No abstract

Acute alcohol exposure alters the trafficking and function of many G-protein-coupled receptors (GPCRs) that are associated with aberrant behavioral responses to alcohol. However, the molecular mechanisms underlying alcohol-induced changes in GPCR function remain unclear. ␤-Arrestin is a key player involved in the regulation of GPCR internalization and thus controls the magnitude and duration of GPCR signaling. Although ␤-arrestin levels are influenced by various drugs of abuse, the effect of alcohol exposure on ␤-arrestin expression and ␤-arrestin-mediated GPCR trafficking is poorly understood. Here, we found that acute ethanol exposure increases ␤-arrestin2 degradation via its increased ubiquitination in neuroblastoma-2a (N2A) cells and rat prefrontal cortex (PFC). ␤-Arrestin2 ubiquitination was likely mediated by the E3 ligase MDM2 homolog (MDM2), indicated by an increased coupling between ␤-arrestin2 and MDM2 in response to acute ethanol exposure in both N2A cells and rat PFC homogenates. Importantly, ethanol-induced ␤-arrestin2 reduction was reversed by siRNA-mediated MDM2 knockdown or proteasome inhibition in N2A cells, suggesting ␤-arrestin2 degradation is mediated by MDM2 through the proteasomal pathway. Using serotonin 5-HT1A receptors (5-HT1ARs) as a model receptor system, we found that ethanol dose-dependently inhibits 5-HT1AR internalization and that MDM2 knockdown reverses this effect. Moreover, ethanol both reduced ␤-arrestin2 levels and delayed agonist-induced ␤-arrestin2 recruitment to the membrane. We conclude that ␤-arrestin2 dysregulation by ethanol impairs 5-HT1AR trafficking. Our findings reveal a critical molecular mechanism underlying ethanol-induced alterations in GPCR internalization and implicate ␤-arrestin as a potential player mediating behavioral responses to acute alcohol exposure. Acute alcohol exposure produces a wide range of behavioral and neurobiological effects, including feelings of euphoria, loss of coordination, and cognitive impairment, which are thought to be key predictors of vulnerability to alcohol abuse in humans (1-3). This notion is further supported by a recent longitudinal study showing that individuals who exhibit higher sensitivity to the rewarding effect and lower sensitivity to the sedative effect of an initial acute alcohol exposure were more likely to develop symptoms of alcohol use disorders and escalate their alcohol consumption over time (4). Many aspects of behavioral responses to alcohol are mediated by various G-proteincoupled receptors (GPCRs). 2 For example, ethanol-induced locomotor stimulation is mediated by serotonin 5-HT 1A receptors (5-HT 1A Rs) because treatment with 5-HT 1A R agonist (8-OH-DPAT) reduced locomotor activity in response to an acute ethanol exposure (2.5 g/kg, i.p.) (5). Moreover, the sedative effect of ethanol is regulated by metabotropic glutamate receptor 5 (mGluR5) as genetic deletion of mGluR5 or treatment with the mGluR5 antagonist, 2-methyl-6-(phenylethynyl)pyridine, increases the duration of loss of righting reflex in mice a...

Background: The conventionally understood pathogenesis of agminated Spitz nevi includes a mosaic HRAS mutation followed by copy number gains in 11p. However, we have recently observed agminated presentations of fusion-driven melanocytic neoplasms.Methods: We retrieved cases from our database of benign fusion-induced melanocytic neoplasms with an agminated presentation. Both the primary lesion and the secondary lesion were sequenced. TERT-promoter mutational testing and the melanoma fluorescence in situ hybridization assay were also performed.Results: Three cases were included. Two had a PRKCA fusion (partners ATP2B4 and MPZL1) and one had a ZCCHC8::ROS1 fusion. None of the cases met morphologic or molecular criteria for malignancy. There was no evidence of tumor progression in secondary lesions. The same fusion was identified in the primary and secondary lesions. None of the patients developed evidence of nodal or systemic metastasis. Conclusions:We present accumulating evidence that fusion-driven melanocytic neoplasms can present with an agminated presentation. The differential diagnosis of an agminated presentation versus a locally recurrent or potentially locally metastatic tumor is critical, and accurate diagnosis has significant prognostic and therapeutic consequences for the patient. As with HRAS mutations, fusion-driven melanocytic tumors may have an agminated presentation.

Desmoplastic melanomas (DMs) are often challenging to diagnose and ancillary tests, such as immunohistochemistry, have limitations. One challenge is distinguishing DM from benign desmoplastic melanocytic neoplasms. In this study, we explored the utility of next-generation sequencing data in the diagnosis of DMs versus desmoplastic Spitz nevi (DSN) and desmoplastic nevi (DN). We sequenced 47 cases and retrieved 12 additional previously sequenced clinical cases from our dermatopathology database. The 59 total cases were comprised of 21 DMs, 25 DSN, and 13 DN. The DMs had the highest tumor mutation burden at 22 mutations/megabase (m/Mb) versus the DSN (6 m/Mb) and DN (8 m/Mb). Truncating mutations in NF1 resulting in a loss-of-function were exclusive to the DM cohort, identified in 8/21 (38%) cases. Importantly, missense mutations in NF1 were nonspecific and seen with similar frequency in the different cohorts. Other mutations exclusive to the DMs included truncating mutations in TP53, CDKN2A, and ARID2. Among the DSN, 17/25 (68%) had an HRAS mutation or receptor tyrosine kinase fusion consistent with other Spitz tumors. Two cases in the DN cohort had missense mutations in BRAF without additional progression mutations and 2 other cases had mutations in GNAQ, supporting a diagnosis of a sclerosing blue nevus. The remainder of the DN had nonspecific mutations in various signaling pathways with few progression mutations. Overall, our study provides preliminary data that next-generation sequencing data may have the potential to serve as an ancillary diagnostic tool to help differentiate malignant and benign desmoplastic melanocytic neoplasms.