(The Protection Research Of Lithium Chloride In Fly Model Of SCA3/MJD)

Background:Autosomal dominant cerebellar ataxia (ADCA) is an obvious clinically and genetically heterogeneous group of neurodegenerative disorders, mainly manifested by ataxias, whose mainly affected parts are the spinal cord, brain stem and the cerebellum, also called spinocerebellar ataxias, SCAs. To date, according to the neuromuscular website (http://neuromuscular.wustl.edu/ataxia/domatax.html) published on Apr11th,2012, at least33gene types of SCAs have been identified, in which19pathogenic genes have been cloned. Among them, spinocerebellar ataxia type3, also called Machado-Joseph disease, is the most common subtype, which accounts for almost62.09%of SCAs in China. SCA3/MJD is an autosomal dominant neurodegenerative disorder caused by CAG trinucleotide repeat expansion within the coding region of MID1I gene. Normal MJD1contains12-40glutamines near the C-terminus, and abnormal polyglutamine tract expands to51-86glutamines in pathogenic gene MJD1.So far nine neurodegenerative disorders have been found to be caused by abnormal CAG trinucleotide repeat expansion within the open reading frame (ORF) of pathogenic genes, also called polyglutamine (polyQ) diseases, including SCA1, SCA2, SCA3/MJD, SCA6, SCA7, SCA17, huntington disease (HD), spinal-bulbar muscular atrophy (SBMA) and dentatorubral-pallidoluysian atrophy (DRPLA). By now, the pathogenesis of polyQ diseases have not been explained clearly. However there are no effective treatment for these diseases up to now.The ubiquitin-proteasome system and autophagy-lysosomal pathways are the two major degradative pathways responsible for eliminating misfolded and unnecessary proteins in the cell, especially autophagy has a very vital role in HD and other neurodegenerative diseases. Proteasomes predominantly degrade short-lived nuclear and cytosolic proteins. The bulk degradation of cytoplasmic proteins or organelles is mediated largely by macro-autophagy, generally referred to as autophagy. Autophagy way also plays a very important role in the degradation of macromolecular proteins complex and aging organelles and in maintaining the intracellular environment steady state, which is also an important mechanism to adapt to the change of external environment of cells. Recent researches suggest that autophagy way participates in the degradation of related pathogenic proteins and the Pathology and disease process in Alzheimer's disease, Polyglutamine disease (HD, SCA3, DRPLA etc), Parkinson's disease and other neurodegenerative diseases. Mizushima and his colleagues recently underscores the autophagy and neurodegenerative diseases have an closely correlations, and the loss of basic level autophagy resulted in neurodegenerative phenotypes and protein aggregates formation because of the conditional knocking out of the genes (Atg5or Atg7) in the brain of mice, so autophagy might play a key role in removal of physiological misfolding proteins.So up-regulated autophagy may be a strategy to reduce the accumulation of polyglutamine-expanded protein aggregates and protect against mutant protein neurotoxicity in neurodegenerative disease. A large amount of signal molecules involves in regulating autophagy, whose specific adjustment mechanism is still unclear. mTOR(mammalian target of rapamycin) and IP3have now been confirmed as negative adjustment factors of autophagy signal transduction pathways. Autophagy inducers may become a new therapeutic strategy of HD and other neurodegeneratie diseases. Autophagy inducers including:(1) mTOR-dependent autophagy inducers:rapamycin may activate autop-hagy by inhibiting autophagy negative regulation site of mTOR;(2) mTOR-independent autophagy inducers:lithium used in the treatment of bipolar disorders can inhibit the inositol single phosphatase (IM Pase), reduce the level of inositol and IP3to induce autophagy. Lithium has been confirmed that can reduce the accumulation and neurotoxicity of mutated Htt proteins in HD cells and Drosophila models.The removal obstacles of misfolding proteins is the key point of PolyQ disease. Many scholars at home and abroad have confirmed that autophagy inducers can up-regulate autophagy, strengthen the removal of the aggregate-prone proteins of PolyQ diseases, inhibit neurotoxicity of PolyQ proteins, alleviate AD, PD, HD and DRPLA degenerative diseases of the nervous system such as the illness, delay progression of disease. And autophagy inducers can also be used in the treatment of SCA3/MJD, also belonging to PolyQ disease, while home and abroad research reports about this rarely. However, the effects of autophagy inducer on models of SCA3/MJD disease have been tested very rarely at home and abroad. It is an established drug that has been used for several decades for chronic treatment of affective disorders. Lithium treatment protects against polyglutamine-mediated toxicity.Lithium agent (Lithium) has clinical application for many years, which is an established drug that has been used for several decades for chronic treatment of bipolar disorders and is known to pass the blood-brain barrier. For one hand, Lithium agent may by mTOR-independent way up-regulate autophagy to remove aggregate-prone proteins in neurodegeneratie diseases, for another hand, it may inhibit GSK3β, up-regulate β-catenin protein and play a role in anti-apoptosis. So we choose lithium to conduct treatment research in SCA3/MJD Drosophila model.Objective:To further investigate the mechanism of SCA3/MJD disease and examine the therapeutic potential of Lithium chloride in SCA3/MJD cells and transgenic Drosophila models.Methods:1. SCA3/MJD transgenic Drosophila models were constructed by GAL4/UAS system.2. Light microscope and scanning electron microscope (SEM) were used to observing the effect of Lithium chloride on the eye morphology of SCA3/MJD transgenic Drosophila models.3. Observation the effect of Lithium chloride and Rapamycin on the emergence rate, climbing ability and lifespan of SCA3/MJD transgenic Drosophila models.4. Transgenic Drosophila models expressing the protein of MJDtr-Q78and Sgg were constructed by double balance fly and GAL4/UAS system.5. Transgenic Drosophila models expressing the protein of MJDtr-Q78and Sgg.S9E were constructed by double balance fly and GAL4/UAS system.6. Observation of Sgg and Sgg.S9E proteins on SCA3/MJD transgenic Drosophila models using light microscope and scanning electron microscope (SEM).Results:1. Using GAL4/UAS transformation system, SCA3/MJD transgenic Drosophila models were constructed by gmr-GAL4and elav-GAL4 which drive target selective gene expression in developing eyes and neurons, respectively. In no case did expression of MJDtr-Q27protein have a phenotypic effect. But expression of MJDtr-Q78protein caused progressive loss of pigmentation and collapse of the eye, decreasing of climbing ability and shortening of lifespan. The severity of the phenotype was relavant to the expression level and site of MJDtr-Q78protein.2. In SCA3/MJD transgenic Drosophila models, the loss of pigmentation and degeneration of the eyes was mitigated by feeding Lithium chloride(5mM-20mM).3. The climbing ability of SCA3/MJD transgenic Drosophila models was improved by feeding Lithium chloride (P<0.05). Especially,10mM or15mM Lithium chloride could obviously improve it's climbing ability.4. We found that proper dosage of Lithium chloride (5-15mM) could extend the SCA3/MJD transgenic Drosophila lifespan(P<0.01). However too much Lithium chloride (20mM)would shorten the lifespan of both SCA3/MJD transgenic Drosophila and wild-type Drosophila.5. Transgenic Drosophila models expressing the protein of MJDtr-Q78and Sgg or MJDtr-Q78and sgg. S9E were constructed by double balance fly and GAL4/UAS system.6. Sgg expression results in the toxicity of SCA3/MJD transgenic Drosophila model, leading to eye phenotype deterioration.7. Sgg.S9E expression can protect SCA3/MJD transgenic Droso- phila model, leading to eye phenotype deterioration, reducing the pigmentation and necrosis of eyes and alleviating the progression of phenotype of SCA3/MJD transgenic Drosophila model.Conclusion:1. We successfully constructed SCA3/MJD transgenic Drosophila models which selective express human MJDtr-Q78protein in developing eyes or neurons respectively. The transgenic Drosophila models simulated the progresssive pattern of neurodegenerative diseases and confirmed that polyQ protein has toxic effects on SCA3/MJD transgenic Drosophila models.2. For the first time, we find that proper dosage of Lithium chloride has therapeutic potential in SCA3/MJD transgenic Drosophila models by reducing pigment loss and structural damage of eyes, improving eclosion rate, climbing ability and extending lifespan.3. Lithium chloride may protect SCA3/MJD transgenic Drosophila models by inhabiting the activation of Sgg protein (the homologous protein of GSK-3β in Drosophila).