Neurobiology of Disease

The cellular form of the prion protein (PrPC) is known for its involvement in the pathogenesis of prion diseases. Recent research implicates the physiological isoform of PrP in neuronal development, excitability, and synaptic plasticity, as well as in other biological processes. However, its precise function in the development and function of neurons remains poorly understood. Here, we investigated its role during different developmental stages, both in vitro and in vivo, using different PrP knock-out (KO) mouse lines (Prnp-/-). Prion protein KO neurons cultured on microelectrode arrays (MEAs) displayed altered network dynamics compared to wild type cultures, comprising reduced burst frequency, and abnormal spike patterns, indicative of impaired maturation of the synaptic circuitry. These functional alterations were associated with a reduced expression of key presynaptic and postsynaptic proteins, including elements of the SNARE complex and regulators of excitation-inhibition balance. Similar molecular changes were also confirmed in a second Prnp-/- model, suggesting that PrPC is directly involved in these mechanisms regardless of genetic backgrounds. Alterations in neuronal networks were traceable into adulthood: in vivo recordings in adult Prnp-/- mice revealed increased neuronal responses to visual danger stimuli, which correlated with behaviorally increased fear responses to those stimuli. Together, our findings support a critical role for PrPC in the establishment and maintenance of functional neuronal networks, from early developmental stages in vitro to behaviorally mature relevant circuits in vivo, beyond genomic background. These results indicate that PrPC acts as a key regulator of synaptic development and function both in physiological and pathological conditions.

Alzheimers disease (AD) is a neurodegenerative disorder characterised by progressive dementia, brain atrophy, and ultimately death. Using cerebral organoids derived from human induced pluripotent stem cells (hiPSCs) carrying the familial PSEN1 A246E variant, we investigated the temporal relationship between amyloid-{beta} (A{beta}) dysregulation and spontaneous neuronal activity. Multielectrode array recordings from the differentiation day 60 (DD60) to at least DD130 revealed that AD organoids exhibited transient hyperexcitability and hypersynchrony compared with wild-type (WT) controls, followed by a gradual decline in activity. During the enhanced excitability stage, both elevated A{beta}42/40 and A{beta} aggregate size showed positive correlations with the percentage of active electrodes and the global synchrony index (GSI) in AD organoids. These findings indicate that A{beta} dysregulation might contribute to transient network hyperexcitability in early AD. The results also suggest that patient-derived cerebral organoids may serve as a translational model to examine early network dysfunction and inform future investigations of potential A{beta}-induced changes in excitability during the preclinical stages of AD.

Insulin-like growth factor-1 (IGF-1) plays a critical role in neuronal signaling. Disrupted insulin/IGF-1 signaling is implicated in Alzheimers disease, among other conditions, yet its specific influence on glutamate receptor-mediated calcium responses remains unclear. We examined the impacts of IGF-1 on glutamate receptor function in primary rat neurons monitored for intraneuronal calcium following stimulation with glutamate, AMPA, or NMDA/glycine. Pharmacological blockers (CNQX for AMPA receptors, APV for NMDA receptors, and nimodipine for L-type calcium channels) were applied to define receptor-specific contributions. In hippocampal neurons, IGF-1 and insulin altered responses to glutamate in different directions, with IGF-1 tending to evoke and enhanced response. In neocortical neurons, by contrast, IGF-1 consistently reduced glutamate- and AMPA-evoked calcium peaks, suggesting an inhibitory effect on AMPA receptors. To rule out effects on voltage-gated calcium channels downstream of AMPA receptors, we tested effects of IGF-1 on depolarization with potassium chloride; calcium elevation in this case was unaffected by IGF-1. Likewise, IGF-1 did not inhibit responses to NMDA/glycine; and IGF-1 did not affect glutamate responses in the presence of CNQX, a selective AMPA receptor blocker. These findings, combined with the observation that IGF-1 effects persisted in the presence of APV (an NMDA receptor antagonist), indicate that the inhibition of glutamate responses by IGF-1 is mediated by suppression of AMPA receptor activity. IGF-1 may thus contribute to normal neurophysiology, and given the role that glutamate receptors play in excitotoxicity, IGF-1 may confer neuroprotection in the neocortex. Disruption of IGF-1 signaling, as seen in states resembling insulin resistance, may therefore worsen glutamate-driven excitotoxicity and contribute to adverse outcomes.

Mutations in the LRRK2 gene are the most frequent cause of familial Parkinsons disease (PD) whereas common variants are associated with an increased risk for sporadic PD. LRRK2 encodes a multi-domain protein displaying two functional enzymatic activities: GTPase and kinase. Familial LRRK2 mutations have been linked to alterations in its GTPase and kinase activities, elevated substrate phosphorylation, as well as increased neurotoxicity in cell and animal models. In addition to familial mutations, several common LRRK2 coding variants have been associated with PD risk in different ethnic populations. Little is known about how these coding variants modulate the risk of developing PD. In this study, we aim to evaluate whether a collection of LRRK2 coding risk variants (A419V, N551K, R1398H, R1628P, M1646T, S1647T, G2385R) modify the biochemical properties of the LRRK2 protein and LRRK2-mediated neurotoxicity using cell-based assays. With the exception of G2385R, we find that coding risk variants have minimal impact on LRRK2 steady-state protein levels, GTP-binding, phosphorylation at Ser910, Ser935, and Ser1292, and subcellular localization in human cell lines and cultured primary neurons. G2385R reduces the steady-state levels of LRRK2 and exhibits reduced phosphorylation at Ser910/Ser935 and Ser1292, consistent with diminished kinase activity. Notably, however, LRRK2 variants associated with increased PD risk (A419V, R1628P, M1646T, and G2385R) significantly elevate the levels of LRRK2-mediated Rab10 phosphorylation by two-fold in cells. In contrast, LRRK2 variants associated with reduced PD risk (N551K, R1398H, and N551K-R1398H) do not alter normal LRRK2-mediated pRab10 levels. Intriguingly, we find that both PD-risk and PD-protective LRRK2 variants can respond normally to kinase activation by different lysosomal stressors (chloroquine, nigericin and monensin) to robustly induce pRab10 levels in cells. The PD risk variant, G2385R LRRK2, significantly inhibits neurite outgrowth in primary cortical neurons compared to wild-type LRRK2. Combining each coding risk variant with the familial G2019S mutation has minimal impact on the elevated kinase activity of G2019S LRRK2 or its capacity to inhibit neurite outgrowth. Our study indicates that LRRK2-dependent Rab phosphorylation represents a relevant readout of PD risk induced by LRRK2 coding variants and demonstrates that the PD risk variant, G2385R LRRK2, creates a hyperactive kinase that can impair neuronal integrity at comparable levels to the effects of G2019S LRRK2.

Friedreich ataxia (FRDA) is a progressive multisystem neurodegenerative disease mostly caused by a homozygous GAA repeat expansion in the FXN gene, leading to deficiency of the protein frataxin. Despite ubiquitous frataxin expression, FRDA pathology is tissue-specific, disproportionately affecting dorsal root ganglia sensory neurons, dentate nuclei of the cerebellum, corticospinal tracts and cardiomyocytes. The molecular basis for this selective vulnerability remains unresolved, suggesting that cell-type specific responses to frataxin deficiency shape disease susceptibility. This incomplete understanding is compounded by the lack of molecular biomarkers that capture FRDA biology beyond frataxin deficiency, thereby limiting therapeutic development and evaluation. Here, we integrated all available human bulk RNA-seq datasets in FRDA (23 datasets across 10 cell types), spanning disease-related (cardiomyocytes, sensory neurons) and relatively FRDA-spared cell types (fibroblasts, lymphoblastoid cells) under a unified analytical framework to identify transcriptional dysregulation underlying selective vulnerability and candidate biomarkers. Meta-analysis revealed recurrent transcriptional perturbations beyond FXN, involving long non-coding RNAs, translational control and cytoskeletal organisation. While shared transcriptional themes were observed, the specific biological programmes engaged were strongly cell-type dependent. The top candidate biomarkers, MYH14, MEG9, and MEG8 showed preferential upregulation in disease-relevant cell types including sensory neurons and cardiomyocytes, supporting their potential relevance to selective vulnerability. Therapeutic responsiveness to these candidates were assessed across RNA-seq datasets from FRDA models exposed to diverse therapeutic strategies, including epigenetic modulation and FXN-targeting approaches, revealing that transcriptional alterations in FRDA are pharmacologically modifiable. To facilitate transparent exploration and reuse of these findings, we developed an interactive FRDA Transcriptomic Atlas, providing a community-accessible resource for investigating gene and pathway-level dysregulation across FRDA studies: https://marniemaddock.github.io/FRDATranscriptomicAtlas/. Together, these findings implicate cell type specific transcriptional programs as potential drivers of selective vulnerability and establish a framework for prioritising biomarkers in FRDA. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/712785v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@123a1d3org.highwire.dtl.DTLVardef@554e49org.highwire.dtl.DTLVardef@86bfb8org.highwire.dtl.DTLVardef@94f66f_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundTau aggregation is the defining feature of tauopathies, however, the mechanisms by which distinct tau strains drive disease-specific responses remain unclear. Existing models largely rely on recombinant tau seeding or tau overexpression, which fail to capture the biochemical diversity of pathological tau. The aim of this study was to develop a robust and reproducible human cell-based model of disease-specific tau pathology and to use this model to determine how tau from unique diseases impact tau accumulation and lysosomal dysfunction. MethodsPatient-derived tau aggregates were enriched from post-mortem brain tissue obtained from sporadic Alzheimers disease (AD), Picks disease (PiD), progressive supranuclear palsy (PSP), and control cases using phosphotungstic acid precipitation. Patient-derived tau preparations were biochemically characterised by immunoblotting and mass spectrometry and normalised for tau content prior to seeding. Patient-derived tau aggregates were seeded into multiple human immortalised cell lines (SH-SY5Y, M03.13, U-87 MG, and U-118 MG cells) and iPSC-derived astrocytes. Tau seeding efficiency, aggregate morphology, and integrity of the autophagy-lysosomal pathway was assessed using quantitative imaging approaches. ResultsPatient-derived tau seeds retained disease-specific phosphorylation patterns and isoform composition and led to reproducible, dose-dependent insoluble tau accumulation in all cell lines tested. Despite equivalent tau input and similar background protein composition, PiD-derived tau had the most aggressive pathological signature, showing the highest number of tau aggregates per cell and inducing system wide disruptions in the autophagy lysosomal system including increased SQSTM1 puncta and lysosomal damage markers. Seeding with AD-derived tau led to a high number of tau aggregates per cell and more specifically depleted the lysosomal protease CTSD and uniquely co-seeded A{beta} pathology. Seeding with PSP-derived tau resulted in only a moderate number of tau aggregates per cell and uniquely caused increased lysosomal biogenesis. ConclusionsTogether, these results demonstrate that intrinsic properties of human tau strains drive disease-specific cellular responses and establish a scalable, physiologically relevant platform for dissecting tau-cell interactions and screening therapeutics across tauopathies.

Altered iron homeostasis has long been implicated in Parkinsons Disease (PD), although the mechanisms have not been clear. Given the critical role of PD-related activating mutations in LRRK2 (leucine-rich repeat protein kinase 2) within membrane trafficking pathways we examined the impact of a homozygous mutant LRRK2G2019S on iron homeostasis within the RAW macrophage cell line with high iron capacity. Proteomics analysis revealed a dysregulation of iron-related proteins in steady state with highly elevated levels of ferritin light chain and a reduction of ferritin heavy chain. LRRK2G2019S mutant cells showed efficient ferritinophagy upon iron chelation, but upon iron overload there was a near complete block in the degradation of the ferritinophagy adaptor NCOA4. These conditions lead to an accumulation of phosphorylated Rab8 at the plasma membrane, which is selectively inhibited by LRRK type II kinase inhibitors. Iron overload then leads to increased oxidative stress and ferroptotic cell death. These data implicate LRRK2 as a key regulator of iron homeostasis and point to the need for an increased focus on the mechanisms of iron dysregulation in PD.

Circadian disruption (CD) is increasingly recognized as a sex-specific risk factor for Alzheimers disease (AD). However, the mechanisms linking CD to AD, and the role of biological sex in this interaction, are unclear. Immunometabolic regulation is extensively circadianly timed, has sex-specific phenotypes, and plays a role in AD. Therefore, we hypothesized that CD affects the timing of immunometabolism, contributing to the sex-specific effects of CD on AD. To demonstrate this, we subjected male and female APP/PS1 mice to chronic disruptive lighting to model circadian disruption, finding CD induced a female-specific reduction in amyloid plaque burden but an increase in the infiltration of peripheral macrophages into the brain. Concomitantly, we found macrophages exhibited CD-associated immune reprogramming, which in females led to altered immunometabolic timing, an increase of macrophages in the activated state, and elevated levels of reactive oxygen species (ROS), supporting a role for immunometabolism in the sex-specific effects of CD in AD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=189 SRC="FIGDIR/small/721994v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@b9a59forg.highwire.dtl.DTLVardef@289219org.highwire.dtl.DTLVardef@18fe804org.highwire.dtl.DTLVardef@c96bb8_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LICircadian disruption reduces A{beta} plaque load specifically in female mice C_LIO_LIPeripheral immune infiltration correlates with reduced A{beta} plaques in females C_LIO_LICircadian disruption coordinates phasing of circadian immunometabolic proteins C_LIO_LIIn females, circadian phase advancement correlates with increased ROS C_LI

Spinal muscular atrophy (SMA) is caused by loss of SMN protein and is increasingly recognized as a multisystem disorder involving molecular pathology beyond motor neurons. Recently, we identified NRF2-KEAP1 signaling as dysregulated in SMA mice. Because NRF2 coordinates transcriptional programs that maintain cellular redox homeostasis and adaptive stress responses, we investigated whether NRF2 signaling is similarly altered in SMA type I patient-derived fibroblasts and whether it can be pharmacologically engaged. Compared with control fibroblasts, SMA fibroblasts displayed reduced basal expression of NRF2 target proteins, including NQO1 and xCT (SLC7A11), along with decreased levels of PGC1. Omaveloxolone (OMAV), a pharmacological NRF2 activator approved for the treatment of Friedreichs ataxia, increased cell viability and upregulated NRF2 target proteins in both control and SMA fibroblasts. Notably, OMAV produced a modest increase in SMN protein abundance and PGC1 levels selectively in SMA cells. Together, these findings support diminished NRF2 pathway output as a feature of SMA fibroblasts and demonstrate that OMAV induces NRF2 target proteins in this human SMA cellular model, consistent with enhanced cytoprotective signaling. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/712434v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1904bfeorg.highwire.dtl.DTLVardef@6d20e2org.highwire.dtl.DTLVardef@89f365org.highwire.dtl.DTLVardef@ca9638_HPS_FORMAT_FIGEXP M_FIG C_FIG

Abnormal beta-band activity (13-30 Hz) within the cortico-basal ganglia network is a hallmark of Parkinsons disease (PD) and is closely linked to motor impairment. Pathological beta activity in the subthalamic nucleus (STN) occurs predominantly as brief, high-amplitude bursts rather than continuous oscillations. Although beta-band coherence between the STN and cortex increases during bursts, it remains unclear whether cortico-STN beta coupling persists outside these bursts. Using intraoperative STN local field potentials and simultaneous cortical electrocorticography from seven patients undergoing deep brain stimulation implantation surgery, cortico-STN beta coupling during burst and non-burst epochs was compared. Coupling was assessed using magnitude-squared coherence and the debiased weighted phase lag index (dwPLI) and compared against surrogate distributions generated by circular time-shifting. Both coupling metrics were significantly elevated during burst epochs relative to non-burst periods. During non-burst epochs, coupling collapsed to surrogate levels, indicating no evidence of sustained synchronization. Time-resolved analyses further demonstrated that elevated coupling was confined to burst epochs. Although a subset of motor cortical contacts exhibited elevated baseline coherence, coupling was less evident using dwPLI. These findings suggest that pathological cortico-STN beta coupling in PD is preferentially expressed during beta bursts rather than sustained across non-burst epochs, with implications for adaptive neuromodulation strategies.

The spinocerebellar ataxia type 5 (SCA5) L253P mutation in {beta}-III-spectrin causes high-affinity actin binding. Here we developed a CRISPR knock-in mouse to determine the in vivo impact of L253P on Purkinje neurons and motor activity, and to establish a model for future testing of SCA5 therapeutics. Significantly, the knock-in mouse shows impaired motor activity on elevated beam assays at 20 weeks. In the cerebellum, L253P causes a subcellular redistribution of {beta}-III-spectrin in Purkinje neurons. This is marked by loss of {beta}-III-spectrin in distal dendrites, accumulation of {beta}-III-spectrin at the plasma membrane of the soma and proximal dendrites, and formation of inclusions in the soma. The inclusions additionally contain F-actin and -II-spectrin, accumulate around the nucleus, form at an early age, and are larger in homozygous {beta}-III-spectrinL253P/L253P compared to heterozygous {beta}-III-spectrinL253P/+ mice. In contrast, neurons of the hippocampus and cerebral cortex, where {beta}-III-spectrin is also known to be expressed, abnormally accumulate {beta}-III-spectrin at the plasma membrane but do not form inclusions. To gain greater insight into disease mechanisms, unbiased proteomics identified over 150 cerebellar proteins that physically associate with {beta}-III-spectrin. Of these, cluster analysis revealed a group of 41 proteins, including glutamate receptors, SERCA2, and CaMKII, linked to synaptic transmission. Thus, the effect of the L253P to alter {beta}-III-spectrin localization, including decreased levels in distal dendrites, is likely associated with a disruption of {beta}-III-spectrin function in postsynaptic signaling. Consistent with this, and in agreement with prior findings in knockout mice, the L253P {beta}-III-spectrin knock-in mouse here shows that CaMKII, a calcium sensor and key mediator of glutamate signaling, is ~2-fold activated. Further, the abundance of EAAT4, a glutamate transporter, is significantly reduced. The L253P knock-in mouse primes future preclinical testing of SCA5 therapeutics, such as small molecule modulators of spectrin-actin binding, and glutamate and calcium signaling pathways.

Sleep abnormalities and dysfunction of gamma band (30-80 Hz) activity generated by parvalbumin (PV) interneurons are early characteristics of Alzheimers disease (AD) which correlate with the severity of amyloid-{beta} deposition (A{beta}) and cognitive impairment. However, the timing of these alterations in vivo with respect to disease progression is unclear. Here, in longitudinal recordings from APP/PS1/PV-cre (AD mice) from 3-6 months, we found reduced sleep slow-wave power (0.5-4 Hz) in hippocampus and medial prefrontal cortex in AD mice as young as 3 months old, compared to non-AD (PV-cre) mice, well before overt pathology. This finding was primarily due to reductions in the NREM delta range (1.5-4 Hz), a hallmark of restorative functions of sleep. In contrast, beta (15-30 Hz) power linked to insomnia was significantly higher across all sleep-wake states. Loss of deep NREM sleep was not compensated by an increase in NREM sleep time, instead NREM sleep during the dark (active) phase was slightly but significantly lower in AD mice. 40-Hz auditory steady-state responses and associated evoked calcium responses of hippocampal PV neurons recorded using fiber photometry were also impaired by 3 months old. However, Y-maze performance in 3- and 6-month-old AD mice was not significantly different from non-AD mice. These results reveal reduced deep sleep and PV-associated 40-Hz activity as very early changes amenable to early intervention occurring prior to cognitive deficits. Furthermore, they establish APP/PS1 mice as a good model to causally test the relationship between sleep, PV neuronal activity and amyloid-mediated pathology.

{gamma}-secretase is a multi-subunit enzyme complex responsible for cleaving hundreds of substrates in diverse cellular contexts. Variation in subunit composition - including the use of alternate catalytic subunits Presenilin 1 (PSEN1) and Presenilin 2 (PSEN2) - results in diverse {gamma}-secretase complexes. Point mutations in PSEN1 and PSEN2 cause familial forms of Alzheimers disease, while loss-of-function mutations in the {gamma}-secretase subunits PSEN1, PSENEN and NCSTN cause acne inversa. To advance therapeutic strategies targeting {gamma}-secretase in Alzheimers disease, a better understanding of individual {gamma}-secretase complexes is required. In this study, we used CRISPR-Cas9 genome engineering to generate PSEN2-knockout iPSCs in order to compare the consequence of PSEN2 knockout versus PSEN1 knockout in iPSC-derived brain cells. In contrast to PSEN1-knockout, PSEN2-knockout did not alter APP cleavage or A{beta} generation in iPSC-neurons, nor did it disrupt Nicastrin maturation. Similarly, PSEN2-knockout had little impact on TREM2 processing in iPSC-microglia. Instead, our data indicate that loss of PSEN2 primarily impacts the endo-lysosomal system in iPSC-neurons, causing an accumulation of early endosome markers and a reduction in lysosomal markers - phenotypes not observed in PSEN1-knockout neurons. Taken together, these findings highlight distinct and non-redundant functions of PSEN1 and PSEN2 in human brain cells, reinforcing findings in animal models and subcellular localisation studies. This work advances our understanding of distinct {gamma}-secretase complex functions and provides insights that will support future therapeutic efforts to inhibit, modulate or stabilise {gamma}-secretase.

Parkinsons disease (PD) is associated with motor impairment and cortical synaptic dysfunction, which involve altered glutamate receptor trafficking, yet the underlying mechanisms remain incompletely understood. VPS26B, a component of the retromer complex, regulates GluA1 recycling in the trans-entorhinal cortex region. However, its role in the primary motor cortex (M1) under Parkinsonian conditions has not been explored. Here, we show that VPS26B levels are reduced in the M1 of an MPTP-induced PD mouse model, accompanied by decreased surface GluA1 and synaptic protein levels. VPS26B overexpression partially attenuated these alterations. In the accelerating rotarod test, VPS26B-deficient mice exhibited unstable motor performance following MPTP administration, whereas VPS26B overexpression was associated with improved performance in both wild-type and knockout mice. These findings suggest that cortical VPS26B may contribute to maintaining glutamate receptor surface expression and synaptic protein levels, especially under Parkinsonian conditions, with potential implications for motor learning.

Background: Mono-allelic Dehydrodolichyl Diphosphate Synthase (DHDDS) variants are associated with juvenile Parkinsonism, developmental delay and seizures. Symptoms are progressive, and various mechanisms, such as defective glycosylation, lysosomal dysfunction and cholesterol accumulation have been hypothesized to underlie disease symptoms. There is no treatment for DHDDS-related disease. Methods: Patient-derived cortical forebrain organoids were created to elucidate disease mechanisms and evaluate potential treatments. In these neuronal models, glycosylation, lipidomics, proteomics, cholesterol/ganglioside accumulation, mitochondrial function and electrophysiological activity were assessed. Finally, we investigated the effects of nicotinamide mononucleotide (NMN), identified through a yeast-based drug screen, in neuronal cell models and in six patients in an off-label, N-of-1, observational series. Results: DHDDS-patient derived organoids showed visual signs of degeneration after four months of culturing. This was accompanied by significant cholesterol accumulation in astrocytes, decreased mitochondrial respiration and loss of deep-layer neurons. In addition, we identified glycosylation abnormalities, showing for the first time that glycosylation in human tissue is affected by monoallelic DHDDS variants. Proteomic analysis revealed altered protein expression of proteins involved in lipid metabolism, cytoskeletal organization and neuronal development. We found that oral Nicotinamide Mononucleotide supplementation led to significant improvement in mitochondrial respiration and electrophysiological parameters in organoids, concurring with clinical improvements in all of the treated patients, particularly regarding their ataxia and tremor. Conclusion: Our findings reveal a progressive phenotype in DHDDS-patient-derived brain organoids, with mitochondrial dysfunction and astrocyte-specific metabolic alterations contributing to disease pathology. Notably, NMN treatment led to clinical improvements in patients with heterozygous DHDDS variants, highlighting its potential as a therapeutic strategy.

Plasma Membrane Calcium ATPase (PMCA) is essential for maintaining intracellular calcium homeostasis. Previously, we used constitutive PMCA downregulation in Drosophila melanogaster dopaminergic neurons as a model to increase intracellular calcium and mimic early neuronal alterations associated with Parkinsons disease. Here, we examined the mechanisms underlying the effects mediated by the conditional, adult-specific downregulation of PMCA in dopaminergic neurons in Drosophila melanogaster, both in vivo and in primary neuronal cultures. Adult-specific conditional silencing of PMCA in dopaminergic neurons reduced lifespan but to a lesser extent than the constitutive model and impaired locomotor performance. At the cellular level, PMCA-downregulated dopaminergic neurons exhibited elevated basal calcium, indicating disrupted calcium regulation. This was associated with a progressive increase in presynaptic vesicles and extracellular dopamine levels, suggesting enhanced neurotransmitter release. Notably, the synaptic active zone structure was preserved, indicating primarily functional rather than structural alterations. In primary neuronal cultures, PMCA downregulation reduced dopaminergic neuron survival and induced transient increases in neurite branching. Together, these findings show that PMCA downregulation leads to calcium dysregulation and presynaptic dysfunction without overt neurodegeneration in vivo, while promoting premature neuronal death in culture, indicating increased vulnerability and supporting a pre-degenerative state in which synaptic alterations precede neuronal loss.

Mutations in SCN1A, which encodes the voltage-gated sodium channel Nav1.1 (I subunit), are the major cause of Dravet syndrome, a severe developmental and epileptic encephalopathy. Although Nav1.1 haploinsufficiency preferentially impairs inhibitory interneuron function, the region-specific contributions of distributed brain circuits to seizure susceptibility, particularly in subcortical structures that have received less attention than the neocortex and hippocampus, remain unclear. Here, we examined the effects of region-specific Nav1.1 deficiency on hyperthermia-induced seizures by selectively deleting Scn1a in the neocortex, nucleus accumbens (NAc), and dorsal striatum (caudate-putamen, CPu) of adult mice using an adeno-associated virus-mediated Cre-loxP approach. Contrary to expectations based on prior cortical studies, homozygous Scn1a deletion in the neocortex produced only modest effects on seizure generalization. In contrast, homozygous deletion in the NAc and CPu induced generalized seizures to varying degrees. Notably, heterozygous Scn1a deletion in the CPu alone was sufficient to trigger generalized seizures, whereas similar manipulations in the neocortex or NAc were not. Seizure threshold temperatures were largely comparable across regions. These findings identify the dorsal striatum as particularly vulnerable to partial Nav1.1 loss and reveal functional heterogeneity within striatal circuits. Our results underscore a previously underappreciated role of striatal inhibitory networks in hyperthermia-induced seizure susceptibility and provide new insights into the circuit mechanisms underlying Dravet syndrome.

Sharp-wave ripple (SWR) oscillations are crucial for memory consolidation and deteriorate in Alzheimers disease (AD). Tau oligomers are suggested to lead to synaptic and neuronal degeneration in AD, but their effects on SWRs are unknown. To study this, we prepared mouse and human hippocampal slices and bath-applied tau oligomer preparations after spontaneous SWR generation. In human slices, acute exposure to tau resulted in decreased ripple duration, whereas in mouse slices it was SWR rate, amplitude, and power that decreased, sparing duration. In a different set of experiments, mouse slices were pre-incubated directly in either tau-ACSF or control-ACSF right after slicing for 2.5-5.5 hours, resulting only in diminished SWR rate. These effects were specific to the presence of {beta}-sheets, as a different tau preparation that lacked {beta}-sheets failed to alter SWRs. This method is therefore suitable to study SWR alterations after short-term exposure to different tau and/or A{beta} species, allows a higher throughput screening of possible therapeutics compared to in vivo animal experiments, and permits direct comparison of SWR alterations in mice and humans.

Huntingtons disease (HD) is a neurodegenerative disorder caused by CAG repeat expansion in the huntingtin (HTT) gene, with longer repeats linked to earlier onset. Somatic CAG expansion, particularly in the striatum, contributes to disease progression and is influenced by HTT biology and genetic modifiers. Modulating somatic expansion is emerging as a promising approach to slow or prevent HD, and mouse models have been crucial for preclinical testing of different therapeutic strategies. The BAC-CAG model, developed on the FVB strain, has been used to study somatic expansion of human expanded HTT. However, comparisons with other key HD mouse models have been limited by differences in genetic background, as many other models are on the C57BL/6 strain. The BAC-CAG model has now been developed on a C57BL/6 background. To determine whether the C57BL/6 BAC-CAG model can be used to study and modulate somatic expansion, we compared CAG expansion in mice on C57BL/6 or FVB backgrounds, with and without intraventricular divalent small interfering RNAs (siRNA) targeting HD modifiers MutS homolog 3 (MSH3) and HTT. Both strains exhibited robust, comparable somatic expansion over two months, which was blocked by MSH3-, but not HTT-, targeted siRNA. RNA sequencing identified gene expression differences primarily in pseudogenes, with no differences in endogenous Htt, human HTT, or mismatch repair genes. These results demonstrate that BAC-CAG mice on a C57BL/6 background exhibit somatic CAG expansion comparable to the validated FVB strain, providing a model to study and preclinically test therapies targeting somatic expansion in HD.

Friedreichs ataxia (FA) is a rare autosomal recessive neurodegenerative disorder caused by reduced expression of frataxin, a mitochondrial protein important for iron-sulfur cluster assembly and mitochondrial homeostasis. Although FA has traditionally been attributed to neuronal dysfunction, increasing evidence suggests that glial cells play a critical role in disease progression, although their contribution remains poorly defined. Using the FXNI151F mouse model, we investigated cell-type-specific metabolic and redox alterations in neurons and glial populations from the cerebrum, cerebellum, and dorsal root ganglia (DRG). Neuronal and glial-enriched fractions were isolated by immunomagnetic separation and analyzed for mitochondrial function, iron metabolism and reactive oxygen species (ROS). The analyses identified the DRG as the most severely affected region, exhibiting early and pronounced mitochondrial respiratory deficits, increased ROS, mitochondrial iron accumulation, lipid peroxidation, and reduced levels of glutathione peroxidase 4 and nuclear factor erythroid 2-related factor 2 in both neuronal and non-neuronal cells. These results highlight the vulnerability of sensory neurons and their supporting satellite glial cells. In contrast, in the cerebrum and cerebellum, astrocytes displayed earlier and more severe alterations than neurons, including impaired respiratory chain efficiency, disrupted complex I-III supercomplex interaction, elevated ROS, and hallmarks of ferroptosis. Neuronal abnormalities emerged later, suggesting that glial dysfunction precedes -or drives- neuronal pathology within the central nervous system. Overall, these findings reveal pronounced region and cell-type-specific vulnerabilities in FA and support the importance of targeting glial mechanisms--particularly iron dysregulation, oxidative stress, and ferroptosis-- as targets for potential therapeutic strategies.