Alpha-synuclein is part of a diverse and highly conserved interaction network that includes PARK9 and manganese toxicity

Abstract

Parkinson's disease (PD), dementia with Lewy bodies and multiple system atrophy, collectively referred to as synucleinopathies, are associated with a diverse group of genetic and environmental susceptibilities. The best studied of these is PD. alpha-Synuclein (alpha-syn) has a key role in the pathogenesis of both familial and sporadic PD, but evidence linking it to other predisposition factors is limited. Here we report a strong genetic interaction between alpha-syn and the yeast ortholog of the PD-linked gene ATP13A2 (also known as PARK9). Dopaminergic neuron loss caused by alpha-syn overexpression in animal and neuronal PD models is rescued by coexpression of PARK9. Further, knockdown of the ATP13A2 ortholog in Caenorhabditis elegans enhances alpha-syn misfolding. These data provide a direct functional connection between alpha-syn and another PD susceptibility locus. Manganese exposure is an environmental risk factor linked to PD and PD-like syndromes. We discovered that yeast PARK9 helps to protect cells from manganese toxicity, revealing a connection between PD genetics (alpha-syn and PARK9) and an environmental risk factor (PARK9 and manganese). Finally, we show that additional genes from our yeast screen, with diverse functions, are potent modifiers of alpha-syn-induced neuron loss in animals, establishing a diverse, highly conserved interaction network for alpha-syn.

Figure 1

Interaction between 〈–syn and the yeast PARK9 homolog. a) Spotting assays with yeast 〈–syn toxicity modifier genes YPT1 and YPK9 showing their ability to suppress toxicity compared to empty vector control. Five-fold serial dilutions of yeast cells were spotted onto glucose (〈–syn expression repressed) or galactose (〈–syn expression induced). b) Deletion of Ypk9 has no effect on 〈–syn toxicity, however deleting the closely related ATPase Spf1 enhances 〈–syn toxicity. c) Synergistic genetic interaction between 〈–syn toxicity modifiers Ypt1 and Ypk9. In a high toxicity (HiTox) 2 copy 〈–syn yeast strain, expression of Ypt1 or Ypk9 alone is not sufficient to rescue toxicity. However, their co-expression restores growth to this strain. d) Ypk9 overexpression eliminates 〈–syn inclusions. 〈–Syn-YFP-expressing cells contain many vesicular inclusions when transformed with an empty vector and these are greatly diminished in cells transformed with a Ypk9 expression plasmid. e) The ability of Ypk9 to suppress the 〈–syn-induced block in ER-Golgi was measured by carboxypeptidase Y (CPY) maturation assay. Ypk9 significantly improved the trafficking of CPY from ER to Golgi.

Figure 2

PARK9 antagonizes α–syn-mediated dopaminergic neuron degeneration in C. elegans. (a, b) Anterior DA neurons in worms expressing P_dat-1::GFP + P_dat-1::α–syn at the day 7 stage. Arrowheads and arrows depict cell bodies and neuronal processes, respectively. WT worms have 6 anterior DA neurons. a) α–Syn toxicity is depicted by the loss of anterior DA neurons. b) DA neurons are protected when P_dat-1::FLAG-W08D2.5 cDNA is co-expressed. c) Quantification of C. elegans PARK9 rescue of α–syn-induced neurodegeneration in 4 independent transgenic lines displaying all six anterior DA neuron. P < 0.05, Student's t test. d) Overexpression of α–syn in P_unc-54::α–syn::GFP results in misfolding and aggregation of α–syn in body wall muscle cells at the young adult stage. e) Co-overexpression of TOR-2, a protein with chaperone activity, attenuates the misfolding of the α–syn::GFP protein. f) The misfolding of α–syn::GFP is enhanced following RNAi targeting W08D2.5.

Figure 3

PARK9 antagonizes α–syn-mediated dopaminergic neuron degeneration in rat primary midbrain neurons. a) Human PARK9 (ATP13A2) protects rat midbrain primary DA neurons from α–synA53T-induced toxicity. Primary rat embryonic midbrain cultures were either mock infected (control) or infected with lentivirus encoding LacZ, ATP13A2 alone, α–synA53T alone or α–synA53T and ATP13A2. Selective loss of DA neurons was determined immunocytochemically by comparing the percentage of MAP2-positive neurons that also stained positive for tyrosine hydroxylase (TH). N ≥ 3, # P < 0.05, ## P < 0.01, ### P < 0.001, one way analysis of variance with Newman-Keuls post-test (α–synA53T vs. control is also ###). b) ATP13A2/PARK9 rescues α–synA53T-induced DA neuron loss in rat primary midbrain cultures. Representative micrographs of cells stained for MAP2 (red) and TH (green). Arrows indicate TH⁺MAP2⁺ DA neurons. Scale bar = 20 μm.

Figure 4

Ypk9 is localized to the vacuole in yeast and PARK9 patient-based mutations affect its ability to rescue α–syn toxicity. a) Fluorescence microscopy to visualize Ypk9 subcellular localization. A chromosomally tagged YFP fusion (YPK9-YFP) localizes to the vacuolar membrane, as does WT GFP-YPK9 expressed from the constitutive GPD promoter. PARK9 patient-based mutations alter YPK9 localization but the ATPase-dead mutant (D781N) does not. GFP-tagged human ATP13A2 also localizes to the vacuole in yeast cells. b) Spotting assays with WT or 〈–syn-expressing cells. WT YPK9 overexpression suppresses 〈–syn toxicity but the two PARK9 patient-based mutant YPK9 proteins as well as the ATPase-dead mutant do not. Expressing mutant YPK9 in WT cells does not inhibit growth, supporting the idea that these are loss-of-function and not dominant negative mutations.

Figure 5

PARK9 protects cells from elevated manganese levels. a) Examples of conditions used to identify the substrate specificity of YPK9. We identified ypk9Δ cells as being sensitive to manganese (Mn²⁺) relative to WT cells. b) The effect of various Mn²⁺ concentrations on ypk9Δ cells grown on rich (YPD) or synthetic (CSM) media. c) Expressing WT YPK9 in ypk9Δ cells is sufficient to rescue Mn²⁺ sensitivity but neither the PARK9 patient-based mutants nor the ATPase-dead mutant are able to rescue. Expressing YPK9 in WT yeast cells makes them more resistant to Mn²⁺ (compare top and bottom spottings). d) YFP- (top panel) and GFP-tagged (bottom panel) Ypk9 fusion proteins used for localization studies are functional because they are able to protect against Mn²⁺ sensitivity. e) ypk9Δ cells also exhibit sensitivity to Mn²⁺ when grown in liquid culture.