guanosine-triphosphate and Epilepsy

The Rab GTPase family comprises ∼70 GTP-binding proteins, functioning in vesicle formation, transport and fusion. They are activated by a conformational change induced by GTP-binding, allowing interactions with downstream effectors. Here, we report five individuals with two recurrent de novo missense mutations in RAB11B; c.64G>A; p.Val22Met in three individuals and c.202G>A; p.Ala68Thr in two individuals. An overlapping neurodevelopmental phenotype, including severe intellectual disability with absent speech, epilepsy, and hypotonia was observed in all affected individuals. Additionally, visual problems, musculoskeletal abnormalities, and microcephaly were present in the majority of cases. Re-evaluation of brain MRI images of four individuals showed a shared distinct brain phenotype, consisting of abnormal white matter (severely decreased volume and abnormal signal), thin corpus callosum, cerebellar vermis hypoplasia, optic nerve hypoplasia and mild ventriculomegaly. To compare the effects of both variants with known inactive GDP- and active GTP-bound RAB11B mutants, we modeled the variants on the three-dimensional protein structure and performed subcellular localization studies. We predicted that both variants alter the GTP/GDP binding pocket and show that they both have localization patterns similar to inactive RAB11B. Evaluation of their influence on the affinity of RAB11B to a series of binary interactors, both effectors and guanine nucleotide exchange factors (GEFs), showed induction of RAB11B binding to the GEF SH3BP5, again similar to inactive RAB11B. In conclusion, we report two recurrent dominant mutations in RAB11B leading to a neurodevelopmental syndrome, likely caused by altered GDP/GTP binding that inactivate the protein and induce GEF binding and protein mislocalization.

Topics: Adolescent; Amino Acid Sequence; Binding Sites; Cerebellar Vermis; Child; Child, Preschool; Corpus Callosum; Epilepsy; Female; Gene Expression; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Intellectual Disability; Magnetic Resonance Imaging; Male; Models, Molecular; Muscle Hypotonia; Mutation; Optic Nerve Diseases; Phenotype; Protein Binding; rab GTP-Binding Proteins; White Matter

Epilepsy is one of the most common and severe neurologic diseases. The mechanisms of epilepsy are still not fully understood. Dock3 (dedicator of cytokinesis 3) is one of the new kinds of guanine-nucleotide exchange factors (GEF) and plays an important role in neuronal synaptic plasticity and cytoskeleton rearrangement; the same mechanisms were also found in epilepsy. However, little is known regarding the expression of Dock3 in the epileptic brain and whether Dock3 interventions affect the epileptic process. In this study, we showed that the expression of Dock3 significantly increased in IE patients and a lithium-pilocarpine epilepsy model compared with the controls. Inhibition of Dock3 by Dock3 shRNA impaired the severity of status epilepticus in the acute stage and decreased the spontaneous recurrent seizures times in the chronic stage of lithium-pilocarpine model and decreased the expression of rac1-GTP. Consistent with decreased expression of Dock3, the latent period in a pentylenetetrazole kindling model also increased. Our results demonstrated that the increased expression of Dock3 in the brain is associated with epileptogenesis and specific inhibition of Dock3 may be a potential target in preventing the development of epilepsy in patients.

Topics: Action Potentials; Adolescent; Adult; Animals; Blotting, Western; Disease Models, Animal; Down-Regulation; Epilepsy; Excitatory Postsynaptic Potentials; Female; Green Fluorescent Proteins; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; Humans; Immunohistochemistry; Lithium; Male; Mice, Inbred C57BL; Mossy Fibers, Hippocampal; Nerve Tissue Proteins; Neurons; Pilocarpine; rac1 GTP-Binding Protein; Rats, Sprague-Dawley; RNA, Small Interfering; Signal Transduction; Young Adult

We present data on the antiepileptic potency of 2-methyl-4-oxo-3H-quinazoline-3-acetyl piperidine (Q5) in juvenile (P9-13) rat hippocampal slices and in particular Q5's action mechanism and target. Q5 (200-500 microM), but not alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/Kainate receptor antagonists blocked low-[Mg2+]-induced seizure-like events (SLE) in the CA3 region. Q5 (100 microM) decreased Glu-induced [35S]guanosine 5'-O-(3-thiotriphosphate) binding enhancement in brain homogenates, without interaction with ionotropic Glu receptor sites and Glu transport. In voltage-clamped CA3 pyramidal cells, Q5 (500 microM) depressed activities of spontaneous excitatory and inhibitory postsynaptic currents without affecting miniature inhibitory currents. Metabotropic Glu receptor (mGluR) subtype antagonists affected network excitability dissimilarly. Intracellular Ca2+ ion transients induced by the mGluR agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) were suppressed by Q5. Agreeing predictions obtained by modelling Q5 binding to different experimental conformations of mGlu1, Q5 was bound partially to an mGluR binding site in the presence of 1mM ACPD. Findings suggest the apparent involvement of a novel phenotype of action or a new mGluR subtype in the specific suppression of epileptiform activity by Q5 through the depression of network excitability.

Topics: Age Factors; Animals; Anticonvulsants; Binding Sites; Calcium Channels; Cell Membrane; Epilepsy; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glutamic Acid; Guanosine Triphosphate; Hippocampus; Male; Nerve Net; Neural Inhibition; Organ Culture Techniques; Patch-Clamp Techniques; Piperidines; Protein Subunits; Pyramidal Cells; Quinazolines; Rats; Rats, Wistar; Receptors, Metabotropic Glutamate; Synaptic Transmission

Topics: Adenosine Diphosphate Ribose; Amygdala; Animals; Autoradiography; Cerebral Cortex; Epilepsy; Guanosine Triphosphate; Kindling, Neurologic; Male; Membrane Proteins; Nitric Oxide; Rats; Rats, Sprague-Dawley; Synaptic Transmission