Neurobiology of Disease

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.

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.

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.

Stress is a commonly reported seizure precipitant and may contribute to the development of psychiatric comorbidities in epilepsy, yet how chronic stress interacts with epileptic circuits remains poorly understood. We investigated the impact of chronic restraint stress on physiological, behavioral, and synaptic outcomes in a mouse model of Dravet syndrome, specifically corticotropin-releasing factor (CRF) neurons in the bed nucleus of the stria terminalis (BNST), a stress-responsive region implicated in epilepsy patients. Chronic restraint stress produced divergent hypothalamic-pituitary-adrenal axis responses, with stressed Dravet syndrome mice exhibiting elevated corticosterone, increased mortality in females, and increased locomotion and anxiety-like behavior. Ex vivo electrophysiological recordings revealed that chronic stress increased spontaneous excitatory event frequency onto BNST CRF neurons in both genotypes and selectively increased sEPSC and sIPSC amplitude in Dravet syndrome mice. Evoked recordings demonstrated genotype-specific effects of stress on glutamatergic transmission in CRF neurons of the DS group. This suggests greater stress-dependent remodeling of spontaneous and evoked synaptic activity in DS. These findings suggest chronic stress may worsen physiological and behavioral outcomes in Dravet syndrome and promote specific maladaptive alterations in BNST CRF circuitry. More broadly, these results suggest that stress interacts with seizure vulnerability and potentially contributes to neuropsychiatric comorbidities and epilepsy.

Disruption of glutamate homeostasis is believed to contribute to the early progression of Alzheimers disease (AD) and associated neurodegeneration. Soluble amyloid-{beta} oligomers impair excitatory amino acid transporters (EAATs), reducing glutamate clearance, while also enhancing glutamate release from neurons and astrocytes. Together, these effects produce persistent glutamatergic dysregulation that disrupts synaptic and network function. Here, we asked whether the effects of EAAT attenuation can be mitigated through ion-channel modulation. TBOA, a selective EAAT inhibitor, was used to model early-stage glutamatergic dysregulation. TBOA reduced the local field potential amplitude of hippocampal sharp-wave ripples (SWRs) in mouse hippocampal slices, suggesting that glutamate accumulation disrupts network synchrony. Calcium imaging further showed that TBOA diminished SWR-associated population calcium transients while promoting spontaneous calcium transients in individual neurons, indicating a shift from coordinated population activity toward disorganized cellular activity. KCNQ-channel openers ML213 and ICA-27243 partially restored the TBOA-induced decline in SWR amplitude. In contrast, similar restorative effects were not observed following modulation of other ion channels, including blockade of AMPA and NMDA receptors or HCN/Ih channels, or activation of large-conductance Ca2+-activated K+ (BK) channels and G-protein-activated inwardly rectifying potassium (GIRK) channels. Together, these findings suggest that KCNQ-channel openers may occupy a unique position in mitigating glutamate-related hyperexcitability during early AD-associated network dysfunction.

Motoneurons are under strong pressure to maintain stable motor output throughout an individual life, through homeostatic regulation of their electrical properties. Dysregulated spinal motoneuron excitability has long been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). Recent work in SOD1G93A mice suggests that the homeostatic response of motoneurons becomes dysregulated as cellular processes are disrupted by the disease, causing fluctuations in motoneuron electrical properties. Yet, few studies directly test whether ALS motoneurons respond differently than wild type motoneurons to a common chronic perturbation. Here, we used in vivo electrophysiology to test whether motoneurons from pre-symptomatic SOD1G93A mice modulate excitability differently than wild type motoneurons in response to the same homeostatic perturbation: chronic inhibition exerted by the benzodiazepine diazepam. Using linear mixed-effects statistical models, we assessed whether diazepam treatment differentially modulated passive properties, firing behavior, spike properties, and/or synaptic inputs in SOD1G93A versus wild type motoneurons. We identified a significant genotype x treatment interaction effect selectively for properties related to passive membrane integration and spike initiation, including membrane time constant, peak input resistance, and recruitment current. In contrast, firing gain, spike waveform characteristics, and synaptic inputs were largely unaffected. These findings indicate that sustained inhibitory perturbation selectively triggered overactive intrinsic compensatory mechanisms in SOD1G93A motoneurons rather than inducing widespread changes in firing or synaptic transmission. Together, our results provide direct evidence for over-active homeostatic control of motoneuron excitability and support a view of motoneuron dysfunction in ALS as a problem of altered feedback regulation rather than simply hyper- or hypo-excitability. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=52 SRC="FIGDIR/small/725609v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@25f125org.highwire.dtl.DTLVardef@faf2c9org.highwire.dtl.DTLVardef@15993a8org.highwire.dtl.DTLVardef@1ed006a_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cognitive impairments significantly impact the daily life of people with Down syndrome (DS). Overinhibition mediated by interneurons in the central nervous system was proposed as a key pathophysiological mechanism. Previous studies demonstrated cognitive rescue in the Ts65Dn mouse model using 5IA, a negative allosteric modulator of the 5 subunit-containing GABAA receptors. Here, we evaluated the effect of this drug in a mouse model carrying a more accurate duplication of the orthologous region to the human chromosome 21, namely the Dp(16)1Yey mouse model. First, we expanded the phenotypic characterization of Dp(16)1Yey mice using translationally more relevant behavioral tests. We confirmed spatial memory deficits in Dp(16)1Yey mice in the Barnes maze, and highlighted robust learning deficits in the pattern dissociation task and impairments in motor coordination. Next, we evaluated the effect of 5IA treatment on cognitive and motor performance. While 5IA treatment improved motor coordination in the Dp(16)1Yey mice, it failed to restore cognitive performance in the Barnes maze or in the pattern dissociation task. These findings could suggest divergent pathophysiological mechanisms between the Dp(16)1Yey and the Ts65Dn models. Potentially, it could explain the limited efficacy of similar pharmacological intervention in clinical trials for DS. Further preclinical studies should prioritize refined behavioral paradigms and probably the use of more complex DS models to enhance the translational potential of candidate therapies.

Efficient transport of membrane proteins through the endosomal network is essential for brain development and function, with perturbation implicated in disease. Deficiencies in Retromer, a key regulator of endosomal transport, have been linked to aging-related neurodegenerative disorders including Alzheimers and Parkinsons disease. To better define the neuroprotective role of Retromer, we have applied cell surface restricted proteomics to identify those integral membrane proteins whose recycling to the plasma membrane is mediated by Retromer and associated cargo adaptors, sorting nexin 3 (SNX3), its paralogue sorting nexin 12 (SNX12), and sorting nexin 27 (SNX27) (data available via ProteomeXchange: PXD078277). By comparing primary rat cortical neurons and astrocytes we have identified several cargoes that require either SNX3/SNX12- or SNX27-Retromer complexes for endosomal recycling, including proteins involved in synapse organisation, synaptic signalling and Alzheimers disease pathology. We highlight that perturbed Retromer function leads to endosomal enlargement, and we establish a key role of SNX27-Retromer in modulating transport of glutamate across both neuronal and astrocytic membranes via recycling of glutamate transporters EAAT3 (SLC1A1) and EAAT1 (SLC1A3) respectively. Our study provides further mechanistic insight into the consequences of Retromer deficiency for neuronal and astrocytic function, offering new avenues of research in the treatment of neurodegenerative disease. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/724903v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@98277forg.highwire.dtl.DTLVardef@1490534org.highwire.dtl.DTLVardef@f4a9feorg.highwire.dtl.DTLVardef@c48402_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO Suppression of Retromer and the sorting nexins (SNX27, SNX3/SNX12) leads to a significant change in the surface proteome of rat cortical neurons and astrocytes. Focusing on the glutamate transporters, SLC1A1 and SLC1A3, we have validated that SNX27-Retromer is required for their trafficking, with SNX27-Retromer suppression in astrocytes leading to a loss of glutamate uptake. C_FIG

Abstract/SummaryRetroelements, including retrotransposons, endogenous retroviruses, and their fragments, as well as rare co-opted or domesticated retroelements, can contribute to neurodegenerative disorders and aging through modulation of gene expression and induction of neuroinflammation. Paternally Expressed Gene 10 (PEG10) is a retroelement-derived human gene that has recently been identified as a putative driver of Amyotrophic Lateral Sclerosis (ALS) and Angelmans Syndrome. PEG10 has been reported to bind nucleic acid and undergoes a complex self-processing pathway that results in gene expression changes when the protein accumulates in cells. Here, we report that PEG10 has selectivity for binding U/G-rich RNAs and influences widespread gene expression changes. PEG10 overexpression mimics the loss of TDP-43 in broad changes to gene expression, including dysregulation of mRNA splicing pathways. Specific changes to mRNA splicing were largely unique between TDP-43 knockdown and PEG10 overexpression, as classic TDP-43 targets including STMN2 were not altered by PEG10. Instead, we identified a unique role for PEG10 in regulating splicing of neuregulin 3 (NRG3), a ligand for the neuronal receptor ERBB4. In SH-SY5Y cells and in human neurons overexpressing PEG10, NRG3 protein levels were decreased along cellular processes, suggesting that these cells are less competent at signaling through the NRG3/ERBB4 axis. Using human patient data, we observed similar changes to NRG3 splicing in UBQLN2-mediated ALS, where PEG10 is accumulated, as well as in some cases of sporadic ALS. In conclusion, the retroelement-derived gene PEG10 plays an unexpected role in regulating splicing of neuronal transcripts, which mimics some of the transcript changes observed in human ALS patient samples. Ultimately, this work has implications for the study of PEG10, and mRNA splicing in neurological diseases associated with elevated PEG10 abundance. HighlightsO_LIPEG10 NC expression influences abundance of transcripts implicated in ALS C_LIO_LIPEG10 NC expression leads to an exon skipping event in neuregulin 3 (NRG3) C_LIO_LINRG3 expression is decreased along dendrites of PEG10 NC expressing human neurons C_LIO_LIExpression of PEG10 NC mimics changes observed in human ALS C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/727000v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@1a957d2org.highwire.dtl.DTLVardef@c4b15corg.highwire.dtl.DTLVardef@15825faorg.highwire.dtl.DTLVardef@25533d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Background: Huntington ' s disease (HD) causes progressive postural control deficits, but how sensory reweighting mechanisms degrade across disease stages remains poorly understood. Objective: To determine whether objective markers of postural sway track disease severity and altered sensory reweighting across the HD spectrum. Methods: Ninety-seven adults (46 {+/-} 14 yrs) were categorized into four groups: 29 with HD, 27 pre-manifest (PM), 28 not at risk (AR-), and 13 age-matched healthy controls (HC). Participants performed three trials of quiet standing with eyes open and eyes closed on a force plate. Results: Manifest HD individuals exhibited greater AP, ML, and total COP sway displacement compared with the PM, AR-, and HC groups. HD and PM groups demonstrated greater instability with eyes closed. COP wavelet power was concentrated below 1 Hz across all groups. The eyes-open to eyes-closed change in 0-1 Hz power predicted total COP sway in HC (68%), AR- (45%), and PM (46%), but this relation was substantially weaker in HD. Conclusions: Progressive weakening of oscillatory-sway coupling distinguishes manifest HD from premanifest stages. PM individuals demonstrate early sensory reweighting deficits that manifest only when vision is removed, while HD individuals show decoupled oscillatory activity that fails to support stable postural regulation. This progressive decoupling may serve as a candidate marker of disease conversion prior to overt motor diagnosis.

Leucine-Rich Repeat Kinase 2 (LRRK2) is a signaling molecule involved in Parkinsons disease pathomechanisms. In disease, the LRRK2 protein displays both a toxic gain of kinase function and a loss of phosphorylation at heterophosphosites found in an extended loop of the LRR domain. RAB GTPases, such as RAB29, have been identified as upstream activators of LRRK2. Indeed, co-expression of LRRK2 with RAB29 induces a hyperactivation of LRRK2 kinase activity, however the role of the LRRK2 heterologous phosphorylation status in its activation remains unknown. Here, our aim was to determine the role of LRRK2 heterologous phosphorylation on its activation by RAB29. Using single and compound phosphodead or phosphomimetic mutants of LRRK2 we show differential sensitivity of LRRK2 phosphomutants to activation by RAB29, with phosphodead mutants being more susceptible to be activated than phosphomimetic mutants. Interestingly, we find that the single phosphodead S910A LRRK2 mutant displays an activation of LRRK2 kinase activity similar to that observed for the compound phosphodead 6xS>A LRRK2 mutant (S860A/S910A/S935A/S955A/S973A/S976A). Time-course analysis revealed that phosphodead mutants displayed higher but also faster activation by RAB29. In addition, both physical interaction between LRRK2 and RAB29 as well as RAB29-induced recruitment of LRRK2 to the trans-Golgi network (TGN) was enhanced by phosphodead compared to phosphomimetic mutants. To confirm effects on native LRRK2, we tested a panel of ten nanobodies targeting LRRK2 that stabilized LRRK2 phosphorylation at varying levels. Nanobodies stabilizing LRRK2 at low S935 phosphorylation levels showed enhanced RAB29-induced activation compared to nanobodies not affecting pS935 LRRK2. Finally, we tested whether LRRK2 heterologous phosphorylation could affect centrosome cohesion deficits, a phenotype that has been linked to LRRK2 hyperactivation, and found that both the phosphodead LRRK2 as well as a nanobody stabilizing dephosphorylated LRRK2 enhanced the centrosome cohesion deficit. Our findings indicate that hyperactivability of LRRK2 is directly related to its heterologous phosphorylation status, with dephosphorylation leading to strong hyperactivation of LRRK2 by upstream activating RABs, and phosphorylated LRRK2 showing the opposite. This implies that strategies favoring LRRK2 phosphorylation will have therapeutic benefit.

Prader-Willi syndrome (PWS) and Schaaf-Yang syndrome (SYS) are neurodevelopmental disorders associated with hypothalamic-pituitary dysregulation. In the pituitary gland, translational control enables rapid peptide hormone production and secretion in response to hypothalamic signals without requiring new mRNA synthesis, yet the mechanisms regulating pituitary translation remain poorly understood. Furthermore, although the PWS-associated gene MAGEL2 has been implicated in neuroendocrine regulation and vesicular trafficking in the hypothalamus, its role in the pituitary gland remains unknown. Initial analysis of previously published pituitary proteomic data revealed enrichment of translation-associated pathways among downregulated proteins in Magel2 KO mice, suggesting translational impairment. Here, we investigated the impact of Magel2 loss on pituitary translatome using polysome profiling and RNA sequencing. We first optimized a polysome profiling workflow for mouse pituitary tissue and established that pooling two to three pituitaries yielded sufficient RNA quality and quantity for downstream analyses. Polysome profiling of WT and Magel2 KO pituitaries revealed no major alterations in global translational activity, as translated and nontranslated fractions were largely unchanged between genotypes. However, transmission electron microscopy revealed a shift toward smaller secretory granule size, indicating altered granule maturation dynamics. To further characterize the pituitary translatome, RNA sequencing was performed on input, monosome, light polysome, and heavy polysome fractions. Clustering analyses identified six distinct translational trajectories across fractions, revealing fraction-specific enrichment of biological pathways. RNAs enriched in heavy polysomes were associated with metabolic and oxidative phosphorylation pathways, whereas monosome-enriched clusters were linked to RNA processing and translation-related functions, suggesting specialized translational regulation within the pituitary. Differential expression analysis demonstrated that translatomic alterations were more pronounced than transcriptomic changes in Magel2 KO pituitaries, with the strongest enrichment observed in heavy polysome fractions. Functional enrichment analyses identified pathways associated with endocrine and metabolic regulation, circadian rhythm, cytoskeleton organization, vesicular trafficking, and RNA regulation, suggesting that translation contributes to pituitary physiological function and patient symptoms. For example, prolactin displayed altered polysome association without changes at the transcript level, consistent with the increased serum prolactin levels observed in Magel2 KO mice and in patients with PWS. Interestingly, the PWS-associated gene Necdin (Ndn) was consistently downregulated across all fractions, which contrasts with previously described compensatory upregulation in the hypothalamus. Together, our findings suggest the involvement of MAGEL2 in pituitary in transcriptional and translational processes and the organization of the secretory pathway and provide the first comprehensive characterization of the mouse pituitary translatome. This work provides new insights into the mechanisms underlying neuroendocrine dysfunction in PWS and SYS and establishes a resource for future studies of translational regulation in neuroendocrine disease.

Recently, we showed that ketoconazole, a known anti-fungal inhibitor of CYP51, stabilized TAR DNA-binding protein 43 (TDP-43) native self-interactions, reduced TDP-43 pathology and rescued TDP-43-induced SREBP2 downregulation. Despite its promising effects, ketoconazole is not viable for repurposing for ALS due to liver toxicity side effects that occur when orally delivered. To address this, we tested the activities of seven additional known azole-based CYP51 inhibitors in order identify a viable alternative to ketoconazole. Using our established TDP-43 mislocalization and aggregation assay in HEK293T cells, we identified posaconazole, an FDA-approved, CNS-penetrant and orally delivered anti-fungal, as the strongest inhibitor of TDP-43 pathology. Posaconazole was able to reduce insoluble TDP-43 and restore SREBP2 levels, outperforming ketoconazole. Mechanism of action (MOA) experiments suggest posaconazole is able to outperform ketoconazole by inducing a significantly stronger activation of autophagy and upregulation of heat shock proteins known to clear TDP-43. Further MOA experiments show that the effects of posaconazole on TDP-43 are dependent on its known ability to lower cellular cholesterol levels. By correlating our experimental results on the eight CYP51 inhibitors tested, we show that predicted affinity towards human CYP51 strongly correlates with the inhibitors ability to lower TDP-43 aggregation and mislocalization. Finally, we tested posaconazole in a low dose sodium arsenite ALS model in iPSC-derived motor neurons, showing that it is efficacious at inhibiting TDP-43 pathology in the nanomolar range. Altogether, these results support the repurposing of posaconazole for ALS/FTD as a means to prevent TDP-43 pathology.

Alzheimers disease (AD) is a neurodegenerative disorder affecting more than 55 million people worldwide, with a diagnosis that remains predominantly clinical and frequently delayed. The electroretinogram (ERG) offers a non-invasive electrophysiological method for detecting retinal dysfunction associated with neurodegeneration; however, it remains unclear whether robust and reliable candidate biomarkers can be extracted from ERG signals beyond conventional amplitude- and latency-based parameters. Here we present a pilot study of a multi-domain signal processing framework applied to ERGs recorded from 46 participants (20 AD patients, 26 controls) with a handheld device (RETeval, LKC Technologies) using sinusoidal (1-50 Hz) and photopic ISCEV protocols. Five complementary techniques were implemented: (i) multiscale fuzzy entropy (MSFuzzyEn); (ii) FFT harmonic analysis; (iii) stimulus-response wavelet time-frequency coherence (WTC); (iv) a novel inter-cycle lag variant of sample entropy (SampEnT), introduced to isolate cycle-to-cycle retinal response consistency independently of stimulus periodicity; and (v) discrete wavelet transform (DWT) for energetic extraction of oscillatory potentials (OPs). Univariate comparisons (Mann-Whitney, Cliffs{delta} , Benjamini-Hochberg FDR) identified seven significant candidate biomarkers (q < 0.05), five with large effect size: AUCfast (|{delta}| = 0.546, q = 0.009), Slopevery-slow (|{delta}| = 0.554, q = 0.007), R14f (|{delta}| = 0.515, q = 0.031), SampEnT (|{delta}| = 0.504, q = 0.019) and WTCR,mean (|{delta}| = 0.531, q = 0.023); and two with medium effect size (OP_amp_sum, band_snr). A logistic regression classifier combining three candidate biomarkers, validated by leave-one-out cross-validation, achieved ROC-AUC = 0.858, sensitivity = 70.0% and specificity = 88.5% (n = 46). These proof-of-concept results demonstrate that multi-domain ERG analysis captures retinal temporal dysfunction signatures in AD that are inaccessible to standard clinical analysis, supporting further investigation of portable ERG devices as a source of non-invasive candidate biomarkers for early AD detection.

There are substantial functional changes in the primary motor cortex (M1) in Parkinsons disease (PD). However, the temporal relationship between midbrain dopaminergic (DA) neurodegeneration, M1 circuit dysfunction, and Parkinsonian motor symptoms remains poorly understood. Using a genetic mouse model of progressive nigrostriatal DA degeneration ("MitoPark" mice), we determine the time course of M1 cellular dysfunction and skilled movement impairment as the midbrain DA neurons gradually degenerate. M1 pyramidal neuronal subtypes were identified using AAV-mediated retrograde labeling. During progressive DA loss, MitoPark mice developed gradually impaired performance in a reach-to-grasp single-food-pellet task. These impairments were detectable at a moderate motor stage of Parkinsonism. In vivo GCaMP6f imaging revealed that impaired skilled movement was associated with reduced cellular activity and movement responsiveness of M1 pyramidal neurons at a moderate motor stage of Parkinsonism. While both the corticospinal (CSp) and intratelencephalic (IT) neurons send glutamatergic inputs to the striatum, only the CSp neurons showed a selective and significant reduction in cellular activity and movement responsiveness during reaches. At the population level, we found that M1 pyramidal neurons include heterogeneous functional clusters with distinct temporal profiles in response to skilled movement. While movement encoding by different functional clusters is longitudinally stable in control mice, it degrades and diverges significantly in MitoPark mice. The impaired stability is further supported by a longitudinal analysis of individual neuronal activity related to movements. Together, these results provide novel insights into the emergence of M1 circuit pathophysiology at cellular and neural population levels during progressive Parkinsonism.

Alzheimer's disease (AD) is characterized by complex alterations in synaptic, glial, neuronal and inflammatory markers. Given its emerging role at the interface of synaptic dysfunction and inflammation, the astrocytic marker GFAP may represent a cross-domain hub linking synaptic, neuronal and inflammatory alterations. Using multivariate and network-based analyses we examined the relationships among cerebrospinal fluid (CSF) biomarkers of astrocytic activation and synaptic failure, inflammation, and neurodegeneration in biologically confirmed AD patients and healthy controls (HC). We studied 60 AD patients and 40 HC. CSF concentrations of Neurogranin, SNAP-25, CAP2, NfL, GFAP, IL-1 , IL-1{beta}, IL-8, MCP-1, TNF were measured. Associations were assessed using Spearman correlations, LASSO regression, and network analysis to characterize multivariate dependency structures. Compared with controls, AD patients showed significantly higher CSF levels of Neurogranin, SNAP-25, CAP2, NfL, GFAP, IL-1{beta}, TNF- .. In AD, synaptic biomarkers were strongly intercorrelated and associated with astroglial activation, inflammatory markers, and tau-related pathology. Network analysis identified GFAP as a cross-domain hub linking synaptic, inflammatory, and neurodegenerative domains in AD. In controls, GFAP was mainly associated with neuronal injury markers. Network-based modelling revealed a disease-related reorganization of biomarker connectivity in AD, with GFAP occupying a central cross-domain position, supporting a systems-level view of AD pathophysiology.

BackgroundAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive motor neuron (MN) loss, muscle atrophy and paralysis. Although traditionally considered a MN-specific disease, accumulating evidence supports a crucial contribution of skeletal muscle pathology to disease onset and progression. Except for specific mutations, to date there is no effective treatment for ALS. FOXO transcription factors regulate programs of atrophy, metabolism and stress response in skeletal muscle, and their inhibition has shown beneficial effects in cellular and Drosophila models of ALS. MethodsIn this study, we investigated whether pharmacological FOXO inhibition (iFOXO) could modify disease progression and muscle pathology in female hSOD1G93A mice. Mice received daily oral administration of iFOXO starting at presymptomatic (P50; n=5 per group) or symptomatic (P90; n=9 mice per group) stages until end-stage. Body weight was monitored longitudinally, and motor performance was evaluated using grip strength and hanging-wire tests. Tibialis anterior and soleus muscles, representing fast- and slow-twitch muscles respectively, were analyzed by histology and immunofluorescence to assess fiber atrophy, fibrosis, lipid accumulation, satellite cell pool and fiber type composition. Quadriceps muscles (n=3 per group) were used for RNA-seq analysis. ResultsWhile histological analyses revealed severe fiber atrophy and increased fibrosis in hSOD1G93A mice, satellite cell numbers were preserved or mildly increased in a muscle and treatment onset dependent manner. iFOXO treatment did not improve motor performance, survival or attenuate muscle atrophy. Transcriptomic profiling indicated that genotype was the predominant driver of gene expression changes, while iFOXO produced only subtle, treatment onset dependent effects on pathways related to oxidative stress responses, mitochondrial function and adaptive metabolism. ConclusionOverall, FOXO inhibition alone showed limited therapeutic benefit in the hSOD1G93A ALS mouse model. These findings highlight the dominant influence of ALS driven molecular alterations over pharmacological modulation and emphasize the need for combinatorial therapeutic strategies targeting multiple disease mechanisms, including those preserving nerve health.

BackgroundHeterozygous mutations in the GBA1 gene encoding the enzyme glucocerebrosidase (GCase) represent the most common genetic risk factor for developing Parkinsons disease (PD). The underlying mechanisms by which GBA1 mutations lead to PD through both loss- and gain-of-function effects remain unclear. There is a strong rationale for the generation and characterisation of a humanised GBA1 mouse model to allow the effect of GBA1 mutations on GCase function to be studied within the context of the human protein. MethodsWe have generated novel humanised mutant GBA-L444P and wild type GBA-WT mouse models using BAC recombineering and site-specific integration allowing the incorporation of the whole GBA1 locus as a transgene, including the endogenous promoter, all exons and introns, and flanking regions. Our experimental design crossed each GBA1 transgene onto a Gba+/- background and included Gba+/- littermate controls in our cohorts, allowing us to explore both the loss- and gain-of-function of GBA1 mutations. We have carried out "deep phenotyping" to characterise these mice by biochemical, stereological and behavioural testing, and assess dopamine release and content using fast-scan cyclic voltammetry and high performance liquid chromatography. ResultsThe GBA-L444P mice showed a significant reduction in GCase activity by 18 months of age and preferentially expressed a high molecular weight form of the GCase protein, likely due to retention in the ER and aberrant glycosylation. The GBA-L444P, but not Gba+/-, mice demonstrated an early and persistent reduction in dorsal striatal dopamine release in the absence of any dopaminergic cell loss or deficits in dopamine synthesis or reuptake, compared to human wild-type controls. GBA-L444P and Gba+/- mice developed an accumulation of oligomeric -synuclein pathology, but only GBA-L444P mice demonstrated subtle but significant changes in behaviour. ConclusionsThe novel humanised GBA-L444P mouse model described here helps to resolve gain- or loss-of-function effects of GBA1 mutations seen in Parkinsons as well as providing a novel set of models to investigate the human protein. Our work demonstrates that changes in dopamine release and behavioural deficits arise from a gain-of-function mechanism, whereas -synuclein pathology arises from GCase loss-of-function.

Purinergic 2X7 receptor (P2X7R) is considered to play a critical role in neurological diseases, including epilepsy, and has also been proposed as a potential marker for neuroinflammation. This study aimed to validate the binding properties of the novel P2X7R radiotracer, [3H]JNJ-64413739, in rat brain using in vitro autoradiography, and additionally to explore spatial and temporal changes in P2X7R binding levels in a rat model of temporal lobe epilepsy using intrahippocampal administration of kainic acid (KA). Saturation of [3H]JNJ-64413739 to brain sections yielded a KD of approximately 3 nM, with full saturation around 10 nM. The radiotracer was displaced with a structurally different P2X7R ligand, JNJ-47965567, indicating high affinity and specificity to rat P2X7R. In post epileptic rats, region-specific [3H]JNJ-64413739 binding revealed a bilateral increase in the hippocampal formation and its subregions few days after status epilepticus, peaking at day 30, and remained stable at this high level until day 90. Similar temporal profiles were identified in subcortical regions such as the thalamus. Interestingly, no change in binding was observed in the temporal and piriform cortices until day 30 where a dramatic increase occurred. Also, in the corpus callosum, significant increase was detected 30 days after the seizure. These results show that P2X7R binding, likely reflecting inflammation, is increased at delayed time points and exhibit region-specific patterns that is different from acute effects. Our findings suggest that P2X7R may contribute to sustained neuroinflammation and may be involved in those changes leading to epileptogenesis and the development of chronic epilepsy. Highlights[3H]JNJ-64413739 binds specifically to the purinergic P2X7 receptor (P2X7R) and saturates in the rat brain. P2X7R binding increases in a region- and time-dependent manner following status epilepticus. P2X7R binding remains elevated during chronic epilepsy in all examined brain regions. P2X7R is considered a link between early seizures and sustained neuroinflammation and epileptogenesis.