Identification of TMEM230 mutations in familial Parkinson's disease

Abstract

Parkinson's disease is the second most common neurodegenerative disorder without effective treatment. It is generally sporadic with unknown etiology. However, genetic studies of rare familial forms have led to the identification of mutations in several genes, which are linked to typical Parkinson's disease or parkinsonian disorders. The pathogenesis of Parkinson's disease remains largely elusive. Here we report a locus for autosomal dominant, clinically typical and Lewy body-confirmed Parkinson's disease on the short arm of chromosome 20 (20pter-p12) and identify TMEM230 as the disease-causing gene. We show that TMEM230 encodes a transmembrane protein of secretory/recycling vesicles, including synaptic vesicles in neurons. Disease-linked TMEM230 mutants impair synaptic vesicle trafficking. Our data provide genetic evidence that a mutant transmembrane protein of synaptic vesicles in neurons is etiologically linked to Parkinson's disease, with implications for understanding the pathogenic mechanism of Parkinson's disease and for developing rational therapies.

Conflict of interest statement

Competing Financial Interests

The authors declare no competing financial interests.

Figures

Figure 1

Mutations of TMEM230 in patients with PD. (a) Evolutionary conservation of mutated amino acids in the TMEM230 protein in different species. Amino acids identical to human TMEM230 are in black letters, and non-identical ones are denoted in red letters. The mutated amino acids are indicated by arrowheads on the top. (b) Predicted putative secondary and tertiary structures. The predominant isoform 2 of TMEM230 is shown. Image is colored by rainbow from N (blue) → C (red) terminus. Two major alpha helix regions are shown on the left. Mutation sites are indicated by arrows. (c) Schematic diagram showing the topology of the TMEM230 protein. Two transmembrane segments (aa 109-129, and aa 142-162) are shown in blue, with the N- and the C-terminal regions exposed to the cytosol. Mutations are indicated by arrows. The X184W and X184PG mutations lead to the C-terminal addition of six and seven amino acids, respectively, with the last five amino acids being identical (HPPHS, blue).

Figure 2

Localization of TMEM230 to synaptic vesicles in neurons. (a-p) Confocal microscopy was performed using transfected (a-h) and non-transfected (i-p) primary neurons from the mouse midbrain. Mouse endogenous (endo-) Tmem230, Syn and Vmat2 were detected with respective antibodies. Representative co-localization signals are indicated by arrows. (e-h) A TMEM230 transfected primary neuron (arrow) shows TMEM230 co-localization with endogenous Vmat2 (dopaminergic neuron); the other transfected neuron (arrowhead) that is TMEM230-positive, but vmat2-negative, suggesting a non-dopaminergic neuron. (m-p) A mouse endogenous Tmem230-positive, but Vmat2-negative (non-dopaminergic neuron) is indicated by an arrowhead. (q) Enrichment of Tmem230 in synaptosomes and synaptic vesicles. Western blot was performed using four fractions isolated from rat brains as indicated. Tmem230 shows a similar enrichment pattern to synaptophysin. (r) Localization of Tmem230 in the presynaptic vesicle pool region. Immunogold electron microscopy was performed using rat brain sections. A representative perforated synapse in rat CA1 region is shown in the center of the image. Presynaptic axonal terminal (Ax) and dendritic spine head (Dsh) are shown. Tmem230-positive gold particles are indicated by an arrow. The presynaptic vesicle pool region and postsynaptic density are indicated by black and green arrowheads, respectively.

Figure 3

Convergence of TMEM230 and VPS35 in vesicle trafficking/recycling to TGN. (a-d) Confocal microscopy was performed using Neuro-2a cells transfected with TMEM230-eGFP construct together with VPS35-myc as indicated. (e-p) Confocal microscopy was performed using mouse primary neurons transfected with TMEM230-eGFP. The mouse endogenous Vps35 was detected with an antibody. Representative images showing the overlap of the signals (arrows) of TMEM230 and mouse endogenous Vps35. (e-h) A less TMEM230 transfected neuron showing Vps35, but only marginal TMEM230-eGFP is indicated by an arrowhead.

Figure 4

Impairment of synaptic vesicle trafficking by PD-linked mutant TMEM230. (a) Fluorescent image of synaptic vesicles in transfected primary neurons. Cultured mouse midbrain primary neurons were co-transfected with the GFP-tagged VAMP2 expressing vector (pEGFP-VAMP2) and each of the TMEM230 tag-free constructs in the pIRES2-ZsGreen1 vector. The pEGFP-VAMP2 was used to label the synaptic vesicles (Supplementary information). White arrowheads mark representative GFP-VAMP2-labeled vesicles quantified in panels b-e. (b) Movement track of the GFP-VAMP2-labeled vesicles. The trafficking of the vesicles in live primary neurons is monitored by confocal microscope and quantified using the Imaris software package. The movement tracks of eight representative vesicles from time-lapse live confocal images (30 frames with 5-second intervals) are shown, with pseudocolored movement tracks marking the position of vesicles at different time points during the 150 seconds. Images shown in panels (a) and (b) are taken at the time point marked on the pseudocolored time scale. (c-e) Quantification of the mean transport speed, track length and displacement length of GFP-VAMP2-labeled vesicles. Vesicles in neurons expressing PD-linked mutants (TMEM230-Y92C, TMEM230-R141L, TMEM230-X184W and TMEM230-X184PG) show impaired movement, as measured by transport speed (c), track length (d) and displacement length (e), when compared to the wild-type (TMEM230-WT). Data are from four independent experiments (1055 moving vesicles in 27 neurons). ** p < 0.01 (Oneway ANOVA test). Error bars, mean ± s.e.m.