Analysis of the microglia transcriptome across the human lifespan using single cell RNA sequencing.

BACKGROUND: Microglia are tissue resident macrophages with a wide range of critically important functions in central nervous system development and homeostasis. METHOD: In this study, we aimed to characterize the transcriptional landscape of ex vivo human microglia across different developmental ages using cells derived from pre-natal, pediatric, adolescent, and adult brain samples. We further confirmed our transcriptional observations using ELISA and RNAscope. RESULTS: We showed that pre-natal microglia have a distinct transcriptional and regulatory signature relative to their post-natal counterparts that includes an upregulation of phagocytic pathways. We confirmed upregulation of CD36, a positive regulator of phagocytosis, in pre-natal samples compared to adult samples in situ. Moreover, we showed adult microglia have more pro-inflammatory signature compared to microglia from other developmental ages. We indicated that adult microglia are more immune responsive by secreting increased levels of pro-inflammatory cytokines in response to LPS treatment compared to the pre-natal microglia. We further validated in situ up-regulation of IL18 and CXCR4 in human adult brain section compared to the pre-natal brain section. Finally, trajectory analysis indicated that the transcriptional signatures adopted by microglia throughout development are in response to a changing brain microenvironment and do not reflect predetermined developmental states. CONCLUSION: In all, this study provides unique insight into the development of human microglia and a useful reference for understanding microglial contribution to developmental and age-related human disease.

Regional and age-related diversity of human mature oligodendrocytes.

Morphological and emerging molecular studies have provided evidence for heterogeneity within the oligodendrocyte population. To address the regional and age-related heterogeneity of human mature oligodendrocytes (MOLs) we applied single-cell RNA sequencing to cells isolated from cortical/subcortical, subventricular zone brain tissue samples, and thoracolumbar spinal cord samples. Unsupervised clustering of cells identified transcriptionally distinct MOL subpopulations across regions. Spinal cord MOLs, but not microglia, exhibited cell-type-specific upregulation of immune-related markers compared to the other adult regions. SVZ MOLs showed an upregulation of select number of development-linked transcription factors compared to other regions; however, pseudotime trajectory analyses did not identify a global developmental difference. Age-related analysis of cortical/subcortical samples indicated that pediatric MOLs, especially from under age 5, retain higher expression of genes linked to development and to immune activity with pseudotime analysis favoring a distinct developmental stage. Our regional and age-related studies indicate heterogeneity of MOL populations in the human CNS that may reflect developmental and environmental influences.

Diverse injury responses of human oligodendrocyte to mediators implicated in multiple sclerosis.

Early multiple sclerosis lesions feature relative preservation of oligodendrocyte cell bodies with dying back retraction of their myelinating processes. Cell loss occurs with disease progression. Putative injury mediators include metabolic stress (low glucose/nutrient), pro-inflammatory mediators (interferon γ and tumour necrosis factor α), and excitotoxins (glutamate). Our objective was to compare the impact of these disease relevant mediators on the injury responses of human mature oligodendrocytes. In the current study, we determined the effects of these mediators on process extension and survival of human brain derived mature oligodendrocytes in vitro and used bulk RNA sequencing to identify distinct effector mechanisms that underlie the responses. All mediators induced significant process retraction of the oligodendrocytes in dissociated cell culture. Only metabolic stress (low glucose/nutrient) conditions resulted in delayed (4-6 days) non-apoptotic cell death. Metabolic effects were associated with induction of the integrated stress response, which can be protective or contribute to cell injury dependent on its level and duration of activation. Addition of Sephin1, an agonist of the integrated stress response induced process retraction under control conditions and further enhanced retraction under metabolic stress conditions. The antagonist ISRIB restored process outgrowth under stress conditions, and if added to already stressed cells, reduced delayed cell death and prolonged the period in which recovery could occur. Inflammatory cytokine functional effects were associated with activation of multiple signalling pathways (including Jak/Stat-1) that regulate process outgrowth, without integrated stress response induction. Glutamate application produced limited transcriptional changes suggesting a contribution of effects directly on cell processes. Our comparative studies indicate the need to consider both the specific injury mediators and the distinct cellular mechanisms of responses to them by human oligodendrocytes to identify effective neuroprotective therapies for multiple sclerosis.

Human Oligodendrocyte Myelination Potential; Relation to Age and Differentiation.

OBJECTIVE: Myelin regeneration in the human central nervous system relies on progenitor cells within the tissue parenchyma, with possible contribution from previously myelinating oligodendrocytes (OLs). In multiple sclerosis, a demyelinating disorder, variables affecting remyelination efficiency include age, severity of initial injury, and progenitor cell properties. Our aim was to investigate the effects of age and differentiation on the myelination potential of human OL lineage cells. METHODS: We derived viable primary OL lineage cells from surgical resections of pediatric and adult brain tissue. Ensheathment capacity using nanofiber assays and transcriptomic profiles from RNA sequencing were compared between A2B5+ antibody-selected progenitors and mature OLs (non-selected cells). RESULTS: We demonstrate that pediatric progenitor and mature cells ensheathed nanofibers more robustly than did adult progenitor and mature cells, respectively. Within both age groups, the percentage of fibers ensheathed and ensheathment length per fiber were greater for A2B5+ progenitors. Gene expression of OL progenitor markers PDGFRA and PTPRZ1 were higher in A2B5+ versus A2B5- cells and in pediatric A2B5+ versus adult A2B5+ cells. The p38 MAP kinases and actin cytoskeleton-associated pathways were upregulated in pediatric cells; both have been shown to regulate OL process outgrowth. Significant upregulation of "cell senescence" genes was detected in pediatric samples; this could reflect their role in development and the increased susceptibility of pediatric OLs to activating cell death responses to stress. INTERPRETATION: Our findings identify specific biological pathways relevant to myelination that are differentially enriched in human pediatric and adult OL lineage cells and suggest potential targets for remyelination enhancing therapies. ANN NEUROL 2022;91:178-191.

Age-related injury responses of human oligodendrocytes to metabolic insults: link to BCL-2 and autophagy pathways.

Myelin destruction and oligodendrocyte (OL) death consequent to metabolic stress is a feature of CNS disorders across the age spectrum. Using cells derived from surgically resected tissue, we demonstrate that young (

Daily Physical Activity in Patients With COPD After Hospital Discharge in a Minority Population.

BACKGROUND: Low physical activity in patients with chronic obstructive pulmonary disease (COPD) is associated with increased morbidity and mortality. To inform the design of a home-based physical activity promotion program for patients with COPD recently discharged from a minority-serving hospital, we conducted a cohort study to evaluate objectively measured daily physical activity and patient-reported outcomes. METHODS: This was a 12-week prospective cohort study of patients with a physician diagnosis of COPD recently hospitalized (≤ 12 weeks) for respiratory symptoms. Daily physical activity was recorded using wrist-based and "clip-on" pedometers, and analyzed as mean daily step counts averaged over 7 days. RESULTS: Twenty-two patients were enrolled a median (interquartile range, [IQR]) of 14 (7 to 29) days after hospital discharge. The median daily step count (IQR) in the first week after enrollment (week 1) was 3710 (1565 to 5129) steps. The median within-person change in daily step count (IQR) from week 1 to week 12 was 314 (-30 to 858) steps (p=0.28). Within-person correlation of week-to-week daily step counts was high (r ≥ 0.75). Time from hospital discharge to enrollment was not correlated with mean daily step counts on week 1 (r= -0.13) and only weakly correlated with change in mean daily step counts from week 1 to week 12 (r=0.37). CONCLUSIONS: Daily physical activity was variable in this cohort of recently hospitalized patients with COPD, but with little within-person change over a 12-week period. These observations highlight the need for flexible physical activity promotion programs addressing the needs of a heterogeneous patient population.

Organotypic and primary neural cultures as models to assess effects of different gold nanostructures on glia and neurons.

Gold nanoparticles (AuNP) have unique physicochemical properties and have been used as delivery vehicles, contrast agents, and therapeutic compounds. Although the effects of AuNPs on peripheral tissues and immortalized cell lines have been extensively characterized, their effects on the central nervous system (CNS) are predominantly unknown. The main objective of the current study was to evaluate how AuNPs of varying sizes (1-100 nm), shapes (clusters, spheres, rods, flowers), and surfaces impact synaptic structures in the hippocampus, a brain structure often affected in neurodegeneration. Using a combination of organotypic hippocampal, as well as, primary neuronal, glial, and astrocytic cultures, we examined AuNPs impact on hippocampal dendritic spine density, internalization in various neural cells, and lysosomal status in astrocytes. Considering that neurons interact with astrocytes, and that lysosomes play a role in dendritic spine status, transcription factor TFEB and abundance of lysosomal marker, LAMP1 were evaluated. Both biomarkers were significantly increased in astrocytes exposed to AuNPs, suggesting that AuNPs not only enter lysosomes, but also increase lysosome biogenesis. Results from our studies show that AuNPs with poly(ethylene glycol) (AuNPs-PEG) or glutathione (AuNP-GSH) surfaces do not substantially decrease hippocampal dendritic spine density. Conversely, AuNPs coated with the detergent, CTAB, significantly decreased total spine density. Interestingly small gold nanoclusters (Au15(SG)13) with GSH reduced spine density, whereas larger gold nanoclusters (Au25(SG)18) with the same ligand did not. Thus, assessment of dendritic morphology, spine densities can reveal subtler changes of neural cells than cell death when exposed to nanoparticles, including AuNPs.

Loss of human ICOSL results in combined immunodeficiency.

Primary immunodeficiencies represent naturally occurring experimental models to decipher human immunobiology. We report a patient with combined immunodeficiency, marked by recurrent respiratory tract and DNA-based viral infections, hypogammaglobulinemia, and panlymphopenia. He also developed moderate neutropenia but without prototypical pyogenic infections. Using whole-exome sequencing, we identified a homozygous mutation in the inducible T cell costimulator ligand gene (ICOSLG; c.657C>G; p.N219K). Whereas WT ICOSL is expressed at the cell surface, the ICOSLN219K mutation abrogates surface localization: mutant protein is retained in the endoplasmic reticulum/Golgi apparatus, which is predicted to result from deleterious conformational and biochemical changes. ICOSLN219K diminished B cell costimulation of T cells, providing a compelling basis for the observed defect in antibody and memory B cell generation. Interestingly, ICOSLN219K also impaired migration of lymphocytes and neutrophils across endothelial cells, which normally express ICOSL. These defects likely contributed to the altered adaptive immunity and neutropenia observed in the patient, respectively. Our study identifies human ICOSLG deficiency as a novel cause of a combined immunodeficiency.