Showing papers by "Garth J. S. Cooper published in 2019"

Regional protein expression in human Alzheimer’s brain correlates with disease severity

Abstract: Alzheimer's disease (AD) is a progressive neurodegenerative disorder that currently affects 36 million people worldwide with no effective treatment available. Development of AD follows a distinctive pattern in the brain and is poorly modelled in animals. Therefore, it is vital to widen the spatial scope of the study of AD and prioritise the study of human brains. Here we show that functionally distinct human brain regions display varying and region-specific changes in protein expression. These changes provide insights into the progression of disease, novel AD-related pathways, the presence of a gradient of protein expression change from less to more affected regions and a possibly protective protein expression profile in the cerebellum. This spatial proteomics analysis provides a framework which can underpin current research and open new avenues to enhance molecular understanding of AD pathophysiology, provide new targets for intervention and broaden the conceptual frameworks for future AD research.

Cerebral Vitamin B5 (D-Pantothenic Acid) Deficiency as a Potential Cause of Metabolic Perturbation and Neurodegeneration in Huntington’s Disease

Abstract: Huntington’s disease (HD) is a neurodegenerative disorder caused by an expanded CAG repeat in exon 1 of the HTT gene. HD usually manifests in mid-life with loss of GABAergic projection neurons from the striatum accompanied by progressive atrophy of the putamen followed by other brain regions, but linkages between the genetics and neurodegeneration are not understood. We measured metabolic perturbations in HD-human brain in a case-control study, identifying pervasive lowering of vitamin B5, the obligatory precursor of coenzyme A (CoA) that is essential for normal intermediary metabolism. Cerebral pantothenate deficiency is a newly-identified metabolic defect in human HD that could potentially: (i) impair neuronal CoA biosynthesis; (ii) stimulate polyol-pathway activity; (iii) impair glycolysis and tricarboxylic acid cycle activity; and (iv) modify brain-urea metabolism. Pantothenate deficiency could lead to neurodegeneration/dementia in HD that might be preventable by treatment with vitamin B5.

Cognitive dysfunction in diabetic rats is prevented by pyridoxamine treatment. A multidisciplinary investigation.

Abstract: Objective The impact of diabetes mellitus on the central nervous system is less widely studied than in the peripheral nervous system, but there is increasing evidence that it elevates the risk of developing cognitive deficits. The aim of this study was to characterize the impact of experimental diabetes on the proteome and metabolome of the hippocampus. We tested the hypothesis that the vitamin B6 isoform pyridoxamine is protective against functional and molecular changes in diabetes. Methods We tested recognition memory using the novel object recognition (NOR) test in streptozotocin (STZ)-induced diabetic, age-matched control, and pyridoxamine- or insulin-treated diabetic male Wistar rats. Comprehensive untargeted metabolomic and proteomic analyses, using gas chromatography-mass spectrometry and iTRAQ-enabled protein quantitation respectively, were utilized to characterize the molecular changes in the hippocampus in diabetes. Results We demonstrated diabetes-specific, long-term (but not short-term) recognition memory impairment and that this deficit was prevented by insulin or pyridoxamine treatment. Metabolomic analysis showed diabetes-associated changes in 13/82 identified metabolites including polyol pathway intermediates glucose (9.2-fold), fructose (4.9-fold) and sorbitol (5.2-fold). We identified and quantified 4807 hippocampal proteins; 806 were significantly altered in diabetes. Pathway analysis revealed significant alterations in cytoskeletal components associated with synaptic plasticity, glutamatergic signaling, oxidative stress, DNA damage and FXR/RXR activation pathways in the diabetic rat hippocampus. Conclusions Our data indicate a protective effect of pyridoxamine against diabetes-induced cognitive deficits, and our comprehensive ‘omics datasets provide insight into the pathogenesis of cognitive dysfunction enabling development of further mechanistic and therapeutic studies.

Tissue-Specific Sample Dilution: An Important Parameter to Optimise Prior to Untargeted LC-MS Metabolomics

Abstract: When developing a sample preparation protocol for LC–MS untargeted metabolomics of a new sample matrix unfamiliar to the laboratory, selection of a suitable injection concentration is rarely described. Here we developed a simple workflow to address this issue prior to untargeted LC–MS metabolomics using pig adipose tissue and liver tissue. Bi-phasic extraction was performed to enable simultaneous optimisation of parameters for analysis of both lipids and polar extracts. A series of diluted pooled samples were analysed by LC–MS and used to evaluate signal linearity. Suitable injected concentrations were determined based on both the number of reproducible features and linear features. With our laboratory settings, the optimum concentrations of tissue mass to reconstitution solvent of liver and adipose tissue lipid fractions were found to be 125 mg/mL and 7.81 mg/mL respectively, producing 2811 (ESI+) and 4326 (ESI−) linear features from liver, 698 (ESI+) and 498 (ESI−) linear features from adipose tissue. For analysis of the polar fraction of both tissues, 250 mg/mL was suitable, producing 403 (ESI+) and 235 (ESI−) linear features from liver, 114 (ESI+) and 108 (ESI−) linear features from adipose tissue. Incorrect reconstitution volumes resulted in either severe overloading or poor linearity in our lipid data, while too dilute polar fractions resulted in a low number of reproducible features (<50) compared to hundreds of reproducible features from the optimum concentration used. Our study highlights on multiple matrices and multiple extract and chromatography types, the critical importance of determining a suitable injected concentration prior to untargeted LC–MS metabolomics, with the described workflow applicable to any matrix and LC–MS system.

Altered metabolic gene expression in the brain of a triprolyl-human amylin transgenic mouse model of type 2 diabetes.

Abstract: Type 2 diabetes mellitus is a major health concern worldwide; however, the molecular mechanism underlying its development is poorly understood. The hormone amylin is postulated to be involved, as human amylin forms amyloid in the pancreases of diabetic patients, and oligomers have been shown to be cytotoxic to β-cells. As rodent amylin is non-amyloidogenic, mice expressing human amylin have been developed to investigate this hypothesis. However, it is not possible to differentiate the effects of amylin overexpression from β-cell loss in these models. We have developed transgenic mice that overexpress [25, 28, 29 triprolyl]human amylin, a non-amyloidogenic variant of amylin, designated the Line 44 model. This model allows us to investigate the effects of chronic overexpression of non-cytotoxic amylin. We characterised this model and found it developed obesity, hyperglycaemia and hyperinsulinaemia. This phenotype was associated with alterations in the expression of genes involved in the amylin, insulin and leptin signalling pathways within the brain. This included genes such as c-Fos (a marker of amylin activation); Socs3 (a leptin inhibitor); and Cart, Pomc and Npy (neuropeptides that control appetite). We also examined Socs3 protein expression and phosphorylated Stat3 to determine if changes at the mRNA level would be reflected at the protein level.

Low Energy Diet-induced and Bariatric Surgery-induced Weight Loss Decreases Branched-chain and Aromatic Amino Acids in Plasma and Tissue (P21-078-19)

Abstract: Objectives Plasma levels of branched-chain amino acids (BCAA) and aromatic amino acids (AAA) phenylalanine (phe) and tyrosine (tyr) have been associated with obesity, insulin resistance and risk of type 2 diabetes. This study aimed to investigate the response of circulating plasma and tissue levels of BCAA and AAA to weight loss, and to correlate the level of these metabolites in plasma and tissue in obese women. Methods 28 obese (mean BMI 46.2 kg/m2) women underwent low energy diet (LED)-induced weight loss (-9.2 ± 4.2 kg) followed by bariatric surgery-induced weight loss (-23.6 ± 2.5 kg). Plasma at baseline (t0), post-LED/pre-surgery (t1) and 6-month post-surgery (t2) as well as biopsies of subcutaneous abdomen adipose tissue (SAfat), superficial thigh adipose tissue (Tfat) and vastus lateralis thigh muscle (Tmuscle) at both t1 and t2 were collected, and profiled using mass spectrometry-based metabolomics approach. Paired t-tests were applied to assess between-timepoint differences, and Pearson correlation used to calculate correlation coefficient of metabolite levels between plasma and tissue. Results Plasma BCAA and AAA were all significantly reduced post-LED at t1 (fold-change of 0.76-0.85 for val, leu, ile, tyr and phe, P < 0.05) and 6-month post-surgery at t2 (fold-change of 0.74-0.85 for val, leu, ile, tyr and phe, P < 0.05) as compared to baseline t0; but not significant between t1 and t2, although trends of decrease were observed. Among the 3 tissue biopsies, only SAfat showed significantly decreased levels of tyr, leu and ile at t2 compared to t1 (fold-change for tyr 0.63, leu 0.66, ile 0.68, P < 0.05). In addition, plasma levels of val and ile were correlated with Tfat levels at both t1 and t2 (r2 = 0.47-0.57), and that of val, ile and leu were correlated with Tmuscle at t1 only (r2 = 0.64-0.67). Conclusions Circulating levels of BCAA and AAA were decreased by weight loss interventions. The decrease following an LED program was sustained after bariatric surgery without further significant decrease. Bariatric surgery also decreased BCAA levels in SAfat; moreover, our data suggested that plasma BCAA levels correlated well with peripheral tissue Tfat and Tmuscle. Funding Sources The New Zealand National Science Challenge High-Value Nutrition program.