Genetic regulation of gene expression and splicing during a 10-year period of human aging.

BACKGROUND: Molecular and cellular changes are intrinsic to aging and age-related diseases. Prior cross-sectional studies have investigated the combined effects of age and genetics on gene expression and alternative splicing; however, there has been no long-term, longitudinal characterization of these molecular changes, especially in older age. RESULTS: We perform RNA sequencing in whole blood from the same individuals at ages 70 and 80 to quantify how gene expression, alternative splicing, and their genetic regulation are altered during this 10-year period of advanced aging at a population and individual level. We observe that individuals are more similar to their own expression profiles later in life than profiles of other individuals their own age. We identify 1291 and 294 genes differentially expressed and alternatively spliced with age, as well as 529 genes with outlying individual trajectories. Further, we observe a strong correlation of genetic effects on expression and splicing between the two ages, with a small subset of tested genes showing a reduction in genetic associations with expression and splicing in older age. CONCLUSIONS: These findings demonstrate that, although the transcriptome and its genetic regulation is mostly stable late in life, a small subset of genes is dynamic and is characterized by a reduction in genetic regulation, most likely due to increasing environmental variance with age.

CRISPR-Cas9-mediated knockout of SPRY2 in human hepatocytes leads to increased glucose uptake and lipid droplet accumulation.

BACKGROUND: The prevalence of obesity and its comorbidities, including type 2 diabetes mellitus (T2DM), is dramatically increasing throughout the world; however, the underlying aetiology is incompletely understood. Genome-wide association studies (GWAS) have identified hundreds of genec susceptibility loci for obesity and T2DM, although the causal genes and mechanisms are largely unknown. SPRY2 is a candidate gene identified in GWAS of body fat percentage and T2DM, and has recently been linked to insulin production in pancreatic ß-cells. In the present study, we aimed to further understand SPRY2 via functional characterisation in HepG2 cells, an in vitro model of human hepatocytes widely used to investigate T2DM and insulin resistance. METHODS: CRISPR-Cas9 genome editing was used to target SPRY2 in HepG2 cells, and the functional consequences of SPRY2 knockout (KO) and overexpression subsequently assessed using glucose uptake and lipid droplet assays, measurement of protein kinase phosphorylation and RNA sequencing. RESULTS: The major functional consequence of SPRY2 KO was a significant increase in glucose uptake, along with elevated lipid droplet accumulation. These changes were attenuated, but not reversed, in cells overexpressing SPRY2. Phosphorylation of protein kinases across key signalling pathways (including Akt and mitogen activated protein kinases) was not altered after SPRY2 KO. Transcriptome profiling in SPRY2 KO and mock (control) cells revealed a number of differentially expressed genes related to cholesterol biosynthesis, cell cycle regulation and cellular signalling pathways. Phospholipase A2 group IIA (PLA2G2A) mRNA level was subsequently validated as significantly upregulated following SPRY2 KO, highlighting this as a potential mediator downstream of SPRY2. CONCLUSION: These findings suggest a role for SPRY2 in glucose and lipid metabolism in hepatocytes and contribute to clarifying the function of this gene in the context of metabolic diseases.

Glucose challenge metabolomics implicates medium-chain acylcarnitines in insulin resistance.

Insulin resistance (IR) predisposes to type 2 diabetes and cardiovascular disease but its causes are incompletely understood. Metabolic challenges like the oral glucose tolerance test (OGTT) can reveal pathogenic mechanisms. We aimed to discover associations of IR with metabolite trajectories during OGTT. In 470 non-diabetic men (age 70.6 ± 0.6 years), plasma samples obtained at 0, 30 and 120 minutes during an OGTT were analyzed by untargeted liquid chromatography-mass spectrometry metabolomics. IR was assessed with the hyperinsulinemic-euglycemic clamp method. We applied age-adjusted linear regression to identify metabolites whose concentration change was related to IR. Nine trajectories, including monounsaturated fatty acids, lysophosphatidylethanolamines and a bile acid, were significantly associated with IR, with the strongest associations observed for medium-chain acylcarnitines C10 and C12, and no associations with L-carnitine or C2-, C8-, C14- or C16-carnitine. Concentrations of C10- and C12-carnitine decreased during OGTT with a blunted decline in participants with worse insulin resistance. Associations persisted after adjustment for obesity, fasting insulin and fasting glucose. In mouse 3T3-L1 adipocytes exposed to different acylcarnitines, we observed blunted insulin-stimulated glucose uptake after treatment with C10- or C12-carnitine. In conclusion, our results identify medium-chain acylcarnitines as possible contributors to IR.

The myeloperoxidase-derived oxidant hypothiocyanous acid inhibits protein tyrosine phosphatases via oxidation of key cysteine residues.

Phosphorylation of protein tyrosine residues is critical to cellular processes, and is regulated by kinases and phosphatases (PTPs). PTPs contain a redox-sensitive active site Cys residue, which is readily oxidized. Myeloperoxidase, released from activated leukocytes, catalyzes thiocyanate ion (SCN(-)) oxidation by H2O2 to form hypothiocyanous acid (HOSCN), an oxidant that targets Cys residues. Dysregulated phosphorylation and elevated MPO levels have been associated with chronic inflammatory diseases where HOSCN can be generated. Previous studies have shown that HOSCN inhibits isolated PTP1B and induces cellular dysfunction in cultured macrophage-like cells. The present study extends this previous work and shows that physiologically-relevant concentrations of HOSCN alter the activity and structure of other members of the wider PTP family (including leukocyte antigen-related PTP, PTP-LAR; T-cell PTP, TC-PTP; CD45 and Src homology phosphatase-1, Shp-1) by targeting Cys residues. Isolated PTP activity, and activity in lysates of human monocyte-derived macrophages (HMDM) was inhibited by 0-100 µM HOSCN with this being accompanied by reversible oxidation of Cys residues, formation of sulfenic acids or sulfenyl-thiocyanates (detected by Western blotting, and LC-MS as dimedone adducts), and structural changes. LC-MS/MS peptide mass-mapping has provided data on the modified Cys residues in PTP-LAR. This study indicates that inflammation-induced oxidants, and particularly myeloperoxidase-derived species, can modulate the activity of multiple members of the PTP superfamily via oxidation of Cys residues to sulfenic acids. This alteration of the balance of PTP/kinase activity may perturb protein phosphorylation and disrupt cell signaling with subsequent induction of apoptosis at sites of inflammation.

Circulating microRNAs as noninvasive diagnostic biomarkers of liver disease in children with cystic fibrosis.

OBJECTIVES: Cystic fibrosis liver disease (CFLD), resulting from progressive hepatobiliary fibrosis, causes significant morbidity and mortality in up to 20% of children with cystic fibrosis (CF). Both pathogenesis and early detection of CFLD are elusive. Current diagnostic procedures to detect early CFLD and stage fibrosis severity are inadequate. Recent studies highlight a role for microRNAs (miRNAs) in the pathogenesis of many diseases and have suggested that serum miRNAs could be used as diagnostic biomarkers. METHODS: We profiled circulating serum miRNA levels in patients with CFLD (n = 52), patients with CF without liver disease (CFnoLD, n = 30), and non-CF pediatric controls (n = 20). Extracted RNA was subjected to polymerase chain reaction (PCR) array of 84 miRNAs detectable in human serum. Seven candidate miRNAs identified were validated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), normalizing data to geNorm-determined stable reference genes, miR-19b and miR-93. RESULTS: miR-122 was significantly elevated in patients with CFLD versus patients with CFnoLD and controls (P < 0.0001). miR-25 (P = 0.0011) and miR-21 (P = 0.0133) were elevated in patients with CFnoLD versus patients with CFLD and controls. CFLD was discriminated by both miR-122 (area under the curve [AUC] 0.71, P = 0.002) and miR-25 (AUC 0.65, P = 0.026). Logistic regression combining 3 miRNAs (-122, -25, -21) was greatly predictive of detecting CFLD (AUC 0.78, P < 0.0001). A combination of 6 miRNAs (-122, -21, -25, -210, -148a, -19a) distinguished F0 from F3-F4 fibrosis (AUC 0.73, P = 0.04), and miR-210 combined with miR-22 distinguished F0 fibrosis from any fibrosis, that is, F1-F4 (AUC 0.72, P = 0.02). CONCLUSIONS: These data provide the first evidence of changes to circulating miRNA levels in CF, suggesting that serum-based miRNA analysis may complement and extend current CFLD screening strategies with potential to predict early hepatic fibrosis.

Myeloperoxidase-derived oxidants rapidly oxidize and disrupt zinc-cysteine/histidine clusters in proteins.

Zinc is an abundant cellular transition metal ion, which binds avidly to protein cysteine (Cys) and histidine (His) residues to form zinc-Cys/His clusters; these play a key role in the function of many proteins (e.g., DNA binding and repair enzymes, transcription factors, nitric oxide synthase). Leukocyte-derived myeloperoxidase generates powerful oxidants including hypochlorous (HOCl), hypobromous (HOBr), and hypothiocyanous (HOSCN) acids from H(2)O(2) and (pseudo)halide ions. Excessive or misplaced formation of these species is associated with cellular dysfunction, apoptosis and necrosis, and multiple inflammatory diseases. HOCl and HOBr react rapidly with sulfur-containing compounds, and HOSCN reacts specifically with thiols. Consequently, we hypothesized that zinc-Cys/His clusters would be targets for these oxidants, and the activity of such enzymes would be perturbed. This hypothesis has been tested using yeast alcohol dehydrogenase (YADH), which contains a well-characterized Zn(1)Cys(2)His(1) cluster. Incubation of YADH with pathologically relevant concentrations of HOSCN, HOCl, and HOBr resulted in rapid oxidation of the protein (rate constants, determined by competition kinetics, for reaction of HOCl and HOSCN with YADH being (3.3±0.9)×10(8) and (2.9±0.4)×10(4) M(-1) s(-1) per YADH monomer, respectively), loss of enzyme activity, Zn(2+) release, changes in protein structure (particularly formation of disulfide cross-links), and oxidation of Cys residues. The loss of enzyme activity correlated with Zn(2+) release, loss of thiols, and changes in protein structure. We conclude that exposure of zinc-Cys/His clusters to inflammatory oxidants can result in impaired protein activity, thiol oxidation, and Zn(2+) release. These reactions may contribute to inflammation-induced tissue damage.

Myeloperoxidase-derived oxidants inhibit sarco/endoplasmic reticulum Ca2+-ATPase activity and perturb Ca2+ homeostasis in human coronary artery endothelial cells.

The sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) plays a critical role in Ca(2+) homeostasis via sequestration of this ion in the sarco/endoplasmic reticulum. The activity of this pump is inhibited by oxidants and impaired in aging tissues and cardiovascular disease. We have shown previously that the myeloperoxidase (MPO)-derived oxidants HOCl and HOSCN target thiols and mediate cellular dysfunction. As SERCA contains Cys residues critical to ATPase activity, we hypothesized that HOCl and HOSCN might inhibit SERCA activity, via thiol oxidation, and increase cytosolic Ca(2+) levels in human coronary artery endothelial cells (HCAEC). Exposure of sarcoplasmic reticulum vesicles to preformed or enzymatically generated HOCl and HOSCN resulted in a concentration-dependent decrease in ATPase activity; this was also inhibited by the SERCA inhibitor thapsigargin. Decomposed HOSCN and incomplete MPO enzyme systems did not decrease activity. Loss of ATPase activity occurred concurrent with oxidation of SERCA Cys residues and protein modification. Exposure of HCAEC, with or without external Ca(2+), to HOSCN or HOCl resulted in a time- and concentration-dependent increase in intracellular Ca(2+) under conditions that did not result in immediate loss of cell viability. Thapsigargin, but not inhibitors of plasma membrane or mitochondrial Ca(2+) pumps/channels, completely attenuated the increase in intracellular Ca(2+) consistent with a critical role for SERCA in maintaining endothelial cell Ca(2+) homeostasis. Angiotensin II pretreatment potentiated the effect of HOSCN at low concentrations. MPO-mediated modulation of intracellular Ca(2+) levels may exacerbate endothelial dysfunction, a key early event in atherosclerosis, and be more marked in smokers because of their higher SCN(-) levels.

Transient receptor potential melastatin 2 expression is increased following experimental traumatic brain injury in rats.

Traumatic brain injury (TBI) elicits a sequence of complex biochemical changes including oxidative stress, oedema, inflammation and excitotoxicity. These factors contribute to the high morbidity and mortality following TBI, although their underlying molecular mechanisms remain poorly understood. Transient receptor potential melastatin 2 (TRPM2) is a non-selective cation channel, highly expressed in the brain and immune cells. Recent studies have implicated TRPM2 channels in processes involving oxidative stress, inflammation and cell death. However, no studies have investigated the role of TRPM2 in TBI pathophysiology. In the present study, we have characterised TRPM2 mRNA and protein expression following experimental TBI. Adult male Sprague Dawley rats were injured using the impact-acceleration model of diffuse TBI with survival times between 5 and 5 days. Real-time RT-PCR (including reference gene validation studies) and semi-quantitative immunohistochemistry were used to quantify TRPM2 mRNA and protein levels, respectively, following TBI. Significant increases in TRPM2 mRNA and protein expression were observed in the cerebral cortex and hippocampus of injured animals, suggesting that TRPM2 may contribute to TBI injury processes such as oxidative stress, inflammation and neuronal death. Further characterisation of how TRPM2 may contribute to TBI pathophysiology is warranted.

Reference genes for normalising gene expression data in collagenase-induced rat intracerebral haemorrhage.

BACKGROUND: The mechanisms of brain injury following intracerebral haemorrhage (ICH) are incompletely understood. Gene expression studies using quantitative real-time RT-PCR following ICH have increased our understanding of these mechanisms, however the inconsistent results observed may be related to inappropriate reference gene selection. Reference genes should be stably expressed across different experimental conditions, however, transcript levels of common reference genes have been shown to vary considerably. Reference gene panels have therefore been proposed to overcome this potential confounder. RESULTS: The present study evaluated the stability of seven candidate reference genes in the striatum and overlying cortex of collagenase-induced ICH in rodents at survival times of 5 and 24 hours. Transcript levels of the candidate reference genes were quantified and ranked in order of stability using geNorm. When our gene of interest, transient receptor potential melastatin 2 (TRPM2), was normalised against each reference gene individually, TRPM2 mRNA levels were highly variable. When normalised to the four most stable reference genes selected for accurate normalisation of data, we found no significant difference between ICH and vehicle rats. CONCLUSION: The panel of reference genes identified in the present study will enable more accurate normalisation of gene expression data in the acute phase of experimental ICH.

Magnesium in acute and chronic brain injury: an update.

While brain free magnesium levels have been shown to decline in a number of acute and chronic brain pathologies, the mechanisms of such decline and the potential for magnesium administration as a therapeutic intervention are still unclear. In acute brain injury, magnesium therapy has failed in recent clinical trials of trauma, presumably because of an intact blood brain barrier at the time of administration reducing central penetration. Under such conditions, magnesium's peripheral effects on cardiovascular parameters may dominate over the central, and potentially neuroprotective, effects of the compound. In contrast, magnesium has been demonstrated to be beneficial in lacunar strokes, albeit that recent animal studies indicate that this effect is without any significant reduction of lesion size. Postnatal magnesium has also been shown to improve neurological outcome in term neonates with perinatal asphyxia, although this may be limited to cases of mild to moderate brain injury; no effect is observed following severe brain injury. Prenatal magnesium has been reported to be beneficial for outcome in very preterm infants, although this may only be at low doses. Combination therapies are also showing promise in experimental studies, with combined magnesium and mild hypothermia as well as magnesium and polyethylene glycol proving effective in ischemic stroke and in spinal cord injury, respectively. With respect to chronic brain injury, recent results indicate that magnesium deficient mice are susceptible to developing Parkinson's disease, which is consistent with earlier findings that magnesium deficiency over a number of generations is associated with the development of Parkinson's disease. The latter was associated with the appearance of variants of the TRPM channels. Our recent studies have shown that Parkinson's disease is associated with reduced TRPM2 and TRPM7 channel mRNA expression. Taken together, a more complete picture is emerging of the role of magnesium in brain injury, its therapeutic potential as well the mechanisms associated with its decline.

Validation of reference genes for normalization of real-time quantitative RT-PCR data in traumatic brain injury.

Careful validation of reference genes used for the normalization of real-time RT-PCR data is required to obtain accurate results regarding gene expression. We evaluated the stability of seven commonly used reference genes in the cerebral cortex and hippocampus of rats 3 days following traumatic brain injury (TBI). HPRT, SDHA, and GUSB were found to be the most stable reference genes in the cerebral cortex, whereas B2MG, TBP, and GAPDH were the most stable in the hippocampus. The use of three reference genes was determined to be the optimal number for accurate normalization of data. To illustrate this point, when our gene of interest, substance P (SP), was normalized against the three most stable reference genes in both brain areas, we found no significant difference between injured and uninjured rats at the 3-day time point. However, when our SP data were normalized to each reference gene individually, SP mRNA level was highly variable depending on the reference gene chosen. The results of the present study highlight the importance of validating reference genes to be used for real-time RT-PCR analysis. The use of the most stable reference genes presented here will allow more accurate normalization of gene expression data in TBI.

Are the transient receptor potential melastatin (TRPM) channels important in magnesium homeostasis following traumatic brain injury?

Traumatic brain injury (TBI) confers a major burden to Western society and effective treatments are urgently required to improve the long-term deficits that inflict TBI survivors. Depletion of intracellular Mg(2+) is a well-known phenomenon occurring after TBI and is associated with poor neurological outcome. However, despite success in pre-clinical experimental studies, therapies utilizing Mg(2+) have not always proven to be clinically effective. Recent evidence implicates members of the transient receptor potential melastatin (TRPM) channel family in processes leading to neuronal cell death following ischemic injury, however, the exact mechanism by which this occurs is not completely understood. Specifically, TRPM7 and TRPM6 are two channels that have been identified as potentially playing a role in regulating Mg(2+) homeostasis, although whether this role in magnesium regulation and neuronal injury is significant is controversial. The purpose of this review is to explore the relationship between TRPM family members and Mg(2+) homeostasis, including their potential involvement in secondary injury processes leading to cell death following TBI.