#METABOLOMICS WORKBENCH hormel101_20170712_144952 DATATRACK_ID:1124 STUDY_ID:ST000795 ANALYSIS_ID:AN001266 PROJECT_ID:PR000575 VERSION 1 CREATED_ON July 13, 2017, 9:32 pm #PROJECT PR:PROJECT_TITLE Mayo Pilot and Feasibility: Targeting Myelin Metabolism to Enhance Recovery of PR:PROJECT_TITLE Function after SCI PR:PROJECT_SUMMARY The loss of myelin, degeneration of the myelin producing oligodendroglia and PR:PROJECT_SUMMARY impaired remyelination are essential features of traumatic spinal cord injury PR:PROJECT_SUMMARY (SCI) that significantly limit patient recovery of function. The lipid rich PR:PROJECT_SUMMARY composition of myelin, including exceptionally high levels of saturated fatty PR:PROJECT_SUMMARY acids, underlie its essential physiological roles, including its structural and PR:PROJECT_SUMMARY signaling properties and electrical insulation of axons to facilitate the PR:PROJECT_SUMMARY conduction of nerve impulses. The myelin sheaths also provide metabolic support PR:PROJECT_SUMMARY to the axons they wrap, and myelin health is therefore essential to the PR:PROJECT_SUMMARY maintenance of axon integrity and function in the brain and spinal cord. The PR:PROJECT_SUMMARY primary goal of this Pilot Proposal to the Mayo Clinic Metabolomics Core is to PR:PROJECT_SUMMARY integrate highly sensitive metabolomics liquid chromatography-tandem mass PR:PROJECT_SUMMARY spectrometry (LC/MS/MS) assays to quantify the lipid composition of the myelin PR:PROJECT_SUMMARY membrane, with our conventional neurobehavioral approaches, enabling us to PR:PROJECT_SUMMARY explore the metabolic basis of new interventions capable of promoting myelin PR:PROJECT_SUMMARY regeneration and restoration of patient function. Metabolomics Core expertise in PR:PROJECT_SUMMARY Magnetic Resonance Spectroscopy (NMR) based evaluation of key metabolites PR:PROJECT_SUMMARY involved in CNS injury and repair (N-acetyl-L-aspartate, choline, myo-inositol, PR:PROJECT_SUMMARY glucose/ glutamine and lactate) will also be applied to strengthen our PR:PROJECT_SUMMARY mechanistic understanding of myelin injury and repair. Specifically, utilizing PR:PROJECT_SUMMARY these innovative approaches we will test a novel hypothesis driven by new PR:PROJECT_SUMMARY preliminary findings that the levels of dietary fatty acids can be optimized PR:PROJECT_SUMMARY alone, or in combination with exercise training, to facilitate myelin PR:PROJECT_SUMMARY regeneration and recovery of neurobehavioral function after injury to the adult PR:PROJECT_SUMMARY spinal cord. In Aim 1, we will determine whether alterations in dietary fat, PR:PROJECT_SUMMARY including saturated fat or omega-3 fatty acids, facilitate restoration of the PR:PROJECT_SUMMARY myelin membrane and metabolite signatures of central nervous system repair after PR:PROJECT_SUMMARY experimental SCI in adult mice. In Aim 2, we will determine whether exercise PR:PROJECT_SUMMARY training alone or in combination with dietary fatty acid supplementation fosters PR:PROJECT_SUMMARY myelin regeneration and recovery of function after experimental SCI. The PR:PROJECT_SUMMARY proposed studies will leverage the expertise of the Mayo Metabolomics Core with PR:PROJECT_SUMMARY that of Dr. Scarisbrick (Mayo) in myelin biology and Dr. Gomez Pinilla (UCLA) in PR:PROJECT_SUMMARY central nervous system plasticity to investigate whether two highly targetable PR:PROJECT_SUMMARY lifestyle variables, that is diet and exercise, can be modulated to improve PR:PROJECT_SUMMARY myelin metabolism and functional recovery after SCI. PR:INSTITUTE Mayo Clinic PR:LAST_NAME Scarisbrick PR:FIRST_NAME Isobel PR:ADDRESS 200 First St. SW, Rochester, Minnesota, 55905, USA PR:EMAIL scarisbrick.isobel@mayo.edu PR:PHONE 507-284-0124 #STUDY ST:STUDY_TITLE Targeted Cholesterol Profiling of Myelin to Enhance Recovery of Function after ST:STUDY_TITLE SCI ST:STUDY_SUMMARY Tissue is from adult mouse spinal cord (SC). We are submitting these samples for ST:STUDY_SUMMARY Untargeted Profiling (unbiased metabolomics assay) and for lipid analysis. The ST:STUDY_SUMMARY lipid assays we request are 1) free fatty acid composition of lipids; 2) free ST:STUDY_SUMMARY fatty acid panel; 3) cholesterol concentration (free and bound); 4) Ceramides, ST:STUDY_SUMMARY including galactosyl and glucosyl; 5) sphingomyelin. The Untargeted profiling is ST:STUDY_SUMMARY our top priority, followed by the lipid assays as listed. All samples were snap ST:STUDY_SUMMARY frozen at the point of harvest and approximate weights are provided. The samples ST:STUDY_SUMMARY are submitted as intact pieces of tissue. There are 20 samples total, n=5 for ST:STUDY_SUMMARY each group that includes LF (low fat diet); HF (high fat diet); HFHS (high fat ST:STUDY_SUMMARY high sucrose diet); and Keto (ketogenic diet). ST:INSTITUTE Mayo Clinic ST:LAST_NAME Scarisbrick ST:FIRST_NAME Isobel ST:ADDRESS 200 First St. SW, Rochester, Minnesota, 55905, USA ST:EMAIL scarisbrick.isobel@mayo.edu ST:PHONE 507-284-0124 #SUBJECT SU:SUBJECT_TYPE Mouse SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - ms6009-1 group:LFD SUBJECT_SAMPLE_FACTORS - ms6009-2 group:LFD SUBJECT_SAMPLE_FACTORS - ms6009-3 group:LFD SUBJECT_SAMPLE_FACTORS - ms6009-4 group:LFD SUBJECT_SAMPLE_FACTORS - ms6009-5 group:LFD SUBJECT_SAMPLE_FACTORS - ms6009-6 group:HFD SUBJECT_SAMPLE_FACTORS - ms6009-7 group:HFD SUBJECT_SAMPLE_FACTORS - ms6009-8 group:HFD SUBJECT_SAMPLE_FACTORS - ms6009-9 group:HFD SUBJECT_SAMPLE_FACTORS - ms6009-10 group:HFD SUBJECT_SAMPLE_FACTORS - ms6009-11 group:HFHS SUBJECT_SAMPLE_FACTORS - ms6009-12 group:HFHS SUBJECT_SAMPLE_FACTORS - ms6009-13 group:HFHS SUBJECT_SAMPLE_FACTORS - ms6009-14 group:HFHS SUBJECT_SAMPLE_FACTORS - ms6009-15 group:HFHS SUBJECT_SAMPLE_FACTORS - ms6009-16 group:Keto SUBJECT_SAMPLE_FACTORS - ms6009-17 group:Keto SUBJECT_SAMPLE_FACTORS - ms6009-18 group:Keto SUBJECT_SAMPLE_FACTORS - ms6009-19 group:Keto SUBJECT_SAMPLE_FACTORS - ms6009-20 group:Keto #COLLECTION CO:COLLECTION_SUMMARY Uninjured or SCI mice will be randomly assigned to one of four groups, LF (low CO:COLLECTION_SUMMARY fat diet); HF (high fat diet); HFHS (high fat high sucrose diet); and Keto CO:COLLECTION_SUMMARY (ketogenic diet). All diets will be obtained from Research Diets, NJ USA43,45. CO:COLLECTION_SUMMARY Dietary fat supplementation will be initiated at 1 week after SCI. This time CO:COLLECTION_SUMMARY point for intervention was chosen to provide a meaningful timeframe for clinical CO:COLLECTION_SUMMARY translation. Also, a 1 week period will allow time for mice to recover prior to CO:COLLECTION_SUMMARY providing access to wheel running (Aim 2). The impact of dietary fat CO:COLLECTION_SUMMARY supplementation on myelin metabolism will be examined after a period of 7 weeks, CO:COLLECTION_SUMMARY including determination of (i) the lipid profile of the myelin membrane using CO:COLLECTION_SUMMARY LC/MS/MS; and (ii) metabolic markers of spinal cord metabolism by NMR. Results CO:COLLECTION_SUMMARY will be correlated with (iii) cellular and molecular markers of spinal cord CO:COLLECTION_SUMMARY pathophysiology including the appearance of OPCs, oligodendroglia and myelin, CO:COLLECTION_SUMMARY axon health, astrogliosis and inflammation; and (iv) the extent of sensorimotor CO:COLLECTION_SUMMARY recovery. (v) In addition, to gauge the impact of the dietary fat on systemic CO:COLLECTION_SUMMARY metabolic status, Insulin and Glucose Resistance Tests will be performed at 7 CO:COLLECTION_SUMMARY weeks. Food intake (g/day) and body weight gain (% initial weight) will be CO:COLLECTION_SUMMARY measured daily until the endpoint of each experiment. Mice will be housed CO:COLLECTION_SUMMARY individually in a temperature-controlled facility with a 12:12-h light-dark CO:COLLECTION_SUMMARY cycle and ad libitum access to each diet and water. A Power Analysis was CO:COLLECTION_SUMMARY performed based on histological outcomes in mice with contusion compression CO:COLLECTION_SUMMARY injury. To detect a difference between groups of 20%, which would very CO:COLLECTION_SUMMARY meaningful, a group size of 8 will be needed to achieve a power of 0.85. An CO:COLLECTION_SUMMARY additional 2 mice per group has been added to account for mortality. The Mayo CO:COLLECTION_SUMMARY Clinic Institutional Animal Care and Use Committee has approved of the proposed CO:COLLECTION_SUMMARY studies. #TREATMENT TR:TREATMENT_SUMMARY To test the hypothesis that optimizing dietary fat will facilitate myelin repair TR:TREATMENT_SUMMARY after SCI, the diet of uninjured adult female C57BL6/J mice (12 week, 22-25g, TR:TREATMENT_SUMMARY Jackson), or those with experimental contusion-compression SCI of the TR:TREATMENT_SUMMARY lumbosacral spinal cord (L2-L3) (Fejota Clip 3g Force, applied for 30s)32,47 TR:TREATMENT_SUMMARY will be supplemented with saturated fat. The 3g Clip produces moderate SCI TR:TREATMENT_SUMMARY including demyelination and clinical impairment and we recently published a TR:TREATMENT_SUMMARY detailed methodology. At 1 week after injury, the 3g injured mice are expected TR:TREATMENT_SUMMARY to have an average Basso Mouse Scale score (BMS)=5 on a 9 point scale such that TR:TREATMENT_SUMMARY they have frequent plantar stepping with no or some coordination. This level of TR:TREATMENT_SUMMARY impairment was chosen to provide a sufficient window to observe recovery, and to TR:TREATMENT_SUMMARY be at a level compatible with examination of exercise training by wheel running TR:TREATMENT_SUMMARY (Aim 2). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY "Cholesterol of mouse spinal cord Lipids will be quantified in myelin isolated SP:SAMPLEPREP_SUMMARY in high yield and purity by subcellular fractionation from the lumbosacral SP:SAMPLEPREP_SUMMARY spinal cord. While there are no absolutely ‘myelin-specific’ lipids, SP:SAMPLEPREP_SUMMARY galactocerebroside is the most typical of myelin in the adult nervous system SP:SAMPLEPREP_SUMMARY being directly proportional to the amount of myelin. Sulfatide is another SP:SAMPLEPREP_SUMMARY galactolipid enriched in myelin. Together with cholesterol, these form 78% of SP:SAMPLEPREP_SUMMARY the total amount of lipid in the myelin membrane and each will be quantified SP:SAMPLEPREP_SUMMARY using LC/MS/MS. A highly sensitive assay for galactocerebroside was recently SP:SAMPLEPREP_SUMMARY established by the Mayo Metabolomics Core and can be implemented immediately. SP:SAMPLEPREP_SUMMARY The LC/MS/MS panel for free fatty acids, including the very long chain fatty SP:SAMPLEPREP_SUMMARY acids found in myelin is also routinely performed by the Core. Cholesterol will SP:SAMPLEPREP_SUMMARY be quantified using an NMR-based approach by the Mayo Dept. of Laboratory SP:SAMPLEPREP_SUMMARY Medicine Clinical Core. Additionally, we have a plan in place with the SP:SAMPLEPREP_SUMMARY Metabolomics Core to develop LC/MS/MS assays for sulfatide and sphingomyelin SP:SAMPLEPREP_SUMMARY during the Pilot proposal. Having quantitative assays for each of these key SP:SAMPLEPREP_SUMMARY myelin lipids will facilitate our goal to comprehensively profile myelin lipid SP:SAMPLEPREP_SUMMARY metabolism and will form foundational assays for a future NIH grant focused on SP:SAMPLEPREP_SUMMARY myelin metabolism." #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Agilent 7890A CH:COLUMN_NAME Agilent HP5-MS (30m × 0.25mm, 0.25 um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 5975C MS:INSTRUMENT_TYPE Single quadrupole MS:MS_TYPE EI MS:ION_MODE POSITIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS ug/mg tissue MS_METABOLITE_DATA_START Samples ms6009-1 ms6009-2 ms6009-3 ms6009-4 ms6009-5 ms6009-6 ms6009-7 ms6009-8 ms6009-9 ms6009-10 ms6009-11 ms6009-12 ms6009-13 ms6009-14 ms6009-15 ms6009-16 ms6009-17 ms6009-18 ms6009-19 ms6009-20 Factors group:LFD group:LFD group:LFD group:LFD group:LFD group:HFD group:HFD group:HFD group:HFD group:HFD group:HFHS group:HFHS group:HFHS group:HFHS group:HFHS group:Keto group:Keto group:Keto group:Keto group:Keto Total Cholesterol 5.19 6.40 10.62 7.96 10.48 6.13 7.46 7.17 8.94 14.98 7.45 7.84 6.39 10.86 10.25 10.38 15.97 10.56 12.40 4.16 Free Cholesterol 5.35 6.85 10.75 8.18 10.11 6.17 7.59 6.96 8.63 14.94 7.39 7.60 6.15 10.62 10.05 10.25 15.77 10.47 11.98 3.45 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Total Cholesterol Free Cholesterol METABOLITES_END #END