Frontiers in Molecular Neuroscience

Pigment epithelial-derived factor (PEDF) is a multifunctional protein produced predominantly by the retinal pigment epithelium and expressed in many tissues, including the brain, highlighting its participation in crucial processes, such as neuroprotection and angiogenesis. Some neurodevelopmental disorders, such as ASD, are characterized by neurodevelopmental abnormalities, including altered neurite formation, spine formation, and neuronal activities. Many efforts have been made to resolve NDDs, but until now, some symptoms remain untargeted. PEDF is involved in many steps of neurodevelopment. The treatment of PEDF peptide might improve the outcome of NDD symptoms by altering neuronal morphologies. We used PEDF peptides that contain different functional domains to study the effect of administering PEDF peptides on neuronal morphology in a prenatal valproic acid (VPA)-exposed mouse model. We identified that the treatment with PEDF peptides rectified the abnormalities in neurite formation and spine formation in VPA-exposed cortical neurons. In vitro calcium imaging showed abnormalities in the spontaneous activity in VPA-exposed cortical neurons. Treatment of a short PEDF peptide normalized intracellular calcium response to the control level. Accordingly, PEDF peptides have the prospect of serving as potential treatments for patients with neurodevelopmental disorders, such as ASD.

Spinal cord injury causes irreversible neurological deficits in mammals; yet zebrafish achieve complete functional recovery through molecular mechanisms that remain poorly defined. In this study we emphasized on a critical miRNA-mediated regulation of Ependymo-radial glial (ERG) cell proliferation in zebrafish spinal cord. Using next-generation sequencing we constructed a spatiotemporal miRNA profile across multiple post-injury time points and identified dre-miR-N1 as a novel injury-responsive miRNA involved in ERG proliferation among several differentially expressed novel miRNAs. Fluorescent in situ hybridization confirmed its robust lesion-site expression and gain-of-function analysis demonstrated that dre-miR-N1 significantly impaired functional recovery. Target prediction and validation unexpectedly identified the odorant receptor gene or42a1 as a high-confidence target and a combinatorial approach of miRNA gain-of-function and or42a1 loss-of-function showed that dre-miR-N1 modulates the proliferative behaviour of or42a1-expressing ERG cells under both homeostatic and injury conditions. These findings uncover a previously unrecognized miRNA-odorant receptor axis governing injury-induced ERG cell expansion establishing a novel molecular framework for endogenous neural regeneration in zebrafish.

Lysosomal trafficking and homeostasis are biological functions that are pivotal for DRG neurons, given their metabolic demands and extremely long axons. Previous studies indicate that lysosomal signaling is altered in a mouse model of chemotherapy-induced peripheral neuropathy (CIPN) and that blocking mitogen activated protein kinase-associated kinase (MNK1/2) signaling can alleviate pain behaviors in CIPN. Here, we investigated lysosome dynamics and lysosome-associated signaling in a mouse model of CIPN induced by paclitaxel (PTX), a chemotherapeutic agent used for various types of cancer. Using spinning disk super-resolution microscope (SPINSR), we demonstrate that PTX treatment in vivo causes reduced lysosome motility observed in vitro. PTX likewise drives the accumulation of Sequestosome 1 (SQSTM1), also known as P62, in cultured mouse DRG neurons, indicating lysosomal dysfunction in DRG neurons. The transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, was also upregulated in the nucleus of cultured mouse DRG neurons treated with PTX. In line with this, increased lysosomal-associated membrane protein 1 (LAMP1) expression was observed in PTX-treated mice. Given that our previous work demonstrated PTX treatment increases MNK1/2-eIF4E signaling in DRG neurons, we examined whether MNK1/2 inhibition could rescue lysosomal dysfunction. Treatment with Tomivosertib (eFT508), a potent MNK1/2 inhibitor, restored P62 levels in DRG neurons of PTX-treated mice and reduced TFEB in DRG treated in vitro. To establish translation relevance, we further show that PTX elevates phosphorylated eiF4E (p-eIF4E) in human DRG neurons, and concurrent eFT508 administration attenuates this effect. Collectively, these findings indicated that PTX disrupts lysosome trafficking and biogenesis, and that MNK inhibition with eFT508 restores lysosomal signaling and can serve as a neuroprotective strategy for CIPN.

CaMKII promoter is widely used to label and manipulate hippocampal pyramidal neurons via transgenic mouse lines or viral approaches. While it targets most excitatory neurons, a small subset remains unlabeled and often overlooked. We present an AAV-based strategy combined with CaMKII-driven Cre expression to access and study this remaining population. Furthermore, we provide a detailed protocol for in-house AAV production, targeted stereotaxic delivery, and functional validation of targeted neurons through slice electrophysiology and behavior. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=194 HEIGHT=200 SRC="FIGDIR/small/723440v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@3a31ccorg.highwire.dtl.DTLVardef@9b7e90org.highwire.dtl.DTLVardef@92297borg.highwire.dtl.DTLVardef@1e159eb_HPS_FORMAT_FIGEXP M_FIG C_FIG

Gene therapy may provide a way to restore inner ear function to deaf and dizzy patients. The mouse is a crucial model system for functional genomics because of the numerous genetic models for hearing loss and inner ear dysfunction. Using an advance generation, E1-/E3-/E2b-(preterminal protein-/polymerase-) Type 5 Adenovirus, we investigated several routes of virus microinjection to determine which were most reproducible in targeting the endolymphatic fluid compartment of the cochlea. We found that when adenovirus is injected via the round window, transduced cells are found only adjacent to the scala tympani and not in the organ of Corti, suggesting that Adenovirus is unable to penetrate the basilar membrane or bony wall of the modiolus. Delivery to the cochlea via the semicircular canals is also inefficient. In contrast, our new method, via a stylomastoid foramen cochleostomy, increases the likelihood of adenovirus gene transfer to the scala media including cells in the organ of Corti and stria vascularis while preserving some hearing. The ability to target delivery of virus and other therapeutic reagents to specific inner ear fluid compartments will facilitate in vivo testing of candidate molecules implicated in multiple aspects of inner ear physiology and regeneration.

S-acylation is a reversible posttranslational lipid modification important in the nervous system that dynamically regulates protein localization and function. Aberrant S-acylation has been implicated in several neurological conditions. While several de-S-acylases (deacylases hereafter) have been identified, little is known regarding their expression and localization in the brain and in neurons. Here, we characterized the expression, localization, and S-acylation of cytosolic deacylases, including acyl-protein thioesterases APT, APT2, and APT1L and /{beta} hydrolase domain-containing proteins ABHD7, ABHD10, ABHD13, ABHD16A, and ABHD17A-C. Mouse brain RNA sequencing data revealed high expression of Lypla1/APT1, Lypla2/APT2, Ephx4/ABHD7, Abhd16a, and Abhd17A-C in the brain, whereas Lyplal1/APT1L, Abhd10, and Abhd13 were expressed at very low levels. Protein analysis demonstrated region-specific expression, with expression of APT1 and ABHD16A highest in the cerebellum and APT2 highest in the hippocampus, with all three highly expressed in cultured hippocampal neurons. Deacylases were observed distributed throughout neurons on punctate structures, with APT2 and ABHD17C to the Golgi by immunocytochemistry. Finally, all ten cytosolic deacylases are themselves S-acylated. These data characterizing deacylase expression, localization, and S-acylation in neural contexts, provides a foundation for future studies investigating deacylase neuronal functions and potential roles in neurological disease.

Local protein synthesis in neurons occurs in both axons and dendrites and plays a central role in synaptic function. High-throughput-based sequencing and imaging studies have demonstrated the presence and translation of synaptically localised mRNAs. However, quantification of activity-dependent translation dynamics at synapses at the transcriptome-wide scale remains limited. Here, we apply ribosome profiling to synapse-enriched fractions (synaptoneurosomes) derived from rat cortical tissue following stimulation with the group 1 mGluR agonist DHPG. DHPG stimulation induced translation of mRNAs involved in synaptic processes, including synaptic vesicle exocytosis and axo-dendritic transport. Notably, translation of ribosomal protein mRNAs was upregulated upon mGluR activation, consistent with the expected increase in de novo protein synthesis. Together, these results demonstrate the use of ribosome profiling to capture changes in local mRNA translation from isolated preparations.

Mutations in chromosome alignment maintaining phosphoprotein 1 (CHAMP1) have been linked to neurodevelopmental disorders characterized by intellectual disability, developmental delay, and autism spectrum disorder. However, the cellular and electrophysiological mechanisms by which CHAMP1 mutations disrupt human neuronal development remain poorly understood. In the present study, we used patient-derived induced pluripotent stem cells (iPSCs) carrying two pathogenic CHAMP1 mutations and generated neural progenitor cells (NPCs) and excitatory neurons to investigate the effects of each mutation on neuronal maturation and function, DNA repair, and gene expression. Proliferative capacity and DNA repair dysfunction operate in a CHAMP1 dose-dependent manner. Whole-cell patch-clamp electrophysiology revealed that CHAMP1 mutant neurons exhibit significant alterations in intrinsic membrane properties during early developmental stages, including depolarized resting membrane potential, reduced action potential firing, and impaired waveform kinetics. These functional deficits were accompanied by reduced sodium and potassium current densities, suggesting impaired ion channel accumulation during neuronal maturation. Furthermore, recordings of spontaneous excitatory postsynaptic currents indicated altered synaptic activity and reduced proportions of synaptically active neurons. Morphological analyses revealed deficits in neurite outgrowth and branching, consistent with delayed neuronal maturation. Single-nucleus transcriptomic profiling further revealed delayed developmental trajectories and mutation-specific dysregulation of synaptic gene programs enriched for autism, ADHD, and epilepsy risk genes. Together, these findings demonstrate that CHAMP1 mutations disrupt multiple aspects of neuronal development, including homologous recombination (HR) dysfunction in NPCs, membrane excitability, ion channel function, and synaptic connectivity. Our results provide insights into the neurobiological consequences of CHAMP1 mutations and establish patient-derived neurons as a platform to investigate cellular pathophysiology and potential therapeutic strategies for CHAMP1-associated neurodevelopmental disorders.

Congenital myasthenic syndromes (CMS) are rare inherited diseases of the neuromuscular junction (NMJ). There are 40 identified CMS genes, but many patients go without genetic diagnosis, which suggests new genes have yet to be discovered and characterised. Here, we describe an optogenetic approach to study fatigable muscle weakness and NMJ function in larval zebrafish to facilitate screening approaches for uncovering novel CMS genes. Using blue-light illumination of spinal motoneurons that express channelrhodopsin-2 (ChR2) to induce muscle contraction, we measure motor defects at the behavioural, synaptic, and genetic level through a novel behavioural assay, standard whole-cell electrophysiology of individual muscle fibers and a customized NMJ gene panel. We employ this approach in synaptotagmin-2 (syt2) morphant zebrafish, an identified CMS gene model, to validate its usefulness. Our customized optogenetic behavioural assay successfully demonstrates reduced, fatigable, locomotor response during repeated activation of spinal motoneurons. Whole-cell electrophysiology recordings of optogenetically-elicited endplate currents in muscle fibers reveal similarities to altered properties of NMJ function in syt2 morphants reported in other studies using the standard paired motoneuron-muscle electrophysiology technique. Finally, we develop a genetic panel of CMS and NMJ-related genes to characterize the expression landscape of syt2 morphants to elucidate potential pathomechanisms and novel therapeutic targets. We propose that this three-tiered approach successfully links behaviour, synaptic motor function, and genetic expression and can be used as a tool in the screening of novel genes associated with CMS.

Human sensory neuron models are an important resource for studying pain mechanisms and axon injury and repair. Current systems are limited by accessibility, scalability, or incomplete functional maturation. The HD10.6 human dorsal root ganglion-derived immortalised cell line represents a promising alternative; however, its maturation trajectory and suitability for disease modelling remain incompletely defined. Here, we performed a longitudinal, multi-modal characterisation of HD10.6 cells during differentiation over 28 days. Bulk RNA sequencing revealed progressive transcriptional remodelling, with temporal up-regulation of neuronal and nociceptor-associated gene programmes, including ion channels implicated in pain signalling. Protein-level analyses confirmed increased expression of key nociceptor markers and neuropeptides, including TRPV1, Nav1.7, Nav1.8 and CGRP. Functional assays demonstrated the emergence of sensory neuron-like properties over time. Calcium imaging revealed increasing responsiveness to capsaicin, allyl isothiocyanate, ,{beta}-MeATP, and prostaglandin E2, while patch-clamp electrophysiology at DIV 21 after maturation showed repetitive firing of action potential and, most importantly, exhibited TTX-Resistant sodium currents. These findings establish a temporal relationship between transcriptional changes and functional competence. Finally, we evaluated the utility of HD10.6 neurons for modelling axon degeneration. Treatment with vacor-induced robust neurite degeneration, which was attenuated by pharmacological inhibition of SARM1, demonstrating engagement of conserved axon degeneration pathways. Together, our findings define the progressive maturation of HD10.6 sensory neurons and establish this system as a scalable human platform for studying nociceptor biology and SARM1-dependent axon degeneration.

Estrogen-related receptor gamma (ESRRG), an orphan nuclear receptor with structural homology to the classical estrogen receptors, is widely recognised as a key metabolic regulator involved in mitochondrial, synaptic, and ion-homeostatic pathways. Previous clinical studies suggest a link between ESRRG and auditory function; for example, ESRRG has been associated with susceptibility to age-related hearing loss in women and implicated in congenital hearing loss. However, the biological mechanisms by which ESRRG may mediate hearing function remain largely unknown. Here, using a combination of in vivo auditory physiological recordings, immunofluorescence analyses, single hair-cell electrophysiology, and transcriptomic approaches, we characterise the phenotype of a new inner-ear conditional Esrrg knockout (Esrrg-cKO) to investigate the role of Esrrg in the auditory system. We found that Esrrg-cKO mice of both sexes develop early-onset hearing loss, as evidenced by elevated auditory brainstem response thresholds and reduced wave 1 amplitudes from two weeks of age. These auditory deficits arise from a combination of early-onset cochlear neuronal and innervation malformations, together with inner hair cell synaptic defects and delayed myelination that persist into adulthood. Furthermore, distortion product otoacoustic emissions and endocochlear potential recordings are normal in Esrrg-cKO mice, and although sensory hair cells are preserved, IHCs retain immature biophysical properties. These findings are consistent with auditory neuropathy, and together with our comparative transcriptome analyses, indicate that Esrrg is an essential molecular driver of normal cochlear innervation and maturation.

Trauma to the spinal cord disrupts the blood-spinal cord barrier and triggers a secondary injury cascade characterized by inflammation and progressive neuronal and glial cell death. Therapeutic cytokines and growth factors have shown promise as a treatment in preclinical studies, though their clinical translation is limited by short protein half-lives and the need for invasive intraspinal administration. Lipid nanoparticle-mediated delivery of mRNA offers an alternative strategy that enables transient protein production. Here, we investigated whether intravenously administered mRNA-lipid nanoparticles could leverage the injury-induced disruption of the blood-spinal cord barrier to access the injured spinal cord for local transgene expression. After spinal cord injury in a rat, lipid nanoparticles loaded with reporter mRNA were administered intravenously, and transgene expression was quantified in the spinal cord and peripheral organs. Intravenous delivery within a 6-hours post-injury resulted in local transgene expression in the injured spinal cord, demonstrating that mRNA-lipid nanoparticles cross the disrupted blood-spinal cord barrier. Transgene expression was observed in astrocytes, oligodendrocytes, microglia, and neurons, detected within 3 hours and remained elevated for up to 5 days post-injury. These findings demonstrate that systemic mRNA-lipid nanoparticles delivery exploit transient blood-spinal cord barrier disruption to achieve local gene expression in the injured spinal cord.

The human olfactory epithelium (OE) represents a lifelong source of neural progenitor cells and has been proposed as an accessible model to investigate molecular alterations associated with neurodevelopmental disorders in postnatal individuals. Globose basal cells are considered the immediate neuronal progenitors within the OE, and several studies have attempted to culture these cells from nasal exfoliates. However, the actual contribution of neurogenic lineages in these cultures remains largely unquantified. Here, we cultured human nasal explants using an established protocol and characterized the resulting cell populations by immunohistochemistry and single-cell RNA sequencing. Integration with primary in vivo OE datasets revealed that these cultures are predominantly composed of mesenchymal-like cells, with limited representation of globose basal cells and neurons, and low expression of canonical neuronal markers. Using curated gene sets associated with neurodevelopmental disorders and malformations of cortical development, we assessed the extent to which disease-relevant transcriptional programs are captured in OE-derived cultures. While disease-associated genes are enriched in neurogenic lineages in vivo, their representation in mesenchymal cells is reduced. Together, our results challenge the assumption that standard OE culture systems faithfully model neurogenic compartments and suggest that current approaches may need refinement to recover neurogenic lineages.

Benzo[a]pyrene (BaP), a representative polycyclic aromatic hydrocarbon (PAH), is a widespread environmental toxicant and potent ligand of the aryl hydrocarbon receptor (AHR). Yet, how early developmental exposure to BaP influences human neurodevelopment remains poorly understood. We first examined AHR expression dynamics during human embryonic stem cell (ESC)-derived cerebral organoid development and found that AHR expression was highest at the ESC stage and declined during subsequent differentiation, suggesting a potential window of heightened susceptibility to AHR-mediated environmental perturbations. Based on this observation, ESCs were exposed to BaP (0.1, 1 M) for 7 days prior to organoid generation. BaP exposure did not alter proliferation, cell death, or global transcription of ESCs but increased expression of a subset of AHR target genes. Remarkably, however, organoids derived from BaP-exposed ESCs exhibited profound morphological defects resulting from premature neurogenesis, characterized by disrupted neural rosette organization, reduced EOMES intermediate progenitors, and increased BCL11B neurons. Pharmacological inhibition of AHR with CH-223191 attenuated AHR activation and rescued the progenitor-neuron imbalance. These findings identify AHR signaling as a critical upstream mediator of BaP-induced developmental neurotoxicity and highlight the vulnerability of early pluripotent stages to environmental insults.

Neural stem cell (NSC) transplantation is a promising strategy for repairing the injured spinal cord, but transplanted cells typically require immunosuppressive therapy to prevent rejection, even for induced pluripotent stem cell (iPSC)-derived autologous grafts. However, the effects of immunosuppressive drugs on neurite outgrowth and axonal regeneration, processes critical for neural circuit reconstruction, have not been fully characterized. In this study, we tested nine clinically relevant immunosuppressants on human iPSC-derived neurons and primary human spinal cord NSCs in vitro at concentrations approximating clinical exposure levels. The drug panel included FK-506 (tacrolimus), cyclosporine A (CsA), rapamycin, belatacept (Nulojix), etanercept (Enbrel), mycophenolate mofetil (CellCept), cyclophosphamide (Cytoxan), prednisone, and azathioprine (Imuran). Neurite outgrowth was quantified via automated high-content imaging. Multiple agents, including CsA, Imuran, Nulojix, and CellCept, induced significant reductions in neurite outgrowth in a cell type- and dose-dependent manner, with CsA producing the most robust and consistent inhibition across both cell lines. In contrast, FK-506 showed no significant effect on neurite extension at clinically relevant concentrations. Consistent with the in vitro results, human neural progenitor cell grafts in a rodent spinal cord injury model exhibited significantly reduced graft-derived axon extension in the host spinal cord when hosts were treated with CsA rather than FK-506. These findings demonstrate that immunosuppressant choice can profoundly influence neural graft integration and axonal regeneration. Our study underscores the importance of preclinical evaluation of immunosuppressive regimens and suggests that selecting agents such as FK-506 over CsA may improve outcomes in future stem cell-based therapeutic trials for spinal cord injury and related disorders of the central nervous system.

Schizophrenia (SCZ) is a severe and debilitating neurodevelopmental disorder with lifelong impact on everyday life. Disruptions in synapse functions play a key role in its complex and poorly understood etiological and pathological mechanisms. Here, we investigated both the molecular composition and the spontaneous and stimulated functional properties of synapses in neural organoids from SCZ individuals. Air-liquid interface forebrain organoids (ALI-FOs) were generated from induced pluripotent stem cells (iPSCs) derived from three individuals with SCZ and three healthy controls. At day 170 synaptosomes were enriched and analyzed by data-independent acquisition mass spectrometry to profile the proteome, alongside with TMT-labeled phosphoproteomics both before and after acute KCl-induced depolarization. In parallel, we characterized the PTMome of the surrounding cellular environment, comprising phosphorylation, peptides with free and reversibly modified cysteines, and sialylated N-linked glycopeptides. Functional glutamatergic and GABAergic activity was assessed using calcium imaging to capture spontaneous neuronal signaling. Both conditions exhibited mature synaptic structures, while growth cones were observed only in SCZ-derived ALI-FOs, indicative of ongoing or delayed synaptogenesis. Proteomic analysis of synaptosome preparations revealed 358 differentially regulated proteins between SCZ and controls and 125 phophoproteins with altered phosphorylation, which clustered into three major categories: (1) synaptogenesis and synapse signaling; (2) cytoskeleton and cell junctions; (3) growth cone dynamics and neurite outgrowth. Analysis of the PTMs in the surrounding cellular environment revealed regulation of key regulatory mechanisms in 526 proteins, supporting the synaptic alterations observed. Notably, components of the Wnt signaling pathway were consistently dysregulated across both the synaptosome preparation and the PTMome in SCZ-derived ALIFOs as compared to controls. Depolarization-induced phospho-signaling revealed SCZ-specific response enriched in synaptic vesicle trafficking pathways. Together, these findings provide new insights into early synaptic alterations in SCZ, highlighting changes not only in protein composition, but more in protein regulatory mechanisms underlying synaptic signaling.

Duchenne muscular dystrophy (DMD) is a X-linked disorder caused by mutations in the DMD gene, which disrupts production of multiple isoforms of dystrophin in multiple organs namely muscle, heart and brain. While progressive muscle disease and cardiomyopathy are the hallmarks of DMD, over 40% of individuals also experience significant neurobehavioral comorbidities, including autism spectrum disorder (ASD), attention deficit hyperactivity disorder (ADHD), obsessive compulsive disorder (OCD) and intellectual disability. These deficits are linked to the loss of brain isoforms and approximately 90% of DMD individuals have loss of either Dp427 or both Dp427 and Dp140 in the brain. We studied the mdx52 mouse model, which lacks these isoforms and exhibits severe fear and anxiety-like behaviours, suitable to evaluate the therapeutic efficacy on neuro-comorbidities after delivering a neuronal-targeted adeno-associated virus (AAV) micro-dystrophin ({micro}Dys) therapy. We compared two delivery routes intravenous (IV) and intracerebroventricular (ICV) in neonatal mdx52 male mice to assess impact on an extensive range of neurobehavioural aspects including emotional reactivity, neurocognitive, OCD and motor coordination deficits. While both routes successfully reduced emotional reactivity and anxiety-related behaviours, IV delivery emerged as the superior therapeutic strategy addressing a more comprehensive spectrum of DMD related brain co-morbidities. Critically, significant improvements in cognitive deficits and OCD-like behaviours were achieved only through IV delivery. This was associated with a widespread lower transduction pattern across the brain, including hindbrain and cerebellum, which were less effectively targeted by ICV injection, although forebrain transduction with ICV delivery was higher. Brain {micro}Dys expression successfully restored dystrophin interactors dystroglycan, syntrophin and pre- and post-synaptic functional interactors VGLUT1, gephyrin, GABAAR with both delivery methods. These results demonstrate that while ICV gene therapy results in improved emotional reactivity and anxiety-related behaviour in the mdx52, only the systemic, neuronal-targeted gene therapy efficiently transduced the central nervous system restoring neuronal synaptic functional complexes of both Dp427 and 140 isoforms and simultaneously restored peripheral NMJ dystrophin deficiency. Beyond cognitive restoration, while both routes improved aspects of gait on CatWalk XT, only IV delivery significantly enhanced motor coordination on the Beam walk and, unexpectedly, normalised grip strength. This was specifically linked to the selective expression of {micro}Dys at neuromuscular junctions (NMJs), which corrected post-synaptic electrophysiological dysfunction of mdx52 mice. Our findings establish a significant foundation for incorporating brain-directed strategies into the future therapeutic approaches for DMD, offering a holistic approach to treating DMD as a multisystemic disease.

In contrast to many other developing systems, in which axon pathfinding and synaptogenesis are separated in time, the pioneering axon of the individually identifiable caudal primary motor neuron in embryonic zebrafish forms en passant synapses during its stereotypical ventral growth. How simultaneous synaptic differentiation and axon pathfinding are coordinated is not fully understood. Here we ask what the role of the tac1 gene, coding for the synaptic tachykinin neuropeptides, is in this unique axon differentiation process. The gene is expressed during axon outgrowth and its disruption results in increased branch length of CaP axons and subtle morphological defects of the pre-synapse. These abnormalities are accompanied by a robust [~]1.5-fold increase in motor neuron activity and in spontaneous early contractions in tac1-deficient embryos. Furthermore, pharmacological inhibition of the tachykinin receptor (Tacr1) leads to altered CaP axonal morphology, mimicking the axonal phenotype observed in tac1-deficient zebrafish. These findings suggest that tachykinin neuropeptides modulate formation and activity of en passant synapses and prevent aberrant axon branching during growth of zebrafish motor axons. HIGHLIGHTS- tac1 refines CaP primary motor axon development in zebrafish - Loss of tac1 disrupts presynaptic maturation at the horizontal myoseptum - tac1 mutants show elevated motor neuron activity and spontaneous contractions

The cerebellar nuclei form the main output structures of the cerebellum and are composed of a deeply conserved set of cell types. Two excitatory cell classes, Class-A and -B, are present in each cerebellar nucleus and mediate all excitatory output of the cerebellum. To provide genetic access to these cell types, here we identified Acan as a marker gene for Class-B cells and generated a knock-in Acan-P2A-Cre mouse line. We demonstrate that this Acan-Cre line selectively labels Class-B neurons in the cerebellar nuclei and validate its use in viral projection tracing. This new mouse line provides a valuable genetic tool to study cerebellar nuclei organization and function.

Endosomal sorting complexes required for transport (ESCRT) regulate membrane protein trafficking through coordinated cargo selection and endosomal processing, yet their contribution to neurotransmitter receptor sorting remains to be defined. Here, we examined how modulation of distinct complex components influences the surface expression of excitatory and inhibitory neurotransmitter receptors. Using surface biotinylation and imaging approaches in heterologous cells and primary neurons, we altered tumour susceptibility gene 101 (TSG101), a core complex I component, and vacuolar protein sorting-associated protein 4A (VPS4a), an ATPase required for complex III disassembly. Reduction of tumour susceptibility gene 101 increased receptor association with early endosomes and enhanced receptor surface localisation, whereas disruption of VPS4A promoted receptor accumulation within late endosomal compartments and impaired degradative progression. Inhibitory receptor subtypes displayed variable sensitivity. Together, these findings demonstrate that endosomal sorting complex components regulate receptor surface expression through stage-specific trafficking mechanisms associated with altered receptor recycling and degradative processing. Graphical abstractDistinct ESCRT components regulate neurotransmitter receptor trafficking through stage-specific control of the endosomal pathway. ESCRT-I disruption promotes early endosomal retention and recycling, whereas ESCRT-III impairment causes late endosomal accumulation and reduced degradation, together increasing receptor surface expression (created using Biorender). O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/732891v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@1fe66b9org.highwire.dtl.DTLVardef@10a29d7org.highwire.dtl.DTLVardef@4109c4org.highwire.dtl.DTLVardef@1e84f19_HPS_FORMAT_FIGEXP M_FIG C_FIG