# PYK2 in the dorsal striatum of Huntington's disease R6/2 mouse model

Omar Al-Massadi, Mélody Labarchède, Benoit de Pins, Sophie Longueville, Albert Giralt, Theano Irinopoulou, Mythili Savariradjane, Enejda Subashi, Silvia Ginés, Jocelyne Caboche, et al.

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To cite this version:

Omar Al-Massadi, Mélody Labarchède, Benoit de Pins, Sophie Longueville, Albert Giralt, et al. PYK2 in the dorsal striatum of Huntington's disease R6/2 mouse model. Neurobiology of Disease, In press, pp.106840. 10.1016/j.nbd.2025.106840. hal-04963950

HAL Id: hal-04963950

https://hal.sorbonne-universite.fr/hal-04963950v1

Submitted on 24 Feb 2025

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PYK2 in the dorsal striatum of Huntington's disease R6/2 mouse model

Omar Al-Massadi<sup>a,b,c*</sup>, Mélody Labarchède<sup>a,b,c,d,#</sup>, Benoit de Pins<sup>a,b,c,#</sup>, Sophie Longueville<sup>a,b,c</sup>, Albert Girault<sup>a,b,c,e,f,g,h</sup>, Theano Irinopoulou<sup>a,b,c</sup>, Mythili Savariradjane<sup>a,b,c</sup>, Enejda Subashi<sup>b,i,j,k</sup>, Silvia Ginés<sup>e,f,g,h</sup>, Jocelyne Caboche<sup>b,i,j,k</sup>, Louise-Laure Mariani<sup>d</sup>, Sandrine Betuing<sup>b,i,j,k</sup>, Jean-Antoine Girault<sup>a,b,c,d*</sup>

a- Inserm UMR-S 1270, Paris 75005, France

b- Sorbonne Université, Faculty of Sciences and Engineering, Paris 75005, France

c- Institut du Fer à Moulin, 17 rue du Fer à Moulin, Paris 75005, France

d- Institut du Cerveau, Paris Brain Institute, ICM, Inserm, CNRS, Sorbonne Université, Paris, France.

e- *Departament de Biomedicina, Facultat de Medicina i Ciències de la Salut, Institut de Neurociències*, Universitat de Barcelona, 08036 Barcelona, Spain

f- *Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas*, Madrid, Spain

g- *Institut d'Investigacions Biomèdiques August Pi i Sunyer*, Barcelona, Spain

h- Production and Validation Center of Advanced Therapies (Creatio), University of Barcelona, Barcelona, Spain.

i- Neuroscience Paris Seine, Institut de Biologie Paris-Seine, Paris, France.

j- CNRS UMR8246, Paris, France

k- INSERM U1130, Paris, France

*Corresponding authors: Omar Al-Massadi [omar.al-massadi.iglesias@sergas.es](mailto:omar.al-massadi.iglesias@sergas.es) and Jean-Antoine Girault [jean-antoine.girault@inserm.fr](mailto:jean-antoine.girault@inserm.fr) (to whom correspondence should be addressed at the time of submission)

# These authors (ML and BdP) contributed equally.
**Current addresses:**

- Omar Al Massadi: Translacional Endocrinology group, Endocrinology section, Instituto de Investigación Sanitaria de Santiago de Compostela, Complexo Hospitalario Universitario de Santiago (IDIS/CHUS), Santiago de Compostela, Spain and CIBER Fisiopatología de la Obesidad y Nutrición (CIBERobn), Av Monforte de Lemos3-5, 28029, Madrid, Spain

- Benoit de Pins, Department of Biology, University of Naples “Federico II”, Naples, Italy

Key words: movement disorder, non-receptor tyrosine kinase, caudate-putamen

**Highlights**

* Huntington's disease is a lethal genetic disease altering striatum, cortex, and hippocampus

* Restoring PYK2 levels in hippocampus improved hippocampal phenotype of Huntington mouse model

* We show that PYK2 levels are decreased in the striatum of R6/2 mice and human patients

* Viral expression of PYK2 limited to the striatum has only a small effect on R6/2 mouse model phenotype

* AAV serotypes and promoters influence striatal interneurons transduction

**Keywords:**

Huntington's disease; Striatum; Caudate-putamen; PYK2; non-receptor tyrosine kinase.

**Abbreviations**

**AAV**, adeno-associated virus; **AD**, Alzheimer's disease; **ANOVA**, analysis of variance; **CAG**, cytosine-adenosine-guanosine; **DARPP-32**, dopamine- and cAMP-regulated phosphoprotein with an apparent molecular weight of 32 kDa; **DS**, dorsal striatum; **GWAS**, genome-wide association study; **HD**, Huntington's disease; **KO**, knockout; **PBS**, phosphate-buffered saline;
**PBS-T**, PBS with Triton X-100; **PSD-95**, post-synaptic density protein with an apparent molecular weight of 95 kDa; **PTK2B**, protein tyrosine kinase 2B, gene coding for PYK2; **PV**, parvalbumin; **PYK2**, proline-rich tyrosine kinase 2; **RT-qPCR**, real time quantitative polymerase chain reaction; **SPN**, striatal/spiny projection neuron; **SST**, somatostatin; **STEP**, striatal-enriched tyrosine phosphatase; **VMAT2**, vesicular monoamine transporter 2; **WT**, wild type.

**Abstract**

Huntington's disease (HD) is a devastating disease due to autosomal dominant mutation in the *HTT* gene. Its pathophysiology involves multiple molecular alterations including transcriptional defects. We previously showed that in HD patients and mouse model, the protein levels of the non-receptor tyrosine kinase PYK2 were decreased in the hippocampus and that viral expression of PYK2 improved the hippocampal phenotype. Here, we investigated the possible contribution of PYK2 in the striatum, a brain region particularly altered in HD. PYK2 mRNA levels were decreased in the striatum and hippocampus of R6/2 mice, a severe HD model. Striatal PYK2 protein levels were also decreased in R6/2 mice and human patients. PYK2 knockout by itself did not result in motor symptoms observed in HD mouse models. We examined whether PYK2 deficiency participated in the R6/2 mice phenotype by expressing PYK2 in their dorsal striatum using AAV vectors. With an AAV1/Camk2a promoter, we did not observe significant improvement of body weight, clasping, motor activity and coordination (rotarod) alterations observed in R6/2 mice. With an AAV9/SYN1 promoter we found a slightly higher body weight and a trend to better rotarod performance. Both viruses similarly transduced striatal projection neurons and somatostatin-positive interneurons but only AAV9/SYN1 led to PYK2 expression in cholinergic and parvalbumin-positive interneurons. Expression of PYK2 in cholinergic interneurons may contribute to the slight effects observed. We conclude that PYK2 mRNA and protein levels are decreased in the striatum as in hippocampus of HD patients and mouse models. However, in contrast to hippocampus, striatal viral expression of PYK2 has only a minor effect on the R6/2 model striatal phenotype.
# 1. Introduction

Huntington's disease (HD) is an autosomal dominant inherited disease caused by trinucleotide repeat expansion in the Huntingtin gene (*HTT*) (Collaborative, 1993). The prevalence of HD is 3-12 cases per 100,000 people of European ancestry (Evans et al., 2013; Pringsheim et al., 2012). HD patients display a broad symptomatology affecting cognition and behavior. These patients also develop major motor impairments including gait disturbances, lack of coordination, choreic and jerky body movements. Another hallmark of the disease is the loss of body weight despite efforts to maintain a high calorie diet (Marder et al., 2009). Although great progress has been achieved in our understanding of the cause of HD and its molecular and cellular mechanisms (Mariani et al., 2016; Saudou and Humbert, 2016), therapeutic options are limited. Inhibitors of the vesicular monoamine transporter VMAT2 can improve movement disorders (Koch et al., 2020), but no disease-modifying treatment is available for patients, despite great effort of the research community (Estevez-Fraga et al., 2024). Thus, the identification of new molecular targets that could help in the fight against HD is urgently desired.

Since the discovery of the *HTT* gene mutation numerous genetic mouse models were genetically engineered (Figiel et al., 2012; Heng et al., 2008; Pouladi et al., 2013). Many motor and cognitive deficits endured by HD patients are recapitulated in these preclinical mouse models, which are useful tools to uncover pathophysiology and to develop potential therapeutic strategies (Crook and Housman, 2011; Gil and Rego, 2009; Switonski et al., 2012). Two genetic mouse lines commonly used to study HD are the R6/1 and R6/2 mice. These mice are transgenic for the first exon of the human *HTT* gene with amplified CAG repeats (Brooks and Dunnett, 2015; Farshim and Bates, 2018; Stricker-Shaver et al., 2018). The R6/2 line displays an accelerated phenotype and develops neurological impairments, including loss of coordination, abnormal gait, hind limb clasping behavior, loss of body weight, and premature death (Brooks and Dunnett, 2015; Farshim and Bates, 2018; Stricker-Shaver et al., 2018). R6/2 mice develop symptoms at the age of 5 weeks and can live up to 14 weeks whereas the R6/1 mice have a longer life expectancy and less severe symptomatology (Brooks and Dunnett, 2015; Farshim and Bates, 2018; Stricker-Shaver et al., 2018).
The most prominent neuronal change in HD patients is a neuronal loss in the dorsal striatum (DS) (Vonsattel and DiFiglia, 1998) also observed in mouse models (Klapstein et al., 2001; Levine et al., 1999). The DS receives information from all areas of the cerebral cortex and its role in motor behavior is altered in HD (Estrada-Sanchez and Rebec, 2013). Striatal projection neurons (SPNs) are medium-size spiny GABAergic neurons, which form the vast majority of striatal neurons [about 95% in rodents and 80-85% in humans (Lecumberri et al., 2018)]. SPNs are particularly vulnerable in HD, undergoing progressive degeneration and massive cell loss over the course of the disease (Klapstein et al., 2001; Levine et al., 1999; Vonsattel and DiFiglia, 1998). Therefore, the DS appears as a key target area in the disease.

PYK2 is a non-receptor tyrosine kinase that can be activated by $Ca^{2+}$ and is highly expressed in the brain, specifically in forebrain principal neurons [(Menegon et al., 1999) and review in (de Pins et al., 2021)]. Previous studies determined that PYK2 is involved in synaptic plasticity (Bartos et al., 2010; Giralt et al., 2017; Huang et al., 2001; Mastrolia et al., 2021) and its gene, *PTK2B*, is a risk locus for Alzheimer's disease (Lambert et al., 2013). Our group previously showed that i) PYK2 protein levels are decreased in the hippocampus of HD patients and R6/1 mice; ii) *Ptk2b* knockout (PYK2-KO) mice hippocampal phenotype resembles that of R6/1 mice; iii) PYK2 viral expression in the hippocampus of R6/1 mice improves memory deficits and some synaptic markers (Giralt et al., 2017).

Given the key role of the striatum in HD and our previous data suggesting a contribution of PYK2 deficit in the HD hippocampal phenotype, we examined the possible alteration of PYK2 in the DS. We measured PYK2 mRNA and protein in R6/2 mice and patients, we studied the motor function of PYK2-KO mice and we investigated the effects of AAV-mediated PYK2 expression in the DS of R6/2 mice.

# 2. Material and Methods

## 2.1. Mice
Wild type (WT) mice were male C57BL/6JRj mice provided by Janvier Laboratories (France). R6/2 [B6CBA-Tg (HDexon1) 62Gpb/1J] mice, which express exon 1 of the human mutant Huntington's disease gene containing 150 CAG repeats, under the control of the human HTT (IT15) promoter, were obtained from Jackson Laboratories, which produces them by crossing ovarian transplant hemizygous females with B6CBAFI/J males. Mice used in this study were males and females from the first offspring and the genotype was verified by polymerase chain reaction (PCR) using genomic DNA extracted from the tail. The CAG repeat length varies in the progeny from the first generation (https://www.chdifoundation.org/wp-content/uploads/HD_Field_Guide_040414.pdf) and can be considered to be around 150 CAG for all the mice that were used in the study. PYK2-KO male mice, which bear a deletion of exons 15 to 18 in the *Ptk2B* gene which codes PYK2, were produced by our laboratory as previously described (Giralt et al., 2017; Giralt et al., 2016). WT littermates and mutant mice from either sex were fed ad libitum and housed in groups under conditions of controlled temperature (23°C) and illumination (12-hour light/12-hour dark cycle). Animal experiments and handling was in accordance with ethical guidelines of the Declaration of Helsinki and NIH, (1985-revised publication no. 85-23, European Community Guidelines), and French Agriculture and Forestry Ministry guidelines for handling animals (decree 87849, license A 75-05-22) and approval of the Charles Darwin ethical committee (APAFIS#8861-2016111620082809).

## 2.2. Viral constructs and stereotactic injection

For expression of PYK2 in the DS, 4-week-old R6/2 and WT mice were stereotactically injected with AAV vectors. Groups of animals received AAV1.Camk2a-eGFP-2A-mPTK2B (referred to below as AAV1/Camk2a-PYK2) expressing PYK2 and GFP separated by a T2A cleavable link (Vector Biolabs Malvern, PA, USA). As control we used AAV9.Camk2a-eGFP.WPRE.rBG (AAV9/Camk2a-GFP) expressing only GFP (Vector Biolabs Malvern, PA, USA). In other experiments we used AAV9-hSYN1-eGFP-2A-mPTKB2B (AAV9/SYN1-PYK2) which express both GFP and PYK2 and as control AAV9/SYN1-GFP, all from University of Pennsylvania Perelman School of Medicine AAV core facility. Mice were anesthetized by an intraperitoneal injection of ketamine, 15 mg kg⁻¹, and xylazine, 3 mg kg⁻¹, and placed in a stereotactic frame (Kopf
Instruments). The DS was targeted bilaterally as previously reported (Boussicault et al., 2016), using a 32-gauge needle connected to a 1-ml syringe (Neuro-Syringe, Hamilton) at stereotactic coordinates 0.5 mm rostral to the bregma, 2.1 mm lateral to midline, and 3.35 mm ventral to the skull surface. The rate of injection was 0.15 µl min⁻¹ for 13 min with a total volume of 2 µl injected per striatum. After AAV infusion the needle was kept in place for an additional 5 minutes. After the procedure, the incision was closed with black braided non-absorbable silk suture (Ethicon, Puerto Rico USA). Mice were placed 2 h for careful monitoring in a heated cage until they recovered from anesthesia and then returned to their home cage for 2 weeks before starting analyses of behavior. Analgesia was achieved during the two days following surgery by subcutaneous injections of a non-steroidal anti-inflammatory drug (flunixin, 4 mg kg⁻¹, Cronyxin, Bimeda).

## 2.3. Behavioral phenotyping

**Open Field.** The open field apparatus was a dimly lit (60 lx) white square arena (40 × 40 × 40 cm in length, width, and height respectively). Animals were placed at the center and allowed to freely explore for 15 min. Spontaneous locomotor activity was continuously recorded with a video tracker (Viewpoint, Newcastle Upon Tyne, UK) and analyzed using a software that automatically detects rodent movements and behaviors (Giralt et al., 2017).

**Circular maze.** Locomotor activity was measured in a circular corridor with four infrared photoelectric beams placed at 90° angles (Imetronic, Pessac, France) in a low luminosity environment. Mice were placed in the maze for 90 min and the spontaneous locomotion measured in ¼ turns (consecutive interruptions of two adjacent beams) and the number of rearings in 5-min time bins.

**Balance beam.** The balance beam test is a sensorimotor integration test evaluating balance and motor skills. The test measures the time taken by the mouse to reach a safe enclosed platform, walking over a narrow and elevated beam. A bright light illuminates the start area and a close 20 x 20 cm square escape box is located at one end of the beam. During the two consecutive days of testing, each mouse was given two trials with a minimum of 10 min between the trials.
One day before the tests, a habituation session was given to familiarize the mouse with the beam. For each trial, the mouse was placed in the center of the beam facing one of the platforms and then released. Latency to traverse the beam was recorded.

**Vertical Pole Test.** The vertical pole test measures the balance, motor coordination, and vertical orientation capability of mice. The mouse was placed head downward in the middle of a round wooden pole (50-cm height, 1-cm diameter). The pole was wrapped with cloth tape for improved traction and preventing sliding. The pole was held in horizontal position, and then was gradually shifted to a vertical position. The time until the mouse stayed or walked up or down the pole were measured with a stopwatch. The cut off time of the test was 60 seconds to avoid exhaustion. The time of latency to fall was calculated for each mouse. All mice were habituated to the task one day before the test, followed by a one-day test. During testing, the following scores were applied: falling from the pole immediately or when trying to turn or descend at any time (scored 0); 0-20 seconds of latency (scored 1); 21-40 seconds of latency (scored 2); 41-60 seconds of latency (scored 3); more than 60 seconds of latency (scored 4); sliding down the pole immediately (scored 5); freezing and never descending the pole (scored 6).

**Rotarod.** Rotarod was used to evaluate motor coordination (Boussicault et al., 2016). Mice were tested on three consecutive days, each daily session including a 5-min training trial at 4 rpm on the rotarod apparatus (LE8200, Bioseb). One hour later, the animals were tested for three consecutive accelerating trials of 5 min with the rotarod speed linearly increasing from 4 to 45 rpm over 300 s. The latency to fall from the rod and the speed at fall were recorded for each trial. Mice remaining on the rod for more than 300 s were removed and their time scored as 300 s. Data from the training trial were not included.

**Hind limb clasping.** Mice were suspended by the tail for 30 s and the clasping phenotype was graded according to the following scale (Guyenet et al., 2010): score 0, no clasping (hind limbs consistently splayed outward, away from the abdomen); score 1, one hind limb retracted toward the abdomen for more than 50% of the time; score 2, both hind limbs partially retracted toward the abdomen for more than 50% of the time suspended; score 3, hind limbs entirely retracted and touching the abdomen for more than 50% of the time suspended.
## 2.4. Immunohistofluorescence

Mice received a lethal dose of pentobarbital (500 mg kg⁻¹, i.p., Dolethal, Vetoquinol, France) and perfused transcardially with 40 g L⁻¹ paraformaldehyde in 0.1 M sodium phosphate buffer pH 7.4. Brains were either post-fixed overnight at 4°C and cut into 30 µm-thick free-floating coronal sections with a vibrating microtome (Leica, VT1000) or post-fixed for 2 hours and cut into 30 µm-thick with freezing microtome (Microm Microtech, HM450). All the following steps were done with gentle shaking. Sections were washed three times in phosphate-buffered saline (PBS), permeabilized in PBS-T (PBS + 3 µl mL⁻¹ Triton X-100) and 30 µl mL⁻¹ normal goat serum (Pierce Biotechnology, Rockford, IL, USA) or bovine serum albumin (50 g L⁻¹, Sigma-Aldrich, #A3912) for 60 min at room temperature, and washed three times. Brain slices were incubated 12-48 hours at 4°C in the presence of primary antibodies in PBS-T: rabbit polyclonal antibodies for PYK2 (1/500, Sigma #07M4755, Chemical Co., St Louis, MO, USA) or parvalbumin (PV, 1/400, Swant, #PV27a), chicken recombinant monoclonal GFP antibody (Abcam, EPR14104, ab300643), mouse monoclonal antibodies for DARPP-32 (1/5,000, gift from Paul Greengard, The Rockefeller University, New York, NY, USA) or for somatostatin (1/100, Santa-Cruz, #sc-55565), and goat polyclonal choline-acetyl-transferase antibodies (ChAT, 1/100, Merck-Millipore, #AB144P). Sections were then washed three times and incubated for 2 h at room temperature with fluorescent secondary antibodies combined as indicated in figure legends: Cy3 goat anti-rabbit (1/200) and/or AlexaFluor 488 goat anti-mouse (1/200), and/or 488 donkey anti-chicken (1/1,000), all three from Jackson ImmunoResearch, West Grove, PA, USA) and/or 555 donkey anti-rabbit (1/1,000) and/or 647 donkey anti-goat (1/1,000, both from ThermoFisher scientific, A32794). No signal was detected in control sections incubated in the absence of the primary antibody.

## 2.5. RT-qPCR

RNA was extracted using Trizol® reagent (Invitrogen) according to the manufacturer's instructions. The RNA was quantified with a Nanodrop 1000 spectrophotometer. Five hundred ng of total RNA were used for each reaction. Reverse transcription, cDNA synthesis was performed using the SuperScript™ First-Strand synthesis System (Invitrogen) and random
primers. Negative control reactions, containing all reagents except the sample were used to ensure specificity of the PCR amplification. Real-time polymerase chain reaction was carried out using a fluorescent temperature cycler (TaqMan®; Applied Biosystems, Foster City, CA, USA) following the manufacturer's instructions. The PYK2-specific oligonucleotide primers were: forward GGGGAAAAACAACCTGTCAGC, reverse GCTGGATGTTGGGCCCTATT. Results were quantified and normalized to a house-keeping gene with delta-delta-CT (ddCT) method and expressed in comparison with the average value for the control group. All samples were run in duplicate and the average values calculated.

## 2.6. Immunoblot analysis

*Mouse brain samples.* Immunoblotting was performed as described (Giralt et al., 2017; Giralt et al., 2018; Montalban et al., 2019). Briefly, animals were killed by CO₂ inhalation and the DS rapidly dissected on ice. Protein lysates from DS (10 µg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred onto nitrocellulose membranes (GE Healthcare, LC, UK). Membranes were probed with rabbit polyclonal antibodies for PYK2 (074M4755, Sigma, 1/1,000). After several washes in TBS-Tween, blots were incubated with secondary anti-rabbit or anti-mouse IgG IRdye800CW coupled or anti-mouse IgG IRdye700DX-coupled antibodies (1/2,000, Rockland Immunochemicals, USA).

*Post-mortem human brain.* Postmortem putamen tissue samples from control and HD patients were obtained from the Neurological Tissue Bank (Biobanc-HC-IDIBAPS), Barcelona, Spain with the approval of the local ethics committee at recruitment and an informed written consent (Martin-Flores et al., 2020). They included six controls (two females, four males aged 81.2 ± 5.0 years, mean ± SD, post-mortem interval, 11.9 ± 8.8 h) and six patients with HD grades 1-3 (all males, age 68.2 ± 12.3 years, post-mortem intervals, 8.8 ± 4.2 h, see **Table 1**).

*Immunoblot quantification.* Secondary antibody binding was detected by Odyssey infrared imaging apparatus (Li-Cor Inc., Lincoln, NE, USA). We used two corrective steps to allow comparison of protein samples: i) to avoid errors in amounts of loaded proteins or lane artifacts, the signal for the relevant protein bands was first normalized to either actin or tubulin (detected
in the same sample lane on the same membrane with appropriate antibodies); ii) Because not all samples could be run on the same gels, results were then expressed as a % of the mean of WT (in mice samples) or controls (in human brain samples) in the same gel/membrane, to allow comparison between them.

## 2.7. Statistical analyses

Analyses were done using GraphPad Prism 6. Normality of data distribution was checked with d'Agostino-Pearson and Shapiro-Wilk tests, and non-parametric analysis used if the distribution was significantly different from normal or data set sample number was too small (<7). Two-group comparisons were done with two-tailed unpaired Student's t test and 3 groups analyzed using one- or two-way ANOVA, followed by Holm-Sidak's post hoc test for multiple comparisons. Non-parametric comparisons of 2 groups were done with Mann-Whitney's test and 3 groups or more with Kruskal-Wallis' test followed by Dunn or Mann-Whitney's test as indicated. Significance threshold was 0.05. Detailed statistical results are presented in **Supplementary Table 1**.

# 3. Results

## 3.1. PYK2 mRNA levels are decreased in the hippocampus and striatum but not in the cerebral cortex of R6/2 mice

The expression of PYK2 mRNA in the brain was previously investigated by in situ hybridization (Menegon et al., 1999). To obtain more quantitative results, we performed RT-PCR using samples from hippocampus, striatum, and cerebral cortex of 3-month-old WT male mice. We confirmed the data of Menegon et al., who found that the hippocampus is the brain region with the highest PYK2 mRNA concentration (Figure 1A). PYK2 mRNA levels in the striatum were intermediate between those in the cerebral cortex and in the hippocampus (Figure 1A, Supplementary Table 1). These measurements indicated that PYK2 may play a role in the striatum in the context of HD. We then investigated whether PYK2 mRNA levels were altered in the R6/2 HD mouse model and measured PYK2 mRNA using RT-qPCR in the cerebral cortex,
hippocampus, and striatum of these mice at various ages. In the cortex of 12-week-old R6/2 mice, PYK2 mRNA levels did not differ significantly from those in WT controls (Figure 1B, Supplementary Table 1) and we did not investigate younger ages. We measured PYK2 mRNA levels in the hippocampus and striatum at 4, 6, and 12 weeks of age (Figures 1C-D). In both regions PYK2 mRNA levels were decreased in R6/2 mice at 6 and 12 weeks as compared to WT mice, but not at 4 weeks (Figure 1C-D, Supplementary Table 1). These data showed an early loss of PYK2 transcripts in the striatum and hippocampus of R6/2 mice but not in the cortex, although the three brain regions display metabolic alterations (Mochel et al., 2012). They also indicated that the previously reported deficit in PYK2 protein in the hippocampus of HD R6/1 mouse model (Giralt et al., 2017) is likely to result, from a decrease in its mRNA.

## 3.2. Striatal PYK2 protein levels are decreased in the striatum of R6/2 mice and HD patients

Because changes in mRNA levels can be inconsistent with the levels of the corresponding protein, we investigated PYK2 protein levels in the striatum of R6/2 mice using immunoblotting. PYK2 protein levels were not changed at 6 weeks of age but decreased at 9 and 12 weeks (Figure 2A-B, Supplementary Table 1). We then measured PYK2 protein levels in post-mortem samples of the putamen of patients with HD compared with controls with no striatal neurodegenerative disease (Table 1). PYK2 levels were reduced in the HD samples compared to controls (controls, 100 ± 9.7%, HD, 63.6 ± 11.8%, mean ± SEM, 6 subjects per group, 2-tailed Mann-Whitney's test, p = 0.04, Figure 2C-D, Supplementary Table 1). These results opened the possibility of a potential contribution of striatal PYK2 deficit in the pathophysiology and symptomatology of HD, as previously observed in the hippocampus (Giralt et al., 2017).

## 3.3. PYK2-KO mice exhibit normal motor behavior

Because PYK2 mRNA levels were decreased in the striatum of R6/2 mice and PYK2 protein in both patients and mice, we next examined whether the absence of PYK2 was able by itself to induce motor deficits. For this purpose, we used 3-4 month-old PYK2-KO male mice and first measured their locomotor activity in an open field. PYK2-KO mice travelled the same distance as WT littermates (Figure 3A, Supplementary Table 1). We then compared the sensorimotor
coordination of PYK2-KO and WT littermate mice in the balance beam, with two trials for each mouse on two consecutive days (Figure 3B, Supplementary Table 1). We did not observe any difference between the two genotypes. We also assessed the PYK2-KO and WT littermate mice in the vertical pole test, which evaluates balance, motor coordination, and vertical orientation capabilities. Compared to WT mice, PYK2-KO mice did not exhibit deficits in this test (Figure 3C, Supplementary Table 1). We finally looked for a characteristic response exhibited by HD animal models, hind limb clasping (Boussicault et al., 2016; Mangiarini et al., 1996). We did not observe this response in PYK2-KO mice (Figure 3D). The absence of patent motor phenotype in PYK2-KO animals was consistent with our previous observation of normal performance of PYK2-KO mice on the accelerating rotarod (de Pins et al., 2020) and showed that the complete lack of this protein is not sufficient to visibly alter motor function in young mice. These results suggested that the partial decrease in PYK2 mRNA and protein levels observed in the striatum of HD mouse models or patients is unlikely to generate by itself the motor alterations they display. Yet, they do not rule out the possibility that the PYK2 deficit synergizes with other molecular defects in HD and plays an aggravating role.

**3.4. Effects of AAV1-mediated PYK2 expression under Camk2a promoter in R6/2 mice**

Because the expression levels of PYK2 in the striatum of R6/2 mice and in the putamen of HD patients were decreased (Figure 2) and because re-expression of PYK2 in the hippocampus had beneficial effects on the symptoms linked to this brain region (Giralt et al., 2017), we asked whether expressing PYK2 in the striatum of R6/2 mice, could partly improve their phenotype. We used an AAV1 expressing either both PYK2 and GFP as separate proteins (AAV1/Camk2a-PYK2) or GFP alone (AAV1/Camk2a-GFP) under a Camk2a promoter. An example is shown in Fig. 4A. We then carried out a bilateral stereotactic injection in the DS of R6/2 mice of AAV1/Camk2a-PYK2 expressing PYK2 and GFP (R6/2-Camk2a-PYK2 group) or GFP alone (R6/2-Camk2a-GFP group) (Supplementary Figure 1A). As control we injected AAV1/Camk2a-GFP in WT mice (WT-Camk2a-GFP group). We measured the body weight in the three groups of mice and found a progressive decrease in the R6/2 mice compared to WT, starting at about 8 weeks, independently of the injected AAV (Figure 4B, Supplementary Table 1). The weekly body weight
change was also decreased in the two R6/2 groups compared to WT and became negative (weight loss) around 10 weeks (Figure 4C, Supplementary Table 1). When we examined hind limb clasping in 6, 9, and 12 week-old mice, we observed this response in both R6/2-Camk2a-GFP and R6/2-Camk2a-PYK2 groups, but not in WT-Camk2a-GFP mice, which did not display this response (Figure 4D). We also measured the horizontal locomotion of the three groups of mice in a circular corridor maze (Figure 4E). The general locomotor activity was not significantly altered at 6 weeks of age, but a slight decrease was visible in 9-week old R6/2 mouse groups (significant in R6/2-Camk2a-GFP group, Figure 4E, Supplementary Table 1). The rearing movements were significantly decreased at 9 weeks in R6/2-PYK2 mice (Figure 4F, Supplementary Table 1). We assessed the potential effects of PYK2 transduction on motor coordination, using the accelerating rotarod (Figure 4G-H). The latency to fall (Figure 4G, Supplementary Table 1) and the speed at fall (Figure 4H, Supplementary Table 1) were dramatically decreased in R6/2 mice as compared to WT. After 8 weeks of age, the performance of AAV-PYK2-transduced mice appeared slightly better for both measurements than that of AAV-GFP-transduced mice, but the difference was not significant (Supplementary Table 1). Overall, our results in R6/2 mice with AAV1/Camk2a-PYK2 transduction in the DS did not show any significant improvement in behavior compared with AAV1/Camk2a-GFP.

### 3.5. Effects of AAV9-mediated PYK2 expression under SYN1 promoter in R6/2 mice

Because of the lack of clear effects of PYK2 viral expression under the control of Camk2a promoter, we then used an AAV9, in which PYK2 expression was controlled by the human SYN1 promoter (Figure 5A, Supplementary Figure 1B). We studied the effects of bilateral striatal stereotactic injection of AAV9/SYN1-PYK2 on the R6/2 mice phenotype by comparing three groups of mice, R6/2 mice injected with AAV9/SYN1-GFP (R6/2-SYN1-GFP group) or AAV9/SYN1-PYK2 (R6/2-SYN1-PYK2 group), and WT mice injected with AAV9/SYN1-GFP as control (WT-SYN1-GFP group). We monitored the body weight and observed a significant decrease in R6/2-SYN1-GFP mice compared to WT mice at 10, 11, and 12 weeks of age (Figure 5B, Supplementary Table 1). In contrast, compared to WT the body weight of R6/2-SYN1-PYK2 mice was decreased only at 12 weeks. The body weight of R6/2-SYN1-PYK2 mice was significantly higher than that of
R6/2-SYN1-GFP mice at 10, 11 and 12 weeks (Figure 5B, Supplementary Table 1). The weekly body weight change decreased in the 2 groups of R6/2 mice after 8 weeks and became a weight loss at 9 weeks in R6/2-SYN1-GFP mice and at 11 weeks in R6/2-SYN1-PYK2 mice (Figure 5C). This indicated a possible delay in the weight loss in SYN1-PYK2-transduced R6/2 mice. The clasping score was increased in R6/2 mice without improvement in R6/2-SYN1-PYK2 mice (Figure 5D). We did not observe any consistent difference in the locomotion and rearing behavior in the circular corridor maze (Figure 5E-F), possibly due to a large variability between animals in this test. The performance of R6/2 mice in the rotarod test, evaluated by the latency to fall (Figure 5G) and speed at fall (Figure 5H), was dramatically altered in R6/2 groups compared to WT mice. However, this alteration appeared to be slightly delayed and consistently less profound in R6/2-SYN1-PYK2 mice than in R6/2-SYN1-GFP mice, without reaching a statistically significant difference between these two groups (Figure 5H, Supplementary Table 1). To determine whether there was any effect of the AAV9/SYN1-PYK2 injection on SPNs in R6/2 mice we carried out a semi-quantitative evaluation of DARPP-32, a marker of these neurons, in both mutant and WT mice (Supplementary Figure 2). We used mice injected 8 weeks before on one side with AAV9/SYN1-PYK2 and on the other side with AAV9/SYN1-GFP. We compared the striatal DARPP-32 immunofluorescence intensity in the injected area of the two sides in the same brain sections. We found it was similar in WT mice (PYK2-injected/GFP-injected ratio, 1.07 ± 0.03 %) but higher in R6/2 mice (1.31 ± 0.09, different from WT by Mann-Whitney's test, p = 0.029, n = 4, Supplementary Figure 2). This result suggested a positive effect of PYK2 viral expression in R6/2 mice on SPNs that express DARPP-32.

**3.6. Similarities and differences between AAV1/Camk2a and AAV9/SYN1 vectors in striatal neurons PYK2 transduction**

Because of the slight differences in the phenotype of R6/2 mice expressing PYK2 with the two viral vectors, we compared the cell types transduced by the two viruses. WT mice were injected with either vector, and the expression of GFP was compared to that of cell type-specific immunomarkers (Figure 6A-B). Both vectors expressed GFP and PYK2, but to identify transduced cells we used GFP labeling and not PYK2 to avoid confusion with the endogenous
protein. In all cases GFP expression was restricted to neurons and most of the neurons
expressing GFP were DARPP-32-positive SPNs (AAV1/Camk2a, 97.5 ± 0.5 %, 6 sections from 4
mice, AAV9/SYN1, 95.4 ± 1.0 %, 8 sections from 4 mice, means ± SEM, **Figure 6A** and **6C**). In
mice injected with AAV1/Camk2a, very few cells expressing GFP were cholinergic interneurons
(choline acetyl transferase-[ChAT]-positive, 0.19 ± 0.09 % of transduced cells, n = 8 sections
from 2 mice, **Figure 6A** and **6C**). In contrast, some cells transduced with AAV9/SYN1 were ChAT-
positive (1.8 ± 0.09 %, n = 8 sections from 2 mice, **Figure 6A** and **6C**), corresponding to the
expected proportion of cholinergic interneurons in the DS (Zhou et al., 2002). We also used two
markers of distinct subpopulations of GABA interneurons (Tepper et al., 2018; Tepper et al.,
2010), parvalbumin (PV) and somatostatin (SST). PV was detected in very few cells transduced
with AAV1/Camk2a (0.24 ± 0.1 %, 4 sections from 2 mice) but in more cells transduced with
AAV9/SYN1 (2.8 ± 0.4 %, 4 sections from 2 mice, **Figure 6B-C**). Somatostatin (SST)
immunoreactivity was detected in a similar proportion of cells transduced with either virus
(AAV1/Camk2a, 1.6 ± 0.4 %, AAV9/SYN1, 1.9 ± 0.3 %, 4 sections from 2 mice, **Figure 6B-C**).

Because interneurons represent only a few percent of the total number of neurons in
the striatum (Tepper et al., 2010), we calculated the proportion of neurons which expressed
GFP within each identified neuronal population, in the area transduced by the viral vector
(Figure 6D, Supplementary Table 1). With the AAV1/Camk2a vector, 9 ± 4 % of the ChAT-
positive and 7 ± 2 % of the PV-positive neurons were transduced (8 and 4 sections from 2 mice,
respectively, Figure 6D). In contrast, with the AAV9/SYN1 vector 81 ± 5 % of the ChAT-positive
and 91 ± 5 % of the PV-positive neurons were transduced, Figure 6D). Among the SST-positive
neurons, most were transduced with either viral vector (AAV1/Camk2a, 86.9 ± 4.7 %,
AAV9/SYN1, 90.2 ± 7.1 %, Figure 6D).

These results indicated that both viral vectors led to a similar expression of GFP-PYK2 in
SPNs and in SST-positive interneurons, whereas only AAV9/SYN1 transduced a high proportion
of the cholinergic interneurons and PV-positive GABAergic interneurons. These differences may
account for some of the differences in the biological effects of the two viruses.
# 4. Discussion

## 4.1. PYK2 protein and mRNA levels are decreased in the striatum of R6/2 mice as well as PYK2 protein in HD patients

PYK2 is a non-receptor tyrosine kinase highly expressed in the forebrain principal neurons [review in (de Pins et al., 2021)]. We show that its mRNA levels are higher in the hippocampus than in the cerebral cortex, and intermediate in the striatum, in agreement with previous in situ hybridization and immunoblotting data (Menegon et al., 1999). We reported that PYK2 levels are low in the hippocampus of R6/1 HD mouse model and human patients, indicating that PYK2 may participate in the pathophysiology of HD in the hippocampus (Giralt et al., 2017). Our present results provide the first information about the possible role of PYK2 in striatal HD-like phenotype. As in hippocampus, the striatal levels of PYK2 protein were decreased in R6/2 mice and HD patients. Here we provide evidence in a mouse HD model that the protein deficit is likely to be the consequence of decreased mRNA levels in both hippocampus and striatum. This observation is in agreement with the role of huntingtin in regulating transcription, among many other processes (Saudou and Humbert, 2016). Accordingly alteration of many transcripts were reported in the striatum of HD patients (Hodges et al., 2006). Additional mechanisms, not explored in our study, could potentially contribute to the decrease in PYK2 protein including the Ca²⁺-activated protease calpain, which cleaves PYK2 (Marzia et al., 2006), and is known to be activated in HD cells (Incebacak Eltemur et al., 2022; Paoletti et al., 2008). Previous studies have investigated phosphoproteomics in the striatum of HD mouse models, focusing on the cyclic nucleotides-related effects of phosphodiesterase 10A (PDE10A) inhibition (Beaumont et al., 2016) and on environmental enrichment (Mees et al., 2022). But neither of these studies reported information about PYK2 or its potential substrates. Thus, our work provides the first information about a neuronal-enriched tyrosine kinase and indicates that phosphotyrosine-targeted phosphoproteomics would be an interesting approach in HD models.

## 4.2. The absence of PYK2 protein in KO mice does not mimic HD striatal phenotype
The hippocampal phenotype of PYK2-KO mice was partly similar to that of R6/1 mice, an
observation which led to the hypothesis of a contribution of PYK2 deficit in the HD-like
hippocampal phenotype (Giralt et al., 2017). Therefore we explored whether the striatal
decrease in PYK2 levels could contribute in the striatal-related HD-like symptoms in young mice.
We did not observe such phenotype in the complete absence of PYK2. Accordingly we
previously reported that PYK2-KO mice do not display any deficit in the accelerating rotarod task
(de Pins et al., 2020). These results ruled out the possibility that PYK2 deficit by itself could be a
major contributor in HD-like striatal manifestations at a young age, although we did not explore
whether such deficits could occur at an older age.

4.3. Viral expression of PYK2 in the striatum of R6/2 mice has a slight positive effect

Because we hypothesized that PYK2 deficit in striatal neurons could contribute to the HD phenotype, not as a major primary mechanism but only in conjunction with the other molecular alterations, we investigated whether viral expression of PYK2 in the DS could improve the symptomatology. We had previously shown that hippocampal viral expression of PYK2 rescued some deficits including Y-maze spontaneous alternation and novel object location tests as well as dendritic spine markers, whereas other parameters such as synaptic plasticity were not improved (Giralt et al., 2017). In the current study we used two different AAV vectors, AAV1-Camk2a-eGFP-2A-mPTK2B (AAV1/Camk2a-PYK2) and AAV9-hSYN1-eGFP-2A-mPTKB2B (AAV9/SYN1-PYK2). Although none of them resulted in a massive overexpression, they increased PYK2 immunofluorescence within the transduced area. There was no significant effect of AAV1/Camk2a-PYK2 on weight loss or motor phenotype, in spite of a noticeable trend. In contrast we observed a slight improvement of several parameters following bilateral injection of AAV9/SYN1-PYK2 compared to AAV-SYN1-GFP. The difference was significant for weight loss and a positive trend was visible in the rotarod performance. These results suggested a minor contribution of PYK2 deficit in the striatum. In addition, a slight increase in DARPP-32 immunofluorescence was observed in the AAV9/SYN1-PYK2-transduced area indicating a possible positive effect on the biology of striatal projection neurons.
The differences between the two viral vectors may be related to the different promoters (mouse *Camk2a* vs. human *SYN1*) and/or the AAV serotypes (AAV1 vs AAV9) because it was suggested that neuronal transduction in the striatum is better with AAV9 than AAV1 (Kofoed et al., 2022). When we compared the two viral vectors we found that the overall transduction levels were similar and that in both cases the vast majority of transduced neurons were DARPP-32-positive SPNs. Although transduction of SPNs and of SST-positive GABAergic interneurons was similar with the two vectors, the AAV9 with a human *SYN1* promoter led to a much higher proportion of transduced cells among cholinergic interneurons and PV-positive GABAergic interneurons. This pronounced difference is worth noticing because, although cholinergic interneurons are relatively spared in HD, they have been recently shown to be the site of CAG-expansions in human patients (Matlik et al., 2024) and to contribute to circuit dysfunction in a mouse model of the disease (Pancani et al., 2023). Moreover, they are functionally altered in the R6/2 model (Crevier-Sorbo et al., 2020; Holley et al., 2015) that we used in our study. Interestingly, deletion of the vesicular acetylcholine transporter in the dorsal striatum alters eating behavior with a weight loss in mice (Favier et al., 2020). It is therefore possible that PYK2 overexpression in cholinergic interneurons in R6/2 mice, in the AAV9/SYN1 line but not in the AAV1/Camk2a line, had a positive effect on weight by reverting alterations in these neurons.

### 4.4. PYK2 multiple roles in various neurodegenerative and psychiatric conditions

PYK2 emerges as a multifaceted regulator of many pathophysiological processes in the brain [see (de Pins et al., 2021)]. The current results combined with other studies over the last years allow to better position PYK2 contribution in neuropsychiatric disorders. In HD, PYK2 may contribute to the hippocampal phenotype (Giralt et al., 2017) but the results reported here indicate that it plays a much lesser role in the striatal phenotype. In Alzheimer's disease (AD), GWAS meta-analyses showed that the locus of PYK2 gene, *PTK2B*, is associated with an increased risk for late onset AD (Lambert et al., 2013), an association confirmed in several other studies (Beecham et al., 2014; Jiao et al., 2015; Lawingco et al., 2021; Nettiksimmons et al., 2016; Schwatzentruber et al., 2021; Wang et al., 2015). In animal models of AD, PYK2 appears to have dual roles. While it contributes to the amyloid cascade leading to neurodegeneration
(Salazar et al., 2019), it can have a protective role in some mouse models (Giralt et al., 2018; Kilinc et al., 2020) and may suppress Tau-related phenotypes (Brody et al., 2022). The role of PYK2 has also been documented in psychiatric conditions, mostly in animal models. Deletion of PYK2 in the amygdala improves some aspects of depressive-like symptoms induced by chronic unpredictable mild stress (Montalban et al., 2019), whereas enhancing PYK2 expression in lateral septum neurons can have antidepressant effects (Sheehan et al., 2003). PYK2 levels in hippocampus modulate specific social behaviors, possibly through modulation of mitochondrial function, and their increase may contribute to schizophrenic-like symptoms (Lopez-Molina et al., 2022; Lopez-Molina et al., 2024). In the context of addiction, PYK2 is induced by chronic cocaine in the striatum and frontal cortex (Freeman et al., 2002; Freeman et al., 2001). In nucleus accumbens neurons expressing D1 receptors, PYK2 is necessary for acute locomotor effects of cocaine but not for its plasticity-related manifestations, locomotor sensitization or conditioned place preference (de Pins et al., 2020). In the orbitofrontal cortex, PYK2 overexpression prevents the spine loss and behavioral alterations induced by cocaine (Whyte et al., 2021). Therefore depending on the brain structures, pathologies, and animal models, PYK2 can play positive or negative roles in various neuropsychiatric conditions. In addition, PYK2 is known to be an important contributor to the pathology of various cancers including breast cancer [review in (Gil-Henn et al., 2023)] and search for selective pharmacological inhibitors of PYK2 is an active area. Therefore further study of PYK2 in physiology and pathological conditions is important to balance the potential benefits and risks of its enhancement or inhibition in any of them.

**4.5. Limitations of the study, therapeutic implications, and conclusions**

Our study has some limitations. First, the R6/2 mouse model we used displays a severe phenotype, which starts at an early age. This may have limited potential beneficial effects of PYK2 viral expression as compared to the milder R6/1 model used in the hippocampal study (Giralt et al., 2017) or to knock-in models such as zQ175 (Kacher et al., 2019; Menalled et al., 2012), in which it would be interesting to study the role of striatal PYK2. A second series of limiting factors included the moderate expression of PYK2 induced by the two viral vectors, its
spatial limitation and the timing of the restoration. A third limitation is that we did not include a group of WT mice transduced with GFP-PYK2. This control was considered to be unnecessary because our previous experience with these vectors did not suggest specific effects of moderate GFP-PYK2 expression on the type of phenotype investigated here. Another point to consider is that a decrease in the expression of PYK2 (or any other protein) could be a homeostatic or coincidental response with positive effects on neuronal function or survival. Our experiments were not designed to detect such protective effects of PYK2 downregulation (e.g., we did not test the effects of further decreasing PYK2 levels). Although we cannot fully rule out this possibility, it appears unlikely because no worsening was observed by restoring PYK2 expression.

Our results support a more prominent role of PYK2 in the hippocampus than in the striatum of HD models. This may be due to the higher expression levels of PYK2 in the hippocampus, where its deletion leads to noticeable alterations (Giralt et al., 2017) although less modifications were reported in a different mouse line (Salazar et al., 2019). Yet, an indirect support of a contribution of striatal PYK2 is provided by the beneficial effects on motor coordination in R6/1 mice of genetic deletion of the striatal-enriched tyrosine phosphatase, STEP (Garcia-Forn et al., 2018), which dephosphorylates and functionally antagonizes PYK2 [see (de Pins et al., 2021)]. Although these results point to a minor role of PYK2 they do not support the possibility of a sufficient therapeutic benefit of direct PYK2 manipulation in the striatum of HD patients. An intervention at the level of STEP, which has other relevant targets in addition to PYK2, might be more promising.

In conclusion, our study reveals a PYK2 deficit in the striatum of HD patients and R6/2 mouse model. It provides evidence that this defect results, at least in part, from decreased mRNA levels presumably linked to the transcriptional alterations observed in this disease. The genetic deletion of PYK2 did not result in a motor phenotype by itself. Viral expression of PYK2 in the striatum with an AAV9/SYN1 vector led to a slight improvement of the R6/2 mice body weight phenotype. This effect may be related to the transduction of cholinergic interneurons with this vector, a possibility that would deserve further exploration. Our results show that
striatal PYK2 is not necessary for motor behavior and is likely to play only a minor role in the striatal pathophysiology of the HD R6/2 mouse model used. They contribute to the evaluation of the multifaceted roles of PYK2 in mouse models of neurological and psychiatric diseases.

## Funding

This work was supported by INSERM, Sorbonne Université and grants to JAG from Inserm-Transfert COPOC, Agence Nationale de la Recherche (ANR-19-CE16-0020 and ANR-20-JPW2-0003-03), and the Fondation pour la recherche médicale (FRM, EQU201903007844), and grants to OA-M from Instituto de Salud Carlos III (ISCIII, grants PI21/01216 and PI24/00916) and co-funded by the European Union, GAIN-XUNTA Galicia Proyectos de Excelencia (IN607D-2022-07). Omar Al-Massadi is currently supported by a research contract “Miguel Servet” (CP20/00146) from ISCIII, co-financed by the European Social Fund (FSE). CIBERobn is an initiative of ISCIII in Spain, supported by FEDER funds. BdP was supported in part by FRM (FDT201805005390).

## Declaration of Competing Interest

Two of the authors (JAG and AG) hold a European and international patent on the possible benefit of PYK2 activation in neurodegenerative diseases, which is not licensed.

## CRediT authorship contribution statement

Omar Al-Massadi: Conceptualization, Formal analysis, Investigation, Methodology, Visualization, Writing - original draft. Mélody Labarchède: Formal analysis, Investigation, Methodology. Benoit de Pins: Formal analysis, Investigation, Methodology. Sophie Longueville: Formal analysis, Investigation, Methodology. Albert Giralt: Investigation, Methodology, Resources. Theano Eirinopolou: Methodology, Formal analysis, Visualization. Mythili Savariradjane: Investigation, Methodology, Visualization. Enejda Subashi: Investigation,
Methodology. Silvia Ginés: Resources. Jocelyne Caboche: Resources, Supervision. Louise-Laure Mariani: Conceptualization, Methodology, Investigation, Resources, Supervision, Writing review & editing. Sandrine Betuing: Conceptualization, Methodology, Resources, Supervision. Jean-Antoine Girault: Conceptualization, Data curation, Formal analysis, Funding acquisition, Project administration, Resources, Supervision, Validation, Writing - review & editing.

**Data availability**

Data will be made available on request.

**Acknowledgments**

We thank the *Mouse breeding and phenotyping facility* for animal care and *Cell and Tissue Imaging facility* of the *Institut du Fer à Moulin* where images were acquired.
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<table><caption>Table 1: Human post-mortem samples</caption><thead><tr><th colspan="4">Control patients</th></tr><tr><th>Sex</th><th>Age (years)</th><th>Neuropathology</th><th>PMI</th></tr></thead><tbody><tr><td>M</td><td>76</td><td>Argyrophilic grain pathology stage I</td><td>11 h 30 min</td></tr><tr><td>M</td><td>78</td><td>Incidental Lewy body pathology stage I</td><td>06 h 00 min</td></tr><tr><td>M</td><td>83</td><td>Argyrophilic grain pathology I + contralat. meningioma</td><td>13 h 00 min</td></tr><tr><td>M</td><td>79</td><td>Contralat. infarct + Argyrophilic grain pathology stage II</td><td>05 h 00 min</td></tr><tr><td>F</td><td>81</td><td>None</td><td>23 h 30 min</td></tr><tr><td>F</td><td>90</td><td>Small vessel pathology</td><td>12 h 20 min</td></tr></tbody></table>

<table><thead><tr><th colspan="6">Huntington disease patients</th></tr><tr><th>Sex</th><th>Age (years)</th><th>Age at diagnosis</th><th>Grade</th><th>CAG#</th><th>PMI</th></tr></thead><tbody><tr><td>M</td><td>53</td><td>43</td><td>3</td><td>45</td><td>07 h 00 min</td></tr><tr><td>M</td><td>85</td><td>-</td><td>3</td><td>40</td><td>05 h 30 min</td></tr><tr><td>M</td><td>55</td><td>38</td><td>3</td><td>48</td><td>15 h 00 min</td></tr><tr><td>M</td><td>72</td><td>57</td><td>2</td><td>-</td><td>13 h 10 min</td></tr><tr><td>M</td><td>76</td><td>65</td><td>2</td><td>41</td><td>06 h 00 min</td></tr><tr><td>M</td><td>68</td><td>53</td><td>2-3</td><td>42</td><td>06 h 10 min</td></tr></tbody></table>

PMI, post mortem interval.

Mann and Whitney test between controls and HD, age U = 5.5, p = 0.045, PMI, U = 16.5, p = 0.85.
**Figure 1: PYK2 mRNA levels are decreased in hippocampus and striatum of R6/2 mice.**

PYK2 mRNA levels were quantified by RT-qPCR. A) PYK2 mRNA in the hippocampus, striatum and cerebral cortex of 12-week old WT male C57Bl/6 mice (6-8 animals per group). Data are expressed as % of the mean in hippocampus, Kruskal-Wallis test, p<0.0001, Dunn's multiple comparisons test. B) PYK2 mRNA levels in the cerebral cortex of 12-week WT and R6/2 mice (6-7 animals per group). Mann Whitney test, not significant (ns). C) PYK2 mRNA levels in the hippocampus of WT and R6/2 mice at the indicated ages (6-10 animals per group). Kruskal-Wallis test, p = 0.0085, Mann-Whitney test, 4 w, ns, 6 w, p = 0.036, 12 w, p = 0.02. D) PYK2 mRNA levels in the DS of WT and R6/2 mice at the indicated ages (4-8 animals per group). Kruskal-Wallis test, p = 0.0017, Mann-Whitney test, 4 w, ns, 6 w, p = 0.0047, 12 w, p = 0.0043. (C-D) Data are expressed as % of the mean in WT at each age. (A-D) Means ± SEM are indicated. * p < 0.05, ** p < 0.01. Detailed statistical results in **Supplementary Table 1**. Data shown in A are the same as for the corresponding region in B, WT, C, WT 12 weeks and D, WT 12 weeks.

**Figure 2: PYK2 protein levels are decreased in the striatum of R6/2 mice and HD patients.**

A) PYK2 protein levels were measured by immunoblotting in the striatum of WT and R6/2 mice at the indicated ages. β-actin was used as a loading control. B) Data such as shown in (A) were quantified by densitometry (5-9 animals per group). PYK2 levels were normalized to the loading control and expressed as % of the mean in WT at each age. Kruskal-Wallis test, p = 0.0007. Mann-Whitney test, 4 w, ns, 6 w, p = 0.0012, 12 w, p = 0.0006. C) PYK2 protein levels were measured by immunoblotting in the putamen of human subjects deceased with HD or with other conditions (Table 1). α-tubulin was used as a loading control. Data were quantified, normalized and expressed as % of the mean in controls (6 controls and 6 HD). Mann-Whitney test, p = 0.041. (A-B) Means ± SEM are indicated,* p < 0.05, ** p < 0.01, *** p < 0.001. Detailed statistical results in **Supplementary Table 1**. See **Supplementary Figure 3** for full length blots used in this Figure.

**Figure 3: Motor behavior is not altered in PYK2-KO mice.**

A) The spontaneous locomotor activity of 3-6-month-old male WT and PYK2-KO littermate mice was studied by video tracking for 15 min in an open field. Unpaired 2-tailed Student's t test, ns (9-10 mice per group). B) Motor skill was evaluated as the time to reach the escape zone on a balance beam during 2 trials on 2 consecutive days (6-8 mice per group). Kruskal-Wallis test, p = 0.0007, Dunn multiple
comparisons test, no difference between genotypes. C) Motor skill was evaluated on a vertical pole by visually scoring (8 mice per group). Mann-Whitney test, ns. D) Examples of hind limb position when mice were held by the tail showing the absence of clasping in WT and PYK2-KO mice. (A-C) Means ± SEM are indicated. Detailed statistical results in **Supplementary Table 1**.

<caption>**Figure 4: Motor function of R6/2 mice injected with AAV-Camk2a-PYK2 in the DS.**</caption>

A) AAV1/Camk2a-PYK2, expressing PYK2 and GFP, was stereotactically injected into one striatum of a WT mouse. Coronal brain section were immunostained for GFP. B-C) R6/2 mice received a bilateral injection of either AAV1/Camk2a-PYK2 or AAV1/Camk2a-GFP in the DS and WT mice a bilateral injection of AAV1/Camk2a-GFP. (B) Their body weight was measured every week. At each age statistical analysis was done with Kruskall-Wallis test: 4-11 weeks, not significant (ns), 12 weeks, p = 0.0043. (C) Same data as in B, plotted as weekly weight change from 8 to 12 weeks. Kruskal-Wallis test: 8-9 weeks, ns, week 10 p = 0.0007, week 11, p = 0.0025, week 12, p = 0.05. D) Clasping responses were scored in the 3 groups at the indicated ages (4-9 mice per group and age). Kruskal-Wallis test, p = 0.025. Horizontal bars are medians. E-F) The motor behavior of the 3 groups of mice was studied in a circular maze at 6 and 9 weeks (7-10 mice per group and age). (E) General locomotor activity was measured as the number of beam breaks. Kruskal-Wallis test, 6 weeks, ns, 9 weeks p = 0.018. (F) The number of rearings was counted. Kruskal-Wallis test, 6 weeks, p = 0.038, 9 weeks p = 0.002. G-H) The motor coordination was evaluated every week from 6 to 11 weeks (w) in a rotarod test by measuring the latency to fall (G) and the speed at fall (H) (4-14 mice per group and age). Kruskal-Wallis test at each age, (G) 6 w, p = 0.0007, 7 w, p = 0.0006, 8 w p<10⁻⁴, 9 w p<10⁻⁴, 10 w, p = 0.013, 11 w, p = 0.0091, (H) 6 w, p = 0.0047, 7 w, p<10⁻⁴, 8 w p<10⁻⁴, 9 w p<10⁻⁴, 10 w, p = 0.0028, 11 w, p = 0.0011. Means ± SEM (B, C, E-H) or medians (D) are indicated. (B-H) Post hoc Dunn's multiple comparisons test. Significant differences are indicated by asterisks. Green, R6/2 AAV-SYN1-GFP vs WT AAV-SYN1-GFP, violet, R6/2 AAV-SYN1-PYK2 vs WT AAV-SYN1-GFP, * p<0.05, ** p<0.01, *** p<0.001, **** p<10⁻⁴. Detailed statistical results in **Supplementary Table 1**.

<caption>**Figure 5: Motor function of R6/2 mice injected with AAV-SYN1-PYK2 in the DS.**</caption>

A) AAV-SYN1-PYK2, expressing PYK2 and GFP, was stereotactically injected in one striatum of a WT mouse. Coronal brain section were immunostained for GFP. B-C) R6/2 mice received a bilateral injection of either AAV1/SYN1-PYK2 or AAV1/SYN1-GFP in the DS and WT mice a bilateral injection of AAV1/SYN1-GFP. (B) Their body weight was measured every week (6 mice per group). At every age statistical analysis
was done with Kruskall-Wallis test: 4-9 weeks, not significant (ns), 10 weeks, p = 0.0055, 11 weeks, p = 0.0015, 12 weeks, p < 10⁻⁴. (C) Same data as in **B**, plotted as weekly weight change from 8 to 12 weeks.
Kruskall-Wallis test: week 8, ns, week 9, p = 0.01, week 10, ns, week 11, p = 0.039, week 12, p= 0.0045.
**D**) Clasping responses were scored in the 3 groups at the indicated ages (6 mice per group). Kruskal-Wallis test, p < 10⁻⁴. Difference between groups with Dunn's multiple comparisons test are indicated.
Horizontal bars are medians. **E-F)** The locomotion of the 3 groups of mice (6 mice per group) was studied in a circular maze at the indicated ages (6 mice per group at every age). **(E)** General locomotor activity was expressed as the number of ¼ turns. Kruskal-Wallis test, 6 w, ns, 9 w, ns, 12 w, p = 0.033. **(F)** The number of rearings was counted. Kruskal-Wallis test, (F) The number of rearings was counted. Kruskal-Wallis test, 6 weeks, ns, 9 weeks, p = 0.034, 12 weeks ns. **G-H)** The motor coordination was evaluated every week from 6 to 12 weeks in a rotarod test by measuring the latency to fall (**G**) and the speed at fall (**H**). Two-way repeated measures ANOVA (6 mice per group at every age), (**G**) interaction, F<sub>(12,90)</sub> = 1.94, p = 0.04, age, F<sub>(6,90)</sub> = 2.93, p = 0.01, mouse group, F<sub>(2,15)</sub> = 19.8, p < 10<sup>-4</sup>, (**H**) interaction, F<sub>(12,90)</sub> = 4.74, p < 10<sup>-4</sup>, age, F<sub>(6,90)</sub> = 7.84, p < 10<sup>-4</sup>, mouse group, F<sub>(2,15)</sub> = 14.56, p = 0.0003. **(B, C, E-H)** Means ± SEM are indicated. Asterisks indicate significant differences with Dunn's (B-E) or Holm-Sidak's (G,H) multiple comparisons test. Asterisks: green R6/2 AAV-SYN1-GFP vs WT AAV-SYN1-GFP, violet, R6/2 AAV-SYN1-PYK2 vs WT AAV-SYN1-GFP, black, R6/2 AAV-SYN1-PYK2 vs R6/2 AAV-SYN1-GFP,* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 10<sup>-4</sup>. Detailed statistical results in **Supplementary Table 1**.

<caption>**Figure 6: Comparison of cell types transduced by AAV1/Camk2a and AAV9/SYN1 vectors.**</caption>

**A-B)** WT male mice received a bilateral injection of either AAV1/Camk2a-GFP-PYK2 or AAV9/SYN1-GFP-PYK2 in the DS. Transduced cells were identified by GFP immunofluorescence and double labeling with (A) antibodies for DARPP-32 (a marker of SPNs) and choline-acetyl transferase (ChAT, a marker of cholinergic interneurons) or (B) antibodies for parvalbumin (PV) or for somatostatin (SST), markers of two different types of GABAergic interneurons. Scale bar, 20 µm. (C) For each viral vector the number of cells expressing each immunomarker was counted within GFP-positive transduced cells and expressed as a % of total number of GFP-positive cells. Box and whisker plots (boxes indicate the median with upper and lower quartiles, whiskers min and max values). For each marker, AAV1/Camk2a vs AAV9/SYN1 comparison with Mann-Whitney's test, * p < 0.05, *** p < 0.001. (D) The proportion of transduced cells was estimated within each immuno-identified population by dividing the number of cells double-immunolabeled for GFP and for the population-specific marker by the total number of cells of the population within the virally transduced area, and expressed as a percentage. Bars are means +/- SEM.
For each marker, AAV1/Camk2a vs AAV9/SYN1 comparison with Mann-Whitney's test, * p <0.05, **** p < 10⁻⁴.