Prenatal diagnosis of spinocerebellar ataxia type 3/Machado-Joseph disease in China's Mainland A case report

Spinocerebellar ataxia type 3/Machado-Joseph disease （SCA3/MJD） is a progressive, currently untreatable and ultimately fatal ataxic disorder that belongs to the group of neurological disorders known as CAG-repeat or polyglutamine diseases. Here, we present the first prenatal diagnosis of SCA3/MJD in China's Mainland in a woman who was known to carry an expanded CAG-trinucleotide repeat in the MJD1 gene. After evaluating motivation and psychological tolerance of the couple, amniocentesis was performed after 14 weeks of gestation. Polymerase chain reactions followed by T-vector cloning and direct sequencing were employed to evaluate the CAG-repeat number of the fetal MJD1 gene. We identified a truncated CAG expansion of 78 repeats in the MJD1 gene of the fetus compared with 81 repeats in his mother.

Clinical manifestations and gene mutation in a case of Machado-Joseph disease

This study reports a case of a 75-year-old female Machado-Joseph disease patient exhibiting unstable walking and inaccurate hand holding for 8 months, which progressively worsened. Physical examination on admission showed cerebellar ataxia and a history of hypertension. Crania MRI demonstrated cerebellar and brain stem atrophy. Gene analysis showed abnormal amplification of the CAG trinucleotide repeat in exon 10 of the ataxin-3 （ATXN3） gene, resulting in 70-81 CAG repeats in the patient, with a significant positive family history.

Emerging Concepts of Pathogenesis and Comprehensive Therapeutic Strategies for Spinocerebellar Ataxia Type 3

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph Disease (MJD), is an autosomal dominant neurodegenerative disorder that predominantly involves the cerebellar, pyramidal, extrapyramidal, motor neuron and oculomotor systems. SCA3 presents strong phenotypic heterogeneity and its causative mutation of SCA3 consists of an expansion of a CAG tract in exon 10 of the ATXN3 gene, situated at 14q32.1. The ATXN3 gene is ubiquitously expressed in neuronal and non-neuronal tissues, and also participates in cellular protein quality control pathways. Mutated ATXN3 alleles present about 45 to 87CAG repeats, which result in an expanded polyglutamine tract in ataxin-3. After mutation, the polyQ tract reaches the pathological threshold (about 50 glutamine residues);the protein is considered that it might gain a neurotoxic function through some unclear mechanisms. We reviewed the literature on the pathogenesis and therapeutic strategies of spinocerebellar ataxia type 3 patients. Conversion of the expanded protein is possible by enhancing protein refolding and degradation or preventing proteolytic cleavage and prevents the protein to reach the site of toxicity by altering its ability to translocate between the nucleus and cytoplasm. Proteasomal degradation and enhancing autophagic aggregate clearance are currently proposed remarkable therapy. In spite of extensive research, the molecular mechanisms of cellular toxicity resulting from mutant ataxin-3 remain no preventive treatment is currently available. These therapeutic strategies might be able to improve sign symptoms of SCA3 as well as slow the disease progression.

Casein kinase 2 interacts with and phosphorylates ataxin-3

Objective Machado-Joseph disease （MJD）/Spinocerebellar ataxia type 3 （SCA3） is an autosomal dominant neurodegenerative disorder caused by an expansion of polyglutamine tract near the C-terminus of the MJD1 gene product, ataxin-3. The precise mechanism of the MJD/SCA3 pathogenesis remains unclear. A growing body of evidence demonstrates that phosphorylation plays an important role in the pathogenesis of many neurodegenerative diseases. However, few kinases are known to phosphorylate ataxin-3. The present study is to explore whether ataxin-3 is a substrate of casein kinase 2 （CK2）. Methods The interaction between ataxin-3 and CK2 was identified by glutathione S-transferase （GST） pull-down assay and co-immunoprecipition assay. The phosphorylation of ataxin-3 by CK2 was measured by in vitro phosphorylation assays. Results （1） Both wild type and expanded ataxin-3 interacted with CK2α and CK2β in vitro. （2） In 293 cells, both wild type and expanded ataxin-3 interacted with CK2β, but not CK2α. （3） CK2 phosphorylated wild type and expanded ataxin-3. Conclusion Ataxin-3 is a substrate of protein kinase CK2.

PolyQ-expanded ataxin-3 interacts with full-length ataxin-3 in a polyQ length-dependent manner

Objective Machado-Joseph disease （MJD）, also known as spinocerebellar ataxia type 3 （SCA3）, is a dominant neurodegenerative disorder caused by an expansion of the polyglutamine （polyQ） tract in MJD-1 gene product, ataxin-3 （AT3）. This disease is characterized by the formation of intraneuronal inclusions, but the mechanism underlying their formation is still poorly understood. The present study is to explore the relationship between wild type （WT） AT3 and polyQ expanded AT3. Methods Mouse neuroblastoma （N2a） cells or HEK293 cells were co-transfected with WTAT3 and different truncated forms of expanded AT3. The expressions of WT AT3 and the truncated forms of expanded AT3 were detected by Western blotting, and observed by an inverted fluorescent microscope. The interactions between AT3 and different truncated forms of expanded AT3 were detected by immunoprecipitation and GST pull-down assays. Results Using fluorescent microscope, we observed that the truncated forms of expanded AT3 aggregate in transfected cells, and the full-length WT AT3 is recruited onto the aggregates. However, no aggregates were observed in cells transfected with the truncated forms of WT AT3. Immunoprecipitation and GST pull-down analyses indicate that WT AT3 interacts with the truncated AT3 in a polyQ length-dependent manner. Conclusion WT AT3 deposits in the aggregation that was formed by polyQ expanded AT3, which suggests that the formation of AT3 aggregation may affect the normal function of WT AT3 and increase polyQ protein toxicity in MJD.

MJD1基因真核表达载体的构建及其在SH-SY5Y细胞中的表达

目的:分别构建野生型MJD1以及CAG三核苷酸重复扩展突变型MJD1的真核表达载体;明确atax-in-3的多聚谷氨酰胺扩展突变是否可导致细胞核内蛋白聚合体形成。方法:分别以pAS2-1-M JD20Q和pAS2-1-M JD68Q为模板,通过PCR扩展野生型MJD1以及CAG三核苷酸重复扩展突变型MJD1的编码序列。PCR产物经BamH I和Hind III双酶切后连入pcDNA3.1-Myc-H is(-)B,通过酶切以及DNA测序鉴定重组质粒pcDNA3.1-Myc-H is(-)B-M JD20Q和pcDNA3.1-Myc-H is(-)B-M JD68Q。重组质粒转染SH-SY5Y细胞,通过W estern印迹验证ataxin-3的表达,应用激光共聚焦显微镜观察转染细胞的核内蛋白聚合体形成情况。结果:酶切和DNA测序证实目的基因已被克隆入pcDNA3.1-Myc-H is(-)B;其在转染细胞中的表达亦经W estern印迹得以验证;SH-SY5Y细胞转染pcDNA3.1-Myc-H is(-)B-M JD68Q后可见核内蛋白聚合体形成,而转染pcDNA3.1-Myc-H is(-)B-M JD20Q后未见核内蛋白聚合体形成。结论:成功构建了MJD1的真核表达质粒;ataxin-3的多聚谷氨酰胺扩展突变可导致细胞核内蛋白聚合体形成。