Abnormally increased surface expression of AMPA receptors in the cerebellum, cortex and striatum of Cln3 mice

Abstract

Mutations in the CLN3 gene cause a fatal neurodegenerative disorder, juvenile CLN3 disease. Exploring the cause of the motor coordination deficit in the Cln3−/− mouse model of the disease we have previously found that attenuation of AMPA receptor activity in 1-month-old Cln3−/− mice significantly improves their motor coordination [20]. To elucidate the mechanism of the abnormally increased AMPA receptor function in Cln3−/− mice, we examined the surface expression of AMPA receptors using surface cross-linking in brain slices from 1-month-old wild type (WT) and Cln3−/− mice. In surface cross-linked brain samples, Western blotting for AMPA receptor subunits revealed significantly increased surface levels of GluA1 and GluA2 in the cerebellum, and of GluA2 in the cortex and striatum of Cln3−/− mice as compared to WT mice. Expression levels of the GluA4 subunit were similar in the cerebellum of WT and Cln3−/− mice. While intracellular GluA1 levels in the WT and Cln3−/− cerebellum or cortex were similar, the intracellular expression of GluA1 in the Cln3−/− striatum was decreased to 56% of the WT level. Our results show a prominent increase in AMPA receptor surface expression in the brain of Cln3−/− mice and suggest that CLN3 is involved in the regulation of AMPA receptor surface expression.

Ionotropic Glutamate Receptors in Epilepsy: A Review Focusing on AMPA and NMDA Receptors

“…Similarly, neurons from a transgenic animal model of juvenile Batten disease (also called CLN3 disease) have been shown to be susceptible to excitotoxicity [126]. Kovács et al investigated the cause of this increased glutamate sensitivity by evaluating AMPA receptor expression at the neuronal surface in the CLN3 mouse model, and reported increased surface expression of AMPA receptors in various areas of the brain [127]; furthermore, the behavioral phenotype of the CLN3 model was ameliorated by the AMPA antagonist EGIS-8332 [128]. Additionally, in an electrophysiological study of brain slices from a mouse model of Niemann-Pick type C disease, D'Arcangelo et al demonstrated impairment of AMPA-induced receptor internalization [129], and, correspondingly, another study reported increased surface expression of GluA2-containing AMPA receptors in neurons derived from Niemann-Pick type C1 patient-specific induced pluripotent stem cells [130].…”

Lack of specificity of antibodies raised against CLN3, the lysosomal/endosomal transmembrane protein mutated in juvenile Batten disease

“…First, we tested a rabbit polyclonal antibody raised against the full-length human CLN3 (Abnova, recommended dilution: 1:500–1:1000; dilution used in the present study: 1:500), a mouse monoclonal antibody raised against the full-length human CLN3 with a glutathione S-transferase (GST) tag (Abnova, recommended dilution: 1:200–1:1000; dilution used in the present study: 1:500), and a rabbit polyclonal antibody produced against a synthetic peptide derived from within residues 400–438 of human CLN3 (Abcam; applied in the recommended dilution of 1:700) using protein extracts of surface cross-linked cerebellum and cortex tissue samples from 1-month-old WT and Cln3 −/− male mice. These protein extracts were prepared by sonication in a lysis buffer containing 500 mM NaCl and 0.1% NP-40 substitute, and we have used aliquots of these samples in immunoblot experiments to successfully measure the intracellular and surface expression of AMPA and NMDA receptor subunits [ 43 ]. To optimize the solubilization of hydrophobic membrane proteins (such as CLN3) for gel electrophoresis, protein extracts (60 µg) were incubated with SDS/PAGE reducing sample buffer at various temperatures (37°C for 30 min, 65°C for 15 min, and 100°C for 10 min), and urea-containing sample buffer (4 M, 65°C for 15 min) was also tested.…”

“…Protein extracts were prepared as we previously described [ 42 , 43 ]. Briefly, the surface cross-linked tissue slices in 1.5-ml microtubes were homogenized by sonication in ice-cold lysis buffer [25 mM HEPES (pH 7.4), 500 mM NaCl, 2 mM EDTA, 20 mM NaF, 1 mM sodium orthovanadate, 0.1% NP-40 substitute, 1 mM DTT, protease inhibitor cocktail, and phosphatase inhibitor cocktail (Sigma)].…”

An Autistic Female with Refractory Juvenile Myoclonic Epilepsy and Sequelae of Neonatal Cerebellar Insults, Effectively Treated with Perampanel

“…In neonates, the cerebellum is still developing and perinatal insults to the cerebellum may result in dysfunction of the tissue [14] and induce the overexpression of AMPA receptors on cerebellar neurons, which cause cerebellar seizures and myoclonic seizures, as are observed in Batten disease [15]. PER acts on the AMPA receptors of cerebellar neuronal cell membranes to decrease the excitable transmission and reduce the seizure activities [16][17].…”