Susan Voglmaier - US grants

DESCRIPTION (provided by applicant): The changes in synaptic strength that underlie behavioral phenomena such as learning, memory and addiction, are usually attributed to postsynaptic changes in receptor sensitivity, but they may also reflect presynaptic regulation of the amount of transmitter released. The amount of glutamate per vesicle available for exocytotic release depends on its transport from the cytoplasm into vesicles by a family of vesicular glutamate transporter proteins, VGLUTs. VGLUT1 and 2, which account for the exocytotic release of glutamate by essentially all well-established excitatory neurons, exhibit a mutually exclusive pattern of expression in adult brain that correlates with the probability of transmitter release and the potential for plasticity. VGLUT1 also contains several protein-protein interaction domains not found in VGLUT2. The long-term goal of this K08 Mentored Clinical Scientist Development Award proposal is to understand how differences in the trafficking of VGLUT isoforms contribute to the differences in glutamate release observed at different synapses. The strategy is to study the role of C-terminal sequences in activity and trafficking of the different VGLUT isoforms. We have found that one of the polyproline motifs in the C-terminus of VGLUT1 interacts with proteins involved in synaptic vesicle recycling. We will characterize these interactions and determine whether they regulate glutamate transport by influencing vesicle filling, trafficking, or other aspects of the vesicle cycle. In addition, we will characterize and identify protein interactions with two other internalization motifs in VGLUT1. Together, these motifs may contribute to differences in the speed of vesicle filling, exocytosis and endocytosis in glutamate signaling. Increasing evidence implicates glutamate hypofunction in the pathophysiology of schizophrenia, which may involve changes in glutamate release. Thus, an understanding of the regulation of glutamate storage and release may be important in the targeting of therapeutic agents for schizophrenia

Dysfunction of glutamatergic neurotransmission is implicated in many neuropsychiatric disorders, including schizophrenia, epilepsy and autism. Although postsynaptic receptors have received the most attention, presynaptic mechanisms controlling glutamate release are also promising therapeutic targets, but have been less amenable to study. Glutamate release by synaptic vesicle exocytosis depends on glutamate packaging and recycling mediated by vesicular glutamate transporters (VGLUTs). VGLUT1 and 2 isoforms exhibit complementary expression in adult brain that distinguishes cortical (VGLUT1) and subcortical (VGLUT2) connections. Using genetically encoded optical reporters of glutamate transmission, VGLUT1 and 2-pHluorins, we have characterized the isoform-specific sorting signals and protein interactions that mediate differences in VGLUT1 and 2 trafficking. The involvement of proteins previously not associated with synaptic vesicle proteins may suggest novel mechanisms for vesicle recycling. Presynaptic signaling networks upstream of isoform- specific VGLUT trafficking present an opportunity to differentially modulate glutamate release in discrete brain pathways, and identify novel therapeutic targets to normalize brain circuits in neuropsychiatric disease. These mechanisms may also differentially depend on neuronal firing rate, offering the possibility of dampening excess activity while allowing normal physiological transmission to proceed. The long-term goal of the proposed research is to understand how membrane trafficking of individual vesicular proteins influences the protein composition of synaptic vesicles, the maintenance of synaptic vesicle pools, and the release of transmitter by specific circuits. The strategy of this proposal is to study signaling pathways upstream from isoform-specific VGLUT synaptic vesicle recycling. The specific aims of this proposal are designed to study the regulation of trafficking of vesicular glutamate transporters by 1) characterizing the modulation of VGLUT1 recycling by ubiquitin ligase interactions, 2) characterizing modulation of VGLUT2 recycling by inositol hexakisphosphate kinases, and 3) characterizing how isoform-specific trafficking changes over synapse development. As key mediators of synaptic transmission, these vesicular proteins and the factors that modulate their expression, localization and activity can dramatically influence neurotransmitter release, making them promising therapeutic targets. Regulation of neurotransmitter release may be an important approach to therapeutic intervention. The molecular machinery offers new targets for the development of better treatments for neuropsychiatric disorders.

DESCRIPTION (provided by applicant): Although dramatic advances have occurred in basic neuroscience and molecular genetics, the utilization of such knowledge in understanding the pathophysiology of mental illness and mechanisms of treatment may be hindered by a dearth of physician scientists trained to apply it to key problems of mental diseases. This application is a competitive renewal of a program that seeks to increase the number and quality of basic and clinical research investigators in mental health through proactive and collaborative mentorship and targeted research support and to facilitate the development of translational research through the collaborative involvement and mentorship of prospective trainees by researchers active in basic neuroscience and clinical behavioral research. Specifically, the program provides support for involvement in psychiatric research training and activity beginning in the PGY 1 year and continuing incrementally through the PGY 4 and, if indicated, in the PGY-5/6 years. The allocation of training support to research residents is determined annually on a competitive basis. In addition to stipend support of individual trainees, the program provides internal funding to support resident research projects, professional travel, and formal course work in methodology, statistics and research ethics. Each trainee is assigned a career mentor who assists them in identifying a research mentor and provides support and advice about ongoing issues related to career development. An individualized training plan (ITP) is developed and reviewed yearly, and a research project is developed in PGY 2 and conducted in PGY 3 & 4. In conjunction with formal coursework and hands-on research experience, networking and career development opportunities are emphasized, as is the successful integration of clinical and research training. Research residents participate in ongoing research related journal clubs, including a Virtual Journal Club that includes research residents from other California training sites, and is co-organized by UCSD, as well as in two annual research retreats: one, a Departmental retreat in which all research trainees and faculty are invited to present a summary of their recent work, and the other, an interinstitutional Research Retreat, rotating between UCSD and UCSF. Participation in a Workshop on Responsible Conduct of Research is also required. The success of the program in facilitating independent research scientist careers will be assessed through a series of subjective and objective instruments constructed to assay changes in attitudes, knowledge, and skills of the trainees in the course of their research training, and their entry into research fellowships, and their subsequent record of publication and training grant submissions. The career development of women and minority trainees in psychiatric research is specifically emphasized.

DESCRIPTION (provided by applicant): Schizophrenia and autism are devastating, complex brain disorders whose causes are poorly understood. Extensive genetic evidence links autism and schizophrenia to mutations in the cell adhesion molecule neuroligin. Disease-linked mutations alter neuroligin levels at the postsynaptic membrane. Abnormal neuroligin levels are associated with immature synapse formation, including immaturity of mechanisms to release neurotransmitter and recycle synaptic vesicles. These findings raise the possibility of treating autism and schizophrenia by targeting mechanisms of vesicular release and recycling to effect the mature presynaptic phenotype. We will use fluorescent markers of synaptic vesicle exocytosis to study the effect of varying neuroligin concentrations on synapse development and function. To model abnormal neuroligin concentration, we will attach neuroligin to coverslips in microislands roughly the size of a synapse and culture dissociated hippocampal neurons on them. Previous studies have shown that neuroligin is sufficient to induce neurons to form presynaptic boutons. Our preliminary experiments suggest that neurons indeed form presynaptic specializations on neuroligin-patterned glass. Unlike current technologies, this novel cell culture substrate allows us to control postsynaptic neuroligin levels precisely and disentangle neuroligin's effects on presynaptic and postsynaptic development. The geometry of the presynaptic terminals that form against the glass will allow us to apply high-resolution total internal reflection (TIRF) microscopy as well as epifluorescence at the same terminals. The work proposed here would be the first application of TIRF to the presynaptic compartment of maturing small central neurons. The detailed view of the presynaptic terminal offered by TIRF imaging has broad implications for understanding normal and pathological presynaptic function. The technology described here could be further developed to rapidly screen therapeutic agents that target abnormal synapse development, vesicle recycling, and neurotransmitter release.