Disease-associated astrocyte epigenetic memory promotes CNS pathology.

Disease-associated astrocyte subsets contribute to the pathology of neurologic diseases, including multiple sclerosis and experimental autoimmune encephalomyelitis1-8 (EAE), an experimental model for multiple sclerosis. However, little is known about the stability of these astrocyte subsets and their ability to integrate past stimulation events. Here we report the identification of an epigenetically controlled memory astrocyte subset that exhibits exacerbated pro-inflammatory responses upon rechallenge. Specifically, using a combination of single-cell RNA sequencing, assay for transposase-accessible chromatin with sequencing, chromatin immunoprecipitation with sequencing, focused interrogation of cells by nucleic acid detection and sequencing, and cell-specific in vivo CRISPR-Cas9-based genetic perturbation studies we established that astrocyte memory is controlled by the metabolic enzyme ATP-citrate lyase (ACLY), which produces acetyl coenzyme A (acetyl-CoA) that is used by histone acetyltransferase p300 to control chromatin accessibility. The number of ACLY+p300+ memory astrocytes is increased in acute and chronic EAE models, and their genetic inactivation ameliorated EAE. We also detected the pro-inflammatory memory phenotype in human astrocytes in vitro; single-cell RNA sequencing and immunohistochemistry studies detected increased numbers of ACLY+p300+ astrocytes in chronic multiple sclerosis lesions. In summary, these studies define an epigenetically controlled memory astrocyte subset that promotes CNS pathology in EAE and, potentially, multiple sclerosis. These findings may guide novel therapeutic approaches for multiple sclerosis and other neurologic diseases.

Disease-associated astrocyte epigenetic memory promotes CNS pathology.

Astrocytes play important roles in the central nervous system (CNS) physiology and pathology. Indeed, astrocyte subsets defined by specific transcriptional activation states contribute to the pathology of neurologic diseases, including multiple sclerosis (MS) and its pre-clinical model experimental autoimmune encephalomyelitis (EAE) 1-8 . However, little is known about the stability of these disease-associated astrocyte subsets, their regulation, and whether they integrate past stimulation events to respond to subsequent challenges. Here, we describe the identification of an epigenetically controlled memory astrocyte subset which exhibits exacerbated pro-inflammatory responses upon re-challenge. Specifically, using a combination of single-cell RNA sequencing (scRNA-seq), assay for transposase-accessible chromatin with sequencing (ATAC-seq), chromatin immunoprecipitation with sequencing (ChIP-seq), focused interrogation of cells by nucleic acid detection and sequencing (FIND-seq), and cell-specific in vivo CRISPR/Cas9-based genetic perturbation studies we established that astrocyte memory is controlled by the metabolic enzyme ATP citrate lyase (ACLY), which produces acetyl coenzyme A (acetyl-CoA) used by the histone acetyltransferase p300 to control chromatin accessibility. ACLY + p300 + memory astrocytes are increased in acute and chronic EAE models; the genetic targeting of ACLY + p300 + astrocytes using CRISPR/Cas9 ameliorated EAE. We also detected responses consistent with a pro-inflammatory memory phenotype in human astrocytes in vitro ; scRNA-seq and immunohistochemistry studies detected increased ACLY + p300 + astrocytes in chronic MS lesions. In summary, these studies define an epigenetically controlled memory astrocyte subset that promotes CNS pathology in EAE and, potentially, MS. These findings may guide novel therapeutic approaches for MS and other neurologic diseases.

Lactate limits CNS autoimmunity by stabilizing HIF-1α in dendritic cells.

Dendritic cells (DCs) have a role in the development and activation of self-reactive pathogenic T cells1,2. Genetic variants that are associated with the function of DCs have been linked to autoimmune disorders3,4, and DCs are therefore attractive therapeutic targets for such diseases. However, developing DC-targeted therapies for autoimmunity requires identification of the mechanisms that regulate DC function. Here, using single-cell and bulk transcriptional and metabolic analyses in combination with cell-specific gene perturbation studies, we identify a regulatory loop of negative feedback that operates in DCs to limit immunopathology. Specifically, we find that lactate, produced by activated DCs and other immune cells, boosts the expression of NDUFA4L2 through a mechanism mediated by hypoxia-inducible factor 1α (HIF-1α). NDUFA4L2 limits the production of mitochondrial reactive oxygen species that activate XBP1-driven transcriptional modules in DCs that are involved in the control of pathogenic autoimmune T cells. We also engineer a probiotic that produces lactate and suppresses T cell autoimmunity through the activation of HIF-1α-NDUFA4L2 signalling in DCs. In summary, we identify an immunometabolic pathway that regulates DC function, and develop a synthetic probiotic for its therapeutic activation.

Engineered probiotics limit CNS autoimmunity by stabilizing HIF-1α in dendritic cells.

Dendritic cells (DCs) control the generation of self-reactive pathogenic T cells. Thus, DCs are considered attractive therapeutic targets for autoimmune diseases. Using single-cell and bulk transcriptional and metabolic analyses in combination with cell-specific gene perturbation studies we identified a negative feedback regulatory pathway that operates in DCs to limit immunopathology. Specifically, we found that lactate, produced by activated DCs and other immune cells, boosts NDUFA4L2 expression through a mechanism mediated by HIF-1α. NDUFA4L2 limits the production of mitochondrial reactive oxygen species that activate XBP1-driven transcriptional modules in DCs involved in the control of pathogenic autoimmune T cells. Moreover, we engineered a probiotic that produces lactate and suppresses T-cell autoimmunity in the central nervous system via the activation of HIF-1α/NDUFA4L2 signaling in DCs. In summary, we identified an immunometabolic pathway that regulates DC function, and developed a synthetic probiotic for its therapeutic activation.

Droplet-based forward genetic screening of astrocyte-microglia cross-talk.

Cell-cell interactions in the central nervous system play important roles in neurologic diseases. However, little is known about the specific molecular pathways involved, and methods for their systematic identification are limited. Here, we developed a forward genetic screening platform that combines CRISPR-Cas9 perturbations, cell coculture in picoliter droplets, and microfluidic-based fluorescence-activated droplet sorting to identify mechanisms of cell-cell communication. We used SPEAC-seq (systematic perturbation of encapsulated associated cells followed by sequencing), in combination with in vivo genetic perturbations, to identify microglia-produced amphiregulin as a suppressor of disease-promoting astrocyte responses in multiple sclerosis preclinical models and clinical samples. Thus, SPEAC-seq enables the high-throughput systematic identification of cell-cell communication mechanisms.

Induction of an effective anti-Amyloid-ß humoral response in aged mice.

Aging-related decline in immune functions, termed immunosenescence, is a primary cause of reduced protective responses to vaccines in the elderly, due to impaired induction of cellular and humoral responses to new antigens (Ag), especially if the response is T cell dependent. The result is a more severe morbidity following infections, more prolonged and frequent hospitalization, and a higher mortality rate than in the general population. Therefore, there is an increasing need to develop vaccination strategies that overcome immunosenescence, especially for aging-related diseases such as Alzheimer's disease (AD). Here we report a new vaccination strategy harnessing memory-based immunity, which is less affected by aging. We found that aged C57BL/6 and 5xFAD mice exhibit a dramatic reduction in anti-Amyloid-ß (Aß) antibody (Ab) production. We aimed to reverse this process by inducing memory response at a young age. To this end, young mice were primed with the vaccine carrier Hepatitis B surface antigen (HBsAg). At an advanced age, these mice were immunized with an Aß1-11 fused to HBsAg. This vaccination scheme elicited a markedly higher Aß-specific antibody titer than vaccinating aged unprimed mice with the same construct. Importantly, this vaccine strategy more efficiently reduced cerebral Aß levels and altered microglial phenotype. Overall, we provide evidence that priming with an exogenous Ag carrier can overcome impaired humoral responses to self-antigens in the elderly, paving the route for a potent immunotherapy to AD.

Immune Dysregulation and the Increased Risk of Complications and Mortality Following Respiratory Tract Infections in Adults With Down Syndrome.

The risk of severe outcomes following respiratory tract infections is significantly increased in individuals over 60 years, especially in those with chronic medical conditions, i.e., hypertension, diabetes, cardiovascular disease, dementia, chronic respiratory disease, and cancer. Down Syndrome (DS), the most prevalent intellectual disability, is caused by trisomy-21 in ~1:750 live births worldwide. Over the past few decades, a substantial body of evidence has accumulated, pointing at the occurrence of alterations, impairments, and subsequently dysfunction of the various components of the immune system in individuals with DS. This associates with increased vulnerability to respiratory tract infections in this population, such as the influenza virus, respiratory syncytial virus, SARS-CoV-2 (COVID-19), and bacterial pneumonias. To emphasize this link, here we comprehensively review the immunobiology of DS and its contribution to higher susceptibility to severe illness and mortality from respiratory tract infections.

Therapeutic B-cell depletion reverses progression of Alzheimer's disease.

The function of B cells in Alzheimer's disease (AD) is not fully understood. While immunoglobulins that target amyloid beta (Aß) may interfere with plaque formation and hence progression of the disease, B cells may contribute beyond merely producing immunoglobulins. Here we show that AD is associated with accumulation of activated B cells in circulation, and with infiltration of B cells into the brain parenchyma, resulting in immunoglobulin deposits around Aß plaques. Using three different murine transgenic models, we provide counterintuitive evidence that the AD progression requires B cells. Despite expression of the AD-fostering transgenes, the loss of B cells alone is sufficient to reduce Aß plaque burden and disease-associated microglia. It reverses behavioral and memory deficits and restores TGFß+ microglia, respectively. Moreover, therapeutic depletion of B cells at the onset of the disease retards AD progression in mice, suggesting that targeting B cells may also benefit AD patients.

Maternal antibodies facilitate Amyloid-ß clearance by activating Fc-receptor-Syk-mediated phagocytosis.

Maternal antibodies (MAbs) protect against infections in immunologically-immature neonates. Maternally transferred immunity may also be harnessed to target diseases associated with endogenous protein misfolding and aggregation, such as Alzheimer's disease (AD) and AD-pathology in Down syndrome (DS). While familial early-onset AD (fEOAD) is associated with autosomal dominant mutations in the APP, PSEN1,2 genes, promoting cerebral Amyloid-ß (Aß) deposition, DS features a life-long overexpression of the APP and DYRK1A genes, leading to a cognitive decline mediated by Aß overproduction and tau hyperphosphorylation. Although no prenatal screening for fEOAD-related mutations is in clinical practice, DS can be diagnosed in utero. We hypothesized that anti-Aß MAbs might promote the removal of early Aß accumulation in the central nervous system of human APP-expressing mice. To this end, a DNA-vaccine expressing Aß1-11 was delivered to wild-type female mice, followed by mating with 5xFAD males, which exhibit early Aß plaque formation. MAbs reduce the offspring's cortical Aß levels 4 months after antibodies were undetectable, along with alleviating short-term memory deficits. MAbs elicit a long-term shift in microglial phenotype in a mechanism involving activation of the FcγR1/Syk/Cofilin pathway. These data suggest that maternal immunization can alleviate cognitive decline mediated by early Aß deposition, as occurs in EOAD and DS.

Specific Susceptibility to COVID-19 in Adults with Down Syndrome.

The current SARS-CoV-2 outbreak, which causes COVID-19, is particularly devastating for individuals with chronic medical conditions, in particular those with Down Syndrome (DS) who often exhibit a higher prevalence of respiratory tract infections, immune dysregulation and potential complications. The incidence of Alzheimer's disease (AD) is much higher in DS than in the general population, possibly increasing further the risk of COVID-19 infection and its complications. Here we provide a biological overview with regard to specific susceptibility of individuals with DS to SARS-CoV-2 infection as well as data from a recent survey on the prevalence of COVID-19 among them. We see an urgent need to protect people with DS, especially those with AD, from COVID-19 and future pandemics and focus on developing protective measures, which also include interventions by health systems worldwide for reducing the negative social effects of long-term isolation and increased periods of hospitalization.

A modified Barnes maze for an accurate assessment of spatial learning in mice.

BACKGROUND: The Morris water maze (MWM) and the Barnes maze (BM) are among the most widely-used paradigms for assessing spatial learning in rodents, with specific advantages and disadvantages for each apparatus. Compared with the intense water-related stress exerted during the MWM, the BM exhibits a milder light-induced stress, while suffering from biasing animals towards non-spatial strategies such as serial search, a heuristic non-spatial search strategy. To overcome this problem, we have developed a modified Barnes maze (MBM) apparatus that recapitulates natural environments more accurately without inducing undesirable exploration strategy bias. NEW METHOD: Apparatus. A circular 122 cm-wide table with 40 randomly placed holes. One target hole is leading to an escape chamber. Task. Three target locations were examined, varying in their distance from the center. C57BL6/j male mice were given three trials per day to find the target. Following acquisition, a probe test was performed by removing the escape chamber. RESULTS: Spatial-encoding-depended reduction in latency to reach the target was observed, along with improvement in path efficiency with test progress. Mice tested with peripheral and distal targets outperformed mice tested with a central target. A robust exploration pattern was identified in the probe test. COMPARISON WITH EXISTING METHOD: The MBM mimics natural environment to a higher degree of accuracy than the BM, without eliciting bias towards non-spatial searching strategies. CONCLUSIONS: Spatial learning in the MBM is a target-location sensitive process, providing flexibility in task difficulty. Along with overcoming biases towards non-spatial strategies, the MBM represents an improvement over the well-validated BM.

L-Lactate Promotes Adult Hippocampal Neurogenesis.

Neurogenesis, the formation of new neurons in the adult brain, is important for memory formation and extinction. One of the most studied external interventions that affect the rate of adult neurogenesis is physical exercise. Physical exercise promotes adult neurogenesis via several factors including brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF). Here, we identified L-lactate, a physical exercise-induced metabolite, as a factor that promotes adult hippocampal neurogenesis. While prolonged exposure to L-lactate promoted neurogenesis, no beneficial effect was exerted on cognitive learning and memory. Systemic pharmacological blocking of monocarboxylate transporter 2 (MCT2), which transports L-lactate to the brain, prevented lactate-induced neurogenesis, while 3,5-dihydroxybenzoic acid (3,5-DHBA), an agonist for the lactate-receptor hydroxycarboxylic acid receptor 1 (HCAR1), did not affect adult neurogenesis. These data suggest that L-lactate partially mediates the effect of physical exercise on adult neurogenesis, but not cognition, in a MCT2-dependent manner.

Restoring microglial and astroglial homeostasis using DNA immunization in a Down Syndrome mouse model.

Down Syndrome (DS), the most common cause of genetic intellectual disability, is characterized by over-expression of the APP and DYRK1A genes, located on the triplicated chromosome 21. This chromosomal abnormality leads to a cognitive decline mediated by Amyloid-ß (Aß) overproduction and tau hyper-phosphorylation as early as the age of 40. In this study, we used the Ts65Dn mouse model of DS to evaluate the beneficial effect of a DNA vaccination against the Aß1-11 fragment, in ameliorating Aß-related neuropathology and rescue of cognitive and behavioral abilities. Anti-Aß1-11 vaccination induced antibody production and facilitated clearance of soluble oligomers and small extracellular inclusions of Aß from the hippocampus and cortex of Ts65Dn mice. This was correlated with reduced neurodegeneration and restoration of the homeostatic phenotype of microglial and astroglial cells. Vaccinated Ts65Dn mice performed better in spatial-learning tasks, exhibited reduced motor hyperactivity typical for this strain, and restored short-term memory abilities. Our findings support the hypothesis that DS individuals may benefit from active immunotherapy against Aß from a young age by slowing the progression of dementia.

A protocol for quantitative analysis of murine and human amyloid-ß1-40 and 1-42.

BACKGROUND: Amyloid-ß (Aß), a hallmark of Alzheimer's disease (AD), has long been a focus of basic and translation research in AD. Quantification and dissociation of the Aß fractions in their soluble and insoluble forms, is a key factor in numerous AD studies. NEW METHOD: Here we provide a generalized sandwich-enzyme-linked-immuno-sorbent-assay (sELISA) protocol for quantification of human and murine Aß1-40 and Aß1-42 and dissociation of these peptides to their soluble-oligomeric and insoluble-fibrillar forms. RESULTS: We have validated the levels of soluble and insoluble Aß1-40 and Aß1-42 in the 5XFAD AD and the Ts65Dn Down-Syndrome (DS) mouse models in both the cortex, hippocampus and blood as follows: (1) blood levels of Aß1-40 and Aß1-42 are elevated in both mouse strains. (2) 5XFAD mice exhibit elevated soluble and insoluble Aß1-40 in cortical and hippocampal tissues, soluble Aß1-42 in the hippocampus, and insoluble Aß1-42in both cortical and hippocampal tissues (3) Ts65Dn mice exhibit elevated levels of Aß1-40 in the cortex. COMPARISON WITH EXISTING METHODS: Several methodologies have been proposed for the high throughput measure of Aß, including HPLC-mass-spectrometry, micro-immunoelectrodes, immunoprecipitation and ELISA. Although commercial sELISA kits are widely used, herein, we describe a more accessible and cost-effective in-house protocol enabling to measure either human or murine, soluble and insoluble Aß1-40 and Aß1-42 levels. CONCLUSIONS: We provide a streamlined and accessible protocol for the assessment of soluble and insoluble Aß1-40 and Aß1-42 levels from mouse or human origins, enabling a higher accessibility for researchers in the field to generate reliable Aß-related measurements.

Sirt6 alters adult hippocampal neurogenesis.

Sirtuins are pleiotropic NAD+ dependent histone deacetylases involved in metabolism, DNA damage repair, inflammation and stress resistance. SIRT6, a member of the sirtuin family, regulates the process of normal aging and increases the lifespan of male mice over-expressing Sirt6 by 15%. Neurogenesis, the formation of new neurons within the hippocampus of adult mammals, involves several complex stages including stem cell proliferation, differentiation, migration and network integration. During aging, the number of newly generated neurons continuously declines, and this is correlated with a decline in neuronal plasticity and cognitive behavior. In this study we investigated the involvement of SIRT6 in adult hippocampal neurogenesis. Mice over-expressing Sirt6 exhibit increased numbers of young neurons and decreased numbers of mature neurons, without affecting glial differentiation. This implies of an involvement of SIRT6 in neuronal differentiation and maturation within the hippocampus. This work adds to the expanding body of knowledge on the regulatory mechanisms underlying adult hippocampal neurogenesis, and describes novel roles for SIRT6 as a regulator of cell fate during adult hippocampal neurogenesis.

Toll-like receptor 3 deficiency decreases epileptogenesis in a pilocarpine model of SE-induced epilepsy in mice.

OBJECTIVE: Epilepsy affects 60 million people worldwide. Despite the development of antiepileptic drugs, up to 35% of patients are drug refractory with uncontrollable seizures. Toll-like receptors (TLRs) are central components of the nonspecific innate inflammatory response. Because TLR3 was recently implicated in neuronal plasticity, we hypothesized that it may contribute to the development of epilepsy after status epilepticus (SE). METHODS: To test the involvement of TLR3 in epileptogenesis, we used the pilocarpine model for SE in TLR3-deficient mice and their respective wild-type controls. In this model, a single SE event leads to spontaneous recurrent seizures (SRS). Two weeks after SE, mice were implanted with wireless electroencephalography (EEG) transmitters for up to 1 month. The impact of TLR3 deficiency on SE was assessed using separate cohorts of mice regarding EEG activity, seizure progression, hippocampal microglial distribution, and expression of the proinflammatory cytokines tumor necrosis factor (TNF)α and interferon (IFN)ß. RESULTS: Our data indicate that TLR3 deficiency reduced SRS, microglial activation, and the levels of the proinflammatory cytokines TNFα and IFNß, and increased survival following SE. SIGNIFICANCE: This study reveals novel insights into the pathophysiology of epilepsy and the contribution of TLR3 to disease progression. Our results identify the TLR3 pathway as a potential future therapeutic target in SE.