Neurobiology of Disease

Pathogenic variants in KCNB1 are associated with a neurodevelopmental disorder spectrum that includes global developmental delays, cognitive impairment, abnormal electroencephalogram (EEG) patterns, and epilepsy with variable age of onset and severity. Additionally, there are prominent behavioral disturbances, including hyperactivity, aggression, and features of autism spectrum disorder. The most frequently identified recurrent variant is KCNB1-p.R306C, a missense variant located within the S4 voltage-sensing transmembrane domain. Individuals with the R306C variant exhibit mild to severe developmental delays, behavioral disorders, and a diverse spectrum of seizures. Previous in vitro characterization of R306C described loss of voltage sensitivity and cooperativity of the sensor and inhibition of repetitive firing. Existing Kcnb1 mouse models include dominant negative missense variants, as well as knockout and frameshifts alleles. While all models recapitulate key features of KCNB1 encephalopathy, mice with dominant negative alleles were more severely affected. In contrast to existing loss-of-function and dominant-negative variants, KCNB1-p.R306C does not affect channel expression, but rather affects voltage-sensing. Thus, modeling R306C in mice provides a novel opportunity to explore impacts of a voltage-sensing mutation in Kcnb1. Using CRISPR/Cas9 genome editing, we generated the Kcnb1R306C mouse model and characterized the molecular and phenotypic effects. Heterozygous and homozygous R306C mice exhibited pronounced hyperactivity, altered susceptibility to flurothyl and kainic acid induced-seizures, and frequent, long runs of spike wave discharges on EEG. This novel model of channel dysfunction in Kcnb1 provides an additional, valuable tool to study KCNB1 encephalopathies. Furthermore, this allelic series of Kcnb1 mouse models will provide a unique platform to evaluate targeted therapies.

Alzheimers disease (AD) is a devastating neurodegenerative condition that affects memory and cognition, characterized by neuronal loss and currently lacking a cure. Mutations in PSEN1 (Presenilin 1) are among the most common causes of early-onset familial AD (fAD). While changes in neuronal excitability are believed to be early indicators of AD progression, the link between PSEN1 mutations and neuronal excitability remains to be fully elucidated. This study examined induced pluripotent stem cell (iPSC)-derived NGN2 induced neurons (iNs) from fAD patients with PSEN1 mutations S290C or A246E, alongside CRISPR-corrected isogenic cell lines, to investigate early changes in excitability. Electrophysiological profiling revealed reduced excitability in both PSEN1 mutant iNs compared to their isogenic controls. Neurons bearing S290C and A246E mutations exhibited divergent passive membrane properties compared to isogenic controls, suggesting distinct effects of PSEN1 mutations on neuronal excitability. Additionally, both PSEN1 backgrounds exhibited higher current density of voltage-gated potassium (Kv) channels relative to their isogenic iNs, while displaying comparable voltage-gated sodium (Nav) channel current density. This suggests that the Nav/Kv imbalance contributes to impaired neuronal firing in fAD iNs. Deciphering these early cellular and molecular changes in AD is crucial for understanding the disease pathogenesis.

Rapid removal of glutamate from the sites of glutamate release is an essential step in excitatory synaptic transmission. Despite many years of research, the molecular mechanisms underlying the intracellular regulation of glutamate transport at tripartite synapses have remained unclear. This limits the options for pharmacological treatment of motor disorders associated with glutamate excitotoxicity. Therefore, using the Q175 mouse model of Huntingtons disease (HD), we explored the effects of structural changes in the astrocytic excitatory amino acid transporter type 2 (EAAT2). We report that expression of a C-terminal-modified variant of EAAT2 can alleviate the symptoms of hypokinesia in mice with already advanced HD. At a cellular level, this beneficial outcome correlated with faster synaptic glutamate clearance, higher astrocytic glutamate uptake and larger amounts of native EAAT2 protein. Proteomics data indicate a partial reversal of HD-induced changes in the EAAT2 interactor spectrum. Thus, astrocytic glutamate transport remains a target for therapeutic intervention.

Increased neurofilament light (NfL) protein in biofluids is reflective of neurodegeneration and has gained interest as a biomarker across neurodegenerative diseases. In spinocerebellar ataxia type 3 (SCA3), the most common dominantly inherited ataxia, patients exhibit progressive NfL increases in peripheral blood when becoming symptomatic, remaining stably elevated throughout further disease course. However, progressive NfL changes are not yet validated in relevant preclinical SCA3 animal models, hindering its application as a biomarker during therapeutic development. We used ultra-sensitive single-molecule array (Simoa) to measure blood NfL over disease progression in the YACQ84 mouse, assessing relationships with measures of disease severity including age, CAG repeat size, and magnetic resonance spectroscopy. We show that YACQ84 mice exhibit increased blood NfL, concomitant with ataxia-related motor deficits and correlated with neurometabolite abnormalities. Our findings establish natural history progression of NfL increases in the preclinical YACQ84 mouse, further supporting the utility of blood NfL as a peripheral neurodegeneration biomarker and informing coinciding timelines of different measures of SCA3 pathogenesis. Summary statementPeripheral blood of SCA3 YACQ84 mice exhibits increased abundance of neuronal-specific NfL protein directly associating with disease progression, providing an accessible disease biofluid biomarker to interrogate in preclinical therapeutic studies.

ObjectiveDravet Syndrome (DS) is a catastrophic form of paediatric epilepsy associated with multiple comorbidities mainly caused by mutations in the SCN1A gene. DS progresses in three different phases termed febrile, worsening and stabilization stage. Mice that are haploinsufficient for Scn1a faithfully model each stage of DS, although various aspects have not been fully described, including the temporal appearance and sex differences of the epilepsy and comorbidities. The aim of the present study was to investigate the epilepsy landscape according to the progression of DS and the long-term co-morbidities in the Scn1a(+/-)tm1Kea DS mouse line that are not fully understood yet. MethodsMale and female F1.Scn1a(+/+) and F1.Scn1a(+/-)tm1Kea mice were assessed in the hyperthermia model or monitored by video electroencephalogram (vEEG) and wireless video-EEG according to the respective stage of DS. Long-term comorbidities were investigated through a battery of behaviour assessments in [~]6 month-old mice. ResultsAt P18, F1.Scn1a(+/-)tm1Kea mice showed the expected sensitivity to hyperthermia-induced seizures. Between P21 and P28, EEG recordings in F1.Scn1a(+/-)tm1Kea mice combined with video monitoring revealed a high frequency of SRS and SUDEP. Power spectral analyses of background EEG activity also revealed that low EEG power in multiple frequency bands was associated with SUDEP risk in F1.Scn1a(+/-)tm1Kea mice during the worsening stage of DS. Later, SRS and SUDEP rates stabilized and then declined in F1.Scn1a(+/-)tm1kea mice. SRS and SUDEP in F1.Scn1a(+/-)tm1kea mice displayed variations with the time of day and sex, with female mice displaying higher numbers of seizures and greater SUDEP risk. F1.Scn1a(+/-)tm1kea mice [~]6 month- old displayed fewer behavioural impairments than expected including hyperactivity, impaired exploratory behaviour and poor nest building performance. SignificanceThese results reveal new features of this model that will optimize use and selection of phenotype assays for future studies on the mechanisms, diagnosis, and treatment of DS. Key point boxO_LIScn1a(+/-)tm1kea DS mouse model faithfully reproduces the three stages of DS C_LIO_LISex of F1.Scn1a(+/-)tm1kea mice influences the epilepsy phenotype C_LIO_LIF1.Scn1a(+/-)tm1kea develop some of the long-term comorbidities of DS C_LI

Epilepsies are multifaceted neurological disorders characterized by abnormal brain activity, e.g., caused by imbalanced synaptic excitation and inhibition. The neural extracellular matrix (ECM) is dynamically modulated by physiological and pathophysiological activity and critically involved in controlling the brains excitability. We used different epilepsy models, i.e. mice lacking the presynaptic scaffolding protein Bassoon at excitatory, inhibitory or all synapse types as genetic models for rapidly generalizing early-onset epilepsy, and intra-hippocampal kainate injection, a model for acquired temporal lobe epilepsy, to study the relationship between epileptic seizures and ECM composition. Electroencephalogram recordings revealed Bassoon deletion at excitatory or inhibitory synapses having diverse effects on epilepsy-related phenotypes. While constitutive Bsn mutants and GABAergic neuron-specific knockouts (BsnDlx5/6cKO) displayed severe epilepsy with more and stronger seizures than kainate-injected animals, mutants lacking Bassoon solely in excitatory forebrain neurons (BsnEmx1cKO) showed only mild impairments. By semiquantitative immunoblotting and immunohistochemistry we show model-specific patterns of neural ECM remodeling, and we also demonstrate significant upregulation of the ECM receptor CD44 in null and BsnDlx5/6cKO mutants. ECM-associated WFA-binding chondroitin sulfates were strongly augmented in seizure models. Strikingly, Brevican, Neurocan, Aggrecan and link protein Hapln1 levels reliably predicted seizure properties across models, suggesting a link between ECM state and epileptic phenotype.

Approximately 22% of Alzheimers disease (AD) patients suffer from seizures, and the co-occurrence of seizures and epileptiform activity exacerbate AD pathology and related cognitive deficits. Hence seizures may be a targetable component of AD progression. As epileptogenesis is associated with changes in neuronal excitatory: inhibitory (E:I) balance, we hypothesized that decreased markers of inhibition relative to those of excitation would be present in AD patients and exacerbated further when seizures were a comorbidity. We similarly hypothesized that an E:I imbalance would be present in five times familial AD (5XFAD) mice and augmented following pentylenetetrazol (PTZ) seizure kindling. AD temporal cortical tissue from patients with or without seizure history and brain tissue from 5XFAD mice were examined for changes in several markers of E:I balance, including the inhibitory GABAA receptor, the chloride cotransporters, sodium potassium chloride cotransporter 1 (NKCC1) and potassium chloride cotransporter 2 (KCC2), and the excitatory NMDA and AMPA type glutamate receptors. We found that AD patients had decreased GABAA receptor subunits and those with comorbid seizures had worsened cognitive and functional scores, and increased in NKCC1/KCC2 ratios, indicative of depolarizing GABA responses. The E:I imbalance appears to occur early in the disease course, as patch clamp recordings from CA1 neurons in hippocampal slices from prodromal 5XFAD mice showed decreased GABAergic inhibitory transmission and increased intrinsic excitability. In addition, seizure induction in prodromal 5XFAD mice further dysregulated NKCC1/KCC2, and altered the excitatory AMPA glutamate receptor protein expression, with a reduction in GluA2 subunit, indicative of calcium permeable-receptors. Finally, we found that chronic treatment with the mTORC1 inhibitor, rapamycin, at doses we have previously shown to attenuate seizure-induced -amyloid pathology and cognitive deficits, could reverse aspects of E:I imbalance in these mice. Our data demonstrate novel mechanisms of interaction between AD and epilepsy and indicate that FDA-approved mTOR inhibitors hold therapeutic promise for AD patients with a seizure history.

ObjectiveGenetic variations in proteins of the mechanistic target of rapamycin (mTOR) pathway cause a spectrum of neurodevelopmental disorders often associated with brain malformations and with intractable epilepsy. The mTORopathies are characterized by hyperactive mTOR pathway and comprise tuberous sclerosis complex (TSC) and focal cortical dysplasia (FCD) type II. How hyperactive mTOR translates into abnormal neuronal activity and hypersynchronous network remains to be better understood. Previously, the role of upregulated GluN2C-containing glutamate- gated NMDA receptors (NMDARs) has been demonstrated for germline defects in the TSC genes. Here, we questioned whether this mechanism would expand to other mTORopathies in the different context of a somatic genetic variation of the MTOR protein recurrently found in FCD type II. MethodsWe used a rat model of FCD created by in utero electroporation of neural progenitors of dorsal telencephalon with expression vectors encoding either the wild-type or the pathogenic MTOR variant (p.S2215F). In this mosaic configuration, patch-clamp whole-cell recordings of the electroporated, spiny stellate neurons and extracellular recordings of the electroporated areas were performed in neocortical slices. Selective inhibitors were used to target mTOR activity and GluN2C- mediated currents. ResultsNeurons expressing the mutant protein displayed an excessive activation of GluN2C NMDAR-mediated spontaneous excitatory post-synaptic currents. GluN2C-dependent increase in spontaneous spiking activity was detected in the area of electroporated neurons in the mutant condition and was restricted to a critical time-window between postnatal days P9 and P20. SignificanceSomatic MTOR pathogenic variant recurrently found in FCD type II resulted in overactivation of GluN2C-mediated NMDARs in neocortices of rat pups. The related and time- restricted hyperexcitability was sensitive to subunit GluN2C-specific blockade. Our study suggests that GluN2C-related pathomechanisms might be shared in common by mTOR pathway-related cortical dysplasia. Key pointsO_LIExcessive activation of GluN2C NMDAR-mediated currents in spiny stellate neurons expressing FCD-causing MTOR somatic variation C_LIO_LIGluN2C-dependent increase in spontaneous spiking activity in rat somatosensory cortex containing mutant MTOR-expressing neurons C_LIO_LIGluN2C-dependent excessive network activity is time-restricted to a critical period between P9 and P20 C_LI

Drug-resistant epilepsy (DRE) is a prevalent problem in children that can lead to abnormal development and various psychiatric comorbidities. Malformations of cortical development (MCD) include focal cortical dysplasia, tuberous sclerosis complex and hemimegalencephaly, which are the most common pathologies among children who undergo surgical resection for treatment of DRE. These disorders share many histopathological features, including dyslamination of the cerebral cortex and enlarged neuronal somata. Recently, genetic mutations in the mammalian target of rapamycin (mTOR) signaling cascade have been shown to underpin most MCDs. Rodent models, including the RhebCA model, recapitulate histologic and physiologic aspects of human DRE. However, there have been few studies characterizing the developmental time point of the histological changes seen in MCDs. In this study, we use in utero electroporation to upregulate the Rheb protein (directly upstream of mTOR) in a focal area of the neocortex. We demonstrate that mTOR dysregulation leads to focal dyslamination and increased neuronal size that is histologically similar to MCD, which correlates to spontaneous recurrent seizures. We used immunohistochemistry to investigate neuronal lamination at several time points during development between E18 and P21 and show early differences in lamination that persisted through development. Furthermore, the increased axonal length associated with mTOR upregulation occurs early in development. Our study provides a time frame for the initial development of abnormal neuronal migration and cellular growth that occurs in MCDs, and our data supports that these anatomical changes may contribute to the formation of epileptic networks.

Cortical-striatal synaptic dysfunction, including enhanced toxic signaling by extrasynaptic N-methyl-D-aspartate receptors (eNMDARs), precedes neurodegeneration in Huntington disease (HD). A previous study showed Activin A, whose transcription is upregulated by calcium influx via synaptic NMDARs, suppresses eNMDAR signaling. Therefore, we examined the role of Activin A in the YAC128 HD mouse model, comparing it to wild-type controls. We found decreased Activin A secretion in YAC128 cortical-striatal co-cultures, while Activin A overexpression in this model rescued altered eNMDAR expression. Striatal overexpression of Activin A in vivo improved motor learning on the rotarod task, and normalized striatal neuronal eNMDAR-mediated currents, membrane capacitance and spontaneous excitatory postsynaptic current frequency in the YAC128 mice. These results support the therapeutic potential of Activin A signaling and targeting eNMDARs to restore striatal neuronal health and ameliorate behavioral deficits in HD.

Sodium channel inhibitors have been reported to protect against a range of neuroinflammatory and neurodegenerative diseases. Here the effect of chronic administration of two Na+ channel inhibitors with different mechanisms of action, phenytoin and GS967 are tested in mouse models of different stages of Alzheimers disease. Subtle changes in the distribution of plaque sizes were observed in AppNLGF/NLGF mouse at 3 months of age, after being fed control or drug-supplemented chow from weaning onwards, with phenytoin treatment resulting in a significant increase in the frequency of the smallest plaques and a decrease in large plaques. The later pathology of neurofibrillary tangles was studied, in old age, by supplementing the food of transgenic mice with a P301L mutation in Tau. Chronic administration of Na+ inhibitors from 15 months of age resulted in a decrease in the density of MC1-positive neurofibrillary tangles, possibly due to effects on microglial Na+ channels. The density of microglial cells was strongly correlated with the density of neurofibrillary tangles but only in mice treated with the Na+ inhibitors.

GALNT2-CDG is a multi-system genetic disorder due to biallelic pathogenic mutations in GALNT2, which encodes a ubiquitously expressed Golgi-localized glycosyltransferase that initiates mucin-type O-glycosylation. Affected individuals exhibit dysmorphic facial features, short stature, decreased HDL-C, and notable impairments in brain function. GALNT2-CDG patients show global developmental delay without speech development, childhood epilepsy, autistic-like features, and white-matter brain abnormalities. The extent of O-glycosylation in brain development and function remains poorly understood. To address this question, we selectively ablated Galnt2 from pan-neuronal cells in the brain and found that conditional knockout mice exhibit deficits across numerous behavioral domains, including locomotion, motor coordination, sociability, learning, and memory, as well as experience spontaneous seizures, recapitulating characteristic neurological manifestations of GALNT2-CDG. Given the catalytic activity of GALNT2 to initiate mucin-type O-glycosylation, we used glycoproteomics to identify disrupted O-glycosylation in synaptosomes purified from cortical tissues. We ascertained a non-redundant, isoform-specific contribution of GALNT2 to the cortical synaptosomal O-glycoproteome, identifying candidate glycoproteins and disrupted O-glycosites that accompany behavioral abnormalities in knockout mice. These findings demonstrate functional impact of O-glycosylation in neurons, implicating roles of O-glycosylation in diverse molecular and cellular pathways related to neuronal function and provide new opportunities to gain insights into the neurological pathophysiology of GALNT2-CDG.

RationaleNorthern epilepsy belongs to a group of genetically diverse lysosomal storage diseases, the neuronal ceroid lipofuscinoses (NCLs). A characteristic feature of NCL pathology is the accumulation of autofluorescent ceroid lipofuscin in the central nervous system. Northern epilepsy is a late-infantile-onset disease. Patients develop normally until 5-10 years old, when they first present with general tonic-clonic seizures, followed by progressive cognitive impairment and a decline in motor skills. Northern epilepsy is caused by a missense variant in CLN8, causing a p.R24G amino acid substitution. CLN8 deficiency has been studied traditionally using motor neuron degeneration (mnd) mice, which carry a spontaneous frame shift variant in the murine orthologue Cln8, which is not known to exist in humans. MethodsWe have generated the first Cln8 p.R24G mouse model (Cln8R24G) using CRISPR/Cas9. Phenotyping analysis of the mice was conducted using behavioral and histopathological studies focusing on the brain and retina. ResultsAt birth, Cln8R24G KI mice were viable and asymptomatic. As the mice aged, a progressive accumulation of autofluorescent ceroid lipofuscin containing the mitochondrial ATP synthase subunit C was evident in different brain regions and retinal layers. Health monitoring revealed that mutant mice developed progressive but mild motor symptoms around 7 months of age. Spontaneous epileptic seizures, like those observed in Northern epilepsy patients, were detected and recorded. An increase in FosB staining intensity, reflecting neuronal hyperactivity, was observed in hippocampal CA1-CA3 pyramidal and dentate granule cells and correlated well with the intensity of seizure activity. Neuroinflammation was evident at 4 months and increased dramatically with age, mainly in the thalamic VPN/VPL nuclei and moderately in the cortex. Neurodegeneration was most prominent in the VPN/VPL thalamic nuclei. ConclusionsWe have generated the Cln8R24G mouse model that genocopies, for the first time, the pathogenic CLN8 variant present in patients with Northern epilepsy. These mice phenocopy major clinical features of the human disease, including mild motor impairment and, unlike the mnd mice, spontaneous generalized tonic-clonic seizures. We hypothesize that our mouse model will open new possibilities for developing and testing targeted treatment options for Northern epilepsy and, more broadly, for early-onset neurodegenerative disorders associated with epilepsy.

Alzheimers disease is increasingly recognized as affecting not only the central nervous system but also the autonomic nervous system, comprising the enteric nervous system, and thus the gut. Given the structural and functional similarities between the enteric and central nervous system, including susceptibility to A{beta}, this study investigated the early and acute effects of monomeric A{beta} on primary enteric neurons in vitro and on intestinal motility ex vivo. Acute A{beta} application caused marked neuronal hyperexcitability, with increased spike frequencies and calcium influx, and led to enhanced intestinal contractility without altering frequency or timing. Prolonged 72-hour exposure did not induce cell death or apoptosis but significantly reduced neurite outgrowth and decreased synaptic markers and beta II tubulin. These findings suggest that acute A{beta} primarily drives the excitability of enteric neurons and intestinal motility, while prolonged exposure subsequently leads to structural and synaptic changes. Overall, the study points to early dysfunction of the enteric nervous system in Alzheimers disease and highlights the gut as a potential target for early interventions. Synopsis O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=190 SRC="FIGDIR/small/678722v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@18b5292org.highwire.dtl.DTLVardef@1da0ecforg.highwire.dtl.DTLVardef@1023b09org.highwire.dtl.DTLVardef@171e3fb_HPS_FORMAT_FIGEXP M_FIG C_FIG The pathology of Alzheimers disease is increasingly recognized in the enteric nervous system (ENS), suggesting an early contribution of the gut to the onset of the disease. This study investigated the acute and short-term effects of amyloid-{beta} (A{beta}) on intestinal motility and enteric neurons to elucidate early functional and structural changes beyond the brain. O_LIAcute A{beta} exposure enhanced intestinal contractility ex vivo. C_LIO_LIEnteric neurons showed hyperexcitability and increased Ca{superscript 2} influx. C_LIO_LIAfter 72 h, neuronal viability and apoptosis remained unaffected. C_LIO_LIProlonged exposure reduced neurite outgrowth and {beta}III-tubulin expression. C_LIO_LISynaptic density was significantly decreased. C_LI

Pannexin-1 channels have garnered attention for their implications in neurodevelopment, potentially having a dual role in mediating a delicate balance between cell death and survival. However, a comprehensive understanding of the underlying molecular and cellular mechanisms and Panx1s potential protective functions throughout neurodevelopment remains to be determined. Zebrafish larvae with loss of Pannexin-1a function were subjected to an acute exposure to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Early-life changes in larvae induced by uncoupling of oxidative phosphorylation were investigated by a computational Gene Set Expression Analysis of RNA-seq data and experimental testing of light-stimulated locomotor behavior, cell death, and bioelectrical properties of local field potentials in the ascending visual pathway. A KEGG pathway analysis underscored Panx1as regulatory influence on neurodevelopment. Targeting Panx1a caused a deregulation of oxidative phosphorylation, glycolysis, reactive oxygen production, hypoxia, unfolded protein response pathways, and reduced extracellular ATP. Further, Panx1a ablation enhanced the transcriptional activation of 5 AMP-activated protein kinase (AMPK) kinase, a cellular energy sensor activated by falling energy status, largely to activate glucose and fatty acid uptake and oxidation when cellular energy is low. The activation of the AMPK pathway in Panx1a knock-out larvae correlated with the stimulation of the mammalian target of rapamycin (mTORC1) pathway that controls cellular metabolism, catabolism, immune responses, autophagy, survival, proliferation, and migration, to maintain cellular homeostasis. The differential expression of mTORC1 pathway genes associated with autophagy, and apoptosis signaling pathways. The resultant cell death was pronounced in the pallium and tectum regions. The loss of cells interrelated with a trans-synaptic a loss of synaptic neurotransmitter receptor and ion channel/transporter expression. Local field potential recordings in the optic tectum and pallium demonstrated that Panx1as involvement in modulating local neuronal networks was altered. Collectively, the results shed light on the impacts of acute MPTP treatment on locomotor behavior, transcriptomic shifts, metabolic disturbances, and the pivotal role of Panx1a in cell death. These insights enhance our comprehension of the intricate molecular and cellular mechanisms underpinning neurodevelopment, with implications for potential therapeutic strategies targeting Panx1 channels in autism and Alzheimers disease. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=172 HEIGHT=200 SRC="FIGDIR/small/590821v2_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1a8688eorg.highwire.dtl.DTLVardef@16849a3org.highwire.dtl.DTLVardef@1ba8298org.highwire.dtl.DTLVardef@1d3efff_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIA genetic model was combined with the neurotoxin MPTP to explore the roles of zebrafish Pannexin-1 channels in neurodevelopment under oxidative stress conditions. C_LIO_LIA potential beneficial impact of targeting Panx1a is superseded by synaptic plasticity loss, dysfunctional mitochondrial metabolism, and cell death pathway activation. C_LIO_LILoss of Panx1a amplifies AMPK/mTORC1 pathway activation of cell death pathways. C_LIO_LIA role of Panx1a as a regulator of energy and synaptic homeostasis was identified. C_LI

Spinocerebellar ataxia type 1 (SCA1) and Huntingtons disease (HD), are motor diseases caused by CAG expansions in ATXN1 and HTT, where SCA1 shows prominent cerebellar neurodegeneration and HD shows prominent striatal neurodegeneration, particularly in the Medium Spiny Neurons (MSNs). Since human and mouse studies demonstrate progressive striatal vulnerability in SCA1, we examined age-dependent molecular, cellular and functional striatal attributes in SCA1 (f-ATXN1146Q/2Q) knockin mice, by assessing RNA-sequencing, immunohistochemistry and electrophysiology. Striatal mRNAs are downregulated in SCA1 mice, many in common with HD mice, and specificity in MSNs is supported by the rescue of transcriptomic dysregulation with deletion of mutant Ataxin1 from MSNs. Immunohistochemistry assessed dopamine receptor 1 (D1R) and 2 (D2R) expression in indirect and direct MSNs. In HD mice (HttQ175/Q7), expression of both D1R and D2R proteins in MSNs decreased with age in parallel with their RNA levels. In the SCA1 mouse striatum, D1R protein expression decreased with age as seen in murine HD striatum. In contrast, while D2R protein level was decreased similar to D1R protein at 5-weeks of age, by 40-weeks expression of D2R protein recovered to levels recorded in WT mice. Electrophysiological assessment showed a reduction of excitatory synaptic transmission in SCA1 mouse MSNs, indicating functional deficits early in disease. In contrast to cerebellar and many other aspects of SCA1 pathology known to depend on proper nuclear localization of ATXN1 with an expanded polyglutamine, mutating ATXN1s nuclear localization failed to correct striatal MSN RNA and protein downregulations, indicating a difference in how ATXN1 exerts its pathological effects between the cerebellum and the striatum. Together, these data provide a molecular and cellular basis of striatal pathology in SCA1.

Brain metabolites, detectable by magnetic resonance spectroscopy (MRS), have been examined as potential biomarkers in Huntingtons Disease (HD). In this study, the RQ140 and BACHD transgenic mouse models of HD were used to investigate the relative sensitivity of the metabolite profiling and the brain volumetry to characterize mouse HD. Magnetic resonance imaging (MRI) and 1H MRS data were acquired at 9.4 T from the transgenic mice and wild-type littermates every 3 months until death. Brain shrinkage was detectable in striatum of both mouse models at 12 months compared to littermates. In Q140 mice, increases in PCr and Gln occurred in striatum prior to cortex. Myo-inositol was significantly elevated in both regions from an early age. Lac, Ala and PE decreased in Q140 striatum. Tau increased in Q140 cortex. Metabolite changes in the BACHD cortex and striatum were minimal with a striatal decrease in Lac being most prominent, consistent with a dearth of ubiquitin and 1C2 positive aggregates detected in those regions. Binary logistical regression models generated from the Q140 metabolite data were able to predict the presence of disease in the BACHD striatal and previously published R6/2 metabolite data. Thus, neurochemical changes precede volume shrinkage and become potential biomarkers for HD mouse models Introduction

In Parkinsons disease (PD) misfolded alpha-synuclein (aSyn) accumulates in the substantia nigra, where dopaminergic neurons are progressively lost. The mechanisms underlying aSyn pathology are still unclear but hypothesized to involve the autophagy-lysosome pathway (ALP). LRRK2 mutations are a major cause of familial and sporadic PD, hyperactivate kinase activity and its pharmacological inhibition reduces pS129-aSyn inclusions. We observed selective downregulation of the novel PD risk factor RIT2 in G2019S-LRRK2 expressing cells. Here we studied whether RIT2 could modulate LRRK2 kinase activity. RIT2 overexpression in G2019S-LRRK2 cells rescued ALP abnormalities and diminished aSyn inclusions. In vivo, viral mediated overexpression of RIT2 conferred neuroprotection against AAV-A53T-aSyn. Furthermore, RIT2 overexpression prevented the A53T-aSyn-dependent increase of LRRK2 kinase activity in vivo. Our data indicate that RIT2 inhibits overactive LRRK2 to ameliorate ALP impairment and counteract aSyn aggregation and related deficits. Targeting RIT2 could represent a novel strategy to combat neuropathology in familial and idiopathic PD.

Eye muscles and the motor neurons in the innervating cranial nerve nuclei are relatively spared in human ALS, and likewise, these cranial motor neurons are spared of SOD1YFP aggregation in a transgenic mouse model of SOD1-linked ALS, G85R SOD1YFP. RNA profiling of mouse oculomotor (CN3) neurons (resistant) vs hypoglossal (CN12) and spinal cord motor neurons (susceptible) from nontransgenic mice identified differentially expressed channel and receptor genes. A number were evaluated for effects on survival of the ALS strain by transgenesis or knockout to emulate the relative RNA level in oculomotor neurons. Transgenesis of Thy1.2-driven cDNA for mouse Kcnn1, a potassium channel subunit, extended the median days of survival time to paralysis of mutant G85R SOD1YFP mice by up to 100%, associated with absence of fluorescent aggregates; extended the median time to paralysis of G93A SOD1 mice by up to 55%; and extended the median time to endstage motor disease of a Thy1.2-driven alpha-synuclein transgenic strain by up to greater than 100%. The overexpressed Kcnn1 subunit was diffusely cytoplasmic in motor neurons and found to induce a multifaceted stress response as judged by RNAseq and immunostaining, including ER stress response, mitochondrial stress response, and an integrated stress response. Like other potassium channel subunits, Kcnn1 subunit is likely targeted to the ER, but as reported earlier in rodent Kcnn1-transfected cultured cells, in the absence of Kcnn2 with which to co-assemble, Kcnn1 is channel-inactive and is diffusely cytoplasmic. Thus, a nonassembled and potentially misfolded state of overexpressed Kcnn1 targeted to the ER of neurons may explain the stress responses, which in the mutant SOD1 and A53T alpha-synuclein mice, protect against the pathogenic proteins. Major neurodegenerative diseases, including Alzheimers Disease and Parkinsons Disease, are associated with the accumulation of characteristic proteins, Abeta/Tau and alpha-synuclein in AD and PD, respectively, that misfold, aggregate, and in many cases form amyloid fibrils (e.g. Long and Holtzman, 2019; Sierksma et al, 2020; Tanner et al, 2024). Such pathogenic behavior is associated with malfunction/death of specific neuronal populations, producing consequent clinical symptoms. It seems counterintuitive to observe proteinopathy as a major facet of these diseases considering that there is generally a quality control machinery in all cells, consisting of effectors - molecular chaperones, ubiquitin/proteasomal components, and autophagy/lysosome components - governed by a "sensor" circuitry - e.g. UPR, ISR, HSF - that can detect such misbehavior and induce protective responses. While neurons may be particularly susceptible because they are postmitotic and unable to distribute damaging protein species to daughter cells as a protective means, it has remained unclear whether the endogenous sensor/effector pathways can be induced sufficiently in vivo so as to mediate protection. Here, we report that neuronal overexpression of a potassium channel subunit, mouse Kcnn1, in two different transgenic mouse neurodegenerative models, protects against aggregation and cell loss by apparent induction of multiple stress response pathways, substantially extending survival of the mice.

Advances in genome sequencing have identified over 1300 mutations in the SCN1A sodium channel gene that result in genetic epilepsies. However, how individual mutations within SCN1A produce seizures remains elusive for most mutations. Previous work from our lab has shown that the K1270T (KT) mutation, which is linked to GEFS+ (Genetic Epilepsy with Febrile Seizure plus) in humans, causes reduced firing of GABAergic neurons in a Drosophila knock-in model. To examine the effect of this mutation in mammals, we introduced the equivalent KT mutation into the mouse Scn1a (Scn1aKT) gene using CRISPR/Cas9. Mouse lines carrying this mutation were examined in two widely used genetic backgrounds, C57BL/6NJ and 129x1/SvJ. In both backgrounds, homozygous mutants had spontaneous seizures and died by postnatal day 23. There was no difference in the lifespan of mice heterozygous for the mutation in either background when compared to wild-type littermates up to 6 months. Heterozygous mutants had heat-induced seizures at ~42 deg. Celsius, a temperature that did not induce seizures in wild-type littermates. In acute hippocampal slices, current-clamp recordings revealed a significant depolarized shift in action potential threshold and reduced action potential amplitude in parvalbumin-expressing inhibitory interneurons in Scn1aKT/+ mice. There was no change in the firing properties of excitatory CA1 pyramidal neurons. Our results indicate that Scn1aKT/+ mice develop seizures, and impaired action potential firing of inhibitory interneurons in Scn1aKT/+ mice may produce hyperexcitability in the hippocampus.