Probes for INS

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.

Relaxin-3 is a highly conserved neuropeptide abundantly expressed in neurons of the nucleus incertus (NI), which project to nodes of the septohippocampal system (SHS) including the medial septum/diagonal band of Broca (MS/DB) and dorsal hippocampus, as well as to limbic circuits. High densities of the Gi/o-protein-coupled receptor for relaxin-3, known as relaxin-family peptide-3 receptor (RXFP3) are expressed throughout the SHS, further suggesting a role for relaxin-3/RXFP3 signaling in modulating learning and memory processes that occur within these networks. Therefore, this study sought to gain further anatomical and functional insights into relaxin-3/RXFP3 signaling in the mouse MS/DB. Using Cre/LoxP recombination methods, we assessed locomotion, exploratory behavior, and spatial learning and long-term reference memory in adult C57BL/6J Rxfp3 (loxP/loxP) mice with targeted depletion of Rxfp3 in the MS/DB. Following prior injection of an AAV((1/2))-Cre-IRES-eGFP vector into the MS/DB to delete/deplete Rxfp3 mRNA/RXFP3 protein, mice tested in a Morris water maze (MWM) displayed an impairment in allocentric spatial learning during acquisition, as well as an impairment in long-term reference memory on probe day. However, RXFP3-depleted and control mice displayed similar motor activity in a locomotor cell and exploratory behavior in a large open-field (LOF) test. A quantitative characterization using multiplex, fluorescent in situ hybridization (ISH) identified a high level of co-localization of Rxfp3 mRNA and vesicular GABA transporter (vGAT) mRNA in MS and DB neurons (~87% and ~95% co-expression, respectively). Rxfp3 mRNA was also detected, to a correspondingly lesser extent, in vesicular glutamate transporter 2 (vGlut2) mRNA-containing neurons in MS and DB (~13% and ~5% co-expression, respectively). Similarly, a qualitative assessment of the MS/DB region, identified Rxfp3 mRNA in neurons that expressed parvalbumin (PV) mRNA (reflecting hippocampally-projecting GABA neurons), whereas choline acetyltransferase mRNA-positive (acetylcholine) neurons lacked Rxfp3 mRNA. These data are consistent with a qualitative immunohistochemical analysis that revealed relaxin-3-immunoreactive nerve fibers in close apposition with PV-immunoreactive neurons in the MS/DB. Together these studies suggest relaxin-3/RXFP3 signaling in the MS/DB plays a role in modulating specific learning and long-term memory associated behaviors in adult mice via effects on GABAergic neuron populations known for their involvement in modulating hippocampal theta rhythm and associated cognitive processes.

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.

Oncostatin M (OSM) is one of the least studied cytokines in the interleukin-6 family especially considering that its expression correlates with hallmarks of chronic itch, rheumatoid arthritis, irritable bowel syndrome, and more recently neuropathic pain. This gap in knowledge is attributed to numerous species differences in the protein structure of OSM, and its receptor usage both of which affect physiological function. Here we uncover some of these discrepancies across mouse, rat, and human models, further underpinning the importance of studying OSM in human context. We characterized the receptors expression profile of OSMR in human dorsal root ganglia (hDRG) from healthy organ donors and confirmed its presence in small-diameter neurons and surrounding glial-like cells via RNAScope in situ hybridization. To investigate OSM-mediated signaling in hDRG, we treated acutely sliced explants with 10ng/ml OSM for 30min and immunoassayed with markers of translation regulation via the Mitogen activated protein kiNase interacting Kinase (MNK) pathway and its downstream target eukaryotic translation Initiation Factor 4E (eIF4E). We noted significant increases in the p-eIF4E intensity signal in small-diameter neurons and glial-like cells suggesting that OSM activates MNK-eIF4E signaling in these cell types. Our findings cumulatively suggest that blocking OSM signaling in hDRG may attenuate nociceptive hyperexcitability and presents a viable therapeutic target for the treatment of pain. NIH NS065926, NIH NS111929.

Estrogen receptor (ER) α-expressing neurons in the ventrolateral area of the ventromedial hypothalamus (VMHvl) are implicated in the control of many behaviors and physiological processes, some of which are sex-specific. Recently, three sex-differentiated ERα subpopulations have been discovered in the VMHvl marked by co-expression with tachikinin1 (Tac1), reprimo (Rprm), or prodynorphin (Pdyn), that may subserve specific functions. These markers show sex differences in adulthood: females have many more Tac1/Esr1 and Rprm/Esr1 co-expressing cells, while males have more Pdyn/Esr1 cells. In this study, we sought to understand the development of these sex differences and pinpoint the sex-differentiating signal. We examined developmental changes in the number of Esr1 cells co-expressing Tac1, Rprm or Pdyn using single-molecule in situ hybridization. We found that both sexes have similarly high numbers of Tac1/Esr1 and Rprm/Esr1 cells at birth, but newborn males have many more Pdyn/Esr1 cells than females. However, the number of cells with Tac1/Esr1 and Rprm/Esr1 co-expression markedly decreases by weaning in males, but not females, leading to sex differences in neurochemical expression. Female mice administered testosterone at birth have expression patterns akin to male mice. Thus, a substantial neurochemical reorganization of the VMHvl occurs in males between birth and weaning that likely underlies the previously reported sex differences in behavioral and physiological responses to estrogens in adulthood.

Advances in antiretroviral therapy have prolonged the life of people living with HIV and diminished the level of virus in these individuals. Yet, HIV quickly rebounds after disruption and/or cessation of treatment due to significant cellular and anatomical reservoirs for HIV, which underscores the challenge for HIV cure strategies. The central nervous system (CNS), in particular, is seeded with HIV within 1-2 weeks of infection and is a reservoir for HIV. In this review, we address the paradigm of HIV reservoirs in the CNS and the relevant cell types, including astrocytes and microglia, that have been shown to harbor viral infection even with antiretroviral treatment. In particular, we focus on developmental aspects of astrocytes and microglia that lead to their susceptibility to infection, and how HIV infection propagates among these cells. We also address challenges of measuring the HIV latent reservoir, advances in viral detection assays, and how curative strategies have evolved in regard to the CNS reservoir. Current curative strategies still require optimization to reduce or eliminate the HIV CNS reservoir, and may also contribute to levels of neuroinflammation that lead to cognitive decline. With this in mind, the latent HIV reservoir in the brain should remain a prominent focus when assessing treatment options and overall viral burden in the clinic, especially in the context of HIV-associated neurocognitive disorders (HAND).

Significant variation in human papillomavirus (HPV) prevalence in oropharyngeal squamous cell carcinoma (OPSCC) across countries ranging from 11% in Brazil to 74% in New Zealand has been reported earlier. The aim of this study was to systematically review the most recently published studies on the occurrence of HPV in OPSCC globally. PubMed and Embase were systematically searched for articles assessing the occurrence of HPV+ OPSCC published between January 2016 and May 2021. Studies with a study period including 2015 and the following years were included. Both HPV DNA and/or p16 were accepted as indicators of HPV+ OPSCC. 31 studies were enrolled comprising 49,564 patients with OPSCC (range 12-42,024 patients per study) from 26 different countries covering all continents. The lowest occurrences of HPV+ OPSCC were observed in India (0%) and Spain (10%) and the highest occurrences were observed in Lebanon (85%) and Sweden (70%). We observed great variation in HPV prevalence in OPSCC worldwide varying from 0% to 85%. The highest occurrences of HPV+ OPSCC were found in general in Northern European countries, USA, Lebanon, China, and South Korea. We observed a trend of increase in HPV-positivity, indicating a mounting burden of HPV+ OPSCC.