High expression and anterograde axonal transport of aminoterminal sonic hedgehog in the adult hamster brain

Abstract

Sonic hedgehog (SHH) is considered to play an important role in tissue induction and patterning during development, particularly in determining neuronal cell fate in the ventral neural tube and in the embryonic forebrain. SHH precursor is autoproteolytically cleaved to an aminoterminal fragment (SHHN) which retains all known SHH biological activities. Here, we demonstrate the expression of a 22-kDa SHHN immunoreactive peptide in developing and adult hamster brain regions using a rabbit antiserum directed against a mouse SHHN fragment. Interestingly, SHHN was developmentally regulated with the highest expression observed in the adult brain, was resistant to Triton X-100 solubilization at 4 degrees C and partitioned with the raft component ganglioside GM1 during density gradient centrifugation. In rat brain, Shh transcripts were identified by double in situ hybridization in GABAergic neurons located in various basal forebrain nuclei including globus pallidus, ventral pallidum, medial septum-diagonal band complex, magnocellular preoptic nucleus and in cerebellar Purkinje cells as well as in motoneurons of several cranial nerve nuclei and of the spinal cord. We show that radiolabelled SHHN peptides are synthesized in the adult hamster retina and are transported axonally along the optic nerve to the superior colliculus in vivo. Our data indicate that SHHN is associated with cholesterol rich raft-like microdomains and anterogradely transported in the adult brain, and suggest that the roles of this extracellular protein are more diverse than originally thought.

Prenatal inhibition of the tryptophan–kynurenine pathway alters synaptic plasticity and protein expression in the rat hippocampus

“…SHH is expressed largely in fully differentiated neurons (Sims, 2009) and has most frequently been studied in relation to early cell proliferation and migration (Palma et al, 2005;Traiffort et al, 2001Traiffort et al, , 2010, although it has a major role to play in the mutual attraction between growing neurites and their targets both in nervous system development and in the early phases of regeneration after tissue injury (Hor and Tang, 2010;Angot et al, 2008;Traiffort et al, 2010). The change in SHH therefore may be a key factor in the altered expression of other proteins and in the functional changes demonstrated here.…”

Modeling and Mutagenesis of the Binding Site of Calhex 231, a Novel Negative Allosteric Modulator of the Extracellular Ca2+-sensing Receptor

“…Briefly, 4 g of pcDNA3 plasmid containing human WT or mutant CaSR DNA were supplemented with 6 g of pRK5 plasmid and used to transfect 10 6 cells in a total volume of 300 l of electroporation buffer (50 (6) was amplified by PCR, inserted into a glutathione S-transferase (GST) fusion pGEX-4T-1 vector (Amersham Biosciences), and sequenced (Eurogentec). The production of a GST fusion protein in Escherichia coli BL21 was then carried out (29), and 100 g were injected into a rabbit to generate 141Ab antiserum.…”

Functional Characterization of Sonic Hedgehog Mutations Associated with Holoprosencephaly

“…Synthesis and secretion of the protein were further monitored by Western blot analysis using two polyclonal antisera recognizing specifically SHH-N (167Ab) (42) or SHH-C (1229Ab) (Fig. 1).…”

“…Media and cellular homogenates were separated on 12.5% acrylamide gel, blotted on nitrocellulose membrane, probed 2 h with specific polyclonal antisera 167Ab (1/1000) directed to mouse Shh-N (42) or 1229Ab (1/1000) directed to the human SHH-C peptide 288-302 and kindly provided by A. Galdes and K. P. Williams (Biogen, Cambridge, MA). Immunoreactivity was revealed as described (42). The effective quantity of loaded protein was determined by probing the blots with a 1/500 dilution of the mouse monoclonal IgG2a ␣-actin antibody (AC-40) (Sigma).…”