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Abnormal tau protein accumulation selectively affects distinct brain regions and specific neuron and glia populations in tau-related dementias like Alzheimer's disease (AD), frontotemporal dementia (FTD, Pick's disease type), and Progressive supranuclear palsy (PSP). The regulatory mechanisms governing cell-type vulnerability remain unclear. In a cross-disorder single-nucleus analysis, we examined 663,896 nuclei, assessing chromatin accessibility in three brain regions (motor cortex, visual cortex and insular cortex) across PSP, AD, and FTD in 40 individuals. Integrating genetic data with single-nucleus RNA sequencing, we identified cell-type-specific cis-regulatory elements (CREs) influencing disease-associated transcriptional changes. Connecting GWAS signals to cell types enabled precise mapping of risk-associated genetic elements to disorder-specific gene regulatory events, accounting for dynamic gene regulation in the diseased brain. Disorder enriched CRE modules were primarily activated in PSP astrocytes and FTD microglia. PSP exhibited increased astrocyte involvement, identified by distinct changes in astrocytes (ast.C1). In contrast, FTD featured microglia with suppressed GRN expression (mg.C4). Differentially accessible gene regulatory elements in mg.C4 uniquely and significantly contributed to FTD risk heritability across microglia. Disease dynamic chromatin accessibility peaks and gene enhancer elements, especially in astrocytes, accounted for more AD and PSP heritability, while FTD heritability predominantly involved microglial upregulated peaks. This study enhances our understanding of disease heritability, reinforcing the multicellular model of neurodegeneration. Glial cell types interacting with disease-specific elements form disorder-specific networks, deepening knowledge of genomic and cellular mechanisms in tau-associated neurodegeneration.

Astrocytes play important roles in neurological disorders such as stroke, injury, and neurodegeneration. Most knowledge on astrocyte biology is based on studies of mouse models and the similarities and differences between human and mouse astrocytes are insufficiently characterized, presenting a barrier in translational research. Based on analyses of acutely purified astrocytes, serum-free cultures of primary astrocytes, and xenografted chimeric mice, we find extensive conservation in astrocytic gene expression between human and mouse samples. However, the genes involved in defense response and metabolism show species-specific differences. Human astrocytes exhibit greater susceptibility to oxidative stress than mouse astrocytes, due to differences in mitochondrial physiology and detoxification pathways. In addition, we find that mouse but not human astrocytes activate a molecular program for neural repair under hypoxia, whereas human but not mouse astrocytes activate the antigen presentation pathway under inflammatory conditions. Here, we show species-dependent properties of astrocytes, which can be informative for improving translation from mouse models to humans. Astrocytes are important players in brain development, homeostasis, and disease. Here, the authors compare the transcriptional profiles of human and mouse astrocytes. They report species-specific susceptibility to oxidative stress and response to hypoxic and inflammatory conditions.

Alzheimer’s disease (AD) is a multi-stage neurodegenerative disorder characterized by beta-amyloid accumulation, hyperphosphorylated Tau deposits, neurodegeneration, neuroinflammation, and cognitive impairment. Recent studies implicate CD8 T cells as neuroimmune responders to the accumulation of AD pathology in the brain and potential contributors to toxic neuroinflammation. However, more evidence is needed to understand lymphocytes in disease, including their functional states, molecular mediators, and interacting cell types in diseased brain tissue. The scarcity of lymphocytes in brain tissue samples has limited the unbiased profiling of disease-associated cell types, cell states, drug targets, and relationships to common AD genetic risk variants based on transcriptomic analyses. However, using recent large-scale, high-quality single-nuclear sequencing datasets from over 84 Alzheimer’s disease and control cases, we leverage single-nuclear RNAseq data from 800 lymphocytes collected from 70 individuals to complete unbiased molecular profiling. We demonstrate that effector memory CD8 T cells are the major lymphocyte subclass enriched in the brain tissues of individuals with AD dementia. We define disease-enriched interactions involving CD8 T cells and multiple brain cell subclasses including two distinct microglial disease states that correlate, respectively, to beta-amyloid and tau pathology. We find that beta-amyloid-associated microglia are a major hub of multicellular cross-talk gained in disease, including interactions involving both vulnerable neuronal subtypes and CD8 T cells. We reproduce prior reports that amyloid-response microglia are depleted in APOE4 carriers. Overall, these human-based studies provide additional support for the potential relevance of effector memory CD8 T cells as a lymphocyte population of interest in AD dementia and provide new candidate interacting partners and drug targets for further functional study.

O-GlcNAc glycosylation (or O-GlcNAcylation) is a dynamic, inducible posttranslational modification found on proteins associated with neurodegenerative diseases such as α-synuclein, amyloid precursor protein, and tau. Deletion of the O-GlcNAc transferase (ogt) gene responsible for the modification causes early postnatal lethality in mice, complicating efforts to study O-GlcNAcylation in mature neurons and to understand its roles in disease. Here, we report that forebrain-specific loss of OGT in adult mice leads to progressive neurodegeneration, including widespread neuronal cell death, neuroinflammation, increased production of hyperphosphorylated tau and amyloidogenic Aβ-peptides, and memory deficits. Furthermore, we show that human cortical brain tissue from Alzheimer’s disease patients has significantly reduced levels of OGT protein expression compared with cortical tissue from control individuals. Together, these studies indicate that O-GlcNAcylation regulates pathways critical for the maintenance of neuronal health and suggest that dysfunctional O-GlcNAc signaling may be an important contributor to neurodegenerative diseases.

Next-generation sequencing technologies allow for rapid and inexpensive large-scale genomic analysis, creating unprecedented opportunities to integrate genomic data into the clinical diagnosis and management of neurological disorders. However, the scale and complexity of these data make them difficult to interpret and require the use of sophisticated bioinformatics applied to extensive datasets, including whole exome and genome sequences. Detailed analysis of genetic data has shown that accurate phenotype information is essential for correct interpretation of genetic variants and might necessitate re-evaluation of the patient in some cases. A multidisciplinary approach that incorporates bioinformatics, clinical evaluation, and human genetics can help to address these challenges. However, despite numerous studies that show the efficacy of next-generation sequencing in establishing molecular diagnoses, pathogenic mutations are generally identified in fewer than half of all patients with genetic neurological disorders, exposing considerable gaps in the understanding of the human genome and providing opportunities to focus research on improving the usefulness of genomics in clinical practice. Looking forward, the emergence of precision health in neurological care will increasingly apply genomic data analysis to pharmacogenetics, preventive medicine, and patient-targeted therapies.

Glycosylation is a well-known post-translational modification, but identifying specific roles for the attached glycans is often challenging. The identification and investigation of a new O -GlcNAc site on the transcription factor CREB provides insights into how glycosylation works together with phosphorylation to coordinate neural function. The transcription factor cyclic AMP–response element binding protein (CREB) is a key regulator of many neuronal processes, including brain development, circadian rhythm and long-term memory. Studies of CREB have focused on its phosphorylation, although the diversity of CREB functions in the brain suggests additional forms of regulation. Here we expand on a chemoenzymatic strategy for quantifying glycosylation stoichiometries to characterize the functional roles of CREB glycosylation in neurons. We show that CREB is dynamically modified with an O-linked β- N -acetyl- D -glucosamine sugar in response to neuronal activity and that glycosylation represses CREB-dependent transcription by impairing its association with CREB-regulated transcription coactivator (CRTC; also known as transducer of regulated CREB activity). Blocking glycosylation of CREB alters cellular function and behavioral plasticity, enhancing both axonal and dendritic growth and long-term memory consolidation. Our findings demonstrate a new role for O-glycosylation in memory formation and provide a mechanistic understanding of how glycosylation contributes to critical neuronal functions. Moreover, we identify a previously unknown mechanism for the regulation of activity-dependent gene expression, neural development and memory.

Microglia responses to Aβ and tau pathology and the dysregulation of the microglial role in synaptic function may determine the onset and course of Alzheimer's disease (AD). While significant work has been performed in mouse models, we still lack a complete understanding of physiological and pathological microglial states and functions in human AD brain. For immunoblotting of brain homogenates against multiple microglial markers, and flow cytometry (FC) analysis of synaptosomal fractions (SNAP25/CD47/Aβ(10G4)/phospho-tau(AT8)), 49 cryopreserved human parietal cortex samples were categorized into four groups: low pathology control (LPC), high Aβ control (HAC), high pathology control (HPC), and AD. Selected microglia markers were assessed in the snRNAseq dataset (Rexach et al., 2023, bioRxiv) and validated by immunohistochemistry (IHC) analysis. In the LPC group, only 22.5% of Aβ-positive (+)/p-tau-negative (-) synaptosomes expressed CD47 on their surface, with a significant progressive increase in the percentage (%) of CD47+ events in the Aβ+/p-tau- group across disease stages, reaching 53.4% in the AD group. Surprisingly, Aβ+/tau+ synaptosomes expressed the highest CD47 levels, suggesting protection from elimination. Higher levels of P2ry12 and CD206 were found in the LPC group compared to all other groups in brain homogenates. Interestingly, the levels of P2ry12, CD206, and Axl1 were significantly higher, and the level of Clec7a was significantly lower in the HPC group compared to the AD group. Linear regression modeling revealed a significant negative association between P2ry12 and synaptic tau pathology, as well as CD47 expression in p-tau-positive synaptosomes. Interestingly, in primary tauopathy microglia (but not AD microglia), IKFZ1-regulated genes, including P2ry12, were upregulated according to snRNAseq and around p-tau-bearing neurons by IHC. Our data suggest that in normal aging, synaptic Aβ accumulation leads to a diminishing of synaptic CD47 ('don't eat me') signal, accelerating the clearance of malfunctioning Aβ but not p-tau positive synapses. Upregulation of P2ry12 and CD206, accompanied by downregulation of Clec7a, may represent an early and potentially protective response to pre-tangle tau pathology. Later in disease progression, P2ry12 reduction may drive a more reactive, phagocytic phenotype in response to tau pathology, leading to an increase in synaptic CD47 levels and synaptic pathology accumulation.

Current methods to investigate glycosylation allow the identification of modification sites but provide limited additional information. A new strategy using polymers to label specific sugars now shows a huge variety in the occupancy of known glycosylation sites as well as unexpected interplay between post-translational modifications. Mechanistic studies of O -GlcNAc glycosylation have been limited by an inability to monitor the glycosylation stoichiometries of proteins obtained from cells. Here we describe a powerful method to visualize the O -GlcNAc–modified protein subpopulation using resolvable polyethylene glycol mass tags. This approach enables rapid quantification of in vivo glycosylation levels on endogenous proteins without the need for protein purification, advanced instrumentation or expensive radiolabels. In addition, it establishes the glycosylation state (for example, mono-, di-, tri-) of proteins, providing information regarding overall O -GlcNAc site occupancy that cannot be obtained using mass spectrometry. Finally, we apply this strategy to rapidly assess the complex interplay between glycosylation and phosphorylation and discover an unexpected reverse 'yin-yang' relationship on the transcriptional repressor MeCP2 that was undetectable by traditional methods. We anticipate that this mass-tagging strategy will advance our understanding of O -GlcNAc glycosylation, as well as other post-translational modifications and poorly understood glycosylation motifs.

Progranulin (PGRN) is a secreted glycoprotein, the expression of which is linked to several neurodegenerative diseases. Although its specific function is still unclear, several studies have linked it with lysosomal functions and immune system regulation. Here, we have explored the role of PGRN in peripheral and central immune system homeostasis by investigating the consequences of PGRN deficiency on adaptive and innate immune cell populations. First, we used gene co-expression network analysis of published data to test the hypothesis that has a critical role in regulating the activation status of immune cell populations in both central and peripheral compartments. To investigate the extent to which PGRN-deficiency resulted in immune dysregulation, we performed deep immunophenotyping by flow cytometry of 19-24-month old male and female deficient mice (PGRN KO) and littermate -sufficient controls (WT). Male PGRN KO mice exhibited a lower abundance of microglial cells with higher MHC-II expression, increased CD44 expression on monocytes in the brain, and more CNS-associated CD8 T cells compared to WT mice. Furthermore, we observed an increase in CD44 on CD8 T cells in the peripheral blood. Female PGRN KO mice also had fewer microglia compared to WT mice, and we also observed reduced expression of MHC-II on brain monocytes. Additionally, we found an increase in Ly-6C monocyte frequency and decreased CD44 expression on CD8 and CD4 T cells in PGRN KO female blood. Given that , which encodes for the lysosomal protein Glycoprotein non-metastatic melanoma protein B, has been reported to be upregulated in PGRN KO mice, we investigated changes in GPNMB protein expression associated with PGRN deficits and found that GPNMB is modulated in myeloid cells in a sex-specific manner. Our data suggest that PGRN and GPNMB jointly regulate the peripheral and the central immune system in a sex-specific manner; thus, understanding their associated mechanisms could pave the way for developing new neuroprotective strategies to modulate central and peripheral inflammation to lower risk for neurodegenerative diseases and possibly delay or halt progression.

The addition of the monosaccharide β- N -acetyl- D -glucosamine to proteins ( O -GlcNAc glycosylation) is an intracellular, post-translational modification that shares features with phosphorylation. Understanding the cellular mechanisms and signaling pathways that regulate O -GlcNAc glycosylation has been challenging because of the difficulty of detecting and quantifying the modification. Here, we describe a new strategy for monitoring the dynamics of O -GlcNAc glycosylation using quantitative mass spectrometry-based proteomics. Our method, which we have termed quantitative isotopic and chemoenzymatic tagging (QUIC-Tag), combines selective, chemoenzymatic tagging of O -GlcNAc proteins with an efficient isotopic labeling strategy. Using the method, we detect changes in O -GlcNAc glycosylation on several proteins involved in the regulation of transcription and mRNA translocation. We also provide the first evidence that O -GlcNAc glycosylation is dynamically modulated by excitatory stimulation of the brain in vivo . Finally, we use electron-transfer dissociation mass spectrometry to identify exact sites of O -GlcNAc modification. Together, our studies suggest that O -GlcNAc glycosylation occurs reversibly in neurons and, akin to phosphorylation, may have important roles in mediating the communication between neurons.