Probes for CX3CR1

Cerebral small vessel disease (cSVD) constitutes a major risk factor for dementia. Monocytes play important roles in cerebrovascular disorders. Herein, we aimed to investigate the contribution of non-classical C-X3-C motif chemokine receptor (CX3CR)1 monocytes to cSVD pathobiology and therapy. To this end, we generated chimeric mice in which CX3CR1 in non-classical monocytes was either functional (CX3CR1GFP/+) or dysfunctional (CX3CR1GFP/GFP). cSVD was induced in mice via the micro-occlusion of cerebral arterioles, and novel immunomodulatory approaches targeting CX3CR1 monocyte production were used. Our findings demonstrate that CX3CR1GFP/+ monocytes transiently infiltrated the ipsilateral hippocampus and were recruited to the microinfarcts 7 days after cSVD, inversely associated with neuronal degeneration and blood-brain barrier (BBB) disruption. Dysfunctional CX3CR1GFP/GFP monocytes failed to infiltrate the injured hippocampus and were associated with exacerbated microinfarctions and accelerated cognitive decline, accompanied with an impaired microvascular structure. Pharmacological stimulation of CX3CR1GFP/+ monocyte generation attenuated neuronal loss and improved cognitive functions by promoting microvascular function and preserving cerebral blood flow (CBF). These changes were associated with elevated levels of pro-angiogenic factors and matrix stabilizers in the blood circulation. The results indicate that non-classical CX3CR1 monocytes promote neurovascular repair after cSVD and constitute a promising target for the development of new therapies.

The multilayered meninges surrounding the brain and spinal cord harbor distinct immune cell populations with prominent roles in health and diseases. Here we present an optimized protocol for RNA fluorescence in situ hybridization (RNA FISH) in meningeal whole mounts, allowing the visualization of gene expression. We also describe the combination of this protocol with immunohistochemistry for simultaneous visualization of mRNA and proteins. This protocol can be used for assessing spatial gene expression within the meninges.

Impairment of microglial clearance activity contributes to beta-amyloid (A?) pathology in Alzheimer's disease (AD). While the transcriptome profile of microglia directs microglial functions, how the microglial transcriptome can be regulated to alleviate AD pathology is largely unknown. Here, we show that injection of interleukin (IL)-33 in an AD transgenic mouse model ameliorates A? pathology by reprogramming microglial epigenetic and transcriptomic profiles to induce a microglial subpopulation with enhanced phagocytic activity. These IL-33-responsive microglia (IL-33RMs) express a distinct transcriptome signature that is highlighted by increased major histocompatibility complex class II genes and restored homeostatic signature genes. IL-33-induced remodeling of chromatin accessibility and PU.1 transcription factor binding at the signature genes of IL-33RM control their transcriptome reprogramming. Specifically, disrupting PU.1-DNA interaction abolishes the microglial state transition and A? clearance that is induced by IL-33. Thus, we define a PU.1-dependent transcriptional pathway that drives the IL-33-induced functional state transition of microglia, resulting in enhanced A? clearance

Kidneys are thought to express eight different connexin isoforms (i.e., Cx 26, 30, 32, 37, 40, 43, 45, and 46), which form either hemichannels or gap junctions serving to intercellular communication and functional synchronization. Proper function of connexins has already been shown to be crucial for regulation of renal hemodynamics and renin secretion, and there is also growing evidence for connexins to play a role in pathologic conditions such as renal fibrosis or diabetic nephropathy. Therefore, exact intrarenal localization of the different connexin isoforms gains particular interest. Until now intrarenal expression of connexins has mainly been examined by immunohistochemistry, which in part generated conflicting results depending on antibodies and fixation protocols used. In this work, we used fluorescent RNAscope as an alternative technical approach to localize renal connexin mRNAs in healthy mouse kidneys. Addition of RNAscope probes for cell type specific mRNAs was used to assign connexin mRNA signals to specific cell types. We hereby found Cx26 mRNA strongly expressed in proximal tubules, Cx30 mRNA was selectively detected in the urothelium, and Cx32 mRNA was found in proximal tubules and to a lesser extent also in collecting ducts. Cx37 mRNA was mainly associated with vascular endothelium, Cx40 mRNA was largely found in glomerular mesangial and less in vascular endothelial cells, Cx43 mRNA was sparsely expressed by interstitial cells of all kidney zones, and Cx45 mRNA was predominantly found in smooth muscle cell layers of both blood vessels and ureter as well as in mesangial and interstitial (fibroblastic) cells. Cx46 mRNA could not be detected. In summary our results essentially confirm previous data on connexin expression in the renal vasculature and in glomeruli. In addition, they demonstrate strong connexin gene expression in proximal tubules, and they suggest significant connexin expression in resident tubulointerstitial cells.