A Single-Cell Transcriptomic Analysis of the Mouse Hippocampus After Voluntary Exercise.

Exercise has been recognized as a beneficial factor for cognitive health, particularly in relation to the hippocampus, a vital brain region responsible for learning and memory. Previous research has demonstrated that exercise-mediated improvement of learning and memory in humans and rodents correlates with increased adult neurogenesis and processes related to enhanced synaptic plasticity. Nevertheless, the underlying molecular mechanisms are not fully understood. With the aim to further elucidate these mechanisms, we provide a comprehensive dataset of the mouse hippocampal transcriptome at the single-cell level after 4 weeks of voluntary wheel-running. Our analysis provides a number of interesting observations. For example, the results suggest that exercise affects adult neurogenesis by accelerating the maturation of a subpopulation of Prdm16-expressing neurons. Moreover, we uncover the existence of an intricate crosstalk among multiple vital signaling pathways such as NF-κB, Wnt/ß-catenin, Notch, and retinoic acid (RA) pathways altered upon exercise in a specific cluster of excitatory neurons within the Cornu Ammonis (CA) region of the hippocampus. In conclusion, our study provides an important resource dataset and sheds further light on the molecular changes induced by exercise in the hippocampus. These findings have implications for developing targeted interventions aimed at optimizing cognitive health and preventing age-related cognitive decline.

Conserved reduction of m6A RNA modifications during aging and neurodegeneration is linked to changes in synaptic transcripts.

N6-methyladenosine (m6A) regulates mRNA metabolism. While it has been implicated in the development of the mammalian brain and in cognition, the role of m6A in synaptic plasticity, especially during cognitive decline, is not fully understood. In this study, we employed methylated RNA immunoprecipitation sequencing to obtain the m6A epitranscriptome of the hippocampal subregions CA1, CA3, and the dentate gyrus and the anterior cingulate cortex (ACC) in young and aged mice. We observed a decrease in m6A levels in aged animals. Comparative analysis of cingulate cortex (CC) brain tissue from cognitively intact human subjects and Alzheimer's disease (AD) patients showed decreased m6A RNA methylation in AD patients. m6A changes common to brains of aged mice and AD patients were found in transcripts linked to synaptic function including calcium/calmodulin-dependent protein kinase 2 (CAMKII) and AMPA-selective glutamate receptor 1 (Glua1). We used proximity ligation assays to show that reduced m6A levels result in decreased synaptic protein synthesis as exemplified by CAMKII and GLUA1. Moreover, reduced m6A levels impaired synaptic function. Our results suggest that m6A RNA methylation controls synaptic protein synthesis and may play a role in cognitive decline associated with aging and AD.

Impact of Industrially Affected Soil on Humans: A Soil-Human and Soil-Plant-Human Exposure Assessment.

Heavy metal (HM) contaminated soil can affect human health via ingestion of foodstuffs, inhalation of soil dust, and skin contact of soil. This study estimates the level of some heavy metals in soils of industrial areas, and their exposures to human body via dietary intake of vegetables and other pathways. Mean concentrations of Cr, Fe, Cu, Zn, As and Pb in the studied soil were found to be 61.27, 27,274, 42.36, 9.77, 28.08 and 13.69 mg/kg, respectively, while in vegetables the respective values were 0.53, 119.59, 9.76, 7.14, 1.34 and 2.69 mg/kg. Multivariate statistical analysis revealed that Fe, Cu, Zn, and Pb originated from lithogenic sources, while Cr and As are derived from anthropogenic sources. A moderate enrichment was noted by Cr, As, and Pb in the entire sampling site, indicating a progressive depletion of soil quality. The bioaccumulation factor (BCF) value for all the vegetables was recorded as BCF < 1; however, the metal pollution index (MPI) stipulates moderately high value of heavy metal accumulation in the vegetable samples. Hazard Index (HI) of >0.1 was estimated for adults but >1 for children by direct soil exposure, whereas HI < 1 for both children and adults via dietary intake of vegetables. Estimated Total carcinogenic risk (TCR) value due to soil exposure showed safe for adults but unsafe for children, while both the population groups were found to be safe via food consumption. Children are found more vulnerable receptors than adults, and health risks (carcinogenic and non-carcinogenic) via direct soil exposure proved unsafe. Overall, this study can be used as a reference for similar types of studies to evaluate heavy metal contaminated soil impact on the population of Bangladesh and other countries as well.

Postnatal expression of the lysine methyltransferase SETD1B is essential for learning and the regulation of neuron-enriched genes.

In mammals, histone 3 lysine 4 methylation (H3K4me) is mediated by six different lysine methyltransferases. Among these enzymes, SETD1B (SET domain containing 1b) has been linked to syndromic intellectual disability in human subjects, but its role in the mammalian postnatal brain has not been studied yet. Here, we employ mice deficient for Setd1b in excitatory neurons of the postnatal forebrain, and combine neuron-specific ChIP-seq and RNA-seq approaches to elucidate its role in neuronal gene expression. We observe that Setd1b controls the expression of a set of genes with a broad H3K4me3 peak at their promoters, enriched for neuron-specific genes linked to learning and memory function. Comparative analyses in mice with conditional deletion of Kmt2a and Kmt2b histone methyltransferases show that SETD1B plays a more pronounced and potent role in regulating such genes. Moreover, postnatal loss of Setd1b leads to severe learning impairment, suggesting that SETD1B-dependent regulation of H3K4me levels in postnatal neurons is critical for cognitive function.

Long-term caffeine treatment of Alzheimer mouse models ameliorates behavioural deficits and neuron loss and promotes cellular and molecular markers of neurogenesis.

Epidemiological studies indicate that the consumption of caffeine, the most commonly ingested psychoactive substance found in coffee, tea or soft drinks, reduces the risk of developing Alzheimer's disease (AD). Previous treatment studies with transgenic AD mouse models reported a reduced amyloid plaque load and an amelioration of behavioral deficits. It has been further shown that moderate doses of caffeine have the potential to attenuate the health burden in preclinical mouse models of a variety of brain disorders (reviewed in Cunha in J Neurochem 139:1019-1055, 2016). In the current study, we assessed whether long-term caffeine consumption affected hippocampal neuron loss and associated behavioral deficits in the Tg4-42 mouse model of AD. Treatment over a 4-month period reduced hippocampal neuron loss, rescued learning and memory deficits, and ameliorated impaired neurogenesis. Neuron-specific RNA sequencing analysis in the hippocampus revealed an altered expression profile distinguished by the up-regulation of genes linked to synaptic function and processes, and to neural progenitor proliferation. Treatment of 5xFAD mice, which develop prominent amyloid pathology, with the same paradigm also rescued behavioral deficits but did not affect extracellular amyloid-ß (Aß) levels or amyloid precursor protein (APP) processing. These findings challenge previous assumptions that caffeine is anti-amyloidogenic and indicate that the promotion of neurogenesis might play a role in its beneficial effects.

Interferon-driven brain phenotype in a mouse model of RNaseT2 deficient leukoencephalopathy.

Infantile-onset RNaseT2 deficient leukoencephalopathy is characterised by cystic brain lesions, multifocal white matter alterations, cerebral atrophy, and severe psychomotor impairment. The phenotype is similar to congenital cytomegalovirus brain infection and overlaps with type I interferonopathies, suggesting a role for innate immunity in its pathophysiology. To date, pathophysiological studies have been hindered by the lack of mouse models recapitulating the neuroinflammatory encephalopathy found in patients. In this study, we generated Rnaset2-/- mice using CRISPR/Cas9-mediated genome editing. Rnaset2-/- mice demonstrate upregulation of interferon-stimulated genes and concurrent IFNAR1-dependent neuroinflammation, with infiltration of CD8+ effector memory T cells and inflammatory monocytes into the grey and white matter. Single nuclei RNA sequencing reveals homeostatic dysfunctions in glial cells and neurons and provide important insights into the mechanisms of hippocampal-accentuated brain atrophy and cognitive impairment. The Rnaset2-/- mice may allow the study of CNS damage associated with RNaseT2 deficiency and may be used for the investigation of potential therapies.

H3 acetylation selectively promotes basal progenitor proliferation and neocortex expansion.

Increase in the size of human neocortex­acquired in evolution­accounts for the unique cognitive capacity of humans. This expansion reflects the evolutionarily enhanced proliferative ability of basal progenitors (BPs), including the basal radial glia and basal intermediate progenitors (bIPs) in mammalian cortex, which may have been acquired through epigenetic alterations in BPs. However, how the epigenome in BPs differs across species is not known. Here, we report that histone H3 acetylation is a key epigenetic regulation in bIP amplification and cortical expansion. Through epigenetic profiling of sorted bIPs, we show that histone H3 lysine 9 acetylation (H3K9ac) is low in murine bIPs and high in human bIPs. Elevated H3K9ac preferentially increases bIP proliferation, increasing the size and folding of the normally smooth mouse neocortex. H3K9ac drives bIP amplification by increasing expression of the evolutionarily regulated gene, Trnp1, in developing cortex. Our findings demonstrate a previously unknown mechanism that controls cortical architecture.

Molecular Profiling Reveals Involvement of ESCO2 in Intermediate Progenitor Cell Maintenance in the Developing Mouse Cortex.

Intermediate progenitor cells (IPCs) are neocortical neuronal precursors. Although IPCs play crucial roles in corticogenesis, their molecular features remain largely unknown. In this study, we aimed to characterize the molecular profile of IPCs. We isolated TBR2-positive (+) IPCs and TBR2-negative (-) cell populations in the developing mouse cortex. Comparative genome-wide gene expression analysis of TBR2+ IPCs versus TBR2- cells revealed differences in key factors involved in chromatid segregation, cell-cycle regulation, transcriptional regulation, and cell signaling. Notably, mutation of many IPC genes in human has led to intellectual disability and caused a wide range of cortical malformations, including microcephaly and agenesis of corpus callosum. Loss-of-function experiments in cortex-specific mutants of Esco2, one of the novel IPC genes, demonstrate its critical role in IPC maintenance, and substantiate the identification of a central genetic determinant of IPC biogenesis. Our data provide novel molecular characteristics of IPCs in the developing mouse cortex.

Intranuclear immunostaining-based FACS protocol from embryonic cortical tissue.

Cell sorting can be used to purify cell populations for cell type-specific molecular probing. Fluorescence-activated cell sorting (FACS) coupled with high-throughput sequencing affords molecular signature identification for specific cell types. FACS has many challenges that limit comprehensive cell purification from the brain, leading to incomplete molecular characterization. Here, we present the intranuclear immunostaining-based FACS protocol with several modified steps, which allows optimized nuclei/cell sorting from mouse or human embryonic cortical tissue for distinct downstream molecular investigation of basal intermediate progenitors.

Epigenetic gene expression links heart failure to memory impairment.

In current clinical practice, care of diseased patients is often restricted to separated disciplines. However, such an organ-centered approach is not always suitable. For example, cognitive dysfunction is a severe burden in heart failure patients. Moreover, these patients have an increased risk for age-associated dementias. The underlying molecular mechanisms are presently unknown, and thus, corresponding therapeutic strategies to improve cognition in heart failure patients are missing. Using mice as model organisms, we show that heart failure leads to specific changes in hippocampal gene expression, a brain region intimately linked to cognition. These changes reflect increased cellular stress pathways which eventually lead to loss of neuronal euchromatin and reduced expression of a hippocampal gene cluster essential for cognition. Consequently, mice suffering from heart failure exhibit impaired memory function. These pathological changes are ameliorated via the administration of a drug that promotes neuronal euchromatin formation. Our study provides first insight to the molecular processes by which heart failure contributes to neuronal dysfunction and point to novel therapeutic avenues to treat cognitive defects in heart failure patients.

TIP60/KAT5 is required for neuronal viability in hippocampal CA1.

Aberrant histone acetylation contributes to age-dependent cognitive decline and neurodegenerative diseases. We analyze the function of lysine acetyltransferase TIP60/KAT5 in neurons of the hippocampus using an inducible mouse model. TIP60-deficiency in the adult forebrain leads within days to extensive transcriptional dysfunction characterized by the presence of a neurodegeneration-related signature in CA1. Cell cycle- and immunity-related genes are upregulated while learning- and neuronal plasticity-related genes are downregulated. The dysregulated genes seen under TIP60-deficiency overlap with those in the well-characterized CK-p25 neurodegeneration model. We found that H4K12 is hypoacetylated at the transcriptional start sites of those genes whose expression is dampened in TIP60-deficient mice. Transcriptional dysregulation is followed over a period of weeks by activation of Caspase 3 and fragmentation of ß-actin in CA1 neurites, eventually leading to severe neuronal loss. TIP60-deficient mice also develop mild memory impairment. These phenotypes point to a central role of TIP60 in transcriptional networks that are critical for neuronal viability.

Precisely measured protein lifetimes in the mouse brain reveal differences across tissues and subcellular fractions.

The turnover of brain proteins is critical for organism survival, and its perturbations are linked to pathology. Nevertheless, protein lifetimes have been difficult to obtain in vivo. They are readily measured in vitro by feeding cells with isotopically labeled amino acids, followed by mass spectrometry analyses. In vivo proteins are generated from at least two sources: labeled amino acids from the diet, and non-labeled amino acids from the degradation of pre-existing proteins. This renders measurements difficult. Here we solved this problem rigorously with a workflow that combines mouse in vivo isotopic labeling, mass spectrometry, and mathematical modeling. We also established several independent approaches to test and validate the results. This enabled us to measure the accurate lifetimes of ~3500 brain proteins. The high precision of our data provided a large set of biologically significant observations, including pathway-, organelle-, organ-, or cell-specific effects, along with a comprehensive catalog of extremely long-lived proteins (ELLPs).

Epigenetic Regulation by BAF Complexes Limits Neural Stem Cell Proliferation by Suppressing Wnt Signaling in Late Embryonic Development.

During early cortical development, neural stem cells (NSCs) divide symmetrically to expand the progenitor pool, whereas, in later stages, NSCs divide asymmetrically to self-renew and produce other cell types. The timely switch from such proliferative to differentiative division critically determines progenitor and neuron numbers. However, the mechanisms that limit proliferative division in late cortical development are not fully understood. Here, we show that the BAF (mSWI/SNF) complexes restrict proliferative competence and promote neuronal differentiation in late corticogenesis. Inactivation of BAF complexes leads to H3K27me3-linked silencing of neuronal differentiation-related genes, with concurrent H3K4me2-mediated activation of proliferation-associated genes via de-repression of Wnt signaling. Notably, the deletion of BAF complexes increased proliferation of neuroepithelial cell-like NSCs, impaired neuronal differentiation, and exerted a Wnt-dependent effect on neocortical and hippocampal development. Thus, these results demonstrate that BAF complexes act as both activators and repressors to control global epigenetic and gene expression programs in late corticogenesis.

KMT2A and KMT2B Mediate Memory Function by Affecting Distinct Genomic Regions.

Kmt2a and Kmt2b are H3K4 methyltransferases of the Set1/Trithorax class. We have recently shown the importance of Kmt2b for learning and memory. Here, we report that Kmt2a is also important in memory formation. We compare the decrease in H3K4 methylation and de-regulation of gene expression in hippocampal neurons of mice with knockdown of either Kmt2a or Kmt2b. Kmt2a and Kmt2b control largely distinct genomic regions and different molecular pathways linked to neuronal plasticity. Finally, we show that the decrease in H3K4 methylation resulting from Kmt2a knockdown partially recapitulates the pattern previously reported in CK-p25 mice, a model for neurodegeneration and memory impairment. Our findings point to the distinct functions of even closely related histone-modifying enzymes and provide essential insight for the development of more efficient and specific epigenetic therapies against brain diseases.

Prediction of epitope-based peptides for the utility of vaccine development from fusion and glycoprotein of nipah virus using in silico approach.

This study aims to design epitope-based peptides for the utility of vaccine development by targeting glycoprotein G and envelope protein F of Nipah virus (NiV) that, respectively, facilitate attachment and fusion of NiV with host cells. Using various databases and tools, immune parameters of conserved sequence(s) from G and F proteins of different isolates of NiV were tested to predict probable epitope(s). Binding analyses of the peptides with MHC class-I and class-II molecules, epitope conservancy, population coverage, and linear B cell epitope prediction were analyzed. Predicted peptides interacted with seven or more MHC alleles and illustrated population coverage of more than 99% and 95%, for G and F proteins, respectively. The predicted class-I nonamers, SLIDTSSTI and EWISIVPNF, superimposed on the putative decameric B cell epitopes, were also identified as core sequences of the most probable class-II 15-mer peptides GPKVSLIDTSSTITI and EWISIVPNFILVRNT. These peptides were further validated for their binding to specific HLA alleles using in silico docking technique. Our in silico analysis suggested that the predicted epitopes, either GPKVSLIDTSSTITI or EWISIVPNFILVRNT, could be a better choice as universal vaccine component against NiV irrespective of different isolates which may elicit both humoral and cell-mediated immunity.