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.

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 Parkinson's 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.

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.

{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.

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 is caused by expansion of a CAG repeat in the human HTT gene, producing a mutant huntingtin protein that misfolds and forms intracellular aggregates. Although Huntingtons disease is primarily characterized as a neurodegenerative disorder, mutant huntingtin is ubiquitously expressed, and peripheral tissues such as skeletal muscle exhibit pathological abnormalities. To define the muscle-intrinsic consequences of pathogenic huntingtin expression, we expressed caspase-6 truncated pathogenic human huntingtin in body wall muscle of Drosophila melanogaster larvae and performed quantitative structural and functional analyses. Aggregate analysis revealed that fluorescence intensity increased with aggregate size while aggregate morphology became more irregular. Delaying transgene expression until later stages of larval development dramatically reduced aggregate number, demonstrating a strong temporal dependence of aggregate formation. Myonuclei were enlarged, misshapen, and exhibited significantly reduced fluorescence intensity, consistent with altered chromatin organization. Notably, huntingtin aggregates were observed within the nucleus, indicating that nuclear proteostasis is directly perturbed by pathogenic huntingtin in muscle cells. Despite these intracellular defects, muscle fiber shape and sarcomere organization were preserved, suggesting that contractile apparatus assembly is not overtly disrupted. In contrast, mitochondrial organization was severely affected, with extensive mitochondrial aggregation throughout muscle fibers, consistent with altered organelle homeostasis. Functional analyses demonstrated that pathogenic huntingtin expression significantly impaired neuromuscular performance. Larvae exhibited reduced excitatory junctional potentials and diminished muscle contractile force, indicating compromised synaptic transmission and muscle function. Together, these findings demonstrate that pathogenic human huntingtin expression in skeletal muscle is sufficient to drive widespread protein aggregation, nuclear and mitochondrial abnormalities, and functional deficits despite the absence of overt structural changes. Our results highlight the importance of muscle-intrinsic pathogenic mechanisms and provide a quantitative framework for understanding how mutant huntingtin disrupts cellular organization and physiology outside the nervous system.

Although a differential vulnerability of dopaminergic neurons to degeneration based on their specific location within the dorsal and ventral tiers of the substantia nigra pars compacta (SNcD and SNcV, respectively) has long been postulated, the underlying mechanisms sustaining these tier-specific differences remain poorly understood. Here, upon inducing a viral-mediated enhancement of neuromelanin (NMel) accumulation within dopaminergic neurons in non-human primates, the distribution of Lewy body-like inclusions (LBs) was analyzed within identified SNcD and SNcV neurons, together with their intracellular NMel levels. Results showed that the vast majority of intracytoplasmic inclusions were found in SNcV neurons, and indeed correlated to higher pigmentation levels. By contrast, only very few LBs were found in calbindin-positive neurons of the SNcD, which in parallel exhibited very low levels of NMel accumulation. These results postulate an additive effect made of a tier-specific location of LB burden together with high pigmentation levels as synergistic drivers sustaining the preferential vulnerability of SNcV dopaminergic neurons. Moreover, the evidence obtained here supported that NMel accumulation beyond a given threshold triggers the aggregation of endogenous -Syn in the form of LBs; therefore, approaches intended to reduce pigmentation levels in SNcV neurons would likely induce a neuroprotective effect by preventing the subsequent aggregation of -Syn.

Mutations in several genes are known to cause familial forms of Parkinsons disease (PD), including mutations in the vacuolar protein sorting 35 ortholog (VPS35) gene linked to late-onset, autosomal dominant PD. VPS35 encodes a core subunit of the retromer complex which functions in endosomal sorting and recycling. It remains unclear how the pathogenic D620N mutation in VPS35 disrupts retromer function to induce neurodegeneration in PD. Using cell-and rodent-based models expressing D620N VPS35, we performed interactome proteomics to identify alterations underlying the pathogenic effects of D620N VPS35 in PD. Using overexpression of VPS35 variants in HEK-293T cells, we conducted tandem affinity purification (TAP) or co-immunoprecipitation (co-IP) with protein chemical crosslinking to determine the native and non-native protein interactomes of wild-type (WT) and D620N VPS35, respectively. Notably, we can confirm the reduced interaction of D620N VPS35 with components of the WASH complex. Additionally, using a viral-mediated gene transfer model of human D620N VPS35 overexpression in adult rat brain, we identify the first brain-specific protein interactome of VPS35. These overexpression models reveal remarkably similar interaction profiles of WT and D620N VPS35, suggesting that the D620N mutation has a subtle effect on the overall VPS35 protein interactome. We also conducted proteomic analysis of brain tissue from a D620N VPS35 knockin (KI) mouse model that expresses VPS35 at endogenous levels. Using co-IP from hemi-brain or striatal extracts of WT and D620N VPS35 KI mice, we reveal a high degree of similarity between the brain interactomes of WT and D620N VPS35, further suggesting a subtle effect of the D620N mutation on VPS35 protein interactions. Notably, in both hemi-brain and striatum, we find a selective decrease in the interaction of two known interactors, TBC1D5 and VPS29, with D620N VPS35. We also performed global proteomic analysis of striatal tissue from D620N VPS35 KI mice and reveal a high degree of similarity between WT and D620N, further suggesting a subtle effect of this mutation. Together, our study provides a comprehensive evaluation of the VPS35 protein interactome and reveals a selective effect of the PD-linked D620N mutation in mammalian cells and brain. Our study provides key insight into the mechanisms of retromer dysfunction in VPS35-linked PD.

The Alzheimers Amyloid Precursor Protein (APP) has determinant roles in neuronal development and function, both in its full-length conformation and as some of its proteolytic peptides, particularly secreted (s)APPa. Given that APP phosphorylation tightly regulates its trafficking, proteolysis, and protein-protein binding, it consequently affects several APP functions. The S655 residue, located in the basolateral sorting motif YTSI at APP C-terminus has been observed to be phosphorylated in mature full-length APP and its C-terminal fragments. Previously observed to modify APPs protein interactions, resulting in altered endolysosomal trafficking, andincreased half-life and sAPPa generation, phosphoS655 APP has potential to modulate APP-mediated neuronal differentiation. To study the phosphoS655 differential interactome relevant for neuronal differentiation, SH-SY5Y cells expressing Wt or S655 phosphomutants APP-GFP were differentiated at two time points. APP-GFP and their respective interacting partners were immunoprecipitated using GFP-trap, and interactors identified by mass spectrometry. Both dephospho and phosphoS655 interactomes were generally enriched in similar processes, primarily RNA processing and translation, as well as signal transduction, metabolism, and cytoskeleton remodeling. The smaller phosphoS655 interactome contributes for functional specialization via binding to e.g. FUBP3, ELAVL4, ATXN2, Tubulin, INA. Several of these specific binding partners are known to promote neurite outgrowth and likely underlie our experimental observation that phosphoS655 APP promotes neuritogenesis, particularly the formation of longer neuritic extensions. These results are not only important for the body of knowledge on this Alzheimers disease core protein, but may also aid in future therapies against this disease.

Background and PurposePathogenic aggregation and propagation of seed-competent TAU assemblies drive tauopathies. MAPT P301 mutations accelerate aggregation and enhance seed competence, yet pharmacological strategies selectively targeting these pathogenic species remain limited. We investigated whether the clinically approved catechol-O-methyltransferase inhibitors tolcapone (TOL) and entacapone (ENT) preferentially modulate mutant TAU aggregation and seeding. Experimental ApproachTOL and ENT effects on TAU aggregation were evaluated via cell-free assays, surface plasmon resonance (SPR), and in silico docking. Functional consequences of compound-modified fibrils were assessed in mutant TAU-expressing SH-SY5Y cells. Translational relevance was examined in human induced pluripotent stem cell (hiPSC)-derived neurons exposed to pathogenic K18 fibrils, followed by post-seeding compound treatment. Key ResultsBoth compounds dose-dependently inhibited TAU aggregation, exhibiting greater potency, stronger SPR binding affinities, and more favorable computed interaction energies for P301S mutant versus wild-type TAU. Fibrils formed with TOL or ENT induced less downstream TAU oligomerization and phosphorylation in SH-SY5Y cells, with TOL showing superior protection. In hiPSC-derived neurons, post-seeding treatment with either compound decreased fibril-induced, sarkosyl-insoluble TAU aggregation and phosphorylation without overt cytotoxicity. Conclusion and ImplicationsTOL and ENT preferentially inhibit the aggregation and seeding of pathogenic P301 mutant TAU. This supports mutation-focused pharmacological strategies and highlights catechol scaffolds as viable starting points for the development of disease-modifying therapeutics. Future research must determine the precise interaction mechanisms with aggregation intermediates and evaluate in vivo efficacy in animal models.

Electroconvulsive therapy (ECT) is a highly effective treatment for several psychiatric disorders, though its biological mechanisms remain unclear. Its therapeutic action has traditionally been attributed to the generalized seizure ECT induces. However, this view is challenged by the recent finding that electroconvulsive stimulation (ECS) can trigger a cortical spreading depression (CSD). Because CSD triggers massive intracellular molecular changes, we hypothesized that it could be a key mediator of ECTs therapeutic, plasticity-inducing effects. We observed similar neuronal oscillations following ECS in mice and patients undergoing ECT. We show that CSD drives increased expression of the immediate early gene Fos, a key marker of neuronal plasticity, and is associated with factors that predict positive ECT therapeutic outcome. Our results suggest that the therapeutic efficacy of ECT may be mediated by CSD. This challenges the seizure-centric model and implies that CSD, a currently unmonitored neurophysiological event, may serve as a more relevant biomarker for predicting and optimizing therapeutic outcomes of ECT.

Polyhydramnios, Megalencephaly, and Symptomatic Epilepsy syndrome (PMSE/STRADA-related disorder) is a rare neurodevelopmental disorder characterized by megalencephaly (ME), early-onset drug-resistant epilepsy, neurocognitive impairment, and high early mortality, often due to status epilepticus. PMSE is caused by a multi-exon deletion in STRADA, encoding STRADA, which regulates the mechanistic target of rapamycin (mTOR) pathway. GABAergic inhibitory interneurons (INs) critically modulate the excitatory:inhibitory balance in cortical and hippocampal networks, and IN deficits contribute to epileptogenesis in several epileptic encephalopathies. However, no studies have investigated INs in PMSE. We used a multimodal approach to study INs in a Strada-/- mouse model engineered with the same causative 5-exon deletion identified in human PMSE. We demonstrate that Strada/STRADA loss causes a reduction of INs in the somatosensory cortex and a corresponding increase in the striatum, representative of remnant ganglionic eminence progenitor origin, in Strada-/- mice and a single PMSE brain tissue specimen. RNA sequencing comparing wildtype to Strada-/- cortex and striatum corroborated these findings, revealing increased IN-related gene expression (e.g., Dlx2) in the striatum and decreased IN-related gene expression (e.g., Pvalb) in the developing cortex. Cytoskeletal (e.g., Tpp3, Kank4, Map1a) and mTOR-associated genes (e.g., Rictor, Cryab) are differentially expressed in the developing cortex, mature striatum, and mature cortex of Strada-/- mice. Functional validation confirmed enlarged INs in mouse and human Strada/STRADA-deficient brain and enhanced S6 phosphorylation in Strada-/- striatum. Together, these findings suggest STRADA/Strada loss contributes to failed IN migration -- the first such report in a developmental, mTOR-associated megalencephaly syndrome -- highlighting INs as a therapeutic target for seizure prevention in PMSE. Key PointsO_LI- Reduced numbers of cortical inhibitory interneurons were observed in the cerebral cortex of Strada-/- mice, with striatal interneuron aggregation C_LIO_LI- Reduced numbers of cortical inhibitory interneurons, with an aggregation in striatum, were observed in human PMSE brain, supporting the observations in Strada-/- mouse C_LIO_LI- Transcriptomic analysis in Strada-/- mice reveals evidence of early developmental interneuron and cytoskeletal dysfunction C_LIO_LI- We introduce a loss of cortical interneurons as a salient feature of PMSE developmental pathogenesis, potentially contributing to a loss of inhibitory modulation C_LIO_LI- This is the first study proposing interneuron migration impairment in the developmental pathogenesis of an mTOR-associated megalencephaly syndrome C_LI

Mutations in DOCK7 have been identified in individuals with epileptic encephalopathies. Given that epileptic encephalopathies are a set of disorders that result in seizure activity and associated cognitive and behavioral impairments, we investigated the role of Dock7 in seizure susceptibility and flurothyl kindling using the repeated flurothyl seizure model in mice. Male and female Dock7+/+ and Dock7{bigtriangleup}ex3-4/{bigtriangleup}ex3-4 mice were subjected to 8 daily flurothyl exposures (kindling, induction phase) followed by a 28-day incubation period and a subsequent flurothyl rechallenge (retest). No significant differences were observed in baseline myoclonic jerk or generalized seizure thresholds between genotypes or sexes. However, over the kindling period, male Dock7{bigtriangleup}ex3-4/{bigtriangleup}ex3-4 mice exhibited slightly higher myoclonic jerk and generalized seizure thresholds compared to Dock7+/+ males across trials. Female mice showed similar trends, but the differences were only significant for generalized seizure thresholds. Following the 28-day incubation period and flurothyl retest, male mice of both genotypes maintained their seizure thresholds upon retest. Dock7+/+ female mice showed increased myoclonic jerk and generalized seizure thresholds during retest, while Dock7{bigtriangleup}ex3-4/{bigtriangleup}ex3-4 females maintained their thresholds. A key finding was the emergence of more severe forebrain[->]brainstem seizures upon flurothyl retest in a significant percentage of mice across all groups. However, the proportion of mice developing these seizures did not differ significantly between genotypes. Although DOCK7 mutations have been linked to human epileptic encephalopathies and neurodevelopmental dysfunction, we find that Dock7{bigtriangleup}ex3-4/{bigtriangleup}ex3-4 male and female mice do not show heightened excitability or seizure susceptibilities using the repeated flurothyl seizure model. HighlightsO_LIDock7{bigtriangleup}ex3-4/{bigtriangleup}ex3-4 mice show slightly higher seizure thresholds during flurothyl kindling C_LIO_LIDock7{bigtriangleup}ex3-4/{bigtriangleup}ex3-4 mice do not exhibit heightened seizure susceptibility upon retest. C_LIO_LIForebrain-brainstem seizures emerged upon retest regardless of Dock7 genotype. C_LI

D-Aspartate (D-Asp) is an endogenous D-amino acid that exhibits a pronounced developmental peak in the mammalian brain, suggesting a potential regulatory role in glutamatergic signaling and neurodevelopment. Disruption of D-Asp homeostasis has been associated with neuropsychiatric disorders characterized by early-life circuit vulnerability, including schizophrenia and autism spectrum disorders. However, its functional impact to hippocampal physiology remains incompletely defined. Here, we investigated how constitutive D-Asp depletion affects synaptic function in the hippocampal CA1 region of Ddo-knock-in (Ddo-KI) mice, in which zygotic overexpression of the D-Asp-degrading enzyme, D-aspartate oxidase (DASPO), results in embryonic and persistent D-Asp deficiency. Electrophysiological recordings were performed in acute hippocampal slices from male and female mice at postnatal day 30 (P30) and day 60 (P60). Basal synaptic transmission, assessed through paired-pulse ratio and spontaneous excitatory/inhibitory events, was unaltered between genotypes, indicating preserved presynaptic release probability and overall excitation/inhibition balance. In contrast, NMDA receptor (NMDAR)-dependent synaptic plasticity was selectively altered, as theta-burst stimulation induced significantly greater long-term potentiation (LTP) in juvenile P30 Ddo-KI mice, whereas this difference was no longer observed at P60. Consistently, patch-clamp recordings revealed a reduced AMPAR/NMDAR ratio in P30 Ddo-KI males, suggesting an increased relative contribution of NMDAR-mediated currents. Importantly, acute bath application of exogenous D-Asp restored LTP to wild-type levels, demonstrating rapid reversibility and supporting a model of homeostatic receptor rebalancing rather than irreversible circuit alterations. Biochemical assays confirmed significantly increased DASPO activity and reduced D-Asp levels in Ddo-KI mice. However, these parameters remained stable between P30 and P60, indicating that the age-dependent plasticity phenotype is unlikely to arise from progressive biochemical changes. Together, these findings indicate that developmental D-Asp deficiency induces a transient, juvenile-specific alteration characterized by enhanced NMDAR-dependent synaptic plasticity, which can be rapidly normalized upon D-Asp re-exposure.

Early detection of disease progression using clinically-relevant biomarkers in animal models is important for mechanistic studies and for developing therapeutics in neurodegenerative diseases including Alzheimers disease (AD). The preclinical stage of AD, when amyloid-{beta} (A{beta}) starts to accumulate before cognitive decline, provides a critical window for disease modification. In humans, decreases in cerebrospinal fluid (CSF) A{beta}42 and the A{beta}42/A{beta}40 ratio in preclinical AD are considered to reflect the preferential sequestration of aggregation-prone A{beta}42 into {beta}-sheet-rich deposition in the brain, with corresponding changes being detectable in plasma. However, the extent to which these biomarker-pathology relationships are recapitulated in AD model mice remains incompletely defined. Here we show that CSF and plasma A{beta}42 and the A{beta}42/A{beta}40 ratio decline with age in parallel with the progression of {beta}-sheet-rich A{beta} deposition in preclinical 5XFAD mice, one of the most widely used AD mouse models, as assessed through monthly profiling of these biomarkers. Notably, the CSF A{beta}42/A{beta}40 ratio showed a negative correlation with {beta}-sheet-rich A{beta} deposition in the brain, whereas CSF A{beta}40 did not show a comparable association. In addition, the plasma A{beta}42/A{beta}40 ratio showed a positive correlation with the CSF A{beta}42/A{beta}40 ratio, suggesting that the plasma A{beta}42/A{beta}40 ratio may also reflect brain A{beta} deposition in this model. The strength of these correlations differed by sex, suggesting that sex-dependent differences in the A{beta} kinetics in this model may influence how closely fluid biomarkers reflect pathological progression. These findings support the potential utility of fluid A{beta} as a pathology-linked, translatable biomarker in preclinical 5XFAD mice. Highlights- Fluid A{beta} biomarkers are associated with early A{beta} deposition in preclinical 5XFAD mice. - The CSF A{beta}42/A{beta}40 ratio negatively correlates with {beta}-sheet-rich brain A{beta} deposition. - The plasma A{beta}42/A{beta}40 ratio positively correlates with the CSF A{beta}42/A{beta}40 ratio. - Monthly profiling defines fluid A{beta} biomarker dynamics in preclinical 5XFAD mice. - Sex differences may affect biomarker-pathology relationships in these mice.

Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis involves dynamic changes in glutamatergic signalling. Magnetic resonance spectroscopy can monitor these changes but lacks temporal resolution and cell-type specificity. We investigated whether urinary astrocyte-derived extracellular vesicles (ADEVs) could serve as a non-invasive proxy for brain receptor dynamics. We prospectively collected longitudinal urine and cerebrospinal fluid (CSF) samples from a 30- 35-year-old female patient during 34 days of treatment. We isolated ADEVs using a specific protocol and measured GluN1 protein levels. A 30-35-year-old healthy female provided control samples. Wavelet transform analysis of the patient's GluN1 time series revealed two distinct patterns. First, a low-frequency trend showed declining GluN1 levels over the treatment period, which mirrored the reduction in CSF GluN1 concentrations. Second, a high-frequency oscillation appeared to be coupled with methotrexate infusions, with GluN1 peaks occurring approximately 48 hours after each dose. This secondary increase may reflect drug-induced p53 activation, which promotes the exosomal release of internalised receptors. These findings suggest that urinary ADEVs provide a feasible and informative method to monitor real-time molecular fluxes in the brain.

Spinal muscular atrophy (SMA), traditionally defined as a neuromuscular disorder characterized by degeneration of lower motor neurons, is increasingly recognized as a multi-organ disease. SMA is caused by deficiency of the survival motor neuron (SMN) protein below a critical threshold required for cellular homeostasis. While motor neurons are particularly vulnerable, the ubiquitous expression and fundamental functions of SMN result in widespread perturbations across multiple tissues. Here, we generated a label-free quantitative proteomics atlas of spinal cord, heart, and gastrocnemius muscle from wild-type, heterozygous, and SMA mice at the symptomatic stage, including cohorts treated, at postnatal day 1 (P1), with a systemic suboptimal dose of SMN antisense oligonucleotides (SMN-ASOs), resulting in partial SMN restoration. SMN deficiency induced pronounced, tissue-specific proteome remodeling, with peripheral tissues exhibiting broader molecular alterations than spinal cord. Cross-tissue analyses revealed limited overlap, although heart and muscle showed partial convergence in metabolic and mitochondrial-associated pathways. SMN-ASO treatment partially repositioned these proteomes toward control states; however, restoration was incomplete and strongly tissue-dependent, with persistent dysregulation of mitochondrial and metabolic pathways. These findings demonstrate that SMN deficiency drives systemic yet heterogeneous proteome remodeling and that partial SMN restoration does not fully reverse established molecular alterations. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/715402v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@114d73borg.highwire.dtl.DTLVardef@13e8c13org.highwire.dtl.DTLVardef@15e4ba0org.highwire.dtl.DTLVardef@1b70fb8_HPS_FORMAT_FIGEXP M_FIG C_FIG