The Effect Of Luteolin On Toxicity And Degradation Of Mutant Huntingtin And Its Underlying Mechanism

Huntington's disease (HD) is a hereditary progressive neurodegenerative disorder caused by expansion of CAG repeats in the first exon of HD gene that encodes huntingtin (Htt) protein. Mutation of HD gene leads to huntingtin protein misfolding and aggregation in the cytoplasm and the nucleus of the neurons in the brains of HD patients and animal models. It has been proposed that mutant huntingtin aggregates are toxic due to the sequestration of vital cellular components. The exact pathological mechanisms determining disease onset and progression remain unclear, however, impaired function of ubiquitin-proteasome system (UPS), defective autophagy function, oxidative stress, apoptosis, are also implicated for the disease pathogenesis mechanism. Accumulating evidences suggest that oxidative stress could promote mutant Htt aggregation, causing cell death by impairing UPS function, indicating that using of the antioxidant agents will be a promising strategy to reduce mutant Htt aggregates, preventing from mutant Htt toxicity. The intense search for small-molecular compounds that may modulate HD pathology has advanced the analysis of specific dietary substances from plants and herb.Luteolin, tetrahydroxyflavone, is a one kind of flavonoids found mainly in many types of plants including medicinal herbs, being considered as main anti-inflammatory, antioxidant and free radical scavenger. Luteolin consumption has been found to be able to improve spatial working memory and restore expression of inflammatory markers in aged mouse hippocampus, indicating its beneficial neuroprotection. In this study we examined the effect of luteolin on mutant Htt toxicity and its mechanism. Our results suggested that luteolin could reduce mutant huntingtin toxicity not only due to its antioxidative activity but, more importantly, also through promoting proteasomal and autophagy degradation of mutant huntingtin and heat shock factor 1 (HSF-1)-mediated upregulation of heat shock proteinsLuteolin protects against toxicity in N2a cells expressing mutant huntingting and improves symptoms of HD transgenic mice. To assess neuroprotection of luteolin against mutant Htt toxicity, the N2a cells transfected with normal (20Q) or mutant (160Q) Htt were treated with different concentrations of the luteolin, and the cell viability was evaluated with MTT assay. Treatment with luteolin resulted in dose-dependent inhibition of decrease in cell viability by mutant Htt. Further PI staining and detection of activated caspase 3 with Western blotting showed that luteolin could significantly reduce percentage of PI-stained cells and dose-dependently decrease level of activated caspase 3 in mutant Htt-transfected cells. Compared with those of untreated HD transgenic (Tg) mice that express the first 171 aa of Htt with 82 glutamines, the HD symptoms including body weight loss, limb clasping, lifespan abbreviation and motor dysfunction in Tg mice treated with intraperitoneal injection of luteolin were significantly improved. The results demonstrated that luteolin had protection against mutant Htt cytotoxicity in both mutant Htt-expressing cells and HD transgenic mice.Luteolin reduces both aggregated and soluble mutant Htt in N2a cells expressing mutant huntingtin and the brain of HD transgenic mice. To assess whether luteolin could reduce the mutant Htt aggregates, the change in amount of mutant Htt aggregates in the luteolin treated cells expressing mutant Htt and HD mice brains was detected. The cells expressing 160Q Htt and Tg mouse brains showed time-or age-dependent accumulation of the mutant Htt aggregates. After treatment with luteolin, the amount of both aggregated and soluble mutant Htt in both 160Q Htt transfected cells and Tg mouse brains was dramatically decreased. RT-PCR analysis did not detect any effect of luteolin on expression of mRNA for transfected mutant Htt in 160Q cells. Thus, the reduction of mutant Htt by luteolin indicates that degradation of mutant Htt was upregulated, or that aggregation of mutant Htt was inhibited, or both.Reduction of mutant huntingtin aggregates by luteolin is independent of activation of Nrf2 pathway. Oxidative stress has been found to be caused by mutant Htt, which in turn leads to aggregation of mutant Htt. To see whether reduction in Htt aggregates is resulted from antioxidation of luteolin, the role of nuclear factor erythroid-2 related factor 2 (Nrf2), the transcription factor that regulates antioxidant responsive elements (ARE), in reduction of mutant Htt aggregates by luteolin was examined. Western blotting analysis for expression levels of Nrf2 and its downstream target, heme oxygenase 1 (HO-1), showed that mutant Htt could elevate levels of Nrf2 and HO-1, and that luteolin treatment could slightly increase levels of Nrf2 and HO-1 in 20Q cells but greatly increase Nrf2 and HO-1 levels in 160Q cells, which was even higher than that in 160Q cells without luteolin treatment. Interestingly, in cells expressing 160Q Htt, the inhibition of mutant Htt aggregate accumulation and/or promotion of mutant Htt degradation by luteolin in 160Q cells was scarcely blocked by knocking down of Nrf2 with Nrf2 specific small interfering RNA (Nrf2 SiRNA). It appears that the effect of luteolin on mutant Htt aggregate accumulation is independent of the Nrf2 pathway activation.Luteolin enhances proteasomal and autophagy degradation of mutant huntingtin. To see the potential involvement of proteasome in the degradation of mutant Htt by luteolin, firstly, we checked if proteasome inhibitor MG132 could block the action of luteolin on mutant Htt. Western blot analysis showed that, addition of MG132 to 160Q transfected cells treated with luteolin blocked the decrease in soluble form of mutant Htt but did not block the decrease in the aggregated form. Next, the effect of luteolin on UPS function was assessed using N2a cells stably expressing a reporter of UPS function, which contains a short degron, CL1, fused to the COOH-terminus of GFP, called GFPu-cells. Consistent with previous study,160Q transfection in GFPu-cells significantly increased the fluorescent intensity and protein level of GFPu, indicating inhibition of UPS function by mutant Htt. Treatment with luteolin slightly decreased the fluorescent intensity and protein level of GFPu in 20Q GFPu-cells but dramatically inhibited increase in those in 160Q GFPu-cells. Furtherly, measurement of proteasome activity (chymotrypsin-like) using a fluorogenic peptide substrate assay showed that luteolin could activate proteasome in 20Q and 160Q cells with stronger activation in 160Q cells than in 20Q cells though 160Q Htt did not inhibit proteasome activity. It is thus suggested that luteolin can promote proteasome to degradate soluble mutan. Since MG132 did not block the decrease in the aggregated form in luteolin treated 160Q cells, autophagy is likely be involved in luteolin-promoted degradation of aggregated mutant Htt. As expected, addition of autophagy inhibitor chloroquine to 160Q cells treated with luteolin obviously blocked the decrease in the aggregated form, and the decrease in the soluble form was slightly inhibited. Moreover, in 160Q Htt-expressing N2a cells, conversion of microtubule-associated protein 1 light chain-3 (LC3)-â… to LC3-â…¡, the indicator for autophagy activiation, was increased by treatment with luteolin. Thus, luteolin can promote degradation of mutant Htt aggregates through inducing activation of autophagy.Luteolin up-regulates expression of the heat shock proteins through activation of HSF-1. The level of soluble mutant Htt in 160Q Htt cells treated with luteolin and MG132 was higher not only than that in 160Q Htt cells without treatment but also than that in 160Q Htt cells treated with MG132 only. Furthermore, in 160Q Htt cells treated with luteolin and chloroquine, the level of aggregated mutant Htt was lower than that in 160Q Htt cells treated with chloroquine only, and the level of soluble form was higher than that in 160Q Htt cells treated with luteolin only. Therefore, it was suggested that luteolin might inhibit soluble mutant Htt to aggregate through other mechanism. Molecular chaperons, such as HSP40, HSP70, HSP105, can inhibit mutant Htt to aggregate through refolding them. To determine whether reduction of the mutant Htt aggregates after luteolin treatment might be caused by increased expression of molecular chaperons, we treated the cells transfected with 20Q or 160Q Htt and examined the expression level of HSP40, HSP70 and HSP105. Transfection of 160Q Htt caused decrease in expression level of these chaperons. Luteolin treatment slightly increased their expression in 20Q Htt transfected cells and restored expression of these chaperones in 160Q Htt transfected cells. The expression of HSP40, HSP70 and HSP105 are transcriptionally upregulated by active HSF-1 the function of which is regulated by chaperone HSP90. HSP90 inactivates HSF-1 through binding to and detaining HSF-1 in the cytoplasm. Inhibition of HSP90 leads to release of HSF-1 from HSP90 complex, which results in its phosphorylation, activation and translocation to the nucleus where it initiates heat shock protein expression. Luteolin has been found to be able to inhibit HSP90. To know if the upregulation of HSP40, HSP70 and HSP105 expression by luteolin is resulted from activation of HSF-1 through inhibition of HSP90 by luteolin, we checked if luteolin treatment could cause phosphorylation and nuclear translocation of HSF-1. The activation of HSF-1 was demonstrated by the results that luteolin treatment increased phosphorylation and nuclear localization of HSF-1 in 160Q Htt-expressing cells. Consistently, degradation of HSP90 was increased by luteolin since the protein level of HSP90 was decreased while its mRNA level was elevated after treating 20Q Htt-or 160Q Htt-expressing cells with luteolin, suggesting inhibition of HSP90 by luteolin (HSP90, when inhibited, is separated from HSF-1, becoming unstable).Taken together, luteolin can reduce mutant Htt through multiple system:promoting degradation of soluble mutant Htt by proteasome system, inducing activity of autophagy system to degrade aggregated form of mutant Htt, and activating HSF-1 via inhibition of HSP90 to upregulate expression of heat shock proteins, which, in turn, inhibits aggregation of mutant Htt. The role of luteolin in reducing mutant Htt could be the important contribution to the suppression of mutant Htt toxicity. These findings show neuroprotection of luteolin, providing a new perspective for using luteolin to treat HD and other polyglutamine neurodegenerative disorders.