in-situ-visualization-axon-growth-growth-cone-dynamics-acute-ex-vivo

<em>In Situ</em> Visualization of Axon Growth and Growth Cone Dynamics in Acute <em>Ex Vivo</em> Embryonic Brain Slice Cultures

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones ...

Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE)

Functional recovery can occur after incomplete spinal cord injury. Takeoka et al. now report that such recovery relies on muscle spindle feedback that is necessary for neuronal circuit remodeling, suggesting novel targets to restore motor functions following spinal cord injuries. 

A new "spin" on recovery after spinal cord injury.

After central nervous system (CNS) injury, inhibitory factors in the lesion scar and poor axon growth potential prevent axon regeneration. Microtubule stabilization reduces scarring and promotes axon growth. However, the cellular mechanisms of this dual effect remain unclear. Here, delayed systemic administration of a blood-brain barrier-permeable microtubule-stabilizing drug, epothilone B (epoB), decreased scarring after rodent spinal cord injury (SCI) by abrogating polarization and directed migration of scar-forming fibroblasts. Conversely, epothilone B reactivated neuronal polarization by inducing concerted microtubule polymerization into the axon tip, which propelled axon growth through an inhibitory environment. Together, these drug-elicited effects promoted axon regeneration and improved motor function after SCI. With recent clinical approval, epothilones hold promise for clinical use after CNS injury.

Axonal regeneration. Systemic administration of epothilone B promotes axon regeneration after spinal cord injury.

The growth and migration of neurons require continuous remodelling of the neuronal cytoskeleton, providing a versatile cellular framework for force generation and guided movement, in addition to structural support. Actin filaments and microtubules are central to the dynamic action of the cytoskeleton and rapid advances in imaging technologies are enabling ever more detailed visualisation of the dynamic intracellular networks that they form. However, these filaments do not act individually and an expanding body of evidence emphasises the importance of actin-microtubule crosstalk in orchestrating cytoskeletal dynamics. Here, we summarise our current understanding of the structure and dynamics of actin and microtubules in isolation, before reviewing both the mechanisms and the molecular players involved in mediating actin-microtubule crosstalk in neurons. 

Coordinating neuronal actin-microtubule dynamics.

The microtubule cytoskeleton is a major determinant in neuronal polarity. In this issue of Neuron, van Beuningen et al. (2015) now report that TRIM46 forms parallel microtubule bundles in the proximal axon and reveal that it is crucial for the establishment and maintenance of neuronal polarity.

Microtubule Organization in the Axon: TRIM46 Determines the Orientation.

Injuries to the adult CNS often result in permanent disabilities because neurons lose the ability to regenerate their axon during development. Here, whole transcriptome sequencing and bioinformatics analysis followed by gain- and loss-of-function experiments identified Cacna2d2, the gene encoding the Alpha2delta2 subunit of voltage-gated calcium channels (VGCCs), as a developmental switch that limits axon growth and regeneration. Cacna2d2 gene deletion or silencing promoted axon growth in vitro. In vivo, Alpha2delta2 pharmacological blockade through Pregabalin (PGB) administration enhanced axon regeneration in adult mice after spinal cord injury (SCI). As PGB is already an established treatment for a wide range of neurological disorders, our findings suggest that targeting Alpha2delta2 may be a novel treatment strategy to promote structural plasticity and regeneration following CNS trauma.

The Calcium Channel Subunit Alpha2delta2 Suppresses Axon Regeneration in the Adult CNS.

Axon regeneration and neuronal tissue repair varies across animal lineages as well as in the mammalian central and peripheral nervous systems. While the peripheral nervous system retains the ability to self-repair, the majority of axons in the adult mammalian central nervous system (CNS) fail to reactivate intrinsic growth programs after injury. Recent findings, however, suggest that long-distance axon regeneration, neuronal circuit assembly and recovery of functions in the adult mammalian CNS are possible. Here, we discuss our current knowledge of the cell signaling pathways and networks controlling axon regeneration. In addition, we outline a number of combinatorial strategies that include among others microtubule-based treatments to foster regeneration and functional connectivity after CNS trauma.

Spatial and temporal arrangement of neuronal intrinsic and extrinsic mechanisms controlling axon regeneration.

In the adult mammalian central nervous system (CNS), neurons typically fail to regenerate their axons after injury. During development, by contrast, neurons extend axons effectively. A variety of intracellular mechanisms mediate this difference, including changes in gene expression, the ability to form a growth cone, differences in mitochondrial function/axonal transport and the efficacy of synaptic transmission. In turn, these intracellular processes are linked to extracellular differences between the developing and adult CNS. During development, the extracellular environment directs axon growth and circuit formation. In adulthood, by contrast, extracellular factors, such as myelin and the extracellular matrix, restrict axon growth. Here, we discuss whether the reactivation of developmental processes can elicit axon regeneration in the injured CNS.

Can injured adult CNS axons regenerate by recapitulating development?

Central nervous system (CNS) injuries cause permanent impairments of sensorimotor functions as mature neurons fail to regenerate their severed axons. The poor intrinsic growth capacity of adult CNS neurons and the formation of an inhibitory lesion scar are key impediments to axon regeneration. Systemic administration of the microtubule stabilizing agent epothilone B promotes axon regeneration and recovery of motor function by activating the intrinsic axonal growth machinery and by reducing the inhibitory fibrotic lesion scar. Thus, epothilones hold clinical promise as potential therapeutics for spinal cord injury. Here we tested the efficacy of epothilone D, an epothilone B analog with a superior safety profile. By using liquid chromatography and mass spectrometry (LC/MS), we found adequate CNS penetration and distribution of epothilone D after systemic administration, confirming the suitability of the drug for non-invasive CNS treatment. Systemic administration of epothilone D reduced inhibitory fibrotic scarring, promoted regrowth of injured raphespinal fibers and improved walking function after mid-thoracic spinal cord contusion injury in adult rats. These results confirm that systemic administration of epothilones is a valuable therapeutic strategy for CNS regeneration and repair after injury and provides a further advance for potential clinical translation.

Systemic administration of epothilone D improves functional recovery of walking after rat spinal cord contusion injury.

Recent years have seen cytoskeleton dynamics emerging as a key player in axon regeneration. The cytoskeleton, in particular microtubules and actin, ensures the growth of neuronal processes and maintains the singular, highly polarized shape of neurons. Following injury, adult central axons are tipped by a dystrophic structure, the retraction bulb, which prevents their regeneration. Abnormal cytoskeleton dynamics are responsible for the formation of this growth-incompetent structure but pharmacologically modulating cytoskeleton dynamics of injured axons can transform this structure into a growth-competent growth cone. The cytoskeleton also drives the migration of scar-forming cells after an injury. Targeting its dynamics modifies the composition of the inhibitory environment formed by scar tissue and renders it more permissive for regenerating axons. Hence, cytoskeleton dynamics represent an appealing target to promote axon regeneration. As some of cytoskeleton-targeting drugs are used in the clinics for other purposes, they hold the promise to be used as a basis for a regenerative therapy after a spinal cord injury.

Cytoskeleton dynamics in axon regeneration.

After an injury in the adult mammalian central nervous system (CNS), lesioned axons fail to regenerate. This failure to regenerate contrasts with axons' remarkable potential to grow during embryonic development and after an injury in the peripheral nervous system (PNS). Several intracellular mechanisms-including cytoskeletal dynamics, axonal transport and trafficking, signaling and transcription of regenerative programs, and epigenetic modifications-control axon regeneration. In this review, we describe how manipulation of intrinsic mechanisms elicits a regenerative response in different organisms and how strategies are implemented to form the basis of a future regenerative treatment after CNS injury.

Axon Regeneration in the Central Nervous System: Facing the Challenges from the Inside.

Reproducibility - The key towards clinical implementation of spinal cord injury treatments?

Axons in the adult mammalian brain and spinal cord fail to regenerate upon lesion. In vivo imaging serves as a tool to investigate the immediate response of axons to injury and how the same injured axons behave over time. Here, we describe the dynamic changes that injured sensory axons undergo and methods of imaging them in vivo. First, we explain how sensory axons in the dorsal column of the adult mouse spinal cord respond to axotomy. Then, we highlight practical considerations for implementing two-photon based in vivo imaging of these axons. Finally, we describe future directions for this technique, including the possibility of in vivo imaging of subcellular dynamics within the axon.

Imaging in vivo dynamics of sensory axon responses to CNS injury.

Here we present a protocol for analyses of axon regeneration and density in unsectioned adult mouse spinal cord. This includes methods for injury and tracing of dorsal column sensory and corticospinal axons; clearing and staining of unsectioned spinal cord; visualization of axon degeneration and regeneration in cleared and uncleared specimens using two-photon microscopy; and either manual or semi-automatic analysis of axon density and regeneration in 3D space using Imaris and ImageJ software. This protocol can be used to elucidate the molecular and cellular mechanisms underlying nervous system degeneration and regeneration and to establish the therapeutic efficacy of candidate neuroregenerative treatments. Because tissue sectioning is not required, this protocol enables unambiguous evaluation of regeneration and greatly accelerates the speed at which analyses can be conducted. Surgical procedures take <30 min per mouse, with a wait period of 2 weeks between axonal injury and tracing and 2-8 weeks between tracing and tissue processing. Clearing and immunolabeling take ~1-2 weeks, depending on the size of the sample. Imaging and analysis can be performed in 1 d. All these procedures can be accomplished by a competent graduate student or experienced technician.

High-resolution 3D imaging and analysis of axon regeneration in unsectioned spinal cord with or without tissue clearing.

Injured axons fail to regenerate in the adult CNS, which contrasts with their vigorous growth during embryonic development. We explored the potential of re-initiating axon extension after injury by reactivating the molecular mechanisms that drive morphogenetic transformation of neurons during development. Genetic loss- and gain-of-function experiments followed by time-lapse microscopy, in vivo imaging, and whole-mount analysis show that axon regeneration is fueled by elevated actin turnover. Actin depolymerizing factor (ADF)/cofilin controls actin turnover to sustain axon regeneration after spinal cord injury through its actin-severing activity. This pinpoints ADF/cofilin as a key regulator of axon growth competence, irrespective of developmental stage. These findings reveal the central role of actin dynamics regulation in this process and elucidate a core mechanism underlying axon growth after CNS trauma. Thereby, neurons maintain the capacity to stimulate developmental programs during adult life, expanding their potential for plasticity. Thus, actin turnover is a key process for future regenerative interventions.

ADF/Cofilin-Mediated Actin Turnover Promotes Axon Regeneration in the Adult CNS.

The specification of an axon and its subsequent outgrowth are key steps during neuronal polarization, a prerequisite to wire the brain. The Rho-guanosine triphosphatase (GTPase) RhoA is believed to be a central player in these processes. However, its physiological role has remained undefined. Here, genetic loss- and gain-of-function experiments combined with time-lapse microscopy, cell culture, and in vivo analysis show that RhoA is not involved in axon specification but confines the initiation of neuronal polarization and axon outgrowth during development. Biochemical analysis and super-resolution microscopy together with molecular and pharmacological manipulations reveal that RhoA restrains axon growth by activating myosin-II-mediated actin arc formation in the growth cone to prevent microtubules from protruding toward the leading edge. Through this mechanism, RhoA regulates the duration of axon growth and pause phases, thus controlling the tightly timed extension of developing axons. Thereby, this work unravels physiologically relevant players coordinating actin-microtubule interactions during axon growth.

RhoA Controls Axon Extension Independent of Specification in the Developing Brain.

After its initial development, the nervous system matures to connect and shape the neuronal circuitry and to keep it functional in humans for decades. Here we conceptualize neuronal maturation as a research field that will have, we would argue, a strong impact on understanding the healthy and diseased nervous system. Identifying the key mechanisms underlying neuronal maturation has the potential to reverse this process in adulthood, thereby facilitating regeneration.

Neuronal Maturation: Challenges and Opportunities in a Nascent Field.

During development of the central nervous system (CNS), neurons polarize and rapidly extend their axons to assemble neuronal circuits. The growth cone leads the axon to its target and drives axon growth. Here, we explored the mechanisms underlying axon growth in three dimensions. Live in situ imaging and super-resolution microscopy combined with pharmacological and molecular manipulations as well as biophysical force measurements revealed that growth cones extend CNS axons independent of pulling forces on their substrates and without the need for adhesions in three-dimensional (3D) environments. In 3D, microtubules grow unrestrained from the actomyosin cytoskeleton into the growth cone leading edge to enable rapid axon extension. Axons extend and polarize even in adhesion-inert matrices. Thus, CNS neurons use amoeboid mechanisms to drive axon growth. Together with our understanding that adult CNS axons regenerate by reactivating developmental processes, our findings illuminate how cytoskeletal manipulations enable axon regeneration in the adult CNS.

Axon Growth of CNS Neurons in Three Dimensions Is Amoeboid and Independent of Adhesions.

In this issue of Neuron, Wang et al. demonstrate that both cell-intrinsic and -extrinsic factors restrict the myelination of newly regenerated axons. Pharmalogical targeting of GPR17 signaling in oligodendrocyte precursor cells (OPCs) and microglial inhibition of oligodendrocyte maturation together promote robust myelination of regenerated axons after CNS injury.

Growing Myelin around Regenerated Axons after CNS Injury.

Editorial overview: Molecular neuroscience.

An inhibitory extracellular milieu and neuron-intrinsic processes prevent axons from regenerating in the adult central nervous system (CNS). Here we show how the two aspects are interwoven. Genetic loss-of-function experiments determine that the small GTPase RhoA relays extracellular inhibitory signals to the cytoskeleton by adapting mechanisms set in place during neuronal polarization. In response to extracellular inhibitors, neuronal RhoA restricts axon regeneration by activating myosin II to compact actin and, thereby, restrain microtubule protrusion. However, astrocytic RhoA restricts injury-induced astrogliosis through myosin II independent of microtubules by activating Yes-activated protein (YAP) signaling. Cell-type-specific deletion in spinal-cord-injured mice shows that neuronal RhoA activation prevents axon regeneration, whereas astrocytic RhoA is beneficial for regenerating axons. These data demonstrate how extracellular inhibitors regulate axon regeneration, shed light on the capacity of reactive astrocytes to be growth inhibitory after CNS injury, and reveal cell-specific RhoA targeting as a promising therapeutic avenue.

Frank Bradke

Laboratory of Axon Growth and Regeneration

Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE)

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

Frank Bradke

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Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE)

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

Laboratory of Axon Growth and Regeneration