Molecular clearance of ataxin-3 is regulated by a mammalian E4

Insoluble aggregates of polyglutamine-containing proteins are usually conjugated with ubiquitin in neurons of individuals with polyglutamine diseases. We now show that ataxin-3, in which the abnormal expansion of a polyglutamine tract is responsible for spinocerebellar ataxia type 3 (SCA3), undergoes ubiquitylation and degradation by the proteasome. Mammalian E4B (UFD2a), a ubiquitin chain assembly factor (E4), copurified with the polyubiquitylation activity for ataxin-3. E4B interacted with, and thereby mediated polyubiquitylation of, ataxin-3. Expression of E4B promoted degradation of a pathological form of ataxin-3. In contrast, a dominant-negative mutant of E4B inhibited degradation of this form of ataxin-3, resulting in the formation of intracellular aggregates. In a Drosophila model of SCA3, expression of E4B suppressed the neurodegeneration induced by an ataxin-3 mutant. These observations suggest that E4 is a rate-limiting factor in the degradation of pathological forms of ataxin-3, and that targeted expression of E4B is a potential gene therapy for SCA3.     

Comparative Genetics of Functional Trinucleotide Tandem Repeats in Humans and Apes

Several human neurodegenerative disorders are caused by the expansion of polymorphic trinucleotide repeat regions. Many of these loci are functional short tandem repeats (STRs) located in brain-expressed genes, and their study is thus relevant from both a medical and an evolutionary point of view. The aims of our study are to infer the comparative pattern of variation and evolution of this set of loci in order to show species-specific features in this group of STRs and on their potential for expansion (therefore, an insight into evolutionary medicine) and to unravel whether any human-specific feature may be identified in brain-expressed genes involved in human disease. We analyzed the variability of the normal range of seven expanding STR CAG/CTG loci (SCA1, SCA2, SCA3-MJD, SCA6, SCA8, SCA12, and DRPLA) and two nonexpanding polymorphic CAG loci (KCNN3 and NCOA3) in humans, chimpanzees, gorillas, and orangutans. The study showed a general conservation of the repetitive tract and of the polymorphism in the four species and high heterogeneity among loci distributions. Humans present slightly larger alleles than the rest of species but a more relevant difference appears in variability levels: Humans are the species with the largest variance, although only for the expanding loci, suggesting a relationship between variability levels and expansion potential. The sequence analysis shows high levels of sequence conservation among species, a lack of correspondence between interruption patterns and variability levels, and signs of conservative selective pressure for some of the STR loci. Only two loci (SCA1 and SCA8) show a human specific distribution, with larger alleles than the rest of species. This could account, at the same time, for a human-specific trait and a predisposition to disease through expansion.This article contains online supplementary material.     

Electromyographic (EMG) Biofeedback in the Comprehensive Treatment of Central Pain and Ataxic Tremor Following Thalamic Stroke

Peripheral pain and ataxic tremor can appear suddenly following thalamic stroke and can significantly alter a patient's psychological, social, and physical functioning. The present paper reports the case of a 70-year-old Caucasian female who sustained an acute left posterior cerebral artery infarction involving the thalamus and left mesiotemporal regions. She subsequently developed Central Poststroke Pain and ataxic movement of her right arm and hand in addition to a significant right-side claudication. She was treated over 16 weeks (6 weeks of EMG biofeedback and 10 weeks of psychotherapy) with a combination of EMG biofeedback, progressive muscle relaxation, behavioral pain coping skills training, Forced Use Therapy, and Cognitive Behavioral Therap 7 years after her initial cerebral accident. The case demonstrates the utility of biofeedback when combined as part of a comprehensive treatment program to address the multiple complications associated with thalamic stroke.     

Loss of Usp14 results in reduced levels of ubiquitin in ataxia mice

The ataxia (ax(J)) mutation is a spontaneous recessive mutation that results in reduced expression of ubiquitin-specific protease 14, Usp14. Mice homozygous for the ax(J) mutation are retarded for growth and exhibit several behavioral disorders, including a resting tremor and hindlimb paralysis. Although pathological defects appear to be limited to the central nervous system, reduction of Usp14 expression was widespread in the ax(J) mice. Usp14 co-fractionated with proteasomes isolated from livers and brains of wild-type mice. Proteasomes isolated from the ax(J) brains still possessed deubiquitinating activity and were functionally competent to hydrolyze 20S proteasomal substrates in vitro. However, the levels of monomeric ubiquitin were reduced approximately 35% in most of the ax(J) tissues examined. These results indicate that Usp14 functions to maintain the cellular levels of monomeric ubiquitin in mammalian cells, and that alterations in the levels of ubiquitin may contribute to neurological disease.     

Ion channel regulation by β-secretase BACE1 – enzymatic and non-enzymatic effects beyond Alzheimer's disease

β-site APP-cleaving enzyme 1 (BACE1) has become infamous for its pivotal role in the pathogenesis of Alzheimer's disease (AD). Consequently, BACE1 represents a prime target in drug development. Despite its detrimental involvement in AD, it should be quite obvious that BACE1 is not primarily present in the brain to drive mental decline. In fact, additional functions have been identified. In this review, we focus on the regulation of ion channels, specifically voltage-gated sodium and KCNQ potassium channels, by BACE1. These studies provide evidence for a highly unexpected feature in the functional repertoire of BACE1. Although capable of cleaving accessory channel subunits, BACE1 exerts many of its physiologically significant effects through direct, non-enzymatic interactions with main channel subunits. We discuss how the underlying mechanisms can be conceived and develop scenarios how the regulation of ion conductances by BACE1 might shape electric activity in the intact and diseased brain and heart.     

A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA

MicroRNAs (miRNAs) are key regulators of gene expression. In the brain, vital processes like neurodevelopment and neuronal functions depend on the correct expression of microRNAs. Perturbation of microRNAs in the brain can be used to model neurodegenerative diseases by modulating neuronal cell death. Currently, stereotactic injection is used to deliver miRNA knockdown agents to specific location in the brain. Here, we discuss strategies to design antagomirs against miRNA with locked nucleotide modifications (LNA). Subsequently describe a method for brain specific delivery of antagomirs, uniformly across different regions of the brain. This method is simple and widely applicable since it overcomes the surgery, associated injury and limitation of local delivery in stereotactic injections. We prepared a complex of neurotropic, cell-penetrating peptide Rabies Virus Glycoprotein (RVG) with antagomir against miRNA-29 and injected through tail vein, to specifically deliver in the brain. The antagomir design incorporated features that allow specific targeting of the miRNA and formation of non-covalent complexes with the peptide. The knock-down of the miRNA in neuronal cells, resulted in apoptotic cell death and associated behavioural defects. Thus, the method can be used for acute models of neuro-degeneration through the perturbation of miRNAs.     

Deferiprone and idebenone rescue frataxin depletion phenotypes in a Drosophila model of Friedreich's ataxia

Friedreich's ataxia (FRDA), the most common inherited ataxia, is a neurodegenerative disease caused by a reduction in the levels of the mitochondrial protein frataxin, the function of which remains a controversial matter. Several therapeutic approaches are being developed to increase frataxin expression and reduce the intramitochondrial iron aggregates and oxidative damage found in this disease. In this study, we tested separately the response of a Drosophila RNAi model of FRDA ( Llorens et al., 2007) to treatment with the iron chelator deferiprone (DFP) and the antioxidant idebenone (IDE), which are both in clinical trials. The FRDA flies have a shortened life span and impaired motor coordination, and these phenotypes are more pronounced in oxidative stress conditions. In addition, under hyperoxia, the activity of the mitochondrial enzyme aconitase is strongly reduced in the FRDA flies. This study reports that DFP and IDE improve the life span and motor ability of frataxin-depleted flies. We show that DFP eliminates the excess of labile iron in the mitochondria and thus prevents the toxicity induced by iron accumulation. IDE treatment rescues aconitase activity in hyperoxic conditions. These results validate the use of our Drosophila model of FRDA to screen for therapeutic molecules to treat this disease.     

Screening for the presence of FMR1 premutation alleles in women with fibromyalgia

Fibromyalgia is a chronic condition characterized by widespread pain, fatigue, non-restorative sleep and cognitive difficulties that affects 2–4% of the general population. Recently a possible relationship between the FMR1 premutation and fibromyalgia has been pointed out. In attempt to gather more data we screened for the FMR1 CGG expansion 700 DNA samples from unrelated fibromyalgia patients. This data might be useful for evaluating the incorporation of this test in rheumatologic procedures for women with fibromyalgia. The observed frequency of FMR1 premutation carriers (3 of 700, 0.4%) is not significantly different from the estimated rate in the general female population (1/250–1/400) (P = 0.539, P = 0.716). Clinical examination of the FMR1 premutation carriers identified revealed that all of them had important neurological symptoms with regard to muscular symptoms, neurocognitive alterations and neurovegetative impairments. With regard to other clinical aspects of the disease the cases apparently did not differ from the average fibromyalgia patients. On the basis of our results an FMR1 screening among fibromyalgia female patients would not be recommended. However it would be worthwhile to further evaluate the different clinical presentations that fibromyalgia patients might present based on their FMR1 premutation carrier status.     

小鼠frataxin抗体的制备及应用

弗里德赖希共济失调(Friedreich ataxia,FRDA)是一种常染色体隐性遗传性疾病,由frataxin(FXN)第一个内含子中GAA重复扩增导致其表达量减少所致。FXN是一种线粒体蛋白,可以调节细胞中铁的代谢,参与铁硫簇和血红素的合成,去除氧化应激等。前期研究发现FRDA病人受累组织有特异性表达的FXN亚型蛋白。为了检测小鼠不同组织中Fxn亚型蛋白的表达,首先要获得具有高度特异性和灵敏性的小鼠Fxn抗体。利用PCR技术扩增小鼠Fxn基因,通过酶切、连接、转化等常规分子克隆方法构建重组原核表达质粒pET24(+)-mFxn,经转化大肠杆菌BL21(DE3),表达可溶性含有his6标记的融合蛋白。该蛋白不含Fxn氨基端77个氨基酸的信号肽,含有130个氨基酸,理论分子量为14.38 kDa。表达的蛋白经Ni-NTA柱和连续梯度离心纯化,获得目的蛋白作为抗原;免疫新西兰大白兔制备抗血清并用硫酸铵沉淀初步纯化得到多克隆抗体。经Western blotting和免疫荧光分析测试,所获得的抗体能够特异性识别细胞内源Fxn,也可应用于组织的免疫沉淀和免疫荧光。这是首次报道利用鼠源Fxn作为免疫原制备的具有高度特异性和灵敏性的Fxn抗体,为深入研究小鼠frataxin亚型蛋白的存在和功能奠定了基础。