| Background:Mitochondrial encephalomyopathy is a metabolic disease in which the mitochondrial structure and function are abnormal due to mitochondrial DNA(mtDNA)or nuclear DNA mutation,resulting in oxidative phosphorylation disorder and insufficient energy synthesis,and thereby leading to brain and muscle damage.Mitochondrial encephalopathy,lactic acidosis,and stroke-like episodes(MELAS)syndrome is a multisystem mitochondrial disorder with broad manifestations including stroke-like episodes,dementia,epilepsy,lactic acidemia,myopathy,recurrent headaches,hearing impairment,diabetes,and short stature.MELAS syndrome is one of the most frequent maternally inherited mitochondrial disorders.This disease is preferentially caused by point mutations in the mitochondrial MT-TL1 gene,which encodes the mitochondrial tRNA Leu(UUR).In approximately 80%of cases,MELAS syndrome is associated with m.3243A>G mutation and other frequent mutations are m.3271T>C,m.3291T>C and m.3252A>G.Although the most common mutation associated with MELAS syndrome is the m.3243A>G mutation in the MT-TLI gene,an increasing number of reports on mitochondrial DNA coding regions'mutations,especially in mitochondrial DNA-encoded NADH dehydrogenase(ND)subunit genes of the respiratory chain complex I,have been published recently.Objective:In this study,14 patients with MELAS caused by mtDNA mutation encoding mitochondrial respiratory chain complex I subunit were selected from the neuromuscular pathology laboratory of Qilu hospital of shandong university,who were diagnosed and treated from January 2006 to February 2019.We collected detailed medical history,physical examination and nervous system physical examination of 14 patients,and conducted various auxiliary examinations.We also performed muscle biopsy and mitochondrial gene sequencing.Histochemical staining and immunohistochemical staining were performed on frozen muscle specimens.Materials and methods:All the 14 cases were diagnosed with MELAS by the Research Institute of Neuromuscular and Neurodegenerative Disease of Shandong University during 2006.1-2019.2 due to mutations in the ND subunit gene encoded by the respiratory chain complex I mitochondrial DNA.All the 14 patients underwent muscle biopsy,serologic test,MRI and Whole mitochondrial DNA sequencing.All the medical records of the patients were obtained from the neurology department of Qilu Hospital.We surmmarize the clinical features,neuroimaging characteristics,laboratory findings,courses and outcomes of the patients.Results:Among the 14 patients,6 were female and 8 were male.The age of onset was 4-47 years,with 6 cases of pre-adult onset and 8 cases of post-adult onset,with a median age of 20.5 years old.The patients were aged 6-57 years when diagnosis,with a median age of 27 years.The interval between onset and diagnosis of the 14 patients was 1-12 years,among which 5 patients were 1 year,2 patients were 3 years,8 patients were over 5 years,the longest was 12 years,and the average was 6 years.The major clinical manifestations of the 14 patients were seizures,impaired vision,limb weakness,headache,vomiting,hearing loss,mental retardation,mental retardation,extraocular palsy and short stature.Of the 14 patients,2 patients existed family history of matrilineal inheritance(2/14),while 8 cases exhibited short stature(8/14).Five cases complained paroxysmal headache(5/14).In all 14 cases,12 cases were shown as epileptic seizure with disturbance of consciousness(12/14),1 case showed with myoclonic seizures(1/14).Muscle weakness were involved in 9 cases(9/14),5 cases complained unilateral limb muscle weakness(5/14),4 cases showed bilateral limbs involvement(4/14).Besides,6 cases complained intelligence impairment(6/14),7 cases showed with decreased vision(7/14),while 5 cases exhibited hearing loss(5/14).Three patients presented with mental abnormalities(3/14),two with extraocular muscles paralysis(2/14),and one with developmental delay(1/14).Blood lactic acid was detected in 10 of the 14 patients,and blood lactic acid was increased in 7 patients,with a lactic acid value of 2.3-4.7mmol/l and a median of 2.75mmol/l.Cerebrospinal fluid(CSF)lactic acid was detected in 4 patients,and the CSF lactic acid value was increased in all 4 patients,ranging from 2.3 mmol/1 to 7.6mmol/l,with a median of 4.15mmol/l.No hyperglycemia was observed in all the 14 patients.Muscle biopsies were performed in 14 patients while 13 patients were taken from biceps brachii and 1 patient was taken from quadriceps femorns.Frozen section staining was performed after muscle biopsy.The results of 10 cases showed myogenic pathological damage changes and 4 cases were normal.In the H-E staining,the size of muscle fibers was different in 10 patients.Basophilic muscle fibers were detected in 3 patients.Two patients showed mild endomysium hyperplasia.Necrotic muscle fibers were detected in 1 patient.In MGT staining,typical or atypical ragged red fibers.(RRF)were observed in 7 patients,and typical RRF were observed in 3 patients while atypical RRF were detected in 4 patients.In SDH staiming,typical or atypical ragged blue fiber(RBF)was observed in 6 patients,and typical RBF was observed in 4 patient while atypical RBF were detected in 2 patients.Enhancement of succinate dehydrogenase reaction(SSV)in blood vessels was observed in 8 patients.In COX staining,total COX enzyme deficiency was seen in 2 cases.Blue fibers were observed im 2 patients with COX and SDH double staining.ORO staining revealed slight elevation of lipid droplets in some muscle fibers in 1 patient.MRI or CT was performed in all the 14 cases.The affected areas in neuroimaging do not correspond to classic vascular distribution,are asymmetric,involve predominantly the temporal,parietal,and occipital lobes,and can be restricted to cortical areas or involve subcortical white matter.Frontal lobe,brainstem,cerebellum,basal ganglia and thalamus were occasionally involved.In the 14 cases,7 involved the temporal lobe,7 involved the parietal lobe,11 involved the occipital lobe,4 involved the frontal lobe,4 involved the basal ganglia and 4 involved the thalamus.In addition,2 patients had involvement of the brainstem and two had involvement of the cerebellum.Mitochondrial DNA analysis Next-generation sequencing of the whole mitochondrial genome detected several mutations in MT-ND Subunits of Complex I of our patients.Most of our cases carried mutations in MT-ND5 while we revealed a heteroplasmic m.13513G>A mutation in 4 patients and m.13046T>C mutation in patient 10.Patient 10 also harbored a m.15077 G>A homozygous mutations that usually result in deafness.m.10158T>C mutation in MT-ND3 was detected in 2 patients.There were 4 cases harbored mutations in MT-ND4,two was m.11777A>C,one was n.11138A>G and one was m.l 1406T>A mutation.The remained three cases contain m.10191 T>C mutation in MT-ND3,m.14487T>C mutation in MT-ND6,m.14453G>A mutation in MT-ND6 respectively.The proportion of mutant DNA ranged from 53 to 97%in muscle samples of all patients.In patient 4,a novel MT-ND6 mutation,m.14487T>C mutation,causing M63V substitution in the MT-ND6 of complex I of the mitochondrial respiratory chain,was first reported in MELAS cases.The mutation was heteroplasmic in muscle and urnne with different mutational loads as the mutation were 97%heteroplasmic iin muscle and 48%heteroplasmic in urine.However,no heteroplasmic sequence varnants were found in blood sample from the patient.In addition,we found two new mutation that have not been reported.Conclusions:1.The main clinical manifestations of the cases in this group are seizures,visual impairment,physical weakness,headache,vomiting,hearing impairment,mental impairment,stunting,mental disorders,extraocular paralysis and short stature.The main body convulsions and visual impairment,muscle weakness.2.The pathological features of this group were lighter,typical RRF was rare,and more of them were enhanced by the succinic acid dehydrogenase reaction.3.The central nervous system image of the patients in this group is heavy and can involve multiple cortices.At the same time,brain stem and cerebellar lesions can occur.4.The patients with MT-NDs mutaltions usually present more severe change in neuroimaging while demonstrate milder myopathic changes in muscle biopsy.Therefore,although costly and time-consuming,sequencing of the whole mtDNA is still necessary for these patients and MT-NDs mutations should not be ignored in MELAS.Background:Mitochondria are an important organelle in eukaryotic cells.They are not only energy processing plants,but also participate in apoptosis,regulate cell calcium balance,and many important biochemical decomposition and synthesis processes of cells.In the physiological state,maintaining the number and quality of mitochondria requires the balance of mitochondrial biogenesis and mitophagy,that is,maintaining the mitochondrial homeostasis.They are a pair of opposing and closely related processes.The former can continuously produce new mitochondria while the latter can remove abnormal mitochondria.The biogenesis of mitochondria is very complicated,and the fission and fusion processes of mitochondria are closely related to the mitophagy process.Mitochondria are highly dynamic organelles that continue to divide and fuse within cells.This process helps cells adapt to different physiological states to meet the needs of the body.On the one hand,mitochondrial division is the process of mitochondrial proliferation,which plays a key role in ensuring that mitochondria are transmitted to daughter cells after cell division and filling vacancies caused by aging mitochondria;On the other hand,mitochondrial fusion can partially offset mitochondrial damage caused by various factors.The balance between mitochondrial biogenesis and mitophagy maintains the function of mitochondria in normal cells.However,in the muscle specimens of patients with mitochondrial encephalomyopathy,there are often a large number of abnormal mitochondria that accumulate under the myofilm or between myofibrils.Ragged red fibers(RRF)can be seen in the pathological staining of modified Gomori trichrome.The unusually swollen mitochondria can be seen under electron microscopy.The accumulation indicates that mitochondrial homeostasis is unbalanced.However,the specific molecular mechanism behind it remains unclear.Peroxisome proliferator activation receptor gamma coactivation factor 1?(PGC-1?)is an important regulating factor in cells and plays a significant role in the process of cell material metabolism and mitochondrial biogenesis.On the one hand,as a nuclear protein,it can promote the expression of mitochondrial proteins encoded by nDNA and promote the proliferation of mitochondria;On the other hand,more and more studies have found that PGC-1 a is also distributed in mitochondria.It forms A protein complex with mitochondrial transcription factor A(TFAM)to promote the expression of mtDNA.Recent studies have found that PGC-la also plays an important role in mitophagy.In the process of skeletal muscle formation,PGC-la can inhibit ROS-induced mitochondrial autophagy.In the process of muscle atrophy pathology,PGC-1? plays an important role in regulating mitophagy.However,in mitochondrial encephalomyopathy,how does PGC-la to regulate the process of mitochondrial autophagy lacks relevant research reports.Objective:Taking specimens of patients with mitochondrial encephalomyopathy and HEK-293 cells as the research object,through a comprehensive analysis of autophagy signal networks in mitochondrial disease,to study the mechanism of PGC-la mediated mitochondrial homeostasis in mitochondrial disease,and to explore the pathogenesis of mitochondrial disease.Methods:1)Analysis of the expression of PGC-la protein and autophagy related protein in muscle tissue of MELAS patients carrying A3243G mutations by Western bolt;2)H-E staining,MGT staining,and immunohistochemical staining of PGC-la and P-S6 antibody were performed on muscle tissue specimens of MELAS patients to observe the expression of PGC-la protein in RRF;3)To explore the mechanism of mitochondrial homeostasis mediated by PGC-la and its molecular regulatory network.The expression of PGC-la in HEK-293 cells was increased by using PGC-la over-expression of slow virus,and the expression of PGC-la in HEK-293 cells was reduced by siRNA.Results:1)The quantitative analysis of Western blot found that the PGC-la in muscle tissue of MELAS patients was significantly increased compared to the normal control group.However,compared with the normal control group,there was no significant difference in the content of autophagic related proteins LC3,P62,Beclinl,BNIP3,Parkin,and P nk.2)HE staining,MGT staining,and immunohistochemical staining were performed on MELAS muscle tissue specimens and PGC-1? was found to be significantly expressed in RRF.3)The autophagy pathway was suppressed in PGC-la over-expression HEK-293 cells,and there was no significant difference in autophagy pathway proteins in HEK-293 cells with siRNA interference.4)The mTOR pathway is activated in the PGC-la over-expressed HEK-293 cells.Conclusion:The expression of PGC-la increased in the muscle of patients carried m.3243A>Gmutation and PGC-1? accumulated in the abnormal mitochondria,which indicated that the abnonnal expression of PGC-la promoted excessive activation of mitochondria in the process of mitochondrial biosynthesis.However the autophagy protein marker was not significantly up-regulated in the muscle of these patients,suggesting that the abnormal mitochondria did not activate mitophagy,causing the accumulation of abnormal mitochondria in the cells of patients.In vitro experiments,the expression of various proteins associated with autophagy in the HEK-293 cells expressed in PGC-la was reduced,indicating that PGC-la inhibited mitochondrial autophagy.Meanwhile,mTOR pathway was activated in hek-293 cells overexpressed by PGC-la,suggesting that PGC-la inhibited mitochondrial autophagy by activating mTOR pathway. |
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