#METABOLOMICS WORKBENCH hormel101_20170712_151748 DATATRACK_ID:1127 STUDY_ID:ST000798 ANALYSIS_ID:AN001269 PROJECT_ID:PR000575 VERSION 1 CREATED_ON July 13, 2017, 9:43 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 Sphinomyelin Panel of Myelin to Enhance Recovery of Function after 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 - ms6012-1 group:LFD SUBJECT_SAMPLE_FACTORS - ms6012-2 group:LFD SUBJECT_SAMPLE_FACTORS - ms6012-3 group:LFD SUBJECT_SAMPLE_FACTORS - ms6012-4 group:LFD SUBJECT_SAMPLE_FACTORS - ms6012-5 group:LFD SUBJECT_SAMPLE_FACTORS - ms6012-6 group:HFD SUBJECT_SAMPLE_FACTORS - ms6012-7 group:HFD SUBJECT_SAMPLE_FACTORS - ms6012-8 group:HFD SUBJECT_SAMPLE_FACTORS - ms6012-9 group:HFD SUBJECT_SAMPLE_FACTORS - ms6012-10 group:HFD SUBJECT_SAMPLE_FACTORS - ms6012-11 group:HFHS SUBJECT_SAMPLE_FACTORS - ms6012-12 group:HFHS SUBJECT_SAMPLE_FACTORS - ms6012-13 group:HFHS SUBJECT_SAMPLE_FACTORS - ms6012-14 group:HFHS SUBJECT_SAMPLE_FACTORS - ms6012-15 group:HFHS SUBJECT_SAMPLE_FACTORS - ms6012-16 group:Keto SUBJECT_SAMPLE_FACTORS - ms6012-17 group:Keto SUBJECT_SAMPLE_FACTORS - ms6012-18 group:Keto SUBJECT_SAMPLE_FACTORS - ms6012-19 group:Keto SUBJECT_SAMPLE_FACTORS - ms6012-20 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 "Sphingomyelin concentrations of mouse spinal cord Lipids will be quantified in SP:SAMPLEPREP_SUMMARY myelin isolated in high yield and purity by subcellular fractionation from the SP:SAMPLEPREP_SUMMARY lumbosacral spinal cord. While there are no absolutely ‘myelin-specific’ SP:SAMPLEPREP_SUMMARY lipids, galactocerebroside is the most typical of myelin in the adult nervous SP:SAMPLEPREP_SUMMARY system 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 Reversed phase CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Waters Acquity BEH C8 (150 x 2mm, 1.7um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Quantiva QQQ MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE POSITIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS ng/vial MS_METABOLITE_DATA_START Samples ms6012-1 ms6012-2 ms6012-3 ms6012-4 ms6012-5 ms6012-6 ms6012-7 ms6012-8 ms6012-9 ms6012-10 ms6012-11 ms6012-12 ms6012-13 ms6012-14 ms6012-15 ms6012-16 ms6012-17 ms6012-18 ms6012-19 ms6012-20 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 SM-C16 89.8 105.7 155.9 94.9 103.5 141.9 99.0 125.0 144.1 220.9 99.3 119.7 103.3 148.5 149.3 132.4 142.9 132.9 133.9 81.1 SM-C18:1 16.2 28.0 32.1 24.1 27.1 25.1 14.0 29.8 37.5 57.7 20.7 23.8 23.8 35.4 36.0 31.4 50.0 26.6 32.1 14.4 SM-C18:0 696 916 1281 813 903 1119 709 1012 1270 1701 747 894 808 1241 1211 1139 1451 1059 1098 624 SM-C24:1 1844 2003 3622 1897 2001 3023 2131 2555 3336 3870 1783 2245 2018 2927 2975 3044 2887 2807 2590 1506 SM-C24:0 364.6 344.5 658.6 369.3 394.2 562.7 419.7 455.3 581.4 652.5 357.8 433.1 399.0 562.6 572.2 595.0 528.0 555.4 517.5 331.5 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name SM-C16 SM-C18:1 SM-C18:0 SM-C24:1 SM-C24:0 METABOLITES_END #END