Molecular Neurodegeneration

Alzheimers disease (AD) is characterized by amyloid plaques and neurofibrillary tangles in addition to neuroinflammation and changes in brain lipid metabolism. Recent findings have demonstrated that microglia are key drivers of neurodegeneration in tauopathy mouse models. A subset of microglia referred to as disease-associated microglia (DAM) display gene signatures signifying changes in proinflammatory signaling and lipid metabolism in mouse models of amyloid and tau pathology. Ch25h is a DAM gene encoding cholesterol 25-hydroxylase that produces 25-hydroxycholesterol (25HC), a known modulator of inflammation as well as lipid metabolism. However, whether Ch25h influences tau-mediated neuroinflammation and neurodegeneration is unknown. Here, we show that in the absence of Ch25h and the resultant reduction in 25HC there is strikingly reduced age-dependent neurodegeneration and neuroinflammation in the hippocampus and entorhinal/piriform cortex of PS19 mice, which express the P301S mutant human tau transgene. Transcriptomic analyses of bulk hippocampal tissue and single nuclei revealed that Ch25h deficiency in PS19 mice strongly suppressed proinflammatory cytokine and chemokine signaling in microglia and restored sterol synthesis. Our results suggest a key role for Ch25h/25HC in potentiating proinflammatory signaling to promote tau-mediated neurodegeneration. Ch25h may represent a novel therapeutic target for primary tauopathies, AD, and other neuroinflammatory diseases.

Microglia are the primary immune cells of the central nervous system and play a crucial role in maintaining brain homeostasis. In common neurodegenerative diseases such as Alzheimers disease and Parkinsons disease (PD), early and sustained microglial activation has been shown to precede neuronal loss, with elevated levels of microglia-derived inflammatory mediators detected in affected brain regions. In contrast, little is known about the role of microglia in rare neurodegenerative disorders. One such disorder is {beta}-propeller protein-associated neurodegeneration (BPAN), a common subtype of neurodegeneration with brain iron accumulation (NBIA). BPAN shares pathological features with PD, including iron accumulation and selective loss of dopaminergic neurons in the substantia nigra, and is caused by mutations in the WD repeat domain 45 (WDR45) gene encoding an autophagy protein also called WIPI4. However, the pathological role of mutant WDR45 in BPAN and the possible contribution of microglia remain unresolved. We generated the first BPAN patient microglia model system using induced pluripotent stem cells (iPSCs) to identify immune-related alterations and immunomodulatory signaling changes in a disease-relevant context. Integrated transcriptomic and proteomic profiling of iPSC-derived microglia from BPAN patients revealed a consistent shift from a homeostatic to a reactive, disease-associated state. Transcriptomic analysis showed disruption of core microglial pathways, including immune activation, stress response, and autophagy, consistent with a chronic pro-inflammatory phenotype. Complementary secretome analysis identified impaired lysosomal function and increased antigen presentation pathways, further supporting persistent microglial activation. Together this suggests that dysfunctional microglial states may contribute to BPAN pathogenesis. Our findings lay the groundwork for advancing immunomodulatory research in BPAN and may open new avenues for therapeutic development targeting microglial dysfunction.

Sex-specific genetic regulation of cerebrospinal fluid (CSF) protein levels may contribute to differential vulnerability to neurodegenerative diseases. To systematically identify sex differences in the genetic regulation of CSF proteome and their link to neurodegeneration, we performed sex-stratified pQTL analysis of 6,361 proteins in 1,713 males and 1,640 females, separately. We identified 1,729 pQTLs significant in either sex. They included 407 sex-specific pQTLs (genetic regulation in only one sex) and 159 sex-biased pQTLs (regulation in both sexes, but with different magnitudes of regulation between sexes). The HLA locus on chromosome 6 and the APOE locus on chromosome 19, two known pleiotropic regions, regulated several proteins in a sex-dependent way. Pathway enrichment revealed several biological processes that were shared and distinctive of sex. Using proteome-wide association study (PWAS) and colocalization, we identified 22 proteins associated and colocalized with AD risk loci. TMEM106B and ACE proteins were identified in only one sex. Four proteins were associated and colocalized with PD risk loci. These findings provide insights into dissecting the underlying mechanisms contributing to sex differences in neurodegeneration.

Intraneuronal aggregates of the microtubule-associated protein tau play a pivotal role in Alzheimers disease and several other neurodegenerative syndromes. Anti-tau antibodies can reduce pathology in mouse models of neurodegeneration and are currently tested in humans. Here, we performed a large-scale seroepidemiological search for anti-tau IgG autoantibodies ({tau}) on 40,497 human plasma samples. High-titer {tau}+ individuals were surprisingly prevalent, with hospital patients being three times more likely to be {tau}+ (EC50 [&ge;] 26; a nominal dilution of > 1/64) than healthy blood donors (4.8% vs 1.6%). The prevalence increased with age over 70 years-old (RR 1.26, 95%CI 1.11-1.43, P<0.001) and was higher for women (RR 1.20, 95%CI 1.07-1.39, P=0.002). The autoantibodies bound selectively to tau, inhibited tau aggregation in vitro, and interfered with tau detection in plasma samples. No association was found between {tau} autoantibodies and neurological disorders. Instead, tau autoreactivity showed a significant association with kidney and urinary disorders (adjusted RR 1.27, 95%CI 1.10-1.45, P=0.001 and 1.40, 95%CI 1.20-1.63, P<0.001, respectively). These results suggest a previously unrecognized association between {tau} autoimmunity and extraneural diseases.

C9orf72 hexanucleotide repeat expansion (HRE) is a major genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. The role of microglia in these C9orf72 HRE-associated diseases is understudied. To elucidate effects of C9orf72 HRE on microglia, we have characterized human induced pluripotent stem cell-derived microglia (iMG) from behavioral variant frontotemporal dementia (bvFTD) patients carrying the C9orf72 HRE. C9orf72 HRE iMG were compared to iMG from healthy controls and sporadic bvFTD patients. The phenotypes of iMG were analyzed using bulk RNA sequencing, biochemical and immunofluorescence analyses, and live cell imaging. C9orf72 HRE-carrying iMG showed nuclear RNA foci and poly-GP dipeptide repeat proteins but no decreased C9orf72 mRNA or protein expression. TDP-43 pathology was absent from all bvFTD iMG. As compared to healthy control iMG, quantitative immunofluorescence analyses indicated that all bvFTD iMG had reduced number, size, and intensity of LAMP2-A-positive vesicles. C9orf72 HRE-carrying iMG additionally showed decreased number, size, and intensity of p62/SQSTM1-positive vesicles. These changes were accompanied by increased phagocytic activity of the C9orf72 HRE-carrying iMG. Serum starvation increased phagocytic activity also in the iMG of sporadic bvFTD patients. RNA sequencing revealed that iMG of C9orf72 HRE-carrying bvFTD patients as compared to the iMG of sporadic bvFTD patients showed differential gene expression in pathways related to RNA and protein regulation and mitochondrial metabolism. Our data suggest potential alterations in the autophagosomal/lysosomal pathways in bvFTD patient iMG, which are further reinforced by the C9orf72 HRE and functionally manifest as increased phagocytic activity.

Neuroinflammation is a pathological hallmark of Alzheimers disease and related neurodegenerative diseases. However, signaling molecules that regulate glial activation status are not fully understood. Microtubule affinity-regulating kinase 2 (MARK2) has been implicated in both immune responses and AD pathology. Here, we report that MARK2 negatively regulates glial immune responses, which protects against neurodegeneration. We found that MARK2 knockdown in the BV2 murine microglial cell line enhanced IL-6 expression in response to LPS. MARK2 knockdown enhanced IL-6 expression induced by TLR7 agonist but not stimulation of RLR pathways and cGAS-STING. In the brains of PS19 tauopathy mice, MARK2 was elevated in homeostatic microglia but reduced in activated microglia. In Drosophila expressing human tau in the retina, expression of AMP downstream of the Toll pathway in the pigment glia enhances degeneration of photoreceptor neurons. Glial knockdown of Par-1, the Drosophila ortholog of MARK2, enhanced Toll-mediated AMP expression and neurodegeneration, whereas overexpression of Par-1 in the pigment glia suppressed them. These results suggest that MARK2/Par-1 in glia negatively regulates Toll pathway-driven inflammation and protects against tau-induced neurodegeneration. These findings provide insight into the molecular underpinnings of glial inflammation in neurodegenerative conditions and highlight MARK2 as a potential therapeutic target for modulating neuroinflammatory responses.

Alzheimers (AD) and Parkinson disease (PD) pathology often co-occur. Amyloid-{beta} and phosphorylated tau are found in 30-50% of idiopathic PD cases, while -synuclein inclusions are present in 50% of AD cases. These co-pathologies are linked to increased mortality and earlier onset of cognitive decline. Immune activation is a hallmark of these neurodegenerative diseases, but current models primarily examine each pathology in isolation. How these co-pathologies drive inflammation and neuronal loss remains poorly understood. We therefore developed a mouse model combining tau, amyloid-{beta}, and -synuclein. We found that co-pathologies synergistically trigger an amplified neuroimmune response, with expanded populations of CD4+ and CD8+ tissue-resident memory T cells and CD68+ microglia, compared to single pathologies. These changes were abundant in the hippocampus and cortex, regions with elevated protein pathology load and enhanced neuronal loss. Our findings demonstrate that co-pathologies enhance proteinopathy and synergistically enhance immune activation and neurodegeneration, suggesting that combinatorial therapeutic strategies that target both co-pathologies and inflammation, may be disease modifying. SummaryWebster et al. demonstrate that co-occurring Alzheimers and Parkinson disease protein pathologies, common in cognitively impaired patient populations, amplify proteinopathy and synergistically enhance CNS neuroinflammatory responses and neurodegeneration. This work supports the need for combinatorial therapeutic strategies and positions neuroinflammation as an important link for co-pathology enhanced neurodegeneration.

Microglia, the innate immune cells of the brain, are activated by damage or disease. In mouse models of amyotrophic lateral sclerosis (ALS), microglia shift from neurotrophic to neurotoxic states with disease progression. It remains unclear how human microglia change relative to the TAR DNA-binding protein 43 (TDP-43) aggregation that occurs in 97% of ALS cases. Here we examine spatial relationships between microglial activation and TDP-43 pathology in brain tissue from people with ALS and from a TDP-43-driven ALS mouse model. Post-mortem human brain tissue from the Neurological Foundation Human Brain Bank was obtained from 10 control and 10 ALS cases in parallel with brain tissue from a bigenic NFFH-tTA/tetO-hTDP-43{Delta}NLS (rNLS) mouse model of ALS at disease onset, early disease, and late disease stages. The spatiotemporal relationship between microglial activation and ALS pathology was determined by investigating microglial functional marker expression in brain regions with low and high TDP-43 burden at end-stage human disease: hippocampus and motor cortex, respectively. Sections were immunohistochemically labelled with a two-round multiplexed antibody panel against; microglial functional markers (L-ferritin, HLA-DR, CD74, CD68, and Iba1), a neuronal marker (NeuN), an astrocyte marker (GFAP), and pathological phosphorylated TDP-43 (pTDP-43). Single-cell levels of microglial functional markers were quantified using custom analysis pipelines and mapped to anatomical regions and ALS pathology. We identified a significant increase in microglial Iba1 and CD68 expression in the human ALS motor cortex, with microglial CD68 being significantly correlated with pTDP-43 pathology load. We also identified two subpopulations of microglia enriched in the ALS motor cortex that were defined by high L-ferritin expression. A similar pattern of microglial changes was observed in the rNLS mouse, with an increase first in CD68 and then in L-ferritin expression, with both occurring only after pTDP-43 inclusions were detectable. Our data strongly suggest that microglia are phagocytic at early-stage ALS but transition to a dysfunctional state at end-stage disease, and that these functional states are driven by pTDP-43 aggregation. Overall, these findings enhance our understanding of microglial phenotypes and function in ALS.

Common and rare variants in the LRRK2 locus are associated with Parkinsons disease (PD) risk, but the downstream effects of these variants on protein levels remains unknown. We performed comprehensive proteogenomic analyses using the largest aptamer-based CSF proteomics study to date (7,006 aptamers (6,138 unique proteins) in 3,107 individuals). We identified eleven independent SNPs in the LRRK2 locus associated with the levels of 26 proteins as well as PD risk. Of these, only eleven proteins have been previously associated with PD risk (e.g., GRN or GPNMB). Proteome-wide association study (PWAS) analyses suggested that the levels of ten of those proteins were genetically correlated with PD risk and seven were validated in the PPMI cohort. Mendelian randomization analyses identified five proteins (GPNMB, GRN, HLA-DQA2, LCT, and CD68) causal for PD and nominate one more (ITGB2). These 26 proteins were enriched for microglia-specific proteins and trafficking pathways (both lysosome and intracellular). This study not only demonstrates that protein phenome-wide association studies (PheWAS) and trans-protein quantitative trail loci (pQTL) analyses are powerful for identifying novel protein interactions in an unbiased manner, but also that LRRK2 is linked with the regulation of PD-associated proteins that are enriched in microglial cells and specific lysosomal pathways.

Brain inflammation contributes to the pathogenesis of neurodegenerative diseases such as Alzheimers disease (AD). Glucose hypometabolism and glial activation are pathological features seen in AD brains; however, the connection between the two is not fully understood. Using a Drosophila model of AD, we identified that glucose metabolism in glia plays a critical role in neuroinflammation under disease conditions. Expression of human Tau in the retinal cells, including photoreceptor neurons and pigment glia, causes photoreceptor degeneration accompanied by inclusion formation and swelling of the lamina cortex. We found that inclusions are formed by glial phagocytosis, and swelling of the laminal cortex correlates with the expression of antimicrobial peptides. Co-expression of human glucose transporter 3 (GLUT3) with Tau in the retina does not affect tau levels but suppresses these inflammatory responses and photoreceptor degeneration. We also found that expression of GLUT3, specifically in the pigment glia, is sufficient to suppress inflammatory phenotypes and mitigate photoreceptor degeneration in the tau-expressing retina. Our results suggest that glial glucose metabolism contributes to inflammatory responses and neurodegeneration in tauopathy. Summary StatementGlucose uptake into pigment glia suppresses inflammatory responses and photoreceptor degeneration in the fly model of tauopathy.

Hexanucleotide repeat expansion (HRE) in the non-coding region of the gene C9orf72 is the most prevalent mutation in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The C9orf72 HRE contributes to neuron degeneration in ALS/FTD through both cell- autonomous mechanisms and non-cell autonomous disease processes involving glial cells such as microglia. The molecular mechanisms underlying the contribution of C9orf72-HRE microglia to neuron death in ALS/FTD remain to be fully elucidated. In this study, we generated microglia from human C9orf72-HRE and isogenic iPSCs using three different microglia derivation methods. RNA sequencing analysis reveals a cell-autonomous dysregulation of extracellular matrix (ECM) genes and genes involved in pathways underlying inflammasome activation in C9orf72-HRE microglia. In agreement with elevated expression of inflammasome components, conditioned media from C9orf72-HRE microglia enhance the death of C9orf72- HRE motor neurons implicating microglia-secreted molecules in non-cell autonomous mechanisms of C9orf72 HRE pathology. These findings suggest that aberrant activation of inflammasome-mediated mechanisms in C9orf72-HRE microglia results in a pro-inflammatory phenotype that contributes to non-cell autonomous mechanisms of motor neuron degeneration in ALS/FTD. SummaryThis study describes phenotypic alterations in C9orf72-ALS/FTD microglia implicating extracellular matrix remodeling and inflammasome activation in microglia-mediated neurodegeneration in ALS/FTD.

TAR DNA binding protein 43 (TDP-43) is the core pathogenic protein across a spectrum of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) cases, but its pathological deposition shows regional selectivity, with abundant TDP-43 positive aggregates in the frontal cortex and spinal cord, and a much lower burden in the cerebellum. In health, TDP-43 expression in the cerebellum is higher than in cortex, hence what underpins this differential vulnerability to TDP-43 aggregation is unclear. Here we demonstrate that in healthy C57Bl/6J mice not only is TDP-43 expression higher in the cerebellum than the cortex, but that this expression difference is driven primarily by differences in cytoplasmic load. Mass spectrometry analysis of TDP-43 pull downs from the cortex and cerebellum of healthy mice identified TDP-43 interactors across a number of core functional pathways, with numerous differences between the two brain regions. Data are available via ProteomeXchange with identifier PXD062532. Notably, there were more interactors identified in both nuclear and cytoplasmic fractions within the cortex than the cerebellum. Putative interactions with four core paraspeckle proteins; SFPQ, NONO, FUS and PSPC1 were confirmed using immunoprecipitation and western blot analysis. Follow up validation using proximity ligation assay showed abundant perinuclear cytoplasmic interactions between TDP-43 and all four paraspeckle proteins in both large motor cortex neurons and purkinje cells, with significantly reduced nuclear interactions detected in the motor cortex for SFPQ, FUS and PSPC1. These findings suggest that TDP-43-protein interactions markedly differ between the TDP-43 pathogenesis vulnerable cortex and relatively resistant cerebellum and exploring these differences may yield new insight into disease mechanisms within ALS/FTD.

Loss-of-function mutations in Progranulin (GRN) cause neuronal ceroid lipofuscinosis (NCL) and hereditary frontotemporal dementia, presumably through lysosomal dysfunction. Lysosomes are key metabolic organelles whose functions vary widely depending on their cell type of origin. These functional variations are driven by the lysosomal proteome, yet whether progranulin deficiency alters the lysosomal composition of the mammalian brain in a cell type-specific manner has not been tested. To answer this unknown, we used cell type-specific LysoIP to perform tandem-mass-tag mass-spectrometry and detected distinct aberrant proteomic signatures in progranulin-deficient astrocytes, neurons, and microglia, indicating cell type-specific dysregulation of key lysosomal proteins with crucial functions in sphingolipid metabolism and lysosome organization. These proteins markedly differed from progranulin-deficient RNAseq data sets, suggesting progranulin regulates lysosomal composition through post-translational mechanisms including the sorting of nascent proteins to the lysosome. Validation experiments confirmed that Mfsd8 and Ppt1, proteins whose mutations on their own cause NCL, were essentially absent from progranulin-deficient neuronal and microglial lysosomes, respectively. Our findings demonstrate the protein composition of lysosomes are uniquely sensitive to progranulin deficiency in a cell type-specific manner and that progranulin may function as an essential hub for endolysosomal homeostasis.

TDP-43 pathology is a defining feature of Limbic-Predominant Age-Related TDP-43 Encephalopathy neuropathologic change (LATE-NC) and is frequently comorbid with Alzheimers disease neuropathologic change (ADNC). However, the molecular consequences of co-occurring LATE-NC and ADNC pathology (TDP-43, {beta}-amyloid, and tau protein pathologies) remain unclear. Here, we conducted a comparative biochemical, molecular, and proteomic analysis of hippocampal tissue from 90 individuals spanning control, LATE-NC, ADNC, and ADNC+LATE-NC groups to assess the impact of cryptic exon (CE) inclusion, phosphorylated TDP-43 pathology (pTDP-43), and AD-related pathologies ({beta}-amyloid, and tau) on the proteome. ADNC+LATE-NC cases exhibited the highest burden of CE inclusion as quantified by measuring the levels of known TDP-43 regulated CEs within eight transcripts: STMN2, UNC13A, ELAVL3, KALRN, ARHGAP32, CAMK2B, PFKP, and SYT7. While CE levels correlated with pTDP-43 pathology, they were more strongly correlated with each other, suggesting that the molecular signature of CE inclusion may serve as a more sensitive measure of TDP-43 dysfunction than pTDP-43 pathology alone. Unbiased classification based on the relative abundance of these eight CEs stratified individual cases into low, intermediate, and high CE burden subtypes, largely independent of {beta}-amyloid and tau pathology. Proteome-wide correlation analysis revealed a bias toward reduced protein levels from genes harboring TDP-43-regulated CEs in cases with high cumulative CE burden. Notably, proteins significantly decreased under high CE burden included canonical STMN2, ELAVL3, and KALRN, as well as kinesin proteins that are genetically associated with amyotrophic lateral sclerosis. Co-expression network analysis identified both shared and distinct biological processes across CE subtypes and pathways associated with pTDP-43, tau, {beta}-amyloid pathologies, and CE accumulation in the hippocampus. Protein modules associated with TDP-43 loss of function were prioritized by integrating proteomic data from TDP-43-depleted human neurons with the hippocampal co-expression network. Specifically, we observed decreased endosomal vesicle, microtubule-binding, and synaptic modules, alongside an increase in RNA-binding modules. These results provide new insights into the proteomic impact of CE burden across the spectrum of LATE and AD pathological severity, highlighting the molecular consequences of TDP-43 dysfunction in neurodegenerative disease

BackgroundDisease-modifying therapies for tauopathies like Alzheimers disease have targeted Tau hyperphosphorylation and aggregation, as both pathological manifestations are implicated in Tau-mediated toxicity. However, the relative contributions of these pathology-linked changes to Tau neurotoxicity remain unclear. MethodsLeveraging the genetic tractability of Drosophila, we generated multiple inducible human Tau transgenes with altered phosphorylation status and/or aggregation propensity. Their individual and combined impact was tested in vivo by quantifying Tau accumulation and neurodegenerative phenotypes in the aging fly nervous system. ResultsWe report that phospho-mimicking Tau (hTau2N4RE14) induced profound neurodegeneration, supporting a neurotoxic role for phosphorylation. However, when we rendered hTau2N4RE14 aggregation incompetent, by deleting the 306VQIVYK311 motif in the microtubule-binding region, neurotoxicity was abolished. Moreover, a peptide inhibitor targeting this motif efficaciously reduced Tau toxicity in aging Drosophila. ConclusionNeurodegeneration mediated by Tau hyperphosphorylation is gated via at least one aggregation-mediating motif on the protein. This highlights the primacy of blocking Tau aggregation in therapy, perhaps without the need to clear phosphorylated species.

Progranulin (PGRN) deficiency is linked to neurodegenerative diseases including frontotemporal dementia, Alzheimers disease, Parkinsons disease, and neuronal ceroid lipofuscinosis. Proper PGRN levels are critical to maintain brain health and neuronal survival, however the function of PGRN is not well understood. PGRN is composed of 7.5 tandem repeat domains, called granulins, and is proteolytically processed into individual granulins inside the lysosome. The neuroprotective effects of full-length PGRN are well-documented, but the role of granulins is still unclear. Here we report, for the first time, that expression of single granulins is sufficient to rescue the full spectrum of disease pathology in mice with complete PGRN deficiency (Grn-/-). Specifically, rAAV delivery of either human granulin-2 or granulin-4 to Grn-/- mouse brain ameliorates lysosome dysfunction, lipid dysregulation, microgliosis, and lipofuscinosis similar to full-length PGRN. These findings support the idea that individual granulins are the functional units of PGRN, likely mediate neuroprotection within the lysosome, and highlight their importance for developing therapeutics to treat FTD-GRN and other neurodegenerative diseases.

GRN haploinsufficiency causes frontotemporal lobar degeneration and results in microglial hyperactivation, lysosomal dysfunction and TDP-43 deposition. To understand the contribution of microglial hyperactivation to pathology we evaluated genetic and pharmacological approaches suppressing TREM2 dependent transition of microglia from a homeostatic to a disease associated state. Trem2 deficiency in Grn KO mice led to a reduction of microglia activation. To explore antibody-mediated pharmacological modulation of TREM2-dependent microglial states, we identified antagonistic TREM2 antibodies. Treatment of macrophages from GRN-FTLD patients with these antibodies allowed a complete rescue of elevated levels of TREM2 together with increased shedding and reduction of TREM2 signaling. Furthermore, antibody-treated PGRN deficient hiMGL showed dampened microglial hyperactivation, reduced TREM2 signaling and phagocytic activity, however, lack of rescue of lysosomal dysfunction. Similarly, lysosomal dysfunction, lipid dysregulation and glucose hypometabolism of Grn KO mice were not rescued by TREM2 ablation. Furthermore, NfL, a biomarker for neurodegeneration, was elevated in the Grn/Trem2 KO. These findings suggest that microglia hyperactivation is not necessarily contributing to neurotoxicity, and instead demonstrates that TREM2 exhibits neuroprotective potential in this model.

TDP-43 proteinopathy is a neuropathological hallmark of nearly all amyotrophic lateral sclerosis (ALS) and approximately half of frontotemporal dementia (FTD) cases. Nuclear loss of TDP-43 leads to widespread RNA misprocessing, such as the inclusion of cryptic exons that are no longer repressed by TDP-43. Notably, in-frame cryptic exons encode novel cryptic peptides that can be detected in biofluids, including that found in the HDGFL2 transcript. Here, we quantified HDGFL2 cryptic peptide and neurofilament light chain (NfL) in paired cerebrospinal fluid (CSF) and plasma samples from ALS and FTD patients. Cryptic HDGFL2 peptide was detected in the CSF of ALS patients, whereas no significant differences were observed between genetic and behavioral FTD subgroups. In contrast, NfL levels were elevated in both ALS and FTD, although this biomarker does not reflect TDP-43 pathology. Notably, NfL:HDGFL2 cryptic peptide ratio outperformed either marker alone in discriminating ALS and FTD cases from controls, achieving high specificity. Moreover, this ratio correlated with disease progression in ALS, suggesting added prognostic value. Collectively, our findings support the NfL:HDGFL2 cryptic peptide ratio as a promising fluid biomarker that integrates neurodegeneration with TDP-43 dysfunction, potentially improving diagnostic accuracy, disease stratification, and longitudinal monitoring in TDP-43-associated neurodegenerative disorders.

BackgroundNeuroinflammation and pathologic -synuclein (-syn) aggregation cooperate to drive dopaminergic neurodegeneration in Parkinsons disease, but the glial receptors that couple extracellular -syn to inflammatory cascades remain incompletely defined. Microglia express higher levels of lymphocyte activation gene 3 (Lag3) than neurons, yet the contribution of microglial Lag3 to -syn recognition, glial crosstalk, and neurodegeneration is unknown. MethodsBiochemical binding assays, live-cell imaging, cytokine profiling, and neuron-microglia-astrocyte co-culture paradigms were used to define Lag3-dependent -syn preformed fibril (PFF) binding, uptake, and microglial activation. To interrogate in vivo function, microglia-specific Lag3 conditional knockout mice (Lag3L/L-Cx3cr1CreER) and littermate controls received unilateral intrastriatal -syn PFF injections, followed by histological, biochemical, and behavioral assessments of -syn pathology, gliosis, nigrostriatal integrity, and motor performance. Results-syn PFFs bound microglial Lag3 with high specificity and nanomolar affinity and required Lag3 for efficient fibril internalization and induction of proinflammatory cytokines. Microglial Lag3 deficiency markedly blunted -syn PFF-evoked microglial activation, prevented cytokine-driven conversion of astrocytes into neurotoxic reactive A1 astrocytes, and abolished astrocyte-dependent neuronal death in vitro. In vivo, microglia-specific Lag3 deletion reduced cortical, striatal, and substantia nigra pS129 -syn pathology, suppressed microgliosis and A1 astrocyte induction, preserved substantia nigra dopaminergic neurons and striatal dopamine transporter/tyrosine hydroxylase expression, and ameliorated -syn PFF-induced motor deficits. ConclusionsThis study identifies microglial Lag3 as a key receptor linking extracellular -syn PFF recognition to inflammatory amplification, neurotoxic reactive A1 astrocyte conversion, and dopaminergic neurodegeneration. Together with prior work on neuronal Lag3, these findings support a cell-type-specific dual-axis model in which neuronal Lag3 mediates -syn propagation while microglial Lag3 drives glia-dependent neurotoxicity, positioning Lag3 as a promising precision therapeutic target in -synucleinopathies.

Comorbid proteinopathies are observed in many neurodegenerative disorders including Alzheimers disease (AD), increase with age, and influence clinical outcomes, yet the mechanisms remain ill-defined. Here, we show that reduction of progranulin (PGRN), a lysosomal protein associated with TDP-43 proteinopathy, also increases tau inclusions, causes concomitant accumulation of -synuclein and worsens mortality and disinhibited behaviors in tauopathy mice. The increased inclusions paradoxically protect against spatial memory deficit and hippocampal neurodegeneration. PGRN reduction with tau pathology attenuates activity of {beta}-glucocerebrosidase (GCase), a protein previously associated with synucleinopathy, while increasing glucosylceramide (GlcCer)-positive tau inclusions. In neuronal culture, GCase inhibition enhances tau aggregation induced by AD-tau. Furthermore, purified GlcCer directly promotes tau aggregation in vitro. Neurofibrillary tangles in human tauopathies are also GlcCer-immunoreactive. Thus, in addition to TDP-43, PGRN regulates tau- and synucleinopathies via GCase and GlcCer. A lysosomal PGRN-GCase pathway may be a common therapeutic target for age-related comorbid proteinopathies.