bioRxiv Subject Collection: Neuroscience's Journal

Inhibition of autophagy-lysosomal function exacerbates microglial and monocyte lipid metabolism reprograming and dysfunction after brain injury
CNS has an overall higher level of lipids than all tissues except adipose and contains up to 25% of total body cholesterol. Recent data demonstrate a complex crosstalk between lipid metabolism and inflammation, suggesting potential contribution of the lipid-rich brain environment to neuroinflammation. While recent data support the importance of brain lipid environment to inflammatory changes observed in age related chronic neurodegenerative diseases, in vivo interactions between lipid environment, lipid metabolism and neuroinflammation in acute brain disease and injury remain poorly understood. Here we utilize a mouse model of traumatic brain injury (TBI) to demonstrate that acute neurotrauma leads to widespread lipid metabolism reprograming in all microglial and brain associated and infiltrating monocyte populations. Additionally, we identify unique microglial and monocyte populations with higher degree of lipid metabolism reprograming and pronounced accumulation of neutral storage lipids, including cholesteryl esters and triglycerides. These lipids accumulate not only in lipid droplets but also in the microglial and monocyte lysosomes and are associated with lysosomal dysfunction and inhibition of autophagy after TBI. Our data indicate that lipid accumulation in these cells is the result of altered lipid handling rather than lipid synthesis and is triggered by phagocytosis of lipid-rich myelin debris generated after TBI. Finally, we use mice with autophagy defects in microglia and monocytes to demonstrate that further inhibition of autophagy leads to more pronounced lipid metabolism reprograming and exacerbated cellular lipid accumulation. Our data suggest a pathological feedback loop, where lipid phagocytosis causes inhibition of autophagy-lysosomal function, which in turn exacerbates cellular lipid retention, reprograming and inflammation.

REST/NRSF phosphorylation by CaMKIV regulates its transcriptional repressor activity and half-life
REST is a repressor of a large cluster of neural genes containing RE1 motifs. In neurons, REST homeostatically regulates intrinsic excitability and synaptic transmission, However, whether REST can be regulated by Ca2+ and Ca2+-activated kinases is unknown. Here, we investigated Ca2+/calmodulin-dependent protein kinases (CaMKs) as upstream regulators of REST fate and activity. We show that REST is phosphorylated by CaMKIV at a main Ser-322 site located within the linker between the 5th and the 6th N-terminal Zn-finger domains. Phosphomimic REST mutant in Serine-322 decreased REST repressor activity and caused its transition from nucleus to cytosol, followed by degradation. Molecular dynamics simulations of the phosphomimic N-terminal REST and the DNA RE1 sequence revealed a sharp decrease in the stability of the REST-RE1 binding interface. Moreover, the homeostatic effects of CaMKIV on the amplitude of excitatory synaptic currents were inhibited by the genetic deletion of REST. The results demonstrate that REST phosphorylation by CaMKIV regulates the repressor activity of REST on neural genes and has a crucial role in the homeostatic regulation of REST levels.

Multimodal imaging of human fetal brain development at the mesoscopic scale using 11.7 T ex vivo MRI
We present the first release of p-HCP (Prenatal Human Connectome Patterns), an imaging dataset of human fetal brain development covering the second half of gestation. This dataset was acquired ex vivo using magnetic resonance imaging (MRI) at ultra high field (11.7~teslas), and includes whole-hemisphere T2-weighted images at 100 {micro}m isotropic resolution, quantitative relaxometry (T1, T2, and T2*), and high angular resolution diffusion-weighted images for multiple b-values at 200 {micro}m. Brains larger than the workspace of the small-bore scanner were sectioned into blocks, acquired blockwise, and digitally reconstructed using a dedicated semi-automatic method. This initial data release includes three gestational ages (18, 27, and 31 post-conceptional weeks) with a complete set of anatomical images, relaxometry maps, and diffusion-based microstructure measurements. This dataset offers new opportunities to investigate neurodevelopmental processes that have not yet been explored with full three-dimensional coverage at this resolution by MRI, and may serve as a multimodal mesoscopic reference template for the fetal brain.

Modeling neurodegenerative diseases in Drosophila is conditioned by stress resistance and gut microbiome composition of the reference line
Drosophila is widely used to study the pathological mechanisms of human diseases in vivo, including metabolic and neurological disorders. In these models, disease-induced alterations in locomotion and stress resistance are generally monitored in comparison to healthy control flies, such the white-eyed strain w1118, used as a reference for normal physiology and behavior. Here we compared two independent w1118 lines and found that they differed strikingly in their susceptibility to oxidative stress and nutrient starvation, and less markedly in their locomotor performance. Interestingly, modulating the gut microbiome by rearing these flies under axenic conditions increased oxidative stress resistance of the more susceptible, but not the more resistant line, while it had no effect on starvation resistance for both lines. We also found that the stress-sensitive line had higher levels of Clostridiales bacteria and of the intracellular endosymbiont Wolbachia in the gut microbiota, as well as lower expression levels of immune effectors (antimicrobial peptides and lysozymes) in the head and gut. Both lines nevertheless showed similar susceptibility to pathogenic bacterial infections. In a transgenic Parkinson's disease model, the stress-resistant background strongly attenuated the progressive locomotor defects induced by pan-neuronal expression of human mutant -synuclein, but intriguingly not when -synuclein expression was restricted to a subset of brain dopaminergic neurons in the protocerebral anterior medial (PAM) cluster. These results suggest that taking into account unapparent features of the reference lines could improve the reproducibility and consistency of neurodegenerative disease models in Drosophila.

An Innate Immune Receptor Toll-1 converts chronic light stress into glial phagocytosis
Chronic stress can cause progressive neuronal degeneration, yet the molecular mechanisms linking stress sensing to neuroimmune responses remain elusive. In this study, a Drosophila model of chronic light-induced stress, we show that photoreceptor neurons accumulate reactive oxygen species (ROS) and exhibit Toll-1 activation, in association with Spz ligands and receptor endocytosis. Toll-1 activation in neurons promotes axonal degeneration by inducing expression of the glial phagocytic receptor Draper (Drpr), leading to the engulfment of stressed axons. Genetic interaction analyses indicate that Toll-1 functions upstream of Drpr in a stress-responsive signaling cascade. Importantly, blocking either Toll-1 or Drpr attenuates axon loss under light stress, highlighting their essential roles. Our findings reveal a neuron-glia communication axis in which neuronal innate immune signaling instructs glial phagocytosis, converting sustained environmental stress into structural degeneration. This study provides a mechanistic framework for sterile neurodegeneration and offers insights into how immune receptors regulate nervous system integrity under non-infectious conditions.

ALCOHOL AUGMENTS SPONTANEOUS GABAERGIC TRANSMISSION AND ACTION POTENTIAL FIRING IN IMMATURE MURINE CEREBELLAR GOLGI CELLS, LEADING TO ENHANCED INHIBITORY INPUT ONTO CEREBELLAR GRANULE CELLS
Golgi cells (GoCs) are cerebellar inhibitory interneurons that provide both phasic and tonic GABAergic input to cerebellar granule cells. They receive inhibitory control from Lugaro cells, other GoCs, and cerebellar nuclear inhibitory neurons via GABAergic and glycinergic inputs. Although fetal alcohol exposure is known to impair cerebellar function, its impact on developing GoC physiology remains unclear. We investigated the acute effects of ethanol on GABA-A receptor-mediated transmission in GoCs during the mouse equivalent of the human third trimester, a critical window for inhibitory circuit formation. To identify GoCs, we used VGAT-Venus transgenic mice, in which the vesicular GABA transporter promoter drives expression of the Venus fluorescent protein. Whole-cell patch-clamp and loose-patch recordings from postnatal day (P) 6-10 mice revealed that ethanol exposure dose-dependently increased the frequency of action potential-dependent GABA-A receptor-mediated spontaneous postsynaptic currents (sPSCs) in GoCs. While ethanol produced variable effects on GoC firing rates, it more consistently enhanced GABA-A-sPSC frequency in granule cells. We also examined expression of the K+-Cl- cotransporter 2 (KCC2), a chloride exporter whose developmental upregulation drives the shift in GABAA receptor signaling from excitatory to inhibitory. Immunohistochemical analysis at P6 showed that GoCs express low levels of KCC2, suggesting that GABA-A receptor-mediated currents may remain depolarizing in a subset of GoCs. This property could contribute to ethanol-induced disruption of cerebellar circuit maturation. Together, these findings provide new insight into the cellular mechanisms by which ethanol perturbs inhibitory circuit development in the cerebellum.

Layer-specific glutamatergic inputs and Parvalbumin interneurons modulate early life stress induced alterations in prefrontal glutamate release during fear conditioning in pre-adolescent rats
Exposure to early life stress (ELS) can exert long-lasting impacts on emotional regulation. The corticolimbic system including the basolateral amygdala (BLA), ventral hippocampus (vHIP), and the medial prefrontal cortex (mPFC) plays a key role in fear learning. Using the limited bedding paradigm (LB), we examined the functional consequences of ELS on excitatory and inhibitory tone in the prelimbic (PL) mPFC after fear conditioning in rats. In adults, LB exposure enhanced in vivo glutamate release in the PL mPFC during fear conditioning in male, but not female offspring. In contrast, the glutamate response to fear conditioning was diminished in LB-exposed pre-adolescent males, but not females. We investigated whether reduced glutamatergic inputs and/or elevated inhibitory tone might contribute to the diminished glutamate response in the mPFC following LB in pre-adolescent male rats. Indeed, we found that LB exposure specifically increased the activation of PV, but not SST interneurons in layer V, but not layer II/III of the PL mPFC in fear-exposed pre-adolescent males. Presynaptic glutamate release probability was reduced by LB exposure in layer V, but increased in layer II/III of the PL mPFC. These functional changes might be related to the LB-induced alterations in the bilaminar distribution of BLA and vHIP projections to the PL mPFC we observed in pre-adolescent males. Overall, our findings suggest that ELS modifies glutamate release and PL mPFC function during fear conditioning in a sex- and age- dependent fashion, likely through layer-specific shifts in excitation/inhibition balance.

Saturation, Task Error, and Feedback Timing Shape Early Implicit Adaptation
Motor adaptation is essential for maintaining coordination and precision in daily activities. Implicit motor adaptation - adaptation that occurs without conscious awareness - is thought to be primarily driven by sensory prediction errors. Here, we investigated how rapidly these unconscious changes in reaching behavior emerge as a function of error magnitude and the availability of task error signals. To this end, we employed a single-trial learning (STL) paradigm within a classical visuomotor rotation task. Participants made center-out reaching movements to either small (dot) or large (arc) targets while experiencing single perturbation trials with cursor rotations ranging from 1 to 90 deg, each followed by an aligned washout trial. By manipulating target size, we systematically modulated the presence of task error while holding sensory prediction error constant. We further compared these early implicit changes with those observed during standard prolonged adaptation to a fixed 20-deg rotation across >100 trials. Our results show that implicit adaptation emerges rapidly, even after a single exposure to small perturbations, and follows a saturating, fixed-rate response profile. Importantly, the magnitude of single-trial adaptation was greater when task error was present (small targets) compared with conditions in which only sensory prediction error was available (large targets). Moreover, STL-derived parameters moderately predicted the initial phase of adaptation during prolonged learning, suggesting that STL captures core dynamics of early implicit processes. These findings provide new insight into the mechanistic principles governing implicit motor adaptation. By identifying the parameters that drive early-stage error-based learning, this work refines current models of sensorimotor learning and highlights potential strategies for designing targeted training or rehabilitation protocols that leverage rapid adaptation processes to enhance motor performance and recovery.

A common cross-species atlas of cortical gray matter
Comparative neuroscience requires a framework that enables consistent anatomical comparison across species with widely differing brain size, folding, and specialization. We introduce a cross-species hierarchical atlas spanning rodents, nonhuman primates, and humans, built upon population-averaged minimal deformation templates (MDTs) to ensure anatomically consistent alignment. The atlas defines homologous cortical and subcortical regions through landmark-guided boundaries and multimodal nonlinear registration. By aligning each species to its MDT and harmonizing anatomical definitions across species, the framework supports robust comparisons of brain organization at multiple levels of granularity. Validation against species-specific atlases confirmed strong regional correspondence and systematic scaling across species. Released as a freely available resource, this common atlas provides a unified coordinate system to support comparative imaging, developmental studies, and cross-species connectomics.

Human-specific features of the cerebellum and ZP2-regulated synapse development
Understanding the unique features of the human brain compared to non-human primates has long intrigued humankind. The cerebellum refines motor coordination and cognitive functions, contributing to the evolutionary development of human adaptability and dexterity. To identify shared and divergent features across primates, we conducted single-nucleus transcriptomic and chromatin accessibility profiling of the adult cerebellar cortex in humans, chimpanzees, macaques, and marmosets. We revealed human-specific transcriptomic and regulatory features, particularly those involved in synaptogenesis. Notably, we identified an enrichment of the sperm receptor zona pellucida glycoprotein 2 (ZP2) and its potential interactors, known for their roles in gamete interaction, in human granule cells. Experimental data show that ZP2 expression in human granule cells is induced by pontine mossy fibers, reducing synaptic proteins at pontocerebellar glomerular synapses, and decreasing cerebellar neuron electrophysiological activity. This unexpected co-option of ZP2 in human-specific synapse regulation provides insights into the evolutionary specialization of the human cerebellum.

Developmental and Stress-Induced Effects on 5-HT3R-Expressing Interneurons within Auditory Cortex
Early life stress (ELS) is a well-known predictor of neuropsychiatric disease and contributes to the development of sensory processing deficits that persist throughout life. Organisms are particularly susceptible to the deleterious effects of stress during critical periods, when neuroplasticity is heightened, and initial representations of the sensory environment are mapped to cortex. When ELS is induced during the auditory cortical (ACx) critical period, it impairs both neural and behavioral responses to a variety of auditory stimuli that rely on temporal processing. Mechanisms by which ELS may alter critical period plasticity are of particular interest in understanding ELS-related pathology, including the 5-HT3R interneuron system, which has been implicated in regulating neural activity during critical periods. Here we examined two principal subpopulations of interneurons in primary ACx: VIP and NDNF cells, which account for a majority of cortical neurons expressing 5-HT3R. The expression of the Htr3a gene during normal development and under ELS conditions was quantified using multiplex fluorescent in situ hybridization. We show that densities of cells expressing NDNF and VIP decrease following ear opening and across the ACx critical period, and that ELS results in the maintenance of elevated cell densities compared to age-matched controls. Further, Htr3a in VIP neurons is developmentally upregulated, and its expression is further increased by ELS beyond normal physiologic levels. Stress-induced shifts in these serotonergic interneurons may contribute to deficits that arise in auditory cortical and perceptual responses via effects on local cortical circuitry.

Readout and delayed transmission of initial afferent V1 activity in decisions about stimulus contrast
Initial afferent activation of V1, indexed by the C1 component of the human VEP, is often considered to be a rudimentary stage of visual processing, operating mostly as a conduit for later stages with limited cognitive penetrability. The full suite of visual analysis entails activity across several visual areas and feedback from later areas to earlier ones. This raises the question of whether the early sensory representation indexed by the C1 is readout for perceptual decisions or whether it is passed over in favour of more advanced representations. To address this question, we asked whether the C1 would predict time-pressured stimulus contrast comparisons independently of physical stimulus conditions, a phenomenon known as choice probability. We found that the C1 did this for a narrow range of response times, indicative of decision readout since the C1 is a transient signal. This effect could not be accounted for by stimulus differences, choice history, or any other choice-predictive signal that we could identify in either the time or frequency domain, either before or after target onset. It also preceded the onset of evidence-dependent decision formation estimated from the centroparietal positivity by tens of milliseconds, together providing an approximate timeline of early evidence readout and its delayed impact on the decision.

EEG Phase Slips Reveal Detailed Brain Activity Patterns of Novice Vipassana Meditators During Decision Making Tasks
Our study adopted a novel approach, utilizing four biomarkers, namely EEG potentials, their first-order derivatives, and phase slip rates derived from each, to discern the differences between novice Vipassana (NVP) subjects and non-meditator controls (NMC). Phase slip rates are discontinuities in instantaneous phase that represent cortical phase transitions, indicating a significant change in the overall brain state. We employed 128-channel EEG data from eight NMC and eight NVP subjects, collected during a gamified protocol, to investigate object identification within a visual oddball paradigm. EEG was continuously acquired during the first 50 trials for each subject. We retained 44 artifact-free trials per subject (the minimum common across all subjects) and computed within-subject averages. The EEGs were then averaged over NMC and NVP subjects. The EEG was filtered in the alpha band, and the phase was extracted using the Hilbert transform, unwrapped, and the phase slip rates were computed. A montage layout of electrodes was used to make the spatiotemporal plots of biomarkers. Our findings revealed that the spatiotemporal profiles of all four biomarkers differed significantly between the two groups of subjects. Furthermore, the NVP subjects demonstrated faster object recognition. These results not only provide a unique method to use phase slip rates to quantify cognitive differences between NMC and NVP subjects but also present a promising set of biomarkers for quantitative task-based EEG analyses.

Muscle Stiffness, Not Force, Limits Human Force Production
Humans have a remarkable ability to manage physical interaction despite a tremendously complex musculoskeletal system, comparatively slow muscles and slow neural communication. Most physical interaction tasks require force generation, and that can compromise stability. To ensure stability, muscles are required to produce a certain mechanical impedance (especially stiffness and damping). In this work we investigate the human limits of force generation and test whether and how those limits are constrained by our ability to stabilize the task. Moreover, we also study how strongly limb configuration - known to play a key role in transmitting muscle tension to hand force - matters in the generation of force. We devised an experiment in which healthy individuals performed a maximum voluntary pushing task using a custom-designed gimbal apparatus which allowed us to conditionally change the ability to transmit torque at the wrist, thus enabling or disabling stabilization via wrist pronation-supination, ulnar-radial deviation, and flexion-extension. The experiment was repeated in two different arm configurations (preferred and undesired) to assess the effect of limb configuration. The results showed: (i) a statistically significant and substantial decrease in maximum output force with less wrist ability to generate stabilizing torque, and (ii) a significant but minimal difference in maximum pushing force between the two arm configurations. Interestingly, subjects unexpectedly improved their maximum pushing performance between pre- and post-experiment, suggesting a negligible effect of fatigue and a possible effect of learning. Our findings confirm our hypothesis that force production appears to be limited by stiffness production. Muscles do not primarily generate force, but generate mechanical impedance.

Adaptation of motor control strategies and physiological arousal during repeated blocks of split-belt walking
Physiological arousal, mediated by the autonomic nervous system (ANS), is known to co-modulate with adaptation of motor control strategies, governed by the central nervous system (CNS), in response to repeated standing perturbations. However, adaptation of the ANS physiological arousal response during repeat exposure to walking challenges remains unknown. This study examines the physiological arousal response (electrodermal activation, EDA) and motor control strategy of gait during a single session of repeated exposure to blocks of split-belt walking. Twenty young adults completed three repeated blocks (3.5 min each) of split-belt walking (2:1 speed ratio) alternating with three blocks of tied-belt walking. Step length symmetry (SLS), EDA, bilateral tibialis anterior (TA) and gastrocnemius medialis (GM) muscle activation and ground reaction forces (GRFs) were measured. For each walking block, the first (early) and last (late) 15 strides were analyzed. A linear mixed-effects model (LMM) tested the effect of repeated blocks and phases (early/late) on SLS. Statistical parametric mapping (SPM) examined patterns of within-block changes in EDA, muscle activation, and GRFs. The greatest within-block adaptation of SLS occurred during first exposure to split-belt walking (S1; p<.001). Similarly, the largest magnitude of within-block adaptation in muscle activation, GRFs, and EDA occurred during S1 (p<.05). Attenuated EDA responses together with lower magnitude of motor adaptation in subsequent split blocks 2 and 3 were observed, indicating savings across the ANS and CNS. Taken together, these findings demonstrate that the ANS-mediated physiological arousal response modulates alongside the CNS-driven locomotor adaptation to repeated exposure to blocks of split-belt walking.

Trial-by-trial fluctuations in decision criterion shape confidence
Many of the choices we make are accompanied by a sense of confidence. Within classical Signal Detection Theory (SDT), confidence is conceptualized as the absolute distance between a decision variable and a decision criterion. The decision criterion is traditionally modelled as being stable over an experimental session. However, recent work challenges the notion of a static decision criterion, suggesting instead that the criterion undergoes trial-by-trial fluctuations. Combining SDT theory and model simulations, we predict that fluctuations in the decision criterion shape confidence. In 15 human decision-making datasets, trial-by-trial estimates of decision criterion were obtained with the Hierarchical Model for Fluctuations in Criterion (hMFC). Across all datasets, we confirmed our pre-registered hypothesis that confidence is shaped by single-trial criterion state. This effect was found in 14 out of 15 individual datasets, indicating a robust pattern across a variety of task paradigms and confidence reporting scales. Going beyond self-report, the shaping of confidence by criterion fluctuations was replicated in an implicit measure of confidence, RTs, and in two key neurophysiological markers, pupil-linked arousal and a neural signature of confidence. Our results demonstrate that variability in confidence, which has traditionally been treated as noise, actually reflects genuine sensitivity to the current state of the (fluctuating) decision criterion.

Recent social experience alters song behavior in Drosophila
Innate behaviors are hardwired into the nervous system, allowing animals to produce them instinctively and without prior learning. This includes social behaviors (e.g., aggression, courtship) in most animals. If and how learning shapes such innate behaviors has not been systematically explored. Here, we investigate learning from social feedback in Drosophila. We use closed-loop optogenetics to systematically perturb the social experience of individual flies during courtship, and find that altered feedback changes the male fly's innate courtship strategy, opening the door for investigating the neural and molecular basis of social plasticity in flies.

Early Diagnosis of Parkinson via Transient Beta Frequencies in a Delayed Van der Pol Model
Biological motor circuits are shaped by pathway-specific delays that generate history-dependent oscillations and short-lived transients not captured by memoryless models. We develop a delayed Van der Pol in which the delay ratio r acts as a control parameter for Hopf bifurcation and internal resonance and we pair it with an auditable, short-window signal-processing pipeline tailored to early beta activity. The pipeline combines Short-Time Fourier Transform (STFT), Continuous Wavelet Transform (CWT) ridge tracking, kernel-density estimates (KDE) of the dominant frequency, and algorithmic beta-burst detection under fixed, shared parameters. Transient burden is quantified by a metric triplet with a software-implementable attenuation rule and complemented by an early-detection index-the Transient Persistence Time. Across methods, we observe a monotonic attenuation of short-lived beta transients as r approaches a critical band. A reliable observation window of 0-0.35 s captures compact early packets at low r, progressive ridge stabilization in CWT, KDE narrowing over time, and near-elimination of bursts for near critical r, consistent with a transition to sustained narrowband rhythm. Clinically, these measures enable stage inference, a bedside Move-Stop protocol for rapid readout, and actionable policies for adaptive DBS and medication titration that minimize transient burden without inducing rigid locking. The fixed parameterization and audit-ready outputs support reproducibility and multi-center deployment. This framework advances data-driven, precision neuromodulation by targeting early transients before pathological synchrony becomes entrenched.

APOE4 promotes cerebrovascular fibrosis and amyloid deposition via a pericyte-to-myofibroblast transition
Cerebrovascular disease is a prominent but poorly understood component of Alzheimer's disease (AD). The strongest genetic risk factor for AD, APOE4, is associated with multiple cerebrovascular pathologies, including vascular amyloid accumulation and fibrosis. APOE4 also renders the cerebrovasculature fragile to the point where the only disease-modifying AD therapeutic, anti-amyloid monoclonal antibodies, are warned against use in APOE4 carriers due to a high risk of hemorrhage and edema. To determine how APOE4 impacts cerebrovascular cells and pathogenesis, we generated a high-resolution single-cell transcriptomics atlas of human cerebrovasculature from APOE4 carriers and non-carriers. In APOE4 individuals, we found that the number of microvascular pericytes was significantly reduced. Notably, loss of pericytes in APOE4 carriers was accompanied by the emergence of a unique population of mural cells characterized by high expression of contraction and extracellular matrix (ECM) genes, hallmarks of myofibroblasts. Immunostaining revealed the presence of myofibroblasts surrounding the microvasculature in APOE4 human hippocampi that were absent in age-matched APOE3/3 controls, even in cases of AD. Myofibroblast presence coincided with a significant increase in fibronectin and amyloid surrounding the vasculature. Myofibroblasts were also present around the microvasculature of aged APOE4/4 but not APOE3/3 mice, suggesting myofibroblast appearance is a direct effect of the APOE4 genotype and not a technical artifact of processing human tissue. To determine the mechanisms and functional implications of APOE4-mediated myofibroblast, we leveraged a vascularized human brain tissue (miBrain) derived from induced pluripotent stem cells. Similar to the post-mortem human brain, APOE4/4 miBrains showed significantly reduced microvascular pericyte coverage, coinciding with the emergence of myofibroblasts co-expressing ECM and contractile genes. Lineage tracing and genetic mixing experiments confirmed that the myofibroblasts emerge from APOE4 pericytes and secrete fibronectin-rich ECM. Knocking down fibronectin in APOE4 mural cells significantly reduced vascular amyloid accumulation. To determine how myofibroblast-like cells arise in the APOE4 brain, we performed computational analysis (NicheNet) on our post-mortem human single-cell transcriptomics cerebrovascular atlas. This predicted that TGF-{beta} is the top causal driver of the pericyte-to-myofibroblast transition. Consistent with this prediction, we found that TGF-{beta} ligands in astrocytes and receptors in mural cells are significantly upregulated compared to APOE3 controls. Chemical and genetic inhibition of TGF-{beta} signaling in APOE4 miBrains significantly reduced myofibroblast presence, while concurrently increasing pericyte microvascular coverage and ultimately lowering vascular fibrosis and amyloid accumulation to APOE3 levels. Using a comprehensive three-pronged approach incorporating analysis of human post-mortem brain, APOE humanized mice, and human iPSC-derived vascularized brain tissue, we demonstrate that APOE4 in mouse and human brain tissue causes increased TGF-{beta} signaling, promoting a pericyte-to-myofibroblast transition that leads to vascular fibrosis and amyloid deposition. This provides critical insight into the mechanisms underlying APOE4 cerebrovascular dysfunction, highlighting new diagnostic and therapeutic strategies for a major AD risk population.

3D MINFLUX combined with DNA-PAINT resolves the arrangement of Bassoon at active zones
Neurotransmitter release and membrane retrieval at active zones require precise spatial and temporal coordination relying on an intricate molecular machinery. However, the exact nano-structural organization of this machinery is not yet fully elucidated. Here, we used 3D MINFLUX combined with both spectral demixing and DNA-PAINT to analyze the positioning of the scaffolding protein Bassoon at presynaptic active zones of glutamatergic spine synapses of hippocampal neurons, achieving a localization precision of 5 nm in 3D. This approach allowed us to visualize directly the distribution of N-terminal and the C-terminal regions of Bassoon, and demonstrates that Bassoon exhibits an orientation at the active zone, where the C-terminal region is directed toward the synaptic cleft and the N-terminal region towards synaptic vesicles. Having demonstrated this spatial configuration in an exemplary fashion for endogenous and recombinant Bassoon molecules, our study paves the way towards molecular-scale resolution analysis of other prominent proteins of the presynaptic release machinery.

Elevated IL-1 beta plasma levels, altered platelet activation and cardiac remodeling lead to moderately decreased LV function in Alzheimer transgenic mice after myocardial ischemia and reperfusion
Introduction: Neurodegeneration and dementia are key factors in Alzheimers disease (AD). The deposition of amyloid-beta into senile plaques in the brain parenchyma and in cerebral vessels known as cerebral amyloid angiopathy (CAA) are the main clinical parameters of AD. Acute myocardial infarction (AMI) and AD share a comparable pathophysiology. In detail, AMI survivors have a higher risk of vascular dementia and AD patients with AMI face a poor prognosis with a high rate of major adverse cardiovascular events. High blood levels of amyloid-beta 40 are identified in patients with high risk for cardiovascular death. However, the underlying mechanisms and the consequences of AMI in AD patients are unclear to date. Methods: AD transgenic APP23 mice were analysed in an experimental AMI using the closed-chest model. Results: APP23 mice displayed significantly decreased left ventricular function as detected by FS/MPI (fractional shortening/myocardial performance index) after 24 h and 3 weeks after ligation of the LAD compared to WT controls. No differences have been observed in infarct and scar size. However, the analysis of cardiac remodeling after 3 weeks showed an altered composition of the collagen tissue of the scar with elevated tight but reduced fine collagen in APP23 mice. Altered scar formation was accompanied by elevated degranulation of platelets following activation of the collagen receptor GPVI. Conclusion: The here presented results suggest that AD patients are at higher risk for cardiac damage after AMI that might increase the risk for cardiovascular death. This implies the need of a personalized therapy of AMI in AD patients.

The 5-HT1A receptor agonist NLX-112 rescues motor swimming deficits in Spinocerebellar Ataxia type 3 mice
Introduction: Spinocerebellar ataxia 3 (SCA3) is a rare neurodegenerative disorder which causes progressive motor disturbances. There is no approved drug treatment but selective activation of serotonin 5-HT1A receptors may be a promising therapeutic strategy to attenuate ataxia symptoms. Methods: NLX-112, a highly selective 5-HT1A full agonist, was tested in the CMVMJD135 transgenic mouse model of SCA3. NLX-112 (1.25 and 5 mg/kg/day) was administered BID intraperitoneally for 14 weeks starting when the mice were 12 weeks of age, i.e., after ataxia signs had become established. The motor swimming test (MST), where mice are required to swim to a raised platform, was used to evaluate the motor behavior of SCA3 mice and their performance was compared with that of wild-type (WT) mice. Results: Both doses of NLX-112 were well tolerated by the SCA3 mice, as assessed by welfare parameters. In the MST, the latency of SCA3 mice to reach the platform was significantly longer than that of WT mice. However, when SCA3 mice were treated with either 1.25 or 5 mg/kg/day of NLX-112, they showed robust improvement of motor performance, with swimming latencies which were similar to those of WT mice. This effect of NLX-112 was maintained throughout the period of the study. Conclusions: The improved motor function of SCA3 mice when treated with NLX-112 supports its investigation as a drug candidate for the treatment of ataxia and related movement disorders.

Analyses of exon 4a structure reveal unique properties of Big tau
Tau is a microtubule-associated protein that modulates the dynamic properties of microtubules and is involved in neurodegenerative diseases known as tauopathies. Tau is expressed as multiple low molecular weight (LMW) isoforms in most neurons of the central nervous system but only as a high molecular weight isoform in neurons of the peripheral nervous system and in a few types of central neurons. Big tau is defined by the inclusion of the alternatively spliced exon 4a, which adds about 250 amino acids to the domain of tau that projects away from the microtubule. Despite low sequence conservation of exon 4a, its length remains remarkably consistent across vertebrates. Here, we analyzed the charge distribution, hydrophobicity, and aggregation propensity of the human sequences of LMW tau, Big tau and the stretch of amino acids encoded by exon 4a. The exon 4a amino acids display a pronounced net negative charge (acidic/basic ratio = 1.30), a consistently hydrophilic composition (average Kyte-Doolittle score = -0.9259) and low {beta}-sheet content of 4.78%. This contrasts with LMW tau, which is more hydrophobic (-0.8930) and contains extended aggregation-prone motifs within the microtubule-binding domain including high {beta}-sheet content of 17.33%. The inclusion of exon 4a in Big tau shifts the global hydrophobicity to intermediate values (-0.9036) and reduces predicted {beta}-sheet content to 13.14%, suggesting decreased aggregation potential. Evolutionary analyses across mammals, birds, and amphibians (human, rat, zebra finch, frog) confirms the minimal sequence identity (16-24% identity in non-mammals) and conserved exon size but show preservation of net negative charge (acidic/basic ratio 1.3-2.3), indicating convergent retention of charge-based properties. Hydrophilicity was also broadly conserved, though less invariant across species. These results demonstrate that exon 4a introduces a highly acidic, hydrophilic module that counterbalances the aggregation-prone domains of LMW tau. The conservation of size and structural properties of the exon-4a-encoded stretch of amino acids, despite sequence divergence, implies strong evolutionary pressure to maintain biophysical properties that counteract pathogenic misfolding.

miR-146a is a Pleiotropic Regulator of Motor Neuron Degeneration
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting motor neurons. Here, we have profiled motor neuron microRNAs (miRNAs) during motor neuron degeneration in vivo to gain a better understanding of ALS pathophysiology. We demonstrate that one miRNA, miR-146a, is downregulated in diseased motor neurons despite upregulation in bulk tissue. Genetic deletion of miR-146a significantly extended survival in SOD1G93A mice with heterozygous animals demonstrating the largest benefit. A corresponding reduction in spinal cord gliosis but not motor neuron loss was observed. Finally, we observed that a proportion of miR-146a knockout animals develop spontaneous paralysis, motor neuron loss and chronic neuroinflammation with advanced age. Together these findings demonstrate that a single miRNA influences multiple aspects of motor neuron disease and highlights the complex role for neuroinflammation in ALS pathogenesis.

A neuroendocrine principle: Pancreatic islets actively shape sympathetic innervation
Survival critically depends on maintaining blood glucose to provide essential energy, especially during emergencies such as the fight-or-flight response, when timely glucose control via neural integration is vital. However, pancreatic islets constitute only a small fraction of the pancreas and are dispersed throughout the organ, raising the fundamental question of how the nervous system coordinates synchronized control of multiple islets. Using whole-organ clearing and 3D imaging, we mapped pancreatic sympathetic innervation, revealing specialized anatomical integration between sympathetic nerves and islets. Transplanted islets intrinsically attracted sympathetic nerves independent of their native environment. Chronic islet injury disrupted sympathetic innervation and impaired nerve regeneration after denervation. Sympathetic denervation markedly elevated islet-derived Reg2 and Reg3{beta}; administration of these proteins accelerated sympathetic regeneration and improved islet graft function. Our findings identify an islet-sympathetic architecture actively maintained by islets, uncovering an endocrine-driven mechanism for neural regulation, highlighting Reg2 and Reg3{beta} as therapeutic candidates for diabetes management.

Shaker it OFF: Biophysical Characterization of an Inactivating Potassium Conductance Mediating Object Segmentation in a Collision-Detecting Neuron
Homologous ion channels are expressed in sensory neurons across species where they shape responses to varied sensory stimuli, on a wide range of timescales. In grasshoppers, visual detection of looming stimuli which simulate an approaching predator involves the sub-cellular expression pattern of a newly characterized Shaker channel in a single identified neuron, discovered using a newly sequenced genome and single cell RNA sequencing. This channel shapes selectivity for approaching threats and was characterized with electrophysiological recordings, RNA interference, pharmacology and biophysical compartmental simulations. Our results explain how a slowly activating and inactivating potassium conductance common in neuronal dendrites contributes to visual object segmentation and implements a complex neural computation within a single neuron.

Characterization of the Lipidome of Neurons in Mouse Brain Nuclei using Imaging Mass Spectrometry
Understanding the molecular composition of the brain at cellular level is essential for deciphering the metabolic alterations associated with brain diseases. Furthermore, the different prevalence of some neurological diseases between males and fe-males highlight the importance of incorporating gender factor in such studies. Here, we demonstrate that using imaging mass spectrometry in negative polarity it is possible to isolate and characterize the lipidome of specific neuronal populations in the mouse brain, including the locus coeruleus (LC), mesencephalic neurons and the substantia nigra pars compacta (SNc). Neuronal identity was validated through immunofluorescence on adjacent serial sections. Comparative analysis revealed that each neuronal population presents a distinct and well-defined lipidic profile, with differences extending across all lipid classes analyzed. Regarding sex-based differences, we found discrete differences in phosphatidylcholine/phosphatidylethanolamine-ether, phosphatidylinositol and sphingomyelin LC neurons. Lipidomic differences were more pronounced in mesencephalic neurons, whereas no significant sex-defendant differences were observed in SNc lipid composition. These findings lay the groundwork for future studies aimed at identifying lipid metabolic dysregulations in the context of neurodegenerative diseases.

Exploring Brain State Changes Through Reaction Time in a Simultaneity Judgment Task: A Pilot Study on Multisensory Integration
Problem statement: Multisensory Integration (MSI), the brains ability to merge information from different sensory modalities, relies on the Temporal Binding Window (TBW)-the interval during which stimuli are most likely to be perceived as simultaneous. Beyond its role in perception, MSI may also reflect changes in brain state as task difficulty evolves. In this pilot study, we aimed to explore whether behavioral metrics, particularly reaction time (RT), can capture these brain state changes during a Simultaneity Judgment (SJ) task. Understanding this relationship is critical for developing adaptive Brain-Computer Interfaces (BCIs) that rely on real-time cognitive and perceptual feedback. Approach: To assess how temporal alignment influences both perception and underlying brain state, we employed a Two-Alternative Forced Choice (2AFC) SJ task using audiovisual stimuli. After estimating individual simultaneity thresholds, we implemented an adaptive task that gradually increased difficulty by manipulating the stimulus onset asynchrony (SOA) closer to the TBW. We hypothesized that as perceptual uncertainty increased, RTs would reflect evolving cognitive states, serving as an observable indicator of underlying neural activity shifts. Materials and Methods: Two participants were tested with consistent auditory (500 Hz tone) and visual (faded white circle) stimuli. The TE phase used a staircase procedure to determine personalized TBWs. In the subsequent adaptive test phase, SOAs were progressively modulated around these thresholds to induce varying levels of perceptual ambiguity. Behavioral responses were analyzed using psychometric curve fitting, confusion matrices, and detailed RT evolution across trials. Results and Discussion: Participant 1 showed clear RT modulation near the TBW, suggesting heightened cognitive load and decision uncertainty - a potential signature of brain state transitions. In contrast, Participant 2 exhibited less sensitivity, possibly due to suboptimal thresholding. These preliminary findings suggest that RT holds promise as a behavioral correlate of perceptual state; however, further refinement and standardization of the task are needed before it can be reliably scaled to larger and more controlled studies of MSI-linked brain dynamics.

Axo-Axonic Synapses on Descending Neurons in the Drosophila Ventral Nerve Cord
Axo-axonic synapses can veto, amplify, or synchronize spikes, yet their circuit-scale logic is unknown. Using the complete electron-microscopy connectome of the adult male Drosophila ventral nerve cord (MANC v1.2.1), we charted every axo-axonic input onto the 1,314 descending neurons that carry brain commands to the body. Only 1.0 % of the 861,591 possible Descending-Descending neuron pairs form such contacts, but when present, synaptic weight grows linearly with partner number regardless of transmitter identity. High-degree neurons integrate the network without forming a rich-club core. Large-scale simulations singled out an octet of ascending neurons (AN08B098) whose axo-axonic input to the Giant Fiber descending neurons (DNp01) predicted modulation of escape control. Immunostaining confirms their cholinergic identity, while optogenetic activation confirmed that this excitatory cohort increases Giant Fiber excitability, validating connectome-derived rules. Our work delivers the first map of axo-axonic wiring and constraints for models of fast motor control.

Redox regulation of neuroinflammatory pathways contributes to damage in Alzheimers disease brain
The mechanism(s) whereby redox stress mediates aberrant immune signaling in age-related neurological disorders remains largely unknown. Normally, the innate immune system mounts a robust response to infectious stimuli. However, unintentional activation by host-derived factors, such as aggregated proteins associated with neurodegenerative disorders or by cytoplasmic genomic or mitochondrial DNA, can elicit aberrant immune responses. One such immune response is represented by the cytosolic GMP-AMP synthase stimulator of interferon genes (cGAS-STING) pathway. Using redox chemical biology and mass spectrometry approaches, we identified S-nitrosylation of STING cysteine 148 as a novel posttranslational redox modification underlying aberrant type 1 interferon signaling in Alzheimers disease (AD). Critically, we observed S-nitrosylated STING (SNOSTING) in postmortem human AD brains, in hiPSC-derived microglia (hiMG) exposed to amyloid-{beta} (A{beta})/-synuclein (Syn) aggregates, and in 5xFAD transgenic mice. Mechanistically, our findings reveal that STING S-nitrosylation is critical in initiating signaling cascades by promoting the formation of disulfide-bonded STING oligomers. This leads to neuroinflammation early in the course of disease in vivo in 5xFAD mice with consequent synaptic loss. Collectively, our research supports the role of SNO-STING in neuroinflammation associated with AD, and points to a novel druggable cysteine residue in STING to prevent this S nitrosylation reaction with its inherent inflammatory response.

The interaction between aging and DNA damage influences the accumulation of APP in Alzheimer's disease
DNA damage is a major driver of the aging process. The progression of Alzheimer's disease (AD) worsens the situation as evidenced by the finding that the neurons of the AD brain have elevated levels of DNA damage. AD is also associated with the amyloid precursor protein (APP) known for its proteolytic peptide fragment, A{beta}. In the current work we find that these three elements - age, DNA damage, and APP - are associated with each other and present evidence in the mouse brain supporting the hypothesis that DNA damage is the likely cause of the increased APP levels. Using the TUNEL reaction to track DNA damage, we show that the TUNEL staining increases significantly with age (6 months to 24 months). Next, we used immunostaining of intracellular APP to show that it too increases over this same time period. To separate correlation from causality we repeated these analyses using tissue from mice genetically deficient in the ATM (ataxia-telangiectasia mutated) kinase, an enzyme associated with part of the DNA damage repair mechanism. TUNEL signal in neurons of 6-month Atm-/- animals was equivalent to that seen in 24-month wild type. Significantly, the APP signal in the Atm-/- cells increased in consort and the relationship between DNA damage and APP was found in both neuronal and non-neuronal cells. These results suggest that the loss of genomic integrity associated with aging is a cellular stressor that increases the levels of APP and thus increases vulnerability to the pathogenesis of AD.

MiR-34a deficiency enhances nucleic acid sensing and type I IFN signaling in a mouse model of Alzheimer's disease
MiR-34a is implicated in aging, cell senescence, inflammation, and neurodegenerative diseases. In order to investigate the role of miR-34a in Alzheimer's disease (AD), we produced an AD mouse model, Tg-SwDI mice, with whole body/constitutive miR-34a knockout (KO). MiR-34a KO improved long-term memory in Tg-SwDI mice, which was associated with decreases in the ratio of insoluble A{beta}42 to A{beta}40 and with increases in soluble and insoluble A{beta}40 in the cerebral cortex. Anti-Iba1 immunofluorescence revealed increases in activated microglia. Bulk RNA-sequencing of the hippocampus followed by a gene set enrichment analysis (Enrichr) identified '' cellular response to type I interferon '' and '' type I interferon signaling pathway'' as the most prominent gene sets in miR-34a KO Tg-SwDI mice compared to miR-34a wild-type Tg-SwDI mice. Many interferon-stimulated genes (ISGs) that characterize interferon responsive microglia (IRM) were upregulated in miR-34a KO Tg-SwDI mice. MiR-34a knockdown strongly enhanced ISGs expression in TLR7 ligand-stimulated BV2 microglia. These results suggest that miR-34a inhibits the transition of microglia to the IRM state that may modulate synaptic and cognitive functions in neurodegenerative diseases and aging.

Interactions of the LINE-1 encoded ORF1p with proteins and chromatin converge on a role in neuronal physiology
Retrotransposons are emerging as novel regulators of embryonic and brain development. We recently demonstrated that the LINE-1 encoded protein ORF1p is abundantly expressed in adult mouse and human neurons, although its function remains unclear. Here, we characterize the ORF1p interactome in differentiated mouse and human neurons using mass spectrometry and identify novel partners implicated in gene regulation and neuron-specific processes. ORF1p localizes not only to neuronal nuclei, where it associates with chromatin under steady-state conditions, but also to neurites, supporting a role in neuronal physiology. To further explore its nuclear functions, we sorted human post-mortem neurons with high or low nuclear ORF1p levels and performed ORF1p knockdown in cultured human neurons, followed by chromatin accessibility assays. Both approaches revealed consistent patterns of differential chromatin accessibility dependent on ORF1p. Loss of ORF1p also led to the downregulation of long, neuron-specific genes and altered neurite morphology. Together, these findings point to a physiological role of ORF1p in post-mitotic neurons, mediated through converging interactions with proteins and chromatin.

TDP-43 loss of function drives aberrant splicing in Parkinson's disease
While mRNA splicing dysregulation is a well-established contributor to neurodegeneration in disorders such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), its role in Parkinson's disease (PD) remains underexplored. Here, we analyse transcriptomic data from >500 post-mortem human brain samples from individuals with and without PD to show that splicing alterations are frequently detected. Differentially spliced genes were significantly more enriched for those causally-implicated in both PD and ALS than genes that were differentially expressed. Furthermore, we observed a strong association between these splicing alterations and dysfunction of the RNA-binding protein (RBP), TAR DNA-binding protein 43 (TDP-43). Strikingly, genes and exon junctions affected by TDP-43 knockdown overlapped significantly with those dysregulated across brain regions in PD. In brains from individuals with the LRRK2 c.6055G>A (p.G2019S) mutation, the most common genetic cause of PD, we also observed significant enrichment of TDP-43-dependent splicing changes. This finding was corroborated in human pluripotent stem cell-derived midbrain dopaminergic neurons and a LRRK2 p.G2019S knock-in mouse model, where reduced nuclear TDP-43 levels evidenced the well-recognised loss-of-function mechanism contributing to splicing dysregulation. By leveraging our RNA-based analyses we predicted TDP-43-dependent novel peptide sequences and validated their existence within human LRRK2 mutation mDNs, while also demonstrating an overall loss of protein and mRNA expression in mis-spliced genes. Collectively, our findings reveal that PD is marked by extensive splicing dysregulation dependent on TDP-43, making TDP-43 a promising new therapeutic target in PD.