Molecular Neurodegeneration

ObjectiveTDP-43 proteinopathies, including amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration with TDP-43 (FTLD-TDP), and limbic-predominant age-related TDP-43 encephalopathy, encompass a spectrum of clinical and neuropathological traits. Despite mounting evidence for shared genetic risk across TDP-43 proteinopathies, the modifiers of individual-level traits are unknown. We aimed to identify polygenic contributions to trait heterogeneity across TDP-43 proteinopathies. MethodsWe used weighted correlation analysis of GWAS summary statistics for ALS, FTLD-TDP, and hippocampal sclerosis of aging (HS-Aging) to identify data-driven clusters of highly correlated single nucleotide polymorphisms (SNPs). We performed gene ontology enrichment analysis for each identified cluster. We derived cluster-specific polygenic scores and evaluated their association with clinical and neuropathological traits in an independently evaluated sample of individuals who met neuropathological and/or genetic criteria for FTLD-TDP or ALS (n=260). ResultsWe identified 5 distinct data-driven clusters, including 3 GWAS phenotype-specific clusters (FTLD-TDP, ALS, HS-Aging) and 2 clusters representing the overlap between a pair of GWAS phenotypes (ALS-FTLD and FTLD-HS). Pathway analysis revealed biologically meaningful associations including distinct GWAS phenotype-specific processes within clusters. Cluster-specific ALS and FTLD-TDP polygenic risk each associated with individual-level clinical traits, even within the context of autosomal dominant mutation carriers, where higher ALS polygenic risk associated with neuromuscular impairment and higher FTLD-TDP polygenic risk associated with cognitive-behavioral impairment. Moreover, higher FTLD-TDP polygenic risk associated with higher TDP-43 burden within characteristic FTLD-TDP brain regions. InterpretationWe suggest that there are polygenic modifiers of clinical and neuropathological traits across TDP-43 proteinopathies that may contribute to individual-level differences, including likelihood for developing FTLD or ALS.

Synucleinopathies, including Parkinsons disease, are neurodegenerative diseases characterized by intracellular inclusions containing the amyloidogenic protein alpha-synuclein. While classically considered to be brain disorders, increasing evidence suggests involvement of the gut, with alpha-synuclein aggregates potentially propagating to the brain via the vagus nerve. Evidence also suggests that the vermiform appendix is particularly susceptible to alpha-synuclein aggregation, and appendectomy impacts the onset of Parkinsons disease. However, the mechanisms underlying the aggregation of alpha-synuclein in the vermiform appendix remains poorly understood. To explore this, we assessed aggregation properties in postmortem appendix tissues from healthy controls and synucleinopathy patients using the alpha-synuclein seed amplification assay (alpha-synuclein-SAA) and performed total RNA sequencing alongside differential bisulfite-hybridization-based DNA methylation analysis in the same tissues to investigate the molecular underpinnings. Moreover, we determined alpha-synuclein cleavage patterns by cataloging soluble alpha-synuclein proteoforms from postmortem substantia nigra and post-surgical appendix tissues using top-down mass spectrometry (TD-MS). Alpha-synuclein-SAA was positive in appendix samples for 68.75% of synucleinopathy patients and 6.6% of controls. Genomic profiling revealed dysregulated expression of genes linked to protein folding/degradation, immune/inflammatory responses, and ciliary dynamics in synucleinopathy appendix tissues. TD-MS identified 65 distinct alpha-synuclein proteoforms in the substantia nigra and appendix, with 9 unique to the appendix. Further, in silico modeling revealed higher aggregation propensity of alpha-synuclein proteoforms in the appendix versus substantia nigra. Together, our findings suggest that a tissue environment of alpha-synuclein dysproteostasis in the appendix has the potential to contribute to the development of synucleinopathies. One Sentence SummaryAppendixes from synucleinopathy patients show altered gene expression, unique -syn proteoforms, and higher aggregation propensity than substantia nigra.

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.

Progranulin (PGRN) deficiency is a common hallmark in frontotemporal dementia (FTD) patients with granulin (GRN) mutations (FTD-GRN). Previous studies by our group and others have observed that reduced PGRN perturbs lysosomal function and microglial activation, which is believed to accelerate neurodegeneration in FTD-GRN patients. Lysosomal function is intrinsically linked to lipid metabolism, and evidence suggests that GRN deficiency can alter lipid profiles in the brain. Unfortunately, studies to date have focused on whole brain or cortical extracts, limiting our ability to assess cell type-dependent changes in lipid metabolism under disease conditions. Here, we employed lipidomic analysis specifically within the pontine microglia of Grn knock-out (KO) mice, a cell population that was previously linked to disease phenotypes in this model. We observed a significant reduction in the endolysosomal lipid bis(monoacylglycero)phosphate (BMP) and the lipid metabolite phosphatidylethanolamine (PE); these microglial-specific lipid alterations mirror previous whole-brain findings, suggesting that similar changes may occur across multiple cell types in the brain. We also detected a significant increase in the myelin-composing factor galactosylceramide (GalCer), which may reflect an aberrant accumulation of myelin debris within microglia that arises due to defective lysosomal clearance. Notably, lipid perturbations were exacerbated with age within Grn KO microglia, suggesting that changes in lipid metabolism are both age- and genotype-dependent in this model. Together, our results support our hypothesis that PGRN acts as a master regulator of critical microglial processes - including lysosomal function, lipid metabolism, and the regulation of myelination - in an age-dependent manner.

Leucine-rich repeat kinase 2 (LRRK2) variants are the most common cause of inherited Parkinsons disease (PD), and the hyperactivity of the LRRK2 variants represent a validated drug target for PD. The penetration of common LRRK2 variants is incomplete, underscoring the need for molecular biomarkers that predict disease onset and guide therapeutics development. Here, we analyzed large datasets of cerebrospinal fluid (CSF) and urinary proteomics from the Parkinsons Progression Markers Initiative (PPMI) and identified distinct lysosomal and immune protein signatures as potential biomarkers for LRRK2-linked PD (LRRK2 PD). Longitudinal analysis revealed that levels of specific lysosomal and immune proteins remained elevated in CSF during the prodromal phase but declined following clinical symptom onset. Furthermore, examination of multiple brain cell types from Lrrk2 mutant mice carrying disease variant (G2019S) showed heightened secretion of lysosomal proteins in microglia and astrocytes, but not neurons, supporting a glial origin and intrinsic LRRK2 mutant activity responsible for the elevated CSF lysosomal proteins. Furthermore, proteomics analysis of urine from humanized LRRK2G2019Stransgenic mice identified lysosome and glycosphingolipid protein signatures shared with human LRRK2 PD patients. Collectively, our integrated proteomics reveals dynamic changes of functional biofluid signatures for LRRK2 PD, which enables the determination of biomarkers for early disease onset. The humanized LRRK2G2019S mice provide a valuable platform for biomarker refinement and therapeutic development. One Sentence SummaryIntegrated human and mouse proteomic analyses identify dynamic lysosomal and immune biofluid signatures, possibly of glial origin, as functional biomarkers of LRRK2-linked Parkinsons disease progression, supported by a novel humanized LRRK2G2019S mouse model that recapitulates key urinary biomarker profiles.

BackgroundNeuromelanin (NM) is a pigment that progressively accumulates with age in catecholaminergic neurons, particularly in the substantia nigra, ventral tegmental area, and locus coeruleus. These neuronal populations are especially vulnerable to degeneration in Parkinsons disease (PD). Elevated intracellular NM levels have been linked to neurodegeneration and PD-like phenotypes in experimental models. However, the molecular mechanisms underlying NM-induced pathology remain poorly understood, as human studies cannot disentangle the specific effects of NM accumulation from those of normal aging. MethodsWe performed transcriptomic microarray analysis on laser-captured catecholaminergic neurons and regions (substantia nigra, ventral tegmental area, locus coeruleus) from NM-producing transgenic mice (tgNM) and NM-free wild-type controls across different ages, and compared them to data from postmortem human brain tissue. One of the molecular targets identified, GPNMB, was validated in mouse and human tissue, and functionally tested in vivo. ResultsWe identified region- and age-dependent transcriptional changes associated with progressive NM accumulation. NM consistently upregulated neuroinflammatory pathways with enrichment of disease-associated microglial genes, while downregulating transcription, translation, and mitochondrial functions. Locus coeruleus exhibited the earliest and strongest transcriptional alterations, whereas substantia nigra and ventral tegmental area showed a later-onset, age-progressive transcriptional dysfunction. Neuron-specific analyses revealed that many changes originated within NM-containing neurons rather than being solely glial-driven. NM-driven transcriptional profiles in mice strongly correlated with postmortem data from PD patients, underscoring their translational relevance. Among molecular targets, the glycoprotein GPNMB was consistently upregulated in NM-containing neurons and validated at RNA and protein levels in both NM-producing transgenic mice and human PD brains. Functional experiments demonstrated that GPNMB overexpression attenuated NM-linked dopaminergic neurodegeneration and improved motor performance in mice. ConclusionThis study provides a comprehensive in vivo characterization of NM-specific transcriptomic changes in catecholaminergic neurons, showing that NM accumulation drives neuroinflammatory and neurodegenerative programs. Our results support that the neuroinflammatory changes observed in tgNM mice and in human PD represent early pathological events that precede overt neurodegeneration. The disease-associated gene GPNMB emerged as a conserved NM-induced factor with protective properties, highlighting its potential as a therapeutic target in PD and aging-related neurodegeneration.

Chronic neurodegenerative diseases, such as tauopathies, cause major metabolic changes in the brain affecting gene expression at both the transcriptional and translational levels. Our understanding of how regulation of translation changes with disease has focused on mRNA and its translational regulatory factors, RNA binding proteins, and microRNAs, despite clear evidence for translational and post-translational dysfunction in ADRD and tauopathies. The neurobiology of tRNA has only recently begun to be studied, but the impact of chronic neurodegenerative diseases on tRNA biology and translational dysfunction is largely unknown. We have previously shown that the tRNA pool and tRNA modifications behave as a system to regulate the cellular stress response by undergoing stress-specific reprogramming and causing selective translation of mRNAs from codon-biased stress response genes. Here we tested this stress-induced tRNA reprogramming and codon-biased translation system in the response to mutant tau expression by performing mass spectrometric quantification of [~]8500 proteins and 49 tRNA modifications, AQRNA-seq analysis of 222 cytosolic and mitochondrial tRNAs and other small RNAs, and informatic analysis of codon usage patterns in >23,000 protein-coding genes. Analysis of these datasets revealed that aging and tauopathy elicit major adaptation of the tRNA transcriptome and epitranscriptome as well as corresponding evidence of a program of translation of families of codon-biased genes for aging and disease responsive proteins. The mitochondrial tRNA transcriptome showed a strong response to aging and disease with 21 of the 22 mt-tRNAs showing age and disease-linked increases in expression, accompanied by mitochondria-specific modifications such as ms2i6A and f5C. Surprisingly, there were few significant changes in the 203 cytosolic tRNA isodecoders. However, a 10-fold increase tRNA isodecoder tRNA-Arg-TCT-5-1 was accompanied by increased translation of proteins encoded by genes highly enriched in its AGA cognate codon. These changes in tRNA biology are mirrored by strongly biased use of synonymous codons among the most highly upregulated and downregulated proteins in the P301S MAPT mice. Taken together these findings suggest the aging and disease brain produces an integrated response for translational control that is highly integrated with changes in tRNA biology.

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.

Microglia are central regulators of neuroinflammation in Alzheimers disease (AD), yet how metabolic states modulate function remains unclear. Here we show that microglia from the APPNL-G-F mouse model revealed upregulation of glycolytic enzymes coinciding with onset of microglial activation. Surprisingly, this glycolytic shift occurred alongside reduced expression of glucose transporters, suggesting that extracellular glucose may not be the primary fuel source, implicating glycogenolysis as the potential metabolic driver. Consistent with this, significant microglial glycogen accumulation was noted in late disease, when cells exhibited features of metabolic exhaustion and functional impairment. Pharmacological inhibition of glycogenolysis blunted microglia responses to Abeta aggregates and markedly reduced Abeta uptake, confirming a functional role for glycogen metabolism in shaping microglial states. Together, these findings identify glycogen as a central regulator of microglial metabolic health and function, highlighting glycogen homeostasis as a potential therapeutic target for promoting Abeta clearance and preserving protective microglial functions in AD.

Neuronal dysfunction has been extensively studied as a central feature of neurodegenerative tauopathies. However, across neurodegenerative diseases, there is strong evidence for active involvement of immune cells like microglia in driving disease pathophysiology. Here, we demonstrate that tau mRNA and protein are expressed in microglia in human brains and in human induced pluripotent stem cell (iPSC)-derived microglia like cells (iMGLs). Using iMGLs harboring the MAPT IVS10+16 mutation and isogenic controls, we demonstrate that a tau mutation is sufficient to alter microglial transcriptional states. We discovered that MAPT IVS10+16 microglia exhibit cytoskeletal abnormalities, stalled phagocytosis, disrupted TREM2/TYROBP networks, and altered metabolism. Additionally, we found that secretory factors from MAPT IVS10+16 iMGLs impact neuronal health, reducing synaptic density in neurons. Key features observed in vitro were recapitulated in human brain tissue and cerebrospinal fluid from MAPT mutations carriers. Together, our findings that MAPT IVS10+16 drives cell-intrinsic dysfunction in microglia that impacts neuronal health has major implications for development of therapeutic strategies.

Microglial phagocytosis is an energetically demanding process that plays a critical role in the removal of toxic aggregates of beta amyloid (A{beta}) in Alzheimers disease (AD). Recent evidence indicates that metabolic programming may breakdown in microglia in AD, thereby disrupting this important protective function. The mechanisms coordinating mitochondrial metabolism to fuel phagocytosis in microglia remain poorly understood, however. Here we demonstrate that mitochondrial displacement of the glucose metabolizing enzyme, hexokinase-II (HK) regulates microglial metabolism and phagocytosis, and that deletion of the translocator protein (TSPO) inhibits this. TSPO is a PET-visible inflammatory biomarker and therapeutic target in AD, previously shown to regulate microglial metabolism via an unknown mechanism. Using RNAseq and proteomic analyses, we found TSPO function in the brain to be linked with the regulation of mitochondrial bioenergetics, lipid metabolism and phagocytosis. In cultured microglia, TSPO deletion was associated with elevated mitochondrial recruitment of HK, which was associated with a switch to non-oxidative glucose metabolism, reduced mitochondrial energy production, lipid storage and impaired phagocytosis. Consistent with in vitro findings, TSPO expression was also associated with phagocytic microglia in both AD brain and AD mice. Conversely, TSPO deletion in AD mice reduced phagocytic microglia and exacerbated amyloid accumulation. Based on these findings we propose that microglial TSPO functions as an immunometabolic brake via regulation of mitochondrial HK recruitment, preventing hyperglycolysis and promoting phagocytosis in AD. Further, we demonstrate that targeting mitochondrial HK may offer a novel immunotherapeutic approach to promote microglial phagocytosis in AD.

Brain-derived extracellular vesicles (EVs) play an active role in Alzheimers disease (AD), relaying important physiological information about their host tissues. Circulating EVs are protected from degradation, making them attractive AD biomarkers. However, it is unclear how circulating EVs relate to EVs isolated from disease-vulnerable brain regions. We developed a novel method for collecting EVs from the hippocampal interstitial fluid (ISF) of live mice. EVs (EVISF) were isolated via ultracentrifugation and characterized by nanoparticle tracking analysis, immunogold labeling, and flow cytometry. Mass spectrometry and proteomic analyses were performed on EVISF cargo. EVISF were 40-150 nm in size and expressed CD63, CD9, and CD81. Using a model of cerebral amyloidosis (e.g. APPswe,PSEN1dE9 mice), we found protein concentration increased but protein diversity decreased with A{beta} deposition. Genotype, age, and A{beta} deposition modulated proteostasis- and immunometabolic-related pathways. Changes in the microglial EVISF proteome were sexually dimorphic and associated with a differential response of plaque associated microglia. We found that female APP/PS1 mice have more amyloid plaques, less plaque associated microglia, and a less robust- and diverse-EVISF microglial proteome. Thus, in vivo microdialysis is a novel technique for collecting EVISF and offers a unique opportunity to explore the role of EVs in AD. Graphical AbstractHippocampal EVISF response to amyloid beta (A{beta}) is sexually dimorphic and related to the microglial EVISF proteome. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/532133v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1a275d6org.highwire.dtl.DTLVardef@e63da5org.highwire.dtl.DTLVardef@1d93cecorg.highwire.dtl.DTLVardef@12ed0db_HPS_FORMAT_FIGEXP M_FIG C_FIG

The microtubule-associated protein tau aggregates in multiple neurodegenerative diseases, causing inflammation and changing the inflammatory signature of microglia by unknown mechanisms. We have shown that microglia phagocytose live neurons containing tau aggregates cultured from P301S tau transgenic mice due to neuronal tau aggregate-induced exposure of the eat me signal phosphatidylserine. Here we show that after phagocytosis, microglia become hypophagocytic while releasing seed-competent insoluble tau aggregates. These microglia activate acidic {beta}-galactosidase, and release senescence-associated cytokines and matrix remodeling enzymes alongside tau, indicating a senescent phenotype. In particular, the marked NF{kappa}B-induced activation of matrix metalloprotease 3 (MMP3/stromelysinl) was replicated in the brains of P301S mutant tau transgenic mice, and in human brains from tauopathy patients. These data show that microglia that have been activated to ingest live neurons with tau aggregates behave hormetically, becoming hypofunctional while acting as vectors of tau aggregate spreading.

Microglia play diverse pathophysiological roles in Alzheimers disease (AD), with genetic susceptibility factors skewing microglial cell function to influence AD risk. CD33 is an immunomodulatory receptor associated with AD susceptibility through a single nucleotide polymorphism that modulates mRNA splicing, skewing protein expression from a long protein isoform (CD33M) to a short isoform (CD33m). Understanding how human CD33 isoforms differentially impact microglial cell function in vivo has been challenging due to functional divergence of CD33 between mice and humans. We address this challenge by studying transgenic mice expressing either of the human CD33 isoforms crossed with the 5XFAD mouse model of amyloidosis and find that human CD33 isoforms have opposing effects on the response of microglia to amyloid-{beta} (A{beta}) deposition. Mice expressing CD33M have increased A{beta} levels, mo7re diffuse plaques, fewer disease-associated microglia, and more dystrophic neurites compared to control 5XFAD mice. Conversely, CD33m promotes plaque compaction and microglia-plaque contacts, and minimizes neuritic plaque pathology, highlighting an AD protective role for this isoform. Protective phenotypes driven by CD33m are detected at an earlier timepoint compared to the more aggressive pathology in CD33M mice that appears at a later timepoint, suggesting that CD33m has a more prominent impact on microglia cell function at earlier stages of disease progression. In addition to divergent roles in modulating phagocytosis, scRNAseq and proteomics analyses demonstrate that CD33m+ microglia upregulate nestin, an intermediate filament involved in cell migration, at plaque contact sites. Overall, our work provides new functional insights into how CD33, as a top genetic susceptibility factor for AD, modulates microglial cell function.

Alzheimer's disease (AD) drives widespread molecular changes beyond the brain that are increasingly detectable in plasma. To map plasma proteomic signatures of AD in a broadly unbiased manner with high depth and reproducibility, we profiled plasma from 214 individuals spanning cognitively normal controls, mild cognitive impairment, and AD using microbead-based enrichment and data-independent acquisition mass spectrometry (DIA-MS). We reliably quantified 5,823 proteins across samples, and network analysis identified 29 plasma modules enriched for functions related to lipid metabolism, extracellular matrix remodeling, immune signaling, mitochondrial function, and proteostasis. Several modules were associated with cognition, APOE4, sex, race, and cerebrospinal fluid (CSF) amyloid and tau biomarkers. Among 129 individuals with paired CSF and plasma biomarker measurements, over 1,500 proteins differed between CSF biomarker positive and negative groups, including amyloid-linked matrisome proteins such as SMOC1, FRZB, SPON1 and CTHRC1. A 10-protein plasma panel classified CSF biomarker positivity with performance similar to plasma pTau217 (AUC = 0.91), and combining both improved accuracy (AUC = 0.99). Integration with a human brain proteomic network revealed that two-thirds of plasma modules were preserved in brain, with many AD-altered modules changing concordantly across compartments. This study establishes a scalable DIA-MS plasma proteomics platform that captures systemic and brain-linked AD biology and identifies complementary biomarkers beyond phosphorylated tau.

Frontotemporal lobar degeneration (FTLD) and Alzheimers disease (AD) differ in their clinical features and genetic etiologies but share progressive cognitive decline. Emerging evidence implicates lipid dysregulation in neurodegeneration, but its extent across FTLD subtypes and how it compares to AD are unclear. Here, we performed integrated lipidomic and proteomic analyses of matched frontal (disease-vulnerable) and occipital (relatively spared) post-mortem cortices from individuals with genetic and sporadic FTLD-TDP, FTLD-tau (Picks disease, PiD), AD, and controls. FTLD and AD exhibited convergent lipid alterations, including reduced levels of cardiolipins and phosphatidylethanolamines, alongside increased gangliosides, diacylglycerols, cholesterol esters, acylcarnitines, and coenzyme Q, with generally greater changes in FTLD frontal cortex. FTLD displayed additional alterations, including reductions in bis(monoacylglycerol)phosphate, ceramides, phosphatidylserines, phosphatidylinositols, and sulfatides. These lipid changes were accompanied by proteomic alterations involving lysosomal proteins, phospholipases, phospholipid remodeling enzymes, and fatty acid oxidation pathways. Although lipidomic and proteomic signatures were broadly shared across FTLD subtypes, GRN associated FTLD-TDP and PiD showed the most extensive alterations. Triglycerides were selectively reduced in PiD in association with decreased DGAT1 expression, whereas cholesterol esters were elevated across all subtypes except C9orf72 associated FTLD-TDP. These findings identify shared disruptions in lipid homeostasis and lysosomal lipid metabolism across FTLD and AD, highlighting convergent metabolic pathways underlying neurodegeneration.

Synucleinopathies are neurodegenerative diseases characterized by pathological inclusions called "Lewy pathology" (LP) that consist of aggregated alpha-synuclein predominantly phosphorylated at serine 129 (PSER129). Despite the importance for understanding disease, little is known about the endogenous function of PSER129 or why it accumulates in disease. Here we conducted several observational studies using a sensitive tyramide signal amplification (TSA) technique to determine PSER129 distribution and function in the non-diseased mammalian brain. In wild-type non-diseased mice, PSER129 was detected in the olfactory bulb (OB) and several brain regions across the neuroaxis (i.e., OB to brain stem). In contrast, PSER129 immunoreactivity was not observed in any brain region of alpha-synuclein knockout mice. We found evidence of PSER129 positive structures in OB mitral cells of non-diseased mice, rats, non-human primates, and healthy humans. Using TSA multiplex fluorescent labeling we show that PSER129 positive punctate structures occur within inactive (i.e., cfos negative) T-box transcription factor 21 (TBX21) positive mitral cells and PSER129 within these cells was spatially associated with PK-resistant alpha-synuclein. Ubiquitin was found in PSER129 mitral cells but was not closely associated with PSER129. Biotinylation by antigen recognition (BAR) identified 125 PSER129-interacting proteins in the OB of healthy mice, which were significantly enriched for presynaptic vesicle trafficking/recycling, SNARE, fatty acid oxidation, oxidative phosphorylation, and RNA binding. TSA multiplex labeling confirmed the physical association of BAR identified protein Ywhag with PSER129 in the OB and in other regions across the neuroaxis. We conclude that PSER129 accumulates in mitral cells of the healthy OB as part of alpha-synuclein normal cellular functions. Incidental LP has been reported in the OB, and therefore we speculate that for synucleinopathies either; the disease processes begin locally in OB mitral cells or a systemic disease process is most apparent in the OB because the natural tendency to accumulate PSER129. Significance StatementMultiple lines of evidence have suggested that the disease process in some synucleinopathies begins in the olfactory bulb. Here we demonstrated that disease-associated phosphorylated alpha-synuclein preferentially occurs in mitral cells of the healthy mammalian olfactory bulb. We identified the protein interactome of phosphorylated alpha-synuclein in the healthy mouse olfactory bulb and established phosphorylated alpha-synuclein associates with presynaptic glutamatergic vesicles, SNARE machinery, and RNA metabolism machinery. Our data implicates olfactory bulb mitral cells in synucleinopathy pathogenesis. These findings advance our understanding of synucleinopathy disease origins and set the stage for new experimental models to interrogate the pathogenesis of synucleinopathies.

Cerebral amyloid angiopathy (CAA), a major vascular contributor to cognitive decline, is present in 85-95% of Alzheimers disease (AD) patients. Despite its high prevalence, the mechanisms by which CAA contributes to neurodegeneration remain poorly understood. Triggering receptor expressed on myeloid cells 2 (TREM2), an innate immune receptor expressed exclusively by microglia, regulates activation, phagocytosis, and amyloid clearance, thereby shaping neuroinflammation. Loss-of-function mutations in TREM2 markedly increase AD risk, but its role in CAA pathology remains unknown. To investigate this, we crossed the Familial Danish Dementia (Tg-FDD) mouse model, which accumulates robust vascular amyloid, with TREM2 knockout (TREM2KO) mice to generate Tg-FDD/TREM2KO animals. Histological and transcriptomic analyses revealed region-specific effects of TREM2 deficiency. In the cortex, TREM2 loss markedly reduced vascular amyloid deposition, accompanied by decreased tau pathology. In contrast, in the cerebellum, TREM2 deletion exacerbated vascular amyloid accumulation, promoted astrogliosis, and enhanced tau pathology. Transcriptomic profiling further identified distinct neuroinflammatory signatures between cortex and cerebellum, particularly in cytokine signaling, matrix remodeling, and lipid metabolism. Together, these findings demonstrate that TREM2 deficiency leads to region-specific effects on CAA, revealing extensive regional variability in vascular amyloid pathology and underscoring the importance of considering these differences when developing TREM2-based therapies.

BackgroundProcesses that drive Alzheimers disease pathogenesis have long been considered to occur within the central nervous system, yet recent studies have bolstered the possibility that changes in the periphery may be relevant to the disease process. Accumulating evidence has suggested that proteins changing in the blood may be reliable indicators of disease within the brain. Recent advances in geroscience have identified potential mechanisms of blood-brain communication that modulate brain function in ways that could be harnessed for therapy. While blood-borne proteins associated with either youth or old age have been targeted to restore function to the aged brain, it remains unclear whether other dysfunctional systemic states can be exploited for similar benefits. Here we investigate whether APOE allelic variation or presence of brain amyloid are associated with distinct proteomic changes within the systemic environment and what molecular processes are associated with these changes. MethodsUsing the SOMAscan assay, we measured 1,305 plasma proteins from 53 homozygous APOE3 and APOE4 subjects (mean age = 68 years; minimum = 54 years) who exhibited no cognitive impairment, some of whom can be categorized as harboring cerebral amyloid based on cerebrospinal fluid A{beta}42 measurements. Using the Dream R package for linear mixed effects modeling, we investigated possible contributions of either the APOE-{varepsilon}4 allele or amyloid positivity to changes in the plasma proteome. Ontology-based pathway and module trait correlation analyses were performed to understand disrupted pathways that vary based on APOE genotype or amyloid positivity. ResultsWe found that expression of the APOE-{varepsilon}4 allele produced distinct changes in the composition of the plasma proteome. Using both pathway enrichment analysis and weighted gene co-expression network analysis, we found that plasma proteins associated with APOE4 expression were linked to pathways related to atherosclerosis, lipid transport, the extracellular matrix, and synaptogenesis signaling. Independent of APOE4, we found that cognitively normal, amyloid-positive subjects exhibit distinct plasma proteome signatures associated with pathways previously linked to AD pathology, relative to amyloid-negative controls. Harboring brain amyloid was associated with plasma proteomic changes linked to dysfunction in blood-brain barrier and other neural cell types. Our results indicate that changes in the plasma proteome are related to possession of AD risk alleles, as well as the presence of amyloid pathology in subjects prior to the onset of symptoms. This work highlights the possibility that pathways in the systemic environment in certain risk contexts may be plausible targets to explore for modulating disease.

Tau pathology is a central driver of neurodegeneration, yet the molecular mechanisms linking tau accumulation to neuroinflammation, metabolic failure, and white matter degeneration remain incompletely understood. Galectin-3 (Gal3) is an inflammation-associated lectin expressed by activated microglia and has been implicated in neurodegenerative disease progression. Here, we investigated whether Gal3 modulates tau-driven pathology across cellular, molecular, and systems levels. Using the P301S tauopathy mouse model with genetic deletion of Gal3, we show that Gal3 loss robustly attenuates tau pathology across vulnerable brain regions, including cortex, hippocampus, and piriform-entorhinal cortex. Gal3 deletion reduced hyperphosphorylated and pathological tau species, normalized tau kinase signaling, and restored mitochondrial and vesicular trafficking pathways disrupted by tau accumulation. Proteomic and phosphoproteomic analyses revealed widespread normalization of tau-associated immune, metabolic, and trafficking pathways, with Tau-Gal3KO mice clustering closely with wild-type controls. In parallel, Gal3 deletion markedly reduced microglial activation and Gal3-positive inflammatory signatures, preserved white matter integrity, prevented axonal degeneration, and normalized oligodendrocyte and myelin abnormalities. Functionally, Gal3 deficiency enhanced microglial myelin phagocytosis and lysosomal degradation both in vitro and in vivo, suggesting improved clearance of myelin debris under inflammatory stress. Cell-type-specific analyses further revealed restoration of mitochondrial complex I subunit expression in both excitatory neurons and parvalbumin-positive interneurons. Importantly, translational studies in human iPSC-derived neurons demonstrated that extracellular Gal3 exacerbates tau hyperphosphorylation and aggregation following tau seeding, effects that were reversed by pharmacological Gal3 inhibition. Together, these findings identify Galectin-3 as a central upstream regulator linking tau pathology to neuroinflammation, proteomic dysregulation, mitochondrial dysfunction, and white matter degeneration. Targeting Gal3 represents a promising therapeutic strategy to mitigate tau-driven neurodegenerative processes.