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1.
Cytoskeleton (Hoboken) ; 73(9): 477-97, 2016 Sep.
Article in English | MEDLINE | ID: mdl-26873625

ABSTRACT

Cytoskeletal abnormalities and synaptic loss, typical of both familial and sporadic Alzheimer disease (AD), are induced by diverse stresses such as neuroinflammation, oxidative stress, and energetic stress, each of which may be initiated or enhanced by proinflammatory cytokines or amyloid-ß (Aß) peptides. Extracellular Aß-containing plaques and intracellular phospho-tau-containing neurofibrillary tangles are postmortem pathologies required to confirm AD and have been the focus of most studies. However, AD brain, but not normal brain, also have increased levels of cytoplasmic rod-shaped bundles of filaments composed of ADF/cofilin-actin in a 1:1 complex (rods). Cofilin, the major ADF/cofilin isoform in mammalian neurons, severs actin filaments at low cofilin/actin ratios and stabilizes filaments at high cofilin/actin ratios. It binds cooperatively to ADP-actin subunits in F-actin. Cofilin is activated by dephosphorylation and may be oxidized in stressed neurons to form disulfide-linked dimers, required for bundling cofilin-actin filaments into stable rods. Rods form within neurites causing synaptic dysfunction by sequestering cofilin, disrupting normal actin dynamics, blocking transport, and exacerbating mitochondrial membrane potential loss. Aß and proinflammatory cytokines induce rods through a cellular prion protein-dependent activation of NADPH oxidase and production of reactive oxygen species. Here we review recent advances in our understanding of cofilin biochemistry, rod formation, and the development of cognitive deficits. We will then discuss rod formation as a molecular pathway for synapse loss that may be common between all three prominent current AD hypotheses, thus making rods an attractive therapeutic target. © 2016 Wiley Periodicals, Inc.


Subject(s)
Actin Cytoskeleton/metabolism , Actin Depolymerizing Factors/metabolism , Actins/metabolism , Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Actin Cytoskeleton/pathology , Adenosine Diphosphate/metabolism , Alzheimer Disease/pathology , Animals , Biological Transport, Active , Humans , Protein Multimerization
2.
PLoS One ; 8(12): e83609, 2013.
Article in English | MEDLINE | ID: mdl-24391794

ABSTRACT

Filament bundles (rods) of cofilin and actin (1:1) form in neurites of stressed neurons where they inhibit synaptic function. Live-cell imaging of rod formation is hampered by the fact that overexpression of a chimera of wild type cofilin with a fluorescent protein causes formation of spontaneous and persistent rods, which is exacerbated by the photostress of imaging. The study of rod induction in living cells calls for a rod reporter that does not cause spontaneous rods. From a study in which single cofilin surface residues were mutated, we identified a mutant, cofilinR21Q, which when fused with monomeric Red Fluorescent Protein (mRFP) and expressed several fold above endogenous cofilin, does not induce spontaneous rods even during the photostress of imaging. CofilinR21Q-mRFP only incorporates into rods when they form from endogenous proteins in stressed cells. In neurons, cofilinR21Q-mRFP reports on rods formed from endogenous cofilin and induced by all modes tested thus far. Rods have a half-life of 30-60 min upon removal of the inducer. Vesicle transport in neurites is arrested upon treatments that form rods and recovers as rods disappear. CofilinR21Q-mRFP is a genetically encoded rod reporter that is useful in live cell imaging studies of induced rod formation, including rod dynamics, and kinetics of rod elimination.


Subject(s)
Actin Depolymerizing Factors/metabolism , Actins/metabolism , Neurons/metabolism , Actin Depolymerizing Factors/chemistry , Actin Depolymerizing Factors/genetics , Actins/chemistry , Actins/genetics , Animals , Cell Line, Tumor , Computer Systems , Genes, Reporter , HeLa Cells , Humans , LLC-PK1 Cells , Luminescent Proteins/chemistry , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Models, Molecular , Multiprotein Complexes/chemistry , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Mutagenesis, Site-Directed , Protein Structure, Quaternary , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Single-Cell Analysis , Swine , Synapses/metabolism , Red Fluorescent Protein
3.
J Neurosci ; 32(19): 6670-81, 2012 May 09.
Article in English | MEDLINE | ID: mdl-22573689

ABSTRACT

Rod-shaped aggregates ("rods"), containing equimolar actin and the actin dynamizing protein cofilin, appear in neurons following a wide variety of potentially oxidative stress: simulated microischemia, cofilin overexpression, and exposure to peroxide, excess glutamate, or the dimer/trimer forms of amyloid-ß peptide (Aßd/t), the most synaptotoxic Aß species. These rods are initially reversible and neuroprotective, but if they persist in neurites, the synapses degenerate without neurons dying. Herein we report evidence that rod formation depends on the generation of intermolecular disulfide bonds in cofilin. Of four Cys-to-Ala cofilin mutations expressed in rat E18 hippocampal neurons, only the mutant incapable of forming intermolecular bonds (CC39,147AA) has significantly reduced ability to incorporate into rods. Rod regions show unusually high oxidation levels. Rods, isolated from stressed neurons, contain dithiothreitol-sensitive multimeric forms of cofilin, predominantly dimer. Oligomerization of cofilin in cells represents one more mechanism for regulating the actin dynamizing activity of cofilin and probably underlies synaptic loss.


Subject(s)
Actin Depolymerizing Factors/metabolism , Actins/physiology , Disulfides/metabolism , Neurodegenerative Diseases/metabolism , Actin Depolymerizing Factors/chemistry , Actin Depolymerizing Factors/genetics , Animals , Cell Line, Tumor , Cells, Cultured , Chickens , Disulfides/chemistry , Female , Humans , Male , Mice , Neurodegenerative Diseases/genetics , Oxidation-Reduction , Protein Multimerization/genetics , Rats , Swine
4.
Adv Neurobiol ; 5: 201-234, 2011.
Article in English | MEDLINE | ID: mdl-35547659

ABSTRACT

Abnormal regulation of the actin cytoskeleton results in several pathological conditions affecting primarily the nervous system. Those of genetic origin arise during development, but others manifest later in life. Actin regulation is also affected profoundly by environmental factors that can have sustained consequences for the nervous system. Those consequences follow from the fact that the actin cytoskeleton is essential for a multitude of cell biological functions ranging from neuronal migration in cortical development and dendritic spine formation to NMDA receptor activity in learning and alcoholism. Improper regulation of actin, causing aggregation, can contribute to the neurodegeneration of amyloidopathies, such as Down's syndrome and Alzheimer's disease. Much progress has been made in understanding the molecular basis of these diseases.

5.
F1000 Biol Rep ; 2: 62, 2010 Aug 19.
Article in English | MEDLINE | ID: mdl-21173851

ABSTRACT

In collaboration or competition with many other actin-binding proteins, the actin-depolymerizing factor/cofilins integrate transmembrane signals to coordinate the spatial and temporal organization of actin filament assembly/disassembly (dynamics). In addition, newly discovered effects of these proteins in lipid metabolism, gene regulation, and apoptosis suggest that their roles go well beyond regulating the cytoskeleton.

6.
Trends Cell Biol ; 20(4): 187-95, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20133134

ABSTRACT

Recent findings have significantly expanded our understanding of the regulation of actin-depolymerizing factor (ADF)/cofilin proteins and the profound multifaceted impact that these well-established regulators of actin dynamics have on cell biology. In this review we discuss new aspects of previously documented regulation, such as phosphorylation, but also cover novel recently established modes of regulation and functions of ADF (also known as destrin)/cofilin. We now understand that their activity responds to a vast array of inputs far greater than previously appreciated and that these proteins not only feed back to the crucially important dynamics of actin, but also to apoptosis cascades, phospholipid metabolism, and gene expression. We argue that this ability to respond to physiological changes by modulating those same changes makes the ADF/cofilin protein family a homeostatic regulator or 'functional node' in cell biology.


Subject(s)
Actin Depolymerizing Factors/metabolism , Actins/metabolism , Destrin/metabolism , Fibroblasts/metabolism , Neurons/metabolism , Actin Depolymerizing Factors/chemistry , Actin Depolymerizing Factors/genetics , Animals , Destrin/chemistry , Destrin/genetics , Fibroblasts/cytology , Gene Expression Regulation , HeLa Cells/cytology , HeLa Cells/metabolism , Humans , Mice , Models, Molecular , Neurons/cytology
7.
Curr Biol ; 20(8): R360-2, 2010 Apr 27.
Article in English | MEDLINE | ID: mdl-21749956

ABSTRACT

Mical, a redox enzyme, binds the cytoplasmic domain of the semaphorin receptor plexin A and mediates semaphorin-signaled collapse of the actin cytoskeleton. Recent work now shows that Mical's ability to bind actin filaments and destabilize them in a NADPH-dependent manner is responsible for semaphorin 1a's effects.


Subject(s)
DNA-Binding Proteins/metabolism , Neurons/physiology , Oxidation-Reduction , Signal Transduction/physiology , Animals , Drosophila/anatomy & histology , Drosophila/physiology , Growth Cones/metabolism
9.
Am J Physiol Cell Physiol ; 291(5): C828-39, 2006 Nov.
Article in English | MEDLINE | ID: mdl-16738008

ABSTRACT

When neurons in culture are transiently stressed by inhibition of ATP synthesis, they rapidly form within their neurites rodlike actin inclusions that disappear when the insult is removed. Oxidative stress, excitotoxic insults, and amyloid beta-peptide oligomers also induce rods. Immunostaining of neurites indicates that these rods also contain the majority of the actin filament dynamizing proteins, actin-depolymerizing factor (ADF) and cofilin (AC). If the rods reappear within 24 h after the stress is removed, the neurite degenerates distal to the rod but with no increase in neuronal death. Here, rods were generated in cultured rat E18 hippocampal cells by overexpression of a green fluorescent protein chimera of AC. Surprisingly, we have found that, for a short period (approximately 60 min) immediately after initial rod formation, the loss of mitochondrial membrane potential (Delta Psi(m)) and ATP in neurites with rods is slower than in neurites without them. The Delta Psi(m) was monitored with the fluorescent dye tetramethylrhodamine methyl ester, and ATP was monitored with the fluorescent ion indicator mag-fura 2. Actin in rods is less dynamic than is filamentous actin in other cytoskeletal structures. Because Delta Psi(m) depends on cellular ATP and because ATP hydrolysis associated with actin filament turnover is responsible for a large fraction of neuronal energy consumption (approximately 50%), the formation of rods transiently protects neurites by slowing filament turnover and its associated ATP hydrolysis.


Subject(s)
Actins/metabolism , Adenosine Triphosphate/metabolism , Destrin/metabolism , Membrane Potentials/physiology , Mitochondrial Membranes/metabolism , Neurons/metabolism , Neurons/pathology , Animals , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cells, Cultured , Fluorescence Recovery After Photobleaching , Hippocampus/cytology , Hippocampus/drug effects , Humans , Membrane Potentials/drug effects , Mitochondrial Membranes/drug effects , Neurons/drug effects , Protein Transport/drug effects , Pseudopodia/drug effects , Rats , Recombinant Fusion Proteins/metabolism , Thiazolidines/pharmacology , Xenopus laevis
11.
Biochemistry ; 43(22): 7127-42, 2004 Jun 08.
Article in English | MEDLINE | ID: mdl-15170350

ABSTRACT

The actin depolymerizing factor (ADF)/cofilins are an essential group of proteins that are important regulators of actin filament turnover in vivo. Although protists and yeasts express only a single member of this family, metazoans express two or more members in many cell types. In cells expressing both ADF and cofilin, differences have been reported in the regulation of their expression, their pH sensitivity, and their intracellular distribution. Each member has qualitatively similar interactions with actin, but quantitative differences have been noted. Here we compared quantitative differences between chick ADF and chick cofilin using several assays that measure G-actin binding, actin filament length distribution, and assembly/disassembly dynamics. Quantitative differences were measured in the critical concentrations of the complexes required for assembly, in the effects of nucleotide and divalent metal on actin monomer binding, in pH-dependent severing, in enhancement of filament minus end off-rates, and in steady-state filament length distributions generated in similar mixtures. Some of these assays were used to compare the activities of several ADF/cofilins from across phylogeny, most of which fall into one of two groups based upon their behavior. The ADF-like group has higher affinities for Mg(2+)-ATP-G-actin than the cofilin-like group and a greater pH-dependent depolymerizing activity.


Subject(s)
Actin Cytoskeleton/metabolism , Actins/metabolism , Microfilament Proteins/metabolism , Actin Depolymerizing Factors , Actins/chemistry , Actins/genetics , Animals , Brain/metabolism , Chickens , Destrin , Gelsolin/metabolism , Humans , Hydrogen-Ion Concentration , In Vitro Techniques , Microfilament Proteins/chemistry , Microfilament Proteins/genetics
13.
Methods Cell Biol ; 71: 37-50, 2003.
Article in English | MEDLINE | ID: mdl-12884685

ABSTRACT

This chapter describes the function and development of the ciliary ganglion, the potential of ciliary ganglion neurons as a cell biological tool, and their dissection, dissociation, and culturing. Ciliary ganglion neurons grow unusually rapidly on a laminin-based substratum and develop large, thin calyx terminals in culture in less than 12 h. The two neuronal classes present in the cultures can be identified by size alone. The limited number of ganglia per animal renders this ganglion a poor choice for biochemical studies based on the extraction of cultured cells. However, they are ideally suited for studies based on single-cell observation, particularly investigation of presynaptic mechanisms using fluorescence microscopy.


Subject(s)
Cell Culture Techniques/methods , Cells, Cultured/cytology , Dissection/methods , Ganglia, Parasympathetic/cytology , Neurons/cytology , Animals , Cell Culture Techniques/instrumentation , Cell Differentiation/physiology , Cells, Cultured/physiology , Chick Embryo , Culture Media/pharmacology , Dissection/instrumentation , Ganglia, Parasympathetic/physiology , Oculomotor Nerve/cytology , Oculomotor Nerve/physiology , Presynaptic Terminals/physiology , Presynaptic Terminals/ultrastructure , Synaptic Transmission/physiology
14.
Methods Cell Biol ; 71: 387-416, 2003.
Article in English | MEDLINE | ID: mdl-12884701

ABSTRACT

Adenoviruses infect a wide range of cell types, do not require integration into the host cell genome, and can be produced as replication-deficient viruses capable of expressing transgenes behind any desired promoter. Thus, they are ideal for use in expressing transgenes in the postmitotic neuron. This chapter describes simplifications in the protocols for making recombinant adenoviruses and their use in expressing transgenes in primary neurons of several different types.


Subject(s)
Adenoviridae/genetics , Genetic Vectors/genetics , Neurons/virology , Transfection/methods , Transgenes/genetics , Animals , Cell Culture Techniques/methods , Cells, Cultured/cytology , Cells, Cultured/physiology , Cells, Cultured/virology , Gene Expression Regulation/genetics , Humans , Neurons/cytology , Neurons/physiology , Virus Replication/genetics
15.
J Neurosci ; 23(1): 1-6, 2003 Jan 01.
Article in English | MEDLINE | ID: mdl-12514193

ABSTRACT

In cultured chick ciliary neurons, when ATP synthesis is inhibited, ATP depletion is reduced approximately 50% by slowing actin filament turnover with jasplakinolide or latrunculin A. Jasplakinolide inhibits actin disassembly, and latrunculin A prevents actin assembly by sequestering actin monomers. Cytochalasin D, which allows assembly-disassembly, but only at pointed ends, is less effective in conserving ATP. Ouabain, an Na(+)-K(+)-ATPase inhibitor, and jasplakinolide both prevent approximately 50% of the ATP loss. When applied together, they completely prevent ATP loss over a period of 20 min, suggesting that filament stabilization reduces ATP consumption by decreasing actin-ATP hydrolysis directly rather than indirectly by modulating the activity of Na(+)-K(+)-ATPase, a major energy consumer.


Subject(s)
Actin Cytoskeleton/metabolism , Actins/metabolism , Adenosine Triphosphate/metabolism , Depsipeptides , Neurons/metabolism , Actin Cytoskeleton/drug effects , Actin Cytoskeleton/ultrastructure , Animals , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Calcium/analysis , Cell Hypoxia , Cells, Cultured , Chick Embryo , Energy Metabolism , Enzyme Inhibitors/pharmacology , Hydrolysis , Neurons/cytology , Neurons/drug effects , Ouabain/pharmacology , Peptides, Cyclic/pharmacology , Sodium/analysis , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors , Thiazoles/pharmacology , Thiazolidines
16.
Radiat Res ; 157(1): 26-31, 2002 Jan.
Article in English | MEDLINE | ID: mdl-11754638

ABSTRACT

It has been known for many years that caffeine reduces or eliminates the G2-phase cell cycle delay normally seen in human HeLa cells or Chinese hamster ovary (CHO) cells after exposure to X or gamma rays. In light of our recent demonstration of a consistent difference between human normal and tumor cells in a G2-phase checkpoint response in the presence of microtubule-active drugs, we examined the effect of caffeine on the G2-phase delays after exposure to gamma rays for cells of three human normal cell lines (GM2149, GM4626, AG1522) and three human tumor cell lines (HeLa, MCF7, OVGI). The G2-phase delays after a dose of 1 Gy were similar for all six cell lines. In agreement with the above-mentioned reports for HeLa and CHO cells, we also observed that the G2-phase delays were eliminated by caffeine in the tumor cell lines. In sharp contrast, caffeine did not eliminate or even reduce the gamma-ray-induced G2-phase delays in any of the human normal cell lines. Since caffeine has several effects in cells, including the inhibition of cAMP and cGMP phosphodiesterases, as well as causing a release of Ca(++) from intracellular stores, we evaluated the effects of other drugs affecting these processes on radiation-induced G2-phase delays in the tumor cell lines. Drugs that inhibit cAMP or cGMP phosphodiesterases did not eliminate the radiation-induced G2-phase delay either separately or in combination. The ability of caffeine to eliminate radiation-induced G2-phase delay was, however, partially reduced by ryanodine and eliminated by thapsigargin, both of which can modulate intracellular calcium, but by different mechanisms. To determine if caffeine was acting through the release of calcium from intracellular stores, calcium was monitored in living cells using a fluorescent calcium indicator, furaII, before and after the addition of caffeine. No calcium release was seen after the addition of caffeine in either OVGI tumor cells or GM2149 normal cells, even though a large calcium release was measured in parallel experiments with ciliary neurons. Thus it is likely that caffeine is eliminating the radiation-induced G2-phase delay through a Ca(++)-independent mechanism, such as the inhibition of a cell cycle-regulating kinase.


Subject(s)
Caffeine/pharmacology , G2 Phase/drug effects , Gamma Rays/adverse effects , Neoplastic Stem Cells/drug effects , 3',5'-Cyclic-AMP Phosphodiesterases/antagonists & inhibitors , 3',5'-Cyclic-GMP Phosphodiesterases/antagonists & inhibitors , 4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone/pharmacology , Animals , Breast Neoplasms/pathology , Calcium Channel Blockers/pharmacology , Calcium Signaling/drug effects , Carcinoma/pathology , Chickens , Demecolcine/pharmacology , Drug Resistance , Female , Fibroblasts/cytology , Fibroblasts/drug effects , Fibroblasts/radiation effects , G2 Phase/radiation effects , HeLa Cells/cytology , HeLa Cells/drug effects , HeLa Cells/radiation effects , Humans , Mitosis/drug effects , Mitosis/radiation effects , Neoplastic Stem Cells/cytology , Neoplastic Stem Cells/radiation effects , Neurons/drug effects , Ovarian Neoplasms/pathology , Phosphodiesterase Inhibitors/pharmacology , Quinazolines/pharmacology , Radiation Tolerance/drug effects , Ryanodine/pharmacology , Ryanodine Receptor Calcium Release Channel/drug effects , Thapsigargin/pharmacology , Tumor Cells, Cultured/cytology , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/radiation effects
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