| Amyotrophic lateral sclerosis(ALS)is the most common type of adult motor neuron disease,first described by Charcot in 1869.The difference between ALS and other motor neuron diseases is that the degeneration occurs both in the lower and upper motor neurons.The main neuropathological features of ALS include extensive loss of spinal anterior horn and motor neurons in the brain stem,cortical spinal cord degeneration and the degeneration and loss of the large motor pyramidal neurons in primary motor cortex.The prevalence of ALS is about 5-7/100,000,most cases of ALS are sporadic ALS(sALS),and 5-10%are familial ALS(fALS).Whether the patient is sporadic or familial,progressive muscle weakness and muscle atrophy can occur.Progressive muscle weakness can begin at the proximal or distal extremities of the upper or lower extremities and may involve all muscles,including those associated with breathing,speech,and swallowing.Most of the patients died of respiratory failure after they are diagnosed 2-5 years later.In the early stages of ALS,the different modes of involvement of lower and upper motor neurons make the diagnosis of ALS challenging.Because the initial symptoms of ALS are similar to other spinal disorders,neuropathies,and neurological syndromes,diagnostic delay is one of the major problems.Because of the absence of specific disease markers,the establishment of clinical diagnostic criteria for ALS is based on the presence and distribution of lower and upper motor neurons lesions and requires the use of exclusion procedures to confirm the diagnosis.Laboratory tests,neuroimaging,and muscle biopsy may all help in the differential diagnosis and elimination of the disesases which mimick ALS.Once other disease processes are excluded,electrodiagnostic evaluation is critical to establishing definitive diagnosis of ALS.Moreover,the electrodiagnostic evaluation is useful not only in the diagnosis of ALS,but also in monitoring the progression of the disease and the response to the intervention and in determining the prognosis.Therefore,currently the diagnosis of ALS relies mainly on the combination of clinical manifestations and neurophysiological examinations.The most common mutations identified in ALS are the SOD1,the RNA-binding protein-encoding genes TARDBP,FUS,and the most recently recognized C9orf72 gene.In 2011,the pathogenic repeated amplification of GGGGCC,a noncoding region of the C9orf72 gene,was discovered and reported in patients with ALS and frontotemporal dementia.GGGGCC amplification in the C9orf72 gene was recognized by the studies of risk haplotypes at the 9p21 locus.Repeated primer PCR was used for sequencing,and it was concluded that repeat amplification of more than 30 was associated with ALS.Compared with non-C9orf72 ALS cases,C9orf72-related ALS has its clinical characteristics.The proportion of C9orf72 ALS accompanied by frontotemporal dementia was significantly higher.C9orf72 ALS is prone to cognitive dysfunction and mental behavior abnormalities,with increasing proportion of the medulla oblongata segment as the initial symptoms,the incidence of age in advance of 1.8-5.0 years,worsening motor symptoms,and the course of shortening 5.7-12.0 months,which shows its stronger disease process.Furthermore,C9orf72 disease spectrum also includes primary lateral sclerosis,progressive muscular atrophy,Parkinsonism/Parkinson’s disease,Huntington’s disease,Alzheimer’s disease,and so on.In view of the large number of patients carrying C9orf72 gene mutations,the extensive spectrum of C9orf72-related diseases and the multifaceted and complex ALS mediated by C9orf72,the C7orf72 gene is becoming a research hotspot in recent years.Nuclear factor kappa B(NF-κB)is an inducible transcription factor in lymphocytes that affects almost all cell types in the body and plays an important role in inflammation,immune response,cell cycle and cell survival.In the central nervous system,NF-κB transcription factor is a key participant in many physiological processes,such as neurogenesis,neurite formation,and synaptic plasticity.NF-κB is constitutively active,involved in neuronal damage and neuroprotection.NF-κB in synapses is present in a latent form and can only be transported to neuronal nuclei when it is activated.Immunohistochemistry confirmed that NF-κB was activated in the glial of the familial and sporadic ALS patients.Studies had shown that SOD1-G93A microglia in NF-κB-dependent mechanism in vivo and in vitro would induce the death of motor neuron.Part I Clinical and electrophysiological characteristics of amyotrophic lateral sclerosisObjectives:By analyzing the clinical and electrophysiological data of ALS,the clinician will be provided with the intuitive and in-depth understanding of the disease.Thus patients can get early diagnosis as soon as possible in order to obtain the correct treatment so that the life span of patients can be prolonged and the quality of life can be improved.Methods:We collected 73 patients from January 2009 to June 2016 in our hospital,which accorded with the diagnostic standard of revised El Escorial.The diagnostic grades were definite and probable diagnosis clinically.The patients’ ages,sites of onset,sites of involvement and main symptoms and signs were collected.Motor conduction velocity(MCV),sensory conduction velocity(SCV),repetitive nerve stimulation(RNS)and needle electrode electromyography(EMG)were measured.The clinical and electrophysiological characteristics of ALS were summarized and analyzed retrospectively.Results:A total of 73 patients was collected,including 42 males(57.5%),31 females(42.5%),and the ratio was 1.4:1.The mean age of onset was(51.7 ± 12.4)years old,and the peak age was 51-60 years old.The highest rate of onset site was cervical segment(56.2%),followed by lumbosacral segment(24.6%)and medullary segment(19.2%).Among the symptoms and signs of lower motor neuron,muscle weakness(97.3%)and muscle atrophy(76.7%)were the most common.The total abnormal rate of motor nerve conduction was 45.9%,but the mean value of the distal latency,amplitude and conduction velocity of the motor nerves was in the normal range.The ratio of the amplitude of the compound muscle action potential(CMAP)and the cutoff value of the median nerve was(1.06 ± 0.74),and that of the ulnar nerve was(1.49 ± 0.81).The ulnar nerve was greater than the median nerve(P=0.000).The total abnormal rate of sensory nerve conduction was 2.3%,but the mean value of the amplitude and conduction velocity of the sensory nerves was in the normal range.The mean decremental CMAP amplitudes of abductor pollicis brevis(dominated by median nerve)and abductor digiti minimi(dominated by ulnar nerve)were(9.07%±8.68%)and(2.89%± 2.47%),respectively,and the decremental amplitude of abductor pollicis brevis was significantly larger than that of abductor digiti minimi(P= 0.000).In the median nerve,the CMAP decrement was negatively correlated with the amplitude of CMAP(R=-0.357,P = 0.000),while in the ulnar nerve,no correlation was observed(P= 0.223).The needle EMG showed that the incidence of the spontaneous potentials,fibrillation potentials/positive sharp waves and fasciculation potentials of the tongue muscles(79.1%,65.1%and 58.1%,respectively)were significantly higher than that of the sternocleidomastoid muscles(48.8%,30.2%and 32.6%)(P=0.004,0.001 and 0.017,respectively).The frequency of the spontaneous potentials of the tongue muscles in patients with bulbar symptoms and limb-only symptoms were 84.2%and 75.0%,respectively.There was no statistically significant differencebetween the two groups(P = 0.708).Conclusions:ALS is more common in men than in women.It occurs in the elderly,and the peak incidence is of 51-60 years of age.The onset of cervical segment is the most common,followed by lumbosacral and medullary segment.In symptoms of lower motor neurons,muscle weakness is the most frequent,followed by muscle atrophy.Some patients will appear abnormal motor conductions,among which decreased amplitude of CMAP is the most common.Abnormal sensory conductions are rare,indicating that the disease has little effect on the sensory conduction pathway;if abnormal,it can not completely rule out ALS,and should be noted to exclude other peripheral neuropathies.Our study shows that the decremental motor response to RNS differs between the median and ulnar nerves.The greater CMAP decrement in the median nerve was attributed to preferential involvement of the abductor pollicis brevis in the pathophysiology of ALS.Decremental CMAP may be caused by neuronal conduction instability in newly sprouted nerve terminals.In assessing the clinical and subclinical involvement of the tongue muscle,the EMG of the tongue needle electrode EMG is a valuable electrophysiological method,regardless of whether it is a bulbar onset or a limb-onset ALS,but we should pay attention to the method of operation.Part II Biological function analysis of the cell model and mechanism of mutant gene C9orf72 in ALSObjectives:The purpose of this study was to construct C9orf72-induced ALS cell model and to analyze its biological function;to detect whether C9orf72 leads to neuronal damage through the NF-κB signaling pathway.Methods:The mutant gene of C9orf72 was obtained in vitro by chemical synthesis method.Then the eukaryotic expression vector was constructed and transfected into NSC-34 cells by cationic liposome transfection.The transcriptome and proteome were examined using RT-PCR and Western blots,respectively,to confirm that the cell model was successfully constructed.Using immunocytochemical methods,the morphology of neuronal cells was examined.Cell oxidative stress was detected using the MDA method.NF-κB signal pathway activity was detected in the experimental group and the control group by RT-PCR and Western blots.SiRNA-NF-KB was transfected into NSC-34 cells.The molecular changes of NF-κB signaling pathways in the experimental group and control group were studied by using Western blots.According to the changes of these protein molecules,the molecules mechanisms of C9orf72-induced ALS were elucidated.Results:A successful cell model of transfection with pcDNA3.1(+)-HCATCATC ACCATCACCAT)(GGGGCC)30 was obtained.Immunocytochemistry showed that compared with the control group,after transfection of pcDNA3.1(+)-(CATCATCACCATCACCAT)(GGGGCC)30,the cell body became rounded and the process of axon was shortened.MDA results showed that the transfection group,the normal group and the empty group were(2.92±0.14)nmol/mgprot,(1.63±0.12)nmol/mgprot and(1.82±0.21)nmol/mgprot,respectively.The content of MDA in the cells of the transfection group was significantly higher than that in the other two groups(P<0.05).The normal cells and the pcDNA3.1(+)group used as the control group,after transfection of pcDNA3.1(+)-(CATCATCACCATCACCAT)(GGGGCC)for 24 hours,the expression of p-NF-KB protein in the transfected group was significantly higher than that in the control group(P<0.05).The expression of NF-κB in siRNA-NF-KB transfection group was significantly inhibited 24 hours after siRNA-NF-κB transfection compared with siRNA transfection group(P<0.05).However,the protein expression of C9orf72 did not show changes.The content of MDA in normal and empty vector group,pcDNA3.1(+)-CATCATCACCATCACCAT(GGGGCC)30 transfected group and siRNA-NF-kB transfected group were(1.72±0.22)nmol/mgprot,(1.86±0.37)nmol/mgprot,(3.12±0.28)nmol/mgprot,(1.75±0.18)nmol/mgprot,respectively.The statistical analysis showed that the content of MDA in cells transfected with pcDNA3.1(+)-(CATCATCACCATCACCAT)(GGGGCC)30 was significantly higher than that of the other three groups(P<0.05).Compared with normal group and empty vector group,MDA content of siRNA-NF-kB transfection group had no significant difference(P>0.05).Conclusions:C9orf72-inducted ALS cell model was successfully constructed.The 30 repetitive GGGGCC in C9orfp2 gene has toxic damage to neuron-like cells.C9orf72 affects neuronal damage through the downstream NF-κB signaling pathway. |
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