The mechanisms by which neurotrophins regulate growth cone motility are unclear. We investigated ... more The mechanisms by which neurotrophins regulate growth cone motility are unclear. We investigated the role of the p75 neurotrophin receptor (p75 NTR) in mediating neurotrophin-induced increases in filopodial length. Our data demonstrate that neurotrophin binding to p75 NTR 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 p75 NTR negatively regulates filopodia length. Furthermore, p75 NTR 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...
It is well established that CNS axons fail to regenerate, undergo retrograde dieback, and form dy... more It is well established that CNS axons fail to regenerate, undergo retrograde dieback, and form dystrophic growth cones due to both intrinsic and extrinsic factors. We sought to investigate the role of axonal mitochondria in the axonal response to injury. A viral vector (AAV) containing a mitochondrially targeted fluorescent protein (mitoDsRed) as well as fluorescently tagged LC3 (GFP-LC3), an autophagosomal marker, was injected into the primary motor cortex, to label the corticospinal tract (CST), of adult rats. The axons of the CST were then injured by dorsal column lesion at C4-C5. We found that mitochondria in injured CST axons near the injury site are fragmented and fragmentation of mitochondria persists for 2 weeks before returning to pre-injury lengths. Fragmented mitochondria have consistently been shown to be dysfunctional and detrimental to cellular health. Inhibition of Drp1, the GTPase responsible for mitochondrial fission, using a specific pharmacological inhibitor (mDiv...
Nerve Growth Factor (NGF) promotes the elaboration of axonal filopodia and branches through PI3K-... more Nerve Growth Factor (NGF) promotes the elaboration of axonal filopodia and branches through PI3K-Akt. NGF activates the TrkA receptor resulting in an initial transient high amplitude burst of PI3K-Akt signaling followed by a maintained lower steady state, hereafter referred to as initiation and steady state phases. Akt initially undergoes phosphorylation at T308 followed by phosphorylation at S473, resulting in maximal kinase activation. We report that during the initiation phase the localization of PI3K signaling, reported by visualizing sites of PIP3 formation, and Akt signaling, reflected by Akt phosphorylation at T308, correlates with the positioning of axonal mitochondria. Mitochondrial oxidative phosphorylation but not glycolysis is required for Akt phosphorylation at T308. In contrast, the phosphorylation of Akt at S473 is not spatially associated with mitochondria and is dependent on both oxidative phosphorylation and glycolysis. Under NGF steady state conditions, maintenanc...
SummaryDual Leucine-zipper Kinase (DLK, a MAP3K) mediates neuronal responses to diverse injuries ... more SummaryDual Leucine-zipper Kinase (DLK, a MAP3K) mediates neuronal responses to diverse injuries and insults via c-Jun N-terminal Kinase (JNK) family Mitogen-activated Protein Kinases (MAPKs). It is unclear why DLK couples to JNKs in mammalian neurons versus other MAPKs, especially because some invertebrate DLK orthologs couple instead to the related p38 family MAPKs. Here we identify two mechanisms that potentially explain this DLK-JNK coupling. First, neural-specific JNK3, but not p38-MAPK, catalyzes positive feedback phosphorylation of DLK that further activates DLK and locks the DLK-JNK3 module in a highly active state. Furthermore, the pro-degenerative JNK2 and JNK3, but not the related JNK1, are endogenously palmitoylated. Moreover, palmitoylation targets both DLK and JNK3 to the same axonal vesicles and JNK3 palmitoylation is essential for pro-degenerative axonal retrograde signaling in vivo. These findings provide insights into DLK-JNK signaling relevant to multiple neuropat...
Following injury, sensory axons locally translate mRNAs that encode proteins needed for the respo... more Following injury, sensory axons locally translate mRNAs that encode proteins needed for the response to injury, locally and through retrograde signaling, and for regeneration. In this study, we addressed the mechanism and role of axotomy-induced intra-axonal translation of the ER chaperone Calreticulin. In vivo peripheral nerve injury increased Calreticulin levels in sensory axons. Using an in vitro model system of sensory neurons amenable to mechanistic dissection we provide evidence that axotomy induces local translation of Calreticulin through PERK (protein kinase RNA-like endoplasmic reticulum kinase) mediated phosphorylation of eIF2α by a mechanism that requires both 5' and 3'UTRs (untranslated regions) elements in Calreticulin mRNA. ShRNA mediated depletion of Calreticulin or inhibition of PERK signaling increased axon retraction following axotomy. In contrast, expression of axonally targeted, but not somatically restricted, Calreticulin mRNA decreased retraction and promoted axon regeneration following axotomy in vitro. Collectively, these data indicate that the intra-axonal translation of Calreticulin in response to axotomy serves to minimize the ensuing retraction, and overexpression of axonally targeted Calreticulin mRNA promotes axon regeneration.
Mitochondria shape cytosolic calcium ([Ca]) transients and utilize the mitochondrial Ca ([Ca]) in... more Mitochondria shape cytosolic calcium ([Ca]) transients and utilize the mitochondrial Ca ([Ca]) in exchange for bioenergetics output. Conversely, dysregulated [Ca] causes [Ca] overload and induces permeability transition pore and cell death. Ablation of MCU-mediated Ca uptake exhibited elevated [Ca] and failed to prevent stress-induced cell death. The mechanisms for these effects remain elusive. Here, we report that mitochondria undergo a cytosolic Ca-induced shape change that is distinct from mitochondrial fission and swelling. [Ca] elevation, but not MCU-mediated Ca uptake, appears to be essential for the process we term mitochondrial shape transition (MiST). MiST is mediated by the mitochondrial protein Miro1 through its EF-hand domain 1 in multiple cell types. Moreover, Ca-dependent disruption of Miro1/KIF5B/tubulin complex is determined by Miro1 EF1 domain. Functionally, Miro1-dependent MiST is essential for autophagy/mitophagy that is attenuated in Miro1 EF1 mutants. Thus, Miro...
Chondroitin sulfate proteoglycans (CSPGs) inhibit the formation of axon collateral branches. The ... more Chondroitin sulfate proteoglycans (CSPGs) inhibit the formation of axon collateral branches. The regulation of the axonal cytoskeleton and mitochondria are important components of the mechanism of branching. Actin-dependent axonal plasticity, reflected in the dynamics of axonal actin patches and filopodia, is greatest along segments of the axon populated by mitochondria. It is reported that CSPGs partially depolarize the membrane potential of axonal mitochondria, which impairs the dynamics of the axonal actin cytoskeleton and decreases the formation and duration of axonal filopodia, the first steps in the mechanism of branching. The effects of CSPGs on actin cytoskeletal dynamics are specific to axon segments populated by mitochondria. In contrast, CSPGs do not affect the microtubule content of axons, or the localization of microtubules into axonal filopodia, a required step in the mechanism of branch formation. It is also reported that CSPGs decrease the mitochondria-dependent axon...
Neurons begin life as spherical cells. A major hallmark of neuronal development is the formation ... more Neurons begin life as spherical cells. A major hallmark of neuronal development is the formation of elongating processes from the cell body which subsequently differentiate into dendrites and the axon. The formation and later development of neuronal processes is achieved through the concerted organization of actin filaments and microtubules. Here, we review the literature regarding recent advances in the understanding of cytoskeletal interactions in neurons focusing on the initiation of processes from neuronal cell bodies and the collateral branching of axons. The complex crosstalk between cytoskeletal elements is mediated by a cohort of proteins that either bind both cytoskeletal systems or allow one to regulate the other. Recent studies have highlighted the importance of microtubule plus-tip proteins in the regulation of the dynamics and organization of actin filaments, while also providing a mechanism for the subcellular capture and guidance of microtubule tips by actin filaments...
Molecular and cellular neurosciences, Jan 27, 2017
The formation of axon collateral branches from the pre-existing shafts of axons is an important a... more The formation of axon collateral branches from the pre-existing shafts of axons is an important aspect of neurodevelopment and the response of the nervous system to injury. This article provides an overview of the role of the cytoskeleton and signaling mechanisms in the formation of axon collateral branches. Both the actin filament and microtubule components of the cytoskeleton are required for the formation of axon branches. Recent work has begun to shed light on how these two elements of the cytoskeleton are integrated by proteins that functionally or physically link the cytoskeleton. While a number of signaling pathways have been determined as having a role in the formation of axon branches, the complexity of the downstream mechanisms and links to specific signaling pathways remain to be fully determined. The regulation of intra-axonal protein synthesis and organelle function are also emerging as components of signal-induced axon branching. Although much has been learned in the l...
Drebrin is a cytoskeleton-associated protein which can interact with both actin filaments and the... more Drebrin is a cytoskeleton-associated protein which can interact with both actin filaments and the tips of microtubules. Its roles have been studied mostly in dendrites, and the functions of drebrin in axons are less well understood. In this work we analyzed the role of drebrin, through shRNA-mediated depletion and over-expression, in the collateral branching of chicken embryonic sensory axons. We report that drebrin promotes the formation of axonal filopodia and collateral branches in vivo and in vitro. Live imaging of cytoskeletal dynamics revealed that drebrin promotes the formation of filopodia from precursor structures termed axonal actin patches. Endogenous drebrin localizes to actin patches and depletion studies indicate that drebrin contributes to the development of patches. In filopodia, endogenous drebrin localizes to the proximal portion of the filopodium. Drebrin was found to promote the stability of axonal filopodia and the entry of microtubule plus tips into axonal filopodia. The effects of drebrin on the stabilization of filopodia are independent of its effects on promoting microtubule targeting to filopodia. Inhibition of myosin II induces a redistribution of endogenous drebrin distally into filopodia, and further increases branching in drebrin overexpressing neurons. Finally, a 30 minute treatment with the branch inducing signal nerve growth factor increases the levels of axonal drebrin. The current study determines the specific roles of drebrin in the regulation of the axonal cytoskeleton, and provides evidence that drebrin contributes to the coordination of the actin and microtubule cytoskeleton during the initial stages of axon branching. This article is protected by copyright. All rights reserved.
Proceedings of the National Academy of Sciences, 2015
Dual leucine-zipper kinase (DLK) is critical for axon-to-soma retrograde signaling following nerv... more Dual leucine-zipper kinase (DLK) is critical for axon-to-soma retrograde signaling following nerve injury. However, it is unknown how DLK, a predicted soluble kinase, conveys long-distance signals and why homologous kinases cannot compensate for loss of DLK. Here, we report that DLK, but not homologous kinases, is palmitoylated at a conserved site adjacent to its kinase domain. Using short-hairpin RNA knockdown/rescue, we find that palmitoylation is critical for DLK-dependent retrograde signaling in sensory axons. This functional importance is because of three novel cellular and molecular roles of palmitoylation, which targets DLK to trafficking vesicles, is required to assemble DLK signaling complexes and, unexpectedly, is essential for DLK’s kinase activity. By simultaneously controlling DLK localization, interactions, and activity, palmitoylation ensures that only vesicle-bound DLK is active in neurons. These findings explain how DLK specifically mediates nerve injury responses a...
ABSTRACT We present an implemented method for automated quantification of neuronal beading. Beadi... more ABSTRACT We present an implemented method for automated quantification of neuronal beading. Beading is the “morphological hallmark” of axonal injury and is present in many neurological disease conditions as well. In vitro models of injury result in beading which has previously been difficult to quantify, and therefore difficult to use as a measurement of the degree of axonal injury. The beading program was designed to quantify beading intensity in single axons and output a unitless beading number that represents the size and number of beads in relation to the length and width of the axon. The program was used to analyze a sample set of images and was successful in demonstrating a quantifiable difference between injured neurons and sham controls. Bland-Altman analysis was used to assess agreement between the number of beads counted by the program and those counted manually.
The protrusion of filopodia and lamellipodia from the surface of neurons is fundamental to axon e... more The protrusion of filopodia and lamellipodia from the surface of neurons is fundamental to axon extension, guidance, the formation of axon branches, and synaptic structures. Protrusion is driven by the polymerization and controlled organization of actin filament arrays. This chapter provides an overview of the basic mechanisms operative during protrusion. Mechanisms underlying the suppression of protrusive activity are also discussed
Neurons are arguably the most complex cells in nature and are characterized by a complex, dynamic... more Neurons are arguably the most complex cells in nature and are characterized by a complex, dynamic, and highly polarized morphology. Actin and its regulatory proteins are the most abundant set of proteins within cells and form one of the major cytoskeletal systems, the actin filament cytoskeleton. While much has been learned about the roles of the actin cytoskeleton in non-neuronal cells, our understanding of the full spectrum of the functions of actin in neurons is far from complete. This book is intended to provide the neuroscience community with an introduction to the interface between the actin cytoskeleton and the myriad of issues fundamental to the understanding of nervous system function. The book covers the neurobiology of actin ranging from basic cellular organization and function to the roles of actin in the health and disease states of the nervous system. This chapter provides a primer on actin intended to serve the reader as background and review for the rest of the chapters.
Vunjak-Novakovic/Culture of Cells for Tissue Engineering, 2005
This chapter represents the joint effort of three distinct laboratories to describe their differe... more This chapter represents the joint effort of three distinct laboratories to describe their different approaches and methodologies for engineering neuroendocrine/neuronal tissues for the possible treatment of neurodegenerative diseases, such as Parkinson disease, which are related to the loss of catecholaminergic neurons. In the first part, Lelkes and Unsworth discuss the generation of three-dimensional catecholaminergic, adrenal medullary organoids with PC12 pheochromocytoma cells. In the second part, Saporta and Cameron ...
The localized debundling of the axonal microtubule array and the entry of microtubules into axona... more The localized debundling of the axonal microtubule array and the entry of microtubules into axonal filopodia are two defining features of collateral branching. We report that nerve growth factor (NGF), a branch inducing signal, increases the frequency of microtubule debundling along the axon shaft of chicken embryonic sensory neurons. Sites of debundling correlate strongly with the localized targeting of microtubules into filopodia. Platinum replica electron microscopy suggests physical interactions between debundled microtubules and axonal actin filaments. However, as evidenced by depolymerization of actin filaments and inhibition of myosin II, actomyosin force generation does not promote debundling. In contrast, loss of actin filaments or inhibition of myosin II activity promotes debundling, indicating that axonal actomyosin forces suppress debundling. MAP1B is a microtubule associated protein that represses axon branching. Following treatment with NGF, microtubules penetrating fi...
The mechanisms by which neurotrophins regulate growth cone motility are unclear. We investigated ... more The mechanisms by which neurotrophins regulate growth cone motility are unclear. We investigated the role of the p75 neurotrophin receptor (p75 NTR) in mediating neurotrophin-induced increases in filopodial length. Our data demonstrate that neurotrophin binding to p75 NTR 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 p75 NTR negatively regulates filopodia length. Furthermore, p75 NTR 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...
It is well established that CNS axons fail to regenerate, undergo retrograde dieback, and form dy... more It is well established that CNS axons fail to regenerate, undergo retrograde dieback, and form dystrophic growth cones due to both intrinsic and extrinsic factors. We sought to investigate the role of axonal mitochondria in the axonal response to injury. A viral vector (AAV) containing a mitochondrially targeted fluorescent protein (mitoDsRed) as well as fluorescently tagged LC3 (GFP-LC3), an autophagosomal marker, was injected into the primary motor cortex, to label the corticospinal tract (CST), of adult rats. The axons of the CST were then injured by dorsal column lesion at C4-C5. We found that mitochondria in injured CST axons near the injury site are fragmented and fragmentation of mitochondria persists for 2 weeks before returning to pre-injury lengths. Fragmented mitochondria have consistently been shown to be dysfunctional and detrimental to cellular health. Inhibition of Drp1, the GTPase responsible for mitochondrial fission, using a specific pharmacological inhibitor (mDiv...
Nerve Growth Factor (NGF) promotes the elaboration of axonal filopodia and branches through PI3K-... more Nerve Growth Factor (NGF) promotes the elaboration of axonal filopodia and branches through PI3K-Akt. NGF activates the TrkA receptor resulting in an initial transient high amplitude burst of PI3K-Akt signaling followed by a maintained lower steady state, hereafter referred to as initiation and steady state phases. Akt initially undergoes phosphorylation at T308 followed by phosphorylation at S473, resulting in maximal kinase activation. We report that during the initiation phase the localization of PI3K signaling, reported by visualizing sites of PIP3 formation, and Akt signaling, reflected by Akt phosphorylation at T308, correlates with the positioning of axonal mitochondria. Mitochondrial oxidative phosphorylation but not glycolysis is required for Akt phosphorylation at T308. In contrast, the phosphorylation of Akt at S473 is not spatially associated with mitochondria and is dependent on both oxidative phosphorylation and glycolysis. Under NGF steady state conditions, maintenanc...
SummaryDual Leucine-zipper Kinase (DLK, a MAP3K) mediates neuronal responses to diverse injuries ... more SummaryDual Leucine-zipper Kinase (DLK, a MAP3K) mediates neuronal responses to diverse injuries and insults via c-Jun N-terminal Kinase (JNK) family Mitogen-activated Protein Kinases (MAPKs). It is unclear why DLK couples to JNKs in mammalian neurons versus other MAPKs, especially because some invertebrate DLK orthologs couple instead to the related p38 family MAPKs. Here we identify two mechanisms that potentially explain this DLK-JNK coupling. First, neural-specific JNK3, but not p38-MAPK, catalyzes positive feedback phosphorylation of DLK that further activates DLK and locks the DLK-JNK3 module in a highly active state. Furthermore, the pro-degenerative JNK2 and JNK3, but not the related JNK1, are endogenously palmitoylated. Moreover, palmitoylation targets both DLK and JNK3 to the same axonal vesicles and JNK3 palmitoylation is essential for pro-degenerative axonal retrograde signaling in vivo. These findings provide insights into DLK-JNK signaling relevant to multiple neuropat...
Following injury, sensory axons locally translate mRNAs that encode proteins needed for the respo... more Following injury, sensory axons locally translate mRNAs that encode proteins needed for the response to injury, locally and through retrograde signaling, and for regeneration. In this study, we addressed the mechanism and role of axotomy-induced intra-axonal translation of the ER chaperone Calreticulin. In vivo peripheral nerve injury increased Calreticulin levels in sensory axons. Using an in vitro model system of sensory neurons amenable to mechanistic dissection we provide evidence that axotomy induces local translation of Calreticulin through PERK (protein kinase RNA-like endoplasmic reticulum kinase) mediated phosphorylation of eIF2α by a mechanism that requires both 5' and 3'UTRs (untranslated regions) elements in Calreticulin mRNA. ShRNA mediated depletion of Calreticulin or inhibition of PERK signaling increased axon retraction following axotomy. In contrast, expression of axonally targeted, but not somatically restricted, Calreticulin mRNA decreased retraction and promoted axon regeneration following axotomy in vitro. Collectively, these data indicate that the intra-axonal translation of Calreticulin in response to axotomy serves to minimize the ensuing retraction, and overexpression of axonally targeted Calreticulin mRNA promotes axon regeneration.
Mitochondria shape cytosolic calcium ([Ca]) transients and utilize the mitochondrial Ca ([Ca]) in... more Mitochondria shape cytosolic calcium ([Ca]) transients and utilize the mitochondrial Ca ([Ca]) in exchange for bioenergetics output. Conversely, dysregulated [Ca] causes [Ca] overload and induces permeability transition pore and cell death. Ablation of MCU-mediated Ca uptake exhibited elevated [Ca] and failed to prevent stress-induced cell death. The mechanisms for these effects remain elusive. Here, we report that mitochondria undergo a cytosolic Ca-induced shape change that is distinct from mitochondrial fission and swelling. [Ca] elevation, but not MCU-mediated Ca uptake, appears to be essential for the process we term mitochondrial shape transition (MiST). MiST is mediated by the mitochondrial protein Miro1 through its EF-hand domain 1 in multiple cell types. Moreover, Ca-dependent disruption of Miro1/KIF5B/tubulin complex is determined by Miro1 EF1 domain. Functionally, Miro1-dependent MiST is essential for autophagy/mitophagy that is attenuated in Miro1 EF1 mutants. Thus, Miro...
Chondroitin sulfate proteoglycans (CSPGs) inhibit the formation of axon collateral branches. The ... more Chondroitin sulfate proteoglycans (CSPGs) inhibit the formation of axon collateral branches. The regulation of the axonal cytoskeleton and mitochondria are important components of the mechanism of branching. Actin-dependent axonal plasticity, reflected in the dynamics of axonal actin patches and filopodia, is greatest along segments of the axon populated by mitochondria. It is reported that CSPGs partially depolarize the membrane potential of axonal mitochondria, which impairs the dynamics of the axonal actin cytoskeleton and decreases the formation and duration of axonal filopodia, the first steps in the mechanism of branching. The effects of CSPGs on actin cytoskeletal dynamics are specific to axon segments populated by mitochondria. In contrast, CSPGs do not affect the microtubule content of axons, or the localization of microtubules into axonal filopodia, a required step in the mechanism of branch formation. It is also reported that CSPGs decrease the mitochondria-dependent axon...
Neurons begin life as spherical cells. A major hallmark of neuronal development is the formation ... more Neurons begin life as spherical cells. A major hallmark of neuronal development is the formation of elongating processes from the cell body which subsequently differentiate into dendrites and the axon. The formation and later development of neuronal processes is achieved through the concerted organization of actin filaments and microtubules. Here, we review the literature regarding recent advances in the understanding of cytoskeletal interactions in neurons focusing on the initiation of processes from neuronal cell bodies and the collateral branching of axons. The complex crosstalk between cytoskeletal elements is mediated by a cohort of proteins that either bind both cytoskeletal systems or allow one to regulate the other. Recent studies have highlighted the importance of microtubule plus-tip proteins in the regulation of the dynamics and organization of actin filaments, while also providing a mechanism for the subcellular capture and guidance of microtubule tips by actin filaments...
Molecular and cellular neurosciences, Jan 27, 2017
The formation of axon collateral branches from the pre-existing shafts of axons is an important a... more The formation of axon collateral branches from the pre-existing shafts of axons is an important aspect of neurodevelopment and the response of the nervous system to injury. This article provides an overview of the role of the cytoskeleton and signaling mechanisms in the formation of axon collateral branches. Both the actin filament and microtubule components of the cytoskeleton are required for the formation of axon branches. Recent work has begun to shed light on how these two elements of the cytoskeleton are integrated by proteins that functionally or physically link the cytoskeleton. While a number of signaling pathways have been determined as having a role in the formation of axon branches, the complexity of the downstream mechanisms and links to specific signaling pathways remain to be fully determined. The regulation of intra-axonal protein synthesis and organelle function are also emerging as components of signal-induced axon branching. Although much has been learned in the l...
Drebrin is a cytoskeleton-associated protein which can interact with both actin filaments and the... more Drebrin is a cytoskeleton-associated protein which can interact with both actin filaments and the tips of microtubules. Its roles have been studied mostly in dendrites, and the functions of drebrin in axons are less well understood. In this work we analyzed the role of drebrin, through shRNA-mediated depletion and over-expression, in the collateral branching of chicken embryonic sensory axons. We report that drebrin promotes the formation of axonal filopodia and collateral branches in vivo and in vitro. Live imaging of cytoskeletal dynamics revealed that drebrin promotes the formation of filopodia from precursor structures termed axonal actin patches. Endogenous drebrin localizes to actin patches and depletion studies indicate that drebrin contributes to the development of patches. In filopodia, endogenous drebrin localizes to the proximal portion of the filopodium. Drebrin was found to promote the stability of axonal filopodia and the entry of microtubule plus tips into axonal filopodia. The effects of drebrin on the stabilization of filopodia are independent of its effects on promoting microtubule targeting to filopodia. Inhibition of myosin II induces a redistribution of endogenous drebrin distally into filopodia, and further increases branching in drebrin overexpressing neurons. Finally, a 30 minute treatment with the branch inducing signal nerve growth factor increases the levels of axonal drebrin. The current study determines the specific roles of drebrin in the regulation of the axonal cytoskeleton, and provides evidence that drebrin contributes to the coordination of the actin and microtubule cytoskeleton during the initial stages of axon branching. This article is protected by copyright. All rights reserved.
Proceedings of the National Academy of Sciences, 2015
Dual leucine-zipper kinase (DLK) is critical for axon-to-soma retrograde signaling following nerv... more Dual leucine-zipper kinase (DLK) is critical for axon-to-soma retrograde signaling following nerve injury. However, it is unknown how DLK, a predicted soluble kinase, conveys long-distance signals and why homologous kinases cannot compensate for loss of DLK. Here, we report that DLK, but not homologous kinases, is palmitoylated at a conserved site adjacent to its kinase domain. Using short-hairpin RNA knockdown/rescue, we find that palmitoylation is critical for DLK-dependent retrograde signaling in sensory axons. This functional importance is because of three novel cellular and molecular roles of palmitoylation, which targets DLK to trafficking vesicles, is required to assemble DLK signaling complexes and, unexpectedly, is essential for DLK’s kinase activity. By simultaneously controlling DLK localization, interactions, and activity, palmitoylation ensures that only vesicle-bound DLK is active in neurons. These findings explain how DLK specifically mediates nerve injury responses a...
ABSTRACT We present an implemented method for automated quantification of neuronal beading. Beadi... more ABSTRACT We present an implemented method for automated quantification of neuronal beading. Beading is the “morphological hallmark” of axonal injury and is present in many neurological disease conditions as well. In vitro models of injury result in beading which has previously been difficult to quantify, and therefore difficult to use as a measurement of the degree of axonal injury. The beading program was designed to quantify beading intensity in single axons and output a unitless beading number that represents the size and number of beads in relation to the length and width of the axon. The program was used to analyze a sample set of images and was successful in demonstrating a quantifiable difference between injured neurons and sham controls. Bland-Altman analysis was used to assess agreement between the number of beads counted by the program and those counted manually.
The protrusion of filopodia and lamellipodia from the surface of neurons is fundamental to axon e... more The protrusion of filopodia and lamellipodia from the surface of neurons is fundamental to axon extension, guidance, the formation of axon branches, and synaptic structures. Protrusion is driven by the polymerization and controlled organization of actin filament arrays. This chapter provides an overview of the basic mechanisms operative during protrusion. Mechanisms underlying the suppression of protrusive activity are also discussed
Neurons are arguably the most complex cells in nature and are characterized by a complex, dynamic... more Neurons are arguably the most complex cells in nature and are characterized by a complex, dynamic, and highly polarized morphology. Actin and its regulatory proteins are the most abundant set of proteins within cells and form one of the major cytoskeletal systems, the actin filament cytoskeleton. While much has been learned about the roles of the actin cytoskeleton in non-neuronal cells, our understanding of the full spectrum of the functions of actin in neurons is far from complete. This book is intended to provide the neuroscience community with an introduction to the interface between the actin cytoskeleton and the myriad of issues fundamental to the understanding of nervous system function. The book covers the neurobiology of actin ranging from basic cellular organization and function to the roles of actin in the health and disease states of the nervous system. This chapter provides a primer on actin intended to serve the reader as background and review for the rest of the chapters.
Vunjak-Novakovic/Culture of Cells for Tissue Engineering, 2005
This chapter represents the joint effort of three distinct laboratories to describe their differe... more This chapter represents the joint effort of three distinct laboratories to describe their different approaches and methodologies for engineering neuroendocrine/neuronal tissues for the possible treatment of neurodegenerative diseases, such as Parkinson disease, which are related to the loss of catecholaminergic neurons. In the first part, Lelkes and Unsworth discuss the generation of three-dimensional catecholaminergic, adrenal medullary organoids with PC12 pheochromocytoma cells. In the second part, Saporta and Cameron ...
The localized debundling of the axonal microtubule array and the entry of microtubules into axona... more The localized debundling of the axonal microtubule array and the entry of microtubules into axonal filopodia are two defining features of collateral branching. We report that nerve growth factor (NGF), a branch inducing signal, increases the frequency of microtubule debundling along the axon shaft of chicken embryonic sensory neurons. Sites of debundling correlate strongly with the localized targeting of microtubules into filopodia. Platinum replica electron microscopy suggests physical interactions between debundled microtubules and axonal actin filaments. However, as evidenced by depolymerization of actin filaments and inhibition of myosin II, actomyosin force generation does not promote debundling. In contrast, loss of actin filaments or inhibition of myosin II activity promotes debundling, indicating that axonal actomyosin forces suppress debundling. MAP1B is a microtubule associated protein that represses axon branching. Following treatment with NGF, microtubules penetrating fi...
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