Anti-Epileptic Effect of Ganoderma Lucidum Polysaccharides by Inhibition of Intracellular Calcium Accumulation and Stimulation of Expression of CaMKII α in Epileptic Hippocampal Neurons

Purpose

To investigate the mechanism of the anti-epileptic effect of Ganoderma lucidum polysaccharides (GLP), the changes of intracellular calcium and CaMK II α expression in a model of epileptic neurons were investigated.

Method

Primary hippocampal neurons were divided into: 1) Control group, neurons were cultured with Neurobasal medium, for 3 hours; 2) Model group I: neurons were incubated with Mg²⁺ free medium for 3 hours; 3) Model group II: neurons were incubated with Mg²⁺ free medium for 3 hours then cultured with the normal medium for a further 3 hours; 4) GLP group I: neurons were incubated with Mg²⁺ free medium containing GLP (0.375 mg/ml) for 3 hours; 5) GLP group II: neurons were incubated with Mg²⁺ free medium for 3 hours then cultured with a normal culture medium containing GLP for a further 3 hours. The CaMK II α protein expression was assessed by Western-blot. Ca²⁺ turnover in neurons was assessed using Fluo-3/AM which was added into the replacement medium and Ca²⁺ turnover was observed under a laser scanning confocal microscope.

Results

The CaMK II α expression in the model groups was less than in the control groups, however, in the GLP groups, it was higher than that observed in the model group. Ca^{2 +} fluorescence intensity in GLP group I was significantly lower than that in model group I after 30 seconds, while in GLP group II, it was reduced significantly compared to model group II after 5 minutes.

Conclusion

GLP may inhibit calcium overload and promote CaMK II α expression to protect epileptic neurons.     

A β-Lactam Antibiotic Dampens Excitotoxic Inflammatory CNS Damage in a Mouse Model of Multiple Sclerosis

In multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE), impairment of glial “Excitatory Amino Acid Transporters” (EAATs) together with an excess glutamate-release by invading immune cells causes excitotoxic damage of the central nervous system (CNS). In order to identify pathways to dampen excitotoxic inflammatory CNS damage, we assessed the effects of a β-lactam antibiotic, ceftriaxone, reported to enhance expression of glial EAAT2, in “Myelin Oligodendrocyte Glycoprotein” (MOG)-induced EAE. Ceftriaxone profoundly ameliorated the clinical course of murine MOG-induced EAE both under preventive and therapeutic regimens. However, ceftriaxone had impact neither on EAAT2 protein expression levels in several brain areas, nor on the radioactive glutamate uptake capacity in a mixed primary glial cell-culture and the glutamate-induced uptake currents in a mammalian cell line mediated by EAAT2. Moreover, the clinical effect of ceftriaxone was preserved in the presence of the EAAT2-specific transport inhibitor, dihydrokainate, while dihydrokainate alone caused an aggravated EAE course. This demonstrates the need for sufficient glial glutamate uptake upon an excitotoxic autoimmune inflammatory challenge of the CNS and a molecular target of ceftriaxone other than the glutamate transporter. Ceftriaxone treatment indirectly hampered T cell proliferation and proinflammatory INFγ and IL17 secretion through modulation of myelin-antigen presentation by antigen-presenting cells (APCs) e.g. dendritic cells (DCs) and reduced T cell migration into the CNS in vivo. Taken together, we demonstrate, that a β-lactam antibiotic attenuates disease course and severity in a model of autoimmune CNS inflammation. The mechanisms are reduction of T cell activation by modulation of cellular antigen-presentation and impairment of antigen-specific T cell migration into the CNS rather than or modulation of central glutamate homeostasis.     

Post-tetanic Potentiation and Depression in Hippocampal Neurons in a Rat Model of Alzheimer’s Disease: Effects of Teucrium Polium Extract

Neurophysiology - The plant Teucrium polium (T.p.) possesses a wide range of pharmacological activities due to the presence, in particular, of different phytochemicals, phenols and flavonoids. We...     

Involvment of Cytosolic and Mitochondrial GSK-3β in Mitochondrial Dysfunction and Neuronal Cell Death of MPTP/MPP+-Treated Neurons

Aberrant mitochondrial function appears to play a central role in dopaminergic neuronal loss in Parkinson''s disease (PD). 1-methyl-4-phenylpyridinium iodide (MPP⁺), the active metabolite of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), is a selective inhibitor of mitochondrial complex I and is widely used in rodent and cell models to elicit neurochemical alterations associated with PD. Recent findings suggest that Glycogen Synthase Kinase-3β (GSK-3β), a critical activator of neuronal apoptosis, is involved in the dopaminergic cell death. In this study, the role of GSK-3β in modulating MPP⁺-induced mitochondrial dysfunction and neuronal death was examined in vivo, and in two neuronal cell models namely primary cultured and immortalized neurons. In both cell models, MPTP/MPP⁺ treatment caused cell death associated with time- and concentration-dependent activation of GSK-3β, evidenced by the increased level of the active form of the kinase, i.e. GSK-3β phosphorylated at tyrosine 216 residue. Using immunocytochemistry and subcellular fractionation techniques, we showed that GSK-3β partially localized within mitochondria in both neuronal cell models. Moreover, MPP⁺ treatment induced a significant decrease of the specific phospho-Tyr216-GSK-3β labeling in mitochondria concomitantly with an increase into the cytosol. Using two distinct fluorescent probes, we showed that MPP⁺ induced cell death through the depolarization of mitochondrial membrane potential. Inhibition of GSK-3β activity using well-characterized inhibitors, LiCl and kenpaullone, and RNA interference, prevented MPP⁺-induced cell death by blocking mitochondrial membrane potential changes and subsequent caspase-9 and -3 activation. These results indicate that GSK-3β is a critical mediator of MPTP/MPP⁺-induced neurotoxicity through its ability to regulate mitochondrial functions. Inhibition of GSK-3β activity might provide protection against mitochondrial stress-induced cell death.     

α-Actinin-2 Mediates Spine Morphology and Assembly of the Post-Synaptic Density in Hippocampal Neurons

Dendritic spines are micron-sized protrusions that constitute the primary post-synaptic sites of excitatory neurotransmission in the brain. Spines mature from a filopodia-like protrusion into a mushroom-shaped morphology with a post-synaptic density (PSD) at its tip. Modulation of the actin cytoskeleton drives these morphological changes as well as the spine dynamics that underlie learning and memory. Several PSD molecules respond to glutamate receptor activation and relay signals to the underlying actin cytoskeleton to regulate the structural changes in spine and PSD morphology. α-Actinin-2 is an actin filament cross-linker, which localizes to dendritic spines, enriched within the post-synaptic density, and implicated in actin organization. We show that loss of α-actinin-2 in rat hippocampal neurons creates an increased density of immature, filopodia-like protrusions that fail to mature into a mushroom-shaped spine during development. α-Actinin-2 knockdown also prevents the recruitment and stabilization of the PSD in the spine, resulting in failure of synapse formation, and an inability to structurally respond to chemical stimulation of the N-methyl-D-aspartate (NMDA)-type glutamate receptor. The Ca²⁺-insensitive EF-hand motif in α-actinin-2 is necessary for the molecule''s function in regulating spine morphology and PSD assembly, since exchanging it for the similar but Ca²⁺-sensitive domain from α-actinin-4, another α-actinin isoform, inhibits its function. Furthermore, when the Ca²⁺-insensitive domain from α-actinin-2 is inserted into α-actinin-4 and expressed in neurons, it creates mature spines. These observations support a model whereby α-actinin-2, partially through its Ca²⁺-insensitive EF-hand motif, nucleates PSD formation via F-actin organization and modulates spine maturation to mediate synaptogenesis.     

Control of βAR- and N-methyl-D-aspartate (NMDA) Receptor-Dependent cAMP Dynamics in Hippocampal Neurons

Norepinephrine, a neuromodulator that activates β-adrenergic receptors (βARs), facilitates learning and memory as well as the induction of synaptic plasticity in the hippocampus. Several forms of long-term potentiation (LTP) at the Schaffer collateral CA1 synapse require stimulation of both βARs and N-methyl-D-aspartate receptors (NMDARs). To understand the mechanisms mediating the interactions between βAR and NMDAR signaling pathways, we combined FRET imaging of cAMP in hippocampal neuron cultures with spatial mechanistic modeling of signaling pathways in the CA1 pyramidal neuron. Previous work implied that cAMP is synergistically produced in the presence of the βAR agonist isoproterenol and intracellular calcium. In contrast, we show that when application of isoproterenol precedes application of NMDA by several minutes, as is typical of βAR-facilitated LTP experiments, the average amplitude of the cAMP response to NMDA is attenuated compared with the response to NMDA alone. Models simulations suggest that, although the negative feedback loop formed by cAMP, cAMP-dependent protein kinase (PKA), and type 4 phosphodiesterase may be involved in attenuating the cAMP response to NMDA, it is insufficient to explain the range of experimental observations. Instead, attenuation of the cAMP response requires mechanisms upstream of adenylyl cyclase. Our model demonstrates that Gs-to-Gi switching due to PKA phosphorylation of βARs as well as Gi inhibition of type 1 adenylyl cyclase may underlie the experimental observations. This suggests that signaling by β-adrenergic receptors depends on temporal pattern of stimulation, and that switching may represent a novel mechanism for recruiting kinases involved in synaptic plasticity and memory.     

Effects of Modeling of Hypercalcemia and β-Amyloid on Cultured Hippocampal Neurons of Rats

Neurophysiology - Alzheimer’s disease (AD) is the most common type of dementia; it is characterized by accumulations of amyloid (Aβ) plaques and neurofibrillary tangles in the brain....     

Involvement of p38α in Kainate-Induced Seizure and Neuronal Cell Damage

We investigated how p38α mitogen-activated protein kinase (p38) is related to kainate-induced epilepsy and neuronal damages, by using the mice with a single copy disruption of the p38 α gene (p38α^+/?). Mortality rate and seizure score of p38α^+/? mice administered with kainate were significantly reduced compared with the case of wild-type (WT) mice. This was clearly supported by the electroencephalography data in which kainate-induced seizure duration and frequency in the brain of p38α^+/? mice were significantly suppressed compared to those of WT mice. As a consequence of seizure, kainate induced delayed neuronal damages in parallel with astrocytic growth in the hippocampus and ectopic innervation of the mossy fibers into the stratum oriens in the CA3 region of hippocampus in WT mice, whose changes were moderate in p38α^+/? mice. Likewise, kainate-induced phosphorylation of calcium/calmodulin-dependent kinase II in the hippocampus of p38α ^+/? mice was significantly decreased compared to that of WT mice. These results suggest that p38α signaling pathway plays an important role in epileptic seizure and excitotoxicity.     

Activation of Yeast Mitochondrial Translation: Who Is in Charge?

Mitochondrial genome has undergone significant reduction in a course of evolution; however, it still contains a set of protein-encoding genes and requires translational machinery for their expression. Mitochondrial translation is of the prokaryotic type with several remarkable differences. This review is dedicated to one of the most puzzling features of mitochondrial protein synthesis, namely, the system of translational activators, i.e., proteins that specifically regulate translation of individual mitochondrial mRNAs and couple protein biosynthesis with the assembly of mitochondrial respiratory chain complexes. The review does not claim to be a comprehensive analysis of all published data; it is rather focused on the idea of the “core component” of the translational activator system.     

Upregulation of HIF-1α Via Activation of ERK and PI3K Pathway Mediated Protective Response to Microwave-Induced Mitochondrial Injury in Neuron-Like Cells

Microwave-induced learning and memory deficits in animal models have been gaining attention in recent years, largely because of increasing public concerns on growing environmental influences. The data from our group and others have showed that the injury of mitochondria, the major source of cellular adenosine triphosphate (ATP) in primary neurons, could be detected in the neuron cells of microwave-exposed rats. In this study, we provided some insights into the cellular and molecular mechanisms behind mitochondrial injury in PC12 cell-derived neuron-like cells. PC12 cell-derived neuron-like cells were exposed to 30 mW/cm² microwave for 5 min, and damages of mitochondrial ultrastructure could be observed by using transmission electron microscopy. Impairments of mitochondrial function, indicated by decrease of ATP content, reduction of succinate dehydrogenase (SDH) and cytochrome c oxidase (COX) activities, decrease of mitochondrial membrane potential (MMP), and increase of reactive oxygen species (ROS) production, could be detected. We also found that hypoxia-inducible factor-1 (HIF-1α), a key regulator responsible for hypoxic response of the mammalian cells, was upregulated in microwave-exposed neuron-like cells. Furthermore, HIF-1α overexpression protected mitochondria from injury by increasing the ATP contents and MMP, while HIF-1α silence promoted microwave-induced mitochondrial damage. Finally, we demonstrated that both ERK and PI3K signaling activation are required in microwave-induced HIF-1α activation and protective response. In conclusion, we elucidated a regulatory connection between impairments of mitochondrial function and HIF-1α activation in microwave-exposed neuron-like cells. By modulating mitochondrial function and protecting neuron-like cells against microwave-induced mitochondrial injury, HIF-1α represents a promising therapeutic target for microwave radiation injury.     

Role of PGC-1α in Mitochondrial Quality Control in Neurodegenerative Diseases

Neurochemical Research - As one of the major cell organelles responsible for ATP production, it is important that neurons maintain mitochondria with structural and functional integrity; this is...     

Amyloid-β and Proinflammatory Cytokines Utilize a Prion Protein-Dependent Pathway to Activate NADPH Oxidase and Induce Cofilin-Actin Rods in Hippocampal Neurons

Neurites of neurons under acute or chronic stress form bundles of filaments (rods) containing 1∶1 cofilin∶actin, which impair transport and synaptic function. Rods contain disulfide cross-linked cofilin and are induced by treatments resulting in oxidative stress. Rods form rapidly (5–30 min) in >80% of cultured hippocampal or cortical neurons treated with excitotoxic levels of glutamate or energy depleted (hypoxia/ischemia or mitochondrial inhibitors). In contrast, slow rod formation (50% of maximum response in ∼6 h) occurs in a subpopulation (∼20%) of hippocampal neurons upon exposure to soluble human amyloid-β dimer/trimer (Aβd/t) at subnanomolar concentrations. Here we show that proinflammatory cytokines (TNFα, IL-1β, IL-6) also induce rods at the same rate and within the same neuronal population as Aβd/t. Neurons from prion (PrP^C)-null mice form rods in response to glutamate or antimycin A, but not in response to proinflammatory cytokines or Aβd/t. Two pathways inducing rod formation were confirmed by demonstrating that NADPH-oxidase (NOX) activity is required for prion-dependent rod formation, but not for rods induced by glutamate or energy depletion. Surprisingly, overexpression of PrP^C is by itself sufficient to induce rods in over 40% of hippocampal neurons through the NOX-dependent pathway. Persistence of PrP^C-dependent rods requires the continuous activity of NOX. Removing inducers or inhibiting NOX activity in cells containing PrP^C-dependent rods causes rod disappearance with a half-life of about 36 min. Cofilin-actin rods provide a mechanism for synapse loss bridging the amyloid and cytokine hypotheses for Alzheimer disease, and may explain how functionally diverse Aβ-binding membrane proteins induce synaptic dysfunction.