bioRxiv Subject Collection: Neuroscience's Journal
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Wednesday, January 1st, 2025
| Time |
Event |
| 9:31p |
Persistent DNA methylation and downregulation of homeostatic genes in astrocytes after pilocarpine-induced status epilepticus: Implications for epileptogenesis
Epilepsy is a debilitating neurological disorder characterized by recurrent seizures, affecting millions of patients worldwide. Retrospective studies in Temporal lobe epilepsy (TLE) patients have shown a high incidence of an initial precipitating event (IPE) in early childhood followed by a silent period where epileptogenesis occurs to end up in chronic epilepsy. Epileptogenesis, the process through which a normal brain undergoes structural and functional changes leading to epilepsy, remains an enigmatic phenomenon. We hypothesized that epigenetics may be involved in epileptogenesis and specifically astrocytes could be affected by pathological remodeling. To study this process, we used three approaches: The lithium-pilocarpine model of TLE in rats, primary astroglial cultures exposed to epileptogenic HMGB1, and brain tissue samples resected from TLE patients with drug-resistant epilepsy. We found that the IPE achieved by lithium-pilocarpine treatment (127/30 mg/kg IP) induced the hypermethylation of astrocytes at 7-, 21-, and 35 days post-IPE, indicating persistent epigenetic alterations in astrocytes during the epileptogenic period. In addition, we observed the downregulation of homeostatic astroglial genes AQP4; glutamine synthase (GS), and Kir4.1 with increased proinflammatory genes (C3, MAFG) and DNMT expression. These alterations were mimicked in primary astrocyte cultures exposed to the epileptogenic DAMP HMGB1 (500 ng/ml; 18 hours) that also induced the hypermethylation of homeostatic astroglial genes. Astrocytes from TLE patients' brains showed reactive astrogliosis, increased DNA methylation, and downregulation of homeostatic genes Kir4.1 and GS. These findings show that astrocytes are pathologically altered during the epileptogenic period, combining the proinflammatory gain of function with the loss of homeostatic profile. This may sustain the long-term alterations underlining epileptogenesis. | | 10:47p |
Circuit dynamics of approach-avoidance conflict in humans
Debilitating anxiety is pervasive in the modern world. Choices to approach or avoid are common in everyday life and excessive avoidance is a cardinal feature of all anxiety disorders. Here, we used intracranial EEG to define a distributed prefrontal-limbic circuit dynamics supporting approach and avoidance. Presurgical epilepsy patients (n=20) performed an approach-avoidance conflict decision-making task inspired by the arcade game Pac-Man, where participants trade-off real-time harvesting rewards with potential losses from attack. As patients approached increasing rewards and threats, we found evidence of a limbic circuit mediated by increased theta power in the hippocampus, amygdala, orbitofrontal cortex (OFC) and anterior cingulate cortex (ACC), which then drops rapidly during avoidance. Theta band connectivity between these regions increases during approach and falls during avoidance, with OFC serving as a connector in this circuit with high theta coherence across limbic regions, but also with regions outside of the limbic system, including the lateral prefrontal cortex. Importantly, the degree of OFC-driven connectivity predicts how long participants approach, with enhanced network synchronicity extending approach times. Finally, under ghost attack, the system dynamically switches to a sustained increase in high-frequency activity (70-150Hz) in the middle frontal gyrus (MFG), marking the retreat from the ghost. The results provide evidence for a distributed prefrontal-limbic circuit, mediated by theta oscillations, underlying approach-avoidance conflict. | | 10:47p |
Detection of HTTex1p by western blot and immunostaining of HD human and mouse brain using neo-epitope antibody P90 highlights impact of CAG repeat expansion on its size, solubility, and response to MSH3 silencing
HTT1a has been identified in human and mouse HD brain as the pathogenic exon 1 mRNA generated from aberrant splicing between exon 1 and 2 that contributes to aggregate formation and neuronal dysfunction (Sathasivam et al., 2013). Detection of the HTT exon 1 protein (HTTex1p) has been accomplished with surrogate antibodies in fluorescence-based reporter assays (MSD, HTRF), and immunoprecipitation assays, in HD postmortem cerebellum and knock-in mice but direct detection by SDS-PAGE and western blot assay has been lacking. Here proteins in subcellular fractions prepared from human and mouse HD brain were separated by SDS-PAGE and probed by western blot with neo-epitope monoclonal antibodies (P90-1B12 and 11G2) directed to the C-terminal 8 residues of HTTex1p. In human HD putamen and cortex, HTTex1p migrated at 56-60 kD and at higher molecular masses (HMM) consistent with the presence of CAG repeat expansion in HTT1a. HTTex1p in control brain was low or undetectable. Immunofluorescence labeling of human HD cortex using P90-11G2 revealed small aggregates that sparsely populated the neuropil in layers 3 and 5. In caudate putamen of 6 month old HD knock-in mice (Q50, Q80, Q111, Q140 and Q175) HTTex1p migration was inversely correlated with CAG repeat length and appeared as a SDS soluble high molecular mass (HMM) smear in HD Q111, Q140 and Q175 mice but not in Q50 and Q80 mice indicating a CAG repeat size threshold for detecting HTTex1p aggregation. Migration of HTTex1p and HMM smear changed with age in caudate putamen of Q111, Q175 and YAC128 mice. Treating HD Q111 mice with siRNA to MSH3, a modifier of CAG repeat expansion, significantly reduced levels of the HMM smear indicating that the effects of curbing CAG repeat expansion was quantifiable. These results show that P90 antibodies can be used in western blot assays and immunostaining to track and quantify HTTex1p levels, subcellular localization, and solubility. | | 10:47p |
Brain activation for language and its relationship to cognitive and linguistic measures
Language learning and use are complex cognitive skills requiring domain-general cognition and sensori-motor skills, these also being important for numerical and music processing. Previous studies have explored behavioural associations across domains and neural underpinnings of specific abilities, yet less work has examined these questions together in the same participants. We collected data from 152 participants on behavioural measures of language, reading, multilingual experience, cognition, musicality, arithmetic, and motor skills, along with fMRI data during L1 story listening. Participants varied in multilingual language experience and reading aptitudes, including both typical (TRs) and dyslexic readers (DRs). Using multivariate Partial Least Squares correlation, we identified a main component linking cognitive, linguistic, and phonological measures to brain areas underlying lexico/semantics, combinatorial processing, and amodal semantic processing. A second analysis excluding DRs showed closer associations between cognitive/linguistic, literacy, phonological and memory processes within the same brain network as in the full sample. Here, we also isolated additional, complementary components, including one involving speed, automatization and lexical access, linked to auditory and motor brain areas. This suggests greater coherence and more integrated, 'expert' processing in TRs. This work is a first step in exploring complex relationships between language and non-linguistic functions that are important to it. | | 10:47p |
Cell-type specific profiling of human entorhinal cortex at the onset of Alzheimer's disease neuropathology
Neurons located in the layer II of the entorhinal cortex (ECII) are the primary site of pathological tau accumulation and neurodegeneration at preclinical stages of Alzheimer's disease (AD). Exploring the alterations that underlie the early degeneration of these cells is essential to develop therapies that delay disease onset. Here we performed cell-type specific profiling of the EC at the onset of human AD neuropathology. We identify an early response to amyloid pathology by microglia and oligodendrocytes. More importantly, we find that the Reelin signaling pathway is already impaired at this early disease stage, particularly in ECII neurons. This indicates that dysregulation of this pathway, with emerging genetic association with AD, plays a pivotal role in the selective vulnerability of the EC and in the onset of AD neuropathology. | | 10:47p |
Different mechanisms link gain and loss of kinesin functions to axonal degeneration
Axons are the slender, often meter-long projections of neurons that form the biological cables wiring our bodies. Most of these delicate structures must survive for an organism's lifetime, meaning up to a century in humans. Long-term maintenance and sustained functionality of axons requires motor protein-driven transport distributing life-sustaining materials and organelles to places of need. It seems therefore plausible that loss of motor function can cause axon degeneration; however, also gain-of-function conditions were linked to disorders including motor neuron disease or spastic paraplegia. To understand this phenomenon, we studied ~40 genetic manipulations of motor proteins, cargo linkers and regulators of reactive oxygen species in one standardised Drosophila primary neuron system. Using axonal microtubule bundle organisation as a relevant readout reflecting the state of axon integrity, we found that losses of Dynein heavy chain, KIF1A/Unc-104 and KIF5/Kinesin heavy chain (Khc) all cause bundle disintegration in the form of chaotically curled microtubules. Detailed functional studies of Khc and its adaptor proteins revealed that losses of mitochondrial or lysosomal transport cause ROS dyshomeostasis, which is a condition that inducing MT-curling in fly and mouse neurons alike. We find that hyper-activated Khc induces the same MT-curling phenotype, not through ROS but directly through enhanced mechanical forces. Studies with loss of Unc-104 and expression of an ALS-linked mutant form of the human Khc orthologue KIF5A suggest that the two mechanisms apply to motors beyond Khc. We discuss a model which can explain the surprising common outcome of both conditions and examine its relevance for understanding motor-linked neurodegeneration. | | 10:47p |
Cortical responses to conflicting binocular stimuli in mouse primary visual cortex
Binocular vision requires that the brain integrate information coming from each eye. These images are combined (fused) to generate a meaningful composite image. Differences between images, within a range, provide useful information about depth (stereopsis). Interocular disparities that are not effectively combined result in diplopia and rivalry. The neural mechanisms underlying these binocular interactions remain poorly understood. Using a combination of visually evoked potential (VEP) recordings, unit recordings, and 2-photon calcium imaging in the binocular region of mouse primary visual cortex (bV1), we probed the neural mechanisms underlying the processing of two distinct forms of disparate binocular signals. Using a dichoptic display, introduction of a spatial interocular phase disparity in grating stimuli reduced VEP magnitude through decreased neuronal firing in the early phase of the response (40-80 ms after stimulus onset, corresponding to the VEP negativity). Introduction of an interocular orientation disparity also decreased VEP magnitude, but this difference was driven by an increase in firing in the late portion of the visual response (100-200 ms after stimulus onset, corresponding to the VEP positivity). This increase in activity was observed for both regular-spiking (putative excitatory) and fast-spiking (putative parvalbumin-positive inhibitory) units. By contrast, visually evoked calcium responses of somatostatin-positive interneurons decreased with introduction of the interocular orientation disparity. Based on these results, we propose that interocular phase differences largely suppress bV1 responses via feedforward thalamocortical interactions, whereas interocular orientation differences prolong visually evoked activity in bV1 through somatostatin-positive interneuron-mediated disinhibition. | | 10:47p |
Nerve Growth Factor Signaling Tunes Axon Maintenance Protein Abundance and Kinetics of Wallerian Degeneration
Neurotrophic factors are critical for establishing functional connectivity in the nervous system and sustaining neuronal survival through adulthood. As the first neurotrophic factor purified, nerve growth factor (NGF) is extensively studied for its prolific role in axon outgrowth, pruning, and survival. Applying NGF to diseased neuronal tissue is an exciting therapeutic option and understanding how NGF regulates local axon susceptibility to pathological degeneration is critical for exploiting its full potential. Our study identifies surprising connections between NGF signaling and proteostasis of axon maintenance factors. NGF deprivation increases Nmnat2 and Stmn2 protein levels in axon segments with a corresponding delay in Wallerian degeneration. Conversely, acute NGF stimulation reduces local abundance of these axon maintenance factors and accelerates Wallerian degeneration. Pharmacological studies implicate phospholipase C as the key effector in TrkA activation, which drives degradation of palmitoylated Stmn2. While seemingly opposed to neuroprotective activities well-documented for NGF, downregulating Nmnat2 and Stmn2 favors axonal outgrowth over transient hyper-susceptibility to Sarm1-dependent degeneration. This new facet of NGF biology has important implications for axonal remodeling during development and sustained integrity through adulthood. | | 10:47p |
Nurr1 orchestrates claustrum development and functionality
Claustrum is known the most widely interconnected tissue with almost all brain subareas to coordinate multiple cognitive behaviors in spite of the lasting controversy on its role in consciousness formation. However, little is known about the molecular mechanisms underlying its development and behavioral control. Here we show that Nurr1 (Nr4a2) is the key factor orchestrating claustral morphogenesis, functional connectivity and thus behaviors. Nurr1 deficient claustral cells over-migrate into insular cortex, shaping claustrum into an abnormal wing-like structure, and ectopically turn on insular cortical genetic program. Accordingly, functional connectivity associated with claustrum and relevant behaviors are dysregulated by Nurr1 deficiency. Additionally, Nurr1 regulates claustral neuron positioning by suppressing G-protein signaling. | | 11:16p |
Rotationally Stable Dynamics Over Long Timescales Emerge in Neuronal Development
Neuronal networks must balance the need for stable yet flexible dynamics. This is evident during brain development, when synaptic plasticity during critical windows enables adaptability to changing environments whilst ensuring the stability of population dynamics. The emergence of population dynamics that balance stability and flexibility during development is poorly understood. Here, we investigated developmental brain dynamics in larval zebrafish, using in vivo 2-photon imaging to record single-cell activity across major brain regions from 3-8 days post-fertilisation, a highly plastic period in which hunting behaviours are established. Our findings revealed region-specific trajectories in the development of such dynamic regimes: the telencephalon exhibited increased neuronal excitability and long-range correlations, alongside the emergence of scale invariant avalanche dynamics indicative of enhanced flexibility. Conversely, while other regions showed increased state transitions over development, the telencephalon demonstrated a surprising rise in state stability, characterized by slightly longer dwell times and drastically reduced angular velocity in state space. Remarkably, such rotationally stable dynamics persisted up to 5 seconds into the future, indicating the emergence of strong attractors supporting stability over long timescales. Notably, we observed that telencephalon dynamics were maintained near to but not at a phase transition, thus allowing for robust responses while remaining adaptable to novel inputs. Our results highlight regionally-specific trajectories in the relationship between flexibility and stability, illustrating how developing neuronal populations can self-organize to balance these competing demands. |
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