Probes for INS

MET, a pleiotropic receptor tyrosine kinase implicated in autism risk, influences multiple neurodevelopmental processes. There is a knowledge gap, however, in the molecular mechanism through which MET mediates developmental events related to disorder risk. In the neocortex, MET is expressed transiently during periods of peak dendritic outgrowth and synaptogenesis, with expression enriched at developing synapses, consistent with demonstrated roles in dendritic morphogenesis, modulation of spine volume and excitatory synapse development. In a recent co-immunoprecipitation (Co-IP)/mass spectrometry screen, β-catenin was identified as part of the MET interactome in developing neocortical synaptosomes. Here, we investigated the influence of the MET/β-catenin complex in mouse neocortical synaptogenesis. Western blot analysis confirms that MET and β-catenin co-immunoprecipitate, but N-cadherin is not associated with the MET complex. Following stimulation with hepatocyte growth factor (HGF), β-catenin is phosphorylated at tyrosine142 (Y142) and dissociates from MET, accompanied by an increase in β-catenin/N-cadherin and MET/synapsin 1 protein complexes. In neocortical neurons in vitro, proximity ligation assays confirmed close proximity of these proteins. Moreover, in neurons transfected with synaptophysin-GFP, HGF stimulation increases the density of synaptophysin/bassoon (a presynaptic marker) and synaptophysin/PSD95 (a postsynaptic marker) clusters. Mutation of β-catenin at Y142 disrupts the dissociation of the MET/β-catenin complex and prevents the increase in clusters in response to HGF. The data demonstrate a new mechanism for modulation of synapse formation, whereby MET activation induces an alignment of pre- and postsynaptic elements that are necessary for assembly and formation of functional synapses by subsets of neocortical neurons that express MET/β-catenin complex.

Significance Statement: The gene encoding the MET receptor tyrosine kinase is associated with autism spectrum disorder, and influences typical and atypical synapse development and cortical circuit function. The present studies focus on determining potential molecular mechanisms through which the receptor functions in neocortical neurons during synaptogenesis. The findings show that the MET receptor interacts functionally with other proteins also implicated in promoting new synapse assembly, which is reduced upon disruption of the interactions. Thus, in some instances of autism spectrum disorder, disturbances of these molecular interactions may relate to the pathophysiology of cortical circuit development.

Abstract

PURPOSE:

Four consensus molecular subtypes (CMS1-4) of colorectal cancer (CRC) were identified in primary tumors and found to be associated with distinctive biological features and clinical outcomes. Given that distant metastasis largely accounts for CRC-related mortality, we examined the molecular and clinical attributes of CMS in metastatic CRC (mCRC).

EXPERIMENTAL DESIGN:

We developed a CRC-focused Nanostring based CMS classifier that is ideally suited to interrogate archival tissues. We successfully employ this panel in the CMS classification of FFPE tissues from mCRC cohorts, one of which is comprised of paired primary tumors and metastases. Finally, we developed novel mouse implantation models to enable modelling of CRC in vivo at relevant sites.

RESULTS:

Using our classifier we find that the biological hallmarks of mCRC, including CMS, are in general highly similar to those observed in non-metastatic early stage disease. Importantly, our data demonstrate that CMS1 has the worst outcome in relapsed disease, compared to other CMS. Assigning CMS to primary tumors and their matched metastases revealed mostly concordant subtypes between primary and metastasis. Molecular analysis of matched discordant pairs revealed differences in stromal composition at each site. The development of two novel in vivo orthotopic implantation models further reinforces the notion that extrinsic factors may impact on CMS identification in matched primary and metastatic CRC.

CONCLUSION:

We describe the utility of a Nanostring panel for CMS classification of FFPE clinical samples. Our work reveals the impact of intrinsic and extrinsic factors on CRC heterogeneity during disease progression.

Detailed anatomical tracing and mapping of the viscerotopic organization of the vagal motor nuclei has provided insight into autonomic function in health and disease. To further define specific cellular identities, we paired information based on visceral connectivity with a cell-type specific marker of a subpopulation of neurons in the dorsal motor nucleus of the vagus (DMV) and nucleus ambiguus (nAmb) that express the autism-associated MET receptor tyrosine kinase. As gastrointestinal disturbances are common in children with autism spectrum disorder (ASD), we sought to define the relationship between MET-expressing (MET+) neurons in the DMV and nAmb, and the gastrointestinal tract. Using wholemount tissue staining and clearing, or retrograde tracing in a METEGFP transgenic mouse, we identify three novel subpopulations of EGFP+ vagal brainstem neurons: 1) EGFP+ neurons in the nAmb projecting to the esophagus or laryngeal muscles, 2) EGFP+ neurons in the medial DMV projecting to the stomach, and 3) EGFP+ neurons in the lateral DMV projecting to the cecum and/or proximal colon. Expression of the MET ligand, hepatocyte growth factor (HGF), by tissues innervated by vagal motor neurons during fetal development reveal potential sites of HGF-MET interaction. Furthermore, similar cellular expression patterns of MET in the brainstem of both the mouse and nonhuman primate suggest that MET expression at these sites is evolutionarily conserved. Together, the data suggest that MET+ neurons in the brainstem vagal motor nuclei are anatomically positioned to regulate distinct portions of the gastrointestinal tract, with implications for the pathophysiology of gastrointestinal comorbidities of ASD.