Research ReportExpression analysis of neuroleukin, calmodulin, cortactin, and Rho7/Rnd2 in the intact and injured mouse brain
Introduction
Adult mammalian central nervous system (CNS) neurons are unable to regenerate their axons following a trauma in vivo. Numerous factors contribute to the absence of spontaneous regeneration in the CNS. Some of these are attributed to the neurons themselves [5], [37]. Indeed, injured neurons fail to reexpress some of the growth-associated proteins that were expressed earlier in development. For example, axon-growth-promoting cues, such as neurotrophic factors that mediate cell survival and neurite outgrowth during development, are often absent following CNS injury. A second pathway responsible for axonal regeneration failure is attributed to glial scar formed at the site of injury [58]. Several molecules synthesized by oligodendrocytes and astrocytes, such as myelin-related molecules and chondroitin sulfate proteoglycans, have been shown to inhibit axonal outgrowth in the CNS, and some of these are upregulated upon injury [12], [14], [15], [18], [43], [60].
Neonatal CNS neurons, in contrast, are able to re-grow axons and to re-innervate target tissue. However, this regenerative capability disappears during early postnatal development [22], [24], [37], [41], [58], [63].
The inferior colliculus (IC) commissure has been used as an in vitro model to study axonal regeneration [22]. In organotypic cultures, severed IC commissural axons from postnatal day 6 (P6) display a robust regeneration 1 week after the trauma. This regenerative capability across a lesion site is completely lost by P10–12 [24], [26]. In order to search for molecules involved in the developmental process in the IC and which could have a role in axonal regeneration failure observed in the IC at P10, a suppression subtractive hybridization (SSH) was carried out previously between IC tissues from P10 and P6 ([8], Decourt et al., companion paper). cDNAs libraries were constructed and partially sequenced. The sequences were then screened by dot blot procedure.
Among the highly expressed clones in the IC at P10, four particular cDNAs corresponding to neuroleukin, calmodulin I, cortactin, and Rho7 were selected for further investigation. The aim of the present study was to investigate the cellular distribution of these molecules in the intact brain and after a mechanical trauma in vivo. These molecules were selected for their potential involvement in signaling pathways regulating axonal extension. Indeed, NLK acts as an extracellular trophic factor for spinal and sensory neurons during development [20]. Its mRNA has been studied by Northern blot in the chicken brain [28], but its precise cellular distribution in the brain has not yet been investigated. Calmodulin is an intracellular calcium binding protein which is a key component of the calcium signal transduction pathway. Cortactin is a protein regulating the polymerization of actin juxtamembrane cytoskeleton in vitro [68]. The cellular localization of cortactin has been recently described in the rat hippocampus [54]. The present study analyzes its cellular distribution in the mouse brain in vivo. Finally, Rho7 belongs to the Rho-GTPase family implicated in the regulation of actin polymerization, but the protein localization in the brain has not yet been reported in vivo.
Section snippets
Materials and methods
Mice of the C57BL/6 strain were used for this study. The care and use of animals were approved by the “Direction départementale des services vétérinaires de la Gironde (autorisation#3303303)”.
Western blot
For immunohistochemistry, the specificity of the four used antibodies was tested by Western blots using adult mouse brain extracts (Fig. 1). The antibodies labeled single bands at the corresponding molecular weight for neuroleukin (55–60 kDa), calmodulin (15 kDa), and cortactin (75–80 kDa). However, Rho7 antibody revealed two bands, one at the corresponding molecular weight (25 kDa) and a second band at three times the molecular weight (75 kDa) possibly corresponding to the interaction with a
Discussion
The aim of the present study was to analyze the distribution of NLK, calmodulin, cortactin, and Rho7 mRNAs and proteins in both adult and developing brain and upon brain injury. Two weeks after brain injury was chosen for analysis because it is the time course when regenerating axons are expected to grow through or around the lesion site in vivo [62]. By doing this, we can correlate the expression of the four investigated proteins with the presence or absence of axonal regenerating process
Acknowledgments
This study was supported by Association Libre pour la Recherche sur la Moelle Epinière (ALARME). We would like to thank E. Normand and D. Bouchet (CNRS 5091, Bordeaux) for their technical help regarding the in situ hybridization and Western blot experiments, Dr. A. Raz for providing us with the anti-neuroleukin antibody, and Dr. James Saunders for helpful comments on the manuscript.
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