Ann M. Rajnicek

Hardwiring the Brain: Endocannabinoids Shape Neuronal Connectivity

The roles of endocannabinoid signaling during central nervous system development are unknown. We report that CB1 cannabinoid receptors (CB1Rs) are enriched in the axonal growth cones of γ-aminobutyric acid–containing (GABAergic) interneurons in the rodent cortex during late gestation. Endocannabinoids trigger CB1R internalization and elimination from filopodia and induce chemorepulsion and collapse of axonal growth cones of these GABAergic interneurons by activating RhoA. Similarly, endocannabinoids diminish the galvanotropism of Xenopus laevis spinal neurons. These findings, together with the impaired target selection of cortical GABAergic interneurons lacking CB1Rs, identify endocannabinoids as axon guidance cues and demonstrate that endocannabinoid signaling regulates synaptogenesis and target selection in vivo.

Electrical dimensions in cell science

Cells undergo a variety of physiological processes, including division, migration and differentiation, under the influence of endogenous electrical cues, which are generated physiologically and pathologically in the extracellular and sometimes intracellular spaces. These signals are transduced to regulate cell behaviours profoundly, both in vitro and in vivo. Bioelectricity influences cellular processes as fundamental as control of the cell cycle, cell proliferation, cancer-cell migration, electrical signalling in the adult brain, embryonic neuronal cell migration, axon outgrowth, spinal-cord repair, epithelial wound repair, tissue regeneration and establishment of left-right body asymmetry. In addition to direct effects on cells, electrical gradients interact with coexisting extracellular chemical gradients. Indeed, cells can integrate and respond to electrical and chemical cues in combination. This Commentary details how electrical signals control multiple cell behaviours and argues that study of the interplay between combined electrical and chemical gradients is underdeveloped yet necessary.

Has electrical growth cone guidance found its potential

Many neurobiologists spurn the existence and use of direct-current (DC) electric fields (EFs) in nervous system development and regeneration. This is despite direct measurement of EFs in embryos and adults, and evidence that EFs are required for normal development, dramatically influence the rate and direction of nerve growth in vitro, and promote nerve regeneration in vivo. The notion that growth cones use EFs as guidance cues was dismissed partly because there was no convincing evidence that naturally occurring EFs influence nerve growth at the single-cell level in vivo. Recent work indicates that growth cones can be guided by EFs in vivo and, intriguingly, that in vitro guidance by chemotropic gradients and EFs might invoke similar mechanisms. Ongoing clinical trials to assess the effectiveness of DC EFs in promoting the regeneration of human spinal cord could allow EFs to achieve their potential.

A role for L-α-lysophosphatidylinositol and GPR55 in the modulation of migration, orientation and polarization of human breast cancer cells

Background and purpose:  Increased circulating levels of L‐α‐lysophosphatidylinositol (LPI) are associated with cancer and LPI is a potent, ligand for the G‐protein‐coupled receptor GPR55. Here we have assessed the modulation of breast cancer cell migration, orientation and polarization by LPI and GPR55.

Growth cone steering by a physiological electric field requires dynamic microtubules, microfilaments and Rac-mediated filopodial asymmetry

Electric fields (EFs) resembling those in the developing and regenerating nervous systems steer growth cones towards the cathode. Requirements for actin microfilaments, microtubules and their interactions during EF growth cone steering have been presumed, but remain unproven. Here, we demonstrate essential roles for dynamic microfilaments and microtubules in cathode-directed migration. Cathodal turning of growth cones on cultured Xenopus embryonic spinal neurons was attenuated significantly by nanomolar concentrations of the microfilament inhibitor latrunculin, the microtubule-stabilising drug taxol, or the microtubule-destabilising drugs vinblastine or nocodazole. Dynamically, the cathodal bias of filopodia preceded cathodal turning of the growth cone, suggesting an instructive role in EF-induced steering. Lamellipodial asymmetry accompanied turning. Filopodia and lamellipodia are regulated by the GTPases Cdc42 and Rac, respectively, and, as shown in the companion paper in this issue, peptides that selectively prevented effector binding to the CRIB domains of Cdc42 or Rac abolished cathodal growth cone turning during 3 hours of EF exposure. Here, the Rac peptide suppressed lamellipodium formation, increased the number of filopodia, abolished cathodal filopodial orientation, and prevented cathodal steering. The Cdc42 peptide suppressed filopodium formation, increased lamellipodial area and prevented cathodal steering. The cathodal bias of lamellipodia was independent of Cdc42 CRIB activity and was not sufficient for cathodal steering in the absence of filopodia, but the cathodal bias of filopodia through Rac CRIB activity was necessary for cathodal turning. Understanding the mechanism for cathodal growth cone guidance will enhance the emerging clinical effort to stimulate human spinal cord regeneration through EF application.

Temporally and spatially coordinated roles for Rho, Rac, Cdc42 and their effectors in growth cone guidance by a physiological electric field

Although it is known that neuronal growth cones migrate towards the cathode of an applied direct current (DC) electric field (EF), resembling the EF present in the developing nervous system, the underlying mechanism remains unclear. Here, we demonstrate temporally and spatially coordinated roles for the GTPases Rac, Cdc42 and Rho and their effectors. Growth cones of cultured Xenopus embryonic spinal neurons turned towards the cathode but collective inhibition of Rho, Rac and Cdc42 attenuated turning. Selective inhibition of Rho, Cdc42 or Rac signalling revealed temporally distinct roles in steering by an electrical gradient. Rho, Rac and Cdc42 are each essential for turning within the initial 2 hours (early phase). Later, Rho and Cdc42 signals remain important but Rac signalling dominates. The EF increased Rho immunofluorescence anodally. This correlated spatially with collapsed growth cone morphology and reduced anodal migration rates, which were restored by Rho inhibition. These data suggest that anodally increased Rho activity induces local cytoskeletal collapse, biasing growth cone advance cathodally. Collapse might be mediated by the Rho effectors p160 Rho kinase and myosin light chain kinase since their inhibition attenuated early turning. Inhibitors of phosphoinositide 3-kinase, MEK1/2 or p38 mitogen-activated protein kinase (MAPK) did not affect turning behaviour, eliminating them mechanistically. We propose a mechanism whereby Rac and Cdc42 activities dominate cathodally and Rho activity dominates anodally to steer growth cones towards the cathode. The interaction between Rho GTPases, the cytoskeleton and growth cone dynamics is explored in the companion paper published in this issue. Our results complement studies of growth cone guidance by diffusible chemical gradients and suggest that growth cones might interpret these co-existing guidance cues selectively.

Alignment of corneal and lens epithelial cells by co-operative effects of substratum topography and DC electric fields.

Corneal and lens epithelial cells (CECs and LECs) in the eye encounter precisely ordered fibre arrays on the nanoscale in tandem with an endogenous electric field (EF). Prosthetic biomaterials often incorporate topographical features intended to mimic those in situ. However, the cellular basis for control of cell morphology by nanotopography or by an EF is not clear. We examined cell axis alignment in response to substratum nanotopography and a physiological EF separately and in combination. Bovine CECs aligned parallel to substratum nanogrooves (NGs) as shallow as 14 nm but LECs were less sensitive. Actin filaments of both cell types concentrated at substratum ridges so we tested the mechanistic roles of rho, rac and cdc42, molecules that control cytoskeletal organization. CEC alignment to 130 nm deep NGs was prevented by the inhibition of rho, but not by the inhibition of cdc42, rac, or the rho effectors myosin light chain kinase or rho kinase. Conversely, CEC alignment was enhanced by the activation of rho. CECs on planar quartz substrata aligned orthogonal to an EF of 150 mV/mm. Alignment required signalling by cdc42 and rho but not rac, and was accompanied by lamellipodial reorganisation and cell migration toward the cathode. When CECs on vertically oriented NGs were exposed simultaneously to a horizontal EF, they aligned more robustly than to either cue alone and the enhanced alignment required rho signalling. Therefore, nanoscale substratum features and EFs co-operate to control cell axis alignment via rho, and cdc42-mediated intracellular signals, which can be exploited in tissue engineering.

Chronic wound state exacerbated by oxidative stress in Pax6+/-aniridia-related keratopathy

Heterozygosity for the transcription factor PAX6 causes eye disease in humans, characterized by corneal opacity. The molecular aetiology of such disease was investigated using a Pax6+/− mouse model. We found that the barrier function of uninjured Pax6+/− corneas was compromised and that Ca2+–PKC/PLC–ERK/p38 signalling pathways were abnormally activated, mimicking a ‘wounded’ epithelial state. Using proteomic analysis and direct assay for oxidized proteins, Pax6+/− corneas were found to be susceptible to oxidative stress and they exhibited a wound‐healing delay which could be rescued by providing reducing agents such as glutathione. Pax6 protein was oxidized and excluded from the nucleus of stressed corneal epithelial cells, with concomitant loss of corneal epithelial markers and expression of fibroblast/myofibroblast markers. We suggest a chronic wound model for Pax6‐related corneal diseases, in which oxidative stress underlies a positive feedback mechanism by depleting nuclear Pax6, delaying wound healing, and activating cell signalling pathways that lead to metaplasia of the corneal epithelium. The study mechanistically links a relatively minor dosage deficiency of a transcription factor with potentially catastrophic degenerative corneal disease. Copyright

The role of electrical signals in murine corneal wound re-epithelialization†

Ion flow from intact tissue into epithelial wound sites results in lateral electric currents that may represent a major driver of wound healing cell migration. Use of applied electric fields (EF) to promote wound healing is the basis of Medicare‐approved electric stimulation therapy. This study investigated the roles for EFs in wound re‐epithelialization, using the Pax6+/− mouse model of the human ocular surface abnormality aniridic keratopathy (in which wound healing and corneal epithelial cell migration are disrupted). Both wild‐type (WT) and Pax6+/− corneal epithelial cells showed increased migration speeds in response to applied EFs in vitro. However, only Pax6+/+ cells demonstrated consistent directional galvanotaxis towards the cathode, with activation of pSrc signaling, polarized to the leading edges of cells. In vivo, the epithelial wound site normally represents a cathode, but 43% of Pax6+/− corneas exhibited reversed endogenous wound‐induced currents (the wound was an anode). These corneas healed at the same rate as WT. Surprisingly, epithelial migration did not correlate with direction or magnitude of endogenous currents for WT or mutant corneas. Furthermore, during healing in vivo, no polarization of pSrc was observed. We found little evidence that Src‐dependent mechanisms of cell migration, observed in response to applied EFs in vitro, normally exist in vivo. It is concluded that endogenous EFs do not drive long‐term directionality of sustained healing migration in this mouse corneal epithelial model. Ion flow from wounds may nevertheless represent an important component of wound signaling initiation. J. Cell. Physiol. 226: 1544–1553, 2011.

Electric fields are novel determinants of human macrophage functions.

Macrophages are key cells in inflammation and repair, and their activity requires close regulation. The characterization of cues coordinating macrophage function has focused on biologic and soluble mediators, with little known about their responses to physical stimuli, such as the electrical fields that are generated naturally in injured tissue and which accelerate wound healing. To address this gap in understanding, we tested how properties of human monocyte‐derived macrophages are regulated by applied electrical fields, similar in strengths to those established naturally. With the use of live‐cell video microscopy, we show that macrophage migration is directed anodally by electrical fields as low as 5 mV/mm and is electrical field strength dependent, with effects peaking ∼300 mV/mm. Monocytes, as macrophage precursors, migrate in the opposite, cathodal direction. Strikingly, we show for the first time that electrical fields significantly enhance macrophage phagocytic uptake of a variety of targets, including carboxylate beads, apoptotic neutrophils, and the nominal opportunist pathogen Candida albicans, which engage different classes of surface receptors. These electrical field‐induced functional changes are accompanied by clustering of phagocytic receptors, enhanced PI3K and ERK activation, mobilization of intracellular calcium, and actin polarization. Electrical fields also modulate cytokine production selectively and can augment some effects of conventional polarizing stimuli on cytokine secretion. Taken together, electrical signals have been identified as major contributors to the coordination and regulation of important human macrophage functions, including those essential for microbial clearance and healing. Our results open up a new area of research into effects of naturally occurring and clinically applied electrical fields in conditions where macrophage activity is critical.