

<0.001

1165

by one-way ANOVA, asterisks and hashtags indicate statistical significance comparing -/+ injury or

1166

genotypes, respectively. (

B-G

) Confocal stacks of the antennal lobe from 7 day-old flies expressing (

1167

D, & F

) HTT

ex1

Q25- or (

C, E, & G

) HTT

ex1

Q91-V5 in Or83b+ ORNs and endogenously-tagged (

B-C

)

1168

YRab10, (

D-E

) YRab5, or (

F-G

) YRab7 in all cells. Brains were immunostained using YFP (

green

), V5

1169

(

magenta

), and N-Cadherin (

blue

) antibodies. Segmented YRab+ or HTT+ surfaces are shown to the

1170

right of each set of raw fluorescent images. Insets show magnified YFP+ surfaces of interest from each

1171

image. Diffuse wtHTT

ex1

signal was adjusted post-acquisition for increased visibility in panels (

B, D,

and

1172

). Scale bars = 10

μm. (

H-I

) Quantification of YRab+ surfaces (

) and YRab+ surfaces within

0.2μm of

1173

HTT

ex1

+ surfaces (

). Data are shown as mean ± s.e.m.; *

<0.05, **

<0.01, ***

<0.001, ****

<0.0001 by

1174

unpaired two-tailed

-test.

1175

1176

Figure 16. Association of neuronal mHTT aggregates with glial Rab GTPases that label early,

1177

maturing, and late phagosomes.

(

A-F

) Confocal stacks of the antennal lobe from 16-19 day-old flies

1178

JNeurosci Accepted Manuscript

expressing (

A, C, E

) HTT

ex1

Q25- or (

B, D, F

) HTT

ex1

Q91-V5 in Or83b+ ORNs together with (

A-B

) YFP-

1179

Rab10, (

C-D

) YFP-Rab5, or (

E-F

) YFP-Rab7 in repo+ glia. Brains were immunostained with anti-GFP

1180

to amplify YFP-Rab signals. Segmented Rab+ or HTT

ex1

+ surfaces are shown to the right of each set of

1181

raw fluorescence images. Insets show magnified YFP-Rab+ surfaces of interest from each image.

1182

Diffuse wtHTT

ex1

signal was adjusted post-acquisition for increased visibility in panels (

A, C,

and

1183

Scale bars = 10

μm. (

G-I

) Quantification of total YFP-Rab+ surfaces (

), YFP-

Rab+ surfaces ≤0.2 μm

1184

from HTT

ex1

surfaces (

), and mean YFP-Rab+ surface volume (

) in brains expressing HTT

ex1

Q25- or

1185

HTT

ex1

Q91-mCherry. The dark red bars in (

) represent YFP-Rab+ surfaces that co-localized with

1186

mHTT

ex1

. All graphed data are shown as mean ± s.e.m.; *

<0.05, **

<0.01, ***

<0.001, ****

<0.0001

1187

by unpaired two-tailed

-test.

1188

1189

Figure 17. Glial YFP-Rab+ surfaces are closely associated with neuronal mHTT

ex1

aggregates

1190

High magnification confocal stacks showing examples of individual YFP+ surfaces within 0.2 μm of

1191

HTT

ex1

Q91 aggregates from 21-22 day-old adult brains expressing HTT

ex1

Q91-mCherry in Or83b+

1192

ORNs and YFP-Rab10 (

A1-3

), YFP-Rab5 (

B1-2

), or YFP-Rab7 (

C1-2

) in repo+ glia. Brains were

1193

immunostained with anti-GFP to amplify YFP-Rab signals. Scale bars = 1

μm.

1194

1195

Figure 18.

“Traffic jam” model illustrating effects of neuronal mHTT

ex1

aggregates on phagocytic

1196

glia.

mHTT

ex1

aggregates (

magenta circles

) generated in axons cause phagolysosomal defects in

1197

neighboring glial cells. Neuronal mHTT

ex1

aggregates activate glial Draper and Toll-6 signaling

1198

pathways, but impair normal phagocytic responses to injury, including reduced nascent phagosome

1199

formation and decreased numbers of Rab5+ early phagosomes (

red arrow

). A buildup of engulfed

1200

mHTT

ex1

aggregates in glia leads to accumulation of maturing (Rab10+ or Rab7+) phagosomes and

1201

lysosomes (LAMP1+) (

green arrows

), possibly further slowing Draper-dependent engulfment and early

1202

phagosome formation. Defective phagocytic clearance could enhance leak or ejection of some mHTT

ex1

1203

aggregates from phagolysosomes to increase their toxicity and capacity to seed soluble HTT proteins

1204

(

magenta + green circles

1205

JNeurosci Accepted Manuscript

Table 1. Drosophila genotype and source information.

Name

ID (if available)

Reference

Notes

Or67d-QF

Liang et al., 2013

repo-Gal4

RRID:BDSC_7415

Sepp et al., 2001

mz0709-Gal4

Ito et al., 1995

a gift from Marc
Freeman (Vollum
Institute)

alrm-Gal4

RRID:BDSC_67032

orco-Gal4

RRID:BDSC_23292

Or47b-Gal4

RRID:BDSC_9983

QUAS-HTT

ex1

Q25-

mCherry

attP24

Donnelly et al.,
2020

QUAS-HTT

ex1

Q91-

mCherry

attP24

Donnelly et al.,
2020

QUAS-HTT

ex1

Q91-

mCherry

attP3

Donnelly et al.,
2020

QUAS-HTT

ex1

Q91-

mCherry

suHw(attP8)

This study

UAS-HTT

ex1

Q25-GFP

Donnelly et al.,
2020

UAS-HTT

ex1

Q91-GFP

Donnelly et al.,
2020

UAS-GFP

Donnelly et al.,
2020

QUAS-HTT

ex1

Q25-GFP

This study

QUAS-HTT

ex1

Q91-GFP

This study

UAS-mCherry-LGals3

This study

UAS-mCherry-LGals8

This study

QUAS-HTT

ex1

Q25-V5

This study

QUAS-HTT

ex1

Q91-V5

This study

UAS-pHluorin-
HTT

ex1

Q25-tdTomato

This study

UAS-pHluorin-
HTT

ex1

Q91-tdTomato

This study

UAS-mCD8-GFP

RRID:BDSC_5137

UAS-Draper

RNAi

Logan et al., 2012

a gift from Marc
Freeman (Vollum
Institute)

tubP-Gal80

RRID:BDSC_7017

UAS-Drs

RNAi

RRID:BDSC_55391

UAS-PGRP-SA

RNAi

RRID:BDSC_60037

UAS-MMP1

RNAi

RRID:BDSC_31489

UAS-ets21c

RNAi

RRID:BDSC_39069

UAS-mCherry

RNAi

RRID:BDSC_35785

UAS-rel

RNAi

RRID:BDSC_33661,

RRID BDSC_28943

UAS-dl

RNAi

RRID:BDSC_38905,
RRID:BDSC_32934,
RRID:BDSC_34938,
RRID:BDSC_27650,
RRID:BDSC_36650

UAS-NinjurinA

RNAi

RRID:BDSC_50632

RRID:BDSC_51358

UAS-Toll-6

RNAi

RRID:BDSC_56048,
RRID:BDSC_64968

Orco-QF2

RRID:BDSC_91997

TI{TI}Rab10

YFP

RRID:BDSC_62548

TI{TI}Rab5

YFP

RRID:BDSC_62543

TI{TI}Rab7

YFP

RRID:BDSC_62545

UAS-MApHS

Han et al., 2014

a gift from Chun Han
(Cornell University)

Repo-Cas9[6A]

Koreman et al.,
2021

a gift from Chun Han
(Cornell University)

gRNA-drpr(BR)

Sapar et al., 2018

a gift from Chun Han
(Cornell University)

Jra-GFP.FLAG

RRID:BDSC_50755

OK107-Gal4

UAS-GFP-LAMP1

Pulipparacharuvil
et al., 2005

A gift from Helmut
Kramer (UT
Southwestern)

UAS-LAMP1-GFP

RRID:BDSC_42714

nSyb-Gal4

was

removed from stock

UAS-Rbcn-3a

RNAi

RRID:BDSC_34612

UAS-Spinster

RNAi

RRID:BDSC_27702

UAS-Vha100-2

RNAi

RRID:BDSC_64859

UAS-Vha16-1

RNAi

RRID:BDSC_40923

UAS-Vha68-3

RNAi

RRID:BDSC_42954

UAS-YFP-Rab5

RRID:BDSC_9775

UAS-YFP-Rab7

RRID:BDSC_23641

UAS-YFP-Rab10

RRID:BDSC_24097

UAS-YFP-Rab10 (T23N)

RRID:BDSC_9788

UAS-YFP-Rab10 (Q68L)

RRID:BDSC_23259

UAS-Rab7

RNAi

RRID:BDSC_27051

UAS-Rab10

RNAi

RRID:BDSC_26289

UAS-Rab2

RNAi

RRID:BDSC_28701

RRID:BDSC_34922

UAS-Rab6

RNAi

RRID:BDSC_27490

RRID:BDSC_35744

UAS-Rab11

RNAi

RRID:BDSC_27730

UAS-Rab3

RNAi

RRID:BDSC_34655

UAS-Rab14

RNAi

RRID:BDSC_28708

UAS-Rab4

RNAi

RRID:BDSC_33757

UAS-Rab5

RNAi

RRID:BDSC_30518

UAS-Rab8

RNAi

RRID:BDSC_27519

RRID:BDSC_34373

UAS-Rab9

RNAi

RRID:BDSC_42942

RRID:BDSC_34374

UAS-luciferase

RNAi

RRID:BDSC_31603

UAS-Rab9Db

RNAi

RRID:BDSC_38269

UAS-Rab32

RNAi

RRID:BDSC_38956

RRID:BDSC_28002

UAS-Rab18

RNAi

RRID:BDSC_34734

RRID:BDSC_27665

UAS-RabX4

RNAi

RRID:BDSC_44070

UAS-Rab27

RNAi

RRID:BDSC_50537

UAS-RabX2

RNAi

RRID:BDSC_53928

UAS-Rab39

RNAi

RRID:BDSC_53995

UAS-Rab23

RNAi

RRID:BDSC_55352

RRID:BDSC_63689

RRID:BDSC_36091

RRID:BDSC_28025

UAS-Rab35

RNAi

RRID:BDSC_67952

RRID:BDSC_80457

UAS-Rab40

RNAi

RRID:BDSC_80472

UAS-RabX6

RNAi

RRID:BDSC_26281

RRID:BDSC_53252

UAS-Rab19

RNAi

RRID:BDSC_34607

UAS-Rab21

RNAi

RRID:BDSC_29403

UAS-Rab27

RNAi

RRID:BDSC_35774

UAS-Draper-I

Logan et al., 2012

a gift from Marc
Freeman (Vollum
Institute)

Rab10(CRISPR-KO)

Kohrs et al., 2021

a gift from P. Robin
Heisinger (Freie
Universität Berlin)

Table 2. Genotypes of flies used in all figures.

Figure

Genotype

Figure 1A & C-E

Or67d-QF/Y

;

QUAS-HTT

ex1

Q25-mCherry

QUAS-mCD8-GFP

Figure 1B-E

Or67d-QF/Y

;

QUAS-HTT

ex1

Q91-mCherry

QUAS-mCD8-GFP

Figure 2A,D-E

Or67d-QF/Y

;

UAS-HTT

ex1

Q25-V5

QUAS-mCD8-GFP

;

repo-gal4

Figure 2B,D-E

Or67d-QF/Y

;

UAS-HTT

ex1

Q91-V5

QUAS-mCD8-GFP

;

repo-gal4

Figure 2C

+/Y

;

UAS-HTT

ex1

Q25-V5

;

repo-gal4

+ & +/Y

;

UAS-HTT

ex1

Q91-

;

repo-gal4

Figure 3A & C-D

w1118/Y

;

UAS-HTT

ex1

Q25-V5

Or47b-Gal4

;

UAS-MapHS/+

Figure 3B-D

w1118/Y

;

UAS-HTT

ex1

Q91-V5

Or47b-Gal4

;

UAS-MapHS/+

;

Figure 3C

w1118/Y;repo-Cas9

Or47b-Gal4

;

UAS-MapHS/gRNA-draper

Figure 4A & C

w1118/Y

;

UAS-pHluorin-HTT

ex1

Q25-tdTomato/Orco-Gal4

Figure 4B-C

w1118/Y

;

UAS-pHluorin-HTT

ex1

Q91-tdTomato/Orco-Gal4

Figure 4D & F

w1118/Y

;

UAS-pHluorin-HTT

ex1

Q25-tdTomato/+;OK107-Gal4/+

Figure 4E-F

w1118/Y

;

UAS- pHluorin-HTT

ex1

Q91-tdTomato/+;OK107-Gal4/+

Figure 4G & I

W1118/Y;Repo-Cas9/+;+/+

Figure 4H-I

W1118/Y;Repo-Cas9/+;gRNA-draper/+

Figure 4J & L

W1118/Y;Or47b-Gal4/repo-Cas9;+/UAS- UAS-pHluorin-HTT

ex1

Q91-

tdTomato

Figure 4J-L

W1118/Y;Or47b-Gal4/repo-Cas9;gRNA-draper/UAS- UAS-pHluorin-

HTT

ex1

Q91-tdTomato

Figure 5A

w1118

;

UAS-GFP

/+;

orco-Gal4

/+ &

w1118

;

UAS-HTT

ex1

Q25-GFP

/+;

orco-Gal4

/+ &

w1118

;

UAS-HTT

ex1

Q91-GFP

/+;

orco-Gal4

Figure 5C & E

w1118/Y

;

UAS-HTT

ex1

Q25-V5

/+;

orco-Gal4

/Toll6

MIMICGFP

Figure 5D-E

w1118/Y

;

UAS-HTT

ex1

Q91-V5

/+;

orco-Gal4

/Toll6

MIMICGFP

Figure 5F & H

w1118/Y

;

UAS-HTT

ex1

Q25-V5

/+;

orco-Gal4

Jra.GFP

Figure 5G-H

w1118/Y

;

UAS-HTT

ex1

Q91-V5

/+;

orco-Gal4

Jra.GFP

Figure 6A-H

w1118

;

UAS-HTT

ex1

Q25-GFP

/+;

orco-Gal4

/+ &

w1118

;

UAS-HTT

ex1

Q91-GFP

/+;

orco-Gal4

Figure 7A, A, C, E

w1118

; Q

UAS-HTT

ex1

Q25-mCherry

/+;

orco-QF2

Figure 7B-D, F-H

w1118

; Q

UAS-HTT

ex1

Q91-mCherry

/+;

orco-QF2

Figure 8A & C-D

or67d-QF,QUAS-HTT

ex1

Q91-mCherry/+;QUAS-HTT

ex1

Q25-

GFP/+;mz0709-Gal4/+

Figure 8B-D

or67d-QF,QUAS-HTT

ex1

Q91-mCherry/+;QUAS-HTT

ex1

Q25-GFP/+;alrm-

Gal4/+

Figure 9A-B, E

(control)

or67d-QF,QUAS-HTT

ex1

Q91-mCherry/+;+/+;mz0709-Gal4 /+ &

or67d-QF,QUAS-HTT

ex1

Q91-mCherry/+;+/+;mz0709-Gal4/UAS-RNAi-X

& or67d-QF,QUAS-HTT

ex1

Q91-mCherry/+; UAS-RNAi-X /+;mz0709-

Gal4/+

Figure 9C-E

(control)

or67d-QF,QUAS-HTT

ex1

Q91-mCherry/

;+/+;repo-

Gal4,Gal80

/UAS-RNAi-mCherry

or67d-QF,QUAS-HTT

ex1

Q91-

mCherry/

;+/+;repo-Gal4,Gal80

/UAS-RNAi-Ets21c

Figure 10A & C-D & F

w1118/Y

;

UAS-HTT

ex1

Q25-GFP

;

orco-Gal4/+

Figure 10B-C & E-H

w1118/Y

;

UAS-HTT

ex1

Q91-GFP

;

orco-Gal4/+

Figure 11A & G-I

w1118/Y;QUAS-HTTex1Q25-mCherry/UAS-LAMP1-GFP;orco-

QF2/repo-Gal4

Figure 11B-C & G-I

w1118/Y;QUAS-HTTex1Q91-mCherry/UAS-LAMP1-GFP;orco-

QF2/repo-Gal4

Figure 11D & G-I

w1118/Y;QUAS-HTTex1Q25-mCherry/UAS-GFP-LAMP1;orco-

QF2/repo-Gal4

Figure 11E-I

w1118/Y;QUAS-HTTex1Q91-mCherry/UAS-GFP-LAMP1;orco-

QF2/repo-Gal4

Figure 12A-B & E-G

Control:

QUAS-HTT

ex1

Q25-GFP/+/+;repo-Gal4,orco-QF2/UAS-mCherry-

LGals3

QUAS-HTT

ex1

Q91-GFP/+/+;repo-Gal4,orco-QF2/UAS-
mCherry-LGals3

Figure 12C-G

Control:

QUAS-HTT

ex1

Q25-GFP/+/+;repo-Gal4,orco-QF2/UAS-mCherry-

LGals8

QUAS-HTT

ex1

Q91-GFP/+/+;repo-Gal4,orco-QF2/UAS-
mCherry-LGals8

Fig 13A-C

Control

: UAS-HTT

ex1

Q25-GFP/Or67d-QF,QUAS-HTT

ex1

Q91-

mCherry;+/+;repo-Gal4/UAS-RNAi.mCherry

UAS-HTT

ex1

Q25-

GFP/Or67d-QF,QUAS-mHTT-mCherry;+/+;repo-Gal4/UAS-RNAi-X

UAS-HTT

ex1

Q25-GFP//Or67d-QF,QUAS-mHTT-mCherry /+;UAS-RNAi-

; repo-Gal4/+

Figure 14A-C

Or67d-QF,QUAS-HTT

ex1

Q91-mCherry /+

;

QUAS- HTT

ex1

Q25-GFP/UAS-

RNAi-X;repo-Gal4/+ & Or67d-QF,QUAS-HTT

ex1

Q91-mCherry /+

;

QUAS-

HTT

ex1

Q25-GFP/+;repo-Gal4/UAS-RNAi-X

Figure 14D-E

Or67d-QF,UAS-HTTex1Q25-GFP/+ ; GH146-Gal4,QUAS-HTTex1Q91-

mCherry/+

Or67d-QF,UAS-HTTex1Q25-GFP/+ ; GH146-Gal4,QUAS-

HTTex1Q91-mCherry/+ ; drprD5/+

Or67d-QF,UAS-HTTex1Q25-

GFP/rab10{CRISPR-KO} ; GH146-Gal4,QUAS-HTTex1Q91-mCherry/+

Or67d-QF,UAS-HTTex1Q25-GFP/rab10{CRISPR-KO} ; GH146-

Gal4,QUAS-HTTex1Q91-mCherry/+ ; drprD5/+

Or67d-QF,UAS-

HTTex1Q25-GFP/rab10CRISPR ; GH146-Gal4,QUAS-HTTex1Q91-

mCherry/rab14{CRISPR-KO}

Or67d-QF,UAS-HTTex1Q25-

GFP/rab10CRISPR ; GH146-Gal4,QUAS-HTTex1Q91-

mCherry/rab14{CRISPR-KO} ; drprD5/+

Figure 14F

UAS-HTT

ex1

Q25-GFP,QUAS-HTT

ex1

Q91-mCherry/Or67d-QF;+/+;repo-

Gal4/+

UAS-HTT

ex1

Q25-GFP,QUAS-HTT

ex1

Q91-mCherry/Or67d-

QF;+/+;repo-Gal4/UAS-RNAi-Rab10

UAS-HTT

ex1

Q25-GFP,QUAS-

HTT

ex1

Q91-mCherry/Or67d-QF;UAS-draper-I/+;repo-Gal4/UAS-RNAi-

Rab10

Figure 14G

w1118/ w1118;+/+;+/+ (w1118)

Rab10(CRISPR-KO)/w1118;+/+;+/+ &

Rab10(CRISPR-KO)/ Rab10(CRISPR-KO);+/+;+/+

Figure 15A

controls:

w1118

;

UAS-GFP

/+;

orco-Gal4

/+ &

w1118

;

UAS-HTT

ex1

Q25-

GFP

/+;

orco-Gal4

/+ &

w1118

;

UAS-HTT

ex1

Q91-GFP

/+;

orco-Gal4

Figure 15B-C & H-I

TI{TI}Rab10[EYFP]

;

UAS-HTT

ex1

Q25-V5

;

orco-Gal4/+

TI{TI}Rab10[EYFP]

;

UAS-HTT

ex1

Q91-V5

;

orco-Gal4/+

Figure 15D-E & H-I

1118/Y

;

UAS-HTT

ex1

Q25-V5

TI{TI}Rab5[EYFP]

;

orco-Gal4/+

w1118/Y

;

UAS-HTT

ex1

Q91-V5

TI{TI}Rab5[EYFP]

;

orco-Gal4/+

Figure 15F-I

w1118/Y

;

UAS-HTT

ex1

Q25-V5

;

orco-Gal4/TI{TI}Rab7[EYFP]

w1118/Y

;

UAS-HTT

ex1

Q91-V5

;

orco-Gal4/ TI{TI}Rab7[EYFP]

Figure 16A-B & G-I

w1118/Y;QUAS-HTTex125-mCherry/UAS-YFP-Rab10;orco-QF2,repo-

Gal4/+

w1118/Y;QUAS-HTTex1Q91-mCherry/UAS-YFP-Rab10;orco-

QF2,repo-Gal4/+

Figure 16C-D & 16G-I

w1118/Y;QUAS-HTTex125-mCherry/UAS-YFP-Rab5;orco-QF2,repo-

Gal4/+

w1118/Y;QUAS-HTTex1Q91-mCherry/UAS-YFP-Rab5;orco-

QF2,repo-Gal4/+

Figure 16E-I

w1118/Y;QUAS-HTTex125-mCherry/UAS-YFP-Rab7;orco-QF2,repo-

Gal4/+

w1118/Y;QUAS-HTTex1Q91-mCherry/UAS-YFP-Rab7;orco-

QF2,repo-Gal4/+

Figure 17A1-3

w1118/Y;QUAS-HTTex1Q91-mCherry/UAS-YFP-Rab10;orco-QF2,repo-

Gal4/+

Figure 17B1-2

w1118/Y;QUAS-HTTex1Q91-mCherry/UAS-YFP-Rab5;orco-QF2,repo-

Gal4/+

Figure 17C1-2

w1118/Y;QUAS-HTTex1Q91-mCherry/UAS-YFP-Rab7;orco-QF2,repo-

Gal4/+

Table 3. Primer sequences used for qPCR analyses.

Gene Target

Forward (F) and Reverse (R) Primer Sequences

(5’-3’)

rpl32

F: CCGCTTCAAGGGACAGTATCTG

R: ATCTCGCCGCAGTAAACGC

toll-6

F: AATATTGTGGAGTGCTCGGG

R: GCGTTTAAGGCCACTGAAAG

dorsal

F: ATGCGAGCGGTGTTCAGTAA

R: ACGATGCGAAAAGCCAGTCT

relish

F: ACAGGACCGCATATCG

R: GTGGGGTATTTCCGGC

draper-I

F: TGTGATCATGGTTACGGAGGAC

R: CAGCCGGGTGGGCAA

mmp1

F: GAAGGCTCGGACAACGAGT
R: GTCGTTGGACTGGTGATCG

ets21c

F: CAACGACGACGAACCAAAT

R: GTTCGCGTTGGACGAATC

rab10

F: ACATCCGCCAAGTCGAACAT

R: CTGGTTCCGGCGATCGATAA

rab5

F: AGTCCGCTGTGGGCAAGTC

R: CTCCTGGTACTCGTGGAACTGTC

rab7

F: AATTTTGCACGCAACCGCTG

R: GAGTAGCCAATTCGATGGTGC

drosomycin

GCTGTCCTGATGCTGGTGGT

R: CGGAAAGGGACCCTTGTATCTTC

attacin-a

F: CTCGTTTGGATCTGACCAAGG
R: CCATGACCAGCATTGTTGTAG

attacin-d

CGTTGAGGTTGAGATTGCCACT

R: CGGTCCCTCAGTTCGGCATGAC

diptericinA

CCACCGCAGTACCCACTCAAT

CGATGACTGCAAAGCCAAAACCA

metchnikowin

CAGTGCTGGCAGAGCCTCAT

CGTCGGTTAGGATTGAAGGGCGA

Table 4.

Statistical information for the data shown in Figure 1

Figure 1C

Genotype

Age

Days-post

Injury

Values

Number of

animals

Or67d-QF/Y

;

QUAS-

HTT

ex1

Q25-

mCherry

QUAS-

mCD8-GFP

100±2.61

10 (19

ORNs)

62.73±4.85

9 (18 ORNs)

37.02±2.37

8 (15 ORNs)

24.7±1.37

9 (18 ORNs)

100±4.98

10 (19

ORNs)

68.8±3.51

10 (19

ORNs)

41.65±2.44

10 (20

ORNs)

20.66±1.53

10 (20

ORNs)

100±5.33

10 (20

ORNs)

84.48±4.57

9 (17 ORNs)

49.67±2.36

10 (20

ORNs)

28.07±2.00

10 (20

ORNs)

Or67d-QF/Y

;

QUAS-

HTT

ex1

Q91-

mCherry

QUAS-

mCD8-GFP

100±4.88

10 (20

ORNs)

99.87±4.25

9 (18 ORNs)

48.08±2.34

9 (18 ORNs)

20.98±0.94

9 (18 ORNs)

100±3.06

10 (19

ORNs)

95.43±2.61

10 (19

ORNs)

59.14±2.35

10 (20

ORNs)

33.67±1.42

10 (20

ORNs)

100±4.19

10 (20

ORNs)

101.72±4.47 9 (18 ORNs)

67.95±2.87

9 (18 ORNs)

51.84±2.48

10 (20

ORNs)

Comparison between

Genotype

Age

Days-post Injury

Test used

P-value

Test

statistics

Or67d-

QF/Y

;

QUAS-

HTT

ex1

Q25-

mCherry

QUAS-

mCD8-GFP &

Or67d-

QF/Y;QUAS-
HTTex1Q91-

mCherry/QUAS-

mCD8-GFP

Mann-Whitney

=0, df=37

Mann-Whitney

1.77881E-06

=5.76, df=34

Mann-Whitney

0.005444174

=2.99, df=34

Mann-Whitney

0.324663317

=2.23, df=34

Mann-Whitney

=0, df=36

Mann-Whitney

5.25933E-07

=6.09, df=36

Mann-Whitney

8.0017E-06

=5.16, df=38

Mann-Whitney

6.19497E-07

=5.97, df=38

Mann-Whitney

=0, df=38

Mann-Whitney

0.014244052

=2.58, df=33

Mann-Whitney

1.72014E-05

=4.96, df=36

Mann-Whitney

5.94561E-09

=7.46, df=36

Figure 1D

Genotype

Age

Days-post

Injury

Values

Number of

animals

Or67d-QF/Y

;

QUAS-

HTT

ex1

Q25-

mCherry

QUAS-

mCD8-GFP

100±3.40

10 (19

ORNs)

23.07±0.76

9 (18 ORNs)

19.06±0.80

8 (15 ORNs)

17.77±0.29

9 (18 ORNs)

100±3.66

10 (19

ORNs)

86.38±3.74

10 (19

ORNs)

54.51±2.21

10 (20

ORNs)

25.49±1.94

10 (20

ORNs)

100±2.55

10 (20

ORNs)

98.35±3.51

9 (17 ORNs)

58.87±3.66

10 (20

ORNs)

42.48±3.85

10 (20

ORNs)

Or67d-QF/Y

;

QUAS-

HTT

ex1

Q91-

mCherry

QUAS-

mCD8-GFP

100±1.03

10 (20

ORNs)

78.47±0.96

9 (18 ORNs)

27.05±0.60

9 (18 ORNs)

17.87±0.48

9 (18 ORNs)

100+0.42

10 (19

ORNs)

99.71±0.81

10 (19

ORNs)

66.69±1.81

10 (20

ORNs)

38.26±0.88

10 (20

ORNs)

100±0.44

10 (20

ORNs)

100.72±0.44 9 (18 ORNs)

73.68±2.47

9 (18 ORNs)

48.87±1.13

10 (20

ORNs)

Comparison between

Genotype

Age

Days-post

Injury

Test used

P-value

Test

statistics

Or67d-

QF/Y

;

QUAS-

HTT

ex1

Q25-

mCherry

QUAS-

mCD8-GFP &

Or67d-

QF/Y;QUAS-
HTTex1Q91-

mCherry/QUAS-

mCD8-GFP

Mann-

Whitney

=0, df=37

Mann-

Whitney

4.27312E-34

=52.37,

df=34

Mann-

Whitney

1.22166E-09

=8.04, df=34

Mann-

Whitney

0.861294225

=0.18, df=34

Mann-

Whitney

=0, df=36

Mann-

Whitney

0.001633943

=3.41, df=36

Mann-

Whitney

7.52573E-05

=4.44, df=38

Mann-

Whitney

2.75857E-06

=5.50, df=38

Mann-

Whitney

=0, df=38

Mann-

Whitney

0.485795632

=0.44, df=33

Mann-

Whitney

0.001469239

=3.44, df=36

Mann-

Whitney

0.096143222

=1.71, df=36

Figure 1E

Genotype

Age

Values

Number of

animals

Or67d-QF/Y

;

QUAS-

HTT

ex1

Q25-

mCherry

QUAS-

mCD8-GFP

3954.35±103.35

10 (19

ORNs)

4259.41±161.74

10 (19

ORNs)

4341.33±231.44

10 (20

ORNs)

Or67d-QF/Y

;

QUAS-

HTT

ex1

Q91-

mCherry

QUAS-

mCD8-GFP

3840.07±187.53

10 (20

ORNs)

2958.99±90.51

10 (19

ORNs)

1640.67±68.73

10 (20

ORNs)

Comparison between

Genotype

Age

Test used

P-value

Test statistics

Or67d-QF/Y

;

QUAS-

HTT

ex1

Q25-mCherry

QUAS-

mCD8-GFP & Or67d-

QF/Y;QUAS-HTTex1Q91-

mCherry/QUAS-mCD8-GFP

Mann-Whitney

0.602011569

=0.53, df=37

Mann-Whitney

1.43E-06

=5.76, df=36

Mann-Whitney

1.38E-13

=11.19, df=38

Table 5.

Statistical information for the data shown in Figure 2

Figure 2D

Genotype

Days-post

Injury

Values

Number of

animals

Or67d-QF/Y;UAS-

HTTex1Q25-

V5/QUAS-mCD8-

GFP;repo-gal4/+

100±3.61

10 (20 ORNs)

65.08±3.50

10 (19 ORNs)

20.19±2.40

9 (18 ORNs)

Or67d-QF/Y;UAS-

HTTex1Q91-

V5/QUAS-mCD8-

GFP;repo-gal4/+

100±5.62

10 (19 ORNs)

86.67±4.94

10 (19 ORNs)

70.57±5.82

10 (19 ORNs)

Comparison between

Genotype

Days-post

Injury

Test used

P-value

Test statistics

Or67d-QF/Y;UAS-

HTTex1Q25-V5/QUAS-

mCD8-GFP;repo-gal4/+ &

Or67d-QF/Y;UAS-

HTTex1Q91-V5/QUAS-

mCD8-GFP;repo-gal4/+

Mann-Whitney

=0, df=37

Mann-Whitney

0.000708059

=3.71, df=36

Mann-Whitney

4.06913E-09

=9.36, df=35

Figure 2E

Genotype

Values

Number of

animals

Or67d-QF/Y;UAS-

HTTex1Q25-

V5/QUAS-mCD8-

GFP;repo-gal4/+

2142.59±77.34

10 (20 ORNs)

Or67d-QF/Y;UAS-

HTTex1Q91-

V5/QUAS-mCD8-

GFP;repo-gal4/+

1961.28±110.26

10 (19 ORNs)

Comparison between

Genotype

Test used

P-value

Test

statistics

Or67d-QF/Y

;

QUAS-HTT

ex1

Q25-

mCherry

QUAS-mCD8-GFP &

Or67d-QF/Y;QUAS-

HTTex1Q91-mCherry/QUAS-

mCD8-GFP

Mann-

Whitney

0.1415

=1.50, df=37

Table 6.

Statistical information for the data shown in Figure 3

Figure 3C

Genotype

Hours

Post Injury

Values

Number of

animals

w1118/Y;UAS-

HTTex1Q25-

V5/Or47b-Gal4;UAS-

MapHS/+

1±0.01

10 (19

ORNs)

0.79±0.01

10 (20

ORNs)

0.64±0.012

10 (19

ORNs)

w1118/Y;UAS-

HTTex1Q91-

V5/Or47b-Gal4;UAS-

MapHS/+

1±0.019

8 (15 ORNs)

0.84±0.015

10 (20

ORNs)

0.76±0.017

8 (16 ORNs)

w1118/Y;repo-

Cas9/Or47b-

Gal4;UAS-

MapHS/gRNA-draper

1±0.019

9 (18 ORNs)

0.96±0.018

9 (18 ORNs)

0.91±0.018

9 (18 ORNs)

Comparison between

Genotype

Hours

Post-Injury

Test used

P-value

Test

statistics

w1118/Y;UAS-HTTex1Q25-

V5/Or47b-Gal4;UAS-MapHS/+

0 & 14

Mann-Whitney 1.10266E-09

=12.73,

df=37

0 & 25

Mann-Whitney 6.14808E-23

=22.72,

df=36

w1118/Y;UAS-HTTex1Q91-

V5/Or47b-Gal4;UAS-MapHS/+

0 & 14

Mann-Whitney 4.77205E-06

=5.46, df=33

0 & 25

Mann-Whitney 1.96095E-10

=9.53, df=29

w1118/Y;repo-Cas9/Or47b-

Gal4;UAS-MapHS/gRNA-draper

0 & 14

Mann-Whitney 0.077495139

=1.45, df=34

0 & 25

Mann-Whitney 0.001621729

=3.43, df=34

w1118/Y;UAS-HTTex1Q25-

V5/Or47b-Gal4;UAS-MapHS/+

& w1118/Y;UAS-HTTex1Q91-

V5/Or47b-Gal4;UAS-MapHS/+

Mann-Whitney

=0, df=32

Mann-Whitney 0.118030728

=1.60, df=38

Mann-Whitney 0.000223765

=4.14, df=33

w1118/Y;UAS-HTTex1Q25-

V5/Or47b-Gal4;UAS-MapHS/+

& w1118/Y;repo-Cas9/Or47b-

Gal4;UAS-MapHS/gRNA-draper

Mann-Whitney

=0, df=35

Mann-Whitney 9.16593E-14

=11.64,

df=36

Mann-Whitney

1.1284E-12

=10.79,

df=35

w1118/Y;UAS-HTTex1Q91-

V5/Or47b-Gal4;UAS-MapHS/+

& w1118/Y;repo-Cas9/Or47b-

Gal4;UAS-MapHS/gRNA-draper

Mann-Whitney

=0, df=31

Mann-Whitney 1.30161E-07

=6.54, df=36

Mann-Whitney 2.61939E-05

=4.82, df=32

Figure 3D

Genotype

Values

Number of

animals

w1118/Y;UAS-

HTTex1Q25-

V5/Or47b-Gal4;UAS-

MapHS/+

1±0.01

10 (19

ORNs)

w1118/Y;UAS-

HTTex1Q91-

V5/Or47b-Gal4;UAS-

MapHS/+

0.92±0.017 8 (15 ORNs)

Comparison between

Genotype

Test

used

P-value

Test

statistics

w1118/Y;UAS-HTTex1Q25-V5/Or47b-

Gal4;UAS-MapHS/+ & w1118/Y;UAS-

HTTex1Q91-V5/Or47b-Gal4;UAS-MapHS/+

Mann-

Whitney

0.000584277 t=3.82, df=32

Table 7.

Statistical information for the data shown in Figure 4

Figure 4C

Genotype

Values

Number of

animals

w1118/Y;+/+;UAS-pHluorin-

HTTex1Q25-tdTomato/Orco-

Gal4

0.86±0.03

7 (14 ORNs)

w1118/Y;+/+;UAS-pHluorin-

HTTex1Q91-tdTomato/Orco-

Gal4

0.72±0.02

10 (20 ORNs)

Comparison between

Genotype

Test used

P-value

Test

statistics

w1118/Y;+/+;UAS-pHluorin-

HTTex1Q25-tdTomato/Orco-Gal4

& w1118/Y;+/+;UAS-pHluorin-

HTTex1Q91-tdTomato/Orco-Gal4

Mann-

Whitney

0.000304582

=4.05, df=32

Figure 4F

Genotype

Values

Number of

animals

w1118/Y;+/+;UAS-pHluorin-

HTTex1Q25-

tdTomato/+;OK107-Gal4/+

0.91±0.02

10 (20 ORNs)

w1118/Y;+/+;UAS- pHluorin-

HTTex1Q91-

tdTomato/+;OK107-Gal4/+

0.72±0.02

8 (16 ORNs)

Comparison between

Genotype

Test used

P-value

Test

statistics

w1118/Y;+/+;UAS-pHluorin-

HTTex1Q25-tdTomato/Orco-Gal4

& w1118/Y;+/+;UAS-pHluorin-

HTTex1Q91-tdTomato/Orco-Gal4

Mann-

Whitney

3.00E-06

=5.58, df=34

Figure 4I

Genotype

Values

Number of

animals

W1118/Y;Repo-Cas9/+;+/+

104375±5086.8

5 (10 ORNs)

W1118/Y;Repo-

Cas9/+;gRNA-draper/+

14232±2657.3

5 (10 ORNs)

Comparison between

Genotype

Test used

P-value

Test

statistics

W1118/Y;Repo-Cas9/+;+/+ &

W1118/Y;Repo-Cas9/+;gRNA-

draper/+

Mann-

Whitney

2.20E-12

=16.66,

df=18

Figure 4L

Genotype

Values

Number of

animals

W1118/Y;Or47b-

Gal4/repo-Cas9;+/UAS-

UAS-pHluorin-

HTTex1Q91-tdTomato

1±0.032

9 (18 ORNs)

W1118/Y;Or47b-

Gal4/repo-Cas9;gRNA-

draper/UAS- UAS-

pHluorin-HTTex1Q91-

tdTomato

1.16±0.033

7 (14 ORNs)

Comparison between

Genotype

Test used

P-value

Test

statistics

W1118/Y;Or47b-Gal4/repo-

Cas9;+/UAS- UAS-pHluorin-

HTTex1Q91-tdTomato &

W1118/Y;Or47b-Gal4/repo-

Cas9;gRNA-draper/UAS- UAS-

pHluorin-HTTex1Q91-tdTomato

Mann-

Whitney

0.001369107

t=3.53, df=30

Table 8.

Statistical information for the data shown in Figure 5

Figure 5A

qPCR Target

Genotype

Uninjured

(-)

/Injured (+)

Values

Biological

Replicates

drpr-I

w1118; UAS-

GFP/+;orco-Gal4/+

1±0.046

1.2±0.015

w1118; UAS-

HTTex1Q25-

GFP/+;orco-Gal4/+

0.97±0.08

1.16±0.035

w1118; UAS-

HTTex1Q91-

GFP/+;orco-Gal4/+

1.17±0.057

1.07±0.032

toll-6

w1118; UAS-

GFP/+;orco-Gal4/+

1±0.027

1.12±0.018

w1118; UAS-

HTTex1Q25-

GFP/+;orco-Gal4/+

0.97±0.074

1.18±0.055

w1118; UAS-

HTTex1Q91-

GFP/+;orco-Gal4/+

1.20±0.059

0.94±0.057

dorsal

w1118; UAS-

GFP/+;orco-Gal4/+

1±0.27

1.90±0.031

w1118; UAS-

HTTex1Q25-

GFP/+;orco-Gal4/+

1.09±0.11

2.0±0.032

w1118; UAS-

HTTex1Q91-

GFP/+;orco-Gal4/+

1.03±0.053

1.40±0.10

mmp1

w1118; UAS-

GFP/+;orco-Gal4/+

1±0.039

3.66±0.043

w1118; UAS-

HTTex1Q25-

GFP/+;orco-Gal4/+

0.85±0.042

3.52±0.146

w1118; UAS-

HTTex1Q91-

GFP/+;orco-Gal4/+

0.82±0.018

2.28±0.064

relish

w1118; UAS-

GFP/+;orco-Gal4/+

1±0.072

20.06±5.82

w1118; UAS-

HTTex1Q25-

GFP/+;orco-Gal4/+

1.09±0.105

20.71±4.72

w1118; UAS-

HTTex1Q91-

GFP/+;orco-Gal4/+

1.51±0.081

12.16±2.30

ets21c

w1118; UAS-

GFP/+;orco-Gal4/+

1±0.101

29.73±0.543

w1118; UAS-

HTTex1Q25-

GFP/+;orco-Gal4/+

0.86±0.54

30.95±0.813

w1118; UAS-

HTTex1Q91-

GFP/+;orco-Gal4/+

0.69±0.018

23.37±1.129

Comparison between

qPCR

target

Genotype

Uninjured (-)

/Injured (+)

Test

used

P-value

Test

statistics

drpr-I

w1118; UAS-GFP/+;orco-Gal4/+

- & +

Mann-

Whitney 5.88326E-05

=5.40,
df=16

w1118; UAS-HTTex1Q25-

GFP/+;orco-Gal4/+

- & +

Mann-

Whitney 0.008881136

=2.98,
df=16

w1118; UAS-HTTex1Q91-

GFP/+;orco-Gal4/+

- & +

Mann-

Whitney 0.319078973

=1.03,
df=16

toll-6

w1118; UAS-GFP/+;orco-Gal4/+

- & +

Mann-

Whitney 0.004132991

=3.34,
df=16

w1118; UAS-HTTex1Q25-

GFP/+;orco-Gal4/+

- & +

Mann-

Whitney 0.003062499

=3.48,
df=16

w1118; UAS-HTTex1Q91-

GFP/+;orco-Gal4/+

- & +

Mann-

Whitney 0.006210947

=3.15,
df=16

dorsal

w1118; UAS-GFP/+;orco-Gal4/+

- & +

Mann-

Whitney

7.0739E-12

=17.55,

df=16

w1118; UAS-HTTex1Q25-

GFP/+;orco-Gal4/+

- & +

Mann-

Whitney 2.21319E-08

=10.16,

df=16

w1118; UAS-HTTex1Q91-

GFP/+;orco-Gal4/+

- & +

Mann-

Whitney 0.005815647

=3.18,
df=16

mmp1

w1118; UAS-GFP/+;orco-Gal4/+

- & +

Mann-

Whitney

9.1496E-15

=29.97,

df=16

w1118; UAS-HTTex1Q25-

GFP/+;orco-Gal4/+

- & +

Mann-

Whitney

5.9892E-12

=17.75,

df=16

w1118; UAS-HTTex1Q91-

GFP/+;orco-Gal4/+

- & +

Mann-

Whitney

8.86E-11

=14.85,

df=16

relish

w1118; UAS-GFP/+;orco-Gal4/+

- & +

Mann-

Whitney 3.08303E-10

=13.66,

df=16

w1118; UAS-HTTex1Q25-

GFP/+;orco-Gal4/+

- & +

Mann-

Whitney 1.95994E-10

=14.08,

df=16

w1118; UAS-HTTex1Q91-

GFP/+;orco-Gal4/+

- & +

Mann-

Whitney 6.95961E-09

=11.02,

df=16

ets21c

w1118; UAS-GFP/+;orco-Gal4/+

- & +

Mann-

Whitney 2.29846E-19

=52.67,

df=16

w1118; UAS-HTTex1Q25-

GFP/+;orco-Gal4/+

- & +

Mann-

Whitney

1.2254E-15

=30.65,

df=16

w1118; UAS-HTTex1Q91-

GFP/+;orco-Gal4/+

- & +

Mann-

Whitney 5.45362E-12

=17.86,

df=16

drpr-I

w1118; UAS-GFP/+;orco-Gal4/+

& w1118; UAS-HTTex1Q25-

GFP/+;orco-Gal4/+ & w1118;

UAS-HTTex1Q91-GFP/+;orco-

Gal4/+

One-

way

ANOVA

0.004843257

=6.71,
df=24

w1118; UAS-GFP/+;orco-Gal4/+

& w1118; UAS-HTTex1Q25-

GFP/+;orco-Gal4/+ & w1118;

UAS-HTTex1Q91-GFP/+;orco-

Gal4/+

One-

way

ANOVA

0.004881337

=6.93,
df=24

toll-6

w1118; UAS-GFP/+;orco-Gal4/+

& w1118; UAS-HTTex1Q25-

GFP/+;orco-Gal4/+ & w1118;

UAS-HTTex1Q91-GFP/+;orco-

Gal4/+

One-

way

ANOVA

0.003380467

=7.28,
df=24

w1118; UAS-GFP/+;orco-Gal4/+

& w1118; UAS-HTTex1Q25-

GFP/+;orco-Gal4/+ & w1118;

UAS-HTTex1Q91-GFP/+;orco-

Gal4/+

One-

way

ANOVA

0.003657762

=7.15,
df=24

dorsal

w1118; UAS-GFP/+;orco-Gal4/+

& w1118; UAS-HTTex1Q25-

0.595137134

=0.53,
df=24

GFP/+;orco-Gal4/+ & w1118;

UAS-HTTex1Q91-GFP/+;orco-

Gal4/+

One-

way

ANOVA

w1118; UAS-GFP/+;orco-Gal4/+

& w1118; UAS-HTTex1Q25-

GFP/+;orco-Gal4/+ & w1118;

UAS-HTTex1Q91-GFP/+;orco-

Gal4/+

One-

way

ANOVA

2.0958E-06

=23.68,

df=24

mmp1

w1118; UAS-GFP/+;orco-Gal4/+

& w1118; UAS-HTTex1Q25-

GFP/+;orco-Gal4/+ & w1118;

UAS-HTTex1Q91-GFP/+;orco-

Gal4/+

One-

way

ANOVA

0.109035328

=2.43,
df=24

w1118; UAS-GFP/+;orco-Gal4/+

& w1118; UAS-HTTex1Q25-

GFP/+;orco-Gal4/+ & w1118;

UAS-HTTex1Q91-GFP/+;orco-

Gal4/+

One-

way

ANOVA

2.52E-09

=58.70,

df=24

relish

w1118; UAS-GFP/+;orco-Gal4/+

& w1118; UAS-HTTex1Q25-

GFP/+;orco-Gal4/+ & w1118;

UAS-HTTex1Q91-GFP/+;orco-

Gal4/+

One-

way

ANOVA

0.001920734

=8.69,
df=24

w1118; UAS-GFP/+;orco-Gal4/+

& w1118; UAS-HTTex1Q25-

GFP/+;orco-Gal4/+ & w1118;

UAS-HTTex1Q91-GFP/+;orco-

Gal4/+

One-

way

ANOVA

2.00856E-05

=18.91,

df=24

ets21c

w1118; UAS-GFP/+;orco-Gal4/+

& w1118; UAS-HTTex1Q25-

GFP/+;orco-Gal4/+ & w1118;

UAS-HTTex1Q91-GFP/+;orco-

Gal4/+

One-

way

ANOVA

0.01295054

=5.24,
df=24

w1118; UAS-GFP/+;orco-Gal4/+

& w1118; UAS-HTTex1Q25-

GFP/+;orco-Gal4/+ & w1118;

One-

way

ANOVA

0.000148302

=13.01,

df=24

UAS-HTTex1Q91-GFP/+;orco-

Gal4/+

Figure 5E

Genotype

Values

Number of

animals

w1118/Y; UAS-

HTTex1Q25-

V5/+;orco-

Gal4/Toll6(MIMICGFP)

371.84±12.60 10 (19 ORNs)

w1118/Y; UAS-

HTTex1Q91-

V5/+;orco-

Gal4/Toll6(MIMICGFP)

416.4±13.92

8 (15 ORNs)

Comparison between

Genotype

Test used

P-value

Test

statistics

w1118/Y; UAS-

HTTex1Q25-V5/+;orco-

Gal4/Toll6(MIMICGFP) &

w1118/Y; UAS-

HTTex1Q91-V5/+;orco-

Gal4/Toll6(MIMICGFP)

Mann-

Whitney

0.024140756

t=2.37, df=32

Figure 5H

Genotype

Values

Number of

animals

w1118/Y; UAS-

HTTex1Q25-

V5/+;orco-Gal4/Jra-

GFP

361.14±13.73

11 (21 ORNs)

w1118/Y; UAS-

HTTex1Q91-

V5/+;orco-Gal4/Jra-

GFP

491±20.21

8 (15 ORNs)

Comparison between

Genotype

Test used

P-value

Test

statistics

w1118/Y; UAS-

HTTex1Q25-V5/+;orco-

Gal4/Jra-GFP & w1118/Y;

UAS-HTTex1Q91-

V5/+;orco-Gal4/Jra-GFP

Mann-

Whitney

3.68E-06

t=5.51, df=34

Table 9.

Statistical information for the data shown in Figure 6

Figure 6A-E

qPCR Target

Genotype

Uninjured (-)

/Injured (+)

Values

Biological

Replicates

drosomycin

w1118; UAS-

HTTex1Q25-

GFP/+;orco-Gal4/+

1.00±0.18

27.44±2.64

w1118; UAS-

HTTex1Q91-

GFP/+;orco-Gal4/+

3.08±0.81

18.27±1.60

attacinA

w1118; UAS-

HTTex1Q25-

GFP/+;orco-Gal4/+

1.0±0.37

55.02±4.56

w1118; UAS-

HTTex1Q91-

GFP/+;orco-Gal4/+

1.01±0.21

38.15±1.78

attacinD

w1118; UAS-

HTTex1Q25-

GFP/+;orco-Gal4/+

1.00±0.43

8.04±2.63

w1118; UAS-

HTTex1Q91-

GFP/+;orco-Gal4/+

2.69±0.39

9.27±2.80

diptericinA

w1118; UAS-

HTTex1Q25-

GFP/+;orco-Gal4/+

1.00±0.57

9.99±1.3

w1118; UAS-

HTTex1Q91-

GFP/+;orco-Gal4/+

1.09±0.91

8.97±0.58

metchnikowin

w1118; UAS-

HTTex1Q25-

GFP/+;orco-Gal4/+

1.00±0.34

6.78±0.57

w1118; UAS-

HTTex1Q91-

GFP/+;orco-Gal4/+

1.03±0.26

5.96±0.11

Comparison between

qPCR target

Genotype(s)

Uninjured (-)

/Injured (+)

Test

used

P-value

Test

statistics

drosomycin

w1118; UAS-HTTex1Q25-

GFP/+;orco-Gal4/+

- & +

Mann-

Whitney 1.86245E-08

=10.28,

df=16

w1118; UAS-HTTex1Q91-

GFP/+;orco-Gal4/+

- & +

Mann-

Whitney

4.1079E-07

=8.19,
df=16

w1118; UAS-HTTex1Q25-

GFP/+;orco-Gal4/+ &

w1118; UAS-HTTex1Q91-

GFP/+;orco-Gal4/+

- & -

Mann-

Whitney 0.007494373

=3.06,
df=16

w1118; UAS-HTTex1Q25-

GFP/+;orco-Gal4/+ &

w1118; UAS-HTTex1Q91-

GFP/+;orco-Gal4/+

+ & +

Mann-

Whitney 0.005336573

=3.22,
df=16

attacinA

w1118; UAS-HTTex1Q25-

GFP/+;orco-Gal4/+

- & +

Mann-

Whitney

1.9821E-09

=12.03,

df=16

w1118; UAS-HTTex1Q91-

GFP/+;orco-Gal4/+

- & +

Mann-

Whitney

5.2746E-13

=20.79,

df=16

w1118; UAS-HTTex1Q25-

GFP/+;orco-Gal4/+ &

w1118; UAS-HTTex1Q91-

GFP/+;orco-Gal4/+

- & -

Mann-

Whitney 0.356588308

=0.95,
df=16

w1118; UAS-HTTex1Q25-

GFP/+;orco-Gal4/+ &

w1118; UAS-HTTex1Q91-

GFP/+;orco-Gal4/+

+ & +

Mann-

Whitney 0.001888713

=3.71,
df=16

attacinD

w1118; UAS-HTTex1Q25-

GFP/+;orco-Gal4/+

- & +

Mann-

Whitney 0.004980191

=3.25,
df=16

w1118; UAS-HTTex1Q91-

GFP/+;orco-Gal4/+

- & +

Mann-

Whitney 0.009781331

=2.93,
df=16

w1118; UAS-HTTex1Q25-

GFP/+;orco-Gal4/+ &

w1118; UAS-HTTex1Q91-

GFP/+;orco-Gal4/+

- & -

Mann-

Whitney 0.002969363

=3.50,
df=16

w1118; UAS-HTTex1Q25-

GFP/+;orco-Gal4/+ &

w1118; UAS-HTTex1Q91-

GFP/+;orco-Gal4/+

+ & +

Mann-

Whitney 0.726979581

=0.36,
df=16

diptericinA

w1118; UAS-HTTex1Q25-

GFP/+;orco-Gal4/+

- & +

Mann-

Whitney 1.35636E-05

=6.17,
df=16

w1118; UAS-HTTex1Q91-

GFP/+;orco-Gal4/+

- & +

Mann-

Whitney 5.22595E-05

=5.46,
df=16

w1118; UAS-HTTex1Q25-

GFP/+;orco-Gal4/+ &

w1118; UAS-HTTex1Q91-

GFP/+;orco-Gal4/+

- & -

Mann-

Whitney 0.584130106

=0.56,
df=16

w1118; UAS-HTTex1Q25-

GFP/+;orco-Gal4/+ &

w1118; UAS-HTTex1Q91-

GFP/+;orco-Gal4/+

+ & +

Mann-

Whitney 0.258371287

=1.17,
df=16

metchnikowin

w1118; UAS-HTTex1Q25-

GFP/+;orco-Gal4/+

- & +

Mann-

Whitney 1.82923E-07

=8.70,
df=16

w1118; UAS-HTTex1Q91-

GFP/+;orco-Gal4/+

- & +

Mann-

Whitney

9.204E-10

=12.68,

df=16

w1118; UAS-HTTex1Q25-

GFP/+;orco-Gal4/+ &

w1118; UAS-HTTex1Q91-

GFP/+;orco-Gal4/+

- & -

Mann-

Whitney 0.486977912

=0.71,
df=16

w1118; UAS-HTTex1Q25-

GFP/+;orco-Gal4/+ &

w1118; UAS-HTTex1Q91-

GFP/+;orco-Gal4/+

+ & +

Mann-

Whitney 0.227598391

=1.25,
df=16

Table 10.

Statistical information for the data shown in Figure 7

Figure 7C

Genotype

Values

Number of

animals

w1118/Y;QUAS-HTTex1Q25-

mCherry/+;orco-QF2/+

246040±17963 6 (12 ORNs)

w1118/Y;QUAS-HTTex1Q91-

mCherry/+;orco-QF2/+

291157±18097 6 (12 ORNs)

Comparison between

Genotype

Test used

P-value

Test

statistics

w1118/Y;UAS-HTTex1Q25-

GFP/+;orco-Gal4/+ & w1118/Y;UAS-

HTTex1Q91-GFP/+;orco-Gal4/+

Mann-

Whitney

0.0475

=2.10, df=22

Table 11.

Statistical information for the data shown in Figure 8

Figure 8C

Genotype

Values

Number of

animals

or67d-QF,QUAS-HTT

ex1

Q91-

mCherry/+;UAS-HTTex1Q25-

GFP/+;mz0709-Gal4/+

32.5±2.83

8 (16

ORNs)

or67d-QF,QUAS-HTT

ex1

Q91-

mCherry/+;UAS-HTTex1Q25-

GFP/+;alrm-Gal4/+

31.89±3.99

9 (18

ORNs)

Comparison between

Genotype

Test used

P-value

Test statistics

or67d-QF,QUAS-HTTex1Q91-

mCherry/+;UAS-HTTex1Q25-

GFP/+;mz0709-Gal4/+ & or67d-

QF,QUAS-HTTex1Q91-

mCherry/+;UAS-HTTex1Q25-

GFP/+;alrm-Gal4/+

Mann-

Whitney

0.90460363

=0.12, df=32

Figure 8D

Genotype

Values

Number of

animals

or67d-QF,QUAS-HTT

ex1

Q91-

mCherry/+;UAS-HTTex1Q25-

GFP/+;mz0709-Gal4/+

4.88±0.83

8 (16

ORNs)

or67d-QF,QUAS-HTT

ex1

Q91-

mCherry/+;UAS-HTTex1Q25-

GFP/+;alrm-Gal4/+

0.67±0.33

9 (18

ORNs)

Comparison between

Genotype

Test used

P-value

Test statistics

or67d-QF,QUAS-HTTex1Q91-

mCherry/+;UAS-HTTex1Q25-

GFP/+;mz0709-Gal4/+ & or67d-

QF,QUAS-HTTex1Q91-

mCherry/+;UAS-HTTex1Q25-

GFP/+;alrm-Gal4/+

Mann-

Whitney

1.93E-04

=4.21, df=32

Table 12.

Statistical information for the data shown in Figure 9

Figure 9B

Genotype

Values

Number of animals

or67d-QF,QUAS-HTT

ex1

Q91-

mCherry/+;+/+;mz0709-Gal4

57.5±3.71

10 (20 ORNs)

or67d-QF,QUAS-

HTTex1Q91-

mCherry/+;+/+;mz0709-

Gal4/UAS-RNAi-mCherry

58±2.32

10 (20 ORNs)

or67d-QF,QUAS-HTT

ex1

Q91-

mCherry/+;+/+;mz0709-Gal4

/UAS-RNAi-Drpr

81.5±2.18

12 (24 ORNs)

or67d-QF,QUAS-HTT

ex1

Q91-

mCherry/+;UAS-RNAi-Toll-

6(1)/+;mz0709-Gal4 /+

83.4±2.62

8 (16 ORNs)

or67d-QF,QUAS-HTT

ex1

Q91-

mCherry/+;+/+;mz0709-Gal4

/UAS-RNAi-Toll-6(2)

71.3±2.65

10 (20 ORNs)

or67d-QF,QUAS-HTT

ex1

Q91-

mCherry/+;+/+;mz0709-Gal4

/UAS-RNAi-Relish(1)

69.1±2.20

9 (18 ORNs)

or67d-QF,QUAS-HTT

ex1

Q91-

mCherry/+;+/+;mz0709-Gal4

/UAS-RNAi-Relish(2)

69.6±1.71

9 (18 ORNs)

or67d-QF,QUAS-HTT

ex1

Q91-

mCherry/+;UAS-RNAi-

NijA(1)/+;mz0709-Gal4 /+

97.4±4.04

9 (18 ORNs)

or67d-QF,QUAS-HTT

ex1

Q91-

mCherry/+;+/+;mz0709-Gal4

/UAS-RNAi-NijA(2)

71.8±2.71

9 (17 ORNs)

or67d-QF,QUAS-HTT

ex1

Q91-

mCherry/+;UAS-RNAi-

Dorsal(1)/+;mz0709-Gal4 /+

62.9±2.42

10 (20 ORNs)

or67d-QF,QUAS-HTT

ex1

Q91-

mCherry/+;+/+;mz0709-Gal4

/UAS-RNAi-Dorsal(2)

61.4±2.49

8 (16 ORNs)

or67d-QF,QUAS-HTT

ex1

Q91-

mCherry/+;+/+;mz0709-Gal4

/UAS-RNAi-Dorsal(3)

55.3±2.45

8 (16 ORNs)

or67d-QF,QUAS-HTT

ex1

Q91-

mCherry/+;+/+;mz0709-Gal4

/UAS-RNAi-Dorsal(4)

68.6±2.67

10 (20 ORNs)

or67d-QF,QUAS-HTT

ex1

Q91-

mCherry/+;+/+;mz0709-Gal4

/UAS-RNAi-Dorsal(5)

72.1±2.90

9 (18 ORNs)

Comparison between

Genotype

Test used

P-value

Test

statistics

or67d-QF,QUAS-HTTex1Q91-

mCherry/+;+/+;mz0709-Gal4 /+ & or67d-

QF,QUAS-HTTex1Q91-

mCherry/+;+/+;mz0709-Gal4/UAS-RNAi-

mCherry

Mann-Whitney 0.90968447

=0.11, df=38

or67d-QF,QUAS-HTTex1Q91-

mCherry/+;+/+;mz0709-Gal4 /+ & or67d-

QF,QUAS-HTTex1Q91-

mCherry/+;+/+;mz0709-Gal4/UAS-RNAi-

mCherry & or67d-QF,QUAS-HTTex1Q91-

mCherry/+;+/+;mz0709-Gal4 /UAS-RNAi-Drpr

One-Way

ANOVA

2.92E-09

=27.60, df=61

or67d-QF,QUAS-HTTex1Q91-

mCherry/+;+/+;mz0709-Gal4 /+ & or67d-

QF,QUAS-HTTex1Q91-

mCherry/+;+/+;mz0709-Gal4/UAS-RNAi-

mCherry & or67d-QF,QUAS-HTTex1Q91-

One-Way

ANOVA

8.93E-08

=22.39, df=53

mCherry/+;UAS-RNAi-Toll-6(1)/+;mz0709-

Gal4 /+

or67d-QF,QUAS-HTTex1Q91-

mCherry/+;+/+;mz0709-Gal4 /+ & or67d-

QF,QUAS-HTTex1Q91-

mCherry/+;+/+;mz0709-Gal4/UAS-RNAi-

mCherry & or67d-QF,QUAS-HTTex1Q91-

mCherry/+;+/+;mz0709-Gal4 /UAS-RNAi-Toll-

6(2)

One-Way

ANOVA

0.00189093

=7.01, df=57

or67d-QF,QUAS-HTTex1Q91-

mCherry/+;+/+;mz0709-Gal4 /+ & or67d-

QF,QUAS-HTTex1Q91-

mCherry/+;+/+;mz0709-Gal4/UAS-RNAi-

mCherry & or67d-QF,QUAS-HTTex1Q91-

mCherry/+;+/+;mz0709-Gal4 /UAS-RNAi-

Relish(1)

One-Way

ANOVA

0.0098065

=5.04, df=55

or67d-QF,QUAS-HTTex1Q91-

mCherry/+;+/+;mz0709-Gal4 /+ & or67d-

QF,QUAS-HTTex1Q91-

mCherry/+;+/+;mz0709-Gal4/UAS-RNAi-

mCherry & or67d-QF,QUAS-HTTex1Q91-

mCherry/+;+/+;mz0709-Gal4 /UAS-RNAi-

Relish(2)

One-Way

ANOVA

0.00484371

=5.88, df=55

or67d-QF,QUAS-HTTex1Q91-

mCherry/+;+/+;mz0709-Gal4 /+ & or67d-

QF,QUAS-HTTex1Q91-

mCherry/+;+/+;mz0709-Gal4/UAS-RNAi-

mCherry & or67d-QF,QUAS-HTTex1Q91-

mCherry/+;UAS-RNAi-NijA(1)/+;mz0709-Gal4

One-Way

ANOVA

4.20E-12

=43.79, df=55

or67d-QF,QUAS-HTTex1Q91-

mCherry/+;+/+;mz0709-Gal4 /+ & or67d-

QF,QUAS-HTTex1Q91-

mCherry/+;+/+;mz0709-Gal4/UAS-RNAi-

mCherry & or67d-QF,QUAS-HTTex1Q91-

mCherry/+;+/+;mz0709-Gal4 /UAS-RNAi-

NijA(2)

One-Way

ANOVA

0.0022

=6.88, df=55

or67d-QF,QUAS-HTTex1Q91-

mCherry/+;+/+;mz0709-Gal4 /+ & or67d-

QF,QUAS-HTTex1Q91-

mCherry/+;+/+;mz0709-Gal4/UAS-RNAi-

mCherry & or67d-QF,QUAS-HTTex1Q91-

mCherry/+;UAS-RNAi-Dorsal(1)/+;mz0709-

Gal4 /+

One-Way

ANOVA

0.3579969

=1.05, df=57

or67d-QF,QUAS-HTTex1Q91-

mCherry/+;+/+;mz0709-Gal4 /+ & or67d-

QF,QUAS-HTTex1Q91-

mCherry/+;+/+;mz0709-Gal4/UAS-RNAi-

mCherry & or67d-QF,QUAS-HTTex1Q91-

mCherry/+;+/+;mz0709-Gal4 /UAS-RNAi-

Dorsal(2)

One-Way

ANOVA

0.62350113

=0.48, df=53

or67d-QF,QUAS-HTTex1Q91-

mCherry/+;+/+;mz0709-Gal4 /+ & or67d-

QF,QUAS-HTTex1Q91-

mCherry/+;+/+;mz0709-Gal4/UAS-RNAi-

mCherry & or67d-QF,QUAS-HTTex1Q91-

mCherry/+;+/+;mz0709-Gal4 /UAS-RNAi-

Dorsal(3)

One-Way

ANOVA

0.79961967

=0.22, df=53

or67d-QF,QUAS-HTTex1Q91-

mCherry/+;+/+;mz0709-Gal4 /+ & or67d-

QF,QUAS-HTTex1Q91-

mCherry/+;+/+;mz0709-Gal4/UAS-RNAi-

mCherry & or67d-QF,QUAS-HTTex1Q91-

mCherry/+;+/+;mz0709-Gal4 /UAS-RNAi-

Dorsal(4)

One-Way

ANOVA

0.0155839

=4.48, df=57

or67d-QF,QUAS-HTTex1Q91-

mCherry/+;+/+;mz0709-Gal4 /+ & or67d-

QF,QUAS-HTTex1Q91-

mCherry/+;+/+;mz0709-Gal4/UAS-RNAi-

mCherry & or67d-QF,QUAS-HTTex1Q91-

mCherry/+;+/+;mz0709-Gal4 /UAS-RNAi-

Dorsal(5)

One-Way

ANOVA

0.00182238

=7.09, df=55

Figure 9D

Genotype

Temperature

(°C)

Values

Number of

animals

or67d-QF,QUAS-HTT

ex1

Q91-

mCherry/

;+/+;repo-

Gal4,tubPGal80

/UAS-RNAi-

mCherry

128.3±4.67

10 (20 ORNs)

130.8±5.46

10 (20 ORNs)

or67d-QF,QUAS-HTT

ex1

Q91-

mCherry/

;+/+;repo-

Gal4,tubPGal80

/UAS-RNAi-

Ets21c

132.5±2.76

10 (20 ORNs)

194±3.51

10 (20 ORNs)

Comparison between

Genotype

Temperature

Test

used

P-value

Test

statistics

or67d-QF,QUAS-HTTex1Q91-

mCherry/+;+/+;repo-

Gal4,tubPGal80ts/UAS-RNAi-mCherry

18 & 29

Mann-

Whitney

0.729999316

=0.348,

df=38

or67d-QF,QUAS-HTTex1Q91-

mCherry/+;+/+;repo-

Gal4,tubPGal80ts/UAS-RNAi-Ets21c

18 & 29

Mann-

Whitney

2.34E-16

=13.776,

df=38

or67d-QF,QUAS-HTTex1Q91-

mCherry/+;+/+;repo-

Gal4,tubPGal80ts/UAS-RNAi-mCherry

& or67d-QF,QUAS-HTTex1Q91-

mCherry/+;+/+;repo-

Gal4,tubPGal80ts/UAS-RNAi-Ets21c

Mann-

Whitney

0.443719166

=0.774,

df=38

or67d-QF,QUAS-HTTex1Q91-

mCherry/+;+/+;repo-

Gal4,tubPGal80ts/UAS-RNAi-mCherry

& or67d-QF,QUAS-HTTex1Q91-

mCherry/+;+/+;repo-

Gal4,tubPGal80ts/UAS-RNAi-Ets21c

Mann-

Whitney

7.26E-12

=9.730,

df=38

Figure 9E

Genotype

Values

Number of animals

or67d-QF,QUAS-HTT

ex1

Q91-

mCherry/+;+/+;mz0709-Gal4

2.28±0.049

10 (20 ORNs)

or67d-QF,QUAS-

HTTex1Q91-

mCherry/+;+/+;mz0709-

Gal4/UAS-RNAi-mCherry

2.34±0.046

10 (20 ORNs)

or67d-QF,QUAS-HTT

ex1

Q91-

mCherry/+;+/+;mz0709-Gal4

/UAS-RNAi-Relish(1)

2.35±0.032

9 (18 ORNs)

or67d-QF,QUAS-HTT

ex1

Q91-

mCherry/+;+/+;mz0709-Gal4

/UAS-RNAi-Relish(2)

2.51±0.049

9 (18 ORNs)

or67d-QF,QUAS-HTT

ex1

Q91-

mCherry/+;UAS-RNAi-Toll-

6(1)/+;mz0709-Gal4 /+

2.31±0.033

8 (16 ORNs)

or67d-QF,QUAS-HTT

ex1

Q91-

mCherry/+;+/+;mz0709-Gal4

/UAS-RNAi-Toll-6(2)

2.41±0.047

10 (20 ORNs)

or67d-QF,QUAS-HTT

ex1

Q91-

mCherry/+;+/+;mz0709-Gal4

/UAS-RNAi-Drpr

2.41±0.033

12 (24 ORNs)

or67d-QF,QUAS-HTT

ex1

Q91-

mCherry/+;UAS-RNAi-

NijA(1)/+;mz0709-Gal4 /+

2.96±0.044

9 (18 ORNs)

or67d-QF,QUAS-HTT

ex1

Q91-

mCherry/+;+/+;mz0709-Gal4

/UAS-RNAi-NijA(2)

2.78±0.044

9 (17 ORNs)

or67d-QF,QUAS-HTT

ex1

Q91-

mCherry/

;+/+;repo-

Gal4,tubPGal80

/UAS-RNAi-

Ets21c (18°C)

2.26±0.030

10 (20 ORNs)

or67d-QF,QUAS-HTT

ex1

Q91-

mCherry/

;+/+;repo-

Gal4,tubPGal80

/UAS-RNAi-

Ets21c (29°C)

2.32±0.027

10 (20 ORNs)

Comparison between

Genotype

Test used

P-value Test statistics

or67d-QF,QUAS-HTTex1Q91-

mCherry/+;+/+;mz0709-Gal4 /+ & or67d-

QF,QUAS-HTTex1Q91-mCherry/+;+/+;mz0709-

Gal4/UAS-RNAi-mCherry

Mann-

Whitney

0.3780

=0.89, df=38

or67d-QF,QUAS-HTTex1Q91-

mCherry/+;+/+;mz0709-Gal4 /+ & or67d-

QF,QUAS-HTTex1Q91-mCherry/+;+/+;mz0709-

Gal4/UAS-RNAi-mCherry & or67d-QF,QUAS-

HTTex1Q91-mCherry/+;+/+;mz0709-Gal4 /UAS-

RNAi-Relish(1)

One-Way

ANOVA

0.4994

=0.70, df=56

or67d-QF,QUAS-HTTex1Q91-

mCherry/+;+/+;mz0709-Gal4 /+ & or67d-

QF,QUAS-HTTex1Q91-mCherry/+;+/+;mz0709-

Gal4/UAS-RNAi-mCherry & or67d-QF,QUAS-

HTTex1Q91-mCherry/+;+/+;mz0709-Gal4 /UAS-

RNAi-Relish(2)

One-Way

ANOVA

0.0045

=5.96, df=56

or67d-QF,QUAS-HTTex1Q91-

mCherry/+;+/+;mz0709-Gal4 /+ & or67d-

QF,QUAS-HTTex1Q91-mCherry/+;+/+;mz0709-

Gal4/UAS-RNAi-mCherry & or67d-QF,QUAS-

HTTex1Q91-mCherry/+;UAS-RNAi-Toll-

6(1)/+;mz0709-Gal4 /+

One-Way

ANOVA

0.6192

=0.48, df=54

or67d-QF,QUAS-HTTex1Q91-

mCherry/+;+/+;mz0709-Gal4 /+ & or67d-

QF,QUAS-HTTex1Q91-mCherry/+;+/+;mz0709-

Gal4/UAS-RNAi-mCherry & or67d-QF,QUAS-

HTTex1Q91-mCherry/+;+/+;mz0709-Gal4 /UAS-

RNAi-Toll-6(2)

One-Way

ANOVA

0.1614

=1.88, df=58

or67d-QF,QUAS-HTTex1Q91-

mCherry/+;+/+;mz0709-Gal4 /+ & or67d-

QF,QUAS-HTTex1Q91-mCherry/+;+/+;mz0709-

Gal4/UAS-RNAi-mCherry & or67d-QF,QUAS-

HTTex1Q91-mCherry/+;+/+;mz0709-Gal4 /UAS-

RNAi-Drpr

One-Way

ANOVA

0.1032

=2.358, df=62

or67d-QF,QUAS-HTTex1Q91-

mCherry/+;+/+;mz0709-Gal4 /+ & or67d-

QF,QUAS-HTTex1Q91-mCherry/+;+/+;mz0709-

Gal4/UAS-RNAi-mCherry & or67d-QF,QUAS-

HTTex1Q91-mCherry/+;UAS-RNAi-

NijA(1)/+;mz0709-Gal4 /+

One-Way

ANOVA

<0.0001

=65.32, df=54

or67d-QF,QUAS-HTTex1Q91-

mCherry/+;+/+;mz0709-Gal4 /+ & or67d-

QF,QUAS-HTTex1Q91-mCherry/+;+/+;mz0709-

Gal4/UAS-RNAi-mCherry & or67d-QF,QUAS-

HTTex1Q91-mCherry/+;+/+;mz0709-Gal4 /UAS-

RNAi-NijA(2)

One-Way

ANOVA

<0.0001

=34.82, df=55

or67d-QF,QUAS-HTTex1Q91-

mCherry/+;+/+;repo-Gal4,tubPGal80ts/UAS-

RNAi-Ets21c (18°C & 29°C)

Mann-

Whitney

0.1207

=1.59, df=38

Table 13.

Statistical information for the data shown in Figure 10

Figure 10C

Genotype

Values

Number of

animals

w1118/Y;UAS-HTTex1Q25-

GFP/+;orco-Gal4/+

102.92±10.70 7 (14 ORNs)

w1118/Y;UAS-HTTex1Q91-

GFP/+;orco-Gal4/+

995.31±68.89 7 (14 ORNs)

co-localized w1118/Y;UAS-

HTTex1Q25-GFP/+;orco-Gal4/+

0.85±0.30

7 (14 ORNs)

co-localized w1118/Y;UAS-

HTTex1Q91-GFP/+;orco-Gal4/+

509.23±19.83 7 (14 ORNs)

Comparison between

Genotype

Test used

P-value

Test statistics

w1118/Y;UAS-HTTex1Q25-

GFP/+;orco-Gal4/+ &

w1118/Y;UAS-HTTex1Q91-

GFP/+;orco-Gal4/+

Mann-Whitney

2.47E-10

=9.93, df=32

co-localized w1118/Y;UAS-

HTTex1Q25-GFP/+;orco-

Gal4/+ & co-localized

w1118/Y;UAS-HTTex1Q91-

GFP/+;orco-Gal4/+

Mann-Whitney

8.46E-17

=19.06, df=32

Figure 10F

Genotype

Values

Number of

animals

w1118/Y;UAS-HTTex1Q25-

GFP/+;orco-Gal4/+

256.35±21.05 9 (17 ORNs)

w1118/Y;UAS-HTTex1Q91-

GFP/+;orco-Gal4/+

557.24±45.46 9 (17 ORNs)

co-localized w1118/Y;UAS-

HTTex1Q25-GFP/+;orco-Gal4/+

4±0.83

9 (17 ORNs)

co-localized w1118/Y;UAS-

HTTex1Q91-GFP/+;orco-Gal4/+

112.53±20.26 9 (17 ORNs)

Comparison between

Genotype

Test used

P-value

Test statistics

w1118/Y;UAS-HTTex1Q25-

GFP/+;orco-Gal4/+ &

w1118/Y;UAS-HTTex1Q91-

GFP/+;orco-Gal4/+

Mann-Whitney

1.06E-06

=6.33, df=32

co-localized w1118/Y;UAS-

HTTex1Q25-GFP/+;orco-

Gal4/+ & co-localized

w1118/Y;UAS-HTTex1Q91-

GFP/+;orco-Gal4/+

Mann-Whitney

7.12E-06

=5.59, df=32

Table 14.

Statistical information for the data shown in Figure 11

Figure 11G-H

Genotype

Values

Number of

animals

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS -LAMP1-

GFP;orco-QF2/repo-Gal4

898.65±40.26

10 (20

ORNs)

w1118/Y;QUAS-HTTex1Q91-

mCherry/UAS-LAMP1-

GFP;orco-QF2/repo-Gal4

1072.9±60.94

10 (20

ORNs)

co-localized w1118/Y;QUAS-

HTTex1Q25-mCherry/UAS-

LAMP1-GFP;orco-QF2/repo-

Gal4

0.9±0.18

10 (20

ORNs)

co-localized w1118/Y;QUAS-

HTTex1Q91-mCherry/UAS-

LAMP1-GFP;orco-QF2/repo-

Gal4

23.15±3.83

10 (20

ORNs)

Comparison between

Genotype

Test used

P-value

Test statistics

w1118/Y;QUAS-

HTTex1Q25-mCherry/UAS-

LAMP1-GFP;orco-QF2/repo-

Gal4 & w1118/Y;QUAS-

HTTex1Q91-mCherry/UAS-

LAMP1-GFP;orco-QF2/repo-

Gal4

Mann-Whitney

0.022139037

=2.405, df=38

co-localized w1118/Y;QUAS-

HTTex1Q25-mCherry/UAS-

LAMP1-GFP;orco-QF2/repo-

Gal4 & co-localized

w1118/Y;QUAS-

HTTex1Q91-mCherry/UAS-

LAMP1-GFP;orco-QF2/repo-

Gal4

Mann-Whitney

1.06E-06

=6.010, df=38

Figure 11I

Genotype

Values

Number of

animals

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS-LAMP1-

GFP;orco-QF2/repo-Gal4

0.33±0.008

10 (20

ORNs)

w1118/Y;QUAS-HTTex1Q91-

mCherry/UAS-LAMP1-

GFP;orco-QF2/repo-Gal4

0.33±0.015

10 (20

ORNs)

co-localized w1118/Y;QUAS-

HTTex1Q91-mCherry/UAS-

GFP-LAMP1;orco-QF2/repo-

Gal4

0.54±0.0.055

10 (20

ORNs)

Comparison between

Genotype

Test used

P-value

Test statistics

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS-LAMP1-GFP;orco-

QF2/repo-Gal4 &

w1118/Y;QUAS-HTTex1Q91-

mCherry/UAS-LAMP1-GFP;orco-

QF2/repo-Gal4

Mann-Whitney

0.960136447

=0.050, df=38

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS-LAMP1-GFP;orco-

QF2/repo-Gal4 & co-localized

w1118/Y;QUAS-HTTex1Q91-

mCherry/UAS-LAMP1-GFP;orco-

QF2/repo-Gal4

Mann-Whitney

7.51E-04

=3.726, df=38

w1118/Y;QUAS-HTTex1Q91-

mCherry/UAS-LAMP1-GFP;orco-

QF2/repo-Gal4 & co-localized

w1118/Y;QUAS-HTTex1Q91-

mCherry/UAS-LAMP1-GFP;orco-

QF2/repo-Gal4

Mann-Whitney

9.90E-04

=3.626, df=38

Figure 11G-H

Genotype

Values

Number of

animals

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS-GFP-

LAMP1;orco-QF2/repo-Gal4

444.19±50.70

8 (16 ORNs)

w1118/Y;QUAS-HTTex1Q91-

mCherry/UAS-GFP-

LAMP1;orco-QF2/repo-Gal4

736.88±53.90

8 (16 ORNs)

co-localized w1118/Y;QUAS-

HTTex1Q25-mCherry/UAS-

GFP-LAMP1;orco-QF2/repo-

Gal4

0.63±0.29

8 (16 ORNs)

co-localized w1118/Y;QUAS-

HTTex1Q91-mCherry/UAS-

GFP-LAMP1;orco-QF2/repo-

Gal4

30.5±3.24

8 (16 ORNs)

Comparison between

Genotype

Test used

P-value

Test statistics

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS-GFP-

LAMP1;orco-QF2/repo-Gal4

& w1118/Y;QUAS-

HTTex1Q91-mCherry/UAS-

GFP-LAMP1;orco-QF2/repo-

Gal4

Mann-Whitney

0.00096758

=3.66, df=30

co-localized w1118/Y;QUAS-

HTTex1Q25-mCherry/UAS-

GFP-LAMP1;orco-QF2/repo-

Gal4 & co-localized

w1118/Y;QUAS-HTTex1Q91-

mCherry/UAS-GFP-

LAMP1;orco-QF2/repo-Gal4

Mann-Whitney

3.20E-10

=9.18, df=30

Figure 5I

Genotype

Values

Number of

animals

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS-GFP-LAMP1;orco-

QF2/repo-Gal4

0.294±0.011 8 (16 ORNs)

w1118/Y;QUAS-HTTex1Q91-

mCherry/UAS-GFP-LAMP1;orco-

QF2/repo-Gal4

0.432±0.039 8 (16 ORNs)

co-localized w1118/Y;QUAS-

HTTex1Q91-mCherry/UAS-GFP-

LAMP1;orco-QF2/repo-Gal4

0.567±0.058 8 (16 ORNs)

Comparison between

Genotype

Test used

P-value

Test statistics

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS-GFP-

Mann-Whitney

0.002100074

=3.37, df=30

LAMP1;orco-QF2/repo-Gal4

& w1118/Y;QUAS-

HTTex1Q91-mCherry/UAS-

GFP-LAMP1;orco-QF2/repo-

Gal4

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS-GFP-

LAMP1;orco-QF2/repo-Gal4

& co-localized

w1118/Y;QUAS-HTTex1Q91-

mCherry/UAS-GFP-

LAMP1;orco-QF2/repo-Gal4

Mann-Whitney

6.22E-05

=4.65, df=30

w1118/Y;QUAS-

HTTex1Q91-mCherry/UAS-

GFP-LAMP1;orco-QF2/repo-

Gal4 & co-localized

w1118/Y;QUAS-HTTex1Q91-

mCherry/UAS-GFP-

LAMP1;orco-QF2/repo-Gal4

Mann-Whitney

6.25E-02

=1.93, df=30

Table 15.

Statistical information for the data shown in Figure 12

Figure 12E

Genotype

Values

Number of

animals

QUAS-HTTex1Q25-

GFP/+;+/+;orco-QF2,repo-

Gal4/UAS-mCherry-LGals3

566.0±100.3

10 (20 ORNs)

QUAS-HTTex1Q91-

GFP/+;+/+;orco-QF2,repo-

Gal4/UAS-mCherry-LGals3

1072.6±98.56

10 (20 ORNs)

QUAS-HTTex1Q25-

GFP/+;+/+;orco-QF2,repo-

Gal4/UAS-mCherry-LGals8

849.7±113

10 (20 ORNs)

QUAS-HTTex1Q91-

GFP/+;+/+;orco-QF2,repo-

Gal4/UAS-mCherry-LGals8

1271±99.8

10 (20 ORNs)

Comparison between

Genotype

Test used

P-value

Test statistics

QUAS-HTTex1Q25-

GFP/+;+/+;orco-QF2,repo-

Gal4/UAS-mCherry-LGals3 &

QUAS-HTTex1Q91-

GFP/+;+/+;orco-QF2,repo-

Gal4/UAS-mCherry-LGals3

Mann-Whitney

9.16E-04

=3.596,

=38

QUAS-HTTex1Q25-

GFP/+;+/+;orco-QF2,repo-

Gal4/UAS-mCherry-LGals8 &

QUAS-HTTex1Q91-

GFP/+;+/+;orco-QF2,repo-

Gal4/UAS-mCherry-LGals8

Mann-Whitney

8.08E-03

=12.301, df=38

Figure 12F

Genotype

Values

Number of

animals

colocalized QUAS-HTTex1Q25-

GFP/+;+/+;orco-QF2,repo-

Gal4/UAS-mCherry-LGals3

0.95±0.31

10 (20 ORNs)

colocalized QUAS-HTTex1Q91-

GFP/+;+/+;orco-QF2,repo-

Gal4/UAS-mCherry-LGals3

29.35±2.4

10 (20 ORNs)

colocalized QUAS-HTTex1Q25-

GFP/+;+/+;orco-QF2,repo-

Gal4/UAS-mCherry-LGals8

0.18±0.08

10 (20 ORNs)

colocalized QUAS-HTTex1Q91-

GFP/+;+/+;orco-QF2,repo-

Gal4/UAS-mCherry-LGals8

35.25±4.76

10 (20 ORNs)

Comparison between

Genotype

Test used

P-value

Test statistics

colocalized QUAS-HTTex1Q25-

GFP/+;+/+;orco-QF2,repo-

Gal4/UAS-mCherry-LGals3 &

colocalized QUAS-HTTex1Q91-

GFP/+;+/+;orco-QF2,repo-

Gal4/UAS-mCherry-LGals3

Mann-

Whitney

1.57736E-14

=2.796, df=38

colocalized QUAS-HTTex1Q25-

GFP/+;+/+;orco-QF2,repo-

Gal4/UAS-mCherry-LGals8 &

colocalized QUAS-HTTex1Q91-

GFP/+;+/+;orco-QF2,repo-

Gal4/UAS-mCherry-LGals8

Mann-

Whitney

1.81E-11

=9.409, df=38

Figure 12G

Genotype

Values

Number of

animals

QUAS-HTTex1Q25-GFP/+;+/+;orco-

QF2,repo-Gal4/UAS-mCherry-LGals3

.24±0.022

10 (20 ORNs)

QUAS-HTTex1Q91-GFP/+;+/+;orco-

QF2,repo-Gal4/UAS-mCherry-LGals3

0.25±0.014

10 (20 ORNs)

colocalized QUAS-HTTex1Q91-

GFP/+;+/+;orco-QF2,repo-Gal4/UAS-

mCherry-LGals3

0.7±0.044

10 (20 ORNs)

QUAS-HTTex1Q25-GFP/+;+/+;orco-

QF2,repo-Gal4/UAS-mCherry-LGals8

0.18±0.006

10 (20 ORNs)

QUAS-HTTex1Q91-GFP/+;+/+;orco-

QF2,repo-Gal4/UAS-mCherry-LGals8

0.2±0.014

10 (20 ORNs)

colocalized QUAS-HTTex1Q91-

GFP/+;+/+;orco-QF2,repo-Gal4/UAS-

mCherry-LGals8

0.47±0.0.071

10 (20 ORNs)

Comparison between

Genotype

Test used

P-value

Test

statistics

0.560724635

QUAS-HTTex1Q25-GFP/+;+/+;orco-QF2,repo-

Gal4/UAS-mCherry-LGals3 & QUAS-HTTex1Q91-

GFP/+;+/+;orco-QF2,repo-Gal4/UAS-mCherry-

LGals3

Mann-

Whitney

=0.586,

df=38

QUAS-HTTex1Q25-GFP/+;+/+;orco-QF2,repo-

Gal4/UAS-mCherry-LGals3 & colocalized QUAS-

HTTex1Q91-GFP/+;+/+;orco-QF2,repo-Gal4/UAS-

mCherry-LGals3

Mann-

Whitney

1.42E-08

=7.176,

df=38

QUAS-HTTex1Q91-GFP/+;+/+;orco-QF2,repo-

Gal4/UAS-mCherry-LGals3 & colocalized QUAS-

HTTex1Q91-GFP/+;+/+;orco-QF2,repo-Gal4/UAS-

mCherry-LGals3

Mann-

Whitney

9.60E-12

=9.631,

df=38

QUAS-HTTex1Q25-GFP/+;+/+;orco-QF2,repo-

Gal4/UAS-mCherry-LGals8 & QUAS-HTTex1Q91-

GFP/+;+/+;orco-QF2,repo-Gal4/UAS-mCherry-

LGals8

Mann-

Whitney

0.084809466

=1.77,
df=38

QUAS-HTTex1Q25-GFP/+;+/+;orco-QF2,repo-

Gal4/UAS-mCherry-LGals8 & colocalized QUAS-

HTTex1Q91-GFP/+;+/+;orco-QF2,repo-Gal4/UAS-

mCherry-LGals8

Mann-

Whitney

2.95E-06

=5.48,
df=38

QUAS-HTTex1Q91-GFP/+;+/+;orco-QF2,repo-

Gal4/UAS-mCherry-LGals8 & colocalized QUAS-

HTTex1Q91-GFP/+;+/+;orco-QF2,repo-Gal4/UAS-

mCherry-LGals8

Mann-

Whitney

1.06E-03

=3.545,

df=38

Table 16.

Statistical information for the data shown in Figure 13

Figure 13B

Genotype

Values

Number of

animals

UAS-HTT

ex1

Q25-GFP/Or67d-QF,QUAS-HTT

ex1

Q91-

mCherry;+/+;repo-Gal4/UAS-RNAi.mCherry

60.22±3.97

9 (18

ORNs)

UAS-HTT

ex1

Q25-GFP/Or67d-QF,QUAS-HTT

ex1

Q91-

mCherry;+/+;repo-Gal4/UAS-RNAi.Rbcn-3a

52.61±3.17

9 (18

ORNs)

UAS-HTT

ex1

Q25-GFP/Or67d-QF,QUAS-HTT

ex1

Q91-

mCherry;+/+;repo-Gal4/UAS-RNAi.Spinster

56.72±2.99

9 (18

ORNs)

UAS-HTT

ex1

Q25-GFP/Or67d-QF,QUAS-HTT

ex1

Q91-

mCherry;UAS-RNAi.Vha100-2/+;repo-Gal4/+

55.81±2.46

8 (16

ORNs)

UAS-HTT

ex1

Q25-GFP/Or67d-QF,QUAS-HTT

ex1

Q91-

mCherry;UAS-RNAi.Vha16-1/+;repo-Gal4/+

70.38±3.05

9 (18

ORNs)

UAS-HTT

ex1

Q25-GFP/Or67d-QF,QUAS-HTT

ex1

Q91-

mCherry;UAS-RNAi.Vha68-3/+;repo-Gal4/+

51.5±2.95

9 (18

ORNs)

Comparison between

Genotype

Test used

P-value

Test statistics

UAS-HTTex1Q25-GFP/Or67d-QF,QUAS-

HTTex1Q91-mCherry;+/+;repo-Gal4/UAS-

RNAi.mCherry & UAS-HTTex1Q25-

GFP/Or67d-QF,QUAS-HTTex1Q91-

mCherry;+/+;repo-Gal4/UAS-RNAi.Rbcn-3a

Mann-

Whitney

0.1550150

=1.45, df=34

UAS-HTTex1Q25-GFP/Or67d-QF,QUAS-

HTTex1Q91-mCherry;+/+;repo-Gal4/UAS-

RNAi.mCherry & UAS-HTTex1Q25-

GFP/Or67d-QF,QUAS-HTTex1Q91-

mCherry;+/+;repo-Gal4/UAS-RNAi.Spinster

Mann-

Whitney

0.5142619

=0.66, df=34

UAS-HTTex1Q25-GFP/Or67d-QF,QUAS-

HTTex1Q91-mCherry;+/+;repo-Gal4/UAS-

RNAi.mCherry & UAS-HTTex1Q25-

GFP/Or67d-QF,QUAS-HTTex1Q91-

mCherry;UAS-RNAi.Vha100-2/+;repo-Gal4/+

Mann-

Whitney

0.3911078

=0.87, df=32

UAS-HTTex1Q25-GFP/Or67d-QF,QUAS-

HTTex1Q91-mCherry;+/+;repo-Gal4/UAS-

RNAi.mCherry & UAS-HTTex1Q25-

GFP/Or67d-QF,QUAS-HTTex1Q91-

mCherry;UAS-RNAi.Vha16-1/+;repo-Gal4/+

Mann-

Whitney

0.0353079

=2.19, df=34

UAS-HTTex1Q25-GFP/Or67d-QF,QUAS-

HTTex1Q91-mCherry;+/+;repo-Gal4/UAS-

RNAi.mCherry & UAS-HTTex1Q25-

GFP/Or67d-QF,QUAS-HTTex1Q91-

mCherry;UAS-RNAi.Vha68-3/+;repo-Gal4/+

Mann-

Whitney

0.0950188

=1.72, df=34

Figure 13C

Genotype

Values

Number of

animals

UAS-HTT

ex1

Q25-GFP/Or67d-QF,QUAS-HTT

ex1

Q91-

mCherry;+/+;repo-Gal4/UAS-RNAi.mCherry

5.78±0.55

9 (18

ORNs)

UAS-HTT

ex1

Q25-GFP/Or67d-QF,QUAS-HTT

ex1

Q91-

mCherry;+/+;repo-Gal4/UAS-RNAi.Rbcn-3a

7.94±0.70

9 (18

ORNs)

UAS-HTT

ex1

Q25-GFP/Or67d-QF,QUAS-HTT

ex1

Q91-

mCherry;+/+;repo-Gal4/UAS-RNAi.Spinster

7.72±0.75

9 (18

ORNs)

UAS-HTT

ex1

Q25-GFP/Or67d-QF,QUAS-HTT

ex1

Q91-

mCherry;UAS-RNAi.Vha100-2/+;repo-Gal4/+

6.81±0.85

8 (16

ORNs)

UAS-HTT

ex1

Q25-GFP/Or67d-QF,QUAS-HTT

ex1

Q91-

mCherry;UAS-RNAi.Vha16-1/+;repo-Gal4/+

4.69±0.91

9 (18

ORNs)

UAS-HTT

ex1

Q25-GFP/Or67d-QF,QUAS-HTT

ex1

Q91-

mCherry;UAS-RNAi.Vha68-3/+;repo-Gal4/+

9.67±0.83

9 (18

ORNs)

Comparison between

Genotype

Test used

P-value

Test statistics

UAS-HTTex1Q25-GFP/Or67d-QF,QUAS-

HTTex1Q91-mCherry;+/+;repo-Gal4/UAS-

RNAi.mCherry & UAS-HTTex1Q25-

GFP/Or67d-QF,QUAS-HTTex1Q91-

mCherry;+/+;repo-Gal4/UAS-RNAi.Rbcn-3a

Mann-Whitney 0.0207176

=2.43, df=34

UAS-HTTex1Q25-GFP/Or67d-QF,QUAS-

HTTex1Q91-mCherry;+/+;repo-Gal4/UAS-

RNAi.mCherry & UAS-HTTex1Q25-

GFP/Or67d-QF,QUAS-HTTex1Q91-

mCherry;+/+;repo-Gal4/UAS-RNAi.Spinster

Mann-Whitney 0.0441169

=2.09, df=34

UAS-HTTex1Q25-GFP/Or67d-QF,QUAS-

HTTex1Q91-mCherry;+/+;repo-Gal4/UAS-

RNAi.mCherry & UAS-HTTex1Q25-

GFP/Or67d-QF,QUAS-HTTex1Q91-

mCherry;UAS-RNAi.Vha100-2/+;repo-Gal4/+

Mann-Whitney 0.3054543

=1.04, df=32

UAS-HTTex1Q25-GFP/Or67d-QF,QUAS-

HTTex1Q91-mCherry;+/+;repo-Gal4/UAS-

RNAi.mCherry & UAS-HTTex1Q25-

GFP/Or67d-QF,QUAS-HTTex1Q91-

mCherry;UAS-RNAi.Vha16-1/+;repo-Gal4/+

Mann-Whitney 0.3001935

=1.05, df=34

UAS-HTTex1Q25-GFP/Or67d-QF,QUAS-

HTTex1Q91-mCherry;+/+;repo-Gal4/UAS-

RNAi.mCherry & UAS-HTTex1Q25-

GFP/Or67d-QF,QUAS-HTTex1Q91-

mCherry;UAS-RNAi.Vha68-3/+;repo-Gal4/+

Mann-Whitney 0.0004219

=3.91, df=34

Table 17.

Statistical information for the data shown in Figure 14

Figure 14B

Genotype

Label

Mean

SEM

# brain

hemispheres (n)

Or67d-QF,UAS-HTT

ex1

Q25-GFP/QUAS-

HTT

ex1

Q91-mCherry ; +/+ ; repo-Gal4/+

no RNAi

60.880

4.051

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

FFLuc

RNAi

FFLuc

RNAi

69.000

2.662

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

mCherry

RNAi

mCherry

RNAi

59.780

1.722

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

drprRNAi

drpr

RNAi

102.800

4.795

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab2RNAi#1

Rab2

RNAi#1

77.000

3.000

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab2RNAi#2

Rab2

RNAi#2

65.000

3.229

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab3RNAi

Rab3

RNAi

72.500

4.175

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab4RNAi

Rab4

RNAi

75.630

2.129

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab5RNAi

Rab5

RNAi

63.500

3.229

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab6RNAi#1

Rab6

RNAi#1

58.500

3.868

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab6RNAi#2

Rab6

RNAi#2

64.180

3.722

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab7RNAi

Rab7

RNAi

65.380

2.652

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab8RNAi#1

Rab8

RNAi#1

60.250

3.075

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab8RNAi#2

Rab8

RNAi#2

53.900

3.167

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab9RNAi#1

Rab9

RNAi#1

63.850

3.521

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab9RNAi#2

Rab9

RNAi#2

62.000

1.902

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab9DbRNAi

Rab9Db

RNAi

64.810

4.467

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab10RNAi

Rab10

RNAi

68.250

2.462

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab14RNAi

Rab14

RNAi

81.790

2.634

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab18RNAi#1

Rab18

RNAi#1

62.200

4.090

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab18RNAi#2

Rab18

RNAi#2

60.080

1.944

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab19RNAi

Rab19

RNAi

67.000

3.893

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab21RNAi

Rab21

RNAi

66.110

4.436

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab23RNAi#1

Rab23

RNAi#1

60.750

6.723

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab23RNAi#2

Rab23

RNAi#2

61.580

3.619

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab23RNAi#3

Rab23

RNAi#3

64.500

4.387

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab23RNAi#4

Rab23

RNAi#4

66.670

4.246

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab27RNAi

Rab27

RNAi

84.110

5.846

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab32RNAi#1

Rab32

RNAi#1

67.000

2.898

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab32RNAi#2

Rab32

RNAi#2

68.440

4.073

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab35RNAi#1

Rab35

RNAi#1

71.270

3.705

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab35RNAi#2

Rab35

RNAi#2

65.890

1.594

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab39RNAi

Rab39

RNAi

64.140

5.343

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab40RNAi

Rab40

RNAi

65.360

4.551

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rabX2RNAi

RabX2

RNAi

56.440

5.460

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rabX4RNAi

RabX4

RNAi

74.700

5.377

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rabX6RNAi#1

RabX6

RNAi#1

61.000

2.226

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rabX6RNAi#2

RabX6

RNAi#2

63.220

4.471

one-way ANOVA:

F (37, 353) = 5.877

Dunnett's multiple comparisons:

Genotypes

p-value

FFLuc

RNAi

vs. no RNAi

0.9466

FFLuc

RNAi

vs. mCherry

RNAi

0.7814

no RNAi vs. mCherry

RNAi

0.9997

FFLuc

RNAi

vs. drpr

RNAi

<0.0001

FFLuc

RNAi

vs. Rab2

RNAi#1

0.9549

FFLuc

RNAi

vs. Rab2

RNAi#2

0.9991

FFLuc

RNAi

vs. Rab3

RNAi

0.9992

FFLuc

RNAi

vs. Rab4

RNAi

0.9924

FFLuc

RNAi

vs. Rab5

RNAi

0.9985

FFLuc

RNAi

vs. Rab6

RNAi#1

0.6401

FFLuc

RNAi

vs. Rab6

RNAi#2

0.9986

FFLuc

RNAi

vs. Rab7

RNAi

0.9989

FFLuc

RNAi

vs. Rab8

RNAi#1

0.8927

FFLuc

RNAi

vs. Rab8

RNAi#2

0.0489

FFLuc

RNAi

vs. Rab9

RNAi#1

0.9983

FFLuc

RNAi

vs. Rab9

RNAi#2

0.9924

FFLuc

RNAi

vs. Rab9Db

RNAi

0.9986

FFLuc

RNAi

vs. Rab10

RNAi

0.9999

FFLuc

RNAi

vs. Rab14

RNAi

0.0833

FFLuc

RNAi

vs. Rab18

RNAi#1

0.9821

FFLuc

RNAi

vs. Rab18

RNAi#2

0.6838

FFLuc

RNAi

vs. Rab19

RNAi

0.9996

FFLuc

RNAi

vs. Rab21

RNAi

0.9994

FFLuc

RNAi

vs. Rab23

RNAi#1

0.9378

FFLuc

RNAi

vs. Rab23

RNAi#2

0.9158

FFLuc

RNAi

vs. Rab23

RNAi#3

0.9989

FFLuc

RNAi

vs. Rab23

RNAi#4

0.9995

FFLuc

RNAi

vs. Rab27

RNAi

0.0681

FFLuc

RNAi

vs. Rab32

RNAi#1

0.9995

FFLuc

RNAi

vs. Rab32

RNAi#2

0.9999

FFLuc

RNAi

vs. Rab35

RNAi#1

0.9995

FFLuc

RNAi

vs. Rab35

RNAi#2

0.9993

FFLuc

RNAi

vs. Rab39

RNAi

0.9989

FFLuc

RNAi

vs. Rab40

RNAi

0.9991

FFLuc

RNAi

vs. RabX2

RNAi

0.2581

FFLuc

RNAi

vs. RabX4

RNAi

0.9982

FFLuc

RNAi

vs. RabX6

RNAi#1

0.9029

FFLuc

RNAi

vs. RabX6

RNAi#2

0.9983

Figure 14C

Genotype

Label

Mean

SEM

# brain

hemispheres (n)

Or67d-QF,UAS-HTT

ex1

Q25-GFP/QUAS-

HTT

ex1

Q91-mCherry ; +/+ ; repo-Gal4/+

no RNAi

7.125

1.315

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

FFLuc

RNAi

FFLuc

RNAi

6.571

0.638

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

mCherry

RNAi

mCherry

RNAi

6.556

0.915

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

drprRNAi

drpr

RNAi

1.000

0.687

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab2RNAi#1

Rab2

RNAi#1

6.625

1.068

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab2RNAi#2

Rab2

RNAi#2

8.250

1.544

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab3RNAi

Rab3

RNAi

6.750

1.236

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab4RNAi

Rab4

RNAi

6.500

1.000

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab5RNAi

Rab5

RNAi

7.000

1.000

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab6RNAi#1

Rab6

RNAi#1

5.375

1.017

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab6RNAi#2

Rab6

RNAi#2

7.273

0.541

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab7RNAi

Rab7

RNAi

7.938

0.924

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab8RNAi#1

Rab8

RNAi#1

5.750

1.264

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab8RNAi#2

Rab8

RNAi#2

5.700

0.651

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab9RNAi#1

Rab9

RNAi#1

8.385

1.457

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab9RNAi#2

Rab9

RNAi#2

7.143

1.184

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab9DbRNAi

Rab9Db

RNAi

6.563

1.114

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab10RNAi

Rab10

RNAi

1.625

0.778

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab14RNAi

Rab14

RNAi

5.214

1.154

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab18RNAi#1

Rab18

RNAi#1

7.700

1.126

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab18RNAi#2

Rab18

RNAi#2

6.667

0.541

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab19RNAi

Rab19

RNAi

5.500

0.957

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab21RNAi

Rab21

RNAi

5.667

0.972

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab23RNAi#1

Rab23

RNAi#1

8.500

0.535

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab23RNAi#2

Rab23

RNAi#2

14.830 2.055

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab23RNAi#3

Rab23

RNAi#3

8.625

0.999

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab23RNAi#4

Rab23

RNAi#4

4.333

0.624

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab27RNAi

Rab27

RNAi

6.333

0.850

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab32RNAi#1

Rab32

RNAi#1

8.188

1.222

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab32RNAi#2

Rab32

RNAi#2

7.000

0.833

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab35RNAi#1

Rab35

RNAi#1

8.455

1.516

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab35RNAi#2

Rab35

RNAi#2

6.444

1.042

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab39RNAi

Rab39

RNAi

10.860 1.455

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rab40RNAi

Rab40

RNAi

8.818

0.773

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rabX2RNAi

RabX2

RNAi

10.440 1.608

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rabX4RNAi

RabX4

RNAi

7.800

1.590

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rabX6RNAi#1

RabX6

RNAi#1

6.000

0.830

Or67d-QF,UAS-HTTex1Q25-GFP/QUAS-

HTTex1Q91-mCherry ; +/+ ; repo-Gal4/UAS-

rabX6RNAi#2

RabX6

RNAi#2

6.889

1.550

one-way ANOVA:

F (37, 353) = 4.208

Dunnett's multiple comparisons:

Genotypes

p-value

FFLuc

RNAi

vs. no RNAi

0.9996

FFLuc

RNAi

vs. mCherry

RNAi

>0.9999

no RNAi vs. mCherry

RNAi

0.9996

FFLuc

RNAi

vs. drpr

RNAi

0.0005

FFLuc

RNAi

vs. Rab2

RNAi#1

>0.9999

FFLuc

RNAi

vs. Rab2

RNAi#2

0.9985

FFLuc

RNAi

vs. Rab3

RNAi

0.9999

FFLuc

RNAi

vs. Rab4

RNAi

>0.9999

FFLuc

RNAi

vs. Rab5

RNAi

0.9997

FFLuc

RNAi

vs. Rab6

RNAi#1

0.9991

FFLuc

RNAi

vs. Rab6

RNAi#2

0.9994

FFLuc

RNAi

vs. Rab7

RNAi

0.9984

FFLuc

RNAi

vs. Rab8

RNAi#1

0.9994

FFLuc

RNAi

vs. Rab8

RNAi#2

0.9993

FFLuc

RNAi

vs. Rab9

RNAi#1

0.9835

FFLuc

RNAi

vs. Rab9

RNAi#2

0.9996

FFLuc

RNAi

vs. Rab9Db

RNAi

>0.9999

FFLuc

RNAi

vs. Rab10

RNAi

0.0395

FFLuc

RNAi

vs. Rab14

RNAi

0.9985

FFLuc

RNAi

vs. Rab18

RNAi#1

0.999

FFLuc

RNAi

vs. Rab18

RNAi#2

>0.9999

FFLuc

RNAi

vs. Rab19

RNAi

0.9991

FFLuc

RNAi

vs. Rab21

RNAi

0.9993

FFLuc

RNAi

vs. Rab23

RNAi#1

0.9981

FFLuc

RNAi

vs. Rab23

RNAi#2

<0.0001

FFLuc

RNAi

vs. Rab23

RNAi#3

0.9854

FFLuc

RNAi

vs. Rab23

RNAi#4

0.9629

FFLuc

RNAi

vs. Rab27

RNAi

0.9999

FFLuc

RNAi

vs. Rab32

RNAi#1

0.9861

FFLuc

RNAi

vs. Rab32

RNAi#2

0.9997

FFLuc

RNAi

vs. Rab35

RNAi#1

0.9843

FFLuc

RNAi

vs. Rab35

RNAi#2

>0.9999

FFLuc

RNAi

vs. Rab39

RNAi

0.1915

FFLuc

RNAi

vs. Rab40

RNAi

0.9208

FFLuc

RNAi

vs. RabX2

RNAi

0.2039

FFLuc

RNAi

vs. RabX4

RNAi

0.9989

FFLuc

RNAi

vs. RabX6

RNAi#1

0.9996

FFLuc

RNAi

vs. RabX6

RNAi#2

0.9998

Figure 14E

Genotype

Label

Mean

SEM

# brain

hemispheres (n)

Or67d-QF,UAS-HTT

ex1

Q25-GFP/+ ;

GH146-Gal4,QUAS-HTTex1Q91-

mCherry/+

+/+

53.400

3.560

Or67d-QF,UAS-HTT

ex1

Q25-GFP/+ ;

GH146-Gal4,QUAS-HTTex1Q91-

mCherry/+ ; drprD5/+

drpr/+

45.250

3.679

Or67d-QF,UAS-HTT

ex1

Q25-

GFP/rab10

{CRISPR-KO}

; GH146-

Gal4,QUAS-HTTex1Q91-mCherry/+

rab10/+

50.900

3.082

Or67d-QF,UAS-HTT

ex1

Q25-

GFP/rab10

{CRISPR-KO}

; GH146-

Gal4,QUAS-HTTex1Q91-mCherry/+ ;

drprD5/+

rab10/+ drpr/+

46.880

3.340

Or67d-QF,UAS-HTT

ex1

Q25-

GFP/rab10

CRISPR

; GH146-Gal4,QUAS-

HTTex1Q91-mCherry/rab14

{CRISPR-KO}

rab14/+

50.630

4.858

Or67d-QF,UAS-HTT

ex1

Q25-

GFP/rab10

CRISPR

; GH146-Gal4,QUAS-

HTTex1Q91-mCherry/rab14

{CRISPR-KO}

;

drprD5/+

rab14/+ drpr/+

58.250

4.887

one-way ANOVA:

F (5, 60) = 1.195

Tukey's multiple comparisons:

p-value

+/+ vs.

drpr/+

0.5577

+/+ vs.

rab10/+

0.9966

+/+ vs.

rab10/+ drpr/+

0.8511

+/+ vs.

rab14/+

0.9961

+/+ vs.

rab14/+ drpr/+

0.953

drpr/+

vs.

rab10/+

0.9206

drpr/+

vs.

rab10/+ drpr/+

0.9998

drpr/+

vs.

rab14/+

0.9498

drpr/+

vs.

rab14/+ drpr/+

0.2869

rab10/+

vs.

rab10/+ drpr/+

0.988

rab10/+

vs.

rab14/+

>0.9999

rab10/+

vs.

rab14/+ drpr/+

0.8546

rab10/+ drpr/+

vs.

rab14/+

0.9932

rab10/+ drpr/+

vs.

rab14/+

drpr/+

0.5369

rab14/+

vs.

rab14/+ drpr/+

0.8627

Figure 14F

Genotype

Label

Mean

SEM

# brain

hemispheres (n)

Or67d-QF,UAS-HTT

ex1

Q25-GFP/+ ; GH146-

Gal4,QUAS-HTTex1Q91-mCherry/+

+/+

2.850 0.274

Or67d-QF,UAS-HTT

ex1

Q25-GFP/+ ; GH146-

Gal4,QUAS-HTTex1Q91-mCherry/+ ; drprD5/+

drpr/+

3.417 0.484

Or67d-QF,UAS-HTT

ex1

Q25-GFP/rab10

{CRISPR-KO}

;

GH146-Gal4,QUAS-HTTex1Q91-mCherry/+

rab10/+

2.800 0.574

Or67d-QF,UAS-HTT

ex1

Q25-GFP/rab10

{CRISPR-KO}

;

GH146-Gal4,QUAS-HTTex1Q91-mCherry/+ ; drprD5/+

rab10/+

drpr/+

1.000 0.500

Or67d-QF,UAS-HTT

ex1

Q25-GFP/+ ; GH146-

Gal4,QUAS-HTTex1Q91-mCherry/rab14

{CRISPR-KO}

rab14/+

2.625 0.822

Or67d-QF,UAS-HTT

ex1

Q25-GFP/+ ; GH146-

Gal4,QUAS-HTTex1Q91-mCherry/rab14

{CRISPR-KO}

;

drprD5/+

rab14/+

drpr/+

3.000 0.655

one-way ANOVA:

F (5, 60) = 2.225

Tukey's multiple comparisons:

p-value

+/+ vs.

drpr/+

0.9355

+/+ vs.

rab10/+

>0.9999

+/+ vs.

rab10/+ drpr/+

0.0972

+/+ vs.

rab14/+

0.9995

+/+ vs.

rab14/+ drpr/+

>0.9999

drpr/+

vs.

rab10/+

0.9525

drpr/+

vs.

rab10/+ drpr/+

0.026

drpr/+

vs.

rab14/+

0.9002

drpr/+

vs.

rab14/+ drpr/+

0.9937

rab10/+

vs.

rab10/+ drpr/+

0.2148

rab10/+

vs.

rab14/+

>0.9999

rab10/+

vs.

rab14/+ drpr/+

0.9998

rab10/+ drpr/+

vs.

rab14/+

0.377

rab10/+ drpr/+

vs.

rab14/+ drpr/+

0.1684

rab14/+

vs.

rab14/+ drpr/+

0.9975

Figure 14G

Genotype

Values

Number of

animals

UAS-HTTex1Q25-GFP,QUAS-

HTTex1Q91-mCherry/or67d-

QF;+/+;repo-Gal4/+

2.39±0.35

7 (13 ORNs)

UAS-HTTex1Q25-GFP,QUAS-

HTTex1Q91-mCherry/or67d-

QF;+/+;repo-Gal4/UAS-RNAi.Rab10

0.46±0.18

7 (13 ORNs)

UAS-HTTex1Q25-GFP,QUAS-

HTTex1Q91-mCherry/or67d-

QF;UAS-Draper-I/+;repo-Gal4/UAS-

RNAi.Rab10

0.62±0.27

7 (13 ORNs)

Comparison between

Genotype

Test

used

P-value

Test statistics

UAS-HTTex1Q25-GFP,QUAS-HTTex1Q91-

mCherry/or67d-QF;+/+;repo-Gal4/+ & UAS-

HTTex1Q25-GFP,QUAS-HTTex1Q91-

mCherry/or67d-QF;+/+;repo-Gal4/UAS-

RNAi.Rab10

Mann-

Whitney

0.0002

=4.38, df=24

UAS-HTTex1Q25-GFP,QUAS-HTTex1Q91-

mCherry/or67d-QF;+/+;repo-Gal4/+ & UAS-

HTTex1Q25-GFP,QUAS-HTTex1Q91-

mCherry/or67d-QF;UAS-Draper-I/+;repo-

Gal4/UAS-RNAi.Rab10

Mann-

Whitney

0.0008

=3.83, df=24

Table 18.

Statistical information for the data shown in Figure 15

Figure 15A

qPCR

Target

Genotype

Uninjured (-

)

/Injured (+)

Values

Biological

Replicates

rab10

w1118; UAS-GFP/+;orco-Gal4/+

1±0.028

2.38±0.034

w1118; UAS-HTTex1Q25-

GFP/+;orco-Gal4/+

1.02±0.019

2.68±0.079

w1118; UAS-HTTex1Q91-

GFP/+;orco-Gal4/+

1.18±0.018

2.33±0.071

rab5

w1118; UAS-GFP/+;orco-Gal4/+

1±0.028

1.84±0.007

w1118; UAS-HTTex1Q25-

GFP/+;orco-Gal4/+

1.21±0.090

1.81±0.045

w1118; UAS-HTTex1Q91-

GFP/+;orco-Gal4/+

1.17±0.034

1.45±0.061

rab7

w1118; UAS-GFP/+;orco-Gal4/+

1±0.038

1.42±0.045

w1118; UAS-HTTex1Q25-

GFP/+;orco-Gal4/+

1.10±0.050

1.51±0.051

w1118; UAS-HTTex1Q91-

GFP/+;orco-Gal4/+

1.17±0.032

1.12±0.032

Comparison between

qPCR

target

Genotype

Uninjured (-)

/Injured (+)

Test used

P-value

Test

statistics

rab10

w1118; UAS-GFP/+;orco-

Gal4/+

- & +

Mann-Whitney

1.07E-15

=30.91,

df=16

w1118; UAS-HTTex1Q25-

GFP/+;orco-Gal4/+

- & +

Mann-Whitney

5.93E-13

=20.63,

df=16

w1118; UAS-HTTex1Q91-

GFP/+;orco-Gal4/+

- & +

Mann-Whitney

3.74E-11

=15.73,

df=16

rab5

w1118; UAS-GFP/+;orco-

Gal4/+

- & +

Mann-Whitney

6.35E-09

=11.10,

df=16

w1118; UAS-HTTex1Q25-

GFP/+;orco-Gal4/+

- & +

Mann-Whitney 1.86046E-05

=6.00,
df=16

w1118; UAS-HTTex1Q91-

GFP/+;orco-Gal4/+

- & +

Mann-Whitney 0.001571296

=3.85,
df=16

rab7

w1118; UAS-GFP/+;orco-

Gal4/+

- & +

Mann-Whitney

3.04E-06

=6.99,
df=16

w1118; UAS-HTTex1Q25-

GFP/+;orco-Gal4/+

- & +

Mann-Whitney 2.04847E-05

=5.95,
df=16

w1118; UAS-HTTex1Q91-

GFP/+;orco-Gal4/+

- & +

Mann-Whitney 0.130016807

=1.60,
df=16

rab10

w1118; UAS-GFP/+;orco-

Gal4/+ & w1118; UAS-

HTTex1Q25-GFP/+;orco-

Gal4/+ & w1118; UAS-

HTTex1Q91-GFP/+;orco-

Gal4/+

One-way

ANOVA

2.39407E-05

=17.12,

df=24

w1118; UAS-GFP/+;orco-

Gal4/+ & w1118; UAS-

HTTex1Q25-GFP/+;orco-

Gal4/+ & w1118; UAS-

HTTex1Q91-GFP/+;orco-

Gal4/+

One-way

ANOVA

0.130455775

=2.22,
df=24

rab5

w1118; UAS-GFP/+;orco-

Gal4/+ & w1118; UAS-

HTTex1Q25-GFP/+;orco-

Gal4/+ & w1118; UAS-

One-way

ANOVA

0.062735542

=3.06,
df=24

HTTex1Q91-GFP/+;orco-

Gal4/+

w1118; UAS-GFP/+;orco-

Gal4/+ & w1118; UAS-

HTTex1Q25-GFP/+;orco-

Gal4/+ & w1118; UAS-

HTTex1Q91-GFP/+;orco-

Gal4/+

One-way

ANOVA

0.000107536

=13.70,

df=24

rab7

w1118; UAS-GFP/+;orco-

Gal4/+ & w1118; UAS-

HTTex1Q25-GFP/+;orco-

Gal4/+ & w1118; UAS-

HTTex1Q91-GFP/+;orco-

Gal4/+

One-way

ANOVA

0.024538394

=4.34,
df=24

w1118; UAS-GFP/+;orco-

Gal4/+ & w1118; UAS-

HTTex1Q25-GFP/+;orco-

Gal4/+ & w1118; UAS-

HTTex1Q91-GFP/+;orco-

Gal4/+

One-way

ANOVA

1.73E-06

=24.26,

df=24

Figure 15H

Genotype

Values

Number of

animals

TI{TI}Rab10[EYFP]/Y;UAS-

HTTex1Q25-V5/+;orco-Gal4/+

36.25±8.74

8 (16 ORNs)

TI{TI}Rab10[EYFP]/Y;UAS-

HTTex1Q91-V5/+;orco-Gal4/+

96.0±18.67

9 (18 ORNs)

w1118/Y;UAS-HTTex1Q25-

V5/TI{TI}Rab5[EYFP];orco-

Gal4/+

812.06±137.47

8 (16 ORNs)

w1118/Y;UAS-HTTex1Q91-

V5/TI{TI}Rab5[EYFP];orco-

Gal4/+

387.71±82.54

9 (17 ORNs)

w1118/Y

;

UAS-HTT

ex1

Q25-

;

orco-

Gal4/TI{TI}Rab7[EYFP]

317.57±82.54

11 (21 ORNs)

w1118/Y

;

UAS-HTT

ex1

Q91-

;

orco-Gal4/

TI{TI}Rab7[EYFP]

1290.74±218.07

10 (19 ORNs)

Comparison between

Genotype

Test used

P-value

Test statistics

TI{TI}Rab10[EYFP]/Y;UAS-

HTTex1Q25-V5/+;orco-Gal4/+ &

TI{TI}Rab10[EYFP]/Y;UAS-

HTTex1Q91-V5/+;orco-Gal4/+

Mann-Whitney

8.96E-03

=2.78, df=32

w1118/Y;UAS-HTTex1Q25-

V5/TI{TI}Rab5[EYFP];orco-Gal4/+ &

w1118/Y;UAS-HTTex1Q25-

V5/TI{TI}Rab5[EYFP];orco-Gal4/+

Mann-Whitney

5.64E-03

=2.97, df=31

w1118/Y;UAS-HTTex1Q25-

V5/+;orco-Gal4/TI{TI}Rab7[EYFP] &

w1118/Y;UAS-HTTex1Q25-

V5/+;orco-Gal4/TI{TI}Rab7[EYFP]

Mann-Whitney

1.04E-04

=4.33, df=38

Figure 15I

Genotype

Values

Number of

animals

TI{TI}Rab10[EYFP]/Y;UAS-

HTTex1Q25-V5/+;orco-Gal4/+

1.44±0.66

8 (16 ORNs)

TI{TI}Rab10[EYFP]/Y;UAS-

HTTex1Q91-V5/+;orco-Gal4/+

6.83±2.69

9 (18 ORNs)

w1118/Y;UAS-HTTex1Q25-

V5/TI{TI}Rab5[EYFP];orco-Gal4/+

2.39±0.61

8 (16 ORNs)

w1118/Y;UAS-HTTex1Q91-

V5/TI{TI}Rab5[EYFP];orco-Gal4/+

5.17±1.33

9 (17 ORNs)

w1118/Y

;

UAS-HTT

ex1

Q25-

;

orco-Gal4/TI{TI}Rab7[EYFP]

0.96±0.28

11 (21 ORNs)

w1118/Y

;

UAS-HTT

ex1

Q91-

;

orco-Gal4/ TI{TI}Rab7[EYFP]

4.35±0.76

10 (19 ORNs)

Comparison between

Genotype

Test used

P-value

Test statistics

TI{TI}Rab10[EYFP]/Y;UAS-

HTTex1Q25-V5/+;orco-Gal4/+ &

TI{TI}Rab10[EYFP]/Y;UAS-

HTTex1Q91-V5/+;orco-Gal4/+

Mann-Whitney 5.96E-03

=2.95, df=32

w1118/Y;UAS-HTTex1Q25-

V5/TI{TI}Rab5[EYFP];orco-Gal4/+ &

w1118/Y;UAS-HTTex1Q91-

V5/TI{TI}Rab5[EYFP];orco-Gal4/+

Mann-Whitney 3.42E-02

=2.22, df=31

w1118/Y;UAS-HTTex1Q25-

V5/+;orco-Gal4/TI{TI}Rab7[EYFP] &

w1118/Y;UAS-HTTex1Q91-

V5/+;orco-Gal4/TI{TI}Rab7[EYFP]

Mann-Whitney 1.72E-04

=4.17, df=38

Table 19.

Statistical information for the data shown in Figure 16

Figure 16G

Genotype

Values

Number of

animals

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS-YFP-

Rab10(T23N);orco-QF2/repo-

Gal4

30.5±6.82

10 (20 ORNs)

w1118/Y;QUAS-HTTex1Q91-

mCherry/UAS-YFP-

Rab10(T23N);orco-QF2/repo-

Gal4

26.9±6.02

10 (20 ORNs)

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS-YFP-Rab10;orco-

QF2/repo-Gal4

66.7±14.90

10 (20 ORNs)

w1118/Y;QUAS-HTTex1Q91-

mCherry/UAS-YFP-Rab10;orco-

QF2/repo-Gal4

122.7±27.43

10 (20 ORNs)

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS-YFP-

Rab10(Q68L);orco-QF2/repo-

Gal4

109.7±25.85

9 (18 ORNs)

w1118/Y;QUAS-HTTex1Q91-

mCherry/UAS-YFP-

Rab10(Q68L);orco-QF2/repo-

Gal4

162.7±38.35

9 (18 ORNs)

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS-YFP-Rab5;orco-

QF2/repo-Gal4

1105.5±276.38

8 (16 ORNs)

w1118/Y;QUAS-HTTex1Q91-

mCherry/UAS-YFP-Rab5;orco-

QF2/repo-Gal4

791.2±197.80

8 (16 ORNs)

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS-YFP-Rab7;orco-

QF2/repo-Gal4

214.8±48.02

10 (20 ORNs)

w1118/Y;QUAS-HTTex1Q91-

mCherry/UAS-YFP-Rab7;orco-

QF2/repo-Gal4

361.5±80.82

10 (20 ORNs)

Comparison between

Genotype

Test used

P-value

Test statistics

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS-YFP-

Rab10(T23N);orco-QF2/repo-Gal4

& w1118/Y;QUAS-HTTex1Q91-

mCherry/UAS-YFP-

Rab10(T23N);orco-QF2/repo-Gal4

Mann-Whitney

0.153063

=1.46, df=38

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS-YFP-Rab10;orco-

QF2/repo-Gal4 & w1118/Y;QUAS-

HTTex1Q91-mCherry/UAS-YFP-

Rab10;orco-QF2/repo-Gal4

Mann-Whitney

1.97E-03

=3.32, df=38

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS-YFP-

Rab10(Q68L);orco-QF2/repo-Gal4

& w1118/Y;QUAS-HTTex1Q91-

Mann-Whitney

2.74E-03

=3.23, df=34

mCherry/UAS-YFP-

Rab10(Q68L);orco-QF2/repo-Gal4

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS-YFP-Rab5;orco-

QF2/repo-Gal4 & w1118/Y;QUAS-

HTTex1Q91-mCherry/UAS-YFP-

Rab5;orco-QF2/repo-Gal4

Mann-Whitney

6.77E-05

=4.62, df=30

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS-YFP-Rab7;orco-

QF2/repo-Gal4 & w1118/Y;QUAS-

HTTex1Q91-mCherry/UAS-YFP-

Rab7;orco-QF2/repo-Gal4

Mann-Whitney

4.58E-05

=4.60, df=38

Figure 16H

Genotype

Values

Number of

animals

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS-YFP-

Rab10(T23N);orco-QF2/repo-Gal4

0.15±0.03

10 (20 ORNs)

w1118/Y;QUAS-HTTex1Q91-

mCherry/UAS-YFP-

Rab10(T23N);orco-QF2/repo-Gal4

0.45±0.10

10 (20 ORNs)

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS-YFP-Rab10;orco-

QF2/repo-Gal4

0±0

10 (20 ORNs)

w1118/Y;QUAS-HTTex1Q91-

mCherry/UAS-YFP-Rab10;orco-

QF2/repo-Gal4

0.85±0.19

10 (20 ORNs)

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS-YFP-

Rab10(Q68L);orco-QF2/repo-Gal4

0.11±0.03

9 (18 ORNs)

w1118/Y;QUAS-HTTex1Q91-

mCherry/UAS-YFP-

Rab10(Q68L);orco-QF2/repo-Gal4

1.06±0.25

9 (18 ORNs)

0.06±0.02

8 (16 ORNs)

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS-YFP-Rab5;orco-

QF2/repo-Gal4

w1118/Y;QUAS-HTTex1Q91-

mCherry/UAS-YFP-Rab5;orco-

QF2/repo-Gal4

1.81±0.45

8 (16 ORNs)

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS-YFP-Rab7;orco-

QF2/repo-Gal4

0.05±0.01

10 (20 ORNs)

w1118/Y;QUAS-HTTex1Q91-

mCherry/UAS-YFP-Rab7;orco-

QF2/repo-Gal4

4.75±1.06

10 (20 ORNs)

Comparison between

Genotype

Test used

P-value

Test statistics

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS-YFP-

Rab10(T23N);orco-QF2/repo-Gal4

& w1118/Y;QUAS-HTTex1Q91-

mCherry/UAS-YFP-

Rab10(T23N);orco-QF2/repo-Gal4

Mann-Whitney

0.092744

=1.72, df=38

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS-YFP-Rab10;orco-

QF2/repo-Gal4 & w1118/Y;QUAS-

HTTex1Q91-mCherry/UAS-YFP-

Rab10;orco-QF2/repo-Gal4

Mann-Whitney

6.09E-04

=3.73, df=38

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS-YFP-

Rab10(Q68L);orco-QF2/repo-Gal4

& w1118/Y;QUAS-HTTex1Q91-

mCherry/UAS-YFP-

Rab10(Q68L);orco-QF2/repo-Gal4

Mann-Whitney

5.13E-05

=4.63, df=34

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS-YFP-Rab5;orco-

Mann-Whitney

1.14E-04

=4.44, df=30

QF2/repo-Gal4 & w1118/Y;QUAS-

HTTex1Q91-mCherry/UAS-YFP-

Rab5;orco-QF2/repo-Gal4

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS-YFP-Rab7;orco-

QF2/repo-Gal4 & w1118/Y;QUAS-

HTTex1Q91-mCherry/UAS-YFP-

Rab7;orco-QF2/repo-Gal4

Mann-Whitney

3.44E-06

=5.43, df=38

Figure 16I

Genotype

Values

Number of

animals

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS-YFP-

Rab10(T23N);orco-QF2/repo-Gal4

0.03±0.01

10 (20 ORNs)

w1118/Y;QUAS-HTTex1Q91-

mCherry/UAS-YFP-

Rab10(T23N);orco-QF2/repo-Gal4

0.03±0.0.01

10 (20 ORNs)

mHTT+/Rab+ w1118/Y;QUAS-

HTTex1Q91-mCherry/UAS-YFP-

Rab10(T23N);orco-QF2/repo-Gal4

0.007±0.003

10 (20 ORNs)

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS-YFP-Rab10;orco-

QF2/repo-Gal4

0.081±0.02

10 (20 ORNs)

w1118/Y;QUAS-HTTex1Q91-

mCherry/UAS-YFP-Rab10;orco-

QF2/repo-Gal4

0.082±0.02

10 (20 ORNs)

mHTT+/Rab+ w1118/Y;QUAS-

HTTex1Q91-mCherry/UAS-YFP-

Rab10;orco-QF2/repo-Gal4

0.07±0.02

10 (20 ORNs)

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS-YFP-

Rab10(Q68L);orco-QF2/repo-Gal4

0.052±0.01

9 (18 ORNs)

w1118/Y;QUAS-HTTex1Q91-

mCherry/UAS-YFP-

Rab10(Q68L);orco-QF2/repo-Gal4

0.049±0.01

9 (18 ORNs)

mHTT+/Rab+ w1118/Y;QUAS-

HTTex1Q91-mCherry/UAS-YFP-

Rab10(Q68L);orco-QF2/repo-Gal4

0.14±0.04

9 (18 ORNs)

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS-YFP-Rab5;orco-

QF2/repo-Gal4

0.16±0.04

8 (16 ORNs)

w1118/Y;QUAS-HTTex1Q91-

mCherry/UAS-YFP-Rab5;orco-

QF2/repo-Gal4

0.15±0.04

8 (16 ORNs)

mHTT+/Rab+ w1118/Y;QUAS-

HTTex1Q91-mCherry/UAS-YFP-

Rab5;orco-QF2/repo-Gal4

0.27±0.08

8 (16 ORNs)

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS-YFP-Rab7;orco-

QF2/repo-Gal4

0.16±0.04

10 (20 ORNs)

w1118/Y;QUAS-HTTex1Q91-

mCherry/UAS-YFP-Rab7;orco-

QF2/repo-Gal4

0.22±0.05

10 (20 ORNs)

mHTT+/Rab+ w1118/Y;QUAS-

HTTex1Q91-mCherry/UAS-YFP-

Rab7;orco-QF2/repo-Gal4

0.46±0.10

10 (20 ORNs)

Comparison between

Genotype

Test used

P-value

Test

statistics

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS-YFP-

Rab10(T23N);orco-QF2/repo-Gal4 &

w1118/Y;QUAS-HTTex1Q91-

mCherry/UAS-YFP-

Rab10(T23N);orco-QF2/repo-Gal4

Mann-Whitney

0.84565

=0.20, df=38

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS-YFP-

Rab10(T23N);orco-QF2/repo-Gal4 &

w1118/Y;QUAS-HTTex1Q91-

mCherry/UAS-YFP-

Rab10(T23N);orco-QF2/repo-Gal4 &

mHTT+/Rab+ w1118/Y;QUAS-

HTTex1Q91-mCherry/UAS-YFP-

Rab10(T23N);orco-QF2/repo-Gal4

One-way

ANOVA

0.0588

=3.02, df=44

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS-YFP-Rab10;orco-

QF2/repo-Gal4 & w1118/Y;QUAS-

HTTex1Q91-mCherry/UAS-YFP-

Rab10;orco-QF2/repo-Gal4

Mann-Whitney

0.967418

=0.04, df=38

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS-YFP-Rab10;orco-

QF2/repo-Gal4 & w1118/Y;QUAS-

HTTex1Q91-mCherry/UAS-YFP-

Rab10;orco-QF2/repo-Gal4 &

mHTT+/Rab+ w1118/Y;QUAS-

HTTex1Q91-mCherry/UAS-YFP-

Rab10;orco-QF2/repo-Gal4

One-way

ANOVA

0.8321

=0.18, df=49

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS-YFP-

Rab10(Q68L);orco-QF2/repo-Gal4 &

w1118/Y;QUAS-HTTex1Q91-

mCherry/UAS-YFP-

Rab10(Q68L);orco-QF2/repo-Gal4

Mann-Whitney

0.787227

=0.27, df=38

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS-YFP-

Rab10(Q68L);orco-QF2/repo-Gal4 &

One-way

ANOVA

9.00E-04

=8.21, df=47

w1118/Y;QUAS-HTTex1Q91-

mCherry/UAS-YFP-

Rab10(Q68L);orco-QF2/repo-Gal4 &

mHTT+/Rab+ w1118/Y;QUAS-

HTTex1Q91-mCherry/UAS-YFP-

Rab10(Q68L);orco-QF2/repo-Gal4

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS-YFP-Rab5;orco-

QF2/repo-Gal4 & w1118/Y;QUAS-

HTTex1Q91-mCherry/UAS-YFP-

Rab5;orco-QF2/repo-Gal4

Mann-Whitney

0.307296

=1.04, df=30

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS-YFP-Rab5;orco-

QF2/repo-Gal4 & w1118/Y;QUAS-

HTTex1Q91-mCherry/UAS-YFP-

Rab5;orco-QF2/repo-Gal4 &

mHTT+/Rab+ w1118/Y;QUAS-

HTTex1Q91-mCherry/UAS-YFP-

Rab5;orco-QF2/repo-Gal4

One-way

ANOVA

0.1202

=2.24, df=40

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS-YFP-Rab7;orco-

QF2/repo-Gal4 & w1118/Y;QUAS-

HTTex1Q91-mCherry/UAS-YFP-

Rab7;orco-QF2/repo-Gal4

Mann-Whitney

0.034779

=2.19, df=38

w1118/Y;QUAS-HTTex1Q25-

mCherry/UAS-YFP-Rab7;orco-

QF2/repo-Gal4 & w1118/Y;QUAS-

HTTex1Q91-mCherry/UAS-YFP-

Rab7;orco-QF2/repo-Gal4 &

mHTT+/Rab+ w1118/Y;QUAS-

HTTex1Q91-mCherry/UAS-YFP-

Rab7;orco-QF2/repo-Gal4

One-way

ANOVA

2.00E-04

=9.83, df=57