The Role Of Adult Neurogenesis And Mitochondrial Protein Homeostasis In ALS Disease And The Intervention Of Nicotinamide Ribose On ALS Transgenic Mice

Background:Amyotrophic lateral sclerosis(ALS)Amyotrophic lateral sclerosis(ALS)is characterized by progressive degeneration of motor neurons in the spinal cord,brain stem and motor cortex.Motor neuron death leads to muscle weakness and paralysis.Respiratory paralysis,which usually occurs within 3 to 5 years from the date of symptoms,is the main cause of death.About 5-10%of ALS patients have family history(fALS),and most of them are related to dominant gene mutations.The rest of the cases were sporadic(sALS),possibly due to undetermined environmental exposure or gene mutation.The first ALS linked gene identified was superoxide dismutase 1(SOD1).SOD1 gene mutations accounted for 20%of Fas and 1-2%of SALS.Mutations in other genes,such as tar-dna binding protein(TAR-DNA)and fusion sarcoma(FUS),can also be found in fALS and sALS.Nicotinamide adenine dinucleotide(NAD+)is known as the active metabolite type of vitamin B3,which plays a key role in several signal transduction pathways controlling the basic biological process.Redox reactions involve the transfer of reduction equivalents between the oxidation form(NAD+)and reduction form(NADH)of nucleotides.Although this electronic current carrying function is essential for metabolic response and energy generation,it does not cause any net loss of NAD+.In many kinds of enzymatic reactions,NAD+as a co substrate leads to its degradation.Three different enzyme families use NAD+as co substrates:poly(ADP ribose)polymerase(parps),ADP ribocyclase and acetylase(sirtuins).Nicotinamide monophosphate ribosyltransferase(Nampt)-mediated NAD+biosynthesis and NAD+consumptive enzyme utilization are in a sensitive equilibrium.The decrease of NAD+indicates the dysfunction of basic physiological system.The understanding that NAD+homeostasis is susceptible to aging and the process of neurodegenerative diseases is developing,which promotes the development of experiments to determine whether NAD+supplementation of cells or tissues can improve the phenotype of neurodegenerative diseases.Mitochondrial protein homeostasis includes mitochondrial biogenesis,mitochondrial unfolded protein response(UPRmt),mitochondrial autophagy and mitochondrial nuclear communication,and the loss of mitochondrial protein balance has been considered to play an important role in the pathogenesis of age-related diseases.NAD+,as an essential coenzyme involved in energy production and redox metabolism,is closely related to mitochondrial energy metabolism.In many cases,a basic pathway related to mitochondrial damage is the consumption of NAD+,which occurs by initiating the use of cells and mitochondrial NAD+pools,such as activated poly(ADP ribose)polymerase(parps)and cyclic ADP ribohydrolase(CD38).The supplement of NAD+pool can resist carbon stress by increasing the activity of acetylase,or prevent oxidative damage with antioxidants,which is helpful to maintain the protein balance of mitochondria,thus effectively clearing the disease-related toxic protein aggregation.At present,the two drugs approved by the U.S.Food and Drug Administration(FDA)for the treatment of ALS are riluzole and edaravone,but the exact neuroprotective mechanism of these drugs is not clear,and the quality of life has not been significantly improved.The adult neurogenesis in the brain of ALS transgenic mice has obvious changes.In recent years,more and more evidences support that NAD+level is closely related to the survival and regeneration of neural stem cells.Furthermore,nicotinamide ribose(NR)treatment can reduce the accumulation of mitochondrial protein and promote the proliferation of brain neural precursor cells.In addition,the central nervous system of ALS transgenic mice has toxic SOD1 protein wrong aggregation and mitochondrial dysfunction.It is worth noting that enhancing NAD+salvage pathway can restore the killing ability of primary astrocytes expressing ALS linked superoxide dismutase 1,and enhancing NAD+salvage pathway can inhibit the protein toxicity in neurodegenerative yeast model by promoting the clearance of misfolded proteins.On the contrary,the decrease of NAD+level in adult mice leads to the loss of Nampt in the projection neurons,which leads to motor dysfunction,neuronal degeneration and death.These findings strongly suggest that the supplement of NAD+precursor to promote NAD+biosynthesis may be an important candidate target for the treatment of ALS.In conclusion,we speculate that the intervention of NAD+nicotinamide ribose(NR)can promote the neurogenesis of SOD1-G93A ALS transgenic mice,improve the fate of neural stem cells/neural precursor cells(NSCs/NPCs),and clear the accumulation of mitochondrial toxic proteins,so as to maintain mitochondrial protein homeostasis.In order to confirm this hypothesis,the following two aspects are explored in this study as following:Firstly,our research group used iTRAQ technology to compare the proteomics of SOD1-G93A ALS transgenic mice and normal control mice,and found the difference protein of brain lipid binding protein(BLBP)between brain and spinal cord.Because BLBP protein is an important marker of central neural stem cells and radial glial cells,we established SOD1-G93A ALS transgenic mice model,and explored the BLBP protein in SOD 1-G93A by immunofluorescence and protein immunoimprinting experiments The relationship between the expression and distribution of ALS transgenic mouse spinal cord and the proliferation,differentiation and migration of adult neurogenesis neural stem cells/neural precursor cells(NSCs/NPCs);Secondly,at the level of ALS transgenic mice,the effect of NAD+metabolic pathway on neurogenesis and mitochondrial protein homeostasis and its mechanism were studied.Through nicotinamide ribose(NR)In order to observe the effect of NR on neurobehavioral of ALS mice,we used Western blotting to detect the expression of mutated hSOD1 protein,mitochondrial unfolded protein response(UPRmt)related protein,mitochondrial autophagy marker,NAD+metabolism related protein,and immunofluorescence to observe the effect of NR intervention on experimental mice The effect of NSCs/NPCs in neurogenesis area of rat brain;Finally,in order to evaluate the preclinical evidence of mesenchymal stem cells(MSCs)in the treatment of ALS,a meta-analysis and systematic evaluation were carried out to analyze the therapeutic effect of MSCs on ALS,and to determine the parameters related to the therapeutic mode that are different from MSCs' therapeutic effect,so as to provide translational medical evidence for the treatment of ALS.The first part:To explore the relationship between the expression and distribution of BLBP protein and the neurogenesis of adult spinal cord in ALS transgenic miceObjective:to establish SOD1-G93A ALS transgenic mice model,and explore the relationship between the expression and distribution of BLBP protein in the spinal cord of g93a-sodlals transgenic mice and adult neurogenesis neural stem cells/neural precursor cells(NSCs/NPCs).Method:(1)The b6sj1 SOD1-G93A transgenic mice model was constructed and the transgenic mice were screened by PCR;(2)The transgenic mice of SOD1-G93A were divided into three stages:non onset stage(about 60 days),onset stage(about 90 days)and progressive stage(about 120 days).In each group,the same sex,the same age,the same nest negative wild-type control was set up:the pre onset control(about 60 days),the onset control(about 90 days),the progression control(about 120 days);(3)BrdU(5-bromo-2-deoxyuridine)was injected intraperitoneally into the spinal cord of wild-type and SOD 1-G93 A ALS transgenic mice;(4)NSCs/NPCs were labeled by nestin,BrdU,Fox and GFAP,respectively;(5)Under the same field of vision and magnification of the microscope,two pairs of positive cells with different staining were imaged by image processing technology to observe whether there was co immune reaction.The changes of brain lipid binding protein(BLBP)in different anatomical parts and segments of the spinal cord of WT and TG mice were observed and analyzed.The relationship between BLBP and neurons,astrocytes or radial glial cells(RGCs)and NSCs/NPCs were also studied.Result:(1)BLBP positive cells(blbppcs)are widely distributed in almost all anatomic regions,especially in anterior horn,posterior horn,central canal and its surrounding,anterior,lateral and posterior funiculus;(2)In WT mice,blbppcs may be redistributed with age,such as migration from the thoracic to the cervical and/or lumbar segments.With the increase of age,the expression of BLBP in the whole spinal cord increased gradually;(3)In TG mice,blbppcs showed an overall upward trend from onset to progression,but there may also be a redistribution phenomenon of migration from thoracic to cervical and/or lumbar segments;(4)The expression of BLBP protein in the whole spinal cord increased significantly from onset to progression;(5)BLBP protein was expressed in neurons,astrocytes or radial glial cells,neural stem cells/neural precursor cells(NSCs/NPCs)at early and late stages;(6)BLBP protein mainly distributed outside the nucleus,but less in the nucleus;(7)The increase of blbppcs is closely related to the death of nerve cells in TG mice.Conclusion:Our results suggest that the increase of BLBP and redistribution of thoracic,cervical and lumbar segments may be involved in the development of ALS,and may play a role in the pathogenesis of ALS by regulating NSCs/NPCs.The second part:Nicotinamide ribose intervention on SOD1-G93A transgenic mice and its molecular mechanismObjective:At the level of transgenic mice,to explore the neurobehavioral effects of NAD+metabolic pathway on SOD1-G93A ALS transgenic mice and its mechanism.Firstly,he mouse nicotinamide ribose(NR)intervention experiments were conducted to observe the effects of NR on neurobehavioral of ALS mice.Secondly,in order to elucidate its specific molecular mechanism,we used Western blotting to detect the expression changes of mutated hSOD1 protein,mitochondrial unfolded protein response(uprmt)related protein,mitochondrial autophagy marker,NAD+metabolism related protein.Finally,we used immunofluorescence to observe the effects of NR intervention on experimental mice The effect of NSCs/NPCs on the neurogenesis of adult rat brain.Method:The model of B6SJL-Tg(SOD1*G93A)1Gur/J ALS transgenic mice was constructed,which was bred with normal C57BL/6J.The offspring mice were detected and screened by PCR.According to the different purposes of the experiment,the model was divided into two parts:1.The neurobehavioral effects of nicotinamide ribose intervention on SOD1-G93A ALS transgenic mice.(1)Preparation and administration of nicotinamide ribose(NR):NR powder was mixed into sterilized distilled water to prepare NR working solution with final concentration of 20mg/ml,which was placed in the drinking bottle of mice,and the mice were free to drink.According to the amount of drinking water per day,the NR dosage of each mouse was controlled to be 400 mg/kg;(2)Study objects and experimental groups:eight male SOD1-G93A transgenic mice were randomly divided into two groups,four in each group,g93a-nr intervention group:drinking NR drug water(n=4);G93A-vehicle control group:drinking sterile distilled water(n=4);(3)The mice in the intervention group and the control group began to drink sterilized distilled water and NR medicine water for a long time at the age of 50 days and died finally;(4)The body weight,onset time and death date of the two groups were measured.The effects of long-term NR intervention on the motor function of the two groups of mice were evaluated by hanging cage experiment.2.The effect of NR intervention on neurogenesis and mitochondrial protein homeostasis of SOD 1-G93 A ALS transgenic mice.(1)NR drug preparation and administration method:NR powder was mixed into sterilized distilled water to prepare NR working solution with final concentration of 20 mg/ml,which was placed in the drinking bottle of mice,and the mice were free to drink.According to the amount of drinking water per day,the NR dosage of each mouse was controlled to be 400 mg/kg;(2)Subjects and experimental groups:16 male SOD1-G93A transgenic mice were randomly divided into two groups,8 in each group,G93A-NR intervention group:drinking NR drug water(n = 8);G93A-vehicle control group:drinking sterile distilled water(n=8);(3)The mice in the intervention group and the control group began to drink sterilized distilled water and NR medicine water for a long time at the age of 50 days,and were killed at the age of 120 days;(4)Western blotting was used to detect the expression of mutated hSOD1 protein,mitochondrial unfolded protein response(UPRmt)related protein,mitochondrial autophagy related marker,NAD+metabolism related protein;(5)The effect of NR on the proliferation,differentiation and migration of NSCs/NPCs in adult neurogenesis area of mouse brain was detected by immunofluorescence.Result:1.The neurobehavioral effects of NR intervention on SOD 1-G93A ALS transgenic mice.(1)Compared with G93A vehicle control group,NR intervention significantly reduced the body weight of SOD1-G93A ALS transgenic mice between 118 and 133 days(P<0.05),but there was no statistical significance at other time points(P>0.05);(2)Compared with WT vehicle,NR had no significant effect on the body weight of wild-type mice(P>0.05);(3)Compared with G93A-vehicle group,NR intervention did not significantly delay the onset of SOD1-G93A ALS transgenic mice(P>0.05);(4)Compared with G93A-vehicle group,NR intervention had no significant effect on the survival time of SOD1-G93AALS transgenic mice(P>0.05);(5)The cage exercise experiment showed that the transgenic mice in G93A-NR group had better endurance performance in the period of 98-126 days than that in G93A-vehicle group(P<0.05),and there was no significant difference in the rest time(P>0.05).2.Intervention of nicotinamide ribose on neurogenesis and mitochondrial protein homeostasis of SOD1-G93A ALS transgenic mice.(1)Without NR intervention,there was significant expression of hSODl protein in the mitochondria of ALS transgenic mice in G93A-vehicle group,but there was no expression of hSODl protein in the mitochondria of wt-vehicle wild control group;(2)After the treatment of NR,the aggregation level of hSODl toxic protein in mitochondria of G93A-NR group was significantly lower than that of G93A-vehicle group(P<0.05);(3)The levels of lonpl,HSP60 and CLPP in G93A vehicle group were significantly higher than those in WT vehicle group(P<0.05),but there was no significant difference in LC3-? and P6(P>0.05);(4)The expression of lonp1,HSP60 and CLPP in mitochondria of G93A-NR group was significantly higher than that of G93A-vehicle group(P<0.05),but there was no significant difference in the expression of LC3-? and P6(P>0.05);(5)Without NR intervention,the level of NMNAT3 protein in G93A vehicle group was significantly lower than that in WT vehicle group(P<0.05),while the level of NMNAT3 protein in G93 A vehicle group was significantly higher than that in WT vehicle group(P<0.05);(6)Compared with G93A-vehicle group,the expression of NMNAT3 protein in brain tissue of G93A-NR group increased significantly(P<0.05)after NR treatment,but the NMNAT3 protein level in brain tissue of G93A-NR group decreased significantly(P<0.05)compared with G93A-vehicle group;(7)Without NR intervention,the number of vimentin positive cells in the lateral ventricles and olfactory bulbs of G93 A vehicle group was significantly lower than that of WT vehicle group(P<0.05),while the number of vimentin positive cells in the dentate gyrus of G93 A vehicle group was not significantly different from that of WT vehicle group(P>0.05);(8)The number of vimentin positive cells in the lateral ventricles and olfactory bulbs of G93 A-NR group was significantly higher than that of G93 A-vehicle group(P<0.05),but the number of vimentin positive cells in the dentate gyrus of SOD1-G93A transgenic mice was not significantly affected by NR intervention(P>0.05).(9)Without NR intervention,the number of DCX positive cells in the lateral ventricle and olfactory bulb of G93 A vehicle group was significantly lower than that of WT vehicle group,while the number of DCX positive cells in the dentate gyrus of G93A vehicle group was not significantly different from that of WT vehicle group(P>0.05);(10)The number of DCX positive cells in the lateral ventricle,dentate gyrus and olfactory bulb of G93 A-NR group was significantly higher than that of G93 A-vehicle group(P<0.05).Conclusion:(1)NR intervention can reduce the weight of SOD1-G93A ALS transgenic mice;(2)NR intervention can improve the cage lifting ability of SOD1-G93A ALS transgenic mice;(3)NR intervention had no significant effect on the onset time and survival time of SOD1-G93A ALS transgenic mice;(4)The hSODl protein was mistakenly deposited in the mitochondria of brain tissue of SOD1-G93A ALS mice;(5)The mitochondrial protein homeostasis in the brain of SOD1-G93A ALS mice was destroyed;(6)In SOD1-G93A ALS mice model,the main reason for the decrease of NAD+level is the insufficient function of NAD+recovery pathway mediated by Nampt protein;(7)NR supplementation promoted the elimination of hSODl neurotoxic protein in mitochondria of SOD1-G93A ALS mice;(8)After NR intervention,the Nampt related protein level in the brain of SOD1-G93A ALS mice increased significantly,but the expression level of mitochondrial autophagy related protein was not affected;(9)NR intervention promoted the proliferation and migration of NSCs/NPCs in the brain of SOD1-G93A ALS mice;(10)NR intervention may regulate mitochondrial protein balance by activating uprmt signal,and promote adult neurogenesis in the brain of SOD1-G93A ALS mice.NR may be a feasible clinical treatment for ALS and other neurodegenerative diseases.The third part:Preclinical study of mesenchymal stem cell transplantation for amyotrophic lateral sclerosis:systematic evaluation and meta-analysisObjective:To evaluate the quality of preclinical evidence of mesenchymal stem cells(MSCs)in the treatment of amyotrophic lateral sclerosis(ALS),to determine the therapeutic effect of MSCs transplantation on ALS mouse model,and to determine the parameters related to the difference of therapeutic effect of MSCs transplantation on ALS mouse model,so as to provide clinical evidence for the treatment of ALS disease by MSCs transplantation.Method:(1)First,the criteria for inclusion and non inclusion are set:? Inclusion criteria:all randomized controlled studies described in the literature on mesenchymal stem cell transplantation for treatment of ALS mouse model were included.The intervention measures included in each included literature,treatment group:various mesenchymal stem cells,control group:PBS or normal saline.Each included literature should include onset time,disease progression rate,survival time and risk ratio.? Exclusion criteria:we excluded the literature on the use of mature cells or pretreated cells or MSCs conditioned medium for the treatment of ALS models.We also excluded the lack of accurate animal numbers and the lack of randomized controlled literature.(2)The required tests were collected from the following English databases:PubMed,EMBASE and web of science.In addition,the references of related research literature are searched manually as supplement.The key words of the search were"amyotrophic posterior sclerosis","ALS","mesosexual stem cells" and "MSCs",to explore the efficacy and the best treatment mode of MSCs transplantation for ALS animal model.The retrieval time limit is from the establishment of the database to July 2019;(3)Results:four main indexes(age of onset,disease progression deceleration,survival time,risk ratio reduction)and 14 treatment mode related parameters were obtained through specific neurobehavioral evaluation;(4)Stata(version 15.0,statacorp)was used for all statistical analysis.The ratio(or)was used as the effect index,and the standard mean difference(SMD)was used as the effect index for the continuous variable.According to the Q statistics of I2 and Cochran,the population heterogeneity is tested,and the fixed or random effect model is used.If there is heterogeneity,the size of the mixed effect is obtained by using the random effect model which depends on dersimonian and Laird methods.Meta regression analysis was used to further explore the source of heterogeneity.14 parameters related to treatment mode,including MSCs intervention time,cell receptor type,animal gender,MSCs type,cell transmission algebra,genetic modification of transplanted cells,immunogenicity,CSA immunosuppressive drugs,treatment route selection,administration frequency,cell dose,blindness,randomization.The funnel was then used to examine the potential for bias to be published in order to obtain a visual impression.Two tail egger regression was used to reevaluate the data with significant publication bias.If publication bias exists,trim and fill analysis is used to adjust the bias and check whether the effect size affects the final result.Result:(1)Finally,25 articles were included in meta-analysis,including 41 treatment arms and 1048 ALS model rats;(2)The scores of literature quality included ranged from 4 to 6,with an average of 5.01;(3)In order to evaluate the effect of MSCs transplantation on the onset time of ALS model rats,a total of 15 arm data were reported.The results showed that MSCs transplantation significantly delayed the onset of ALS,and the total effect value was(SMD:0.52,95%CI:0.18-0.86).The results of hedges'g Q test showed that there was significant heterogeneity(I2=53.3%,P=0.008).After sensitivity analysis,the heterogeneity decreased(I2=0.0%,P=0.962)after two unsuitable literatures were excluded.The corrected mixed SMD was 0.28(95%CI:0.04-0.53,P=0.022);(4)In order to evaluate the effect of MSCs transplantation on the survival time of ALS model rats,37 arm data were included.The results showed that MSCs transplantation significantly increased the survival time effect of ALS model rats(SMD:0.56,95%CI:0.36-0.76).The results of Hedges' Q test showed that there was significant heterogeneity(I2=52.1%,P=0.000)among the groups.The heterogeneity decreased(I2=33.7%,P=0.029)and the corrected mixed SMD was 0.54(95%CI:0.37-0.72,P=0.000);(5)The risk ratio reduction effect of 35 MSCs transplantation arms was evaluated.The SMD of hazard ratio reduction was 0.48(95%CI:0.39-0.56,P=0.000),and the heterogeneity was low(I2=16.9%,P=0.192);(6)In order to evaluate the effect of MSCs transplantation on the deceleration of disease progression in ALS model rats,32 arm data were included.For disease progression deceleration,the mixed effect value was(SMD:0.73,95%CI:0.33-1.12).Because the heterogeneity among the 32 treatment arms included in the study was large(I2=85.1%,P=0.000),the heterogeneity decreased significantly(I2=58%,P=0.000)and the corrected mixed effect value was 0.25(95%CI:0.02-0.48,P=0.036)after excluding 5 effect values greater than 3.0 treatment arms through the sensitivity analysis;(7)After the evaluation of the effect value and sensitivity analysis,the heterogeneity of the treatment arm is still large.Therefore,we used subgroup analysis and meta-regression analysis to reveal the relationship between 14 treatment mode related parameters and disease deceleration effect value,and further analyzed the source of heterogeneity.The results are as follows:?The gender of animals was related to the decelerating effect of disease progression(adj R2=66.95%,P=0.003).The female had the greatest effect,followed by the mixed type,and then the male(the average effect was 0.723 vs 0.278 vs 0.823);?Whether to use CSA immunosuppressant is also closely related to the effect value of disease progression deceleration(adj R2=61.57%,P=0.003).The use of CSA has a faster disease progression than that of no use(mean effect value:0.305 vs 0.417,suggesting that the use of CSA immunosuppressant may have the effect of nerve element damage;?Blinding and randomization did not affect the decelerating effect of disease progression;(8)The funnel chart of disease onset age showed a roughly symmetrical distribution,suggesting no bias was published.Egger test further confirmed this point,in which the relative p value is 0.397,more than 0.1.Although there was publication bias in the effect values of disease progression deceleration,survival time and risk ratio reduction,the P values related to the two tail egger regression test were 0.016,0.011 and 0.000,respectively.After trimming and filling analysis,these three recalibrated effect values are stable.Conclusion:(1)MSCs cell transplantation significantly improved the onset age,disease progression,survival time and risk ratio of ALS rats;(2)The effect value of disease progression deceleration is closely related to animal sex and the use of CSA immunosuppressant;(3)There was no correlation between therapeutic time,cell receptor type,cell donor type,MSCs type,cell passage,genetic modification,immunity,therapeutic approach,drug administration frequency,cell dose,blinding,randomization and other clinical variables and the therapeutic effect of MSCs cell transplantation on ALS model rats;(4)MSCs cell transplantation has shown promise in the treatment of ALS animals.However,due to the poor quality of research and publication bias,whether there is definite efficacy still needs further study.