sl-327 and Seizures

Recently, we demonstrated that inhibition of ERK1/2 activity by SL-327 treatment blocks seizure behavior in Krushinsky-Molodkina (KM) rats, which was mediated by altering of GABA and glutamate release mechanism in the hippocampus. Basal ganglia representing various subcortical cell groups play a significant role in the regulation of motor activity, including epileptiform seizures..  To verify if nigrostriatal system could be also affected by SL-327 treatment we analyzed the expression of tyrosine hydroxylase, D1 and D2 dopamine receptors, NR2B subunit of NMDA receptor as well as vesicular glutamate transporter VGLUT2 and glutamic acid decarboxylases GAD65/67 in the striatum and substantia nigra of KM rats.. Animals were injected i.p. with SL-327 (50 mg/kg) 60 min before audio stimulation. After audiogenic stimulation the brains of control and SL 327 treated rats were removed for further immunohistochemical and biochemical analysis..  Obtained results demonstrated a decrease activity in synapsin I, and accumulation of VGLUT2 in the striatum after blockade of audiogenic seizure (AGS) by SL 327 that could lead to inhibition of glutamate release. While in the striatum GAD65/67 level was diminished, in the substantia nigra GAD65/67 was increased showing enhanced inhibitory output to the compact part of the substantia nigra. Analysis of dopaminergic system showed a significant reduction of tyrosine hydroxylase activity and expression in the substantia nigra, and decreased D1 and D2 receptor expression in the striatum. In summary, we propose that changes in the nigrostriatal system could be mediated by inhibitory effect of SL 327 on AGS expression.

Topics: Acoustic Stimulation; Aminoacetonitrile; Animals; Auditory Perception; Corpus Striatum; Disease Models, Animal; Female; gamma-Aminobutyric Acid; Glutamic Acid; Male; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neurons; Protein Kinase Inhibitors; Rats; Seizures; Substantia Nigra; Synapsins

Fragile X syndrome (FXS) is a developmental disorder caused by the loss of Fragile X Mental Retardation 1 (FMR1) gene function because of a CGG repeat expansion (> 200 repeats) in the gene. The molecular mechanism(s) linking loss of FMR1 function to the molecular pathology and cognitive/behavioral disability remain unclear. Given the critical role of extracellular signal-regulated kinase (ERK) in synaptic plasticity and neurodevelopment, a number of recent studies have investigated ERK phosphorylation under basal conditions or upon mGluR-induction using neuronal and peripheral tissues from Fmr1 knockout mice and peripheral tissues from FXS patients. However, these reports have presented conflicting results. The current study is the first to focus on the levels of ERK phosphorylation in brain tissue from human FXS patients. In both human brain tissue and brain tissue from Fmr1 knockout mice there was significantly increased phosphorylation of MEK1/2 and ERK. Indeed, treating Fmr1 knockout mice with the MEK1/2 inhibitor SL327 abrogated audiogenic seizure activity, a feature of the Fmr1 knockout mice that replicates the symptom in patients with FXS. These findings suggest that activation of the ERK pathway results in some cardinal cognitive and clinical features in FXS patients and likely have profound translational implications.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Aminoacetonitrile; Animals; Blotting, Western; Child; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Female; Fragile X Mental Retardation Protein; Fragile X Syndrome; Humans; Immunohistochemistry; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Middle Aged; Phosphorylation; Protease Inhibitors; Seizures; Signal Transduction; Young Adult

Synapsin I is a synaptic vesicle-associated protein which is phosphorylated at multiple sites by various kinases. It has been proposed to play a role in the regulation of neurotransmitter release and the organization of cytoskeletal architecture in the presynaptic terminal. To better understand the physiological regulation of its phosphorylation in vivo, we induced acute, reversible neuronal excitation by electroconvulsive treatment (ECT) in rats, and studied its effects on synapsin I phosphorylation at sites 3, 4/5 and 6 by immunoblot analyses of homogenates from hippocampus and parietal cortex using phospho-site-specific antibodies. A decrease in phosphorylation at all sites was observed soon after the electrical stimulation, followed by a large increase in phosphorylation at site 4/5 peaking at 5 min and a moderate increase in phosphorylation at site 6 peaking at 20 min. Systemic injection of SL327, a mitogen-activated protein kinase (MAPK) kinase inhibitor, prior to ECT, suppressed the increase in phospho-site 4/5 level, as well as that in MAPK activity, but not that in phospho-site 6 level. Thus, phosphorylation at site 4/5 of synapsin I has been shown to be regulated by MAPK in vivo.

Topics: Aminoacetonitrile; Animals; Binding Sites; Cyclin-Dependent Kinase 5; Cyclin-Dependent Kinases; Electroshock; Enzyme Activation; Enzyme Inhibitors; Hippocampus; Immunoblotting; Male; Mitogen-Activated Protein Kinases; Neurons; Parietal Lobe; Phosphorylation; Protease Inhibitors; Rats; Rats, Wistar; Seizures; Synapsins

Pilocarpine-induced seizures are mediated by the M(1) subtype of muscarinic acetylcholine receptor (mAChR), but little is known about the signaling mechanisms linking the receptor to seizures. The extracellular signal-regulated kinase (ERK) signaling cascade is activated by M(1) mAChR and is elevated during status epilepticus. Yet, the role of ERK activation prior to seizure has not been evaluated. Here, we examine the role of pilocarpine-induced ERK activation in the induction of seizures in mice by pharmacological and behavioral approaches. We show that pilocarpine induces ERK activation prior to the induction of seizures by both western blot and immunocytochemistry with an antibody to phosphorylated ERK. In addition, we show that the ERK pathway inhibitor SL327 effectively blocks the pilocarpine-induced ERK activation. However, SL327 pretreatment has no effect on the initiation of seizures. In fact, animals treated with SL327 had higher seizure-related mortality than vehicle-treated animals, suggesting activated ERK may serve a protective role during seizures. In addition, ERK inhibition had no effect on the development of the long-term sequelae of status epilepticus (SE), including mossy fiber sprouting, neuronal death and spontaneous recurrent seizures.

Topics: Aminoacetonitrile; Animals; Blotting, Western; Cell Death; Disease Models, Animal; Enzyme Activation; Enzyme Inhibitors; Immunohistochemistry; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Mossy Fibers, Hippocampal; Neurons; Pilocarpine; Protease Inhibitors; Receptors, Muscarinic; Recurrence; Seizures; Status Epilepticus; Survival Rate