Promotion of Neuronal Plasticity by (−)-Epigallocatechin-3-Gallate

The consumption of (−)-epigallocatechin-3-gallate (EGCG), the major polyphenolic compound found in green tea, has been associated with various neurological benefits including cognitive improvement. The physiological basis for this effect is unknown. In this study, we used synaptic transmission between the CA3 and CA1 regions (Schaffer collateral) of the mouse hippocampus to examine the effects of EGCG on neuronal plasticity. We found that the level of high frequency stimulation-evoked long-term potentiation (LTP) was significantly enhanced when hippocampal slices were pre-incubated with 10 μM EGCG for 1 h prior to the experiment. EGCG incubation also enabled hippocampal slices prepared from Ts65Dn mice, a Down syndrome mouse model deficient in LTP, to express LTP to a level comparable to the normal controls. EGCG treatment did not alter the degree of pair-pulse inhibition; therefore, the enhancement effect of EGCG is unlikely to involve the attenuation of this inhibitory mechanism.     

Mechanism of Auxiliary Subunit Modulation of Neuronal α1E Calcium Channels

Voltage-gated calcium channels are composed of a main pore-forming α₁ moiety, and one or more auxiliary subunits (β, α₂δ) that modulate channel properties. Because modulatory properties may vary greatly with different channels, expression systems, and protocols, it is advantageous to study subunit regulation with a uniform experimental strategy. Here, in HEK 293 cells, we examine the expression and activation gating of α_1E calcium channels in combination with a β (β₁–β₄) and/or the α₂δ subunit, exploiting both ionic- and gating-current measurements. Furthermore, to explore whether more than one auxiliary subunit can concomitantly specify gating properties, we investigate the effects of cotransfecting α₂δ with β subunits, of transfecting two different β subunits simultaneously, and of COOH-terminal truncation of α_1E to remove a second β binding site. The main results are as follows. (a) The α₂δ and β subunits modulate α_1E in fundamentally different ways. The sole effect of α₂δ is to increase current density by elevating channel density. By contrast, though β subunits also increase functional channel number, they also enhance maximum open probability (G_max/Q_max) and hyperpolarize the voltage dependence of ionic-current activation and gating-charge movement, all without discernible effect on activation kinetics. Different β isoforms produce nearly indistinguishable effects on activation. However, β subunits produced clear, isoform-specific effects on inactivation properties. (b) All the β subunit effects can be explained by a gating model in which subunits act only on weakly voltage-dependent steps near the open state. (c) We find no clear evidence for simultaneous modulation by two different β subunits. (d) The modulatory features found here for α_1E do not generalize uniformly to other α₁ channel types, as α_1C activation gating shows marked β isoform dependence that is absent for α_1E. Together, these results help to establish a more comprehensive picture of auxiliary-subunit regulation of α_1E calcium channels.     

Neuronal variability: noise or part of the signal?

Sensory, motor and cortical neurons fire impulses or spikes at a regular, but slowly declining, rate in response to a constant current stimulus. Yet, the intervals between spikes often vary randomly during behaviour. Is this variation an unavoidable effect of generating spikes by sensory or synaptic processes ('neural noise') or is it an important part of the 'signal' that is transmitted to other neurons? Here, we mainly discuss this question in relation to sensory and motor processes, as the signals are best identified in such systems, although we also touch on central processes.     

Protective Effects of Curcumin on Amyloid-β-Induced Neuronal Oxidative Damage

To investigate the protective effects of curcumin against amyloid-β (Aβ)-induced neuronal damage. Primary rat cortical neurons were cultured with different treatments of Aβ and curcumin. Neuronal morphologies, viability and damage were assessed. Neuronal oxidative stress was assessed, including extracellular hydrogen peroxide and intracellular reactive oxygen species. The abilities of curcumin to scavenge free radicals and to inhibit Aβ aggregation and β-sheeted formation are further assessed and discussed. Curcumin preserves cell viability, which is decreased by Aβ. The results of changed morphology, released Lactate dehydrogenases and cell viability assays indicate that curcumin protects Aβ-induced neuronal damage. Curcumin depresses Aβ-induced up-regulation of neuronal oxidative stress. The treatment sequence impacts the protective effect of curcumin on Aβ-induced neuronal damage. Curcumin shows a more protective effect on neuronal oxidative damage when curcumin was added into cultured neurons not later than Aβ, especially prior to Aβ. The abilities of curcumin to scavenge free radicals and to inhibit the formation of β-sheeted aggregation are both beneficial to depress Aβ-induced oxidative damage. Curcumin prevents neurons from Aβ-induced oxidative damage, implying the therapeutic usage for the treatment of Alzheimer's disease patients.     

Presence of DNase γ-Like Endonuclease in Nuclei of Neuronal Differentiated PC12 Cells

DNase , which cleaves chromosomal DNA into nucleosomal units (DNA ladder formation), has been suggested to be the critical component of apoptotic machinery. Using rat pheochromocytoma PC12 cells, which are differentiated to sympathetic neurons by nerve growth factor (NGF), we investigated whether DNase -like enzyme is present in neuronal cells and is involved in neuronal cell death. The nuclear auto-digestion assay for DNase catalyzing internucleosomal DNA cleavage revealed that nuclei from neuronal differentiated PC12 cells contain acidic and neutral endonucleases, while nuclei from undifferentiated PC12 cells have only acidic endonuclease. The DNA ladder formation observed in isolated nuclei from neuronal differentiated PC12 cells at neutral pH requires both Ca²⁺ and Mg²⁺, and is sensitive to Zn²⁺. The molecular mass of the neutral endonuclease present in neuronal differentiated PC12 cell nuclei is 32000 as determined by activity gel analysis (zymography). The properties of the neuronal endonuclease present in neuronal differentiated PC12 cell nuclei were similar to those of purified DNase from rat thymocytes and splenocytes. Interestingly, in neuronal differentiated PC12 cells, internucleosomal DNA fragmentation is observed following NGF deprivation, whereas undifferentiated PC12 cells fail to exhibit DNA ladder formation during cell death by serum starvation. These results suggest that the DNase -like endonuclease present in neuronal differentiated PC12 cell nuclei is involved in internucleosomal DNA fragmentation during apoptosis, induced by NGF deprivation.     

The Role of Globo-Series Glycolipids in Neuronal Cell Differentiation—A Review

Alterations in glycolipid composition as well as glycosyltransferase activities during cellular differentiation and growth have been well documented. However, the underlying mechanisms for the regulation of glycolipid expression remain obscure. One of the major obstacles has been the lack of a well defined model system for studying these phenomena. We have chosen PC 12 pheochrom-ocytoma cells as a model because (a) the properties of these cells have been well characterized, and (b) they respond to nerve growth factor (NGF) by differentiating into sympathetic-like neurons and are amenable to well-controlled experimentation. Thus, PC12 cells represent a suitable model for studying changes in glycolipid metabolism in relation to cellular differentiation. We have previously shown that subcloned PC12 cells accumulate a unique series of globo-series neutral glycolipids which are not expressed in parental PC12 cells. This unusual change in glycolipid distribution is accompanied by changes in the activities of specific glycosyltransferases involved in their synthesis and is correlated with neuritogenesis and/or cellular differentiation in this cell line. We have further demonstrated that changes in the glycosyltransferase activities may be modulated by the phosphorylation states of the cells via protein kinase systems. We conclude that these unique globo-series glycolipids may play a functional role in the initiation and/or maintenance of neurite outgrowth in PC12 cells.     

Time-Warp–Invariant Neuronal Processing

Fluctuations in the temporal durations of sensory signals constitute a major source of variability within natural stimulus ensembles. The neuronal mechanisms through which sensory systems can stabilize perception against such fluctuations are largely unknown. An intriguing instantiation of such robustness occurs in human speech perception, which relies critically on temporal acoustic cues that are embedded in signals with highly variable duration. Across different instances of natural speech, auditory cues can undergo temporal warping that ranges from 2-fold compression to 2-fold dilation without significant perceptual impairment. Here, we report that time-warp–invariant neuronal processing can be subserved by the shunting action of synaptic conductances that automatically rescales the effective integration time of postsynaptic neurons. We propose a novel spike-based learning rule for synaptic conductances that adjusts the degree of synaptic shunting to the temporal processing requirements of a given task. Applying this general biophysical mechanism to the example of speech processing, we propose a neuronal network model for time-warp–invariant word discrimination and demonstrate its excellent performance on a standard benchmark speech-recognition task. Our results demonstrate the important functional role of synaptic conductances in spike-based neuronal information processing and learning. The biophysics of temporal integration at neuronal membranes can endow sensory pathways with powerful time-warp–invariant computational capabilities.     

Nuclear Recruitment of Neuronal Nitric-oxide Synthase by α-Syntrophin Is Crucial for the Induction of Mitochondrial Biogenesis

Neuronal nitric-oxide synthase (nNOS) has various splicing variants and different subcellular localizations. nNOS can be found also in the nucleus; however, its exact role in this compartment is still not completely defined. In this report, we demonstrate that the PDZ domain allows the recruitment of nNOS to nuclei, thus favoring local NO production, nuclear protein S-nitrosylation, and induction of mitochondrial biogenesis. In particular, overexpression of PDZ-containing nNOS (nNOSα) increases S-nitrosylated CREB with consequent augmented binding on cAMP response element consensus sequence on peroxisome proliferator-activated receptor γ co-activator (PGC)-1α promoter. The resulting PGC-1α induction is accompanied by the expression of mitochondrial genes (e.g., TFAM, MtCO1) and increased mitochondrial mass. Importantly, full active nNOS lacking PDZ domain (nNOSβ) does not localize in nuclei and fails in inducing the expression of PGC-1α. Moreover, we substantiate that the mitochondrial biogenesis normally accompanying myogenesis is associated with nuclear translocation of nNOS. We demonstrate that α-Syntrophin, which resides in nuclei of myocytes, functions as the upstream mediator of nuclear nNOS translocation and nNOS-dependent mitochondrial biogenesis. Overall, our results indicate that altered nNOS splicing and nuclear localization could be contributing factors in human muscular diseases associated with mitochondrial impairment.     

α-Synuclein and Neuronal Cell Death

Parkinson’s disease (PD) is a progressive neurodegenerative disorder affecting ～1 % of people over the age of 65. Neuropathological hallmarks of PD are prominent loss of dopaminergic (DA) neurons in the substantia nigra and formation of intraneuronal protein inclusions termed Lewy bodies, composed mainly of α-synuclein (αSyn). Missense mutations in αSyn gene giving rise to production of degradation-resistant mutant proteins or multiplication of wild-type αSyn gene allele can cause rare inherited forms of PD. Therefore, the existence of abnormally high amount of αSyn protein is considered responsible for the DA neuronal death in PD. Normally, αSyn protein localizes to presynaptic terminals of neuronal cells, regulating the neurotransmitter release through the modulation of assembly of soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex. On the other hand, of note, pathological examinations on the recipient patients of fetal nigral transplants provided a prion-like cell-to-cell transmission hypothesis for abnormal αSyn. The extracellular αSyn fibrils can internalize to the cells and enhance intracellular formation of protein inclusions, thereby reducing cell viability. These findings suggest that effective removal of abnormal species of αSyn in the extracellular space as well as intracellular compartments can be of therapeutic relevance. In this review, we will focus on αSyn-triggered neuronal cell death and provide possible disease-modifying therapies targeting abnormally accumulating αSyn.     

The “Janus-Faced Role” of Autophagy in Neuronal Sickness: Focus on Neurodegeneration

The mature brain is a highly dynamic organ that constantly changes its organization by destroying and forming new connections. Collectively, these changes are referred to as brain plasticity and are associated with functional changes, such as memory, addiction, and recovery of function after brain damage. Neuronal plasticity is sustained by the fine regulation of protein synthesis and organelle biogenesis and their degradation to ensure efficient turnover. Thus, autophagy, as quality control mechanism of proteins and organelles in neurons, is essential to their physiology and pathology. Here, we review recent several findings proving that defects in autophagy affect neuronal function and impair functional recovery after brain insults, contributing to neurodegeneration, in chronic and acute neurological disorders. Thus, an understanding of the molecular mechanisms by which the autophagy machinery is finely regulated might accelerate the development of therapeutic interventions in many neurological disorders for which no cure is available.     

Recruitment of β-Arrestin into Neuronal Cilia Modulates Somatostatin Receptor Subtype 3 Ciliary Localization

Primary cilia are essential sensory and signaling organelles present on nearly every mammalian cell type. Defects in primary cilia underlie a class of human diseases collectively termed ciliopathies. Primary cilia are restricted subcellular compartments, and specialized mechanisms coordinate the localization of proteins to cilia. Moreover, trafficking of proteins into and out of cilia is required for proper ciliary function, and this process is disrupted in ciliopathies. The somatostatin receptor subtype 3 (Sstr3) is selectively targeted to primary cilia on neurons in the mammalian brain and is implicated in learning and memory. Here, we show that Sstr3 localization to cilia is dynamic and decreases in response to somatostatin treatment. We further show that somatostatin treatment stimulates β-arrestin recruitment into Sstr3-positive cilia and this recruitment can be blocked by mutations in Sstr3 that impact agonist binding or phosphorylation. Importantly, somatostatin treatment fails to decrease Sstr3 ciliary localization in neurons lacking β-arrestin 2. Together, our results implicate β-arrestin in the modulation of Sstr3 ciliary localization and further suggest a role for β-arrestin in the mediation of Sstr3 ciliary signaling.     

Nonlinear Decoding and Asymmetric Representation of Neuronal Input Information by CaMKIIα and Calcineurin

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Clustering of Neuronal K+-Cl? Cotransporters in Lipid Rafts by Tyrosine Phosphorylation

The neuronal K⁺-Cl⁻ cotransporter (KCC2) is a membrane transport protein that extrudes Cl⁻ from neurons and helps maintain low intracellular [Cl⁻] and hyperpolarizing GABAergic synaptic potentials. Depolarizing γ-aminobutyric acid (GABA) responses in neonatal neurons and following various forms of neuronal injury are associated with reduced levels of KCC2 expression. Despite the importance for plasticity of inhibitory transmission, less is known about cellular mechanisms involved in more dynamic changes in KCC2 function. In this study, we investigated the role of tyrosine phosphorylation in KCC2 localization and function in hippocampal neurons and in cultured GT1-7 cells. Mutation to the putative tyrosine phosphorylation site within the long intracellular carboxyl terminus of KCC2(Y1087D) or application of the tyrosine kinase inhibitor genistein shifted the GABA reversal potential (E_GABA) to more depolarized values, indicating reduced KCC2 function. This was associated with a change in the expression pattern of KCC2 from a punctate distribution to a more uniform distribution, suggesting that functional tyrosine-phosphorylated KCC2 forms clusters in restricted membrane domains. Sodium vanadate, a tyrosine phosphatase inhibitor, increased the proportion of KCC2 associated with lipid rafts membrane domains. Loss of tyrosine phosphorylation also reduced oligomerization of KCC2. A loss of the punctuate distribution and oligomerization of KCC2 and a more depolarized E_GABA were seen when the 28-amino-acid carboxyl terminus of KCC2 was deleted. These results indicate that direct tyrosine phosphorylation of KCC2 results in membrane clusters and functional transport activity, suggesting a mechanism by which intracellular Cl⁻ concentrations and GABA responses can be rapidly modulated.The inhibitory neurotransmitters GABA² and glycine activate ionotropic Cl⁻ channels, typically leading to membrane hyperpolarization in the adult central nervous system. The neuronal K⁺-Cl⁻ cotransporter (KCC2) is the principal membrane transport protein that maintains low intracellular [Cl⁻] ([Cl⁻]_i) in mature and healthy neurons to allow such Cl⁻ influx and hyperpolarization. However, in immature neurons and in neurons following various forms of neuronal injury, [Cl⁻]_i is elevated and GABA and glycine can cause membrane depolarization and neuronal excitation (1–3). A reduced expression of KCC2 protein in immature neurons (4) and a decrease of KCC2 expression in response to various pathophysiological conditions, e.g. axotomy (5, 6) and global ischemia (7), are primarily responsible for this increased [Cl⁻]_i and for the depolarizing GABA response.In addition to changes in the expression levels of KCC2 protein, the function of KCC2 can be more dynamically and rapidly modulated by the availability of transport substrates and by various forms of kinase activity. Cl⁻ extrusion is quantitatively regulated by the K⁺ driving force across the membrane (8). Protein kinase C can down-regulate both KCC2 function (9) and surface expression (10). Staurosporine, a broad spectrum kinase inhibitor, produces a rapid up-regulation of KCC2 function in immature neurons (11). Brain-type creatinine kinase binding to KCC2 may also regulate its function (12). Finally, WNK3, by interacting with Ste20-related proline-alanine-rich kinase, prevents the cell swelling-induced activation of KCC2 in Xenopus oocytes (13, 14).KCC2 contains one tyrosine protein kinase phosphorylation consensus site (Tyr-1087) within the long carboxyl terminus in the intracellular region (15). Tyr-1087 is not present in KCC1, another family of KCCs, suggesting that direct tyrosine phosphorylation may uniquely regulate KCC2. The receptor tyrosine kinase, IGF-1, and the soluble tyrosine kinase, Src kinase, activate KCC2 during maturation of hippocampal neurons (16). Oxidative stress decreases the tyrosine phosphorylation of KCC2 and reduces KCC2 function (17). However, just how tyrosine phosphorylation regulates KCC2 function under more physiological conditions is unclear, although modulation of KCC2 has important implications for inhibitory synaptic transmission and neuronal excitability. Furthermore, although KCC2 is uniquely expressed in neurons and may be influenced by the neuronal microenvironment, many of the studies on modulation of KCC function have been done in non-neuronal cell lines, e.g. HEK293 cells, and Xenopus oocytes. In this study, we therefore examined the role and mechanisms of tyrosine phosphorylation in the regulation of KCC2 function in cultured hippocampal neurons and in GT1-7 cells, a brain-derived cell line that possesses many neuronal characteristics but does not express endogenous KCC2 (18, 19) (also, see “Experimental Procedures”). The present study proposes that tyrosine phosphorylation of KCC2 results in clustering within lipid rafts via interactions within the carboxyl terminus of KCC2 and that this clustering results in efficient extrusion of Cl⁻.     

Is Neuronal Injury Caused by Hypoglycemic Coma of the Necrotic or Apoptotic Type?

In this study, we explored if a 30 minute period of hypoglycemic coma yields damage which shows some features associated with apoptosis. To that end, we induced insulin-hypoglycemic coma of 30 min duration, and studied brain tissues after the coma period, and after recovery period of 30 min, 3 h, and 6 h. Histopathological data confirmed neuronal damage in all of the vulnerable neuronal populations. Release of cytochrome c (cyt c), assessed by Western Blot, was observed in the neocortex and caudoputamen after 3 and 6 h of recovery. In these regions, the caspase-like activity increased above control after 6 h of recovery. By laser-scanning confocal microscopy, a clear expression of Bax was observed after 30 min of coma in the superficial layers of the neocortex, reaching a peak after 30 min of recovery. Punctuate immunolabeling surrounding nuclei in soma and dendrites in cortical pyramidal neurons likely represents mitochondria, which suggests that Bax protein assembled at the surface of mitochondria in vulnerable neocortical neurons. It is concluded that although previous morphological data have suggested that cells die by necrosis, neuronal damage after hypoglycemic coma shows some features of apoptosis.     

Inhibitory Effects of Bombusae concretio Salicea on Neuronal Secretion of Alzheimer's β-Amyloid Peptides, a Neurodegenerative Peptide

Alzheimer's disease (AD) is characterized by the age-related deposition of -amyloid (A) 40/42 peptide aggregates in vulnerable brain regions. Multiple levels of evidence implicate a central role for A in the pathophysiology of AD. A is generated by the regulated cleavage of a = 700 amino acid A precursor protein (APP). Full-length APP can undergo proteolytic cleavage either within the A domain to generate secreted sAPP or at the N-terminal and C-terminal domain(s) of A to generate amyloidogenic A peptides. Several epidemiological studies have reported that estrogen replacement therapy protects against the development of AD in postmenopausal women. The aim of this study was to elucidate the antioxidant neuroprotective mechanism of Bombusae concretio Salicea (BC). BC was effective protectants against oxidative glutamate toxicity in the murine neuroblastoma cells (N2a) and human neuroblastoma cells (SK-N-MC). BC exhibited similar protective properties against oxidative glutamate toxicity and H₂O₂ toxicity. BC exhibited an antioxidant activity at approximately 20 g/ml. BC of 5 g/ml was ineffective in preventing the oxidative modification of LDL. The half-maximal effective concentration for BC was 16 g/ml. These results suggested that BC supplementation in elderly men may be protective in the treatment of Alzheimer's disease (AD). We report here that treatment with BC increases the secretion of the nonamyloidogenic APP fragment, sAPP and decreases the secretion of A peptides from N2a cells and rat primary cerebrocortical neurons. These results raise the possibility that BC supplementation in elderly men may be protective in the treatment of AD.     

Involvement of α7 nAChR Signaling Cascade in Epigallocatechin Gallate Suppression of β-Amyloid-Induced Apoptotic Cortical Neuronal Insults

Excessive generation and accumulation of the β-amyloid (Aβ) peptide in selectively vulnerable brain regions is a key pathogenic event in the Alzheimer's disease (AD), while epigallocatechin gallate (EGCG) is a very promising chemical to suppress a variety of Aβ-induced neurodegenerative disorders. However, the precise molecular mechanism of EGCG responsible for protection against neurotoxicity still remains elusive. To validate and further investigate the possible mechanism involved, we explored whether EGCG neuroprotection against neurotoxicity of Aβ is mediated through the α7 nicotinic acetylcholine receptor (α7 nAChR) signaling cascade. It was shown in rat primary cortical neurons that short-term treatment with EGCG significantly attenuated the neurotoxicity of Aβ_1–42, as demonstrated by increased cell viability, reduced number of apoptotic cells, decreased reactive oxygen species (ROS) generation, and downregulated caspase-3 levels after treatment with 25-μM Aβ_1–42. In addition, EGCG markedly strengthened activation of α7nAChR as well as its downstream pathway signaling molecules phosphatidylinositol 3-kinase (PI3K) and Akt, subsequently leading to suppression of Bcl-2 downregulation in Aβ-treated neurons. Conversely, administration of α7nAChR antagonist methyllycaconitine (MLA; 20 μM) to neuronal cultures significantly attenuated the neuroprotection of EGCG against Aβ-induced neurototoxicity, thus presenting new evidence that the α7nAChR activity together with PI3K/Akt transduction signaling may contribute to the molecular mechanism underlying the neuroprotective effects of EGCG against Aβ-induced cell death.