Gene therapy of murine motor neuron disease using adenoviral vectors for neurotrophic factors.

Motor neuron diseases such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy cause progressive paralysis, often leading to premature death. Neurotrophic factors have been suggested as therapeutic agents for motor neuron diseases, but their clinical use as injected recombinant protein was limited by toxicity and/or poor bioavailability. We demonstrate here that adenovirus-mediated gene transfer of neurotrophin-3 (NT-3) can produce substantial therapeutic effects in the mouse mutant pmn (progressive motor neuronopathy). After intramuscular injection of the NT-3 adenoviral vector, pmn mice showed a 50% increase in life span, reduced loss of motor axons and improved neuromuscular function as assessed by electromyography. These results were further improved by coinjecting an adenoviral vector coding for ciliary neurotrophic factor. Therefore, adenovirus-mediated gene transfer of neurotrophic factors offers new prospects for the treatment of motor neuron diseases.

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.

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.

Purification of two astroglial growth factors from bovine brain.

The astroglial growth factor (AGF), which induces a characteristic morphological change in cultured rat astroglial cells and stimulates their proliferation, was purified to homogeneity from bovine brain. Two different methods were used, the second one including heparin-Sepharose affinity chromatography. AGF is actually composed of two factors, AGF1 and AGF2, which both modify the morphology and stimulate the proliferation of the astroglial cells. Several data suggest that the AGFs are similar or possibly identical to the fibroblast growth factors (FGFs) isolated from brain [(1984) Proc. Natl. Acad. Sci. USA 81, 357-361; and 6963-6967]. A specific antiserum against AGFs was raised in mouse.

Uptake and metabolism of choline in primary isolated neurons and glial cells in culture.

The influx and metabolism of choline have been studied in primary cultures of isolated neurons and glial cells from chick embryo dissociated cerebral hemispheres. The results showed a correlation between both influx and metabolism of choline and the exogenous concentrations of choline. When neurons and glial cells were preincubated (10 min) and incubated in Krebs-Ringer phosphate solution with concentrations of choline lower (0.5 ?M) or higher (150 ?M) than the one present in the growth medium, the metabolism of choline, as a function of time, approached saturation following unusual kinetics. This suggests a non steady state of the endocellular concentrations of free choline. Moreover, when both neurons and glial cells were preincubated (10 min) with 50 ?M choline and then incubated (2 min) with various concentrations of choline, only one uptake mechanism was measured, while the preincubation in the absence of choline followed by the incubation of the cells with various concentrations of choline showed the presence of two apparent K(m)'s with different affinities. The results also indicate the capacity of glial cells to incorporate choline suggesting a storage function for the cells.

Primary culture of rat ependymal cells in serum-free defined medium.

We have developed a serum-free chemically defined medium which allows the obtainment of a primary culture highly enriched in ciliated ependymal cells. Serum was never used. Mechanically dissociated neonatal rat brain hemisphere cells were seeded on a fibronectin substratum. Culture medium was minimum Eagle's medium until day 14 in vitro and Waymouth's MD 705/l medium thereafter. Both media were supplemented with insulin (5 micrograms/ml), transferrin (10 micrograms/ml) and fatty acid-free bovine serum albumin (0.5 mg/ml). In these conditions, without addition of growth factors, the culture was enriched in ependymal cells; with the addition of thrombin cell growth was stimulated and moreover a nearly pure culture of ependymal cells was obtained. In this very simple culture medium, cells looked healthy up to two months after seeding and about 75% of the cells were ciliated. In a fraction of these cells the glial fibrillary acidic protein, a protein specific for astroglial cells in the central nervous system, was immunohistochemically revealed. The availability of an almost pure primary culture of ependymal cells will make many studies possible on this cell type, particularly studies on the development of these cells, on the regulation of their genetic expression and on their physiological function.

An immunohistochemical study of two myelin-specific proteins in enriched oligodendroglial cell cultures combined with an autoradiographic investigation using [3H]thymidine.

The aim of the present work was to examine the possible relationship between proliferation and expression of 2 myelin specific proteins in cultured oligodendroglial cells. Mixed cultures of glial cells, from newborn rat brain, containing astroglia and oligodendroglia were grown in 2 different culture media, minimum Eagle's medium and Waymouth's medium both supplemented with 10% calf serum in presence or absence of adult rat brain soluble extract. The proliferative activity of the cells was followed over a 28-day period by autoradiography after radioactive thymidine incorporation. It was found that in cultures grown in Waymouth's medium the proportion of oligodendroglial cells was higher and that proliferation was more active than in minimum Eagle's medium. Addition of brain extract elicited a stimulation of the proliferation of the cells in the 2 basal media. Under all conditions W1 protein appeared earlier than MBP by immunofluorescent visualization. Some oligodendroglial cells synthesizing W1 protein were still able to proliferate. MBP appears to be a marker of a later stage of cell maturation since very few MBP-positive cells incorporated tritiated thymidine. More cells contained MBP in the presence of brain extract. These results suggest that oligodendroglial cell maturation proceeds by steps, the step of W1 protein expression is compatible with proliferation while that of MBP expression appears at the end of the proliferation phase.

Rat amnion membrane matrix as a substratum for regenerating axons from peripheral and central neurons: effects in a silicone chamber model.

An extracellular matrix preparation, the human amnion membrane matrix (hAMM) can serve as a neurite-promoting substratum for cultured peripheral and central neurons, and also as a support for axonal growth in experimentally injured adult brain in vivo. In the present study, we tested similar materials as bridges in a silicone chamber model for the regeneration of sciatic nerve in the adult rat. Since hAMM elicited an inflammatory response, we developed a rat amnion membrane matrix (rAMM), which proved to be an excellent neurite-promoting substratum for cultured ganglionic and spinal cord neurons. The rAMM was coiled and inserted in the 10 mm gap between the two nerve stumps from the silicone chambers. At 16 days after implantation, temporal progress of regeneration was grossly similar as in saline-prefilled control chambers. However, rAMM-prefilled chambers displayed significantly higher number of vessels and a markedly different geometry of the regenerate: an endoneurium, surrounded by a perineurial-like cell layer, was formed outside the largely preserved central portion of the rAMM coil. After longer regeneration times (28 days), a rAMM core was no longer detected, but some rAMM-like materials remained interspersed in the endoneurium. The overall organization of the regenerate and the number of myelinated axons at this time were similar to those of control chambers, although the endoneurial cross-sectional area was larger in the rAMM chambers. One specimen, however, displayed the very patterns for which the experiments were designed, namely an array of numerous, myelinated axons tracing the spiraling spaces between consecutive lamellae of the rAMM coil.

Chemically defined medium for rat astroglial cells in primary culture.

We have developed a serum-free defined medium that supports the growth in primary culture of rat astroglial cells. Cells dissociated from cerebral hemispheres of newborn rats were maintained for 4 days in a basal medium (Waymouth's medium) containing 10% fetal calf serum, which was substituted by a serum-free medium. The basal medium was supplemented with insulin (5 µg/ml) and fatty acid free bovine serum albumin (0.5 mg/ml). Under these conditions the cells proliferate as estimated by cell counts and DNA content; however, growth was less than in Waymouth's medium supplemented with 10% fetal calf serum. In contrast, a very similar morphology was observed between cultures grown in the serum-free or serum-containing media. The serum-free medium allows some maturation of the astroglial cells as shown by the presence of glial fibrillary acidic (GFA) protein, S-100 protein and glutamine synthetase (GS) activity. The astroglial cells can survive and grow in this chemically defined medium for up to 5 weeks. The ability to culture astroglial cells in such a minimal defined medium should facilitate investigations concerning the effects of growth factors on their proliferation and maturation.

Synapse formation and development of neurotransmitter functions in neuronal cells from chick brain cultured in a serum-free, defined medium.

Cells dissociated from cerebral hemispheres of 8-day-old chick embryos were seeded on poly-L-lysine coated Petri dishes in serum-containing medium. After 24 hr the culture medium was switched to a serum-free, chemically defined medium. These cultures contain mainly neuronal cells until day 14, characterized by the presence of acetylcholinesterase activity and neurofilament proteins. After 2 weeks glial cells progressively contaminated the neuronal culture. Cultures were maintained for a period of 4 weeks. From day 6 on numerous synapses with clear vesicles were observed. The activity of choline acetyltransferase remained low throughout the culture period, while GABA levels increased in parallel with synaptogenesis. Our observations indicate that chick cerebral hemisphere neuronal cultures grown in serum-free, chemically defined medium contain GABAergic neurons that undergo maturation.

Morphological and biochemical maturation of rat astroglial cells grown in a chemically defined medium: Influence of an astroglial growth factor.

Astroglial cells from cerebral hemispheres of newborn rats were cultured for 5 days in Waymouth's MD 705/1 medium containing 10% fetal calf serum. Thereafter, cells were grown in a chemically defined medium consisting of basal Waymouth's medium supplemented with insulin (5 µg/ml) and fatty acid free bovine serum albumin (0.5 mg/ml). The cells underwent morphological and biochemical development over a period of 28 days. The changes in the amount of glial fibrillary acidic protein indicated a development of gliofilaments. The level of S100 protein increased during the entire culture period, while glutamine synthetase activity remained low and relatively constant. The addition of an astroglial growth factor, partially purified from bovine brain soluble extract, stimulated the morphological maturation of the astroglial cells. The cells extended cytoplasmic processes and resembled mature astrocytes. At the ultrastructural level an increase in free ribosomes was observed and the intermediate filaments became organized into large bundles. The amount of glial fibrillary acidic protein was not significantly increased, but the level of S100 protein and the glutamine synthetase activity were greatly enhanced. Our results indicate that astroglial cells undergo limited maturation in the chemically defined medium and that this process is positively affected by the astroglial growth factor.

The brain fibroblast growth factor (FGF) is localized in neurons.

The physiological role of fibroblast growth factor (FGF), like many other characterized growth factors, is poorly understood. Therefore, it would be useful to know in which cell type this growth factor is present. Using monoclonal and polyclonal antibodies against FGF, we have localized this molecule in rat brain. Interestingly, FGF is found exclusively in neuronal cells. Since FGF stimulates the proliferation and maturation of glial cells in vitro, its involvement in the interaction between neuronal and glial cells in vivo is likely. A role in the triggering of reactive gliosis can also be hypothesized.

Decrease in the density of orthogonal arrays of particles in membranes of cultured rat astroglial cells by the brain fibroblast growth factor.

The effect of brain-derived basic fibroblast growth factor (bFGF) on the membrane structure of cultured rat astroglial cells was investigated by quantitative freeze-fracture replica examination. In the presence of bFGF the number of the orthogonal arrays of particles (OAP), which are characteristic compounds of the membrane of mammalian astrocytes, is clearly reduced. This finding is discussed in the framework on current views about neuro-glial interactions and their implications in the process of fiber regeneration in the central nervous system.

Ultrastructural localization of fibroblast growth factor in neurons of rat brain.

The distribution of fibroblast growth factor (FGF) at the ultrastructural level in the brain of young (15- and 20-day-old) and adult (3-month-old) rats was investigated by immunocytochemistry. Strong staining was observed in most neurons of the cortex of young rat brain. In the same brain area of adult rat many neurons were also stained intensely, while others were negative. Neurons in the other parts of the brain and especially in the adult rat, were generally more weakly stained. The reaction product was located in the cytoplasm of the neuronal cell bodies and their processes. Astrocytes, oligodendrocytes, microglial cells, meningeal cells, choroïd epithelial cells, ependymal cells and capillary endothelial cells showed no staining.

Brain basic fibroblast growth factor stimulates the release of plasminogen activators by newborn rat cultured astroglial cells.

Basic fibroblast growth factor (bFGF), a growth factor for many cell types including newborn rat astroglial cells, stimulates in a dose-dependent fashion the release of plasminogen activators (PAs) by these cells as measured by the fibrin-overlay method or the Coleman and Green's colorimetric assay. This effect of bFGF on PAs secretion (about 4.5-fold increase at 40 ng/ml bFGF) does not result from an aspecific stimulation of protein secretion by astrocytes and is only partly correlated with the mitogenic activity of bFGF. bFGF was also tested on two clonal glioma cell lines (C6 and LN18). Only one of those cell types (LN18) showed a stimulated PA release in the presence of bFGF. These data are discussed with respect to the putative roles of plasminogen activators in the developing nervous system.

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.

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.

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.