Beneficial effects of r-h-CLU on disease severity in different animal models of peripheral neuropathies.

Clusterin is a protein involved in multiple biological events, including neuronal cytoprotection, membrane recycling and regulation of complement-mediated membrane attack after injury. We investigated the effect of recombinant human clusterin in preclinical models of peripheral neuropathies. Daily treatment with clusterin accelerated the recovery of nerve motor evoked potential parameters after sciatic nerve injury. Prophylactic or therapeutic treatment of experimental autoimmune neuritis rats with clusterin also accelerated the rate of recovery from the disease, associated with remyelination of demyelinated nerve fibers. These data demonstrate that clusterin is capable of ameliorating clinical, neurophysiological and pathological signs in models of peripheral neuropathies.

Phosphatidylinositol 3-kinase activity in murine motoneuron disease: the progressive motor neuropathy mouse.

A murine model of motoneuron disease, the pmn/pmn mouse, shows a reduction in the retrograde transport of fluorescent probes applied directly onto the cut end of sciatic nerve. Brain-derived neurotrophic factor (BDNF), when co-applied with fluorescent tracers, increases the number of retrograde labelled motoneurons. We demonstrate here that spinal cord tissue from pmn/pmn mice had significantly reduced phosphatidylinositol 3-kinase activity and expression in the particulate fraction compared to controls, without changes in the activities or expression of the downstream kinases, protein kinase B/Akt or Erk1. Systemic administration of BDNF augmented phosphatidylinositol 3-kinase specific activity in spinal cord tissue from pmn/pmn and control mice, with a greater elevation in the particulate fractions of pmn/pmn mice than in controls. We examined the effect of inhibitors of phosphatidylinositol 3-kinase and mitogen-activated protein kinase kinase on the retrograde labelling of motoneurons, 24h following the direct application of inhibitors and Fluorogold to the cut end of sciatic nerve in control and pmn/pmn mice (labelling index). The mitogen-activated protein kinase kinase inhibitor PD 98059 had no effect on the labelling index in control or pmn/pmn mice. In the absence of exogenous BDNF, phosphatidylinositol 3-kinase inhibitors reduced the number of labelled motoneurons in control mice, without changing the labelling index in pmn/pmn. Co-application of phosphatidylinositol 3-kinase inhibitors with BDNF to the cut end of sciatic nerve blocked the action of BDNF on retrograde labelling in pmn/pmn mice. These results indicate that the retrograde labelling of motoneurons is mediated by phosphatidylinositol 3-kinase-dependent and -independent pathways. In pmn/pmn mice, phosphatidylinositol 3-kinase activity in spinal neurons is below the level required for optimal retrograde labelling of motoneurons and labelling can be augmented by the administration of growth factors stimulating phosphatidylinositol 3-kinase activity. The data indicate that phosphatidylinositol 3-kinase activity is important in the uptake and/or retrograde transport of substances by motoneurons and is altered in this model of motoneuron diseases.

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.

IAP family proteins delay motoneuron cell death in vivo.

Neuronal apoptosis inhibitory protein (NAIP), and human inhibitors of apoptosis 1 and 2 (HIAP1 and HIAP2) are three members of the mammalian family of antiapoptosis proteins called 'inhibitors of apoptosis' (IAP). These molecules can prevent apoptosis in vitro and the over-expression of NAIP can decrease ischemic damage in the hippocampus. The goal of our experiments was to determine whether administration of NAIP, HIAP1 and HAIP2 could rescue motoneurons following axotomy of a peripheral nerve. In young rats, an adenoviral gene transfer technique was used to deliver and express these proteins in motoneurons; a fluorescent tracer was simultaneously added as a means for quantitatively assessing the rescue of fluorescently labelled motoneurons in serial sections of the lumbar spinal cord. Control experiments using adenoviral vectors (adv) expressing the lacZ gene showed that 14% of the sciatic motoneuron pool could be transfected indicating the existence of a subpopulation of spinal motoneurons susceptible to this class of viral vectors. The administration of an adv-NAIP, adv-HIAP1 and adv-HIAP2 rescued 30-40% of motoneurons at one week after sciatic axotomy. The efficiency of these proteins was similar to that of two neurotrophic factors, ciliary neurotrophic factor and brain-derived neurotrophic factor, administrated by the same viral technique. The effect of the IAP proteins on motoneuron survival decreased with time but was still present after 4 weeks postaxotomy; the duration of the response was dependent upon the viral titre. These experiments demonstrate that IAP family proteins can prevent motoneuron cell death in vivo and may offer a new therapeutic approach for motoneuron diseases.

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.

Differential effects of neurotrophic factors on motoneuron retrograde labeling in a murine model of motoneuron disease.

It has been shown that abnormalities in axonal transport occur in several mouse models with motoneuron degeneration and also in the human disease amyotrophic lateral sclerosis. In this report, we have examined the potential of neurotrophic factors to act on axonal transport properties in a mouse mutant, progressive motor neuronopathy (pmn). This mouse mutant has been characterized as a "dying-back" motoneuronopathy, with a loss of motoneuron cell bodies and motor fibers. Retrograde transport to the spinal cord motoneurons was determined using fluorescent tracers either injected into the gastrocnemius muscle or applied directly onto the cut sciatic nerve. Because the rate of retrograde labeling was significantly reduced in the pmn, we examined the potential of neurotrophic factors to compensate for the impairment. Ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) but not glial-derived neurotrophic factor (GDNF) or nerve growth factor (NGF) were capable of significantly improving the rate of labeling. The differential effects of these factors agree with previous studies showing that molecules that promote cell survival do not necessarily compensate for axonal deficiency. Because impairment of axonal properties appears as an early event in motoneuron pathology, our results may have important clinical implications in the treatment of motoneuron diseases.

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.

Injury-induced synthesis and release of apolipoprotein E and clusterin from rat neural cells.

Apolipoproteins in the brain have assumed major clinical importance since it was shown that one of the allelic forms of apolipoprotein E, apoE-4, is a risk factor for Alzheimer's disease. Using tissue culture of embryonic rat spinal cord, we examined the effect of neuronal injury on the up-regulation of two apolipoproteins, apolipoprotein E and clusterin (apoJ). In order to study the influence of neuronal cells, we exploited the specific neurotoxic effect of elevated glutamate on these cells. Overstimulation by excess glutamate induced neuronal degeneration as assessed by morphological and biochemical criteria, notably the activity of choline acetyltransferase, which serves as a marker for cholinergic neurons. High concentrations of glutamate increased mRNA synthesis and the production and secretion of both apolipoprotein E and clusterin protein. Both neuronal cell death and release of the peptides were calcium-dependent and could be blocked by the NMDA receptor antagonist MK-801. Immunohistochemical data revealed the presence of clusterin in both neuronal and non-neuronal cells whereas apolipoprotein E was mainly expressed in non-neuronal cells. The results are suggestive of concerted up-regulation of apolipoprotein E and clusterin when neural cells are subjected to injury.

Antibodies directed against the beta 1-integrin subunit and peptides containing the IKVAV sequence of laminin perturb neurite outgrowth of peripheral neurons on immature spinal cord substrata.

Neuron-substratum interactions regulating axon growth in the developing central nervous system of the rat have been studied by means of an in vitro bioassay: the tissue section culture. We have previously shown that purified chicken sensory or sympathetic neurons grown on natural substrata consisting of cryostat sections of neonatal rat spinal cord elaborate numerous long neurites [Sagot et al. (1991) Brain Res. 543, 25-35]. Perturbation experiments, in which neuron-substratum interactions are modified by antibodies and peptides, have allowed us to analyse some of the molecular determinants which control neurite outgrowth in this system. Antibodies directed against the beta 1-integrin subunit, one of the neuronal receptors for extracellular matrix molecules, reduced the percentage of growing neurons by about 30% and the length of neurites by about 50%. In contrast, antibodies directed against laminin-1 or fibronectin, two extracellular matrix proteins transiently expressed in various areas of the developing central nervous system, were unable to block neurite outgrowth. Paradoxically, a peptide containing the IKVAV sequence, which mimics an active sequence of the laminin alpha 1 chain responsible for neurite extension, also blocked neurite outgrowth on neonatal spinal cord substrata. These results indicate that integrin receptors containing the beta 1 subunit may play a role in regulating axon growth in the developing nervous system. Among the putative extracellular matrix ligands for these receptors, laminin and fibronectin do not appear as prominent candidates in the neonatal spinal cord. However, our data also suggest that the developing central nervous system may contain neurite outgrowth-promoting proteins carrying the IKVAV sequence, different from laminin-1.

GDNF slows loss of motoneurons but not axonal degeneration or premature death of pmn/pmn mice.

Glial cell line-derived neurotrophic factor (GDNF), a member of the TG F-beta superfamily, has been shown to be a highly potent neurotrophic factor that enhances survival of various neuronal cell types including motoneurons. To assess its therapeutic potential in treating neurodegenerative diseases such as amyotrophic lateral sclerosis, we treated mutant mice displaying motoneuron degeneration (progressive motor neuropathy; pmn) with encapsulated GDNF-secreting cells. Effects of GDNF treatment on pmn/pmn mice were compared with previous results obtained with ciliary neurotrophic factor (CNTF) [Sagot Y, Tan SA, Baetge E, Schmalbruch H, Kato AC, Aebischer P (1995) Eur J Neurosci 7:1313-1322]. In contrast to CNTF, GDNF did not increase the lifespan of pmn/pmn mice. However, GDNF significantly reduced the loss of facial motoneurons by 50%, a value similar to what was observed when CNTF was administered to the pmn/pmn mice. Surprisingly, myelinated axon counts revealed that GDNF had no effect on nerve degeneration. Therefore, despite its potential in rescuing motoneuron cell bodies, the inability of GDNF to prevent nerve degeneration in pmn/pmn mice suggests that its usefulness in the treatment of motor neuron diseases may be restricted to cotreatment with other factors that act on the nerve process.

Bcl-2 overexpression prevents motoneuron cell body loss but not axonal degeneration in a mouse model of a neurodegenerative disease.

Bcl-2 and its analogs protect different classes of neurons from apoptosis in several experimental situations. These proteins may therefore provide a means for treatment of neurodegenerative diseases. We examined the effects of Bcl-2 overexpression in a genetic mouse model with motor neuron disease (progressive motor neuronopathy/pmn). Pmn/pmn mice lose motoneurons and myelinated axons, and die at 6 weeks of age. When these mice were crossed with transgenic mice that overexpress human Bcl-2, there was a rescue of the facial motoneurons with a concomitant restoration of their normal soma size and expression of choline acetyltransferase. However, Bcl-2 overexpression did not prevent degeneration of myelinated axons in the facial and phrenic motor nerves and it did not increase the life span of the animals. Since Bcl-2 acts strictly on neuronal cell body survival without compensating for nerve degeneration in pmn/pmn/bcl-2 mice, this proto-oncogene would not in itself be sufficient for treatment of neurodegenerative diseases where axonal impairment is a major component.

Polymer encapsulated cell lines genetically engineered to release ciliary neurotrophic factor can slow down progressive motor neuronopathy in the mouse.

Ciliary neurotrophic factor (CNTF) has recently generated great interest due to its potential as a therapeutic agent for the treatment of human neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Because the systemic half-life of CNTF is only in the order of a few minutes, continuous delivery of this trophic factor could be attractive or even necessary in the therapy of these diseases. One promising technique involves the polymer encapsulation of cells which have been genetically modified to secrete neurotrophic factors. The polymer capsules can be implanted into animals and effect the slow release of the protein for several months. The encapsulation technique immuno-isolates the foreign cells from host immune cells and at the same time prevents tumour formation by the transplanted cells. In this study, we have used progressive motoneuronopathy (pmn) mice to determine the extent to which encapsulated cell lines secreting CNTF could alter the course of the disease. pmn/pmn homozygotes present severe loss of myelinated motor fibres and a significant reduction of facial motoneuron cell bodies. The mice develop weakness of the hindlimbs and die during the sixth week after birth. We found that CNTF delayed the disease progression by increasing the survival time by 40% and by improving motor function as assessed by three behavioural tests. Moreover, histological counts of the phrenic nerve myelinated axons and facial nucleus motoneurons indicated a significant reduction of motoneuron loss. These results suggest that polymer-encapsulated cells releasing neurotrophic factors may provide a potential delivery system for treating neurodegenerative diseases such as ALS.

Quantitative comparison of the transient rescue effects of neurotrophic factors on axotomized motoneurons in vivo.

A reproducible neuronal degeneration induced by nerve lesion in neonatal rats or mice provides a convenient in vivo assay for testing the survival-promoting activity of putative growth factors on motoneurons. The goal of this study was to compare the rescue effects of the four known neurotrophins [nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4)] and two of the cytokines [ciliary neurotrophic factor (CNTF) and leukaemia inhibitory factor (LIF)] in one particular experimental model of spinal motoneuron degeneration at two different survival times. The sciatic nerve was cut in neonatal rats and the factors were applied onto the nerve stump; bovine serum albumin was used in controls. Simultaneous application of the retrograde tracer fluoro-gold made it possible to count motoneurons specifically in the sciatic pool. One week after lesion, the neurotrophins BDNF, NT-3 and NT-4, but not NGF, equally enhanced motoneuron survival compared to controls; their effects were significantly better than those of the cytokines. However, the rescue from cell death was only transitory because a great number of the motoneurons died during the second week after nerve lesion. Additional BDNF and/or CNTF supplied by repeated subcutaneous injections (1 mg/ml) over 2 weeks could not prevent this delayed motoneuron loss. These results suggest that still other factors or alternative routes of administration may be required for permanent rescue of the lesioned immature motoneurons.

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.

Long-lasting accumulation of hemopexin in permanently transected peripheral nerves and its down-regulation during regeneration.

We have previously demonstrated that hemopexin is present in the intact sciatic nerve and is overproduced in the distal stump after nerve transection (Swerts et al.: J Biol Chem 267:10596-10600, 1992). To get further insight into the function of this hemoprotein in nervous tissue, we have documented long-term changes in hemopexin levels in permanently degenerated (transected) and regenerating (crush-lesioned) sciatic nerves of adult rats, using immunochemical techniques. As early as a couple of days after nerve transection, the amount of hemopexin was raised in the distal stump and at the end of the proximal stump. Similarly, after a crush lesion hemopexin was rapidly increased at the injury site and in the distal part of the nerve. Subsequently, in transected nerves the level of hemopexin rose steadily and remained elevated, representing, three months after injury, over 20 times the amount found in intact contralateral nerves. In contrast, in crush-lesioned nerves, hemopexin level declined progressively in a proximodistal direction and returned to basal values 2 months after injury, together with axonal regeneration. This long-term increase in hemopexin in permanently degenerated nerves and its progressive return to normal levels during nerve regeneration suggests that hemopexin content could be regulated negatively, directly or indirectly, by growing axons. In turn, these results support the idea that hemopexin could be involved in the process of Wallerian degeneration and/or in nerve repair.

Hemopexin is synthesized in peripheral nerves but not in central nervous system and accumulates after axotomy.

In adult mammals, injured axons regrow over long distances in peripheral nerves but fail to do so in the central nervous system. Analysis of molecular components of tissue environments that allow axonal regrowth revealed a dramatic increase in the level of hemopexin, a heme-transporting protein, in long-term axotomized peripheral nerve. In contrast, hemopexin did not accumulate in lesioned optic nerve. Sciatic nerve and skeletal muscle, but not brain, were shown to be sites of synthesis of hemopexin. Thus, hemopexin expression, which can no longer be considered to be liver-specific, correlates with tissular permissivity for axonal regeneration.

Changes in permissivity for neuronal attachment and neurite outgrowth of spinal cord grey and white matters during development: a study with the 'cryoculture' bioassay.

We have used the recently developed cryoculture bioassay (Carbonetto et al., J. Neurosci., 7 (1987) 610-620) to document changes during development of CNS tissular ability to support nerve fiber growth. Neuronal attachment and neurite outgrowth of purified neurons cultured on tissue sections of rat spinal cord at various stages of development were quantified. Nerve fiber growth permissivity increased during embryonic stages, reaching as postnatal days 2-4 (P2-P4) a maximum value, higher than that found on adult PNS tissue sections. This permissivity diminished rapidly thereafter, indicating that early postnatally, the nerve fiber growth supporting ability of the CNS environment shifts abruptly from an increasingly permissive mode to an increasingly non-permissive status. Furthermore, after P4, neurite outgrowth permissivity diminished in parallel on white and grey matters, whereas neuronal attachment declined much more drastically on white matter than on grey matter. This indicates that the progressive loss of spinal cord ability to support nerve fiber growth is attributable to both grey and white matters. In several instances it also appeared that neuronal adhesion was not necessarily followed by a comparable level of nerve fiber growth, suggesting that these two processes could be regulated by different factors.

Astroglial differentiation from neuroepithelial precursor cells of amphibian embryos: an in vivo and in vitro analysis.

Initial development of astroglial phenotype has been studied in vitro in an amphibian embryo (Pleurodeles waltI), to document the differentiation potentialities acquired by neural precursor cells isolated at the early neurula stage. In particular, we sought to determine whether interactions between neuroepithelial cells and the inducing tissue, the chordamesoderm, are required beyond this stage to specify precursor cells along glial lineages. Glial cell differentiation was documented by examining the appearance of glial fibrillary acidic protein (GFAp), a specific marker of astroglial lineages. Cells expressing GFAp-immunoreactivity differentiated rapidly, after 48 hours of culture, from cultivated neural plate cells, irrespective of the presence or absence of the inducing tissue. The widespread expression of Pleurodeles GFAp protein in neural plate cultures, in which CNS precursor cells develop alone in a simple saline medium, showed that prolonged contact with chordamesodermal cells was not necessary for the emergence of the astroglial phenotype. In addition, the initial development of astroglial phenotype has been defined in vivo. The first detectable GFAp-immunoreactivity was visualized in the neural tube of stage-24 embryos, a stage corresponding to 2-3 days in culture, defining radial glial cell end-feet. Thus, dissociation and culture of neural precursor cells did not appear to modify the onset of astroglial differentiation. At stage 32, GFAp-immunoreactivity was observed over the entire length of radial glial fibers and was also evidenced in mitotic cells located in the ventricular zone, suggesting that radial glial cells were not all post-mitotic.