Progress Report for Neurotherapeutics Unit

National Institute Of Neurological Disorders And Stroke

Investigators

Linked publications, trials & patents

Abstract

Within the NTDU group, we continue to utilize many automated, medium-throughput cell-based assays to screen small compound collections against rat mixed cortical cultures, human neural stem cells (NSC) and human NSC-derived neurons and astrocytes. We've utilized live cell imaging-based assays with human neurons, as well as developed fluorescence-based assays to probe high content imaging endpoints. These cell-based screening assays, in 384 well plate format, have been successfully employed in multiple screens with multiple investigators. We continue to produce and purify a number of recombinant proteins, such as HERV-K Envelope proteins and HIV Tat protein for our own studies and provide that Tat protein to extramural researchers. With the Section of Infections of the Nervous System (SINS), we have continued to work on multiple research projects. We continue to characterize the neurotoxicity resulting from the expression of the HERV-K env protein in NSC and human neurons, and have already identified some compounds and proteins which attenuate HERVK env mediated neurotoxicity. Exogenously added recombinant HERV-K Env protein also elicits significant neurotoxicity. Microelectrode array electrophysiologic studies have provided a functional endpoint to these studies. Specific anti-HERV-K envelope antibodies can block the envelope mediated neurotoxicity, as well as neurotoxic envelope protein contained in CSF samples from ALS patients. The HIV Tat antagonist program has incorporated direct label-free binding assays, including protein thermal shift assays, as well as Tat-dependent selective LTR activation assays to complete the screening of more than 2000 compounds to identify new lead Tat inhibitors. The resultant "hit" compounds continue to be characterized for potency and biopharmaceutical properties, as well as activity against HIV viral replication. Additional Tat antagonist identification and characterization has been completed by collaboration with NCATS scientists. The NTDU has continued multiple studies in collaboration with other intramural investigators. We continue to work with the Sibley laboratory to characterize the dopamine D3 receptor modulators that they have identified, using our live cell imaging platforms for quantitative analysis. This work includes working with C elegans models as well as iPSC derived neuronal cultures. We continue working with three clinical investigators, Drs. Kareem Zaghloul, Prashant Chittiboina and Peter Williamson on new research projects with their respective laboratories. We are collaborating with Dr. Wei Lu laboratory to characterize the activity of GABA A receptors with their newly identified modulator protein Shisa7. Interaction of GABA A Receptors with Shisa changed the binding activity of a number of GABA ligands. Over a thousand compounds have been screened and characterized as GABA A receptor modulators. We continue to work closely with the Dr. Francis McMahon laboratory to generate fluorescently labeled NSC and neuronal cultures from control and neuropsychiatric patients, to characterize their morphologic changes in response to stressors or toxic challenges. We have characterized the neuronal growth and development of human iPSC-derived neurons with the laboratory of Dr. Michael Ward. We have initiated a drug screening initiative to characterize multiple individual neuronal populations with selective protein knockouts in order to more fully characterize their role(s) in neurodegenerative disorders. Cell line generation NeuroTherapeutics Development Unit (NTDU): As part of its role in facilitating translational research, the NTDU develops cell lines in human neural stem cells (NSCs) for both internal and collaborative use. Lines have been developed to ubiquitously express the tdTomato fluorescent protein to aid in conducting screening assays. Collaborations between the NTDU and intramural and extramural groups has led to the development of more than ten such NSC lines that will make it possible to conduct medium throughput screens aimed at targeting several neurological diseases including bipolar disorder and Alzheimer diseases. Additionally, the NTDU has also generated additional cell specific lines for internal use that are also available to collaborators. These lines use cell specific promoters to express tdTomato in neurons and astrocytes and could have also have broad utility for conducting screens in distinct neural cells.