Unit: Retroviruses and Gene Transfer - INSERM U622

Director: Jean Michel Heard

Topics 1: Neurodegeneration associated to lysosomal storage diseases. Pathophysiology and therapeutic approaches are investigated. Topics 2: Neural stem cells. Forced expression of transcription factors is used to reprogramming stem cell fate. Topics 3: Study of the mechanisms by which the homeogene barhl2 participates to the interpretation of the cell position information in the developing neural plate.

Topics 1: Neurodegeneration associated to lysosomal storage diseases.

Lysosomal storage diseases are inborn error of the metabolism with frequent and often devestating neurological manifestations in young children. Genetics defects have been well characterised, as well as their biochemical consequences on the catabolic functions of lysosomes. However, little is know about the links between biochemical disorders, neuronal dysfunctions and clinical manifestations, for which there is currently no treatment. We study two models of mucopolysacchridoses in which the accumulating catabolites are partially degraded and hypersulfated heparan sulfates. We attempt to elucidate how these accumulating compounds induce microglia activation and neurodegeneration. Studies are performed in cultured cells and mice, including mouse strains genetically invalidated for one or several relevant functions. We examine 1) how interaction between oligosaccharides derived from heparan sulfates triggers cell activation by interacting with receptors, including molecules involved in the innate immune response; 2) we analyse activated signalling pathways in various cell types: 3) we examine consequences on the control of intracellular trafficking, axonal transport and myelination.

Extracellular supply of the missing enzyme to deficient cells in the brain represents a realistic treatment for the neurological manifestations of mucoplysaccharidoses provided that the enzyme can be delivered to the central nervous system. We documented the efficacy and the safety of therapeutic approaches based on the stereotactic injection of adeno-associated vectors coding for the missing enzyme in the brain of deficient mice or deficient dogs. Therapeutic trials are being prepared for investigation in children affected by Sanfilippo syndrome or Hurler syndrome.

Topics 2: Neural stem cells.

Neural stem cells survival, proliferation, migration, differentiation and maturation are controlled by a combination of external environmental signals and the genetic competency of target cells to respond to them. Determinants of motor neuron differentiation have been identified in the developing embryo, including the soluble factors retinoic acid (RA) and Sonic Hedgehog (Shh), whose concentration determines whether homeodomain or bHLH transcription factors specifically inducing motor neuron differentiation will be expressed.

We study the fate of neural stem cells from fetal or adult origin grown as neurospheres in which expression of transcription factors of the motor neuron pathway (Pax6, Nkx6.1, Olig2, Ngn2, HB9) has been induced by lentivirus-mediated gene transfer. Expression of multiple markers associated to motor neuron differentiation are studied by quantitative PCR and immunolabeling. We saw that the forced expression of HB9 alone affected neuronal differentiation but was not sufficient to induce motor neuron generation in vitro or after stem cell transplantation in the rat spinal cord. The combined expression of Ngn2 and HB9 induced the expression of other transcription factors important for motor neuron fate in target cells and conferred the capacity to produce motor neurons in response to RA and Shh. Simultaneous expression of Nkx6.1 amplified this phenomenon.

We also studied the fate of fetal neural stem cells following transplantation in the facial motor nucleus in the mouse with the aim to observed axonal growth. For that purpose, a predegenerated peripheral nerve is inserted directly in the motor nucleus. We showed that transplantation in this environment affects stem cell differentiation. After 2 months, the number of transplanted cells expressing cholinergic markers was higher at this location than when cells were transplanted in the striatum, and higher with than without inserted peripheral nerve graft. These cells grew cholinergic prolongations in the nerve graft.

Topics 3: barhl2 controlled apoptosis in forebrain development

Apoptosis, a process that rapidly removes superfluous and potentially dangerous cells, is implemented by an evolutionarily conserved molecular programme. Targeted disruption of effector molecules of the apoptotic pathway has demonstrated the occurrence and magnitude of early programmed cell death (EPCD), which affects proliferating cells, as is most dramatically observed in the central nervous system (CNS). Although the effector mechanisms controlling apoptotic processes appear to be conserved, the genetic controls for EPCD induction have not been elucidated. Survival of vertebrate cells is under the control of secreted factors, in particular Sonic Hedgehog (Shh). We have established that Barhl2, a highly conserved homeodomain protein, promotes apoptosis during Xenopus neurodevelopment by acting as a transcriptional repressor. Our findings show that the barhl2 gene is part of a pathway regulating cell survival and that it could be part of the Shh pathway. Mutations in the Shh pathway have been described in 25% of human sporadic medulloblastomas, tumours that arise in the cerebellum by the erratic proliferation of granular precursor cells. Barhl2 and barhl1, its closest vertebrate homologue, are co-expressed in these cells, and specific destruction of the barhl1 locus demonstrates its role in the maintenance of cerebellar neurons. Our projects are focused on 1) the analysis of genetic interactions between barhl2 and gli3, an effector of the Shh pathway, through a microarray approach; 2) the identification of molecular connections between these two pathways and the apoptotic machinery during early neural plate development in Xenopus and in granular precursor cells of the cerebellum; and 3) deciphering barhl2 function in diencephalic patterning.

Our work will provide important information on the signaling networks that control the survival of neural precursor cells, a process that plays an important and unexplained role during CNS morphogenesis and moreover, one that is deregulated in cancer cells.

Photos : In vitro generation of motor neurons from neural stem cells treated with a combination of three lentiviral vector coding for HB9, Ngn2 and Nkx6.1, respectively. Cholinesterasic activity is revealed by Karnovsky's staining.

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

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