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.
|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|