Noggin and Sonic hedgehog are involved in compensatory changes within the motoneuron-depleted mouse spinal cord Rosario Gulino ⁎, Massimo Gulisano Department of Bio-Medical Sciences, Section of Physiology, University of Catania, Viale Andrea Doria 6, I95125 Catania, Italy abstract article info Article history: Received 29 March 2013 Received in revised form 22 May 2013 Accepted 25 June 2013 Available online 13 July 2013 Keywords: Cholera toxin-B saporin Glutamate receptor Morphogen Noggin Regeneration Sonic hedgehog Spinal cord Synaptic plasticity Sonic hedgehog and Noggin are morphogenetic factors involved in neural induction and ventralization of the neural tube, but recent findings suggest that they could participate in regeneration and functional recovery after injury. Here, in order to verify if these mechanisms could occur in the spinal cord and involve synaptic plas- ticity, we measured the expression levels of Sonic hedgehog, Noggin, Choline Acetyltransferase, Synapsin-I and Glutamate receptor subunits (GluR1, GluR2, GluR4), in a motoneuron-depleted mouse spinal cord lesion model obtained by intramuscular injection of Cholera toxin-B saporin. The lesion caused differential expression changes of the analyzed proteins. Moreover, motor performance was found correlated with Sonic hedgehog and Noggin expression in lesioned animals. The results also suggest that Sonic hedgehog could collaborate in modu- lating synaptic plasticity. Together, these findings confirm that the injured mammalian spinal cord has intrinsic potential for repair and that some proteins classically involved in development, such as Sonic hedgehog and Nog- gin could have important roles in regeneration and functional restoration, by mechanisms including synaptic plasticity. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Noggin, encoded by the NOG gene, is a secreted glycoprotein involved in the embryonic morphogenesis. In particular, Noggin is expressed in the Spemann organizer and induces neural tissue by act- ing as an inhibitor of bone morphogenetic proteins [1–3]. Sonic hedge- hog (Shh) is a morphogen involved in the ventralization of the neural tube [4–7]. Both proteins collaborate in the development and pattern- ing of the neuraxis [2,8–10]. Recent findings have shown that some proteins classically involved in neural tissue morphogenesis could also act as regulators of neurogenesis and/or neuroplasticity, in the adult nervous system [11–14]. Interestingly, we have reported that Shh could be also involved in spinal cord plasticity [15,16]. So far, little is known about the possible role of Noggin in the adult spinal cord, and the role of Shh needs to be further clarified. It has been found that Noggin stimulates neural stem cell proliferation in the hippocam- pus [17] and modulates hippocampal plasticity and spatial learning [18,19]. In the present study, the expression levels of Noggin and Shh, as well as several proteins directly involved in synaptic function, such as Choline Acetyltransferase (ChAT), Synapsin-I and the AMPA recep- tor subunits GluR1, GluR2 and GluR4, were measured by western blot- ting in a previously established model of motoneuron-depleted mouse spinal cord [15,16]. In order to verify some possible mechanisms by which Noggin and Shh could be functionally related to the spontane- ous spinal cord plastic changes and the resulting functional recovery, we used multivariate regression models to correlate protein expres- sion levels with each other, as well as with the motor performance of motoneuron-depleted animals. 2. Experimental procedures Young adult male mice (n = 47) (Charles River, Strain 129, 5 weeks aged) were used. Animal care and handling were carried out in accordance with the EU Directive 86/609/EEC. All experiments have also been approved by our institutions. All efforts were made to minimize the number of animals used and their suffering. Surgical procedures were performed under deep anesthesia where necessary (10 mg ketamine/2 mg xylazine per 100 g body weight, i.p.). 2.1. Neurotoxic lesion Motoneuron depletion was induced by injection of the retrograde- ly trasported, ribosome-inactivating toxin, Cholera toxin-B saporin (Advanced Targeting Systems, San Diego, CA, USA) into the medial and lateral gastrocnemius muscles at a dose of 3.0 μg/2.0 μl PBS per muscle, as described previously [15]. After the bilateral injection of Cholera toxin-B saporin, mice were allowed to survive for either one week (LES-1wk, n = 10) or one month (LES-1mo, n = 9). Other ani- mals received an equal volume of Cholera toxin-B-only vehicle, and they were then sacrificed at the same time-points as lesioned animals Journal of the Neurological Sciences 332 (2013) 102–109 ⁎ Corresponding author. Tel.: +39 095 7384268; fax: +39 095 7384217. E-mail address: sarogulino@gmail.com (R. Gulino). 0022-510X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jns.2013.06.029 Contents lists available at SciVerse ScienceDirect Journal of the Neurological Sciences journal homepage: www.elsevier.com/locate/jns