Probes for INS

The Neuregulin (NRG) family of ErbB ligands is comprised of numerous variants originating from the use of different genes, alternative promoters and splice variants. NRGs have generally been thought to be transported to axons and presynaptic terminals where they signal via ErbB3/4 receptors in paracrine or juxtacrine mode. However, we recently demonstrated that unprocessed pro-NRG2 accumulates on cell bodies and proximal dendrites, and that NMDAR activity is required for shedding of its ectodomain by metalloproteinases. Here we systematically investigated the subcellular distribution and processing of major NRG isoforms in rat hippocampal neurons. We show that NRG1 isotypes I and II, which like NRG2 are single-pass transmembrane proteins with an Ig-like domain, share the same subcellular distribution and ectodomain shedding properties. We furthermore show that NRG3, like CRD-NRG1, is a dual-pass transmembrane protein that harbors a second transmembrane domain near its amino-terminus. Both NRG3 and CRD-NRG1 cluster on axons through juxtacrine interactions with ErbB4 present on GABAergic interneurons. Interestingly, while single-pass NRGs accumulate as unprocessed pro-forms, axonal puncta of CRD-NRG1 and NRG3 are comprised of processed protein. Mutations of CRD-NRG1 and NRG3 that render them resistant to BACE cleavage, as well as BACE inhibition, result in the loss of axonal puncta and in the accumulation of unprocessed proforms in neuronal soma. Together, these results define two groups of NRGs with distinct membrane topologies and fundamentally different targeting and processing properties in central neurons. The implications of this functional diversity for the regulation of neuronal processes by the NRG/ErbB pathway are discussed.SIGNIFICANCE STATEMENTNumerous Neuregulins are generated through the use of different genes, promoters and alternative splicing, but the functional significance of this evolutionary conserved diversity remains poorly understood. Here we show that NRGs can be categorized by their membrane topologies. Single-pass Neuregulins such as NRG1 types I/II and NRG2 accumulate as unprocessed pro-forms on cell bodies, and their ectodomains are shed by metalloproteinases in response to NMDA receptor activation. By contrast, dual-pass CRD-NRG1 and NRG3 are constitutively processed by BACE and accumulate on axons where they interact with ErbB4 in juxtacrine mode. These findings reveal a previously unknown functional relationship between membrane topology, protein processing and subcellular distribution, and suggest that single- and dual-pass NRGs regulate neuronal functions in fundamentally different ways.

Shank2 is an abundant postsynaptic scaffolding protein implicated in neurodevelopmental and psychiatric disorders, including autism spectrum disorders (ASD). Deletion of Shank2 in mice has been shown to induce social deficits, repetitive behaviors, and hyperactivity, but the identity of the cell types that contribute to these phenotypes has remained unclear. Here, we report a conditional mouse line with a Shank2 deletion restricted to parvalbumin (PV)-positive neurons (Pv-Cre;Shank2fl/fl mice). These mice display moderate hyperactivity in both novel and familiar environments and enhanced self-grooming in novel, but not familiar, environments. In contrast, they showed normal levels of social interaction, anxiety-like behavior, and learning and memory. Basal brain rhythms in Pv-Cre;Shank2fl/fl mice, measured by electroencephalography, were normal, but susceptibility to pentylenetetrazole (PTZ)-induced seizures was decreased. These results suggest that Shank2 deletion in PV-positive neurons leads to hyperactivity, enhanced self-grooming and suppressed brain excitation.

Gene expression analysis is essential for understanding the rich repertoire of cellular functions. With the development of sensitive molecular tools such as single-cell RNA sequencing, extensive gene expression data can be obtained and analyzed from various tissues. Single-molecule fluorescence in situ hybridization (smFISH) has emerged as a powerful complementary tool for single-cell genomics studies because of its ability to map and quantify the spatial distributions of single mRNAs at the subcellular level in their native tissue. Here, we present a detailed method to study the copy numbers and spatial localizations of single mRNAs in the cochlea and inferior colliculus. First, we demonstrate that smFISH can be performed successfully in adult cochlear tissue after decalcification. Second, we show that the smFISH signals can be detected with high specificity. Third, we adapt an automated transcript analysis pipeline to quantify and identify single mRNAs in a cell-specific manner. Lastly, we show that our method can be used to study possible correlations between transcriptional and translational activities of single genes. Thus, we have developed a detailed smFISH protocol that can be used to study the expression of single mRNAs in specific cell types of the peripheral and central auditory systems.

Increased testing for human papillomavirus (HPV) in oropharyngeal carcinomas has broadened the range of HPV-associated malignancies identified at this site. While HPV-related oropharyngeal non-keratinizing squamous cell carcinomas (SCC) are known to have a better prognosis than their non-HPV counterparts, HPV positivity may not alter the aggressive nature of HPV-associated small cell neuroendocrine carcinomas (SCNEC). We report a unique case of a mixed non-keratinizing type HPV-associated tonsillar SCC with SCNEC differentiation, and provide a comparison with the rare reported cases of such mixed carcinomas in the literature. Our patient is only the second such case positive for HPV genotype 18 and the only case in which this HPV-related mixed tonsillar tumor occurred in a patient with small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL). The case discussion supports the concept that HPV positivity does not confer a better prognosis in such mixed non-keratinizing type SCC with SCNEC. Our report also alerts pathologists to the need to evaluate for the possibility of a coexisting neuroendocrine component when oropharyngeal squamous cell carcinoma (OPSCC) is diagnosed, as its presence will affect the patients’ clinical management and prognosis