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  Director : Jean Michel Heard (jmheard@pasteur.fr)


  abstract

 

We investigate strategies aimed at repairing lesions in the central nervous system (CNS). Gene therapy is used to deliver a soluble corrective factor in laboratory and large animal models of neurodegeneration associated to lysosomal storage diseases, with the perspective of human application. Models have been created in which the modalities of the regeneration of efferent and afferent connections of resident motor neurons or of transplanted stem cells giving rise to new motor neurons can be explored. The group of B. Durand studies the effectors of barhl2, a homeogene involved in neural plate patterning.



  report

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The notion that repair strategies could be proposed for treating CNS lesions in the future emerged from various observations made over the last decade, including fetal cell graft in Parkinson's disease patients, evidence for neurogenesis in adult mammals, isolation of neural stem cells from various part of the adult mammalian CNS. At the same time, gene transfer vectors capable of inducing the expression of foreign genes into CNS cells became available.

A first type of repair strategy for the CNS relies on the delivery of soluble factors that could prevent cell death or stimulate axonal regeneration. Local sources of soluble factors can be created by genetically modifying resident cells with gene transfer vectors. We approach this first type of strategy by delivering gene transfer vectors coding for the missing enzyme in models of lysosomal storage diseases, which induce dramatic neurodegeneration in children.

A second type of strategy would consist in replacing lost cells. A requisite is that CNS had sufficient plasticity in the adult to incorporate exogenous cells within the pre-existing architecture. It is postulated that neural progenitors might do so, either spontaneously, or after genetic reprogrammation. Successful achievement will require combining cell transplantation with interventions aimed at favoring plasticity. We approach this second type of strategies in models of motor pathway repair in rodents.

Knowledge about tools and targets that would make manipulations of neural progenitors cell fate in adults possible comes from studies of embryonic developmental pathways. B. Durand studies genetic regulators governing neural plate patterning in vertebrates.

Investigation and treatment of neurodegeneration in mucopolysaccharidosis (MPS)

Previous results obtained by N. Desmaris and A. Cressant in mouse models of Hurler syndrome (MPSI) and Sanfilippo syndrome (MPSIIIb) indicate that a single stereotactic injection of AAV2 vectors in the brain is sufficient for correcting pathology in the entire encephalon, preventing neuronal cell death and improving behavioral performances.

In the perspective of human trials, we are currently examining the therapeutic efficacy and safety of vector delivery and enzyme secretion in large animal brains, according to a clinically relevant protocol. AAV2 as well as new AAV serotypes are used. Experiments in affected MPSI dogs allow to draw brain maps of vector distribution, enzyme spreading and disease correction. A similar protocol, including behavioral testing and MRI, is used for tolerance studies in normal macaques. This is a collaborative program with the team of P. Moullier in Nantes, the Ecole Nationale Vétérinaire de Nantes, a neuropediatrician (Pr. M. Tardieu) and neurosurgeons (Prs. M. Tadié and Y. Lajat).

Mechanisms responsible for lysosomal enzyme spreading in the brain are not known. F. Chen examines enzymes transport along axons. Experiments are performed with lysosomal enzyme/GFP fusion proteins and time-lapse microscopy on cultured neurons. In vivo administration in deficient mice allows marking of brain areas in which enzyme delivery is associated with improved behavior. We also introduce enzyme modifications aimed at increasing spreading in brain tissues, such as fusion with the HIV-1 Tat protein.

Repair strategies for motor pathways

Motricity is controlled at the cortical, sub-cortical and spinal levels. Motor pathways can undergo limited reorganization at each level, allowing partial adaptive compensation after lesion. We explore repair strategies at the spinal cord level. S. Liu is a neurosurgeon who described surgical methods for the reconstruction of motor pathways after spinal cord section at the thoraco-lumbar boundary in rats and primates. Methods are based on the reconnection of lumbar ventral roots to thoracic motor neurons. Surgery induces neuro-regeneration, which results in placing hind limb muscles under the control of thoracic motor neurons that are a priori unable to govern locomotion. Experimental models are thus created in which the development of efferent and afferent connections of transplanted motor neurons can be investigated in adult rats, as well as the conditions for anatomical and functional re-organizations leading to the return of locomotion behaviors.

T. Bréjot documented the kinetics of thoracic motor neuron axonal regrowth toward hind limb muscles after surgery. Combining surgery with cell transplantation in the ventral horn, similar investigations are performed by D. Bohl in the rat using transplanted neural stem cells. A. Nosjean approaches the same question in the mouse, using embryonic stem cells, such as mouse models of motor neuron disorders can be explored in the future. Transplanted cells are either left unmodified, or exposed to differentiation inducing factors or genetically reprogrammed.

Consistently with the reconnection of hind limb muscles to thoracic motor neurons, return of motricity in treated animals is not adequate for locomotion. With the aim to improve function, D. Bohl, S. Liu and A. Cressant use locomotion training and intra-spinal secretion of neurotrophic factors in order to induce local anatomical re-organization.

Neural differentiation

Homeobox proteins are transcriptional regulators that induce specific target gene expression controlling a developmental pathway. The identity of different domains and segments of the CNS depends on sets of homeobox proteins and other gene regulatory proteins that control the transcription of specific target genes. In the proencephalic neural plate, patterns of homeobox genes expression can be related to primary morphogenetic processes that organize the histological primordia of the embryonic CNS into a grid of domains. At later stages, patterning relies on organizing centers. Transverse rings of specialized neuroepithelia for the brain, as well as floor plate and roof plate for the spinal cord, provide sources of secreted patterning molecules like Shh, Fgf8 and BMP, which establishe regional identity and neuronal fate in adjacent domains of the neural tube.

B. Durand recently isolated a new homeobox gene in xenopus and mice, called barhl2. barhl2 pattern of expression and phenotype in xenopus suggest a role in dorso-ventral patterning of the neural tube. Using morphological and molecular criteria, barhl2 appeared to mimic BMP and FGF signaling, and thus likely represents a target for the patterning effect of these factors. barhl2 is a transcriptional repressor specifically expressed in the dorsal diencephalon, the dorsal part of the retina and the roof plate of the neural tube at early stages of development. Its expression has a dorsalizing effect. Studies of the cellular and molecular mechanisms underlying the function of barhl2 are performed to identify genetic regulators downstream and upstream of this gene.

Other project

In collaboration with the laboratory of C. Petit, B. Delprat explores the potentials of gene transfer into the cochlea in mouse models of hearing deficiency.

Keywords: gene therapy, stem cells, nervous system, lysosomal storage diseases, motricity, homoegenes



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
  Renée Communal,communal@pasteur.fr Delphine Bohl CR2 INSERM, dbohl@pasteur.fr

Béatrice Durand CR1 CNRS, bdurand@pasteur.fr

Jean Michel Heard DR1 INSERM ,jmheard@pasteur.fr

Song Liu CR1 INSERM ,songliu@pasteur.fr

Anne Nosjean CR1 CNRS, nosjean@pasteur.fr

Arnaud Cressant Stage post-doctoral ,cressant@pasteur.fr

Benjamin Delprat Stage post-doctoral ,delprat@pasteur.fr

Thomas Bréjot Thèsard Paris VII ,tbrejot@pasteur.fr

Fengtian Chen Thèsard Paris VII, ftchen@pasteur.fr

Nathalie Desmaris Technicienne Supérieure 1D ,nchodan@pasteur.fr

Sébastien Franconi Technicien Supérieur 1D ,franconi@pasteur.fr


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