Menas Beshara

Introduction. 1 in every 100,000 babies are born with a subset of Batten disease, a collective term used to describe a family of 13 inherited neurodegenerative lysosomal storage disorders. Batten Disease Type 3, also referred to as CLN3, is caused by a mutation in the CLN3 gene which codes for the CLN3 protein. The neurogenerative symptoms have recently been attributed to malfunctioning astrocytes which ultimately impact the health of neurons, rather than neurons themselves being the origin of pathogenesis. Impaired Ca2+ signaling in malfunctioned astrocytes leads to increased presynaptic glutamate concentrations which results in glutamate excitotoxicity and an impaired blood brain barrier via swelling of astrocyte end feet. Methods. Astrocytes and neurons were collected from wildtype and CLN3 -/- mice then loaded with a Ca2+ indicator dye. Ca2+ oscillations were measured at rest and after glutamate exposure. Astrocytes obtained from the cerebral cortices of rats were dissociated to individual cells and exposed to different concentrations of glutamate and DHPG, a glutamate agonist, for 48 hours. Then, immunofluorescent staining for AQP4 was carried by incubating the cells with polyclonal antibody against AQP4. Astrocyte end feet size were quantified using CLN3 -/-, +/-, and +/+ mice, and a hoechst dye was used to assess BBB function. Ibuprofen and lamotrigine were administered daily to symptomatic cln3 deficient mice for a 3-month period. The treatment began when the mice reached 6 months of age to mimic the juvenile onset observed in CLN3 batten disease. The Rotarod and Vertical pole tests were utilized to analyze motor skill, balance, motor coordination, and vertical orientation. Results. Decreased Ca2+ oscillations were observed in cln3 -/- astrocytes. Decreased cytosolic Ca2+ concentrations post-glutamate exposure was recorded in cln3 -/- astrocytes. DHPG induced astrocyte swelling and AQP4 to the same extent as 1 mM glutamate. Fenobam, a glutamate antagonist, reversed DHPG-induced cell swelling. In WT cells, hypotonic-induced Hoechst penetration was evident. In cln3 -/- mice, minimal penetration was detected. Ibuprofen and lamotrigine-treated cln3 -/- mice performed significantly better than untreated cln3 -/- mice on both tests. Conclusion. The neurodegeneration of CLN3 Juvenille Batten disease is largely associated with astrocyte Ca+ signaling malfunction resulting in an impaired blood brain barrier and excitotoxity. The ability of Ibuprofen, an anti-inflammatory medication, to improve symptoms specifically supports the astrocytic-induced blood brain barrier dysfunction mechanism of neuronal cell death. Correspondingly, lamotrigine is an anti-epileptic medication which inhibits presynaptic glutamate release. It’s ability in improving symptoms supports the mechanism correlating increased presynaptic glutamate concentrations to glutamate excitotoxicity.

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