Probes for INS

The ventrolateral periaqueductal gray (vlPAG) constitutes a major descending pain modulatory system and is a crucial site for opioid-induced analgesia. A number of previous studies have demonstrated that glutamate and GABA play critical opposing roles in nociceptive processing in the vlPAG. It has been suggested that glutamatergic neurotransmission exerts antinociceptive effects, whereas GABAergic neurotransmission exert pro-nociceptive effects on pain transmission, through descending pathways. The inability to exclusively manipulate subpopulations of neurons in the PAG has prevented direct testing of this hypothesis. Here we demonstrate the different contributions of genetically-defined glutamatergic and GABAergic vlPAG neurons in nociceptive processing by employing cell type-specific chemogenetic approaches in mice. Global chemogenetic manipulation of vlPAG neuronal activity suggests that vlPAG neural circuits exert tonic suppression of nociception, consistent with previous pharmacological and electrophysiological studies. However, selective modulation of GABAergic or glutamatergic neurons demonstrates an inverse regulation of nociceptive behaviors by these cell populations. Selective chemogenetic activation of glutamatergic neurons, or inhibition of GABAergic neurons, in vlPAG suppresses nociception. In contrast, inhibition of glutamatergic neurons, or activation of GABAergic neurons, in vlPAG facilitates nociception. Our findings provide direct experimental support for a model in which excitatory and inhibitory neurons in the PAG bidirectionally modulate nociception.

Significance Statement The PAG is a midbrain region critical for the modulation of pain. However, the roles played by the distinct cell types within the PAG in nociceptive processing are poorly understood. This work addresses the divergent roles of glutamatergic and GABAergic PAG neuronal subpopulations in nociceptive processing. We demonstrate that activation of glutamatergic neurons or inhibition of GABAergic neurons suppresses nociception. Whereas inhibition of glutamatergic neuronal activity or activation of GABAergic neuronal activity potentiates nociception. This report identifies distinct roles for these neuronal populations in modulating nociceptive processing.

Telomerase consists of at least two essential elements, an RNA component hTR or TERC that contains the template for telomere DNA addition, and a catalytic reverse transcriptase (TERT). While expression of TERT has been considered the key rate limiting component for telomerase activity, increasing evidence suggests an important role for the regulation of TERC in telomere maintenance and perhaps other functions in human cancer. By using three orthogonal methods including RNAseq, RT-qPCR, and an analytically validated chromogenic RNA in situ hybridization assay, we report consistent overexpression of TERC in prostate cancer. This overexpression occurs at the precursor stage (e.g. high grade prostatic intraepithelial neoplasia or PIN), and persists throughout all stages of disease progression. Levels of TERC correlate with levels of MYC (a known driver of prostate cancer) in clinical samples and we also show the following: forced reductions of MYC result in decreased TERC levels in 8 cancer cell lines (prostate, lung, breast, and colorectal); forced overexpression of MYC in PCa cell lines, and in the mouse prostate, results in increased TERC levels; human TERC promoter activity is decreased after MYC silencing; and MYC occupies the TERC locus as assessed by chromatin immunoprecipitation (ChIP). Finally, we show that knockdown of TERC by siRNA results in reduced proliferation of prostate cancer cell lines. These studies indicate that TERC is consistently overexpressed in all stages of prostatic adenocarcinoma, and its expression is regulated by MYC. These findings nominate TERC as a novel prostate cancer biomarker and therapeutic target.

The circadian transcription factor neuronal PAS domain 2 (NPAS2) is linked to psychiatric disorders associated with altered reward sensitivity. The expression of Npas2 is preferentially enriched in the mammalian forebrain, including the nucleus accumbens (NAc), a major neural substrate of motivated and reward behavior. Previously, we demonstrated that down-regulation of NPAS2 in the NAc reduces the conditioned behavioral response to cocaine in mice. We also showed that Npas2 is preferentially enriched in dopamine receptor 1 containing medium spiny neurons (D1R-MSNs) of the striatum. To extend these studies, we investigated the impact of NPAS2 disruption on accumbal excitatory synaptic transmission and strength, along with the behavioral sensitivity to cocaine reward in a cell-type specific manner. Viral-mediated knockdown of Npas2 in the NAc of male and female C57BL/6J mice increased the excitatory drive onto MSNs. Using Drd1a-tdTomato mice in combination with viral knockdown, we determined these synaptic adaptations were specific to D1R-MSNs relative to non-D1R-MSNs. Interestingly, NAc-specific knockdown of Npas2 blocked cocaine-induced enhancement of synaptic strength and glutamatergic transmission specifically onto D1R-MSNs. Lastly, we designed, validated, and employed a novel Cre-inducible short-hairpin RNA virus for MSN-subtype specific knockdown of Npas2 Cell-type specific Npas2 knockdown in D1R-MSNs, but not D2R-MSNs, in the NAc reduced cocaine conditioned place preference. Together, our results demonstrate that NPAS2 regulates excitatory synapses of D1R-MSNs in the NAc and cocaine reward-related behavior.SIGNIFICANCE STATEMENTDrug addiction is a widespread public health concern often comorbid with other psychiatric disorders. Disruptions of the circadian clock can predispose or exacerbate substance abuse in vulnerable individuals. We demonstrate a role for the core circadian protein, NPAS2, in mediating glutamatergic neurotransmission at medium spiny neurons (MSNs) in the nucleus accumbens (NAc), a region critical for reward processing. We find that NPAS2 negatively regulates functional excitatory synaptic plasticity in the NAc and is necessary for cocaine-induced plastic changes in MSNs expressing the dopamine 1 receptor (D1R). We further demonstrate disruption of NPAS2 in D1R-MSNs produces augmented cocaine preference. These findings highlight the significance of cell-type specificity in mechanisms underlying reward regulation by NPAS2 and extend our knowledge of its function.

Kinase activation by chromosomal translocations is a common mechanism that drives tumorigenesis in spitzoid neoplasms. To explore the landscape of fusion transcripts in these tumors, we performed whole-transcriptome sequencing using formalin-fixed, paraffin-embedded (FFPE) tissues in malignant or biologically indeterminate spitzoid tumors from 7 patients (age 2-14 years). RNA sequence libraries enriched for coding regions were prepared and the sequencing was analyzed by a novel assembly-based algorithm designed for detecting complex fusions. In addition, tumor samples were screened for hotspot TERT promoter mutations, and telomerase expression was assessed by TERT mRNA in situ hybridization (ISH). Two patients had widespread metastasis and subsequently died of disease, and 5 patients had a benign clinical course on limited follow-up (mean: 30 months). RNA sequencing and TERT mRNA ISH were successful in six tumors and unsuccessful in one disseminating tumor because of low RNA quality. RNA sequencing identified a kinase fusion in five of the six sequenced tumors: TPM3-NTRK1 (2 tumors), complex rearrangements involving TPM3, ALK, and IL6R (1 tumor), BAIAP2L1-BRAF (1 tumor), and EML4-BRAF (1 disseminating tumor). All predicted chimeric transcripts were expressed at high levels and contained the intact kinase domain. In addition, two tumors each contained a second fusion gene, ARID1B-SNX9 or PTPRZ1-NFAM1. The detected chimeric genes were validated by home-brew break-apart or fusion fluorescence in situ hybridization (FISH). The two disseminating tumors each harbored the TERT promoter -124C>T (Chr 5:1,295,228 hg19 coordinate) mutation, whereas the remaining five tumors retained the wild-type gene. The presence of the -124C>T mutation correlated with telomerase expression by TERT mRNA ISH. In summary, we demonstrated complex fusion transcripts and novel partner genes for BRAF by RNA sequencing of FFPE samples. The diversity of gene fusions demonstrated by RNA sequencing defines the molecular heterogeneity of spitzoid neoplasms.

Telomerase is reactivated in most cancers and is possibly an early driver event in melanoma. Our aim was to test a novel in situ hybridization technique, RNAscope, for the detection of human telomerase reverse transcriptase (hTERT) mRNA in archival formalin-fixed, paraffin-embedded (FFPE) tissue and to compare the mRNA expression of melanomas and benign naevi. Furthermore, we wanted to see if hTERT mRNA could be a diagnostic or prognostic marker of melanoma. In situ hybridization for the detection of hTERT mRNA was performed on FFPE tissue of 17 melanomas and 13 benign naevi. We found a significant difference in the expression of hTERT mRNA between melanomas and benign naevi (P<0.001) and the expression of hTERT mRNA correlated with Breslow thickness (ρ=0.56, P=0.0205) and the Ki67 proliferation index (ρ=0.72, P=0.001). This study showed that RNAscope was a reliable in situ hybridization method for the detection of hTERT mRNA in FFPE tissue of melanomas and benign naevi. hTERT mRNA was more abundantly expressed in melanomas compared with benign naevi, but cannot be used solely as a diagnostic marker due to an overlap in expression. The hTERT mRNA expression in melanomas correlated with the prognostic markers Breslow thickness and the Ki67 index indicating a prognostic potential of hTERT mRNA.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited.