Amy J. Gleichman, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): Neural paraneoplastic syndromes arise when an immune response to a tumor cross reacts with a protein in the nervous system, causing alterations of neuronal function. Recently, our laboratory in collaboration with the laboratory of Dr. Josep Dalmau (Department of Neurology, Hospital of the University of Pennsylvania) has helped characterize a new paraneoplastic syndrome, anti-N-methyl-D-aspartate receptor (a-NMDAR) encephalitis. This syndrome develops in patients with teratomas that express NMDARs, who then present with a variety of neurological problems including amnesia, psychosis, and autonomic distress. We have found over 40 patients in 3 years, indicating that this disorder frequently goes undiagnosed and untreated, probably resulting in death from autonomic instability. Patients recover with plasmapheresis, indicating antibody involvement. While we have previously shown that these patients make a-NMDA receptor antibodies, it is unclear how these antibodies mediate this phenotype. NMDAR hypofunction has previously been implicated in the pathogenesis of schizophrenia; based on the similarities of the clinical phenomena, it [unreadable] is possible that a-NMDAR encephalitis represents an autoimmune disorder that mimics the neurological [unreadable] changes occurring in schizophrenia. I propose to investigate the mechanisms by which NMDAR antibodies trigger these neurological problems, which may provide broader insights into the general mechanisms of schizophrenia. To do so, I will use whole-cell patch clamp and Ca2+ imaging to determine if these antibodies have a direct effect on channel function. These antibodies could also change in the number of channels on the cell surface or the location of those channels, possibly by disrupting an interaction with an anchoring protein. I will examine this possibility with cell-surface labeling techniques, primarily biotinylations. Finally, I will map the main immunogen of the receptor by transfecting HEK293 cells with mutant NMDAR subunits that are missing portions of the receptor, as well as chimeric receptors. Finding the epitope could help develop an immunodiagnostic test, preventing unnecessary deaths. PUBLIC HEALTH RELEVANCE: One of the major theories of the cause of schizophrenia is hypofunction of the NMDA receptor. Anti-NMDAR encephalitis, therefore, in which individuals make antibodies to the NMDAR that then cause psychological symptoms similar to schizophrenia, presents a unique opportunity for studying the specific contribution of this receptor. In addition to this possible mechanistic connection to schizophrenia, however, this disease was only recently discovered and seems to frequently go undiagnosed and therefore untreated. Developing a better test for this condition, which this research could help do, would help solve this problem. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Stroke is a leading cause of death and disability, yet there are few treatment options available to enhance stroke recovery. While it has long been known that astrocyte morphology changes in a graded fashion after stroke, little is known about the exact changes astrocytes undergo or the ways in which these responses may be manipulated to promote recovery. Reactive astrocytes have been considered as simple scar-forming cells in CNS injury. However, astrocytes have distinct effects on recovery depending on timing, location and astrocyte phenotype in some injury models, but this has not been studied in stroke. Here, I propose to identify phenotypic subsets of astrocytes and manipulate key astrocyte-secreted proteins within those subsets to determine molecular mechanisms of astrocyte responses to stroke, and their impact on tissue repair. Aim 1 will examine the morphologic, phenotypic, and transcriptomic changes astrocytes undergo, in order to identify subpopulations of astrocytes with functional roles in tissue repair. To explore astrocyte morphology, one of several astrocyte-specific lentiviral vectors that drive expression of fluorophores will be injected in the peri-infarct cortex. These fluorescent proteins diffuse throughout the cell, clearly delinating the full architecture of the astrocyte and allowing a detaied analysis of changes in astrocyte length, polarity, complexity, and domain size. Phenotypic analysis will be conducted using specific astrocytic markers that reveal different aspects of astrocytic function that are related to tissue repair. Finally, the different transcriptomic change that different zones of astrocytes undergo will be determined using a line of mice in which tagged ribosomes are expressed in an astrocyte-specific manner combined with laser capture microscopy. In Aim 2, astrocyte-secreted molecules that affect synapse formation and neural repair will be altered in subpopulation-specific fashions. Astrocyte-secreted proteins that promote neural repair are a newly identified and growing class of proteins about which little is known in stroke; their regional specificity is likely to play a crucial role in their action. Thereare three main known classes of protein that are secreted from astrocytes and affect synapse formation and neuronal repair: 1) those that help form synapses, 2) those that antagonize the first class, and 3) those that help synapses become electrically active. Preliminary results suggest that these classes are differentially regulated in different astrocytic subpopulations post stroke. I will first fully analyze the expression patterns of these molecules in different astrocytc subpopulations post-stroke. This information will inform interventional studies. As class 1 and 3 proteins are complementary in action, local delivery of exogenous proteins to those astrocytic subpopulations that do not express them post-stroke may improve recovery. Simultaneously, decreasing expression of class 2 proteins may allow the repair-mediating effects of class 1 and 3 proteins to be revealed. These modifications will be performed using localized delivery of astrocyte-specific delivery paradigms.