Spontaneous shaker rat mutant - a new model for X-linked tremor/ataxia

Abstract

The shaker rat is an X-linked recessive spontaneous model of progressive Purkinje cell (PC) degeneration exhibiting a shaking ataxia and wide stance. Generation of Wistar Furth (WF)/Brown Norwegian (BN) F1 hybrids and genetic mapping of F2 sib-sib offspring using polymorphic markers narrowed the candidate gene region to 26 Mbp denoted by the last recombinant genetic marker DXRat21 at 133 Mbp to qter (the end of the long arm). In the WF background, the shaker mutation has complete penetrance, results in a stereotypic phenotype and there is a narrow window for age of disease onset; by contrast, the F2 hybrid phenotype was more varied, with a later age of onset and likely non-penetrance of the mutation. By deep RNA-sequencing, five variants were found in the candidate region; four were novel without known annotation. One of the variants caused an arginine (R) to cysteine (C) change at codon 35 of the ATPase, Ca(2+) transporting, plasma membrane 3 (Atp2b3) gene encoding PMCA3 that has high expression in the cerebellum. The variant was well supported by hundreds of overlapping reads, and was found in 100% of all affected replicas and 0% of the wild-type (WT) replicas. The mutation segregated with disease in all affected animals and the amino acid change was found in an evolutionarily conserved region of PMCA3. Despite strong genetic evidence for pathogenicity, in vitro analyses of PMCA3(R35C) function did not show any differences to WT PMCA3. Because Atp2b3 mutation leads to congenital ataxia in humans, the identified Atp2b3 missense change in the shaker rat presents a good candidate for the shaker rat phenotype based on genetic criteria, but cannot yet be considered a definite pathogenic variant owing to lack of functional changes.

Keywords: Ataxia; Atp2b3; PMCA3; X-linked; shaker.

Fig. 1.

Significant cerebellar pathology in shaker rats. Three (40 μm) midline cerebellar sections of each group – wild-type (WT) and shaker F1 (50/50 WF/BN) hybrid rats – were analyzed at 2 and 6 months of age (n=3/group). Sections were stained with an antibody to mouse monoclonal anti-calbindin-D-28K (Calb1; 1:500) and counterstained with DAPI. Calb1 staining in Purkinje cells (PCs) was reduced in shaker 2-month-old animals, and was virtually absent in somata and dendrites in PCs of shaker 6-month-old animals. PCs are denoted by arrows. PF, primary fissure.

Fig. 2.

Age-of-onset graph for Wistar Furth (WF) and F2 male offspring. Graph represents 105 WF and 92 F2 males. All male progeny were aged to 36 weeks of age before euthanization. 100% of WF and 95% of F2 mutation carriers exhibited an affected phenotype by 36 weeks of age. ‘Non-penetrant’ was defined as mutation-positive but not exhibiting a phenotype by 36 weeks of age. Reduced penetrance was observed only in the F2 cohort.

Fig. 3.

Characterization of PMCA3. (A) Schematic representation of GFP-tagged PMCA3 clones. GFP-tagged wild-type (WT) PMCA3 or PMCA3^R35C (i, ii) or non-tagged (pAdTrack) WT PMCA3 or PMCA3^R35C (iii, iv) with an independent GFP cassette under the transcriptional control of CMV promoters are shown. The bottom images show a plasmid map delineating restriction enzymes used to clone the insert, and the selecting antibiotic used. (B) Western blot of SH-SY5Y cell lysates following transfection by GFP-tagged PMCA3 plasmids. Control lanes represent untransfected cell extract. Western blot results confirmed the expression of WT PMCA3 or PMCA3^R35C recombinant PMCA3. The experiment was replicated three times. (C) Twelve (10 μm) midline cerebellar sections of each group – WT and shaker WF rats – were analyzed at 9 weeks age (n=2). Sections were stained with an antibody to rabbit polyclonal anti-PMCA3 ATPase (1:2000) and counterstained with DAPI. PMCA3 staining shows no difference between WT and shaker rats. ML, molecular layer; PC, Purkinje cells; GC, granular cell layer.

Fig. 4.

Cytosolic Ca²⁺ measurements in HeLa cells overexpressing the GFP-tagged and untagged wild-type (WT) and R35C mutant PMCA3a variant. HeLa cells were co-transfected with cytAEQ and the expression plasmid for the WT and the R35C mutant GFP-tagged and untagged PMCA3a variant. Average peak values (A,C) and cytosolic Ca²⁺ transients (B,D) were recorded following 100 μM histamine (His) stimulation. Bars in panels A and C represent mean [Ca²⁺] values upon stimulation (μM; ±s.e.m.). Peak values in µM are: 4.20±0.15, n=27 for cytAEQ, 3.97±0.21, n=29 for GFP-tagged WT PMCA3a, and 3.82±0.18, n=28 for GFP-tagged R35C PMCA3a; 3.95±0.23, n=30 for un-tagged WT PMCA3a, and 4.09±0.26, n=29 for un-tagged R35C PMCA3a. The experiment was replicated three times.

Fig. 5.

Cytosolic Ca²⁺ measurements in HeLa cells overexpressing the untagged wild-type (WT) and R35C mutant PMCA3b variant. HeLa cells were co-transfected with cytAEQ and the expression plasmid for the WT and the R35C mutant untagged PMCA3b variant. Average peak values (A) and cytosolic Ca²⁺ transients (B) were recorded following 100 μM histamine (His) stimulation. Bars in panel A represent mean [Ca²⁺] values upon stimulation (μM; ±s.e.m.). Peak values in µM are: 4.50±0.21, n=10 for cytAEQ, 4.16±0.14, n=11 for un-tagged WT PMCA3b, and 4.30±0.20, n=7 for un-tagged R35C PMCA3b. The experiment was replicated three times.

Fig. 6.

Functional complementation assay in K616 yeast cells. Yeast K616 cells were transformed with pYES2-derived vectors carrying one of the following PMCA3b variants: wild type (WT), C-terminally truncated (PMCA3 ΔC_ter), R35C mutant, D465A mutant and W1104A mutant. Exponentially growing cells have been serially diluted and spotted on glucose+CaCl₂ medium (A), galactose+7.5 mM EGTA (B) and galactose+10 mM EGTA (C). Cells carrying empty plasmid or expressing constitutively active PMCA3 ΔC_ter are negative and positive internal controls of the functional assay, respectively. The experiment was replicated three times.