{'fraction_non_alphanumeric': 0.03965832824893228, 'fraction_numerical': 0.0313071995118975, 'mean_word_length': 5.113053613053613, 'pattern_counts': {'":': 0, '<': 8, '<?xml version=': 0, '>': 1, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Restored Fyn Levels in Huntington’s Disease Contributes to Enhanced Synaptic GluN2B-Composed NMDA Receptors and CREB Activity

N-methyl-D-aspartate receptors (NMDARs) are important postsynaptic receptors that contribute to normal synaptic function and cell survival; however, when overactivated, as in Huntington’s disease (HD), NMDARs cause excitotoxicity. HD-affected striatal neurons show altered NMDAR currents and augmented ratio of surface to internal GluN2B-containing NMDARs, with augmented accumulation at extrasynaptic sites. Fyn protein is a member of the Src kinase family (SKF) with an important role in NMDARs phosphorylation and synaptic localization and function; recently, we demonstrated that Fyn is reduced in several HD models. Thus, in this study, we aimed to explore the impact of HD-mediated altered Fyn levels at post-synaptic density (PSD), and their role in distorted NMDARs function and localization, and intracellular neuroprotective pathways in YAC128 mouse primary striatal neurons. We show that reduced synaptic Fyn levels and activity in HD mouse striatal neurons is related to decreased phosphorylation of synaptic GluN2B-composed NMDARs; this occurs concomitantly with augmented extrasynaptic NMDARs activity and currents and reduced cAMP response element-binding protein (CREB) activation, along with induction of cell death pathways. Importantly, expression of a constitutive active form of SKF reestablishes NMDARs localization, phosphorylation, and function at PSD in YAC128 mouse neurons. Enhanced SKF levels and activity also promotes CREB activation and reduces caspase-3 activation in YAC128 mouse striatal neurons. This work supports, for the first time, a relevant role for Fyn protein in PSD modulation, controlling NMDARs synaptic function in HD, and favoring neuroprotective pathways and cell survival. In this respect, Fyn Tyr kinase constitutes an important potential HD therapeutic target directly acting at PSD.


Introduction
Huntington's Disease (HD) is an autosomal dominant progressive neurodegenerative disorder that affects mainly the striatum (caudate and putamen) and later the cortex [1]. HD is characterized by psychiatric and behavioral disturbances, such as motor impairment, and psychiatric symptoms such as obsessive-compulsive disorder, depression and/or anxiety, cognitive decline and weight loss [2,3]. HD is caused by an abnormal expansion of cytosineadenine-guanine (CAG) repeat at the HTT gene, encoding for mutant huntingtin (mHTT) retaining a polyglutamine (polyQ) extension at the N-terminus [4]. mHTT has been associated with protein conformational changes, aggregation, and abnormal protein-protein interactions [2], which cause cytotoxicity, evidenced through changes in gene transcription, synaptic dysfunction, N-methyl-D-aspartate receptors (NMDARs) overactivation, decreased mitochondrial calcium (Ca 2+ ) handling and organelle dysfunction, and increased

YAC128 Mice
YAC128 mice, previously described by Slow and co-authors (2003) [17], express fulllength mutant HTT with 128 CAG repeats from a yeast artificial chromosome (YAC) transgene (RRID: MGI_MGI:3613515). YAC128 (line HD53) and wild-type mice were housed in the animal facility of the Center for Neuroscience and Cell Biology and Faculty of Medicine at the University of Coimbra (Coimbra, Portugal), under controlled temperature (22-23 • C) and a 12 h light/12 h dark cycle with lights on at 07:00 h. Food and water were available ad libitum throughout the experiment. Animal experiments in this study were performed in accordance with the European Community directive (2010/63/EU) and protocols approved by the Faculty of Medicine, University of Coimbra (ORBEA_189_2018/11042018). All efforts were made to minimize animal suffering and to reduce the number of animals used. Animals were used at 3, 6, and 12 months of age.

Primary Striatal Neurons from YAC128 Mice
Primary striatal neurons were prepared as described previously [18], with some minor modifications. At 16 days of gestation, pregnant female mice were sacrificed by cervical dislocation following anesthesia using (RS)-2-chloro-2-(difluoromethoxy)-1,1,1-trifluoroethane. Striata were dissected out from fetal mice and cells were separated by mechanical digestion using a pipette in Ca 2+ -and Mg 2+ -free Hank's balanced salt solution containing 137 mM NaCl, 5.36 mM KCl, 0.44 mM KH 2 PO 4 , 0.34 mM Na 2 HPO 4 .2H 2 O, 5 mM glucose, 1 mM sodium pyruvate, and 10 mM HEPES, at pH 7.2. Cells were plated at a density of 8.4 × 10 4 cells/cm 2 in poly-D-lysine-coated 6-well or 96-well plates, and at a density of 4.2 × 10 4 cells/cm 2 in poly-D-lysine coated glass coverslips for immunocytochemistry. Cells were cultured for 12 days in Neurobasal medium supplemented with 2% B27, 0.5 mM glutamine and 0.12 mg/mL gentamicin, at 95% air and 5% CO 2 . To reduce glia growth, 10 µM of the mitotic inhibitor 5-fluorodeoxyuridine (5-FDU, Sigma, #F0503, St. Louis, MI, USA) was added to the culture after 72 h in culture. One half of the medium was changed with fresh medium without 5-FDU at day 7.

Constructs and Neuron Transfection
Cells were transfected with pLNCX chick SKF Y527F (Addgene, plasmid #13660), empty pLNCX vector, and GFP (Origene, Rockville, ML, USA, GFP; #PS100010). Empty vector pLNCX was obtained from the SKF Y527F plasmid using the restriction enzyme digestion ClaI (BioLabs, #Ro197L) according to manufacturer's protocol. The result of digestion was visualized in 1% agarose gel; the band corresponding to the empty vector was cropped and DNA was extracted using the NucleoSpin ® Gel and PCR Clean-up (Macherey-Nagel, #740609) according to the manufacturer's protocol. Then, the blunt end and cohesive end termini of the resulting empty vector were joined using T4 DNA Ligase enzyme (BioLabs, #M0202S). Transfection was performed at 8 DIV using the Ca 2+ phosphate precipitation method. Briefly, plasmid was diluted in TE (1 mM Tris-HCl pH 7.3, 1 mM EDTA), followed by the addition of CaCl 2 (2.5 M CaCl 2 in 10 mM HEPES, pH 7.2). The DNA solution was carefully added to 2× HEBS (12 mM dextrose, 50 mM HEPES, 10 mM KCl, 280 mM NaCl, and 1.5 mM Na 2 HPO 4 .2H 2 O, pH 7.2) while bubbling air through the solution with a micropipette. The mixture was then incubated for 25 min at room temperature. The precipitates were added dropwise to the coverslips in Neurobasal medium and incubated for 80 min, at 37 • C. The DNA-Ca 2+ -phosphate precipitates were dissolved in freshly made dissolution medium (Neurobasal medium with 20 mM HEPES, pH 6.8) and incubated for 7 min at room temperature. The transfected neurons were then washed with Neurobasal medium and transferred back to their original dishes containing conditioned culture medium.

Sample Preparation and Western Blotting
Total extracts were obtained from primary striatal neurons or striatal or cortical brain areas from YAC128 mice at 3, 6, or 12 months of age. The cells were scraped and brain areas suspended in Ripa buffer (containing 150 mM NaCl, 50 mM Tris HCl, 5 mM EGTA, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate, pH 7.5) supplemented with 100 nM okadaic acid, 1 mM PMSF, 25 mM NaF, 1 mM Na 3 VO 4 , 1 mM DTT, and 1 µg/mL protease inhibitor cocktail (chymostatin, pepstatin A, leupeptin and antipain). Total homogenates were lysed in an ultrasonic bath (UCS 300-THD; at heater power 200 W and frequency 45 kHz) during 10 s and centrifuged at 4 • C for 10 min at 20,800× g to remove cell debris. The supernatant was collected, and protein content was determined using the Bio-Rad protein assay reagent based on the Bradford dye-binding procedure (Bio-Rad, Hercules, CA, USA). Then, protein extracts were denatured with 6× concentrated loading buffer (containing 300 mM Tris-HCl pH 6.8, 12% SDS, 30% glycerol, 600 mM DTT, 0.06% bromophenol blue) at 95 • C, for 5 min. Equivalent amounts of protein samples (

Electrophysiological Recordings
NMDA-induced currents were recorded in transfected primary striatal neurons (DIV 11) at −60 mV by whole-cell patch clamping using an AxonPatch 200B amplifier (Molecular Devices, San Jose, CA, USA). The borosilicate glass micropipettes used had a resistance of 4-6 MΩ and were filled with the following internal solution (in mM): CsMeSO 4 130, CsCl 10, CaCl 2 0.5, EGTA 5, HEPES 10, and NaCl 10 (pH 7.3 adjusted with CsOH). Cells were perfused with extracellular solution containing 140 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl 2 , 10 mM HEPES, and 15 mM glucose supplemented with 10 µM glycine (pH 7.4 adjusted with NaOH). NMDA (100 µM) was diluted in the extracellular solution and rapidly perfused with a six-channel perfusion valve control system VC-77SP/perfusion fast-step SF-77B (Warner Instruments, Hamden, CT, USA). All experiments were performed at RT (22-25 • C). The currents were filtered at 1 kHz (4-pole low-pass Bessel filter) and digitized at a sampling rate of 10 kHz to a personal computer and analyzed with pClamp 10.7 software (Molecular Devices, San Jose, CA, USA).

Measurement of Intracellular Calcium Levels
Twenty-four hours after transfection with empty or empty+ SKF Y527F , primary striatal neurons were incubated in experimental media (in mM: 132 NaCl, 4 KCl, 1 CaCl 2 , 1.2 NaH 2 PO 4 .H 2 O, 1.4 MgCl 2 , 6 Glucose, 10 HEPES, pH 7.4) plus 2 µM Fluo4-AM (Thermo Fisher Sci., #F14201) for 45 min, at 37 • C. Cells were then washed, and the experiment was recorded in experimental media without Mg 2+ and supplemented with glycine (20 µM) and serine (30 µM). Fluo4 fluorescence was monitored before and after exposure to 100 µM NMDA in primary striatal neurons from WT and YAC128 mice, using an Axio Observer Z1 system, a fully motorized inverted widefield microscope (Zeiss, Jena, Germany) equipped with a large stage incubator for temperature and humidity control and EC planneofluar/1.3NA 63x lens. Fluo4 fluorescence was imaged along time at 494 nm excitation and 506 nm emission, respectively. Fluorescence intensities were calculated using Fiji software (Zurich, Switzerland).

Statistical Analyses
Data were analyzed by using Excel (Microsoft, Seattle, WA, USA) and GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA) software, and are expressed as the mean ± S.E.M. of the number of independent experiments or cells indicated in figure legends. Comparisons among multiple groups were performed by one-way ANOVA followed by the Bonferroni or Dunnett's nonparametric Multiple Comparison post-hoc tests or by two-way ANOVA, followed by Sidak's Multiple Comparison as post-hoc test. Unpaired non-parametric Mann-Whitney test was also performed for comparison between two Gaussian populations, when applicable, as described in figure legends. Significance was defined as p < 0.05.

Synaptic and Extrasynaptic GluN2B-Composed NMDAR Are Altered in Early HD Stages
The two most common non-obligatory NMDAR subunits, GluN2B and GluN2A, predominate in the striatum [19] and have differential roles in synaptic plasticity and NMDARs function in adult cortex, as well as different patterns of expression at PSD [20].
Thus, we initially determined the relative levels of GluN2B and GluN2A NMDARs in striatum from YAC128 mouse model at 3, 6, and 12 months of age ( Figure 1A-C). Total GluN2A levels were significantly increased at 6 months of age, but not at 3 or 12 months of age in the striatum of YAC128 mice ( Figure 1A), whereas total GluN2B levels were not significantly altered at 3-12 months of age ( Figure 1B). Additionally, no significant changes were observed in total levels of GluN2B in soma, proximal or distal neurites from YAC128 striatal neuros, compared with WT neurons ( Figure S1).
GluN2B phosphorylation at Tyr1472 by SKF is associated with enrichment of synaptic NMDAR [6]. We observed a significant decrease in Tyr1472 phosphorylation of GluN2B subunit in the striatum of 3-month-old YAC128 mice, a relative presymptomatic stage compared with WT mice ( Figure 1C). Similar results were observed in primary striatal neurons from YAC128 mice ( Figure 1D-F), which may suggest that in early HD stages NMDARs' retention at the synapse is altered, potentially contributing for modified synaptic function in HD.  To confirm these results, we analyzed co-localization of total and phosphorylated levels of GluN2B-containing NMDARs with PSD or non-PSD sites. As described in other published data [5], in the present paper we defined PSD or non-PSD sites through the co-localization or not with PSD-95, respectively, in proximal (<50 µm) or distal (>50 µm) neurites in primary striatal neurons, as identified in [15]. In accordance with published data, YAC128 mouse striatal neurons showed reduced GluN2B-NMDARs levels at the synapse and augmented in the non-synaptic sites ( Figure 1G), both in proximal and distal neurites. Tyr1472 phosphorylation of GluN2B subunit was reduced both in PSD and non-PSD compartments in HD neurons ( Figure 1H), indicating that this NMDARs subunit is less phosphorylated. Additionally, primary striatal neurons showed reduced PSD-95 levels and puncta ( Figure 1I), which suggests decreased PSD number and potential synaptic pruning in HD striatal neurons.

Fyn Total and Phosphorylated Levels Are Reduced in PSD of HD Neurons
Fyn play a relevant role in regulating dendritic spine and synapse formation [21,22]. Furthermore, Fyn regulates learning and memory through phosphorylation of NMDAR subunits, playing an important role in synaptic plasticity [23]. Considering that Fyn phosphorylation of GluN2B at Tyr1472 is associated with enrichment of synaptic NMDARs, when compared with extrasynaptic membrane receptors [6], and we recently observed diminished Fyn in several HD models [15], we further analyzed Fyn total and phosphorylated levels at Tyr416, the latter indicating SKF activation since all the family members share the C-terminal. Firstly, we confirmed reduced Fyn total and phosphorylated/active levels in different neuronal sections, from soma to proximal and distal neurites in YAC128 primary striatal neurons (Figure 2A,B). Considering SKF role in NMDARs regulation, we further assessed Fyn total and phosphorylated levels in PSD and non-PSD portions in proximal and distal neurites of primary striatal neurons. As shown in Figure 2C,D, total and phosphorylated Fyn levels are significantly reduced in HD PSD. Taking into account that inhibition of SKF activity with PP2 decreased NMDAR subunits in synaptic and extrasynaptic membranes [6], reduced Fyn levels and activity are apparently related with reduced synaptic GluN2B Tyr1472 phosphorylation ( Figure 1H). These data suggest that Fyn reduced levels and activity in PSD may contribute to altered NMDARs phosphorylation and potentially their function.

Constitutive Active SKF Reestablishes GluN2B-Composed NMDAR Levels and Activity in PSD
Considering reduced Fyn levels and activation within PSD and the observation that Fyn-mediated phosphorylation of GluN2B at Tyr1472 is related with enrichment of synaptic NMDARs [6], we evaluated the influence of SKF proteins on NMDARs localization and function in HD cells. GluN2B-composed NMDARs total and phosphorylated levels were assessed in striatal primary neurons from YAC128 and WT mice, both in PSD and non-PSD sites following transfection with a constitutively active form of the SKF, SKF Y527F (Figure 3). The SKF Y527F mutation enables a mutationally activated form by locking SKF proteins into the open conformation [24]. Importantly, expression of SKF Y527F in YAC128 primary striatal neurons restored GluN2B Tyr1472 phosphorylated levels in both PSD and non-PSD compartments ( Figure 3E-H) and GluN2B total levels in PSD, while decreasing GluN2B total levels in a non-PSD compartment ( Figure 3A-D) in proximal ( Figure 3A,B,E,F) and distal (Figure 3C,D,G,H) neurites. Additionally, no significant differences were observed in total GluN2B levels, in proximal and distal neurites from YAC128 and WT striatal neurons, before and after transfection with SKF Y527F plasmid ( Figure S2). These data indicate that augmented SKF activity is important for normal GluN2B phosphorylation and possible synaptic enrichment in HD.   To support the hypothesis that HD-mediated SKF reduced levels influence the presence and function of NMDARs in the synapse, we evaluated the NMDARs function in soma, proximal, and distal neurites (Figure 4). In accordance with previous published data [5], we observed augmented NMDAR-mediated current density ( Figure 4A), and activity, as observed by NMDAR-dependent Ca 2+ entry ( Figure 4B) in the soma of YAC128 striatal neurons. This augmented NMDAR-mediated currents and Ca 2+ -entry were partially reduced after SKF Y527F transfection to levels similar to WT neurons, which suggests that augmented NMDARs occur due to augmented extrasynaptic NMDARs function. On other hand, in proximal ( Figure 4C) and distal ( Figure 4D) neurites, we observed reduced NMDARs activity in YAC128 striatal neurons, which was reestablished after expression of SKF Y527F , augmenting NMDA-induced Ca 2+ -entry. Altogether, these data suggest that altered PSD number and reduced SKF levels and function in HD proximal and distal neurites may result in altered established synapses and reduced NMDARs activity. Of relevance, altered NMDARs activity in proximal and distal neurites can be restored by augmenting active SKF levels. As such, these results suggest that restoration of active SKF reduces extrasynaptic GluN2B-composed NMDARs, augmenting GluN2B-NMDARs at PSD and thus restoring normal NMDAR-dependent Ca 2+ entry.

Restoration of Active SKF Enhances CREB Activation and Reduces Apoptotic Pathway Activation
Activated CREB promotes the expression of survival-related genes, including BDNF, which has neuroprotective properties and can rescue neurons from NMDAR blockadeinduced neuronal death [25]. Additionally, several studies showed that extrasynaptic NMDAR's activation is associated with cell death pathways, whereas synaptic NMDAR function is associated to augmented CREB phosphorylation and activation, BDNF transcription, and improved antioxidant defenses [26]. Considering restored synaptic NM-DAR localization and function after expression of active form of SKF, we next evaluated CREB stimulation and apoptotic pathway activation ( Figure 5). As expected, and in accordance with previous published data, we observed reduced CREB protein levels and activation/phosphorylation at Ser133 in YAC128 striatal neuron nuclei ( Figure 5A-C). After SKF Y527F expression, CREB protein levels and activation were restored in the nucleus of YAC128 striatal neurons.    Caspase-3 is a cytosolic effector caspase with a central role in apoptosis, specifically being involved in the progression of neurodegenerative disorders [27]. Accordingly, our data showed augmented cleaved/active caspase-3 in YAC128 mouse neurons in soma ( Figure 5D), proximal ( Figure 5E), and distal ( Figure 5F) neurites. Importantly, increased cleaved/active caspase-3 levels were reduced following SKF Y527F plasmid expression, which validates apoptosis modulation ( Figure 5D-F).
These results suggest that reestablished SKF levels and activity influence NMDARs presence and function at synaptic membrane, which may activate pro-survival pathways through augmented CREB activation. , as well as the levels of cleaved/active caspase-3 (D) in soma, proximal, (E) and distal (F) neurites were evaluated by immunocytochemistry, using confocal microscope and Image J software in WT vs. YAC128 striatal neurons. Confocal images were obtained with a 63× objective in confocal microscope Zeiss LSM 710 (scale bar: 10 μm). Data are presented as the mean ± SEM of 3 to 4 independent experiments considering ~6 wells/condition. Statistical analysis: * p < 0.05,** p < 0.01, and **** p < 0.0001 versus WT or YAC128 (two-way ANOVA, followed by Sidak's Multiple Comparison as posthoc test).

Discussion
NMDARs are important synaptic receptors that contribute to normal synaptic function, cell survival, learning, and memory [28]. Our study shows augmented extrasynaptic , as well as the levels of cleaved/active caspase-3 (D) in soma, proximal, (E) and distal (F) neurites were evaluated by immunocytochemistry, using confocal microscope and Image J software in WT vs. YAC128 striatal neurons. Confocal images were obtained with a 63× objective in confocal microscope Zeiss LSM 710 (scale bar: 10 µm). Data are presented as the mean ± SEM of 3 to 4 independent experiments considering~6 wells/condition. Statistical analysis: * p < 0.05, ** p < 0.01, and **** p < 0.0001 versus WT or YAC128 (two-way ANOVA, followed by Sidak's Multiple Comparison as post-hoc test).

Discussion
NMDARs are important synaptic receptors that contribute to normal synaptic function, cell survival, learning, and memory [28]. Our study shows augmented extrasynaptic GluN2B-composed NMDARs in HD mouse neurons (Figure 1), while expression of a con-stitutive active form of SKF contributed to reestablish NMDARs localization and activity at PSD (Figure 3). Indeed, reduced Fyn activity in YAC128 PSDs (Figure 2) contributes to reduced GluN2B phosphorylation at Tyr1472, which decreases synaptic NMDAR retention and may facilitate movement to extrasynaptic sites ( Figure 4). Apart from restoring NMDARs phosphorylated levels at PSD, enhanced SKF levels and activity promote CREB activation and reduce apoptosis in YAC128 primary striatal neurons ( Figure 5).
Previous studies showed that GluN2B-composed NMDARs function and trafficking are altered in HD [5,[29][30][31]. Importantly, synaptic or extrasynaptic NMDARs activation are related to survival or apoptosis activation, respectively [32]. Striatal neurons showed faster NMDAR trafficking to the surface membrane induced by mHTT [29], whereas this receptor accumulated at extrasynaptic sites of YAC128 mice at early age [5]. Concordantly, our results evidence a reduction in GluN2B-composed NMDARs levels in PSD, whereas these levels were augmented at extrasynaptic sites is YAC128 mouse striatal neurons. Moreover, in accordance with our data, other studies showed augmented NMDARs currents in HD mouse models [5]. However, this alteration is only measured in HD neuronal soma. In contrast, in proximal and distal neurites, altered PSD number, as well as reduced SKF levels and activity may result in abnormal synaptic function in HD, which may explain the reduction in NMDARs activity. Importantly, altered NMDARs function in proximal and distal neurites can be partially restored by augmenting active SKF levels.
Additionally, GluN2B Tyr1472 phosphorylation levels were decreased both in synaptic and extrasynaptic sites, which is in accordance with previous studies. Gladding and coworkers showed increased synaptic STEP activity in YAC128 striatum, correlated with decreased Tyr1472-GluN2B phosphorylation in YAC128 non-PSD and PSD fractions similar to what we observed in the present study, which facilitates NMDAR movement to extrasynaptic sites, reducing synaptic NMDAR retention [16]. Indeed, GluN2B-composed NMDAR Tyr1472 phosphorylation by SKF is associated with enrichment of synaptic NM-DARs [6], indicating that NMDAR lateral diffusion between synaptic and extrasynaptic sites is modulated by Tyr1472 phosphorylation. We previously showed that SKF members, specifically c-Src and Fyn proteins, are reduced in several HD models due to augmented degradation by autophagy [15]. Concordantly with these data, here we show that Fyn total and phosphorylated levels are reduced at both PSD and non-PSD compartments, which may contribute to decrease Tyr1472-GluN2B phosphorylation at PSD and thus reduce synaptic NMDAR retention.
Fyn influence and its fundamental role in PSD has long been recognized. Several studies have previously shown c-Src and Fyn roles in synapse development, plasticity, learning, and memory [22,23]. Takasu and colleagues showed that Ca 2+ influx mediated by NMDARs activation upon its Tyr1472 phosphorylation by SKF resulted in gene transcription required for the remodeling of synaptic connections in excitatory synapses of primary cortical neurons [33]. In the present study we show, for the first time, that expression of a constitutive active form of SKF reestablishes NMDARs localization and function at PSD in YAC128 mouse striatal neurons, restoring neuronal function.
Decreased synaptic NMDARs and augmented extrasynaptic currents are associated with reduced nuclear CREB activation, reduced neuronal expression of the survival factor BDNF, and caused dysfunctional mitochondria, low energy levels, and cell death in HD mouse striatum, before and after phenotype onset [5]. Extrasynaptic NMDARs activity is linked to cell death signaling cascades due to reduced phosphorylation/activation of CREB [26]. Concordantly with previous studies showing decreased CREB activation in striatum of 1 and 4 months of age YAC128 mice [5], our results show reduced nuclear CREB activation in YAC128 primary striatal neurons ( Figure 5A,B). Interestingly, decreased CREB activity and augmented active caspase-3 found in YAC128 striatal neurons were reversed following expression of the constitutive active form of SKF, SKF Y527F ( Figure 5), previously shown by our group to be neuroprotective against mHTT-induced mitochondrial dysfunction and enhanced ROS levels [15].
Our study evidence, for the first time, that Fyn protein has an important role in synaptic GluN2B-composed NMDARs phosphorylation and activity, restoring CREB activation and decreasing caspase-3 levels, indicative of a decrease in cell death by apoptosis in HD. Decreased Fyn PSD co-localization correlates with HD-related reduced Tyr1472 GluN2B phosphorylation and augmented extrasynaptic NMDARs currents, as well as decreased CREB activation and cell death. Interestingly, reestablished active SKF levels partially restored NMDARs currents, NMDAR-dependent Ca 2+ levels, CREB activation, and reduced caspase-3 cleavage. Hence, we describe a potential mechanism involving Fyn restored levels and activity that may constitute a potential HD therapeutic target to promote striatal glutamatergic synaptic function and survival.

Conflicts of Interest:
The authors declare no conflict of interest.