Comparative Analysis of In vivo Endothelial Cell Translatomes Across Central Nervous System Vascular Beds.

Endothelial cells (ECs) display organ- and tissue-specific heterogeneity. In the eye, the retinal and choroidal vascular beds are distinct networks with different molecular and morphological properties that serve location-specific functions, i.e., the former maintaining a tight barrier and the latter, a permeable fenestrated vasculature. Given that retinal health critically relies on the function of these vascular beds and that their dysfunction is implicated in a variety of retinal diseases, a molecular understanding of both physiological and pathophysiological characteristics of these distinct vasculatures is critical. Given their interspersed anatomic distribution among parenchymal cells, the study of EC gene expression, in vivo, has been hampered by the challenge of isolating pure populations of ocular ECs in sufficient quantities for large-scale transcriptomics. To address this challenge, we present a methodological and analytical workflow to facilitate inter-tissue comparisons of the in vivo EC translatome isolated from choroid, retina, and brain using the Cre-inducible NuTRAP flox construct and two widely-used endothelial Cre mouse lines: constitutive Tie2-Cre and tamoxifen-inducible Cdh5-CreERT2. For each Cre line, inter-tissue comparison of TRAP-RNAseq enrichment (TRAP-isolated translatome vs input transcriptome) showed tissue-specific gene enrichments with differential pathway representation. For each mouse model, inter-tissue comparison of the EC translatome (choroid vs brain, choroid vs retina, and brain vs retina) showed over 50% overlap of differentially expressed genes (DEGs) between the three paired comparisons, with differential pathway representation for each tissue. Pathway analysis of DEGs in the Cdh5-NuTRAP vs Tie2-NuTRAP comparison for retina, choroid, and brain predicted inhibition of processes related to myeloid cell function and activation, consistent with more specific targeting of ECs in the Cdh5-NuTRAP than in the Tie2-NuTRAP model which also targets hematopoietic progenitors giving rise to immune cells. Indeed, while TRAP enriches for EC transcripts in both models, myeloid transcripts were also captured in the Tie2-NuTRAP model which was confirmed using cell sorting. We suggest experimental/analytical considerations should be taken when selecting Cre-lines to target ECs.

Flow Cytometry Analysis of Microglial Phenotypes in the Murine Brain During Aging and Disease.

Microglia, the brain's primary resident immune cell, exists in various phenotypic states depending on intrinsic and extrinsic signaling. Distinguishing between these phenotypes can offer valuable biological insights into neurodevelopmental and neurodegenerative processes. Recent advances in single-cell transcriptomic profiling have allowed for increased granularity and better separation of distinct microglial states. While techniques such as immunofluorescence and single-cell RNA sequencing (scRNA-seq) are available to differentiate microglial phenotypes and functions, these methods present notable limitations, including challenging quantification methods, high cost, and advanced analytical techniques. This protocol addresses these limitations by presenting an optimized cell preparation procedure that prevents ex vivo activation and a flow cytometry panel to distinguish four distinct microglial states from murine brain tissue. Following cell preparation, fluorescent antibodies were applied to label 1) homeostatic, 2) disease-associated (DAM), 3) interferon response (IRM), and 4) lipid-droplet accumulating (LDAM) microglia, based on gene markers identified in previous scRNA-Seq studies. Stained cells were analyzed by flow cytometry to assess phenotypic distribution as a function of age and sex. A key advantage of this procedure is its adaptability, allowing the panel provided to be enhanced using additional markers with an appropriate cell analyzer (i.e., Cytek Aurora 5 laser spectral flow cytometer) and interrogating different brain regions or disease models. Additionally, this protocol does not require microglial cell sorting, resulting in a relatively quick and straightforward experiment. Ultimately, this protocol can compare the distribution of microglial phenotypic states between various experimental groups, such as disease state or age, with a lower cost and higher throughput than scRNA-seq. Key features • Analysis of microglial phenotypes from murine brain without the need for cell sorting, imaging, or scRNA-seq. • This protocol can distinguish between homeostatic, disease-associated (DAM), lipid-droplet accumulating (LDAM), and interferon response (IRM) microglia from any murine brain region and/or disease model of interest. • This protocol can be modified to incorporate additional markers of interest or dyes when using a cell analyzer capable of multiple color detections.

Transcriptomic Analysis Reveals That Excessive Thyroid Hormone Signaling Impairs Phototransduction and Mitochondrial Bioenergetics and Induces Cellular Stress in Mouse Cone Photoreceptors.

Thyroid hormone (TH) plays an essential role in cell proliferation, differentiation, and metabolism. Experimental and clinical studies have shown a potential association between TH signaling and retinal degeneration. The suppression of TH signaling protects cone photoreceptors in mouse models of retinal degeneration, whereas excessive TH signaling induces cone degeneration, manifested as reduced light response and a loss of cones. This work investigates the genes/transcriptomic alterations that might be involved in TH-induced cone degeneration in mice using single-cell RNA sequencing (scRNAseq) analysis. One-month-old C57BL/6 mice received triiodothyronine (T3, 20 µg/mL in drinking water) for 4 weeks as a model of hyperthyroidism/excessive TH signaling. At the end of the experiments, retinal cells were dissociated, and cell viability was analyzed before being subjected to scRNAseq. The resulting data were analyzed using the Seurat package and visualized using the Loupe browser. Among 155,866 single cells, we identified 14 cell clusters, representing various retinal cell types, with rod and cone clusters comprising 76% and 4.1% of the total cell population, respectively. Cone cluster transcriptomes demonstrated the most alterations after the T3 treatment, with 450 differentially expressed genes (DEGs), accounting for 38.5% of the total DEGs. Statistically significant changes in the expression of genes in the cone cluster revealed that phototransduction and oxidative phosphorylation were impaired after the T3 treatment, along with mitochondrial dysfunction. A pathway analysis also showed the activation of the sensory neuronal/photoreceptor stress pathways after the T3 treatment. Specifically, the eukaryotic initiation factor-2 signaling pathway and the cAMP response element-binding protein signaling pathway were upregulated. Thus, excessive TH signaling substantially affects cones at the transcriptomic level. The findings from this work provide an insight into how excessive TH signaling induces cone degeneration.

Microglia undergo sex-dimorphic transcriptional and metabolic rewiring during aging.

Microglia, the brain's resident macrophages, maintain brain homeostasis and respond to injury and infection. During aging they undergo functional changes, but the underlying mechanisms and their contributions to neuroprotection versus neurodegeneration are unclear. Previous studies suggested that microglia are sex dimorphic, so we compared microglial aging in mice of both sexes. RNA-sequencing of hippocampal microglia revealed more aging-associated changes in female microglia than male microglia, and more sex differences in old microglia than young microglia. Pathway analyses and subsequent validation assays revealed a stronger AKT-mTOR-HIF1α-driven shift to glycolysis among old female microglia and indicated that C3a production and detection was elevated in old microglia, especially in females. Recombinant C3a induced AKT-mTOR-HIF1α signaling and increased the glycolytic and phagocytic activity of young microglia. Single cell analyses attributed the aging-associated sex dimorphism to more abundant disease-associated microglia (DAM) in old female mice than old male mice, and evaluation of an Alzheimer's Disease mouse model revealed that the metabolic and complement changes are also apparent in the context of neurodegenerative disease and are strongest in the neuroprotective DAM2 subset. Collectively, our data implicate autocrine C3a-C3aR signaling in metabolic reprogramming of microglia to neuroprotective DAM during aging, especially in females, and also in Alzheimer's Disease.

Age- and sex- divergent translatomic responses of the mouse retinal pigmented epithelium.

Aging is the main risk factor for age-related macular degeneration (AMD), a retinal neurodegenerative disease that leads to irreversible blindness, particularly in people over 60 years old. Retinal pigmented epithelium (RPE) atrophy is an AMD hallmark. Genome-wide chromatin accessibility, DNA methylation, and gene expression studies of AMD and control RPE demonstrate epigenomic/transcriptomic changes occur during AMD onset and progression. However, mechanisms by which molecular alterations of normal aging impair RPE function and contribute to AMD pathogenesis are unclear. Here, we specifically interrogate the RPE translatome with advanced age and across sexes in a novel RPE reporter mouse model. We find differential age- and sex- associated transcript expression with overrepresentation of pathways related to inflammation in the RPE. Concordant with impaired RPE function, the phenotypic changes in the aged translatome suggest that aged RPE becomes immunologically active, in both males and females, with some sex-specific signatures, which supports the need for sex representation for in vivo studies.

A single-cell atlas of the aging mouse ovary.

Ovarian aging leads to diminished fertility, dysregulated endocrine signaling and increased chronic disease burden. These effects begin to emerge long before follicular exhaustion. Female humans experience a sharp decline in fertility around 35 years of age, which corresponds to declines in oocyte quality. Despite a growing body of work, the field lacks a comprehensive cellular map of the transcriptomic changes in the aging mouse ovary to identify early drivers of ovarian decline. To fill this gap we performed single-cell RNA sequencing on ovarian tissue from young (3-month-old) and reproductively aged (9-month-old) mice. Our analysis revealed a doubling of immune cells in the aged ovary, with lymphocyte proportions increasing the most, which was confirmed by flow cytometry. We also found an age-related downregulation of collagenase pathways in stromal fibroblasts, which corresponds to rises in ovarian fibrosis. Follicular cells displayed stress-response, immunogenic and fibrotic signaling pathway inductions with aging. This report provides critical insights into mechanisms responsible for ovarian aging phenotypes. The data can be explored interactively via a Shiny-based web application.

Specificity and efficiency of tamoxifen-mediated Cre induction is equivalent regardless of age.

Temporally controlling Cre recombination through tamoxifen (Tam) induction has many advantages for biomedical research. Most studies report early post-natal/juvenile (<2 m.o.) Tam induction, but age-related neurodegeneration and aging studies can require Cre induction in older mice (>12 m.o.). While anecdotally reported as problematic, there are no published comparisons of Tam-mediated Cre induction at early and late ages. Here, microglial-specific Cx3cr1creERT2 mice were crossed to a floxed NuTRAP reporter to compare Cre induction at early (3-6 m.o.) and late (20 m.o.) ages. Specificity and efficiency of microglial labeling at 21-22 m.o. were identical in mice induced with Tam at early and late ages. Age-related microglial translatomic changes were also similar regardless of Tam induction age. Each Cre and flox mouse line should be independently validated, however, these findings demonstrate that Tam-mediated Cre induction can be performed even into older mouse ages and should be generalizable to other inducible Cre models.

VTA dopamine neurons are hyperexcitable in 3xTg-AD mice due to casein kinase 2-dependent SK channel dysfunction.

Alzheimer's disease (AD) patients exhibit neuropsychiatric symptoms that extend beyond classical cognitive deficits, suggesting involvement of subcortical areas. Here, we investigated the role of midbrain dopamine (DA) neurons in AD using the amyloid + tau-driven 3xTg-AD mouse model. We found deficits in reward-based operant learning in AD mice, suggesting possible VTA DA neuron dysregulation. Physiological assessment revealed hyperexcitability and disrupted firing in DA neurons caused by reduced activity of small-conductance calcium-activated potassium (SK) channels. RNA sequencing from contents of single patch-clamped DA neurons (Patch-seq) identified up-regulation of the SK channel modulator casein kinase 2 (CK2). Pharmacological inhibition of CK2 restored SK channel activity and normal firing patterns in 3xTg-AD mice. These findings shed light on a complex interplay between neuropsychiatric symptoms and subcortical circuits in AD, paving the way for novel treatment strategies.

Differential usage of DNA modifications in neurons, astrocytes, and microglia.

BACKGROUND: Cellular identity is determined partly by cell type-specific epigenomic profiles that regulate gene expression. In neuroscience, there is a pressing need to isolate and characterize the epigenomes of specific CNS cell types in health and disease. In this study, we developed an in vivo tagging mouse model (Camk2a-NuTRAP) for paired isolation of neuronal DNA and RNA without cell sorting and then used this model to assess epigenomic regulation, DNA modifications in particular, of gene expression between neurons and glia. RESULTS: After validating the cell-specificity of the Camk2a-NuTRAP model, we performed TRAP-RNA-Seq and INTACT-whole genome oxidative bisulfite sequencing (WGoxBS) to assess the neuronal translatome and epigenome in the hippocampus of young mice (4 months old). WGoxBS findings were validated with enzymatic methyl-Seq (EM-Seq) and nanopore sequencing. Comparing neuronal data to microglial and astrocytic data from NuTRAP models, microglia had the highest global mCG levels followed by astrocytes and then neurons, with the opposite pattern observed for hmCG and mCH. Differentially modified regions between cell types were predominantly found within gene bodies and distal intergenic regions, rather than proximal promoters. Across cell types there was a negative correlation between DNA modifications (mCG, mCH, hmCG) and gene expression at proximal promoters. In contrast, a negative correlation of gene body mCG and a positive relationship between distal promoter and gene body hmCG with gene expression was observed. Furthermore, we identified a neuron-specific inverse relationship between mCH and gene expression across promoter and gene body regions. CONCLUSIONS: Neurons, astrocytes, and microglia demonstrate different genome-wide levels of mCG, hmCG, and mCH that are reproducible across analytical methods. However, modification-gene expression relationships are conserved across cell types. Enrichment of differential modifications across cell types in gene bodies and distal regulatory elements, but not proximal promoters, highlights epigenomic patterning in these regions as potentially greater determinants of cell identity. These findings also demonstrate the importance of differentiating between mC and hmC in neuroepigenomic analyses, as up to 30% of what is conventionally interpreted as mCG can be hmCG, which often has a different relationship to gene expression than mCG.

Consistent specificity and efficiency of tamoxifen-mediated cre induction across ages.

Temporally controlling cre recombination through tamoxifen (Tam) induction has many advantages for biomedical research. Most studies report Tam induction at early post-natal/juvenile (<2 m.o.) mouse ages, but age-related neurodegeneration and aging studies can require cre induction in older mice (>12 m.o.). While anecdotally reported as problematic, there are no published comparisons of Tam mediated cre induction at early and late ages. Here, microglial-specific Cx3cr1 creERT 2 mice were crossed to a floxed NuTRAP reporter to compare cre induction at early (3-6 m.o.) and late (20 m.o.) ages. Specificity and efficiency of microglial labeling at 21-22 m.o. were identical in mice induced with Tam at 3-6 m.o. or 20 m.o. of age. Age-related microglial translatomic changes were also similar regardless of Tam induction age. Each cre and flox mouse line should be validated independently, however, these findings demonstrate that Tam-mediated cre induction can be performed even into older mouse ages.

Characterization of novel mouse models to study the role of necroptosis in aging and age-related diseases.

To study the impact of necroptosis-induced chronic inflammation on age-related diseases and aging, two knockin mouse models (Ripk3-KI and Mlkl-KI) were generated that overexpress two genes involved in necroptosis (Ripk3 or Mlkl) when crossed to Cre transgenic mice. Crossing Ripk3-KI or Mlkl-KI mice to albumin-Cre transgenic mice produced hepatocyte specific hRipk3-KI or hMlkl-KI mice, which express the two transgenes only in the liver. Ripk3 and Mlkl proteins were overexpressed 10- and fourfold, respectively, in the livers of the hRipk3-KI or hMlkl-KI mice. Treating young (2-month) hRipk3-KI or hMlkl-KI mice with carbon tetrachloride (CCl4), a chemical inducer of oxidative stress, resulted in increased necroptosis (Mlkl-oligomers) and inflammation in the liver compared to control mice receiving CCl4. Mlkl-oligomerization also was significantly increased in old (18-month) hRipk3-KI and hMlkl-KI mice compared to old control (Cre negative, Ripk3-KI and Mlkl-KI) mice. The increase in necroptosis was associated with an increase in inflammation, e.g., inflammatory cytokines (TNFα, IL-6) and macrophage markers (F4/80, CD68). Importantly, steatosis (triglycerides) and fibrosis (e.g., picrosirius red staining, hydroxyproline levels, and transcripts for TGFß, Col1α1, and Col3α1) that increase with age were significantly higher in the livers of the old hRipk3-KI or hMlkl-KI mice compared to old control mice. In addition, markers of cellular senescence were significantly increased in the livers of the old hRipk3-KI and hMlkl-KI mice. Thus, the first mouse models have been developed that allow researchers to study the impact of inducing necroptosis in specific cells/tissues on chronic inflammation in aging and age-related diseases.

Microglial senescence contributes to female-biased neuroinflammation in the aging mouse hippocampus: implications for Alzheimer's disease.

BACKGROUND: Microglia, the brain's principal immune cells, have been implicated in the pathogenesis of Alzheimer's disease (AD), a condition shown to affect more females than males. Although sex differences in microglial function and transcriptomic programming have been described across development and in disease models of AD, no studies have comprehensively identified the sex divergences that emerge in the aging mouse hippocampus. Further, existing models of AD generally develop pathology (amyloid plaques and tau tangles) early in life and fail to recapitulate the aged brain environment associated with late-onset AD. Here, we examined and compared transcriptomic and translatomic sex effects in young and old murine hippocampal microglia. METHODS: Hippocampal tissue from C57BL6/N and microglial NuTRAP mice of both sexes were collected at young (5-6 month-old [mo]) and old (22-25 mo) ages. Cell sorting and affinity purification techniques were used to isolate the microglial transcriptome and translatome for RNA-sequencing and differential expression analyses. Flow cytometry, qPCR, and imaging approaches were used to confirm the transcriptomic and translatomic findings. RESULTS: There were marginal sex differences identified in the young hippocampal microglia, with most differentially expressed genes (DEGs) restricted to the sex chromosomes. Both sex chromosomally and autosomally encoded sex differences emerged with aging. These sex DEGs identified at old age were primarily female-biased and enriched in senescent and disease-associated microglial signatures. Normalized gene expression values can be accessed through a searchable web interface ( https://neuroepigenomics.omrf.org/ ). Pathway analyses identified upstream regulators induced to a greater extent in females than in males, including inflammatory mediators IFNG, TNF, and IL1B, as well as AD-risk genes TREM2 and APP. CONCLUSIONS: These data suggest that female microglia adopt disease-associated and senescent phenotypes in the aging mouse hippocampus, even in the absence of disease pathology, to a greater extent than males. This sexually divergent microglial phenotype may explain the difference in susceptibility and disease progression in the case of AD pathology. Future studies will need to explore sex differences in microglial heterogeneity in response to AD pathology and determine how sex-specific regulators (i.e., sex chromosomal or hormonal) elicit these sex effects.

Heterochronic Plasma Transfer: Experimental Design, Considerations, and Technical Challenges.

Experimental approaches such as Heterochronic Plasma Transfer (HPT) provide insights into the aging process and help identify the factors that impact aging, with the aim of developing anti-aging therapies. HPT involves the transfer of plasma from an animal of one age to an animal of a different age and highlights the effects of the systemic environment on aging. Despite its importance as an aging research tool, HPT is not without limitations and HPT experiments across various studies differ in key experimental designs considerations, presenting a challenge in obtaining comparable outcomes. In this review, we examine the caveats and experimental design considerations of HPT as a research tool. We provide insights into plasma preparation procedures, route of administration, dosing regimen, and appropriate controls to assist investigators in achieving their experimental goals.

Microglial MHC-I induction with aging and Alzheimer's is conserved in mouse models and humans.

Major histocompatibility complex I (MHC-I) CNS cellular localization and function is still being determined after previously being thought to be absent from the brain. MHC-I expression has been reported to increase with brain aging in mouse, rat, and human whole tissue analyses, but the cellular localization was undetermined. Neuronal MHC-I is proposed to regulate developmental synapse elimination and tau pathology in Alzheimer's disease (AD). Here, we report that across newly generated and publicly available ribosomal profiling, cell sorting, and single-cell data, microglia are the primary source of classical and non-classical MHC-I in mice and humans. Translating ribosome affinity purification-qPCR analysis of 3-6- and 18-22-month-old (m.o.) mice revealed significant age-related microglial induction of MHC-I pathway genes B2m, H2-D1, H2-K1, H2-M3, H2-Q6, and Tap1 but not in astrocytes and neurons. Across a timecourse (12-23 m.o.), microglial MHC-I gradually increased until 21 m.o. and then accelerated. MHC-I protein was enriched in microglia and increased with aging. Microglial expression, and absence in astrocytes and neurons, of MHC-I-binding leukocyte immunoglobulin-like (Lilrs) and paired immunoglobin-like type 2 (Pilrs) receptor families could enable cell -autonomous MHC-I signaling and increased with aging in mice and humans. Increased microglial MHC-I, Lilrs, and Pilrs were observed in multiple AD mouse models and human AD data across methods and studies. MHC-I expression correlated with p16INK4A, suggesting an association with cellular senescence. Conserved induction of MHC-I, Lilrs, and Pilrs with aging and AD opens the possibility of cell-autonomous MHC-I signaling to regulate microglial reactivation with aging and neurodegeneration.

Differential usage of DNA modifications in neurons, astrocytes, and microglia.

Background: Cellular identity is determined partly by cell type-specific epigenomic profiles that regulate gene expression. In neuroscience, there is a pressing need to isolate and characterize the epigenomes of specific CNS cell types in health and disease. This is especially true as for DNA modifications where most data are derived from bisulfite sequencing that cannot differentiate between DNA methylation and hydroxymethylation. In this study, we developed an in vivo tagging mouse model (Camk2a-NuTRAP) for paired isolation of neuronal DNA and RNA without cell sorting and then used this model to assess epigenomic regulation of gene expression between neurons and glia. Results: After validating the cell-specificity of the Camk2a-NuTRAP model, we performed TRAP-RNA-Seq and INTACT whole genome oxidative bisulfite sequencing to assess the neuronal translatome and epigenome in the hippocampus of young mice (3 months old). These data were then compared to microglial and astrocytic data from NuTRAP models. When comparing the different cell types, microglia had the highest global mCG levels followed by astrocytes and then neurons, with the opposite pattern observed for hmCG and mCH. Differentially modified regions between cell types were predominantly found within gene bodies and distal intergenic regions, with limited differences occurring within proximal promoters. Across cell types there was a negative correlation between DNA modifications (mCG, mCH, hmCG) and gene expression at proximal promoters. In contrast, a negative correlation of mCG with gene expression within the gene body while a positive relationship between distal promoter and gene body hmCG and gene expression was observed. Furthermore, we identified a neuron-specific inverse relationship between mCH and gene expression across promoter and gene body regions. Conclusions: In this study, we identified differential usage of DNA modifications across CNS cell types, and assessed the relationship between DNA modifications and gene expression in neurons and glia. Despite having different global levels, the general modification-gene expression relationship was conserved across cell types. The enrichment of differential modifications in gene bodies and distal regulatory elements, but not proximal promoters, across cell types highlights epigenomic patterning in these regions as potentially greater determinants of cell identity.

A single-cell atlas of the aging murine ovary.

Ovarian aging leads to diminished fertility, dysregulated endocrine signaling, and increased chronic disease burden. These effects begin to emerge long before follicular exhaustion. Around 35 years old, women experience a sharp decline in fertility, corresponding to declines in oocyte quality. Despite a growing body of work, the field lacks a comprehensive cellular map of the transcriptomic changes in the aging ovary to identify early drivers of ovarian decline. To fill this gap, we performed single-cell RNA sequencing on ovarian tissue from young (3-month-old) and reproductively aged (9-month-old) mice. Our analysis revealed a doubling of immune cells in the aged ovary, with lymphocyte proportions increasing the most, which was confirmed by flow cytometry. We also found an age-related downregulation of collagenase pathways in stromal fibroblasts, which corresponds to rises in ovarian fibrosis. Follicular cells displayed stress response, immunogenic, and fibrotic signaling pathway inductions with aging. This report raises provides critical insights into mechanisms responsible for ovarian aging phenotypes.

Absence of Either Ripk3 or Mlkl Reduces Incidence of Hepatocellular Carcinoma Independent of Liver Fibrosis.

Nonalcoholic fatty liver disease (NAFLD) is one of the etiologies that contribute to hepatocellular carcinoma (HCC), and chronic inflammation is one of the proposed mediators of HCC. Because necroptosis is a cell death pathway that induces inflammation, we tested whether necroptosis-induced inflammation contributes to the progression of NAFLD to HCC in a mouse model of diet-induced HCC. Male and female wild-type (WT) mice and mouse models where necroptosis is blocked (Ripk3-/- or Mlkl-/- mice) were fed either a control diet, choline-deficient low-fat diet or choline-deficient high-fat diet. Blocking necroptosis reduced markers of inflammation [proinflammatory cytokines (TNFα, IL6, and IL1ß), F4/80+ve macrophages, CCR2+ve infiltrating monocytes], inflammation-associated oncogenic pathways (JNK, PD-L1/PD-1, ß-catenin), and HCC in male mice. We demonstrate that hepatic necroptosis promotes recruitment and activation of liver macrophages leading to chronic inflammation, which in turn trigger oncogenic pathways leading to the progression of NAFLD to HCC in male mice. Whereas in female mice, blocking necroptosis reduced HCC independent of inflammation. Our data show a sex-specific difference in the development of inflammation, fibrosis, and HCC in WT mice. However, blocking necroptosis reduced HCC in both males and females without altering liver fibrosis. Thus, our study suggests that necroptosis is a valid therapeutic target for NAFLD-mediated HCC. IMPLICATIONS: Necroptosis is a major contributor to hepatic inflammation that drives the progression of NAFLD to HCC and therefore represents a valid target for NAFLD-mediated HCC.

Microglial MHC-I induction with aging and Alzheimer's is conserved in mouse models and humans.

Major Histocompatibility Complex I (MHC-I) CNS cellular localization and function is still being determined after previously being thought to be absent from the brain. MHC-I expression has been reported to increase with brain aging in mouse, rat, and human whole tissue analyses but the cellular localization was undetermined. Neuronal MHC-I is proposed to regulate developmental synapse elimination and tau pathology in Alzheimer's disease (AD). Here we report that across newly generated and publicly available ribosomal profiling, cell sorting, and single-cell data, microglia are the primary source of classical and non-classical MHC-I in mice and humans. Translating Ribosome Affinity Purification-qPCR analysis of 3-6 and 18-22 month old (m.o.) mice revealed significant age-related microglial induction of MHC-I pathway genes B2m , H2-D1 , H2-K1 , H2-M3 , H2-Q6 , and Tap1 but not in astrocytes and neurons. Across a timecourse (12-23 m.o.), microglial MHC-I gradually increased until 21 m.o. and then accelerated. MHC-I protein was enriched in microglia and increased with aging. Microglial expression, and absence in astrocytes and neurons, of MHC-I binding Leukocyte Immunoglobulin-like (Lilrs) and Paired immunoglobin-like type 2 (Pilrs) receptor families could enable cell-autonomous MHC-I signaling and increased with aging in mice and humans. Increased microglial MHC-I, Lilrs, and Pilrs were observed in multiple AD mouse models and human AD data across methods and studies. MHC-I expression correlated with p16INK4A , suggesting an association with cellular senescence. Conserved induction of MHC-I, Lilrs, and Pilrs with aging and AD opens the possibility of cell-autonomous MHC-I signaling to regulate microglial reactivation with aging and neurodegeneration.

Microglial senescence contributes to female-biased neuroinflammation in the aging mouse hippocampus: implications for Alzheimer's disease.

Background: Microglia, the brain's principal immune cells, have been implicated in the pathogenesis of Alzheimer's disease (AD), a condition shown to affect more females than males. Although sex differences in microglial function and transcriptomic programming have been described across development and in disease models of AD, no studies have comprehensively identified the sex divergences that emerge in the aging mouse hippocampus. Further, existing models of AD generally develop pathology (amyloid plaques and tau tangles) early in life and fail to recapitulate the aged brain environment associated with late-onset AD. Here, we examined and compared transcriptomic and translatomic sex effects in young and old murine hippocampal microglia. Methods: Hippocampal tissue from C57BL6/N and microglial NuTRAP mice of both sexes were collected at young (5-6 month-old [mo]) and old (22-25 mo) ages. Cell sorting and affinity purification techniques were used to isolate the microglial transcriptome and translatome for RNA-sequencing and differential expression analyses. Flow cytometry, qPCR, and imaging approaches were used to confirm the transcriptomic and translatomic findings. Results: There were marginal sex differences identified in the young hippocampal microglia, with most differentially expressed genes (DEGs) restricted to the sex chromosomes. Both sex chromosomally-and autosomally-encoded sex differences emerged with aging. These sex DEGs identified at old age were primarily female-biased and enriched in senescent and disease-associated microglial signatures. Normalized gene expression values can be accessed through a searchable web interface ( https://neuroepigenomics.omrf.org/ ). Pathway analyses identified upstream regulators induced to a greater extent in females than in males, including inflammatory mediators IFNG, TNF, and IL1B, as well as AD-risk genes TREM2 and APP. Conclusions: These data suggest that female microglia adopt disease-associated and senescent phenotypes in the aging mouse hippocampus, even in the absence of disease pathology, to a greater extent than males. This sexually divergent microglial phenotype may explain the difference in susceptibility and disease progression in the case of AD pathology. Future studies will need to explore sex differences in microglial heterogeneity in response to AD pathology and determine how sex-specific regulators (i.e., sex chromosomal or hormonal) elicit these sex effects.