Mar 2026· bioRxiv· Vol 534· 0 citations· 106 references
MedicineBiology
TL;DR
A novel epitope-tagged knock-in mouse line, in which two hemagglutinin epitopes were inserted near the N-terminus of the endogenous C1QL3 protein, which enables purification, detection, and subcellular localization of native C1QL3 protein (C1QL3-2HA) with high specificity, eliminating the need for overexpression or custom antibodies.
Abstract
Synapse formation and function are coordinated spatially and temporally by a host of synaptic proteins that regulate neuronal signaling, synapse specificity, and plasticity; many of which are implicated in neuropsychiatric disorders. Members of the C1q/TNF superfamily function as synaptic organizers, shaping synapse assembly and maintenance. Among them, C1QL3 plays a putative role in trans-synaptic adhesion and modulation of synaptic strength, but the lack of a reliable antibody to detect it has severely limited the ability to map its endogenous localization and study its biochemical properties. Here, we present a novel epitope-tagged knock-in mouse line (C1ql32HA), in which two hemagglutinin (HA) epitopes were inserted near the N-terminus of the endogenous C1QL3 protein. This model enables purification, detection, and subcellular localization of native C1QL3 protein (C1QL3-2HA) with high specificity, eliminating the need for overexpression or custom antibodies. We validated that C1ql32HA mice maintain normal mRNA expression, biochemical properties, and behavior. Using native PAGE, we determined the endogenous oligomeric state of C1QL3-2HA. Brain-wide light-sheet microscopy uncovered an expanded neuroanatomical map of C1QL3-2HA expression, including newly identified populations in cortical and subcortical regions as well as the retina. Dual immunohistochemistry confirmed cell type-specific expression patterns, and super-resolution STED microscopy localized C1QL3-2HA to hippocampal mossy fiber synapses, positioned between pre- and post-synaptic markers, supporting its hypothesized role in trans-synaptic complexes. This knock-in mouse line is a powerful tool for studying the anatomical, molecular, and synaptic biology of C1QL3 in all cellular/tissue contexts, enabling future studies into its potential roles in the nervous system and beyond.
Gephyrin is identified as the primary synaptic anchor for PX-RICS and the N-terminal gephyrin-binding region (GBR) engages gephyrin E-domain through conserved hydrophobic interactions, explaining the isoform-specific targeting of PX-RICS (but not RICS) to inhibitory synapses.
Guanhua Bai, Ruifeng Huang, Yinmiao Lian et al.· Proceedings of the National...· 0 citations
Vesicular (synaptic) zinc is a neuromodulator that fine-tunes synaptic transmission and sensory processing across many brain areas, including the brainstem, hippocampus, amygdala, and neocortex. Throughout the central auditory system, synaptic zinc plays a crucial role in modulating neurotransmission as well as baseline and adaptive sound processing. However, the developmental changes in the protein expression levels and localization of the vesicular zinc transporter (ZnT3), which loads synaptic zinc into presynaptic vesicles, remain unknown—due in part to the lack of robust ZnT3 antibodies. To address this question, we used the recently developed and validated ZnT3-HA transgenic mouse line, in which the ZnT3 protein contains a C-terminal HA epitope tag. We performed immunohistochemical staining and confocal microscopy in the central auditory system across development to localize and quantify changes in ZnT3 expression and explore potential colocalization of ZnT3 with the vesicular glutamate and GABA transporters VGLUT1 and VGAT, respectively. We found that ZnT3 expression increased significantly between P7 and P14 in both the dorsal cochlear nucleus (DCN) and the auditory cortex (AC), reaching a stable overall expression level and layer distribution by P21. Both in the DCN and AC, ZnT3 was mainly colocalized with VGLUT1. We did not find any significant levels of ZnT3 expression in either the IC or the auditory thalamus. Together, these results highlight major developmental changes in zinc signaling that may affect synaptic transmission and plasticity, as well as normal and pathological sound processing.
Jesse Weisbord, Christopher L. Cunningham, T. Tzounopoulos et al.· Cells· 0 citations
Neurexins and neuroligins are evolutionarily conserved synaptic adhesion molecules that play essential roles in synapse formation and neural circuit function, with mutations linked to neurodevelopmental disorders such as autism. Here, we combined whole-transcriptome sequencing with phenotypic characterization to define the molecular consequences of neurexin and neuroligin deficiency in Caenorhabditis elegans. Young adult worms carrying allele-specific loss-of-function mutations in nrx-1 (ok1649 and tm1961) or nlg-1 (ok259 and tm474), orthologues for human NRXNs or NLGNs, respectively, were subjected to RNA sequencing and compared with wild-type animals. Mutant strains exhibited impaired growth, altered locomotor activity, increased social aggregation, and reduced ventral nerve cord neuronal integrity. Transcriptomic analysis revealed extensive gene-expression changes, particularly in the nrx-1 (tm1961) allele, with dysregulation of genes involved in cuticle development, neuronal signaling, protein homeostasis, innate immunity, mitochondrial organization, and transcriptional regulation. Gene Ontology and KEGG enrichment analyses identified significant perturbations in developmental, metabolic, stress-response, translational, and synaptic pathways. Together, these findings demonstrate that disruption of neurexin–neuroligin signaling drives transcriptional reprogramming that extends beyond synaptic dysfunction, linking molecular alterations to developmental, behavioral, and neuromorphological abnormalities.
Omamuyouwi M Ijomone, Victor E. Anadu, Toheeb O. Oyerinde et al.· Research Square· 0 citations
The findings implicate disrupted SYTL4-RAB27A-dependent vesicle trafficking in ASD pathogenesis and identify SYTL4 and RAB27A as previously unrecognized contributors to autism-associated synaptic deficits and behavior.
Yang Liao, Shuju Zhang, Xiaolei Zhang et al.· Proceedings of the National...· 0 citations
This study indicates that KIF2C may regulate microtubule dynamics to control deep-layer cortical neuron number and organization and modulate neuronal projections and signaling pathways and provides a foundation for understanding the role of KIF2C in neural development.
While advances in omics profiling rapidly expand the catalog of genes associated with brain activity in health and disease, functional annotation lags far behind. Here, we establish a high-throughput functional genomics platform that couples the calcium-integrating sensor CaMPARI2 with CRISPRi screening in human iPSC-derived neurons. By converting cumulative neuronal activity into a stable, flow cytometry-readable signal, this approach enables systematic interrogation through pooled screening. Using a focused library of memory-associated genes, we recover known regulators and identify TMEM50A, a previously uncharacterized protein, as an essential regulator of neuronal activity. TMEM50A forms a complex with LEPROTL1 and associates with ESCRT-III machinery on multivesicular bodies (MVBs). TMEM50A loss impairs MVBs function, remodels the neuronal surface proteome, reduces synapse density, and alters behavior in mice. This platform enables systematic discovery of neuronal activity regulators and reveals a critical role for TMEM50A-dependent MVB function in maintaining synaptic integrity and behavior.
