Therapeutic Effect And Mechanism Of ScAAV9-hIGF1 Retrograde Axonal Targeting Motor Neurons In ALS Mouse Model

Part one The theory and practice of sc AAV9-h IGF1 retrograde to motor neuronsObjective: AAV9 can retrograde axonal transport and pass the blood brain barrier to transfect neurons of brain and spinal cord. Self-complementary AAV(sc AAV) is more efficiency than the traditional single stranded AAV.To verify whether AAV9-GFP and sc AAV9-h IGF1 virus vectors(1 ×1012vg/ml) can retrograde axonal transport to motor neurons of adult ALS mice by intramuscular injection and continuously express the target proteins.And to establish sc AAV9 and neurotrophic factor based gene therapy for treatment of amyotrophic lateral sclerosis.Methods:1 Adult SOD1G93 A ALS mice(60d old) were intramuscular injected with AAV9-GFP virus vectors(bilateral gastrocnemius, 10ul/muscle, 1 × 1010vg/muscle). Three weeks later, the animals were sacrificed,the expression of green fluorescent protein(GFP) in muscle and spinal cord cryosections was observed directly under the fluorescent microscope.2 Adult SOD1G93 A ALS mice(60d old), randomly assigned for non intervention group(control group, n=15) and AAV9-GFP intervention group(10ul/muscle, i.e. 1 × 1010vg/muscle, masseter muscle, biceps, triceps,intercostal muscles, abdominal and femoral biceps muscle, quadriceps femoris,gastrocnemius muscle, bilateral,n=15). The onset and survival of the mice were observed daily. The expression of GFP protein in end-stage was detected by western blot.3 Adult SOD1G93 A ALS mice(60d old) were randomly intramuscular injected with sc AAV9-h IGF1 virus vectors(bilateral gastrocnemius,10ul/muscle, 1 × 1010vg/muscle). Three weeks later, anesthesia andparaformaldehyde perfusion, h IGF1 was observed in motor neurons of lumbar cord anterior horn by immunofluorescence.4 Adult SOD1G93 A ALS mice(60d old) were randomly intramuscular injected with sc AAV9-h IGF1 virus vectors(bilateral gastrocnemius, 1 ×1010vg/muscle). Three weeks later, the expression of h IGF1 was determined by enzyme-linked immunosorbent assay from the lumber spinal cord and gastrocnemius of mice.Results: 1 Intramuscular injection of AAV9-GFP(10ul/muscle). Three weeks later, frozen sections under a fluorescence microscope, green fluorescent protein(GFP) were visible in gastrocnemius, axons and motor neurons; 2 The onset and survival of AAV9-GFP treated mice and control mice had no significant difference, onset were 96 d, 97 d, respectively; survival were both 120 d. AAV9-GFP does not affect the onset and survival of the mice and no obvious adverse reactions. In end stage the expression of GFP was also observed by western blot; 3 Positive h IGF1 staining was observed at the lumber spinal cord by the cofocal microscophe; 4 The expression of h IGF1 was detected by ELISA, h IGF1 expression of lumbar spinal cord account for35% of the muscle. The transduction efficiency is 26%.Part Two Efficacy of intramuscular sc AAV9-h IGF1 delivery in ALS mouse modelObjective: To find whether intramuscularly injected sc AAV9 encoding human insulin-like growth factor-1 to the h SOD1G93 A mouse model of ALS can delay disease onset, prolong life span, and reduce the loss of motor neurons in the spinal cord of ALS mice.Methods:1 At 60 d and 90 d of age, littermate and copy number-matched mice were randomly divided into sc AAV9-h IGF1 and AAV9-GFP groups. Mice were bilaterally intramuscular injected with vectors by using a Hamilton syringe(masseter, biceps, triceps, intercostal muscle, abdominal muscle,gastrocnemius, biceps femoris muscle, quadriceps, 10μl per muscle, n=15).2 The motor function was assessed weekly by using a rotarod device. Thedate of disease onset was recorded when the mice could not stay on the rod for180 s. Mortality was scored as the age at which the mouse was unable to right itself within 30 s when placed on its back in a supine position. Footprint analyses were performed at 110 d of age. The hind feet were dipped into black finger paint and the front feet were dipped into red finger paint, animals placed on a gangway.3 Excised muscles were rapidly frozen in liquid nitrogen-cooled isopentane. For general morphology, cryostat muscle sections were stained with hematoxylin and eosin(HE) according to standard protocols. And NADH staining and Gomori’s modified trichrome method were used for assessing muscle fibrosis.4 For DAB staining, spinal cord sections with the primary antibodies:rabbit anti-Iba1, mouse anti-SMI32, mouse anti-GFAP. The numbers of SMI32-positive(Motor neurons), GFAP-positive(astrocyte) and Iba1-positive(microglial) cells in the ventral horn area were estimated using Image J software.Results: 1 SOD1G93 A ALS mice usually develop a progressive neurodegenerative disorder, resulting in various degrees of weakness and atrophy of limb musculature at around 90 d of age. Sc AAV9-h IGF1 mice could hold its hind limbs outward at 110 d of age, while AAV9-GFP mice did not hold its hind limbs outward; 2 Footprint showed significant impairment in walking patterns of AAV9-GFP injected mice at 110 d of age. Moreover, we tested body weight and motor function of the mice during disease progression.It showed sc AAV9-h IGF1 mice exhibited better motor function and slowed down the weight loss compared with the age-matched AAV9-GFP mice; 3 We found that disease onset and end stage were significantly delayed in ALS mice treated with sc AAV9-h IGF1 compared to AAV9-GFP treated mice. 60 d treatment male delayed disease onset 24 d, 60 d treatment female delayed disease onset 18 d. 60 d treatment male extend life span 29 d, 60 d treatment female extend life span 24 d, 90 d treatment male extend life span 15 d, 90 d treatment female extend life span 14d; 4 Morphological analysis of muscleswith HE staining showed typical features of muscle degeneration that included atrophic fibers in AAV9-GFP mice. NADH staining showed grouped type I or type II myofibers in the two group mice. MGT staining showed the number of mitochondria in sc AAV9-h IGF1 mice was more than AAV9-GFP mice in the gastrocnemius; 5 Statistic analysis showed that there was an obviously reducing the lost of SMI32-positive neurons in sc AAV9-h IGF1 mice compared with AAV9-GFP mice. IHC showed that there were signi?cantly fewer GFAP-positive glial cells in the lumbar spinal cords of sc AAV9-h IGF1 treated ALS mice compared with the GFP-treated mice. IHC also showed that sc AAV9-h IGF1 treatment signi?cantly reduced Iba1 levels in the lumbar spinal cords of ALS mice at 110 d of age.Part three The protection mechanism of intramuscular injection of sc AAV9-h IGF1 in h SOD1G93 A ALS mouse modelObjective: We found that intramuscular delivery of sc AAV9-h IGF1 remarkably slowed the onset of disease progression and increased survival compare to AAV9-GFP treated mice. However, the protection mechanism is still unclear. It has been reported that IGF1 protects motor neurons through an antiapoptotic and attenuates glial cell-mediated release of tumor necrosis factor-α(TNF-α) and nitric oxide(NO). In this study, we further explore the protection mechanism of intramuscular injection of sc AAV9-h IGF1 in h SOD1G93 A ALS mouse model.Methods:1 Littermates and copy number matched SOD1G93 A mice were randomly divided into sc AAV9-h IGF1 and AAV9-GFP group, 80 d old mice,intramuscular injection bilateral masseter muscle, biceps, triceps, intercostal muscles, abdominal muscles, biceps femoris muscle, quadriceps femoris,gastrocnemius muscle, 10ul/muscle, 14 days after intervention, rapid extracting RNA of lumbar spinal cord, using gene chip to find the different genes.2 Littermates and copy number matched SOD1G93 A mice were randomly divided into sc AAV9-h IGF1 group and AAV9-GFP group, 60 d or 90 d old,intramuscular injection into bilateral masseter, biceps, triceps, intercostal muscles, abdominal muscle, biceps femoris muscle, quadriceps femoris,gastrocnemius, 10ul/muscle, used real-time quantitative PCR to verify the genes, western blot and immunofluorescence to quantitate and positioning the protein at 110 d.3 The anti-apoptotic mechanism of IGF1 was confirmed by immunofluorescence and western blot, including TUNEL, cleaved-caspase 3and 9, BAX and Bcl-2.4 Using of CRISPR-Cas9 technology and target knockdown the central nervous system IGF1 gene, 30 d old SOD1G93 A ALS mice were randomly intrathecal injection of sc AAV9-sg RNA(Lac Z)-cas9 and sc AAV9-sg RNA(IGF1)-cas9. Using real-time PCR, western blotting and immunohistochemistry and immunofluorescence technology further verify selected genes.Results: 1 It was found that DAO m RNA expression was upregulated in sc AAV9-h IGF1 treated mice. Then, we performed real-time PCR to test and verify the result. In addition, we tested the DAO protein though western blot,we found that DAO protein in sc AAV9-h IGF1 mice was increased compared with AAV9-GFP mice. For identification of the type of cell of DAO location in the spinal cord, transverse sections of the lambar spinal cord were colabeled with antibodies directed against DAO and either Neu N, a neuron-specific nuclear protein or GFAP, a marker of astrocytes; Iba1, a marker of microglia.Fluorescence microscope showed that DAO located mainly in neuronal cells;2 It found that D-serine was reduced in sc AAV9-h IGF1 treated mice.AAV9-GFP treated mice exhibited large numbers of cells that were positive for TUNEL and sc AAV9-h IGF1 treated mice had little TUNEL reactivity at110 d of age. In addition, IF showed that cleaved-caspase3 was also decreased in sc AAV9-h IGF1 treated mice compared to the AAV9-GFP mice. We also found that sc AAV9-h IGF1 treated mice had higher levels of phosphorylated Akt and significantly reduced the amount of cleaved-caspase9 and cleaved-caspase3 when compared to AAV-GFP mice. In our study, proteinlevels of Bcl-2 were significantly upregulated in sc AAV9-h IGF1 treated mice,while the expression of BAX was downregulated markedly; 3 IGF-1deficiency mice could not stretch its hind limbs, and muscle atrophy was more serious at 90 d of age. IHC, IF and WB showed that the expression of DAO was down-regulated in IGF-1 knockdown group. At the same time, the D-serine level was up-regulated in IGF-1 knockdown mice. We also tested the expression of cleaved-caspase3 and cleaved-caspase9 raised in IGF-1knockdown group compare to Lac Z group.Conclusions:1 We have showed that a single i.m. injection of sc AAV9-h IGF1 vectors allowed widespread gene transfer to CNS in h SOD1G93 A ALS adult mice;2 Sc AAV9-h IGF1 could improve motor function and delay muscle atrophy, reduce the glial cell reaction and protect motor neurons, delay onset,extend survival by intramuscular injection in SOD1G93 AALS mouse model;3 Sc AAV9-h IGF1 protected motor neurons from apoptosis by upregulating of DAO.