Chaperone mediated autophagy respond to dynein mediated transport inhibition in motor neuron diseases

The presence of protein aggregates in degenerating motoneurons represents a common hallmark of motoneuron diseases (MNDs), including amyotrophic lateral sclerosis (ALS) and spinal and bulbar muscular atrophy (SBMA). Dysfunctions of the protein quality control (PQC) systems have been suggested to contribute to protein aggregation in MNDs. In NSC34 and motoneuron derived from iPS cells, inhibition of retrograde transport of misfolded protein mediated by dynein results in macroautophagy reduction and in co-chaperone BCL2-Associated Athanogene 1 (BAG1) mRNA increase. While dynein mediated transport is necessary for autophagy, BAG1 is responsible for misfolded protein degradation via proteasome. We have demonstrated that exogenous BAG1 overexpression reduced misfolded and aggregated species by proteasome degradation. BAG1 in association with HSPA8/HSC70 protein may also route misfolded proteins to chaperone-mediated autophagy (CMA) for degradation. CMA is a selective catabolic pathway responsible for the degradation of specific cellular proteins containing the KFERQ-like motif. After the recognition of this motif by HSPA8, substrates are translocated to the lysosomal receptor LAMP2A that allows the entry of substrates into the lumen of competent lysosomes. In NSC34 cells, filter trap assay showed a reproducible non-significant reduction of SOD1 and ARpolyQ insoluble species in response to dynein mediated transport inhibition when proteasome was inhibited. Interestingly, dynein mediated transport inhibition reduced ARpolyQ insoluble species, also when macroautophagy and proteasome are both inhibited. Dynein mediated transport inhibition did not modify the mRNA levels of all autophagy markers tested, but significantly increased Lamp2A mRNA and protein levels. Instead, HspA8 mRNA and protein remained unaltered. We demonstrated that dynein mediated transport inhibition reduced the Lamp1 protein levels. By overexpressing alpha-synuclein (SNCA), a well-established CMA substrate, we found that dynein mediated transport inhibition increased its clearance confirming that transport alteration may result in CMA response to clear misfolded protein. Collectively, these data show that inhibition of retrograde transport drastically impairs macroautophagy and this possibly prevents misfolded protein toxicity via CMA. Considering that CMA is a ubiquitous pathway, it is a conceivable that CMA can be used as a potential target to increase the clearance of misfolded proteins.