Molecular and Cellular Neuroscience

Drosophila glutathione S-transferase S1 (GstS1) is a unique member of the glutathione S-transferase family and is considered the ortholog of vertebrate hematopoietic prostaglandin D synthase based on amino acid sequence similarity. A previous forward genetic screen revealed that a null mutation in GstS1 dominantly suppresses genetically induced behavioral hyperexcitability in several seizure-prone Drosophila mutants, including paraShu, a gain-of-function mutant of the voltage-gated sodium channel gene paralytic (para), and eas2, a loss-of-function mutant of the ethanolamine kinase gene easily shocked (eas). Here, using the GAL4/UAS system combined with GstS1 RNAi, we show that hemocyte-specific GstS1 knockdown suppresses hyperexcitable phenotypes in both paraShu and eas2. Notably, suppression of adult paraShu phenotypes required GstS1 knockdown before adult eclosion, suggesting that GstS1 modulates adult neuronal excitability by influencing neural development through neuroimmune interactions. Consistent with this model, hemocyte-specific GstS1 knockdown rescued abnormal dendritic morphology in developing class IV dendritic arborization (C4da) neurons of paraShu larvae. Moreover, paraShu and eas2 larvae exhibited increased numbers of sessile hemocytes surrounding C4da neuron somata, a phenotype reversed by either GstS1 knockdown or dietary supplementation with the {omega}-3 polyunsaturated fatty acid -linolenic acid. Together, these findings reveal a previously unrecognized developmental role for GstS1 in promoting nervous system hyperexcitability through hemocyte-mediated neuroimmune interactions, potentially involving bioactive lipid signaling. Our results highlight neuroimmune crosstalk as an important mechanism linking genetic perturbations to altered neural development and seizure-associated hyperexcitability.

Background: Insufficient oxygen (hypoxia) or complete lack of oxygen (anoxia) causes cellular dysfunction and death in terrestrial animals. As a result, animals have developed homeostatic strategies, including changes in movement strategies to escape and survive stressful environments. Caenorhabditis elegans respond to changes in oxygen by increasing the frequency of reversals (backward movement) and increasing locomotor speed; these behaviors enable them to escape unfavorable environments and move to more suitable ones. The molecular and neural mechanisms underlying behavioral responses to low-oxygen environments are incomplete. Neuropeptides regulate C. elegans behaviors by modifying neural circuit function in response to hypoxia. Loss of function in C. elegans neuropeptide nlp-40 and its receptor aex-2 increased survival to anoxia [1]. However, the cells in which NLP-40 and AEX-2 function for anoxia, as well as the mechanisms underlying responses to anoxia, remain elusive. We hypothesized that NLP-40 and AEX-2 regulate anoxia by altering escape behaviors. Results: We found that neuropeptide NLP-40 functions in cholinergic neurons and AEX-2 functions in cholinergic or GLR-1 interneurons or motor neurons to regulate susceptibility to anoxia. We found that loss of either nlp-40 or aex-2 decreased locomotor activity and reversal frequency early during 48 hours of anoxia, suggesting that nlp-40 and aex-2 are required for the proper execution of these behaviors. We found that neuronal knockdown of nlp-40 recapitulated the behaviors observed in nlp-40 mutant animals and increased survival; however, restoring aex-2 function in glr-1 interneurons or motor neurons was sufficient to rescue behaviors to wild-type and restore vulnerability to anoxia. Conclusions: Overall, our results suggest that NLP-40 and AEX-2 likely function in cholinergic or glr-1 interneurons or motor neurons to regulate escape behaviors during anoxia. Escape behaviors are part of the homeostatic response and are typically protective in the wild; however, in our paradigm, we show that loss of either nlp-40 or aex-2 reduces escape responses and confers protection against 48 hours of anoxia. Collectively, these results highlight a new role for NLP-40 and AEX-2 in anoxia and shed light on mechanisms underlying the adaptive responses to anoxia.

Achromatopsia (ACHM) is a rare autosomal recessive retinal disorder characterized by absent cone photoreceptor function from early life, leading to severe visual impairment. Mutations in genes involved in the cone phototransduction cascade frequently result in elevated cyclic guanosine monophosphate (cGMP) levels and activation of stress pathways, including endoplasmic reticulum (ER) stress and the unfolded protein response. Targeting common downstream mechanisms rather than individual mutations may provide a broadly applicable therapeutic strategy. Here, we investigated whether pharmacological inhibition of valosin-containing protein (VCP), a key regulator of ER and protein homeostasis, can prevent cone degeneration in the spontaneous cone photoreceptor function loss 1 (cpfl1) mouse model of ACHM. Organotypic culture of retinal explants from cpfl1 mice were treated with the selective VCP inhibitor ML240. Cone survival, cell death, opsin expression and localization were assessed by TUNEL assay, immunohistochemistry, and quantitative image analysis. ML240 treatment significantly increased cone density and improved cone opsin expression and trafficking to the outer segments (OSs) in cpfl1 explants compared to controls. Importantly, rhodopsin trafficking in rod photoreceptors was unaffected, indicating that VCP inhibition did not impair normal rod phototransduction. These findings demonstrate that VCP inhibition by ML240 effectively preserves cone photoreceptors and improves cone-specific functional markers in the cpfl1 model. Targeting VCP may represent a mutation-independent therapeutic strategy for preventing cone death in ACHM.

A key pathophysiological hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is loss of nuclear localization and abnormal cytoplasmic aggregation of TAR DNA-binding protein 43 (TDP-43). Upstream factors that trigger the onset and progression of such neurodegenerative diseases are largely unknown. Aging and environmental factors contribute as potential risk factors. But epidemiological evidence suggests psychological stressors such as social isolation and loneliness are also associated with increased risks of neurodegenerative disease [1-8]. We examined the impacts of social isolation on TDP-43 related neurodegeneration. To investigate the potential for causative impact on neurodegeneration, we compared the effects of social isolation versus social enrichment in Drosophila. We utilized an established social isolation paradigm in which flies were either housed alone or in groups as young adults. We found that early-life social isolation acts as a primer to exacerbate the rate of neurodegeneration in response to subsequent induction of pathological levels of TDP-43. Social isolation stress drives more rapid activation of mdg4, an endogenous retrovirus that functionally mediates TDP-43 effects and more aggressive propagation of TDP-43 protein pathology from surface glial cells to nearby neurons. Shortened life span also ensues. We demonstrate that providing isolated flies with visual, olfactory and tactile social cues from conspecifics living behind a divider was insufficient to alleviate these effects of social isolation on neurodegeneration. Our findings have implications for the association between psychological stressors such as loneliness and risk of neurodegenerative diseases in humans and provide a platform to investigate mechanistic underpinnings in animal models.

Axonal projections and precise synapse formation are essential for the establishment of functional neural circuits, and their dysregulation contributes to various psychiatric and neurodegenerative disorders. Although numerous extracellular cues secreted from surrounding tissues regulate axon guidance and synapse formation, the mechanisms by which neurons locally regulate terminal patterning within their projection fields remain poorly understood. Bone morphogenetic protein 4 (BMP4) is a classical morphogen that is involved in early neural tube patterning during development. However, its postnatal function remains unclear. Previous in vitro studies using hippocampal glutamatergic neurons demonstrated that BMP4 can act locally on axons to suppress synapse formation, raising the possibility that BMP4 regulates terminal differentiation after target innervation. Here, we investigated the role of BMP4 in postnatal cholinergic circuit development using cholinergic neuron-specific Bmp4 conditional knockout (Bmp4 cKO) mice, with adult analyses performed in males. Basal forebrain cholinergic neurons project extensively throughout the neocortex and are essential for higher brain functions, including arousal and attention. Bmp4 cKO mice exhibited increased cholinergic axon terminals in the neocortex, hippocampus, and amygdala, as well as increased axonal ramification in the neocortex without affecting cytoarchitectonic organization or cholinergic neuron numbers in the basal forebrain. These findings demonstrate that BMP4 functions as a negative regulator of cholinergic terminal formation and axonal arborization, suggesting that BMP4 may act locally to restrict cholinergic axonal expansion during neural circuit maturation.

Traumatic brain injury (TBI) is one of the world's leading causes of death and disability and a major risk factor for dementias. The primary dementia associated with TBI is chronic traumatic encephalopathy (CTE), a neurodegenerative disease classified as a tauopathy, in which toxic tau molecules lead to disease pathologies and degeneration. The processes that lead to tauopathy and subsequent dementia after TBI remain unclear. Here, we built upon the finding that seizures after TBI may be a mechanism leading to tauopathy, by dissecting the functions of the metabotropic glutamate receptor 5 - cellular prion protein (mGluR5-PrPC) pathway. We delivered TBI to larval in a blast paradigm, and quantified aggregation of Tau via a genetically-encoded Tau-GFP fusion reporter. Zebrafish larvae lacking prp2 (homolog of mammalian cellular Prion Protein, PrPC) displayed a 168% increase in post-traumatic seizures activity after TBI. An mGluR5 agonist (CHPG) reduced post-traumatic seizures, whereas an mGluR5 antagonist (MPEP) increased post-traumatic seizures. Moreover, agonizing mGluR5 reduced tau aggregation and antagonizing mGluR5 increased tau burden. Larvae seizing from convulsants, rather than TBI, were treated with CHPG/MPEP and provided a similar pattern of outcomes, suggesting seizures may be a factor needed for mGluR5 activity to influence tau aggregation. The PrPC-mGluR5 pathway is proposed as one candidate pathomechanism linking TBI to subsequent seizures and tauopathy, and thus it warrants investigation as a target for prophylactic interventions.

Amacrine cells (ACs) are the most diverse neuron in the retina, with more than sixty different subtypes, each contributing to the detection of specific visual features. Despite their importance in visual feature detection, the genetic basis for this diversity is still not well understood. Recent single-cell transcriptomic datasets have identified several potential genes that may regulate cellular diversity, but the underlying mechanisms have not been investigated. For example, the transcription factor Meis2 is selectively expressed in the GABAergic subclass of ACs. In this study, we focused on how Meis2 regulates the development of GABAergic ACs. We hypothesized that Meis2 is required for specification and survival of GABAergic ACs. To test this, we used the Meis2 conditional knockout mouse (Meis2-CKO) where Meis2 was deleted from either the entire retina or specifically from AC precursor cells. The deletion of Meis2 led to a ~60% reduction in total GABAergic ACs in the retina. This loss of GABAergic ACs occurred only after postnatal day 5 (P5), suggesting Meis2 is not required for early specification but may be required for survival. To further investigate cell death and survival, we quantified cleaved caspase-3 positive cells in the retina at several developmental timepoints. In Meis2-CKO mice, we found an increase in cleaved caspase-3 immunolabeling at P5 and P6 timepoints coinciding with the reduction of GABAergic ACs. Further analysis of GABAergic AC subtypes demonstrated a subtype-specific cell death in Meis2-CKO retinas. For example, the total number of dopaminergic (TH) ACs were significantly reduced, whereas starburst (ChAT) ACs remained unchanged. These findings suggest that Meis2 is important for the survival of a subset of GABAergic AC subtypes in the mammalian retina. In addition, Meis2 may control the expression of specific survival factors that prevent apoptosis during developmental cell death in the retina.

3q29 deletion (3q29Del) is a genetic risk variant for autism spectrum disorder and schizophrenia that often results in developmental delays, cognitive disability, and impaired fine motor function. People with 3q29Del have reduced cerebellar volume, which correlates with symptom severity, and many 3q29Del-associated phenotypes also commonly occur after cerebellar injury or dysfunction. However, it is unknown whether the existing 3q29Del mouse model recapitulates the cerebellar dysfunction observed in humans. To characterize cerebellar phenotypes and uncover pathological differences in the 3q29Del mouse model, we investigated cerebellar structure, motor and vocal behaviors, protein expression, and mitochondrial function. We found uniformly reduced cerebellar volume in 3q29Del mice. Behavioral assays revealed vocal impairments in 3q29Del pups, fine motor impairments in adult mice, and reduced social mating calls in adult male mice. Proteomic analysis revealed enrichment of synaptic and mitochondrial proteins among the differentially expressed proteins in 3q29Del cerebellum tissue. Furthermore, mitochondria from 3q29Del mouse cerebellum displayed reduced oxygen flux and increased electron leak. These results recapitulate many human 3q29Del phenotypes in the 3q29Del mouse model and indicate mitochondrial dysfunction as a potential driver of 3q29Del pathology. Our findings also point to cerebellar involvement in 3q29Del phenotypes and provide a foundation for further research on cerebellar development in 3q29Del.

The FoxP family of transcription factors has been linked to neurodevelopmental disorders. FoxP1 and FoxP2 were identified as genes associated with autism spectrum disorders (ASD). FoxP1, FoxP2, and FoxP4 are expressed in many brain regions, including the cerebellum that has been of particular interest in the context of ASD. However, their role in the development of the peripheral nervous system (PNS) has been neglected. In view of the fact that a majority of ASD patients show some signs of sensory perception deficits that could go beyond the central nervous system, we focused on neural circuit formation in the PNS. Our in vivo and in vitro studies revealed a role of all three FoxP transcription factors in neural circuit formation in the hindlimb of the chicken embryo. Silencing of FoxP genes altered motor axon guidance and also impacted neural crest cell migration. This in turn resulted in aberrant formation of dorsal root ganglia and deficits in neural circuits in the PNS. Taken together, our results are in line with the hypothesis that some of the sensory perception deficits in ASD patients, either hyper- or hyposensitivity, are due to aberrant neural circuits in the PNS.

Intracellular neurofibrillary tangles formed from hyperphosphorylated tau and extracellular amyloid plaques containing aggregated A{beta}-peptides, specific cleavage products of the Amyloid Precursor Protein (APP), are the primary histopathological hallmarks of Alzheimers Disease (AD), the leading cause of dementia in humans. However, the initiating steps that lead to these pathologies and early neurodegeneration, and the mechanisms by which tau- and A{beta}-induced effects might be linked remain unclear. Using the prostate-like secondary cell (SC) in Drosophila, we recently showed that A{beta} modulates normal APP- and membrane-associated protein aggregation in the dense-core granule (DCG) compartments of the regulated secretory pathway by interfering with subsequent membrane:DCG dissociation. This disrupts endolysosomal trafficking and propagates the resulting endolysosomal defects to other cells that endocytose the secreted abnormal DCG proteins. Here we show that overexpressing human tau also disrupts DCG aggregation and membrane:DCG dissociation inside SC secretory compartments, leading to increased endolysosomal targeting of these compartments. In a genetic screen, we find that knockdown of cofilin, which encodes an actin-severing protein required for dynamic remodelling of microfilaments, generates a similar phenotype. Consistent with this, overexpression of Cofilin, which is known to suppress tau-induced neurodegeneration in flies, reduces tau-induced DCG defects in SCs. Indeed, we find that Cofilin overexpression also suppresses A{beta}-induced degeneration in the fly eye. We conclude that membrane:DCG aggregate dissociation in DCG compartments is disrupted by both tau- and A{beta}-induced genetic changes that are relevant to AD, and this partially involves inhibition of actin cytoskeleton dynamics. Increasing actin remodelling activity can suppress neurodegeneration induced by both tau and A{beta}, suggesting that this process provides an important functional link between them that might be targeted therapeutically.

Subtle variations in the activity and signaling of PERK, an endoplasmic reticulum kinase, can shift stress response outcomes. PERK has three commonly occurring exonic single-nucleotide variants frequently inherited together as part of the PERK haplotype B (PERK B), which occurs in 28% of the global population and is associated with several chronic neurodegenerative diseases, although its impact on PERK activity remains controversial. We hypothesized that PERK-B was associated with increased activity, altering stress response and stress tolerance, and that minorly but consistently elevated PERK activity, in combination with additional environmental or genetic risk factors, would lead to adverse outcomes. Comparison of stress response between PERK A, the major PERK haplotype, and PERK B in neuronal cultures using novel knock-in PERK-B mice alongside wild-type PERK A mice revealed differential, transient changes. Following ER stress, astrocyte-containing neuronal cultures, but not neuron-only cultures expressing PERK B exhibited similar levels of eIF2 phosphorylation, the canonical PERK substrate at PERK peak activation point (2h), but showed higher levels at 4 to 6 h, consistent with altered kinetics of PERK signaling. PERK-B neuronal cultures pretreated with a low dose PERK-specific activator exhibited increased vulnerability to N-methyl-D-aspartate-induced death compared to PERK-A neuronal cultures, indicating impaired acquired stress tolerance. Additionally, neuron-astrocyte coculture experiments revealed that the observed difference in acquired stress tolerance was dependent on the astrocyte genotype. The association of PERK-B with transient changes in stress response and reduced stress tolerance with higher vulnerability to cellular stress, albeit transient and small/moderate in size, has clinical relevance based on its potential contribution to the overall genetic vulnerability in neurodegenerative diseases.

Chronic social isolation induces persistent behavioral and physiological changes across species. In humans, prolonged social isolation is associated with insomnia and increased risks of cognitive decline, cardiovascular disease, compromised immunity, and premature mortality. Yet whether these consequences are modified by the environment in which social isolation is experienced remains unclear. In particular, can access to social sensory information from conspecifics alleviate the effects of isolation and can various ambient environmental settings modify them? A related question arises during behavioral measurement because behavioral assays themselves impose a new environmental context. Whether these assays simply capture the enduring effects of prior social experience or further modify those effects remains unknown. Using social isolation-induced sleep loss in Drosophila as a model, we systematically examined how the social, spatial, and photoperiodic environments during isolation and behavioral monitoring influence the isolation phenotype. Providing isolated flies with visual, olfactory, or tactile social cues from conspecifics living behind a divider, either individually or in combination, was insufficient to alleviate sleep loss. Spatial confinement exacerbated isolation-induced sleep loss, while winter-like photoperiod in spatially confined Drosophila Activity Monitor assays revealed the clearest progressive decline in sleep during sleep monitoring. Together, these findings demonstrate that social isolation-induced sleep loss is both enduring and dynamic: it persists despite exposure to social cues yet remains sensitive to the environmental context in which isolation is experienced and behavior is measured. More broadly, our study establishes a tractable framework for investigating the environmental modulation of chronic social isolation.

Asthma patients suffer from neuropsychiatric disorders (NPDs) more often than expected and NPD patients have a higher prevalence of smoking, which can lead to asthma. To investigate the genetic mechanisms underlying this complex relationship, we performed an integrative analysis of GWAS summary statistics from four asthma subtypes, 12 NPDs, and six substance use behaviors (SUBs). Asthma shares an important genetic component with post-traumatic stress disorder (PTSD), major depressive disorder, and attention-deficit/hyperactivity disorder (ADHD) that cannot be fully accounted for by smoking. Multi-trait GWAS identified 354 independent variants mapping to 178 pleiotropic genes capturing the core of the shared genetics of asthma, NPD, and SUB. Single-cell transcriptomic analysis across blood, brain, and lung tissues revealed pleiotropic gene overexpression in 57 cell types and three candidate mechanisms linking asthma and NPDs. First, immune-related pleiotropic genes are expressed in microglia, blood and lung immune cells, suggesting shared immune dysfunction. Second, the pleiotropic genes EP300, RERE, and MAML3 regulate NOTCH signaling and are expressed in neurons, microglia, and lymphoid progenitors. Third, pleiotropic genes expressed in ciliated cells of both lung and brain implicate ciliary dysfunction. These findings may help identify new treatment strategies for patients with co-occurring asthma and neuropsychiatric disorders.

Fetal alcohol spectrum disorders (FASD) are caused by prenatal alcohol exposure (PAE), which leads to developmental defects during embryogenesis. Angiogenesis is one facet of development that is heavily dysregulated during PAE and where such impairment is poorly understood. Here we found that expression levels of focal adhesion molecules Tensin 1 (Tns1) and Tensin 2 (Tns2) were significantly reduced by alcohol exposure in brain microvascular endothelial cells (BMVECs). We also found that miR-150-5p, which we previously found was increased by PAE, regulates the expression of Tns1 and Tns2. Dual-luciferase assays demonstrated direct binding of miR-150-5p to the 3UTRs of Tns1 and Tns2. Additionally, we utilized sprouting angiogenesis bead assays to evaluate endothelial cell sprouting in response to ethanol (EtOH) treatment and following Tns1 and/or Tns2 overexpression as well as miR-150-5p modulation. Tns1 and Tns2 rescued both EtOH- and miR-150-5p-mediated deficits in sprouting angiogenesis. Overall, we found that miR-150-5p negatively regulates Tns1 and Tns2 in BMVECs and that augmenting Tns1 and Tns2 expression may reverse the negative effects of EtOH, and subsequent miR-150-5p elevation, thus improving endothelial cell sprouting during cerebrovascular development.

CUB and Sushi domains-containing CSMD proteins are involved in neuron development. Variants in the CSMD1-3 paralogs were GWAS-associated with cognition, attention-deficit hyperactivity disorder (ADHD), schizophrenia and brain structure, suggesting that these genes could be linked to neurodevelopmental disorders (NDD). Indeed, rare biallelic variants in all three CSMD paralogs were recently associated with epilepsy, while rare biallelic variants in CSMD1 were also linked with intellectual disability and cortical malformations. We identified nine individuals from eight families carrying CSMD2 biallelic missense variants in developmental delay/intellectual disability cohorts. Affected individuals presented with autism spectrum disorder (ASD), ADHD and brain anomalies. Consistent with gnomAD metrics that suggest CSMD2 haploinsufficiency, we also report two families segregating monoallelic CSMD2 frameshift variants, which complement a described de novo truncation variant in an ASD proband. We concomitantly report two phenotypically overlapping individuals with de novo CSMD2 missense variants and compare them to twelve de novo CSMD2 cases identified in published trio analyses. UK Biobank analyses showed nominally significant enrichment of ADHD, ASD and neuroticism in volunteers carrying at least one rare CSMD2 variant. In silico modelling and immunofluorescence assays showed that mutated residues in affected individuals with biallelic and de novo missense variants are clustering in two regions and that the encoded proteins showed impaired membrane localization and an increased cytoplasmic aggregation. Zebrafish crispant larvae for the orthologous csmd2 presented with microcephaly, smaller cerebellum, and reduced Purkinje cell size. Larvae also displayed decreased swimming velocity, diminished connectivity between the optic tecta, and abnormal peripheral neuronal branching. We demonstrate that rare, likely pathogenic variants in CSMD2 are associated with both autosomal recessive and dominant forms of NDD further substantiating the functional significance of CSMD paralogs in neurogenesis/synapse formation.

Traumatic brain injury (TBI) is a risk factor for Alzheimer's disease (AD), and both conditions are associated with impaired glucose metabolism and mitochondrial dysfunction. We previously reported time-dependent mitochondrial dysfunction following closed-head injury (CHI) in amyloid precursor protein/presenilin 1 (APP/PS1) knock-in (KI) mice, but whether glucose-derived carbon labeling showed a corresponding temporal pattern remained unknown. We hypothesized that early deficits would involve tricarboxylic acid (TCA)-cycle metabolites, whereas chronic changes persisted upstream at the pyruvate-lactate branchpoint. Here, oral uniformly 13C-labeled glucose ([U-13C]-glucose) tracing and gas chromatography-mass spectrometry (GC-MS)-based isotopologue analysis were used to quantify first-turn glucose-derived carbon fate in cortex and hippocampus. To distinguish the effects of age at injury from those of post-injury interval, mice were analyzed 1 month after injury either at 7 or 12 months of age or 8 months after injury at 12 months of age. The most pronounced alterations occurred 8 months after CHI, when KI mice exhibited increased pyruvate and lactate labeling in the cortex and broader increases in glycolytic labeling in the hippocampus. These findings suggest that in APP/PS1 KI mice, chronic closed-head injury produced a delayed shift in glucose-derived carbon labeling centered on glycolytic and pyruvate/lactate nodes, with loss of hippocampal precursor-product coupling but without broad reduction of first-turn TCA-linked labeling.

Tau is a microtubule-associated protein important for neuronal structure and function, with abnormal phosphorylation associated with neuronal dysfunction and degeneration. While tau pathology has been extensively studied in neurodegenerative diseases and traumatic brain injury, its role in spinal cord injury (SCI) remains unclear. This study investigates changes in the levels of tau isoforms and phosphorylation following a high lateral C2 hemisection (C2Hx) in adult female rats. Western blot analysis was performed on spinal cord tissue collected caudal to the lesion at 4 weeks post-injury. Low-molecular-weight (LMW) tau and high-molecular-weight, Big tau did not differ significantly between injured and control groups. In contrast, phosphorylated tau (p-tau), detected using the AT8 antibody, was significantly elevated after injury, suggesting a shift in tau functional state. Assessment of microtubule stability markers revealed increased acetylated tubulin and decreased tyrosinated tubulin after SCI, indicating a shift toward a more stable cytoskeletal state. Immunohistochemistry was performed on spinal cord sections at 6 weeks post-injury and dorsal root ganglia (DRG) at 8 weeks post-injury. These analyses demonstrated increased p-tau immunoreactivity within the ipsilateral dorsal horn of the injured spinal cord and a higher proportion of p-tau-positive neurons in DRGs from injured animals compared with controls. Together, these findings demonstrate that cervical SCI induces persistent tau phosphorylation without altering total tau levels, and that this modification extends beyond the lesion site into distal spinal regions and beyond the acute phase of injury. These alterations may influence neural plasticity and remodeling in the injured spinal cord.

Abstract Human induced pluripotent stem cells (hiPSCs) provide a translational platform to interrogate the impact of genetic risk factors for various disorders on human biology. It has been a common practice to engineer premature stop codons to model predicted loss-of-function mutations using isogenic hiPSCs, yet effects of mutation position for the same gene and genetic background have been rarely tested systematically and at multiple levels for comparison to those of patient-specific mutations. Ultra-rare, heterozygous, and predicted loss-of-function mutations in the SETD1A gene, which encodes a H3K4 methyltransferase, are highly penetrant genetic risk factors for schizophrenia. Here, we generated a large panel of isogenic heterozygous SETD1A mutant hiPSC lines, including premature stop-codon mutations in one early exon and one late exon in five genetic backgrounds and two patient-specific mutations, to gain insight into molecular, cellular, and functional changes in a disease-relevant and highly pure population of human hippocampal dentate granule cell-like neurons. Our multi-level analyses showed that different SETD1A mutations lead to divergent molecular and cellular phenotypes related to synapses, but convergent neuronal network deficits, while the genetic background influences gene expression with no substantial effects at cellular and network levels. Mechanistic analysis confirmed the early exon patient mutation as loss-of-function, but revealed that the predicted loss-of-function patient mutation in the late exon is likely gain-of-function. Consequently, while all mutations decreased global H3K4me3 levels, deficits were only partially rescued by inhibiting H3K4 demethylase LSD1 for early exon mutations, but not for late exon mutations. Together, these results suggest that highly penetrant mutations can lead to similar cellular and functional phenotypes across different genetic backgrounds, whereas different premature stop-codon mutations could have contrasting impacts due to distinct mechanisms. Our findings have practical implications for applying genome-editing in hiPSCs to model disorders, highlight the value of modeling patient-specific mutations and testing multiple mutations, and suggest the need for tailored mutation-dependent therapeutic strategies.

Metabotropic glutamate receptors (mGluR) are G-protein coupled receptors that regulate neuroplasticity and have been shown to play a role in auditory processing. However, not much is known about the expression of mGluRs during cochlear nucleus development in the context of Fragile X syndrome (FXS). FXS is the most common heritable form of intellectual disability and autism and is associated with auditory processing deficits including hypersensitivity. Audiogenic seizure is a notable feature of the Fmr1 knock-out (KO) mouse model for FXS, in which mGluR5 has been shown to be necessary. In this study, we investigated the expression of eight metabotropic glutamate receptors (mGluRs) in the cochlear nucleus at both young and adult ages and in wildtype and Fmr1 KO mice. While it is established that mGluR1 is the predominantly expressed mGluR in adult cochlear nucleus, we report the following new finding that mGluR5 is the more abundant receptor during early development. The expression of Group II and III mGluRs (2,3,4,6,7,8) was relatively stable during development. We show that mGluR1 and mGluR5 are co-expressed across multiple neuron types. Fmr1 genotype did not have statistically significant impact on the expression pattern of mGluRs in cochlear nucleus. Although hearing onset occurs during this mGluR5 to 1 transition period, mGluR expression patterns were not impacted by cochlear activity. We conclude that there is an mGluR5 is to mGluR1 transition that occurs within cochlear nucleus neurons during the critical period closure that is intrinsic to neuronal development.

Background: Nervous system regeneration requires creation of new neurons and their integration into new and existing networks. How new neurons find their targets and reconnect in the environment of regenerating tissue remains a major gap in knowledge. The planarian Schmidtea mediterranea is well suited to the study of neural regeneration due to the remarkable ability of planarians to regenerate their entire body, including the nervous system. However, we have not yet been able to explore synaptogenesis in regeneration using planarians due to a lack of tools to study synapses. Here we investigate potential players in planarian synapse, focusing on the conserved syntaxin protein family, which functions in neurotransmitter vesicle fusion at the synapse. Results: We found that reduction of either syntaxin-1 (stx1) or syntaxin-binding protein 1-1 (stxbp1-1) decreased locomotion and impeded brain regeneration, specifically in synapse-rich regions. Conclusions: Our observations support STX-1 and STXBP1-1 as bona fide synapse components, providing a starting point for studies neuronal connectivity during homeostatic remodeling and regeneration.