Adenoviral cardiotrophin-1 gene transfer protects pmn mice from progressive motor neuronopathy.

Cardiotrophin-1 (CT-1), an IL-6-related cytokine, causes hypertrophy of cardiac myocytes and has pleiotropic effects on various other cell types, including motoneurons. Here, we analyzed systemic CT-1 effects in progressive motor neuronopathy (pmn) mice that suffer from progressive motoneuronal degeneration, muscle paralysis, and premature death. Administration of an adenoviral CT-1 vector to newborn pmn mice leads to sustained CT-1 expression in the injected muscles and bloodstream, prolonged survival of animals, and improved motor functions. CT-1-treated pmn mice showed a significantly reduced degeneration of facial motoneuron cytons and phrenic nerve myelinated axons. The terminal innervation of skeletal muscle, grossly disturbed in untreated pmn mice, was almost completely preserved in CT-1-treated pmn mice. The remarkable neuroprotection conferred by CT-1 might become clinically relevant if CT-1 side effects, including cardiotoxicity, could be circumvented by a more targeted delivery of this cytokine to the nervous system.

Olesoxime favors oligodendrocyte differentiation through a functional interplay between mitochondria and microtubules.

Multiple sclerosis (MS) is a neurodegenerative disease characterized by episodes of immune attacks and oligodendrocyte death leading to demyelination and progressive functional deficits. New therapeutic strategies are needed to stimulate the spontaneous regenerative process observed in some patients. Spontaneous myelin repair relies on the mobilization and differentiation of endogenous oligodendrocyte progenitors at the lesion site. Olesoxime, a cholesterol-like compound, has been shown to favor oligodendrocyte maturation in culture and promote myelin regeneration in rodents. Here, we study the mode of action of this compound and show that it binds to oligodendrocyte mitochondria, leading to their hyperfilamentation. This is accompanied by a reduction of basal superoxide levels, and accumulation of End Binding Protein 1 (EB1) at growing ends of microtubules. In parallel, we demonstrate that Reactive Oxygen Species (ROS) scavengers also promote oligodendrocyte differentiation, together with increasing mitochondrial filamentation and EB1-dependent microtubule polymerization. Altogether, our data uncover the mechanisms by which olesoxime promotes oligodendrocyte maturation. They also reveal that a bidirectional relationship between mitochondria hyperfilamentation and ROS level modulation controls oligodendrocyte maturation. This study identifies new cellular mechanisms to target for the development of regenerative treatments for MS.

Analytical study of rat retrotransposon VL30 RNA dimerization in vitro and packaging in murine leukemia virus.

A sequence of the rat retrotransposon virus-like 30 S RNA (VL30) located next to the 5' end of the Harvey murine sarcoma virus (HaMSV) genome was recently found to form stable dimeric RNA in vitro and to direct the efficient packaging of VL30-derived recombinant RNAs into MuLV virions. To study the structure-function relationships of the rat VL30 dimerization-encapsidation signal (E/DLS), we have performed biochemical and genetic studies of rat VL30 RNA dimerization in vitro. The results show that temperature and specific cation/RNA interactions are important for VL30 dimerization in vitro. VL30 RNA dimerization is optimal at 55 degrees C and Li+ dramatically enhances the stability of VL30 dimeric RNA. In addition, a genetic analysis of VL30 RNA dimerization reveals that a 5' G-rich sequence is critical for dimer formation and that a UGUCUUGUC repeat contributes to VL30 dimer stability. Interestingly enough, substitution of an A for a G in the 5' G-rich sequence is sufficient to abolish VL30 RNA dimerization in vitro. Taken together, these biochemical and genetic data indicate that dimerization of VL30 RNA involves non-canonical base-pairings and possible purine-purine interactions. Nucleocapsid protein NCp10 of murine leukemia virus (MuLV), a gag-encoded protein that is tightly associated with genomic RNA in the virion core, has been shown to have nucleic acid binding and annealing activities. Here we report that the viral NCp10 protein is able to bind tightly to annealing activities. Here we report that the viral NCp10 protein is able to bind tightly to the retrotransposon VL30 RNA and to activate its dimerization. Moreover, mutations in the 5' G-rich sequence of the VL30 dimerization sequence impaired NCp10 binding to RNA. Recombinant MLV-VL30 vectors with mutations in the VL30 dimerization sequence were constructed. Results obtained in vivo clearly show that the mutations that had a deleterious effect on the packaging of MLV-VL30 retroviral vector in vivo were those that impaired VL30 RNA dimerization and interactions with NCp10 in vitro, even the single mutation in the 5' G-rich region. Therefore, these findings suggest that packaging of VL30 RNA into MuLV virions requires specific interactions between RNA dimerization sequences and viral NC protein molecules.

Protective role of olesoxime against wild-type α-synuclein-induced toxicity in human neuronally differentiated SHSY-5Y cells.

BACKGROUND AND PURPOSE: Parkinson's disease (PD) is usually diagnosed clinically from classical motor symptoms, while definitive diagnosis is made postmortem, based on the presence of Lewy bodies and nigral neuron cell loss. α-Synuclein (ASYN), the main protein component of Lewy bodies, clearly plays a role in the neurodegeneration that characterizes PD. Additionally, mutation in the SNCA gene or copy number variations are associated with some forms of familial PD. Here, the objective of the study was to evaluate whether olesoxime, a promising neuroprotective drug can prevent ASYN-mediated neurotoxicity. EXPERIMENTAL APPROACH: We used here a novel, mechanistically approachable and attractive cellular model based on the inducible overexpression of human wild-type ASYN in neuronally differentiated human neuroblastoma (SHSY-5Y) cells. This model demonstrates gradual cellular degeneration, coinciding temporally with the appearance of soluble and membrane-bound ASYN oligomers and cell death combining both apoptotic and non-apoptotic pathways. KEY RESULTS: Olesoxime fully protected differentiated SHSY-5Y cells from cell death, neurite retraction and cytoplasmic shrinkage induced by moderate ASYN overexpression. This protection was associated with a reduction in cytochrome c release from mitochondria and caspase-9 activation suggesting that olesoxime prevented ASYN toxicity by preserving mitochondrial integrity and function. In addition, olesoxime displayed neurotrophic effects on neuronally differentiated SHSY-5Y cells, independent of ASYN expression, by promoting their differentiation. CONCLUSIONS AND IMPLICATIONS: Because ASYN is a common underlying factor in many cases of PD, olesoxime could be a promising therapy to slow neurodegeneration in PD.

Olesoxime delays muscle denervation, astrogliosis, microglial activation and motoneuron death in an ALS mouse model.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. The pathology is mimicked to a striking degree in transgenic mice carrying familial ALS-linked SOD1 gene mutations. Olesoxime (TRO19622), a novel neuroprotective and reparative compound identified in a high-throughput screen based on motoneuron (MN) survival, delays disease onset and improves survival in mutant SOD1(G93A) mice, a model for ALS. The present study further analyses the cellular basis for the protection provided by olesoxime at the neuromuscular junctions (NMJ) and the spinal cord. Studies were carried out at two disease stages, 60 days, presymptomatic and 104 days, symptomatic. Cohorts of wild type and SOD1(G93A) mice were randomized to receive olesoxime-charged food pellets or normal diet from day 21 onward. Analysis showed that olesoxime initially reduced denervation from 60 to 30% compared to SOD1(G93A) mice fed with control food pellets while at the symptomatic stage only a few NMJs were still preserved. Immunostaining of cryostat sections of the lumbar spinal cord with VAChT to visualize MNs, GFAP for astrocytes and Iba1 for microglial cells showed that olesoxime strongly reduced astrogliosis and microglial activation and prevented MN loss. These studies suggest that olesoxime exerts its protective effect on multiple cell types implicated in the disease process in SOD1(G93A) mice, slowing down muscle denervation, astrogliosis, microglial activation and MN death. A Phase 3 clinical study in ALS patients will determine whether olesoxime could be beneficial for the treatment of ALS.

Therapeutic benefit of ciliary neurotrophic factor in progressive motor neuronopathy depends on the route of delivery.

Ciliary neurotrophic factor (CNTF) has demonstrated therapeutic effects in several mouse mutants with motoneuronal degeneration. However, the poor bioavailability and toxic side effects of recombinant CNTF protein have complicated its use in patients with amyotrophic lateral sclerosis. CNTF gene transfer strategies were developed but faced the question of whether CNTF should be delivered to motoneuron cell bodies or to their axons or muscle targets. To address this issue, we have used an adenoviral vector (AdCNTF) coding for a secretable form of CNTF and compared different routes of its administration in the mouse mutant progressive motor neuronopathy (pmn). Intramuscular, intravenous, and intracerebroventricular injections of AdCNTF or the control vector AdlacZ resulted in transgene expression in skeletal muscle fibers, hepatocytes, and ependymal cells, respectively, as determined by histochemistry and reverse transcription-polymerase chain reaction. AdCNTF intramuscularly treated and intravenously treated pmn mice showed a 25% increase in mean life span and a reduced degeneration of phrenic myelinated nerve fibers, which correlated with elevated CNTF serum bioactivities. In contrast, intracerebroventricular AdCNTF administration did not affect the mean life span or motor axonal degeneration of pmn mice. The differential efficacy of peripheral and central CNTF vector administrations might be of interest for future studies in human motor neuron diseases.

Neuronal targeting of cardiotrophin-1 by coupling with tetanus toxin C fragment.

Cardiotrophin-1 (CT-1) is a potent neurotrophic factor for motoneurons but its clinical use in motor neuron diseases is precluded by side effects on the heart and liver. We explored the possibility of targeting CT-1 to neurons by coupling with the tetanus toxin fragment TTC. Genetic fusion proteins between CT-1 or GFP and TTC were produced in Escherichia coli and assayed in vitro. In contrast to uncoupled CT-1 or GFP, TTC-coupled proteins bound with high affinity to cerebral neurons and spinal cord motoneurons and were rapidly internalized. Glia, hepatocytes, or cardiomyocytes did not show detectable binding or uptake of TTC-coupled proteins. Similar to CT-1, TTC-coupled CT-1 induced IL-6 secretion by KB cells, activated Reg-2 gene expression, and promoted motoneuron survival in a dose-dependent manner. In vivo studies will test whether TTC-coupled CT-1 might be targeted to degenerating spinal cord or brain-stem motoneurons and migrate trans-synaptically to cortical motoneurons, which are also affected in amyotrophic lateral sclerosis.

Protective effects of cardiotrophin-1 adenoviral gene transfer on neuromuscular degeneration in transgenic ALS mice.

Amyotrophic lateral sclerosis (ALS) is mainly a sporadic neurodegenerative disorder characterized by loss of cortical and spinal motoneurons. Some familial ALS cases (FALS) have been linked to dominant mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1). Transgenic mice overexpressing a mutated form of human SOD1 with a Gly93Ala substitution develop progressive muscle wasting and paralysis as a result of spinal motoneuron loss and die at 5 to 6 months. We investigated the effects of neurotrophic factor gene delivery in this FALS model. Intramuscular injection of an adenoviral vector encoding cardiotrophin-1 (CT-1) in SOD1G93A newborn mice resulted in systemic delivery of CT-1, supplying motoneurons with a continuous source of trophic factor. CT-1 delayed the onset of motor impairment as assessed in the rotarod test. Axonal degeneration was slowed and skeletal muscle atrophy was largely reduced by CT-1 treatment. By monitoring the amplitude of the evoked motor response, we showed that the time-course of motor impairment was significantly decreased by CT-1 treatment. Thus, adenovirus-mediated gene transfer of neurotrophic factors might delay neurogenic muscular atrophy and progressive neuromuscular deficiency in ALS patients.