👤 Ashley Warnock

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. Show less

Defects of the CLN3 gene on chromosome 16p12.1 lead to the juvenile form of neuronal ceroid-lipofuscinosis (JNCL, Batten Disease), the most common recessive inherited neurodegenerative disorder in children. Dysregulation of intracellular calcium homeostasis in the absence of a functional CLN3 protein (CLN3P, Battenin) has been linked to synaptic dysfunction and accelerated apoptosis in vulnerable neuronal cells. Prolonged increase of intracellular calcium concentration is considered to be a significant trigger for neuronal apoptosis and cellular loss in JNCL. We examined the potential effect of 41 different calcium channel modulators on intracellular calcium concentration in CLN3 siRNA knock down SH-SY5Y neuroblastoma cells. Six drugs belonging to the group of voltage dependent L-type channel blockers show significant lowering of the increased intracellular calcium levels in CLN3 siRNA knock down cells. Our studies provide important new data suggesting possible beneficial effects of the tested drugs on calcium flux regulated pathways in neuronal cell death. Therapeutic intervention in this untreatable disease will likely require drugs that cross the blood-brain barrier as did all of the positively screened drugs in this study. Better comprehension of the mechanism of neurodegeneration in rare recessive disorders, such as neuronal ceroid-lipofuscinoses, is likely to help to better understand mechanisms involved in more complex genetic neurodegenerative conditions, such as those associated with aging. Show less