Towards gene therapy in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a paralytic neurodegenerative disorder, characterised by a specific loss of motoneurons. Although the exact pathogenesis is largely enigmatic, it is known that glutamate excitotoxicity plays an important role in motoneuron cell death. Glutamate is one of the major neurotransmitters in the central nervous system. Relative high levels of glutamate can lead to motoneuron death by overstimulation of calcium-permeable receptors, resulting in an excess influx of calcium. This can lead to activation of several death-inducing pathways. Therefore, it is of great significance to keep the extracellular glutamate levels at a non-toxic value. Under physiological conditions the glutamate transporter EAAT2, predominantly expressed by astrocytes, accomplishes the majority of the glutamate uptake from the synaptic cleft. In ALS patients and in ALS animal models, however, it is found that EAAT2 protein and functioning is decreased. The aim of the studies described in this thesis was to protect motoneurons from excitotoxicity by overexpressing the glutamate transporter EAAT2 in a localised, long-term manner via gene therapy, both ex vivo and in vivo gene therapy are exploited for this purpose. In the first part of this thesis we examined the possibilities for ex vivo gene therapy. To this end, HEK-cells were genetically engineered to overexpress EAAT2. This resulted in an increase in EAAT2 protein as well as a profuse increase in glutamate uptake compared to wild-type cells. Furthermore, the engineered cells protected primary motoneurons in culture from an glutamate-induced toxicity. Hence, it might be worthwhile to investigate whether ex vivo gene therapy for EAAT2 might be of therapeutic value in amyotrophic lateral sclerosis. In the second part of this thesis we further examined the possibilities for in vivo gene therapy with the use of lentiviral vectors (LVV) to overexpress EAAT2. For this purpose we first examined the cellular pattern of LVV-mediated gene transduction in mouse spinal cord both in vitro and in vivo. Therefore, organotypic mouse spinal cord cultures were treated with LVV-green fluorescent protein (GFP) in vitro and wild-type mice were intraspinally injected with LVV-GFP at the level of vertebra L1. In both situations GFP expression was found to be expressed predominately by astrocytes, while the neuronal cells were virtually not transduced. Next, we examined whether LVV-EAAT2 could be used as a tool to deliver EAAT2 to primary adult human astrocytes from either cortical or spinal post-mortem brain tissue. Concomitant with a marked increase in EAAT2 mRNA and EAAT2 protein, a significant increase in functional glutamate uptake was observed in LVV-EAAT2 treated astrocytic cultures compared to LVV-GFP treated controls. Finally, we examined the neuroprotective efficacy of intraspinally delivered LVV-EAAT2 on the course of the disease in G93A-hSOD1 ALS mice by using motor performance and body weight as clinical parameters. We found that EAAT2 was effectively transduced in the spinal cord, but that disease onset and survival in the LVV-EAAT2 treated mice did not significantly differ from either LVV-GFP treated or naive control mice. We concluded that, although LVV-EAAT2 did not effect the clinical outcome of ALS mice, as such LVV can be successfully used to deliver therapeutic genes into spinal astrocytes.