Jean-Charles Bensadoun

Lentiviral-mediated silencing of SOD1 through RNA interference retards disease onset and progression in a mouse model of ALS

Mutations in Cu/Zn superoxide dismutase (encoded by SOD1), one of the causes of familial amyotrophic lateral sclerosis (ALS), lead to progressive death of motoneurons through a gain-of-function mechanism. RNA interference (RNAi) mediated by viral vectors allows for long-term reduction in gene expression and represents an attractive therapeutic approach for genetic diseases characterized by acquired toxic properties. We report that in SOD1G93A transgenic mice, a model for familial ALS, intraspinal injection of a lentiviral vector that produces RNAi-mediated silencing of SOD1 substantially retards both the onset and the progression rate of the disease.

Self-Inactivating Lentiviral Vectors with Enhanced Transgene Expression as Potential Gene Transfer System in Parkinson's Disease

Glial cell line-derived neurotrophic factor (GDNF) is able to protect dopaminergic neurons against various insults and constitutes therefore a promising candidate for the treatment of Parkinsons disease. Lentiviral vectors that infect quiescent neuronal cells may allow the localized delivery of GDNF, thus avoiding potential side effects related to the activation of other brain structures. To test this hypothesis in a setting ensuring both maximal biosafety and optimal transgene expression, a self-inactivating (SIN) lentiviral vector was modified by insertion of the posttranscriptional regulatory element of the woodchuck hepatitis virus, and particles were produced with a multiply attenuated packaging system. After a single injection of 2 microl of a lacZ-expressing vector (SIN-W-LacZ) in the substantia nigra of adult rats, an average of 40.1 +/- 6.0% of the tyrosine hydroxylase (TH)-positive neurons were transduced as compared with 5.0 +/- 2.1% with the first-generation lentiviral vector. Moreover, the SIN-W vector expressing GDNF under the control of the mouse phosphoglycerate kinase 1 (PGK) promoter was able to protect nigral dopaminergic neurons after medial forebrain bundle axotomy. Expression of hGDNF in the nanogram range was detected in extracts of mesencephalon of animals injected with an SIN-W-PGK-GDNF vector, whereas it was undetectable in animals injected with a control vector. Lentiviral vectors with enhanced expression and safety features further establish the potential use of these vectors for the local delivery of bioactive molecules into defined structures of the central nervous system.

Lentiviral vectors as a gene delivery system in the mouse midbrain : Cellular and behavioral improvements in a 6-OHDA model of Parkinson's disease using GDNF

Local delivery of therapeutic molecules represents one of the limiting factors for the treatment of neurodegenerative disorders. In vivo gene transfer using viral vectors constitutes a powerful strategy to overcome this limitation. The aim of the present study was to validate the lentiviral vector as a gene delivery system in the mouse midbrain in the perspective of screening biotherapeutic molecules in mouse models of Parkinsons disease. A preliminary study with a LacZ-encoding vector injected above the substantia nigra of C57BL/6j mice indicated that lentiviral vectors can infect approximately 40,000 cells and diffuse over long distances. Based on these results, glial cell line-derived neurotrophic factor (GDNF) was assessed as a neuroprotective molecule in a 6-hydroxydopamine model of Parkinsons disease. Lentiviral vectors carrying the cDNA for GDNF or mutated GDNF were unilaterally injected above the substantia nigra of C57BL/6j mice. Two weeks later, the animals were lesioned ipsilaterally with 6-hydroxydopamine into the striatum. Apomorphine-induced rotation was significantly decreased in the GDNF-injected group compared to control animals. Moreover, GDNF efficiently protected 69.5% of the tyrosine hydroxylase-positive cells in the substantia nigra against 6-hydroxydopamine-induced toxicity compared to 33.1% with control mutated GDNF. These data indicate that lentiviral vectors constitute a powerful gene delivery system for the screening of therapeutic molecules in mouse models of Parkinsons disease.

KAP1-Mediated Epigenetic Repression in the Forebrain Modulates Behavioral Vulnerability to Stress

KAP1 is an essential cofactor of KRAB-zinc finger proteins, a family of vertebrate-specific epigenetic repressors of largely unknown functions encoded in the hundreds by the mouse and human genomes. Here, we report that KAP1 is expressed at high levels and necessary for KRAB-mediated repression in mature neurons of the mouse brain. Mice deleted for KAP1 in the adult forebrain exhibit heightened levels of anxiety-like and exploratory activity and stress-induced alterations in spatial learning and memory. In the hippocampus, a small number of genes are dysregulated, including some imprinted genes. Chromatin analyses of the promoters of two genes markedly upregulated in knockout mice reveal decreased histone 3 K9-trimethylation and increased histone 3 and histone 4 acetylation. We propose a model in which the tethering of KAP1-associated chromatin remodeling factors via KRAB-ZFPs epigenetically controls gene expression in the hippocampus, thereby conditioning responses to behavioral stress.

Attenuation of 6-OHDA-induced neurotoxicity in glutathione peroxidase transgenic mice

Normal cellular metabolism produces oxidants which are neutralized within cells by antioxidant enzymes and other antioxidants. An imbalance between oxidants and antioxidants has been postulated to lead to the degeneration of specific populations of neurons in neurodegenerative diseases, e.g. Parkinsons disease. The present study investigates whether overexpression of glutathione peroxidase, the enzyme which metabolizes hydrogen peroxide to water, can prevent or slow down neuronal injury in an animal model of Parkinsons disease. Transgenic mice overexpressing the human glutathione peroxidase gene under the control of the mouse hydroxymethylglutaryl‐coenzyme A promoter and genetically matched control mice were injected intracerebroventricularly with the dopaminergic neurotoxin 6‐hydroxydopamine. Seven days after injection, the number of tyrosine hydroxylase‐positive nigral dopaminergic neurons was decreased by 52.4% and 20.5% in 6‐hydroxydopamine‐injected control and glutathione peroxidase transgenic mice, respectively. Similarly, 3 days after injection of the neurotoxin, striatal dopamine was decreased by 71.2% and 56.5%, respectively. Overexpression of glutathione peroxidase therefore partially protects dopaminergic neurons against 6‐hydroxydopamine‐induced toxicity.

In Vivo Electrical Impedance Spectroscopy of Tissue Reaction to Microelectrode Arrays

The goal of this experiment was to determine the electrical properties of the tissue reaction to implanted microelectrode arrays. We describe a new method of analyzing electrical impedance spectroscopy data to determine the complex impedance of the tissue reaction as a function of postimplantation time. A model is used to extract electrical model parameters of the electrode-tissue interface, and is used to isolate the impedance of the tissue immediately surrounding the microelectrode. The microelectrode arrays consist of microfabricated polyimide probes, incorporating four 50-mum-diameter platinum microelectrodes. The devices were implanted in the primary motor cortex of adult rats, and measurements were performed for 12 weeks. Histology was performed on implants at three time points in one month. Results demonstrate that the tissue reaction causes a rapid increase in bioimpedance over the first 20 days, and then stabilizes. This result is supported by histological data.

Transient striatal delivery of GDNF via encapsulated cells leads to sustained behavioral improvement in a bilateral model of Parkinson disease

Numerous studies have shown the neuroprotective and regenerative benefits of glial cell line-derived neurotrophic factor (GDNF) in animal models of PD. Brain delivery of GDNF can, however, be associated with limiting side-effects in both primates and PD patients, rendering the duration of delivery a critical factor. In the present study, the effects of transient vs. sustained GDNF delivery by encapsulated cells were evaluated in a bilateral animal model, closely mimicking advanced PD. One week following bilateral striatal 6-hydroxydopamine injections in rats, capsules loaded with human fibroblasts genetically engineered to release GDNF were bilaterally implanted in the striatum. GDNF delivery resulted in a significant improvement of movement initiation and swimming performance in the lesioned animals, associated with striatal reinnervation of dopaminergic fibers. To test the sustainability of the behavioral improvement, GDNF-secreting capsules were withdrawn in a subgroup of animals, 7 weeks post-implantation. Strikingly, both the behavioral and morphological improvements were maintained until the sacrifice of the animals 6 weeks post-GDNF withdrawal. The sustained cellular and behavioral benefits after GDNF washout suggest the need for temporary delivery of the trophic factor in PD. Retrievable encapsulated cells represent an attractive delivery tool to achieve this purpose.

Long-term lentiviral-mediated expression of ciliary neurotrophic factor in the striatum of Huntington"s disease transgenic mice

Ciliary neurotrophic factor (CNTF) has been shown to prevent behavioral deficits and striatal degeneration in neurotoxic models of Huntingtons disease (HD), but its effect in a genetic model has not been evaluated. Lentiviral vectors expressing the human CNTF or LacZ reporter gene were therefore injected in the striatum of wild-type (WT) and transgenic mice expressing full-length huntingtin with 72 CAG repeats (YAC72). Behavioral analysis showed increased locomotor activity in 5- to 6-month-old YAC72-LacZ mice compared to WT-LacZ animals. Interestingly, CNTF expression reduced the activity levels of YAC72 mice compared to control animals. In both WT and YAC72 mice, CNTF expression was demonstrated in striatal punches, up to a year after lentiviral injection. Stereological analysis revealed that the number of LacZ and DARPP-32-positive neurons were decreased in YAC72-LacZ mice compared to WT-LacZ animals. Assessment of the benefit of CNTF expression in the YAC72 mice was, however, complicated by a down-regulation of DARPP-32 and to a lesser extent of NeuN in all mice treated with CNTF. The expression of the neuronal marker NADPH-d was unaffected by CNTF, but expression of the astrocytic marker glial fibrillary acidic protein (GFAP) was increased. Finally, a reduction of the number of striatal dark cells was observed in YAC mice treated with CNTF compared to LacZ. These data indicate that sustained striatal expression of CNTF can be achieved with lentiviruses. Further studies are, however, needed to investigate the intracellular signaling pathways mediating the long-term effects of CNTF expression on dopamine signaling, glial cell activation and how these changes may affect HD pathology.

Lentivirus-mediated expression of glutathione peroxidase: neuroprotection in murine models of Parkinson's disease

Reactive oxygen species are considered to contribute to the pathogenesis of Parkinsons disease (PD). In order to study viral vector-mediated overexpression of the antioxidant enzyme glutathione peroxidase (GPX) as a potential neuroprotective approach in both an in vitro and in vivo model of PD, we have developed a lentiviral vector carrying the human GPX1 gene. Neuroblastoma cells infected with this vector showed a 2-fold increase in GPX activity compared to cells infected with a control vector. In addition, overexpression of GPX protected 83.0 +/- 14.2% of these cells against 6-hydroxydopamine (6-OHDA)-induced toxicity, while only 22.9 +/- 4.6% of the cells infected with a control vector survived. Furthermore, lentivirus-mediated expression of GPX1 in nigral dopaminergic neurons in vivo prior to intrastriatal injection of 6-OHDA led to a small, but significant protection of these cells against drug-induced toxicity. These results indicate that antioxidative gene therapy strategies may be relevant for PD.

Lentiviral vectors express chondroitinase ABC in cortical projections and promote sprouting of injured corticospinal axons

Highlights ► Lentiviral vectors can transduce neurons and glia to secrete chondroitinase. ► The active enzyme is secreted from long-distance axon projections from the cerebral cortex. ► Chondroitinase transduction promotes preservation and sprouting of damaged corticospinal axons.