The Effect And Mechanism Of Simvastatin On ALS Cell Model

Amyotrophic lateral sclerosis(ALS)is the most common adult-onset motor neuron disease characterized by selective loss of motor neurons.The loss of motor neurons results in progressive muscle wasting,muscle weakness,fasciculation,muscle spasms,speech disorders,dysphagia,and reflex changes.The sporadic form of ALS(SALS)accounts for approximately 90%-95% of cases,whereas the remaining 5%-10% are familial(FALS).The pathogenesis of ALS is unclear and is generally understood as the result of interactions between genetic mutations and environmental factors.The molecular mechanisms of ALS include oxidative stress,abnormal cytoskeleton,autophagy dysfunction,apoptosis,glutamate excitotoxicity,and immune abnormalities.At present,there is no effective treatment for ALS.The main purpose is to delay disease progression and improve life quality.Mutant SOD1 gene was the first ALS pathogenic gene to be diagnosed,which accounts for approximately 20% of FALS and 2-7% of SALS.Transgenic mice overexpressing the G93 A mutation in human SOD1(h SOD1G93A)are a classic ALS research model.NSC34 cells transfected with h SOD1G93A(NSC34-h SOD1G93 A cells)show SOD1 protein accumulation,lowered viability,a decreased proliferation rate,mitochondrial dysfunction,and greater susceptibility to oxidation-induced cell death;therefore,this cell model have also been widely used as disease model of ALS.Previous studies have shown that ALS patients have metabolic abnormalities.Weight loss during the disease course can aggravate clinical symptoms.High-fat diet can prolong the lifespan of ALS mice.Statins can accelerate disease progression,but there exist conflicting reports.Meanwhile the mechanism of statins on ALS is unclear.It is well known that statins,which are inhibitors of 3-hydroxy-3-methylglutarylcoenzyme A(HMG-Co A)reductase,are widely used in the prevention and treatment of cardio-cerebrovascular diseases due to their powerful ability to lower plasma cholesterol treatment.Therefore,in this study,we first analyzed the cardio-cerebrovascular diseases and related risk factors in ALS patients,and found that some ALS patients had hypercholesterolemia or cardio-cerebrovascular diseases and needed statins.Next,we observed the effects of simvastatin on NSC34-h SOD1G93 A cells(familial ALS cell model),and explored its possible mechanism.Part One Clinical observation of cardio-cerebrovascular diseases and risk factors in patients with ALSObjective: To retrospectively observe and analyze the clinical conditions of cardio-cerebrovascular diseases and related risk factors in patients with ALS.Methods: The clinical data of 150 patients with ALS were collected.Then we analyzed the incidence of cardio-cerebrovascular disease and related risk factors in ALS patients,and compared the age of onset between ALS patients with and without cardio-cerebrovascular diseases and related risk factors.Results:1.ALS is more comman in men than in women.The age of onset is mostly between 51 years and 60 years.The onset sites are most seen in upper limbs.2.The smoking rate of male ALS patients is lower than that of the entire population,and the incidence of coronary heart disease and hypercholesterolemia is higher than that of the entire population.3.The age of onset of ALS patients with coronary heart disease or hypertension is later than that of ALS patients without coronary heart disease or hypertension.4.Some ALS patients have coronary heart disease,cerebral infarction and hypercholesterolemia,they may need to be treated with statins.It is necessary to clarify the effect of statins on ALS.Part Two Simvastatin caused damage to NSC34-h SOD1G93 A cellsObjective: To observe the effect of simvastatin on the viability of NSC34-h SOD1G93 A cells.Methods:1.NSC34 cells stably transfected with the GFP-human SOD1 G93A(h SOD1G93A)plasmid had been established in our laboratory.First,we verified the successful establishment of the cell models.2.CCK-8 and LDH were used to detect the effect of simvastatin on the viability of NSC34-h SOD1G93 A cells.Results:1.Immunofluorescence staining SMI-32,which is specific to motor neurons,confirmed the neuronal properties of the NSC34 cell line.GFP and DAPI fluorescence nearly completely merged.Western blot results showed that NSC34-E cells expressed only GFP,while cells transfected with GFP-h SOD1 WT and GFP-h SOD1G93 A expressed GFP-human SOD1 fusion proteins,confirming the success of the transfections.2.As the concentration of simvastatin increased or the intervention time prolonged,the viability of NSC34-h SOD1G93 A cells gradually decreased,and the LDH release increased,whereas the same simvastatin treatments resulted in a slight but not statistically significant decrease in NSC34-E cells and NSC34-h SOD1 WT cells viability.The results supported that NSC34-h SOD1G93 A cells were susceptible to simvastatin toxicity,simvastatin caused damage to NSC34-h SOD1G93 A cells in a dose-and time-dependent manner.Part Three Simvastatin aggravated autophagic flux impairment in NS-C34-h SOD1G93 A cellsObjective: To observe the autophagic status of NSC34-h SOD1G93A cells and the effect of simvastatin on autophagy of NSC34-h SOD1G93A cells.Methods: We used transmission electron microscopy(TEM)to observe the autophagic vacuoles in NSC34-h SOD1G93 A cells and western blot to detect the protein expression levels of LC3 II/I and P62.Results:1.Compared with NSC34-E cells and NSC34-h SOD1 WT cells,NSC34-h SOD1G93 A cells had increased numbers of autophagic vacuoles.Western blot results showed that LC3 II/I and P62 levels were significantly higer in NSC34-h SOD1G93 A cells than in NSC34-E cells and NSC34-h SOD1 WT cells.Moreover,LC3 II/I and P62 levels were increased markedly all in NSC34-E,NSC34-h SOD1 WT and NSC34-h SOD1G93 A cells treated with bafilomycin A1 respectively.The results indicated that the impairment of autophagic flux is not complete in NSC34-h SOD1G93 A cells.2.After simvastatin treatment,the autophagic vacuoles in NSC34-h SOD1G93 A cells clearly increased,and the expression levels of LC3 II/I and P62 were higher in the treated groups than in the untreated groups in a dose-and time-dependent manner.There were no significant differences in LC3 II/I and P62 levels between simvastatin plus baflumycin A1 and simvastatin or baflumycin A1 alone.These findings implied that simvastatin aggravated the autophagic flux impairment in NSC34-h SOD1G93 A cells.Part Four Simvastatin aggravated impaired autophagic flux in NSC34-h SOD1G93 A cells and caused damage to NSC34-h SOD-1G93A cells through inhibiting GGPP synthesisObjective: To observe whether the mevalonate pathway products can reverse the effect of simvastatin and observe the effect of mevalonate pathway downstream inhibitors on NSC34-h SOD1G93 A cells.Methods:1.Simvastatin combined with mevalonate,cholesterol,FPP,or GGPP was used to incubate NSC34-h SOD1G93 A cells.CCK-8 and LDH were used to detect changes in cell viability.TEM was used to observe changes in the autophagic vacuoles.Western blot was used to detect the protein expression levels of LC3 II/I and P62.2.NSC34-h SOD1G93 A cells were incubated with AY9944,FTI-277,or GGTI-286.Cell viability was detected by CCK-8.Expression levels of LC3 II/I and P62 were detected by western blot.Results:1.The addition of mevalonate,FPP or GGPP could improve the cell damage caused by simvastatin,markly reduce simvastatin-induced increase in autophagic vacuole formation,and reverse the increase in LC3 II/I and P62 levels induced by simvastatin,whereas cholesterol could not reverse the effect of simvastatin.These results suggested that the simvastatin-induced damage to NSC34-h SOD1G93 A cells and aggravation of autophagic flux impairment occurred mainly through the inhibition of isoprenoid FPP or GGPP synthesis instead of cholesterol.2.GGTI-286 induced a significant decrease in cell viability,while AY9944 has no significant effect on cell viability,FTI-277 causes a slight but not statistically significant decrease in cell viability.Moreover,GGTI-286 treatment elevated LC3 II/I and P62 levels in NSC34-h SOD1G93 A cells,indicating that inhibition of GGPP synthesis was the leading mechanism underlying simvastatin-induced effects on NSC34-h SOD1G93 A cells.Conclusions:1.NSC34-h SOD1G93 A cells are more susceptible to the cytotoxicity of simvastatin.Simvastatin caused damage to NSC34-h SOD1G93 A cells in a dose-and time-dependent manner.2.Simvastatin aggravated impaired autophagic flux in NSC34-h SOD1G93 A cells in a dose-and time-dependent manner.3.These effects of simvastatin on NSC34-h SOD1G93 A cells were related to the inhibition of geranylgeranyl pyrophosphate synthesis.4.ALS patients with coronary heart disease,cerebral infarction and hypercholesterolemia should be cautious when using statins.