Journal: Nature Communications

Article Title: RCAN1 links impaired neurotrophin trafficking to aberrant development of the sympathetic nervous system in Down syndrome

doi: 10.1038/ncomms10119

Figure Lengend Snippet: ( a ) Quantitative RT–RCR shows a 2.9-fold increase in RCAN1 mRNA in SCGs from Dp(16)1Yey/+ mice compared with wild-type litter-mates. RCAN1 levels are reduced in Dp(16)1Yey/+:RCAN +/− mice that are diploid for RCAN1 and trisomic for the rest of human chromosome 21 syntenic region on mouse chromosome 16. Results are mean±s.e.m., n =7 mice for each genotype. * P <0.05 different from all other conditions. ( b ) Calcineurin activity is significantly reduced in SCG lysates from P0.5 Dp(16)1Yey/+ mice. RCAN1 reduction improves calcineurin activity in Dp(16)1Yey/+:RCAN +/− mice relative to Dp(16)1Yey/+ litter-mates. Calcineurin phosphatase activity was measured using a colorimetric assay that detects free phosphate released from the calcineurin-specific RII phosphopeptide. Results are mean±s.e.m. from n =6 mice per genotype. ** P <0.01, *** P <0.001. ( c , d ) Reducing RCAN1 gene dosage restores dynamin1 phosphorylation status in Dp(16)1Yey/+ mice. Dp(16)1Yey/+ mice have increased levels of phospho-dynamin1 in sympathetic axons in vivo , that is corrected by removing one copy of RCAN1 in Dp(16)1Yey/+:RCAN +/− mice. Salivary gland lysates from P0.5 wild type, Dp(16)1Yey/+ and Dp(16)1Yey/+:RCAN +/− mice were immunoblotted using phospho-dynamin1 (Ser778) antibody. Immunoblots were stripped and reprobed for total dynamin1 for normalization. ( d ) Densitometric quantification of phospho-dynamin1 (Ser778) after treatments as described in c . Values are expressed relative to wild type. Results are means±s.e.m. from n =7 mice per genotype. * P <0.05, ** P <0.01. ( e – j ) Reducing RCAN1 gene dosage rescues defective Trk receptor endocytosis in Dp(16)1Yey/+ mice. Scale bar, 5 μm and 10 μm for axons and cell bodies, respectively. ( k , l ) Quantification of internalized Trk in cell bodies and axons. 40–50 cells were analysed per condition per experiment. Quantification is represented as fold-change relative to wild-type neurons with no ligand. Results are the mean±s.e.m. from at least five independent experiments. NS, not significant, * P <0.05, ** P <0.01, *** P <0.001. Statistical analyses done by one-way ANOVA and Tukey's post-hoc test for a , b , d , and two-way ANOVA and Bonferroni post-hoc test for k , l . Full-length blot scans are shown in .

Article Snippet: Lysates were prepared by laemmli sample buffer (62.5 mM Tris-HCl, 2% SDS, 10% Glycerol, 5% 2-mercaptoethanol, 0.01% bromophenol blue) and subjected to immunoblotting with the phospho-dynamin1 (P-Ser 778, 1:5,000; Imgenex) and dynamin1 (1:1,000; Abcam) antibodies.

Techniques: Activity Assay, Colorimetric Assay, Phospho-proteomics, In Vivo, Western Blot

Journal: Nature Communications

Article Title: RCAN1 links impaired neurotrophin trafficking to aberrant development of the sympathetic nervous system in Down syndrome

doi: 10.1038/ncomms10119

Figure Lengend Snippet: ( a ) In situ hybridization shows endogenous expression of RCAN1 mRNA in the developing mouse superior cervical ganglia at P0.5. Sense control is shown in b . Scale bar: 100 μm. ( c – e ) RCAN1 protein is localized to both cell bodies ( d ) and axons ( e ) of cultured sympathetic rat neurons as detected using a RCAN1 antibody. Staining with pre-immune serum control is shown in c . Scale bar, 10 μm for c , d and 5 μm for e . ( f ) A cell surface biotinylation assay shows that adenoviral overexpression of RCAN1.4 attenuates NGF-dependent TrkA internalization in cultured rat sympathetic neurons. Membrane proteins were subjected to cell-surface biotinylation. Internalized TrkA receptors were detected by surface stripping of biotin, neutravidin precipitation and TrkA immunoblotting. Supernatants were probed for p85 for normalization of protein amounts. ( g ) Densitometric quantification of internalized TrkA. Results are means±s.e.m. from four independent experiments. * P <0.05 significantly different from all other conditions. ( h ) Uptake of biotin-labelled transferrin (biotin-Tfn) is unaffected by RCAN1 overexpression in rat sympathetic neuron cultures. After internalization at 37 °C and acid washes to remove surface-bound transferrin, internalized biotin-Tfn was detected in neuronal lysates by neutravidin precipitation and immunoblotting using a transferrin antibody. Supernatants were probed for p85 for normalization of protein amounts. ( i ) Densitometric quantification of internalized biotin-Tfn. Results are means±s.e.m. from five independent experiments. * P <0.05 significantly different from corresponding controls at 4 °C. ( j ) NGF stimulation results in dephosphorylation of dynamin1, that is abrogated by excess RCAN1. Neuronal lysates were immunoblotted using a phospho-Ser778 dynamin antibody. Immunoblots were stripped and reprobed for total dynamin1 for normalization. ( k ) Densitometric quantification of phospho-dynamin1 levels normalized to total dynamin1 levels. All values are expressed relative to the no neurotrophin treatment in GFP-expressing neurons. Results are means±s.e.m. from seven independent experiments. * P <0.05 significantly different from all other conditions. Statistical analyses by two-way ANOVA and Bonferroni post-hoc test for g , i , k . Full-length blot scans are shown in .

Article Snippet: Lysates were prepared by laemmli sample buffer (62.5 mM Tris-HCl, 2% SDS, 10% Glycerol, 5% 2-mercaptoethanol, 0.01% bromophenol blue) and subjected to immunoblotting with the phospho-dynamin1 (P-Ser 778, 1:5,000; Imgenex) and dynamin1 (1:1,000; Abcam) antibodies.

Techniques: In Situ Hybridization, Expressing, Control, Cell Culture, Staining, Cell Surface Biotinylation Assay, Over Expression, Membrane, Stripping Membranes, Western Blot, De-Phosphorylation Assay