Journal: Nature Communications

Article Title: Neuronal differentiation is associated with a redox-regulated increase of copper flow to the secretory pathway

doi: 10.1038/ncomms10640

Figure Lengend Snippet: ( a ) Immunostaining of PAM (green) in a chicken spinal cord (HH stage 20 embryo). Traverse sections of chick embryos were analysed. Differentiated cells in marginal zones were identified by postmitotic marker isl1/2 (red). Scale bar, 50 μm. ( b ) Intensity profile along the line indicated by the arrow in a shows overlap between PAM and Isl1/2 expression. Medial and lateral borders are represented as M and L, respevtively. ( c ) Schematic of differentiation of SH-SY5Y cells by sequential treatments with retinoic acid (DIF RA) and BDNF (DIF BDNF). ( d ) Differentiation is associated with upregulation of genes involved in copper fluxes to mitochondria and, especially, the secretory pathway. The mRNA levels were determined by ΔΔCt analysis using GAPDH as a reference gene and normalized to non-differentiated cells (red line). Each value is presented as mean±s.d. ( n =3). ( e ) Protein levels for ATP7A and Atox1 increase upon differentiation. Equal amount of protein was loaded to each lane. Neuronal differentiation was verified by upregulation of MAP2. ( f ) Cellular copper content in differentiated SH-SY5Y cells (BDNF) is higher than in non-differentiated (ND) cells. Copper amounts were determined by atomic absorption and normalized to protein amounts. Data from three independent measurements. ( g , h ) ATP7A is localized within TGN and vesicular structures which are distinct from endosomal or lysosomal compartments. Coimmunostaining with TGN was shown as a representative image (see for other images). Nucleus was stained with DAPI (blue). Scale bar, 10 μm. Colocalization of ATP7A and various markers were evaluated using Pearson's product-moment coefficients. Three replicate samples were prepared and analysed. Each value is presented as mean±s.d. ( n =3).

Article Snippet: Primary antibodies used in cell staining were mouse monoclonal anti-FLAG M2 clone (Sigma, F1804), rabbit polyclonal anti-ATP7A (Hycult Biotech, HP8040), mouse monoclonal anti-ATP7A (Santa Cruz, 376467), mouse monoclonal anti-EEA1 (BD Biosciences, 610456), mouse monoclonal anti-Rab11 (BD Biosciences, 610656), mouse monoclonal anti-Lamp1 (developmental studies hybridoma bank, H4A3-s), and sheep monoclonal anti-TGN46 (Gene Tex, GTX74290).

Techniques: Immunostaining, Marker, Expressing, Staining

Journal: Disease Models & Mechanisms

Article Title: The Menkes and Wilson disease genes counteract in copper toxicosis in Labrador retrievers: a new canine model for copper-metabolism disorders

doi: 10.1242/dmm.020263

Figure Lengend Snippet: Manhattan plots for hepatic copper score in Labrador retrievers. (A) Manhattan plot of hepatic copper score in 235 Labrador retrievers shows a genome-wide association signal at chromosome 22. (B) Manhattan plot of chromosome 22 shows that the signal comprises an LD block comprising the first 10 Mb of the chromosome. The arrow indicates the location of ATP7B . (C) Mapping results for hepatic copper score at the X chromosome in male dogs shows an association signal at position X:60203319-60356690 (CanFam 3.1). The arrows indicate the location of ATP7A . (D) Mapping results for hepatic copper score at the X chromosome in females shows no substantial association. Note that the solid line indicates the boundary for suggestive genome-wide association at a significance level of P =5×10 −5 . The dotted line indicates the boundary used for determination of the crucial regions at P =5×10 −4 .

Article Snippet: Antibodies were obtained from the following sources: anti-ATP7A (Hycult Biotech, Plymouth Meeting, PA, USA), anti-Human Golgin-97 Mouse Monoclonal CDF4 (Molecular Probes, Paisley, UK), anti-GFP and anti-VAP-A from M.A.

Techniques: GWAS, Blocking Assay

Journal: Disease Models & Mechanisms

Article Title: The Menkes and Wilson disease genes counteract in copper toxicosis in Labrador retrievers: a new canine model for copper-metabolism disorders

doi: 10.1242/dmm.020263

Figure Lengend Snippet: Hepatic histological copper score in relation to ATP7A and ATP7B genotype in male and female Labrador retrievers

Article Snippet: Antibodies were obtained from the following sources: anti-ATP7A (Hycult Biotech, Plymouth Meeting, PA, USA), anti-Human Golgin-97 Mouse Monoclonal CDF4 (Molecular Probes, Paisley, UK), anti-GFP and anti-VAP-A from M.A.

Techniques:

Journal: Disease Models & Mechanisms

Article Title: The Menkes and Wilson disease genes counteract in copper toxicosis in Labrador retrievers: a new canine model for copper-metabolism disorders

doi: 10.1242/dmm.020263

Figure Lengend Snippet: Protein domains of ATP7A and ATP7B involved in copper toxicosis in Labrador retrievers. Overview of the ATP7A (A) and ATP7B (B) proteins with the N-terminus, metal-binding domains (MBDs), actuator domain (A), nucleotide-binding domain (N), phosphorylation domain (P) and the C-terminus. The green asterisk indicates the position of the mutations. (A) Alignment of the region containing ATP7A T327I (in green) shows a strong conservation of this amino acid position in the human, rat, mouse, cow, cat and horse. The copper-binding site XMXCXXC (boxed), predicted α-helix (red) and β-sheets (blue) are indicated. (B) Alignment of the region containing ATP7B R1453Q (in green) shows a strong conservation of this amino acid position in the human, rat, mouse, cow, cat and horse. The predicted α-helix (red) and β-sheets (blue) are indicated.

Article Snippet: Antibodies were obtained from the following sources: anti-ATP7A (Hycult Biotech, Plymouth Meeting, PA, USA), anti-Human Golgin-97 Mouse Monoclonal CDF4 (Molecular Probes, Paisley, UK), anti-GFP and anti-VAP-A from M.A.

Techniques: Binding Assay

Journal: Disease Models & Mechanisms

Article Title: The Menkes and Wilson disease genes counteract in copper toxicosis in Labrador retrievers: a new canine model for copper-metabolism disorders

doi: 10.1242/dmm.020263

Figure Lengend Snippet: Copper accumulation and retention in canine dermal fibroblasts. (A) Copper influx in canine fibroblasts. Dermal fibroblasts derived from ATP7A:p.Ile327 (ATP7A T327I ) dogs showed significantly more uptake of 64 Cu than dermal fibroblasts derived from dogs with ATP7A:p.Thr327 (ATP7A WT ). The overall difference in copper accumulation was 86% (95% confidence interval 25-176%, P =6.1×10 −3 ). The overall increase in 64 Cu was compared to time point 6 h. After 22 h, the estimated increase in copper was 45% (95% confidence interval 27-64%, P <1.0×10 −4 ), and after 30 h this was 72% (95% confidence interval 45-104%, P <1.0×10 −4 ). Dots represent mean values, and standard deviation is represented by the error bars. (B) Copper efflux in canine fibroblasts. Dermal fibroblasts from ATP7A T327I dogs showed significantly more retention of copper than dermal fibroblasts derived from dogs with ATP7A WT . The overall average export rate in ATP7A T327I was significantly lower than in ATP7A WT ( P =0.011). Dots represent mean percentages, and standard deviation is represented by the error bars. (C) Copper influx in human fibroblasts. Dermal fibroblasts from a human with Menkes disease showed considerably more uptake of copper than dermal fibroblasts derived from a human with ATP7A wild type. (D) Copper efflux in human fibroblasts. Dermal fibroblasts from a human with Menkes disease showed considerably more retention of copper than dermal fibroblasts derived from a person with ATP7A wild type.

Article Snippet: Antibodies were obtained from the following sources: anti-ATP7A (Hycult Biotech, Plymouth Meeting, PA, USA), anti-Human Golgin-97 Mouse Monoclonal CDF4 (Molecular Probes, Paisley, UK), anti-GFP and anti-VAP-A from M.A.

Techniques: Derivative Assay, Standard Deviation

Journal: Stem Cell Research & Therapy

Article Title: Impaired osteogenesis in Menkes disease-derived induced pluripotent stem cells

doi: 10.1186/s13287-015-0147-5

Figure Lengend Snippet: Genetic information of Menkes patients

Article Snippet: The primary antibodies used in this study were as follows: SMAD2 (Cell Signaling); p-SMAD2 (Cell Signaling); ACTIN (Santa Cruz Biotechnology); ATP7A (Hycult Biotech, Uden, Netherlands); SMAD1 (Cell Signaling); and p-SMAD1 (Cell Signaling).

Techniques: Mutagenesis

Journal: Stem Cell Research & Therapy

Article Title: Impaired osteogenesis in Menkes disease-derived induced pluripotent stem cells

doi: 10.1186/s13287-015-0147-5

Figure Lengend Snippet: Impaired osteogenesis in MD-MSCs. a Representative images of ALP activity in WT- and MD-MSCs during OB differentiation. ALP activity was observed as red granules. D, days after osteogenesis induction. b Representative images of alizarin red S staining in WT- and MD-MSCs during OB differentiation. Alizarin red S staining presented as red granules. c Representative images of Von Kossa staining in WT- and MD-MSCs during OB differentiation. Von Kossa staining was observed as black dots. Scale bars = 500 μm. d Relative expression of osteogenic genes RUNX2 , OPN , and OCN in MD-MSCs during osteogenesis. The data are presented as the mean ± SE (n = 3). e, f Effects of ATP7A knock-down on osteogenesis in WT-MSCs. e Representative images of ALP activity, alizarin red S staining, and Von Kossa staining after transfection of siRNAs targeting ATP7A gene. Scramble siRNA (si-SCR) was also transfected in WT-MSCs as a control. f Relative expression of RUNX2 , OPN , and OCN after ATP7A knockdown. The data are represented as the mean ± SE (n = 3). * p < 0.05, ** p < 0.01. ALP alkaline phosphatase, D day of differentiation, MD1/2 Menkes disease patient 1/2, WT wild type

Article Snippet: The primary antibodies used in this study were as follows: SMAD2 (Cell Signaling); p-SMAD2 (Cell Signaling); ACTIN (Santa Cruz Biotechnology); ATP7A (Hycult Biotech, Uden, Netherlands); SMAD1 (Cell Signaling); and p-SMAD1 (Cell Signaling).

Techniques: Activity Assay, Staining, Expressing, Transfection