CLN3 disease (Spielmeyer-Vogt-Sjogren-Batten disease) is a severe pediatric neurodegenerative disorder for which there is currently no effective treatment. The disease is characterized by progressive neuronal death, which may be triggered by abnormal intracellular calcium levels leading to neuronal apoptosis. Previously, we demonstrated reversal of the calcium effect in a neuroblastoma cell line using amlodipine and other calcium channel antagonists. In the present studies, we developed a CLN3 siRNA-inhibited primary rat neuron model to further study etoposide-induced calcium changes and apoptosis in CLN3 disease followed by recovery experiments with amlodipine. Our results show that intracellular calcium is significantly elevated in siRNA-inhibited cortical neurons after potassium chloride-induced depolarization. We were also able to show that amlodipine, a predominantly L-type dihydropyrimidine calcium channel antagonist can reverse the aberrant calcium elevations in this model of the disease. We performed an in situ TUNEL assay following etoposide-exposure to siRNA inhibited primary neurons, and apoptotic nuclei were detected providing additional evidence that increased neuronal apoptosis is associated with increased calcium levels. Amlodipine also reduced the absolute number of apoptotic cells in this experimental model.
Keywords: Apoptosis; Batten disease; CLN; Calcium signaling; GFP; L-type calcium channel antagonists; NCL; Primary neurons; ceroid-lipofuscinosis neuronal (gene symbol); green fluorescent protein; neuronal ceroid-lipofuscinoses; siRNA; small inhibiting RNA.
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