Synemin isoforms in astroglial and neuronal cells from human central nervous system.

The intermediate filament (IF) synemin gene encodes three IF proteins (H 180, M 150, L 41 kDa) with overlapping distributions. Synemin M was present early with vimentin and nestin. Synemin H was found later in the nervous system and mesodermic derivatives concomitantly with angiogenesis and the migration of neural crest cells. Synemin L appeared later in neurons. A series of in vitro cell cultures were done to identify the linkage between synemin isoforms and specific cell types of the central nervous system (CNS). The neurons and glia from the brains of humans and rats were cultured and double immunostaining done with antibodies against the H/M or L synemin isoforms and neural cell types (betaIII-tubulin or NeuN) or astrocyte intermediate filaments (GFAP or vimentin). In neurons of the CNS, synemin H/M were co-expressed with GFAP, vimentin or nestin in glial cells, whereas synemin L was found in neurons.

Different expression of synemin isoforms in glia and neurons during nervous system development.

The synemin gene encodes proteins belonging to the intermediate filament family. These proteins confer resistance to mechanical stress and modulate cell shape. Three synemin isoforms, of 180 (H), 150 (M) and 41 (L) kDa, are produced by alternative splicing of the pre-mRNA and are regulated differently during development. The three isoforms differ in their C-terminal tail domains, while their IF rod domains are identical. Synemins H/M occurred together with nestin and vimentin in glial progenitors during the early differentiation of the developing mouse central nervous system. They are later found in GFAP-labeled cells. In contrast, the L isoform appeared only in neurons, together with neurofilaments and betaIII-tubulin in the brain after birth. However, synemin L appeared from E13 in the peripheral nervous system, where it was confined to the neurons of spinal ganglia. In the meantime, the synemin H/M isoforms were found in both the neurons and Schwann cells of the sensorial ganglia from E11. Tissue fractionation and purification of IFs from adult mouse spinal cord revealed that the synemin L isoform binds to neurofilaments associated with the membrane compartment. This report describes the synthesis of the three synemin isoforms by selective cell types, and their temporal and spatial distributions. Mechanisms specific to neurons and glia probably control the splicing of the common synemin mRNA and the synthesis of each synemin isoform.

Blood borne macrophages are essential for the triggering of muscle regeneration following muscle transplant.

The transplantation of satellite cells may constitute a strategy for rebuilding muscle fibres in inherited myopathies. However, its development requires a great understanding of the role of environmental signals in the regenerative process. It is therefore essential to identify the key events triggering and controlling this process in vivo. We investigated whether macrophages play a key role in the course of the regenerative process using skeletal muscle transplants from transgenic pHuDes-nls-LacZ mice. Before grafting, transplants were conditioned with macrophage inflammatory protein 1-beta (MIP 1-beta; stimulating the macrophages infiltration or vascular endothelial growth factor (VEGF) stimulating angiogenesis). Treatment of transplants with MIP 1-beta and VEGF both accelerated and augmented monocyte-macrophage infiltration and satellite cell differentiation and/or proliferation, as compared to controls. In addition, VEGF treatment enhanced the number of newly formed myotubes. When a complete depletion of host monocyte-macrophages was experimentally induced, no regeneration occurred in transplants. Our data suggest that the presence of blood borne macrophages is required for triggering the earliest events of skeletal muscle regeneration. The understanding of macrophage behaviour after muscle injury should allow us to develop future strategies of satellite cell transplantation as a treatment for muscular dystrophies.

Computerised dystrophic muscle simulator: prospecting potential therapeutic strategies for muscle dystrophies using a virtual experimental model.

Inherited muscle diseases are often characterised by widespread muscle damage in the body, limiting the clinical relevance of cell or gene therapy based upon direct injections into muscles. Recent studies have shown, however, that cells originating from the bone marrow are able to target necrosis-regeneration sites as they occur and, in addition, may also participate in the muscle regeneration after undergoing myogenic differentiation. Here, we present a computerised dystrophic muscle simulator that allows the prospecting of different scenarios of both disease evolution and appropriate employment of blood-borne cells as therapeutic shuttles. It provides the option of examining their use either to transfer a healthy gene into the tissue or to impart substances designed to boost its regeneration. One of the major advantages of this tool is that it offers the opportunity of visualising and composing therapeutic strategies in virtual paradigms in which severe clinical situations, not necessarily available in animal models, can be created. The dystrophic muscle simulator is freely accessible via the Genethon web site (www.genethon.fr), and in the online version via http:@www.wiley.co.uk/genmed.

Cell engineering for muscle gene therapy: Extemporaneous production of retroviral vector packaging macrophages using defective herpes simplex virus type 1 vectors harbouring gag, pol, env genes.

Gene therapy as a treatment for neuromuscular diseases is an ever-developing concept based on the use of DNA as the therapeutic agent. In the search for appropriate strategies a bottleneck exists, however, concerning the targeting of vectors carrying the therapeutic gene, to all pathologic sites. These diseases are often characterised by multiple widespread lesions spread over a large area, rendering administration by local injection into tissues, clinically irrelevant. With this in mind, we have proposed that circulating cells (monocytes/macrophages), which home naturally to inflammatory lesions, characteristic of degenerating muscle, could be used as shuttles able to track down every damaged site, and deliver there a corrective gene. Our aim is to mobilise a corrective gene from these infiltrating monocyte-macrophages, into muscle cells, a process of in situ cell to cell gene transfer which could be accomplished using a retroviral vector, since the regeneration process involves the proliferation of muscle precursors before they fuse to form replacement fibres. For this, monocyte-macrophages must be engineered into 'packaging cells' containing both the replication deficient retrovirus carrying the gene of interest and an helper genome (gag-pol-env) needed for its assembly and secretion. Here, we have transduced a monocyte cell line using a defective murine Moloney leukemia retrovirus carrying the LacZ reporter gene. This provided us with a platform to investigate the possibility of gag-pol-env vector driven packaging of the defective retrovirus by macrophages. We show that an herpes simplex virus type I amplicon harbouring the Moloney gag, pol, env sequences is able to rescue the defective retrovirus vector from macrophages, allowing gene transfer into muscle precursor cells. After fusion, these cells gave rise to genetically modified myotubes in vitro.

Phenotypic alteration of astrocytes induced by ciliary neurotrophic factor in the intact adult brain, As revealed by adenovirus-mediated gene transfer.

Synthesis of the ciliary neurotrophic factor (CNTF) and its specific receptor (CNTFRalpha) is widespread in the intact CNS, but potential biological roles for this system remain elusive. Contradictory results have been obtained concerning a possible effect on the morphological and biochemical phenotype of astrocytes. To reassess this question, we have taken advantage of adenovirus-mediated gene transfer into the rat brain to obtain the local release of CNTF. Stereotaxic administration of CNTF recombinant adenovirus vectors into the striatum led to phenotypic changes in astrocytes located in regions that were related axonally to striatal neurons at the injection site. Astrocytes appeared hypertrophied and displayed an increase in both GFAP and CNTF immunoreactivity. This response was observed up to 5 weeks after injection, the longest time studied. It was not observed after the administration of a control vector. The methodology used in the present study, allowing us to analyze the effect of the factor in areas remote from the injection site, has provided conclusive evidence that CNTF affects the astroglial phenotype in the intact CNS. The characteristics of these effects may explain why contradictory results have been obtained previously, because this signaling system seems to have a low efficiency and therefore requires a high local concentration of the factor close to the target cells. One might speculate as to the involvement of a CNTF astroglio-astroglial signaling system in the organized response of a population of astrocytes to changes in CNS homeostasis detected locally, even by a single cell.

Adenovirus-mediated gene transfer to the brain: methodological assessment.

The purpose of this short review is to analyse major advantages and limitations of the adenovirus (Ad), specifically with relevance to its use as a vector for gene transfer to the brain. The characteristics of Ad transduction include: the relative absence of cell type specificity; the limited spatial spread of the virus; and the long-term expression of the transgene. In the central nervous system, in contrast to that which occurs in other organs, Ad transduction in the adult does not systematically provoke cell death. Nevertheless, a proportion of the transduced cells do die, and this represents a conspicuous problem. Mechanisms leading to cell death in the brain may include immune rejection and inflammation-related toxicity, although this would not explain all of the results, and direct toxicity related to either inappropriate preparation or the transduction itself. Taking into account uncertainties concerning the innocuousness of Ad transduction, it may seem unwise to envisage Ad gene therapy for diseases that are not life-threatening and/or benefit from adequate drug or surgical treatments (e.g. Parkinson's disease or epilepsy). Ad vectors may not be easily used either in diseases displaying major immune dysfunction (e.g. multiple sclerosis). In contrast, malignant brain tumors and numerous neurodegenerative diseases (such as Huntington's, Alzheimer's diseases or amyotrophic lateral sclerosis) are directly life-threatening and deprived of any adequate treatment. They may be appropriate targets for Ad-mediated gene therapy, once both the vector and the gene of interest have been defined and optimized.