Neurobiology of Disease

Subtle variations in the activity and signaling of PERK, an endoplasmic reticulum kinase, can shift stress response outcomes. PERK has three commonly occurring exonic single-nucleotide variants frequently inherited together as part of the PERK haplotype B (PERK B), which occurs in 28% of the global population and is associated with several chronic neurodegenerative diseases, although its impact on PERK activity remains controversial. We hypothesized that PERK-B was associated with increased activity, altering stress response and stress tolerance, and that minorly but consistently elevated PERK activity, in combination with additional environmental or genetic risk factors, would lead to adverse outcomes. Comparison of stress response between PERK A, the major PERK haplotype, and PERK B in neuronal cultures using novel knock-in PERK-B mice alongside wild-type PERK A mice revealed differential, transient changes. Following ER stress, astrocyte-containing neuronal cultures, but not neuron-only cultures expressing PERK B exhibited similar levels of eIF2 phosphorylation, the canonical PERK substrate at PERK peak activation point (2h), but showed higher levels at 4 to 6 h, consistent with altered kinetics of PERK signaling. PERK-B neuronal cultures pretreated with a low dose PERK-specific activator exhibited increased vulnerability to N-methyl-D-aspartate-induced death compared to PERK-A neuronal cultures, indicating impaired acquired stress tolerance. Additionally, neuron-astrocyte coculture experiments revealed that the observed difference in acquired stress tolerance was dependent on the astrocyte genotype. The association of PERK-B with transient changes in stress response and reduced stress tolerance with higher vulnerability to cellular stress, albeit transient and small/moderate in size, has clinical relevance based on its potential contribution to the overall genetic vulnerability in neurodegenerative diseases.

Background: Dysregulated innate immune activation is a key feature of Parkinson disease (PD), but the upstream triggers and cell-type-specific mechanisms remain unclear. Double-stranded RNA (dsRNA) has emerged as a potential driver of innate immune responses. Adenosine deaminase acting on RNA 1 (ADAR1), particularly its interferon-inducible p150 isoform, edits dsRNA to limit aberrant immune activation. Whether this dsRNA-ADAR1 axis contributes to neuroinflammation in PD in a cell-type-specific manner is unknown. Methods: We performed immunofluorescence on post-mortem cingulate cortex tissue from PD cases and controls to localise dsRNA, ADAR1 p150 and pathological alpha-synuclein protein. We integrated these findings with single-nucleus RNA sequencing data from three independent human brain datasets. An eight-module gene scoring framework was applied to assess dsRNA processing, sensing, and downstream innate immune pathways in neurons and astrocytes. Results: In PD cortex, the proportion of dsRNA-positive astrocytes was higher than in controls, whereas neuronal dsRNA positivity did not differ between groups. ADAR1 p150 positivity increased in both astrocytes and neurons, and the proportion of ADAR1 p150 positive astrocytes correlated with astrocytic -synuclein burden. Transcriptomic analyses showed that neurons were enriched for dsRNA editing and processing machinery, whereas astrocytes exhibited higher expression of dsRNA sensing pathways, innate immune responses, and transposable element control. While overall pathway activity did not differ between PD and controls after covariate adjustment, network analyses revealed cell-type-specific coupling patterns. Notably, complement gene expression showed a strong and selective association with dsRNA-related pathways in astrocytes, whereas interferon and NF-{kappa}B signalling were shared across cell types. In both cell types, ADAR1-associated gene programmes were enriched for oxidative phosphorylation and PD-related pathways. Conclusions: These findings identify astrocytes as a central site of relative dsRNA accumulation and ADAR1 p150 induction in the PD cortex. The selective coupling of dsRNA sensing to complement activation in astrocytes highlights a cell-type-specific innate immune pathway that is constitutively present rather than induced by disease. Together, our protein-level and network-level findings converge on a coherent and testable model in which a constitutive astrocytic circuit is engaged by disease-associated dsRNA accumulation, with potential relevance for disease progression and therapeutic targeting.

Deletion of the microtubule-associated protein, tau reduces seizure burden in rodent models of genetic epilepsies and suppresses evoked seizures. In the intrahippocampal kainate (IHK) model of acquired temporal lobe epilepsy (TLE), mice lacking tau expression develop spontaneous seizures, but at a lower rate than controls. Mechanisms by which tau deletion modifies the development of acquired TLE are not well understood, but a modification of the synaptic reorganization that accompanies epileptogenesis may contribute to modified seizure expression. Increased inhibitory synaptic input to dorsal dentate granule cells (DGCs) contralateral to the focal kainate injection results after development of TLE in tau-/- mice, but inhibitory synaptic reorganization distal to the kainate lesion is undefined in this model. We therefore investigated how development of TLE modifies excitability and synaptic transmission in DGCs of the ventral dentate gyrus, distant from the kainate lesion. Our results show that development of TLE increases evoked action potential firing of ventral DGCs (vDGCs) in mice that develop TLE, demonstrating an effect of TLE development on excitability of DGCs distant from the focal injury. After development of TLE, DGCs from tau-/- mice received increased inhibitory synaptic input bilaterally, suggesting that tau expression suppresses inhibitory synaptic reorganization in DGCs. Optogenetic stimulation of interneurons in tau-/- mice revealed increased inhibitory synaptic input to vDGCs and an increase in inhibitory convergence onto vDGCs once epileptogenesis occurred. Therefore, tau deletion modifies spontaneous seizure expression after epileptogenesis in mice, potentially involving increased synaptic inhibition of DGCs throughout the dentate gyrus.

Traumatic brain injury (TBI) is one of the world's leading causes of death and disability and a major risk factor for dementias. The primary dementia associated with TBI is chronic traumatic encephalopathy (CTE), a neurodegenerative disease classified as a tauopathy, in which toxic tau molecules lead to disease pathologies and degeneration. The processes that lead to tauopathy and subsequent dementia after TBI remain unclear. Here, we built upon the finding that seizures after TBI may be a mechanism leading to tauopathy, by dissecting the functions of the metabotropic glutamate receptor 5 - cellular prion protein (mGluR5-PrPC) pathway. We delivered TBI to larval in a blast paradigm, and quantified aggregation of Tau via a genetically-encoded Tau-GFP fusion reporter. Zebrafish larvae lacking prp2 (homolog of mammalian cellular Prion Protein, PrPC) displayed a 168% increase in post-traumatic seizures activity after TBI. An mGluR5 agonist (CHPG) reduced post-traumatic seizures, whereas an mGluR5 antagonist (MPEP) increased post-traumatic seizures. Moreover, agonizing mGluR5 reduced tau aggregation and antagonizing mGluR5 increased tau burden. Larvae seizing from convulsants, rather than TBI, were treated with CHPG/MPEP and provided a similar pattern of outcomes, suggesting seizures may be a factor needed for mGluR5 activity to influence tau aggregation. The PrPC-mGluR5 pathway is proposed as one candidate pathomechanism linking TBI to subsequent seizures and tauopathy, and thus it warrants investigation as a target for prophylactic interventions.

Epilepsy is increasingly recognized as a disorder involving both neuronal and vascular dysfunction. While connexin signaling has been implicated in epileptogenesis, the contribution of vascular connexins to seizure associated cerebrovascular pathology remains poorly understood. Connexin40 (Cx40) is an endothelial gap junction protein that plays a crucial role in vascular communication and blood-flow regulation. Seizures induce dynamic changes in cerebral perfusion and oxygenation, including prolonged postictal hypoperfusion/hypoxia. To determine whether Cx40 influences postictal hypoxia following focal seizures, we examined seizure characteristics and postictal oxygen dynamics in Cx40 knockout (Cx40-/-) mice using an established focal hippocampal seizure model. Electrically kindled seizures were elicited in wild-type and Cx40-/- mice, and local hippocampal tissue oxygenation was continuously monitored before and after seizure induction. Seizure duration did not differ between genotypes, indicating comparable seizure severity. Interestingly, Cx40 deletion altered the temporal pattern of postictal oxygen recovery, producing greater early hypoxia and a delayed secondary rebound in pO2 despite similar peak oxygen levels and overall hypoxic burden. These findings demonstrate that loss of Cx40 selectively alters the temporal profile of postictal oxygen dynamics without affecting seizure duration. Taken together, the results suggest that endothelial gap junctional communication contributes to postictal vascular recovery and identify Cx40 as a potential modulator of seizure associated neurovascular dysfunction.

Huntington&'s disease (HD) is characterized by progressive accumulation of mutant huntingtin (mHTT), neurodegeneration and motor dysfunction. Increasing evidence implicates impaired glymphatic function and loss of perivascular aquaporin 4 (AQP4) polarization in HD, suggesting that defective brain waste clearance may contribute to disease pathogenesis. Whether restoration of AQP4&-mediated glymphatic transport can ameliorate HD pathology remains unknown. We tested whether genetic restoration of AQP4 improves glymphatic function and HD&-associated phenotypes in heterozygous zQ175 knock-in mice. At 2 months of age, premanifest zQ175 mice received a single retro-orbital injection of BBB-penetrant AAV9&-PHP.eB encoding the AQP4 (M1 isoform). Glymphatic transport was assessed by intracisternal injection of fluorescent BSA 647 and quantification of parenchymal influx and drainage to the deep cervical lymph nodes. Perivascular AQP4 localization, mHTT aggregation, gliosis, DARPP32 immunoreactivity, CSF neurofilament light chain (NfL) and total Tau, structural brain volumes by 11.7T MRI, and motor behavior were assessed longitudinally through 12 months of age. AAV9&-PHP.eB&-AQP4 produced widespread brain expression and restored perivascular AQP4 localization in the striatum and cortex of zQ175 mice. AQP4 overexpression significantly increased glymphatic tracer influx into brain parenchyma and BSA-647 drainage to the deep cervical lymph nodes. At 12 months, AQP4 restoration reduced CSF NfL and total Tau concentrations, mHTT aggregate number and mean aggregate size, and striatal Iba1 positive microglia and GFAP-positive astrocytes (P < 0.05). AQP4 overexpression also preserved striatal DARPP32 immunoreactivity and significantly attenuated striatal atrophy measured by MRI. Female zQ175 mice showed improved balance-beam performance and locomotor activity, while effects on motor performance in males did not reach statistical significance. AQP4 overexpression attenuated body-weight loss in both sexes. No detectable structural, behavioral or body-weight abnormalities were observed following sustained AQP4 overexpression in wild-type mice. In summary, restoration of AQP4 enhances glymphatic transport and is associated with reduced proteopathic, neuroinflammatory and neurodegenerative phenotypes in zQ175 HD mice. These findings provide proof-of-concept that impaired glymphatic function is a modifiable component of HD pathology and support AQP4&-mediated restoration of brain waste clearance pathways as a potential disease-modifying therapeutic strategy.

Wolfram syndrome (WS) is a rare, life-limiting progressive neurodegenerative disorder caused by pathogenic variants in the WFS1 gene. It is a prototype endoplasmic reticulum (ER) stress disorder, and the mechanisms underlying neurological phenotypes in WS remain incompletely understood. Although WFS1 protein is abundant in the hippocampus, the role of this region in WS remains relatively understudied. Here we characterize hippocampal function across scales in mice carrying a patient orthologous Wfs1 variant (p.W542*) on both alleles. Male mutants lose excitatory synapses, and dissociated hippocampal neurons are markedly hyperexcitable yet generate no increase in network bursting and no shift in network criticality, indicating that their additional firing is not recruited into coordinated population activity. This finding translates in vivo wherein intracranial electroencephalogram (EEG) recordings show no epileptiform activity or seizures in either sex, with no hippocampal astrogliosis, alongside a male-specific reduction in hippocampal low-gamma power. Additionally, EEG demonstrates a sex-specific disruption of sleep, which parallels a sex-divergent synaptic phenotype. Mutant male mice show impaired hippocampus-dependent fear memory and reduced voluntary motor activity. Taken together, we propose that cellular hyperexcitability in the WS hippocampus does not translate into increased network bursting, potentially due to disproportionate loss of excitatory synapses. This dissociation may provide a functional substrate for the neurological features of WS.

Friedreich ataxia (FRDA) is an inherited multisystem disorder caused by a GAA repeat expansion in the FXN gene and consequent frataxin deficiency, causing progressive neurodegeneration and heart disease. Clinical studies have documented autonomic dysfunction in FRDA, including elevated resting heart rate, raised plasma catecholamines and a high burden of urinary, bowel and sexual symptoms. Consistent with this, autopsy studies show degeneration of the cardiac conduction system and its associated ganglia in FRDA. Whether these features are intrinsic to FRDA autonomic neurons has been untestable without a human cellular model. Here, a human model of FRDA autonomic neurons was established by differentiating induced pluripotent stem cells (iPSCs) from individuals with FRDA and from their CRISPR/Cas9-corrected isogenic controls. FRDA autonomic neurons expressed canonical autonomic lineage markers and retained reduced FXN mRNA and frataxin protein levels. Electrophysiological analysis revealed pronounced hyperexcitability, with an approximately five-fold increase in spike number, shortened interspike intervals, and longer bursts. FRDA autonomic neurons also exhibited increased cell death, elevated mitochondrial reactive oxygen species, and a shift from networked to tubular mitochondrial morphology. Bulk RNA sequencing of all three isogenic pairs revealed loss of extracellular matrix programs and activation of stress response pathways. The top differentially expressed gene was MEG3, a maternally imprinted long noncoding RNA, which was consistently upregulated across all three isogenic pairs. MEG3 sits within the imprinted 14q32 DLK1-DIO3 locus, whose maternally expressed noncoding transcripts were co-ordinately de-repressed in FRDA autonomic neurons. Competitive gene-set testing of 43 imprinted genes across 20 domains showed this to be specific to 14q32: no other imprinted domain was co-ordinately altered. Together, these findings establish hyperexcitability of autonomic neurons as a cell autonomous consequence of frataxin deficiency, accompanied by mitochondrial dysfunction. This provides a cellular basis for the increased sympathetic tone observed in clinical studies.

Absence epilepsy is a generalized non-convulsive epilepsy characterized by recurrent absence seizures (ASs) and enhanced GABAA receptor-mediated tonic inhibition of thalamocortical (TC) neurons has been shown to lead to ASs. Serotonergic signalling is an important modulator of these seizures, yet the contribution of specific 5-HT receptor subtypes remains incompletely understood. Here, we investigated whether endogenous serotonergic signalling through 5-HT2A receptors (5-HT2ARs) contributes to pathological tonic inhibition and ASs expression. Patch-clamp recordings were obtained from ventrobasal (VB) TC neurons of Genetic Absence Epilepsy Rats from Strasbourg (GAERS) and non-epileptic control (NEC) rats. The selective 5-HT2AR antagonist MDL11,939 had no effect on tonic GABA-A currents in NEC TC neurons but significantly reduced the enhanced tonic current in GAERS neurons, revealing a disease-dependent contribution of endogenous 5-HT2AR signalling. MDL11,939 administered bilaterally into the VB of freely moving GAERS by reverse microdialysis did not alter ASs. This dissociation between the modulation of a disease-associated cellular mechanism and seizure outcome suggests that the previously reported reduction of ASs by systemic 5-HT2AR blockade may involve mechanisms outside the VB.

Alternating hemiplegia of childhood (AHC) is a rare neurodevelopmental disorder primarily caused by heterozygous de novo mutations in the ATP1A3 gene, which encodes the neuron-specific catalytic 3 subunit of the P-type Na/K-ATPase. Among these, the E815K variant is the second most frequent ATP1A3 mutation in AHC and is associated with the most severe clinical phenotypes, characterized by frequent epileptic and dystonic episodes and profound developmental impairment, making it a key target for mechanistic study. Although E815K was shown to disrupt pump function in heterologous systems and animal models, its impact on human neurons remain poorly understood. To investigate its disease-relevant pathophysiology, we generated induced pluripotent stem cells (iPSCs) from an AHC patient carrying the E815K mutation and differentiated them into mature neurons. Patient-derived neurons displayed normal differentiation and synaptogenesis, with preserved evoked synaptic transmission. However, 3-specific Na/K pump currents were nearly abolished, whereas 1 activity remained intact. This selective dysfunction was associated with a depolarized resting membrane potential, reduced input resistance, and diminished intrinsic excitability. Patient neurons generated fewer action potentials in response to depolarizing current steps, displayed altered firing patterns, and showed significantly decreased voltage-gated sodium current densities, while potassium conductance remained unaffected. Taken together, these results indicate that the ATP1A3 E815K mutation disrupts intracellular sodium homeostasis and consequently modifies the excitability of human neurons. Overall, these findings provide mechanistic insight into the molecular pathophysiology underlying this AHC variant and thereby establish a patient-derived neuronal model which serves as a platform for future drug screening approaches and therapeutic investigation.

Axons of substantia nigra dopaminergic neurons "die back" in Parkinson's disease (PD), exhibiting early degeneration relative to cell bodies, a key feature also observed in PD models. A critical step towards understanding the process of axonal neurodegeneration is to determine the physiological properties of dopaminergic axons that are altered during the early stages of cellular dysfunction. Here, we compared the electrophysiology and calcium dynamics of dopaminergic axons in the MitoPark model of PD with age-matched controls. Our experiments show a pronounced reduction in action potential (AP)-evoked calcium signals in MitoPark axons that emerges well before the onset of dopaminergic fiber loss and motor deficits. In addition, calcium imaging and direct axon recording revealed deficits in AP propagation accompanied by alterations in the axonal AP waveform. Therefore, our experimental findings identify the progression of axon-specific physiological deficits that occur long before neurodegeneration, more precisely defining the period that precedes axonal loss.

Variants at the CD2AP locus, which encodes a branched-actin regulator, are associated with late-onset Alzheimer's disease (AD), and CD2AP-deficient mice show increased blood-brain barrier (BBB) permeability. CD2AP is enriched in endothelial cells, but its function there is poorly understood. In endothelium, branched actin networks maintain cell-cell junctions and are also required for immune cells to cross through individual endothelial cells (transcellular transendothelial migration, TEM). We previously showed that the Arp2/3 activators N-WASP and WAVE2 contribute to these processes, but the capping protein (CP)-interacting proteins involved are unknown. Here we tested whether CD2AP, a CP-interacting protein recruited to cell-cell contacts, regulates both junction integrity and TEM. In primary human microvascular endothelial cells, shRNA knockdown of CD2AP increased TNF-induced monolayer permeability, measured by transendothelial electrical resistance, and altered VE-cadherin junction morphology. Unexpectedly, on soft hydrogels the same knockdown reduced TEM of NK-92 MI cells, lowering the proportion of transcellular events. Thus, loss of CD2AP weakens the endothelial barrier yet impairs a distinct, actin-dependent route of immune-cell passage. These findings suggest that CD2AP risk variants may affect the BBB through more than permeability, and they identify endothelial immune-cell trafficking as a testable link between CD2AP and AD.

Spinal muscular atrophy (SMA) is usually considered a lower motor neuron disease; however, growing evidence from both experimental models and clinical studies indicates that SMN deficiency affects the whole CNS, beyond the spinal and brainstem motor system. Here, we investigated the temporal progression and selective vulnerability of cortical projection neurons in the SMN{Delta}7 mouse model at early (P5) and late (P11) symptomatic stages. Combining subtype-specific immunohistochemistry, DiI tracing associated with morphometric reconstruction, and dendritic spine analysis, we identified stage- and subtype-dependent cortical alterations. At P11, corticospinal and infragranular callosal projection neurons were selectively reduced in number and showed marked dendritic remodeling and altered excitatory synaptic organization, whereas corticothalamic neurons and supragranular SATB2-positive populations were comparatively preserved. Notably, cortical abnormalities were already detectable at P5, before overt neuronal loss: indeed, corticospinal neurons exhibited reduced soma size and dendritic complexity, while dendritic spine density remained preserved, but spine morphology was shifted toward immature phenotypes. These early maturation defects persisted during disease progression and preceded the reduction in spine density observed at P11. Together, our findings identify cortical projection neurons as selectively vulnerable to constitutive SMN deficiency and suggest that impaired neuronal and synaptic maturation represents an early feature of cortical pathology preceding subtype-specific neurodegeneration. These results expand the pathophysiological spectrum of SMA beyond lower motor neurons and highlight early brain alterations as a potential component of disease progression.

Parkinsons disease is a neurodegenerative disorder characterized by altered subcortical-cortical connectivity, leading to motor and cognitive deficits. Saccadic eye movement abnormalities are among the earliest and most persistent markers of the disease, serving as sensitive indicators of disrupted oculomotor and cognitive control. While changes in average saccadic behavioral measures are widely reported in Parkinson's disease, intra-individual variability in saccadic performance and the underlying network dysfunction remain poorly understood. Here, we quantified intra-individual saccadic variability in early-stage Parkinsons disease and identified its functional correlates within the resting-state saccadic network. We analyzed data from 91 patients with early-stage Parkinsons disease and 46 healthy controls who underwent oculomotor testing and resting-state functional magnetic resonance imaging. First, at the behavioral level, we assessed intra-individual variability in saccadic performance within each group using the coefficient of variation of reaction time and normalized peak velocity across trials. Second, at the brain level, we assessed group differences in static and dynamic functional connectivity across 39 regions comprising the saccadic network. We examined the relationship between network connectivity and saccadic variability using partial least squares analysis. We also quantified network organization using graph-theory measures of global efficiency and clustering coefficient. Finally, we tested the relevance of intra-individual saccadic variability and network features in distinguishing patients from healthy controls using a multivariate Mahalanobis classifier. Our results showed that, compared to HC, patients had higher intra-individual variability in saccadic behavioral measures of reaction time (pHolm = 0.012) and normalized peak velocity (pHolm = 0.012). Interestingly, patients had (i) reduced static and dynamic connectivity within the saccadic network and (ii) weaker associations between network connectivity and intra-individual saccadic variability of specifically normalized peak velocity. These results were particularly driven by regions within the cognitive saccade network. The classifier that integrated intra-individual saccadic variability and network connectivity achieved 95.62% accuracy in distinguishing patients from healthy controls, with dynamic connectivity contributing more strongly to classification performance than static connectivity. We demonstrate that elevated intra-individual variability of saccades, underpinned by altered functional connectivity within cognitive-oculomotor networks, is a prominent feature of early-stage Parkinsons disease. Emerging as a promising physiological marker of disease pathophysiology, intra-individual variability offers a powerful new framework to refine early diagnosis and improve the tracking of neurodegeneration before severe clinical milestones are reached.

Alexander disease (AxD) is a rare genetic leukodystrophy caused by dominant mutations in the gene coding for the astrocytic intermediate filament protein Glial Fibrillary Acidic Protein (GFAP). Understanding disease mechanisms is complicated by the fact that mutant GFAP promotes further GFAP accumulation, making it difficult to distinguish mutation-specific effects from those caused by elevated GFAP levels. Here, we performed mass spectrometry-based single cell proteomics to investigate proteomic alterations associated with increasing levels of wildtype GFAP and a common AxD-causing mutant GFAP R239C. By analyzing individual cells spanning a broad range of GFAP expression levels, we separated dosage-dependent effects from mutation-specific responses. Cells expressing high levels of wildtype GFAP increasingly resembled R239C-expressing cells at the proteomic level, supporting a role for GFAP accumulation in disease pathogenesis. In contrast, mutant GFAP specifically correlated with increased mitochondrial antioxidant enzymes and remodeling of the proteasome tailored toward ATP- and ubiquitin-independent degradation of oxidized proteins. Integration with proteomics data from patient-derived cortical organoids revealed increased abundance of nuclear-encoded mitochondrial proteins and respiratory chain assembly factors without corresponding changes in mitochondrially encoded proteins, suggesting impaired electron transport chain (ETC) assembly. Consistent with this, AxD cells displayed reduced oxygen consumption, increased glycolytic activity, enhanced utilization of the cytosolic part of the glycerol-3-phosphate (G3P) shuttle, and accumulation of triacylglycerides and lipid droplets. We propose that ETC dysfunction promotes partial uncoupling of the G3P shuttle, allowing sustained regeneration of cytosolic NAD to support glycolysis while diverting G3P toward lipid synthesis and storage. By regenerating NAD through the G3P shuttle rather than lactate fermentation, AxD astrocytes may sustain glycolytic flux while preserving lactate production, an important metabolic substrate for neighboring neurons. Together, our findings identify ETC dysfunction as a central feature of AxD and suggest that metabolic adaptation through the G3P shuttle links mitochondrial dysfunction to glycolytic reprogramming and lipid accumulation. More broadly, these results provide new insight into how astrocyte dysfunction drives molecular pathways implicated in neurodegeneration.

Experimental models of Parkinson's disease (PD) traditionally involve direct perturbations of the functioning of dopamine (DA) neurons. However, PD is increasingly recognized as multifactorial in origin and involving both cell-autonomous vulnerability factors and non-cell-autonomous triggers including inflammatory signals deriving from bacterial or viral infections. Notably, genes associated with early-onset familial PD as PRKN (Parkin) and PINK1, are ubiquitously expressed and have been implicated in immune regulation, further supporting a role for inflammation in disease etiology. In the present study, we tested the hypothesis that alternate Polyinosinic:polycytidylic acid (Poly(I:C)), mimicking viral infection, and lipopolysaccharides (LPS), mimicking bacterial infection, may induce sufficient brain inflammation to impair the DA system in Parkin-deficient mice and recapitulate some of the systemic features of PD. We developed a protocol with alternating Poly(I:C) and LPS, inducing acute peripheral inflammation in both genotypes, with consistent body weight loss and an increase of fecal lipocalin-2. Modest levels of neuroinflammation were present six months after the treatment, with higher levels of the microglial marker Iba1 in the dorsal striatum and reduced levels of the astrocyte marker GFAP in the mesencephalon, potentially indicating reduced neuronal support in the longer term. Finally, we find that this alternating Poly(I:C)/LPS treatment does not induce dopaminergic denervation or motor dysfunctions, even in Parkin KO mice. Taken together, our findings suggest that repeated short-term exposure to pathogen-derived signals is insufficient to induce DA system impairment in young adult Parkin deficient mice. Instead, induction of PD-like pathology may require a convergence of factors, including a prolonged or recurrent inflammatory burden, aging, genetic susceptibility, and potentially additional environmental or cellular stressors, that together create a pathogenic "perfect storm". Thus, in its current form, the paradigm developed in the present study should not be regarded as a fully representative model of PD. Rather, it provides proof of concept that temporally alternating inflammatory stimuli can shape non-motor symptom-like phenotypes and underscores the need for future animal model research to incorporate interacting risk factors, disease duration, and age-dependent vulnerability.

The dentate gyrus (DG) is a critical regulator of cortical input processing in the hippocampus, supporting spatial navigation and memory processing. In epilepsy, the DG undergoes extensive structural and functional reorganization that diminishes its ability to filter afferent inputs, resulting in enhanced hippocampal hyperexcitability, seizures, and cognitive impairment. Yet, how DG network dynamics evolve over time in the epileptic brain is not fully understood. Here, we investigated DG network excitability, feedback inhibition, and throughput in rats 1 week, 8 weeks, and 4 months after pilocarpine-induced status epilepticus (SE), and examined the role of presynaptic GABAB receptor-mediated modulation of DG activity. Recording from urethane-anesthetized rats in vivo, we show a persistent increase in excitation-spike (E-S) coupling after SE. In contrast, feedback inhibition measured by short-duration paired-pulse ratios (PPR at 20ms, 40 ms and 60ms intervals) was significantly increased 1 and 8 weeks after SE, but was indistinguishable from controls by 4 months. Notably, we found that dentate spike (DS) frequency remained persistently elevated up to 10 months post-SE. Selective inhibition of presynaptic GABAB receptors in the DG using CGP36216 further enhanced E-S coupling at 1 and 8 weeks in post-SE rats without affecting age-matched controls. CGP36216 did not alter E-S coupling in rats 4 months after SE, revealing a homeostatic role for basal presynaptic GABAB signaling in restraining network excitability early after SE. CGP36216 robustly increased PPR at 20ms across all groups without altering DS frequency, suggesting that GABAB receptors boost DG feedback inhibition. Mechanistically, CGP36216 reduced single stimulus-evoked inhibitory postsynaptic currents (eIPSCs) in dentate granule cells without altering excitatory transmission after SE. Together, these findings support a disinhibitory role for presynaptic GABAB receptors, which bolsters synaptic inhibition to transiently counter post-SE hyperexcitability, and offers a targetable mechanism for therapeutic intervention during epileptogenesis.

Gaucher disease is a rare lysosomal storage disorder caused by autosomal recessive mutations in the GBA1 gene, encoding the lysosomal enzyme glucocerebrosidase. Gaucher disease is classified in 3 different subtypes depending on the presence and severity of neurological involvement, with type 2 resulting in fatal early-onset neuropathology and patients exhibiting developmental delays, seizures and early death. Studies investigating disease mechanisms of neuronopathic Gaucher disease are mainly based on animal models and focus predominantly on late neuronal phenotypes. Here, we established healthy control and Gaucher disease patient-derived iPSC lines and engineered them to obtain isogenic control and disease lines. Using these lines, we generated cortical and subpallial brain organoids in which we identified early-onset lipid dysregulation in form of glucosylceramide accumulation, highly elevated glucosylsphingosine, and a later increase in ganglioside levels, recapitulating clinical findings. Furthermore, single-cell transcriptomic profiling uncovered novel phenotypes in both cortical and subpallial forebrain organoids. Subpallial alterations consisted of an early increase in migrating interneurons in subpallial organoids, which upregulated cholesterol metabolism. Cortical alterations showed early upregulation of mitochondrial genes and a downregulation of proliferation, with a subsequent switch from GABAergic to glutamatergic neuron fate with a striking increase in gene expression related to the synaptic assembly. Functional assays demonstrated a marked hyperexcitability of cortical organoids and reduced response to GABA-A receptor blockage in Gaucher disease. Additional 2D neuronal network models confirmed the organoid data and showed that both glutamatergic and GABAergic neurons contribute to the phenotype, with hyperexcitability of Gaucher glutamatergic neurons and incapacity of Gaucher GABAergic neurons to balance the excessive excitation. This alteration represents a clinically significant phenotype as many patients exhibit an excitation/inhibition imbalance leading to treatment-resistant seizures, hastening their decline. In conclusion, our defined human models of Gaucher disease identify novel and clear phenotypes that can be used for drug screening or aid in development of new therapeutic strategies to ameliorate Gaucher disease.

Pathological tau disrupts neuronal activity and brain function in Alzheimer s disease and other tauopathies. However, the underlying mechanisms for neuronal network dysfunction remain largely unknown. Using human inducible pluripotent stem cell (iPSC) - derived neurons, we show that the familial MAPT P301L mutation leads to neuronal network hyperexcitability. Imbalanced excitation and inhibition were attributable to neuronal subtype-specific tau pathology that was associated with different alterations of tau interactomes in excitatory versus inhibitory neurons. Suppressing enhanced excitatory synaptic transmission or blocking Ca2+ influx resulting from enhanced P301L tau/AHNAK interaction in inhibitory neurons rescued the neuronal network hyperexcitability. This study expands our understanding on how disease-causing tau mutations alter the excitatory/inhibitory balance, leading to network dysfunction in human neurons.

Intracellular neurofibrillary tangles formed from hyperphosphorylated tau and extracellular amyloid plaques containing aggregated A{beta}-peptides, specific cleavage products of the Amyloid Precursor Protein (APP), are the primary histopathological hallmarks of Alzheimers Disease (AD), the leading cause of dementia in humans. However, the initiating steps that lead to these pathologies and early neurodegeneration, and the mechanisms by which tau- and A{beta}-induced effects might be linked remain unclear. Using the prostate-like secondary cell (SC) in Drosophila, we recently showed that A{beta} modulates normal APP- and membrane-associated protein aggregation in the dense-core granule (DCG) compartments of the regulated secretory pathway by interfering with subsequent membrane:DCG dissociation. This disrupts endolysosomal trafficking and propagates the resulting endolysosomal defects to other cells that endocytose the secreted abnormal DCG proteins. Here we show that overexpressing human tau also disrupts DCG aggregation and membrane:DCG dissociation inside SC secretory compartments, leading to increased endolysosomal targeting of these compartments. In a genetic screen, we find that knockdown of cofilin, which encodes an actin-severing protein required for dynamic remodelling of microfilaments, generates a similar phenotype. Consistent with this, overexpression of Cofilin, which is known to suppress tau-induced neurodegeneration in flies, reduces tau-induced DCG defects in SCs. Indeed, we find that Cofilin overexpression also suppresses A{beta}-induced degeneration in the fly eye. We conclude that membrane:DCG aggregate dissociation in DCG compartments is disrupted by both tau- and A{beta}-induced genetic changes that are relevant to AD, and this partially involves inhibition of actin cytoskeleton dynamics. Increasing actin remodelling activity can suppress neurodegeneration induced by both tau and A{beta}, suggesting that this process provides an important functional link between them that might be targeted therapeutically.