AAV‐mediated expression of wild‐type and ALS‐linked mutant VAPB selectively triggers death of motoneurons through a Ca²⁺‐dependent ER‐associated pathway

Abstract

J. Neurochem. (2010) 114, 795–809. Abstract A dominant mutation in the gene coding for the vesicle‐associated membrane protein‐associated protein B (VAPB) was associated with amyotrophic lateral sclerosis, a fatal paralytic disorder characterized by the selective loss of motoneurons in the brain and spinal cord. Adeno‐associated viral vectors that we show to transduce up to 90% of motoneurons in vitro were used to model VAPB‐associated neurodegenerative process. We observed that Adeno‐associated viral‐mediated over‐expression of both wild‐type and mutated form of human VAPB selectively induces death of primary motoneurons, albeit with different kinetics. We provide evidence that ER stress and impaired homeostatic regulation of calcium (Ca2+) are implicated in the death process. Finally, we found that completion of the motoneuron death program triggered by the over‐expression of wild‐type and mutant VAPB implicates calpains, caspase 12 and 3. Our viral‐based in vitro model, which recapitulates the selective vulnerability of motoneurons to the presence of mutant VAPB and also to VAPB gene dosage effect, identifies aberrant Ca2+ signals and ER‐derived death pathways as important events in the motoneuron degenerative process.

Endoplasmic Reticulum Ca2+Handling in Excitable Cells in Health and Disease

“…A dominantly inherited mutation in the vesicle-associated membrane proteinassociated protein B (VAPB) is responsible for some cases of ALS. Expression of mutant VAPB in motor neurons results in ER stress and dysregulation of ER and cellular Ca 2ϩ homeostasis, and this abnormal Ca 2ϩ handling plays a pivotal role in the death of motor neurons caused by the mutant VAPB (Langou et al, 2010). Collectively, the available data suggest a role for excessive elevation of intracellular Ca 2ϩ levels in the degeneration of neurons in ALS, although the contribution of specific alterations in ER Ca 2ϩ handling systems in this disease is unknown.…”

IFNγ triggers a LIGHT-dependent selective death of motoneurons contributing to the non-cell-autonomous effects of mutant SOD1

“…Analysis of nuclear mean fluorescence intensity was done on 100-200 motoneurons per culture condition. 22 Western blot. Neurons were plated at the density of 20 000 cells/cm 2 in 6-cm diameter dishes containing corresponding complemented neurobasal medium (see above).…”

“…14 We first determined the phosphorylation levels of nuclear p38 by quantitative confocal miscroscopy. 15,22 Treatment of motoneurons with sLIGHT, as well as sFasL, resulted in a significant increase in levels of p38 phosphorylation (Figure 2b and Supplementary Figure 2a). SB203580, a pyridinyl imidazole inhibitor of p38, prevented phosphorylation of p38, consistent with its inhibitory effect on the autophosphorylation-dependent activation of p38.…”

Neuron-Microglia Interactions in Motor Neuron Degeneration. The Inflammatory Hypothesis in Amyotrophic Lateral Sclerosis Revisited

“…Different VAPB mutations combined with other ALS-relevant mutations, like those in C9orf72 [57,58] [59,60].…”