Molecular Neurodegeneration

SummaryFrontotemporal dementia (FTD), a leading cause of young-onset dementia, is characterized by progressive behavioral and cognitive decline associated with frontotemporal cortical atrophy. Nearly 40% of cases exhibit tauopathy, yet the molecular drivers of tau aggregation leading to synaptic dysfunction remain poorly understood. Here, we investigated whether Phospholipase D1 (PLD1, a lipid signaling enzyme), implicated in Alzheimers disease (AD), and amyotrophic lateral sclerosis (ALS), contributes to tau pathology dependent synaptic deficits in FTD. Postmortem temporal (BA38) and frontal (BA9) cortices from clinically diagnosed FTD and age-matched control subjects were analyzed using fluorescence-assisted single synaptosome long-term potentiation (FASS-LTP), immunofluorescence, proximity ligation assays (PLA), and PLD1-interactome proteomics. FASS-LTP revealed markedly reduced glutamatergic potentiation in BA38 and BA9 crude synaptoneurosomes from FTD brains compared to controls. Western blotting demonstrated elevated PLD1 expression in both crude synaptoneurosomal and cytosolic fractions from FTD subjects in BA38, but not BA9. Bielschowsky staining confirmed increased Pick body burden in FTD temporal cortex. Immunofluorescence and PLA showed robust PLD1 co-localization with total tau (HT7), hyperphosphorylated tau (AT8), and acetylated tau oligomers (TOMA2), indicating a strong spatial association between PLD1 and pathological tau species. PLD1 also exhibited enhanced co-localization with astrocytic GFAP and synaptic markers (PSD95, Nrx1{beta}), suggesting compartmentalized involvement in glial and synaptic remodeling. Proteomic profiling of PLD1-associated complexes revealed compartment-specific alterations with cytosolic fractions enriched for metabolic enzymes, stress-response proteins, and GFAP, while crude synaptoneurosomal fractions showed depletion of presynaptic scaffolds, vesicle-trafficking regulators, and proteostasis components. Cross-compartment integration indicated that over one-third of proteins were redistributed from synapses to cytosol, consistent with trafficking and degradative impairments. Gene Ontology analysis highlighted lipid metabolism, astrocyte activation, and proteasome dysfunction as dominant pathways. Collectively, these findings identify PLD1 as a critical mediator of synaptic dysfunction and tau pathology in FTD, acting through astroglial activation and disrupting synaptic proteostasis. This study provides the human clinical relevance towards PLD1 attenuation as a therapeutic target for FTD and related tauopathies to mitigate tau-driven neurodegeneration and restore synaptic integrity.

Pathogenic variants in leucine-rich repeat kinase 2 (LRRK2), vacuolar protein sorting 35 (VPS35), and RAB32 cause dominantly inherited parkinsonism, indistinguishable from idiopathic late-onset Parkinsons disease (PD). All three causes constitutively activate LRRK2 kinase activity to augment immune responses, enhancing immunity to fight pathogens, but similar mechanisms in the brain increase the vulnerability of dopaminergic neurons to degeneration. Although VPS35 p.D620N possess the highest constitutive increase in LRRK2 kinase activity among known variants in LRRK2 or RAB32, its effects on the immune system remain poorly understood. LRRK2 and Rab32 are highly expressed in myeloid cells including microglia; thus we examined the transcriptomic and functional consequences of Vps35 p.D620N in knock-in mice (VKI). Microglia were isolated from brains of six-month-old VKI mice and were analyzed via single-cell RNA sequencing. Differential gene expression highlighted pathways involved in antimicrobial humoral immune response, lysosomal stress sensing, and phagocytosis. Notably, genes of S100 family proteins, along with lipocalin 2 (Lcn2), were significantly upregulated, and those measures were complimented by immunohistochemistry and quantitative PCR. In contrast, pathways involved in synaptic transmission, neuronal development, and homeostatic immune signaling were downregulated. Peripheral stimulation with lipopolysaccharide amplified microglial activation and phagocytic markers in wildtype mice, and VKI mice also display enhanced morphological activation and increased synaptic engulfment. Collectively, Vps35 p.D620N drives a chronic pro-inflammatory microglial phenotype characterized by heightened innate immune signaling, lysosomal stress, and enhanced phagocytic activity. VKI microglia are sensitized to peripheral immune challenges and may promote synaptic remodeling and neurodegenerative vulnerability in PD. These results provide mechanistic insight into how retromer dysfunction and LRRK2 kinase hyperactivity intersect with microglial biology to influence PD pathogenesis.

BackgroundLactamase {beta} (LACTB) is a serine {beta}-lactamase-like mitochondrial enzyme associated with cancer progression, obesity, and lipid metabolism. LACTB is located in an Alzheimers Disease (AD) risk locus and has been associated with AD in a proteomic study. MethodsWe performed Mendelian Randomization (MR) analysis to estimate the association between LACTB expression, succinylcarnitine levels, and AD risk. We generated LACTB knock-down (KD) THP1 macrophages, LACTB knock-out (KO) iPSC-derived microglia and LACTB enzymatically-dead (ED) mice. The impact of LACTB loss-of-function in myeloid cells was characterized via transcriptomics, metabolomics, lipidomics, and functional assays. Finally, human LACTB KO microglia precursors were xenotransplanted into the brains of mice with amyloid pathology to assess in vivo interactions with amyloid plaques. ResultsMR analyses revealed that lower LACTB expression in myeloid cells may lead to reduced AD risk and higher levels of succinylcarnitine, a metabolite associated with AD risk. We identified LACTB as a primary enzyme responsible for succinylcarnitine hydrolysis. Transcriptional and functional studies showed that loss of LACTB enhances OXPHOS, and reduces protein synthesis and triglycerides. LACTB expression was upregulated following interferon or TNF stimulation, and its loss modified efferocytosis- related functions under inflammatory conditions. In vivo, xenotransplanted human LACTB KO microglia exhibited enhanced association with amyloid plaques. ConclusionsOur findings define a previously unrecognized axis linking LACTB and succinylcarnitine to myeloid cell function and AD susceptibility. Given the druggability of LACTB and the potential for succinylcarnitine to serve as a translational biomarker, this enzyme represents a promising therapeutic target for modulation of neuroinflammation in AD.

Parkinsons Disease (PD) is the second most common neurodegenerative disease, with many cases being attributed to environmental contaminant exposures. Paraquat (PQ), is a pesticide and environmental neurotoxicant that has been strongly associated with increased risk of PD. PQ is known to be a weak inhibitor of complex I of the electron transport chain, and while its acute toxicity is well understood, the underlying mechanism by which PQ exposure contributes to PD pathophysiology remains unclear. Additionally, the mechanism of PQ neurotoxicity has yet to be effectively compared and related to genetic forms of PD. Given that PD is a heterogeneous disease with both genetic and environmental determinants, we sought to systematically compare the proteomic changes that occur in different genetic and environmental models of PD. In this study, we leveraged untargeted omics approaches to differentiate between systemic, peripheral, and CNS-specific changes in the proteome. We did this by performing a comparative proteomic analysis on the heads and bodies of Drosophila models of PQ ingestion and neuronal -synuclein expression in males. Additionally, we validated the findings with metabolomic analysis of male and female brain stems from a murine PQ inhalation model using C57BL/6J mice. Our findings indicate shared dysregulated pathways across all models, highlighting similar mechanisms of action. Specifically, we identified a glia-specific role in purine nucleotide metabolism upstream of inosine catabolism, which may protect against PQ neurotoxicity. This work identifies potential early points for biomarker detection and potential targets for drug intervention. Significance StatementNeurodegenerative diseases such as Parkinsons disease (PD) pose a growing public health burden, yet disease-modifying therapies remain limited due to lack of mechanistic understanding and disease heterogeneity. Both genetic and environmental factors contribute to PD, complicating the identification of shared therapeutic targets. Here, we identify a convergent pathway common to genetic and environmental models of Parkinsonism that not only affects the brain but also systemically. Using integrated metabolomics, proteomics, and genome-scale metabolic modeling, we demonstrate that purine metabolism is dysregulated across models. Reverse genetic screening of key enzymes in this pathway mitigates locomotor deficits induced by neurotoxic pesticide exposure in Drosophila. These findings reveal a shared metabolic vulnerability in PD and highlight purine metabolism as a potential therapeutic target.

SORL1, the gene encoding the SORLA protein, has arisen as a potential therapeutic target for Alzheimers disease (AD). Studies suggest that restoring SORLA function or its trafficking pathways, particularly the SORLA-retromer recycling system, may offer a promising strategy to slow or halt AD progression. While both rare and common SORL1 variants have been associated with increased AD risk, recent evidence suggests a potential involvement of SORL1 in other neurodegenerative conditions. This study assessed the contribution of SORL1 genetic variation to the risk of AD, related dementias (RD), and Parkinsons disease (PD) using data from six large-scale biobanks, comprising 15,043 AD, 9,943 RD, and 42,763 PD cases, along with 111,969 controls across 11 ancestries. We identified 53 potentially disease-related SORL1 variants (CADD score > 20, MAC [≥] 2, annotated as protein-altering or splicing, and with the mutated allele present only in cases), including 41 novel and 12 previously reported variants. Three were found across multiple ancestries. Overall, 13 variants were found in AD-related cohorts, 5 in RD cohorts, and 35 in PD cohorts. Association analysis identified 10 nominally significant variants associated with AD and 5 with PD. The replication of multiple SORL1 variants across neurodegenerative diseases and ancestrally diverse populations underscores its potential broad genetic contribution to neurodegeneration and reinforces its relevance across distinct clinical phenotypes. Gene-based burden analysis did not reveal any significant cumulative effect of SORL1 variants in the populations tested. A family-based analysis identified a rare predicted-damaging variant in two East Asian families (11:121478242:G:A, p.R176Q) and two variants in two families of European ancestry (11:121514222:A:C, p.N371T; 11:121545392:G:A, p.V672M) that show some evidence of segregation in PD families. Although these variants were slightly more frequent in unrelated PD cases vs. controls, none of them showed statistically significant enrichment in PD, likely due to their very low frequency. Overall, our results extend the understanding of SORL1 beyond AD, suggesting a broader role in neurodegeneration and emphasizing the need for diverse population studies when evaluating genetic risk.

Microglia, resident immune cells of the brain, are important players in neurodegeneration. While microglial activation is a hallmark of many neurodegenerative diseases, the specific role of microglia intrinsic factors in microglial activation and disease pathogenesis remains unknown. Spinocerebellar ataxia type-1 (SCA1) is an inherited autosomal dominant neurodegenerative disease characterized by severe neuronal loss and early microglial activation in the cerebellum. SCA1 is caused by CAG repeat expansion in the ubiquitously expressed ATAXIN1 (ATXN1) gene. Using human microglia differentiated from SCA1 patient derived iPSCs, we found that mutant ATXN1 is sufficient to alter morphology, gene and protein expression in human microglia in a cell-autonomous manner. Moreover, compared to controls, human SCA1 microglia exhibited increased phagocytosis and pro-inflammatory cytokine production, indicating an immune priming. To determine the extent to which mutant ATXN1 in microglia contributes to SCA1 pathogenesis and behavioral symptoms, we removed mutant ATXN1 from microglia and macrophages in a novel conditional SCA1 mouse model, f-ATXN1146Q/2Q mice. Microglial mutant ATXN1 reduction led to a marked correction in microglia phenotype, in particular in the transcriptomic signature of interferon type 1 mediated immune response, reduced microglial density and resulted in smaller microglia with reduced branching in the cerebellum. Pathology of Purkinje neurons and cerebellar astrogliosis were also ameliorated. Utilizing a battery of behavioral tests, we found that microglia and macrophage mutant ATXN1 reduction ameliorated cognitive, mood, and motor deficits in SCA1 mice. Together, these results indicate that mutant ATXN1 directly impacts microglial phenotype in SCA1, contributing to SCA1 pathology and behavioral deficits.

Frontotemporal lobar degeneration with tau inclusions (FTLD-tau) comprise a class of fatal heterogeneous neurodegenerative diseases. Approximately 10% arise from pathogenic MAPT mutations and often cause severe, early-onset disease with pathology that is distinct yet partially overlapping with sporadic cases. Here, we evaluated post-mortem tissue from a patient with FTLD-tau due to MAPT S305I showing neuropathology most consistent with argyrophilic grain disease (AGD), a prevalent limbic tauopathy of aging. Structures determined by cryo-electron microscopy reveal tau filament folds that differ from those found in sporadic AGD or other tauopathies and feature a 4-layer architecture stabilized by the Ile substitution within its core. Comparative structural analysis reveals conserved motifs are shared among AGD, corticobasal degeneration, and MAPT P301T. A well-defined density stacks along a cationic cleft, indicative of a bound RNA-like polyanion or small-molecule. In vitro analysis shows the S305I mutation promotes fibrilization relative to normal tau. These results demonstrate that MAPT S305I stabilizes a distinct aggregation-prone tau fold that likely contributes to disease pathology and heterogeneity beyond its known splicing defects, and underscore potential limitations of using the most pathologically similar genetic form as a model for sporadic FTLD-tau.

BackgroundHuntingtons disease (HD) is a neurodegenerative disorder caused by an abnormal polyglutamine expansion in mutant huntingtin (mHTT) and is characterized by movement dysfunction and neuronal loss. Siglecs, a family of sialic acid-binding proteins, are expressed on brain microglia and implicated in Alzheimers disease. Sialic acids are abundant in mammalian brains and cap the termini of the glycocalyx of various brain cells. Alterations in sialoglycans or Siglecs may affect interactions between microglia and other brain cells. However, the roles of Siglecs in HD have not been investigated. MethodsWe profiled Siglecs in postmortem caudate nucleus samples from HD subjects and in a mouse model of HD (R6/2) using RT-qPCR and mass cytometry analyses. CD22 functions in microglia were evaluated using a microglial cell line (BV2) and primary microglia. Native ligands for microglial CD22 were assessed via glycomic profiling and flow cytometry. Regulation of CD22 ligands in astrocytes was investigated in an astrocytic cell line (C8-D1A) and primary astrocytes. The role of CD22 in HD was examined by genetic deletion in HD mice, followed by behavioral analyses and pathological evaluation with immunofluorescence staining and MRI. ResultsUpregulation of CD22 in microglia, observed in the brains of patients and mice with HD, impairs microglial phagocytosis via ITIM-ITAM signaling crosstalk. This CD22 upregulation was driven by chronic oxidative stress, as antioxidant treatment (N-acetylcysteine) markedly normalized CD22 levels. CD22 ligand, 2,6-sialylated-6-sulfo-LacNAc, primarily expressed by astrocytes, was significantly reduced in HD mice. mHTT, but not wild-type HTT, suppressed ligand synthesis in astrocytes under elevated oxidative stress, allowing more CD22 on the microglial surface to inhibit phagocytosis. Treatment with a neutralizing antibody or ligand-enriched extracellular vesicles depleted surface CD22 and restored the phagocytic function of microglia. Genetic deletion of CD22 in HD mice improved rotarod performance, reduced mHTT inclusion burden, increased Darpp32 expression, and alleviated brain atrophy, supporting the concept that CD22-mediated inhibition of microglial phagocytosis contributes to HD pathogenesis. ConclusionOur findings suggest that CD22 acts as a checkpoint-like regulator that restrains microglial phagocytosis and contributes to HD progression when astrocyte-microglia communication is impaired, thereby highlighting CD22 as a promising therapeutic target.

Parkinsons disease (PD) exhibits well-established sex differences in prevalence and clinical phenotypes, yet the underlying molecular mechanisms remain largely elusive. Here, we conducted a comprehensive sex-stratified multi-omic integration to identify sex-specific causal proteins and biological pathways in PD. We performed gene-based association analysis, transcriptome-wide association studies (TWAS), and proteome-wide Mendelian randomization (PWMR) with colocalization analysis using GWAS summary statistics from the International PD Genetics Consortium (IPDGC; 12,054 male cases/11,999 controls; 7,384 female cases/12,389 controls) for sex-stratified analyses and Global Parkinsons Genetics Program (GP2; 34,933 cases/31,009 controls) for sex-combined analyses. Prioritized candidates were further evaluated through MR with brain expression quantitative trait loci (eQTLs) from MetaBrain and differential protein abundance analysis using the Global Neurodegeneration Proteomics Consortium (GNPC; 704 PD cases/5,629 controls in plasma; 78 cases/1,411 controls in cerebrospinal fluid). Additionally, pathway enrichment analysis was performed for prioritized molecules. Integration across three analytical layers prioritized 102 molecular candidates across 31 unique loci, significant from multiple analyses. Of these, eleven genes reached significance across all three layers, including SNCA, MAPT, and CTSB significant in both sexes; CD160, GPNMB, and LRRC37A2 as male-predominant; STX4 and PRSS53 as female-predominant; and BST1, SCARB2, and LGALS3 significant only in sex-combined analysis. In males, CD160 emerged as a novel candidate with convergent evidence across all three analyses and colocalization, while L3MBTL2 was identified as a novel risk gene from gene-based association and TWAS analyses. In females, STX4 and PRSS53 at the 16p11.2 locus showed female-predominant associations. Pathway enrichment analysis revealed innate immune and SUMOylation pathways in males, with CD160 and L3MBTL2 as key contributors respectively, contrasting with WDR5-mediated chromatin remodeling in females. Brain eQTL-based MR confirmed significant associations for 69 of 86 testable candidates (80.2%) in at least one tissue. Protein abundance analysis confirmed sex-specific patterns, and several candidates showed discordant directions between genetically predicted causal effects and observed protein abundance -- including male-specific plasma elevation of CD160 and female-specific patterns for STX4 -- underscoring the distinction between causal risk mechanisms and disease-state molecular changes. These findings demonstrate that PD is a molecularly heterogeneous disorder with sexually dimorphic pathogenic drivers. While shared axes such as lysosomal dysfunction and vesicle trafficking disruption exist, the divergence into male-specific immune dysregulation and female-specific chromatin remodeling suggests that the primary triggers of neurodegeneration differ by sex. Our results underscore the necessity of sex-stratified approaches in biomarker discovery and the development of precision therapeutic strategies for PD.

BackgroundFilamentation induced by cAMP domain-containing protein (FICD) is an endoplasmic reticulum (ER)-resident adenylyltransferase that catalyzes protein AMPylation, a post-translational modification. Although FICD-mediated AMPylation has been linked to the fine-tuning of proteostasis and neuronal integrity, its role in neurodegenerative diseases characterized by protein dyshomeostasis remains unclear. Parkinsons disease (PD) is defined by dopaminergic neurodegeneration and aggregation of -synuclein (aSyn) as a consequence of impaired protein homeostasis. We therefore investigated whether dysregulated FICD-mediated AMPylation contributes to PD pathogenesis. MethodsWe combined analyses of human post-mortem PD brain tissue with complementary models, including midbrain dopaminergic neurons derived from human induced pluripotent stem cells (hiPSCs) of a PD patient carrying an SNCA gene duplication and its isogenic gene dosage-corrected control line, transgenic mouse models of synucleinopathy, and an aSyn-overexpressing H4 neuroglioma cell model. Genetic and pharmacological modulation of FICD activity was integrated with multi-proteomic approaches, including chemical proteomics-based AMPylation profiling, stable isotope labelling with amino acids in cell culture-based global protein turnover analysis, and whole-proteome profiling to identify AMPylation-associated molecular pathways. ResultsFICD was preferentially expressed in dopaminergic neurons and was upregulated in SNCA duplication PD patient-derived neurons, as well as in the basal ganglia of PD post-mortem brains and synucleinopathy mice. Despite this overall increase, the proportion of FICD-expressing dopaminergic neurons was reduced under PD conditions, suggesting selective vulnerability of dopaminergic neurons to FICD. Mechanistically, FICD selectively AMPylated lysosomal proteins, thereby linking AMPylation to the regulation of degradative pathways. Moreover, hyperactivation of FICD-induced AMPylation triggered ER stress, impaired lysosomal function, reduced protein turnover, and ultimately promoted aSyn aggregation and apoptotic cell death. Importantly, pharmacological inhibition of AMPylation reversed aSyn pathology and neurite degeneration in PD patient-derived neurons. ConclusionsWe identify the pathological relevance of FICD-mediated AMPylation in PD-related neurodegeneration and its contribution to aSyn aggregation through a bidirectional interplay with aSyn pathology. Our findings support FICD-mediated AMPylation as a defining molecular switch regulating intracellular protein homeostasis in PD and highlight the FICD-AMPylation pathway as a potential therapeutic target for restoring aSyn pathology and mitigating disease progression.

In Alzheimers disease (AD) astrocytes become reactive, displaying hypertrophic morphology, increased expression of intermediate filament proteins GFAP and Vimentin and impaired homeostatic support to neurons. However, the contribution of reactive astrocytes to AD progression, particularly the role of cytoskeletal hypertrophy, remains unclear. Here, we investigate whether astrocyte intermediate filaments actively contribute to early AD progression. We show that astrogliosis appears as early as at 3 months in APP/PS1 mice, preceding amyloid-{beta} plaque deposition, and is characterized by a strong upregulation of GFAP and Vimentin. Genetic ablation of GFAP and Vimentin attenuated astrogliosis, as evidenced by the absence of hypertrophy of astrocyte processes and restored expression of glutamine synthetase and other proteins altered in reactive astrocytes in AD. Importantly, GFAP and Vimentin deletion prevented cognitive decline in 4-month old male and female mice, independently of amyloid plaque pathology or microglial reactivity. Mass-spectrometry based proteomics of the dorsal hippocampus revealed a downregulation of synaptic proteins and dysregulation of ribosomal and RNA-binding proteins in APP/PS1 mice, both of which were rescued by GFAP and Vimentin deletion. Using astrocyte-specific CRISPR-Cas9-mediated knockout of GFAP and Vimentin, we further demonstrate translation impairments in AD astrocytes, and that GFAP and Vimentin deletion restores this impaired astrocytic translation. Together, our findings identify intermediate filament proteins GFAP and Vimentin as active regulators of astrocyte protein synthesis, and reveal a previously unrecognized mechanism by which reactive astrocytes contribute to early cognitive dysfunction in AD. This highlights these astrocyte intermediate filaments as promising therapeutic targets to counteract reactive astrocyte-driven cognitive decline in the early stages of Alzheimers disease.

The microglial CD33 gene is overexpressed in the brains of individuals with Alzheimers disease, and multiple CD33 genetic variants are directly linked to disease risk. Here, we investigate CD33 function in induced human microglial cells by increasing or decreasing CD33 levels with AAV6-mediated gene transfer and with CD33-targeting siRNA, respectively. Phagocytosis of oligomeric amyloid beta into live microglia was doubled by reducing CD33 levels, with concomitant increases in secreted TREM2 and in TREM2 activation as assayed by phosphorylation of downstream SYK. Increasing CD33 had opposing effects on microglial activity, decreasing amyloid beta uptake by approximately half, reducing LPS-induced increases in IL-10, and decreasing the efficacy of a TREM2-activating antibody. Experiments combining siRNA and AAV6 demonstrated a linear relationship between CD33 protein and amyloid beta uptake. Results show that CD33 largely governs multiple microglial functions associated with Alzheimers disease and affects both pharmacological and physiological activation of TREM2.

Alzheimers disease (AD) is a progressive neurodegenerative disorder with a rapidly increasing global prevalence. Current pharmacological interventions offer symptomatic relief but do not modify disease progression. Secretome-based therapeutics have emerged as a potential disease-modifying strategy, given their capacity to influence multiple pathological pathways, including amyloid burden, reactive gliosis, and neuronal survival. Early clinical studies support the safety and potential efficacy of these approaches, indicating mechanisms involving neuroprotection, neurodegeneration, and modulation of neuroinflammation, processes central to AD pathology. In the present study, we investigated the therapeutic efficacy of multipotent stromal cell (MSC)-derived secretomes produced by a specific platform (Secretomix) in two distinct mouse models of neurodegenerative disease: An AD model characterized by amyloid pathology, and a motor neurone disease (MND) model exhibiting TDP-43 protein aggregation. Administration of the MSC secretome resulted in a positive modulation of the behavioural phenotype in the AD model, and reduction in the rate of decline of motor co-ordination (attenuated the progression of motor deficits) in the MND model. In the latter, these functional benefits were accompanied by a measurable reduction in neuroinflammatory responses but without direct alteration of standard neuropathological markers. Additionally, ex vivo assays using human peripheral blood demonstrated broad anti-inflammatory activity of the MSC secretome, providing a potential mechanistic basis for the in vivo observations. Collectively, these findings support further investigation of MSC-derived secretomes as a promising therapeutic approach for neurodegenerative disorders, with relevance across proteinopathies characterised by distinct molecular pathways. Significance StatementHere we demonstrate the efficacy of a stem cell secretome in ameliorating cognitive and behavioural phenotypes in different models of neurodegeneration. These models represent distinct neuropathological features that are unaffected by stem cell secretome treatment but share common features of modulation of inflammation post stem cell secretome treatment. This study highlights the therapeutic potential of stem cell secretomes in the treatment of neurodegenerative conditions with an already existing neuropathology.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive loss of motor function. We have developed a Drosophila model of ALS8 (VAPBP58S) using CRISPR/Cas9 genome editing. VAPB is an ER-based adapter protein associated with and regulating intracellular membrane:membrane contact sites. VAPBP58S flies show progressive age-dependent motor deficits and a shortened lifespan, paralleling features of the human disease. VAPBP58S brains exhibit age-dependent neuroinflammation, as measured by whole-transcriptome quantitative mRNA sequencing, suggesting a broad, low-grade enhancement in signalling in multiple (Toll, IMD, Jak-STAT and Jun-kinase) immune pathways. We implicate glial cells in the brain as the site of brain inflammation and identify Drosophila Fos (Kayak) as a key modulator of age-dependent inflammation. In accordance, we find that overexpression of wild-type kayak or its dominant-active variant kayakK357R in glia reduces inflammation and, concomitantly, improves motor function. In contrast, knockdown of glial kayak accelerates age-dependent deterioration of motor function and enhances neuroinflammation. Our study underscores the roles of glial-modulated brain inflammation in dictating ALS8 progression and identifies kayak as a central negative regulator of neuroinflammation in disease. Summary StatementWe uncover definitive evidence for age-dependent neuroinflammation, originating from glial cells and regulated by Fos, as a key mechanism underlying Amyotrophic Lateral Sclerosis 8.

Amyloid Precursor Protein (APP) has been reported to partially localize to mitochondria, and mitochondrial dysfunction is a key feature of Alzheimers disease; however, the mechanisms linking APP to mitochondrial functions remain incompletely defined. In this study, we identified an interaction between APP and phosphoglycerate mutase family member 5 (PGAM5), a mitochondrial protein phosphatase. We confirmed their endogenous interaction in mouse brain tissue and determined that APP and PGAM5 are both present at mitochondria-ER contact sites (MERCS) and. Using in vitro binding assays, we demonstrate a direct interaction between the linker region of APP and a region of PGAM5 that includes the Kelch-like ECH-associated protein 1 (Keap-1) binding domain. PGAM5 is known to anchor a portion of Nuclear factor erythroid 2 p45-related factor 2 (Nrf2) through Keap1 at the outer mitochondrial membrane and regulates mitochondrial respiration and stress responses. We found that the Nrf2-regulated genes Hmox1 (Heme oxygenase-1) and Nqo1 (NADH:quinone oxidoreductase 1), which are involved in mitochondrial respiration, are downregulated in APP KO astrocytes. Accordingly, mitochondria isolated from the brains of APP knockout (KO) mice have impaired substrate-specific respiration and electron transport chain (ETC) function. Together, these findings suggest that APP supports mitochondrial respiration by binding to PGAM5 and modulating Keap1-Nrf2 signaling.

BackgroundAmyotrophic lateral sclerosis (ALS) is clinically heterogeneous, and genetic modifiers may drive molecular endophenotypes without obvious clinical stratification. The apolipoprotein E {varepsilon}4 (APOE {varepsilon}4) allele is a major Alzheimers disease risk allele, but its biological impact in ALS remains unclear. MethodsUsing the Answer ALS cohort, longitudinal motor, cognitive, and neuropsychiatric measures were modelled using mixed-effects approaches. Patient induced pluripotent stem cell-derived motor neuron multiomics (chromatin accessibility, transcriptomics, and proteomics) were analysed using supervised machine learning. Plasma SomaScan profiling was used to derive an APOE {varepsilon}4-associated protein signature and to test its stability across serial visits, biological pathway enrichment, and associations with clinical progression. ResultsAPOE {varepsilon}4 carriage was not associated with baseline severity or rate of functional decline and showed no consistent effects on cognitive or neuropsychiatric trajectories. Motor neuron multiomic profiles similarly demonstrated no reproducible APOE {varepsilon}4 signal and did not reliably classify genotype. In contrast, plasma proteomics identified an APOE {varepsilon}4 protein signature that classified {varepsilon}4 status with high accuracy in ALS (AUC 0.98) and non-ALS motor neuron disease (AUC 0.86) and was enriched for immune and inflammatory biology. This systemic signature was highly stable across repeated sampling, indicating a persistent genotype-associated state. Within this plasma endophenotype, a small set of proteins tracked functional decline and a composite score stratified fast versus slow progression. Baseline composite scores were elevated in APOE {varepsilon}4 carriers in both ALS and neurologically unimpaired controls, consistent with a stable systemic shift detectable beyond overt disease status. ConclusionsAPOE {varepsilon}4 defines a persistent, immune-enriched systemic proteomic endophenotype in ALS that is not captured by clinical trajectories or motor neuron-only profiling yet relates to disease progression. Plasma-based, genotype-informed endophenotyping offers a translational pathway for biomarker stratification and therapeutic prioritisation in ALS and potentially other heterogeneous neurodegenerative disorders.

Recent genetic studies have underscored the central role of microglia in orchestrating neurodegenerative pathology, particularly in Alzheimers disease (AD). However, contributions of other cell types are complex and poorly understood. Studying specific genetic mutations related to the disease is limited by the lack of robust in vitro models. To address this, we investigated the impact of the high-risk AD-associated TREM2-R47H variant using iPSC-derived forebrain organoids co-cultured with microglia, comparing mutant and isogenic control lines. Organoids were cultured up to 173 days, co-cultured with microglia and profiled using single-cell transcriptomics, bulk RNA sequencing, and confocal microscopy. Our findings demonstrate that the mutant co-culture model exhibits AD-specific signatures in vitro. Notably, confocal imaging revealed that control microglia internalized phosphorylated-Tau throughout the tissue, while R47H microglia showed no such uptake. Single-cell and bulk RNA profiling uncovered alterations in gene expression associated with oxidative phosphorylation, lysosomal activity and pathways of neurodegeneration. Interestingly, signs of neurodegeneration appeared as early as day 139 in variant organoids cultured without microglia, whereas wild-type (WT) organoids did not exhibit comparable changes at the whole-organoid level. By day 163, robust neurodegenerative profiles spanning neuronal and glial populations were evident exclusively in TREM2-R47H samples. These data suggest that the TREM2-R47H variant impairs microglial clearance of pathological proteins and may affect broader cellular networks beyond microglia, challenging current assumptions about its role and opening avenues for redefining AD pathogenesis.

Increased lysosomal stress responses (LSR) are commonly implicated in the pathogenesis of neurodegenerative disorders including HIV-1-associated neurocognitive disorders (HAND). The HIV-1 envelope glycoprotein gp120 causes LSR, increases levels of ferrous iron (Fe2+) in the cytosol and in mitochondria, disrupts the reactive species interactome (RSI), and increases neural cell death. Here, we report that TRPML1, an endolysosome redox-sensitive cation channel, is mechanistically involved in gp120-induced neurotoxicity. TRPML1 was activated by gp120-induced increases in cytosolic reactive oxygen species (ROS) and resulted in release of Fe2+ from endolysosomes in levels sufficient to increase cytosolic levels of Fe2+ and ROS as well as decrease levels of hydrogen sulfide (H2S). Reduced glutathione normally buffers intracellular Fe2+, but gp120 decreased endolysosome glutathione levels and disrupted this regulatory control mechanism thereby promoting TRPML1-mediated Fe2+ efflux from endolysosomes. TRPML1 redox activation led to changes to the RSI in endolysosomes including increased ROS, lipid peroxidation, nitric oxide, and sulfane sulfur as well as decreased H2S. These changes were accompanied by increased cysteine oxidation of luminal proteins and endolysosome deacidification. Pharmacological inhibition of TRPML1 or knocking down expression levels of TRPML prevented these effects. Thus, our findings suggest that TRPML1 redox activation controls gp120-induced endolysosome dysfunction and iron/redox imbalance, and further implicates TRPML1 in the pathogenesis of HAND.

Tauopathies are characterised by progressive deterioration of brain regions due to abnormal accumulation of the microtubule-associated protein tau (MAPT). Alternative splicing of MAPT pre-mRNA results in six tau isoforms, which are classified into two groups depending on the number of microtubule-binding domain repeats (3R vs 4R). Although many tauopathies are 3R or 4R-specific, the relative contributions of individual isoforms to neurotoxicity remain incompletely understood. To systematically characterise differences in tau isoform toxicity, we created a novel set of Drosophila lines expressing equivalent amounts of the six human tau isoforms (hTau) at levels sufficient to induce visible phenotypes. Using a variety of assays including survival, negative geotaxis and tissue-level or cell-type-specific degeneration, we found that hTau isoform toxicity is not uniform across different biological contexts. Despite generally higher toxicity of 4R isoforms compared to 3R, the effects of individual hTau isoforms varied with the temporal window of expression, tissue type, and neuronal identity. Restricting hTau expression to small homogeneous neuronal populations enabled detailed analysis of isoform-specific degeneration. Neurons previously observed to be vulnerable or resilient to hTau toxicity exhibited differences in the onset and progression of degeneration, suggesting that resilience may be an early and transitory state, with most or all neurons eventually succumbing to tau toxicity over time. Notably, these differences in toxicity were not readily explained by variations in hTau abundance and phosphorylation. Together, our findings demonstrate that tau toxicity is highly context-dependent, clearly isoform-specific, and shaped by interactions between tau and its cellular environment.

Heterozygous mutations in the progranulin (PGRN) encoding gene GRN cause frontotemporal dementia (FTD), whereas homozygous GRN mutations lead to neuronal ceroid lipofuscinosis (NCL). However, the mechanisms underlying neurodegeneration due to PGRN deficiency remain unclear. In the aged brains or under neurodegenerative conditions, the accumulation of lipid droplets (LDs) in microglia contributes to cellular dysfunction and pro-inflammatory responses, exacerbating neurodegenerative pathology. Here, we investigated how PGRN deficiency induces LD accumulation in microglia. We found that PGRN ablation significantly upregulated triglycerol-3-phosphate acyltransferase 3 (GPAT3), the first and rate-limiting enzyme for triacylglycerol biosynthesis, and increased LD formation in both Grn-/- BV2 microglial cells and Grn-/-mouse brains. Mechanistic study revealed that PGRN deficiency upregulates GPAT3 via stimulating signal transduction and activator of transcription factor 3 (STAT3) signaling. Silencing Gpat3, inhibiting GPAT3 activity with the small molecule inhibitor FSG67, or PGRN restoration reduced LD accumulation and rescued the cytotoxicity of Grn-/- microglia conditional medium (MCM) toward N2a cells. Notably, intraperitoneal administration of FSG67 or AAV-MG1.2-mCx3cr1-mGrn-eGFP-mediated microglia-specific gene therapy reduced microglial LDs and ameliorated behavior phenotypes in Grn-/- mice. Our study elucidates the role of PGRN loss in microglial lipid homeostasis and identifies the STAT3-GPAT3 axis as a potential therapeutic target for LD-associated neurodegenerative diseases.