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     Retroviruses and Gene Transfer - INSERM U622


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


  abstract

 

Topic 1: Neurodegeneration associated to lysosomal storage diseases. Studies on pathophysiology (inflammation, axonal transport) and therapeutics (gene therapy for the delivery of the missing enzyme). Topic 2: Neural stem cells. Genetic reprogramming (forced expression of transcription factors). Topic 3: studies on the role of the Barhl2 gene during development, cancerogenesis and neurodegenerative processes.



  report

cale

Topic 1: Neurodegeneration associated to lysosomal storage diseases (F. Chen, A. Cressant, N. Desmaris, S. Vitry).

Lysosomal storage diseases are inborn errors of the metabolism characterised by the high frequency of neurological manifestations, which are often severe with early onset in childhood. Genetic basis (most often a loss of function) are well documented, as well as the consequences on lysosomal catabolic functions. Mechanisms leading from the biochemical defect to neuropathology are however still obscure and so far inaccessible to therapeutics. Our studies focus on two models of mucopolysaccharidoses in which partially degraded and hypersulfated heparan sulfates accumulate. We investigate links between the accumulation of these compounds, microglial and astrocytic activation and neurodegeneration. Experiments are performed in in vitro models and in affected mice or dogs. Aims are to decipher mechanisms leading to chronic inflammation in the central nervous system (CNS) and their consequences with regards to neurotoxicity and axonal transport.

The delivery of the missing enzyme is a plausible treatment for these diseases. However, access to the CNS implies crossing or bypassing the blood-brain barrier. We demonstrated that gene therapy allows to reach that goal in affected mice and dogs. The creation of continuous sources of enzyme can be achieved through the injection of adeno-associated vectors in the striatum (AAV1, 2 or 5). We showed that enzyme delivery to the entire brain could be achieved through a single AAV5 vector injection in mice and only 4 injections in dogs. A collaborative network involving neuropediatricians, neurosurgeons and veterinarians was set up with the aim to complete the ongoing preclinical studies in large animals and to design a therapeutic trial for affected children. This program is supported by the AFM, the INSERM and the Institut Pasteur.

Topic 2: Neural stem cells (S. Blanchard, D. Bohl, T. Bréjot, S. Liu, A. Nosjean, M. Hocquemiller).

The survival, multiplication, differentiation and migration of neural stem cells, as well as the development of synaptic connections, depend on environmental signals and on the genetically-determined competence of target cells to interpret these signals. Extrinsic and intrinsic factors acting on the formation and installation of motoneurons in the ventral horn of the spinal cord have been identified. They include soluble factors like retinoic acid (RA) or Sonic Hedgehog (Shh), which concentration determines the expression of various sets of homeotic or bHLH transcription factors.

A goal of this program is to examine the fate of neural stem cells isolated from fetal tissues and amplified as neurospheres following exposition of these cells to soluble factors or to lentiviral vectors able to induce the forced expression of transcription factors involved in the motor neuron differentiation pathway (Pax6, Nkx6.1, Olig2, Ngn2, HB9, Islet1). The expression of multiple markers associated to motor neuron differentiation was studied by real-time quantitative PCR and immunostaining. Forced expression of the bHLH transcription factor Ngn2 was the only situation in which neural stem cell cell fate was modified in vitro, leading to massive differentiation to cell expressing neuronal markers, whereas astrocyte and oligodendrocyte generation was inhibited. Further characterisation of these cells and of their fate in vitro and upon in vivo transplantation in the adult spinal cord or hippocampus are currently investigated.

Similar studies are ongoing with mouse embryonic stem (ES) cells. Selection of cells expressing the transcription factor Sox1 after the generation of embryoid bodies allows to isolate a subpopulation essentially forming neuronal cells upon differentiation. These cells differentiate into motor neurons in vitro when exposed to RA and Shh. There are now transplanted into the facial motor nucleus in mice with the aim to assess their capacity to form motor neurons and motor axons in vivo.

Topic 3 : studies on the role of the Barhl2 gene during development, cancerogenesis and neurodegenerative processes (B. Durand, N. Duval, N. Offner).

Early programmed cell death (EPCD) affects proliferating and newly differentiated cells in vertebrates, and most dramatically cells of the central nervous system (CNS). We characterized a new function for a highly conserved homeodomain protein Barhl2 in Xenopus neurodevelopment. Barhl2 promotes apoptosis in Xenopus neuroectoderm and mesoderm, acting as a transcriptional repressor. The pro-apoptotic activity of Barhl2 is essential during normal neural plate formation. It limits the size of the midline organising centre and plays an important role in the establishment of the pro-encephalic area. Analysis in Xenopus embryos and in cell culture demonstrated that Barhl2-induced apoptosis involves caspase activation and can be inhibited by Bcl2.

We study barhl2 function(s) and the regulation of barhl2 expression and activity in the early neural plate and neural tube. We showed that barhl2 controls the commitment of neuroectodermal cells towards apoptosis. Preliminary data support the hypothesis that Barhl2 participates to signalling pathways downstream of the Patch and Smothen receptors that mediate Shh activity. Alteration of the Shh signalling pathway is frequent in cancers, including meduloblastoma. It may account for abnormal cell proliferation. The association of abnormal response to both mitogenic and apoptotic stimuli is common in malignant processes. We are currently investigating the implication of Barhl2 in the Shh pathway and we examine a possible link between Barhl2 loos-of-function and carcinogenesis. The hypothesis according to which Barhl2 gain-of-function would be associated to neurodegenerative processes is also considered.

This work will provide crucial information on the extent, regulation and function of apoptosis, an underestimated aspect of brain morphogenesis. Learning whether and how extra-cellular signals control apoptosis during embryogenesis is a major issue towards understanding deregulation of apoptosis associated to cancer and neurodegeneration.

Photo : Overexpression of barhl2 induces apoptosis in neural cell plate cells. TUNEL staining was performed at stage 15 on Xenopus embryo injected in one dorsal blastomere at the 4-cell stage with Barhl2 encoding RNA. An increase number of TUNEL-positive cells is visible on the injected side.

Keywords: gene therapy, neurodegeneration, lysosomal storage diseases, neural stem cells, viral vectors, development of the nervous system, sonin hedgehog, Barh, homeotic genes



  publications

puce Publications 2004 of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
  Chantal Brulet, rtg-sec@pasteur.fr Delphine Bohl CR1 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

Nathalie Duval CR IP natoche@pasteur.fr

Arnaud Cressant Stage post-doctoral cressant@pasteur.fr

Nicolas Offner Stage post-doctoral offner@pasteur.fr

Sandrine Vitry Stage post-doctoral svitry@pasteur.fr

Thomas Bréjot Thèsard Paris VII tbrejot@pasteur.fr

Fengtian Chen Thèsard Paris VII ftchen@pasteur.fr

Michael Hocquemiller Etudiant M2 Paris VII michoc@pasteur.fr

Nathalie Desmaris Technicienne Supérieure 2D nchodan@pasteur.fr

Stéphane Blanchard Technicien Supérieur 2D sblancha@pasteur.fr


Activity Reports 2004 - Institut Pasteur
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