Analysis Of Serum GFAP/HDAC/HAT/miRNA In SCA3/MJD Patients

Chapter 1 Analysis of serum levels of GFAP/HDAC/HAT in SCA3/MJD patientsHereditary spinocerebellar ataxia (SCA) are a group of common neurodegenerative diseases of high clinical and genetic heterogeneity, with mainly clinical manifestations of ataxia and dysmetria, mostly by autosomal dominant inherited pattern, also being sporadic. Currently, there are more than 30 subtypes, of which the Machado-Joseph disease/ SCA3 was the most common subtype in the Hans of China which was apppriximately 50%.The pathogenic gene MJD1 has CAG trinucleotide repeats in its 3'end of protein coding region. The normal repeats are about 12-40 times, while SCA3/MJD patients can have up to 51-86 repeats. The more the CAG repeats, the earlier the age of onset, and the more severe the symptoms. Ataxin-3, encoded by MJD1,with an abnormaly expanded polyQ peptide in the carboxyl terminus aggregates selectively in specific areas of the nervous system (cerebellum, brain stem, spinal cord and so on), contributes into the formation of neuronal intranuclear inclusions (NⅡs),which lead to neuron death.Up to date, nine polyQ expansion has been found,which were caused by expanded polyQ mutant proteins, leading to hereditary neurodegenerative disease, also being known as polyQ diseases, including SCA1, SCA2, SCA3/MJD, SCA6, SCA7, SCA17, and huntington disease (HD), spinal bulbar muscular atrophy (SBMA), dentatorubral-pallidoluysian atrophy(DRPLA).Current studies have clearly showed that abnormal expansion of mutant polyQ protein were cytotoxic which triggered the occurrence and development of these diseases. Although the exact mechanism of neuronal damage is still unknown, recent research has found that abnormal transcription may involve in the pathogenesis of polyQ diseases, and mutant polyQ with abnormal expansion inhibited the transcription, suggesting that transcriptional repression mechanism may be one of the common mechanisms of polyQ diseases.Histone acetylation is of epigenetic modification, causing changes in chromatin structure and transcriptional regulation. Several studies have now successfully manipulated that only the enzymatic activity of histone acetyltransferases (HATs) and histone deacetylases (HDACs) keep normal dynamic balanced could the gene transcription regulation be normally maintained in neuronal vitality. It is now increasingly becoming clear that acetylation balance is greatly impaired during neurodegenerative conditions. And both deacetylases and hyperacetylases can induce neuronal death. Our study mainly discuss the role of unbalanced acetylation of histone and non-histone in the pathogenesis of polyQ diseases and the serum level of acetylation in SCA3/MJD patients.GFAP is the hallmark protein of astrocyte, involving in myelin formation and maintenance of blood-brain barrier integrity in the central nervous system. After the nervous system got damaged, GFAP was quickly expressed, which regulated the local micro-environment, assisting in nervous rehabilitation. It has been confirmed that GFAP could get through blood brain barrier. Therefore, the degree of GFAP-positive astrocytes responding to neural injury could be assessed by detecting the serum level of GFAP.At present, there is no related reports about the differences on expression levels of HDAC, HAT, GFAP in peripheral serum between SCA3/MJD patients and the controls. ObjectiveTo make confirmed diagnosis of SCA3/MJD through molecular genetic detection for those who were clinically diagnozed as SCA. To investigate changes in serum levels of HDAC,HAT,GFAP in paitents with SCA3/MJD and its clinical relevance.MethodsSubjects and blood samplesAll SCA patients were clinically diagnosed according to Harding criteria. After being diagnosed by genetic detection, one hundred SCA3/MJD patients were choosed for study, as well as one hundred healthy controls, after being informed consent. The two groups are both matched in gender and age. Blood was drawn from SCA3/MJD patients and controls, then was processed within 4-6 hours. Serum samples were stored at-80℃until analysis. The project was approved by the Ethics Committee of central south university.Genetic analysisPolymerase chain reaction (PCR),8% denaturing polyacrylam-idegel electrophoresis and recombinant DNA technology by T-vector clone and direct sequencing were evaluated for detection of the CAG-repeat of MJD1 for the patients being clinically diagnozed as SCA. Then one hundred SCA3/MJD patients of 18-50 years were choosed and assessed by SARA score and ICARS score.ELISAThree kits including Human GFAP Immunoassay Kits (#K15431), Human HDAC Immunoassay Kits (#K17255) and Human HAT I-mmunoassay Kits (#K17845) were all provided by RB Corporation and applied respectively to detect concentrations of GFAP, HDAC and HAT in serums of SCA3/MJD patients. In our study, all samples were detected twice in the same plate. Statistical analysisSPSS 16.0 and Excel 2007 software were used for data processing and statistical analysis. Serum GFAP, HDAC and HAT concentrations were recorded as means±standard deviation. Serum GFAP, HDAC and HAT concentrations were nonnormal distribution data.Wilcoxon rank test were used to analyze their concentrations. Differences between groups were analyzed using ANOVA. Spearman test was used to analyze the relevance about serum GFAP and HAT concentration respectivly with CAG repeats, age, ICARS scores and SARA scores of SCA3/MJD patients.Results1) Genetic analysisSCA3/MJD patients in our study has been genetically confirmed and the exact CAG repeats of them were also found, ranging from 61～85 that was determined by DNA directly sequencing.2) Serum GFAP, HDAC, HAT concentrations in controls and SCA3/MJD patientsWe detected soluble GFAP, HDAC, HAT in serums of all subjects. The serum level of GFAP was significantly higher in patients with SCA3/MJD[(8.864±4.328)×102ng/ml] than that in healthy normal controls[(3.929±2.380)×102ng/ml, Z=99.12, P<0.05]. There was no significant difference in serum HDAC between patients with SCA3/MJD[(1.636±1.039)×103ng/ml] and controls [(1.594±0.768)×103ng/ml, Z=0.11, P>0.05]. The serum level of HAT was significantly higher in patients with SCA3/MJD [(3.563±1.899)×102ng/ml] than that in healthy controls [(2.738±1.737)×102ng/ml, Z=10.12, P<0.05].3) Relevance analysis between serum GFAP concentration and CAG repeats, ages, ICARS scores and SARA scores of SCA3 /MJD patients.No relevance was found between serum GFAP concentration and CAG repeats, ages, ICARS scores and SARA scores of SCA3 /MJD patients (P>0.05).4) Relevance analysis between serum HAT concentration and CAG repeats, ages, ICARS scores and SARA scores of SCA3 /MJD patients.No relevance was found between serum HAT concentration and CAG repeast, ages, ICARS scores and SARA scores of SCA3 /MJD patients (P>0.05).Conclusions1. There may exist global higher levels of acetylation in SCA3/MJD patients, suggesting that hyperacetylation may be involved in the pathogenesis of SCA3/MJD;2. There has obviously neuronal damage in the central nervous system of SCA3/MJD patients, and serum GFAP can be used as non-specific marker for measurements of injury severity of SCA3/MJD patients. MicroRNAs (miRNAs) are a class of endogenous, small noncoding RNAs of 19-25 nucleotides (21-23 nt) in length. Knowing as a new class of non-protein genetic expression regulator factor, recent studies focus on its function in the pathogenesis of neurodegeneration, especially for polyQ diseases. MicroRNAs (miRNAs) which regulate gene expression negatively, as well as positively, act as post-transcriptional regulators of gene expression in diverse cellular and developmental processes. Many miRNAs are expressed specifically in central nervous system, where they play a part in differentiation, neuronal survival, and potentially also in plasticity and learning. The absence of miRNAs in a variety of specific neurons can lead to progressive loss of these neurons and behavioral defects reminiscent of the phenotypes seen in the pathologies of neurodegenerative diseases.Spinocerebellar ataxia (spinocerebellar ataxia, SCA) is one of the common inherited neurodegenerative diseases threatening the human nervous system. Spinocerebellar ataxia type 3 (SCA3/MJD) is the most common one. Ataxin-3 is a kind of cytoplasmic protein encoded by pathogenic gene MJD1, containing polyglutamine (polyQ). Ataxin-3 containing abnormal expansion of mutant polyQ would get into the nucleus to form nuclear inclusion bodies and accumulate together, leading to neuron death. Dicer is a key enzyme in the process of miRNA mature. Bilen et al.found that the cytotoxicity induced by ataxin-3 was significantly enhanced after the dicer gene was knockouted in HeLa cells. While supplying with all the miRNA fragments, the toxicity in HeLa cells will be reduced. So miRNA may have neuroprotective effects for SCA3/MJD, but the specific neuroprotective miRNA isoforms and its specific functional pathway in SCA3/MJD is still unclear.Spinocerebellar ataxia type 1 (SCA1) is another common subtype among SCAs. Depletion of Dicer by using Purkinje cell-specific Pcp2 promotor-driven crerecombinase from mouse Purkinje neurons did not impair cell function and survival in young mice (eight-week-old). However, in 13-week-old mice, the Purkinje neurons progressively degenerated and cell death started to occur. At the same age, the mice developed a slight tremor and mild ataxia, both of which became more severe with advancing age.Therefore, miRNAs may have a protective function for SCAl,while the exact targets and pathway which miRNAs act are still unclear.In recent years,various studies have confirmed that miRNA exist stably and specificially in peripheral serum, also being present in urine, saliva, cerebrospinal fluid, amniotic fluid and other human body fluids, which could reflect the characteristics of physiological and pathological processes of the body dynamically. At present, there have not yet reports about clear differences of miRNA expression level in peripheral serum between SCA3/MJD patients and healthy controls at home and abroad.Objective To screen the differentially expressed microRNAs (miRNAs) in serum of SCA3/MJD and healthy controls using miRNA microarray and to explore the relationship between pathogenesis and miRNAs aberrantly exp ressed in SCA3/MJD,trying to find specificially-expressed miRNAs in serum of SCA3/MJD patients as its biomarkers.Methods①Total RNA, including the small RNAs, was isolated using TRIzol and miRNeasy mini kit according to manufacturer's instruction.②Hybridization was carried out on miRNA miRCURYTM LNA Array for 9 SCA3/MJD and 6 healthy controls.③Real-time quantitative RT-PCR was performed to confirm results obtained by microarray analysis for 35 SCA3/MJD and 25 healthy controls.Results Microarray screening showed that there were 26 miRNAs which were significantly downregulated and 20 miRNAs upregulated. Four miRNAs have been identified,three of them were down-regulated and the remaining one were up-regulated in serum of SCA3/MJD.The above four miRNA,as well as miR-181 b and miR-32* which were predicted to be related with SCA3/MJD according to miRNA databases,were all confirmed by microarray analysis and real-time RT-PCR.Conclusion 1. miR-181b, miR-29a, miR-125b, miR-25, miR-32* and miR-34b may be involved in the pathogenesis of SCA3-/MJD, of which miR-181b, miR-29a, miR-125b and miR-25 are downregulated, while miR-32* and miR-34b are upregulated.2. miR-181b, miR-29a, miR-125b, miR-25, miR-32* and miR-34b may be promising molecular biomarkers helping for further research in pathogenesis of SCA3/MJD.