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bioRxiv2026

Design and Validation of New Primers for Specific and Sensitive Real-time PCR Detection and Quantification of Seven Botulinum Encoding Genes (Serotype A-G) of Clostridium botulinum

Botulinum neurotoxins (BoNTs) comprise a highly diverse group of seven serotypes (from A-G) and over 40 subtypes worldwide. Previous primer- and probe-based nucleic acid amplification tests (NAATs) for detection of BoNT encoding genes are challenged by high levels of nucleotide polymorphism both across and within subtypes. In this study, multiple BoNT gene sequences were aligned to identify highly conserved regions for the design of new primers that enable the detection of all seven serotypes under the same conditions. Specific primer sets were designed and validated using in silico, conventional and real-time PCR with constructed plasmids carrying the target fragments and spiked food matrices. The established procedure achieved highly specific and sensitive detection of BoNT serotypes A-G with sensitivity of 10 copies/reaction and a total turnaround time of approximately 1.5 hours. The procedure also eliminated the carryover PCR product by using uracil-N-glycosylase in combination with dUTP in the assay reaction mix. This study provides an alternative NAAT with higher coverage and compliments the traditional mouse bioassays in enhancing global botulism surveillance capabilities.

molecular biology↗

bioRxiv2026

From Bile Acids to a Gas-Producing Microbiome Phenotype: A Novel Mechanism of Host-Microbiome Communication

Background Microbiome-derived metabolites regulate host physiology, yet bacterial gaseous metabolites remain largely overlooked. Traditionally regarded as fermentation end-products, bacterial gases may act as biologically active mediators of host-microbiome communication. We hypothesized that bile acids regulate bacterial gaseous metabolism and influence host epithelial responses. Methods A high gas-producing clinical Escherichia coli isolate from a patient with moderately severe acute pancreatitis was cultured with selected primary and secondary bile acids. Gas production was assessed by pressure measurements, GC-TCD and GC-MS. Biological activity was evaluated by indirect exposure of Caco-2 and PANC-1 epithelial cells, followed by apoptosis/necrosis assays and whole-transcriptome RNA sequencing. Results Bile acids markedly reshaped bacterial gaseous metabolism. Cholic acid and deoxycholic acid promoted intense gas production, whereas chenodeoxycholic acid almost completely abolished it. Despite minimal apoptosis and necrosis, bacterial gaseous metabolites induced extensive transcriptional remodeling. Caco-2 cells showed stronger responses than PANC-1 cells, particularly to deoxycholic acid-derived gases, involving inflammatory signaling, extracellular matrix remodeling, epithelial plasticity, stress responses, and cancer-associated genes including PTGS2, MMP1, PLAUR, NR4A2, and SERPINE1. PANC-1 cells exhibited a more restricted response involving oxidative stress, proteostasis, and autophagy-associated pathways. Conclusions Our findings indicate that bacterial gases are a previously underrecognized class of microbiome-derived signaling molecules capable of modulating host gene expression independently of direct bacterial contact. We identify a gas-producing microbiome phenotype regulated by bile acid composition, linking microbial metabolism with epithelial signaling. These findings expand the concept of host-microbiome communication and provide a framework for investigating bacterial gaseous metabolites in intestinal and pancreatic diseases.

microbiology↗

bioRxiv2026

PhageTAILor leverages machine learning for phage tail-like elements detection and classification in plant-associated bacteria

Phage tail-like elements (PTEs) -- tailocins, bacterial type VI secretion systems (T6SS), and extracellular contractile injection systems (eCIS) -- are contractile nanomachines that bacteria use to kill their neighbors and compete within their micro-ecosystems. PTEs help shape microbial community composition. Most PTE detection tools only detect a single PTE class. Moreover, most tailocin detection methods are largely restricted to Pseudomonas, leaving a key part of tailocin diversity uncharacterized. In this work, we present PhageTAILor (https://github.com/hjcho-bio/PhageTAILor), an integrative and fully automated pipeline that detects and classifies prophages and 3 PTE classes from bacterial genomes. PhageTAILor combines a 6-detector homology-based candidate search (geNomad, tail-gene, PHROGs-tail, SecReT6, eCIStem, and a divergence-tolerant tail-HMM detector) with a LightGBM classifier comprising 1 multiclass and 3 binary heads, trained on 6,501 bacterial genomes carrying 13,082 prophages and PTEs. A phylogeny-free feature matrix used in our model keeps predictions reproducible between model construction and user inference. PhageTAILor performs strongly at the genome level and generalizes beyond its Pseudomonas-rich training set. On a 76-strain cross-clade benchmark, PhageTAILor detected tailocins at F1 = 0.955. Furthermore, it identified 12 of 13 experimentally validated tailocins spanning five genera versus 2 of 13 for a Pseudomonas-restricted tool TattleTail. PhageTAILor also demonstrated sensitivity equivalent to viral detection tool geNomad while avoiding its higher false-positive rate. Applied to 7,925 plant- and soil-associated bacterial isolates, PhageTAILor showed that prophages in the phyllosphere and tailocins in plant-associated bacteria, whereas eCIS are enriched in soil. PhageTAILor is distributed as an open-source, modular pipeline with a command-line interface.

microbiology↗

bioRxiv2026

Layer 5 anterior cingulate cortical neurons engage dorsolateral periaqueductal gray excitatory neurons to facilitate the affective component of pain

Pain is a conscious perceptual experience characterized by its aversive quality and consequent motivation to quench pain perception. The anterior cingulate cortex (ACC) critically contributes to the emotional dimension of pain. In both humans and rodents, ACC neural activity increases during acute and chronic pain, whereas ACC lesioning or excitability reduction decreases emotional reactivity during pain. However, the ACC is connected to many brain regions and is engaged during experiences beyond pain. Thus, it remains unclear through which circuit mechanisms the ACC shapes pain experience, and how specific those circuits are to nociception. Here, we show that excitatory input from the ACC to the dorsolateral periaqueductal gray (dlPAG) facilitates the affective-motivational dimension of pain. We first examined ACC[->]dlPAG connectivity using histology, optogenetics, and electrophysiology. We found that the axons of layer 5 ACC neurons terminate in the dlPAG and monosynaptically excite Slc17a6+ (VGLUT2-expressing) dlPAG neurons. Second, we genetically targeted ACC[->]dlPAG neurons with viral vectors to express the inhibitory DREADD hM4Di and then exposed the animals to an array of pain tests. We found that, across acute and chronic pain states, inhibition of the ACC[->]dlPAG pathway reduced affective-motivational but not reflexive pain behaviors. Third, we used fiber photometry to record neural calcium activity in the ACC in behaving mice and found that ACC[->]dlPAG neurons are engaged during a broad array of aversive experiences, rather than exclusively during pain, and exhibit task-specific activity patterns. Collectively, these results uncover the direct contribution of ACC[->]dlPAG neural activity to pain unpleasantness and the necessity of this pathway for generating aversive behavioral responses in general, rather than specifically for encoding the unpleasant quality of noxious stimuli.

neuroscience↗

bioRxiv2026

A reproducibility-audit framework for generalizable versus dataset-specific molecular transition boundaries in Alzheimer's disease

Molecular staging of Alzheimer's disease (AD) increasingly defines transition boundaries along single-cell pseudo-progression trajectories, yet whether such boundaries reproduce across brain regions, cohorts and molecular modalities is rarely tested. We present a permutation-controlled audit that combines nine boundary-detection algorithms with a fixed marker panel and four orthogonal reproducibility axes-algorithmic consensus, region, cohort and modality. On synthetic data with planted ground-truth boundaries the audit reaches 100% sensitivity and 94% specificity, rejecting four distinct artefact classes each by a different axis. Applied to the Seattle Alzheimer's Disease Brain Cell Atlas middle temporal gyrus, it localizes a transition that is robust across algorithms and recovered in most cell types but does not generalize: its leading marker is attenuated or absent in prefrontal cortex, entorhinal cortex and cerebrospinal fluid, and an apparent cross-region conservation of glial metabolic genes proves to be a global-expression offset rather than a shared program. The same audit nonetheless certifies an externally validated marker (astrocytic PTGDS) as reproducible across regions and modalities, showing that it separates generalizable anchors from dataset-specific ones rather than rejecting all signals. We provide this four-axis audit as a transferable, code-available standard to apply before a trajectory boundary is read as a biological stage, in AD and other progressive proteinopathies.

neuroscience↗

bioRxiv2026

Sympathetic activation and the force-frequency relationship in heart failure with reduced ejection fraction

Background Limited heart rate rise contributes to reduced exercise tolerance for people who have heart failure with reduced ejection fraction (HFrEF), yet rate-adaptive pacing does not improve functional capacity due to an attenuated force-frequency relationship (FFR). How the FFR relates to total peripheral resistance and sympathetic tone in HFrEF is unknown. Methods In a prospective, observational study, participants with HFrEF and controls underwent an incremental pacing protocol, during which heart rate was increased from 50 to 140 beats per minute. At each heart rate increment LV contractility was measured by echocardiography to determine the FFR, as well as continuous beat-to-beat measurement of systolic and diastolic blood pressures with a plethysmography device to determine cardiac output, total peripheral resistance and blood pressure variability (BPV). A microneurography study was then conducted to measure muscle sympathetic nerve activity (MSNA) during incremental pacing. Results A total of 157 participants with HFrEF and 55 controls (mean age 71.1{+/-}1.4 years, 172 (81.1%) male) underwent the pacing protocol. We observed single units in seven of 11 participants who participated in the microneurography study. In both groups, LV contractility and cardiac output increased until the peak of the FFR, after which these declined. We observed a reduction in total peripheral resistance, blood pressure variability, MSNA frequency and incidence coinciding with the peak of the FFR, beyond which these increased. Whilst these relationships were present in both groups, they were more evident in participants with HFrEF. Conclusions For people with HFrEF there is a bidirectional relationship between heart rate and sympathetic activation, with a nadir of sympathetic tone occurring at the peak of the FFR. Both excessively low and high heart rates are accompanied by greater sympathetic activation. Taken together, these data suggest that optimal heart rate targets for HFrEF are likely to be individual.

physiology↗

bioRxiv2026

Pallidal beta oscillations underlying locomotor adaptation in Parkinsons disease

BackgroundLocomotor adaptation is essential for adjusting walking patterns to complex environments. This study investigated locomotor adaptation deficits in people with Parkinsons disease (PD) and examined oscillatory activity in the globus pallidus internus (GPi) during walking adaptation. We hypothesized that elevated beta-band activity in the GPi is associated with reduced locomotor adaptability in PD. MethodsTwelve PD patients with GPi deep brain stimulation (DBS) (eleven bilateral and one unilateral) were included. Local field potentials (LFPs) were recorded from DBS electrodes during split-belt treadmill walking. Patients were tested in the medication-off, DBS-off state. Locomotor adaptation was measured as the change in step length asymmetry during split-belt walking, with smaller changes indicating greater adaptation deficits. ResultsWe found that GPi high beta (20-30 Hz) and low gamma (30-60 Hz) oscillations were modulated during split-belt walking. Compared to adapters, non-adapters showed decreased movement-related beta suppression during walking. Across participants, beta activity in the GPi contralateral to the fast leg was negatively associated with adaptation magnitude (Spearmans {rho} = -0.65 to -0.75). ConclusionsGPi oscillations are dynamically modulated during locomotor adaptation in PD. Increased beta activity may underlie impaired sensorimotor adaptation during walking. These findings provide novel insight into basal ganglia mechanisms of gait adaptation in PD and suggest that elevated GPi beta activity may serve as a marker of locomotor adaptation deficits.

neuroscience↗

bioRxiv2026

The circadian system is affected by Alzheimers disease independently from amyloid beta deposits

Circadian disruption, notably sleep disturbances, serves as an early indicator of Alzheimers disease (AD), preceding cognitive symptoms like memory loss. The suprachiasmatic nucleus (SCN) governs biological rhythms and receives direct retinal input via melanopsin-expressing retinal ganglion cells (mRGCs) to synchronize with environmental light cycles. The anatomical and functional basis for circadian disruption in AD remains unclear. Here, we explored the multi-level relationships between gene expression, the SCN connectome, and regulations of sleep and circadian rhythms in the APP/PS1 mouse model. The sleep architecture of APP/PS1 mice displayed significantly reduced rapid eye movement sleep (REM), associated with a reduced daily core body temperature amplitude and locomotor hyperactivity. Lastly, APP/PS1 mice showed an impaired response to acute light pulse stimulation and present hyperactivity of mRGCs at a young age and hypoactivity of these cells at older ages. These physiological functions are known to be, at least in part, regulated by the SCN, the main target of mRGCs. We noted several modifications in SCN connectomics using serial blockface electron microscopy (SBEM), including a reduction of the dendro-dendritic chemical synapse (DDCS) network that receives a large part of the retinal input and is thought to be crucial for synchronicity between SCN neurons. In addition, we observed multiple signs of dystrophy, including modifications of the shape of dendrites and cell soma, accumulation of aggregated lysosomes, and swelling of axons. At the same time, we investigated the changes in gene expression using spatial transcriptomics. The SCN presents changes in the expression of genes associated with synapse formation, cell adhesion, and neurite growth. These results suggest that, despite the absence of amyloid plaques in the ventral hypothalamus, the SCN of APP/PS1 mice still undergo profound gene expression changes, impacting connectomics and physiological functions. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=157 SRC="FIGDIR/small/744599v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@ceedb0org.highwire.dtl.DTLVardef@156cfaaorg.highwire.dtl.DTLVardef@5bc262org.highwire.dtl.DTLVardef@36df4d_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2026

AmPair: automating housekeeping-gene primer design for species-level metataxonomics

Amplicon sequencing of the 16S rRNA gene is the most widely used approach for profiling bacterial communities, but its taxonomic resolution is typically limited to the genus level. Many species carry multiple divergent 16S rRNA alleles that overlap across species boundaries, an ambiguity that even full-length, long-read sequencing cannot fully resolve. Shotgun metagenomics achieves species-level resolution but remains costly, particularly when only a single genus is of interest. Amplicon sequencing of rapidly evolving, protein-coding housekeeping genes offers a cost-effective alternative, yet no tool exists to identify suitable primer sets for a given target taxon. Here we present AmPair, a Snakemake pipeline that, given a target genus and one or more candidate housekeeping genes, designs and ranks primer pairs binding conserved regions while flanking a variable region capable of species-level discrimination, and validates them in silico across all available genomes. Using the genus Bacillus and the housekeeping gene tuf as a case study, the primer set recommended by AmPair amplified 99% of 2,392 genomes; only 0.04% carried multiple alleles and none showed inter-species allele overlap, compared with 91.41% and 69.49%, respectively, for the standard 16S rRNA V1-V9 region. Applied to a Bacillus community profiled by Nanopore sequencing, the same primers resolved closely related species. AmPair thus offers a generalizable and accessible route to species-level community profiling.

bioinformatics↗

bioRxiv2026

Chemosensory ERP Suggest Peripherally Driven Olfactory-Trigeminal Interactions in Healthy Older Adults

The olfactory and intranasal trigeminal systems interact closely, influencing chemosensory perception, yet the mechanisms underlying their interaction remain poorly understood and have been studied mainly in young adults. We aimed to characterize olfactory-trigeminal interactions in aging by comparing electrophysiological and behavioral responses under ipsilateral and contralateral olfactory-trigeminal co-stimulation, to determine the relative contributions of peripheral and central mechanisms. Using chemosensory event-related potentials and a localization task, 44 healthy older adults (66.3 {+/-} 4.6 years; 29 women) were tested under four conditions: pure trigeminal (carbon dioxide; CO2), pure olfactory (2-phenylethanol; PEA), ipsilateral co-stimulation (PEA+CO2 in the same nostril), and contralateral co-stimulation (PEA+CO2 in opposite nostrils). Ipsilateral, but not contralateral olfactory-trigeminal co-stimulation, improved trigeminal localization performance and induced larger late positive component amplitudes. Together, these findings suggest that olfactory-trigeminal interactions are driven primarily by peripheral rather than central mechanisms. This study also provides normative CSERP data for healthy older adults.

neuroscience↗

bioRxiv2026

Meso2EM: a cross-scale CLEM workflow linking mesoscale functional imaging to targeted electron microscopy

Meso2EM is a correlative light and electron microscopy workflow that transfers neurons selected from mesoscale functional images to targeted electron microscopy. We recorded Ca{superscript 2} signals from layer 2/3 neurons across a contiguous 3 x 3 mm cortical field in awake mice and reidentified a selected neuron after fixation and tangential sectioning. Lectin-labeled vascular architecture served as a shared landmark across in vivo two-photon imaging, confocal microscopy, laboratory micro-CT of resin-embedded tissue, and block-surface scanning electron microscopy, guiding focused-ion-beam scanning electron microscopy to the target cell body. The same progressive-targeting principle also supported serial ATUM-SEM reconstruction of an in vivo-tracked dendrite and serial transmission electron microscopy of optically selected dendrites from a patch-clamp-recorded Martinotti cell. Meso2EM therefore provides a practical route for preserving target identity across large changes in scale and specimen state while restricting electron-microscopy acquisition to a selected region.

neuroscience↗

bioRxiv2026

Proteolytic Remodeling of Cargo Receptor Networks by RHBDL4 Tunes Secretory Pathway Flux

Cargo receptors are central organizers of the secretory pathway, yet the mechanisms controlling their abundance remain poorly understood. The endoplasmic reticulum (ER)-resident intramembrane protease RHBDL4 promotes substrate turnover via a non-canonical branch of ER-associated degradation and has recently been implicated in regulating secretory pathway components. We previously identified the p24 cargo receptor TMED7 as an RHBDL4 substrate, suggesting that cargo receptor turnover contributes to secretory pathway regulation. Here, quantitative proteomics identify members of the ER-Golgi intermediate compartment (ERGIC) cargo receptor family as endogenous RHBDL4 substrates, demonstrating that RHBDL4 targets multiple cargo receptor families within the early secretory pathway. Accordingly, RHBDL4 modulates multiple ERGIC-dependent transport pathways. In addition, unbiased secretome analysis reveals increased secretion of lysosomal precursor proteins upon RHBDL4 ablation. Mechanistically, we show that this phenotype is mediated, at least in part, by RHBDL4-dependent cleavage of the lysosomal cargo receptor sortilin/SORT1. Together, these findings identify cargo receptors as a major class of RHBDL4 substrates and establish proteolytic remodeling of cargo receptor networks as a mechanism for regulating secretory pathway flux.

cell biology↗

bioRxiv2026

A mutation-agnostic and allele-specific ASO strategy demonstrates potent functional rescue and retinal preservation in RHO-linked retinitis pigmentosa

Autosomal dominant retinitis pigmentosa (adRP) caused by RHO mutations is a leading form of inherited retinal degeneration. Extensive allelic heterogeneity of RHO pathogenic variants limits the translational applicability of mutation-specific gene therapies. To address this, we developed SNARE (SNP-guided Silencing of Aberrant RHO Expression), a mutation-independent, allele-specific antisense oligonucleotide (ASO) strategy. SNARE selectively suppresses mutant RHO transcripts by targeting the common, benign c.-26A/G single-nucleotide polymorphism (SNP) as an allelic discriminator. Candidate gapmer ASOs were screened in engineered reporter lines and validated in patient-derived retinal organoids, identifying RHOligo-A as the lead c.-26A-targeting candidate. In vitro, RHOligo-A achieved robust, preferential knockdown of the target allele, improving RHO localization in retinal organoids, and demonstrated a favorable safety profile with minimal transcriptomic off-target effects and no detectable immunostimulatory activity. Subsequent validation in a novel, humanized RHOP347L/WT mouse model, achieved sustained c.-26A-linked allele-selective suppression, retinal structure preservation, and significantly restored visual function, upon a single intravitreal administration. These findings establish RHOligo-A and SNARE as a scalable, mutation-independent therapeutic platform with strong translational potential and substantial clinical reach for RHO-associated adRP.

genetics↗

bioRxiv2026

CD36 phosphorylation alters the thrombospondin binding site and reduces internal cavity accessibility and volume

The cluster of differentiation 36 (CD36) is a membrane protein with broad physiological roles in health and disease, and its function is regulated in part by phosphorylation. Experimental evidence shows that phosphorylation of Thr92 reduces CD36 affinity for thrombospondin-1 (TSP-1), binding of which initiates antiangiogenic signaling, whereas phosphorylation of Ser237 decreases CD36-mediated fatty acid uptake, with implications for energy metabolism. However, the only available crystal structure of CD36 lacks phosphorylation, and the molecular mechanisms by which phosphorylation regulates CD36 function remain largely unknown. This study provides an atomically detailed computational characterization of CD36 in unphosphorylated and dual phosphorylated states, using molecular dynamics simulations with a total sampling time of 30 microseconds in combination with Markov state models. We present, to our knowledge, the first evidence of a cryptic pocket on CD36 surface that is formed by phosphorylation. This cryptic surface pocket and a loop spanning residues 121-131 form a high affinity binding site for TSP-1 derived ligands, shifting their binding away from the canonical site. We propose that this altered binding provides a molecular basis for the disruption of antiangiogenic signaling upon CD36 phosphorylation. Additionally, our data indicate that, phosphorylation increases helicity and compaction within the helix-loop region spanning residues 296-331, narrowing one of the entrances to the internal cavity and reducing its overall volume. These conformational changes provide a potential mechanistic explanation for the decrease in fatty acid uptake upon CD36 phosphorylation. Our findings provide structural insights that may inform the future design of CD36 modulators and emphasize the importance of targeting phosphorylation induced CD36 conformations in angiogenic and metabolic diseases.

biophysics↗

bioRxiv2026

Ancient Somatosensory Circuit Architectures Employ Flexible Molecular Strategies

The extent to which conserved neural circuit architectures depend on shared molecular specification programs remains unclear. Here, we address this question by examining the somatosensory system of the little skate, Leucoraja erinacea, an early-diverging vertebrate that retains ancestral features of both finned and limb-based body plans. We show that core features of somatosensory circuit organization, including laminar organization of the spinal cord and dorsally restricted targeting of sensory afferents, are deeply conserved. Unexpectedly, the molecular programs specifying dorsal root ganglion (DRG) sensory subtypes diverge extensively from those of mammals. Although DRG neuron subtype specification and spinal connectivity rely on target-derived cues, skates employ distinct neurotrophin receptor and transcription factor identity codes. These findings support a model in which conserved spinal circuit architectures provide a stable scaffold that leverages flexible sensory neuron specification programs, enabling the evolutionary diversification of vertebrate somatosensory systems. HighlightsO_LIIntegrated analysis of spinal cord and DRG neuronal diversity in Leucoraja erinacea C_LIO_LILaminar organization of the dorsal spinal cord is an ancestral vertebrate feature C_LIO_LIDivergent neurotrophin receptor and transcription factor codes in sensory neurons C_LIO_LIConserved target-dependent regulation of sensory identity and connectivity C_LI

neuroscience↗

bioRxiv2026

Increased activity in the somatosensory and insular cortex during the transition from acute to chronic neuropathic pain

Pathophysiological mechanisms underlying the transition from acute to chronic neuropathic pain remain incompletely understood. The somatosensory and insular cortices are key cortical components of the pain matrix. We examined changes in activation of these cortical regions during the transition from acute to chronic neuropathic pain. The right sciatic nerve was ligated in activity reporter TRAP mice using standard procedures. Mechanical allodynia was confirmed after CCI or sham surgery using von Frey monofilaments applied to the hind paws. To label active neurons, 4-hydroxytamoxifen was administered to separate cohorts at 1, 3, and 6 weeks following nerve ligation. Passive tissue clearing of brain sections and confocal imaging was used to assess active neurons. Progressive reduction of ipsilateral hind paw in CCI mice indicated mechanical allodynia development. CCI mice showed robust neuronal activation in the bilateral somatosensory and insular cortices. The somatosensory cortical activation peaked at 3 weeks post-CCI, whereas insular cortical activity increased during the transition from acute to chronic neuropathic pain. These studies revealed that CCI induced progressive mechanical allodynia and distinct temporal patterns of cortical neuronal activation, with transient peak neuronal activity in the somatosensory cortex and sustained, increasing activation in the insular cortex during acute-to-chronic pain transformation.

neuroscience↗

bioRxiv2026

Synaptic adhesion molecule signaling is activated and organized by tyrosine phosphorylation-induced biomolecular condensate formation

The precise formation of synapses ensures the proper wiring and function of nervous systems. Specific synapse formation is controlled by synaptic adhesion molecules, which link pre- and post-synaptic cells. Despite this central role, details of how adhesion molecules organize and signal intracellularly to build core synaptic structures are limited. Here, we identify multiple tyrosine phosphorylation sites on the cytoplasmic tail of the C. elegans SYG-1 synaptic adhesion molecule that are critical to initiate presynapse formation. We determine that SRC-1 and SRC-2 tyrosine kinases are redundantly responsible for SYG-1 phosphorylation and are consequently critical for presynapse assembly. The phosphorylated population of SYG-1 localizes in clusters within a larger SYG-1 pool and these clusters mark sites of presynaptic active zone assembly. Reconstitution of SYG-1 clusters in vitro with SH2-domain adapters and WSP-1 reveals a dynamic biomolecular condensate-forming system. Blocking phosphotyrosine adapters and condensate formation in vivo results in the loss of SYG-1 clusters, defective presynapse formation, and compromised neurotransmission. We conclude that phosphorylation of a subpopulation of synaptic adhesion molecules activates and organizes them into condensate-based clusters to initiate presynapse formation.

neuroscience↗

bioRxiv2026

Molecular and functional profiling distinguishes PACS1 syndrome variant from PACS1 loss-of-function in iNeurons

PACS1 syndrome is a rare neurodevelopmental disorder caused by a recurrent de novo missense variant (p.R203W) in the PACS1 protein. However, it remains unclear whether the p.R203W variant acts through a loss-of-function or alternative mechanism. Here, we used isogenic iPSC-derived neurons (iNs) to directly compare the effects of PACS1 p.R203W to complete loss of PACS1 function. Using a combination of proteomic, biochemical and electrophysiological approaches, we identified molecular and functional phenotypes associated with each genotype. While PACS1(+/R203W) and PACS1(-/-) iNs shared phenotypic abnormalities, the overall molecular and functional consequences of the p.R203W variant were distinct from those caused by PACS1 deficiency. Notably, PACS1(+/R203W) presented with unique proteomic and kinase signaling signatures and a shift in stimulus dependent excitability. These findings demonstrate that PACS1 syndrome is not caused by a simple loss of function and instead support a non-loss-of-function mechanism. Lastly, our interactome analysis suggests that the p.R203W variant retains aspects of canonical PACS1 function while acquiring novel molecular interactions that could contribute to PACS1 syndrome pathogenesis. Altogether, these findings provide a framework for future mechanistic studies and therapeutic development in PACS1 syndrome. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/747101v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@d1522corg.highwire.dtl.DTLVardef@69e4dforg.highwire.dtl.DTLVardef@30eebcorg.highwire.dtl.DTLVardef@899b9d_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗