PFTK1 kinase regulates axogenesis during development via RhoA activation.

Cyclin Dependent Kinase family members include members of the non-cell cycle CDK, such as PFTK1/Eip63E. Eip63E expresses primarily in postnatal and adult nervous system in Drosophila melanogaster but its role in CNS development remains unknown. We sought to understand its role in the CNS by studying the fly ventral nerve cord during development. Eip63E regulates axogenesis in neurons and its deficiency leads to neuronal defects. We describe a functional interaction between Eip63E and Rho1. Studies in cultured cortical neurons from PFTK1 knockout mice, confirmed that PFTK1 plays a role in axonal outgrowth and its deficiency resulted in faster growing axons. We demonstrate that GDP bound RhoA is a substrate of PFTK1 and this phosphorylation resulted in higher activity of RhoA. In conclusion, our work represents the first steps in the characterization of the neuronal functions of PFTK1 and points to RhoA activation in the regulation of PFTK1 mediated axogenesis.

Lipopolysaccharide From E. coli Increases Glutamate-Induced Disturbances of Calcium Homeostasis, the Functional State of Mitochondria, and the Death of Cultured Cortical Neurons

Lipopolysaccharide (LPS), a fragment of the bacterial cell wall, specifically interacting with protein complexes on the cell surface, can induce the production of pro-inflammatory and apoptotic signaling molecules, leading to the damage and death of brain cells. Similar effects have been noted in stroke and traumatic brain injury, when the leading factor of death is glutamate (Glu) excitotoxicity too. But being an amphiphilic molecule with a significant hydrophobic moiety and a large hydrophilic region, LPS can also non-specifically bind to the plasma membrane, altering its properties. In the present work, we studied the effect of LPS from Escherichia coli alone and in combination with the hyperstimulation of Glu-receptors on the functional state of mitochondria and Ca2+ homeostasis, oxygen consumption and the cell survival in primary cultures from the rats brain cerebellum and cortex. In both types of cultures, LPS (0.1–10 μg/ml) did not change the intracellular free Ca2+ concentration ([Ca2+]i) in resting neurons but slowed down the median of the decrease in [Ca2+]i on 14% and recovery of the mitochondrial potential (ΔΨm) after Glu removal. LPS did not affect the basal oxygen consumption rate (OCR) of cortical neurons; however, it did decrease the acute OCR during Glu and LPS coapplication. Evaluation of the cell culture survival using vital dyes and the MTT assay showed that LPS (10 μg/ml) and Glu (33 μM) reduced jointly and separately the proportion of live cortical neurons, but there was no synergism or additive action. LPS-effects was dependent on the type of culture, that may be related to both the properties of neurons and the different ratio between neurons and glial cells in cultures. The rapid manifestation of these effects may be the consequence of the direct effect of LPS on the rheological properties of the cell membrane.

Insulin and α-Tocopherol Enhance the Protective Effect of Each Other on Brain Cortical Neurons under Oxidative Stress Conditions and in Rat Two-Vessel Forebrain Ischemia/Reperfusion Injury

Clinical trials show that insulin administered intranasally is a promising drug to treat neurodegenerative diseases, but at high doses its use may result in cerebral insulin resistance. Identifying compounds which could enhance the protective effects of insulin, may be helpful to reduce its effective dose. Our aim was thus to study the efficiency of combined use of insulin and α-tocopherol (α-T) to increase the viability of cultured cortical neurons under oxidative stress conditions and to normalize the metabolic disturbances caused by free radical reaction activation in brain cortex of rats with two-vessel forebrain ischemia/reperfusion injury. Immunoblotting, flow cytometry, colorimetric, and fluorometric techniques were used. α-T enhanced the protective and antioxidative effects of insulin on neurons in oxidative stress, their effects were additive. At the late stages of oxidative stress, the combined action of insulin and α-T increased Akt-kinase activity, inactivated GSK-3beta and normalized ERK1/2 activity in cortical neurons, it was more effective than either drug action. In the brain cortex, ischemia/reperfusion increased the lipid peroxidation product content and caused Na+,K+-ATPase oxidative inactivation. Co-administration of insulin (intranasally, 0.25 IU/rat) and α-T (orally, 50 mg/kg) led to a more pronounced normalization of the levels of Schiff bases, conjugated dienes and trienes and Na+,K+-ATPase activity than administration of each drug alone. Thus, α-T enhances the protective effects of insulin on cultured cortical neurons in oxidative stress and in the brain cortex of rats with cerebral ischemia/reperfusion injury.

Loop Diuretics Inhibit Ischemia-Induced Intracellular Ca2+ Overload in Neurons via the Inhibition of Voltage-Gated Ca2+ and Na+ Channels

One consequence of ischemic stroke is disruption of intracellular ionic homeostasis. Intracellular overload of both Na+ and Ca2+ has been linked to neuronal death in this pathophysiological state. The etiology of ionic imbalances resulting from stroke-induced ischemia and acidosis includes the dysregulation of multiple plasma membrane transport proteins, such as increased activity of sodium-potassium-chloride cotransporter-1 (NKCC-1). Experiments using NKCC1 antagonists, bumetanide (BMN) and ethacrynic acid (EA), were carried out to determine if inhibition of this cotransporter affects Na+ and Ca2+ overload observed following in vitro ischemia-acidosis. Fluorometric Ca2+ and Na+ measurements were performed using cultured cortical neurons, and measurements of whole-cell membrane currents were used to determine target(s) of BMN and EA, other than the electroneutral NKCC-1. Both BMN and EA depressed ischemia-acidosis induced [Ca2+]i overload without appreciably reducing [Na+]i increases. Voltage-gated Ca2+ channels were inhibited by both BMN and EA with half-maximal inhibitory concentration (IC50) values of 4 and 36 μM, respectively. Similarly, voltage-gated Na+ channels were blocked by BMN and EA with IC50 values of 13 and 30 μM, respectively. However, neither BMN nor EA affected currents mediated by acid-sensing ion channels or ionotropic glutamatergic receptors, both of which are known to produce [Ca2+]i overload following ischemia. Data suggest that loop diuretics effectively inhibit voltage-gated Ca2+ and Na+ channels at clinically relevant concentrations, and block of these channels by these compounds likely contributes to their clinical effects. Importantly, inhibition of these channels, and not NKCC1, by loop diuretics reduces [Ca2+]i overload in neurons during ischemia-acidosis, and thus BMN and EA could potentially be used therapeutically to lessen injury following ischemic stroke.

Fluorescent Analysis Reveals that Insulin Diminishes Superoxide Increase in Neurons Induced with Toxic Glutamate Treatment

Glutamate excitotoxicity is implicated in the pathogenesis of many disorders, including stroke, traumatic brain injury, and Alzheimer’s disease, for which central insulin resistance is a comorbid condition. Massive glutamate release primarily through ionotropic N-methyl-D-aspartate receptors (NMDARs) causes a sustained rise in [Ca2+]i, followed by mitochondrial depolarization and an increase in intracellular O2• (superoxide) production. Recently, we found that insulin protected neurons against excitotoxicity by diminishing the delayed calcium deregulation (DCD), However, a role of insulin in superoxide production in excitotoxicity still needs to be clarified. The present study is aimed to investigate the effects of insulin on glutamate-evoked superoxide generation and DCD using the fluorescent indicators dihydroethidium, MitoSOX Red, and Fura-FF in rats cultured cortical neurons. We found that insulin significantly diminished both the intracellular and mitochondrial superoxide production in neurons exposed to glutamate and there was a strong linear correlation between [Ca2+]i and intracellular superoxide. MK 801, an inhibitor of NMDAR-gated Ca2+ influx, completely abrogated the glutamate effects in both the presence and absence of insulin. In experiments on sister cultures, insulin diminishes neuronal death. Thus, collectively, data obtained suggest that insulin diminishes glutamate-induced superoxide production in neurons via fall of [Ca2+]i increased and thereby improves viability of neurons

Neuroprotective effect of selumetinib on acrolein-induced neurotoxicity

Abnormal accumulation of acrolein, an α, β unsaturated aldehyde has been reported as one pathological cause of the CNS neurodegenerative diseases. In the present study, the neuroprotective effect of selumetinib (a MEK–ERK inhibitor) on acrolein-induced neurotoxicity was investigated in vitro using primary cultured cortical neurons. Incubation of acrolein consistently increased phosphorylated ERK levels. Co-treatment of selumetinib blocked acrolein-induced ERK phosphorylation. Furthermore, selumetinib reduced acrolein-induced increases in heme oxygenase-1 (a redox-regulated chaperone protein) and its transcriptional factor, Nrf-2 as well as FDP-lysine (acrolein-lysine adducts) and α-synuclein aggregation (a pathological biomarker of neurodegeneration). Morphologically, selumetinib attenuated acrolein-induced damage in neurite outgrowth, including neuritic beading and neurite discontinuation. Moreover, selumetinib prevented acrolein-induced programmed cell death via decreasing active caspase 3 (a hallmark of apoptosis) as well as RIP (receptor-interacting protein) 1 and RIP3 (biomarkers for necroptosis). In conclusion, our study showed that selumetinib inhibited acrolein-activated Nrf-2-HO-1 pathway, acrolein-induced protein conjugation and aggregation as well as damage in neurite outgrowth and cell death, suggesting that selumetinib, a MEK–ERK inhibitor, may be a potential neuroprotective agent against acrolein-induced neurotoxicity in the CNS neurodegenerative diseases.