Microglial activation has been closely associated with accelerated ALS disease progression. However, specific microglial pathways that regulate microglial activation and ALS disease progression remain limitedly understood. Here, we determined the role of Clec7a (or Dectin-1), a core signature gene of disease-associated microglia (DAM) in ALS, in regulating microglial activation and ALS disease progression. Our spinal cord scRNA-Seq results found that Clec7a deficiency specifically attenuated microglial neuroimmune gene expression in SOD1G93A mice and human ALS. In addition, in vivo two-photon imaging of human (h) TDP43 phagocytosis by microglia in the cortex showed that Clec7a deficiency promotes microglial phagocytosis of pathological hTDP43 by enhancing microglial process dynamics. Subsequent survival analysis further showed that selective deletion of Clec7a in microglia mitigates motor neuron degeneration and delays disease progression in SOD1G93A ALS mice. Together, our results establish that Clec7a is a key regulator in shaping disease microglial functions and promotes disease progression in ALS.

Spinal cord injury (SCI) leads to severe neurological and functional impairments, yet reliable biomarkers for assessing injury severity and predicting recovery remain limited. Cerebrospinal fluid (CSF) is in direct contact with the central nervous system and provides a valuable source for detecting molecular changes after SCI. Although exosomal microRNAs (miRNAs) and proteins are increasingly recognized as mediators of intercellular communication, the role of human CSF exosomes in SCI has not been systematically investigated. To identify exosome-based biomarkers and potential therapeutic targets, we analyzed CSF and serum exosomes from patients with acute SCI using RNA sequencing and proteomic profiling. Weighted gene co-expression network analysis identified 6 gene modules significantly associated with injury severity and neurological recovery at 3 months. Proteomic analysis revealed a 5-protein panel that distinguished complete from incomplete SCI and a 4-protein panel that predicted neurological improvement. Additionally, 15 CSF-specific and 9 serum-specific exosomal miRNAs were identified independent of injury severity. Among 10 tested miRNAs associated with neurological recovery, 7 regulated astrocyte proliferation, and 6 promoted neurite extension and synapse formation. Overall, this study provides a comprehensive characterization of CSF exosomal miRNAs and proteins in human SCI and identifies molecular signatures associated with injury severity and recovery.

Abstract Retinitis Pigmentosa is a chronic retinal degenerative disease characterized by the gradual loss of rod, and later, cone photoreceptors until the individual is completely blind. Regeneration of photoreceptors from endogenous progenitor cells is a possible therapeutic approach, but mammals do not do this naturally. Mammalian models can be induced to generate retinal progenitors from Müller glial cells, but there has been limited success in rod photoreceptor specific regeneration. Unlike mammals, zebrafish have the natural ability to regenerate neurons after injury or disease and can provide insight into the molecular mechanisms of regeneration. In this study, we used a zebrafish model of Retinitis Pigmentosa to investigate the class of progenitors responsible for rod photoreceptor regeneration in the context of chronic disease. Using bioinformatic analyses of single-cell RNA sequencing datasets, we identified master regulator genes responsible for proliferation of retinal progenitors, differentiation of progenitors into rod photoreceptors, and maturation of the new rod photoreceptors. Using transient knockdown of gene expression in adult regenerating retina we determined that e2f1, e2f2, e2f3 and aurkb are critical for proliferation of progenitors, and prdm1a is critical for differentiation of progenitors into rod photoreceptors. This study provides a list of master regulators responsible for the specific regeneration of rod photoreceptors during chronic retinal degeneration.

Neuronal extracellular vesicles (microvesicles and exosomes) are emerging secreted vesicular signals that play important roles in the CNS. Currently, little is known about how glutamatergic signalling affects the subcellular localisation of exosome precursor intraluminal vesicles (ILVs), microRNA (miR) packaging into ILVs and in vivo spreading of neuronal EVs. By selectively labelling ILVs and exosomes (but not plasma membrane-derived MVs) with GFP-tagged human CD63 (hCD63-GFP) in cortical neurons, we found that glutamate stimulation significantly redistributes subcellular localisation of hCD63-GFP+ ILVs, especially decreasing its co-localisation with multi-vesicular body (MVB) marker Rab7 while substantially promoting EV secretion. Interestingly, glutamate stimulation only modestly alters EV miR profiles based on small RNA sequencing. Subsequent in vivo cortical neuronal DREADD activation leads to significantly more widespread hCD63-GFP+ area in hCD63-GFPf/+ mice, consistently supporting the stimulatory effect of glutamatergic activation on neuronal EV secretion and spreading. Moreover, in situ localisation of hCD63-GFP+ ILVs and hCD63-GFP+ secreted exosomes from specialised HB9+ and DAT+ neurons were also illustrated in the CNS. Taken together, our results demonstrated that glutamate activity stimulates neuronal exosome secretion and spreading in vitro and in vivo, but only modestly affects miR cargo packaging in neuronal exosomes.

Neuronal exosomes are emerging secreted signals that play important roles in the CNS. Currently little is known about how glutamatergic signaling affects the subcellular localization of exosome precursor intraluminal vesicles (ILVs), microRNA (miR) packaging into ILVs, and in vivo neuronal exosome spreading. By selectively labeling ILVs and exosomes with GFP-tagged human CD63 (hCD63-GFP) in cortical neurons, we found that glutamate stimulation significantly redistributes subcellular localization of hCD63-GFP+ ILVs especially decreases its co-localization with multi-vesicular body (MVB) marker Rab7 while substantially promoting exosome secretion. Interestingly, glutamate stimulation only modestly alters exosomal miR profiles based on small RNA sequencing. Subsequent in vivo cortical neuronal DREADD activation leads to significantly more widespread hCD63-GFP+ area in hCD63-GFPf/+ mice, consistently supporting the stimulatory effect of glutamatergic activation on neuronal exosome secretion and spreading. Moreover, in situ localization of hCD63-GFP+ ILVs and secreted exosomes from specialized Hb9+ and DAT+ neurons were also illustrated in the CNS. ### Competing Interest Statement The authors have declared no competing interest.

Circadian rhythms are internal biological rhythms driving temporal tissue-specific, metabolic programs. Loss of the circadian transcription factor BMAL1 in the paraventricular nucleus (PVN) of the hypothalamus reveals its importance in metabolic rhythms, but its functions in individual PVN cells are poorly understood. Here, loss of BMAL1 in the PVN results in arrhythmicity of processes controlling energy balance and alters peripheral diurnal gene expression. BMAL1 chromatin immunoprecipitation sequencing (ChIP-seq) and single-nucleus RNA sequencing (snRNA-seq) reveal its temporal regulation of target genes, including oxytocin (OXT), and restoring circulating OXT peaks in BMAL1-PVN knockout (KO) mice rescues absent activity rhythms. While glutamatergic neurons undergo day/night changes in expression of genes involved in cell morphogenesis, astrocytes and oligodendrocytes show gene expression changes in cytoskeletal organization and oxidative phosphorylation. Collectively, our findings show diurnal gene regulation in neuronal and non-neuronal PVN cells and that BMAL1 contributes to diurnal OXT secretion, which is important for systemic diurnal rhythms.

Alzheimer's disease (AD) is characterized by the presence of two hallmark pathologies: the accumulation of Amyloid beta (Aβ) and tau proteins in the brain. There is a growing body of evidence suggesting that astrocytes, a type of glial cell in the brain, play crucial roles in clearing Aβ and binding to tau proteins. However, due to the heterogeneity of astrocytes, the specific roles of different astrocyte subpopulations in response to Aβ and tau remain unclear. To enhance the understanding of astrocyte subpopulations in AD, we investigated astrocyte lineage cells based on single-nuclei transcriptomic data obtained from both human and mouse samples. We characterized the diversity of astrocytes and identified global and subpopulation-specific transcriptomic changes between control and AD samples. Our findings revealed the existence of a specific astrocyte subpopulation marked by low levels of GFAP and the presence of AQP4 and CD63 expression, which showed functional enrichment in Aβ clearance and tau protein binding, and diminished in AD. We verified this type of astrocytes in mouse models and in AD patient brain samples. Furthermore, our research also unveiled significant alterations of the ligand-receptor interactions between astrocytes and other cell types. These changes underscore the complex interplay between astrocytes and neighboring cells in the context of AD. Overall, our work gives insights into astrocyte heterogeneity in the context of AD and reveals a distinct astrocyte subpopulation that holds potential for therapeutic interventions in AD. Targeting specific astrocyte subpopulations may offer new avenues for the development of novel treatments for AD.

BACKGROUND:Normalization is a critical step in the analysis of single-cell RNA-sequencing (scRNA-seq) datasets. Its main goal is to make gene counts comparable within and between cells. To do so, normalization methods must account for technical and biological variability. Numerous normalization methods have been developed addressing different sources of dispersion and making specific assumptions about the count data.MAIN BODY:The selection of a normalization method has a direct impact on downstream analysis, for example differential gene expression and cluster identification. Thus, the objective of this review is to guide the reader in making an informed decision on the most appropriate normalization method to use. To this aim, we first give an overview of the different single cell sequencing platforms and methods commonly used including isolation and library preparation protocols. Next, we discuss the inherent sources of variability of scRNA-seq datasets. We describe the categories of normalization methods and include examples of each. We also delineate imputation and batch-effect correction methods. Furthermore, we describe data-driven metrics commonly used to evaluate the performance of normalization methods. We also discuss common scRNA-seq methods and toolkits used for integrated data analysis.CONCLUSIONS:According to the correction performed, normalization methods can be broadly classified as within and between-sample algorithms. Moreover, with respect to the mathematical model used, normalization methods can further be classified into: global scaling methods, generalized linear models, mixed methods, and machine learning-based methods. Each of these methods depict pros and cons and make different statistical assumptions. However, there is no better performing normalization method. Instead, metrics such as silhouette width, K-nearest neighbor batch-effect test, or Highly Variable Genes are recommended to assess the performance of normalization methods.

>Glial progenitor cells were reported to have the capacity to generate various types of cells in both the central nervous system(CNS) and peripheral nervous system.Glial progenitor cells can respond to diverse environmental signals and transform into distinct populations,each serving specific functions.Notably,the adult spinal cord hosts various populations of glial progenitors,a region integral to the central nervous system.During development,glial progenitors express glial fibrillary acidic protein(GFAP;Dimou and Gotz,2014).However,the specific identities of GFAP-expressing progenitors in the adult spinal cord were not thoroughly investigated.

Recent studies have revealed the heterogeneous nature of astrocytes; however, how diverse constituents of astrocyte-lineage cells are regulated in adult spinal cord after injury and contribute to regeneration remains elusive. We perform single-cell RNA sequencing of GFAP-expressing cells from sub-chronic spinal cord injury models and identify and compare with the subpopulations in acute-stage data. We find subpopulations with distinct functional enrichment and their identities defined by subpopulation-specific transcription factors and regulons. Immunohistochemistry, RNAscope experiments, and quantification by stereology verify the mo-lecular signature, location, and morphology of potential resident neural progenitors or neural stem cells in the adult spinal cord before and after injury and uncover the populations of the intermediate cells enriched in neuronal genes that could potentially transition into other subpopulations. This study has expanded the knowl-edge of the heterogeneity and cell state transition of glial progenitors in adult spinal cord before and after injury.

A hallmark of inherited retinal degenerative diseases such as retinitis pigmentosa (RP) is progressive structural and functional remodeling of the remaining retinal cells as photoreceptors degenerate. Extensive remodeling of the retina stands as a barrier for the successful implementation of strategies to restore vision. To understand the molecular basis of remodeling, we performed analyses of single-cell transcriptome data from adult zebrafish retina of wild type AB strain (WT) and a P23H mutant rhodopsin transgenic model of RP with continuous degeneration and regeneration. Retinas from both female and male fish were pooled to generate each library, combining data from both sexes. We provide a benchmark atlas of retinal cell type transcriptomes in zebrafish and insight into how each retinal cell type is affected in the P23H model. Oxidative stress is found throughout the retina, with increases in reliance on oxidative metabolism and glycolysis in the affected rods as well as cones, bipolar cells, and retinal ganglion cells. There is also transcriptional evidence for widespread synaptic remodeling and enhancement of glutamatergic transmission in the inner retina. Notably, changes in circadian rhythm regulation are detected in cones, bipolar cells, and retinal pigmented epithelium. We also identify the transcriptomic signatures of retinal progenitor cells and newly formed rods essential for the regenerative process. This comprehensive transcriptomic analysis provides a molecular road map to understand how the retina remodels in the context of chronic retinal degeneration with ongoing regeneration.

It is widely believed that hematopoiesis after birth is established by hematopoietic stem cells (HSCs) in the bone marrow and that HSC-independent hematopoiesis is limited only to primitive erythro-myeloid cells and tissue-resident innate immune cells arising in the embryo. Here, surprisingly, we find that significant percentages of lymphocytes are not derived from HSCs, even in 1-year-old mice. Instead, multiple waves of hematopoiesis occur from embryonic day 7.5 (E7.5) to E11.5 endothelial cells, which simultaneously produce HSCs and lymphoid progenitors that constitute many layers of adaptive T and B lymphocytes in adult mice. Additionally, HSC lineage tracing reveals that the contribution of fetal liver HSCs to peritoneal B-1a cells is minimal and that the majority of B-1a cells are HSC independent. Our discovery of extensive HSC-independent lymphocytes in adult mice attests to the complex blood developmental dynamics spanning the embryo-to-adult transition and challenges the paradigm of HSCs exclusively underpinning the postnatal immune system.

Inherited retinal degenerative diseases such as Retinitis Pigmentosa (RP) result in progressive loss of photoreceptors until an individual is completely blind. A hallmark of these diseases is progressive structural and functional remodeling of the remaining retinal neurons as rod photoreceptors are lost. While many studies focus on regenerative or bionic therapies to restore vision, extensive remodeling of retinal cell types throughout the course of retinal degenerative diseases stands as a barrier for successful implementation of these strategies. As a window onto the molecular basis of remodeling, we have performed a comparative analysis of single-cell transcriptome data from adult Zebrafish retina of wild-type and a P23H mutant rhodopsin model of RP. In addition to providing a benchmark atlas of retinal cell type transcriptomes in the wild-type adult Zebrafish retina, we find transcriptional changes in essentially all retinal cell types in the P23H model. Increased oxidative stress is evident not only in the rods but also in cones, retinal pigmented epithelium (RPE) and to a lesser extent in amacrine and bipolar cells. Metabolic changes increasing oxidative metabolism and glycolysis are found in rods and cones, while evidence of increased activity of the mitochondrial electron transport chain is found in retinal ganglion cells (RGCs). Evidence of synaptic remodeling is found throughout the retina, with changes to increase synaptic transmission in photoreceptors and bipolar cells, increased ionotropic glutamate receptors in amacrine and ganglion cells, and dendritic and axon remodeling throughout. Surprisingly, RPE, cones and bipolar cells in the P23H retinas also have increased expression of genes involved in circadian rhythm regulation. While this model system undergoes continuous regeneration, ongoing remodeling impacts the entire retina. This comprehensive transcriptomic analysis provides a molecular road map to understand how the retina remodels in the context of chronic retinal degeneration with ongoing regeneration.

Opioid abuse can cause brain alterations, including neuroinflammation and increased risk for ischemic stroke. Cancer studies show that opioids affect angiogenesis and endothelial cell proliferation. However, human brain studies are scarce, and the molecular mechanisms of opioid-induced neurotoxicity are unclear. This study comprehensively interrogated the transcriptomic and proteomic profiles of the dorsolateral prefrontal cortex (BA9) in subjects with opioid use disorder (OUD), to investigate the molecular effects of opioid abuse in the human brain.

Alzheimer's disease (AD) is a progressive neurodegenerative disease and is the most common cause of dementia in an aging population. The majority of research effort has focused on the role of neurons in neurodegeneration and current therapies have limited ability to slow disease progression. Recently more attention has been given to the role of astrocytes in the process of neurodegeneration. Specifically, reactive astrocytes have both advantageous and adverse effects during neurodegeneration. The ability to isolate and depict astrocyte phenotype has been challenging. However, with the recent development of single-cell sequencing technologies researchers are provided with the resource to delineate specific biomarkers associated with reactive astrocytes in AD. In this review, we will focus on the role of astrocytes in normal conditions and the pathological development of AD. We will further review recent developments in the understanding of astrocyte heterogeneity and associated biomarkers. A better understanding of astrocyte contributions and phenotypic changes in AD can ultimately lead to more effective therapeutic targets.

Spinal cord injury disrupts ascending and descending neural signals causing sensory and motor dysfunction. Neuromodulation with electrical stimulation is used in both clinical and research settings to induce neural plasticity and improve functional recovery following spinal trauma. However, the mechanisms by which electrical stimulation affects recovery remain unclear. In this study we examined the effects of cortical electrical stimulation following injury on transcription at several levels of the central nervous system. We performed a unilateral, incomplete cervical spinal contusion injury in rats and delivered stimulation for one week to the contralesional motor cortex to activate the corticospinal tract and other pathways. RNA was purified from bilateral subcortical white matter and 3 levels of the spinal cord. Here we provide the complete data set in the hope that it will be useful for researchers studying electrical stimulation as a therapy to improve recovery from the deficits associated with spinal cord injury.

Spinal cord injury (SCI) is one of the most devastating neural injuries without effective therapeutic solutions. Astrocytes are the predominant component of the scar. Understanding the complex contributions of reactive astrocytes to SCI pathophysiologies is fundamentally important for developing therapeutic strategies. We have studied the molecular changes in the injury environment and the astrocyte-specific responses by astrocyte purification from injured spinal cords from acute to chronic stages. In addition to protein-coding genes, we have systematically analyzed the expression profiles of long non-coding RNAs (lncRNAs) (>200 bp), which are regulatory RNAs that play important roles in the CNS. We have identified a highly conserved lncRNA, Zeb2os, and demonstrated using functional assays that it plays an important role in reactive astrogliosis through the Zeb2os/Zeb2/Stat3 axis. These studies provide valuable insights into the molecular basis of reactive astrogliosis and fill the knowledge gap regarding the function(s) of lncRNAs in astrogliosis and SCI.