A Mild PUM1 Mutation Is Associated with Adult-Onset Ataxia, whereas Haploinsufficiency Causes Developmental Delay and Seizures

Abstract

Certain mutations can cause proteins to accumulate in neurons, leading to neurodegeneration. We recently showed, however, that upregulation of a wild-type protein, Ataxin1, caused by haploinsufficiency of its repressor, the RNA-binding protein Pumilio1 (PUM1), also causes neurodegeneration in mice. We therefore searched for human patients with PUM1 mutations. We identified eleven individuals with either PUM1 deletions or de novo missense variants who suffer a developmental syndrome (Pumilio1-associated developmental disability, ataxia, and seizure; PADDAS). We also identified a milder missense mutation in a family with adult-onset ataxia with incomplete penetrance (Pumilio1-related cerebellar ataxia, PRCA). Studies in patient-derived cells revealed that the missense mutations reduced PUM1 protein levels by ∼25% in the adult-onset cases and by ∼50% in the infantile-onset cases; levels of known PUM1 targets increased accordingly. Changes in protein levels thus track with phenotypic severity, and identifying posttranscriptional modulators of protein expression should identify new candidate disease genes.

Keywords: Ataxin-1; PADDAS; PRCA; Pumilio1; RNA-binding proteins; ataxia; chromosome 1p35.2; copy number variants; developmental delay; intellectual disability; seizures.

Figure 1. Deletions and mutations in PUM1 identified in early- and late-onset diseases

(A) Deletions spanning PUM1 on chromosome 1p35.2 (shown in red) were identified in nine patients with developmental disability; Mb, megabases. Dashed lines indicate the minimal region spanning PUM1. (B) Schematic of the PUM1 protein. Low-complexity regions are shown as purple boxes and PUM1 homology domains (HDs) as orange boxes. Locations of the PUM1 mutations in subjects 10, 11 and Family X are indicated. (C) Pedigree shows autosomal dominant inheritance of adult-onset ataxia in Family X. White and black denote unaffected and affected individuals, respectively; squares indicate males and circles indicate females; diamonds and numbers indicate the respective offspring; a line through the box indicates the individual is deceased. Subjects 12–18, who have been sequenced, are numbered in the order in which they were identified; DNA was not available from affected individuals without a subject number. S17 (asterisk) carries the familial mutation but does not have reported ataxia. The square box with dots (the great-grandfather of the proband) is a deceased individual who began using a walker in his 30s or 40s. The red arrow indicates the proband (Subject 12). (D) Protein alignment and comparison of the affected PUM1 residues compared to 21 organisms from human to Drosophila melanogaster. Different colors highlight degree of conservation: yellow for full conservation, light blue if conserved in all but one organism, and gray if more than one organism does not share the same amino acid. The human PUM1 amino acid sequence is used here as the reference protein. See also Figure S1 and Table S1, S2 and S3.

Figure 2. Magnetic Resonance Imaging (MRI) for subjects 10, 11, 12 and 13

Representative sagittal and transverse MRI images show normal imaging for Subject 10, an enlarged fourth ventricle with elevation and shortening of the vermis in Subject 11, and cerebellar atrophy in Subjects 12 and 13 (Family X). See also Figure S2.

Figure 3. Missense mutations decrease PUM1 stability in patient-derived cells

(A) Representative western blot and (B) left panel: quantification of protein levels of PUM1 and its targets in patient-derived fibroblast cells from Subject 11 (PADDAS), compared to three age-matched fibroblast control cell lines. PUM1 levels are about 50–60% lower than in healthy controls. Right panel: RNA quantification of PUM1 and its targets in fibroblasts from Subject 11 compared to three age-matched fibroblast control cell lines. FEV, which does not have PUM1 binding sites in its 3′UTR, served as a negative control. (C) Representative western blot and (D) left panel: quantification of PUM1 and its targets in patient-derived lymphoblastoid cell lines from Subjects 12 and 13 (late-onset ataxia), compared to three age-matched lymphoblastoid control cell lines, showing a PUM1 decrease of about 25%. Right panel: quantification of mRNA of PUM1 and its targets in patient-derived lymphoblasts from Subjects 12 and 13 compared to three age-matched lymphoblast controls. FEV again served as a negative control. All experiments were performed six times, blinded to genotype (data represent mean ± SEM). Data were normalized to GAPDH protein or mRNA levels, as appropriate. p values were calculated by Student’s t test, *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S3.

Figure 4. The missense mutations alter neuronal morphology and impair PUM1’s ability to suppress its targets

(A) Protein levels in HEK293T cells transfected with different amounts (from zero to 2μg) of either WT or mutant PUM1. Cells transfected with PUM1 bearing a mutation in the PUM-HD (either R1139W or T1035S) cannot suppress levels of ATXN1 and E2F3, two well-known PUM targets. See Figure S4A, B and C for the relative quantification. (B) mRNA quantification from HEK293T cells transfected as described in A, showing that PUM1 mRNA repression is lost when the mutation falls inside the PUM-HD. Empty vector transfection was used as a negative control for A and B. All experiments were performed in triplicate (data represent mean ± SEM); p values were calculated by Student’s t test. Data in A and B were normalized to GAPDH protein or mRNA levels, respectively. (C) Left panel: Sholl analysis of primary mouse hippocampal neurons after overexpression of either WT or PUM1 mutants quantified the number of intersections at various distances from the soma (left panel). Right panel: representative images of dendritic branching as influenced by overexpression of wild-type or mutant PUM1. Empty vector served as a negative control. Data represent mean ± SEM from 38–44 neurons per transfection. Statistical analysis was performed by two-way ANOVA with Tukey’s multiple comparisons test. *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S4.

Figure 5. Pum1 mutant mice manifest spontaneous seizures, abnormal EEG and cerebellar hypoplasia

(A) Pum1^+/− mice (n = 12) began to manifest spontaneous seizures at 22 weeks of age (68%, 11 out of 16). See also Video S5. Data are shown here as cumulative events. (B) Representative EEG traces. The Pum1^+/− mice (n = 4) predominantly showed prolonged hyperexcitability discharges in the neocortex. Generalized epileptiform spikes typically lasted over 10s, indicating a state of subclinical seizure. Neither hyperexcitability discharges nor electrographic seizures were observed in any of the recorded brain regions in wild-type littermates (n = 4). The scale bars are for both WT and Pum1^+/− mice. (C) Nissl staining at 5 weeks of age shows that Pum1^−/− mice (n = 6) had cerebellar hypoplasia (as confirmed by cerebellar weight, left bottom panel) in comparison to WT and Pum1^+/− mice. Scale bars 200 μm. *p < 0.05; **p < 0.01; ***p < 0.001.