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Date of Award

Spring 2011

Author's School

College of Arts & Sciences

Author's Department/Program

Biology

Abstract

Alzheimer’s Disease (AD), the most common cause of dementia, is a neurodegenerative disorder characterized by loss of memory, progressive cognitive decline and eventual death. The pathological hallmark of the disease is the accumulation of amyloid plaques in the extracellular space in the brain. Amyloid plaques are composed of tightly aggregated amyloid beta peptide (Aβ). Aβ is a proteolytic fragment of the Amyloid Precursor Protein (APP), a transmembrane protein, which resides on the plasma membranes of neurons. In order for APP to be proteolytically cleaved into Aβ, it must be internalized into the cell and trafficked to endosomes where specific proteases can cleave APP. A recent Genome Wide Association Studies (GWAS) reported that a single nucleotide polymorphism in the PICALM gene was significantly associated with AD. PICALM (phosphatidylinositol clathrin assembly lymphoid-myeloid leukemia) is a protein that is involved in clathrin-mediated endocytosis. Due to its critical role in endocytosis, we hypothesized that PICALM might play a role in APP endocytosis and subsequent Aβ generation. Because PICALM gene deletion results in embryonic lethality due to brain maldevelopment, we took a gene knockdown approach using shRNA. Knockdown and overexpression of PICALM were performed in Neuroblastoma N2a-APP cells. APP internalization was measured after labeling cell surface proteins with biotin. Western blotting was used to detect internalized APP. Extracellular Aβ levels were measured using ELISA from conditioned medium. PICALM co-localized with APP in intracellular vesicles of N2a-APP cells only after endocytosis was initiated. PICALM knockdown in N2a-APP cells resulted in reduced APP internalization and Aβ generation, as measured in conditioned medium. Conversely, PICALM overexpression increased APP internalization and Aβ production. In vivo, PICALM expression was altered using AAV-8 viral gene transfer of PICALM shRNA or PICALM cDNA into the hippocampus of 6 month old APP/PS1 mice. Aβ concentration and amyloid plaque load were measured 4 months later. PICALM knockdown decreased PBS-soluble Aβ levels and amyloid plaque load in the hippocampus. Conversely, overexpression of PICALM increased Aβ levels and amyloid plaque load. These data indicate that PICALM, an adaptor protein involved in clathrin-mediated endocytosis, regulates APP internalization and subsequent Aβ generation. Furthermore, PICALM plays a role in amyloid plaque pathogenesis and may provide a novel therapeutic target for AD.

Language

English (en)

Advisor/Committee Chair

Lee

Second Advisor

Xiao