| Part I:The Pathophysiological Studies of RR-MADD by Generating a Novel Etfdh(h)c.250 G>A Knock in Mice ModelBackgroundMultiple acyl-coenzyme A dehydrogenation deficiency(MADD),also known as glutaricaceduria type Ⅱ,is an autosomal recessive fatty acid metabolism disorder.The clinical features of MADD are heterogeneous and have been classified as neonatal-onset form with or without congenital anomalies,as well as late-onset form.The late-onset form of MADD,characterized by muscle weakness,exercise intolerance,hepatomegaly,episodes of vomiting,hypoglycemia and metabolic acidosis,is the most common type in Chinese patients.A considerable number of late onset MADD cases have been reported to be dramatically responsive to high dosage riboflavin supplementation.Such patients are referred to as riboflavin-responsive MADD(RR-MADD).Since first reported in 2007,approximately 80%of the RR-MADD were proved to be caused by mutations of the ETFDH gene.ETFDH encodes electron transfer flavoprotein-ubiquinone oxidoreductase(ETF-QO),which is an enzyme that transfers electrons from electron-transferring flavoprotein in the mitochondrial matrix to the ubiquinone pool located in the inner mitochondrial membrane.Up to now,more than 80 ETFDH mutations have been reported.Most patients with the mutation of c.G250A are homozygous,which indicates the possibility of a founder effect in this mutation.In 2012,Olsen’s group demonstrated the protein folding defects hypothesis in a human HEK-293 cell expressing the variant ETF-QO proteins;the variant ETF-QO proteins associated with RR-MADD caused folding defects,which resulted in electron leakage and increased reactive oxygen species.Furthermore,her group showed that FAD can correct the chaperone-mediated folding and conformational stabilization of variant ETF-QO protein caused by ETFDH missense mutations.Despite these advances in understanding the molecular mechanism of RR-MADD,there are still some things that cannot be easily explained by ETFDH mutation alone.Before the ETFDH mutations were confirmed,low mitochondrial and cellular concentration of FMN and FAD had been found in muscles of non-genetically confirmed RR-MADD patients.Recently,some riboflavin transporter genes as well as the FAD synthase gene were reported as the pathogenic genes for RR-MADD.The evidence further emphasizes the role of FAD homeostasis disturbance in the pathogenesis of RR-MADD.However,the relationship between ETFDH mutation and FAD concentration has not yet been confirmed.Moreover,it is interesting that in the clinical practice,a one-month course of riboflavin therapy will correct the symptoms caused by lipid metabolic disturbances,such as muscle weakness and exercise intolerance.In the present study,we generated knock-in mice carrying the human p.A84T(c.250G>A)mutation in Etfdh gene.After fed a special diet for 5 weeks,the homozygote mice developed features similar to RR-MADD patients.Subsequently,we performed several cellular and molecular analyses on these mice to clarify the molecular and pharmacotherapeutic mechanism of RR-MADD.Method:1.Creation of the targeting vector carrying the single point mutation.The targeting vector was created to insert a single point mutation,mice c.G247A encoding the p.A83Tmutation(equivalent to human c.G250A,p.A84T),into exon 3 of the murine Etfdh gene.2.The vector was then electroporated into W4 ES cells,followed by positive selection with G418 and negative selection with GANC.The ES cell colonies were screened for correct gene targeting by Southern blot.3.Generation of the chimeras.One successfully targeted clone was injected into C57BL/6 blastocysts,which was then implanted into pseudo-pregnant females to generate chimeras.4.Generation of the F1 mice.Male chimeras were bred with female wild type C57BL/6 mice,and offspring with germline transmission of the Etfdh(h)A84T(Neo)allele were identified by PCR of genomic DNA.Male Etfdh+(g)/A84T(Neo)mice were bred with female CMV-Cre transgenic mice to excise the Neo cassette.5.Generation of homozygote mice carrying ETFDH c.250G>A(p.A84T)mutation.Etfdh+/(h)A84T(KI/WT,heterozygote)mice were crossed to generate Etfdh+/+(WT/WT,wild type),Etfdh+/(h)A84T(KI/WT,heterozygote),and Etfdh(h)A84T/(h)A84T(KI/K1,homozygote)mice,whose genotypes were again identified by PCR of genomic DNA.6.Diet composition and supplementation.At the age of 18-20 weeks,the WT/WT and KI/KI mice were randomly divided in 4 groups and fed 4 different diets:AIN-93G diet(93G),vitamin B2 deficiency diet(B2D),high fat diet(HF)and high fat with vitamin B2 deficiency diet(HF-B2D).All groups received water ad libitum.7.Observation of the body weight and the survival time.Body weight was tested at the same time each week.The survival curve was made after 8 weeks observation.8.Behavioral tests include rota rod test and open field test.The rota rod test consisted of three 5-min trials,all of which were at a constant speed of 30 rpm,with a 5-min break between trials.The time to fall was measured in each trial and the data was expressed as a percentage of not-fall trial for each test.In open field test,the number of lines that the mice crossed in 3min was tested to access the motor ability.9.Acylcarnitine analysis in mice blood.Blood acylcarnitine was measured by tandem mass spectrometry(MS/MS).10.Histology.The paraformaldehyde-fixed livers,brains,kidney and heartswere stained with H&E.The gastrocnemius specimens were stained with H&E,ORO and SDH.11.Using real-time quantitative PCR to test mRNA copy number of Etfdh in muscles and livers of mice in different diets groups.12.Using western blot to evaluate ETF-QO protein amount in muscles and livers of mice in different diets groups.13.Using HPLC to test the FAD concentration in muscles and livers of mice in different diets groups。Results:1.We generated knock-in mice carrying the Etfdh(h)c.250G>A(p.A84T)mutation successfully.Identification of genotypes of 294 offspring from crossing KI/WT mice revealed that there was no statistical difference between the observed frequency versus the expected Mendelian ratio of the three genotypes of WT/WT,KI/WT,and KI/KI,indicating that this mutation was not embryonic lethal.2.Heterozygote mice exhibit normal phenotypes when fed by normal food.3.KI/KI mice in HF-B2D diet group developed characteristics similar to those observed in RR-MADD patients.KI/KI mice in HF-B2D diet group(KI/KI-HF-B2D)showed weight loss,shorten of survival time,medium and long chain acylcarnitine elevation,abnormal in muscle biopsy,hepatocyte steatosis and hepatomegaly.Neither KI/KI mice in other diets groups nor WT/WT mice in HF-B2D group develpoed such characterization.4.KI/KI-HF-B2D mice experienced a significant recovery after fed by high-dose riboflavin.Restoration of body weight and recovery of movement ability were obvious after 21 days riboflavin supplement.Acylcarnitine patterns in blood reverted to normal.Hepatomegaly and hepatocyte steatosis disappeared.The abnormalities in muscle pathology were corrected.5.The real-time PCR results revealed no marked variation in Etfdh mRNA levels between KI/KI and WT/WT cohorts in any diet groups.ETF-QO protein amounts for KI/KI mice tissues were mildly lower than that of WT/WT mice when fed with food containing a normal.amount of riboflavin(93G and HF diets).However,in the riboflavin deficiency diet groups(93G-B2D and HF-B2D diet groups),ETF-QO protein amounts in KI/KI mice decreased significantly when compared with WT/WT littermates.After given riboflavin for 21 days,ETF-QO protein amount in KI/KI-HF-B2D mice increased although it was still lower than that in WT/WT.6.The HPLC results showed that,when riboflavin was depleted(in 93G-B2D and HF-B2D groups),FAD concentration in muscles and livers decreased in both KI/KI and WT/WT mice.Interestingly,in the HF-B2D diet group,FAD concentration in KI/KI tissues decreased further and was significantly lower than that in WT/WT in the same group,which emphasized the role of FAO stress in the FAD concentration fluctuation in vivo.After fed with riboflavin for 21 days,both KI/KI and WT/WT cohorts had obviously elevated FAD concentrations.Discussion:Here,we made knock-in mice with an Etfdh(h)p.84A>T mutation,which give us a chance to investigate the pathophysiological machenism of RR-MADD in vivo.Finally,homozygous mice manifested characteristics resembling phenotypic human MADD patients and showed dramatic responses to riboflavin treatment.In our research,HF-B2D diet,which creates a low-riboflavin,high-FAO-stress condition,act as trigger for the onset of diseased state.It has been proved that subclinical riboflavin deficiency is common in human beings,epically in adolescent girls.In addition,high-FAO-stress can be caused by cold,starvation and illness.Therefore,the low-riboflavin high-FAO-stress condition can be quite common in humans.Pateinets who carry ETFDH mutation would not developed RR-MADD symptoms until the low-riboflavin,high-FAO-stress condition happened.Our result,in vivo,indirectly confirmed the hypothesis of previous research:the ETF-QO protein synthesis was not affected and shouldn’t be the culprit of ETF-QO protein reduction.The decrease in ETF-QO protein might possibly be due to a faster degradation of mutant ETF-QO proteins.The defective FAD binding to ETF-QO in RR-MADD patients should be explained on the basis of a subtly altered protein structure that partially,but not entirely,hampers the affinity of FAD to flavoproteins.Interestingly,the FAD levels in KI/KI mice were lower than controls at the disease inducement condition,which indicated a conditional shortage of FAD in MADD with ETFDH mutation.Although FAD concentration decreased in tissues from all mice under riboflavin deficiency diet(93G-B2D and HF-B2D)groups,it decreased further in the muscles and livers of KI/KI-HF-B2D mice,which were the only ones to develop MADD syndrome.Recently,the pathogenic gene for RR-MADD has extended to riboflavin transporter genes and FAD synthase gene.All of this suggests a potential unified mechanism of RR-MADD onset;FAD homeostasis disturbance.With riboflavin supplementation,FAD concentration increased,and improved the folding of flavoproteins via increased opportunity to directly participate in the folding process,or by affecting chaperones involved in protein maturation.Alternatively,an increase in FAD-binding flavoproteins after riboflavin supplementation could release more FAD to mitochondrial matrix during degradation,keeping a larger circulating FAD pool even after discontinuation of riboflavin treatment.This may partially explain why riboflavin supplementation for RR-MADD patients is required for only two or three months but not the whole lifetime.PartⅡ:The Pathophysiological Studies of Cylindrical Spirals in Skeletal MusclesBackgroundCylindrical spirals(CSs)are rare but distinct structures in skeletal muscle fibers.To date,CSs have been identified in muscle biopsies from 16 patients.The abnormal accumulations of CSs are present almost exclusively in the subsarcolemma of type II fibers.They appear basophilic with hematoxylin&eosin(H&E)stain,bright red with modified trichrome(mGT)stain,and strongly or faintly colored with nicotinamide adenine dinucleotide-tetrazolium reductase(NADH-TR)stain,but unreactive to myofibrillar adenosine triphosphatase(ATPase).On electron microscopy(EM),CSs are composed of 6-30 lamellae wrapped around a central core containing glycogen granules.CSs have been noted to be associated with various clinical manifestations.The presence of CSs is therefore considered to be non-specific and presumably reactive to metabolic disturbances in muscle fibers.However,muscle-related symptoms in these cases are relatively consistent.Moreover,the association with familial disorders implicated a role of CSs in some disorders particularly those with a genetic component.The origin and composition of CSs remain unknown.Some studies indicated that CSs may derive from the sarcoplasmic reticulum(SR)proliferation.In the present study,we described CSs in muscle biopsies from two Chinese siblings,and performed immunohistochemistry to prove that the CSs originate from the sarcoplasmic reticulum.Methods1.2 present patients,2 patients with the diagnosis of tubular aggregates myopathy and 2 subjects with no neuromuscular disorders were included in this study.2.Muscle sections were stained with H&E,mGT,NADH-TR,SDH,CCO,PAS and ATPase(pH at 4.3),according to the standard staining protocols.3.For immunohistochemistry,the employed primary antibodies were anti-SERCA1,calsequestrin,RYR1,emerin,myosin,GM130,dystrophin,LC 3,LAMP2,and desmin.4.Electron microscopy test.5.Genomic DNA-based PCR amplifications and sequence analysis of STIM1,ST1M2 and ORAI1 genes in the patients were performed.Results1.We report the first Chinese sibling with cylindrical spirals in muscle fibers.Patient 1 and patient 2 are siblings,both presented with progressive muscle weakness and exercise intolerance.The muscle weakness started in the lower limbs and later progressed to upper limbs,from the proximal to distal.Serum CK were not elevated.Electromyography suggested myogenic changes.CT scan of the lower limbs revealed atrophy of the left gluteus maximus and vastus lateralis,as well as an abnormal density of bilateral tibialis anteriors and gastrocnemius.2.The muscle biopsies from both patients showed abnormal accumulation in muscle fibers.These accumulations stained bright red with mGT,faintly with NADH-TR,lightly with PAS,but not reactive to myosin ATPase,SDH,and CCO stains.3.Ultrastructural examination of the muscle specimens from both patients revealed clusters of spiral laminated cylindrical structures,suggestive of CSs.4.Immunohistochemical studies of biopsied muscles revealed the subsarcolemmal immunoreactivity for SERCA1 in the longitudinal SR,but no immunoreactivity for calsequestrin in the terminal cisternae or RyR1 in the junctional SR.In comparison,the muscles biopsied from two patients with TAs showed the immunoreactivity not only for SERCA]but also for the other SR proteins including calsequestrin and RyR1.Our results suggest that CSs may originate only from the longitudinal SR,while TAs are composed of both the junctional and longitudinal SR.5.Genetic studies revealed no disease-associated mutations in STIM1、STIM2 or ORA11 in these 2 patients.DiscussionThe present study is the first to demonstrate the accumulation of CSs as the major pathological abnormality in Chinese patients with a neuromuscular disorder,and the first to provide immunohistochemical evidence that CSs may originate from the longitudinal SR.Despite some histochemical resemblance between CSs and TAs,our present study has identified the differences in constituents between these 2 structures.CSs are highly immunoreactive for the longitudinal SR protein SERCA1,with the absence of immunoreactivity for the junctional SR protein RYR1 and calsequestrin,which indicates their origin from the longitudinal SR.In comparison,TAs are immunoreactive for the other SR segments proteins,including SERCA1,RyR1 and calsequestrin,which suggests their derivation from the whole SR.Immunochemical staining with antibodies against calsequestrin and RYR1 can help to distinguish these two pathological alterations.Interestingly,despite the consistent morphology with H&E and mGT staining,there have been variable NADH activities of CSs in previously published cases.We have reviewed the previously published cases with CSs,compared to the present 2 cases,with a particular focus on NADH-TR staining and clinicopathological correlation.All 10 cases(including the present 2 cases)have been classified into 2 subgroups according to the activities of NADH:CSs with low NADH activity,and CSs with high NADH activity.Of particular interest is that all 6 cases with low NADH activity in CSs suffer from muscle weakness with or without myalgia,in the absence of cramps and increased muscle tone.In contrast,among the other 4 patients with high NADH activity,only one has episodic weakness and myalgia,while 2 complain of cramp or increased muscle tone,and the other is asymptomatic.It has been noted that,in the skeletal muscle,NADH could stimulate the nearby normal RyR1 channel activity and inspire Ca2+ release,which would further result in the muscle contraction.This physiologic mechanism may explain that the patients with low NADH activity present with muscle weakness while the patients with high NADH activity manifest cramps or myotonia.In our present cases,the relationship between NADH activity and their clinical phenotypes further suggests a role of NADH in the pathogenesis of disorders with CSs,which may also provide an important clue to its therapy. |
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