Anne Messer - US grants

The basic questions which this project is dealing with concern the genetic and epigenetic control mechanisms which specify the numbers and types of cells that are destined to interact in the mature cerebellum. A hypothesis that cerebellar Purkinje cells control both the onset and cessation of proliferation of cells in the external granule layer, mediate the timing signals of hormones, and signal the onset of some differentiated functions in both neurons and glial cells is tested using neurological mutants which exhibit degeneration of Purkinje cells and/or granule cells, congenital hypothyroid mutants, and cerebellar cells in culture. The general approach is to go back and forth between cell cultures and mutants, using the former to examine effects of signals on isolated cells, and interactions between more limited cell populations and cell type ratios than can be found in vivo. The mutants serve as sources of defined, reproducible perturbations of the developing system in vivo, and can be used to generate hypotheses which can then be further tested in vitro. This combination of studies at several levels allows a long-term goal of integrating these cerebellar events with more general chains of developmental events as the more primary effects of the mutations are examined. Specific experiments proposed examine the role of Purkinje cell signals vs. (or in conjunction with) hormonal signals, and interactions with other developing cell types, both in vitro and in vivo. The basic knowledge gained about cell interactions and hormone signaling should be applicable to brain areas other than the cerebellum. Clinically, cerebellar dysfunction in hypothyroidism is an important problem even with current early detection and treatment regimens, and knowledge of a mechanism of action in a hereditary neurodegenerative disease may be of value in understanding human counterparts.

DESCRIPTION (provided by applicant): The specific goal of this developmental project is to optimize and characterize conformation-specific single-chain Fv (scFv) antibodies as tools to recognize and manipulate misfolded neurodegeneration proteins intracellularly. We have developed unique capabilities that enable us to generate scFvs that target specific protein morphologies and to test their function intracellularly. Given the known structural commonality of proteins involved in Alzheimer's, Parkinson's, Huntington's, prion, and ALS diseases, these reagents will allow investigators working on these various diseases to distinguish between oligomeric and fibrillar forms of aggregates in a cellular context. It will also allow redirection of specific forms of proteins to other intracellular compartments to test hypotheses of where the most critical site of action resides. This is a multiple-PI application. The anti-alpha-synuclein oligomeric and anti- fibrillar scFvs are currently being selected in the laboratory of Dr. Michael Sierks, at ASU. Much of the first year will be devoted to efficient rapid screening of intracellular expression in neuronal cell lines by Dr. Anne Messer, Wadsworth Center in Albany, NY, followed immediately by the more labor-intensive engineering of affinity and stability of the most promising scFvs. By the second year, the majority of the work will consist of detailed biological studies done in Albany, with further protein interactions and optimization as needed at ASU. PUBLIC HEALTH REVELANCE: Neurodegenerative diseases are taking an increasing human and financial toll as our population ages. Engineered antibody therapeutics are already in use for cancer and infectious disease, and offer an exciting new platform for neurological disease drug discovery and therapeutics. This project specifically targets the consequences of protein misfolding in Parkinson's disease, with direct applications to related diseases such as Alzheimer's, Huntington's, and ALS.