Probes for INS

The neurons in the rostral ventromedial medulla (RVM) play a major role in pain modulation. We have previously shown that early-life noxious bladder stimuli in rats resulted in an overall spinal GABAergic disinhibition and a long-lasting bladder/colon sensitization when tested in adulthood. However, the neuromolecular alterations within RVM neurons in the pathophysiology of early life bladder inflammation have not been elucidated. In this study, we have identified and characterized RVM neurons that are synaptically linked to the bladder and colon and examined the effect of neonatal bladder inflammation on molecular expressions of these neurons. A transient bladder inflammation was induced by intravesicular instillation of protamine sulfate and zymosan during postnatal days 14 through 16 (P14-16) followed by pseudorabies virus PRV-152 and PRV-614 injections into the bladder and colon, respectively, on postnatal day P60. Tissues were examined 96 hours post-inoculation for serotonergic, GABAergic, and enkephalinergic expressions using In situ Hybridization and/or Immunohistochemistry techniques. The results revealed that >50% of RVM neurons that are synaptically connected to the bladder (i.e., PRV-152+) were GABAergic, 40% enkephalinergic, and about 14% expressing serotonergic marker TpH2. Neonatal cystitis resulted in a significant increase in converging neurons in RVM receiving dual synaptic inputs from the bladder and colon. In addition, neonatal cystitis significantly downregulated GABA transporter VGAT with a concomitant increase in TpH2 expression in bladder-linked RVM neurons suggesting an alteration in supraspinal signaling. These alterations of synaptic connectivity and GABAergic/serotonergic expressions in RVM neurons may contribute to bladder pain modulation and cross-organ visceral sensitivity. This article is protected by

Receptor activator of NF-κB (RANK) expression is increased in podocytes of patients with diabetic nephropathy. However, the relevance of RANK to diabetic nephropathy pathobiology remains unclear. Here, to evaluate the role of podocyte RANK in the development of diabetic nephropathy, we generated a mouse model of podocyte-specific RANK depletion (RANK-/-Cre T), and a model of podocyte-specific RANK overexpression (RANK TG), and induced diabetes in these mice with streptozotocin. We found that podocyte RANK depletion alleviated albuminuria, mesangial matrix expansion, and basement membrane thickening, while RANK overexpression aggravated these indices in streptozotocin-treated mice. Moreover, streptozotocin-triggered oxidative stress was increased in RANK overexpression, but decreased in the RANK depleted mice. Particularly, the expression of NADPH oxidase 4, and its obligate partner, P22phox, were enhanced in RANK overexpression, but reduced in RANK depleted mice. In parallel, the transcription factor p65 was increased in the podocyte nuclei of RANK overexpressing mice but decreased in the RANK depleted mice. The relevant findings were largely replicated with high glucose-treated podocytes in vitro. Mechanistically, p65 could bind to the promoter regions of NADPH oxidase 4 and P22phox, and increased their respective gene promoter activity in podocytes, dependent on the levels of RANK. Taken together, these findings suggested that high glucose induced RANK in podocytes and caused the increase of NADPH oxidase 4 and P22phox via p65, possibly together with the cytokines TNF- α, MAC-2 and IL-1 β, resulting in podocyte injury. Thus, we found that podocyte RANK was induced in the diabetic milieu and RANK mediated the development of diabetic nephropathy, likely by promoting glomerular oxidative stress and proinflammatory cytokine production.

Background Sexual dimorphism in depression is well documented. Women and men differ in the prevalence, symptom presentation, and responses to antidepressant treatment. Methods Chronic social defeat stress protocol was used to induce depression-like behavior in mice. Multiplex cytokine assay was used to assess peripheral inflammation. RNA-seq was conducted to assess for gene expression regulation in the PFC. RNAscope combined with immunohistochemistry was used to identify cell-specific expression of CCL5 receptors in the brain. Results Characterization of peripheral inflammation in female mice revealed positive correlation between susceptibility and plasma levels of CCL5, but not with IL-6 which was associated with stress susceptibility in male mice. RNA-seq analysis of PFC revealed that CCR5, one of the major receptors for CCL5 was significantly higher in defeat stressed female mice compared to the control mice. However, this increase was not seen in stressed male mice. We found that the expression of CCR5 is mainly in the microglia. Treatment with CCR5 antagonist significantly attenuated CSDS-induced depression-like behavior in female mice. Conclusions Higher level of CCL5 was also reported in human subjects with MDD and higher levels of peripheral CCL5 in women compared to men. Cross-examination with human MDD RNA-seq data showed that in female MDD subjects, the level of CCR5 in the ventromedial PFC was 2.8 fold higher compared to the control subjects and this increase was not seen in male MDD subjects. The human data supports our finding, strongly implicating sexual dimorphic interactions between CCL5/CCR5 expression and depression. CCL5/CCR5 signaling may be potential target for treating female depression.

Significance Molecules regulated by neuronal activity are necessary for circuits to adapt to changing inputs. Specific classical major histocompatibility class I (MHCI) molecules play roles in circuit and synaptic plasticity, but the function of most members of this family remains unexplored in brain. Here, we show that a nonclassical MHCI molecule, Qa-1 (H2-T23), is expressed in a subset of excitatory neurons and regulated by visually driven activity in the cerebral cortex. Moreover, CD94/NKG2 heterodimers, cognate receptors for Qa-1, are expressed in microglia. A functional interaction between Qa-1 and CD94/NKG2 is necessary for regulating the magnitude of ocular dominance plasticity during the critical period in the visual cortex, implying an interaction in which activity-dependent changes in neurons may be monitored by microglia.

Obesity and high-fat diet (HFD) consumption result in hypothalamic inflammation and metabolic dysfunction. While the TLR4 activation by dietary fats is a well-characterized pathway involved in the neuronal and glial inflammation, the role of its accessory proteins in diet-induced hypothalamic inflammation remains unknown. Here, we demonstrate that the knockdown of TLR4-interactor with leucine-rich repeats (Tril), a functional component of TLR4, resulted in reduced hypothalamic inflammation, increased whole-body energy expenditure, improved the systemic glucose tolerance and protection from diet-induced obesity. The POMC-specific knockdown of Tril resulted in decreased body fat, decreased white adipose tissue inflammation and a trend toward increased leptin signaling in POMC neurons. Thus, Tril was identified as a new component of the complex mechanisms that promote hypothalamic dysfunction in experimental obesity and its inhibition in the hypothalamus may represent a novel target for obesity treatment.

The dorsal vagal complex (DVC) in the hindbrain, composed of the area postrema, nucleus of the solitary tract, and dorsal motor nucleus of the vagus, plays a critical role in modulating satiety. The incretins glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) act directly in the brain to modulate feeding, and receptors for both are expressed in the DVC. Given the impressive clinical responses to pharmacologic manipulation of incretin signaling, understanding the central mechanisms by which incretins alter metabolism and energy balance is of critical importance. Here, we review recent single-cell approaches used to detect molecular signatures of GLP-1 and GIP receptor-expressing cells in the DVC. In addition, we discuss how current advancements in single-cell transcriptomics, epigenetics, spatial transcriptomics, and circuit mapping techniques have the potential to further characterize incretin receptor circuits in the hindbrain.

Localization of mRNAs at the front of migrating cells is a widely used mechanism that functionally supports efficient cell movement. It is observed in single cells on two-dimensional surfaces, as well as in multicellular three-dimensional (3D) structures and in tissue in vivo. 3D multicellular cultures can reveal how the topology of the extracellular matrix and cell-cell contacts influence subcellular mRNA distributions. Here we describe a method for mRNA imaging in an inducible system of collective cancer cell invasion. MDA-MB-231 cancer cell spheroids are embedded in Matrigel, induced to invade, and processed to image mRNAs with single-molecule sensitivity. An analysis algorithm is used to quantify and compare mRNA distributions at the front of invasive leader cells. The approach can be easily adapted and applied to analyze RNA distributions in additional settings where cells polarize along a linear axis.

PURPOSE : ADOA is the most common inherited optic neuropathy, starting in the first decade of life and resulting in severe and progressive visual decline due to loss of RGCs. Most patients harbor loss-of-function mutations in the _OPA1 _gene that lead to haploinsufficiency. Reduced OPA1 protein levels result in impaired mitochondrial function in RGCs leading to cell death. Currently, there is no treatment for patients with ADOA. Targeted Augmentation of Nuclear Gene Output (TANGO) ASOs, such as STK-002, reduce the inclusion of a non-productive, alternatively spliced exon in _OPA1, _and leverage the wild-type allele to increase productive _OPA1_ mRNA and protein. We previously demonstrated that TANGO ASOs can increase OPA1 protein levels in human cell lines, rabbit retina, and ADOA patient fibroblasts. In this study, we evaluated ASO localization and OPA1 protein levels in the retina following intravitreal administration of STK-002 to NHPs.

Recent contradictory data has renewed discussion regarding the existence of adult hippocampal neurogenesis (AHN) in humans, i.e., the continued production of new neurons in the brain after birth. The present review revisits the debate of AHN in humans from a historical point of view in the face of contradictory evidence, analyzing the methods employed to investigate this phenomenon. Thus, to date, of the 57 studies performed in humans that we reviewed, 84% (48) concluded in favor of the presence of newborn neurons in the human adult hippocampus. Besides quality of the tissue (such as postmortem intervals below 26hours as well as tissue conservation and fixation), considerations for assessing and quantify AHN in the human brain require the use of stereology and toxicological analyses of clinical data of the patient.

Bladder Cancer is the 9th most common malignancy in the world. Transitional cell carcinoma (TCC) is the most common of bladder cancers, occurring in 90% of cases. There has been no great model established to study TCC in vitro. In this study, we explore urine-derived, 3-dimensional, canine TCC organoids as a possible model to study TCC in vitro. After establishing the cell line, we subjected the 3-D cells to RNA in situ hybridization (RNAish) and cell viability assays. Overall, 3-D cell culture from urine samples of TCC diagnosed canines expressed RNA biomarkers in a similar manner to parent tumors via RNAish and showed more sensitivity to Cisplatin treatment when compared to 2-D human TCC cells. With further experimentation, canine TCC organoids could be an ideal model to study TCC in vitro.

Chronic stress is a well-known risk factor for the development of hypertension. However, the underlying mechanisms remain unclear. Corticotropin-releasing hormone (CRH) neurons in the central nucleus of the amygdala (CeA) are involved in the autonomic responses to chronic stress. Here, we determined the role of CeA-CRH neurons in chronic stress-induced hypertension.Borderline hypertensive rats (BHRs) and Wistar-Kyoto (WKY) rats were subjected to chronic unpredictable stress (CUS). Firing activity and M-currents of CeA-CRH neurons were assessed, and a CRH-Cre-directed chemogenetic approach was used to suppress CeA-CRH neurons. CUS induced a sustained elevation of arterial blood pressure (ABP) and heart rate (HR) in BHRs, while in WKY rats, CUS-induced increases in ABP and HR quickly returned to baseline levels after CUS ended. CeA-CRH neurons displayed significantly higher firing activities in CUS-treated BHRs than unstressed BHRs. Selectively suppressing CeA-CRH neurons by chemogenetic approach attenuated CUS-induced hypertension and decreased elevated sympathetic outflow in CUS-treated BHRs. Also, CUS significantly decreased protein and mRNA levels of Kv7.2 and Kv7.3 channels in the CeA of BHRs. M-currents in CeA-CRH neurons were significantly decreased in CUS-treated BHRs compared with unstressed BHRs. Blocking Kv7 channel with its blocker XE-991 increased the excitability of CeA-CRH neurons in unstressed BHRs but not in CUS-treated BHRs. Microinjection of XE-991 into the CeA increased sympathetic outflow and ABP in unstressed BHRs but not in CUS-treated BHRs.CeA-CRH neurons are required for chronic stress-induced sustained hypertension. The hyperactivity of CeA-CRH neurons may be due to impaired Kv7 channel activity, which represents a new mechanism involved in chronic stress-induced hypertension.We found that hyperactivity of CRH neurons in the CeA, likely due to diminished Kv7 channel activity, play a major role in the development of chronic stress-induced hypertension. Our study suggests that CRH neurons in the brain may be targeted for treating chronic stress-induced hypertension. Thus, increasing Kv7 channel activity or overexpressing Kv7 channels in the CeA may reduce stress-induced hypertension. Further studies are needed to delineate how chronic stress diminishes Kv7 channel activity in the brain.

The mammalian epidermis undergoes constant renewal, replenished by a pool of stem cells and terminal differentiation of their progeny. This is accompanied by changes in gene expression and morphology that are orchestrated, in part, by epigenetic modifiers. Here, we define the role of the histone acetyltransferase KAT2A in epidermal homeostasis and provide a comparative analysis that reveals key functional divergence with its paralog KAT2B. In contrast to the reported function of KAT2B in epidermal differentiation, KAT2A supports the undifferentiated state in keratinocytes. RNA-seq analysis of KAT2A- and KAT2B- depleted keratinocytes revealed dysregulated epidermal differentiation. Depletion of KAT2A led to premature expression of epidermal differentiation genes in the absence of inductive signals, whereas loss of KAT2B delayed differentiation. KAT2A acetyltransferase activity was indispensable in regulating epidermal differentiation gene expression. The metazoan-specific N terminus of KAT2A was also required to support its function in keratinocytes. We further showed that the interplay between KAT2A- and KAT2B-mediated regulation was important for normal cutaneous wound healing in vivo. Overall, these findings reveal a distinct mechanism in which keratinocytes use a pair of highly homologous histone acetyltransferases to support divergent functions in self-renewal and differentiation processes.