Chemical Modulation of Chaperone-Mediated Autophagy by Novel Retinoic Acid Derivatives.

摘要

Chaperone-mediated autophagy (CMA) contributes to cellular quality control and the cellular response to stress through the selective degradation of cytosolic proteins in lysosomes. Decrease in CMA activity occurs in aging and in age-related disorders (i.e. neurodegenerative diseases, diabetes, etc.). Although prevention of this age-dependent decline through genetic manipulation in mouse has proven beneficial, chemical modulation of CMA is not currently possible, due in part to the lack of information on the signaling mechanisms that modulate this pathway. In this work, we have identified that signaling through the retinoic acid receptor alpha inhibits CMA and have used computerized molecular dynamics simulations along with structure activity relationship design chemistry, to develop synthetic derivatives of all-trans-retinoic acid to specifically neutralize this inhibitory effect. We demonstrate that chemical enhancement of CMA with these novel compounds protects cells from oxidative stress and from proteotoxicity, supporting their potential therapeutic value in conditions in which reduced CMA contributes to cell malfunctioning and disease.;The goal of this thesis research was to determine how retinoic acid signaling functions in CMA regulation and to design small molecules that could function as activators or inhibitors of CMA.;In the first part of this study, we have focused on the RARα because it is important in oxidative stress, neurodegeneration and neurodevelopment, conditions in which CMA activity also changes. Here, we have completed the following studies: We analyzed the consequences of a decrease or increase of RA-mediated signaling on autophagy. To this purpose we have generated stable cell lines knocked-down for the RARa (decreased signaling) and treated cells with all-trans-retinoic acid (ATRA) (increased signaling). We have found that reduction of signaling through RARa reduces macroautophagy activity and increases CMA, whereas treatment with ATRA has the opposite effects. The inhibitory mechanism of ATRA on CMA was dependent on RARa whereas the effect on macroautophagy seemed to involve additional RAR's.;In the second part of these studies, our previous results motivated us to attempt designing compounds that by targeting RARa could help separate their effect on the different autophagic pathways. Therefore, in this part we focused on the design and synthesis of a library of 29 compounds targeting the RARa. 1. Primary screening: In the initially screening for lead molecules, we used NIH 3T3 mouse fibroblasts in culture and tested the effect of each of the compounds on: CMA using a photo-switchable CMA reporter. 2. Total intracellular protein degradation by metabolic labeling and pulse and chase experiments. 3. Macroautophagy by analysis of LC3-flux.;These studies have allowed us to narrow the list of compounds with a stimulatory effect only on CMA but not on macroautophagy from 29 to 3 target molecules.;Validation studies: We analyzed the effect of the CMA-modifiers on RARa knock-down (KD) cells to determine that the effect on CMA was through RARa signaling and in LAMP-2A KD cells to determine that the effect was specifically through CMA.;Mechanism of action: We have attempted to investigate the mechanism of action by which the RARa targeting compounds modulate CMA. First, we have experimentally determined that, as anticipated from our previous results, the three lead molecules function as antagonist of signaling through the RARa, and we verified that they do not affect RXR signaling. To determine the basis behind their stimulatory effect on CMA, we have first discarded that their effect is caused by inducing oxidative stress thereby activating CMA, or by directly acting on the lysosomal compartment. The three lead compounds have a very discrete upregulating effect on the expression of CMA components such as LAMP-2A.;Practical application: We have demonstrated a protective effect of the novel CMA modulators in NIH 3T3 cells against stressors that require maximal activation of this pathway, such as oxidative stress or proteotoxicity, and validated that this effect depends on RARa and active CMA.;Overall, this study contributes to the advancement and our understanding of modulation of Chaperone-Mediated Autophagy. Our findings could potentially impact the field of autophagy and contribute to the therapeutic treatment of neurodegenerative diseases such Parkinson's disease and aging. Future studies will determine if the target compounds generated in our studies could potentially be used in clinical trials.