Zn²⁺ dyshomeostasis caused by loss of ATP13A2/PARK9 leads to lysosomal dysfunction and alpha-synuclein accumulation - PubMed (original) (raw)

. 2014 Jun 1;23(11):2791-801.

doi: 10.1093/hmg/ddt572. Epub 2013 Dec 13.

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Zn²⁺ dyshomeostasis caused by loss of ATP13A2/PARK9 leads to lysosomal dysfunction and alpha-synuclein accumulation

Taiji Tsunemi et al. Hum Mol Genet. 2014.

Abstract

Mutations in ATP13A2 (PARK9) cause Kufor-Rakeb syndrome (KRS) characterized by juvenile-onset parkinsonism, pyramidal signs and dementia. PARK9 belongs to type 5 P-type ATPase with its putative function as a cation transporter. Loss of PARK9 leads to lysosomal dysfunction and subsequent α-synuclein (α-Syn) accumulation. However, the mechanistic link between PARK9 and lysosomal dysfunction remains unclear. Here, we found that patient fibroblasts expressing mutant PARK9 or primary neurons with silenced PARK9 exhibited increased sensitivity to extracellular zinc (Zn(2+)). This effect was rescued with the Zn(2+) chelators clioquinol or TPEN. PARK9-deficient cells showed decreased lysosomal sequestration of Zn(2+) and increased expression of zinc transporters. Importantly, increased concentrations of Zn(2+) (Zn(2+) stress) resulted in lysosomal dysfunction that was partially restored by expression of wild-type PARK9. Zn(2+) stress also caused increased expression of α-Syn and consequently decreased activity of the lysosomal enzyme glucocerebrosidase. Together, these data suggest that PARK9 loss of function leads to dyshomeostasis of intracellular Zn(2+) that in turn contributes to lysosomal dysfunction and accumulation of α-Syn. It will be of interest to examine whether therapeutic lowering of zinc may prove beneficial for patients with KRS.

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Figures

Figure 1.

Figure 1.

PARK9 mutant fibroblasts and primary cortical neurons are sensitive to zinc (Zn2+). (A) LDH release from fibroblasts cultured in a medium containing one of the cations (Cu2+, Fe2+, Mn2+, Ni2+ or Zn2+, concentration is 100 μ

m

) for 24 h (n = 3, *P < 0.005, **P < 0.001). (B) Toxicity of Zn2+ was attenuated by adding Zn2+ chelator, clioquinol (2 μ

m

) in a medium (n = 3, *P < 0.01, **P < 0.03, ***P < 0.001). (C) LDH release from primary cortical neurons (PCN) cultured in the presence of cations (n = 3, *P < 0.005). (D) Toxicity of Zn2+ was attenuated by adding Zn2+ chelator, TPEN (N,N,_N_‘,N'-Tetrakis(2-pyridylmethyl)ethylenediamine) (1 μ

m

) (n = 3, *P < 0.05, **P < 0.005). The values are the mean ± SEM.

Figure 2.

Figure 2.

PARK9 mutation alters Zn2+ distribution in fibroblasts. (A) The representative confocal live-cell images of fibroblasts stained with FluoZin-3 (green) and LysoTracker Red (red). Two wild-type (WT1 and WT2) and two PARK9 mutant (MUT1 and MUT2) fibroblast lines were stained before (left) or after treatment with 100 μ

m

Zn2+ for 1 h (right). Scale bars indicate 20 μm. (B) Quantification analysis of LysoTracker Red and FluoZin-3 area per total cell area before (left) and after Zn2+ incubation (right). Left, upper, quantification analysis of LysoTracker Red-positive area per total cell area (n = 10, *P < 0.01, **P < 0.001). Middle, quantification of FluoZin-3-positive area per total cell area (n = 10, *P < 0.005). Bottom, quantification of FluoZin-3-positive area per LysoTracker Red-positive area (n = 10, *P < 0.05). Right, upper, quantification of LysoTracker Red-positive area per total cell area (n = 10, *P < 0.05, **P < 0.03). Middle, quantification of FluoZin-3-positive area per total cell area. Bottom, quantification analysis of FluoZin-3-positive area per LysoTracker Red-positive area (n = 10, *P < 0.03, **P < 0.005). The values are the mean ± SEM.

Figure 3.

Figure 3.

Loss of PARK9 induces expression of zinc transporters and metallothionein proteins. (A) The mRNA expression of indicated zinc transporters in WT and MUT fibroblasts (n = 3, *P < 0.05, **P < 0.03). The expression of zinc transporters in MUT fibroblasts is divided by that in WT fibroblasts. (B) The mRNA expression of MT-III in scrambled- (Scrb) and PARK9 shRNA-treated primary cortical neurons (PCNs) (n = 3, *P < 0.001). The expression of MT-III in PARK9-treated PCNs is divided by that in Scrb-treated PCNs. (C) The PARK9 mRNA expression in PCNs cultured in the presence of 100 μ

m

Zn2+ (n = 3, *P < 0.001). The expression of PARK9 in PCNs cultured the medium containing 100 μ

m

Zn2+ is divided by that in PCNs cultured the normal medium. The values are the mean ± SEM.

Figure 4.

Figure 4.

Zinc dyshomeostasis leads to impaired lysosomal function. (A–E) PARK9 expression affects lysosomal proteolysis that is impaired by Zn2+. Lysosomal proteolysis in control, PARK9-depleted or PARK9 over-expressed PCNs cultured in a medium containing either 0 μ

m

(A), 1 μ

m

(B), 10 μ

m

(C) or 100 μ

m

Zn2+ (D). Lysosomal proteolysis is calculated by subtracting lysosomal inhibitors (2.5 m

m

NH4Cl and 50 μ

m

leupeptin) sensitive proteolysis from total proteolysis at 8, 20 and 28 h after chase. (E) Quantification of lysosomal proteolysis is calculated from the area under the curve (n = 3, *P < 0.05, **P < 0.005). (F) Dual-emission ratiometric measurement of lysosomal pH using LysoSensor Yellow/Blue DND-160. The effect of Zn2+ on lysosomal pH in Scrb or PARK9 shRNA treated PCNs is shown (n = 3, *P < 0.05, **P < 0.005). The values are the mean ± SEM.

Figure 5.

Figure 5.

Loss of PARK9 enhances zinc-mediated α-Syn accumulation and reduction of lysosomal hydrolases activities. (A) Upper left: western blot analysis of the effect of Zn2+ on α-Syn expression in PCNs treated with Scrb or PARK9 shRNA. NSE was used as loading control. MW is shown as kDa. Bottom left: the results of densitometric analysis of α-Syn oligomers which were detected with syn 505 (*P < 0.01). Upper right: the results of densitometric analysis of α-Syn monomers which were detected with C-20 (*P < 0.01). (B) Glucocerebrosidase (GCase) activities in lysosome-enriched fractions extracted from PCNs (n = 3, *P < 0.005). (C) Acid sphingomyelinase (aSMase) activities in lysosome-enriched fractions extracted from PCNs (n = 3, *P < 0.03).

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References

    1. Najim al-Din A.S., Wriekat A., Mubaidin A., Dasouki M., Hiari M. Pallido-pyramidal degeneration, supranuclear upgaze paresis and dementia: Kufor-Rakeb syndrome. Acta Neurol. Scand. 1994;89:347–352. - PubMed
    1. Ramirez A., Heimbach A., Grundemann J., Stiller B., Hampshire D., Cid L.P., Goebel I., Mubaidin A.F., Wriekat A.L., Roeper J., et al. Hereditary parkinsonism with dementia is caused by mutations in ATP13A2, encoding a lysosomal type 5 P-type ATPase. Nat. Genet. 2006;38:1184–1191. - PubMed
    1. Park J.S., Mehta P., Cooper A.A., Veivers D., Heimbach A., Stiller B., Kubisch C., Fung V.S., Krainc D., Mackay-Sim A., et al. Pathogenic effects of novel mutations in the P-type ATPase ATP13A2 (PARK9) causing Kufor-Rakeb syndrome, a form of early-onset parkinsonism. Hum. Mutat. 2011;32:956–964. - PubMed
    1. Podhajska A., Musso A., Trancikova A., Stafa K., Moser R., Sonnay S., Glauser L., Moore D.J. Common pathogenic effects of missense mutations in the P-type ATPase ATP13A2 (PARK9) associated with early-onset parkinsonism. PLoS ONE. 2012;7:e39942. - PMC - PubMed
    1. Ramonet D., Podhajska A., Stafa K., Sonnay S., Trancikova A., Tsika E., Pletnikova O., Troncoso J.C., Glauser L., Moore D.J. PARK9-associated ATP13A2 localizes to intracellular acidic vesicles and regulates cation homeostasis and neuronal integrity. Hum. Mol. Genet. 2012;21:1725–1743. - PMC - PubMed

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