Specific features of chronic astrocyte gliosis after experimental central nervous system (CNS) xenografting and in Wobbler neurological mutant CNS.

This study sets out to compare and contrast the astrocyte reaction in two unrelated experimental designs both resulting in marked chronic astrogliosis and natural motoneuron death in the wobbler mutant mouse and brain damage in the context of transplantation of xenogeneic embryonic CNS tissue into the striatum of newborn mice. The combined use of GFAP-labeling and confocal imaging allows the morphological comparison between these two different types of astrogliosis. Our findings demonstrate that, in mice, after tissue transplantation in the striatum, gliosis is not restricted to the regions of damage: it occurs not only near the site of transplantation, the striatum, but also in more distant regions of the CNS and particularly in the spinal cord. In the wobbler mutant mouse, a strong gliosis is observed in the spinal cord, site of motoneuronal cell loss. However, moderate astrocytic reaction (increased GFAP-immunoreactivity) can also be found in other wobbler CNS regions, remote from the spinal cord. In the wobbler ventral horn, where neurons degenerate, the hypertrophied reactive astrocytes exhibit a dramatic increase of glial fibrils and surround the motoneuron cell bodies, occupying most of the motoneuron environment. The striking and specific presence of hypertrophic astrocytes in wobbler mice accompanied by a dramatic increase of glial fibrils located in the vicinity of motoneuron cell bodies suggests that short astrogliosis fills the space left by degenerating motoneurons and interferes with their survival. In the spinal cord of xenografted mice, chronic astrogliosis is also observed, but only glial processes without hypertrophied cell bodies are found in the neuronal micro-environment. It is tempting to speculate that gliosis in the wobbler spinal cord, the local accumulation of astrocyte cell bodies, and high density of astrocytic processes may interfere with the diffusion of neuroactive substances in gliotic tissue, some of which are neurotoxic, and cooperate or even trigger neuronal death.

Influence of factors secreted by wobbler astrocytes on neuronal and motoneuronal survival.

During late postnatal development, mice with the autosomal recessive wobbler mutation (wr/wr) develop motoneuron degeneration associated with astrogliosis in the spinal cord. In vitro, primary wobbler astrocytes are also affected, exhibiting abnormal cell-cell contacts. To characterize further the wobbler disease, we investigated the in vitro effects of wobbler astrocytes on primary neuronal cultures from the spinal cords of 15-day-old wild-type mouse and rat embryos. Cocultures with the wobbler astrocytes, or direct addition of wobbler astrocyte-conditioned medium, led to a decrease in neuron number in primary mixed neuronal cultures, containing motoneurons and interneuron-like cells. In contrast, wobbler astrocyte-conditioned medium enhanced survival of highly purified motoneurons. These in vitro results suggest the possibility that wobbler astrocytes act not on motoneurons directly but, rather, through other spinal neurons to induce motoneuron degeneration in the wobbler disease.

Astrocyte proliferation induced by wobbler astrocyte conditioned medium is blocked by tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) neutralizing antibodies in vitro.

The wobbler mutant mouse (wr/wr) displays motoneuron degeneration and astrocyte reactivity in the spinal cord. We have previously reported that, in vitro, primary wobbler astrocytes display morphological and biochemical changes. In this report, we show that wobbler astrocyte conditioned medium enhances the in vitro proliferation of normal neonatal primary astrocytes. This stimulated proliferation is correlated with high levels of IL1-beta and TNF-alpha cytokines in the conditioned medium of wobbler astrocytes. Neutralizing antibodies directed against both IL1-beta and TNF-alpha block the wobbler astrocyte conditioned medium-enhanced astrocyte proliferation. Moreover, IL1-beta and TNF-alpha mRNAs are elevated in the wobbler spinal cord. All these data suggest that diffusible IL1-beta and TNF-alpha are involved in the processus of astrogliosis observed in the wobbler spinal cord.

[Gliotoxic factor and multiple sclerosis]. / Un facteur gliotoxique et la sclérose en plaques.

Multiple sclerosis in a disease of the central nervous system characterized by perivascular and periventricular lesions of the myelin and immune cell infiltrates and increased permeability of the blood-brain barrier. We have found a cytotoxic factor of the cerebrospinal fluid (CSF) specific for multiple sclerosis patients which has 2 main characteristic effects in vitro on primary or immortalized astrocyte cultures: (1) disruption of the gliofilament network of the cells; and (2) apoptotic cell death induction. Moreover, in vivo, intraventricular injections of minute amounts of partially purified gliotoxic factor in adult rats have striking effects on both the morphology and general organization of astrocytes in the entire brain and the permeability characteristics of the blood brain barrier, which becomes leaky to immunoglobulins. These pathological effects are strongly similar to some of the neuropathological findings reported during the course of MS--They suggest an entirely new hypothesis to explain the active stage of the disease: the presence of a new factor of unknown extrinsic (viral) or intrinsic (cellular) origin, able to disorganize the glial cytoskeleton and glial cell differentiation. This factor is then able to provoke glial cell death. Such glial cell death may result in both demyelination and increased blood brain barrier permeability. Both in vitro and in vivo studies strongly support the idea that this gliotoxic factor plays a central role in the pathogenesis of MS, making its full identification a critical theme for MS research.

The motoneuron degeneration in the wobbler mouse is independent of the overexpression of a Bcl2 transgene in neurons.

The wobbler mouse mutation, an autosomal recessive mutation, leads to motoneuron degeneration in early post-natal development. Transgenic mice in which neurons overexpress human bcl2 transgene have been generated: the overexpression of bcl2 reduces the neuron loss during naturally occurring and experimentally-induced cell deaths. In the present study, we generate mice co-expressing the wobbler mutant gene and the bcl2 transgene in order to determine the effects of Bcl2 overexpression on the neurodegenerative disorders of the wobbler mouse. The clinical signs of the disease (weakness, tremor, small size) as well as biochemical and histological parameters (choline acetyltransferase (ChAT) activity in muscles, gliosis in spinal cord) are similar in bcl2 positive and negative wobbler mice. These results point to the fact that the neuron-specific expression of the human bcl2 transgene does not correct the effects of the wobbler mutation.

Excess extracellular and low intracellular glutamate in poorly differentiating wobbler astrocytes and astrocyte recovery in glutamine-depleted culture medium.

The wobbler mouse develops an inherited motoneuronal degeneration of unknown origin in the spinal cord. Primary cultures of adult wobbler spinal cord astrocytes display abnormal morphological characteristics with fewer processes and paucity of cell-cell contacts. We have searched for a possible involvement of glutamate and glutamine intra- and extracellular accumulations in vitro in the abnormal differentiation of mutant astrocytes. We have found significantly higher glutamate and glutamine concentrations in the culture media of mutant astrocytes over a 3-day period compared with normal control astrocytes. Moreover, intracellular glutamate concentrations decreased substantially in mutant astrocytes, but intracellular glutamine concentrations remained unchanged. Furthermore, decreasing initial glutamine concentrations in the culture medium (glutamine-depleted medium) led to the recovery of normal extra- and intracellular concentrations of glutamate and recovery of quasi-normal morphological differentiation and increased cell-cell contacts, leading to an essentially normal looking astrocyte network after 3 days of culture. Under these conditions, which lead to recovery, the only remaining abnormality was the higher glutamine extracellular concentration attained in the originally depleted glutamine media. These findings suggest that mechanisms regulating glutamate/glutamine synthesis and/or influx/efflux are defective in wobbler astrocytes, leading to metabolic imbalance and possible cytotoxic effects characterized by disturbed intercellular networks and poor differentiation.

Astrocytosis in wobbler mouse spinal cord involves a population of astrocytes which is glutamine synthetase-negative.

Mice affected by the wobbler mutation are characterized by a muscular atrophy associated with motoneuron degeneration. As soon as the first clinical signs of the disease appear, reactive astrocytes, strongly glial fibrillary acidic protein (GFAP)-positive, are observed in the spinal cord grey matter. They become prevalent at all levels with disease progression. Immunostaining of glutamine synthetase (GS) shows that these reactive astrocytes are never GS-positive. The activity and protein amounts of GS remain normal in wobbler spinal cord although astrocytosis develops. Thus, gliosis in the wobbler mouse seems to involve a subpopulation of astrocytes, which is strongly GFAP-positive but GS-negative.

Abnormal astrocyte differentiation and defective cellular interactions in wobbler mouse spinal cord.

The wobbler mutation is inherited as an autosomal recessive trait and displays a muscular atrophy associated with motoneuron degeneration in early postnatal development. It has been shown that the level of glial fibrillary acidic protein (GFAP) is greatly increased in the spinal cord of wobbler mice. We performed immunocytochemical analyses combined with confocal microscopy to study the developmental distribution of GFAP-positive astrocytes in the spinal cord of wobbler mice during the course of the disease, and in primary cultures of adult wobbler spinal cord astrocytes. Many changes in the number and distribution of astrocytes were observed in the wobbler mice from 1-10 months post-partum. Strongly GFAP-positive astrocytes are present in small number in the anterior horn by 1 month. They increase in number and are observed in the entire spinal cord grey and white matters by 2-10 months. These reactive astrocytes have thick, short, extensively branched processes which contrast with the long, unbranched processes observed in control mice. The wobbler astrocyte processes are oriented perpendicular to the surface of the spinal cord, which contrasts with the normal parallel, concentric orientation. No expansion of astrocyte processes exit from the white matter towards the grey matter. Moreover, the surface of the wobbler spinal cord beneath the meninges displays a dramatic decrease of interdigitating processes, end feet and flattened cell bodies of astrocytes that form a disorganized layer. In vitro, mutant astrocytes have morphological characteristics similar to those in vivo and, in particular, develop short, thick, branched processes. These mutant astrocytes in cultures do not contact one another, whereas normal mature cultures show an increased incidence of cell-cell contacts between long processes. The increase of astrocyte reactivity associated with these modifications in astrocytic process arrangement may reflect an important primary event in the course of the wobbler disease rather than a non-specific response to motoneuronal death.

Effects of calcium ion on neurite outgrowth of rat spinal cord neurons in vitro: the role of non-neuronal cells in regulating neurite sprouting.

The interactions of nerve cells with their environment and other cells are specific to different stages of cellular differentiation. Neurite outgrowth was measured from cultured spinal cord neurons under the influence of different Ca2+ concentrations. We used fluorodeoxyuridine (FuDr), an antimitotic agent which reduces significantly the proportion of non-neuronal cells in spinal cord cell cultures, to examine the effects of non-neuronal cells on neurite outgrowth. Spinal cord neurons responded to changes in their environment by means of two types of neurite outgrowth: sprouting and elongation. The concurrent presence of non-neuronal cells led to increased sprouting of neurites in certain ionic environments, thus lending support to the idea that non-neuronal cells release diffusible factors which influence sprouting and guide neurite outgrowth.

In vitro evidence for a neurite growth-promoting activity in Trembler mouse serum.

Basal lamina components, such as heparan sulfate proteoglycan (HSPG) and laminin play an important role in neuritic outgrowth for CNS and PNS neurons in culture. The mutant mouse 'Trembler' is characterized by hypomyelinization and production of an excess of basal lamina layers around Schwann cells in peripheral nerves. In order to analyse whether or not the serum of the mutant animals contains neurite outgrowth-promoting factors, we cultured rat spinal cord neurons in the presence of Trembler serum. Under these conditions, the outgrowth of neurites was increased approx. 2 times as compared to control serum. Trembler serum induces neuritic outgrowth characterized both by an increase in number of primary neurites emerging from the nerve cell body as well as by an increase in peripheral branching of neurites. To characterize the factors implicated in this increase we added antibodies directed against HSPG or laminin to the mutant serum. As a result, the increase in neuritic outgrowth was reduced or abolished in both cases. Trembler effect on neurite growth disappeared when the number of the non-neuronal cells was reduced, suggesting that the mutant serum did not act directly on neurons but by the intermediary action of non-neuronal cells.

Effects of trembler mouse serum and laminin on oligodendrocyte proliferation and differentiation in culture.

Oligodendrocytes in primary cultures derived from rat embryo spinal cord were examined in control medium and in Trembler mouse serum (TMS)-supplemented medium. The oligodendrocytes were identified on the basis of the synthesis and surface expression of galactocerebrosides revealed by a monoclonal antibody directed against this component. We noticed two effects of TMS compared to control mouse serum. First, our results revealed that in TMS medium there is a mitogenic response of galactocerebroside (GalC)-positive cells. Second, in the presence of TMS, oligodendrocytes do not develop processes as they do in the presence of normal mouse serum. When laminin, a basal lamina component was added to TMS medium, GalC+ oligodendrocytes decreased in number and differentiation was normal. Possible explanations of the effects of TMS and laminin on oligodendrocyte proliferation and differentiation are discussed.

Heparan sulfate proteoglycan and laminin mediate two different types of neurite outgrowth.

Spinal cord neurons cultured in vitro have been shown to respond to changes in their environment by means of 2 different types of neurite outgrowth: (1) neurite elongation and (2) emergence and branching of newly formed neurites. Culture of spinal cord neurons with heparan sulfate proteoglycan (HSPG) medium resulted in a 3-fold increase in neurite elongation compared to the control. Extensive branching was seen when neurons were cultured in laminin-supplemented culture medium. HSPG-induced elongation and laminin-induced branching of neurites were blocked by specific anti-HSPG and antilaminin sera, respectively. Furthermore, laminin antibodies did not inhibit neurite elongation and HSPG antibodies did not block neurite branching. Conditioned medium from primary embryonic rat muscle cultures (MCM) mimicked the effects of both HSPG and laminin on neurite outgrowth. Immunoprecipitation with anti-HSPG and antilaminin antibodies demonstrated that MCM contains these 2 basal lamina components. Our observations suggest that HSPG and laminin might be highly effective molecules for promoting neurite outgrowth of rat spinal cord neurons in vitro.

Simultaneous demonstration of 3 synaptic markers: nerve endings, localization of AChE and AChR clusters at neuromuscular junctions of the rat in vitro.

Our triple labelling method allows the AChE and AChR distributions and the nerve endings to be stained in the same preparation. This method which provides a detailed visualization of the synaptic contacts and is capable of revealing a weak AChE activity is particularly valuable in studies of synaptogenesis.

[In vitro study of the action of isaxonine on the growth of rat spinal cord neurons (author's transl)]. / Etude in vitro de l'action de l'isaxonine sur la croissance des neurones de la moelle épinière de rat.

Dissociated spinal cord cells were cultured in appropriate culture medium. In these conditions the cell bodies tended to emit neurites and to interconnect. Grown with isaxonine, spinal cord neurons form larger aggregates, neurites number is higher, connection between cellular groups is more larger, and branching of neurites is more distal. Future research will try to verify the assumption of an increase in neuromuscular contacts under the influence of isaxonine.