Glutamate is the main excitatory neurotransmitter in the brain, acting through ionotropic receptors such as AMPA receptors (AMPARs). While their neuronal role is well established, AMPAR function in astrocytes remains poorly understood, especially in the hippocampus. In this study, we investigated the expression and functional properties of astrocytic AMPARs, focusing on their capacity to mediate calcium signaling and inter-cellular communication. Using astrocyte primary cultures, we confirmed the expression of GluA1 and GluA2 subunits, along with detectable levels of GluA4. Calcium imaging revealed that pharmacological AMPAR stimulation elicited intracellular Ca²⁺ signals in a subset of astrocytes which were markedly potentiated when desensitization was impaired. Notably, some of these responses persisted without extracellular Na⁺, compatible with the presence of functional calcium-permeable AMPARs (CP-AMPARs) in hippocampal astrocytes. However, sodium was required for full amplification of the Ca²⁺ signals, suggesting a synergistic role of Na⁺ and Ca²⁺ influx. Pharmacological inhibition of GluA2-lacking AMPARs reduced the responses, supporting the coexistence of GluA2-lacking and GluA2-containing subtypes. Sniffer-calcium assays suggested that strong AMPAR stimulation can engage ATP-dependent signaling, facilitating calcium wave propagation. Altogether, our results reveal functional and heterogeneous AMPAR populations in hippocampal astrocytes. Further investigations will elucidate their physiological roles, localization, and contribution to neuroglial interactions under physiological and pathological conditions.

Huntington’s disease (HD) is an inherited, fatal neurodegenerative disorder caused by expanded CAG repeats in the Huntingtin gene, leading to progressive motor, cognitive and psychiatric impairment. Despite its monogenic origin, HD pathogenesis is multifactorial, with convergent contributions from mitochondrial dysfunction, oxidative stress, synaptic failure, and chronic neuroinflammation, which drive neuronal vulnerability and degeneration, particularly within the striatum. Current clinical management remains exclusively symptomatic and fails to halt disease progression, highlighting a critical unmet need for strategies targeting fundamental pathogenic mechanisms. Cortistatin, a neuropeptide expressed in the nervous and immune systems, exhibits potent immunomodulatory properties and has recently been implicated in the regulation of mitochondrial function. Notably, cortistatin deficiency is associated with exacerbated systemic and central inflammation, suggesting that impaired cortistatin signaling may contribute to neurodegeneration. However, its role in HD pathophysiology remains unexplored. We performed a comprehensive reanalysis of publicly available transcriptomic datasets from HD patients to assess cortistatin expression, followed by validation in experimental HD models. Wild-type and cortistatin-deficient mice treated with 3-nitropropionic acid served as pharmacological HD models, enabling evaluation of cortistatin-dependent disease severity. Behavioral assessments, glial and oxidative markers, and immune factors were evaluated to determine neurological dysfunction and inflammatory responses. Complementary in vitro studies were conducted in striatal neurons expressing mutant huntingtin to examine mitochondrial integrity, inflammatory signaling, metabolic function, and mitochondria-endoplasmic reticulum interactions. Cortistatin expression was significantly reduced in postmortem HD human brains and across experimental HD models. Cortistatin deficiency exacerbated motor deficits, neuropathological alterations, inflammatory activation, and neuronal vulnerability in HD context. At the cellular level, reduced cortistatin expression was accompanied by amplified inflammatory signaling, disrupted mitochondrial integrity, impaired mitochondria-endoplasmic reticulum interactions, and increased oxidative stress. Conversely, exogenous cortistatin administration attenuated inflammatory mediator production, preserved mitochondrial structure, and improved redox balance in mutant huntingtin-expressing striatal neurons. Our findings identify cortistatin deficiency as a previously unrecognized contributor to HD pathogenesis and establish cortistatin as a key modulator of neuroinflammation and mitochondrial homeostasis. These results support cortistatin-based strategies as a promising disease-modifying therapeutic avenue for HD and related neurodegenerative disorders characterized by inflammatory activation and mitochondrial impairment.

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.

Dysregulation of the endocannabinoid system (eCBS) and the loss of CB1 receptors (CB1R) in the basal ganglia are well-established hallmarks of Huntington’s disease (HD). As a result, significant research efforts have focused on targeting the eCBS to alleviate motor disturbances associated with the disease. Beyond its role in motor control, the eCBS is a complex signaling network critically involved in regulating learning and memory. Despite this, the potential involvement of eCBS dysfunction in the cognitive decline characteristic of HD, often manifested well before motor dysfunction, has remained largely unexplored. CB1R expression in the hippocampus was evaluated in both human HD samples and HD mouse models (R6/1 and HdhQ7/Q111 models, including both sexes) using Western blotting, immunohistochemistry, and radioligand binding assays. To restore CB1R function, CB1R agonist WIN-55212–2 was systemically administered, or viral vectors encoding CB1R were locally infused into the hippocampus of HD mice. A multidisciplinary approach combining behavioral, biochemical, electrophysiological, and morphological analyses, was employed to investigate the molecular mechanisms underlying the effects of CB1R activation in the context of HD-related cognitive dysfunction. In both human HD samples and HD mouse models, CB1R protein levels were reduced in the hippocampus, accompanied by structural synaptic alterations and impairment in spatial, recognition and working memory. Moreover, hippocampal depolarization-induced suppression of inhibition was significantly disrupted in R6/1 mice. Administration of WIN-55212–2 successfully restored these synaptic and cognitive deficits. Immunohistochemical analysis revealed that the CB1R decrease was specifically localized to GABAergic interneurons within the hippocampus. Notably, targeted restoration of CB1R expression in these interneurons via viral vector delivery was sufficient to rescue hippocampal-dependent memory deficits in HD mice. This study suggests that impaired CB1R function in hippocampal GABAergic interneurons contributes to memory dysfunction in HD.

Deficits in mitochondrial bioenergetics and dynamics are strongly implicated in the selective vulnerability of striatal neurons in Huntington´s disease. Beyond these neuron-intrinsic factor, increasing evidence suggest that non-neuronal mechanisms, particularly astrocytic dysfunction involving disrupted homeostasis and metabolic support also contribute to disease progression. These findings underscore the critical role of metabolic crosstalk between neurons and astrocytes in maintaining striatal integrity. However, it remains unclear whether this impaired communication affects the transfer of mitochondria from astrocytes to striatal neurons, a potential metabolic support mechanism that may be compromised in Huntington´s Disease. Primary striatal astrocytes were obtained from wild-type and R6/1 mice to investigate mitochondrial dynamics. Expression levels of key mitochondrial fusion and fission proteins were quantified by Western blotting and RT-PCR. Mitochondria morphology, oxidative stress and membrane potential were assessed using confocal microscopy following staining with mitochondria-specific dyes. Mitochondrial respiration was measured using the Oxygraph-2k respirometer system (Oroboros Instruments). Transmitophagy was evaluated by confocal imaging after labeling astrocytic mitochondria with Mitotracker dyes. To assess the functional impact of mitochondrial transfer on neurons, Sholl analysis, neuronal death and oxidative stress levels were quantified using specific fluorogenic probes. Striatal astrocytes from HD mice exhibited a significant increase in mitochondrial fission, and mitochondrial oxidative stress, mirroring alterations previously reported in striatal neurons. Analysis of mitochondrial oxygen consumption rate (OCR) revealed elevated respiration activity and enhanced ATP-linked respiration, indicative of a hypermetabolic state. Concurrently, increased lactate production suggested a shift toward dysregulated astrocytic energy metabolism. These mitochondrial alterations were functionally detrimental: astrocytic mitochondria derived from HD mice when taken up by striatal neurons via transmitophagy, led to reduced neuronal branching and disrupted oxidative homeostasis. Our findings demonstrate that striatal astrocytes from HD mice exhibit a hypermetabolic phenotype, characterized by increased mitochondrial respiration, disrupted mitochondrial dynamics, and elevated mitochondrial oxidative stress. Importantly, we identify a novel mechanism of astrocyte-neuron interaction involving the transfer of dysfunctional mitochondria from astrocytes to neurons. The uptake of these compromised mitochondria by striatal neurons results in reduced neuronal branching and increased reactive oxygen species (ROS) production. Collectively, these results highlight the pathological relevance of impaired astrocyte-to-neuron mitochondrial transfer and emphasize the contributory role of astrocytic dysfunction in Huntington´s disease progression.

Synapse-to-nucleus signaling regulates activity-dependent synaptic plasticity underlying memory by linking N-methyl-D-aspartate (NMDA) glutamate receptors (GluN) to gene transcription mediated by the transcription factor cAMP-response element binding protein (CREB), but the underlying gene programs mediating potentiation at excitatory synapses are unknown. Here, we analyzed genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) datasets of mouse and human CREB and the synaptonuclear factor CREB-regulated transcription coactivator1 (CRTC1) to identify relevant target genes and biological pathways coupling neuronal activity to synaptic function/plasticity. Our analyses indicate that CRTC1 specifically couples neuronal activity with synaptic plasticity by binding to conserved promoters of CREB target genes comprising inducible transcription factors (including c-fos, Crem, Npas4 and Nr4a1-3), and neuronal excitability and plasticity genes, including Ntrk2, Homer1, Dlg4 (PSD-95) and the NMDA receptor subunit Grin1 (GluN1). CRTC1/CREB target genes were highly enriched in gene ontology (GO) nuclear terms, including several members of the CREB family, and transcriptional modulators and repressors. Interestingly, GO enrichment and protein-protein interaction (PPI) network analyses revealed that genes mediating synapse-to-nucleus signaling (including most known synaptonuclear factors and direct interacting modulators) are collectively regulated by CREB/CRTC1, and that protein kinase C (PKC) is a key interactor of the CRTC1/14-3-3 complex at synapses. In agreement with these in silico analyses, we show that CRTC1 regulates synaptic activity-dependent phosphorylation and synaptic recruitment of GluN1 mediated by PKC in hippocampal neurons, and that PKC activation reverses NMDA receptor-mediated currents and long-term potentiation (LTP) deficits caused by CRTC1 silencing in the hippocampus. Consistent with genomics and functional data, morphological and behavioral analyses show crucial roles of CRTC1 on dendritic spine structure, plasticity, and hippocampal-dependent associative memory. Our results support a model in which neuronal activity and synaptic inputs are integrated in the nucleus through conserved CREB/CRTC1-regulated transcriptional programs sustaining global synapse-to-nucleus signaling pathways impacting on synaptic plasticity and memory. ### Competing Interest Statement The authors have declared no competing interest.

In Huntington's disease (HD), aberrant processing of huntingtin (HTT) mRNA produces HTT1a transcripts that encode the pathogenic HTT exon 1 protein. The mechanisms behind HTT1a production are not fully understood. Considering the role of m6A in RNA processing and splicing, we investigated its involvement in HTT1a generation. Here, we show that m6A methylation is increased before the cryptic poly(A) sites (IpA1 and IpA2) within the huntingtin RNA in the striatum of Hdh+/Q111 mice and human HD samples. We further assessed m6A's role in mutant Htt mRNA processing by pharmacological inhibition and knockdown of METTL3, as well as targeted demethylation of Htt intron 1 using a dCas13-ALKBH5 system in HD mouse cells. Our data reveal that Htt1a transcript levels are regulated by both METTL3 and the methylation status of Htt intron 1. They also show that m6A methylation in intron 1 depends on expanded CAG repeats. Our findings highlight a potential role for m6A in aberrant splicing of Htt mRNA.

A combination of Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD) at non-psychoactive doses was previously demonstrated to reduce cognitive decline in APP/PS1 mice, an animal model of Alzheimer's disease (AD). However, the neurobiological substrates underlying these therapeutic properties of Δ9-THC and CBD are not fully understood. Considering that dysregulation of glutamatergic activity contributes to cognitive impairment in AD, the present study evaluates the hypothesis that the combination of these two natural cannabinoids might reverse the alterations in glutamate dynamics within the hippocampus of this animal model of AD. Interestingly, our findings reveal that chronic treatment with Δ9-THC and CBD, but not with any of them alone, reduces extracellular glutamate levels and the basal excitability of the hippocampus in APP/PS1 mice. These effects are not related to significant changes in the function and structure of glutamate synapses, as no relevant changes in synaptic plasticity, glutamate signaling or in the levels of key components of these synapses were observed in cannabinoid-treated mice. Our data instead indicate that these cannabinoid effects are associated with the control of glutamate uptake and/or to the regulation of the hippocampal network. Taken together, these results support the potential therapeutic properties of combining these natural cannabinoids against the excitotoxicity that occurs in AD brains.

ABSTRACT Huntington’s disease (HD) is a dominantly inherited neurodegenerative disorder caused by an expanded, somatically unstable CAG repeat in the first exon of the huntingtin gene ( HTT) . In the presence of an expanded CAG repeat, huntingtin mRNA undergoes an aberrant processing that generates HTT1a transcripts with exon 1 and intron 1 sequences, which encodes the aggregation-prone and pathogenic HTTexon 1 protein. The regulatory mechanisms that contribute to the production of HTT1a are not fully understood. In a previous transcriptome-wide m6A landscape study performed in Hdh +/Q111 knock-in mice, we have found that the proximal region of intron 1 to exon1-intron 1 splice site in Htt RNA is highly modified by m6A. Several pieces of evidence have demonstrated that m6A is involved in RNA processing and splicing. Therefore, in this study we set out to explore the impact of m6A RNA modifications in the generation of Htt1a . We show in the striatum of Hdh +/Q111 mice that m6A is enriched in intronic sequences 5’ to the cryptic poly (A) sites (IpA1 and IpA2) at 680 and 1145 bp into intron 1 as well as in Htt1a polyadenylated mRNA. We also verified the presence of specific m6A-modified sites near the 5’ exon1-intron1 splice donor site. Intronic HTT m6A methylation was recapitulated in human samples showing a significantly increased methylation ratio in HD putamen post-mortem samples and in HD fibroblast cell lines from pre-symptomatic and symptomatic patients. In order to test the hypothesis that the m6A modification is involved in mutant Htt RNA processing, we performed a pharmacological inhibition of METTL3 and a targeted demethylation of Htt intron 1 in HD cells using a dCas13-ALKBH5 system. We found that Htt1a transcript levels in HD cells are regulated by METTL3 and by methylation status in Htt intron 1. Site-specific manipulation with an RNA editing system resulted in decreased expression levels of Htt1a , which was accompanied by a reduction in DNA damage, a major hallmark in HD. Finally, we propose that m6A methylation in intron 1 is likely dependent on the expanded CAG repeats. These findings provide insight into the role of m6A in the generation of the aberrantly spliced mutant Htt transcripts with important implications for therapeutic strategies.

Increasing evidence indicates that a key factor in neurodegenerative diseases is the activation of the unfolded protein response (UPR) caused by an accumulation of misfolded proteins in the endoplasmic reticulum (ER stress). Particularly, in Huntington's disease (HD) mutant huntingtin (mHtt) toxicity involves disruption of the ER-associated degradation pathway and loss of the ER protein homeostasis leading to neuronal dysfunction and degeneration. Besides the role of the UPR in regulating cell survival and death, studies that demonstrate the contribution of sustained UPR activation, particularly of PERK signaling, in memory disturbances and synaptic plasticity deficiencies are emerging. Given the contribution of hippocampal dysfunction to emotional and cognitive deficits seen in HD, we have analyzed the involvement of ER stress in HD memory alterations. We have demonstrated that at early disease stages, ER stress activation manifested as an increase in GRP78 and CHOP is observed in the hippocampus of R6/1 mice. Genetic reduction of GRP78 expression resulted in preventing hippocampal-dependent memory alterations but no motor deficits. Accordingly, hippocampal neuropathology namely, dendritic spine loss and accumulation of mHtt aggregates was ameliorated by GRP78 reduction. To elucidate the signaling pathways, we found that the inactivation of PERK by GSK2606414 restored spatial and recognition memories in R6/1 mice and rescued dendritic spine density in CA1 pyramidal neurons and protein levels of some specific immediate early genes. Our study unveils the critical role of the GRP78/PERK axis in memory impairment in HD mice and suggests the modulation of PERK activation as a novel therapeutic target for HD intervention.

Huntington’s disease (HD) is a dominantly inherited neurodegenerative disorder caused by an expanded, somatically unstable CAG repeat in the first exon of the huntingtin gene ( HTT) . In the presence of an expanded CAG repeat, huntingtin mRNA undergoes an aberrant processing that generates HTT1a transcripts with exon 1 and intron 1 sequences, which encodes the aggregation-prone and pathogenic HTTexon 1 protein. The regulatory mechanisms that contribute to the production of HTT1a are not fully understood. In a previous transcriptome-wide m6A landscape study performed in Hdh+/Q111 knock-in mice, we have found that the proximal region of intron 1 to exon1-intron 1 splice site in Htt RNA is highly modified by m6A. Several pieces of evidence have demonstrated that m6A is involved in RNA processing and splicing. Therefore, in this study we set out to explore the impact of m6A RNA modifications in the generation of Htt1a . We show in the striatum of Hdh+/Q111 mice that m6A is enriched in intronic sequences 5’ to the cryptic poly (A) sites (IpA1 and IpA2) at 680 and 1145 bp into intron 1 as well as in Htt1a polyadenylated mRNA. We also verified the presence of specific m6A-modified sites near the 5’ exon1-intron1 splice donor site. Intronic HTT m6A methylation was recapitulated in human samples showing a significantly increased methylation ratio in HD putamen post-mortem samples and in HD fibroblast cell lines from pre-symptomatic and symptomatic patients. In order to test the hypothesis that the m6A modification is involved in mutant Htt RNA processing, we performed a pharmacological inhibition of METTL3 and a targeted demethylation of Htt intron 1 in HD cells using a dCas13-ALKBH5 system. We found that Htt1a transcript levels in HD cells are regulated by METTL3 and by methylation status in Htt intron 1. Site-specific manipulation with an RNA editing system resulted in decreased expression levels of Htt1a , which was accompanied by a reduction in DNA damage, a major hallmark in HD. Finally, we propose that m6A methylation in intron 1 is likely dependent on the expanded CAG repeats. These findings provide insight into the role of m6A in the generation of the aberrantly spliced mutant Htt transcripts with important implications for therapeutic strategies.### Competing Interest StatementThe authors have declared no competing interest.

Increasing evidence indicates that a key factor in neurodegenerative diseases is the activation of the unfolded protein response (UPR) caused by an accumulation of misfolded proteins in the endoplasmic reticulum (ER stress). Particularly, in Huntington's disease (HD) mutant huntingtin (mHtt) toxicity involves disruption of the ER-associated degradation pathway and loss of the ER protein homeostasis leading to neuronal dysfunction and degeneration. Besides the role of the UPR in regulating cell survival and death, studies that demonstrate the contribution of sustained UPR activation, particularly of PERK signaling, in memory disturbances and synaptic plasticity deficiencies are emerging. Given the contribution of hippocampal dysfunction to emotional and cognitive deficits seen in HD, we have analyzed the involvement of ER stress in HD memory alterations. We have demonstrated that at early disease stages, ER stress activation manifested as an increase in GRP78 and CHOP is observed in the hippocampus of R6/1 mice. Genetic reduction of GRP78 expression resulted in preventing hippocampal-dependent memory alterations but no motor deficits. Accordingly, hippocampal neuropathology namely, dendritic spine loss and accumulation of mHtt aggregates was ameliorated by GRP78 reduction. To elucidate the signaling pathways, we found that the inactivation of PERK by GSK2606414 restored spatial and recognition memories in R6/1 mice and rescued dendritic spine density in CA1 pyramidal neurons and protein levels of some specific immediate early genes. Our study unveils the critical role of the GRP78/PERK axis in memory impairment in HD mice and suggests the modulation of PERK activation as a novel therapeutic target for HD intervention.

Background Neuron-glia crosstalk implies the release of different factors that may influence both cell types and modulate synaptic transmission and neuronal function. However, most of our knowledge about the molecular mechanisms underlying HD pathology has been gained from studies conducted in neurons. Reduced neuronal BDNF secretion and altered expression of neuronal BDNF receptors has been associated with diminished striatal neurotrophic support. However, astrocytes are also an important source of BDNF. In fact, compromised exocytosis of BDNF vesicles has been reported in primary HD astrocytes. ARMS/kidins220 is a multi-functional scaffolding protein highly expressed in the CNS that has emerged as a novel player of neuronal activity and linked to the regulation of BDNF signalling. Aims Given that reduced BDNF secretion has been associated with an aberrant increase in ARMS expression in the HD mouse brain, our goal is to analyse whether astrocytic ARMS expression is also altered in HD and its possible role in the secretome of astrocytes. Methods The current project is performed on striatal primary astrocytes, adult isolated astrocytes from R6/1 mice and hiPSCs-derived astrocytes. Results Results from our lab have demonstrated increased ARMS expression in HD primary striatal astrocytes compared to controls. Notably, injection of AAV2/5-GFAP-shARMS-GFP in the striatum of R6/1 mice significantly ameliorated motor learning and coordination deficits suggesting an important role of astrocytic ARMS in motor alterations in HD mice. Conclusions Taken together, our results suggest an abnormal regulation of astrocytic ARMS in HD that could be modulating astrocyte’s proper function and the activity and vulnerability of striatal neurons.

Background The ‘exon 1 fragment’ of mutant HTT has been postulated as a key driver of neurotoxicity in Huntington´s Disease. It can be generated by proteolytic processing as well as by incomplete splicing of expanded-CAG-repeat HTT RNA. Although regulatory mechanisms that influence the amount of HTTexon1 production by incomplete splicing have been described, the impact of RNA modifications on the post-transcriptional regulation of HTT is unknown. Aims To unravel the mechanisms involving N6-methyladenosine (m6A) in the post-transcriptional regulation of HTT. Methods We performed MeRIP 3’ RACE analysis for cryptic poly A sites to measure splicing in the striatum of HdhQ111/Q7 Knock in (KI) mice. We identified methylation motifs in both human and mouse models using a MazF-qPCR approach. Finally, we studied in vitro association between m6A modifications in the intronic region of mutant Htt and the generation of Htt exon 1 by targeting RNA demethylation in mutant Htt using a CRISPR/Cas13-based approach. Results We have detected that the proximal region of intron 1 in Htt mRNA is heavily modified by m6A in HdhQ111/Q7 KI mice and human samples. Here we demonstrate in the striatum of KI mice m6A an enrichment in intronic sequences 5´ to the cryptic poly (A) sites but not in Full Length transcripts. Targeting RNA demethylation in mutant HTT, we showed that demethylation of the proximal region of Httexon1 leads to a significant reduction in Httexon1 transcripts levels. Conclusions Our results support the involvement of m6A in the generation of aberrantly spliced mutant Htt transcripts.

Pyk2 is a non-receptor tyrosine kinase enriched in hippocampal neurons, which can be activated by calcium-dependent mechanisms. In neurons, Pyk2 is mostly localised in the cytosol and dendritic shafts but can translocate to spines and/or to the nucleus. Here, we explore the function of a new localisation of Pyk2 in mitochondria-associated membranes (MAMs), a subdomain of ER-mitochondria surface that acts as a signalling hub in calcium regulation. To test the role of Pyk2 in MAMs’ calcium transport, we used full Pyk2 knockout mice (Pyk2−/−) for in vivo and in vitro studies. Here we report that Pyk2−/− hippocampal neurons present increased ER-mitochondrial contacts along with defective calcium homeostasis. We also show how the absence of Pyk2 modulates mitochondrial dynamics and morphology. Taken all together, our results point out that Pyk2 could be highly relevant in the modulation of ER-mitochondria calcium efflux, affecting in turn mitochondrial function.