Corinne Schmalenbach

Astroglia—Neuron interactions that promote long-term neuronal survival

Besides intrinsic determinants of cell growth, epigenetic signals have been proposed to regulate development and maintenance of neurons. Here we provide evidence that cerebral astrocytes contribute significantly to the set of environmental influences that are required for long-term survival of neurons derived from the mammalian central nervous system. Cerebral astrocytes in serum-free culture express diffusible and non-diffusible neuron-supporting signals, including cell-adhesive neurite growth-promoting glycoproteins, diffusible neurotrophic factors as well as membrane-bound molecules that mediate cell contact interactions. The combination and synergistic interaction of these environmental signals markedly enhance the survival of brain neurons. While astroglia-derived cell-adhesive substrates that include a high molecular weight complex consisting of laminin beta-chains and proteoglycan (Matthiessen et al., 1989) stimulate neurite outgrowth, they fail to enhance long-term neuronal survival when additional neurotrophic and cell-contact interactions are lacking. Astrocytes release a diffusible neurotrophic activity that, when permanently applied, maintains long-term survival of central neurons in culture. The soluble neurotrophic activity seems to interact synergistically with cell-bound signals which are also required for long-term survival and which are expressed by astrocytes and neurons, but not by fibroblasts. Among neurons from different brain areas, such as hippocampus, cerebral cortex and septum, regional differences in their responsiveness to the astroglial neurotrophic activity have been observed.

Influence of elevated expression of rat wild-type PMP22 and its mutant PMP22Trembler on cell growth of NIH3T3 fibroblasts

Abstract.The peripheral myelin gene PMP22 is the rat and human homologue of the murine growth-arrest-specific gene gas3. The biological function of PMP22 is unknown, but recent progress in the analysis of rat Schwann cells expressing altered levels of PMP22 revealed that one role of PMP22 is as a negative growth modulator. We have investigated the influence of rat PMP22 (rPMP22) and a mutant of PMP22 (rPMP22Tr) resembling the murine trembler mutation on cell growth of retrovirus-vector-infected mouse NIH3T3 cells. Transduced cells carrying the two different sense constructs expressed rPMP22 and rPMP22Tr mRNAs and proteins. Elevated levels of rPMP22 and rPMP22Tr significantly reduced fibroblast growth as judged by proliferation assays. Despite a negative modulatory influence of rPMP22 and rPMP22Tr on cell proliferation, cell cycle analyses by flow cytometry did not reveal an influence of rPMP22 or rPMP22Tr on the synchronous progression of resting NIH3T3 cells from G0 into S phase. However, cell cycle analyses by flow cytometry of asynchronously dividing cultures demonstrated that the expression of rPMP22 and rPMP22Tr increased the fraction of cells in the G1 phase of the cell cycle. Furthermore, cell death analyses revealed that, in contrast to control cells and cells carrying the rPMP22Tr construct, a significantly increased fraction of NIH3T3 cells expressing rPMP22 exit the proliferation compartment showing hallmarks of programmed cell death. These results indicate that (i) rPMP22 and rPMP22Tr act as negative modulators of proliferation in murine fibroblasts probably through extension of the G1 phase of the cell cycle and (ii) rPMP22 but not rPMP22Tr promotes programmed death of these cells.

Studies on the effects of altered PMP22 expression during myelination in vitro

Severe inherited dysmyelinating diseases of the peripheral nervous system, the Charcot‐Marie‐Tooth type1A disease (CMT1A) and the hereditary neuropathy with liability to pressure palsies (HNPP) are associated with a large DNA duplication or deletion of a chromosomal region containing the peripheral myelin protein 22 (PMP22) gene. It has been suggested that a gene dosage effect involving PMP22 is responsible for the pathological phenotype. We investigated if altered PMP22 expression affects the onset of myelin formation and the ultrastructure of myelin. Rat Schwann cell cultures were stably infected with recombinant retrovirus vectors harboring the rat PMP22 cDNA in sense or antisense orientation. Schwann cells over‐ or underexpressing PMP22 were cocultured with purified DRG neurons under conditions that promote myelination. We examined PMP22 expression and localization in the myelin forming cultures by RT‐PCR, immunohistochemistry and confocal microscopy, and we analyzed myelin ultrastructure by electron microscopy. Our results demonstrate that abnormal levels of PMP22 expression do not impair the early stages of myelination and membrane compaction and do not interfere with the expression of other myelin genes. Our observations further indicate that PMP22 is involved more in controlling myelin thickness and stability than in the events determining the initial steps of myelin formation. J. Neurosci. Res. 48:31–42, 1997.

Identification of Meningeal Cell Released Neurite Promoting Activities for Embryonic Hippocampal Neurons

Abstract: Primary cultures of meningeal cells from embryonic rat cerebra secrete neurite growth‐inducing components into serum‐free culture medium. This conditioned medium (CM) was analyzed by FPLC and immunochemical and enzymatic treatments and tested for neurite promoting activity (NPA) in a quantitative bioassay using hippocampal neurons from embryonic rat. By immunoprecipitation or specific adsorption we identified laminin (LN)‐proteoglycan complexes and fibronectin (FN), respectively, as the major neurite promoting components within meningeal cell CM. The LN‐proteoglycan complexes and their NPA were sensitive to chondroitinase (chondroitin ABC lyase, EC 4.2.2.4) and to a smaller extent to heparitinase (heparitin sulfate lyase, EC 4.2.2.8). Minor fractions of the total NPA in CM correlated with free LN and a putative but not yet characterized FN‐proteoglycan complex.

Glial support of CNS neuronal survival, neurite growth and regeneration

In an attempt to identify specific molecular and cellular requirements necessary to support long-term maintenance and differentiation of central neurons we have identified laminin-HSPG and free fibronectin as two major neurite promoting substrate adhesion factors released by immature cerebral astrocytes in serum-free culture. Astrocytes further secrete diffusible neurotrophic protein factor(s) which are permanently required for survival of cultured neurons from various brain regions. However, both the presence of substrate-bound neurite-promoting factors and diffusible neurotrophic activities were not sufficient to support long-term maintenance of central neurons in culture. Cell contact-mediated interactions which appear to be cell type-restricted (e.g. to neurons and astrocytes, but not to fibroblasts) are further required for neuronal stabilization. The implantation of immature astroglial cells into the injured adult CNS should provide a supportive environmental condition for damaged neurons to enhance their recovery and stimulate regenerative responses.

GABA activated chloride currents in cultured rat hippocampal and septal region neurons can be inhibited by curare and atropine.

In order to study the influence of curare and atropine on the gamma-aminobutyric acid (GABA)-evoked chloride current, we have investigated cultures of hippocampal and septal region neurons from embryonic rats (E18). The neurons were cultured under the trophic influence of spatially separated astrocytes in serum-free medium. By means of the patch clamp technique, the excitable cells displayed GABA induced chloride currents within 1-15 days in vitro. In both cultures picrotoxin or bicuculline as well as curare or atropine reversibly inhibited the chloride current in a dose dependent manner. We conclude that curare and atropine are not specifically anti-cholinergic for cultured central neurons. Our results provide evidence for common ligand binding properties shared by GABAA and acetylcholine (ACh) receptors supporting the recent concept of a receptor superfamily.

Electrophysiological properties of rat septal region neurons during development in culture

We report on the development of membrane properties of septal region neurons from embryonic rats in serum-free culture during 1-25 days in vitro (DIV). Na(+)-dependent action potentials could be evoked within 1 day after plating and 3 different types of outward current were observed by means of the patch-clamp technique: IK, IA and IC. In some neurons the neurotransmitter GABA evoked a chloride current after 2 DIV. In addition a cationic current elicited by glutamate appeared after 4 DIV. Within 8-12 DIV virtually all neurons were sensitive to both GABA and glutamate. Spontaneous action potentials and postsynaptic potentials occurred after 7-10 DIV but cultured septal neurons did not generate any pacemaker-like activity.

Retrovirus-Mediated Gene Transfer of PMP22 in Schwann Cells: Studies on Cell Growth

The recent discovery of the novel peripheral myelin protein PMP22 and the association of mutations of this gene with dominant inherited peripheral neuropathies in mice and man has raised particular interest in the biological function of this gene (for review: Lemke, 1993; Suter et al., 1993). Due to the separate occasions leading to the identification of this gene different names have been given for PMP22 cDNAs in the literature. The reported cDNA sequences are: gas3 (growth arrest-specific gene 3; Schneider et al., 1988), CD25 (crush denervated cDNA clone 25; Spreyer et al., 1991) SR13 (sciatic nerve regeneration clone 13; Welcher et al., 1991) or PMP22 (peripheral myelin protein 22kDa; Snipes et al, 1992). All sequences are variants of the murine growth arrest-specific gene gas3, which was originally cloned from NIH3T3 fibroblasts as one of a set of genes induced when these cells are growth-arrested (Schneider et al., 1988; Manfioletti et al., 1990). The demonstration that gas3 is also a prominent Schwann cell gene product came from the independent cloning of cDNAs that are either induced or repressed following rat sciatic nerve crush or transection (Spreyer et al., 1991; Welcher et al., 1991). It turned out that PMP22 expression is axonally regulated like the major myelin genes. The level of expression is high in fully differentiated Schwann cells, declines rapidly upon Schwann cell dedifferentiation and proliferation as a result of axonal degeneration after nerve injury, and is upregulated upon regeneration (Kuhn et al., 1993).