Simmaron Research Inc

THE CENTRAL ROLE OF MTOR IN ME/CFS PATHOGENESIS. - PROJECT SUMMARY. MYALGIC ENCEPHALOMYELITIS OR CHRONIC FATIGUE SYNDROME (ME/CFS) IS A MULTISYSTEM CHRONIC ILLNESS CHARACTERIZED BY SEVERE MUSCLE FATIGUE, PAIN, DIZZINESS, AND BRAIN FOG. MANY PATIENTS WITH ME/CFS EXPERIENCE POST-EXERTIONAL MALAISE (PEM), WHICH IS CHARACTERIZED BY SEVERE MUSCLE FATIGUE, PAIN, AND COGNITIVE IMPAIRMENT AFTER PHYSICAL OR MENTAL EXERCISE. DESPITE INTENSE INVESTIGATION, THE MOLECULAR MECHANISM OF THIS DEBILITATING CONDITION IS STILL UNKNOWN. RECENTLY, WE REPORTED THAT THE CHRONIC ACTIVATION OF MAMMALIAN TARGET OF RAPAMYCIN (MTOR) MAY PLAY A CRITICAL ROLE IN THE PATHOGENESIS OF PEM. FIRST, THE CHRONIC ADMINISTRATION OF SELECTIVE MTOR AGONIST MHY1485 WAS OBSERVED TO INDUCE SEVERE SPONTANEOUS AND POST-EXERTIONAL MUSCLE FATIGUE AFTER A SINGLE SESSION-TREADMILL EXERCISE. SECOND, HISTOPATHOLOGICAL ANALYSES OF SKELETAL MUSCLE REVEALED THAT THERE WAS A STRONG INFILTRATION OF MONONUCLEAR CELLS IN THE VASCULATURE OF MUSCLE TISSUE UPON CHRONIC INDUCTION OF THE MTOR ACTIVATION. FURTHER ANALYSIS REVEALED THAT THESE MONONUCLEAR CELLS ARE MACROPHAGES STRONGLY EXPRESSED INFLAMMATORY SURFACE MARKER CD40 AND ENZYME INOS SUGGESTING THE POTENTIAL ROLE OF MTOR ACTIVATION IN THE INFILTRATION AND POLARIZATION OF MΦ TO ITS INFLAMMATORY MODE (M1MΦ) . THIRD, WE OBSERVED THAT THE ACTIVATION OF MTOR DIRECTLY INHIBITED THE AUTOPHAGY PROCESS VIA SUPPRESSING THE ACTIVITY OF EARLY AUTOPHAGY PROTEIN ATG13 IN THESE MICE. AUTOPHAGY IS A QUALITY CONTROL PROCESS FOR MITOCHONDRIAL HEALTH AND FUNCTION. ACCORDINGLY, THE GENETIC MUTATION OF THE EARLY AUTOPHAGY GENE ATG13 FOLLOWED BY THE AUTOPHAGY IMPAIRMENT STIMULATED SEVERE SPONTANEOUS AND POST-EXERTIONAL MUSCLE FATIGUE. THE SUBSEQUENT MOLECULAR ANALYSES ALSO REVEALED THAT THE GENETIC ABLATION OF ATG13 CAUSED SEVERE INFILTRATION OF INFLAMMATORY MACROPHAGES IN THE VASCULATURE OF SKELETAL MUSCLE. FINALLY, WE OBSERVED THAT THE CHRONIC ACTIVATION OF MTOR INDUCED THE ACTIVATION OF A NUCLEAR TRANSCRIPTION FACTOR NAMED STAT3 CAUSING THE SECRETION OF INFLAMMATORY MEDIATORS SUCH AS IL6 AND RANTES. THEREFORE, WE HYPOTHESIZE THAT THE ACTIVATION OF MTOR PLAYS A CENTRAL ROLE IN THE PATHOGENESIS OF PEM. THE CENTRAL OBJECTIVE WILL BE STUDIED IN THREE SPECIFIC AIMS. IN AIM1, WE WILL STUDY THE EFFECT OF MTOR ACTIVATION ON THE POLARIZATION OF M1MΦ IN PEM PATHOGENESIS. HERE WE APPLIED A NOVEL OXYGEN-DEFICIENT CELL CULTURE MODEL, IN WHICH MΦ CELLS WILL BE TREATED WITH PLASMA SAMPLES OF ME/CFS PATIENTS AND ME/CFS PATIENTS TREATED WITH 6MG/KG/WEEK RAPAMYCIN FOR 30 DAYS (T1). IN AIM 2, WE WILL STUDY THE MOLECULAR MECHANISM ON HOW THE MTOR ACTIVATION POLARIZES M1 PHENOTYPE. THE ROLE OF MTOR ON ATG13 INACTIVATION WILL BE EVALUATED IN A NOVEL ATG13∆MUSCLE STRAIN, IN WHICH ATG13 GENE IS SELECTIVELY KNOCKED OUT IN MUSCLE. WE ALSO DEVELOPED A NEW ELISA KIT TO STUDY MTOR MEDIATED SERINE 258 PHOSPHORYLATION OF ATG13. OUR PROPOSED RESEARCH WILL FURTHER INVESTIGATE HOW THE MTOR-MEDIATED ACTIVATION OF STAT3 EMPLOYS THE POLARIZATION OF M1MΦ CELLS. UNDER AIM3, WE PROPOSE A COMPREHENSIVE METABOLOMIC STUDY TO INVESTIGATE THE ROLE OF MTOR ACTIVATION IN REGULATING THE PURINE AND GLUCOSE METABOLISM IN ME/CFS CASES, WHICH POTENTIALLY REGULATES THE POLARIZATION OF M1MΦ CELLS.

ATG13: A NEW PLAYER IN ME/CFS - PROJECT SUMMARY: MYALGIC ENCEPHALOMYELITIS AND CHRONIC FATIGUE SYNDROME (ME/CFS) IS A MULTISYSTEM CHRONIC ILLNESS CHARACTERIZED BY EXTREME FATIGUE, MUSCLE WEAKNESS, MUSCLE PAIN, DIZZINESS, A COGNITIVE DEFICIT OF ATTENTION, AND DEPRESSION. DESPITE INTENSE INVESTIGATION, THE MOLECULAR MECHANISM OF THIS DISEASE IS STILL UNKNOWN. OUR RECENT FINDING SUGGESTS THAT THE FUNCTIONAL INACTIVATION OF ATG13, AN ESSENTIAL PROTEIN OF CELLULAR AUTOPHAGY, CONTRIBUTES TO THE PATHOGENESIS OF ME/CFS. TO FURTHER CHARACTERIZE THE ROLE OF ATG13 IN THE PATHOGENESIS OF ME/CFS, A RELIABLE DISEASE MODEL IS REQUIRED THAT EXHIBITS SOME OF THE CARDINAL DISEASE SYMPTOMS SUCH AS POST-EXERTIONAL MALAISE (PEM) AND ORTHOSTATIC INTOLERANCE (OI). PEM IS CHARACTERIZED BY SEVERE MUSCLE FATIGUE AND PAINS IMMEDIATELY AFTER EXERCISE, WHEREAS OI IS CHARACTERIZED BY THE SUDDEN DROP IN BLOOD PRESSURE IN AN UPRIGHT CONDITION. OUR PRELIMINARY RESULTS SUGGEST THAT MHY1485, AN AGONIST OF MAMMALIAN TARGET OF RAPAMYCIN (MTOR), INACTIVATES ATG13-DEPENDENT AUTOPHAGY AND INDUCES SEVERE FATIGUE AND PEM IN 3-4 WEEKS OLD FEMALE MICE. THEREFORE, TO ESTABLISH THE DIRECT ROLE OF ATG13 IN ME/CFS PATHOGENESIS, OUR OVERALL HYPOTHESIS IS THAT ATG13 DEPLETION WILL DISPLAY A SPONTANEOUS PEM PATHOLOGY. IN AIM1, WE WILL GENERATE A MOUSE STRAIN WITH MUSCLE-SPECIFIC KNOCK-OUT OF THE ATG13 GENE (ATG13MUSCLE). A 20-MINUTE ACUTE TREADMILL RUNNING PROTOCOL AND 2-DAYS CPET TESTS WOULD BE ADOPTED IN THESE MICE FOLLOWED BY SIMULTANEOUS MEASUREMENT OF M- WAVE AMPLITUDE THROUGH EMG RECORDING, DECREASED MOBILITY RECORDED BY STOELTING ANY-MAZE TRACKING SOFTWARE, MUSCLE PAIN, STRESS, AND NEUROCOGNITIVE IMPAIRMENT OF ATTENTION (AIM 1.1). MOLECULAR ANALYSIS OF MITOCHONDRIAL IMPAIRMENT IN ENERGY METABOLISM, CALCIUM HOMEOSTASIS, APOPTOSIS, AND ANAEROBIC GLYCOLYSIS WOULD BE EVALUATED IN THESE MICE (AIM 1.2). UNDER AIM2, WE WOULD SEE IF NEURAL MUTATION OF ATG13 IN MYELINATING CELLS OF THE BRAIN (ATG13BRAIN) OR ABLATION OF ATG13 IN SENSORY NEURONS (ATG13PNS) CONTRIBUTES TO THE PATHOGENESIS OF ME/CFS SUCH AS AUTONOMIC DYSFUNCTION, THE COGNITIVE DEFICIT OF ATTENTION, ANXIETY, AND PAIN. BRIEFLY, ATG13BRAIN AND ATG13PNS MICE WOULD BE GENERATED AND THEN ANALYZED FOR CHRONIC FATIGUE, FOLLOWED BY A 2-DAYS CPET TEST. AFTER ANOTHER 3-4 WEEKS, MUSCLE FATIGUE, PAIN, STRESS, AND NEUROCOGNITIVE IMPAIRMENT (AIM 2.1) WOULD BE EVALUATED. SINCE CHRONIC INFLAMMATION IS ONE OF THE CARDINAL SYMPTOMS OF ME/CFS, WE WOULD ASSESS PERIPHERAL INFLAMMATION OF CD4 TH1 CELL ACTIVATION IN THE SPLEEN- AND LYMPH NODE-DERIVED MONONUCLEAR CELLS (AIM 2.2). CENTRAL INFLAMMATION WILL BE ASSESSED BY MONITORING THE ACTIVATION OF NF-B, INOS, AND RANTES PRODUCTION IN THE BRAIN AND SPINAL CORD (AIM 2.2). IF SUCCESSFUL, OUR CURRENT PROPOSAL IDENTIFIES THE FIRST MOUSE MODEL TO STUDY ME/CFS AND ME/CFS- ASSOCIATED PEM.