| BackgroundMultiple sclerosis (MS) is one of the most common demyelinating diseases of the central nervous system (CNS), which often affects young adults aged 20 to 40. In MS, cognitive impairment is the common concomitant, with prevalence rates ranging from 43% to 70%.Fllow-up studies have found that the cognitive dysfunction of MS becomes more and more serious with the time. Cognitive impairment detrimentally affects many aspects of daily life in MS patient populations, including learning and working. Despite the high incidence of cognitive impairment in MS patients, the data indicate that most of the pharmacological symptomatic treatments for MS have no cognitive benefits, and there is no effective treatment aimed at recovering the cognitive impairment. Autopsy studies and imaging studies have observed myelin impairment in the brain regions involved in cognition, and imaging studies also find that there is a close relationship between myelin impairment and cognitive decline in MS. However, whether myelin impairment can be the target of treatment to cognitive dysfunction in MS is still unclear.LINGO-1 (Leucine rich repeat and Ig domain containing NOGO receptor interacting protein 1) is an important transmembrane protein that is specifically expressed in oligodendrocytes and neurons in the CNS; it is a key inhibitor of oligodendrocyte precursor cells (OPCs) differentiation and myelination. Attenuation of LINGO-1 function with the LINGO-1 antibody facilitates OPCs differentiation and myelination. However, whether the LINGO-1 antibody could effectively restore the cognitive impairment in MS is still unknown.In this research, we observed the behaviors of two common animal models of MS(experimental autoimmune encephalomyelitis mice and cuprizone-fed mice) and used the western blot and immunofluorescence to detecte the levels of myelin associated proteins and key structural and functional proteins in neuronal axons. The purposes of this research includes: 1. To detecte the behaviors of the MS models and find whether demyelination is in the brain associated with cognition; 2. To find whether LINGO-1 antibody can improve the cognition of the MS models and recover the pathological damage. It could provide the evidence to demonstrate that LINGO-1 antagonism may be an effective approach to the treatment of the cognitive impairment in MS patients.Part 1 Establish and optimize the model of experimental autoimmune encephalomyelitis in miceObjective: To find the appropriate dosage of drugs inducing experimental autoimmune encephalomyelitis (EAE), establish and optimize EAE mice, which can be used for cognitive behavioral research.Methods: Different doses of myelin oligodendrocyte glycoprotein (MOG35-55), together with different doses of inactivation of mycobacterium tuberculosis (H37RA) and pertussis toxin, were used to immunize the mice and induced the EAE model. After immunized, the clinical disease severity of EAE mice was measured using the standard EAE grading clinical score daily. The open field test was used to detect the locomotion of mice.Results: The model of EAE was successfully induced with different doses of MOG35-55, H37RA and pertussis toxin. Compared with the EAE mice induced with high-dose of drugs, the mice with low-dose of drugs (25 μg MOG35-55 per mouse, 100 μg H37RA per mouse, 200 ng pertussis toxin per mouse) inducing had low clinical score. And they displayed normal locomotion compared with the control mice [day 16,8.885 ±0.772 cm/s (EAE) VS 8.933 ± 0.567 cm/s(control),P>0.05; day 31, 11.130±0.630 cm/s (EAE) VS 10.670±0.959 cm/s (control),P>0.05;day 55, 7.686±0.428 cm/s (EAE) VS 8.313±0.918 cm/s (control),P>0.05].Conclusion: The EAE mice induced with low-dose of drugs have low clinical score and normal locomotion. And they could be used in the cognitive behavioral research of demyelination disease,such as multiple sclerosis.Part 2 The behavior and pathology of experimental allergic encephalomyelitis in miceObjective: to investigate whether experimental allergic encephalomyelitis (EAE) mice have the spatial learning and memory disorders, and depression and anxiety-like behaviors; to explore whether there is myelin injury in brain related to cognitive function in EAE mice.Methods: 25 μg MOG35-55 per mouse, 100 μg H37RA per mouse, and 200 ng pertussis toxin per mouse were used to induce EAE mice; After immunized, the elevated plus maze (EPM), open field test, sucrose preference test and Morris water maze test (MWT) were tested in both the early(day 13) and late (day 55) stages of EAE; After one batch of the behavior tests, the EAE mice and age-matched controls were killed; The brains were dissected from the skulls, and six different brain areas, including the prefrontal cortex, cingulate, caudate putamen (striatum), fimbria-fornix,dorsal hippocampus and parahippocampal cortex (PHC), were dissected and the level of the myelin basic protein (MBP) was detected by western blot.Results: The results showed that the EAE mice have no anxiety and depression-like behavior in the two periods (P>0.05). It also displayed no significantly cognitive decline in the EAE mice in the early stage (P>0.05). However, in the late stage, the performance of the EAE mice in MWT was significantly worse compared with the control animals. The latency to the hidden platform in the EAE mice was longer than in the control mice, especially in the first day (P<0.05).Furthermore, on the detecting day, a defect in the spatial memory test is observed in the EAE mice,and the percentage of the distance in the target quadrant is significantly shorter than that of controls (P<0.05). The results of western blot showed that there was a significant decrease of MBP in the PHC and fimbria-fornix of EAE mice (P<0.05).Conclusion: Deficits in learning and memory of EAE mice occurred in the late stages of the disease rather than in the early stages. During the course of the disease, the EAE mice did not display anxiety and depression-like behaviors. And in EAE mice, demyelination was detected in the PHC and fimbria-fornix, which are involved in memory acquisition and maintenance. It infers that myelin damage in the PHC and fimbria-fornix may be the cause of the learning and memory disorders in EAE mice.Part 3 LINGO-1 antibody ameliorates myelin impairment and spatial memory deficits in experimental autoimmune encephalomyelitis miceObjective: to assess whether LINGO-1 antibody could recover the behavior changes of experimental autoimmune encephalomyelitis (EAE) mice and underlying pathology in the brain.Methods: 25 μg MOG35-55 per mouse, 100 μg H37RA per mouse, and 200 ng pertussis toxin per mouse were used to induce EAE mice; 10 mg/kg LINGO-1 antibody was intraperitoneally injucted once every six days from day 14 to the end of the behavior tests; The elevated plus maze(EPM), open field test, sucrose preference test and Morris water maze test (MWT) in behavior tests were detected at the beginning of day 50; After the behavior tests, mice were killed and the parahippocampal cortex (PHC) and fimbria-fornix were used to detecte the level of myelin associated protein, axonal structural and functional proteins, and proteins in PI3K/AKT/m-TOR signaling pathway through western blot and immunofluorescence.Results: After LINGO-1 antibody intervention, there was no significant difference among the three groups of mice in the EPM, open field test, and sucrose preference test (P>0.05). In the Morris water maze the mean escape latency of the EAE mice was significantly longer than the controls (P<0.05),whereas that of the EAE mice with systemic administration of the LINGO-1 antibody was significantly shorter compared with the mice that were not treated (P<0.05). On the sixth day, the distance around the platform covered by the EAE mice was significantly less than that of the control mice (P<0.05),whereas the LINGO-1 antibody-treated EAE mice covered more distance around the platform than the EAE mice, but this difference was not significant, and the distance covered by the LINGO-1 antibody-treated EAE mice was similar to that of the control mice (P>0.05). EAE mice had lower MBP than Controls in parahippocanpal cortex and fimbria-fornix (P<0.05). Concurrently, the reduction of Kinesin light chain (KLC),a protein of anterograde transport, was observed in parahippocanpal cortex of EAE mice (P<0.01). After systemic administration of LINGO-1 antibody, the level of MBP and KLC was increased in PHC(P<0.05). However, the level of MBP and KLC was not increased in fimbria-fornix after treatment of LINGO-1 antibody (P>0.05). Furthermore, p-Akt and p-mTOR, which correlates with increased kinase activity, increased simultaneously in parahippocanpal cortex of EAE mice with treatment (P<0.05).Conclusion: LINGO-1 Antibody could promote remyelination through activating the AKT/mTOR signaling pathway, a key pathway involved in myelination, to improve the memory of EAE mice. It inferred that LINGO-1 antibody may be an effective drug to ameliorate the cognitive impairment of demyelinating diseases in CNS.Part 4 LINGO-1 antibody ameliorates the pathology of the dorsal hippocampus in cuprizone-fed miceObjective: to investigate whether cuprizone-fed mice have the spatial learning and memory disorders, and depression and anxiety-like behaviors; to explore the pathology in cuprizone-fed mice; to assess whether LINGO-1 antibody could recover the behavior changes of cuprizone-fed mice and underlying pathology in the brain.Methods: Adult (C57BL/6, eight-week-old, male) mice were fed with 0.2% (w/w) cuprizone in ground breeder chow for about ten weeks; LINGO-1 antibody treatment was begun in the third week and the mice in the treatment group received intraperitoneal injections of 10 mg/kg LINGO-1 antibody once every six days; The elevated plus maze, open field test, sucrose preference test, and Morris water maze were tested among the control group, cuprizine-fed group and LINGO-1 antibody treated cuprizine-fed group at weeks 9; Mice were killed after the behavioral tests and the dorsal hippocampus was detected the expression of myelin proteins,axonal transport-associated proteins, axonal structural proteins, synapse-associated proteins, tau and beta amyloid through western blot and immunofluorescence.Results: The mild impairment of learning ability was observed in cuprizone-fed mice (P<0.05),accompanied by the decreased expression of myelin basic protein (MBP) in the dorsal hippocampus (P<0.05). And LINGO-1 antibody improved learning ability and partly restored MBP level (P<0.05). Furthermore, the level of kinesin light chain (KLC), neurofilament light chain (NFL) and neurofilament heavy chain (NF200) was declined in demyelinated hippocampus(P<0.05),which could be partly improved by treatment with LINGO-1 antibody. However, the level of beta amyloid and hyperphosphorylation of tau was similar amonge the three groups(P>0.05).Conclusion: Myelin injure may involve in the mild impairment of learning ability in cuprizone-fed mice. LINGO-1 antibody could ameliorate the cognitive impairment through promoting remyelination. Demyelination might lead to the impairment of neuronal transport, but not cause increased level of hyperphosphorylated tau and beta amyloid. Our research demonstrates remyelination might be an effective pathway to recover the function of neuronal axons and cognition in MS. |
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