Dynamics In Neurons Research Articles

MicroTUB(B3)ules and Brain Development

The microtubule network is crucial for the developing nervous system, and mutations in tubulin-encoding genes disrupt neuronal migration. Tischfield et al. (2010) now report that mutations in the tubulin-encoding gene TUBB3 have a striking impact on microtubule dynamics in neurons, resulting in a diverse set of disease symptoms.

Three-dimensional Computational Modeling of Cellular Calcium Dynamics in Healthy and Pathological Neurons

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Neuroprotective Effects of Selective N-Type VGCC Blockade on Stretch-Injury-Induced Calcium Dynamics in Cortical Neurons

Acute elevation in intracellular calcium ([Ca(2+)](i)) following traumatic brain injury (TBI) can trigger cellular mechanisms leading to neuronal dysfunction and death. The mechanisms underlying these processes are not completely understood, but calcium influx through N-type voltage-gated calcium channels (VGCCs) appears to play a central role. The present study examined the time course of [Ca(2+)](i) flux, glutamate release, and loss of cell viability following injury using an in vitro neuronal-glial cortical cell-culture model of TBI. The effects of N-channel blockade with SNX-185 (e.g. omega-conotoxin TVIA) before or after injury were also examined. Neuronal injury produced a transient elevation in [Ca(2+)](i), increased glutamate release, and resulted in neuronal and glial death. SNX-185 administered before or immediately after cell injury reduced glutamate release and increased the survival of neurons and astrocytes, whereas delayed treatment did not improve cell survival but significantly facilitated the return of [Ca(2+)](i) to baseline levels. The new findings that N-type VGCCs are critically involved in injury-induced glutamate release and recovery of [Ca(2+)](i) argue for continued investigation of this treatment strategy for the clinical management of TBI. In particular, SNX-185 may represent an effective class of drugs that can significantly protect injured neurons from the secondary insults that commonly occur after TBI.

Use of NAD(P)H and flavoprotein autofluorescence transients to probe neuron and astrocyte responses to synaptic activation

Synaptic stimulation in brain slices is accompanied by changes in tissue autofluorescence, which are a consequence of changes in tissue metabolism. Autofluorescence excited by ultraviolet light has been most extensively studied, and is due to reduced pyridine nucleotides (NADH and NADPH, collectively termed NAD(P)H). Stimulation generates a characteristic compound NAD(P)H response, comprising an initial fluorescence decrease and then an overshooting increase that slowly recovers to baseline levels. Evoked NAD(P)H transients are relatively easy to record, do not require the addition of exogenous indicators and have good signal-noise ratios. These characteristics make NAD(P)H imaging methods very useful for tracking the spread of neuronal activity in complex brain tissues, however the cellular basis of synaptically-evoked autofluorescence transients has been the subject of recent debate. Of particular importance is the question of whether signals are due primarily to changes in neuronal mitochondrial function, and/or whether astrocyte metabolism triggered by glutamate uptake may be a significant contributor to the overshooting NAD(P)H fluorescence increases. This mini-review addresses the subcellular origins of NAD(P)H autofluorescence and the evidence for mitochondrial and glycolytic contributions to compound transients. It is concluded that there is no direct evidence for a contribution to NAD(P)H signals from glycolysis in astrocytes following synaptic glutamate uptake. In contrast, multiple lines of evidence, including from complimentary flavoprotein autofluorescence signals, imply that mitochondrial NADH dynamics in neurons dominate compound evoked NAD(P)H transients. These signals are thus appropriate for studies of mitochondrial function and dysfunction in brain slices, in addition to providing robust maps of postsynaptic neuronal activation following physiological activation.

Calcium Involvement in Regulation of Neuronal Bursting in Disinhibited Neuronal Networks: Insights from Calcium Studies in a Spherical Cell Model

Cytosolic calcium is involved in the regulation of many intracellular processes. Intracellular calcium may therefore potentially affect the behavior of both single neurons and synaptically connected neuronal assemblies. In computer model studies, we investigated calcium dynamics in spherical neurons during periods of recurrent neuronal bursting that were simulated in a disinhibited neuronal network. The model takes into account calcium influx via voltage-gated calcium channels, extrusion through the cell membrane, and binding to two different buffers representing fixed and mobile endogenous calcium buffers. Throughout the duration of the simulated recurrent neuronal bursting, the concentration of free fixed buffers shows a hyperbolic decrease in time at a rate that is not uniform inside a neuron. Recurrent calcium influxes associated with bursting lead to the formation of gradients in the concentration of the fixed buffer in the radial direction, and are accompanied by the redistribution of mobile buffers acting to compensate for these gradients. Simulated intracellular calcium transients have a slow component characterized by a gradual increase in the calcium baseline level that reaches a plateau 120–200 s after the onset of recurrent bursting. Using this model, we demonstrate what we believe is a novel mechanism of regulation of network excitability that occurs in conditions of prolonged and recurrent neuronal bursting in disinhibited networks. This mechanism is expressed via interaction of calcium clearance systems inside neurons with calcium-dependent potassium regulation of neuronal excitability in membranes. This is a network phenomenon because it arises largely by synaptic interactions. Therefore, it can serve as a network safety mechanism to prevent excessive and uncontrolled neuronal firing resulting from the lack of inhibition or after acute suppression of the inhibitory drive.

Calcium Signaling in Carassius Cerebellar Neurons: Role of the Mitochondria

We studied the involvement of the mitochondria playing the role of a calcium store in the control of calcium exchange in cerebellar neurons of a fish species tolerant to hypoxia, crucian (Carassius gibelio). In our experiments we used an ionophore, CCCP, that blocked accumulation of calcium by the above organelles. The intracellular concentration of free Ca2+ ([Ca2+] і ) was measured using a calcium-sensitive dye, Fura-2AM, and the microfluorescent technique. We found that cerebellar neurons of Carassius gibelio possess a well-expressed system clearing the cytoplasm from excessive Ca2+, and the mitochondria are actively involved in this process. Under conditions of suppression of the process of accumulation of calcium by the mitochondria under the action of CCCP, the amplitude of calcium transients increased by about 50%. In addition, the decay phase of depolarization-induced intracellular calcium transients was slowed down considerably. Therefore, our experiments are indicative of the significant role of the mitochondria in the control of calcium dynamics in cerebellar neurons of Carassius gibelio in the course of functional activity of these cells.

Investigating Sub-Spine Actin Dynamics in Rat Hippocampal Neurons with Super-Resolution Optical Imaging

Morphological changes in dendritic spines represent an important mechanism for synaptic plasticity which is postulated to underlie the vital cognitive phenomena of learning and memory. These morphological changes are driven by the dynamic actin cytoskeleton that is present in dendritic spines. The study of actin dynamics in these spines traditionally has been hindered by the small size of the spine. In this study, we utilize a photo-activation localization microscopy (PALM)–based single-molecule tracking technique to analyze F-actin movements with ∼30-nm resolution in cultured hippocampal neurons. We were able to observe the kinematic (physical motion of actin filaments, i.e., retrograde flow) and kinetic (F-actin turn-over) dynamics of F-actin at the single-filament level in dendritic spines. We found that F-actin in dendritic spines exhibits highly heterogeneous kinematic dynamics at the individual filament level, with simultaneous actin flows in both retrograde and anterograde directions. At the ensemble level, movements of filaments integrate into a net retrograde flow of ∼138 nm/min. These results suggest a weakly polarized F-actin network that consists of mostly short filaments in dendritic spines.

Inositol 1,4,5-Trisphosphate 3-Kinase A Functions As a Scaffold for Synaptic Rac Signaling

Activity-dependent alterations of synaptic contacts are crucial for synaptic plasticity. The formation of new dendritic spines and synapses is known to require actin cytoskeletal reorganization specifically during neural activation phases. Yet the site-specific and time-dependent mechanisms modulating actin dynamics in mature neurons are not well understood. In this study, we show that actin dynamics in spines is regulated by a Rac anchoring and targeting function of inositol 1,4,5-trisphosphate 3-kinase A (IP(3)K-A), independent of its kinase activity. On neural activation, IP(3)K-A bound directly to activated Rac1 and recruited it to the actin cytoskeleton in the postsynaptic area. This focal targeting of activated Rac1 induced spine formation through actin dynamics downstream of Rac signaling. Consistent with the scaffolding role of IP(3)K-A, IP(3)K-A knock-out mice exhibited defects in accumulation of PAK1 by long-term potentiation-inducing stimulation. This deficiency resulted in a reduction in the reorganization of actin cytoskeletal structures in the synaptic area of dentate gyrus. Moreover, IP(3)K-A knock-out mice showed deficits of synaptic plasticity in perforant path and in hippocampal-dependent memory performances. These data support a novel model in which IP(3)K-A is critical for the spatial and temporal regulation of spine actin remodeling, synaptic plasticity, and learning and memory via an activity-dependent Rac scaffolding mechanism.

Molecular interaction of α-synuclein with tubulin influences on the polymerization of microtubule in vitro and structure of microtubule in cells

Microtubule dynamics is essential for many vital cellular processes such as in intracellular transport, metabolism, and cell division. Evidences demonstrate that α-synuclein may associate with microtubular cytoskeleton and its major component, tubulin. In the present study, the molecular interaction between α-synuclein and tubulin was confirmed by GST pull-down assay and co-immunoprecipitation. The interacting regions within α-synuclein with tubulin were mapped at the residues 60-100 of α-synuclein that is critical for the binding activity with tubulin. Microtubule assembly assays and sedimentation tests demonstrated that α-synuclein influenced the polymerization of tubulin in vitro, revealing an interacting region-dependent feature. Confocal microscopy detected that exposures of α-synuclein proteins inhibited microtubule formation in the cultured cells, with a length-dependent phenomenon. Our data highlight a potential role of α-synuclein in regulating the microtubule dynamics in neurons. The association of α-synuclein with tubulin may further provide insight into the biological and pathophysiological function of synuclein.

Mutant SOD1 in neuronal mitochondria causes toxicity and mitochondrial dynamics abnormalities

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder characterized by motor neuron degeneration. Mutations in Cu,Zn-superoxide dismutase (SOD1) are responsible for 20% of familial ALS cases via a toxic gain of function. In mutant SOD1 transgenic mice, mitochondria of spinal motor neurons develop abnormal morphology, bioenergetic defects and degeneration, which are presumably implicated in disease pathogenesis. SOD1 is mostly a cytosolic protein, but a substantial portion is associated with organelles, including mitochondria, where it localizes predominantly in the intermembrane space (IMS). However, whether mitochondrial mutant SOD1 contributes to disease pathogenesis remains to be elucidated. We have generated NSC34 motor neuronal cell lines expressing wild-type or mutant SOD1 containing a cleavable IMS targeting signal to directly investigate the pathogenic role of mutant SOD1 in mitochondria. We show that mitochondrially-targeted SOD1 localizes to the IMS, where it is enzymatically active. We prove that mutant IMS-targeted SOD1 causes neuronal toxicity under metabolic and oxidative stress conditions. Furthermore, we demonstrate for the first time neurite mitochondrial fragmentation and impaired mitochondrial dynamics in motor neurons expressing IMS mutant SOD1. These defects are associated with impaired maintenance of neuritic processes. Our findings demonstrate that mutant SOD1 localized in the IMS is sufficient to determine mitochondrial abnormalities and neuronal toxicity, and contributes to ALS pathogenesis.

The JNK pathway amplifies and drives subcellular changes in tau phosphorylation

Neurofibrillary tangles composed of hyperphosphorylated tau are a major hallmark of Alzheimer's Disease. This phosphorylated tau may be a root cause of the disorder and therefore understanding its regulation is important for therapeutic intervention. To model this pathology, Okadaic acid (OA) has been used in primary cultured hippocampal neurons to investigate effects on tau, and the role of the JNK pathway in tau phosphorylation. The use of high content screening has allowed us to quantitatively assess the profound spatiotemporal profile of these proteins, finding dramatic and inhibitable changes. Furthermore, in vitro phosphorylation experiments show that the JNK3 isoform, which is predominantly expressed in the brain, can strongly autophosphorylate itself. This has profound implications on the importance of JNK3 in the CNS and its ability to sustain signaling both towards tau and other apoptotic targets. Together these data provide novel insights into the JNK pathway and a high resolution perspective on how this pathway influences tau phosphorylation and dynamics in neurons.

Burst firing regulates correlated activity in neurons

Understanding how sensory information is encoded by populations of neurons is complicated by the fact that neurons display variability in their responses to repeated presentations of the same stimulus. A further complication comes from the fact that these variabilities can be correlated. In fact, the role of correlations in neural variabilities (noise correlations) has been the focus of much debate in recent years as even a small amount of correlation between a pair of neurons can have dramatic effects on information transmission by neural populations [1]. Recent experimental evidence has shown that burst firing can modulate the amount of noise correlations displayed by neural populations [2]. Here we investigate the role of intrinsic bursting dynamics in model neurons receiving correlated input. These model neurons transition from a tonic firing regime to a bursting regime as the amount of depolarizing current is varied. We find that, given an input correlation c, the output correlation R between neural pairs in the network is less when both neurons are in bursting regime than when they are in the tonic regime. Our theoretical results are supported by experimental results obtained from a slice preparation. We show that intrinsic burst dynamics can decorrelate neural populations and therefore regulate their information transmission properties.

Dynein motor contributes to stress granule dynamics in primary neurons

Mobilization and translation of mRNAs are essential for cellular response to environmental stress, and important to neuronal survive and activity. Studies have shown mRNAs accumulation in stress granules (SGs) in stressed cells. We previously reported the formation of SGs in embryonal carcinoma cells can be regulated by kinases such as focal adhesion kinase (FAK) and RNA binding proteins such as Grb7. However, the formation and disassembly of SGs in neurons remains unclear. In this study, we used arsenite to induce cellular oxidative stress in primary neuronal cultures, and found SGs formation in both cell bodies and neurites of stressed primary spinal cord neurons. By employing siRNA dockdown, we discovered that dynein motor subunit localizes in SG, and is important for SG assembly in neurons. Under stress, dynein motor subunit also facilitates translational repression and enhances the formation and integrity of SG in neurons. By blocking the energy source of dynein motor, both the formation and disassembly of SG are attenuated. These findings demonstrate, for the first time, that dynein motor complex plays a critical role in the formation of neuronal SGs, as well as translation of certain mRNAs. This work was supported by DA11190, DA11806, DK54733, DK60521, K02‐DA13926 to L.‐N. W.

Facilitating neural dynamics for delay compensation: A road to predictive neural dynamics?

Goal-directed behavior is a hallmark of cognition. An important prerequisite to goal-directed behavior is that of prediction. In order to establish a goal and devise a plan, one needs to see into the future and predict possible future events. Our earlier work has suggested that compensation mechanisms for neuronal transmission delay may have led to a preliminary form of prediction. In that work, facilitating neuronal dynamics was found to be effective in overcoming delay (the Facilitating Activation Network model, or FAN). The extrapolative property of the delay compensation mechanism can be considered as prediction for incoming signals (predicting the present based on the past). The previous FAN model turns out to have a limitation especially when longer delay needs to be compensated, which requires higher facilitation rates than FAN's normal range. We derived an improved facilitating dynamics at the neuronal level to overcome this limitation. In this paper, we tested our proposed approach in controllers for 2D pole balancing, where the new approach was shown to perform better than the previous FAN model. Next, we investigated the differential utilization of facilitating dynamics in sensory vs. motor neurons and found that motor neurons utilize the facilitating dynamics more than the sensory neurons. These findings are expected to help us better understand the role of facilitating dynamics in delay compensation, and its potential development into prediction, a necessary condition for goal-directed behavior.

In Situ Mitochondrial Ca2+ Buffering Differences of Intact Neurons and Astrocytes from Cortex and Striatum

The striatum is particularly vulnerable to neurological disorders, such as Huntington disease. Previous studies, with nonsynaptic mitochondria isolated from cortical and striatal homogenates, suggest that striatal mitochondria are highly vulnerable to Ca(2+) loads, possibly influencing striatal vulnerability. However, whether and how neuronal and glial mitochondria from cortex and striatum differ in Ca(2+) vulnerability remains unknown. We test this hypothesis using a novel strategy allowing comparisons of mitochondrial Ca(2+) buffering capacity in cortical and striatal neuron-astrocyte co-cultures. We provide original evidence that mitochondria not only in neurons but also in astrocytes from striatal origin exhibit a decreased Ca(2+) buffering capacity when compared with cortical counterparts. The decreased mitochondrial Ca(2+) buffering capacity in striatal versus cortical astrocytes does not stem from variation in mitochondrial concentration or in the rate of intracellular Ca(2+) elevation, being mechanistically linked to an increased propensity to undergo cyclosporin A (CsA)-sensitive permeability transition. Indeed, 1 microm CsA selectively increased the mitochondrial Ca(2+) buffering capacity of striatal astrocytes, without modifying that of neurons or cortical astrocytes. Neither thapsigargin nor FK506 modified mitochondrial Ca(2+) buffering differences between cell types, excluding a predominant contribution of endoplasmic reticulum or calcineurin. These results provide additional insight into the mechanisms of striatal vulnerability, showing that the increased Ca(2+) vulnerability of striatal versus cortical mitochondria resides in both intact neurons and astrocytes, thus positioning the striatum at greater risk for disturbed neuron-astrocyte interactions. Also, the selective effect of CsA over striatal astrocytes suggests that in vivo neuronal sheltering with this compound may indirectly result from astrocytic protection.

Dynein motor contributes to stress granule dynamics in primary neurons

Mobilization and translation of mRNAs, two important events believed to involve stress granules (SGs), in neurons are important for their survival and activities. However, the formation and disassembly of SGs in neurons remains unclear. By using an arsenite-induced neuronal stress model of rat primary spinal cord neuron cultures, we demonstrate the formation of SGs that contain common SG components and RNAs in both stressed neuronal cell bodies and their neurites. By employing small interfering RNA (siRNA) knockdown, we discovered that dynein motor subunit localizes in SG, and is important for SG assembly in neurons. Under stress, dynein motor subunit also facilitates translational repression and enhances the formation and integrity of SG in neurons. By blocking the energy source of dynein motor, both the formation and disassembly of SG are attenuated. These findings demonstrate, for the first time, that dynein motor complex plays a critical role in the dynamics of neuronal SGs, as well as translation of certain mRNAs.

アポリポ蛋白Eと脳内コレステロール代謝

Although, studies of lipid metabolism have been focusing on that in the systemic circulation using nonneuronalcells for decades, knowledge regarding cholesterol metabolism in the brain, the most lipid–rich organs, is very limit-ed. The cholesterol metabolism in the brain is supposed to be independently and uniquely regulated, because brainis segregated from the systemic circulation by the blood–brain barrier. Thus, the cholesterol being transported inthe systemic circulation by serum lipoprotein cannot be available to the brain.It has been shown that cholesterolsupplied as a lipoprotein complex, i.e., high–density–lipoprotein (HDL), is critical for the maturation of synapses andthe maintenance of synaptic plasticity.Interestingly, apolipoprotein E (apoE)is the major apolipoprotein generatingHDL in the brain, which is supplied to neurons via apoE receptors. The discovery that possession of apoE alleleε4is a strong risk factor for Alzheimer’s disease (AD)leads us to focus on the role of cholesterol in the pathogenesis ofAD. Accumulating epidemiological and biological evidence suggests the link between the serum cholesterol leveland the development of AD, and the potential therapeutic effectiveness of statins for AD and mild cognitive impair-ment (MCI), whereas other lines of evidence show controversial results. Cholesterol is known to interact with amy-loidβ–protein (Aβ)in a reciprocal manner: cellular cholesterol levels modulate Aβgeneration, while, Aβalters cho-lesterol dynamics in neurons, leading to tauopathy.In this review, the relationship between the cholesterol levels inserum or cerebrospinal fluid (CSF)and the induction of AD is discussed. The mechanism(s), if this is the case, ofhow cholesterol in the central nervous system (CNS)is involved in the induction of pathologies of AD includingAβgeneration and tauopathy, and how statins prevent it are also discussed.

Amyloid-β overproduction causes abnormal mitochondrial dynamics via differential modulation of mitochondrial fission/fusion proteins

Mitochondrial dysfunction is a prominent feature of Alzheimer disease but the underlying mechanism is unclear. In this study, we investigated the effect of amyloid precursor protein (APP) and amyloid beta on mitochondrial dynamics in neurons. Confocal and electron microscopic analysis demonstrated that approximately 40% M17 cells overexpressing WT APP (APPwt M17 cells) and more than 80% M17 cells overexpressing APPswe mutant (APPswe M17 cells) displayed alterations in mitochondrial morphology and distribution. Specifically, mitochondria exhibited a fragmented structure and an abnormal distribution accumulating around the perinuclear area. These mitochondrial changes were abolished by treatment with beta-site APP-cleaving enzyme inhibitor IV. From a functional perspective, APP overexpression affected mitochondria at multiple levels, including elevating reactive oxygen species levels, decreasing mitochondrial membrane potential, and reducing ATP production, and also caused neuronal dysfunction such as differentiation deficiency upon retinoic acid treatment. At the molecular level, levels of dynamin-like protein 1 and OPA1 were significantly decreased whereas levels of Fis1 were significantly increased in APPwt and APPswe M17 cells. Notably, overexpression of dynamin-like protein 1 in these cells rescued the abnormal mitochondrial distribution and differentiation deficiency, but failed to rescue mitochondrial fragmentation and functional parameters, whereas overexpression of OPA1 rescued mitochondrial fragmentation and functional parameters, but failed to restore normal mitochondrial distribution. Overexpression of APP or Abeta-derived diffusible ligand treatment also led to mitochondrial fragmentation and reduced mitochondrial coverage in neuronal processes in differentiated primary hippocampal neurons. Based on these data, we concluded that APP, through amyloid beta production, causes an imbalance of mitochondrial fission/fusion that results in mitochondrial fragmentation and abnormal distribution, which contributes to mitochondrial and neuronal dysfunction.

Abelson tyrosine kinase links PDGFbeta receptor activation to cytoskeletal regulation of NMDA receptors in CA1 hippocampal neurons

BackgroundWe have previously demonstrated that PDGF receptor activation indirectly inhibits N-methyl-D-aspartate (NMDA) currents by modifying the cytoskeleton. PDGF receptor ligand is also neuroprotective in hippocampal slices and cultured neurons. PDGF receptors are tyrosine kinases that control a variety of signal transduction pathways including those mediated by PLCγ. In fibroblasts Src and another non-receptor tyrosine kinase, Abelson kinase (Abl), control PDGF receptor regulation of cytoskeletal dynamics. The mechanism whereby PDGF receptor regulates cytoskeletal dynamics in central neurons remains poorly understood.ResultsIntracellular applications of active Abl, but not heat-inactivated Abl, decreased NMDA-evoked currents in isolated hippocampal neurons. This mimics the effects of PDGF receptor activation in these neurons. The Abl kinase inhibitor, STI571, blocked the inhibition of NMDA currents by Abl. We demonstrate that PDGF receptors can activate Abl kinase in hippocampal neurons via mechanisms similar to those observed previously in fibroblasts. Furthermore, PDGFβ receptor activation alters the subcellular localization of Abl. Abl kinase is linked to actin cytoskeletal dynamics in many systems. We show that the inhibition of NMDA receptor currents by Abl kinase is blocked by the inclusion of the Rho kinase inhibitor, Y-27632, and that activation of Abl correlates with an increase in ROCK tyrosine phosphorylation.ConclusionThis study demonstrates that PDGFβ receptors act via an interaction with Abl kinase and Rho kinase to regulated cytoskeletal regulation of NMDA receptor channels in CA1 pyramidal neurons.

A critical role of Rho-kinase ROCK2 in the regulation of spine and synaptic function

The actin cytoskeleton is critically involved in the regulation of the dendritic spine and synaptic properties, but the molecular mechanisms underlying actin dynamics in neurons are poorly defined. We took genetic approaches to create and analyze knockout mice specifically lacking ROCK2, a protein kinase that directly interacts with and is activated by the Rho GTPases, the central mediator of actin reorganization. We demonstrated that while these knockout mice were normal in gross brain anatomy, they were impaired in both basal synaptic transmission and hippocampal long-term potentiation (LTP). Consistent with the electrophysiological deficits, the ROCK2 knockout neurons showed deficits in spine properties, synapse density, the actin cytoskeleton, and the actin-binding protein cofilin. These results indicate that ROCK2/cofilin signaling is critical in the regulation of neuronal actin, spine morphology and synaptic function.