No abstract

Neuronal loss, gliosis and axonal sprouting in the hippocampal formation are characteristics of the syndrome of mesial temporal sclerosis (MTS). In the post-status epilepticus (SE) rat model of spontaneous seizures these features of the MTS syndrome can be reproduced. To get a global view of the changes in gene expression in the hippocampus we applied serial analysis of gene expression (SAGE) during the early phase of epileptogenesis (latent period), prior to the onset of the first spontaneous seizure. A total of 10 000 SAGE tags were analyzed per experimental group, resulting in 5053 (SE) and 5918 (control group) unique tags (genes), each representing a specific mRNA transcript. Of these, 92 genes were differentially expressed in the hippocampus of post-SE rats in comparison to controls. These genes appeared to be mainly associated with ribosomal proteins, protein processing, axonal growth and glial proliferation proteins. Verification of two of the differentially expressed genes by in situ hybridization confirmed the changes found by SAGE. Histological analysis of hippocampal sections obtained 8 days after SE showed extensive cell loss, mossy fibre sprouting and gliosis in hippocampal sub regions. This study identifies new high-abundant genes that may play an important role in post-SE epileptogenesis.

The rab family of GTP-binding proteins regulates membrane transport between intracellular compartments. The major rab protein in brain, rab3A, associates with synaptic vesicles. However, rab3A was shown to regulate the fusion probability of synaptic vesicles, rather than their transport and docking. We tested whether rab3A has a transport function by analyzing synaptic vesicle distribution and exocytosis in rab3A null-mutant mice. Rab3A deletion did not affect the number of vesicles and their distribution in resting nerve terminals. The secretion response upon a single depolarization was also unaffected. In normal mice, a depolarization pulse in the presence of Ca(2+) induces an accumulation of vesicles close to and docked at the active zone (recruitment). Rab3A deletion completely abolished this activity-dependent recruitment, without affecting the total number of vesicles. Concomitantly, the secretion response in the rab3A-deficient terminals recovered slowly and incompletely after exhaustive stimulation, and the replenishment of docked vesicles after exhaustive stimulation was also impaired in the absence of rab3A. These data indicate that rab3A has a function upstream of vesicle fusion in the activity-dependent transport of synaptic vesicles to and their docking at the active zone.

Munc18-1 is a mammalian member of the SEC1 protein family implicated in neuronal secretion. Its sequence contains several consensus sites for phosphorylation by protein kinase C (PKC), a kinase known to enhance secretion. We have characterized the phosphorylation of the synaptic munc18-1 pool by endogenous, presynaptic PKC-isoforms. In isolated rat brain nerve terminals, munc18-1 was almost completely nonphosphorylated. Its phosphorylation state increased by 250% on inhibition of endogenous phosphatases and by 1500% on additional, direct PKC activation using phorbol esters. K+-evoked depolarization also increased munc18-1 phosphorylation, by 50% within 5 s in a Ca2+-dependent manner. Munc18-1 phosphorylation in nerve terminals was blocked by PKC inhibitors. Activation of endogenous PKC in nerve terminals inhibited the interaction of synaptic munc18-1 with its binding partner syntaxin-1A by 50%. Munc18-1 antisera precipitated 80% of native, brain-derived munc18-1 from salt solutions, but only 12% from synaptosomal lysates, together with 6% synaptic syntaxin-1A/B; these amounts were not changed by PKC activation. In this 12%, the phosphate incorporation per mole of munc18 was four-fold lower than the total pool. We conclude that the synaptic munc18-1 pool can be readily and rapidly phosphorylated by endogenous presynaptic PKC isoforms. A high constitutive phosphatase activity keeps its basal phosphorylation state low so that PKC activation can increase the phosphorylation state dramatically. These phosphorylation dynamics and the effects on the interaction with syntaxin-1A make munc18-1 a prominent candidate to account for PKC-dependent enhancement of secretion.