Jacques Mallet

PREVENTION OF MOTONEURON DEATH BY ADENOVIRUS-MEDIATED NEUROTROPHIC FACTORS

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of motoneurons, and has no effective treatment. Experimental studies in rodents have shown that motoneurons respond to a variety of molecules including brain‐derived neurotrophic factor (BDNF), and the glial‐cell line‐derived neurotrophic factor (GDNF). Here we investigated the neuroprotective effect of these growth factors, encoded by an adenovirus, on the death of axotomized facial motoneurons in newborn rats. We used a new gene therapy strategy that involves gene transfer to motoneurons by intramuscular injection of an adenoviral vector, which is retrogradely transported from injected target muscle (Finiels et al.,: NeuroReport 7:373–378, 1995). A significant increased survival of motoneurons was observed in animals pretreated with adenovirus encoding BDNF (34.5%, P < 0.05) or GDNF (41.9%, P < 0.05) 1 week after axotomy.

Induction of Neuronal Apoptosis by Excitotoxins Associated with Long-Lasting Increase of 12-O-Tetradecanoylphorbol 13-Acetate-Responsive Element-Binding Activity

Abstract: We show that excitotoxic cell death, which is associated with pathological neurodegenerative processes, can display morphological and biochemical features characteristic of apoptosis, a mode of cell death typical of physiological neuronal elimination during development. Cortical neurons cultured in the absence of serum, stimulated with NMDA, glutamate, or quisqualate after 3–5 days in vitro, showed significant degeneration. This death was blocked by 1 µM MK‐801, indicating that it was mediated by the activation of NMDA receptors. Dying cells displayed an apoptotic morphology, characterized by cytoplasm and chromatin condensation. No internucleosomal DNA degradation was observed, confirming that morphological changes of apoptosis can be dissociated from DNA laddering. Inhibitors of protein or RNA synthesis abolished cell death, and the protective effect of cycloheximide was similar when the drug was applied 2 h before or 8 h after glutamate. These experiments suggest the participation of active gene transcription in the mechanism of death. We thus analyzed the modulation of transcription factors in dying cells using electrophoretic mobility shift assays. The level of factors binding to the 12‐O‐tetradecanoylphorbol 13‐acetate‐responsive element (TRE) displayed a late and sustained increase preceding neuronal death, which was not found for factors complexing the Sp1 P, Oct, and USF binding sites. These results raise the possibility that apoptosis is one of the mechanisms of death in the pathologies linked to excitotoxicity and that activation of TRE‐binding factors could play a role in these processes.

Adenovirus-mediated suicide gene therapy in an in vitro model of reactive gliosis

Adenovirus‐mediated herpes simplex thymidine kinase/ganciclovir (HSV‐tk/GCV) system has been demonstrated to be efficient for the treatment of experimental brain tumors. However, no study has been directed to the elimination of proliferating cellular populations in other pathological conditions. In this study we used this suicide gene approach in a primary culture of astrocytes, as a model of reactive gliosis, in order to evaluate its efficiency as a therapeutic strategy for post‐traumatic astrogliosis in vivo. First, we evaluated the peak of astrocytic proliferation to characterize our model. Second, the efficiency of adenovirus‐mediated lacZ gene transfer is shown to be dependent on vector multiplicity of infection (MOI). As expected, the cells transfected with the HSV‐tk gene showed an increase in sensibility to GCV compared with cells transfected with lacZ gene. Finally, an unexpected interaction between the adenoviral vector and bromodeoxyuridine (BrdU) or [3H]‐Thymidine ([3H]‐Thy) was evidenced in transfected cultures, whose interpretation is discussed. The present study demonstrates that a recombinant adenoviral vector carrying the tk gene confers to in vitro cultured astrocytes a cytotoxic sensibility to GCV, and that this system constitutes a potentially efficient tool to eliminate the hyperplasia of astrocytes following injury to the central nervous system in vivo. GLIA 25:293–303, 1999.