Early signs of motoneuron vulnerability in a disease model system: Characterization of transverse slice cultures of spinal cord isolated from embryonic ALS mice.

Mutations in the SOD1 gene are associated with familial amyotrophic lateral sclerosis. The mechanisms by which these mutations lead to cell loss within the spinal cord ventral horns are unknown. In the present report we used the G93A transgenic mouse model of amyotrophic lateral sclerosis to develop and characterize an in vitro tool for the investigation of subtle alterations of spinal tissue prior to frank neuronal degeneration. To this aim, we developed organotypic slice cultures from wild type and G93A embryonic spinal cords. We combined immunocytochemistry and electron microscopy techniques to compare wild type and G93A spinal cord tissues after 14 days of growth under standard in vitro conditions. By SMI32 and choline acetyl transferase immunostaining, the distribution and morphology of motoneurons were compared in the two culture groups. Wild type and mutant cultures displayed no differences in the analyzed parameters as well as in the number of motoneurons. Similar results were observed when glial fibrillary acidic protein and myelin basic protein-positive cells were examined. Cell types within the G93A slice underwent maturation and slices could be maintained in culture for at least 3 weeks when prepared from embryos. Electron microscopy investigation confirmed the absence of early signs of mitochondria vacuolization or protein aggregate formation in G93A ventral horns. However, a significantly different ratio between inhibitory and excitatory synapses was present in G93A cultures, when compared with wild type ones, suggesting the expression of subtle synaptic dysfunction in G93A cultured tissue. When compared with controls, G93A motoneurons exhibited increased vulnerability to AMPA glutamate receptor-mediated excitotoxic stress prior to clear disease appearance. This in vitro disease model may thus represent a valuable tool to test early mechanisms contributing to motoneuron degeneration and potential therapeutic molecular interventions.

Interneurons transiently express the ERG K+ channels during development of mouse spinal networks in vitro.

During spinal cord maturation neuronal excitability gradually differentiates to meet different functional demands. Spontaneous activity, appearing early during spinal development, is regulated by the expression pattern of ion channels in individual neurons. While emerging excitability of embryonic motoneurons has been widely investigated, little is known about that of spinal interneurons. Voltage-dependent K+ channels are a heterogeneous class of ion channels that accomplish several functions. Recently voltage-dependent K+ channels encoded by erg subfamily genes (ERG channels) were shown to modulate excitability in immature neurons of mouse and quail. We investigated the expression of ERG channels in immature spinal interneurons, using organotypic embryonic cultures of mouse spinal cord after 1 and 2 weeks of development in vitro. We report here that all the genes of the erg family known so far (erg1a, erg1b, erg2, erg3) are expressed in embryonic spinal cultures. We demonstrate for the first time that three ERG proteins (ERG1A, ERG2 and ERG3) are co-expressed in the same neuronal population, and display a spatio-temporal distribution in the spinal slices. ERG immuno-positive cells, representing mainly GABAergic interneurons, were present in large numbers at early stages of development, while declining later, with a ventral to dorsal gradient. Patch clamp recordings confirmed these data, showing that ventral interneurons expressed functional ERG currents only transiently. Similar expression of the erg genes was observed at comparable ages in vivo. The role of ERG currents in regulating neuronal excitability during the earliest phases of spinal circuitry development will be examined in future studies.

Spinal circuits formation: a study of developmentally regulated markers in organotypic cultures of embryonic mouse spinal cord.

In this study, we have addressed the issue of neural circuit formation using the mouse spinal cord as a model system. Our primary objective was to assess the suitability of organotypic cultures from embryonic mouse spinal cord to investigate, during critical periods of spinal network formation, the role of the local spinal cellular environment in promoting circuit development and refinement. These cultures offer the great advantage over other in vitro systems, of preserving the basic cytoarchitecture and the dorsal-ventral orientation of the spinal segment from which they are derived [Eur J Neurosci 14 (2001) 903; Eur J Neurosci 16 (2002) 2123]. Long-term embryonic spinal cultures were developed and analyzed at sequential times in vitro, namely after 1, 2, and 3 weeks. Spatial and temporal regulation of neuronal and non-neuronal markers was investigated by immunocytochemical and Western blotting analysis using antibodies against: a) the non-phosphorylated epitope of neurofilament H (SMI32 antibody); b) the enzyme choline acetyltransferase, to localize motoneurons and cholinergic interneurons; c) the enzyme glutamic acid decarboxylase 67, to identify GABAergic interneurons; d) human eag-related gene (HERG) K(+) channels, which appear to be involved in early stages of neuronal and muscle development; e) glial fibrillary acidic protein, to identify mature astrocytes; f) myelin basic protein, to identify the onset of myelination by oligodendrocytes. To examine the development of muscle acetylcholine receptors clusters in vitro, we incubated live cultures with tetramethylrhodamine isothiocyanate-labeled alpha-bungarotoxin, and we subsequently immunostained them with SMI32 or with anti-myosin antibodies. Our results indicate that the developmental pattern of expression of these markers in organotypic cultures shows close similarities to the one observed in vivo. Therefore, spinal organotypic cultures provide a useful in vitro model system to study several aspects of neurogenesis, gliogenesis, muscle innervation, and synaptogenesis.

Homeostatic plasticity induced by chronic block of AMPA/kainate receptors modulates the generation of rhythmic bursting in rat spinal cord organotypic cultures.

Generation of spontaneous rhythmic activity is a distinct feature of developing spinal networks. We report that rat embryo organotypic spinal cultures contain the basic circuits responsible for pattern generation. In this preparation rhythmic activity can be recorded from ventral interneurons and is developmentally regulated. When chronically grown in the presence of an AMPA/kainate receptor blocker, this circuit expresses long-term plasticity consisting largely of increased frequency of fast synaptic activity and reduction in slow GABAergic events. We examined whether, once this form of homeostatic plasticity is established, the network could still exhibit rhythmicity with properties similar to controls. Control or chronically treated ventral interneurons spontaneously generated (with similar probability) irregular, network-driven bursts over a background of ongoing synaptic activity. In control cultures increasing network excitability by strychnine plus bicuculline, or by raising [K(+)](o), induced rapid-onset, regular rhythmic bursts. In treated cultures the same pharmacological block of Cl(-)-mediated transmission or high-K(+) application also induced regular patterned activity, although significantly faster and, in the case of high K(+), characterized by slow onset due to postsynaptic current summation. Enhancing GABAergic transmission by pentobarbital surprisingly accelerated the high-K(+) rhythm of control cells (though depressing background activity), whereas it slowed it down in chronically treated cells. This contrasting effect of pentobarbital suggests that, to preserve bursting ability, chronic slices developed a distinct GABAergic inhibitory control on over-expressed bursting circuits. Conversely, in control slices GABAergic transmission depressed spontaneous activity but it facilitated bursting frequency. Thus, even after homeostatic rearrangement, developing mammalian spinal networks still generate rhythmic activity.

Transport and localization elements in myelin basic protein mRNA.

Myelin basic protein (MBP) mRNA is localized to myelin produced by oligodendrocytes of the central nervous system. MBP mRNA microinjected into oligodendrocytes in primary culture is assembled into granules in the perikaryon, transported along the processes, and localized to the myelin compartment. In this work, microinjection of various deleted and chimeric RNAs was used to delineate regions in MBP mRNA that are required for transport and localization in oligodendrocytes. The results indicate that transport requires a 21-nucleotide sequence, termed the RNA transport signal (RTS), in the 3' UTR of MBP mRNA. Homologous sequences are present in several other localized mRNAs, suggesting that the RTS represents a general transport signal in a variety of different cell types. Insertion of the RTS from MBP mRNA into nontransported mRNAs, causes the RNA to be transported to the oligodendrocyte processes. Localization of mRNA to the myelin compartment requires an additional element, termed the RNA localization region (RLR), contained between nucleotide 1,130 and 1, 473 in the 3' UTR of MBP mRNA. Computer analysis predicts that this region contains a stable secondary structure. If the coding region of the mRNA is deleted, the RLR is no longer required for localization, and the region between nucleotide 667 and 953, containing the RTS, is sufficient for both RNA transport and localization. Thus, localization of coding RNA is RLR dependent, and localization of noncoding RNA is RLR independent, suggesting that they are localized by different pathways.

Transport and localization of exogenous myelin basic protein mRNA microinjected into oligodendrocytes.

We have studied transport and localization of MBP mRNA in oligodendrocytes in culture by microinjecting labeled mRNA into living cells and analyzing the intracellular distribution of the injected RNA by confocal microscopy. Injected mRNA initially appears dispersed in the perikaryon. Within minutes, the RNA forms granules which, in the case of MBP mRNA, are transported down the processes to the periphery of the cell where the distribution again becomes dispersed. In situ hybridization shows that endogenous MBP mRNA in oligodendrocytes also appears as granules in the perikaryon and processes and dispersed in the peripheral membranes. The granules are not released by extraction with non-ionic detergent, indicating that they are associated with the cytoskeletal matrix. Three dimensional visualization indicates that MBP mRNA granules are often aligned in tracks along microtubules traversing the cytoplasm and processes. Several distinct patterns of granule movement are observed. Granules in the processes undergo sustained directional movement with a velocity of approximately 0.2 micron/s. Granules at branch points undergo oscillatory motion with a mean displacement of 0.1 micron/s. Granules in the periphery of the cell circulate randomly with a mean displacement of approximately 1 micron/s. The results are discussed in terms of a multi-step pathway for transport and localization of MBP mRNA in oligodendrocytes. This work represents the first characterization of intracellular movement of mRNA in living cells, and the first description of the role of RNA granules in transport and localization of mRNA in cells.

Transient reversion of O4+ GalC- oligodendrocyte progenitor development in response to the phorbol ester TPA.

The physiological importance of protein kinase C during oligodendrocyte progenitor maturation was investigated by analyzing the effects of the protein kinase C activator phorbol 12-myristate 13-acetate (TPA) on the morphology, proliferation, and differentiation of oligodendrocytes at sequential stages of development. Monoclonal antibodies A2B5 and O4 were used to identify the A2B5+O4- and the A2B5+O4+ galactocerebroside- progenitor stages. Anti-galactocerebroside and anti-myelin basic protein were used to identify mature, post-mitotic oligodendrocytes. Proliferation was measured by bromodeoxyuridine incorporation. Within 24 hr after addition, TPA induced a down-regulation of the O4 antigen in OL progenitors, and an increase of expression of the intermediate filament protein vimentin, leading to a phenotypic reversion from the vimentin-A2B5+O4+ phenotype to the less mature vimentin+A2B5+O4- stage. Concomitantly, TPA induced an increase in the number of bromodeoxyuridine-labeled oligodendrocyte progenitors and extensive process elongation. The response of O4+ progenitors was transient. Even with continued exposure to TPA, by 4 days after TPA addition the reverted cells ceased proliferation, reacquired O4 immunoreactivity, became vimentin-negative, and began to express galactocerebroside and myelin basic protein, and to display the complex, highly branched morphology characteristic of terminally differentiated oligodendrocytes. These results indicate that modulation of protein kinase C activity by TPA induces a transient reversion of O4+ progenitors to less mature O4- cells, causing a transient inhibition of terminal differentiation. The relationship of these data to similar responses of the OL lineage to specific growth factors and implications for remyelination after pathologic injury are discussed.

Characterization of secretory protein translocation: ribosome-membrane interaction in endoplasmic reticulum.

Secretory proteins are synthesized on ribosomes bound to the membrane of the endoplasmic reticulum (ER). After the selection of polysomes synthesizing secretory proteins and their direction to the membrane of the ER via signal recognition particle (SRP) and docking protein respectively, the polysomes become bound to the ER membrane via an unknown, protein-mediated mechanism. To identify proteins involved in protein translocation, beyond the (SRP-docking protein-mediated) recognition step, controlled proteolysis was used to functionally inactivate rough microsomes that had previously been depleted of docking protein. As the membranes were treated with increasing levels of protease, they lost their ability to be functionally reconstituted with the active cytoplasmic fragment of docking protein (DPf). This functional inactivation did not correlate with a loss of either signal peptidase activity, nor with the ability of the DPf to reassociate with the membrane. It did correlate, however, with a loss of the ability of the microsomes to bind ribosomes. Ribophorins are putative ribosome-binding proteins. Immunoblots developed with monoclonal antibodies against canine ribophorins I and II demonstrated that no correlation exists between the protease-induced inability to bind ribosomes and the integrity of the ribophorins. Ribophorin I was 85% resistant and ribophorin II 100% resistant to the levels of protease needed to totally eliminate ribosome binding. Moreover, no direct association was found between ribophorins and ribosomes; upon detergent solubilization at low salt concentrations, ribophorins could be sedimented in the presence or absence of ribosomes. Finally, the alkylating agent N-ethylmaleimide was shown to be capable of inhibiting translocation (beyond the SRP-docking protein-mediated recognition step), but had no affect on the ability of ribosomes to bind to ER membranes. We conclude that potentially two additional proteinaceous components, as yet unidentified, are involved in protein translocation. One is protease sensitive and possibly involved in ribosome binding, the other is N-ethylmaleimide sensitive and of unknown function.

A structural and functional analysis of the docking protein. Characterization of active domains by proteolysis and specific antibodies.

Docking protein is a 73-kDa integral membrane protein of the rough endoplasmic reticulum. It is essential for translocation of nascent secretory proteins into the lumen of the endoplasmic reticulum. Monoclonal and polyclonal antibodies have been generated which, in conjunction with limited proteolysis, have been used to characterize several subspecies of docking protein. These proteolytic fragments have been analyzed with respect to the various functions ascribed to docking protein which can be assayed in vitro. Proteolytic digestion of membrane-associated or of affinity-purified intact docking protein showed that: elastase cleavage generates a 59-kDa soluble fragment and one of 14 kDa which contains the membrane anchoring domain; trypsin as well as endogenous proteolysis generates a 46-kDa fragment, leaving a 27-kDa domain containing the membrane anchor. This 27-kDa fragment can be reduced to a 13- and a 14-kDa piece by elastase digestion. The characteristics of these various subspecies were examined. The 59-kDa soluble fragment, which can reconstitute full translocation activity to docking protein-depleted microsomes (Meyer, D. I., and Dobberstein, B. (1980) J. Cell Biol. 87, 503-508) was capable of releasing a signal recognition particle-mediated translation arrest. The 46-kDa fragment was neither able to reassociate with nor to reconstitute the activity of docking protein-depleted microsomes. Moreover this fragment was unable to release a signal recognition particle-mediated arrest. This suggests that the 13-kDa fragment (the difference between 46 and 59 kDa) is both essential for association with the membrane, and for the release of translation arrests.