Probes for INS

Somatosensory low threshold mechanoreceptors (LTMRs) sense innocuous mechanical forces, largely through specialized axon termini termed sensory nerve endings, where the mechanotransduction process initiates upon activation of mechanotransducers. In humans, a subset of sensory nerve endings is enlarged, forming bulb-like expansions, termed bulbous nerve endings. There is no in vitro human model to study these neuronal endings. Piezo2 is the main mechanotransducer found in LTMRs. Recent evidence shows that Piezo1, the other mechanotransducer considered absent in dorsal root ganglia (DRG), is expressed at low level in somatosensory neurons. We established a differentiation protocol to generate, from iPSC-derived neuronal precursor cells, human LTMR recapitulating bulbous sensory nerve endings and heterogeneous expression of Piezo1 and Piezo2. The derived neurons express LTMR-specific genes, convert mechanical stimuli into electrical signals and have specialized axon termini that morphologically resemble bulbous nerve endings. Piezo2 is concentrated within these enlarged axon termini. Some derived neurons express low level Piezo1, and a subset co-express both channels. Thus, we generated a unique, iPSCs-derived human model that can be used to investigate the physiology of bulbous sensory nerve endings, and the role of Piezo1 and 2 during mechanosensation.

Spatial gene expression, achieved classically through in situ hybridization, is a fundamental tool for topographic phenotyping of cell types in the nervous system. Newly developed techniques allow for visualization of multiple mRNAs at single-cell resolution and greatly expand the ability to link gene expression to tissue topography, yet there are challenges in efficient quantification and analysis of these high-dimensional datasets. We have therefore developed the single-cell automated multiplex pipeline for RNA (SCAMPR), facilitating rapid and accurate segmentation of neuronal cell bodies using a dual immunohistochemistry-RNAscope protocol and quantification of low- and high-abundance mRNA signals using open-source image processing and automated segmentation tools. Proof of principle using SCAMPR focused on spatial mapping of gene expression by peripheral (vagal nodose) and central (visual cortex) neurons. The analytical effectiveness of SCAMPR is demonstrated by identifying the impact of early life stress on gene expression in vagal neuron subtypes.

Cognitive impairment caused by systemic chemotherapy is a critical question that perplexes the effective implementation of clinical treatment, but related molecular events are poorly understood. Herein, we show that bortezomib exposure leads to microglia activation and cognitive impairment, this occurs along with decreased nuclear translocation of TFEB (transcription factor EB), which is linked to macroautophagy/autophagy disorder, STAT3 (signal transducer and activator of transcription 3) phosphorylation and IL23A (interleukin 23 subunit alpha) expression. Pharmacological enhancement of TFEB nuclear translocation by digoxin restores lysosomal function and reduces STAT3-dependent endothelial IL23A secretion. As a consequence, we found that brain endothelial-specific ablation of Il23a ameliorated both microglia activation and cognitive dysfunction. Thus, the endothelial TFEB-STAT3-IL23A axis in the brain represents a critical cellular event for initiating bortezomib-mediated aberrant microglial activation and synapse engulfment. Our results suggest the reversal of TFEB nuclear translocation may provide a novel therapeutic approach to prevent symptoms of cognitive dysfunction during clinical use of bortezomib.Abbreviations: AAV: adeno-associated virus; BBB: blood-brain barrier; BTZ: bortezomib; DG: digoxin; DGs: dentate gyrus; DLG4/PSD95: discs large MAGUK scaffold protein 4; HBMECs: human brain microvascular endothelial cells; HP: hippocampus; IL23A: interleukin 23 subunit alpha; MBVECs: mouse brain vascular endothelial cells; mPFC: medial prefrontal cortex; NORT: novel object recognition test; OLT: object location test; PLX5622: 6-fluoro-N-([5-fluoro-2-methoxypyridin-3-yl]methyl)-5-(5-methyl-1H-pyrrolo[2,3-b]pyridin-3- yl)methyl; PPP3/calcineurin: protein phosphatase 3; SBEs: STAT3 binding elements; shRNA: small hairpin RNA; SLC17A7/VGLUT1: solute carrier family 17 member 7; SLC32A1/VGAT: solute carrier family 32 member 1; STAT3: signal transducer and activator of transcription 3, TFEB: transcription factor EB; Ub: ubiquitin.