Juvenile Neuronal Ceroid Lipofuscinosis (JNCL) is the most common pediatric neurodegenerative disease caused by a mutation in the CLN3 gene. Children with JNCL experience progressive visual, cognitive, and motor deterioration with a decreased life expectancy (late teens-early 20s). There is currently no cure for JNCL, making research to identify novel drug targets crucial to extend the survival and quality of life of afflicted patients. Previous studies have shown that astrocyte activation precedes and predicts regions of neuronal loss in JNCL, suggesting a defect in supportive glial functions. Glutamate levels are elevated in the JNCL brain and neuronal loss is thought to occur, in part, via glutamate excitotoxicity. Currently, little is known about aberrant glutamate-glutamine cycling in astrocytes and no reports investigating neuron-astrocyte cross-talk or regulation in JNCL exist. Our prior studies revealed a significant decrease in glutamine synthetase coupled with altered expression of the glutamate transporters in CLN3?ex7/8 astrocytes both in vitro and in vivo. This proposal will utilize mice harboring a 1.02 kb mutation spanning exons 7 and 8 of CLN3 (CLN3?ex7/8) to study glutamate regulatory pathways both in vivo and in vitro. My preliminary data indicates that primary CLN3?ex7/8 astrocytes display decreased glutamate transporter expression and activity when exposed to stimuli present in the JNCL brain. By extension, this disruption in glutamate homeostasis in the context of CLN3 mutation threatens to disrupt neuron-astrocyte signaling pathways, which if chronically perturbed can induce neuron excitotoxicty. Indeed, my preliminary studies have demonstrated reduced Ca2+ signaling in CLN3?ex7/8 astrocytes concomitant with heightened Ca2+ transients in CLN3?ex7/8 neurons, suggesting that disruptions in glutamate cycling interrupt critical homeostatic cell signaling networks. Concurrent with impaired Ca2+ signaling, preliminary electrophysiology conducted on hippocampus slice cultures from 30-day-old mice show that CLN3?ex7/8 mice have higher population spike amplitude and field excitatory post-synaptic potentials. This data indicates that the CLN3 mutation causes an increase in neuron activation that would potentate elevated glutamate release. Collectively, these findings formulated the hypothesis that the CLN3 mutation perturbs glutamate cycling pathways, which potentially creates a toxic cellular milieu, culminating in neuron death.
The Specific Aims of this proposal are: 1) To identify the mechanisms responsible for astrocyte glutamate dysregulation and altered astrocyte-neuronal signaling networks in the context of CLN3 mutation; and 2) To evaluate whether the S100B synthesis inhibitor ONO-2506 can limit neurotoxicity in CLN3?ex7/8 mice by enhancing glutamate transporter expression and glutamate uptake. Establishing key regulatory mechanisms of glutamate and astrocyte-neuron cross-talk in JNCL may unveil novel therapeutic targets to extend the quality-of-life for children suffering from this devastating neurodegenerative disease.
Juvenile Neuronal Ceroid Lipofuscinosis is a lysosomal storage disease caused by a mutation in CLN3 that leads to progressive visual, cognitive, and motor deterioration with a decreased life expectancy (late teens- early 20s). This proposal will investigate disruptions in key metabolic pathways responsible for glutamate regulation in astrocytes and neurons, which are implicated in excessive glutamate levels and neuron excitotoxicity in JNCL. The S100B inhibitor ONO-2506 will be examined for its ability to restore critical glutamate homeostatic pathways in CLN3 mutant mice with the long-term goal of exploring its utility as a potential therapeutic to extend the quality of life for children suffering from JNCL.
