Rab7 induces clearance of α‐synuclein aggregates

Abstract

Parkinson's disease can be caused by mutations in the α-synuclein gene and is characterized by aggregates of α-synuclein protein. Aggregates are degraded by the autophago-lysosomal pathway. Since Rab7 has been shown to regulate trafficking of late endosomes and autophagosomes, we hypothesized that over-expressing Rab7 might be beneficial in Parkinson's disease. To test this hypothesis, we expressed the pathogenic A53T mutant of α-synuclein in HEK293 cells and Drosophila melanogaster. In HEK293 cells, EGFP-Rab7-decorated vesicles contain α-synuclein. Rab7 over-expression reduced the percentage of cells with α-synuclein particles and the amount of α-synuclein protein. Time-lapse microscopy confirmed that particles frequently disappeared with Rab7 over-expression. Clearance of α-synuclein is explained by the increased occurrence of acidified α-synuclein vesicles with Rab7 over-expression, presumably representing autolysosomes. Rab7 over-expression reduced apoptosis and the percentage of dead cells in trypan blue staining. In the fly model, Rab7 rescued the locomotor deficit induced by neuronal expression of A53T-α-synuclein. These beneficial effects were not produced by Rab7 missense mutations causing Charcot Marie Tooth neuropathy, or by the related GTPases Rab5, Rab9, or Rab23. Using mass spectrometry, we identified Rab7 in neuromelanin granules purified from human substantia nigra, indicating that Rab7 might be involved in the biogenesis of these possibly protective, autophagosome-like organelles in dopaminergic neurons. Taken together, Rab7 increased the clearance of α-synuclein aggregates, reduced cell death, and rescued the phenotype in a fly model of Parkinson's disease. These findings indicate that Rab7 is rate-limiting for aggregate clearance, and that Rab7 activation may offer a therapeutic strategy for Parkinson's disease. Cells over-expressing aggregation-prone A53T alpha-synuclein develop cytoplasmic aggregates mimicking changes observed in Parkinson's disease. When following cells in time-lapse microscopy, some few cells are able to remove these aggregates (Opazo et al. 2008). We now show that the percentage of cells clearing all aggregates from their cytosol is greatly increased with Rab7 over-expression, indicating that availability of Rab7 is rate-limiting for autophagic clearance of aggregates. The functional significance of this effect in neurons was confirmed in a Drosophila melanogaster model of Parkinson's disease.

FYCO1 mediates clearance of α‐synuclein aggregates through a Rab7‐dependent mechanism

“…As described previously (Dinter et al . ), coexpression of Rab7 with α‐synuclein increased the percentage of cells with a homogenous distribution of fluorescence, that is without a‐synuclein aggregates. Coexpression of RILP decreased the percentage of cells with a homogenous distribution (Fig.…”

“…; Dinter et al . ) to determine differences between α‐synuclein variants and the effects of therapeutic manipulations. Under control conditions, about 40% of cells were in the homogenous category.…”

“…Bands were quantified using the Software BioDocAnalyze (Biometra, Göttingen, Germany) as described previously (Dinter et al . ).…”

“…; Dinter et al . ). Cells were grown in 24‐well plates with glass bottom (Cat# 662892 Greiner bio‐one, Frickenhausen, Germany).…”

“…The mCherry version was described in Dinter et al . . GFP‐tagged human Rab7A (WT and T22N in pEGFP‐C1) were gifts from Richard Pagano (Addgene plasmids #12605 and 12660).…”