The Effects Of Isorhamnetin On The Cytotoxicity Of Mutant Huntingtin And The Underlying Mechanism

Huntington’s disease (HD) is a neurodegenerative genetic disorder affects muscle coordination and leads to cognitive decline and dementia. It is an inherited disease, caused by a CAG triplet repeat expansion in the gene encoding huntingtin (Htt), for which till now there is no effective disease-slowing treatment. HD is produced by an expanded CAG trinucleotide repeat in exon1of huntingtin (Htt) gene. This mutation leads to a progressive neurodegeneration and cell death of striatal medium spiny neurons, which is associated with striatal atrophy, and resulting in increased length of a polyglutamine (polyQ) stretch in Huntingtin protein (htt). Pathological symptoms appear with polyQ repeat>37glutamine residues. Although the causative gene has been found, the exact mechanisms of the pathogenesis are still unknown. The function of htt in humans is unclear. It interacts with proteins which are involved in transcription, cell signaling and intracellular transporting. In humans the disruption of normal gene does not cause disease. It is currently concluded that the disease is not caused by inadequate production of htt, but by a gain of toxic function of mutant htt. Broad approaches are under study to attempt to slow the progression of Huntington’s disease including:reducing production of the mutant protein; improving cells’ ability to survive its diverse harmful effects; and replacing lost neurons.Considerable research activity has been directed towards understanding common pathological mechanisms that might be amenable to therapeutic intervention. There is a large amount of in vitro and in vivo evidence suggesting that oxidative stress occurs during neurodegeneration, and, as a result, ameliorating oxidative stress with antioxidant drugs has been a major focus of therapeutic research. A second hallmark of neurodegeneration is the accumulation and aggregation of misfolded pathogenic proteins.Flavonoids are a large group of secondary plant metabolites with more than6000different compounds described to date. Numerous observations on flavonoids, both in vivo and in vitro, demonstrate a wide variety of physiological and biological effects. Biological functions, such as antiinflammatory, antiallergic, antiviral, anticarcinogenic and antiproliferative activities, have long been recognized. Several flavonoids have been demonstrated to have an antimutagenic effect on various mutagens or carcinogens. Flavonoids comprise the most common group of polyphenolic compounds in the human diet and exert a multiplicity of neuroprotective actions, including a potential to protect neurons against injury induced by neurotoxins; an ability to suppress neuroinflammation; and the potential to promote memory, learning and cognitive function.In this work we tried to find the neuroprotective effect of one of flavonoids named isorhamnetin on the neuron cells and its role in huntington’s disease. Isorhamnetin is a flavonoid with four hydroxyl groups, it is highly consumed in the Greek and Danish traditional diet, and it can be found in the Chinese sea buckthorn juice.In this in vitro study, the aim was to assess whether isorhamnetin could protect neuronal cells from the effect of mutant huntingtin gene damages, and to specify the possible mechanism through which it can do this action. Therefore, in this study, the possible mechanisms through which isorhamnetin exerts its protective effects was evaluated in cellular system. In addition, its effect on mutant htt beside several proteins and apoptotic enzymes was examined by western blot analysis.Isorhamnetin protects against toxicity in N2a cells expressing mutant huntingtin. We used trypan blue staining of N2a cells to assess the viability mediated by isorhamnetin treatment through evaluation of the improvements in cell viability and increasing in the cell survival in mutant huntingtin cells after treatment with isorhamnetin. The cells expressing the normal huntingtin (20Q htt) or the mutant huntingtin (160Q htt) were treated with different concentrations of isorhamnetin, and then the viability of the cells was assessed48 hours after the time of transfection. The non-viable cells can be seen as stained cells whereas viable cells excluded the stain. It was found that the non-viable cells are lower when isorhamnetin was added compared with untreated mutant and wild type htt cells. Mutant htt cells treated with isorhamnetin were found to have a significant increase in relative cell viability in comparison with untreated mutant htt cells. This result indicates that isorhamnetin has a high protective effect against toxicity caused by mutant htt, therefore different concentrations of isorhamnetin were used for further assessment of its efficiency against huntingtin aggregates.Since many evidences revealed that caspase-3is an important mediator of apoptosis’ so we detected active caspase-3level in treated and untreated mutant htt cells. Western blotting analysis revealed that the untreated160Q mutant htt cells had an increased level of the pro-apoptotic active caspase-3in comparison with treated ones. It was found that treatment with isorhamnetin at80ng/ml inhibits the active caspase-3in the mutant htt cells, but increased again at160ng/ml, which indicates that isorhamnetin has an anti-apoptotic activity at a certain concentration.Isorhamnetin reduces both aggregated and soluble mutant htt in N2a cells expressing mutant huntingtin. It was found that there was a significant reduction in the accumulation of aggregates of mutant htt protein when isorhamnetin added in comparison to control cells (without isorhamnetin). This was assessed through fluorescent microscopy and western blotting analysis. It was found that there was a significant reduction in mutant htt aggregates in cell cultures treated with isorhamnetin compared with the untreated cultures. It was also obvious that in N2a cell cultures treated with isorhamnetin in different concentrations, the mutant htt aggregates were reduced in a concentration dependent manner, the more the concentration the less the mutant htt aggregates. This indicates that this medicine has a protective function in reducing the mutant huntingtin aggregates which is thought to play a part in the pathogenesis of Huntington’s disease.Western blotting analysis showed that there was a reduction in both soluble and aggregated forms of htt in the treated group compared to the untreated cultures, and this reduction was directly proportional to the increase in concentration of the medicine. Reduction of mutant huntingtin aggregates by isorhamnetin is related to activation of Nrf2pathway. Many reports proved that the beneficial activities of antioxidant agents might contribute to the decrease of htt aggregation and toxicity. It was found that isorhamnetin can enhance the anti-oxidative defense system. So to assess whether isorhamnetin can protect the mutant htt cells against oxidative stress, the antioxidant Nrf2-ARE pathway activation was detected in the mutant htt160Q cells treated with isorhamnetin or vehicle. Western blotting analysis revealed that isorhamnetin had significantly increased the protein level of Nrf2(which is considered as the master regulator of neuronal adaptation to an oxidant environment) in comparison with untreated control cells. It was also found that isorhamnetin had also increased the protein level of HO-1(which is a cytoprotective enzyme that plays a critical role in defending the body against oxidant-induced injury during inflammatory processes) in comparison with untreated control cells. These results indicate that isorhamnetin can activate the Nrf2-ARE pathway, which is considered as a protective mechanism against oxidative stress; hence it can play a role in decreasing htt aggregation and toxicity.Isorhamnetin enhances both autophagy lysosomal and proteasomal degradation of soluble and aggregated mutant huntingtin. Misfolded proteins are primarily cleared in cells by two systems:the ubiquitin-proteasome system (UPS) and autophagy. Since both the UPS and autophagy are found to clear mutant htt, so comparing their abilities to remove misfolded proteins is important. To determine whether the UPS activation or the autophagy or both were involved in the clearance of mutant htt mediated by the isorhamnetin treatment, the cells expressing the N-terminal mutant htt160Q were treated with the UPS inhibitor MG132or the autophagy inhibitor3MA and then treated with isorhamnetin or vehicle. It was found that MG132has attenuated the effect of isorhamnetin on reducing the aggregated mutant htt protein but not on soluble mutant htt. This result indicates that isorhamnetin treatment can reduce the aggregated mutant htt protein through activation of the UPS, which might be triggered by the antioxidant function of this medicine. It was also shown that3MA has not attenuated the effect of isorhamnetin on reducing the aggregated mutant htt protein, but it has attenuated the effect of isorhamnetin on reducing the soluble mutant htt protein. This result indicates that isorhamnetin treatment can reduce the soluble form of mutant htt through autophagy pathway. These results suggest that isorhamnetin enhance the degradation of the insoluble mutant htt aggregates through the UPS, and the degradation of the soluble mutant htt through the autophagy pathway. The antioxidant properties of the drug may be responsible for activation of these pathways.Collectively, isorhamnetin protects against toxicity in N2a cells expressing mutant huntingtin by increasing cell viability which indicates increase in cell resistance against toxicity mediated by mutant htt. It can also reduce both aggregated and soluble mutant huntingtin in N2a cells expressing mutant huntingtin either directly or through its reducing effect on caspase3. This medicine has a role in activation of the NF-E2-related factor2-antioxidant response element (Nrf2-ARE) antioxidant pathway which can protect the mutant htt cells from the oxidative stress induced by mutant htt. Finally it can enhance the degradation of the insoluble mutant htt aggregates through the Ubiquitin Proteasome System (UPS), and the degradation of the soluble mutant htt through the autophagy pathway.From the above observations we can conclude that Isorhamnetin (as a type of flavonoids) is regarded as a novel therapeutic agent in the prevention and treatment of Huntingtons disease.Since isorhamnetin can protect against mutant htt toxicity in cell lines, it is an excellent candidate for further in vivo therapeutic trials.