Ranjita Betarbet, PhD - US grants

[unreadable] DESCRIPTION (provided by applicant): In an effort to delineate the mechanisms that cause the formation of inclusions and induce dopamine cell death in PD, we propose to examine a novel protein, RING-Finger Protein 11 (RNF11) and its interactions with environmental toxins associated with PD pathogenesis. Aim 1: To test that (a) RNF11 is an E3 ubiquitin ligase and (b) that RNF11 is a membrane-associated protein expressed in dopaminergic neurons. Rationale: The RING-domain is essential for recognition of E2 ubiquitin-conjugating enzyme and ubiquitination of spec- ific substrates. Expression of RNF11 in dopaminergic neurons will be suggestive of its site of E3 ubiquitin ligase activity and its role in PD. Approach: We will examine the autoubiquitination properties of RNF11, and its interaction with other proteins using GST pull down assays to determine its substrate/s in cultured cells. We will examine regional, cellular and subcellular distribution of RNF11 in normal rat and human brain by using (a) immunocytochemistry at light and electron microscope levels and (b) sub-cellular fractionation protocols to determine its site of activity. Aim 2: To test the hypothesis that RNF11 has a neuroprotective function and oxidative modification(s) of RNF11 as a result of exposure to environmental toxins will compromise its neuroprotective function. Rationale: As a component of the proteasomal degradation pathway, RNF11 will facilitate or promote clearance of proteins and increase cell viability. Exposure to environmental toxins and oxidative modifications of RNF11 could impair RNF11's role as a neuroprotectant. Approach: In cell culture systems we will examine (A) the role of RNF11 on proteasomal activity and cell survival following (i) exposure to oxidizing agents and (ii) genetic alterations including mutant RNF11 (B) Effects of structural modifications of RNF11 's subcellular distribution and E3 ligase activity and on cell viability. Aim 3: To test the hypothesis that neuroprotective function of RNF11 is compromised in PD due to depletion of functional RNF11. Rationale: Modifications of RNF11 or sequestration of RNF11 in cytoplasmic inclusions will reduce the levels of functional RNF11 and compromise its neuroprotective function. Approach: We will examine changes in RNF11 in (a) animal models of PD ie., rotenone model in rats and a-synuclein transgenic mice and (b) in autopsy brain tissue from PD patients. This study will determine a role for RNF11 and how it confers protection against dopamine cell death seen in Parkinson's disease. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is the most common cause of dementia in aging humans, currently afflicting approximately 5 million Americans. Research recently has shown that an important feature of AD is the abnormal accumulation of specific proteins in the brain, and that the aggregation of the protein A[unreadable] is a primary event. However, how this process of aggregation is initiated, and how the aggregates spread from one region to another, remains uncertain. The goal of our research is to understand the cellular and molecular processes that initiate the pathogenesis of AD. Our previous studies found that the deposition of A[unreadable] can be induced, or seeded, in the brains of transgenic mouse models of AD by the infusion of dilute, A[unreadable]-rich brain extracts containing aggregated A[unreadable]. Very recently we found that A[unreadable] aggregation can be seeded by injections of A[unreadable]-rich brain extracts into the abdominal cavity of mice. Preliminary data implicate mononuclear phagocytes (macrophages) as the vectors of the seeds, in that A[unreadable]-seed- laden macrophages enter the circulation following the intraperitoneal injection of seed. However, direct evidence for the cellular transport of the seeds into the brain is lacking. The objective of this project is to clarify the role of macrophages in disseminating the seeds for A[unreadable] aggregation using a transgenic mouse model and in vitro models of the processing of seeds by macrophages. In the vast majority of AD cases, the factors that precipitate protein aggregation remain unknown. Understanding the cellular mechanisms underlying induced A[unreadable] aggregation and spread could eventually point the way to new therapies for Alzheimer's disease and other debilitating brain disorders of the elderly. PUBLIC HEALTH RELEVANCE: This application is the first attempt to investigate the role of macrophages in the processing and trafficking of pathogenic A[unreadable] seeds. The key question that we will address is whether macrophages act as vectors for the spread of A[unreadable]-amyloidosis by phagocytosing, translocating and releasing corruptive protein aggregates. We will approach this question by investigating the functional and pathologic consequences of the phagocytic ingestion of A[unreadable] in cell culture and transgenic mouse models. The impact of these studies is that they will yield novel insights into the origins of protein aggregation in idiopathic AD and cerebral [unreadable]-amyloid angiopathy, and thus will help to illuminate the fundamental mechanisms by which neurodegenerative proteopathies are initiated and propagated in the brain. A fuller understanding of templated protein corruption in living systems could open new pathways to the treatment some of the most devastating degenerative disorders of old age.