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.

Enhanced neuronal Met signalling levels in ALS mice delay disease onset.

Signalling by receptor tyrosine kinases (RTKs) coordinates basic cellular processes during development and in adulthood. Whereas aberrant RTK signalling can lead to cancer, reactivation of RTKs is often found following stress or cell damage. This has led to the common belief that RTKs can counteract degenerative processes and so strategies to exploit them for therapy have been extensively explored. An understanding of how RTK stimuli act at cellular levels is needed, however, to evaluate their mechanism of therapeutic action. In this study, we genetically explored the biological and functional significance of enhanced signalling by the Met RTK in neurons, in the context of a neurodegenerative disease. Conditional met-transgenic mice, namely Rosa26(LacZ-stop-Met), have been engineered to trigger increased Met signalling in a temporal and tissue-specific regulated manner. Enhancing Met levels in neurons does not affect either motor neuron (MN) development or maintenance. In contrast, increased neuronal Met in amyotrophic lateral sclerosis (ALS) mice prolongs life span, retards MN loss, and ameliorates motor performance, by selectively delaying disease onset. Thus, our studies highlight the properties of RTKs to counteract toxic signals in a disease characterized by dysfunction of multiple cell types by acting in MNs. Moreover, they emphasize the relevance of genetically assessing the effectiveness of agents targeting neurons during ALS evolution.

IFNγ triggers a LIGHT-dependent selective death of motoneurons contributing to the non-cell-autonomous effects of mutant SOD1.

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease that primarily affects motoneurons in the brain and spinal cord. Dominant mutations in superoxide dismutase-1 (SOD1) cause a familial form of ALS. Mutant SOD1-damaged glial cells contribute to ALS pathogenesis by releasing neurotoxic factors, but the mechanistic basis of the motoneuron-specific elimination is poorly understood. Here, we describe a motoneuron-selective death pathway triggered by activation of lymphotoxin-ß receptor (LT-ßR) by LIGHT, and operating by a novel signaling scheme. We show that astrocytes expressing mutant SOD1 mediate the selective death of motoneurons through the proinflammatory cytokine interferon-γ (IFNγ), which activates the LIGHT-LT-ßR death pathway. The expression of LIGHT and LT-ßR by motoneurons in vivo correlates with the preferential expression of IFNγ by motoneurons and astrocytes at disease onset and symptomatic stage in ALS mice. Importantly, the genetic ablation of Light in an ALS mouse model retards progression, but not onset, of the disease and increases lifespan. We propose that IFNγ contributes to a cross-talk between motoneurons and astrocytes causing the selective loss of some motoneurons following activation of the LIGHT-induced death pathway.

Trim17, a novel E3 ubiquitin-ligase, initiates neuronal apoptosis.

Accumulating data indicate that the ubiquitin-proteasome system controls apoptosis by regulating the level and the function of key regulatory proteins. In this study, we identified Trim17, a member of the TRIM/RBCC protein family, as one of the critical E3 ubiquitin ligases involved in the control of neuronal apoptosis upstream of mitochondria. We show that expression of Trim17 is increased both at the mRNA and protein level in several in vitro models of transcription-dependent neuronal apoptosis. Expression of Trim17 is controlled by the PI3K/Akt/GSK3 pathway in cerebellar granule neurons (CGN). Moreover, the Trim17 protein is expressed in vivo, in apoptotic neurons that naturally die during post-natal cerebellar development. Overexpression of active Trim17 in primary CGN was sufficient to induce the intrinsic pathway of apoptosis in survival conditions. This pro-apoptotic effect was abolished in Bax(-/-) neurons and depended on the E3 activity of Trim17 conferred by its RING domain. Furthermore, knock-down of endogenous Trim17 and overexpression of dominant-negative mutants of Trim17 blocked trophic factor withdrawal-induced apoptosis both in CGN and in sympathetic neurons. Collectively, our data are the first to assign a cellular function to Trim17 by showing that its E3 activity is both necessary and sufficient for the initiation of neuronal apoptosis.

Chronic activation in presymptomatic amyotrophic lateral sclerosis (ALS) mice of a feedback loop involving Fas, Daxx, and FasL.

The reasons for the cellular specificity and slow progression of motoneuron diseases such as ALS are still poorly understood. We previously described a motoneuron-specific cell death pathway downstream of the Fas death receptor, in which synthesis of nitric oxide (NO) is an obligate step. Motoneurons from ALS model mice expressing mutant SOD1 showed increased susceptibility to exogenous NO as compared with controls. Here, we report a signaling mechanism whereby NO leads to death of mutant, but not control, motoneurons. Unexpectedly, exogenous NO triggers expression of Fas ligand (FasL) in cultured motoneurons. In mutant SOD1(G93A) and SOD1(G85R), but not in control motoneurons, this up-regulation results in activation of Fas, leading through Daxx to phosphorylation of p38 and further NO synthesis. This Fas/NO feedback amplification loop is required for motoneuron death in vitro. In vivo, mutant SOD1(G93A) and SOD1(G85R) mice show increased numbers of positive motoneurons and Daxx nuclear bodies weeks before disease onset. Moreover, FasL up-regulation is reduced in the presence of transgenic dominant-negative Daxx. We propose that chronic low-level activation of the Fas/NO feedback loop may underlie the motoneuron loss that characterizes familial ALS and may help to explain its slowly progressive nature.

Death of motoneurons induced by trophic deprivation or by excitotoxicity is not prevented by overexpression of SMN.

The telomeric copy of the survival motor neuron gene (SMN1) is deleted or mutated in all spinal muscular atrophy (SMA) patients and these patients present mainly a loss in spinal motoneurons. Although studies performed in HeLa cells suggest that SMN may be involved in the biogenesis and possibly in recycling of spliceosomal small nuclear ribonucleoproteins (snRNPs), no link has been established between this function and the consequence of the absence of SMN in the specific loss of motoneurons. We attempted to answer the question of whether SMN plays a direct role in motoneuron survival by transducing cultured motoneurons with lentiviral vectors coding either for an antisense Smn mRNA or for full-length or truncated forms of SMN. We studied their effect on survival under different anti- or proapoptotic culture conditions. Our results show that increased levels of SMN are unable to protect motoneurons from death induced by trophic deprivation or by excitotoxicity. These results suggest that SMN is not a survival factor per se for motoneurons. In addition, overexpression of a truncated form of SMN shown to induce a modified subcellular localization and to exert a dominant-negative effect on snRNP biogenesis and RNA splicing in HeLa cells was ineffective in modifying both localization and survival in motoneurons.

Expression and subcellular localization of two isoforms of the survival motor neuron protein in different cell types.

The survival motor neuron (SMN) gene is deleted or mutated in over 98% of spinal muscular atrophy patients who show specific motoneuron loss. By performing transfection experiments with rat smn cDNA, we show that two isoforms of SMN with Mr of 32 kDa and 35 kDa are produced by the same cDNA. In cultured motoneurons, both forms colocalize in coiled bodies and not in GEMS bodies as shown for HeLa cells. Subcellular fractionation of cells acutely dissociated from rat embryonic ventral spinal cord shows that the two SMN isoforms have a different subcellular localization, namely, that the 32 kDa isoform is enriched in the cytosol, whereas the 35 kDa isoform is segregating in the microsomal fraction. We show that the 35 kDa isoform of SMN is part of an insoluble complex but is absent from the cytoplasmic membranes and from the mitochondria. Immunostaining studies show that neither SMN isoform colocalizes with Bcl-2, the mitochondrial antiapoptotic protein suggested to bind to SMN in HeLa cells. Our results show that the isoforms of SMN protein have different subcellular localization and may therefore play independent biological roles. Moreover, the absence of colocalization of SMN with Bcl-2 in motoneurons suggests that some of the interactors of SMN may vary depending on the cell type, and this underscores the importance of identifying motoneuron-specific SMN interactors.

Efficient gene transfer and expression of biologically active glial cell line-derived neurotrophic factor in rat motoneurons transduced wit lentiviral vectors.

Several studies have shown the ability of human immunodeficiency virus type 1 (HIV1)-based lentiviral vectors to infect nondividing brain and retinal neurons with high efficiency and long-term expression of the transduced gene. We show that purified embryonic motoneurons can be efficiently (>95%) transduced in culture using an HIV1-based lentiviral vector encoding LacZ. Expression of beta-galactosidase was observed for at least 9 days in these conditions. Furthermore, motoneurons transduced with a lentiviral vector expressing glial cell line-derived neurotrophic factor survived in the absence of additional trophic support, showing that the overexpressed protein was biologically active. Our results demonstrate the potential of lentiviral vectors in studying the biological effects of proteins expressed in motoneurons and in the development of future gene therapy for motoneuron diseases.

FGF9: a motoneuron survival factor expressed by medial thoracic and sacral motoneurons.

In the nervous system, fibroblast growth factor-9 (FGF9) is produced mainly by neurons. By whole-mount in situ hybridization, on embryonic rat spinal cord, we observed Fgf9 expression in a subpopulation of motoneurons located in the thoracic and sacral regions of the median motor column that innervate the axial muscles. Furthermore, FGF9 prevented death of purified rat and chicken motoneurons in culture in the same concentration range as FGF2. The targets of FGF9 are more restricted than that of the other FGFs, however, because conversely to FGF1 or FGF2, FGF9 had only weak or inexistent survival effects on chicken ciliary neurons or rat DRG. FGF9 may therefore play a role as an autocrine/paracrine survival factor for motoneurons.

Active killing of neurons during development and following stress: a role for p75(NTR) and Fas?

Evidence for active triggering of neuronal death continues to accumulate. The transmembrane receptors p75(NTR) and Fas can trigger (and in some cases are required for) programmed cell death of the neurons that express them, through signalling pathways that are regulated by a variety of cytoplasmic effectors. Neuronal death induced by trophic deprivation often requires Fas signalling, further blurring the boundaries between naturally occurring and stress-induced neuronal death.

Programmed cell death of embryonic motoneurons triggered through the Fas death receptor.

About 50% of spinal motoneurons undergo programmed cell death (PCD) after target contact, but little is known about how this process is initiated. Embryonic motoneurons coexpress the death receptor Fas and its ligand FasL at the stage at which PCD is about to begin. In the absence of trophic factors, many motoneurons die in culture within 2 d. Most (75%) of these were saved by Fas-Fc receptor body, which blocks interactions between Fas and FasL, or by the caspase-8 inhibitor tetrapeptide IETD. Therefore, activation of Fas by endogenous FasL underlies cell death induced by trophic deprivation. In the presence of neurotrophic factors, exogenous Fas activators such as soluble FasL or anti-Fas antibodies triggered PCD of 40-50% of purified motoneurons over the following 3-5 d; this treatment led to activation of caspase-3, and was blocked by IETD. Sensitivity to Fas activation is regulated: motoneurons cultured for 3 d with neurotrophic factors became completely resistant. Levels of Fas expressed by motoneurons varied little, but FasL was upregulated in the absence of neurotrophic factors. Motoneurons resistant to Fas activation expressed high levels of FLICE-inhibitory protein (FLIP), an endogenous inhibitor of caspase-8 activation. Our results suggest that Fas can act as a driving force for motoneuron PCD, and raise the possibility that active triggering of PCD may contribute to motoneuron loss during normal development and/or in pathological situations.

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.

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.

Subcellular distribution of survival motor neuron (SMN) protein: possible involvement in nucleocytoplasmic and dendritic transport.

Spinal muscular atrophy (SMA) is among the most common recessive autosomal diseases and is characterized by the loss of spinal motor neurons. A gene termed 'Survival of Motor Neurons' (SMN) has been identified as the SMA-determining gene. Recent work indicates the involvement of the SMN protein and its associated protein SIP1 in spliceosomal snRNP biogenesis. However, the function of SMN remains unknown. Here, we have studied the subcellular localization of SMN in the rat spinal cord and more generally in the central nervous system (CNS), by light fluorescence and electron microscopy. SMN immunoreactivity (IR) was found in the different regions of the spinal cord but also in various regions of the CNS such as the brainstem, cerebellum, thalamus, cortex and hippocampus. In most neurons, we observed a speckled labelling of the cytoplasm and a discontinuous staining of the nuclear envelope. For some neurons (e.g. brainstem nuclei, dentate gyrus, cortex: layer V) and, in particular in motoneurons, SMN-IR was also present as prominent nuclear dot-like-structures. In these nuclear dots, SMN colocalized with SIP1 and with fibrillarin, a marker of coiled bodies. Ultrastructural studies in the anterior horn of the spinal cord confirmed the presence of SMN in the coiled bodies and also revealed the protein at the external side of nuclear pores complexes, in association with polyribosomes, and in dendrites, associated with microtubules. These localizations suggest that, in addition to its involvement in the spliceosome biogenesis, the SMN protein could also play a part in nucleocytoplasmic and dendritic transport.

Cardiotrophin-1 requires LIFRbeta to promote survival of mouse motoneurons purified by a novel technique.

The cytokines ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) signal through a receptor complex formed between two transmembrane proteins, gp130 and LIFRbeta. In addition, CNTF also uses a ligand-binding component which is anchored to the cell membrane. In the case of cardiotrophin-1 (CT-1), LIFRbeta is also required in cardiomyocytes, but this has not been proven in neurons, and published data suggest that motoneurons may use a different receptor complex. We used Lifrbeta knockout mice to assess the requirement for this receptor component in the signal transduction of CT-1 in motoneurons. To study purified motoneurons from such mutants, we have developed a method allowing for isolation of highly purified mouse motoneurons. This protocol is based on the immunoaffinity purification of motoneurons using antibodies against the extracellular domain of the neurotrophin receptor, p75, followed by cell sorting using magnetic microbeads. We show that CNTF, LIF, and CT-1 are unable to promote the survival of motoneurons derived from homozygous Lifrbeta-/- mutant embryos. Thus, LIFRbeta is absolutely required to transduce the CT-1 survival signal in motoneurons.

Adenovirus-mediated transfer of the neurotrophin-3 gene into skeletal muscle of pmn mice: therapeutic effects and mechanisms of action.

Several neurotrophic factors (CNTF, BDNF, IGF-1) have been suggested for the treatment of motor neuron diseases. In ALS patients, however, the repeated subcutaneous injection of these factors as recombinant proteins is complicated by their toxicity or poor bioavailability. We have constructed an adenovirus vector coding for neurotrophin-3 (AdNT-3) allowing for stable and/or targeted delivery of NT-3 to motoneurons. The intramuscular administration of this vector was tested in the mouse mutant pmn (progressive motor neuronopathy). AdNT-3-treated pmn mice showed prolonged lifespan, improved neuromuscular function, reduced motor axonal degeneration and efficient reinnervation of muscle fibres. NT-3 protein and also adenovirus vectors, when injected into muscle, can be transported by motoneurons via retrograde axonal transport to their cell bodies in the spinal cord. Using ELISA and RT-PCR analyses in muscle, spinal cord and serum of AdNT-3-treated pmn mice, we have investigated the contribution of these processes to the observed therapeutic effects. Our results suggest that most if not all therapeutic benefit was due to the continuous systemic liberation of adenoviral NT-3. Therefore, viral gene therapy vectors auch as adenoviruses, AAVs, lentiviruses and new types of gene transfer not based on viral vectors that allow for efficient in vivo liberation of neurotrophic factors have potential for the future treatment of human motor neuron diseases.

Survival motor neuron (SMN) protein in rat is expressed as different molecular forms and is developmentally regulated.

Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by a progressive degeneration of motoneurons in spinal cord and brainstem. The telomeric copy of a duplicated gene termed survival motor neuron (smn), which maps to chromosome 5q13, has been found to be deleted in most patients. The encoded gene product is a novel protein which recently has been shown to accumulate in specific nuclear organelles (gemini of coiled bodies, GEMS), and to play a part in the formation of the spliceosome complex. We have cloned and sequenced the rat smn cDNA. Antibodies generated against an N-terminus peptide recognized a main protein of 32 kDa in immunoblots of rat embryonic tissue extracts. Minor bands of 35 kDa, 45 kDa and, in perinatal muscle, of 24 kDa were also specifically detected, indicating that SMN is expressed as different molecular forms. Subcellular fractionation indicated that the 32 kDa form is mainly soluble, while the 35 kDa and 45 kDa products segregate to the microsomal-mitochondrial fraction. SMN protein is highly regulated during development: expression is high in embryonic tissues (central nervous system, muscle, lung and liver), and then progressively decreases to very low levels in most tissues of the adult. The demonstration of different molecular forms of SMN along with its developmental regulation may help to understand the contribution of this protein in the appearance of SMA phenotype.

Calpain inhibitors, but not caspase inhibitors, prevent actin proteolysis and DNA fragmentation during apoptosis.

Apoptosis, or programmed cell death, involves a cascade of regulatory events leading to the activation of specific proteases. However, the key substrates for these proteases remain to be identified. We previously demonstrated that levels of five unidentified polypeptides were specifically increased in neurons from embryonic chicken ciliary ganglia undergoing apoptosis by trophic deprivation. Here we show by microsequencing of two of these polypeptides that they are fragments of actin. One of them represents cleavage of actin at the site of interaction with DNase I. The same actin fragments are also found at early stages of apoptosis in chicken and rat dorsal root ganglion neurons, chicken spinal motoneurons and rat thymocytes. Actin fragmentation may play a role in the apoptotic process, since calpain inhibitors I and II both inhibit neuronal death and suppress actin fragmentation. In contrast, caspase (ICE family) inhibitors, though effective in delaying neuronal death, do not prevent actin cleavage or DNA fragmentation. These results indicate a key role for calpain-like proteases in neuronal programmed cell death and suggest that actin fragmentation in the cell is correlated with subsequent DNA fragmentation.

Trophic action of pharmacological substances with a guanidine group on mouse neuroblastoma cells and chick ganglionic neurons in culture.

A series of substances (designated CTQ compounds) with a guanidine group have been synthesized and tested for their ability to promote neuronal survival and neurite outgrowth. Mouse neuroblastoma clonal cell lines grown in serum-containing medium for 10 days as well as primary cultures of embryonic chicken ganglion neurons grown in serum-free defined medium for 1 or 2 days have been used for the experiments. Among the various CTQ compounds (CTQ1-CTQ20) tested, only CTQ8 exerted positive neurotrophic effects on these peripheral neuronal cells. At a concentration of 10(-4) M, CTQ8 enhanced neuritogenesis of neuroblastoma cells. However, the most striking influence of CTQ8 was its promoting effect (6- to 10-fold) on the survival of chicken ciliary and dorsal root ganglionic neurons at concentrations ranging from 10(-3) M to 5 x 10(-4) M.