Doctor of Philosophy (PhD)
Pharmacology and Experimental Neuroscience
Tammy L. Kielian
Juvenile Neuronal Ceroid Lipofuscinosis (JNCL) is a lysosomal storage disease caused by autosomal recessive mutations in CLN3. Neuronal loss is thought to occur via glutamate excitotoxicity; however, little is known about neuron-astrocyte glutamate regulation in JNCL. We discovered that Cln3Δex7/8 astrocytes have significantly lower basal spontaneous Ca2+ oscillations and decreased responses to glutamate, indicating a disrupted signaling network. Cln3Δex7/8 astrocytes also displayed significantly lower basal mitochondrial respiration and ATP production, suggesting impaired metabolic functions. Concurrent with diminished astrocyte metabolism and Ca2+ signaling, Cln3Δex7/8 neurons were hyper-responsive to glutamate stimulation. These studies suggest that CLN3 loss disrupts neuronal synaptic activity causing increased glutamate release, which astrocytes are unequipped to regulate due to reductions in Ca2+ signaling and energy metabolism. Therefore, a therapeutic capable of targeting several molecular deficits associated with JNCL would be an ideal. Cyclic AMP (cAMP) is a second messenger with pleiotropic effects, including regulating neuroinflammation, glutamate transporter expression, and neuron survival. We investigated whether three distinct phosphodiesterase-4 (PDE4) inhibitors could mitigate disease pathology in the Cln3Δex7/8 mouse model. cAMP levels were significantly reduced in the Cln3Δex7/8 brain, which were restored by PF-06266047. PDE4 inhibitors significantly improved motor function, attenuated glial activation and lysosomal pathology, and restored glutamate transporter expression with no evidence of toxicity. These studies revealed neuroprotective effects of PDE4 inhibitors in Cln3Δex7/8 mice and support their therapeutic potential. However, PDE4 inhibitors do not provide a long-term treatment for JNCL because the CLN3 mutation is still present. Therefore, we explored a gene delivery approach for JNCL utilizing two self-complementary AAV9 (scAAV9) constructs with the MeCP2 and β-actin promoters to drive low versus high transgene expression and to test efficacy in the Cln3Δex7/8 model. Only the scAAV9/MeCP2-hCLN3 construct corrected motor deficits and attenuated glia activation and lysosomal pathology, possibly resulting from preferential viral promoter usage by neurons. This is the first demonstration of a systemic delivery route to restore CLN3 in vivo using scAAV9 and highlights the importance of promoter selection for disease modification. Collectively, these projects provide novel insights into CLN3 pathology and identify two promising modes of therapeutic advancement for this fatal lysosomal storage disease.
Bosch, Megan Elizabeth, "Novel therapeutic approaches for Juvenile Neuronal Ceroid Lipofuscinosis (CLN3)" (2017). Theses & Dissertations. 227.