bioRxiv Subject Collection: Neuroscience's Journal

Endocytome profiling uncovers cell-surface protein dynamics underlying neuronal connectivity
Endocytosis actively remodels the neuronal surface proteome to drive diverse cellular processes, yet its global extent and circuit-level consequences have defied comprehensive interrogation. Here, we introduce endocytome profiling: a systematic, cell-type-specific approach for mapping cell-surface protein (CSP) dynamics in situ. Quantitative proteomic analysis of developing olfactory receptor neuron (ORN) axons generated an endocytic atlas comprising over 1,100 proteins and revealed the extent to which the surface proteome is remodeled to meet distinct developmental demands. Targeted interrogation of a junctional CSP showed that its endosome-to-surface ratio is precisely balanced to enable developmental axon pruning while preserving mature axon integrity. Multi-omic integration uncovered wide-spread transcellular signaling and identified a growth factor secreted by neighboring neurons to direct ORN axon targeting via endocytic regulation of its receptor. Endocytome profiling thus provides unprecedented access to cell-surface proteome dynamics and offers a powerful platform for dissecting proteome remodeling across diverse cell types and contexts.

Selective changes in cortical cholinergic signaling during learning
Cognition relies on the function of local and long-range neural circuits in the neocortex, which are dynamically regulated by neuromodulatory signals including acetylcholine (ACh). Recent work suggests that the role of acetylcholine varies across cortical areas and behavioral states. In addition, the precise role of cholinergic signaling during learning and plasticity remains unclear. We performed simultaneous, dual-color mesoscopic imaging of ACh and calcium signaling in the neocortex of awake mice across multiple stages of visual task learning to identify learning-related plasticity in neural activity and cholinergic signaling. Using a novel Pattern Reconstruction and Interpretation with a Structured Multimodal transformer (PRISMT) model combining masked autoencoding with multimodal causal attention, we identified key cortical regions that reorganize with learning. We found that cholinergic signaling exhibited spatiotemporally selective plasticity in frontal cortical subnetworks during visual perceptual learning. Overall, our findings demonstrate the utility of transformer models for generating biologically interpretable insight into large multimodal datasets and link long-term functional reorganization of cortical networks to enhanced task performance.

Higher baseline alpha power is associated with faster responses in visual search
Visual search models have long emphasised that task-relevant items must be prioritized for optimal performance. While it is known that search efficiency also benefits from active distractor inhibition, the underlying neuronal mechanisms are debated. Neuronal alpha oscillations (7-14 Hz) have been associated with functional inhibition of cortical excitability, as well as distractor suppression in spatial attention and visual working memory tasks. We therefore hypothesised that alpha oscillations similarly support the deselection of distractors in visual search. Using Magnetoencephalography (MEG), we here show that high alpha power before the onset of a complex search display is associated with faster search performance. Crucially, we used a General Linear Model (GLM) approach to control for confounds between alpha power and task duration, ruling out that this result was merely driven by practice effects paired with increased fatigue over time. In addition to spontaneous oscillatory activity, we quantified the cortical excitability to colours of the search stimuli based on Rapid Invisible Frequency Tagging (RIFT) responses. In contrast to our initial hypothesis, increased pre-search alpha power did not correlate with the RIFT response, providing no direct evidence for feature-specific inhibition of distracting stimuli by alpha. Our findings challenge the traditional view of alpha oscillations reducing visual processing, showing instead that increased occipital alpha power can enhance performance in a visual task. We propose that the increase in alpha power may reflect increased top-down control supporting visual search

Hierarchical coding of local and global positions in episodic memory for naturalistic sequences
Life unfolds continuously across multiple timescales, yet how the brain encodes hierarchical event sequences remains poorly understood. Using magnetoencephalography, we investigated the neural coding of hierarchical positions in naturalistic sequence memory. Participants viewed video sequences with a nested structure and later recalled their serial order. Behavioral analyses revealed transposition errors for items occupying identical global or local position. Multivariate classification and subspace analyses revealed separable neural representations of global and local positions. Local positions were better decoded in high-dimensional space, whereas global positions exhibited greater pattern separability in low-dimensional space. Brain regions encoding global positions exhibited longer neural timescales and reduced sensitivity to low-level event transitions compared to regions encoding local positions. These findings support a parallel hierarchical organization of content and context, a novel framework that enables integration of item and order across hierarchical levels and provides principles for hierarchical representations in both human memory and artificial intelligence.

Stable clique membership in mouse societies requires oxytocin-enabled social sensory states
The ability to form stable de novo relationships in complex environments is essential for social functioning and is impaired in severe psychiatric disorders including autism. Yet, the neurobiological basis and cognitive processes enabling the formation of stable bonds in larger groups remain poorly understood, thereby limiting our ability to develop effective therapies. Here, we establish a semi-naturalistic model of clique formation in mouse societies, where individuals are tracked longitudinally from massive video data. Small, stable rich-clubs develop within these mouse social networks. Consistent with human rich-clubs, these cohesive cliques tended to have high social rank and exerted influence on non-members. Interestingly, neither prior rich-club-membership in a different group nor kinship facilitated entry into rich-clubs. Mimicking sparse population genetics, we probed the open question whether a subtle neuro-cognitive phenotype, namely impaired induction of social sensory processing states by cortical oxytocin signaling, disrupts higher-order social bonding in these complex social environments. Despite preserved social motivation, mice with alterations in this oxytocin subsystem failed to join rich-clubs. They approached group members less consistently, and connections from others towards them fluctuated more as well. This reciprocal disorganization highlights how interactional dynamics within social networks can amplify individual-level deficits, consistent with models of emergent properties of social behavior. These findings underscore the role of oxytocin in tuning sensory systems into a social processing state. Its dysfunction affects an individual's ability to establish stable relationships in complex social networks, with profound implications for social functioning deficits in psychiatric disorders.

Striatal visual responses increase prior to visuomotor learning
The cortex and basal ganglia exhibit interdependent changes during learning. However, it is not clear whether plasticity occurs in sequence or concurrently across these structures. To address this question, we simultaneously recorded cortical and striatal activity while training mice on a visuomotor association task, which involved turning a wheel to move a stimulus from a cue location to a target location. Prior to the development of learned behavior, visual responses increased in the visual-recipient striatum. This was followed by the emergence of stimulus responses in both the medial prefrontal cortex (mPFC) and mPFC-recipient striatum from the onset of learned behavior. All of these regions also exhibited increased responses to stimuli in the rewarded target position, but while the visual-recipient striatum was non-selective between cue and target stimuli, the mPFC and mPFC-recipient striatum switched from being target-stimulus responsive before learning to being cue-stimulus responsive after learning. Our results suggest that sensorimotor learning involves routing stimulus information first to the sensory striatum, and then to frontal motor circuits.

CEREBRAL GLUCOSE UTILISATION DURING MUSICAL EMOTIONS: A MULTIMODAL FUNCTIONAL PET/MRI STUDY.
Functional magnetic resonance imaging (fMRI) studies have demonstrated music-induced activation of the blood-oxygen-level-dependent (BOLD) signal across brain networks associated with auditory perception, motor control, and emotion. However, BOLD-fMRI reflects vascular responses that may not fully capture underlying neural activity. Here, we used simultaneous [18F]fluorodeoxyglucose (FDG) functional positron emission tomography (fPET) and fMRI to examine glucose metabolism closely linked to neural activity, alongside hemodynamic responses during pleasurable music listening. Thirty-five female participants listened to self-selected pleasurable music and control stimuli while undergoing 90-minute PET-MRI scans. fPET revealed music-evoked increase in glucose consumption in auditory and motor cortices, as well as reward-related regions, including the nucleus accumbens (NAcc), caudate, insula, and orbitofrontal cortex. The fPET and fMRI results showed substantial overlap though some discrepancies were also observed. Notably, the NAcc exhibited increased glucose consumption in fPET but showed no activation in fMRI. Conversely, deactivation of the default mode network during music processing was only observed with fMRI. These results highlight the complementary nature of neurometabolic and neurovascular processes and offer novel insights into their dynamics during the processing of aesthetic rewards.

Early Metabolic Alterations in Cerebrospinal Fluid Fatty Acid Profiles Linked to Cognitive Decline and All-Cause Dementia
Background: While All-cause dementia (ACD) may often be characterized by the abnormal deposition of extracellular {beta}-amyloid (A{beta}) in the brain cortex and hyperphosphorylated tau (p-tau) as neurofibrillary tangles intracellularly, there is a need to identify early metabolic changes that may accompany these pathological changes. Objective: This study evaluated the predictive value of fatty acids in cerebrospinal fluid (CSF) fractions in differentiating cognitively unimpaired (CU), mild cognitive impairment (MCI), and ACD participants. Methods: CSF fatty acid profiles were analyzed from CU (n=68), MCI (n=38), and ACD (n=37) individuals aged 77.3 plus-or-minus sign 7.7 years, sourced from the Huntington Medical Research Institutes (HMRI). Multivariable binary logistic regression identified the most effective CSF fatty acid biomarkers for distinguishing CU, MCI, and ACD groups. The top-performing CSF fatty acid biomarkers were combined with A{beta}42, tau, and the A{beta}42/tau ratio to evaluate their collective diagnostic performance. The model was adjusted for covariates, including age, sex, smoking status, hypertension, diabetes, and APOE genotype. Receiver operating characteristic (ROC) curves were generated for the top 10 CSF fatty acids ranked by area under the curve (AUC), sensitivity, and specificity, and their significance was assessed using the DeLong test. Results: The top 10 fatty acids in CSF fractions demonstrated superior discrimination between CU individuals and those with MCI compared to traditional markers such as A{beta}42, tau, and the A{beta}42/tau ratio. Furthermore, incorporating a panel of these fatty acid biomarkers alongside A{beta}42/tau significantly improved diagnostic accuracy. Age, sex, smoking, hypertension, APOE genotype, and diabetes did not significantly influence the model's performance. Conclusion: This study suggests that changes in fatty acid metabolism occur in the early stages of ACD pathology. Thus, strategies that regulate fatty acid metabolism may prevent cognitive decline in an older population.

Helicobacter pylori infection and α-synuclein pathology drive parallel neurodegenerative pathways in the substantia nigra
Parkinson's disease is a common neurodegenerative disease related to both genetic and environmental insults. Epidemiological studies have linked Helicobacter pylori (H. pylori) infection to Parkinson's disease risk, but the underlying mechanisms of this association remain unclear. In this study, we investigate whether chronic infection with a pathogenic H. pylori strain can induce -synuclein aggregation or neurodegeneration, and whether infection clearance mitigates these effects. We also assessed whether H. pylori infection exacerbates -synuclein pathology and neuron loss when combined with seeding of -synuclein pathology. We find that chronic H. pylori infection induces a sustained immune response in the gut and plasma that leads to mild brain inflammation and dopaminergic neuron loss, independent of -synuclein pathology. These effects are attenuated by eradication of the infection. In mice with -synuclein pathology induced by pre-formed fibrils, H. pylori did not further exacerbate the extent of pathology or neuronal death. Together, these results suggest that H. pylori infection can lead to neurodegeneration through inflammatory mechanisms independent of -synuclein aggregation. Our findings offer mechanistic insights into how pathogens influence the risk and progression of Parkinson's disease.

Song familiarity relies on evidence accumulation
Familiarity judgements are thought to involve evidence accumulation, a decision-making process in which information is gathered over time until a threshold is reached. Previous work has identified a scalp-recorded signature of evidence accumulation called the central-parietal positivity (CPP). We built on this previous work and recorded electroencephalography (EEG) while participants listened to several melodies, instructing participants to respond as soon as the song felt familiar. A prominent CPP was noted, time-locked to decisions. We then used linear regression to unmix overlapping neural responses and observed a stimulus-locked indicator of evidence accumulation that increased in amplitude just prior to a familiarity decision. This result suggests that song familiarity relies on evidence accumulation, with individual notes in a familiar song acting as "evidence".

Comparative snRNAseq study of C9orf72, SOD1, and sALS spinal cord
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by the loss of motor neurons, yet the cell-type specific molecular alterations within the spinal cords are not well characterized. In this study, we conducted deep molecular profiling of spinal cord tissues donated by people living with sporadic, C9orf72, and SOD1-ALS using single-nucleus RNA sequencing (snRNAseq). We observed numerous distinct gene expression patterns and enriched pathways among ALS types. However, when focusing on common features, we identified activation of stress-response and inflammatory pathways in specific microglia subtypes, as well as disrupted vesicle transport and synaptic function in a ventral inhibitory neuronal subtype. Notably, CPLX3, a SNARE regulator, was uniquely expressed in alpha-motor neurons and was commonly downregulated across all ALS types. While this study uncovers the molecular heterogeneity underlying ALS, it also highlights shared pathways within specific cell types, especially in the ventral inhibitory neurons that have been less explored in ALS research.

Modified tau leads to neuronal and global ribosome dysfunction in C. elegans model of tauopathy
Alzheimer's disease (AD) is a neurodegenerative disease characterized by an early loss of memory formation which requires protein synthesis. Tau is an intrinsically disordered protein and is subject to extensive post-translational modifications (PTMs). Some PTMs have been shown to alter localization of tau and allow tau to disrupt protein translation. Protein interactome studies indicate that tau might interact with ribosomal proteins. Therefore, we hypothesized that tau is causing ribosomal dysfunction as an early event and this interaction is dependent on tau's PTMs. To test this, we used a C. elegans strain expressing single copy insertion of human tau as well as two of the most frequent modified versions of tau in mechanosensory neurons. With our assay to measure translation, we showed that in our T231 phosphorylation mimetic strain, there was a significant decrease in neuronal translation. This mimetic strain also showed a significant decrease in median lifespan and locomotion. Unexpectedly, in all our Tau-expressing strains, we detected a significant decrease in whole worm translation, suggesting a possible role of tau to influence translation in other tissues in worm. Our in vitro, in vivo and ex vivo efforts to demonstrate tau-ribosome association via fluorescent polysome profiling have shown that there is no direct association between tau and the ribosome. Ribosome dysfunction caused by modified tau could be an early event in AD pathology before the pathological hallmarks appear.

Slow-varying normalization explains auditory steady-state masking interactions in human EEG
The inherent ability of sensory neurons to entrain to modulations in the temporal structure of an auditory stimulus gives rise to the auditory steady-state response (ASSR). Simultaneous presentation of multiple acoustic stimuli by frequency tagging them to generate ASSRs at different frequencies is routinely employed for hearing threshold determination and cognitive studies. However, the nature of ASSR interactions as a function of competing modulation frequencies in the absence of overt behaviour and the underlying neural mechanisms have not been well studied. Such interactions have previously been studied with visual stimuli that generate steady-state visually evoked potentials (SSVEPs) and explained using a normalization model. Here, we tested whether similar interactions are observed in the auditory domain, and if so, can be explained using a similar model. We played sinusoidally amplitude-modulated stimuli simultaneously modulated at two frequencies while we recorded a 64-channel EEG from subjects who passively listened to these sounds with closed eyes. We used multiple modulation frequencies and depths to characterize the ASSR modulation masking response profile. We observed that modulation frequencies closer to each other suppress the ASSR strongly than frequencies that are farther apart, similar to the interaction observed in the visual domain using SSVEPs. The observed suppression was captured by a slow-varying normalization model, which was initially used to explain SSVEP interactions. We obtained a band-pass shaped suppression profile with a low-pass cutoff that matched to that observed for SSVEP interactions. Our findings highlight the universality of the normalization model in accounting for masking interactions across modalities.

Transdiagnostic symptomatology amidst real-world environmental uncertainty: a cross-sectional and cross-lagged panel network analysis
Uncertainty is a potential driver of poor mental health outcomes, and uncertainty is mounting globally across many domains of daily life. However, it remains unclear how anxiety and depression symptoms emerge in response to uncertainty in these real-life contexts. Capitalising on the COVID-19 pandemic as a naturalistic experiment, we investigate how heightened real-world environmental uncertainty interacts with intolerance of uncertainty (IU) to drive mental health symptoms. We collected self-reported data on symptoms of anxiety, depression and IU from 301 participants for two time points. We performed cross-sectional network analyses to identify influential contributors and established a cross-lagged panel network (CLPN) to investigate longitudinal effects. Anxiety, depression and IU symptoms were higher when real-world uncertainty was higher (t2) compared to when it was lower (t1). Network analyses revealed the same three symptom clusters at both timepoints, however worry became more central to the network at t2. In the CLPN, unlike anxiety and depression symptoms, IU symptoms had the highest autoregressive values, meaning IU at t2 was well predicted by IU at t1. The item "inhibition of behaviour due to doubt" positively predicted both anxiety and anhedonia. This study highlights the relevance of worry during higher real-world uncertainty and the predictive value of predispositional IU. Our findings further suggest behavioural inhibition to be a potential target to alleviate internalising psychopathology. During ever-increasing fluctuations in global uncertainty, we provide novel insights into the temporal relationships of highly prevalent psychopathology, which might inform support strategies for people with existing mental health vulnerabilities.

Nanophotonic neural probes for in vivo photostimulation, electrophysiology, and microfluidic delivery
Implantable silicon neural probes with integrated optical emitters and electrodes are emerging tools for simultaneous optogenetic stimulation and electrophysiological recording in deep brain regions. In parallel, neural probes with microfluidic channels have been developed for localized drug delivery and neurochemical sampling. However, thus far, such fluidic probes have lacked optical and electrical functionalities or been limited to a low number of optical emitters and/or electrodes, constraining their utility in multimodal investigations of neural circuits. Here, we introduce foundry-fabricated silicon nanophotonic neural probes with monolithically integrated microfluidics. Each probe has 16 silicon nitride grating coupler emitters, 18 titanium nitride microelectrodes, and one embedded microfluidic channel. We evaluate the photonic, electrophysiological, and microfluidic functionalities in vivo in optogenetic, blue-light-sensitive mice. With our multifunctional neural probes, we demonstrate local suppression of epileptic seizure activity (induced by microfluidic injection of 4-aminopyridine) using photostimulation. Through foundry-compatible microfluidics integration, this work advances the versatility of nanophotonic neural probes and presents new possibilities for multimodal neuroscience experiments leveraging this scalable neurotechnology.

Variation in Synaptic Inputs Drives Functional Versatility for Sound Encoding in Globular Bushy Cells
Synaptic convergence is fundamental to neuronal circuit function, enabling diverse processing such as coincidence detection and reliable signal transmission. In sensory systems, the architecture of convergence and strengths of synaptic inputs are pivotal for extracting distinct features of the sensory stimulus. In auditory system, globular bushy cells (GBC) in the cochlear nucleus receive multiple axosomatic endbulb of Held terminals from the auditory nerves, which vary considerably in size, even among inputs targeting the same cell. However, the functional consequences of this input strength variation for sound encoding remain unclear. Here, we investigated how synaptic input variation shapes sound encoding in GBC of Mongolian gerbils, using in vitro conductance-clamp recordings and computational modeling, in which synaptic inputs with varying strengths were simulated. We found that input variation critically shapes GBC sound encoding by influencing temporal precision and firing rates. Low input variation allows GBC to act as coincidence detectors, thereby improving temporal precision. Conversely, high input variation increases overall firing rates and enhances the encoding of amplitude modulations, albeit at the expense of temporal precision. These findings suggest that inherent endbulb strength heterogeneity allows GBC to operate along a functional continuum. This variation in input strength may provide the basis for generating diverse information streams to downstream targets including the medial nucleus of the trapezoid body (MNTB).

Measuring partner synchrony during salsa dancing and its relationship to changes in psychosocial domains
Background: Dance is a promising rehabilitation adjunct but understanding the mechanisms through which dance improves physical and psychosocial well-being is necessary for implementation. This study investigated the feasibility of a measure of one possible mechanism: partner synchrony. Secondary objectives were to examine the relationships between partner synchrony, instructor ratings, and changes in psychosocial variables. Methods: Participants wore an Inertial Measurement Unit (IMU) sensor during a salsa class that was video recorded. Partner synchrony was quantified with correlations of x, y, and z-axis acceleration between dancing pairs. Psychosocial variables were measured pre- and post-class. Salsa instructors rated partner synchrony from video recordings. Feasibility parameters included percentage of participants with data collected and sensor comfort. Pre-post changes were analyzed with Wilcoxon ranked tests and relationships were analyzed with Spearman correlations. Results: Data was collected for 23/24 (96%) participants and 17/24 (81%) reported the sensor was comfortable. All psychosocial variables improved from pre- to post- class. Partner synchrony was significantly associated with instructor ratings and change in positive and negative affect. Conclusions: An IMU-based measure of dance partner synchrony is feasible, associated with ratings by dance instructors and related to changes in mood. The partner synchrony measure can be used to advance our understanding of dance as a therapeutic tool after its reliability is investigated.

Vascular draining confounds laminar decoding in fMRI
Laminar fMRI using GE-BOLD is vulnerable to spatial blurring from intracortical veins, while multivariate pattern analysis (MVPA) is often assumed to mitigate these biases. Yet, this assumption has not been systematically investigated. We thus developed a mechanistic laminar response model that simulates voxel-wise patterns across cortical depths, incorporating a vascular draining model. We conducted simulations in which the ground-truth signal originated in a single, several, or across all layers, and applied standard MVPA decoding before and after deconvolution of the draining effect. Decoding accuracies were consistently influenced by draining veins: deep-origin signals yielded above-chance decoding in superficial layers, and null scenarios produced false positives in middle or deep layers. Vascular deconvolution enhanced specificity in single-layer cases but did not resolve ambiguities in null decoding profiles. Simulating six thinner layers improved decoding accuracies, especially in the deconvolved signal scenarios. These findings demonstrate that multivariate techniques are not inherently immune to vascular biases, but also demonstrate that careful modeling can help correct draining effects.

Stuck on you! Social brain stimulation increases the cognitive effort required to return to the egocentric perspective
Flexible switching between self and other perspectives is critical for adaptive social cognition and is thought to rely on the dynamic regulation of self-other representations. Although neuroimaging implicates the dorsomedial prefrontal cortex (dmPFC) and right temporoparietal junction (rTPJ) in perspective-taking, causal evidence for their specific contributions to perspective switching is lacking. Here, we applied focal transcranial direct current stimulation (f-tDCS) to the dmPFC and rTPJ while participants completed a visual perspective-taking task requiring switches between egocentric and altercentric viewpoints. Anodal stimulation to either site selectively increased the cognitive cost of switching back to the egocentric-perspective, without affecting switches into the altercentric-perspective. Rather than facilitating re-engagement with self-referential processing, stimulation enhanced altercentric persistence or impaired disengagement from the altercentric perspective. These findings provide novel causal evidence that both the dmPFC and rTPJ are involved in regulating the inhibition and updating of self-other representations during perspective switching. Results suggest that stimulation of these hubs may disrupt efficient realignment to the self, highlighting their role in maintaining an altercentric cognitive state. Future studies are required to uncover the precise neural computations that account for the comparable behavioural outcomes observed across distinct social brain hubs.

Mice wiggle a wheel to boost the salience of low visual contrast stimuli
From the Welsh tidy mouse to the New York City pizza rat, movement reveals rodent intelligence. Akin to humans shaking a computer mouse to find the cursor on a screen, we show that head-fixed mice develop an active sensing strategy in a visual perceptual decision-making task (The International Brian Laboratory, 2021). We demonstrate that mice wiggle a wheel that controls the movement of a stimulus during low visual stimulus contrast trials. When animals wiggle, the low visual stimulus contrast accuracy increases by 6.0% (Pearson correlation, r=0.900, p=0.038, N=5 group means computed from 213 mice). Moreover, mice wiggle the wheel at a speed that corresponds to a visual stimulus temporal frequency (11.52 {+/-} 2.45 Hz) demonstrated to maximize contrast sensitivity in a Go/No-Go task (Umino et al, 2018). These findings suggest that mice wiggle a wheel to boost the salience of low visual contrast stimuli.

Molecular aging is the main driver of Parkinson's Disease
Aging as well as the presence of a-synuclein (a-syn) oligomers in the brain are indisputably linked to Parkinsons disease (PD). A central concept of geroscience is that the biological processes of aging drive the onset of aging-associated diseases. The extent to which the biological processes of aging directly contribute to PD and the inter-relationship with a-syn oligomers for the onset of PD symptoms remains unclear. Using an inducible a-syn oligomer mouse model of PD, we demonstrate that the induction of PD associated a-syn oligomers for the same timespan caused PD associated symptoms only in aged, but not in young mice. Biochemical studies revealed that a-syn oligomer formation precedes motor decline in these aged mice, and age together with a-syn expression determine the motor phenotype. Single-nucleus RNA sequencing (snRNA-seq) identified a PD disease signature that was particularly linked to basal ganglia neurons (BGNs) and was in part shared with an aging transcriptional signature. PD symptoms, as well as the PD Signature, were significantly altered by a short-term pharmacological attenuation of the activity of the small RhoGTPase CDC42 in already aged animals with PD symptoms. Attenuation of activity of CDC42 is known to target the general biological processes of aging. Interestingly, the intervention did not affect the amount of a-syn oligomers in the animals, while still improving phenotypes. Together, the data demonstrates that the biological processes of aging are a major causative driver for the onset of PD in the a-syn model of PD.

Molecular Architecture and Function Mechanism of Tri-heteromeric GluN1-N2-N3A NMDA Receptors
N-methyl-D-aspartate receptors (NMDARs) play a pivotal role in brain development and synaptic function. Previous studies have focused on GluN1-N2 (2A-2D) and GluN1-N3 (3A and 3B) di-heteromeric (di-) NMDARs, leaving the activation mechanism and stoichiometry of GluN1-N2-N3 tri-heteromeric (tri-) NMDARs largely unexplored. In this study, we employed a two-step affinity-tagged chromatography approach to purify recombinantly expressed GluN1-N2A-N3A tri-NMDARs and determined their cryo-EM structure. Based on the proteoliposome single-channel recording, we discovered GluN1-N2A-N3A can be activated upon co-binding of glycine and glutamate, exhibiting two distinct conductance levels. Furthermore, leveraging structural-based click-chemistry, we introduced photo-crosslinker p-azido-phenylalanine (AzF) into the N-terminal domain of GluN2A and GluN2B, enabling the crosslinking with GluN3A subunit both in vitro and in vivo. These findings provide molecular insights into the subunit arrangement, native architecture and activation mechanism of GluN1-N2-N3A tri-NMDARs and also highlight the complexity of NMDAR assembly and function in the brain.