Regulation of axonal and dendritic growth by the extracellular calcium-sensing

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1 Regulation of axonal and dendritic growth by the extracellular calcium-sensing receptor (CaSR). Thomas N. Vizard, Gerard W. O Keeffe, Humberto Gutierrez, Claudine H. Kos, Daniela Riccardi, Alun M. Davies Supplementary Figure 1. CaSR protein is expressed in the developing SCG. Western blot analysis showing CaSR protein immunoreactive bands at ~140 kd and 160 kd (representing the partially and fully glycosylated monomeric forms of the receptor, respectively) in E16, E18 and P1 SCG lysates showing barely detectable protein at E16, a high level of expression at E18 and some decrease at P1. Equal loading is demonstrated by the detection of β-iii tubulin from the same samples.

2 Supplementary Figure 2. CaSR protein is localised in E18 SCG neurons. Labelling of a typical E18 SCG neuron with a C-terminus anti-casr polyclonal antibody after 24 hours in culture. The neuron was double labelled with an anti-β-iii tubulin antibody. Scale bar = 20 μm.

3 Supplementary Figure 3. Modulation of CaSR function/expression does not affect neuronal survival. Quantification of survival of (a) E18 SCG neurons grown in a range of Ca 2+ o concentrations after 24 hr, (b) E18 SCG neurons grown in 0.7 mm [Ca 2+ ] o with and without 10 nm of the calcimimetic NPS-R 467 after 24 hr, (c) E18 SCG neurons grown in 2.3 mm [Ca 2+ ] o with and without 10 nm of the calcilytic NPS after 24 hr, (d) E18 SCG neurons transfected with either dominant-negative CaSR (DNCaSR) or control (CTR) plasmids after 48 hr, (e) P1 SCG transfected with either wild-type CaSR (WTCaSR) or control (CTR) plasmids after 48 hr, (f) E18 SCG neurons from Casr+/+ (WT), Casr+/ _ (HET) and Casr _ / _ (KO) mice grown in 2.3 mm [Ca 2+ ] o after 24 hr. Mean ± sem of data from at least 3 separate experiments in all cases.

4 Supplementary Figure 4. Marginal activation of CaSR at 0.7 mm Ca 2+ o. (a) Total neurite length and (b) Sholl profiles of SCG neurons from E18 wild-type (WT) or Casr _ / _ (KO) mice cultured for 24 hrs in medium containing 0.7 mm (wild-type) or 2.3 mm (wild-type and Casr _ / _ ) [Ca 2+ ] o. Mean ± sem of data from 254 and 630 neurons per condition from 4 to 11 separate experiments.

5 Supplementary Figure 5. CaSR _ deficient neurons do not respond to NPS R-467. Total neurite length and Sholl profiles of SCG neurons from E18 Casr _ / _ mice cultured for 24 hrs in medium containing 0.7 mm [Ca 2+ ] o in the absence (CTR) or presence of 10 nm NPS R-467 (Calcimimetic). Mean ± sem of data from 109 and 121 neurons per condition from two separate experiments.

6 Supplementary Figure 6. Genetic loss of CaSR does not affect neuronal survival. Percent survival of E18 SCG neurons from Casr+/+ (WT) and Casr _ / _ (KO) mice grown for 24 hrs with a range of NGF concentrations in medium containing 2.3mM [Ca 2+ ] o and no caspase inhibitor. Mean ± sem of data from 3 separate experiments.

7 Supplementary Figure 7. SCG of WT and CaSR _ deficient mice are indistinguishable at P1. Immunohistochemistry revealing no difference in tyrosine hydroxylase immunofluorescence in the SCG of P1 Casr+/+ (WT) and Casr _ / _ (KO) mice (a). Scale bar = 100 μm. Mean neuronal nuclear diameter (b) and mean SCG volume (c) in P1 Casr+/+ (WT), Casr+/ _ (HET) and Casr _ / _ (KO) littermates. Mean ± sem of data from 3 mice of each genotype.

8 Supplementary Figure 8. CaSR protein is expressed in post-natal hippocampus. RT- PCR detection of the full-length Casr transcript (584 bp) in P4 mouse hippocampus and the full length and exon 5-deficient Casr transcript (354 bp) in kidney obtained from Casr+/ _ mice (HET), used as positive control 34 ( _ RT = no reverse transcriptase negative control) (a). CaSR immunopositive cells in P4 hippocampal cultures stained with an N-terminus anti-casr polyclonal antibody (neurons were double labelled with anti-βiii tubulin) (b). Scale bar = 50 μm.

9 Supplementary Figure 9. WT or CaSR _ deficient SCG neurons do not express the exon5 less splice variant of the CaSR. RT-PCR detection of the full-length CaSR transcript (584 bp) in P1 Casr+/+ (WT), Casr+/ _ (HET) but not Casr _ / _ (KO) SCG. Expression of the exon 5-deficient CaSR transcript (354 bp) was not detected in the SCG of any genotype. Both transcripts were amplified from the positive control tissue (kidney of Casr+/ _ mice 33 ). β-actin was amplified from the same samples as a positive reverse transcription control, and no-reverse transcriptase was used as negative control ( _ RT).