{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002928","ANALYSIS_ID":"AN004802","VERSION":"1","CREATED_ON":"July 20, 2023, 9:25 am"},

"PROJECT":{"PROJECT_TITLE":"The major TMEM106B dementia risk allele affects TMEM106B protein levels, fibril formation, and myelin lipid homeostasis in the ageing human hippocampus","PROJECT_SUMMARY":"Background: The risk for dementia increases exponentially from the seventh decade of life. Identifying and understanding the biochemical changes that sensitize the ageing brain to neurodegeneration will provide new opportunities for dementia prevention and treatment. This study aimed to determine how ageing and major genetic risk factors for dementia affect the hippocampal proteome and lipidome of neurologically-normal humans over the age of 65. The hippocampus was chosen as it is highly susceptible to atrophy with ageing and in several neurodegenerative diseases. Methods: Mass spectrometry-based proteomic and lipidomic analysis of CA1 hippocampus samples from 74 neurologically normal human donors, aged 66 – 104, was used in combination with multiple regression models and gene set enrichment analysis to identify age-dependent changes in the proteome and lipidome. ANOVA was used to test the effect of major dementia risk alleles in the TMEM106B and APOE genes on the hippocampal proteome and lipidome, adjusting for age, gender, and post-mortem interval. Fibrillar C-terminal TMEM106B fragments were isolated using sarkosyl fractionation and quantified by immunoblotting. Results: Forty proteins were associated with age at false discovery rate-corrected P<0.05, including proteins that regulate cell adhesion, the cytoskeleton, amino acid and lipid metabolism, and ribosomal subunits. TMEM106B, a regulator of lysosomal and oligodendrocyte function, was regulated with greatest effect size. The increase in TMEM106B levels with ageing was specific to carriers of the rs1990622-A allele in the TMEM106B gene that increases risk for frontotemporal dementia, Alzheimer’s disease, Parkinson’s disease, and hippocampal sclerosis with ageing. Rs1990622-A was also associated with higher TMEM106B fibril content. Hippocampal lipids were not significantly affected by APOE genotype, however levels of myelin-enriched sulfatides and hexosylceramides were significantly lower, and polyunsaturated phospholipids were higher, in rs1990622-A carriers after controlling for APOE genotype. Conclusions: Our study demonstrates that TMEM106B protein abundance is increased with brain ageing in humans, establishes that dementia risk allele rs1990622-A predisposes to TMEM106B fibril formation in the hippocampus, and provides the first evidence that rs1990622-A affects brain lipid homeostasis, particularly myelin lipids. Our data suggests that TMEM106B is one of a growing list of major dementia risk genes that affect glial lipid metabolism.","INSTITUTE":"University of Sydney","DEPARTMENT":"Faculty of Medicine and Health","LAST_NAME":"Lee","FIRST_NAME":"JUN YUP","ADDRESS":"John Hopkins Dr, Camperdown NSW 2050","EMAIL":"jlee4934@uni.sydney.edu.au","PHONE":"0401042970"},

"STUDY":{"STUDY_TITLE":"The major TMEM106B dementia risk allele affects TMEM106B protein levels, fibril formation, and myelin lipid homeostasis in the ageing human hippocampus","STUDY_SUMMARY":"Background: The risk for dementia increases exponentially from the seventh decade of life. Identifying and understanding the biochemical changes that sensitize the ageing brain to neurodegeneration will provide new opportunities for dementia prevention and treatment. This study aimed to determine how ageing and major genetic risk factors for dementia affect the hippocampal proteome and lipidome of neurologically-normal humans over the age of 65. The hippocampus was chosen as it is highly susceptible to atrophy with ageing and in several neurodegenerative diseases. Methods: Mass spectrometry-based proteomic and lipidomic analysis of CA1 hippocampus samples from 74 neurologically normal human donors, aged 66 – 104, was used in combination with multiple regression models and gene set enrichment analysis to identify age-dependent changes in the proteome and lipidome. ANOVA was used to test the effect of major dementia risk alleles in the TMEM106B and APOE genes on the hippocampal proteome and lipidome, adjusting for age, gender, and post-mortem interval. Fibrillar C-terminal TMEM106B fragments were isolated using sarkosyl fractionation and quantified by immunoblotting. Results: Forty proteins were associated with age at false discovery rate-corrected P<0.05, including proteins that regulate cell adhesion, the cytoskeleton, amino acid and lipid metabolism, and ribosomal subunits. TMEM106B, a regulator of lysosomal and oligodendrocyte function, was regulated with greatest effect size. The increase in TMEM106B levels with ageing was specific to carriers of the rs1990622-A allele in the TMEM106B gene that increases risk for frontotemporal dementia, Alzheimer’s disease, Parkinson’s disease, and hippocampal sclerosis with ageing. Rs1990622-A was also associated with higher TMEM106B fibril content. Hippocampal lipids were not significantly affected by APOE genotype, however levels of myelin-enriched sulfatides and hexosylceramides were significantly lower, and polyunsaturated phospholipids were higher, in rs1990622-A carriers after controlling for APOE genotype. Conclusions: Our study demonstrates that TMEM106B protein abundance is increased with brain ageing in humans, establishes that dementia risk allele rs1990622-A predisposes to TMEM106B fibril formation in the hippocampus, and provides the first evidence that rs1990622-A affects brain lipid homeostasis, particularly myelin lipids. Our data suggests that TMEM106B is one of a growing list of major dementia risk genes that affect glial lipid metabolism.","INSTITUTE":"University of Sydney","LAST_NAME":"Lee","FIRST_NAME":"JUN YUP","ADDRESS":"John Hopkins Dr, Camperdown NSW 2050","EMAIL":"jlee4934@uni.sydney.edu.au","PHONE":"0401042970"},

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"SUBJECT_SAMPLE_FACTORS":[
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},
{
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},
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},
{
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},
{
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},
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},
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},
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{
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{
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{
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},
{
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{
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},
{
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},
{
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},
{
"Subject ID":"-",
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},
{
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},
{
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"Additional sample data":{"Sex":"M","Age":"88","PMI":"9","RAW_FILE_NAME":"1","RAW_FILE_NAME":"73_pos.raw","RAW_FILE_NAME":"73_neg.raw"}
},
{
"Subject ID":"-",
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"Additional sample data":{"Sex":"F","Age":"81","PMI":"28","RAW_FILE_NAME":"2","RAW_FILE_NAME":"74_pos.raw","RAW_FILE_NAME":"74_neg.raw"}
},
{
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"Factors":{"APOE genotype":"e3/e3","TMEM SNP":"A/A"},
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},
{
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"Sample ID":"76",
"Factors":{"APOE genotype":"e3/e3","TMEM SNP":"A/A"},
"Additional sample data":{"Sex":"M","Age":"73","PMI":"39","RAW_FILE_NAME":"1","RAW_FILE_NAME":"76_pos.raw","RAW_FILE_NAME":"76_neg.raw"}
},
{
"Subject ID":"-",
"Sample ID":"77",
"Factors":{"APOE genotype":"e3/e3","TMEM SNP":"NA"},
"Additional sample data":{"Sex":"M","Age":"86","PMI":"15","RAW_FILE_NAME":"1","RAW_FILE_NAME":"77_pos.raw","RAW_FILE_NAME":"77_neg.raw"}
},
{
"Subject ID":"-",
"Sample ID":"78",
"Factors":{"APOE genotype":"e3/e3","TMEM SNP":"A/G"},
"Additional sample data":{"Sex":"F","Age":"80","PMI":"21","RAW_FILE_NAME":"1","RAW_FILE_NAME":"78_pos.raw","RAW_FILE_NAME":"78_neg.raw"}
},
{
"Subject ID":"-",
"Sample ID":"79",
"Factors":{"APOE genotype":"e3/e3","TMEM SNP":"G/G"},
"Additional sample data":{"Sex":"M","Age":"83","PMI":"47","RAW_FILE_NAME":"0","RAW_FILE_NAME":"79_pos.raw","RAW_FILE_NAME":"79_neg.raw"}
},
{
"Subject ID":"-",
"Sample ID":"80",
"Factors":{"APOE genotype":"e2/e3","TMEM SNP":"A/G"},
"Additional sample data":{"Sex":"M","Age":"85","PMI":"25","RAW_FILE_NAME":"2","RAW_FILE_NAME":"80_pos.raw","RAW_FILE_NAME":"80_neg.raw"}
},
{
"Subject ID":"-",
"Sample ID":"12",
"Factors":{"APOE genotype":"e3/e3","TMEM SNP":"A/G"},
"Additional sample data":{"Sex":"F","Age":"77","PMI":"18","RAW_FILE_NAME":"3","RAW_FILE_NAME":"12_pos.raw","RAW_FILE_NAME":"12_neg.raw"}
},
{
"Subject ID":"-",
"Sample ID":"57",
"Factors":{"APOE genotype":"e3/e3","TMEM SNP":"G/G"},
"Additional sample data":{"Sex":"M","Age":"69","PMI":"14","RAW_FILE_NAME":"0","RAW_FILE_NAME":"57_pos.raw","RAW_FILE_NAME":"57_neg.raw"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Homogenates were prepared by placing 10-20 mg of frozen tissue into 500 L of 20 mM Hepes pH 7.4, 10 mM KCl, EDTA-free cOmplete protease inhibitor cocktail (Roche), 1 mM dithiothreitol, and 3 mM β-glycerophosphate, and ultrasonicating for 5 min (30 s on/30 s off) at 4 °C in a Qsonica Q800R2 sonicating bath. Homogenates were cleared by centrifugation at 1000g for 10 min at 4 °C, and the supernatant was stored at -80 °C in 100 µL aliquots. Total protein concentrations were determined using the Bradford assay (Bio-Rad). Lipids were extracted from hippocampal homogenates (~100 µg protein) using a one phase butanol-methanol (BUME) (1:1 v/v) procedure [34]. The following internal standards were added to each sample: 5 nmoles of d19:0/19:0 PC, 2 nmoles of d18:1/17:0 SM, d18:1/12:0 HexCer, 17:0/17:0 PS, 17:0/17:0 PE, 17:0/17:0/17:0 TG, 17:0/17:0 PG, 14:0/14:0/14:0/14:0 cardiolipin, 17:0 cholesteryl ester, 1 nmole of 17:0/17:0 PA, d18:1/15:0-d7 PI, cholesterol-d7, 0.5 nmoles of d18:1/17:0 ST, d18:1/17:0 ceramide, 17:1 LPE, 17:1 LPS, 17:0 LPC, 18:1-d7 monoacylglycerol, d18:1/15:0-d7 diacylglycerol, d18:1/12:0 Hex2Cer, and 0.2 nmoles of 17:1 sphingosine, 17:1 sphingosine 1-phosphate, d3-16:0 acylcarnitine, 17:0 LPA.","SAMPLE_TYPE":"Brain"},

"TREATMENT":{"TREATMENT_SUMMARY":"No treatment. This study investigated the effects of age, TMEM106B rs1990622 SNP, and APOE genotype on the lipidome of CA1 hippocampus from physiologically ageing individuals."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Lipids were extracted from hippocampal homogenates (~100 µg protein) using a one phase butanol-methanol (BUME) (1:1 v/v) procedure [34]. The following internal standards were added to each sample: 5 nmoles of d19:0/19:0 PC, 2 nmoles of d18:1/17:0 SM, d18:1/12:0 HexCer, 17:0/17:0 PS, 17:0/17:0 PE, 17:0/17:0/17:0 TG, 17:0/17:0 PG, 14:0/14:0/14:0/14:0 cardiolipin, 17:0 cholesteryl ester, 1 nmole of 17:0/17:0 PA, d18:1/15:0-d7 PI, cholesterol-d7, 0.5 nmoles of d18:1/17:0 ST, d18:1/17:0 ceramide, 17:1 LPE, 17:1 LPS, 17:0 LPC, 18:1-d7 monoacylglycerol, d18:1/15:0-d7 diacylglycerol, d18:1/12:0 Hex2Cer, and 0.2 nmoles of 17:1 sphingosine, 17:1 sphingosine 1-phosphate, d3-16:0 acylcarnitine, 17:0 LPA."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Thermo Vanquish","COLUMN_NAME":"Waters ACQUITY UPLC BEH Amide (100 x 2.1mm,1.7um)","SOLVENT_A":"60% acetonitrile/40% water; 10 mM ammonium formate; 0.1% formic acid","SOLVENT_B":"10% acetonitrile/90% isopropanol; 10 mM ammonium formate; 0.1% formic acid","FLOW_GRADIENT":"Run time was 25 min using a binary gradient starting at 20% B for 3 min, increasing to 45% B from 3 - 5.5 min, then to 65% B from 5.5 - 8 min, then to 85% B from 8 - 13 min, then to 100% B from 13 - 14 min. The gradient was held at 100% B from 14 - 20 min, then decreased to 20% B and held to 25 min.","FLOW_RATE":"0.28 ml/min","COLUMN_TEMPERATURE":"45"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive HF-X Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"UNSPECIFIED","MS_COMMENTS":"Each sample injected twice, once in positive and once in negative."},

"MS_METABOLITE_DATA":{
"Units":"% Total",

"Data":[{"Metabolite":"LPA 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18:0","1":"0.008021568","2":"0.011526061","5":"0.010242958","6":"0.011194011","7":"0.011436461","8":"0.010389926","9":"0.017198621","10":"0.010220923","11":"0.014456121","13":"0.007381477","14":"0.00871408","15":"0.006506105","16":"0.009721544","17":"0.009827112","18":"0.011013222","19":"0.01053503","20":"0.010949058","21":"0.006655583","22":"0.011357585","23":"0.011562506","24":"0.007684504","25":"0.009694462","26":"0.012428031","27":"0.008342216","28":"0.008880426","29":"0.007814254","30":"0.009689974","31":"0.011609598","32":"0.00945288","33":"0.008812279","34":"0.010188896","35":"0.010312781","36":"0.007854586","37":"0.007906661","38":"0.010906771","39":"0.009135886","41":"0.009668496","42":"0.008386608","43":"0.010933554","44":"0.01090911","45":"0.010597259","46":"0.011475107","47":"0.008194769","48":"0.012244699","49":"0.009143366","50":"0.008857178","51":"0.009239073","52":"0.009586143","53":"0.012319833","54":"0.012397789","55":"0.008269416","56":"0.009771703","58":"0.006651003","59":"0.009337803","60":"0.010035566","61":"0.008761808","62":"0.007347851","63":"0.008417721","64":"0.006692675","65":"0.007509358","66":"0.005075756","67":"0.010605628","68":"0.00993032","69":"0.009894292","70":"0.008924874","71":"0.008191806","73":"0.010709719","74":"0.012364117","75":"0.014468236","76":"0.007691835","77":"0.011564126","78":"0.010423919","79":"0.007635414","80":"0.00951523","12":"0.01176175","57":"NA"},{"Metabolite":"LPA 18:1","1":"0.005170675","2":"0.005604433","5":"0.006592908","6":"0.003857701","7":"0.005080853","8":"0.008287315","9":"0.004808641","10":"0.004789437","11":"0.006847817","13":"0.005030624","14":"0.004219079","15":"0.003862023","16":"0.003842158","17":"0.010311235","18":"0.005391343","19":"0.005422889","20":"0.004364388","21":"0.007683987","22":"0.005442012","23":"0.007009028","24":"0.004775513","25":"0.004175344","26":"0.004540524","27":"0.008394982","28":"0.004242133","29":"0.005073881","30":"0.003506802","31":"0.003643754","32":"0.004600843","33":"0.005398009","34":"0.007600326","35":"0.004947669","36":"0.006734262","37":"0.00718697","38":"0.005703276","39":"0.004316601","41":"0.006666082","42":"0.003903153","43":"0.004223196","44":"0.008045203","45":"0.004547486","46":"0.008981403","47":"0.005874689","48":"0.007478973","49":"0.008033384","50":"0.007294653","51":"0.006242603","52":"0.006802548","53":"0.006150823","54":"0.007071084","55":"0.005170612","56":"0.006162138","58":"0.005266354","59":"0.004496769","60":"0.00562806","61":"0.007803938","62":"0.006451517","63":"0.00550684","64":"0.003799018","65":"0.003408521","66":"0.011804666","67":"0.002928005","68":"0.004164325","69":"0.004391661","70":"0.003719876","71":"0.003643836","73":"0.008033825","74":"0.00579276","75":"0.006610906","76":"0.006220201","77":"0.004567963","78":"0.003487716","79":"0.008497125","80":"0.00447131","12":"0.010295996","57":"0.00209851"},{"Metabolite":"LPA 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}

}