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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 invitro. 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.

Ho et al. demonstrate that old HSCs are extremely resistant to bloodborne systemic rejuvenation approaches and long-term exposure to a young BM niche microenvironment, maintaining a cell-intrinsic aged state. They also show that young HSCs are not affected by old blood or blood-derived factors, an important finding for transplantations in older patients and other emerging anti-aging interventions., While young blood can restore many aged tissues, its effects on the aged blood system itself and old hematopoietic stem cells (HSCs) have not been determined. Here, we used transplantation, parabiosis, plasma transfer, exercise, calorie restriction, and aging mutant mice to understand the effects of age-regulated systemic factors on HSCs and their bone marrow (BM) niche. We found that neither exposure to young blood, nor long-term residence in young niches after parabiont separation, nor direct heterochronic transplantation had any observable rejuvenating effects on old HSCs. Likewise, exercise and calorie restriction did not improve old HSC function, nor old BM niches. Conversely, young HSCs were not affected by systemic pro-aging conditions, and HSC function was not impacted by mutations influencing organismal aging in established long-lived or progeroid genetic models. Therefore, the blood system that carries factors with either rejuvenating or pro-aging properties for many other tissues is itself refractory to those factors., Graphical Abstract

Introduction Mucopolysaccharidosis type 7 (MPS7) is a lysosomal storage disorder typically fatal in utero. Postnatal enzyme replacement therapy (ERT) to replace missing glucuronidase (GUS) does not penetrate the blood-brain barrier (BBB). We investigated whether in utero ERT (IUERT) specifically targets brain microglia (natural GUS storehouses and key brain inflammation mediators) and whether in utero hematopoietic stem cell transplantation (IUHSCT) results in microglial engraftment as a strategy for permanent correction. Methods We performed IUERT by injecting GUS into MPS7 fetuses mid-gestation, and analyzed tissue homogenates (via colorimetric substrate) and brain microglia (via flow cytometry) for enzyme activity after 4-7 days. We performed IUHSCT by transplanting HSCs mid-gestation from CX3CR1-GFP donors. We examined blood, bone marrow, and brain for engraftment. We assessed brain inflammation by staining for CD68. We performed RNA sequencing to characterize engrafted microglia. Results IUERT resulted in detectable brain GUS activity. Flow cytometry showed that GUS activity after IUERT was near wild-type levels, and brains harvested in adulthood had decreased inflammation via CD68 immunohistochemistry. IUHSCT resulted in multilineage engraftment of hematopoietic cells in blood and bone. Confocal microscopy revealed multifocal engraftment of donor-derived microglia. RNA sequencing indicated that engrafted microglia were nearly identical to endogenous microglia. MPS7 chimeras had evidence of reduced brain inflammation near donor microglia. Conclusion Both IUERT and IUHSCT are complementary treatment modalities that can penetrate the BBB and ameliorate neurologic manifestations of diseases such as MPS7. These results lay the foundation for future studies using in utero molecular therapies for MPS7 as well as other storage disorders.

The aged systemic milieu promotes cellular and cognitive impairments in the hippocampus. Here, we report that aging of the hematopoietic system directly contributes to the pro‐aging effects of old blood on cognition. Using a heterochronic hematopoietic stem cell (HSC) transplantation model (in which the blood of young mice is reconstituted with old HSCs), we find that exposure to an old hematopoietic system inhibits hippocampal neurogenesis, decreases synaptic marker expression, and impairs cognition. We identify a number of factors elevated in the blood of young mice reconstituted with old HSCs, of which cyclophilin A (CyPA) acts as a pro‐aging factor. Increased systemic levels of CyPA impair cognition in young mice, while inhibition of CyPA in aged mice improves cognition. Together, these data identify age‐related changes in the hematopoietic system as drivers of hippocampal aging., The aged systemic milieu promotes cellular and cognitive impairments in the hippocampus. Here, we report that heterochronic hematopoietic stem cell (HSC) transplantation impairs hippocampal neurogenesis, decreases synaptic marker expression, and elicits age‐related cognitive impairments in young mice. We identify a number of factors that are elevated in the blood of young mice reconstituted with old HSCs, of which cyclophilin A (CyPA) acts as a pro‐aging factor. Increased systemic levels of CyPA impair cognition in young mice, while inhibition of CyPA in aged mice improves cognition.

Plasma transfers exercise benefit in mice Exercise has a broad range of beneficial healthful effects. Horowitz et al. tested whether the beneficial effects of exercise on neurogenesis in the brain and improved cognition in aged mice could be transferred in plasma (blood without its cellular components) from one mouse to another (see the Perspective by Ansere and Freeman). Indeed, aged mice that received plasma from young or old mice that had exercised showed beneficial effects in their brains without hitting the treadmill. The authors identified glycosylphosphatidylinositol-specific phospholipase D1 as a factor in plasma that might, in part, mediate this favorable effect. Science , this issue p. 167 ; see also p. 144

Mucopolysaccharidosis type VII (MPS7) is a lysosomal storage disorder (LSD) resulting from mutations in the β-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.

Summary The perinatal period is a critical window for distribution of innate tissue-resident immune cells within developing organs. Despite epidemiologic evidence implicating the early-life environment in the risk for allergy, temporally controlled lineage tracing of group 2 innate lymphoid cells (ILC2s) during this period remains unstudied. Using complementary fate-mapping approaches and reporters for ILC2 activation, we show that ILC2s appeared in multiple organs during late gestation like tissue macrophages, but, unlike the latter, a majority of peripheral ILC2 pools were generated de novo during the postnatal window. This period was accompanied by systemic ILC2 priming and acquisition of tissue-specific transcriptomes. Although perinatal ILC2s were variably replaced across tissues with age, the dramatic increases in tissue ILC2s following helminth infection were mediated through local expansion independent of de novo generation by bone marrow hematopoiesis. We provide comprehensive temporally controlled fate mapping of an innate lymphocyte subset with notable nuances as compared to tissue macrophage ontogeny.

Proteins of the major histocompatibility complex class I (MHC I), predominantly known for antigen presentation in the immune system, have recently been shown to be necessary for developmental neural refinement and adult synaptic plasticity. However, their roles in nonneuronal cell populations in the brain remain largely unexplored. Here, we identify classical MHC I molecule H2-Kb as a negative regulator of proliferation in neural stem and progenitor cells (NSPCs). Using genetic knockout mouse models and in vivo viral-mediated RNA interference (RNAi) and overexpression, we delineate a role for H2-Kb in negatively regulating NSPC proliferation and adult hippocampal neurogenesis. Transcriptomic analysis of H2-Kb knockout NSPCs, in combination with in vitro RNAi, overexpression, and pharmacological approaches, further revealed that H2-Kb inhibits cell proliferation by dampening signaling pathways downstream of fibroblast growth factor receptor 1 (Fgfr1). These findings identify H2-Kb as a critical regulator of cell proliferation through the modulation of growth factor signaling., This study identifies a non-canonical role for the classical Major Histocompatibility Complex (MHC) class I molecule H2-Kb as a negative regulator of neural stem and progenitor cell proliferation, independent of its function in the immune system.

Aging results in impaired neurogenesis in the two neurogenic niches of the adult mammalian brain, the dentate gyrus of the hippocampus and the subventricular zone of the lateral ventricle. While significant work has characterized intrinsic cellular changes that contribute to this decline, it is increasingly apparent that the systemic environment also represents a critical driver of brain aging. Indeed, emerging studies utilizing the model of heterochronic parabiosis have revealed that immune-related molecular and cellular changes in the aging systemic environment negatively regulate adult neurogenesis. Interestingly, these studies have also demonstrated that age-related decline in neurogenesis can be ameliorated by exposure to the young systemic environment. While this burgeoning field of research is increasingly garnering interest, as yet, the precise mechanisms driving either the pro-aging effects of aged blood or the rejuvenating effects of young blood remain to be thoroughly defined. Here, we review how age-related changes in blood, blood-borne factors, and peripheral immune cells contribute to the age-related decline in adult neurogenesis in the mammalian brain, and posit both direct neural stem cell and indirect neurogenic niche-mediated mechanisms.

Mucopolysaccharidosis type 7 (MPS7) is a lysosomal storage disorder resulting in multi-organ dysfunction and in utero demise. Post-natal enzyme replacement therapy (ERT) and hematopoietic stem cell transplantation (HSCT) are unable to address neurologic manifestations because of the blood-brain barrier (BBB). We study whether in utero ERT (IUERT) or HSCT (IUHSCT) can penetrate the BBB, targeting microglia as they are enriched in the missing enzyme. Prior IUERT work showed neurologic benefit - we now aim to analyze the extent of microglial engraftment, and to provide a permanent enzyme source with IUHSCT. We treated MPS7 fetuses at E14.5 with IUERT or IUHSCT. Following injection of missing glucuronidase (GUS), performed flow cytometry for enzyme activity on brain at E18. Controls included GUS injection in adult mice. We stained brains for CD68 (to quantify microglial inflammation). We bred CX3CR1-GFP donor mice (engrafted microglia express CX3CR1 and are therefore green on histology) and transplanted fetal liver mononuclear cells, initially into wild-type mice. Chimeric brains then underwent immunofluorescent staining for microglial markers. We injected 46 fetuses with enzyme or PBS control. 6 of 7 treated homozygous fetuses had detectable enzyme activity (24.6 ± 9.5% of microglia, some with wild-type activity levels). Accordingly, CD68 staining showed decreased brain inflammation. In contrast, 6 adult homozygous mice were treated, and none developed detectable brain GUS activity. We transplanted 5 litters of wild-type mice, and 9 mice engrafted (8.5±2.2% multilineage chimerism in blood and bone marrow). In chimeric mice at 3 weeks, transplanted stem cells co-stained with GFP and Iba1, indicating engraftment into the brain as microglia. These experiments suggest that IUERT can penetrate the BBB and decrease brain inflammation. IUHSCT results in microglial engraftment and increasing chimerism may further improve results. These findings support in utero therapy for MPS7, for which we are currently developing a clinical protocol.

During ageing, microglia acquire a phenotype that may negatively affect brain function. Here we show that ageing microglial phenotype is largely imposed by interferon type I (IFN-I) chronically present in aged brain milieu. Overexpression of IFN-β in the CNS of adult wild-type mice, but not of mice lacking IFN-I receptor on their microglia, induces an ageing-like transcriptional microglial signature, and impairs cognitive performance. Furthermore, we demonstrate that age-related IFN-I milieu downregulates microglial myocyte-specific enhancer factor 2C (Mef2C). Immune challenge in mice lacking Mef2C in microglia results in an exaggerated microglial response and has an adverse effect on mice behaviour. Overall, our data indicate that the chronic presence of IFN-I in the brain microenvironment, which negatively affects cognitive function, is mediated via modulation of microglial activity. These findings may shed new light on other neurological conditions characterized by elevated IFN-I signalling in the brain., Microglia cells in the brain regulate immune responses, but in ageing can negatively affect brain function. Here the authors show that the chronic presence of type I interferon in aged mouse brain impedes cognitive ability by altering microglia transcriptome and limiting Mef2C, a microglia ‘off’ signal.

Aging drives cognitive and regenerative impairments in the adult brain, increasing susceptibility to neurodegenerative disorders in healthy individuals. Experiments using heterochronic parabiosis, in which the circulatory systems of young and old animals are joined, indicate that circulating pro-aging factors in old blood drive aging phenotypes in the brain. Here we identify β2-microglobulin (B2M), a component of major histocompatibility complex class 1 (MHC I) molecules, as a circulating factor that negatively regulates cognitive and regenerative function in the adult hippocampus in an age-dependent manner. B2M is elevated in the blood of aging humans and mice, and it is increased within the hippocampus of aged mice and young heterochronic parabionts. Exogenous B2M injected systemically, or locally in the hippocampus, impairs hippocampal-dependent cognitive function and neurogenesis in young mice. The negative effects of B2M and heterochronic parabiosis are, in part, mitigated in the hippocampus of young transporter associated with antigen processing 1 (Tap1)-deficient mice with reduced cell surface expression of MHC I. The absence of endogenous B2M expression abrogates age-related cognitive decline and enhances neurogenesis in aged mice. Our data indicate that systemic B2M accumulation in aging blood promotes age-related cognitive dysfunction and impairs neurogenesis, in part via MHC I, suggesting that B2M may be targeted therapeutically in old age.

As human lifespan increases, a greater fraction of the population is suffering from age-related cognitive impairments, making it important to elucidate a means to combat the effects of aging. Here we report that exposure of an aged animal to young blood can counteract and reverse pre-existing effects of brain aging at the molecular, structural, functional and cognitive level. Genome-wide microarray analysis of heterochronic parabionts--in which circulatory systems of young and aged animals are connected--identified synaptic plasticity-related transcriptional changes in the hippocampus of aged mice. Dendritic spine density of mature neurons increased and synaptic plasticity improved in the hippocampus of aged heterochronic parabionts. At the cognitive level, systemic administration of young blood plasma into aged mice improved age-related cognitive impairments in both contextual fear conditioning and spatial learning and memory. Structural and cognitive enhancements elicited by exposure to young blood are mediated, in part, by activation of the cyclic AMP response element binding protein (Creb) in the aged hippocampus. Our data indicate that exposure of aged mice to young blood late in life is capable of rejuvenating synaptic plasticity and improving cognitive function.

T-705 (Favipiravir) is a broad-spectrum antiviral molecule currently in late stage clinical development for the treatment of influenza virus infection. Although it is believed that T-705 potency is mediated by its ribofuranosyl triphosphate (T-705 RTP) metabolite that could be mutagenic, the exact molecular interaction with the polymerase of influenza A virus (IAVpol) has not been elucidated. Here, we developed a biochemical assay to measure the kinetics of nucleotide incorporation by IAVpol in the elongation mode. In this assay, T-705 RTP was recognized by IAVpol as an efficient substrate for incorporation to the RNA both as a guanosine and an adenosine analog. Compared to natural GTP and ATP, the discrimination of T-705 RTP was about 19- and 30-fold, respectively. Although the single incorporation of the ribonucleotide monophosphate form of T-705 did not efficiently block RNA synthesis, two consecutive incorporation events prevented further primer extension. In comparison, 3'-deoxy GTP caused immediate chain termination but was incorporated less efficiently by the enzyme, with a discrimination of 4,900-fold relative to natural GTP. Collectively, these results provide the first detailed biochemical characterization to evaluate the substrate efficiency and the inhibition potency of nucleotide analogs against influenza virus polymerase. The combination of ambiguous base-pairing with low discrimination of T-705 RTP provides a mechanistic basis for the in vitro mutagenic effect of T-705 towards influenza virus.