Expression of Caytaxin protein in Cayman Ataxia mouse models correlates with phenotype severity

Abstract

Caytaxin is a highly-conserved protein, which is encoded by the Atcay/ATCAY gene. Mutations in Atcay/ATCAY have been identified as causative of cerebellar disorders such as the rare hereditary disease Cayman ataxia in humans, generalized dystonia in the dystonic (dt) rat, and marked motor defects in three ataxic mouse lines. While several lines of evidence suggest that Caytaxin plays a critical role in maintaining nervous system processes, the physiological function of Caytaxin has not been fully characterized. In the study presented here, we generated novel specific monoclonal antibodies against full-length Caytaxin to examine endogenous Caytaxin expression in wild type and Atcay mutant mouse lines. Caytaxin protein is absent from brain tissues in the two severely ataxic Atcay(jit) (jittery) and Atcay(swd) (sidewinder) mutant lines, and markedly decreased in the mildly ataxic/dystonic Atcay(ji-hes) (hesitant) line, indicating a correlation between Caytaxin expression and disease severity. As the expression of wild type human Caytaxin in mutant sidewinder and jittery mice rescues the ataxic phenotype, Caytaxin's physiological function appears to be conserved between the human and mouse orthologs. Across multiple species and in several neuronal cell lines Caytaxin is expressed as several protein isoforms, the two largest of which are caused by the usage of conserved methionine translation start sites. The work described in this manuscript presents an initial characterization of the Caytaxin protein and its expression in wild type and several mutant mouse models. Utilizing these animal models of human Cayman Ataxia will now allow an in-depth analysis to elucidate Caytaxin's role in maintaining normal neuronal function.

Figure 1. Caytaxin protein expression in Atcay mouse mutants.

(A) Western blot depicting Caytaxin expression in sidewinder littermates using anti-Caytaxin mAb 8F4, developed with DAB. Lane 1, wild type (wt/wt); lane 2, heterozygote (swd/wt); lane 3, homozygote (swd/swd). (B) Represents a segment of the blot shown in (A) that was probed with an antibody specific for beta-actin. (C) Caytaxin expression in Atcay mutant mouse lines using anti-Caytaxin mAb 1E2, developed with DAB. Lane 1, wildtype (wt/wt); lane 2, hesitant mutatnt (hes/hes); lane 3, jittery mutant (ji/ji); lane 4, sidewinder mutant (swd/swd). (D) Blot from (B) re-probed with mAb 1E2 and developed with ECL. (E) Displays a segment of the immunoblot shown in (C) and (D) that was probed with an antibody against beta-actin. All lanes contain 60 µg total protein from frozen mouse brain lysates.

Figure 2. Caytaxin protein expression in various species and cell lines.

Each lane contains 60 µg total protein. (A) Lane 1, total Drosophila embryo extract; lane 2, Zebrafish nervous system; lane 3, Xenopus laevis adult brain; lane 4, chicken brain; and lane 5, mouse adult brain. Probed with anti-Caytaxin mAb 1E2 and developed with ECL. (B) Total protein lysates from frozen cell line pellets. Lane 1, AtT20 (mouse corticotroph pituitary tumor); lane 2, PC12 (rat pheochromocytoma); lane 3, C6 (rat astrocytoma); lane 4, 132-1N1 (human glioma); lane 5, A172 (human glioma); lane 6, N2A (mouse neuroblastoma); lane 7, SH-SY5Y (human neuroblastoma); and lane 8, wild type mouse brain. Probed with anti-Caytaxin mAb 8F4 and developed with DAB.

Figure 3. In vitro translation of wild type and mutant Atcay/ATCAY cDNAs.

(A) shows an autoradiogram of Caytaxin protein that was produced from cDNA by in vitro transcription/translation as described in Materials and Methods. Lane 1, T7 RNA Polymerase, no DNA; lane 2, SP6 RNA polymerase, no DNA; lane 3, T7 RNA polymerase, mouse Atcay cDNA; lane 4, T7 RNA polymerase, human ATCAY cDNA; lane 5, SP6 RNA polymerase, mouse Atcay cDNA; and lane 6, SP6 RNA polymerase, human ATCAY cDNA. (B) Western blot from an adjacent part of the gel depicted in (A), incubated with anti-Caytaxin mAb 8F4 and stained with ECL. Lane 1, 30 µg wild type mouse brain and lane 2, 1×10⁶ human neuroblastoma SHSY5Y cells. (C) Caytaxin protein from methionine mutant cDNA constructs by in vitro transcription/translation using SP6 RNA polymerase. Lane 1, wild type mouse Atcay cDNA; land 2, no DNA; lane 3, mutation of first methionine; lane 4, mutation of second methionine; and lane 5, mutation of both methionine residues. (D) Nucleotide and protein amino acid sequence comparison between mouse and human Caytaxin. The two conserved methionine residues (Met1 and Met 10), which are separated by 8 amino acids, are marked in bold.

Figure 4. Caytaxin protein expression throughout mouse brain development.

Each lane contains 30 µg of total brain protein extract obtained from wt/wt sidewinder mice at post-natal ages Day 1 (lane 1), Day 7 (lane 2), Day 10 (lane 3), Day 14 (lane 4), Day 21 (lane 5), 1 month (lane 6), 2 months (lane 7), 3 months (lane 8), 4 months (lane 9), 6 months (lane 10), 8 months (lane 11), and 10 months (lane 12). (A) Western blot probed with anti-Caytaxin mAb 8F4 and re-probed with anti-β-tubulin as a loading control (B). (C) Protein band density calculations described in materials and methods. Densities were averaged among all mice per time point (n = 3, except at 10 months where n = 2). Caytaxin was normalized to β-tubulin and plotted relative to average density at 3 months. P-values generated using Student’s t-test. Error bars generated based on standard distribution from the mean of each group.

Figure 5. Caytaxin protein expression and functional rescue in transgenic human ATCAY BAC⁺ mice.

(A) Western blot with 30 µg protein loaded, probed with anti-Caytaxin mAb 8F4 and developed using ECL. Lane 1, wild type mouse brain; lane 2, human SH-SY5Y cell lysate; lane 3, total brain protein from ji/ji ATCAY BAC⁺; and lane 4, swd/swd ATCAY BAC⁺. (B) Shows a segment of the immunoblot shown in (A) that was probed with an antibody specific for beta-actin. (C) Average time on a rotating rod per day, over 3 days. Wild type control +/swd BAC- mice (light grey bars, n = 7), and transgenic rescue swd/swd BAC⁺ mice (dark grey bars, n = 10). P-values between the control and the rescued group performance and between Day 1 and Day 3 are indicated above the columns.