High glucose treatment does not affect APP transcription.

<p>The 2.94 kb human APP promoter was transfected into SH-SY5Ycells and treated with high glucose for 24 hours (<b>A</b>) and 48 hours (<b>B</b>). 2.5 mM glucose serves as control. Luciferase assasy was performed.High glucose treatment did not affect APP promoter activity. All the promoter data shown are results of 4 independent experiments, with each condition performed in triplicates. The values are expressed as mean±S.E.M. n = 4, by ANOVA. SH-SY5Y cells were treated with different concentration of glucose for 24 hours(<b>C</b>) and 48 hours (<b>E</b>). RNA was extracted and APP mRNA level was measured by semi-quantitative PCR with specific primers. β-actin served as an internal control. 24-hourand 48-hourtreatment of high glucose did not significantly affect APP mRNA. Quantification of full-length APP after 24-hour treatment of high glucose (<b>D</b>)The values are expressed as mean±S.E.M, n = 7, by ANOVA. Quantification of full-length APP after 48-hour treatment of high glucose (<b>F</b>)The values are expressed as mean±S.E.M, n = 5, by ANOVA.</p

RCAN1.4 overexpression exacerbates calcium overloading-induced neuronal apoptosis.

<p>(<b>A</b>) RCAN1.4 overexpression reduced cell survival. SH-SY5Y cells transfected with empty vector pcDNA4mychisA or pcDNA4-RCAN1.4mychis were treated with 2.5 uM A23187. MTS assay was used to indicate the viability of SH-SY5Y cells. Values represent mean ± SEM, n = 3, *P<0. 05 by ANOVA. (<b>B</b>) and (<b>C</b>) RCAN1.4 overexpression exacerbated cell apoptosis. SH-SY5Y cells transfected with empty vector pcDNA4mychisA and pcDNA4-RCAN1.4mychis were treated with vehicle control solution (B) or 2.5 uM A23187and (C). TUNEL staining was used to indicate cell apoptosis (green color). Nuclei were counterstained with DAPI (blue color). (<b>D</b>) Quantification of (B) and (C). Values represent means ± SE (n = 3), *P<0. 05 by ANOVA.</p

High glucose treatment inhibits APP protein degradation.

<p>20E2 cells were treated with 5.5 mM, 10 mM or 25 mM glucose for 24 hours, and the cell lysates were analyzed by Western blot (<b>A</b>). Full-length APP was detected by C20 antibody. β-actin, serving as internal control, was detected by AC-15 antibody. The level of APP protein was quantified by Image J (<b>B</b>). The values are expressed as mean±S.E.M. n = 3,*p<0.0001, by ANOVA<b>.</b> For APP degradation experiment, 20E2 cells were treated with culturing media containing 100 ug/ml CHX along with 5.5 mM or 10 mM glucose. The cell lysates were harvested at 0, 15, 30 or 60 minutes after treatment and analyzed by Western blot (<b>C, D</b>).Quantification of APP protein by Image J (<b>E</b>). APP protein level was plotted as a percentage of the amount at 0 minute. The values are expressed as mean±S.E.M. n = 3,*p<0.001, by two-way ANOVA. The APP degradation experiment was also conducted using SH-SY5Y cells treated with 100 ug/ml CHX along with 5.5 mM, 10 mM or 25 mM glucose for 30 minutes. The cell lysates were analyzed by Western blot (<b>F</b>).The level of APP protein was quantified by Image J(<b>G</b>) andwas plotted as a percentage of the amount at 0 minute. The values are expressed as mean±S.E.M. n = 3,*p<0.05, by ANOVA.</p

High glucose treatment increases C99 and Aβ₄₀ production.

<p>The 20E2 cells were cultured and treated with different concentrations of glucose for 24 hours. Media containing 5.5 mM glucose served as control. The cell lysates were analyzed by western blot (<b>A</b>).C99 was detected by C20 antibody. β-actin, serving as internal control, was detected by AC-15 antibody. Quantification of C99 after 24-hour treatment of high glucose in 20E2 cells (<b>B</b>) The values are expressed as mean±S.E.M, n = 3,*p<0.001 by ANOVA The level of Aβ₄₀ in conditioned media of 20E2 cells was measured by ELISA(<b>C</b>). The values are expressed as mean±S.E.M, n = 4, *p<0.001, by ANOVA.</p

Two alternative promoters distinctly control RCAN1 expression.

<p>(<b>A</b>) Genomic organization of the <i>RCAN1</i> gene. <i>RCAN1</i> gene has seven exons and six introns. The first four exons are alternatively spliced and the last three exons are constitutive. There are two promoters and two translation initiation codons in the <i>RCAN1</i> gene, in the 5′UTR of exon 1 and 5′UTR of exon 4 respectively. TSS, transcription start site. E stands for exon. (<b>B</b>) Human <i>RCAN1</i> isoform 4 promoter sequence. A 1200 bp fragment of the 5′ flanking region of human RCAN1 exon 4 was amplified from a human genomic library. Thymine +1 represents the major transcription start site. Positions of some of the unique and common restriction enzymes are indicated in italics and boldface. Putative transcription factor binding sites are underlined in boldface. The codon of the first nine amino acids of exon 4 is indicated. (<b>C</b>) 1200-bp fragment upstream of the exon 4 had significant promoter activity. 1200-bp fragment upstream of the exon 4 was cloned into pGL3-Basic to generate the pDE4Luc luciferase reporter plasmid. pDE4Luc was transfected into HEK293 cells. pGL3-Basic was used as negative control. Luciferase activity was measured 24 hours after transfection. Values represent means ± SE (n = 4), *P<0.05 by student's t-test. (<b>D</b>) Compared to the RCAN1 exon 1 promoter pRCAN1Luc-A, RCAN1 exon 4 promoter pDE4Luc had a higher promoter activity in C6 cells but a lower activity in N2A cells. RCAN1 promoter constructs pDE4Luc and pRCAN1Luc-A were transfected into C6 and N2A cells. pGL3-Basic was used as negative control. Luciferase activity was measured 24 hours after transfection by a luminometer. <i>Renilla</i> luciferase activity was used to normalize transfection efficiency. Values represent means ± SE (n = 4), *P<0.05 by student's t-test. (<b>E</b>) Calcium ionophore A23187 significantly increased pDE4Luc activity. HEK293 cells transfected with pDE4Luc were treated with 2.5 µM A23187 for 12 hours. Luciferase assay was used to measure the promoter activity. pGL3-Basic was used as negative control. <i>Renilla</i> luciferase activity was used to normalize transfection efficiency. Values represent means ± SE (n = 4), *P<0. 05 by student's t-test.</p

Caspase-3 mediates the neurotoxic effect of RCAN1.4 and calcium overloading.

<p>(<b>A</b>) RCAN1.4 overexpression increased caspase-3 activation. SH-SY5Y cells transfected with empty vector pcDNA4mychisA (Vector) and pcDNA4-RCAN1.4mychis (RCAN1) were treated with 2.5 uM A23187 for 12 hours. 100 µg cell lysates were separated in a 16% tricine SDS-PAGE gel. Procaspase-3 and cleaved caspase-3 were detected with anti-caspase-3 antibody from Sigma. Myc-tagged RCAN1.4 was detected by 9E10 antibody. β-actin served as loading control. (<b>B</b>) Quantification of (A). The ratio of cleaved caspase-3 to procaspase-3 was calculated. Values represent means ± SE (n = 3), *P<0. 05 by ANOVA.</p

High glucose treatment increases full-length APP protein level.

<p>SH-SY5Y cells were treated with different concentration of glucose for 24 hours (<b>A</b>), and 48 hours(<b>C</b>) with2.5 mM glucose treatment serving as control. The cell lysates were analyzed by Western blot. Full-length APP was detected by C20 antibody. β-actin, serving as internal control, was detected by AC-15 antibody. 24-hour and 48-hour of high glucose treatment significantly increased full-length APP protein level.Quantification of full-length APP after 24-hour treatment of high glucose in SH-SY5Y cells (<b>B</b>). The values are expressed as mean±S.E.M, n = 4,*p<0.05 by ANOVA. Quantification of full-length APP after 48-hour treatment of high glucose in SH-SY5Y cells (<b>D</b>) The values are expressed as mean±S.E.M, n = 3,*p<0.05 by ANOVA.</p

Table_2.DOCX

<p>Serum uric acid (SUA), as the end product of purine metabolism, has proven emerging roles in human disorders. Here based on a sample of 379 middle and old-aged Chinese twin pairs, we aimed to explore the magnitude of genetic impact on SUA variation by performing sex-limitation twin modeling analyses and further detect specific genetic variants related to SUA by conducting a genome-wide association study. Monozygotic (MZ) twin correlation for SUA level (rMZ = 0.56) was larger than for dizygotic (DZ) twin correlation (rDZ = 0.39). The common effects sex-limitation model provided the best fit with additive genetic parameter (A) accounting for 46.3%, common or shared environmental parameter (C) accounting for 26.3% and unique/nonshared environmental parameter (E) accounting for 27.5% for females and 29.9, 33.1, and 37.0% for males, respectively. Although no SUA-related genetic variants reached genome-wide significance level, 25 SNPs were suggestive of association (P < 1 × 10⁻⁵). Most of the SNPs were located in an intronic region and detected to have regulatory effects on gene transcription. The cell-type specific enhancer of skeletal muscle was detected which has been reported to implicate SUA. Two promising genetic regions on chromosome 17 around rs2253277 and chromosome 14 around rs11621523 were found. Gene-based analysis found 167 genes nominally associated with SUA level (P < 0.05), including PTGR2, ENTPD5, well-known SLC2A9, etc. Enrichment analysis identified one pathway of transmembrane transport of small molecules and 20 GO gene sets involving in ion transport, transmembrane transporter activity, hydrolase activity acting on acid anhydrides, etc. In conclusion, SUA shows moderate heritability in women and low heritability in men in the Chinese population and genetic variations are significantly involved in functional genes and regulatory domains that mediate SUA level. Our findings provide clues to further elucidate molecular physiology of SUA homeostasis and identify new diagnostic biomarkers and therapeutic targets for hyperuricemia and gout.</p

Table_1.DOCX

<p>Serum uric acid (SUA), as the end product of purine metabolism, has proven emerging roles in human disorders. Here based on a sample of 379 middle and old-aged Chinese twin pairs, we aimed to explore the magnitude of genetic impact on SUA variation by performing sex-limitation twin modeling analyses and further detect specific genetic variants related to SUA by conducting a genome-wide association study. Monozygotic (MZ) twin correlation for SUA level (rMZ = 0.56) was larger than for dizygotic (DZ) twin correlation (rDZ = 0.39). The common effects sex-limitation model provided the best fit with additive genetic parameter (A) accounting for 46.3%, common or shared environmental parameter (C) accounting for 26.3% and unique/nonshared environmental parameter (E) accounting for 27.5% for females and 29.9, 33.1, and 37.0% for males, respectively. Although no SUA-related genetic variants reached genome-wide significance level, 25 SNPs were suggestive of association (P < 1 × 10⁻⁵). Most of the SNPs were located in an intronic region and detected to have regulatory effects on gene transcription. The cell-type specific enhancer of skeletal muscle was detected which has been reported to implicate SUA. Two promising genetic regions on chromosome 17 around rs2253277 and chromosome 14 around rs11621523 were found. Gene-based analysis found 167 genes nominally associated with SUA level (P < 0.05), including PTGR2, ENTPD5, well-known SLC2A9, etc. Enrichment analysis identified one pathway of transmembrane transport of small molecules and 20 GO gene sets involving in ion transport, transmembrane transporter activity, hydrolase activity acting on acid anhydrides, etc. In conclusion, SUA shows moderate heritability in women and low heritability in men in the Chinese population and genetic variations are significantly involved in functional genes and regulatory domains that mediate SUA level. Our findings provide clues to further elucidate molecular physiology of SUA homeostasis and identify new diagnostic biomarkers and therapeutic targets for hyperuricemia and gout.</p

Table_3.DOCX

<p>Serum uric acid (SUA), as the end product of purine metabolism, has proven emerging roles in human disorders. Here based on a sample of 379 middle and old-aged Chinese twin pairs, we aimed to explore the magnitude of genetic impact on SUA variation by performing sex-limitation twin modeling analyses and further detect specific genetic variants related to SUA by conducting a genome-wide association study. Monozygotic (MZ) twin correlation for SUA level (rMZ = 0.56) was larger than for dizygotic (DZ) twin correlation (rDZ = 0.39). The common effects sex-limitation model provided the best fit with additive genetic parameter (A) accounting for 46.3%, common or shared environmental parameter (C) accounting for 26.3% and unique/nonshared environmental parameter (E) accounting for 27.5% for females and 29.9, 33.1, and 37.0% for males, respectively. Although no SUA-related genetic variants reached genome-wide significance level, 25 SNPs were suggestive of association (P < 1 × 10⁻⁵). Most of the SNPs were located in an intronic region and detected to have regulatory effects on gene transcription. The cell-type specific enhancer of skeletal muscle was detected which has been reported to implicate SUA. Two promising genetic regions on chromosome 17 around rs2253277 and chromosome 14 around rs11621523 were found. Gene-based analysis found 167 genes nominally associated with SUA level (P < 0.05), including PTGR2, ENTPD5, well-known SLC2A9, etc. Enrichment analysis identified one pathway of transmembrane transport of small molecules and 20 GO gene sets involving in ion transport, transmembrane transporter activity, hydrolase activity acting on acid anhydrides, etc. In conclusion, SUA shows moderate heritability in women and low heritability in men in the Chinese population and genetic variations are significantly involved in functional genes and regulatory domains that mediate SUA level. Our findings provide clues to further elucidate molecular physiology of SUA homeostasis and identify new diagnostic biomarkers and therapeutic targets for hyperuricemia and gout.</p