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Alternative splicing contributes to phenotypic diversity at multiple biological scales, and its dysregulation is implicated in both ageing and age-associated diseases in human. Cross-tissue variability in splicing further complicates its links to age-associated phenotypes and elucidating these links requires a comprehensive map of age-associated splicing changes across multiple tissues. Here, we generate such a map by analyzing ~8500 RNA-seq samples across 48 tissues in 544 individuals. Employing a stringent model controlling for multiple confounders, we identify 49,869 tissue-specific age-associated splicing events of 7 distinct types. We find that genome-wide splicing profile is a better predictor of biological age than the gene and transcript expression profiles, and furthermore, age-associated splicing provides additional independent contribution to age-associated complex diseases. We show that the age-associated splicing changes may be explained, in part, by concomitant age-associated changes of the upstream splicing factors. Finally, we show that our splicing-based model of age can successfully predict the relative ages of cells in 8 of the 10 paired longitudinal data as well as in 2 sets of cell passage data. Our study presents the first systematic investigation of age-associated splicing changes across tissues, and further strengthening the links between age-associated splicing and age-associated diseases.

Objective: To explore the effects of exercise and resveratrol on retinol binding protein 4( RBP4) mRNA and protein expression in visceral adipose tissue, plasma RBP4 concentration and blood glucose and insulin sensitivity of aged obese rats., Methods: To establish elderly obese SD rat model, 6 aged rats of natural growth were selected normal group, 24 obese rats were randomly divided into four groups: obesity control group, exercise group, resveratrol group, exercise combined with resveratrol group. For Resveratrol treatment, each rat was fed 52. 5 mg/( kg d), 5 times/week, continuous intervention for 8 weeks. After intervention, measured blood glucose, insulin sensitivity, visceral adipose tissue RBP4 mRNA expression( qRT-PCR), protein expression( Western blot) and plasma RBP4 concentration( ELISA)., Results: After 8week intervention, ( 1) RBP4 mRNA expression: The obesity group( 2. 63 ± 0. 45) was higher than that of the normal group( 2. 10 ± 0. 15)( P < 0. 05). The resveratrol group( 1. 84 ± 0. 33), exercise group( 1. 91 ± 0. 15), and exercise combined with resveratrol group( 2. 131 ± 0. 111) were lower than that of the obesity group( P < 0. 05, P < 0. 01). ( 2) RBP4 protein expression: The obesity group( 1. 346 ± 0. 025) was higher than that of the normal group( 1. 196 ± 0. 017)( P < 0. 01). The exercise group( 1. 025 ± 0. 006)was lower than that of the obesity group( P < 0. 01), The resveratrol group( 0. 735 ±0. 015) and exercise combined with resveratrol group( 0. 701 ± 0. 018) were lower than that of the exercise group( P < 0. 01). The exercise combined with resveratrol group was lower than that of resveratrol group( P < 0. 05). ( 3) Plasma RBP4 concentration: The obesity group [( 16. 00 ± 1. 54) μg/L]was higher than that of the normal group [( 13. 02± 2. 20) μg/L]( P < 0. 01). The exercise group [( 14. 76 ± 1. 56) μg/L] was lower than that of the obesity group, but the difference was not statistically significant. The resveratrol group [( 13. 59 ± 0. 07) μg/L]and exercise combined with resveratrol group[( 12. 98 ± 1. 69) μg/L] were lower than that of the obesity group( P < 0. 05, P <0. 01). ( 4) Blood glucose: The obesity group [( 17. 93 ± 6. 09) mmol/L]was higher than that of the normal group [( 11. 64 ± 3. 57) mmol/L ]( P < 0. 01). The exercise group [( 13. 36 ± 1. 82) mmol/L]was lower than that of the obesity group( P < 0. 05), the resveratrol group [( 15. 24 ± 2. 19) mmol/L] and exercise combined with resveratrol group [( 13. 95 ± 2. 26) mmol/L] were lower than that of the obesity group, but the difference was not statistically significant( P > 0. 05). ( 5) Insulin sensitivity: The obesity group( 0. 37 ± 0. 02) was lower than that of the normal group( 0. 39 ± 0. 02)( P < 0. 05). The resveratrol group( 0. 38 ± 0. 01) and the exercise group( 0. 39 ± 0. 02)were higher than that of the obesity group, but the difference was not statistically significant, the exercise combined with resveratrol group( 0. 39 ± 0. 15) was higher than that of the obesity group( P < 0. 05). Above indexes( except protein expression) were no significant difference between the each intervene groups( P > 0. 05)., Conclusion: RBP4 mRNA expression and protein expression in the visceral adipose tissue of aged obese rats, plasma RBP4 concentration and blood glucose were increased, and insulin sensitivity was decreased. Exercise and resveratrol could reduce the expression of RBP4 mRNA and protein expression and plasma RBP4 concentration. The simple exercise could significantly reduce the blood glucose and exercise combined with resveratrol could significantlyimprove insulin sensitivity. The decrease in protein expression, plasma RBP4 concentration and improve insulin sensitivity, exercise combined with resveratrol showed synergy.

Objective: To investigate effects of different intensity exercise prescription on visceral adipose tissue-derived serine protease inhibitor( Vaspin) mRNA and protein expression in visceral adipose tissue and plasma Vaspin concentration of elderly obese rats., Methods: SD rats as three stage of fattening divided the growth period, middle-aged and elderly, to establish rats model of elderly obesity, 6 natural growing aged rats were selected as blank control group, 18 elderly obese rats were randomly divided into 3 groups, 6 rats in each group: obese control group, low intensity exercise group, moderate intensity exercise group. Exercise prescription were 12 m /min × 15 min for each group, 4groups / times, rest between groups 5min, 60 min/time. 15 m/min×15 min for each group, 4 groups / times, rest between groups 5min, 60 min / time. Vaspin mRNA expression was measured by qRT-PCR and Vaspin protein expression was measured by WB in visceral adipose tissue and plasma Vaspin concentration was measured by ELISA after exercise., Results: After exercise intervention, (1) Vaspin mRNA(×103) expression:Obese control group(25. 761±11. 581) was higher than the control group(12. 161±5. 648). (P<0. 01), low intensity exercise group and moderate intensity exercise group((10. 482±2. 668), (13. 478±5. 115)) were lower than obese control group (P<0. 01), moderate intensity exercise group was higher than low intensity exercise group, but the difference was not statistically significant (P>0. 05). (2) Vaspin protein expression: Obese control group was higher than the control group (P<0. 01), low intensity exercise group was lower than obese control group (P<0. 05), but moderate intensity exercise group was higher than obese control group (P<0. 01). (3) Plasma Vaspin concentration: Obese control group((25. 000±3. 767) ng/mL) was higher than the control group((23. 765±3. 045) ng/m L), low intensity exercise group and moderate intensity exercise group((25. 637±5. 240) ng/mL and (26. 422±3. 4175) ng/mL) was higher than obese control group, but the difference were not statistically significant( P >0. 05). ( 4) Blood glucose and insulin sensitivity index: Blood glucose of obese control group was higher than the control group( P < 0. 01). Insulin sensitivity index of obese control group was lower than the control group, but the difference was not statistically significant( P > 0. 05). Blood glucose of low and moderate intensity exercise groups were lower than obese control group( P < 0. 05), but insulin sensitivity index of low and moderate intensity exercise groups were higher than obese control group( P < 0. 01)., Conclusion: Vaspin mRNA and protein expression in visceral adipose tissue and plasma Vaspin concentration of elderly obese rats are higher. Low intensity exercise can reduce Vaspin mRNA and protein expression, but increase exercise intensity may improve Vaspin mRNA and protein expression in visceral adipose tissue and plasma Vaspin concentration of elderly obese rats.

Objective: To exploer effects of exercise and resveratrol on visceral fat resistin expression and plasma resistin concentration in elderly obese rats., Methods: SD rats as three stage of fattening divided the growth period, middle-aged and elderly, toestablish rats model of elderly obesity. 6 natural growing aged rats were selected as blank control group( C). 24 elderly obese rats were randomly divided into 4 groups, 6 rats in each group: obese control group( OC), supplementary resveratrol group( OR), exercise group( OE), exercise combined with resveratrol group( OER). Running speed and time of animal were 15 m/min×15 min for each group, 4 groups/times, rest between groups5 min, 60min/time. For resveratrol treatment, each rat was fed 52. 5mg/kg, one time/day, 5 times/week, for 8 weeks. The content of blood glucose was measured, the insulin resistance was calculated, testing the expression of resistin mRNA in visceral adipose tissue and plasma Resistin concentration., Results: Body weight, visceral fat mass, body fat ratio, and blood glucose, and insulin resistance of OC group were higher than C group( P < 0. 01, P < 0. 05), Resistin mRNA×10~3 expression in visceral adipose tissue and plasma concentration of OC group(( 29. 80 ± 4. 91) μg/L and( 4. 30 ± 1. 93) μg/L, repectively) were higher than C group(( 23. 09 ± 6. 12) μg/L and( 3. 49 ± 0. 42) μg/L, repectively), but the difference was not statistically significant. Body weight, visceral fat mass, body fat rate, and blood glucose, and insulin resistance of OE group and OER group were lower than OC group( P < 0. 01, P < 0. 05), of OR group were lower than OC group, of OER group were lower than OE group, but the difference was not statistically significant. Resistin mRNA×10~3 expression in visceral adipose tissue of OR group( 21. 63 ±4. 91) was lower than OC group( P < 0. 05), of OE group and OER group(( 28. 21 ± 4. 80), ( 25. 22 ± 11. 12), respectively) were lower than OC group, but the difference was not statistically significant. Plasma resistin concentration of OR group and OE group and OER group(( 3. 71 ± 1. 01), ( 3. 29 ± 1. 19) and( 2. 99 ± 0. 44) μg/L, respectively), were lower than OC group, of OER group were lower than OE group, of OE group were lower than OR group, but the difference was not statistically significant., Conclusion: Elderly obesity result in insulin resistance and high blood glucose, resistin expression in visceral adipose tissueand and plasma resistin concentration is higher. In elderly obese rats, simple exercise and exercise combined with resveratrol can significantly reduce weight, visceral fat mass, body fat ratio, and blood glucose content, and insulin resistance, but that are less affected on resistin expression in visceral fat and plasma resistin concentration. Simple add resveratrol can reduce resistin mRNA expression in visceral adipose tissue.

Purpose: The urinary glucose tetrasaccharide, Glcα1-6Glcα1-4Glcα1-4Glc (Glc4), is a biomarker of glycogen accumulation and tissue damage and is elevated in patients with Pompe disease. We report baseline urinary Glc4 concentrations for patients with classic infantile-onset or late-onset Pompe disease, and those with a pseudodeficiency of acid alpha-glucosidase (GAA), identified through newborn screening (NBS) in Taiwan., Methods: Infants identified through NBS with (1) classic infantile-onset Pompe disease (NBS-IOPD) (n = 7) defined as patients with evidence for hypertrophic cardiomyopathy by EKG, X-ray, and echocardiogram, (2) a late-onset phenotype (NBS-LOPD) (n = 13) defined as patients without evidence for cardiomyopathy, (3) a GAA pseudodeficiency (n = 58), and (4) one patient with LOPD diagnosed in infancy due to family history were consented to the study. Four infants diagnosed after the onset of clinical symptoms (CLIN-IOPD) were included for comparison. Glc4 concentrations in dried urine samples on filter paper were determined using tandem mass spectrometry., Results: Baseline Glc4 concentrations were at or above the 90th centile of the age-matched reference range for the NBS-IOPD cohort. The median Glc4 level for this group was lower than that of the CLIN-IOPD group, although not at the level of significance (p = 0.07), but was significantly higher than that of the NBS-LOPD group (p < 0.05). Baseline Glc4 was not elevated for the NBS-LOPD and GAA pseudodeficiency cohorts and remained low for late-onset patients that did not require treatment before the age of three years., Conclusion: Baseline urinary Glc4 is elevated in neonates with infantile-onset Pompe disease identified through NBS.

Hutchinson-Gilford progeria syndrome (HGPS) is a severe human premature aging disorder caused by a lamin A mutant named progerin. Death occurs at a mean age of 13 y from cardiovascular problems. Previous studies revealed loss of vascular smooth muscle cells (SMCs) in the media of large arteries in a patient with HGPS and two mouse models, suggesting a causal connection between the SMC loss and cardiovascular malfunction. However, the mechanisms of how progerin leads to massive SMC loss are unknown. In this study, using SMCs differentiated from HGPS induced pluripotent stem cells, we show that HGPS SMCs exhibit a profound proliferative defect, which is primarily caused by caspase-independent cell death. Importantly, progerin accumulation stimulates a powerful suppression of PARP1 and consequently triggers an activation of the error-prone nonhomologous end joining response. As a result, most HGPS SMCs exhibit prolonged mitosis and die of mitotic catastrophe. This study demonstrates a critical role of PARP1 in mediating SMC loss in patients with HGPS and elucidates a molecular pathway underlying the progressive SMC loss in progeria.

The A- and B-type lamins are nuclear intermediate filament proteins in eukaryotic cells with a broad range of functions, including the organization of nuclear architecture and interaction with proteins in many cellular functions. Over 180 disease-causing mutations, termed 'laminopathies,' have been mapped throughout LMNA, the gene for A-type lamins in humans. Laminopathies can range from muscular dystrophies, cardiomyopathy, to Hutchinson-Gilford progeria syndrome. A number of mouse lines carrying some of the same mutations as those resulting in human diseases have been established. These LMNA-related mouse models have provided valuable insights into the functions of lamin A biogenesis and the roles of individual A-type lamins during tissue development. This review groups these LMNA-related mouse models into three categories: null mutants, point mutants, and progeroid mutants. We compare their phenotypes and discuss their potential implications in laminopathies and aging., (© 2012 The Authors Aging Cell © 2012 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.)

Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disease that is frequently caused by a de novo point mutation at position 1824 in LMNA. This mutation activates a cryptic splice donor site in exon 11, and leads to an in-frame deletion within the prelamin A mRNA and the production of a dominant-negative lamin A protein, known as progerin. Here we show that primary HGPS skin fibroblasts experience genome-wide correlated alterations in patterns of H3K27me3 deposition, DNA-lamin A/C associations, and, at late passages, genome-wide loss of spatial compartmentalization of active and inactive chromatin domains. We further demonstrate that the H3K27me3 changes associate with gene expression alterations in HGPS cells. Our results support a model that the accumulation of progerin in the nuclear lamina leads to altered H3K27me3 marks in heterochromatin, possibly through the down-regulation of EZH2, and disrupts heterochromatin-lamina interactions. These changes may result in transcriptional misregulation and eventually trigger the global loss of spatial chromatin compartmentalization in late passage HGPS fibroblasts.

Mucopolysaccharidosis type VI (MPS VI, Maroteaux-Lamy syndrome, MIM 253200 ) is an autosomal recessive lysosomal storage disease (LSD) caused by decreased activity of arylsulfatase B (N-acetylgalactosamine 4-sulfatase) enzyme resulting in dermatan sulfate accumulation; mucopolysaccharidosis type IVA (MPS IVA, Morquio syndrome A, MIM 253000 ) by decreased activity of N-acetylgalactosamine 6-sulfatase enzyme resulting in accumulation of keratan sulfate. Clinical symptoms include coarse facial features, joint stiffness, hepatosplenomegaly, hip osteonecrosis, and dysostosis multiplex. MPS IVA symptoms are similar but with joint hypermobility.With suspicion of MPS disease, clinicians request urine studies for quantitative and qualitative glycosaminoglycans (GAGs). Diagnosis is confirmed by decreased enzyme activity in leukocytes or cultured skin fibroblasts. Further confirmation is obtained with identification of two mutations in the ARSB gene for MPS VI or mutations in the GALNS gene for MPS IVA.We report slowly progressing patients, one with MPS VI and two with MPS IVA, who presented with skeletal changes and hip findings resembling Legg-Calvé-Perthes disease or spondyloepiphyseal dysplasia and normal/near normal urine GAG levels. The urine analysis data presented suggest that present screening techniques for MPS are inadequate in milder patients and result in delayed or missed diagnoses. The patients presented in this paper emphasize the importance of enzymatic and molecular testing.

Rationale: Acylcarnitines are routinely analyzed by electrospray ionization tandem mass spectrometry (ESI-MS/MS) both in clinical diagnostic and public health newborn screening laboratories from plasma and dried whole blood spots (DBS) on filter paper. The use of DBS as a convenient method of collecting and storing samples for subsequent analysis of various biomolecules is increasing, thus prompting the development of new devices to recover and quantify such analytes in an automated manner., Methods: Acylcarnitines were extracted directly from DBS using a novel autosampler that sequentially loads DBS cards into a pneumatic clamp and then pumps a fixed volume of solvent containing appropriate internal standards through a section of the DBS card directly into a triple quadrupole mass spectrometer via ESI. Plasma was first spiked with internal standard then spotted onto filter paper for analysis., Results: Acylcarnitines were analyzed in DBS, and both free and total carnitine were assayed in dried plasma spots (DPS). Results using the new autosampling technique were of equal quality to those obtained by punching a 3-mm diameter disk from a DBS or DPS, then extracting and analyzing the target analytes from conventional 96-well microtiter plates, with far reduced time per sample. Recovery for most analytes was >60% and reproducibility was generally within 20% (CV)., Conclusions: The simplicity and robustness of the DBS autosampler make it an attractive alternative to conventional methods of analyzing DBS specimens, thus saving time and labor costs, especially in high-throughput applications. Although the method as described is for direct infusion analysis, the autosampler is easily coupled to column hardware for applications requiring liquid chromatography/mass spectrometry (LC/MS)., (Copyright © 2012 John Wiley & Sons, Ltd.)

Defining disease severity in patients with Pompe disease is important for prognosis and monitoring the response to therapies. Current approaches include qualitative and quantitative assessments of the disease burden, and clinical measures of the impact of the disease on affected systems. The aims of this manuscript were to review a noninvasive urinary glucose tetrasaccharide biomarker of glycogen storage, and to discuss advances in imaging techniques for determining the disease burden in Pompe disease. The glucose tetrasaccharide, Glcα1-6Glcα1-4Glcα1-4Glc (Glc(4) ), is a glycogen-derived limit dextrin that correlates with the extent of glycogen accumulation in skeletal muscle. As such, it is more useful than traditional biomarkers of tissue damage, such as CK and AST, for monitoring the response to enzyme replacement therapy in patients with Pompe disease. Glc(4) is also useful as an adjunctive diagnostic test for Pompe disease when performed in conjunction with acid alpha-glucosidase activity measurements. Review of clinical records of 208 patients evaluated for Pompe disease by this approach showed Glc(4) had 94% sensitivity and 84% specificity for Pompe disease. We propose Glc(4) is useful as an overall measure of disease burden, but does not provide information on the location and distribution of excess glycogen accumulation. In this manuscript we also review magnetic resonance spectroscopy and imaging techniques as alternative, noninvasive tools for quantifying glycogen and detailing changes, such as fibrofatty muscle degeneration, in specific muscle groups in Pompe disease. These techniques show promise as a means of monitoring disease progression and the response to treatment in Pompe disease. © 2012 Wiley Periodicals, Inc., (Copyright © 2012 Wiley Periodicals, Inc.)

Background: Infantile-onset glycogen storage disease type II (GSD-II; Pompe disease; MIM 232300) causes death early in childhood from cardiorespiratory failure in the absence of effective treatment, whereas late-onset Pompe disease causes a progressive skeletal myopathy. The limitations of enzyme replacement therapy could potentially be addressed with adeno-associated virus (AAV) vector-mediated gene therapy., Methods: AAV vectors containing tissue-specific regulatory cassettes, either liver-specific or muscle-specific, were administered to 12- and 17-month-old Pompe disease mice to evaluate the efficacy of gene therapy in advanced Pompe disease. Biochemical correction was evaluated through acid alpha-glucosidase (GAA) activity and glycogen content analyses of the heart and skeletal muscle. Western blotting, urinary biomarker, and Rotarod performance were evaluated after vector administration., Results: The AAV vector containing the liver-specific regulatory cassette secreted high-level human GAA into the blood and corrected glycogen storage in the heart and diaphragm. The biochemical correction of the heart and diaphragm was associated with efficacy, as reflected by increased Rotarod performance; however, the clearance of glycogen from skeletal muscles was relatively impaired compared to in younger Pompe disease mice. An alternative vector containing a muscle-specific regulatory cassette transduced skeletal muscle with high efficiency, but also failed to achieve complete clearance of accumulated glycogen. Decreased transduction of the heart and liver in older mice, especially in females, was implicated as a cause for reduced efficacy in advanced Pompe disease., Conclusions: The impaired efficacy of AAV vector-mediated gene therapy in old Pompe disease mice emphasizes the need for early treatment to achieve full efficacy.

Glycogen storage disease type II (GSD-II; Pompe disease; MIM 232300) is an inherited muscular dystrophy caused by deficiency in the activity of the lysosomal enzyme acid alpha-glucosidase (GAA). We hypothesized that chimeric GAA containing an alternative signal peptide could increase the secretion of GAA from transduced cells and enhance the receptor-mediated uptake of GAA in striated muscle. The relative secretion of chimeric GAA from transfected 293 cells increased up to 26-fold. Receptor-mediated uptake of secreted, chimeric GAA corrected cultured GSD-II patient cells. High-level hGAA was sustained in the plasma of GSD-II mice for 24 weeks following administration of an AAV2/8 vector encoding chimeric GAA; furthermore, GAA activity was increased and glycogen content was significantly reduced in striated muscle and in the brain. Administration of only 1 x 10(10) vector particles increased GAA activity in the heart and diaphragm for >18 weeks, whereas 3 x 10(10) vector particles increased GAA activity and reduced glycogen content in the heart, diaphragm, and quadriceps. Furthermore, an AAV2/2 vector encoding chimeric GAA produced secreted hGAA for >12 weeks in the majority of treated GSD-II mice. Thus, chimeric, highly secreted GAA enhanced the efficacy of AAV vector-mediated gene therapy in GSD-II mice.