2024.01.17.575386v1.full-html.html

bioRxiv preprint

Abstract

The c-Jun N-terminal kinase 3 (JNK3) is a stress-responsive protein kinase primarily expressed in

the central nervous system (CNS). JNK3 exhibits nuanced neurological activities, such as roles in

behavior, circadian rhythms, and neurotransmission, but JNK3 is also implicated in cell death and

neurodegeneration. Despite the critical role of JNK3 in neurophysiology and pathology, its

localization in the brain is not fully understood due to a paucity of tools to distinguish JNK3 from

other isoforms. While previous functional and histological studies suggest locales for JNK3 in the

CNS, a comprehensive and higher resolution of JNK3 distribution and abundance remained

elusive. Here, we sought to define the anatomical and cellular distribution of JNK3 in adult mouse

brains. Data reveal the highest levels of JNK3 and pJNK3 were found in the cortex and the

hippocampus. JNK3 possessed neuron-type selectivity as JNK3 was present in GABAergic,

cholinergic, and dopaminergic neurons, but was not detectable in VGLUT-1-positive

glutamatergic neurons and astrocytes

in vivo

. Intriguingly, higher JNK3 signals were found in

motor neurons and relevant nuclei in the cortex, basal ganglia, brainstem, and spinal cord. While

JNK3 was primarily observed in the cytosol of neurons in the cortex and the hippocampus, JNK3

appeared commonly within the nucleus in the brainstem. These distinctions suggest the potential

for significant differences between JNK3 actions in distinct brain regions and cell types. Our

results provide a significant improvement over previous reports of JNK3 spatial organization in

the adult CNS and support continued investigation of JNK3’s role in neurophysiology and

pathophysiology.

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Introduction

The c-Jun N-terminal kinases (JNKs) are vital members of the mitogen-activated kinase

(MAPK) family and play crucial roles within the central nervous system (CNS). These include but

are not limited to, cell differentiation and proliferation, axonal growth and transport, and neuronal

cell death [1-5]. The JNK family comprises three distinct isoforms JNK1, JNK2, and JNK3, which

are further distinguished from one another by more than ten unique splice variants [6]. While JNK1

and JNK2 are ubiquitously expressed across all tissue types [5], JNK3 is primarily localized in the

brain, with minor traces found in the testis and heart [7-9]. Intriguingly, whole-body gene ablation

Jnk3

in mice only appears to confer neuroprotection to stress-induced dysfunction and death

[10-13]. To gain insights into why JNK3 is readily present in the CNS, but only seemingly

responsive to stress, a more descriptive anatomical description of JNK3 distribution within the

CNS may be required.

To date, the primary narrative regarding JNK3 activities, as with other JNK isoforms, has

focused on interrogating its role in neuron death and in the pathogenesis of neurodegenerative

diseases [1-3, 14, 15]. Indeed, early studies into JNK3 selective events in the CNS, were based on

the generation of

Jnk3

-/-

mice that had no distinguishable phenotype from wild-type mice except

when challenged by kainic acid [10]. The kainic acid-exposed

Jnk3

-/-

mice exhibited reduced

hippocampal neuron loss than their wild-type counterparts [10], thus implicating JNK3 in neuronal

apoptosis. The association of JNK3 and neuronal apoptosis was strengthened through a series of

studies on cell lines and cultured neurons identifying JNK3 activation as an early cell death

response in neurotrophic withdrawal [9, 16], arsenite exposure [17, 18], and other neurologically

relevant stressors [19-22]. Thus, it was not surprising when elevated phospho-JNK levels were

found in the post-mortem brains of patients diagnosed with neurodegenerative diseases [23-25].

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Recent preclinical studies in Alzheimer’s disease (AD) and Parkinson’s disease (PD) models

illustrate that induction of JNK3-related activities may lead to neuronal cell death [11, 23, 24, 26-

32]. In AD, JNK3 has been shown to phosphorylate the microtubule‐associated protein, tau, which

is hyperphosphorylated in AD leading to the formation of neurofibrillary tangles [33-35].

Additionally, isoform-selective inhibition of JNK3 in the 3xTg-AD mouse model resulted in

reduced tau phosphorylation and improved cognitive function [36]. In various models of

neurodegenerative diseases, JNK3-selective inhibitors protect neurons against neurotoxin insults

and genetic induction of symptoms [32, 37-39]. Consequently, sustained JNK3 activity may lead

to neuron loss (reviewed in [26] and [5]).

The activities of JNK3 in neuronal physiology continue to emerge. Specifically, JNK3 may

contribute to neuronal morphology, as JNK3 overexpression in nerve growth factor (NGF)-treated

pheochromocytoma (PC12) cells leads to enhanced dendrite sprouting [40]. Complementary to

dendritic architecture maintenance, JNK3 may play crucial roles in synaptic neurotransmission, as

JNK3 is involved in the assembly and disassembly of microtubules by phosphorylating SCG10, a

neuronal growth-associated protein [15]; additionally, JNK3 has been found on vesicular

structures in axons [41], wherein, it may engage in the phosphoregulation of dynein and kinesin

[41-43]. Accordingly, early studies into JNK3 subcellular localization found the protein on

vesicles [44] placing it in close proximity to these motor proteins. Potential neurological

contributions of JNK3 have also been described in knockout mice.

Jnk3

-/-

mice were found to have

diminished neurogenesis in adult mice evidenced by a decrease in cells positive for neural

precursor markers in the dentate gyrus [45]. Additionally,

Jnk3

-/-

mice exhibited behavioral

phenotypes in a battery of tests [46]; specifically,

Jnk3

-/-

mice had a lower distance moved and

decreased center crossing in the elevated plus maze compared to control animals, while in the

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Morris water maze,

Jnk3

-/-

mice trended toward longer training times, increased times in the border

zone, and increased total times than control animals [46]. Recent studies now suggest a putative

role for JNK3 in metabolic regulation and homeostasis broadening the impact of JNK3 in the CNS

[47]. Taken together, these results indicate the possibility of nominal JNK3 activities supporting

basal neurological functions.

Although these findings underscore the significance of JNK3 within the brain, a

comprehensive understanding of JNK3’s precise localization remains elusive. Initial studies

employing the p493F12 antibody for immunohistochemical staining in AD human brains along

with

in situ

hybridization revealed that JNK3 is prominently expressed in the cortex, hippocampus,

subcortical regions, and the brainstem, with a weaker signal detected in the spinal cord. This study

further suggested that the localization of JNK3 was primarily neuronal [8], but lacked the context

of a healthy brain. Subsequent research using

in situ

hybridization on adult male Sprague-Dawley

rats illustrated high JNK3 expression in the cerebral cortex and hippocampus, with a lesser degree

in the striatum. In the hippocampus, high JNK3 expression was reported predominantly within

neuron-like cell bodies in CA1, CA3, the subiculum, and the dentate gyrus, while moderate

staining was observed in the habenula [48]. Most recently, radiolabeled-aminopyrazoles were

developed as isoform-selective probes for activated JNK3 (pJNK3), which were tested for

selectivity in a Parkinson’s disease mouse model (Mutator/Park2

-/-

mice) [49]. These probes found

more pJNK3 in the hippocampus and the cortex of the C57BL6/J mice with other regions

exhibiting relatively low levels. However, when compared to the 9-month-old Mutator/Park2

-/-

mice, higher pJNK3 concentrations were detected in the ventral midbrain, suggesting that disease

progression may correlate with pJNK3 levels in the ventral midbrain. Despite these advances, basal

JNK3 localization in a healthy adult mouse brain remains to be extensively explored with validated

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JNK3-selective reagents with areas like the brainstem and the spinal cord yet to be thoroughly

examined.

The primary aim of this study is to identify JNK3 localization within healthy adult male and

female mouse brains. Using a knockout-validated, isoform selective antibody, we report the

immunohistochemical and immunofluorescent localization of JNK3 in various regions of the brain

and cervical spinal cord and compare histological protein levels to pJNK3/JNK3 levels in dissected

brain regions. Furthermore, we identify JNK3-containing neuronal cell types, thereby contributing

to a more comprehensive understanding of JNK3's nature in the healthy adult brain.

Experimental Procedures

Materials.

General laboratory reagents and cell culture reagents were purchased from

ThermoFisher Scientific (Waltham, MA). Immunohistochemical reagents were purchased from

Vector Labs, Inc. (Newark, CA). Western blotting reagents were purchased from Bio-Rad

Laboratories (Hercules, CA) and Li-COR, Inc. (Lincoln, NE).

Animal care.

All animal care and experimental procedures were performed in compliance with

the Institutional Animal Care and Use Committee (IACUC) guidelines at Florida International

University. The subjects of the study were 3-month-old C57BL6/J mice (n=4/sex) and 12-month-

old C57BL6/J mice, both males (n=9) and females (n=10). The mice underwent two types of

procedures. For some mice, transcardial perfusion with formalin was performed for tissue fixation.

Briefly, mice were deeply anesthetized with isoflurane and then transcardially perfused with

phosphate-buffered saline (PBS), followed by 10% formalinin. The brains were carefully dissected

out, post-fixed in 10% formalin for one week and stored in 30% sucrose solution containing 0.1%

sodium azide until sectioning on microtome Brains were either sectioned sagittaly or coronally on

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the Thermo Scientific Microm HM 450 sliding microtome (Walldorf, Germany) into 30 um

sections and stored in 96 well plates containing anti-freeze solution. For the other mice, brain

regions were dissected for fresh freezing. Mice were euthanized following IACUC-approved

procedures. Cerebellum, brainstem, hippocampus, midbrain, striatum, and cortex were quickly and

carefully dissected. The brain regions were immediately frozen in dry ice or liquid nitrogen. The

frozen tissue samples were then stored at -80°C until homogenizing.

Immunohistochemical (IHC) Staining.

Tissues were thoroughly washed six times with

phosphate-buffered saline (PBS) (Gibco #21600-010) for 5 minutes. Next, the tissues were treated

with a solution containing 2.5% hydrogen peroxide (30%

)

, 75% methanol, and 22.5% deionized

water for 20 minutes on an orbital shaker. Afterward, the tissues were blocked using a PBS solution

containing 15% bovine serum albumin (BSA) (Sigma-Aldrich # A7030), 4% normal goat serum

(Vector #S-1000), and 0.6% Triton X-100 which was allowed to shake for an hour on an orbital

shaker. Subsequently, the primary antibody (Table 1) was diluted with PBS and added to the tissue

sections for overnight incubation at 4°C on orbital shaker. Following incubation, tissue sections

were washed five times with PBS for 5 minutes. The goat anti-rabbit (Vector Laboratories #BA-

1000), or goat anti-mouse (Vector Laboratories #BA-9200) secondary antibodies, conjugated to

horseradish peroxidase (HRP), was then added and incubated for 1 hour at room temperature on

an orbital shaker. After incubation, tissue sections were washed another five times with PBS for 5

minutes. The sections were then incubated with an avidin-biotin complex (ABC) solution (Vector

Laboratories #PK-4000) which was prepared as suggested by the manufacturer for 1 hour at room

temperature on an orbital shaker. Sections were washed five times with PBS for 5 minutes.

Visualization of the target protein was achieved by adding 3,3'-diaminobenzidine (DAB) solution

(Vector Laboratories #SK-4100), and the reaction was stopped by placing the sections in PBS.

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Afterward, the tissue sections were mounted on glass slides and air-dried. The sections were then

rehydrated through a series of ethanol washes (30%,50%,75%,95%, and 100%) and cleared using

CitriSolv (Decon Laboratories # 1601). Finally, the tissue sections were coverslipped using DPX

mounting medium (Electron Microscopy Sciences #13512), and the slides were allowed to dry

completely before microscopic examination on BZ-X All-in-One Fluorescence microscope

(Keyence, Itasca, IL).

Immunofluorescent (IF) Staining.

Tissues were washed overnight with PBS at 4°C shaking on

the orbital shaker. Next day, tissues were then permeabilized with PBS solution containing 0.5%

Triton X-100 for 20 minutes on an orbital shaker. Following permeabilization, tissues were washed

three times with PBS for 10 minutes each. Then tissues were blocked using a solution containing

PBS with 10% bovine serum albumin (BSA) and 0.1% Triton X-100 for 1 hour at room

temperature shaking on an orbital shaker. Primary antibodies (Table 1), diluted in PBS with 1%

BSA, was added to the tissue sections and incubated overnight at 4°C on an orbital shaker. The

next day, tissue sections were allowed to equilibrate at room temperature for 1 hour on an orbital

shaker. Afterward, tissue sections were washed three times with PBS containing 0.1% Triton X-

100. Goat anti-Rabbit IgG Alexa Fluor Plus 488 highly absorbed (Thermo Scientific #A32731) or

goat anti-mouse IgG Alexa Fluor Plus 555 highly absorbed (Thermo Scientific #A32727)

secondary antibodies, diluted in PBS with 1% BSA, were added to the tissue sections and

incubated for 1 hour at room temperature on an orbital shaker. Subsequently, tissue sections were

washed twice with PBS containing 0.1% Triton X-100 for 5 minutes each, followed by two washes

with PBS for 10 minutes each. The tissue sections were then mounted on glass slides, allowed to

air-dry, and a hydrophobic barrier was drawn around the tissue. If dual staining was not performed,

NeuroTrace Red (Thermo Scientific #N21482), diluted 1:100 in PBS, was added to the sections

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and incubated for 30 minutes on an orbital shaker. After incubation, tissue sections were washed

three times with PBS for 10 minutes each. TrueBlack (Biotium #23007) was applied for 10 minutes

and the tissue sections were coverslipped using ProLong Antifade mounting medium containing

DAPI (Thermo Scientific #P36935) to counterstain nuclei. The stained sections were then allowed

to dry completely before microscopic examination on BZ-X All-in-One Fluorescence microscope

(Keyence, Itasca, IL).

Regional immunoreactivity strength score for JNK3.

Eight matching sections were selected from

each animal and subjected to immunohistochemical staining for JNK3 (DAB) as described

previously [50]. Utilizing a BZ-X All-in-One microscope (Keyence, Itasca, IL), a single observer

scored the immunoreactivity strength to JNK3. The scoring rubric applied ranged from 0-3: 0

signified the absence of reactivity, 1 represented mild reactivity, 2 corresponded to moderate

reactivity, and 3 indicated high reactivity. A visual representation of this scoring method can be

viewed in Figure 3. An average of 3 animals for each sex was take for regions quantified. To

provide comprehensive, visual representations, spatial mappings were created with Adobe

Photoshop 2023. These mappings, presented as a six-color gradient scale, encompass eight brain

atlas sections [50]. The sections were adapted from the Allen Mouse Brain Atlas (Allen Institute

of Brain Science, 2011) and Paxinos and Franklin's "The Mouse Brain in Stereotaxic Coordinates,"

Fifth Edition [50].

Western Blotting.

Fluorescent semiquantitative western blotting was performed on brain

cerebellum, brainstem, hippocampus, midbrain, striatum and cortex obtained from 12-month-old

male and female C57BL/6J mice. The brain tissues were homogenized using N-PER lysis buffer

(Thermo Scientific #87792) supplemented with protease and phosphatase inhibitors without

EDTA (Thermo Scientific #1861281). Protein concentration was determined by performing a

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bicinchoninic acid (BCA) assay (Thermo Scientific #23225) following the manufacturer's

protocol. Equal amounts of protein (20



g) samples were then subjected to SDS-PAGE. Proteins

were transferred onto nitrocellulose membranes using a BioRad TurboTransfer system. The

membranes were blocked with 5% non-fat milk in Tris-buffered saline (TBS) for 1 hour at room

temperature rocking. Primary antibodies (Table 1), diluted in TBS containing 0.1% Tween20

(TBST), were added to the membranes and incubated overnight at 4°C rocking. Following primary

antibody incubation, membranes were washed three times for 7 minutes each with TBST. Anti-

rabbit IgG (Cell Signaling #5151S) and anti-mouse IgG (Cell Signaling #5470S) fluorophore-

conjugated secondary antibodies were then applied to the membranes and incubated for 1 hour at

room temperature rocking. After secondary antibody incubation, membranes were washed three

times for 7 minutes each with TBST. Fluorescent signals were detected and captured using

Odyssey CLx Imaging System. Images of the western blots were imported directly into the Empiria

Software (LI-COR Biosciences, Lincoln, NE). Subsequently, these images were quantified in

relation to housekeeping gene expression or total protein stain.

Phos-tag Western Blotting.

For Phos-tag Western blotting, equal amounts (20



g) of previously

prepared brain lysate samples were loaded into SuperSep Phos-tag pre-cast gels (FUJIFILM Wako

Chemicals # 198-17981) and subjected to electrophoresis. Following electrophoresis, the gels were

washed three times for 20 minutes each with transfer buffer containing 20% methanol, 10% stock

transfer buffer (30g TrisBase and 144g Glycine in 1 littler deionized water), 70% deionized water,

and 10 mM EDTA. The gels underwent a final wash with transfer buffer to eliminate any residual

EDTA. Next, proteins were transferred from the gel to a nitrocellulose membrane using a

conventional wet transfer method as described by SuperSep Phos-tag precasted gels. The

membrane was then washed with TBS for 10 minutes. This was followed by drying the membrane

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in 37°C bench incubator for 10 minutes and subsequent rehydration using TBS. Total protein

staining was then carried out on the membrane using the REVERT Total Protein Stain (LI-COR

Biosciences) #926-11010 following the manufacturer's instructions. The membrane was imaged

using the Odyssey CLx Imaging System. The membrane was blocked using 5% non-fat milk in

TBS for 1 hour rocking at room temperature. The membrane was then incubated with a JNK3

primary antibody, dissolved in TBST containing 5% BSA, and left overnight at 4°C rocking. The

following day, the membrane was washed thrice with TBST for 7 minutes each. Secondary

antibodies were added to the membrane and rocked for 1 hour at room temperature. Following the

incubation, the membrane was washed again three times with 1x TBST for 7 minutes each. The

membrane was then imaged again using the Odyssey CLx Imaging System. The images were

quantified and normalized to the total protein stain using Empiria Studio Software to determine

relative protein expression levels. Images of the Western blots were imported directly into the

Empiria Software (LI-COR Biosciences, Lincoln, NE). Subsequently, these images were

quantified in relation to total protein staining and fold-changes were obtained.

Cell Culture.

Cerebral tissues were collected from mixed-sex C57BL/6J pups on postnatal day

(PND) 0–1 and dissected for primary cortical neuron culture preparation. Dissociation of cortical

cells was achieved using 1mg/mL of papain and 0.5U/mL of dispase. Post-dissociation, cells were

washed, triturated, pelleted, and subsequently resuspended in the culture medium. The resultant

cell suspension was seeded onto poly-D-lysine-coated 6-well plates at an average density of 1x10

cells per well. Cells were cultured in a neurobasal medium, enriched with B-27 plus, 50 IU of

penicillin, 50mg/mL of streptomycin, and 2 mM of L-glutamine. Half of the medium was refreshed

24 hours post-isolation. After four days, half of the medium was substituted with a culture medium

supplemented with 2% B-27 plus supplement and 5μM of cytosine β-d-arabinofuranoside (Ara-C;

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Sigma; #C1768). The culture medium was then changed every three days. For microglia and

astrocytes culture, brains were also collected from mixed C57BL/6J pups on PND 0-3, and then

homogenized in a solution of 0.25% trypsin with EDTA. The homogenized cell suspension was

relocated to a T175 flask containing DMEM medium, supplemented with 10% FBS, 2mM l-

glutamine, 1mM Na-Pyruvate, 100



M non-essential amino acids, and 1% penicillin and

streptomycin. On day 7, the culture medium within the flask was replaced. By day 14, mature

microglial cells were isolated from astrocytes using EasySep Mouse CD11b Positive Selection Kit

II (Stemcell Technologies #18970A,) and subsequently seeded into p60 plates. The cells were

harvested using a RIPA buffer containing protease and phosphate inhibitors. Lysates were left on

ice for 30 minutes, homogenized, and centrifuged to gather the supernatant. All samples were

stored at -80

C until further use.

Statistical Analyses.

For western blots, fold values which were obtained from Empiria Software

were then incorporated into the GraphPad Prism 9 Software, where a 2-way ANOVA was executed

to analyze differences related to age, brain region, and sex.

Results

JNK3 localization in adult male and female mouse brain.

Immunohistochemical (IHC) staining

was employed to visualize the relative abundance of JNK3 within different brain regions of 12-

month-old male and female C57BL6/J mice using a knockout-validated isoform-selective antibody

(Supplemental Figure 1). JNK3 was consistently identifiable across all brain regions in sagittal

brain sections, using DAB IHC-staining, with notably enriched concentrations in the cortex and

hippocampus (Figure 1). JNK3 abundance was clear in the primary somatosensory cortex (Figure

1A) and the primary motor cortex (Figure 1B) particularly. In Figure 1C, robust JNK3 staining

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was noticed in the hippocampal formation. Additional areas with enriched JNK3 localization

included the piriform cortex (Figure 1D), the olfactory tubercle (Figure 1E), and the areas of the

thalamus (Figure 1F). Within these images, higher magnification (20x and 60x) images illustrate

that the JNK3 distribution appears to be predominantly in neurons in these regions (Figure 1).

JNK3 localization in the brainstem of adult mouse brain.

IHC staining was employed on sagittal

slices of C57BL6/J mouse brains to analyze JNK3 distribution in specific regions of the brainstem

(Figure 2). JNK3 staining was observed throughout the brainstem, albeit more diffuse than in the

cortical and hippocampal regions (Figure 1). The JNK3 distribution in the brainstem was

consistent more with specific nuclei. Specifically, JNK3 prominently appears in the spinal

trigeminal nucleus (Figure 2A), the intermediate reticular nucleus (Figure 2B), the facial nucleus

(Figure 2C), the motor trigeminal nucleus (Figure 2D), the vestibular nucleus (Figure 2E), and the

interposed cerebellar nucleus (Figure 2F). Significant staining was also observed in the cerebellar

cortex of the anterior and posterior lobes (Figure 2F). As in other brain regions, JNK3 localization

at higher magnification appears to be almost exclusively neuronal consistent with previous

histological reports [7, 8].

Qualitative anaysis of JNK3 levels in the adult mouse brain and cervical spinal cord.

To semi-

quantify JNK3 abundance in each region, mouse brains were coronally sectioned and IHC-stained

with DAB. Based on the relative intensities of JNK3 signal in these sections, JNK3

immunoreactivity was characterized as low (Figure 3A), moderate (Figure 3B), or high (Figure

3C). These benchmarks were then used to form a scored rubric of JNK3 abundance across the

brain. From the anterior to the posterior, high JNK3 immunoreactivity was noted in the anterior

cortical regions, including the agranular insular cortex and dorsal peduncular cortex (Table 2).

Higher immunoreactivity was observed in the cingulate cortex, primary and secondary motor

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cortices, primary and secondary somatosensory cortices, and the piriform cortex (Table 2, Figure

4A-C). High JNK3 immunoreactivity was also evident in the olfactory tubercle (Table 2, Figure

4A-B). Contrariwise, the striatum and the globus pallidus in the same region exhibited low JNK3

immunoreactivity (Table 2, Figure 4A-C). The immunoreactivity for JNK3 was consistent

throughout the striatum, with the internal globus pallidus demonstrating slightly higher

immunoreactivity for JNK3 than the external globus pallidus (Figure 4C). Transitioning into the

temporal lobe, the auditory cortex and other cortical regions showed high immunoreactivity for

JNK3 (Table 2, Figure 4C-D). The hippocampus demonstrated significantly high JNK3

immunoreactivity, particularly in the CA1-CA3 regions and the dentate gyrus; whereas, the

subiculum had very low JNK3 immunoreactivity (Table 2, Figure 4C-D). Near the hippocampus,

the thalamus and hypothalamus were observable (Table 2, Figure 4C), both displaying moderate

to high immunoreactivity for JNK3. Consistently, most staining in the thalamus was observed in

the mediodorsal region, with slightly less in the ventral lateromedial region (Figure 4C). However,

the amygdala, found within this region, had notably low immunoreactivity for JNK3 (Table 2,

Figure 4D). The nearby habenular nucleus also showed considerable JNK3 immunoreactivity

(Table 1, Figure 4C). As we moved closer to the brainstem, the cerebellar lobes and fissures were

visible, although they displayed very low reactivity for JNK3. Within the anterior region of the

brainstem, the midbrain, JNK3 exhibited high immunoreactivity in the cerebellar peduncle and

motor trigeminal nucleus (Table 2; Figure 4E-F). JNK3 also showed high immunoreactivity in the

olivary nucleus, pontine reticular nucleus, and raphe nucleus (Table 2; Figure 4E-F). Furthermore,

JNK3 demonstrated moderate immunoreactivity in the cuneiform nucleus and low reactivity in the

reticulotegmental nucleus (Table 2, Figure 4E-F). Transitioning into the posterior part of the

brainstem, the medulla oblongata, JNK3 also demonstrated significantly high immunoreactivity in

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motor-associated areas, including the vagus nerve nucleus, hypoglossal nucleus, lateral reticular

nucleus, and inferior olive nucleus (Table 2; Figure 4G). The cuneate nucleus also displayed high

JNK3 immunoreactivity, albeit slightly less than the other regions (Table 2, Figure 4G). In the

pons region of the brainstem, JNK3 displayed high immunoreactivity overall. This was particularly

evident in the facial nucleus, cerebellar nucleus, vestibular nucleus, granular insular cortex,

gigantocellular reticular nucleus, spinal trigeminal nucleus, and cochlear nucleus (Table 2, Figure

4F). The immunoreactivity observed in the cervical spine displayed marked similarity to that of

the brainstem; wherein, JNK3 localization in the cervical spinal cord (Figure 4H) was also

consistently high across nuclei in the grey matter but was absent in the lateral regions (anterior and

posterior horns) and the funiculus (Figure 4H). Lastly, no significant sex differences were observed

between male and female brains in terms of JNK3 distribution.

JNK3 co-localization with various neuron types.

JNK3 distribution in our current results and in

other histological studies has been described as primarily neuronal [7, 8]. To dissect the types of

neurons that may express JNK3, co-immunofluorescent (co-IF) staining was performed with

neuron-selective markers/antibodies and the JNK3 antibody. Across all regions of the C57BL6/J

mouse brain, JNK3 exhibited extensive co-localization with NeuroTrace Red staining (Figure 5A).

Antibodies for selective neuronal markers were used to discern the JNK3-positive neuron species.

In the brainstem, an overlay of JNK3 and the motor neuronal marker, transcription factor islet 1

(Isl1), emerged throughout the region (Figure 5B). This co-localization was observed in areas

critical for motor function, including the vestibular nuclei, pontine reticular nucleus, and all cranial

nerve nuclei. Similar Isl1 localization also became apparent in the spinal cord, particularly in the

anterior sections containing motor neurons. JNK3 also displayed co-localization with the

cholinergic neuronal marker, choline acetyltransferase (ChAT), especially in regions densely

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populated with cholinergic neurons, such as the habenular nucleus (Figure 5C) and the cuneate

nucleus in the brainstem. In dopaminergic neuron-rich regions, including the substantia nigra, co-

localization of JNK3 with the dopaminergic neuronal marker, tyrosine hydroxylase (TH), also

became evident (Figure 5D). The examination of JNK3 and glutamate decarboxylase (GAD67), a

GABAergic marker, possessed overlapping signals that appeared across all brain regions, with

higher intensity areas of the hippocampus and cortex shown in Figure 5E. In contrast, a lack of co-

localization between JNK3 and the glutamatergic neuronal marker, vesicular glutamate transporter

1 (VGLUT1), was noted, even in areas known for a high concentration of glutamatergic neurons,

such as the hippocampus (Figure 5E). These findings collectively suggest that JNK3 is localized

in most types of neurons, but not all. The subcellular localization of JNK3 varied across different

regions of the brain. Primarily, JNK3 appeared to be distributed throughout the entire cell body,

whereas in the brainstem and spinal cord, JNK3 localization was more nuclear (Figure 5A).

JNK3 protein levels in adult male and female brain.

To corroborate the immunohistochemical

and immunofluorescent findings, the protein levels across multiple regions of the C57BL6/J mouse

brains were analyzed. Initially, a comparison of 3-month-old (the age of previous histological

studies [7, 8]) and 12-month-old male mice revealed no significant differences in JNK3 levels

were detected between the two age groups (Supplemental Figure 2). In 12-month-old mice,

significantly higher JNK3 protein levels in the hippocampus and cortex compared to the

cerebellum (Figure 6A). On the other hand, regions such as the brainstem, midbrain, and striatum

exhibited JNK3 protein levels comparable to those in the cerebellum (Figure 6A). Semi-

quantification of the protein levels was performed to illustrate any differences among the brain

regions (Figure 6B). In addition, a sex-based comparison between male and female mice revealed

no significant differences in JNK3 protein levels.

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Phosphorylated JNK and JNK3 abundance in adult male and female brain.

Prevailing research

suggests that elevated JNK3 activity may contribute to the induction of apoptosis; meanwhile,

studies implicating of JNK3 in neuron morphology, neurotransmission, and behavior would

indicate that elevated JNK3 activity in the healthy brain could illustrate areas that require JNK3

for normal neurophysiology. Thus, we used traditional and Phos-Tag© western blot analyses with

total JNK antibodies and the JNK3-selective antibody to measure the abundance of JNK3 in

dissected brain regions. A slight elevation of total JNK protein levels was observed in the

brainstem and midbrain, while a decrease was observed in the hippocampus and striatum (Figure

7A). In contrast, the levels of phosphorylated JNK were mildly higher in the brainstem, while

remaining consistent across all other regions (Figure 7A). Quantification of phosphorylated JNK3

in the brains of mice by Phos-Tag© western blotting revealed a higher proportion of

phosphorylated JNK3 species in the hippocampus and cortex (Figure 7A & B). However, the

brainstem presented fewer phosphorylated JNK3 species (Figure 7A & B). Upon further analysis

of total JNK and phosphorylated JNK proteins, no statistically significant sex-based differences

emerged between male and female mice regarding phosphorylated JNK, pJNK, and pJNK3 in the

brain (Figure 7B). Thus, the presence of active JNK3 can be found at distinct concentrations within

the brain.

JNK3 concentrations within astrocytes and in primary CNS cells.

Recent studies have suggested

that the expression of JNK3 may extend beyond neurons to glial cells, such as astrocytes and

oligodendrocytes [33, 51-53]. To compare JNK3 abundance in other prominent cell types in the

brain, primary neurons, astrocytes, and microglia were cultured from C57BL/6J mice. JNK3 was

found in the cultured neurons and astrocytes (Figure 8A); meanwhile, JNK3 was not detectable in

primary microglia (Figure 8A).Further examination of potential co-localization of JNK3 with

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astrocytes employed glial fibrillary acidic protein (GFAP) in IHC staining (Figure 8B). However,

alignment of JNK3 staining with astrocyte localization did not occur in adjacent brain slices,

indicating the absence of JNK3 in regions rich with astrocytes (Figure 8B). Cell morphologies in

these regions are noted in Supplemental Figure 3 for GFAP-positive and JNK3-positive cells. This

result may suggest that the prevailing JNK3 species in the healthy adult brain may be affiliated

with neurons, as suggested by previous studies.

Discussion

JNK3 is emerging as not only a central element of neurodegenerative disease pathophysiology,

but also for relevance to normal neurological function. Our study builds upon previous JNK3

histological and neurological studies and provides a greater resolution of JNK3 distribution in the

adult brain to help understand the kinase’s roles within the nervous system. The findings of our

current study demonstrate that JNK3 is distributed throughout the brain in healthy adult C57BL6/J

mice with protein (Figure 6) and phosphoprotein (Figure 7) levels elevated in the cortex and the

hippocampus as observed in previous studies [7, 8, 48]. However, our study uncovers the

enrichment of JNK3 and pJNK3 in areas of the brainstem, cerebellum, and cervical spinal cord not

mentioned in previous histological investigations. Furthermore, the study reaffirms the

predominance of JNK3 in neurons and identifies the specific types and locations of JNK3-positive

neurons. Collectively, these data set the stage to examine the nuanced functions of JNK3 in these

areas of the brain concerning motor function, cognitive abilities, disease progression, and other

neurological processes, but the distribution of JNK3 from our studies may provide insights into

the vulnerability of neurons in distinct brain regions to neurotoxic exposures and hallmark events

in neurodegenerative diseases.

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This study replicated earlier results with the p493F12 antibody, which indicated elevated JNK3

protein expression in the cortex and hippocampus, with diminished presence in the striatum [7, 8].

The cortical and hippocampal localization (Figure 1) corresponds with not only the proposed

involvement of JNK3 in AD and related dementias (ADRD) [25, 26, 37, 38], but it also aligns with

the putative involvement of JNK3 in neurobehavioral outcomes [46, 54]. As mentioned, earlier

Jnk3

-/-

mice exhibit some behavioral deficits in the elevated plus maze and potentially in the Morris

water maze compared to wild-type mice, and these tests require processes affiliated with the

cortical and hippocampal regions suggesting that JNK3 activities may be crucial to basal

neurological function [46]. Our current study finds that pJNK3 levels are elevated in the

hippocampus and cortex of healthy male and female mice (Figure 7) indicating that there is basal

JNK3 activities are contributing to neurophysiology in these regions, which is consistent with

recent pJNK3 radioligand labeling studies [49]. The higher levels of JNK3 activity in the

hippocampal region could also represent a “double-edged sword” to explain why these regions are

vulnerable to neuron loss in AD and other neurodegenerative disorders, as elevated and sustained

levels of pJNK3 have been implicated in these conditions. Perhaps, the physiological dependence

on JNK3 in the cortex and hippocampus, as well as other areas with elevated pJNK3, may lower

the threshold for triggering detrimental JNK3 signaling events. One possible mechanism for this

might be the physiological phosphorylation of Bcl-2 and BH3-only protein members [55] that

could lead to apoptotic priming and polarization of mitochondria and cellular pathways towards

cell death [56]. Additionally, inhibition of mitochondrial JNK signaling in the brain is

neuroprotective

in vitro

and

in vivo

in part by altering the phospho-status of Bcl-2 superfamily

proteins [4, 57]. Indeed, the mitochondrial scaffold for JNK, SH3-binding protein 5 (SH3BP5 or

Sab) is also highly expressed in the hippocampus and contributes to apoptosis and

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neurotransmission [58]. Thus, the tight coupling of JNK3 activity to basal neurophysiology in the

hippocampus and other regions may be a vulnerability when neurotoxic insults arise.

Alternatively, the diminished presence of JNK3 in the striatum (Figure 1, Figure 4, and Table

2), a site of Parkinson’s disease pathology (the loss of dopaminergic terminals [59]), is interesting

given not only the involvement of JNK3 in synapse dysfunction and dendritic pruning [15, 60],

but also many data indicating that JNK3-selective inhibitors protect in part against the loss of

striatal dopamine in preclinical models [11, 26, 49, 61]. The lower levels of JNK3 in the striatum

may speak to a reduced capacity for neuritogenesis. The first description of JNK3’s role in neurite

initiation was demonstrated when

Jnk3

-/-

mice and JNK3-selective inhibitors prevented

neuritogenesis of dorsal root ganglion neurons [62]; JNK3 was later implicated in neurite initiation

in other neurons, including dopaminergic neurons [63, 64]. Our analyses of pJNK3 levels in

striatum did not reveal robust levels of active JNK3, but if the pJNK3 species are restricted to

synaptic terminals or neurites in this large area, the pJNK3 could be diluted from highly

concentrated, but small regions of the area. Another possibility is that JNK3 could be signaling

from dopaminergic soma in the substantia nigra, as midbrain levels of pJNK3 were found in male

and female adult mice (Figure 7), and JNK3 protein levels were high in the substantia nigra (Figure

4) as well. Further, Bales, et al. found that in a preclinical model of PD, pJNK3 levels significantly

increased in the midbrain and in the area of the substantia nigra [49]. These data indicate that the

increase in JNK3 activity for extended duration may be neurodegenerative [26], and the ability to

detect these increased may be a useful way to identify vulnerable brain regions or to monitor

disease progression to better inform treatment of individual patients [49].

Intriguingly, our study indicates that there are moderate JNK3 protein levels in various

subcortical areas, including the thalamus and hypothalamus (Figures 1 & 4 and Table 2). The

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recent implications of JNK3 in metabolic homeostasis and disease [47, 65, 66] illustrate its

possible function in these regions. Specifically, smaller neurons of the supraoptic nucleus

exhibited an modest increase in JNK3 following tetrodotoxin treatment

in vitro

[65]. Additionally,

brain-penetrant JNK inhibitors with partial selectivity for JNK3, namely SR11935, reduced food

intake and body mass in adult mice [67]. It appears that JNK3/JNK2 inhibition sensitized animals

to leptin effects by increasing leptin-mediated activation of signal transducer and activator of

transcription 3 (STAT3) through the downregulation of suppressor of cytokine signaling 3

(SOCS3) [67]. Thus, while there may be therapeutic advantages to targeting JNK3 in neurological

disorders, the specificity (i.e., targeting mitochondrial JNK signaling) and dose of these

prospective interventions will require careful consideration to prevent effects on basal neurological

functions of JNK3.

The distribution of JNK3 was found to span the entire brainstem and extend into the cervical

spinal cord (Figure 2 & 4 and Table 2). Parallel findings were made previously in AD patient

brains; wherein, JNK3 localization was noted not just in the cortical and hippocampal regions, but

also within the brainstem and spinal cord [8]. Intriguingly, JNK3 colocalized with the motor

neuron marker Isl1 in the brainstem and spinal cord with a prevailing nuclear appearance in its

subcellular localization (Figure 5). The presence of JNK3 in motor neurons fits with the

identification of JNK3 in other brain regions implicated in motor functions, such as the motor

cortices, basal ganglia, cerebellum, and brainstem nuclei (Figure 1, 2, and 4). Examining pJNK3

levels in these regions, especially the cortices, cerebellum, and brainstem indicate there is active

JNK3 in these regions with higher levels of activity in the cortex and cerebellum when compared

to the brainstem. Nonetheless, these observations may indicate that JNK3 may have a role in the

regulation of motor functions. Past research has implicated JNK3 activity in neurodegenerative

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conditions associated with motor neurons, such as spinal cord injury, spinal muscular atrophy

(SMA) and amyotrophic lateral sclerosis (ALS) [68-70]. These studies suggest that elevated

pJNK3 initiates motor neuron loss and that inhibition of JNK3 by small molecules or gene ablation

can protect motor neurons [68-71]. The presence of JNK3 and pJNK3 in motor regions is an

noteworthy result given the absence of apparent motor defects in knockout animals or those treated

with JNK3-selective inhibitors [5]; nonetheless, the magnitude of JNK3 levels may be indicative

of an inherent vulnerability of motor neurons in these regions to stress.

Prior research has identified JNK3 as primarily neuronal, specifically apparent in

pyramidal neurons located in the hippocampus [8, 72], yet many JNK3 CNS activities highlighted

here span across distinct neuron types. Moreover, the co-localization of JNK3 with specific

neuronal types has been scarcely documented. Our study reports the co-localization of JNK3 with

a variety of neuronal types, including motor neurons and dopaminergic neurons mentioned earlier,

and cholinergic and GABAergic neurons. In AD, the loss of cholinergic neurons precipitates

disease progression and may coincide with increases in pJNK3 along the course of disease [73].

Similarly, the role for JNK3 in the maintenance and survival of GABAergic neurons may align

with alterations in GABAergic signaling that might contribute to the cognitive impairments seen

in AD [74]. A particularly interesting results was the absence of co-localization between JNK3

and VGLUT1 because of JNK3’s relationships with endosomes and vesicular trafficking [41-44].

Postulation on this result may explain why JNK3 could be a crucial inducer of glutamate

excitotoxicity in non-glutamatergic neurons, and that its minimal expression in VGLUT1-positive

neurons could provide a level of protection. Cell-type-specific studies into the roles of JNK3 in

distinct neurons could provide useful insights into its physiological and pathological activities in

the brain and spinal cord.

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In addition to neurons, astrocytes and oligodendrocytes have been identified as cell types

that may have JNK3 expression within the CNS [52, 53, 75-77]. Our analyses of GFAP in adult

mouse brain section do not align with JNK3 signals. However, primary cultured astrocytes do have

substantial JNK3 concentrations consistent with previous studies implicating JNK3 in cultured

astrocytes [52]. It is important to consider that our studies have been conducted on healthy adult

brains, yet neurodegenerative conditions widely exhibit neuroinflammation and reactive gliosis

[78] that could change the context of astrocytes allowing for JNK3 expression. Perhaps, these

stresses may be similar to some of those in cell culture settings that trigger JNK3 expression in

primary astrocytes. Unfortunately, we were unable to examine whether oligodendrocytes in the

brain tissue possess JNK3 and pJNK3, but the relationship between oligodendrocytes and JNK3-

implicated conditions like multiple sclerosis, suggests a potential interplay may exist. It is

interesting that two CNS-resident glial cells (astrocytes and oligodendrocytes) closely related to

neuronal function and neurotransmission would express JNK3. Studies are ongoing to determine

if JNK3 glial activities may contribute to neurophysiology and disease progression.

In summary, JNK3 is broadly localized throughout a healthy adult mouse brain, with

certain regions, such as the hippocampus and the cortex, displaying higher levels of JNK3

compared to others like the striatum. JNK3 is found in various neuronal cell types, including motor

neurons, cholinergic neurons, dopaminergic neurons, and GABAergic neurons, but it appears to

be absent from VGLUT1-positivve glutamatergic neurons. The activity of JNK3 is heightened in

regions that exhibit high immunoreactivity for JNK3, such as the cortex and the hippocampus. In

cell culture, JNK3 is present in neurons and astrocytes, though interestingly, in the brain, JNK3

does not co-localize with astrocytes. Overall, the insights gleaned from this study offer a higher

resolution of the distribution of JNK3 and pJNK3 activity in the adult mouse brain, connecting

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long-standing observation in the field of neuro-JNK research. Given the inherent differences

between mice and human neuroanatomy and CNS physiology, future studies will focus on

conducting similar studies in human brain sections to define JNK3 and pJNK3 localization and

subcellular distributions. Consequently, once the locations of JNK3 and active JNK3 are

elucidated, this information will deepen our understanding of the potential mechanisms and

vulnerabilities in neurodegenerative conditions, potentially setting the stage for the development

of more effective diagnostic, prognostic, and/or therapeutic approaches.

Acknowledgements

The authors would like to thank the members of the Chambers and Richardson labs for their helpful

comments during the experiments and preparation of this manuscript. We are especially grateful

to Dr. Melissa Edler (Kent State University) for her advice and encouragement during the imaging

quantification and analyses from our study. We would like to thank Dr. Roger J. Davis (University

of Massachusetts Chan Medical School and Howard Hughes Medical Institute) for the kind gift of

the JNK-null MEFs we used to validate the JNK3 antibody for this study. Also, I would like to

thank our Drs. Kim Tieu, Fenfei Leng, Timothy Allen, Hongxia Zhou, and Qin Li Cao for their

helpful comments in the editing of the manuscript. This research was made possible by start-up

funds provided by FIU to J.W.C and a grant from the Michael J. Fox Foundation for Parkinson’s

Disease Research (#1211), and V.G. was supported by a research assistantship from the

Transdisciplinary Biomolecular and Biomedical Sciences Training Program (NIH T32

GM132054-01). JRR was supported, in part, by NIH R01ES033892.

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Figure Legends

Figure 1.

Localization of JNK3 in 12-month-old C57BL6/J male mice with immunohistochemical

(DAB) staining.

Primary somatosensory cortex.

Primary motor cortex.

CA3 of the

hippocampus.

Piriform cortex.

Olfactory tubercle.

Thalamic Region (Lateral 1.68mm).

Increasing levels of magnification are indicated along the left side of the rows as 2x, 20x, and 60x.

Scale bar = 100μm. The black boxes at 2x magnification highlight the areas of magnification for

the 20x and 60x images for each column. Black arrows in the 60x panel identify neurons with

JNK3 positive signal in the above indicated regions.

Figure 2.

Localization of JNK3 in 12-month-old C57BL6/J male mice with immunohistochemical

(DAB) staining.

Spinal trigeminal nucleus.

Intermediate reticular nucleus.

Facial nucleus.

Motor trigeminal nucleus.

Vestibular nucleus.

Interposed cerebellar nucleus. (Lateral

1.68mm) Scale bar = 100μm. The black boxes at 2x magnification highlight the areas of

magnification for the 20x and 60x images for each column. Black arrows in the 60x panel identify

neurons with JNK3 positive signal in the above indicated regions.

Figure 3.

Photomicrographs of JNK3 immunoreactivity (DAB) in the brain of a C57BL/6J mouse

categorizing the strength of reactivity:

0.5 indicates low reactivity

2 indicates moderate

reactivity

3 indicates high reactivity. The black arrows indicate cells possessing the reactivity

described in the qualitative rubric. Scale bar = 100μm.

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Figure 4.

Spatial mapping of JNK3 immunoreactivity in the

frontal cortex (bregma 1.33mm),

striatal (bregma 0.73mm),

thalamic (bregma -1.67mm),

hippocampal (bregma -3.27mm),

midbrain (bregma -4.83mm),

pons (bregma -5.79mm),

medulla oblongata (bregma -

7.19mm), and

spinal cord (330 microns) regions. The expression of JNK3 is similar in males

and females throughout the brain. The map was adapted from

Table 1

. Gray areas were not

quantified. The images and labeling were adapted from Alan Brain Atlas and Paxinos and

Franklin’s The Mouse Brain Atlas.

Figure 5.

Co-localization of JNK3 with neurons in 12-month-old C57BL6/J male mice with

immunofluorescent staining.

JNK3 is present in neurons (NeuroTrace straining) in the facial

nucleus.

JNK3 co-localizes with a motor neuron marker (Isl1) in the hypoglossal nucleus.

JNK3 co-localizes with the cholinergic neuron marker (ChAT) in primary cortex.

JNK3 co-

localizes with dopaminergic neuron marker (TH) in the substantia nigra.

JNK3 co-localizes

with GABAergic neuron marker (GAD67) in the CA3 of the hippocampus.

JNK3 JNK3 does

not co-localize with glutaminergic neuron marker (VGLUT1) in the primary cortex. White arrows

indicate cells exhibiting colocalization of JNK3 and neuron-specific signals. Scale bar = 100μm.

Figure 6. A.

Sex comparison of JNK3, JNK, and pJNK protein levels in different brain regions of

12-month-old C57BL/6J mice.

Analysis of JNK3, JNK, and pJNK protein levels comparing

different brain regions of 12-month-old male and female C57BL/6J mice, with hippocampus and

cortex being significantly higher in males and females. Brain regions were normalized to the

cerebellum and quantified using Licor Empiria Software. Statistical analysis performed on Prism

9. Male n=5 and female n=6.

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Figure 7. A.

Sex comparison of pJNK3 protein levels in different brain regions of 12-month-old

C57BL/6J mice.

Analysis of pJNK3 protein levels comparing different brain regions of 12-

month-old male and female C57BL/6J mice. Brain regions were normalized to the cerebellum and

quantified using Empiria Software. Statistical analysis performed on Prism 9. Male n=4 and female

n=4.

Figure 8. A.

JNK3 abundance in primary cells from C57BL/6J mice. Cell lysates were normalized

to REVERT staining and quantified using Licor Empiria Software. The molecular weight

standards are indicated by L, primary mouse neurons are marked as PMN, Primary mouse

astrocytes are indicated as PMA, and Primary mouse microglia are labeled as PMG. Statistical

analysis performed on Prism 9. n=3.

Localization of GFAP and JNK3 in adjacent coronal

slices of C57BL6/J mice brain:

CA3 of the hippocampus,

ii.

primary motor cortex,

iii.

forceps

minor of the corpus callosum, and

iv.

spinal trigeminal region. The white arrows indicate the cells

possessing either a JNK3 positive or GFAP positive signal. Scale bar = 100μm.

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Figure 6

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Tables

Table 1

Summary of Antibodies

Antigen

Antibody

Cell Type

Dilution

Company/

Catalog #

JNK3 (55A8)

Rabbit monoclonal

antibody to JNK3

clone 55A8

Neurons

WB: 1:1000 IF:

1:500

Cell Signaling, 2305,

RRID: AB_2281744

ChAT (CL3137)

Mouse monoclonal

antibody to ChAT

clone CL3137

Cholinergic neurons

IF: 1:500

Invitrogen, MA5-

31383, RRID:

AB_2787020

VGLUT1 (CL2754)

Mouse monoclonal

antibody to

VGLUT1 clone

CL2754

Glutaminergic

neurons

IF 1:1000

Invitrogen , MA5-

31373, RRID:

AB_2787010

GAD67 (OT13G9)

Mouse monoclonal

antibody to DAG67

clone OT13G9

GABAergic neurons

IF 1:500

Invitrogen, MA5-

24909, RRID:

AB_2723202

Anti-TH Clone LNC1

Mouse monoclonal

antibody to TH clone

LNC1

Dopaminergic

neurons

IF: 1:500

EMD Milipore Corp,

MAB318,

RRID:AB_2201528

ISL1 (1B1)

Mouse monoclonal

antibody to ISL1

clone 1B1

Motor neurons

IF 1:500

Invitrogen, MA5-

15516, RRID:

AB_10982886

GFAP

Mouse Monoclonal

Antibody clone GA5

Astrocytes

IHC 1:1000

Cell Signaling, 3670:

RRID: AB_561049

SAPK/JNK

Rabbit polyclonal

antibody to JNK

Brain Lysates

WB: 1:1000

Cell Signaling, 9252,

RRID: AB_2250373

p-SAPK/JNK

(Thr183/Tyr185) (G9)

Rabbit monoclonal

antibody to

residues Thr183/Tyr

185 of phospho-JNK

Brain Lysates

WB: 1:1000

Cell Signaling, 9255,

RRID: AB_2307321

β-actin (8H10D10)

Mouse monoclonal

actibody to β-actin

clone 8H10D10

Brain Lysates

WB: 1:7000

Cell Signaling, 3700,

RRID: AB_2242334

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Table 2.

JNK3 immunoreactivity in the brains of C57BL/6J 12 months males and females.

The immunoreactivity scale is 3– very high reactivity, 2.5 – high reactivity, 2 – moderate reactivity, 1.5 – low reactivity,

below 1 – very low reactivity. n=3 for each sex group.

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Supplemental Figures and Legends

Supplementa1 Figure 1.

Validation of JNK3-selective antibody.

JNK-null murine embryonic fibroblasts (a kind gift of

Dr. Roger J. Davis) were transfected with empty vector or pCDNA encoding human JNK1



1, JNK2



1, and JNK3



variants. Cell lysates were then subjected to western blot analyses with JNK3-selective antibody (Cell Signaling) and

Total JNK antibody (Cell Signaling).

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Supplementa1 Figure 2.

A. Age comparison of JNK3, JNK, and pJNK protein levels in different brain regions of 3 and

12 months old C57BL/6J mice. B. Analysis of JNK3, JNK, and pJNK protein levels comparing different brain regions of

3 and 12 months old male C57BL/6J mice. Brain regions were normalized to cerebellum and quantified using Empiria

Software. n=3

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Supplementary Figure 3.

Localization of GFAP and JNK3 in adjacent coronal slices of C57BL6/J mice brain. (A)

Localization of GFAP and JNK3 in the spinal trigeminal region (B) Localization of GFAP and JNK3 in the primary motor

cortex. (C) Localization of GFAP and JNK3 in the forceps minor of the corpus callosum. Black arrows identify

representative cells with the respective proteins present. Scale bar = 100 μm.

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