Molecular Neurodegeneration

Parkinsons disease (PD) is characterized by progressive degeneration of nigrostriatal dopamine neurons and synucleinopathy, which is the accumulation of aggregated -synuclein (-syn). Increasing evidence implicates -syn-associated neuroinflammation as a contributor to PD pathogenesis; however, immune mechanisms linking synucleinopathy to neurodegeneration remain incompletely defined. Activation of the complement cascade occurs in PD and other neurodegenerative disorders, but most studies report complement activation after overt neurodegeneration, making it difficult to conclude if complement is directly activated by pathological -syn or secondarily following neurodegeneration. We used the rat -syn preformed fibril (PFF) mode, in vitro complement assays and human postmortem PD tissue to test whether pathological -syn directly activates complement prior to overt neurodegeneration. The -syn PFF model exhibits a protracted pathological time course and distinct temporal separation between peak -syn aggregation and nigrostriatal degeneration; thus we quantified complement expression, activation, and regulation during the aggregation phase. Synucleinopathy induced complement activation prior to nigrostriatal degeneration, including upregulation of components of both the classical (C1qa, C1r, C4b) and alternative (Cfd, Cfb) pathways, the anaphylatoxin (C3aR, C5aR) and phagocytic (CR3) complement receptors, and activation of complement C3. During early synucleinopathy, microglia upregulated C3 which significantly correlated with synucleinopathy burden across several brain regions, including the substantia nigra pars compacta (SNc) and cortex. Concurrently, complement regulatory proteins, including CD55, CD59, neuronal pentraxin-1 (Nptx1), and the neuronal pentraxin receptor were downregulated in the synucleinopathy-affected SNc. Importantly, increased levels of C1q and iC3b along with downregulation of CD55 and NPTX1 were also observed in human postmortem PD SNc, supporting the translational relevance of our findings. Mechanistically, we demonstrate that aggregated, but not monomeric, -syn directly binds C1q and activates the complement cascade in a C1q-dpendent manner. These data provide the first in vivo evidence that synucleinopathy triggers complement activation and dysregulation prior to neurodegeneration.

Metabolic dysfunction and proteinopathy are hallmarks of many neurodegenerative diseases, yet their mechanistic interplay remains poorly understood. Here, we demonstrate that amyloid precursor protein (APP) processing in cortical neurons is disrupted upon loss of Nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2), the NAD-synthesizing enzyme in neurons, resulting in accumulation of APP C-terminal fragments (APP-CTFs). Knockdown (KD) of the NAD hydrolase sterile alpha and TIR motif-containing protein 1 (SARM1) restores APP-CTF levels in NMNAT2 knockout (KO) neurons to wild-type levels, whereas NAD supplementation yields modest rescue. Redox profiling indicates that NMNAT2 loss reduces NAD/NADH redox potential when APP-CTF starts accumulating. Seahorse metabolic flux analysis shows that NMNAT2 deficiency induces early glycolytic impairment, followed by deficits in mitochondrial respiration. Notably, SARM1 KD, but not NAD supplementation, rescues mitochondrial function in NMNAT2 KO neurons. Temporal profiling of NMNAT2 KO neurons revealed a biphasic pattern in APP-CTF accumulation, with an initial gradual increase followed by a marked acceleration, paralleling the transition from an initially small number to a substantially greater number of differentially expressed proteins. Pathway enrichment analysis of proteomic changes suggests JNK/MAPK signaling is upregulated in the early phase, with late-phase downregulation of mitochondrial function and upregulation of endoplasmic reticulum stress and unfolded protein response pathways. Collectively, these findings demonstrate that neuronal NAD depletion drives a progressive, SARM1-dependent disruption of redox homeostasis and proteostasis, resulting in impaired APP processing. The NMNAT2-SARM1 axis emerges as a critical pathway linking metabolic stress to proteinopathy, positioning SARM1 as a key mediator of neurodegenerative dysfunction.

Alzheimers disease (AD) is a progressive neurodegenerative disorder characterized by {beta}-amyloid (A{beta}) accumulation and oxidative stress, with aging being its greatest risk factor. Age-related decline in antioxidant defenses, particularly glutathione (GSH), may increase neuronal vulnerability to A{beta}-mediated toxicity; however, the mechanisms linking redox dysregulation to neuronal death remain incompletely understood. In this study, we investigated how impaired GSH homeostasis influences neuronal susceptibility to A{beta}-associated injury. Human SH-SY5Y neuron-like cells were engineered to express either wild-type APP695 or the familial AD-associated APPSwe/Ind mutant, and intracellular GSH depletion was induced using both pharmacological and genetic approaches. GSH depletion markedly sensitized APPSwe/Ind-expressing cells to cell death, accompanied by increased plasma membrane lipid peroxidation, elevated malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) levels, and enhanced lactate dehydrogenase (LDH) release. This cell death was not prevented by the pan-caspase inhibitor Z-VAD-FMK but was effectively rescued by the ferroptosis inhibitors ferrostatin-1 (Fer-1) and liproxstatin-1 (Lip-1), indicating a ferroptotic mechanism. Similar ferroptotic responses were observed when A{beta} oligomers were combined with intracellular GSH depletion. Mechanistically, A{beta} and GSH depletion synergistically increased transferrin receptor-1 expression and intracellular iron levels while markedly suppressing glutathione peroxidase 4 (GPX4), a central regulator of ferroptosis. Importantly, inhibition of autophagy with bafilomycin A1 restored GPX4 expression and rescued cells from ferroptotic death, suggesting that autophagy-mediated GPX4 degradation contributes to this process. Collectively, our findings demonstrate that GSH dysregulation synergizes with A{beta} to induce lipid peroxidation and ferroptosis in neuron-like cells. These results identify impaired redox homeostasis as a critical driver of neuronal vulnerability in AD and suggest that preserving GSH levels or targeting ferroptotic pathways may offer promising therapeutic strategies for neurodegeneration.

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.

Tauopathies, including Alzheimers disease, involve progressive neurodegeneration and sustained neuroinflammation. We present a multi-compartment transcriptomic atlas of 9.6-month-old PS19 tauopathy mice compared with wild-type (WT) controls (n=8/group), profiling cortical mRNA, cortical non-coding RNA (ncRNA), and plasma small extracellular vesicle (pEV) ncRNA. In the PS19 cortex, mRNA sequencing identified 917 differentially expressed genes (DEGs), with microglial deconvolution revealing a robust transition toward disease-associated microglia (DAM) gene signature and downregulation of genes involved in oxidative phosphorylation and cholesterol biosynthesis relative to WT. Cortical ncRNA profiling identified 466 differentially expressed ncRNAs, primarily circular RNAs (circRNAs; n=331). In pEVs, 822 ncRNAs were differentially abundant, of which 657 circRNAs were identified in PS19 compared to WT mice. Cross-compartment integration demonstrated that pEV miRNA gene targets functionally mirrored genes involved in the brains inflammatory and metabolic failure. We identified a core shared signature of 33 ncRNAs, including miR-5114 (up in brain, down in pEV), circ_0008242 and circ_0002153 (up in brain and pEV), and circ_0007688 (down in brain and pEV) differentially enriched across both brain and periphery in PS19 compared to WT mice. These results demonstrate that the pEV non-coding landscape effectively tracks central tau-mediated changes in the brain transcriptional response. This study identifies circRNAs as the most numerically perturbed ncRNA class and provides a foundation for non-invasive biomarker development in tauopathy.

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.

Astrocytes are key regulators of lipid metabolism, and dysregulated astrocytic lipid processing is implicated in Parkinsons disease (PD) pathogenesis. Our prior genome-wide screens identified ACSBG1, an astrocyte-enriched acyl-CoA synthetase, as a candidate regulator of -synuclein (-Syn) levels. However, how ACSBG1 links lipid reprogramming to inflammatory astrocyte activation and -Syn pathology remains unknown. We compared the transcriptomic, cytokine, and lipid secretomes of TNF- and IL-1 stimulated primary astrocytes from wild-type (WT) and Acsbg1 knockout (KO) mice. In vivo, we crossed Acsbg1 KO mice with a Thy1--Syn PD model to assess behavior, neuroinflammation, synaptic integrity, and -Syn levels. Following cytokine exposure, Acsbg1 KO astrocytes mounted an attenuated inflammatory transcriptional response, secreting significantly fewer inflammatory mediators (e.g., IL-6, RANTES, MIP-3) and less long-chain Sphingosine 20:1 than WT astrocytes. Importantly, exogenous Sphingosine 20:1 or cytokines from WT reactive astrocytes induced neuronal -Syn phosphorylation (pS129). In vivo, Acsbg1 deletion in Thy1--Syn mice reduced astrogliosis, rescued synaptic and behavioral deficits, and decreased total and pS129--Syn. These findings establish ACSBG1 as a key regulator of inflammatory astrocyte signaling that contributes to -Syn phosphorylation via specific cytokine and lipid mediators, identifying ACSBG1 as a novel therapeutic target for modulating astrocyte-neuron communication in PD.

Microglial activation has been closely associated with accelerated ALS disease progression. However, specific microglial pathways that regulate microglial activation and ALS disease progression remain limitedly understood. Here, we determined the role of Clec7a (or Dectin-1), a core signature gene of disease-associated microglia (DAM) in ALS, in regulating microglial activation and ALS disease progression. Our spinal cord scRNA-Seq results found that Clec7a deficiency specifically attenuated microglial neuroimmune gene expression in SOD1G93A mice and human ALS. In addition, in vivo two-photon imaging of human (h) TDP43 phagocytosis by microglia in the cortex showed that Clec7a deficiency promotes microglial phagocytosis of pathological hTDP43 by enhancing microglial process dynamics. Subsequent survival analysis further showed that selective deletion of Clec7a in microglia mitigates motor neuron degeneration and delays disease progression in SOD1G93A ALS mice. Together, our results establish that Clec7a is a key regulator in shaping disease microglial functions and promotes disease progression in ALS.

NOD-like receptor family pyrin domain-containing 3 (NLRP3) is a cytosolic regulator of an inflammasome-mediated innate immune response. In the central nervous system (CNS), NLRP3 inflammasome activation has been implicated in multiple neurodegenerative diseases, yet the mechanisms by which it contributes to disease remain unclear. Here, we investigated the CNS effects of chronic NLRP3 activation using a humanized NLRP3 gain-of-function mouse model (hNLRP3D305N). Bulk brain analyses confirmed constitutive inflammasome activation, widespread cytokine induction, and the increased presence of blood-associated proteins suggestive of dysfunction at CNS border sites and the blood-brain barrier (BBB). Furthermore, cerebrospinal fluid (CSF) neurofilament light chain levels were elevated, indicating neuronal damage. Single-cell RNA-sequencing of CD45+ immune cells in the brain demonstrated that microglia adopt distinct reactive states and that peripheral immune cells infiltrate the CNS, with neutrophils emerging as the predominant infiltrating immune cell type. This finding was confirmed by untargeted bulk brain and CSF proteomics that also suggest neutrophil reactivity. Immunohistochemistry further revealed regional neutrophil entry into the brain parenchyma, concurrent with reactive microglia and engulfment of neutrophils, suggesting functional microglia-neutrophil interactions. Collectively, these findings establish a direct pathogenic role for the NLRP3 inflammasome in the CNS independent of other neurodegeneration-related disease pathologies.

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.

INTRODUCTIONThe slow, age-related development of Alzheimers disease (AD) and inaccessibility of early-stage brain tissue necessitates model studies to understand its origins and progression. Non-human primate models can provide a platform for linking molecular changes to translatable phenotypes. Here, we assess fibroblast lines derived from marmosets with engineered variants in the PSEN1 gene. METHODSFibroblast cultures were obtained from 10 animals and assayed using a NanoString AD gene expression panel and label-free proteomics. We compared mutant expression changes to human AD signatures in human iPSC-derived neurons and postmortem brains to assess disease relevance. RESULTSGene products involved in amyloid-beta interaction and regulation were differentially expressed, providing evidence for the functional relevance of the engineered fibroblasts. Both gene and protein expression changes in the undifferentiated fibroblasts correlated with human iPSCs from AD donors reprogrammed into neuronal lineages, as well as postmortem brains derived from case-control cohorts. Altered expression profiles were noted based on marmoset donor sex and mutation status, highlighting underlying sex-specific biology relevant to Alzheimers disease. DISCUSSIONThese findings demonstrate that disease-relevant pathways and processes are altered in fibroblasts from mutant marmosets, emphasize the complementarity of transcriptomic and proteomic profiling in AD, and provide a roadmap for more advanced molecular studies of AD in aging marmosets and marmoset-derived cell models.

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.

IntroductionApoE4 is the strongest genetic risk factor for Alzheimers disease (AD). Emerging evidence suggests that ApoE4 increases AD risk by disrupting microglial metabolism and function. However, whether ApoE lipidation state contributes to microglial dysfunction remains poorly understood. MethodsHuman microglia were treated with lipid-free or lipid-bound ApoE3 or ApoE4. Label-free live-cell holotomography and global proteomics were used to assess isoform- and lipidation-specific effects on lipid droplet dynamics, mitochondrial morphology, and microglial phenotype. ResultsApoE4 treatment resulted in fewer but enlarged lipid droplets and increased mitochondrial fragmentation compared to ApoE3, effects that were enhanced by lipid-bound ApoE4. Proteomic analyses revealed a strong type I interferon response in cells exposed to lipid-free ApoE, which was exacerbated by lipid-free ApoE4. DiscussionThese findings indicate that lipid-bound ApoE4 drives metabolic reprogramming, whereas lipid-free ApoE4 promotes inflammatory signaling, identifying ApoE lipidation as a critical modifier of ApoE4-associated AD risk.

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.

Astrocytes directly influence neuronal survival and increasingly are understood to contribute to the progression of neurodegenerative diseases including Parkinsons disease (PD). Mitochondrial damage is a hallmark of PD pathology in both neurons and astrocytes. Damaged mitochondria are cleared by PINK1/Parkin-mediated mitophagy; loss-of-function mutations in either PINK1 or Parkin are sufficient to cause PD. Neuronal mitophagy is well-studied, but far less is known about how mitochondrial dysfunction in astrocytes affects neural health. While microglial release of pro-inflammatory cytokines has been shown to induce astrocytes to mount their own inflammatory response, we hypothesize that a more direct pathway is involved, and that mitochondrial damage to astrocytes directly triggers release of proinflammatory cytokines. To address these questions, we treated primary murine cortical astrocytes with oxidative phosphorylation (OXPHOS) inhibitors antimycin A (AA) and oligomycin A (OA) and observed the PINK1-dependent accumulation of Parkin on damaged mitochondria, leading to phospho-ubiquitination of proteins in the outer mitochondrial membrane and the recruitment of the autophagy receptor SQSTM1/p62. To identify transcriptional changes caused by mitochondrial damage and the resulting activation of mitophagic machinery, we performed bulk RNA-sequencing on astrocytes isolated from WT, PINK1-/-, or Parkin-/- mice treated with AA/OA or a vehicle control. In WT astrocytes, TNF- signaling via NF-{kappa}B was the most significantly upregulated pathway following OXPHOS inhibition. OXPHOS inhibitor treatment also stimulated p62 expression, while NF-{kappa}B inhibition prevented this upregulation. Astrocytic secretion of cytokines, including TNF-, was increased following mitochondrial damage; this secretion was dependent on NF-{kappa}B activation and occurred at levels sufficient to induce mitochondrial depolarization in hippocampal neurons. Compared to WT astrocytes, PINK1-/- astrocytes showed a significant reduction in transcriptional signatures associated with TNF- signaling following mitochondrial damage, while Parkin-/- astrocytes exhibited upregulation of both IFN-{gamma} and IFN- signaling. These findings indicate altered inflammatory responses to mitochondrial damage in the absence of functional PINK1 or Parkin. Finally, we analyzed scRNA-sequencing data from substantia nigra astrocytes harvested from human brain tissue from PD-positive or control samples. Distinct clusters comprised predominantly of PD-positive or control astrocytes emerged. Astrocytes in the PD-positive cluster were enriched for NF-{kappa}B, IFN- and IFN-{gamma} responses, consistent with the signaling observed in vitro post-OXPHOS inhibition. Together, these findings identify inflammatory signatures activated by mitochondrial damage in astrocytes, and establish this pathway as a potential contributor to neuroinflammation in PD.

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.

Parkinsons disease (PD) is increasingly recognized as a multisystem disorder, yet the mechanisms linking neurodegeneration with muscle dysfunction remain largely unknown. In this study, using an A53T -synuclein (Syn) transgenic mouse model, we demonstrate coordinated pathological changes across the brain-muscle organs characterized by systemic inflammation, iron accumulation, oxidative stress, and ferroptosis-associated lipid peroxidation. Our quantitative proteomics data revealed dysregulated iron metabolism and ferroptosis in the brain and skeletal muscle. Biochemical validation confirmed increased expression of Transferrin receptor 1 (TFRC), elevated lipid peroxidation, and suppression of antioxidant defenses, including SLC7A11 and GPX4, indicating enhanced ferroptotic susceptibility. Cell-surface proteomics and biophysical assays further revealed that pathological Syn directly interacts with TFRC, promoting iron accumulation and ferroptosis-associated oxidative damage in neuronal and muscle cells. Together, our findings identify ferroptosis as a shared pathological mechanism across the brain and muscle, mediated by the Syn-TFRC interaction, thus linking neurodegeneration and peripheral muscle pathology in PD.

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.