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Generation of a specific antibody against Ca2+/ calmodulin-dependent protein kinase (CaM-KII) which was specifically autophosphorylated at Thr-286 was presented. Peptide Y-66 antibody was synthesized and phosphorylated by CaM-KII. ELISA assay showed specific reaction of purified IgG with phosphorylated Y-66 peptide while immunoblot assay demonstrated specific reaction of the antibody to purified and cytoskeleton form of the autophosphorylated CaM-KII. Results suggested the potential use of this antibody for biological investigations of CaM-KII.

Abstract Background Proneurotrophins and mature neurotrophins elicit opposite effects via the p75 neurotrophin receptor (p75NTR) and Trk tyrosine kinase receptors, respectively; however the molecular roles of proneurotrophins in the CNS are not fully understood. Results Based on two rare single nucleotide polymorphisms (SNPs) of the human brain-derived neurotrophic factor (BDNF) gene, we generated R125M-, R127L- and R125M/R127L-BDNF, which have amino acid substitution(s) near the cleavage site between the pro- and mature-domain of BDNF. Western blot analyses demonstrated that these BDNF variants are poorly cleaved and result in the predominant secretion of proBDNF. Using these cleavage-resistant proBDNF (CR-proBDNF) variants, the molecular and cellular roles of proBDNF on the CNS neurons were examined. First, CR-proBDNF showed normal intracellular distribution and secretion in cultured hippocampal neurons, suggesting that inhibition of proBDNF cleavage does not affect intracellular transportation and secretion of BDNF. Second, we purified recombinant CR-proBDNF and tested its biological effects using cultured CNS neurons. Treatment with CR-proBDNF elicited apoptosis of cultured cerebellar granule neurons (CGNs), while treatment with mature BDNF (matBDNF) promoted cell survival. Third, we examined the effects of CR-proBDNF on neuronal morphology using more than 2-week cultures of basal forebrain cholinergic neurons (BFCNs) and hippocampal neurons. Interestingly, in marked contrast to the action of matBDNF, which increased the number of cholinergic fibers and hippocampal dendritic spines, CR-proBDNF dramatically reduced the number of cholinergic fibers and hippocampal dendritic spines, without affecting the survival of these neurons. Conclusion These results suggest that proBDNF has distinct functions in different populations of CNS neurons and might be responsible for specific physiological cellular processes in the brain.

We have previously shown that repetitive induction of long‐term potentiation (LTP) by glutamate (100 μM, 3 min, three times at 24‐hr intervals) provoked long‐lasting synaptic enhancement accompanied by synaptogenesis in rat hippocampal slice cultures, a phenomenon termed RISE (repetitive LTP‐induced synaptic enhancement). Here, we examined the role of Ca2+‐permeable (CP) AMPA receptors (AMPARs) in the establishment of RISE. We first found a component sensitive to the Joro‐spider toxin (JSTX), a blocker of CP‐AMPARs, in a field EPSP recorded from CA3‐CA1 synapses at 2–3 days after stimulation, but this component was not found for 9–10 days. We also observed that rectification of AMPAR‐mediated current appeared only 2–3 days after stimulation, using a whole‐cell patch clamp recording from CA1 pyramidal neurons. These findings indicate that CP‐AMPAR is transiently expressed in the developing phase of RISE. The blockade of CP‐AMPARs by JSTX for 24 hr at this developing phase inhibited RISE establishment, accompanied by the loss of small synapses at the ultrastructural level. These results suggest that transiently induced CP‐AMPARs play a critical role in synaptogenesis in the developing phase of long‐lasting hippocampal synaptic plasticity, RISE. [ABSTRACT FROM AUTHOR]