Alzheimer Disease (AD) affects over 5 million people in the U.S. alone but our understanding of the mechanisms underlying pathogenesis remains incomplete and there is currently no cure. Recently, we reported that levels of the autophagy-related protein beclin 1 are decreased in AD brain extract. Heterozygous deficiency of beclin 1 in mice results in synaptic loss and neurodegeneration. Reduced beclin 1 levels in microglia impair recycling of the phagocytic receptor CD36, resulting in reduced clearance of amyloid plaques, a pathological hallmark of AD. Given that beclin 1 is expressed in neurons, this novel function of beclin 1 in mammals may affect recycling of receptors important for neuronal health. Neuronal survival depends on trophic factor signaling, and alterations in neurotrophic signaling pathways, including the neuroprotective TGF-? pathway, have been observed in AD. I present data here that shows knock-down of beclin 1, but not the autophagy-related protein Atg7, decreases TGF-? signaling in vitro. Beclin 1 is known to mediate its roles in autophagy and protein sorting in yeast through interaction with two distinct type III phosphatidylinositol-3-kinae (PI3K) complexes. Knock-down of the catalytic subunit of the PI3K results in decreased TGF-? signaling. Furthermore, knock-down of UVRAG, a PI3K complex component specific to protein sorting in yeast, also decreased TGF-? signaling, while knock-down of Atg14, specific to the complex involved in autophagy, has no effect. This suggests a specific role for beclin 1, as part of the PI3K complex containing UVRAG, in regulating TGF-? signaling. The purpose of this proposal is to test the hypothesis that reduced beclin 1 contributes to neurodegeneration, in part, by impairing TGF-? receptor recycling and depriving neurons of protective signaling through this pathway.
In Aim 1, I determine the functional and physical interactions between beclin 1, and the PI3K complexes that mediate TGF-? signaling using a series of mutant constructs. I also characterize the relationship between beclin 1, the TGF-? receptors and PI3-phosphate, the product of the PI3K complex, by live-cell imaging.
In Aim 2, I determine where in the TGF-? signaling pathway beclin 1 functions using a combination of flow cytometry, microscopy, and immunoprecipitation to directly test the effect of beclin 1 KD on TGF-? receptor endocytosis and recycling, and recruitment of downstream signaling molecules.
In Aim 3, I test if beclin 1 KD impairs TGF-? signaling in vivo and prevents TGF-?-mediated neuroprotection against excitotoxicity. These experiments use a combination of bioluminescence imaging and immunohistochemistry to determine the effect of beclin 1 KD on TGF-? signaling and neurodegeneration. Therapeutic approaches targeting the TGF-? pathway in AD are currently under study. Given the effect of reduced beclin 1 levels on TGF-? signaling, it is crucial to understand the mechanisms underlying this process so that we maximize our chance of success in developing therapies for this devastating disease.
The purpose of this proposal is to determine how reduced levels of the protein beclin 1, a change observed in Alzheimer Disease, affects protective signaling in the TGF-? pathway. Current efforts to develop therapeutics to target this pathway in AD aim to identify molecules that mimic TGF-? signaling. Understanding how beclin 1 deficiency affects signaling through TGF-? receptors will not only further our understanding of the mechanisms underlying AD, but also lead to better strategies for drug development.
|O'Brien, Caitlin E; Bonanno, Liana; Zhang, Hui et al. (2015) Beclin 1 regulates neuronal transforming growth factor-? signaling by mediating recycling of the type I receptor ALK5. Mol Neurodegener 10:69|
|Lucin, Kurt M; O'Brien, Caitlin E; Bieri, Gregor et al. (2013) Microglial beclin 1 regulates retromer trafficking and phagocytosis and is impaired in Alzheimer's disease. Neuron 79:873-86|