ADF/Cofilin-actin rods in neurodegenerative diseases.

Dephosphorylation (activation) of cofilin, an actin binding protein, is stimulated by initiators of neuronal dysfunction and degeneration including oxidative stress, excitotoxic glutamate, ischemia, and soluble forms of beta-amyloid peptide (Abeta). Hyperactive cofilin forms rod-shaped cofilin-saturated actin filament bundles (rods). Other proteins are recruited to rods but are not necessary for rod formation. Neuronal cytoplasmic rods accumulate within neurites where they disrupt synaptic function and are a likely cause of synaptic loss without neuronal loss, as occurs early in dementias. Different rod-inducing stimuli target distinct neuronal populations within the hippocampus. Rods form rapidly, often in tandem arrays, in response to stress. They accumulate phosphorylated tau that immunostains for epitopes present in "striated neuropil threads," characteristic of tau pathology in Alzheimer disease (AD) brain. Thus, rods might aid in further tau modifications or assembly into paired helical filaments, the major component of neurofibrillary tangles (NFTs). Rods can occlude neurites and block vesicle transport. Some rod-inducing treatments cause an increase in secreted Abeta. Thus rods may mediate the loss of synapses, production of excess Abeta, and formation of NFTs, all of the pathological hallmarks of AD. Cofilin-actin rods also form within the nucleus of heat-shocked neurons and are cleared from cells expressing wild type huntingtin protein but not in cells expressing mutant or silenced huntingtin, suggesting a role for nuclear rods in Huntington disease (HD). As an early event in the neurodegenerative cascade, rod formation is an ideal target for therapeutic intervention that might be useful in treatment of many different neurological diseases.

Intracellular pH modulation of ADF/cofilin proteins.

The ADF/cofilin (AC) proteins are necessary for the high rates of actin filament turnover seen in vivo. Their regulation is complex enough to underlie the precision in filament dynamics needed by stimulated cells. Disassembly of actin by AC proteins is inhibited in vitro by phosphorylation of ser3 and pH<7.1. This study of Swiss 3T3 cells demonstrates that pH also affects AC behavior in vivo: (1) Wounded cells show pH-dependent AC translocation to alkaline-induced ruffling membrane; (2) The Triton extractable (soluble) ADF from Swiss 3T3 cells decreases from 42+/-4% to 23+/-4% when the intracellular pH (pH(i)) is reduced from 7.4 to 6.6; (3) Covariance and colocalization analyses of immunostained endogenous proteins show that ADF partitions more with monomeric actin and less with polymeric actin when pH(i) increases. However, the distribution of cofilin, a less pH-sensitive AC in vitro, does not change with pH; (4) Only the unphosphorylatable AC mutant (A3), when overexpressed as a GFP chimera, uniquely produces aberrant cellular phenotypes and only if the pH is shifted from 7.1 to 6.6 or 7.4. A mechanism is proposed that explains why AC(A3)-GFP and AC(wt)-GFP chimeras generate different phenotypes in response to pH changes. Phospho-AC levels increase with cell density, and in motile cells, phospho-AC increases with alkalization, suggesting a homeostatic mechanism that compensates for increased AC activity and filament turnover. These results show that the behavior of AC proteins with pH-sensitivity in vitro is affected by pH in vivo.

Regulating actin dynamics in neuronal growth cones by ADF/cofilin and rho family GTPases.

Growth cone motility and navigation in response to extracellular signals are regulated by actin dynamics. To better understand actin involvement in these processes we determined how and in what form actin reaches growth cones, and once there, how actin assembly is regulated. A continuous supply of actin is maintained at the axon tip by slow transport, the mobile component consisting of an unassembled form of actin. Actin is co-transported with actin-binding proteins, including ADF and cofilin, structurally related proteins essential for rapid turnover of actin filaments in vivo. ADF and cofilin activity is regulated through phosphorylation by LIM kinases, downstream effectors of the Rho family of GTPases, Cdc42, Rac and Rho. Attractive and repulsive extracellular guidance cues might locally alter actin dynamics by binding specific GTPase-linked receptors, activating LIM kinases, and subsequently modulating the activity of ADF/cofilin. ADF is enriched in growth cones and is required for neurite outgrowth. In addition, signals that influence growth cone behavior alter ADF/cofilin phosphorylation, and overexpression of ADF enhances neurite outgrowth. Growth promoting effects of laminin are mimicked by expression of constitutively active Cdc42 and blocked by expression of the dominant negative Cdc42. Repulsive effects of myelin and sema3D on growth cones are blocked by expression of constitutively active Rac1 and dominant negative Rac1, respectively. Thus a series of complex pathways must exist for regulating effectors of actin dynamics. The bifurcating nature of the ADF/cofilin phosphorylation pathway may provide the integration necessary for this complex regulation.

Regulating actin-filament dynamics in vivo.

The assembly and disassembly (i.e. turnover) of actin filaments in response to extracellular signals underlie a wide variety of basic cellular processes such as cell division, endocytosis and motility. The bulk turnover of subunits is 100-200 times faster in cells than with pure actin, suggesting a complex regulation in vivo. Significant progress has been made recently in identifying and clarifying the roles of several cellular proteins that coordinately regulate actin-filament turnover.

Actin disassembles reversibly during electrically induced recycling of synaptic vesicles in cultured neurons.

We have studied depolarization-induced regulation of actin assembly in exocytotically active areas of dissociated chick sympathetic neurons. Active areas were identified with the fluorescent dye FM1-43 which labels synaptic vesicles that recycle in these regions. Exocytosis (electrically stimulated) was monitored in real time through depletion of FM1-43 fluorescence. To study depolarization-induced disassembly of actin in the FM1-43-stained regions, the cells were fixed after different periods of depolarization and stained with rhodamine phalloidin, which binds preferentially to the filamentous form of actin. In active regions, actin disassembles and reassembles during continuous 2 min depolarization. Actin disassembly that occurs after the first 25 s of depolarization was detected by a reduction in rhodamine phalloidin staining and confirmed by correlative electron microscopy. Immunogold staining revealed that actin is abundant throughout resting terminals. In some experiments, actin filaments were stabilized by loading cells with unlabelled phalloidin before stimulating secretion. Stabilizing the filaments does not alter the initial release but strongly reduces the release rate at later stages. These data are consistent with a model in which partial disassembly of actin filaments is necessary for facilitating the transport of vesicles within the terminal and reassembly is necessary for limiting that movement.

Actin in emerging neurites is recruited from a monomer pool.

Does actin in the emerging axons of regenerating neurons arise from the assembled or unassembled actin pool in the cell soma? We investigated this question by loading neurons with one of two fluorescently labeled molecules: rhodamine actin (r-actin) and rhodamine phalloidin (r-phalloidin). The assembly behavior of r-actin in vitro was identical to unlabeled actin. R-phalloidin binds tightly only to the filamentous form of actin (F-actin) and stabilizes filaments against disassembly. Hence, r-phalloidin-tagged filaments should be less likely to disassemble than r-actin-tagged filaments. Neurons of 10-d-old chick embryos were loaded with r-actin or r-phalloidin by triturating trypsinized dorsal root ganglia in isotonic sucrose containing the fluorescently tagged molecule. Isolated neurons were plated on glass coverslips in modified L15 medium containing nerve growth factor. Video images of the live cells on a thermoregulated stage were acquired with a computer imaging system. After 24 h in culture, the fluorescence distribution of r-phalloidin and r-actin was examined in live neurons of comparable morphology, neurite outgrowth, and intensity of somal fluorescence. Greater than 90% of the neurons labeled with r-actin (n = 81) contained detectable levels of fluorescence in emerging neurite fibers, often extending to the tip of the growing process. Less than 10% of the neurons labeled with r-phalloidin (n = 53) contained any fluorescence in the neurite fibers. In those that did contain fluorescence, the r-phalloidin usually was confined to the proximal segment of the neurite, and in no case was it found at the growing tip. Confocal microscopy and cooled CCD imaging of fixed neurons showed that all structures that incorporated r-actin or r-phalloidin also stained with bodipy phallacidin. This colocalization confirms the association of rhodamine-tagged species with F-actin. Our data support a model in which actin, needed in early stages of neurite outgrowth, arises from a pool in the soma that is capable of disassembly.

Cycling of actin assembly in synaptosomes and neurotransmitter release.

We have investigated the regulation of actin assembly in whole mouse brain synaptosomes and how that regulation modulates neurotransmitter release. During a 30 s depolarization with high K+, filamentous actin (F-actin) levels, monitored by staining with rhodamine phalloidin, increase dramatically (up to 300% in 3 s), decrease, and increase once again. This F-actin cycling is regulated by pathways both dependent and independent of Ca2+ influx and is markedly affected by exposing synaptosomes to Li+, tetrodotoxin, and diacylglycerol. Measurement of [3H]norepinephrine release from synaptosomes containing entrapped agents that modulate actin assembly (DNAase I or phalloidin) indicates that actin depolymerization is necessary for normal release and that repolymerization limits release.

Properties of purified actin depolymerizing factor from chick brain.

Actin depolymerizing factor (ADF) from 19-day embryonic chick brains has been purified to greater than 98% homogeneity with a yield of 7.2 mg/100 g of brain. Quantitative immunoblotting with a monospecific antibody to ADF indicated that ADF comprises 0.3% of the total brain protein, resulting in an actual purification yield of about 20%. Brain ADF migrates as a single polypeptide of 19,000 kDa on SDS-containing polyacrylamide gels. The molecular weight of the native protein determined from sedimentation equilibrium in buffers containing from 50 to 200 mM KCl is 20,000. The secondary structure of ADF calculated from the circular dichroic spectrum consists of about 22% alpha-helix, 24% beta-sheet, and 18% beta-turn. ADF contains a blocked N-terminus, a single tryptophan residue located about one-third of the way from one end of the protein, and six cysteine residues (all in reduced form in the native protein). All six cysteine residues could be chemically modified with eosinylmaleimide under nondenaturing conditions; however, ADF activity was lost when more than one cysteine residue was modified. ADF microheterogeneity has been observed upon nonequilibrium pH gradient electrophoresis in polyacrylamide gels containing 9 M urea, the major isoform having a pI of congruent to 7.9-8.0. ADF can interact with either monomeric or filamentous actin to give a complex which can be isolated by gel filtration chromatography. Both major and minor isoforms of the ADF are found in the complex. Assembly-competent actin and active ADF can both be recovered from the complex by chromatography on ATP-saturated DEAE-cellulose.(ABSTRACT TRUNCATED AT 250 WORDS)

Depolarization of brain synaptosomes activates opposing factors involved in regulating levels of cytoskeletal actin.

Depolarization of mouse brain synaptosomes elicits transmitter release and modifies factors that regulate cytoskeletal actin (C-actin) levels. We previously reported (Bernstein and Bamburg, J. Neurosci. 1985. 5:2565-2569) that depolarization causes a release of about 25% of the actin associated with the cytoskeleton of synaptosomal lysates. From our current studies we conclude that depolarization only transiently perturbs the balance in opposing factors which regulate C-actin levels in lysates. Prolonged incubation of the lysates permits the actin to reequilibrate so that no difference between C-actin levels of resting and depolarized synaptosomes is observed. Both the initial transient release of actin from the cytoskeleton and its reassociation with the cytoskeleton during prolonged incubation are calcium dependent and involve factors in both the cytoskeletal and soluble fractions. Depolarization initiates modifications that both increase and decrease the C-actin level probably through mechanisms involving calcium sensitive actin binding proteins.

Reorganization of actin in depolarized synaptosomes.

Depolarization of whole brain synaptosomes, which stimulates transmitter release, also affects regulation of the assembly of actin microfilaments. Lysates of depolarized synaptosomes contain 20% less cytoskeletal actin than lysates of unstimulated synaptosomes. Parameters affecting the assembly of actin are modified before lysis, but release of actin from the Triton-insoluble cytoskeleton does not occur until after lysis. Actin released from the cytoskeleton is not precipitated with myosin, indicating that it consists of monomers and/or short oligomers. Synaptosomes were incubated for 12 sec in one of three solutions of identical ionic strength but of different salt mixtures: 75 mM KCl-2 mM CaCl2, 5 mM KCl-2mM CaCl2, or 75 mM KCl-0.1 mM EGTA. Synaptosomes were then lysed in an F-actin stabilizing buffer containing 1% Triton X-100. Control synaptosomes (no incubation) were lysed directly into the same lysis buffer containing one of the three different salt mixtures. The cytoskeletal and noncytoskeletal actin pools were separated 25 sec after lysis by centrifugation at 10(4) X g for 1 min, and the actin in each pool was quantitated by the DNase I inhibition assay. The drop in cytoskeletal actin induced by depolarization is maximized by including Ca2+ in the depolarizing buffer, and it is blocked completely by adding a neutral thiol protease inhibitor, leupeptin, to either the pre- or post-lysis buffer. The drop is also completely reversed by repolarizing the synaptosomes.

Tropomyosin binding to F-actin protects the F-actin from disassembly by brain actin-depolymerizing factor (ADF).

Brain or muscle F-actin is rapidly depolymerized to monomeric actin in vitro by actin-depolymerizing factor, a protein isolated from chick embryo brain. Binding of muscle tropomyosin to muscle F-actin protects the F-actin from depolymerization by this factor. A 8.4/1.0 molar ratio of actin subunits to tropomyosin, achieved by incubation of the F-actin with excess tropomyosin, protects 58% of the F-actin from depolymerization by excess actin-depolymerizing factor for at least 3 hr at 25 degrees C. Thus, actin-depolymerizing factor seems to be specifically directed toward actin filaments lacking tropomyosin.

In vitro labeling of proteins by reductive methylation: application to proteins involved in supramolecular structures.

Actin and tropomyosin, purified from both muscle and brain, and alpha-actinin, purified from muscle, have been labeled in vitro by reductive methylation to specific activities of greater than 10(5) dpm/micrograms protein. Actin so modified bound DNase I and polymerized identically to unmodified actin. Furthermore, the spectral properties of actin did not change after labeling. The interactions of labeled tropomyosin and alpha-actinin with F-actin were nearly identical to those of the unmodified proteins. These modified proteins comigrated with their unmodified counterparts in both SDS-containing polyacrylamide gels and isoelectric focusing gels. The labeled actin was quantitatively extracted from SDS-containing polyacrylamide gels (yield greater than 98% of radioactivity applied demonstrating that all of the radioactivity was protein bound. The reductive methylation procedure worked well at pH 8.0-8.5 in either pyrophosphate buffer or Bicine buffer using formaldehyde with [3H]-sodium borohydride as the reducing agent. The procedure could also be performed at pH 7.0 in phosphate buffer using (14C]-formaldehyde with sodium cyanoborohydride as the reducing agent. Proteins so labeled are ideal for use in quantitative experiments involving protein-protein interactions.