Probes for INS

Rhabdomyosarcoma (RMS) is a pediatric soft tissue sarcoma that histologically resembles embryonic skeletal muscle. RMS occurs throughout the body and an exclusively myogenic origin does not account for RMS occurring in sites devoid of skeletal muscle. We previously described an RMS model activating a conditional constitutively active Smoothened mutant (SmoM2) with aP2-Cre. Using genetic fate mapping, we show SmoM2 expression in Cre-expressing endothelial progenitors results in myogenic transdifferentiation and RMS. We show that endothelium and skeletal muscle within the head and neck arise from Kdr-expressing progenitors, and that hedgehog pathway activation results in aberrant expression of myogenic specification factors as a potential mechanism driving RMS genesis. These findings suggest that RMS can originate from aberrant development of non-myogenic cells.

Glucagon-like peptide-1 (GLP-1) is a physiological regulator of appetite and long-acting GLP-1 receptor agonists (GLP-1RA) lower food intake and bodyweight in both human and animal studies. The effects are mediated through brain GLP-1Rs, and several brain nuclei expressing the GLP-1R may be involved. To date, mapping the complete location of GLP-1R protein in the brain has been challenged by lack of good antibodies and the discrepancy between mRNA and protein especially relevant in neuronal axonal processes. Here, we present a novel and specific monoclonal GLP-1R antibody for immunohistochemistry with murine tissue and show detailed distribution of GLP-1R expression as well as mapping of GLP-1R mRNA by non-radioactive in situ hybridization. Semi-automated image analysis was performed to map the GLP-1R distribution to atlas plates from the Allen Institute of Brain Science (AIBS). The GLP-1R was abundantly expressed in numerous regions including the septal nucleus, the hypothalamus and the brain stem. GLP-1R protein expression was also observed on neuronal projections in brain regions devoid of any mRNA which has not been observed in earlier reports. Taken together, these findings provide new knowledge on GLP-1R expression in neuronal cell bodies and neuronal projections.

The tick-borne flavivirus, Powassan virus (POWV) causes life-threatening encephalitis in humans in North America and Europe. POWV is transmitted by ixodid tick vectors that feed on small to medium-sized mammals, such as Peromyscus leucopus mice, which may serve as either reservoir, bridge or amplification hosts. Intraperitoneal and intracranial inoculation of 4-week old Peromyscus leucopus mice with 103 PFU of POWV did not result in overt clinical signs of disease. However, following intracranial inoculation, infected mice seroconverted to POWV and histopathological examinations revealed that the mice uniformly developed mild lymphocytic perivascular cuffing and microgliosis in the brain and spinal cord from 5 to 15 days post infection (dpi), suggesting an early inflammatory response. In contrast, intracranial inoculation of 4-week old C57BL/6 and BALB/c mice was lethal by 5 dpi. Intraperitoneal inoculation was lethal in BALB/c mice, but 40% (2/5) of C57BL/6 mice survived. We concluded that Peromyscus leucopus mice infected i.c. with a lethal dose of POWV support a limited infection, restricted to the central nervous system and mount an antibody response to the virus. However, they fail to develop clinical signs of disease and are able to control the infection. These results suggest the involvement of restriction factors, and the mechanism by which Peromyscus leucopus mice restrict POWV infection remains under study.

Therapeutic protein delivery using viral vectors has shown promise in preclinical models of Parkinson’s disease (PD) but clinical trial success remains elusive. This may partially be due to a failure to include advanced age as a covariate despite aging being the primary risk factor for PD. We investigated transgene expression following intracerebral injections of recombinant adeno-associated virus pseudotypes 2/2 (rAAV2/2), 2/5 (rAAV2/5), 2/9 (rAAV2/9), and lentivirus (LV) expressing green fluorescent protein (GFP) in aged versus young adult rats. Both rAAV2/2 and rAAV2/5 yielded lower GFP expression following injection to either the aged substantia nigra or striatum. rAAV2/9-mediated GFP expression was deficient in the aged striatonigral system but displayed identical transgene expression between ages in the nigrostriatal system. Young and aged rats displayed equivalent GFP levels following LV injection to the striatonigral system but LV-delivered GFP was deficient in delivering GFP to the aged nigrostriatal system. Notably, age-related transgene expression deficiencies revealed by protein quantitation were poorly predicted by GFP-immunoreactive cell counts. Further, in situ hybridization for the viral CβA promoter revealed surprisingly limited tropism for astrocytes compared to neurons. Our results demonstrate that aging is a critical covariate to consider when designing gene therapy approaches for PD.

Abstract

Recent advances in chemotherapeutics highlight the importance of molecularly-targeted perturbagens. Although these therapies typically address dysregulated cancer cell proteins, there are increasing therapeutic modalities that take into consideration cancer cell-extrinsic factors. Targeting components of tumor stroma such as vascular or immune cells has been shown to represent an efficacious approach in cancer treatment. Cancer-associated fibroblasts (CAFs) exemplify an important stromal component that can be exploited in targeted therapeutics, though their employment in drug discovery campaigns has been relatively minimal due to technical logistics in assaying for CAF-tumor interactions. Here we report a 3-dimensional multi-culture tumor:CAF spheroid phenotypic screening platform that can be applied to high-content drug discovery initiatives. Using a functional genomics approach we systematically profiled 1,024 candidate genes for CAF-intrinsic anti-spheroid activity; identifying several CAF genes important for development and maintenance of tumor:CAF co-culture spheroids. Along with previously reported genes such as WNT, we identify CAF-derived targets such as ARAF and COL3A1 upon which the tumor compartment depends for spheroid development. Specifically, we highlight the G-protein-coupled receptor OGR1 as a unique CAF-specific protein that may represent an attractive drug target for treating colorectal cancer. In vivo, murine colon tumor implants in OGR1 knockout mice displayed delayed tumor growth compared to tumors implanted in wild type littermate controls. These findings demonstrate a robust microphysiological screening approach for identifying new CAF targets that may be applied to drug discovery efforts.