Lack of dystrophin in mdx mice modulates the expression of genes involved in neuron survival and differentiation.

Duchenne muscular dystrophy is an X-linked disease characterized by progressive and lethal muscular wasting. Dystrophic patients, however, are also afflicted by several neurological disorders, the importance of which is generally underestimated. As promising therapies for muscles are currently in clinical trial stages, with the potential to provide an increase in the lifespan of young patients, determination of the genetic and molecular aspects characterizing this complex disease is crucial in order to allow the development of therapeutic approaches specifically designed for the nervous system. In this study, differences in gene expression in the superior cervical ganglion of postnatal day (P)5, P10 and 6-7-week-old wild-type and genetically dystrophic mdx mice were evaluated by DNA microarray analysis. The main aim was to verify whether the lack of dystrophin affected the transcript levels of genes related to different aspects of neuron development and differentiation. Ontological analysis of more than 500 modulated genes showed significant differences in genetic class enrichment at each postnatal date. Upregulated genes mainly fell in the categories of vesicular trafficking, and cytoskeletal and synaptic organization, whereas downregulated genes were associated with axon development, growth factors, intracellular signal transduction, metabolic processes, gene expression regulation, synapse morphogenesis, and nicotinic receptor clustering. These data strongly suggest that the structural and functional alterations previously described in both the autonomic and central nervous systems of mdx mice with respect to wild-type mice and related to crucial aspects of neuron life (i.e. postnatal development, differentiation, and plasticity) result not only from protein post-translational modifications, but also from direct and/or indirect modulation of gene expression.

Lack of dystrophin functionally affects α3ß2/ß4-nicotinic acethylcholine receptors in sympathetic neurons of dystrophic mdx mice.

In the sympathetic superior cervical ganglion (SCG), nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic transmission. We previously demonstrated that in SCG neurons of mdx mice, an animal model for Duchenne muscular dystrophy, lack of dystrophin causes a decrease, compared to the wild-type, in post-synaptic nAChRs containing the α3 subunit associated with ß2 and/or ß4 (α3ß2/ß4-nAChRs), but not in those containing the α7 subunit. Here we show, by whole cell patch-clamp recordings from cultured SCG neurons, that both nicotine and acetylcholine-evoked currents through α3ß2/ß4-nAChRs are significantly reduced in mdx mice compared to the wild-type, while those through α7-nAChR are unaffected. This reduction associates with that of protein levels of α3, ß2 and ß4 subunits. Therefore, we suggest that, in mdx mouse SCG neurons, lack of dystrophin, by specifically affecting membrane stabilization of α3ß2/ß4-nAChRs, could determine an increase in receptor internalization and degradation, with consequent reduction in the fast intraganglionic cholinergic transmission.

Components of the NGF signaling complex are altered in mdx mouse superior cervical ganglion and its target organs.

We previously reported that in the superior cervical ganglion (SCG) of dystrophic mdx mice, which lack full-length dystrophin, there is a loss of neurons projecting to SCG muscular targets, like the iris. Nonetheless, surviving neurons, innervating either iris or submandibular gland (SuGl), a SCG non-muscular target, underwent reduced axon defasciculation and terminal branching. Here we report that, during early post-natal development, levels of pro-apoptotic proNGF in mdx mouse iris, but not in the SuGl, are higher than in the wild-type. This increase, along with reduced levels of NGF receptors (TrkA and p75NTR) in SCG, may be partly responsible for the observed loss of neurons projecting to the iris. These alterations, combined with a reduction in polysialylated-NCAM and neurofilament protein levels in SCG, may also account for reduced axon defasciculation and terminal branching in mdx mouse SCG targets.

Synaptic remodeling induced by axotomy of superior cervical ganglion neurons: Involvement of metalloproteinase-2.

We previously demonstrated the involvement of the dystrophin-dystroglycan (Dys-DG) complex in the stabilization of intraganglionic synapses in rodent superior cervical ganglion (SCG) by investigating changes in the organization of their post-synaptic apparatus induced either by ganglionic neuron axotomy or by the lack of Dys in genetically dystrophic mdx mice, or by the combination of the two. A role of the matrix metalloproteinases (MMPs) MMP-2 and MMP-9 in the degradation of DG and, hence, in disrupting the connection between the extracellular matrix (ECM) and the cortical cytoskeleton, has recently been proposed. We hypothesized that the degradation by MMPs of ECM proteins and DG in ganglionic neurons may be involved in injury-induced synaptic detachment observed in rodent SCG. In this review, we report changes in MMP-2 and in the proteins involved in one of the enzymatic cascades of activation induced by axotomy of rat SCG neurons. This will be preceded by a description of our previous observations that led to investigate the role of MMP-2 in this experimental model.

Axotomy of sympathetic neurons activates the metalloproteinase-2 enzymatic pathway.

We have previously shown that intraganglionic synapse disassembly consequent on superior cervical ganglion (SCG) neuron axotomy was preceded by the loss of the dystroglycan beta subunit (beta-DG) localized at the postsynaptic specializations. Because DG, a transmembrane molecular complex bridging the extracellular matrix to the cortical cytoskeleton, could be a physiological target of metalloproteinases (MMPs) 2 and 9, we investigated their possible involvement in the injury-induced intraganglionic synapse disassembly. In rat SCG, only MMP-2 was present and localized in both neurons and nonneuronal cells. After ganglion neuron axotomy, both MMP-2 activity and protein level increased, whereas the level of its mRNA was unchanged, suggesting prominent MMP-2 posttranslational regulation. mRNA and protein levels of the enzymes involved in the MMP-2 activation pathway, the membrane-type 1-MMP (MT1-MMP), and the tissue inhibitor of metalloproteinase-2 (TIMP-2) also increased after injury with a time course that correlated with that of MMP-2 activation. In addition, postganglionic nerve crush induced an increase in the beta-DG 30-kDa fragment produced by the MMP-dependent degradation of DG. These data suggest that MMP-2 activated during SCG neuron reaction to axotomy may degrade postsynaptic DG, contributing to the disruption of the molecular bridge between pre- and postsynaptic elements and disassembly of the intraganglionic synapses.

Nicotinic acetylcholine receptor subtypes in the rat sympathetic ganglion: pharmacological characterization, subcellular distribution and effect of pre- and postganglionic nerve crush.

Nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic transmission in autonomic ganglia, which innervate and control the activity of most visceral organs. By combining ultrastructural, immunocytochemical, and pharmacological analyses, we characterized the nAChR subtypes in the rat superior cervical ganglion (SCG) and the effect of pre- and postganglionic nerve crush on their number in the ganglion and their distribution at the intraganglionic synapses. Binding with radioactive nicotinic ligands, immunoprecipitation, and immunolocalization experiments revealed the presence of different nAChR subtypes: those containing the alpha3 subunit associated with beta4 and/or beta2 subunits that bind 3H-Epibatidine with high affinity, and those containing the alpha7 subunit that bind 125I-alphaBungarotoxin. After postganglionic nerve crush, the number of nicotinic receptors and immunopositive intraganglionic synapses for each nAChR subunit strongly decreased. Both the number of nAChRs and immunoreactivity recovered 26 days after injury, when regenerating postganglionic fibers had reinnervated the peripheral target organs, as shown by the restoration of tyrosine hydroxylase immunoreactivity in the iris. This observation and the lack of any effect of preganglionic nerve crush on the number of nicotinic receptors suggest that the peripheral targets affect the organization of intraganglionic synapses in adult SCG.

Genetically dystrophic mdx/mdx mice exhibit decreased response to nicotine in passive avoidance.

mdx mice are considered as a genetic homologous of human Duchenne muscular dystrophy. Recent evidence demonstrates that in mouse sympathetic ganglion dystrophin is involved in the stabilization of nicotinic acetylcholine receptor clusters. The purpose of this study was to verify possible effects of dystrophin alterations at the central level. This was assessed by evaluating the response to nicotine administration in mdx and wild-type mice. Thus the effects of post-training nicotine administrations (0.1, 0.25 and 0.5 mg/kg) were tested in mice subjected to a passive avoidance memory task, that measures the ability of mice to remember on test day a shock received 24 h before. Nicotine enhanced memory in wild-type as well as in mdx mice. However, the doses needed to increase memory in mdx were higher than in wild-type. These results are discussed in terms of possible functional changes in central nicotinic acetylcholine receptor in mdx mice.

Effects of acute and repeated daily exposure to hypergravity on spatial learning in mice.

Studies in humans have revealed that exposure to altered gravity may lead to impairments in cognitive functions. The objective of this study was to test whether mice exposed to hypergravity using a centrifuge apparatus showed learning impairments in a spatial learning task. Mice rotating at 1G or at 2G acceleration gravity and non-rotating controls were tested for reactivity to a spatial change after either a single 1 h or five repeated 1 h daily rotations in the centrifuge. While no differences among groups were found in the performance after single exposure to altered gravity, 5 days of repeated exposures to 1G or 2G gravity conditions significantly affected mouse ability to discriminate a new spatial arrangement. Additionally, this effect was stronger in the animals repeatedly exposed to 2G rather than to 1G conditions.

Involvement of the plasminogen enzymatic cascade in the reaction to axotomy of rat sympathetic neurons.

Axotomy of superior cervical ganglion (SCG) neurons is characterized by peripheral regeneration of injured axons and temporary disassembly of the intraganglionic synapses, necessary for synaptic silencing. Both events require remodeling of the extracellular matrix achieved through controlled proteolysis of its components by different enzymatic systems. In this study, we investigate the involvement of the plasminogen enzymatic cascade in the response to axotomy of rat SCG neurons. All components of this proteolytic pathway, tissue plasminogen activator (tPA), plasminogen, membrane receptor annexin II and tPA inhibitor (PAI-1), are constitutively expressed in uninjured SCG and increase significantly after SCG neuron axotomy. Immunolocalization of plasminogen, the key protein converted into the enzymatically active plasmin by tPA, in both neurons and non-neuronal cells indicates that all cell types are involved in the response to axotomy. The time course of activation of tPA/plasmin enzymatic pathway suggests its involvement in both intraganglionic synapse remodeling and axonal regeneration.

Gene expression pathways induced by axotomy and decentralization of rat superior cervical ganglion neurons.

To identify genes potentially involved in remodelling synaptic connections, we induced the temporary detachment of pre- and post-synaptic elements by axotomy or denervation of rat superior cervical ganglion neurons. cDNA microarray analysis followed by stringent selection criteria allowed the identification of a panel of genes whose expression was modulated by axotomy at various time points after injury. Among these genes, 11 were validated by real-time reverse transcriptase-polymerase chain reaction on independently prepared samples after superior cervical ganglion neuron axotomy (1, 3 and 6 days) and compared with the effect of decentralization (8 h, 1 and 3 days). These genes code for extracellular matrix/space [apolipoprotein D (apoD), decorin, collagen alpha1 type I, collagen alpha1 type III] and intermediate filament (vimentin) proteins, for modulators of neurite outgrowth (thrombin receptor, plasminogen activator inhibitor-1, bone morphogenetic protein 4, annexin II and S-100-related protein, clone 42C) and for a nerve cell transcription factor (brain finger protein). Eight of these 11 genes showed significant and persistent modulations after both types of injury. Finally, protein levels of apoD were shown to increase in superior cervical ganglion after axotomy. Our results identify hitherto unrecorded genes responsive to axotomy and decentralization of superior cervical ganglion neurons, and probably involved in synapse formation, remodelling and elimination.

Lack of dystrophin leads to the selective loss of superior cervical ganglion neurons projecting to muscular targets in genetically dystrophic mdx mice.

Autonomic imbalance is a pathological aspect of Duchenne muscular dystrophy. Here, we show that the sympathetic superior cervical ganglion (SCG) of mdx mice, which lack dystrophin (Dp427), has 36% fewer neurons than that of wild-type animals. Cell loss occurs around P10 and affects those neurons innervating muscular targets (heart and iris), which, differently from the submandibular gland (non-muscular target), are precociously damaged by the lack of Dp427. In addition, although we reveal altered axonal defasciculation in the submandibular gland and reduced terminal sprouting in all SCG target organs, poor adrenergic innervation is observed only in the heart and iris. These alterations, detected as early as P5, when neuronal loss has not yet occurred, suggest that in mdx mice the absence of Dp427 directly impairs the axonal growth and terminal sprouting of sympathetic neurons. However, when these intrinsic alterations combine with structural and/or functional damages of muscular targets, neuronal death occurs.

Dystrophin stabilizes alpha 3- but not alpha 7-containing nicotinic acetylcholine receptor subtypes at the postsynaptic apparatus in the mouse superior cervical ganglion.

The nicotinic acetylcholine receptor (nAChR) subtypes were characterized in the superior cervical ganglion (SCG) of wild-type and dystrophin-lacking mdx mice. The binding of Epibatidine and alphaBungarotoxin, ligands for alpha3- and alpha7-containing receptors, respectively, revealed, for each ligand, a single class of high-affinity binding sites, with similar affinity in both wild-type and mdx mice. The Epibatidine-labeled receptors were immunoprecipitated by antibodies against the alpha3, beta2, and beta4 subunits. Immunocytochemistry showed that the percentage of alpha3-, beta2-, and beta4- but not of alpha7-immunopositive postsynaptic specializations was significantly lower in mdx than in wild-type mouse SCG. These observations suggest that the mouse SCG contains nAChRs, stabilized by dystrophin, in which the alpha3 subunit is associated with the beta2 and/or beta4 subunits. Conversely, dystrophin is not involved in the stabilization of the alpha7-containing nAChRs, as the percentage of alpha7-immunopositive synapses is similar in both wild-type and mdx mouse SCG.