PTPA variants and impaired PP2A activity in early-onset parkinsonism with intellectual disability

Abstract

The protein phosphatase 2A complex (PP2A), the major Ser/Thr phosphatase in the brain, is involved in a number of signalling pathways and functions, including the regulation of crucial proteins for neurodegeneration, such as alpha-synuclein, tau and LRRK2. Here, we report the identification of variants in the PTPA/PPP2R4 gene, encoding a major PP2A activator, in two families with early-onset parkinsonism and intellectual disability. We carried out clinical studies and genetic analyses, including genome-wide linkage analysis, whole-exome sequencing, and Sanger sequencing of candidate variants. We next performed functional studies on the disease-associated variants in cultured cells and knock-down of ptpa in Drosophila melanogaster. We first identified a homozygous PTPA variant, c.893T>G (p.Met298Arg), in patients from a South African family with early-onset parkinsonism and intellectual disability. Screening of a large series of additional families yielded a second homozygous variant, c.512C>A (p.Ala171Asp), in a Libyan family with a similar phenotype. Both variants co-segregate with disease in the respective families. The affected subjects display juvenile-onset parkinsonism and intellectual disability. The motor symptoms were responsive to treatment with levodopa and deep brain stimulation of the subthalamic nucleus. In overexpression studies, both the PTPA p.Ala171Asp and p.Met298Arg variants were associated with decreased PTPA RNA stability and decreased PTPA protein levels; the p.Ala171Asp variant additionally displayed decreased PTPA protein stability. Crucially, expression of both variants was associated with decreased PP2A complex levels and impaired PP2A phosphatase activation. PTPA orthologue knock-down in Drosophila neurons induced a significant impairment of locomotion in the climbing test. This defect was age-dependent and fully reversed by L-DOPA treatment. We conclude that bi-allelic missense PTPA variants associated with impaired activation of the PP2A phosphatase cause autosomal recessive early-onset parkinsonism with intellectual disability. Our findings might also provide new insights for understanding the role of the PP2A complex in the pathogenesis of more common forms of neurodegeneration.

Keywords: PP2A; PPP2R4; PTPA; intellectual disability; parkinsonism.

Figure 1

Pedigrees and identified PTPA variants. A and B show the pedigrees of Families 1 and 2, respectively. Filled symbols indicate individuals affected by parkinsonism; unfilled symbols indicate unaffected individuals; arrows denote index cases. AAO = age at onset; ref/var = heterozygous genotype for the corresponding variant; var/var = homozygous variant genotype; ref/ref = wild-type (reference) genotype. (C) PTPA gene schematic representation, including relative positions of the variants identified in this study annotated according to NM_178001. (D) 3D molecular rendering of PTPA, with the relative positions of the variants and the ATP binding and PP2A-C binding sites.

Figure 2

Comparison of protein and RNA levels of wild-type PTPA and the identified PTPA variants p.Ala171Asp PTPA and p.Met298Arg PTPA in cultured cells. (A) Representative western blot of protein extracts from cultured cells transfected with wild-type PTPA (PTPA_WT), p.Ala171Asp PTPA (PTPA_A171D), p.Met298Arg PTPA (PTPA_M298R) and empty vector (Transfection Ctrl). Blots were probed for expression of PTPA and vinculin. (B) Quantification showing a significant decrease in PTPA levels in p.Ala171Asp and p.Met298Arg PTPA-expressing cells (n = 3). Mean (SD); PTPA_WT: 1.00 (0.13); PTPA_A171D: 0.06 (0.01); PTPA_M298R: 0.81 (0.09); transfection control: 0.00 (0.00). (C) RT-qPCR analysis of PTPA in transfected cells, showing a significant decrease in PTPA expression in PTPA_A171D, PTPA_M298R and empty vector-expressing cells. Data represent relative normalized expression of PTPA mRNA. CLK2 and COPS5 were used as reference genes (n = 3). Mean (SD); PTPA_WT: 1.00 (0.00); PTPA_A171D: 0.74 (0.07); PTPA_M298R: 0.77 (0.08); transfection control: 0.00 (0.00). (D) Representative western blot of PTPA in transfected cells treated with cycloheximide (CHX) for the indicated times. (E) Quantification of PTPA over cycloheximide treatment, showing a significant decrease in PTPA_A171D compared to PTPA_WT 2 h after treatment, indicating protein instability. The bars indicate mean PTPA levels per time point as percentage of cells treated at time = 0 h (n = 3). Error bars represent ± SD. Only significant changes *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 are shown.

Figure 3

Effect of wild-type PTPA and PTPA variants p.Ala171Asp and p.Met298Arg expression on PP2A complex levels and phosphatase activity. (A) Representative western blot of protein extracts from cultured cells transfected with wild-type PTPA (PTPA_WT), p.Ala171Asp PTPA (PTPA_A171D), p.Met298Arg PTPA (PTPA_M298R) and empty vector (Transfection Ctrl). Blots were probed for expression of PP2A-C and vinculin (B) Quantification showing a significant decrease in PP2A-C levels in PTPA_A171D, PTPA_M298R and empty vector-expressing cells (n = 3). Mean (SD); PTPA_WT: 1.00 (0.12); PTPA_A171D: 0.67 (0.03); PTPA_M298R: 0.74 (0.03); transfection control: 0.60 (0.09). (C) RT-qPCR analysis of PP2A-C in transfected cells. Quantification shows a mild but significant decrease of PP2A-C levels in PTPA_A171D-expressing cells when compared to PTPA_WT-expressing cells. Data represent relative normalized expression of PP2A-C mRNA. CLK2 and COPS5 were used as reference genes (n = 3). Mean (SD); PTPA_WT: 1.00 (0.00); PTPA_A171D: 0.85 (0.06); PTPA_M298R: 0.93 (0.05); transfection control: 1.02 (0.10). (D) Quantification of Ser/Thr phosphatase activity assay in transfected cells, showing a significant decrease in activity in PTPA_A171D and PTPA_M298R-expressing cells (n = 3). Baseline represents pmoles of released phosphate from cells expressing an empty vector. Mean (SD); PTPA_WT: 2626 (159.70); PTPA_A171D: 1930 (91.24); PTPA_M298R: 2139 (90.59); baseline: 1601.67. (E) Scheme representing PTPA-induced activation of PP2A complex phosphatase function by antagonizing the PP2A inhibitor PME-1, and proposed model where the identified PTPA variants p.Ala171Asp and p.Met298Arg are less efficient in inducing PP2A activation. Error bars represent ± SD. Only significant changes *P < 0.05, **P < 0.01 and ***P < 0.001 are shown.

Figure 4

Effect of ptpa knock-down on D. melanogaster negative geotaxis measured by the climbing test. Comparative climbing test of three different UAS constructs (prkn, ptpa, nsmase) with and without L-DOPA treatment. prkn is used as positive control (significant locomotor impairment). nsmase is used as a negative control (no locomotor impairment). Climbing test performed at Days 10, 20, and 35 represented as proportion of flies that did not reach the 5 cm threshold. No significant difference is observed in locomotor behaviour between ptpa and controls on Day 10. Significant locomotor defect was observed at Days 20 and 35 for the ptpa and prkn lines compared to the negative control nsmase. This defect was significantly corrected with L-DOPA treatment. Significant comparisons are indicated by an asterisk. *P < 0.05. Human and Drosophila orthologues: PRKN (human)/prkn (D. melanogaster); PTPA (human)/ptpa (D. melanogaster); SMPD2 (human)/nsmase (D. melanogaster).