Hans H. Althaus

Nerve growth factor induces proliferation and enhances fiber regeneration in oligodendrocytes isolated from adult pig brain

Mature oligodendrocytes (OL) isolated from adult pig brains start to regenerate their fibers after 4-5 days in vitro (DIV); after 14 DIV a network of OL fibers is formed. Growth factors, of which it was known that they play an important part during proliferation and differentiation of OL progenitor cells, were used to study their influence on the regeneration of mature OL. For this purpose, OL were treated at 6 DIV with different concentrations of various growth factors. At 24 h intervals the [3H]thymidine incorporation was measured and at 8 DIV the OL fiber production evaluated. None of these factors did influence the regenerative process to any significant extent except nerve growth factor (NGF). For the first time it could be shown that NGF enhanced the OL fiber regeneration considerably and induced the proliferation of a subset of OL. These results may have important implications for the remyelinating process in demyelinating diseases such as multiple sclerosis.

Glial cells as targets and producers of neurotrophins

Glial cells fulfill important tasks within the neural network of the central and peripheral nervous systems. The synthesis and secretion of various polypeptidic factors (cytokines) and a number of receptors, with which glial cells are equipped, allow them to communicate with their environment. Evidence has accumulated during recent years that neurotrophins play an important role not only for neurons but also for glial cells. This brief update of some morphological, immunocytochemical, and biochemical characteristics of glial cell lineages conveys our present knowledge about glial cells as targets and producers of neurotrophins under normal and pathological conditions. The chapter discusses the presence of neurotrophin receptors on glial cells, glial cells as producers of neurotrophins, signaling pathways downstream Trk and p75NTR, and the significance of neurotrophins and their receptors for glial cells during development, in cell death and survival, and in neurological disorders.

Negative Impact of Statins on Oligodendrocytes and Myelin Formation In Vitro and In Vivo

Statins are widely prescribed drugs in cardiovascular diseases. Recent studies also demonstrated anti-inflammatory and immunomodulatory properties of statins by modulating the activity of small GTPases. Statins are thus considered as potential therapeutic drug for the inflammatory demyelinating disease multiple sclerosis (MS). However, little is known about the effects of statins on myelin-forming oligodendrocytes. Here, we show that statins hamper process and myelin formation in vitro by interfering with Ras and Rho signaling in mature oligodendrocytes and provide evidence that statins impair ongoing remyelination in vivo. Our findings may have significant implications for the application of statins in MS patients and in other demyelinating diseases of the CNS.

Bulk Separation and Long‐Term Culture of Oligodendrocytes from Adult Pig Brain. I. Morphological Studies

Abstract: A method is described by which oligodendrocytes from adult pig brains can be isolated. It results in a cellular preparation suitable for long‐term culture. The entire procedure can be accomplished within 2–3 h. The purity of oligodendrocytes ranges between 80 and 95% depending on the Percoll gradient used and on the time in vitro. Yields between 2.5 and 4 × 107 cells per brain and plating efficiencies on the order of 60% make the system very useful for biochemical investigations. It was shown by immunocytochemical studies that oligodendrocytes produce extensive networks of processes, some of them having elaborate membranous expansions. Anti‐galactocerebroside (GC) antibodies as well as anti‐myelin basic protein (MBP), anti‐Wolfgram protein (WP), antiglial fibrillary acidic protein (GFAP), and monoclonal antibodies O1 and O4 are used to identify the cell types and to characterize the cellular composition of the cultures. Anti‐GC and O1 are suitable markers for these oligodendrocytes. Both antibodies label similar cells, and the staining intensities are equally strong. In the case of O4, variable staining intensities are observed, and a few additional cells are labeled that are anti‐GC−. After 31/2 weeks in culture, about 60% of the cells can be labeled by anti‐MBP. Here too differences in staining intensities are observed. The anti‐WP stain is too weak to be defined as positive. The percentage of GFAP+ cells lies in the range 15–20% at maximum. Cells were also mixed into collagen gels. This method appears to be more useful for outgrowth and branching of fibers than are monolayer systems. Drawbacks, however, include limited access for the antibodies and poor recovery of undamaged cells with their fibers.

Remyelination in multiple sclerosis : a new role for neurotrophins ?

Multiple sclerosis (MS) is a common neurological disease, which affects young adults. Its course is unpredictable and runs over decades. It is considered as an autoimmune disease, and is neuropathologically characterized by demyelination, variable loss of oligodendroglial cells, and axonal degeneration. Demyelination provides a permitting condition for axonal degeneration, which seems to be causative of permanent neurological deficits. Hence, the current treatment, which works preferentially immunmodulatory, should be complemented by therapeutics, which improves remyelination not only for restoring conduction velocity but also for preventing an irreversible axonal damage. One strategy to achieve this aim would be to promote remyelination by stimulating oligodendroglial cells remaining in MS lesions. While central nervous system neurons were already known to respond to neurotrophins (NT), interactions with glial cells became apparent more recently. In vitro and in vivo studies have shown that NT influence proliferation, differentiation, survival, and regeneration of mature oligodendrocytes and oligodendroglial precursors in favor of a myelin repair. Two in vivo models provided direct evidence that NT can improve remyelination. In addition, their neuroprotective and anti-inflammatory role would support a repair. Hence, a wealth of data point to NT as promising therapeutical candidates.

Glial-Neuronal Communication in Development and Regeneration

At the beginning of this century popular books on the function of the nervous system depicted the brain more mechanistically as a fabrique, which was organized in a number of functional units (1) and supported by pipelines transporting the nutrients.This simplistic view saw mainly the neurons as the predominant elements at work, whereas the glial cells, which rule out the neurons in number by 10:1, had to manage their life as underdogs. Despite the efforts to demonstrate partnerships the hierarchic view prevailed until nowadays (2). Kolliker, Lugaro, Spemann and others outlined already decades ago that the nervous system will only function on the basis of cooperativity: different cellular elements have to fulfill different tasks and have to interact. The guidance of neurons by radial glial cells, the intercellular K+ regulation, the detoxification and the axonal ensheathment by oligodendroglial cells might serve as examples. Research progressed only slowly to support this concept of glial-neuronal interrelationships. A major step forward was achieved by culturing glial cells as mixed or pure populations. This allowed a better characterisation of glial properties. Together with the refinement of the experimental tools at our disposal, we are till now faced with exciting new facets of the glial-neuronal communication in development and regeneration. In the following sections properties of the individual neural cells will be presented and their response to environmental influences described.

Isolation and cultivation of mature oligodendroglial cells

ConclusionThe isolation of mature ODC was improved appreciably during the past five years. It is now possible to maintain ODC cultures for several weeks. This should help to elucidate the functions of ODC in myelination and inflammation and their basic role within the neural network. However, several experimental strategies are needed to answer the manifold open questions addressed to ODC. Among others a very basic one is: what factors direct the glial cell lineage to become an oligodendrocyte or an astrocyte, if a common progenitor cell exists [76, 100].

Nerve growth factor signal transduction in mature pig oligodendrocytes

It has previously been shown that nerve growth factor (NGF) is of functional significance for mature pig oligodendrocytes (OLs) in culture. The present data give evidence for the expression of TrkA, the so‐called high‐affinity NGF receptor, and of p75NTR, the so‐called low‐affinity NGF receptor. TrkA is upregulated during culturing, in contrast to the p75 receptor. Exposure of OLs to NGF induces an autophosphorylation of TrkA via its intrinsic tyrosine kinase. K‐252a inhibits the TrkA autophosphorylation, which reduces the OL process formation to control levels. To the tyrosine‐phosphorylated sites of TrkA several proteins, such as phospholipase C‐γ1, the adaptor protein SHC, the phosphotyrosine phosphatase SH‐PTP2 (SYP) associate via their SH2 phosphotase SH‐PTP2 domain. The association of SHC to TrkA is shown by co‐immunoprecipitation. Indirect evidence for a possible activation of PLC‐γ1 is given by an NGF‐induced increase of oligodendroglial [Ca2+]i. Downstream from TrkA, a mitogen‐activated protein kinase cascade, which includes Erk1 and Erk2, is operating. An in‐gel myelin basic protein kinase assay revealed that NGF activates predominantly Erk1. Finally, it is shown that NGF stimulates expression of c‐fos. J. Neurosci. Res. 50:729–742, 1997.

Oligodendroglial Cells and Neurotrophins: A Polyphonic Cantata in Major and Minor

Oligodendrocytes met neurotrophins in the early 1990s of the last century. Since then, their relationship underwent functional ups and downs partially dependent on the developmental stage of the oligodendroglial cells and the species, from which the cells were derived. This review provides a brief overview of oligodendroglial cells and neurotrophins, characterizes neurotrophin signaling during oligodendroglial development, and discusses the significance of proneurotrophins and sortilin for oligodendroglial death and survival. Furthermore, data are provided that TrkA, the tyrosine kinase competent NGF receptor, is localized to caveolincontaining microdomains on the oligodendroglial plasma membrane; an interplay of caveolin and NGF signaling via TrkA might be of functional importance. Finally, experimental evidence of studies is presented which support the idea that neurotrophins are promising candidates for improving oligodendroglial regeneration and remyelination.

Mature pig oligodendrocytes rapidly process human recombinant pro‐nerve growth factor and do not undergo cell death

The neurotrophin family with its first member, nerve growth factor (NGF), binds two classes of receptors, more specifically to Trk receptors and to a shared p75NTR receptor. It has been shown that proNGF rather than NGF is predominant in the mature central nervous system. A recent finding indicated that a furin‐resistant proNGF preferentially binds to p75NTR, initiating a pro‐apoptotic cascade even in the presence of TrkA. In this context, rodent oligodendrocytes were reported to undergo cell death when exposed to proNGF. We have investigated the effect of a non‐mutated 32 kDa human recombinant proNGF (rhproNGF) on cultured pig oligodendrocytes which express TrkA, p75NTR and sortilin. Pig oligodendrocytes respond to rhproNGF (50 ng/mL) with an enhanced regeneration of their processes as already observed for NGF. Activity of mitogen‐activated protein kinase (MAPK), which plays an important role in oligodendroglial process formation, was increased even when rhproNGF processing was inhibited by the furin inhibitor Decanoyl‐RVKR‐CMK. Similarly, a cleavage‐resistant proNGF (R‐1G) activated MAPK and promoted oligodendroglial process regeneration. High concentrations of rhproNGF (300 ng/mL) did not induce cell death. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis and Western blotting revealed that oligodendrocytes process rhproNGF to NGF. NGF was detected in Western blots of oligodendroglial lysates already 10 min after rhproNGF exposure, followed by a release of NGF into the culture medium. Indirect evidence indicates that rhproNGF processing occurs via an endocytotic route.