Autophagy is a lysosome-dependent cellular catabolic mechanism mediating the turnover of intracellular organelles and long-lived proteins. Reduction of autophagy activity has been shown to lead to the accumulation of misfolded proteins in neurons and may be involved in chronic neurodegenerative diseases such as Huntington's disease and Alzheimer's disease. To explore the mechanism of autophagy and identify small molecules that can activate it, we developed a series of high-throughput image-based screens for small-molecule regulators of autophagy. This series of screens allowed us to distinguish compounds that can truly induce autophagic degradation from those that induce the accumulation of autophagosomes as a result of causing cellular damage or blocking downstream lysosomal functions. Our analyses led to the identification of eight compounds that can induce autophagy and promote long-lived protein degradation. Interestingly, seven of eight compounds are FDA-approved drugs for treatment of human diseases. Furthermore, we show that these compounds can reduce the levels of expanded polyglutamine repeats in cultured cells. Our studies suggest the possibility that some of these drugs may be useful for the treatment of Huntington's and other human diseases associated with the accumulation of misfolded proteins.A utophagy is a cellular catabolic mechanism mediating the turnover of intracellular organelles and proteins through a lysosome-dependent but proteasome-independent degradative pathway (1, 2). An autophagosome sequesters cytoplasmic constituents, such as mitochondria, endoplasmic reticulum, and ribosomes, by forming a double-membrane vesicle. The outer membrane of the autophagosome then fuses with the lysosome in mammalian cells delivering the sequestered content to the lumen of lysosome for degradation. Autophagy is critical for the survival of yeast and mammalian cells under starvation conditions because it functions to recycle intracellular material for macromolecular synthesis and energy production (3).Autophagy occurs in all cells at low basal levels under normal conditions to perform homeostatic functions, but it can be rapidly up-regulated under starvation or stress conditions (3). Elegant genetic analysis has identified 17 genes that are essential for autophagy in yeast (referred to as the ATG genes) (4, 5). In mammalian cells, mTOR kinase, the target of rapamycin, mediates the major inhibitory signal that shuts off autophagy under nutrient-rich conditions (3). On the other hand, mammalian type III PI3-kinase, the homolog of yeast VPS34 and inhibitable by 3-methyladenine (3-MA) (a nonspecific inhibitor of PI3-kinase), is required for the onset of autophagy. In this regard, rapamycin and 3-MA, the most commonly used chemicals to induce and inhibit autophagy, respectively, provide convenient tools to study autophagy.To explore the mechanism of autophagy and identify additional small molecules that can activate it, we developed a high-throughput image-based screen. This system takes advantage of the local...

Aging is a major risk factor for both genetic and sporadic neurodegenerative disorders. However, it is unclear how aging interacts with genetic predispositions to promote neurodegeneration. Here, we investigate how partial loss of function of TBK1, a major genetic cause for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) comorbidity, leads to age-dependent neurodegeneration. We show that TBK1 is an endogenous inhibitor of RIPK1 and the embryonic lethality of Tbk1 mice is dependent on RIPK1 kinase activity. In aging human brains, another endogenous RIPK1 inhibitor, TAK1, exhibits a marked decrease in expression. We show that in Tbk1 mice, the reduced myeloid TAK1 expression promotes all the key hallmarks of ALS/FTD, including neuroinflammation, TDP-43 aggregation, axonal degeneration, neuronal loss, and behavior deficits, which are blocked upon inhibition of RIPK1. Thus, aging facilitates RIPK1 activation by reducing TAK1 expression, which cooperates with genetic risk factors to promote the onset of ALS/FTD.

Necroptosis and ferroptosis are two distinct necrotic cell death modalities with no known common molecular mechanisms. Necroptosis is activated by ligands of death receptors such as tumor necrosis factor-α (TNF-α) under caspase-deficient conditions, whereas ferroptosis is mediated by the accumulation of lipid peroxides upon the depletion/or inhibition of glutathione peroxidase 4 (GPX4). The molecular mechanism that mediates the execution of ferroptosis remains unclear. In this study, we identified 2-amino-5-chloro-N,3-dimethylbenzamide (CDDO), a compound known to inhibit heat shock protein 90 (HSP90), as an inhibitor of necroptosis that could also inhibit ferroptosis. We found that HSP90 defined a common regulatory nodal between necroptosis and ferroptosis. We showed that inhibition of HSP90 by CDDO blocked necroptosis by inhibiting the activation of RIPK1 kinase. Furthermore, we showed that the activation of ferroptosis by erastin increased the levels of lysosome-associated membrane protein 2a to promote chaperone-mediated autophagy (CMA), which, in turn, promoted the degradation of GPX4. Importantly, inhibition of CMA stabilized GPX4 and reduced ferroptosis. Our results suggest that activation of CMA is involved in the execution of ferroptosis.