Alisa E. Shaw

Brain-Derived Neurotrophic Factor Regulation of Retinal Growth Cone Filopodial Dynamics Is Mediated through Actin Depolymerizing Factor/Cofilin

The molecular mechanisms by which neurotrophins regulate growth cone motility are not well understood. This study investigated the signaling involved in transducing BDNF-induced increases of filopodial dynamics. Our results indicate that BDNF regulates filopodial length and number through a Rho kinase-dependent mechanism. Additionally, actin depolymerizing factor (ADF)/cofilin activity is necessary and sufficient to transduce the effects of BDNF. Our data indicate that activation of ADF/cofilin mimics the effects of BDNF on filopodial dynamics, whereas ADF/cofilin inactivity blocks the effects of BDNF. Furthermore, BDNF promotes the activation of ADF/cofilin by reducing the phosphorylation of ADF/cofilin. Although inhibition of myosin II also enhances filopodial length, our results indicate that BDNF signaling is independent of myosin II activity and that the two pathways result in additive effects on filopodial length. Thus, filopodial extension is regulated by at least two independent mechanisms. The BDNF-dependent pathway works via regulation of ADF/cofilin, independently of myosin II activity.

ADF/Cofilin-Mediated Actin Retrograde Flow Directs Neurite Formation in the Developing Brain

Neurites are the characteristic structural element of neurons that will initiate brain connectivity and elaborate information. Early in development, neurons are spherical cells but this symmetry is broken through the initial formation of neurites. This fundamental step is thought to rely on actin and microtubule dynamics. However, it is unclear which aspects of the complex actin behavior control neuritogenesis and which molecular mechanisms are involved. Here, we demonstrate that augmented actin retrograde flow and protrusion dynamics facilitate neurite formation. Our data indicate that a single family of actin regulatory proteins, ADF/Cofilin, provides the required control of actin retrograde flow and dynamics to form neurites. In particular, the F-actin severing activity of ADF/Cofilin organizes space for the protrusion and bundling of microtubules, the backbone of neurites. Our data reveal how ADF/Cofilin organizes the cytoskeleton to drive actin retrograde flow and thus break the spherical shape of neurons.

Growth cone‐like waves transport actin and promote axonogenesis and neurite branching

Axonogenesis involves a shift from uniform delivery of materials to all neurites to preferential delivery to the putative axon, supporting its more rapid extension. Waves, growth cone‐like structures that propagate down the length of neurites, were shown previously to correlate with neurite growth in dissociated cultured hippocampal neurons. Waves are similar to growth cones in their structure, composition and dynamics. Here, we report that waves form in all undifferentiated neurites, but occur more frequently in the future axon during initial neuronal polarization. Moreover, wave frequency and their impact on neurite growth are altered in neurons treated with stimuli that enhance axonogenesis. Coincident with wave arrival, growth cones enlarge and undergo a marked increase in dynamics. Through their engorgement of filopodia along the neurite shaft, waves can induce de novo neurite branching. Actin in waves maintains much of its cohesiveness during transport whereas actin in nonwave regions of the neurite rapidly diffuses as measured by live cell imaging of photoactivated GFP‐actin and photoconversion of Dendra‐actin. Thus, waves represent an alternative axonal transport mechanism for actin. Waves also occur in neurons in organotypic hippocampal slices where they propagate along neurites in the dentate gyrus and the CA regions and induce branching. Taken together, our results indicate that waves are physiologically relevant and contribute to axon growth and branching via the transport of actin and by increasing growth cone dynamics.

ADF/Cofilin Regulates Actomyosin Assembly through Competitive Inhibition of Myosin II Binding to F-Actin

The contractile actin cortex is important for diverse fundamental cell processes, but little is known about how the assembly of F-actin and myosin II motors is regulated. We report that depletion of actin depolymerizing factor (ADF)/cofilin proteins in human cells causes increased contractile cortical actomyosin assembly. Remarkably, our data reveal that the major cellular defects resulting from ADF/cofilin depletion, including cortical F-actin accumulation, were largely due to excessive myosin II activity. We identify that ADF/cofilins from unicellular organisms to humans share a conserved activity to inhibit myosin II binding to F-actin, indicating a mechanistic rationale for our cellular results. Our study establishes an essential requirement for ADF/cofilin proteins in the control of normal cortical contractility and in processes such as mitotic karyokinesis. We propose that ADF/cofilin proteins are necessary for controlling actomyosin assembly and intracellular contractile force generation, a function of equal physiological importance to their established roles in mediating F-actin turnover.

Arp2/3- and cofilin-coordinated actin dynamics is required for insulin-mediated GLUT4 translocation to the surface of muscle cells.

Insulin increases GLUT4 at the muscle cell surface, and this process requires actin remodeling. We show that a dynamic cycle of actin polymerization and severing is induced by insulin, governed by Arp2/3 and dephosphorylation of cofilin, respectively. The cycle is self-perpetuating and is essential for GLUT4 translocation.

Incorporation of Cofilin into Rods Depends on Disulfide Intermolecular Bonds: Implications for Actin Regulation and Neurodegenerative Disease

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-β peptide (Aβd/t), the most synaptotoxic Aβ 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.

Production and use of replication-deficient adenovirus for transgene expression in neurons.

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.

Amyloid-β and proinflammatory cytokines utilize a prion protein-dependent pathway to activate NADPH oxidase and induce cofilin-actin rods in hippocampal neurons.

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

Peptide regulation of cofilin activity in the CNS: A novel therapeutic approach for treatment of multiple neurological disorders

&NA; Cofilin is a ubiquitous protein which cooperates with many other actin‐binding proteins in regulating actin dynamics. Cofilin has essential functions in nervous system development including neuritogenesis, neurite elongation, growth cone pathfinding, dendritic spine formation, and the regulation of neurotransmission and spine function, components of synaptic plasticity essential for learning and memory. Cofilins phosphoregulation is a downstream target of many transmembrane signaling processes, and its misregulation in neurons has been linked in rodent models to many different neurodegenerative and neurological disorders including Alzheimer disease (AD), aggression due to neonatal isolation, autism, manic/bipolar disorder, and sleep deprivation. Cognitive and behavioral deficits of these rodent models have been largely abrogated by modulation of cofilin activity using viral‐mediated, genetic, and/or small molecule or peptide therapeutic approaches. Neuropathic pain in rats from sciatic nerve compression has also been reduced by modulating the cofilin pathway within neurons of the dorsal root ganglia. Neuroinflammation, which occurs following cerebral ischemia/reperfusion, but which also accompanies many other neurodegenerative syndromes, is markedly reduced by peptides targeting specific chemokine receptors, which also modulate cofilin activity. Thus, peptide therapeutics offer potential for cost‐effective treatment of a wide variety of neurological disorders. Here we discuss some recent results from rodent models using therapeutic peptides with a surprising ability to cross the rodent blood brain barrier and alter cofilin activity in brain. We also offer suggestions as to how neuronal‐specific cofilin regulation might be achieved.

A Genetically Encoded Reporter for Real-Time Imaging of Cofilin-Actin Rods in Living Neurons

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.