Probes for INS

Ischemic injury occurs when the brain is deprived of blood flow, preventing cells from receiving essential nutrients. The injury core is the brain region directly deprived and is surrounded by the peri-infarct area, the region with recovery potential. In the peri-infarct area neurons undergo acute loss of dendritic spines, which modifies synaptic plasticity and determines neuronal survival. Astrocytes can be protective or detrimental to the ischemic injury response depending on the specific stage, yet we lack clear understanding of the underlying mechanisms. Chordin-like 1 (Chrdl1) is an astrocyte-secreted protein that promotes synaptic maturation and limits experience-dependent plasticity in the mouse visual cortex. Given this plasticity-limiting function we asked if Chrdl1 regulates the response to ischemic injury, modelled using photothrombosis (PT). We find that Chrdl1 mRNA is upregulated in astrocytes in the peri-infarct area in both acute and sub-acute phases post-PT. To determine the impact of increased Chrdl1 on the response to PT we analyzed Chrdl1 knock-out mice. We find that absence of Chrdl1 prevents ischemia-induced spine loss in the peri-infarct area and reduces cell death in the core, without impacting gliosis. These findings highlight the important role of astrocyte-secreted proteins in regulating structural plasticity in response to brain ischemic injuries.

Astroglial cells are key players in the development and maintenance of neurons and neuronal networks. Astroglia express steroid hormone receptors and show rapid responses to hormonal manipulations. However, despite important sex differences in the cortex and hippocampus, few studies have examined sex differences in astroglial cells in telencephalic development. To characterize the cortical astroglial translatome in male and female mice across postnatal development, we use translating ribosome affinity purification together with RNA sequencing and immunohistochemistry to phenotype astroglia at six developmental time points. Overall, we find two distinct astroglial phenotypes between early (P1-P7) and late development (P14-adult), independent of sex. We also find sex differences in gene expression patterns across development that peak at P7 and appear to result from males reaching a mature astroglial phenotype earlier than females. These developmental sex differences could have an impact on the construction of neuronal networks and windows of vulnerability to perturbations and disease.

In the developing brain, immature synapses contain calcium-permeable AMPA glutamate receptors (AMPARs) that are subsequently replaced with GluA2-containing calcium-impermeable AMPARs as synapses stabilize and mature. Here, we show that this essential switch in AMPARs and neuronal synapse maturation is regulated by astrocytes. Using biochemical fractionation of astrocyte-secreted proteins and mass spectrometry, we identified that astrocyte-secreted chordin-like 1 (Chrdl1) is necessary and sufficient to induce mature GluA2-containing synapses to form. This function of Chrdl1 is independent of its role as an antagonist of bone morphogenetic proteins (BMPs). Chrdl1 expression is restricted to cortical astrocytes in vivo, peaking at the time of the AMPAR switch. Chrdl1 knockout (KO) mice display reduced synaptic GluA2 AMPARs, altered kinetics of synaptic events, and enhanced remodeling in an in vivo plasticity assay. Studies have shown that humans with mutations in Chrdl1 display enhanced learning. Thus astrocytes, via the release of Chrdl1, promote GluA2-dependent synapse maturation and thereby limit synaptic plasticity.

Genomic analyses have revealed heterogeneity among glial progenitor cells (GPCs), but the compartment selectivity of human GPCs (hGPCs) is unclear. Here, we asked if GPCs of human grey and white brain matter are distinct in their architecture and associated gene expression. RNA profiling of NG2-defined hGPCs derived from adult human neocortex and white matter differed in their expression of genes involved in Wnt, NOTCH, BMP and TGFβ signaling, suggesting compartment-selective biases in fate and self-renewal. White matter hGPCs over-expressed the BMP antagonists BAMBI and CHRDL1, suggesting their tonic suppression of astrocytic fate relative to cortical hGPCs, whose relative enrichment of cytoskeletal genes presaged their greater morphological complexity. In human glial chimeric mice, cortical hGPCs assumed larger and more complex morphologies than white matter hGPCs, and both were more complex than their mouse counterparts. These findings suggest that human grey and white matter GPCs comprise context-specific pools with distinct functional biases.

Smooth muscle cells (SMCs) execute important physiological functions in numerous vital organ systems, including the vascular, gastrointestinal, respiratory, and urogenital tracts. SMC differ morphologically and functionally at these different anatomical locations, but the molecular underpinnings of the differences remain poorly understood. Here, using deep single-cell RNA sequencing combined with in situ gene and protein expression analysis in four murine organs-heart, aorta, lung, and colon-we identify a molecular basis for high-level differences among vascular, visceral, and airway SMC, as well as more subtle differences between, for example, SMC in elastic and muscular arteries and zonation of elastic artery SMC along the direction of blood flow. Arterial SMC exhibit extensive organotypic heterogeneity, whereas venous SMC are similar across organs. We further identify a specific SMC subtype within the pulmonary vasculature. This comparative SMC cross-organ resource offers insight into SMC subtypes and their specific functions.

Wnt and Rspondin (RSPO) signaling drives proliferation, and bone morphogenetic protein inhibitors (BMPi) impede differentiation, of intestinal stem cells (ISCs). Here, we identify the mouse ISC niche as a complex, multi-layered structure that encompasses distinct mesenchymal and smooth muscle populations. In young and adult mice, diverse sub-cryptal cells provide redundant ISC-supportive factors; few of these are restricted to single cell types. Niche functions refine during postnatal crypt morphogenesis, in part to oppose the dense aggregation of differentiation-promoting BMP+ sub-epithelial myofibroblasts at crypt-villus junctions. Muscularis mucosae, a specialized muscle layer, first appears during this period and supplements neighboring RSPO and BMPi sources. Components of this developing niche are conserved in human fetuses. The in vivo ablation of mouse postnatal smooth muscle increases BMP signaling activity, potently limiting a pre-weaning burst of crypt fission. Thus, distinct and progressively specialized mesenchymal cells together create the milieu that is required to propagate crypts during rapid organ growth and to sustain adult ISCs.

Helicobacter pylori causes gastric inflammation, gland hyperplasia and is linked to gastric cancer. Here, we studied the interplay between gastric epithelial stem cells and their stromal niche under homeostasis and upon H. pylori infection. We find that gastric epithelial stem cell differentiation is orchestrated by subsets of stromal cells that either produce BMP inhibitors in the gland base, or BMP ligands at the surface. Exposure to BMP ligands promotes a feed-forward loop by inducing Bmp2 expression in the epithelial cells themselves, enforcing rapid lineage commitment to terminally differentiated mucous pit cells. H. pylori leads to a loss of stromal and epithelial Bmp2 expression and increases expression of BMP inhibitors, promoting self-renewal of stem cells and accumulation of gland base cells, which we mechanistically link to IFN-γ signaling. Mice that lack IFN-γ signaling show no alterations of BMP gradient upon infection, while exposure to IFN-γ resembles H. pylori-driven mucosal responses.