A Role For Impaired Autophagy In The Neurodegeneration Of Experimental Autoimmune Encephalomyelitis (EAE)

Objective: Neurodegeneration, along with inflammatorydemyelination, is an important component of multiple sclerosis (MS)pathogenesis, an autoimmune inflammatory disease of the central nervoussystem (CNS). Neurodegeneration occurs during the early stages of MSand is a key indicator of disease progression and irreversible neurologicaldamage in MS patients. The underlying mechanisms ofneurodegeneration in MS remain to be elucidated. Aside frominflammatory neurotoxicity, neurodegeneration is associated with certaininflammation-independent mechanisms such as oxidative stress,axoplasmic Ca2+accumulation, glutamate excitotoxicity, mitochondrialdysfunction, protein aggregation and carbonylation. It is necessary tounderstand fully the mechanisms of neurodegeneration in MS so thatmore efficient therapeutic strategies can be employed.Autophagy, sometimes also known as macroautophagy, is alysosome-mediated degradation pathway through which cells engulf andtransport superfluous or impaired organelles, mis/unfolded proteins andinvading microorganisms to the lysosomes for degradation. Autophagyplays pro-survival role and pro-death double roles, which is a criticalmediator of inflammation and immunity. Autophagy plays an essentialrole in maintaining the health and homeostasis of neurons. A disturbancein autophagy has been associated with numerous neurodegenerativedisorders, including Alzheimer’s disease, Huntington’s disease,Parkinson’s disease, and amyotrophic lateral sclerosis. A recent studyshows that significantly increased Atg5levels were detected in T cellsand the brain tissue of MS patients and EAE mice (a murine model ofMS). This would indicate there is a specific link between autophagy and MS pathology. However, there are no data involving the roles ofautophagy pathways in neurodegeneration of MS.Nowadays autophagic modulation is generally considered to be apromising potential therapeutic target for neurodegenerative diseases.Rapamycin, a mammalian target of rapamycin (mTOR) inhibitor andclassical pharmacological inducer of autophagy, ameliorates the clinicaland pathological progress of MS in patients and EAE mice, but whetherthis promotes survival of neurons remains to be investigated. To date,anti-inflammatory and immune modulating therapies are the major formsof MS treatment. These treatments can effectively alleviate the symptomsin acute phase of MS, but can not improve long-term neurological deficits.It is necessary to understand fully the mechanisms of neurodegenerationin MS so that more efficient therapeutic strategies can be employed.In this study, we explored the potential links between autophagy andneurodegeneration in MS, and assessed the effects of pharmacologicalinduction of autophagy on neuronal cell injury in an in vivo model of MS.We also sought to characterize alterations in activity and to determine thepossible molecular mechanisms of autophagy during neurodegenerationin MS. Using mice with experimental autoimmune encephalomyelitis(EAE), a well-established animal model of MS suitable for the study ofneurodegeneration, we characterized alterations in autophagic activity,and the molecular mechanisms of autophagy.Methods:MiceFemale C57BL/6mice,6-8weeks old, weighted18-20g, werepurchased from Vital River Laboratory Animal Technology Co. Ltd(Beijing,China). Mice were maintained under specific pathogen-free(SPF)conditions at room temperature(24士2℃), with light and dark by12h:12halternate cycles, given free food and drink. All animal work was approvedby the Institutional Animal Care and Use Committee of Hebei MedicalUniversity and the local Experimental Ethics Committee. EAE induction and evaluationEAE was induced in C57BL/6mice by immunization with250μg ofMOGp35–55(lysine Bio-system, XiAn, China). All peptides weredissolved in complete Freund’s adjuvant (Sigma, St Louis, MO, USA)containing4mg/ml of heat-killed mycobacterium tuberculosis H37Ra(Difco Laboratories, Detroit, MI, USA). At day0and48h postimmunization, C57BL/6mice were injected with500ng of pertussis toxin(Alexis, San Diego, CA, USA) in PBS, intraperitoneally (i.p.). Mice wereexamined daily for clinical signs of EAE and scored as follows:0, noparalysis;1, loss of tail tone;2, hindlimb weakness;3, hindlimb paralysis;4, hindlimb and forelimb paralysis;5, moribund or dead.When clinicalsigns were inter-mediate between two grades of the disease,0.5wasadded to the lower score.Experimental group and TreatmentTo investigate autophagic alteration, mice were randomly distributedequally to healthy control group and EAE group. Each group was dividedinto age-matched three subgroups: premorbid (13d), fastigium (20d) andparamastic(30d). Mice were sacrificed at indicated time points and tissuesamples were taken for further analysis. In the experiments withrapamycin, mice were randomized divided into three groups: controlgroup, EAE-Vehicle group and EAE-Rapamycingroup.Rapamycin(sigma)(1mg/kg) i.p. injections were given once daily.EAE-Vehicle mice received a daily injection (i.p.) of distilled water(vehicle). Treatment began on the first day of EAE induction.Autophagic flux analysisFor analysis of autophagic flux in the spinal cord of EAE, mice wererandomly distributed equally to healthy control group, EAE group,control+chloroquine group and EAE+chloroquine group. Chloroquine-treated mice were injected i.p. with chloroquine(Sigma-Aldrich, Milano,Italy)(60mg/kg body weight) in PBS once a day for7days starting atEAE onset (clinical score=1.0). Autophagic flux was determined by the difference in LC3-II protein levels in the absence and presence ofchloroquine.Nissl stainingMice were induced to death by deep anesthesia and cardiacperfusion with4%paraformaldehyde（PFA）(sigma). Lumbar spinal cordsspecimen were taken from both groups, and then performed by routinefixation and paraffin embedding. Continuous sections were made forNissl staining and TUNEL assay. Nissl stain was used to detect neurondamage. Sections were stained with cresyl violet and visualized andphotographed by microscope (Olympus BX51, Tokyo, Japan). MNs werecounted according to the following criteria:(1) neurons located in theanterior horn ventral to the line tangential to the ventral tip of the centralcanal;(2) neurons with a maximum diameter of20μm or larger; and (3)neurons with a distinct nucleolus.TUNEL assayApoptosis was measured by terminal-deoxynucleotidyl-transferase-mediated dUTP-biotin nick end-labeling (TUNEL) assay (Roche,Mannheim, Germany). Spinal cord tissue slides were stained withTUNEL reagents, Alexa fluor568-conjugated-phalloidin and DAPIaccording to the manufacturer’s instructions. Tissue sections wereobserved under the Olympus microscope (Olympus BX51, Tokyo, Japan).TUNEL-positive nuclei were calculated in15randomly selected fields,averaged and expressed as percentage of labeled nuclei within the fields.ImmunofluorescenceThe fixed specimen of lumbar spinal cord at20d after immunizationwere taken and made continuous30μm freezing sections. Rinse sectionsthree times in PBS for5minutes each. Block specimen in10%horseserum in PBS/Triton for60minutes. Then incubate in diluted primaryantibodies overnight at4°C. Rinse three times in PBS for5minutes each.Subsequently the sections were incubated in fluorochrome-conjugatedsecondary antibody diluted in PBS/Triton for1hour at room temperature in dark. Cells were then mounted in mowiol and images were acquiredusing Olympus Fluoview (FV1000, Tokyo, Japan) confocal laserscanning microscope.Western blot analysisThe spinal cords samples were quickly removed and kept in-80°Cfor Western blot analyses. After protein extraction and concentrationdetermination, proteins were separated by electrophoresis using10%Bis-Tris SDS-PAGE gel (Life Technologies), and followed by transfer toa nitrocellulose membrane at100v for2h, blocking for1h with5%milk,and overnight incubation with primary antibodies (phospho-AKT, ATK,phospho-p70S6K, p70S6K, LC3-II, Beclin1, Bax, Bcl-2and GAPDH) at4°C. Following a wash with0.1%Tween in PBS (4×15min), theHRP-conjugated secondary antibody is added for2h at room temperature.The membrane was washed again to remove the unbound antibodies anddetected by western blotting detection system.Statistical AnalysisAll data are expressed as mean±SD. Statistical analyses betweengroups of two were conducted with the unpaired Student t test. Groups ofthree or more were analyzed with one-way ANOVA, followed by theNewman–Keuls multiple comparison test. Data were analyzed usingSPSS13.0software. p <0.05were considered statistically significant.Results:1Up-regulation of LC3-II was observed in the spinal cords of EAEmice compared with those in controls at each time point, in particularafter20days. Levels of Beclin1were down-regulated in the spinal cordsof mice as early as13days post-immunization. Lowest levels werereached at20days post-immunization, before returning to control levelsat30days, as compared with age-matched controls. LC3-II levels in thespinal cords of mice in the control chloroquine group were elevatedcompared with those in the control vehicle group. There were nosignificant changes in LC3-II levels between mice treated with EAE vehicle and EAE chloroquine.2LC3was mainly expressed in neurons, to a lesser extent in matureoligodendrocytes, and barely co-localized with astrocytes in the lumbarspinal cords of EAE mice. At the peak stages of EAE, the intensity ofLC3fluorescent signals with NeuN was significantly stronger than inage-matched control mice.3Compared with the control group, phosphorylation levels of Akt(Ser473) in the spinal cords of EAE mice were slightly decreased at13days post-immunization. Phosphorylation levels were elevated at20days post-immunization, but were then reduce at30days.Thetime-dependent expression of p70S6K (Thr389) phosphorylation in thespinal cords of EAE mice was consistent with that for Aktphosphorylation.Rapamycin treatment resulted in significant increases of LC3-II andBeclin1levels along with reductions in phosphorylation levels of Aktand p70S6K.4Compared with the EAE control group, mice treated withrapamycin exhibited postponed onset of disease symptoms. The severityof EAE was reduced as indicated by lower mean maximum clinicalscores. The numbers of Nissl-stained cells and NeuN-positive neurons inEAE-vehicle and EAE-rapamycin mice were reduce compared with thosein control mice. Mice treated with rapamycin had higher numbers ofNissl-stained cells and NeuN-positive neurons compared with those inEAE-vehicle mice.5Rapamycin treatment obviously up-regulated the anti-apoptosisprotein Bcl-2, with some down-regulation of Bax, a pro-apoptosis protein.At the peak stages of EAE, the ratio of the Bax to Bcl-2was reduced,indicating that cells were resistent to EAE-induced apoptosis.There was a marked increase in the number of apoptotic cells inEAE mice. There was no significant difference in the number ofTUNEL-positive cells between the EAE-rapamycin and EAE-vehicle groups. Our results also showed an elevation in the number ofbcl-2-positive neurons in the spinal cords of rapamycin-treated micecompared with EAE-vehicle mice.Conclusion:1Autophagic flux is impaired in the spinal cords of EAE mice.Increased neuronal autophagosome accumulation in EAE mice,suggesting autophagic deficiency in EAE maybe is associated withneurodegeneration.2Autophagy is regulated via the Akt/mTOR/p70S6K pathway, whilerapamycin can restore autophagy activity in the spinal cords of EAE mice3Rapamycin ameliorated clinical signs, and alleviated neuronalinjury in EAE mice. Rapamycin induces autophagy and promtes mutantprotein clearness and Bax-dependent apoptosis in neuron, and along withthe suppression of inflammatory, which contributed to neuroprotection inthe EAE model.