Gianluca Gallo

Reevaluation of in vitro differentiation protocols for bone marrow stromal cells: Disruption of actin cytoskeleton induces rapid morphological changes and mimics neuronal phenotype

Bone marrow stromal cells (MSC), which represent a population of multipotential mesenchymal stem cells, have been reported to undergo rapid and robust transformation into neuron‐like phenotypes in vitro following treatment with chemical induction medium including dimethyl sulfoxide (DMSO; Woodbury et al. [ 2002 ] J. Neurosci. Res. 96:908). In this study, we confirmed the ability of cultured rat MSC to undergo in vitro osteogenesis, chondrogenesis, and adipogenesis, demonstrating differentiation of these cells to three mesenchymal cell fates. We then evaluated the potential for in vitro neuronal differentiation of these MSC, finding that changes in morphology upon addition of the chemical induction medium were caused by rapid disruption of the actin cytoskeleton. Retraction of the cytoplasm left behind long processes, which, although strikingly resembling neurites, showed essentially no motility and no further elaboration during time‐lapse studies. Similar neurite‐like processes were induced by treating MSC with DMSO only or with actin filament‐depolymerizing agents. Although process formation was accompanied by rapid expression of some neuronal and glial markers, the absence of other essential neuronal proteins pointed toward aberrantly induced gene expression rather than toward a sequence of gene expression as is required for neurogenesis. Moreover, rat dermal fibroblasts responded to neuronal induction by forming similar processes and expressing similar markers. These studies do not rule out the possibility that MSC can differentiate into neurons; however, we do want to caution that in vitro differentiation protocols may have unexpected, misleading effects. A dissection of molecular signaling and commitment events may be necessary to verify the ability of MSC transdifferentiation to neuronal lineages.

Axon growth and recovery of function supported by human bone marrow stromal cells in the injured spinal cord exhibit donor variations

Bone marrow stromal cells (MSC) are non-hematopoietic support cells that can be easily derived from bone marrow aspirates. Human MSC are clinically attractive because they can be expanded to large numbers in culture and reintroduced into patients as autografts or allografts. We grafted human MSC derived from aspirates of four different donors into a subtotal cervical hemisection in adult female rats and found that cells integrated well into the injury site, with little migration away from the graft. Immunocytochemical analysis demonstrated robust axonal growth through the grafts of animals treated with MSC, suggesting that MSC support axonal growth after spinal cord injury (SCI). However, the amount of axon growth through the graft site varied considerably between groups of animals treated with different MSC lots, suggesting that efficacy may be donor-dependent. Similarly, a battery of behavioral tests showed partial recovery in some treatment groups but not others. Using ELISA, we found variations in secretion patterns of selected growth factors and cytokines between different MSC lots. In a dorsal root ganglion explant culture system, we tested efficacy of conditioned medium from three donors and found that average axon lengths increased for all groups compared to control. These results suggest that human MSC produce factors important for mediating axon outgrowth and recovery after SCI but that MSC lots from different donors vary considerably. To qualify MSC lots for future clinical application, such notable differences in donor or lot-lot efficacy highlight the need for establishing adequate characterization, including the development of relevant efficacy assays.

Actin turnover is required to prevent axon retraction driven by endogenous actomyosin contractility

Growth cone motility and guidance depend on the dynamic reorganization of filamentous actin (F-actin). In the growth cone, F-actin undergoes turnover, which is the exchange of actin subunits from existing filaments. However, the function of F-actin turnover is not clear. We used jasplakinolide (jasp), a cell-permeable macrocyclic peptide that inhibits F-actin turnover, to study the role of F-actin turnover in axon extension. Treatment with jasp caused axon retraction, demonstrating that axon extension requires F-actin turnover. The retraction of axons in response to the inhibition of F-actin turnover was dependent on myosin activity and regulated by RhoA and myosin light chain kinase. Significantly, the endogenous myosin-based contractility was sufficient to cause axon retraction, because jasp did not alter myosin activity. Based on these observations, we asked whether guidance cues that cause axon retraction (ephrin-A2) inhibit F-actin turnover. Axon retraction in response to ephrin-A2 correlated with decreased F-actin turnover and required RhoA activity. These observations demonstrate that axon extension depends on an interaction between endogenous myosin-driven contractility and F-actin turnover, and that guidance cues that cause axon retraction inhibit F-actin turnover.

p75 Neurotrophin Receptor Signaling Regulates Growth Cone Filopodial Dynamics through Modulating RhoA Activity

The mechanisms by which neurotrophins regulate growth cone motility are unclear. We investigated the role of the p75 neurotrophin receptor (p75NTR) in mediating neurotrophin-induced increases in filopodial length. Our data demonstrate that neurotrophin binding to p75NTR is necessary and sufficient to regulate filopodial dynamics. Furthermore, retinal and dorsal root ganglion growth cones from p75 mutant mice are insensitive to neurotrophins but display enhanced filopodial lengths comparable with neurotrophin-treated wild-type growth cones. This suggests unoccupied p75NTR negatively regulates filopodia length. Furthermore, p75NTR regulates RhoA activity to mediate filopodial dynamics. Constitutively active RhoA blocks neurotrophin-induced increases in filopodial length, whereas inhibition of RhoA enhances filopodial lengths, similar to neurotrophin treatment. BDNF treatment of retinal neurons results in reduced RhoA activity. Furthermore, p75 mutant neurons display reduced levels of activated RhoA compared with wild-type counterparts, consistent with the enhanced filopodial lengths observed on mutant growth cones. These observations suggest that neurotrophins regulate filopodial dynamics by depressing the activation of RhoA that occurs through p75NTR signaling.

The cytoskeletal and signaling mechanisms of axon collateral branching.

During development, axons are guided to their appropriate targets by a variety of guidance factors. On arriving at their synaptic targets, or while en route, axons form branches. Branches generated de novo from the main axon are termed collateral branches. The generation of axon collateral branches allows individual neurons to make contacts with multiple neurons within a target and with multiple targets. In the adult nervous system, the formation of axon collateral branches is associated with injury and disease states and may contribute to normally occurring plasticity. Collateral branches are initiated by actin filament–based axonal protrusions that subsequently become invaded by microtubules, thereby allowing the branch to mature and continue extending. This article reviews the current knowledge of the cellular mechanisms of the formation of axon collateral branches. The major conclusions of this review are (1) the mechanisms of axon extension and branching are not identical; (2) active suppression of protrusive activity along the axon negatively regulates branching; (3) the earliest steps in the formation of axon branches involve focal activation of signaling pathways within axons, which in turn drive the formation of actin‐based protrusions; and (4) regulation of the microtubule array by microtubule‐associated and severing proteins underlies the development of branches. Linking the activation of signaling pathways to specific proteins that directly regulate the axonal cytoskeleton underlying the formation of collateral branches remains a frontier in the field.

Mitochondria Coordinate Sites of Axon Branching through Localized Intra-axonal Protein Synthesis

The branching of axons is a fundamental aspect of nervous system development and neuroplasticity. We report that branching of sensory axons in the presence of nerve growth factor (NGF) occurs at sites populated by stalled mitochondria. Translational machinery targets to presumptive branching sites, followed by recruitment of mitochondria to these sites. The mitochondria promote branching through ATP generation and the determination of localized hot spots of active axonal mRNA translation, which contribute to actin-dependent aspects of branching. In contrast, mitochondria do not have a role in the regulation of the microtubule cytoskeleton during NGF-induced branching. Collectively, these observations indicate that sensory axons exhibit multiple potential sites of translation, defined by presence of translational machinery, but active translation occurs following the stalling and respiration of mitochondria at these potential sites of translation. This study reveals a local role for axonal mitochondria in the regulation of the actin cytoskeleton and axonal mRNA translation underlying branching.

Axonally Synthesized β-Actin and GAP-43 Proteins Support Distinct Modes of Axonal Growth

Increasing evidence points to the importance of local protein synthesis for axonal growth and responses to axotomy, yet there is little insight into the functions of individual locally synthesized proteins. We recently showed that expression of a reporter mRNA with the axonally localizing β-actin mRNA 3′UTR competes with endogenous β-actin and GAP-43 mRNAs for binding to ZBP1 and axonal localization in adult sensory neurons (Donnelly et al., 2011). Here, we show that the 3′UTR of GAP-43 mRNA can deplete axons of endogenous β-actin mRNA. We took advantage of this 3′UTR competition to address the functions of axonally synthesized β-actin and GAP-43 proteins. In cultured rat neurons, increasing axonal synthesis of β-actin protein while decreasing axonal synthesis of GAP-43 protein resulted in short highly branched axons. Decreasing axonal synthesis of β-actin protein while increasing axonal synthesis of GAP-43 protein resulted in long axons with few branches. siRNA-mediated depletion of overall GAP-43 mRNA from dorsal root ganglia (DRGs) decreased the length of axons, while overall depletion of β-actin mRNA from DRGs decreased the number of axon branches. These deficits in axon growth could be rescued by transfecting with siRNA-resistant constructs encoding β-actin or GAP-43 proteins, but only if the mRNAs were targeted for axonal transport. Finally, in ovo electroporation of axonally targeted GAP-43 mRNA increased length and axonally targeted β-actin mRNA increased branching of sensory axons growing into the chick spinal cord. These studies indicate that axonal translation of β-actin mRNA supports axon branching and axonal translation of GAP-43 mRNA supports elongating growth.

Neurotrophins and the Dynamic Regulation of the Neuronal Cytoskeleton

The morphology of neuronal axons and dendrites is dependent on the dynamics of the cytoskeleton. An understanding of neurodevelopment and adult neuroplasticity must therefore include a detailed description of the intrinsic and extrinsic mechanisms that regulate the organization and dynamics of actin filaments and microtubules. In this paper we review recent advances in the understanding of the dynamic regulation of neuronal morphology by interactions among cytoskeletal components and the regulation of the cytoskeleton by neurotrophins.

The actin nucleating Arp2/3 complex contributes to the formation of axonal filopodia and branches through the regulation of actin patch precursors to filopodia

The emergence of axonal filopodia is the first step in the formation of axon collateral branches. In vitro, axonal filopodia emerge from precursor cytoskeletal structures termed actin patches. However, nothing is known about the cytoskeletal dynamics of the axon leading to the formation of filopodia in the relevant tissue environment. In this study we investigated the role of the actin nucleating Arp2/3 complex in the formation of sensory axon actin patches, filopodia, and branches. By combining in ovo chicken embryo electroporation mediated gene delivery with a novel acute ex vivo spinal cord preparation, we demonstrate that actin patches form along sensory axons and give rise to filopodia in situ. Inhibition of Arp2/3 complex function in vitro and in vivo decreases the number of axonal filopodia. In vitro, Arp2/3 complex subunits and upstream regulators localize to actin patches. Analysis of the organization of actin filaments in actin patches using platinum replica electron microscopy reveals that patches consist of networks of actin filaments, and filaments in axonal filopodia exhibit an organization consistent with the Arp2/3‐based convergent elongation mechanism. Nerve growth factor (NGF) promotes formation of axonal filopodia and branches through phosphoinositide 3‐kinase (PI3K). Inhibition of the Arp2/3 complex impairs NGF/PI3K‐induced formation of axonal actin patches, filopodia, and the formation of collateral branches. Collectively, these data reveal that the Arp2/3 complex contributes to the formation of axon collateral branches through its involvement in the formation of actin patches leading to the emergence of axonal filopodia.

Nerve Growth Factor Induces Axonal Filopodia through Localized Microdomains of Phosphoinositide 3-Kinase Activity That Drive the Formation of Cytoskeletal Precursors to Filopodia

The initiation of axonal filopodia is the first step in the formation of collateral branches and synaptic structures. In sensory neurons, nerve growth factor (NGF) promotes the formation of axonal filopodia and branches. However, the signaling and cytoskeletal mechanisms of NGF-induced initiation of axonal filopodia are not clear. Axonal filopodia arise from precursor axonal cytoskeletal structures termed filamentous actin (F-actin) patches. Patches form spontaneously and are transient. Although filopodia emerge from patches, only a fraction of patches normally gives rise to filopodia. Using chicken sensory neurons and live imaging of enhanced yellow fluorescent protein (eYFP)–actin dynamics, we report that NGF promotes the formation of axonal filopodia by increasing the rate of F-actin patch formation but not the fraction of patches that give rise to filopodia. We also demonstrate that activation of the phosphatidylinositol 3-kinase (PI3K)–Akt pathway is sufficient and required for driving the formation of axonal F-actin patches, filopodia, and axon branches. Using the green fluorescent protein–plekstrin homology domain of Akt, which targets to PI3K-generated phosphatidylinositol-3,4,5-triphosphate (PIP3), we report localized microdomains of PIP3 accumulation that form in synchrony with F-actin patches and that NGF promotes the formation of microdomains of PIP3 and patches. Finally, we find that, in NGF, F-actin patches form in association with axonal mitochondria and oxidative phosphorylation is required for patch formation. This investigation demonstrates that surprisingly NGF promotes formation of axonal filopodia by increasing the formation of cytoskeletal filopodial precursors (patches) through localized microdomains of PI3K signaling but not the emergence of filopodia from patches.