Michael A. Silverman - US grants

DESCRIPTION (provided by applicant): The dendrites of neurons are the primary surface for synaptic input in the central nervous system. To decode synaptic signals, dendrites rely on the proper transport and delivery of cell surface proteins to the dendritic plasma membrane. The goal of this proposal is to understand the cellular and molecular mechanisms that underlie the sorting and transport of endogenous neuronal proteins of physiological importance. Transmembrane proteins are sorted into carrier vesicles in the Golgi apparatus, transported along microtubules, and finally delivered to the plasma membrane. The initial step of membrane protein sorting relies on discrete amino acids in the primary sequence of the membrane protein. The experiments described within will identify new sorting signals in two dendritic G-protein coupled receptors that are relevant to neurological disorders: the D2 Long dopamine receptor and the 5HT1a serotonin receptor. We will also investigate the targeting of neuroligin, a key dendritic molecule in the composition of synapses. We will test whether or not a canonical tyrosine-based sorting signal in the molecule neuroligin is responsible for its dendritic localization, and ask if neuroligin is directly transported to the plasma membrane like other dendritic proteins characterized so far. To perform the described experiments, we will combine molecular and cellular techniques that allow for the direct observation and quantitation of the distribution of membrane proteins in neurons. By expressing wild type and mutant forms of the proteins above in low-density cultured hippocampal neurons we will compare the distribution of proteins using live-cell immunofluoresence, quantitative fluorescence microscopy, and digital image analysis. Green fluorescent protein technology will be employed to directly observe the transport of neuroligin in living neurons. Generally, neuronal polarity describes the morphological and functional differences between axons and dendrites. The establishment and maintenance of neuronal polarity is absolutely critical for nervous system function. There are several examples of human and animal diseases where the underlying cellular defect relates to problems in protein trafficking. Thus, understanding the basic aspects of protein trafficking and nerve cell biology will significantly advance our understanding of the causes of disorders like Lou Gehrig's and Alzheimer's disease.

DESCRIPTION (provided by applicant): This proposal describes a 5-year mentored research project that has the goal of exploring the relationship between disease-protective major histocompatibility complex (MHC)/human leukocyte antigen (HLA) genetic loci, gut commensal microbiota and autoimmune diabetes (T1D). This project will build upon the principal investigator's preliminary data and strong background in immunology and infectious diseases. The comprehensive career development plan combines a strong mentorship team with additional didactic and practical training in gnotobiotic, microbiologic and large data set analyti techniques, which will facilitate the PI's transition into a successful independent investigator. The proposed research will take place in the stimulating research environment of Harvard Medical School within the laboratory of Drs. Diane Mathis and Christophe Benoist. The mentor, Dr. Mathis, is a world-renowned immunologist who has successfully mentored many physician-scientists during her 30-year career. The research and career development of the primary investigator will benefit from the extensive scientific and mentorship resources available to him. The MHC and HLA loci possess the strongest genetic association with T1D, in mice and humans respectively. Some loci such as the MHC class II E molecule offer dominant protection from T1D, but the mechanisms for this protection remains poorly understood. Commensal microbiota also influences the development of the immune system and affects the risk for developing T1D. The goal of this proposal is to explore interactions between gut microbiota, the MHC/HLA loci and the development of T1D in the non-obese diabetic (NOD) strain of mice and its closely related but non-diabetic transgenic line, expressing the protective MHCII E molecule (E?.NOD). Our preliminary data indicate that antibiotic disruption of the gut microbiota induces insulitis in E?.NOD mice. We have also demonstrated maternal transfer of protection from insulitis and T1D from E?.NOD mothers to NOD pups, suggesting transfer of protective microbiota from mother to pup. Therefore, we hypothesize that the diabetes-protective effect of the E molecule reflects an impact on the gut microbiota, which has a secondary influence on the immune system. To explore this hypothesis, we will directly test whether E?.NOD mice possess different microbes than NOD mice, and whether transfer of these microbes protects NOD mice from T1D. We will further identify these protective microbes and identify immune system components in E?.NOD mice that correlate with protection from T1D. To test whether the HLA alleles behave similarly we will explore the microbiota and gut immune system of NOD mice expressing human disease protective or risk-associated HLA alleles in place of their MHC genes. Ultimately, these experiments will shed light on the relationship between MHC and HLA loci, commensal microbiota and autoimmune diabetes. Moreover, identification of immunomodulatory bacteria and their associated immune system targets offers the potential for novel therapies for T1D, and perhaps other autoimmune diseases.