Despite advances in acute care, ischemic stroke remains a major cause of long-term disability. Approaches targeting both neuronal and glial responses are needed to enhance recovery and improve long-term outcome. The complement C3a receptor (C3aR) is a regulator of inflammation with roles in neurodevelopment, neural plasticity, and neurodegeneration. Using mice lacking C3aR (C3aR^–/–) and mice overexpressing C3a in the brain, we uncovered 2 opposing effects of C3aR signaling on functional recovery after ischemic stroke: inhibition in the acute phase and facilitation in the later phase. Peri-infarct astrocyte reactivity was increased and density of microglia reduced in C3aR^–/– mice; C3a overexpression led to the opposite effects. Pharmacological treatment of wild-type mice with intranasal C3a starting 7 days after stroke accelerated recovery of motor function and attenuated astrocyte reactivity without enhancing microgliosis. C3a treatment stimulated global white matter reorganization, increased peri-infarct structural connectivity, and upregulated Igf1 and Thbs4 in the peri-infarct cortex. Thus, C3a treatment from day 7 after stroke exerts positive effects on astrocytes and neuronal connectivity while avoiding the deleterious consequences of C3aR signaling during the acute phase. Intranasal administration of C3aR agonists within a convenient time window holds translational promise to improve outcome after ischemic stroke.

The role of nonneuronal cells in the resolution of cerebral ischemia remains to be fully understood. To decode key molecular and cellular processes that occur after ischemia, we performed spatial and single-cell transcriptomic profiling of the male mouse brain during the first week of injury. Cortical gene expression was severely disrupted, defined by inflammation and cell death in the lesion core, and glial scar formation orchestrated by multiple cell types on the periphery. The glial scar was identified as a zone with intense cell–cell communication, with prominent ApoE-Trem2 signaling pathway modulating microglial activation. For each of the three major glial populations, an inflammatory-responsive state, resembling the reactive states observed in neurodegenerative contexts, was observed. The recovered spectrum of ischemia-induced oligodendrocyte states supports the emerging hypothesis that oligodendrocytes actively respond to and modulate the neuroinflammatory stimulus. The findings are further supported by analysis of other spatial transcriptomic datasets from different mouse models of ischemic brain injury. Collectively, we present a landmark transcriptomic dataset accompanied by interactive visualization that provides a comprehensive view of spatiotemporal organization of processes in the postischemic mouse brain.

Reverse transcription quantitative PCR (RT-qPCR) has delivered significant insights in understanding the gene expression landscape. Thanks to its precision, sensitivity, flexibility, and cost effectiveness, RT-qPCR has also found utility in advanced single-cell analysis. Single-cell RT-qPCR now represents a well-established method, suitable for an efficient screening prior to single-cell RNA sequencing (scRNA-Seq) experiments, or, oppositely, for validation of hypotheses formulated from high-throughput approaches. Here, we aim to provide a comprehensive summary of the scRT-qPCR method by discussing the limitations of single-cell collection methods, describing the importance of reverse transcription, providing recommendations for the preamplification and primer design, and summarizing essential data processing steps. With the detailed protocol attached in the appendix, this tutorial provides a set of guidelines that allow any researcher to perform scRT-qPCR measurements of the highest standard.

NEUROD1 is a transcription factor that helps maintain a mature phenotype of pancreatic β cells. Disruption ofNeurod1during pancreatic development causes severe neonatal diabetes; however, the exact role of NEUROD1 in the differentiation programs of endocrine cells is unknown. Here, we report a crucial role of the NEUROD1 regulatory network in endocrine lineage commitment and differentiation. Mechanistically, transcriptome and chromatin landscape analyses demonstrate thatNeurod1inactivation triggers a downregulation of endocrine differentiation transcription factors and upregulation of non-endocrine genes within theNeurod1-deficient endocrine cell population, disturbing endocrine identity acquisition.Neurod1deficiency altered the H3K27me3 histone modification pattern in promoter regions of differentially expressed genes, which resulted in gene regulatory network changes in the differentiation pathway of endocrine cells, compromising endocrine cell potential, differentiation, and functional properties.

The role of non-neuronal cells in the resolution of cerebral ischemia remains to be fully understood. To decode key cellular processes that occur after ischemia, we performed spatial and single-cell transcriptomic profiling of mouse brain tissue during the first week of injury. Cortical gene expression was severely disrupted, being defined by inflammation and cell death in the lesion core, and glial scar formation on the periphery. For each of the three major glial populations, an inflammatory-responsive state, resembling the reactive states observed in neurodegenerative contexts, was documented. The recovered spectrum of ischemia-induced oligodendrocyte states supports the emerging hypothesis that oligodendrocytes actively respond to and modulate the neuroinflammatory stimulus. Thus, we present a landmark transcriptomic dataset that provides a comprehensive view of spatiotemporal organization of processes in the post-ischemic brain and documents the conservation of glial response in CNS pathology.

Background: Recent technical advances allowing quantification of RNA from single cells are revolutionizing biology and medicine. Currently, almost all single-cell transcriptomic protocols rely on conversion of RNA to cDNA by reverse transcription (RT). However, RT is recognized as highly limiting step due to its inherent variability and suboptimal sensitivity, especially at minute amounts of RNA. Primary factor influencing RT outcome is reverse transcriptase (RTase). Recently, several new RTases with potential to decrease the loss of information during RT have been developed, but the thorough assessment of their performance is missing. Methods: We have compared the performance of 11 RTases in RT-qPCR on single-cell and 100-cell bulk templates using two priming strategies: conventional mixture of random hexamers with oligo(dT)s and reduced concentration of oligo(dT)s mimicking common single-cell RNA-Seq library preparation protocols. Based on the performance, two RTases were further tested in high-throughput single-cell experiment. Results: All RTases tested reverse transcribed low-concentration templates with high accuracy ( R ² > 0.9445 ) but variable reproducibility (median CV _RT = 40.1 %). The most pronounced differences were found in the ability to capture rare transcripts (0 - 90% reaction positivity rate) as well as in the rate of RNA conversion to cDNA (7.3 - 124.5 % absolute yield). Finally, RTase performance and reproducibility across all tested parameters were compared using Z-scores and validity of obtained results was confirmed in a single-cell model experiment. The better performing RTase provided higher positive reaction rate and expression levels and improved resolution in clustering analysis. Conclusions: We performed a comprehensive comparison of 11 RTases in low RNA concentration range and identified two best-performing enzymes (Maxima H-; SuperScript IV). We found that using better-performing enzyme (Maxima H-) over commonly-used below-average performer (SuperScript II) increases the sensitivity of single-cell experiment. Our results provide a reference for the improvement of current single-cell quantification protocols.