MICROGLIA AGING IN THE HIPPOCAMPUS ADVANCES THROUGH INTERMEDIATE STATES THAT DRIVE INFLAMMATORY ACTIVATION AND COGNITIVE DECLINE.

During aging, microglia - the resident macrophages of the brain - exhibit dystrophic phenotypes and contribute to age-related neuroinflammation. While numerous hallmarks of age-related microglia dystrophy have been elucidated, the progression from homeostasis to dysfunction during the aging process remains unresolved. To bridge this gap in knowledge, we undertook complementary cellular and molecular analyses of microglia in the mouse hippocampus across the adult lifespan and in the experimental aging model of heterochronic parabiosis. Single-cell RNA-Seq and pseudotime analysis revealed age-related transcriptional heterogeneity in hippocampal microglia and identified intermediate states of microglial aging that also emerge following heterochronic parabiosis. We tested the functionality of intermediate stress response states via TGFß1 and translational states using pharmacological approaches in vitro to reveal their modulation of the progression to an inflammatory state. Furthermore, we utilized single-cell RNA-Seq in conjunction with an in vivo adult microglia-specific Tgfb1 conditional genetic knockout mouse model, to demonstrate that microglia advancement through intermediate aging states drives inflammatory activation and associated hippocampal-dependent cognitive decline.

Spatial Coding Dysfunction and Network Instability in the Aging Medial Entorhinal Cortex.

Across species, spatial memory declines with age, possibly reflecting altered hippocampal and medial entorhinal cortex (MEC) function. However, the integrity of cellular and network-level spatial coding in aged MEC is unknown. Here, we leveraged in vivo electrophysiology to assess MEC function in young, middle-aged, and aged mice navigating virtual environments. In aged grid cells, we observed impaired stabilization of context-specific spatial firing, correlated with spatial memory deficits. Additionally, aged grid networks shifted firing patterns often but with poor alignment to context changes. Aged spatial firing was also unstable in an unchanging environment. In these same mice, we identified 458 genes differentially expressed with age in MEC, 61 of which had expression correlated with spatial firing stability. These genes were enriched among interneurons and related to synaptic transmission. Together, these findings identify coordinated transcriptomic, cellular, and network changes in MEC implicated in impaired spatial memory in aging.

Loss of neuronal Tet2 enhances hippocampal-dependent cognitive function.

DNA methylation has emerged as a critical modulator of neuronal plasticity and cognitive function. Notwithstanding, the role of enzymes that demethylate DNA remain to be fully explored. Here, we report that loss of ten-eleven translocation methylcytosine dioxygenase 2 (Tet2), which catalyzes oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), in adult neurons enhances cognitive function. In the adult mouse hippocampus, we detected an enrichment of Tet2 in neurons. Viral-mediated neuronal overexpression and RNA interference of Tet2 altered dendritic complexity and synaptic-plasticity-related gene expression in vitro. Overexpression of neuronal Tet2 in adult hippocampus, and loss of Tet2 in adult glutamatergic neurons, resulted in differential hydroxymethylation associated with genes involved in synaptic transmission. Functionally, overexpression of neuronal Tet2 impaired hippocampal-dependent memory, while loss of neuronal Tet2 enhanced memory. Ultimately, these data identify neuronal Tet2 as a molecular target to boost cognitive function.

Tolerance induction and microglial engraftment after fetal therapy without conditioning in mice with Mucopolysaccharidosis type VII.

Mucopolysaccharidosis type VII (MPS7) is a lysosomal storage disorder (LSD) resulting from mutations in the ß-glucuronidase gene, leading to multiorgan dysfunction and fetal demise. While postnatal enzyme replacement therapy (ERT) and hematopoietic stem cell transplantation have resulted in some phenotypic improvements, prenatal treatment might take advantage of a unique developmental window to penetrate the blood-brain barrier or induce tolerance to the missing protein, addressing two important shortcomings of postnatal therapy for multiple LSDs. We performed in utero ERT (IUERT) at E14.5 in MPS7 mice and improved survival of affected mice to birth. IUERT penetrated brain microglia, whereas postnatal administration did not, and neurological testing (after IUERT plus postnatal administration) showed decreased microglial inflammation and improved grip strength in treated mice. IUERT prevented antienzyme antibody development even after multiple repeated postnatal challenges. To test a more durable treatment strategy, we performed in utero hematopoietic stem cell transplantation (IUHCT) using congenic CX3C chemokine receptor 1-green fluorescent protein (CX3CR1-GFP) mice as donors, such that donor-derived microglia are identified by GFP expression. In wild-type recipients, hematopoietic chimerism resulted in microglial engraftment throughout the brain without irradiation or conditioning; the transcriptomes of donor and host microglia were similar. IUHCT in MPS7 mice enabled cross-correction of liver Kupffer cells and improved phenotype in multiple tissues. Engrafted microglia were seen in chimeric mice, with decreased inflammation near donor microglia. These results suggest that fetal therapy with IUERT and/or IUHCT could overcome the shortcomings of current treatment strategies to improve phenotype in MPS7 and other LSDs.

Tet2 Rescues Age-Related Regenerative Decline and Enhances Cognitive Function in the Adult Mouse Brain.

Restoring adult stem cell function provides an exciting approach for rejuvenating the aging brain. However, molecular mechanisms mediating neurogenic rejuvenation remain elusive. Here we report that the enzyme ten eleven translocation methylcytosine dioxygenase 2 (Tet2), which catalyzes the production of 5-hydroxymethylcytosine (5hmC), rescues age-related decline in adult neurogenesis and enhances cognition in mice. We detected a decrease in Tet2 expression and 5hmC levels in the aged hippocampus associated with adult neurogenesis. Mimicking an aged condition in young adults by abrogating Tet2 expression within the hippocampal neurogenic niche, or adult neural stem cells, decreased neurogenesis and impaired learning and memory. In a heterochronic parabiosis rejuvenation model, hippocampal Tet2 expression was restored. Overexpressing Tet2 in the hippocampal neurogenic niche of mature adults increased 5hmC associated with neurogenic processes, offset the precipitous age-related decline in neurogenesis, and enhanced learning and memory. Our data identify Tet2 as a key molecular mediator of neurogenic rejuvenation.

Microglia communication: Parallels between aging and Alzheimer's disease.

Aging alters the functional integrity of the adult brain, driving cognitive impairments and susceptibility to neurodegenerative disorders in healthy individuals. In fact, aging remains the most dominant risk factor for Alzheimer's disease (AD). Recent findings have expanded our understanding of microglia function in the normal aging and AD brain, provoking an appreciation for microglia involvement in remodeling neuronal connections and maintaining brain integrity. This homeostatic function of microglia is achieved in part through the ability of microglia to interact extensively with and rapidly respond to changes in the brain microenvironment to enable adequate phenotypic transformations. Here, we discuss pro-inflammatory drivers of microglia transformation in aging and AD by focusing on the immune-modulatory functions of secreted factors, such as cytokines, complement factors and extracellular vesicles. We highlight the involvement of these secreted factors in aging and AD-associated cellular changes in microglia immune activation, surveillance function, and phagocytosis. Finally, we discuss how pro-inflammatory phenotypic changes associated with altered immune communication could both facilitate and exacerbate impairments in synaptic plasticity and cognitive function observed in the aged and AD brain.

Regulation of X-linked gene expression during early mouse development by Rlim.

Mammalian X-linked gene expression is highly regulated as female cells contain two and male one X chromosome (X). To adjust the X gene dosage between genders, female mouse preimplantation embryos undergo an imprinted form of X chromosome inactivation (iXCI) that requires both Rlim (also known as Rnf12) and the long non-coding RNA Xist. Moreover, it is thought that gene expression from the single active X is upregulated to correct for bi-allelic autosomal (A) gene expression. We have combined mouse genetics with RNA-seq on single mouse embryos to investigate functions of Rlim on the temporal regulation of iXCI and Xist. Our results reveal crucial roles of Rlim for the maintenance of high Xist RNA levels, Xist clouds and X-silencing in female embryos at blastocyst stages, while initial Xist expression appears Rlim-independent. We find further that X/A upregulation is initiated in early male and female preimplantation embryos.

Biogenesis and function of tRNA fragments during sperm maturation and fertilization in mammals.

Several recent studies link parental environments to phenotypes in subsequent generations. In this work, we investigate the mechanism by which paternal diet affects offspring metabolism. Protein restriction in mice affects small RNA (sRNA) levels in mature sperm, with decreased let-7 levels and increased amounts of 5' fragments of glycine transfer RNAs (tRNAs). In testicular sperm, tRNA fragments are scarce but increase in abundance as sperm mature in the epididymis. Epididymosomes (vesicles that fuse with sperm during epididymal transit) carry RNA payloads matching those of mature sperm and can deliver RNAs to immature sperm in vitro. Functionally, tRNA-glycine-GCC fragments repress genes associated with the endogenous retroelement MERVL, in both embryonic stem cells and embryos. Our results shed light on sRNA biogenesis and its dietary regulation during posttesticular sperm maturation, and they also link tRNA fragments to regulation of endogenous retroelements active in the preimplantation embryo.

Genetic and Epigenetic Variation, but Not Diet, Shape the Sperm Methylome.

Paternal diet can impact metabolic phenotypes in offspring, but mechanisms underlying such intergenerational information transfer remain obscure. Here, we interrogate cytosine methylation patterns in sperm obtained from mice consuming one of three diets, generating whole genome methylation maps for four pools of sperm samples and for 12 individual sperm samples, as well as 61 genome-scale methylation maps. We find that "epivariation," either stochastic or due to unknown demographic or environmental factors, was a far stronger contributor to the sperm methylome than was the diet consumed. Variation in cytosine methylation was particularly dramatic over tandem repeat families, including ribosomal DNA (rDNA) repeats, but rDNA methylation was strongly correlated with genetic variation in rDNA copy number and was not influenced by paternal diet. These results identify loci of genetic and epigenetic lability in the mammalian genome but argue against a direct role for sperm cytosine methylation in dietary reprogramming of offspring metabolism.

Paternally induced transgenerational environmental reprogramming of metabolic gene expression in mammals.

Epigenetic information can be inherited through the mammalian germline and represents a plausible transgenerational carrier of environmental information. To test whether transgenerational inheritance of environmental information occurs in mammals, we carried out an expression profiling screen for genes in mice that responded to paternal diet. Offspring of males fed a low-protein diet exhibited elevated hepatic expression of many genes involved in lipid and cholesterol biosynthesis and decreased levels of cholesterol esters, relative to the offspring of males fed a control diet. Epigenomic profiling of offspring livers revealed numerous modest (∼20%) changes in cytosine methylation depending on paternal diet, including reproducible changes in methylation over a likely enhancer for the key lipid regulator Ppara. These results, in conjunction with recent human epidemiological data, indicate that parental diet can affect cholesterol and lipid metabolism in offspring and define a model system to study environmental reprogramming of the heritable epigenome.

Rb regulates interactions between hematopoietic stem cells and their bone marrow microenvironment.

Hematopoiesis is maintained by stem cells (HSCs) that undergo fate decisions by integrating intrinsic and extrinsic signals, with the latter derived from the bone marrow (BM) microenvironment. Cell-cycle regulation can modulate stem cell fate, but it is unknown whether this represents an intrinsic or extrinsic effector of fate decisions. We have investigated the role of the retinoblastoma protein (RB), a central regulator of the cell cycle, in hematopoiesis. Widespread inactivation of RB in the murine hematopoietic system resulted in profound myeloproliferation. HSCs were lost from the BM due to mobilization to extramedullary sites and differentiation. This phenotype was not intrinsic to HSCs, but, rather, was the consequence of an RB-dependent interaction between myeloid-derived cells and the microenvironment. These findings demonstrate that myeloproliferation may result from perturbed interactions between hematopoietic cells and the niche. Therefore, RB extrinsically regulates HSCs by maintaining the capacity of the BM to support normal hematopoiesis and HSCs.

Lyophilization does not inactivate infectious retrovirus in systemically infected bone and tendon allografts.

BACKGROUND: A review of multiple transplantations of human immunodeficiency virus-infected musculoskeletal allografts found that recipients of lyophilized (freeze-dried) bone or tendon from an infected donor all tested negative for human immunodeficiency virus. The finding that 75% of the recipients of fresh-frozen bone from the same donor contracted human immunodeficiency virus has led to speculation that freeze-drying may render retroviral-infected musculoskeletal allografts noninfectious. HYPOTHESIS: Lyophilization does not inactivate retrovirus in systemically infected bone and tendon. STUDY DESIGN: Controlled laboratory study. METHODS: Tendons and cortical bone segments from cats systemically infected with feline leukemia virus were used in this study. Feline embryonic fibroblast cells were cultured in the presence of fresh-frozen or freeze-dried cortical bone or tendon segments. At each passage, feline leukemia virus p27 antigen was measured in media by enzyme-linked immunosorbent assay, and feline leukemia virus (pro)viral nucleic acids were quantified by real-time quantitative polymerase chain reaction in the DNA extracted from cells. RESULTS: Enzyme-linked immunosorbent assay results and quantitative polymerase chain reaction results demonstrated retroviral antigen and proviral DNA in all cultured cell replicates after exposure to fresh-frozen or freeze-dried bones or tendons. CONCLUSION: Freeze-drying (lyophilization) of retroviral-infected cortical bone and tendon does not inactivate retrovirus. CLINICAL RELEVANCE: These results conclusively demonstrate that freeze-drying should not be relied on to inactivate infectious retrovirus in systemically infected musculoskeletal allografts.