Research Projects The role of of a-SMN, the novel SMN1 gene isoform, in the pathogenesis of Spinal Muscular Atrophy (SMA) and in the process of axon growth
The survival motor neuron (SMN) gene is the determining gene of spinal muscular atrophy (SMA), a lethal autosomal recessive disease of childhood characterized by selective motor neuron death (1). Two SMN genes are present in humans: the more telomeric copy SMN1, i.e., the gene responsible for SMA, and the centromeric copy SMN2, which modulates disease severity (2). The SMN1 gene mostly produces the full-length protein (FL-SMN), whereas SMN2 mainly produces a protein isoform lacking exon 7, which is unstable and rapidly degraded (3). The pathogenesis of SMA is still unclear, and the issue of why reduced levels of an ubiquitously expressed protein like SMN eventually leads to selective motor neuron death remains unsolved. In addition to the poor understanding of SMA pathogenesis, no effective therapy is available.
We have recently identified a novel splice variant of the SMN gene, the a-SMN protein (axonal SMN: Setola et al, PNAS 2007) (4), with selective expression in the axons of spinal cord motor neurons. The a-SMN protein is endowed with remarkable properties in inducing axon growth. Indeed, the over-expression of a-SMN dramatically stimulates time-dependent axon growth in both immortalized motor neurons and primary cultured neurons. The axonogenic properties of a-SMN are potentially of great relevance for the development of viral-vector-based strategies for the management of SMA.
The present research project intends to provide on one side compelling evidence that a-SMN is involved in the pathogenesis of SMA, and, on the other, to investigate the cellular and molecular mechanisms underlying the a-SMN-induced axon growth. The project will focus on the following aims:
1) Effect of SMN1 mutations on a-SMN-induced axon growth. SMN1 mutations derived from SMA patients have been already used in in vitro and in vivo assays to verify FL-SMN functional alterations possibly relevant for SMA pathogenesis. These mutations impair specific functional properties of the FL-SMN protein (5). Some intragenic mutations are distributed in the 5’ portion of the SMN1 gene (6), thus altering the structure of the a-SMN transcript and protein. We will verify whether SMN1 mutations (A2G; A111G; E134K) may alter the axonogenic properties of a-SMN. Wild-type and the different point mutated a-SMN constructs tagged at the N-terminus will be transiently over-expressed in NSC34 motor neurons, and the expression of the constructs will be evaluated by means of Western Blot and confocal immunofluorescence (IF). Modifications of different cell parameters induced by the different a-SMN constructs will be quantified and statistically analyzed, with particular attention to axonal arborization and growth. The demonstration that SMA-derived SMN1 mutations may alter the axonogenic properties of a-SMN would represent a further indication of the role of a-SMN in SMA. This part of the project will be conducted with the collaboration of the group led by Enrico Garattini at the “Mario Negri” Institute in Milano.
2) Proteins interacting with a-SMN in the process of axon growth. As a first step in analyzing the molecular processes leading to axonogenesis set in motion by a-SMN, we intend to investigate the partner proteins functionally interacting with a-SMN. We will verify the FL-SMN and SIP1/gemin2 proteins (7) as first potential partners of a-SMN. The gemin2 binding site (located in exon1/2b) is maintained in the a-SMN primary sequence, indicating that the gemin2/a-SMN interaction is at least theorically possible. After co-transfection of N-terminally tagged proteins in NSC34 motor neurons, we will verify the functional association between a-SMN and the other two proteins at the single cell and molecular levels. Confocal IF will evaluate the co-localization patterns and the post-transfection modifications of different cell parameters. Subcellular fractionation and co-immunoprecipitation will evaluate the molecular association of the transfected proteins. Finally, bidimensional Blue Native electrophoresis will verify whether a-SMN and gemin2 are the core of a macromolecular complex acting in axons to promote axonogenesis similarly to what demonstrated for FL-SMN/gemin2 complex in regulating spliceosomal biogenesis. The demonstration of a-SMN/gemin2 functional synergism, as indicated by our preliminary data, would be an important step forward in the dissection of the molecular mechanisms underlying the role of a-SMN in axonogenesis.
3) a-SMN functional knockdown in vitro. Preliminary RT-PCR and confocal IF data have already shown that native a-SMN is indeed expressed in primary cultured neurons and subcellularly localized in axons. We will therefore use RNA interference in primary neuronal cultures to verify whether a-SMN silencing is associated with axon retraction. We will use short hairpin RNAs selectively targeting the intron3 sequence and GFP-expressing vectors to allow visualization of transfected cells. shRNAs reducing the expression of FL-SMN mRNAs will be also selected and employed. The degree of a-SMN or FL-SMN silencing, along with the effect of the different shRNAs on axonal length and cell survival will be verified. These experiments should provide further insights into the normal role of a-SMN in neuronal differentiation and survival. This part of the project will be conducted in collaboration with the group of Chris Henderson at the Motor Neuron Centre of the Columbia University, New York (8).
4) novel therapeutic strategies in transgenic models for both SMA and amyotrophic lateral sclerosis (ALS). We plan to utilize replication incompetent AAV6 vectors expressing the novel a-SMN transcript. These vectors will be injected in peripheral muscles of two different transgenic mice, the G93A mice and Smn-/-/SMN2/Delta7 mice, i.e., the two most widely used transgenic mice for ALS and SMA, respectively. The rational of using injections in the peripheral muscle stands on the retrograde transport of these vectors from the muscular cells to the spinal cord motor neurons. Mice motor behavior and survival rate, as well as the morphology of transduced motor neurons within the spinal cord, will be assessed to verify the functional impact of the transduced SMN proteins. This part of the project will be conducted in collaboration with the group of Patrick Aebischer at EPFL in Lausanne, Switzerland.
References
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3. Lorson and Androphy, Hum Mol Genet 2000, 9:259-65.
4. Setola et al, PNAS 2007, 104:1959-1964.
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8. Raoul et al, Nat Med 2005, 11:423-8.
NMDA receptor alterations in malformations of cortical development: analysis of human patients and experimental animal models
Epilepsy is a group of heterogeneous syndromes affecting more than 50 million people worldwide, and about 30% of epileptic patients are intractable to anti-epileptic drugs. In the last two decades, the widespread use of modern imaging techniques in medical practice has allowed the in vivo diagnosis of an increasing number of patients affected by malformations of cortical development or MCDs. MCDs are abnormalities of the normal cerebral structure originating during ontogenesis from either genetic or acquired impairments of the complex processes by which the brain is eventually built. They are now recognized as a major cause of developmental disabilities and focal drug-resistant epilepsy, and affected patients are frequently subjected to epilepsy surgery for the relief of their intractable seizures.
Despite recent efforts in the electro-clinical characterization and classification of the different types of MCDs, and the report of a few responsible genes in a limited number of affected patients, the issues of why developmental malformations develop, why heterotopic neurons become hyperexcitable, and why epilepsy is drug-resistant in the majority of cases of MCDs, are all largely unexplored issues. Recent data have suggested that alterations of excitatory synaptic activity, particularly mediated by N-Methyl-D-Aspartate (NMDA) receptors, are major determinants of epileptogenesis in MCDs (1-4). On this line, we have recently shown a selective alteration of the NMDA receptor complex in human cerebral heterotopia (5) and in rats prenatally treated with the antiproliferative agent methylazoxymethanol-acetate or MAM (6), i.e., an animal model of developmental human brain malformations (7-8).
On these bases, the present research is aimed at investigating the cellular and molecular mechanisms underlying the origin of the brain malformation and the genesis of epileptic phenomena in both human patients with MCDs and in the MAM-treated rats. The project will focus on the following aims:
1) analysis of synaptic and extrasynaptic NMDA receptors in human focal cortical dysplasia. We plan to analyze human cortical samples obtained from human patients affected by Taylor’s type focal cortical dysplasia (9) undergoing epilepsy surgery. For this goal, human cortical samples ablated for strictly therapeutic reasons will be either immediately fixed by immersion in freshly prepared 4% paraformaldehyde and then used in confocal immunofluorescence experiments; or snap frozen in liquid nitrogen and used in western blot experiments. This combined approach will allow verifying at the single cell level and in different subcellular compartments i) the expression, composition, and localization at post-synaptic or extra-synaptic sites of the different NMDA receptor subunits and associated proteins of the MAGUK family, and ii) the NMDA-mediated down-stream activation of intracellular pathways leading to cell survival or death (10) in the hyperexcitable neurons of the dysplastic cortex.
2) generation of a “double-hit” animal model characterized by brain malformations and spontaneous epilepsy. Since MAM-rats are not characterized by frequently occurring spontaneous seizures, we also plan to induce the occurrence of spontaneous seizures by treating adult MAM rats with intra-peritoneal injections of pilocarpine. Pilocarpine is a muscarinic agent that induces acute epileptic status and, after a silent period of approximately 2 weeks, the occurrence of high frequency spontaneous seizures (11). We plan to verify i) whether the brain and cortical abnormalities induced by MAM in treated rats may increase the susceptibility of the epileptogenic process set in motion by pilocarpine. To this end, double-treated rats will be EEG recorded by means of skull and intracerebral electrodes, and video-monitored for the occurrence, frequency, and phenomenology of seizures. In addition, we will ii) investigate at both the morphology and molecular level (with confocal microscopy, western blot, and RT-PCR analysis) whether and how the pilocarpine treatment could increase the abnormalities of the NMDA receptor complex, already present in the heterotopic neurons of MAM treated rats; and iii) verify if other non-neuronal factors (i.e., blood-brain barrier alterations, inflammatory changes,…) may influence the epileptogenic process.
3) analysis of conventional and novel therapeutic approaches in the “double-hit” MAM/pilocarpine model. Preliminary data obtained in the “double-hit” MAM-pilocarpine model suggest that these rats are indeed more prone to develop spontaneous and recurrent seizures if compared to age-matched rats treated with pilocarpine alone. The development of spontaneous epilepsy is paralleled by alteration in the expression level and subunit composition of NMDA receptor in the dysplastic epileptogenic neurons. We intend to use this model i) to test the efficacy of conventional antiepileptic drugs (i.e., oxcarbazepine, phenytoin, benzodiazepines…) in controlling seizures on one side, and affecting the NMDA receptor alterations and NMDA-dependent downstream intracellular pathways on the other; and ii) to verify whether non-conventional therapeutic approaches (such as anti-inflammatory agents, cell permeable peptides (12) interfering with the intracellular C-terminus of NMDA receptor subunits) may modify seizure susceptibility and intracellular signalling in cortical pyramidal neurons.
References
1. During et al, Science 2000; 287: 1453-60.
2. Ying et al, J Neuropathol Exp Neurol 1998; 57: 47-62.
3. Crino et al, Neurology 2001; 56: 906-913.
4. Moddel et al, Brain Res 2005; 1046: 10-23.
5. Finardi et al, J Neuropathol Exp Neurol. 2006; 65:883-93.
6. Gardoni et al, J Neurophatol Exp Neurol 2003; 62:662-75.
7. Colacitti et al, J Neurophatol Exp Neurol 1999; 58:92-106.
8. Battaglia et al, Epilepsia. 2006; 47: 86-97.
9. Taylor et al, J Neurol Neurosurg Psychiatry. 1971; 34: 369-87.
10. Hardingham et al, Nat Neurosci 2001; 4: 261-7.
11. Leite et al, Epilepsy Res. 2002; 50: 93-103.
12. Cui et al, J Neurosci 2007; 27: 9901-15.
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