IAPs are essential for GDNF-mediated neuroprotective effects in injured motor neurons in vivo.

During embryonic development, and in certain neurodegenerative diseases, neurons die by apoptosis. A new family of anti-apoptotic proteins, termed inhibitors of apoptosis (IAP), suppresses apoptosis through the direct inhibition of caspases. The anti-apoptotic activity of IAPs is inhibited by second mitochondria-derived activator of caspase (Smac)/DIABLO and XAF1 (ref. 8). IAPs, as well as neurotrophic factors, can protect degenerating neurons both in vivo and in vitro. However, the downstream targets of neurotrophic factors have not yet been identified. Here, we demonstrate that XIAP and NAIP, but not HIAP2, are directly involved in the intracellular response to glial cell-derived neurotrophic factor (GDNF). In newborn rats, GDNF regulates endogenous levels of XIAP and NAIP in motor neurons after sciatic nerve axotomy. The inhibition of XIAP or NAIP activity prevents GDNF-mediated neuroprotective effects. These results suggest that XIAP and NAIP are essential for intracellular signalling of GDNF in motor neuron survival.

Intrathecal delivery of CNTF using encapsulated genetically modified xenogeneic cells in amyotrophic lateral sclerosis patients.

Neuronal growth factors hold promise for providing therapeutic benefits in various neurological disorders. As a means of ensuring adequate central nervous system delivery of growth factors and minimizing significant adverse side effects associated with systemic delivery methods, we have developed an ex vivo gene therapy approach for protein delivery using encapsulated genetically modified xenogeneic cells. Ciliary neurotrophic factor (CNTF) has been shown in various rodent models to reduce the motor neuron cell death similar to that seen in amyotrophic lateral sclerosis (ALS). The initial trials focusing on the systemic administration of CNTF for ALS have been discontinued as a result of major side effects, thus preventing determination of the potential efficacy of the molecule. In order to deliver CNTF directly to the nervous system, we conducted a phase I study in which six ALS patients were implanted with polymer capsules containing genetically engineered baby hamster kidney cells releasing approximately 0.5 microgram of human CNTF per day in vitro. The CNTF-releasing implants were surgically placed within the lumbar intrathecal space. Nanogram levels of CNTF were measured within the patients' cerebrospinal fluid (CSF) for at least 17 weeks post-transplantation, whereas it was undetectable before implantation. Intrathecal delivery of CNTF was not associated with the limiting side effects observed with systemic delivery. These results demonstrate that neurotrophic factors can be continuously delivered within the CSF of humans by an ex vivo gene therapy approach, opening new avenues for the treatment of neurological diseases.

Cholinergic differentiation factor (CDF/LIF) promotes survival of isolated rat embryonic motoneurons in vitro.

We present evidence that the cholinergic differentiation factor (CDF), originally purified from cardiac and skeletal muscle cell-conditioned medium and found to be identical to leukemia inhibitory factor (LIF), promotes survival of embryonic day 14 rat motoneurons in vitro. These neurons were retrogradely labeled with the fluorescent tracer Dil and enriched on a density gradient or purified to homogeneity by fluorescence-activated cell sorting. Subnanomolar concentrations of CDF/LIF supported the survival of 85% of the motoneurons that would have died between days 1 and 4 of culture. The enhanced survival was accompanied by a 4-fold increase in choline acetyltransferase (ChAT) activity per culture. CDF/LIF also increased ChAT activity in dorsal spinal cord cultures, but had no detectable effect on ChAT levels in septal or striatal neuronal cultures. For comparison, other neurotrophic molecules were tested on motoneuron cultures. Ciliary neurotrophic factor had effects on motoneuron survival similar to those of CDF/LIF, whereas basic fibroblast growth factor was somewhat less effective. Nerve growth factor had no effect on the survival of rat motoneurons.

Cardiotrophin-1, a cytokine present in embryonic muscle, supports long-term survival of spinal motoneurons.

The muscle-derived factors required for survival of embryonic motoneurons are not clearly identified. Cardiotrophin-1 (CT-1), a cytokine related to ciliary neurotrophic factor (CNTF), is expressed at high levels in embryonic limb bud and is secreted by differentiated myotubes. In vitro, CT-1 kept 43% of purified E14 rat motoneurons alive for 2 weeks (EC50 = 20 pM). In vivo, CT-1 protected neonatal sciatic motoneurons against the effects of axotomy. CT-1 action on motoneurons was inhibited by phosphatidylinositol-specific phospholipase C (PIPLC), suggesting that CT-1 may act through a GPI-linked component. Since no binding of CT-1 to CNTFR alpha was detected, CT-1 may use a novel cytokine receptor alpha subunit. CT-1 may be important in normal motoneuron development and as a potential tool for slowing motoneuron degeneration in human diseases.

Persephin, a novel neurotrophic factor related to GDNF and neurturin.

A novel neurotrophic factor named Persephin that is approximately 40% identical to glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) has been identified using degenerate PCR. Persephin, like GDNF and NTN, promotes the survival of ventral midbrain dopaminergic neurons in culture and prevents their degeneration after 6-hydroxydopamine treatment in vivo. Persephin also supports the survival of motor neurons in culture and in vivo after sciatic nerve axotomy and, like GDNF, promotes ureteric bud branching. However, in contrast to GDNF and NTN, persephin does not support any of the peripheral neurons that were examined. Fibroblasts transfected with Ret and one of the coreceptors GFRalpha-1 or GFRalpha-2 do not respond to persephin, suggesting that persephin utilizes additional, or different, receptor components than GDNF and NTN.

Low intensity exercise attenuates disease progression and stimulates cell proliferation in the spinal cord of a mouse model with progressive motor neuronopathy.

Physical exercise has been shown to stimulate neurogenesis, increase resistance to brain trauma and disease, improve learning and increase levels of growth factors. We show that low intensity exercise has profound effects on the phenotype of a mouse mutant with progressive motor neuronopathy. These animals normally die at 47 days of age due to motoneuron loss and muscle atrophy. When mice undergo low intensity exercise, their lifespan increased by 74%, they exhibited a decreased loss of motoneurons, improved muscle integrity and a twofold increase in proliferating cells in the spinal cord. The molecular mechanism of neuroprotection may be related to insulin-like-growth factor 1 (IGF-1) since injections of antibodies to IGF-1 abrogated the effects of exercise on the increased life-span. Thus IGF-1 may act as a possible "exercise-induced" neuroprotective factor.

[Pathogenic mechanisms of neurodegenerative diseases: amyotrophic lateral sclerosis]. / Mécanismes pathogéniques des maladies neurodégénératives: l'exemple de la sclérose latérale amyotrophique.

Since its description by Charcot in 1869, the mechanism underlying the characteristic selective degeneration and death of motor neurons in amyotrophic lateral sclerosis (ALS) has remained a mystery. There is no effective remedy for this progressive, fatal disorder. Modern genetics have now identified two genes, SODI and ALS2 as primary causes of the disease and has implicated others as potential contributors. These insights have enabled development of model systems to test hypotheses of disease mechanism and potential therapies. Along with errors in the handling of synaptic glutamate and the potential excitotoxic response that it provokes, these model systems underscore the involvement of non-neuronal cells in disease progression and provide new therapeutic strategies.

Motoneuron survival is enhanced in the absence of neuromuscular junction formation in embryos.

Approximately half of the motoneurons produced during development die before birth or shortly after birth. Although it is believed that survival depends on a restricted supply of a trophic sustenance produced by the synaptic target tissue (i.e., muscle), it is unclear whether synapse formation per se is involved in motoneuron survival. To address this issue, we counted cranial motoneurons in a set of mutant mice in which formation of neuromuscular junctions is dramatically impaired (i.e., null mutants for agrin, nerve-derived agrin, rapsyn, and MuSK). We demonstrate that in the absence of synaptogenesis, there is an 18-34% increase in motoneuron survival in the facial, trochlear, trigeminal motor, and hypoglossal nuclei; the highest survival occurred in the MuSK-deficient animals in which synapse formation is most severely compromised. There was no change in the size of the mutant motoneurons as compared with control animals, and the morphology of the mutant motoneurons appeared normal. We postulate that the increased axonal branching observed in these mutants leads to a facilitated "access" of the motoneurons to muscle-derived trophic factors at sites other than synapses or that inactivity increases the production of such factors. Finally, we examined motoneurons in double mutants of CNTFRalpha(-/-) (in which there is a partial loss of motoneurons) and MuSK(-/-) (in which there is an increased survival of motoneurons). The motoneuron numbers in the double mutants parallel those of the single MuSK-deficient mice, indicating that synapse disruption can even overcome the deleterious effect of CNTFRalpha ablation.

Analysis of the retrograde transport of glial cell line-derived neurotrophic factor (GDNF), neurturin, and persephin suggests that in vivo signaling for the GDNF family is GFRalpha coreceptor-specific.

Neurturin (NRTN) and glial cell line-derived neurotrophic factor (GDNF) are members of a family of trophic factors with similar actions in vitro on certain neuronal classes. Retrograde transport of GDNF and NRTN was compared in peripheral sensory, sympathetic, and motor neurons to determine whether in vivo these factors are transported selectively by different neuronal populations. After sciatic nerve injections, NRTN was transported by sensory neurons of the dorsal root ganglion (DRG). Competition studies demonstrated only limited cross-competition between NRTN and GDNF, indicating selective receptor-mediated transport of these factors. By using immunohistochemistry, we identified two populations of NRTN-transporting DRG neurons: a major population of small, RET-positive, IB4-positive, non-TrkA-expressing neurons that also show the ability to transport GDNF and a minor population of calretinin-expressing neurons that fail to transport GDNF. Spinal motor neurons in the adult showed relatively less ability to transport NRTN than to transport GDNF, although NRTN prevented the cell death of neonatal motor neurons in a manner very similar to GDNF (Yan et al., 1995) and persephin (PSPN) (Milbrandt et al., 1998). Last, NRTN, like GDNF, was not transported to sympathetic neurons of the adult superior cervical ganglion (SCG) after injection into the anterior eye chamber. These data reveal a high degree of functional selectivity of GDNF family receptor-alpha (GFRalpha) coreceptor subtypes for NRTN and GDNF in vivo.

Clinical and molecular aspects of motoneurone diseases: animal models, neurotrophic factors and Bcl-2 oncoprotein.

Animal models of motor neurone disease (MND) are being increasingly used for screening molecules with clinical potential. A number of different treatments to decrease the progression of neuronal cell loss have been proposed; these include: Bcl-2 (B-cell leukaemia oncogene-2), neurotrophic factors, glutamate receptor inhibitors and Ca2+ channel antagonists. In this review Yves Sagot, Richard Vejsada and Ann C. Kato focus on the effects of neurotrophic factors and Bcl-2, both of which have been shown to prevent cell death in various experimental paradigms. Studies performed in animal models of MND have confirmed the potential of these molecules to support motoneurone survival. Some of them have been shown to act in synergy and these results are discussed in the context of molecular mechanisms leading to collaborative and synergistic activities, and also with respect to presumptive subpopulations of motoneurones, which express diverse receptors for neurotrophic factors. Finally, the current status of clinical trials for amyotrophic lateral sclerosis using neurotrophic factors will be discussed, as well as recent reports that neurotrophic factors can exert adverse effects on neuronal survival.

Gene therapy for amyotrophic lateral sclerosis (ALS) using a polymer encapsulated xenogenic cell line engineered to secrete hCNTF.

The gene therapy approach presented in this protocol employs a polymer encapsulated, xenogenic, transfected cell line to release human ciliary neurotrophic factor (hCNTF) for the treatment of Amyotrophic Lateral Sclerosis (ALS). A tethered device, containing around 10(6) genetically modified cells surrounded by a semipermeable membrane, is implanted intrathecally; it provides for slow continuous release of hCNTF at a rate of 0.25 to 1.0 micrograms/24 hours. The semipermeable membrane prevents immunologic rejection of the cells and interposes a physical, virally impermeable barrier between cells and host. Moreover, the device and the cells it contains may be retrieved in the event of side effects. A vector containing the human CNTF gene was transfected into a line of baby hamster kidney cells (BHK) with calcium phosphate using a dihydrofolate reductase-based selection vector with a SV40 promoter and contains a HSV-tk killer gene. hCNTF is a potent neurotrophic factor which may have utility for the treatment of ALS. Systemic delivery of hCNTF in humans has been frustrated by peripheral side effects, the molecule's short half life, and its inability to cross the blood-brain barrier. The gene therapy approach described in this protocol is expected to mitigate such difficulties by local intrathecal delivery of a known quantity of continuously-synthesized hCNTF from a retrievable implant.

ALS serum has no effect on three enzymatic activities in cultured human spinal cord neurons.

Dissociated human spinal cord cells were grown in monolayer cultures in the presence of serum from normal controls or from patients with ALS or other neurologic diseases. After 20 to 24 days, the levels of choline acetyltransferase, glutamic acid decarboxylase, and lactate dehydrogenase activities were determined in the cultures. On the basis of these biochemical measurements, there was no detectable difference between the effects of the three types of serum on the cultures.

Effects of amyotrophic lateral sclerosis sera on cultured cholinergic neurons.

Dissociated monolayer cultures of chick ciliary ganglion neurons have been used to study the effects of control and ALS sera. The cultured neurons survive and extend neurites for a minimum of 2 weeks in a standard tissue culture medium that contains 10% heat-inactivated human serum. Three parameters of the neurons have been examined when cultured in control and ALS sera for 8 to 12 days: (1) neuronal survival, (2) activity of the enzyme choline acetyltransferase, and (3) synthesis of 3H-acetylcholine using 3H-choline as precursor. ALS sera cause a small decrease in these three parameters, but this difference is not significant.

Partial purification of lymphoblasts after in vitro immunization increases the yield in Ig-producing hybridomas.

Using in vitro immunization with a human plasma protein (apolipoprotein-A1) as antigen, we have shown that it is possible to prepare more monoclonal antibodies using a ten-fold lower concentration of antigen compared to in vivo immunization procedures (Weech et al., 1985). In addition, we can increase the number of Ig-producing hybridomas after in vitro immunization by a simple one-step separation of the lymphoblasts on a Percoll gradient before the fusion procedure. In order to apply this procedure to in vivo immunization techniques, it is necessary to expand the B-blast/plasma cell population by culturing the spleen cells for 4-6 days before fusion. Only antibodies of the IgM class were produced with the in vitro technique. However, by combining in vivo priming with in vitro immunization, it is possible to produce specific antibodies to both IgG and IgM classes.

Synergistic but transient rescue effects of BDNF and GDNF on axotomized neonatal motoneurons.

Brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF), members of distinct families of polypeptide growth factors, have been shown to support motoneurons under various in vitro and in vivo conditions. We used a model of motoneuron cell death induced by sciatic nerve section in newborn rats and compared the efficacy of BDNF and GDNF administered alone or simultaneously in order to determine whether combinations of neurotrophic proteins can produce more potent motoneuron rescue than individual factors. The factors were administered by different methods, including (i) a single dose on to the transected nerve, (ii) continuous delivery from implanted slow-release polymer rods (BDNF) or encapsulated cells (GDNF), and (iii) repeated systemic injections (BDNF). Irrespective of the method of administration, either factor alone produced rescue effects which dramatically declined at two weeks as compared to one week post-lesion. In contrast, this decrease was significantly reduced when BDNF and GDNF were used simultaneously provided that one factor was applied on to the nerve while the other was continuously released from the rods or capsules. Other combinations in which GDNF was replaced by ciliary neurotrophic factor or axokine-1 failed to reproduce such additive activity. Two conclusions can be made from these experiments. First, when BDNF and GDNF are administered simultaneously but by distinct routes of delivery, their survival-promoting effects on the injured developing motoneurons are potentiated; second, even continuous delivery of each of these trophic factors alone cannot completely abrogate the time-dependent decline in rescue effects in this model of motoneuron cell death.

An orally active anti-apoptotic molecule (CGP 3466B) preserves mitochondria and enhances survival in an animal model of motoneuron disease.

Apoptosis and mitochondrial dysfunction are thought to be involved in the aetiology of neurodegenerative diseases. We have tested an orally active anti-apoptotic molecule (CGP 3466B) that binds to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in an animal model with motoneuron degeneration, i.e. a mouse mutant with progressive motor neuronopathy (pmn). In pmn/pmn mice, CGP 3466B was administered orally (10 - 100 nmol kg(-1)) at the onset of the clinical symptoms (2 weeks after birth). CGP 3466B slowed disease progression as determined by a 57% increase in life-span, preservation of body weight and motor performance. This improvement was accompanied by a decreased loss of motoneurons and motoneuron fibres as well as an increase in retrograde transport. Electron microscopic analysis showed that CGP 3466B protects mitochondria which appear to be selectively disrupted in the motoneurons of pmn/pmn mice. The data support evaluation of CGP 3466B as a potential treatment for motor neuron disease.

Progressive and selective degeneration of motoneurons in a mouse model of SMA.

Spinal muscular atrophy (SMA), an autosomal recessive disorder characterized by the degeneration of motoneurons of the spinal cord and brainstem, results from loss-of-function mutations in the survival motor neuron gene (smn). The goal of these experiments was to analyse axons and cell bodies of motoneurons in different regions of the CNS during disease progression in a mouse model of SMA carrying a deletion of the exon 7 directed to neurons. These experiments demonstrate a progressive loss of motor axons and of motoneurons in the CNS. This is the first study that describes a selective neurodegeneration in this line of mice and underlines the importance of exon 7 in some populations of motoneurons for survival in vivo.

BDNF-mediated rescue of axotomized motor neurones decreases with increasing dose.

Direct application of brain-derived neurotrophic factor (BDNF) to the cut end of axotomized immature motor neurones had only transient survival-promoting effects. Therefore, we have examined whether additional delivery of BDNF with repeated subcutaneous injections (1 mg/ml) could potentiate this short-term rescue of the lesioned sciatic and facial motor neurones in neonatal rats. Direct application of BDNF combined with intermittent (3-day intervals) injections slightly improved motor neurone survival. However, when BDNF was injected daily in addition to the direct application, the number of surviving lesioned motor neurones was markedly reduced. These findings, corroborated by results in embryonic spinal cord cultures, show that a dose-dependent reversal of BDNF-mediated positive effects on motor neurones occurs in vivo.

NGF-induced motoneuron cell death depends on the genetic background and motoneuron sub-type.

Nerve growth factor (NGF) promotes the survival of several neuronal populations, but recently it has also been shown to induce neuronal cell death. Here we report the effects of NGF on lesioned motoneurons. We have analyzed facial and sciatic motoneurons in newborn and adult BALB/c and C57BL/6 mice, in addition to mice deficient in the low-affinity p75 receptor for the neurotrophins (p75NTR). NGF application did not alter survival of lesioned facial motoneurons in any of the strains examined independent of the age of the animals. Only in the adult C57BL/6 mouse strain where the sciatic nerve had been crushed prior to factor application did NGF induce cell death of axotomized sciatic motoneurons. Our results illustrate the importance of the genetic background and the motoneuron sub-type in studies related to cell death and survival of motoneurons in relation to NGF and p75NTR.

Rescue of motoneurons from axotomy-induced cell death by polymer encapsulated cells genetically engineered to release CNTF.

The neurodegenerative disease amyotrophic lateral sclerosis (ALS) results from the progressive loss of motoneurons, leading to death in a few years. Ciliary neurotrophic factor (CNTF), which decreases naturally occurring and axotomy-induced cell death, may result in slowing of motoneuron loss and has been evaluated as a treatment for ALS. Effective administration of this protein to motoneurons may be hampered by the exceedingly short half-life of CNTF, and the inability to deliver effective concentration into the central nervous system after systemic administration in vivo. The constitutive release of CNTF from genetically engineered cells may represent a solution to this delivery problem. In this work, baby hamster kidney (BHK) cells stably tranfected with a chimeric plasmid construct containing the gene for human or mouse CNTF were encapsulated in polymer fibers, which prevents immune rejection and allow long-term survival of the transplanted cells. In vitro bioassays show that the encapsulated transfected cells release bioactive CNTF. In vivo, systemic delivery of human and mouse CNTF from encapsulated cells was observed to rescue 26 and 27% more facial motoneurons, respectively, as compared to capsules containing parent BHK cells 1 wk postaxotomy in neonatal rats. With local application of CNTF on the nerve stump and by systemic delivery through repeated subcutaneous injections, 15 and 13% more rescue effects were observed. These data illustrate the potential of using encapsulated genetically engineered cells to continuously release CNTF to slow down motoneuron degeneration following axotomy and suggest that encapsulated cell delivery of neurotrophic factors may provide a general method for effective administration of therapeutic proteins for the treatment of neurodegenerative diseases.