Altered splicing of ATP6AP2 causes X-linked parkinsonism with spasticity (XPDS)

Abstract

We report a novel gene for a parkinsonian disorder. X-linked parkinsonism with spasticity (XPDS) presents either as typical adult onset Parkinson's disease or earlier onset spasticity followed by parkinsonism. We previously mapped the XPDS gene to a 28 Mb region on Xp11.2-X13.3. Exome sequencing of one affected individual identified five rare variants in this region, of which none was missense, nonsense or frame shift. Using patient-derived cells, we tested the effect of these variants on expression/splicing of the relevant genes. A synonymous variant in ATP6AP2, c.345C>T (p.S115S), markedly increased exon 4 skipping, resulting in the overexpression of a minor splice isoform that produces a protein with internal deletion of 32 amino acids in up to 50% of the total pool, with concomitant reduction of isoforms containing exon 4. ATP6AP2 is an essential accessory component of the vacuolar ATPase required for lysosomal degradative functions and autophagy, a pathway frequently affected in Parkinson's disease. Reduction of the full-size ATP6AP2 transcript in XPDS cells and decreased level of ATP6AP2 protein in XPDS brain may compromise V-ATPase function, as seen with siRNA knockdown in HEK293 cells, and may ultimately be responsible for the pathology. Another synonymous mutation in the same exon, c.321C>T (p.D107D), has a similar molecular defect of exon inclusion and causes X-linked mental retardation Hedera type (MRXSH). Mutations in XPDS and MRXSH alter binding sites for different splicing factors, which may explain the marked differences in age of onset and manifestations.

Figure 1.

Family with X-linked parkinsonism and spasticity (XPDS). The individual tested via exome sequencing is designated with an arrow.

Figure 2.

The silent c.345C>T (p.S115S) mutation results in overexpression of ATP6AP2 splice isoforms that lack exon 4. (A) RT–PCR with primers positioned in exons 3 and 5 amplifies a major 250 bp and a minor 150 bp product in controls; the minor product is highly overexpressed in patients. (B and C) Sequence analysis of splicing products from 250 bp (B) and 150 bp bands (C) confirming exon 4 skipping in the 150 bp band. III-8, IV-1: affected patients from the XPDS pedigree depicted in Fig. 1. NC1, NC2: normal controls. M: molecular weight marker.

Figure 3.

Distribution of ATP6AP2 splice isoforms in humans (A) and mice (B). The shaded box highlights the minor isoform c that corresponds to the Δe4 isoform increased in XPDS patients. (NM), isoform corresponding to RefSeq gene: NM_005765 (human) and NM_027439 (mouse).

Figure 4.

qRT–PCR quantification of ATP6AP2 splice isoforms in XPDS patients and controls. (A) Percentage of the Δe4 ATP6AP2 isoform in blood cells and brain tissues. (B) Relative expression of ATP6AP2 isoforms containing exon 4. (C) Relative expression of total ATP6AP2 transcript as measured with primers for constant exons 8 and 9. (D) Relative expression of Δe4 isoform. Open circles, controls; filled circles, patients. PB, uncultured peripheral blood cells.

Figure 5.

Reduced ATP6AP2 immunostaining in the XPDS brain. Representative immunostaining of brain sections of the XPDS case (A and B) and controls (C and D). In both the frontal cortex (A and C) and striatum (B and D), the XPDS case demonstrated normal distribution but marked reduction in the intensity of ATP6AP2 immunostaining in neurons.

Figure 6.

Knockdown of ATP6AP2 affects V-ATPase function and sensitizes cells to low concentrations of BafA1 (A). Dose–response of HEK293T cells to BafA1 after ATP6AP2 knockdown by siRNA. Forty-eight hours after transfection with three different siRNAs targeting ATP6AP2 (si-803, si-421, si-1978) or negative control siRNA (NC), HEK293T cells were exposed to BafA1 at the concentrations indicated. Cell-viability data are normalized to controls treated with 0.5% DMSO. (B) Autophagy flux measured in three independent experiments. HEK293T cells were transfected with negative control siRNA (NC) or with a pool of ATP6AP2 siRNA (ATP6AP2) and assayed for autophagy for a total of 6 h. BafA1 was added during the last 2 h of incubation. (C). ATP6AP2 deficiency induces accumulation of LC3-positive puncta. Shown is a representative ATP6AP2 siRNA knockdown experiment in HEK293 cells stably expressing the ptfLC3 reporter. Left panel, untransfected cells (NC); right panel, cells 96 h after transfection with a pool of ATP6AP2 siRNA (ATP6AP2); bottom, statistical analysis of puncta counts was performed on untreated (N = 31) and ATP6AP2 siRNA treated cells (N = 21) using a two-tailed t-test (P < 0.0005) for both autolysosomes (red puncta) and autophagosomes (yellow puncta).

Figure 7.

p62 immunostaining of the striatum of a control (A) and XPDS case (B). Note p62 labeling of plaque-like pathology, similar to tau immunostaining previously described in this case (1).