The Involvement Of KDEL Receptor In The Degradation Of Neurodegenerative Disease Proteins Via Autophagy

Neurodegenerative Diseases, including Alzheimer's Disease (AD), Parkinson's Disease (PD), Amyotrophic Lateral Sclerosis (ALS) and Huntington's Disease (HD), are often suffered by the elderly population. One of the typical characteristics of these diseases is the appearance of many intra-or extra-cellular protein aggregates in specific brain regions, such as A(βin AD and Lewy bodies in PD, The mutant disease-related proteins accumulate in the endoplasmic reticular (ER) and cause ER stress. Then the unfolded protein response (UPR) is activated to inhibit the general protein translation, induce the expression of specific ER chaperones, and degrade the misfolded proteins to attenuate the stress. Moreover, cellular signals that lead to cell death are activated in case of chronic or aggravated ER stress.There are two major protein degradative pathways:the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway (ALP). Autophagy is mediated by the autophagosome formation. The original double-membrane phagopore wraps the substrates and forms autophagosome. Then the autophagosome transports to and fuses with lysosome, degradating the substrates by plentiful proteases in the lysosome, Although the mechanisms are still unclear, ER stress can induce autophagy, and autophagy is responsible for the clearance of the neurodegenerative diseases-related proteins, such as a-synuclein A53T, SOD1 G93A and mutant huntingtin.The KDEL receptor selectively recognizes the ER-retained signal to retrotransport the "escaped proteins" from Golgi complex back to the ER. In mammalian cells, the most common signal is the KDEL (Lys-Asp-Glu-Leu) tetrapeptide sequence and many ER-resident chaperones, such as Grp78/BIP, Grp94, and PDI, bear the retrieval motif at their C-terminals. The KDEL receptor, which shuttles between the Golgi and ER to retrieve these target proteins, is involved in the ER quality control and ER stress response. ER stress can induce the chaperones upregulation, while it remains unclear whether the KDEL receptor can be regulated under ER stress condition.In this study, we draw the following conclusions:1. Using RT-PCR, either ER stress activators, such as thapsigargin and tunicamycin, or overexpression of EGFP-tHttQ150 is able to upregulate the mRNA levels of the KDEL receptor in consist with the ER stress marker CHOP upregulation. This suggests the KDEL receptor is also a responsive target against ER stress. 2. The overexpressed KDEL receptor in different cell lines, such as HEK293A and H1299 cells, shows diverse cellular localizations according to the protein expressing level. In some highly expressed cells, the KDEL receptor has a lysosomal localization except for the classical Golgi and ER localizations.3. Overexpression of the KDEL receptor increases the LC3II levels in a dose dependant manner, and promotes the punctuate LC3 formations. Clear double-membraned autophagosomes can be observed under the Transmission Electron Microscope (TEM). These results indicate the increased amount of autophagosomes.The overexpressed KDEL receptor in the EGFP-p62 and EGFP-LC3 stably expressed cell lines decreases the EGFP-p62 protein levels and increases the degraded free GFP protein levels, which indicate the enhanced autophagic activity. BafilomycinAl and CQ can inhibit the fusion of autophagosomes with lysosomes to block the autophagy flux, and can be used to detect the LC3 turnover. The KDEL receptor still increases the LC3II levels under the inhibitors treatments. Therefore, the KDEL receptor is an autophagy inducer.4. The KDEL receptor activates the three MAPKs, JNKs, p38, and ERKs. Inhibition of the MEK1/ERKs signaling leads to the suppression of autophagy induction implied by the LC3Ⅱand EGFP-p62 protein levels. The two other MAPKs family members, JNKs and p38, are not involved in autophagy regulation.5. The loss-of-function mutants of the KDEL receptor, D193N and R169N, fail to induce the MEK1/ERKs activation and autophagy.6. The mutant proteins linked to neurodegenerative diseases, such as tHttQ150, a-synuclein A53T, and SOD1 G93A, can also increase the KDEL receptor mRNA levels. Overexpression of the KDEL receptor promotes the degradation of these proteins, whereas knockdown of the KDEL receptor increases the disease-related protein levels. In the autophagy deficient ATG5 KO MEF cells, the KDEL receptor cannot decrease the protein levels of SOD1 G93A any more.Therefore, our results identify a novel function in association with disease protein degradation of the KDEL receptor as an autophagy inducer through MEK1/ERKs activation.