The Protective Effect To MN And Mechanism Of ScAAV9-hIGF1 In ALS Mice Model Via Intrathecal Injection

Amyotrophic lateral sclerosis(ALS) is a chronic, progressive, fatal neurodegenerative disease. The incidence rate is about 5/100 thousand to 7/100 thousand, and about 90% of patients with sporadic amyotrophic lateral sclerosis(SALS), the others with familial amyotrophic lateral sclerosis(FALS). Clinical manifestations and pathological characteristics of two kinds of patients are similar, which patients reveal symptoms of disease, progressive muscle weakness and atrophy, would eventually died of respiratory failure after 3 to 5 years. There is no effective treatment so far.The etiology of SALS is not clear. 20% patients of FALS were confirmed due to mutations in the Cu/Zn superoxide dismutase 1 gene. G93A-SOD1 transgenic mice(ALS mice) develop the clinical characteristics of human ALS disease and are used as a tool in the study of ALS pathology. High copy number transgenic G93A-SOD1 mice appear hind legs symptoms of motor impairment at about 12 weeks of age and gradually progress, die about 17-20 weeks age. It is considered that the mutant SOD1 protein is toxic due to its misfolding, easy to form aggregates, and resistance to the degradation of short life protein via the ubiquitin proteasome system, and cause ALS disease.Autophagy is a highly conserved cell biological behavior, which can eliminate the long life protein, the misfolding protein and the aging damage orgenelle.In the central nervous system, autophagy is an important way to clear the misfolding proteins in order to maintain the normal physiological function of neurons. Therefore, it is significant to study the effective degradation of mutant SOD1 protein in the treatment of ALS disease.Insulin like growth factor-1 is a similar to insulin in molecular structure, containing 70 amino acids, relative molecular weight of 7 649, produced by endocrine, autocrine and paracrine. IGF-1 is involved in a variety of important physiological functions in the body. One of its important role is to recover the damage of nerve. Application of adeno associated virus serum type 2 carrying human single chain IGF1 gene treats G93A-SOD1 transgenic mice via intramuscular injection through neural inverse axis axoplasmic transport,significantly delaying onset, prolonging the survival time. The study found that IGF1 could activate the autophagy pathway and inhibit apoptosis motor neurons so as to protect the motor neurons in ALS mice. This study did not reported the changes of mutant SOD1 protein levels. We hypothesized that autophagy activation may be involved in down-ragulating pathogenic mutant SOD1 protein levels and reduce the toxicity to motor neurons.We apply the neurotrophic adeno-associated virus serum type 9(AAV9)carrying encoding double stranded human IGF1 gene to treat G93A-SOD1 transgenic mice via intrathecal injection(i.t.) in order to observe the behavior changes and to explore the expressing h IGF1 to affect the expression of mutant SOD1 protein and pathological changes in ALS mice. We also apply CRISPR-Cas9 system to verify. Sc AAV9-sg RNA-IGF1-Cas9 are administrated to G93A-SOD1 transgenic female mice via i.t. to knock down the m IGF1 and to research the expression of mutant SOD1 protein and pathological changes showing the opposite direction or not. Similar report has little seen in the domestic and foreign literature.Part one The effects of behaviors and mutant SOD1 protein in ALS mice via i.t. sc AAV9-h IGF1Objective: We administrated the neuropathic AAV9 carrying encoding double stranded human IGF1 gene in G93A-SOD1 transgenic female mice(60 days of age, 90 days of age) by i.t. to observe the behavior changes of ALS mice, to further research the effects of mutant SOD1 protein expression and pathology after expressing h IGF1 in ALS mice.Methods: We selected G93A-SOD1 transgenic mice as animal model of the ALS, and used the sequences of primers and identification method of G93A-SOD1 transgenic mice via PCR, which provided by the Jackson Laboratory. G93A-SODl positive transgenic female littermates mice were paired as test objects. The G93A-SOD1 transgenic positive female mice(60days of age and 90 days of age) were randomly assigned to the drug treatment group(sc AAV9-h IGF1) via i.t(n=15), the control group(AAV9-GFP)(n=15).In addition to selecting 10 pairs of G93A-SOD1 transgenic female littermate mice(60 day of age)were administrated randomly sc AAV9-h IGF1 or AAV9-GFP via i.t.. Ones of 3 pairs were collected fresh samples, extracted the protein and observated distribution characteristics of expression of h IGF-1by ELISA after i.t. 3 weeks. The lumbar spinal cord oscillation section were observed the expression of h IGF1 by double immunofluorescent staining with4% paraformaldehyde perfusion fixed. 3 pairs of them at the age of 110 days old were observed the pathological changes of lumbar spinal cord by immunohistochemistry and immunofluorescence methods with 4%paraformaldehyde perfusion fixed. 3 pairs of them at the age of 110 days old were used as collecting fresh samples and observed the expression levels of GFAP, CD11 b, SOD1, LC3, P62 through western blot and m RNA transcription levels of SOD1 by RT-PCR.Results: 1 All the female mice in the experiment were verified G93A-SOD1 positive by DNA identification. 2 G93A-SOD1 transgenic mice treated with sc AAV9-h IGF1 or AAV9-GFP were collected fresh samples after i.t. 3 weeks tissue protein extraction, testing by enzyme linked immunosorbent assay. The results show that expression of h IGF1 is in nervous tissue, and concentration of h IGF1 decreases from the lumbar spinal cord, thoracic spinal cord, cervical spinal cord to brain stem. HIGF1 concentration is the highest in the lumbar spinal cord and is the minimum in brain stem. HIGF1 mainly expressed in motor neurons by double immunofluorescent staining transverse sections of the lumbar spinal cord after 4% paraformaldehyde perfusion fixation. 3 G93A-SOD1 transgenic mice treated with sc AAV9-h IGF1 improve the motor function of mice. The median of the onset at 60 days of age treatment compared to control groups delays 20 days. The median of the survival time at 60 days of age treatment group compared to control groups prolongs 44 days. The median of the survival time at 90 days of age treatment group prolongs 38 days compared to control group. It is extremely significant difference compared to the treatment group and the control group by statistical analysis(P﹤0.01). 4 For expressing h IGF1 in G93A-SOD1 transgenic mice,lumbar motor neurons in treatment group spinal cord sections significantly loss less compared with the control group at 110 days of age by immunofluorescence double staining. The hyperplasia of astrocytes and microglial is significantly less in the treatment group compared to the control group. The results of western blot showed that the expression levels of GFAP and CD11 b in the treatment group were significantly lower than those in the control group(P﹤0.05). 5 Expressing h IGF1 in G93A-SOD1 transgenic mice,the results of western blot showed that the expression levels of mutant SOD1 protein and p62 protein in the treatment group are significantly lower than that in the control group at 110 days of age(P ﹤ 0.05). Expression levels of LC3-Ⅱprotein in the treatment group is higher than the control group(P﹤0.05). The lumbar enlargement is homogenized with ultrasound and extracted total RNA using RNA kit for RT-PCR reverse transcription. SOD1 m RNA transcriptional levels had no difference between treatment group and control group(P﹥0.05).Part two To research the protective effect on MN expressing h IGF1 in ALS miceObjective: We administrated sc AAV9-h IGF1 into 60 days of age G93A-SOD1 transgenic female mice via i.t., and studied expression of h IHG1 how to inhibit the proliferation of microglia in playing a protection of motor neurons.Methods: We selected G93A-SOD1 transgenic mice as animal model of the ALS, and used the sequences of primers and identification method of G93A-SOD1 transgenic mice via PCR provided by the Jackson Laboratory. 6 pairs of G93A-SODl positive transgenic female littermates mice were paired as test subjects. 60 day old G93A-SOD1 transgenic female mice randomly assigned to sc AAV9-h IGF1 treatment group and the AAV9-GFP treatment group via i.t.. 3 pairs of ALS mice were given 4% poly formaldehyde solution perfusion fixation at 110 days of age. Expression and distribution of TGF-β,arginase, IKBα, IKKγ, were observed in transverse sections of lumbar spical cord by double-immunofluorescence. The other 3 pairs of ALS mice were collected fresh lumbar spinal cord samples by Western blot analyzing expression of i NOS, TNF-α, TGF-β, arginase, CYLD, IKBα, IKKβ, IKKγ,PP65 at 110 days old.Results: 1 Expressing h IGF1 in G93A-SOD1 transgenic mice, doubleimmunofluorescence staining showed arginase and TGF-β mainly express in neurons, and the fluorescence intensity of treated group is higher than that in the control group at 110 days of age lumbar spinal cord sections. The results of western blot showed that protein levels of arginase, TGF-β were significantly higher in treatment group than those in the control group(P<0.05). 2 Expressing h IGF1 in G93A-SOD1 transgenic mice, the results of western blot showed that protein levels of TNF-α, i NOS were significantly lower in treatment group than those in the control group at 110 days old(P<0.05). 3 Expressing h IGF1 in G93A-SOD1 transgenic mice, double immunofluorescence staining showed that IKBα expressed in neurons and microglias of lumbar spinal cord sections, and the bright fluorescence intensity of the treated group compared to control group. Double-immunofluorescence staining showed that IKKγ was mainly expressed in microglias of lumbar spinal cord sections, the dim fluorescence intensity of the treated group compared to control group at 110 days of age. Western blot gray comparison results showed that expression levels of IKKβ, IKKγ, PP65 in the treated group were significantly lower than those in the control group(P<0.05).Expression of CYLD, IKBα in treatment group were significantly higher than those in the control group(P<0.05).with sc AAV9-sg RNA-IGF1-Cas9 via i.t.Part three Researching effect and mechanism of ALS mice treatedObjective: We administrated G93A-SOD1 transgenic female mice sc AAV9-sg RNA-IGF1-cas9 applying CRISPR-Cas9 system via i.t. to observe the behavior changes and to further study expression of mutant SOD1 protein and NF-κB signaling pathway molecules after knock-down G93A-SOD1 transgenic female mice IGF1 m RNA.Methods: We selected G93A-SOD1 transgenic mice as animal model of the ALS, and used the sequences of primers and identification method of G93A-SOD1 transgenic mice via PCR, which provided by the Jackson Laboratory. G93A-SODl positive transgenic female littermates mice were paired as study subjects. 13 pairs of G93A-SOD1 transgenic littermate female mice at 30 days old were randomly assigned to treat with sc AAV9-sg RNA-IGF1-Cas9 or sc AAV9-sg RNA-lacz-Cas9 via i.t.. 7 pairs of ALS mice were used to observe the changes of behavioral, onset, and survival time. 3 pairs of ALS mice were given 4% poly formaldehyde solution perfusion fixation at 90 days old to observe pathology by immunofluorescence staining IKKβ, PP65 in sections of lumbar spinal. The other 3 pairs of ALS mice were collected fresh lumbar spinal cord samples by Western blot analyzing expression of i NOS, TNF-α, TGF-β, arginase, IGF1, CYLD,IKBα, IKKβ, IKKγ, PP65 at 90 days old.Results: 1 Knockdown G93A-SOD1 transgenic mice IGF1, body weight and score were lower in the treatment group than those in the control group from 70 days of age. Rotarod test showed more poor in the treatment group than in the control group from 80 days of age. Motor function damage occurred in the treatment group earlier. The median of treated group onset shortened 10 days compared to the control group. The median of treatment group survival reduced 16 days compared to the control group. It is much significant difference between groups by statistical analysis(P<0.01). 2Knockdown G93A-SOD1 transgenic mice IGF1, lumbar spinal cord sections were stained SMI32, GFAP and Iba-1 by immunohistochemistry at 90 days old mice. Results showed that motor neuron loss of treatment group compared with the control group were significantly more, and the hyperplasia of astrocytes and microglia in treatment group was significantly more than that in control group. 3 Knockdown G93A-SOD1 transgenic mice IGF1, Western blot results showed that expression levels of mutant SOD1 protein in treatment group were significantly higher than that in the control group at 90 days old(P<0.05). Expression level of m IGF1 and LC3B-II protein in treatment group was significantly lower than that in control group(P<0.05). 4 Knockdown G93A-SOD1 transgenic mice IGF1, the results of western blot showed that the expression levels of arginase, TGF-β, m IGF1 in treatment group were significantly lower than those in the control group at 90 days of age(P<0.05).Double immunofluorescence staining showed Arginase, TGF-β had similar trend. 5 Knockdown of G93A-SOD1 transgenic mice IGF1, the results of western blot showed that expression levels of i NOS, TNF-α protein in treated group were significantly higher than those in the control group at 90 day old(P<0.05). 6 Knockdown G93A-SOD1 transgenic mice IGF1, lumbar spinal cord sections were stained IKKβ, PP65 by double-immunofluorescence at 90 days of age.The results showed that PP65 mainly distributed in microglia, in the treatment group with strong fluorescence intensity than that of the control group. IKKβ distributed in microglia and neurons, showing dim fluorescence in the treatment group than that of the control group. The results of western blotting showed that expression levels of NF-κB signaling pathway positive regulator IKKβ, IKKγ, PP65 in treatment group were significantly higher than those of control group(P<0.05), expression levels of NF-κBsignaling pathway negative regulator factor CYLD and IKBα in treatment group of were significantly lower than those in the control group(P < 0.05).Conclusions:1 By consulting literature and comprehensively analysing experimental results, we believe that such a correlation exists among mutant SOD1 protein,inflammatory factor of i NOS, TNF-α, NF-κB pathway and IGF1, namely expression of h IGF1 in ALS mice promotes the degradation of mutant SOD1 protein and reduces the toxicity of motor neurons, i NOS and TNF-α showing lower expression, inhibiting the proliferation of microglias, down-regulating the expression of NF-κB signaling pathway factor, to further reducing the damage of motor neuron, delaying onset and prolonging survival time of ALS mice.2 Instead, knocking-down IGF1 in ALS mice makes mutant SOD1 degradation to be blocked, causing the expression of mutant SOD1 protein accumulation and serious toxicity, aggravating the damage of motor neurons,showing i NOS, TNF-α high expression and remarkable hyperplasia of microglias, up-regulating the expression of NF-κB signaling pathway factor,further damaging the motor neurons, leading to onset occuring early and survival time shortening in clinical manifestations.3 Based on the above research results, we conclude that the mutant SOD1 protein toxicity causes motor neuron inflammatory responses, triggers upregulation of inflammatory factor, stimulates microglial cells proliferation,leading to inflammatory cascade. They all involve in the occurrence of ALS.However, the signaling pathway remains to be further studied.4 Expression of h IGF1 promotes the mutant SOD1 protein degradation via gene therapy in ALS mice, inhibites the above pathological progress and exerts the satisfactory therapeutic effect. It targets pathogeny of ALS mouse and provides experimental basis for the treatment of clinical ALS diseases.The experiments also show that blocking each link of inflammatory reaction process is a target for treatment of ALS.