The mechanisms by which mutations in FUS and other RNA binding proteins cause ALS and FTD remain ... more The mechanisms by which mutations in FUS and other RNA binding proteins cause ALS and FTD remain controversial. We propose a model in which low-complexity (LC) domains of FUS drive its physiologically reversible assembly into membrane-free, liquid droplet and hydrogel-like structures. ALS/FTD mutations in LC or non-LC domains induce further phase transition into poorly soluble fibrillar hydrogels distinct from conventional amyloids. These assemblies are necessary and sufficient for neurotoxicity in a C. elegans model of FUS-dependent neurodegeneration. They trap other ribonucleoprotein (RNP) granule components and disrupt RNP granule function. One consequence is impairment of new protein synthesis by cytoplasmic RNP granules in axon terminals, where RNP granules regulate local RNA metabolism and translation. Nuclear FUS granules may be similarly affected. Inhibiting formation of these fibrillar hydrogel assemblies mitigates neurotoxicity and suggests a potential therapeutic strategy that may also be applicable to ALS/FTD associated with mutations in other RNA binding proteins.
Cell adhesion molecules play a central role in mediating axonal tract development within the nasc... more Cell adhesion molecules play a central role in mediating axonal tract development within the nascent nervous system. NF-protocadherin (NFPC), a member of the non-clustered protocadherin family, has been shown to regulate retinal ganglion cell (RGC) axon and dendrite initiation, as well as influencing axonal navigation within the mid-optic tract. However, whether NFPC mediates RGC axonal behaviour at other positions within the optic pathway remains unclear. Here we report that NFPC plays an important role in RGC axonogenesis, but not in intraretinal guidance. Moreover, axons with reduced NFPC levels exhibit insensitivity to Netrin-1, an attractive guidance cue expressed at the optic nerve head. Netrin-1 induces rapid turnover of NFPC localized to RGC growth cones, suggesting that the regulation of NFPC protein levels may underlie Netrin-1-mediated entry of RGC axons into the optic nerve head. At the tectum, we further reveal a function for NFPC in controlling RGC axonal entry into th...
Advances in Experimental Medicine and Biology, 2007
During the formation of the nervous system, axonal growth cones navigate through the complex envi... more During the formation of the nervous system, axonal growth cones navigate through the complex environment of the developing embryo to innervate their targets. Growth cones achieve this formidable feat by responding to attractive or repulsive guidance cues expressed at specific points along the trajectory of their growth, which impart the directional information required for accurate pathfinding. While much is known about guidance molecules and their receptors, many questions remain unanswered. Which signal transduction pathways are activated within the growth cone after encountering a guidance cue? How is this related to rearrangement of the growth cone cytoskeleton? Do different cues use different signal transduction pathways? This chapter will review some of the work that has addressed these fundamental questions, with a specific focus on the role of the cyclic nucleotides, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), in axon guidance.
Axon growth is governed by the ability of growth cones to interpret attractive and repulsive guid... more Axon growth is governed by the ability of growth cones to interpret attractive and repulsive guidance cues. Recent studies have shown that secreted signaling molecules known as morphogens can also act as axon guidance cues. Of the large family of Wnt signaling components, only Wnt4 and Wnt5 seem to participate directly in axon guidance. Here we show that secreted Frizzledrelated protein 1 (SFRP1), a proposed Wnt signaling inhibitor, can directly modify and reorient the growth of chick and Xenopus laevis retinal ganglion cell axons. This activity does not require Wnt inhibition and is modulated by extracellular matrix molecules. Intracellularly, SFRP1 function requires G a protein activation, protein synthesis and degradation, and it is modulated by cyclic nucleotide levels. Because SFRP1 interacts with Frizzled-2 (Fz2) and interference with Fz2 expression abolishes growth cone responses to SFRP1, we propose a previously unknown function for this molecule: the ability to guide growth cone movement via the Fz2 receptor.
Regulated protein degradation via the ubiquitin-proteasome system (UPS) plays a central role in b... more Regulated protein degradation via the ubiquitin-proteasome system (UPS) plays a central role in building synaptic connections, yet little is known about either which specific UPS components are involved or UPS targets in neurons. We report that inhibiting the UPS in developing Xenopus retinal ganglion cells (RGCs) with a dominant-negative ubiquitin mutant decreases terminal branching in the tectum but does not affect long-range navigation to the tectum. We identify Nedd4 as a prominently expressed E3 ligase in RGC axon growth cones and show that disrupting its function severely inhibits terminal branching. We further demonstrate that PTEN, a negative regulator of the PI3K pathway, is a key downstream target of Nedd4: not only does Nedd4 regulate PTEN levels in RGC growth cones, but also, the decrease of PTEN rescues the branching defect caused by Nedd4 inhibition. Together our data suggest that Nedd4-regulated PTEN is a key regulator of terminal arborization in vivo.
NF-protocadherin (NFPC)-mediated cell-cell adhesion plays a critical role in vertebrate neural tu... more NF-protocadherin (NFPC)-mediated cell-cell adhesion plays a critical role in vertebrate neural tube formation. NFPC is also expressed during the period of axon tract formation, but little is known about its function in axonogenesis. Here we have tested the role of NFPC and its cytosolic cofactor template-activating factor 1 (TAF1) in the emergence of the Xenopus retinotectal projection. NFPC is expressed in the developing retina and optic pathway and is abundant in growing retinal axons. Inhibition of NFPC function in developing retinal ganglion cells (RGCs) severely reduces axon initiation and elongation and suppresses dendrite genesis. Furthermore, an identical phenotype occurs when TAF1 function is blocked. These data provide evidence that NFPC regulates axon initiation and elongation and indicate a conserved role for TAF1, a transcriptional regulator, as a downstream cytosolic effector of NFPC in RGCs.
The assembly of functional neural circuits in the developing brain requires neurons to extend axo... more The assembly of functional neural circuits in the developing brain requires neurons to extend axons to the correct targets. This in turn requires the navigating tips of axons to respond appropriately to guidance cues present along the axonal pathway, despite being cellular 'outposts' far from the soma. Work over the past few years has demonstrated a critical role for local translation within the axon in this process in vitro, making axon guidance another process that requires spatially localized translation, among others such as synaptic plasticity, cell migration, and cell polarity. This article reviews recent findings in local axonal translation and discusses how new protein synthesis may function in growth cone guidance, with a comparative view toward models of local translation in other systems.
Recent data indicate that local translation in growth cones is critical for axon guidance. Eviden... more Recent data indicate that local translation in growth cones is critical for axon guidance. Evidence from the Drosophila midline axon guidance system suggests that the F-actin-microtubule cross-linker Short stop (Shot) might link the translation machinery to the cytoskeleton in the growth cone. The identification of a complex of translation factors attached to the actin and microtubule networks points to a mechanism by which cytoskeletal dynamics regulate translation in axons and vice versa.
Slits mediate multiple axon guidance decisions, but the mechanisms underlying the responses of gr... more Slits mediate multiple axon guidance decisions, but the mechanisms underlying the responses of growth cones to these cues remain poorly defined. We show here that collapse induced by Slit2-conditioned medium (Slit2-CM) in Xenopus retinal growth cones requires local protein synthesis (PS) and endocytosis. Slit2-CM elicits rapid activation of translation regulators and MAP kinases in growth cones, and inhibition of MAPKs or disruption of heparan sulfate blocks Slit2-CM-induced PS and repulsion. Interestingly, Slit2-CM causes a fast PS-dependent decrease in cytoskeletal F-actin concomitant with a PS-dependent increase in the actin-depolymerizing protein cofilin. Our findings reveal an unexpected link between Slit2 and cofilin in growth cones and suggest that local translation of actin regulatory proteins contributes to repulsion.
Background: Translation in axons is required for growth cone chemotropic responses to many guidan... more Background: Translation in axons is required for growth cone chemotropic responses to many guidance cues. Although locally synthesized proteins are beginning to be identified, how specific mRNAs are selected for translation remains unclear. Control of poly(A) tail length by cytoplasmic polyadenylation element (CPE) binding protein 1 (CPEB1) is a conserved mechanism for mRNAspecific translational regulation that could be involved in regulating translation in axons.
Local protein synthesis regulates the turning of growth cones to guidance cues yet little is know... more Local protein synthesis regulates the turning of growth cones to guidance cues yet little is known about which proteins are synthesized or how they contribute to directional steering. Here we show that β-actin mRNA resides in Xenopus retinal growth cones where it binds to the RNA-binding protein, Vg1RBP. Netrin-1 induces the movement of Vg1RBP granules into filopodia suggesting that it may direct the localization and translation of mRNAs in growth cones. Indeed, a gradient of netrin-1 activates the translation initiation regulator, eIF4E-BP, asymmetrically and triggers a polarized increase in β-actin translation on the near side of the growth cone prior to growth cone turning. Inhibition of β-actin translation abolishes both the asymmetric rise in β-actin and attractive, but not repulsive, turning. Our data suggest that newly synthesized β-actin, concentrated near sites of signal reception, provides the directional bias for polymerizing actin in the direction of an attractive stimulus. stimulation with neurotrophins, such as NT-3, BDNF or NGF, increases the transport of βactin mRNA in both embryonic and adult axons13-15. Asymmetric actin polymerization is thought to play a major role in growth cone turning, since asymmetrical application of cytochalasin, an actin depolymerizing drug, causes repulsive turning16. These findings suggest that asymmetrical β-actin synthesis may be involved in growth cone turning.
It has been proposed that growth cones navigating through gradients adapt to baseline concentrati... more It has been proposed that growth cones navigating through gradients adapt to baseline concentrations of guidance cues. This adaptation process is poorly understood. Using the collapse assay, we show that adaptation in Xenopus laevis retinal growth cones to the guidance cues Sema3A or netrin-1 involves two processes: a fast, ligand-specific desensitization that occurs within 2 min of exposure and is dependent on endocytosis, and a slower, ligand-specific resensitization, which occurs within 5 min and is dependent upon protein synthesis. These two phases of adaptation allow retinal axons to adjust their range of sensitivity to specific guidance cues.
Nerve growth factor induces sensory neuron survival via retrograde signalling from the axon to th... more Nerve growth factor induces sensory neuron survival via retrograde signalling from the axon to the cell body. Local translation of the transcription factor CREB in the axon, followed by its transport to the nucleus, is involved in this process.
We have developed a compartmentalised culture model for the purification of axonal mRNA from embr... more We have developed a compartmentalised culture model for the purification of axonal mRNA from embryonic, neonatal and adult rat dorsal root ganglia. This mRNA was used un-amplified for RT-qPCR. We assayed for the presence of axonal mRNAs encoding molecules known to be involved in axon growth and guidance. mRNAs for β-actin, β-tubulin, and several molecules involved in the control of actin dynamics and signalling during axon growth were found, but mRNAs for microtubule-associated proteins, integrins and cell surface adhesion molecules were absent. Quantification of β-actin mRNA by means of qPCR showed that the transcript is present at the same level in embryonic, newborn and adult axons. Using the photoconvertible reporter Kaede we showed that there is local translation of β-actin in axons, the rate being increased by axotomy. Knock down of β-actin mRNA by RNAi inhibited the regeneration of new axon growth cones after in vitro axotomy, indicating that local translation of actin-related molecules is important for successful axon regeneration.
be investigated in vivo. In this study, we tried to build up the system to observe mesoderm cell ... more be investigated in vivo. In this study, we tried to build up the system to observe mesoderm cell migration in chicken embryo using live imaging.
Slit is a large secreted protein that provides important guidance cues in the developing nervous ... more Slit is a large secreted protein that provides important guidance cues in the developing nervous system and in other organs. Signaling by Slit requires two receptors, Robo transmembrane proteins and heparan sulfate (HS) proteoglycans. How HS controls Slit-Robo signaling is unclear.
In Vitro Cellular & Developmental Biology - Animal, 1996
... JC BURNS, L. McNEILL, C. SHIMIZU, T. MATSUBARA, J.-K. YEE, T. FRIEDMANN, B.KURDI-HAIDAR, E. M... more ... JC BURNS, L. McNEILL, C. SHIMIZU, T. MATSUBARA, J.-K. YEE, T. FRIEDMANN, B.KURDI-HAIDAR, E. MALIWAT, AND CE HOLT 1 ... transferred to 2.5-ml vials, and incubated in Xgal solution [10 mM KaFe(CN)6 , 10 mM K4Fe(CN)6 , and 0.15% x-gal in dimethylsulfoxide ...
The mechanisms by which mutations in FUS and other RNA binding proteins cause ALS and FTD remain ... more The mechanisms by which mutations in FUS and other RNA binding proteins cause ALS and FTD remain controversial. We propose a model in which low-complexity (LC) domains of FUS drive its physiologically reversible assembly into membrane-free, liquid droplet and hydrogel-like structures. ALS/FTD mutations in LC or non-LC domains induce further phase transition into poorly soluble fibrillar hydrogels distinct from conventional amyloids. These assemblies are necessary and sufficient for neurotoxicity in a C. elegans model of FUS-dependent neurodegeneration. They trap other ribonucleoprotein (RNP) granule components and disrupt RNP granule function. One consequence is impairment of new protein synthesis by cytoplasmic RNP granules in axon terminals, where RNP granules regulate local RNA metabolism and translation. Nuclear FUS granules may be similarly affected. Inhibiting formation of these fibrillar hydrogel assemblies mitigates neurotoxicity and suggests a potential therapeutic strategy that may also be applicable to ALS/FTD associated with mutations in other RNA binding proteins.
Cell adhesion molecules play a central role in mediating axonal tract development within the nasc... more Cell adhesion molecules play a central role in mediating axonal tract development within the nascent nervous system. NF-protocadherin (NFPC), a member of the non-clustered protocadherin family, has been shown to regulate retinal ganglion cell (RGC) axon and dendrite initiation, as well as influencing axonal navigation within the mid-optic tract. However, whether NFPC mediates RGC axonal behaviour at other positions within the optic pathway remains unclear. Here we report that NFPC plays an important role in RGC axonogenesis, but not in intraretinal guidance. Moreover, axons with reduced NFPC levels exhibit insensitivity to Netrin-1, an attractive guidance cue expressed at the optic nerve head. Netrin-1 induces rapid turnover of NFPC localized to RGC growth cones, suggesting that the regulation of NFPC protein levels may underlie Netrin-1-mediated entry of RGC axons into the optic nerve head. At the tectum, we further reveal a function for NFPC in controlling RGC axonal entry into th...
Advances in Experimental Medicine and Biology, 2007
During the formation of the nervous system, axonal growth cones navigate through the complex envi... more During the formation of the nervous system, axonal growth cones navigate through the complex environment of the developing embryo to innervate their targets. Growth cones achieve this formidable feat by responding to attractive or repulsive guidance cues expressed at specific points along the trajectory of their growth, which impart the directional information required for accurate pathfinding. While much is known about guidance molecules and their receptors, many questions remain unanswered. Which signal transduction pathways are activated within the growth cone after encountering a guidance cue? How is this related to rearrangement of the growth cone cytoskeleton? Do different cues use different signal transduction pathways? This chapter will review some of the work that has addressed these fundamental questions, with a specific focus on the role of the cyclic nucleotides, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), in axon guidance.
Axon growth is governed by the ability of growth cones to interpret attractive and repulsive guid... more Axon growth is governed by the ability of growth cones to interpret attractive and repulsive guidance cues. Recent studies have shown that secreted signaling molecules known as morphogens can also act as axon guidance cues. Of the large family of Wnt signaling components, only Wnt4 and Wnt5 seem to participate directly in axon guidance. Here we show that secreted Frizzledrelated protein 1 (SFRP1), a proposed Wnt signaling inhibitor, can directly modify and reorient the growth of chick and Xenopus laevis retinal ganglion cell axons. This activity does not require Wnt inhibition and is modulated by extracellular matrix molecules. Intracellularly, SFRP1 function requires G a protein activation, protein synthesis and degradation, and it is modulated by cyclic nucleotide levels. Because SFRP1 interacts with Frizzled-2 (Fz2) and interference with Fz2 expression abolishes growth cone responses to SFRP1, we propose a previously unknown function for this molecule: the ability to guide growth cone movement via the Fz2 receptor.
Regulated protein degradation via the ubiquitin-proteasome system (UPS) plays a central role in b... more Regulated protein degradation via the ubiquitin-proteasome system (UPS) plays a central role in building synaptic connections, yet little is known about either which specific UPS components are involved or UPS targets in neurons. We report that inhibiting the UPS in developing Xenopus retinal ganglion cells (RGCs) with a dominant-negative ubiquitin mutant decreases terminal branching in the tectum but does not affect long-range navigation to the tectum. We identify Nedd4 as a prominently expressed E3 ligase in RGC axon growth cones and show that disrupting its function severely inhibits terminal branching. We further demonstrate that PTEN, a negative regulator of the PI3K pathway, is a key downstream target of Nedd4: not only does Nedd4 regulate PTEN levels in RGC growth cones, but also, the decrease of PTEN rescues the branching defect caused by Nedd4 inhibition. Together our data suggest that Nedd4-regulated PTEN is a key regulator of terminal arborization in vivo.
NF-protocadherin (NFPC)-mediated cell-cell adhesion plays a critical role in vertebrate neural tu... more NF-protocadherin (NFPC)-mediated cell-cell adhesion plays a critical role in vertebrate neural tube formation. NFPC is also expressed during the period of axon tract formation, but little is known about its function in axonogenesis. Here we have tested the role of NFPC and its cytosolic cofactor template-activating factor 1 (TAF1) in the emergence of the Xenopus retinotectal projection. NFPC is expressed in the developing retina and optic pathway and is abundant in growing retinal axons. Inhibition of NFPC function in developing retinal ganglion cells (RGCs) severely reduces axon initiation and elongation and suppresses dendrite genesis. Furthermore, an identical phenotype occurs when TAF1 function is blocked. These data provide evidence that NFPC regulates axon initiation and elongation and indicate a conserved role for TAF1, a transcriptional regulator, as a downstream cytosolic effector of NFPC in RGCs.
The assembly of functional neural circuits in the developing brain requires neurons to extend axo... more The assembly of functional neural circuits in the developing brain requires neurons to extend axons to the correct targets. This in turn requires the navigating tips of axons to respond appropriately to guidance cues present along the axonal pathway, despite being cellular 'outposts' far from the soma. Work over the past few years has demonstrated a critical role for local translation within the axon in this process in vitro, making axon guidance another process that requires spatially localized translation, among others such as synaptic plasticity, cell migration, and cell polarity. This article reviews recent findings in local axonal translation and discusses how new protein synthesis may function in growth cone guidance, with a comparative view toward models of local translation in other systems.
Recent data indicate that local translation in growth cones is critical for axon guidance. Eviden... more Recent data indicate that local translation in growth cones is critical for axon guidance. Evidence from the Drosophila midline axon guidance system suggests that the F-actin-microtubule cross-linker Short stop (Shot) might link the translation machinery to the cytoskeleton in the growth cone. The identification of a complex of translation factors attached to the actin and microtubule networks points to a mechanism by which cytoskeletal dynamics regulate translation in axons and vice versa.
Slits mediate multiple axon guidance decisions, but the mechanisms underlying the responses of gr... more Slits mediate multiple axon guidance decisions, but the mechanisms underlying the responses of growth cones to these cues remain poorly defined. We show here that collapse induced by Slit2-conditioned medium (Slit2-CM) in Xenopus retinal growth cones requires local protein synthesis (PS) and endocytosis. Slit2-CM elicits rapid activation of translation regulators and MAP kinases in growth cones, and inhibition of MAPKs or disruption of heparan sulfate blocks Slit2-CM-induced PS and repulsion. Interestingly, Slit2-CM causes a fast PS-dependent decrease in cytoskeletal F-actin concomitant with a PS-dependent increase in the actin-depolymerizing protein cofilin. Our findings reveal an unexpected link between Slit2 and cofilin in growth cones and suggest that local translation of actin regulatory proteins contributes to repulsion.
Background: Translation in axons is required for growth cone chemotropic responses to many guidan... more Background: Translation in axons is required for growth cone chemotropic responses to many guidance cues. Although locally synthesized proteins are beginning to be identified, how specific mRNAs are selected for translation remains unclear. Control of poly(A) tail length by cytoplasmic polyadenylation element (CPE) binding protein 1 (CPEB1) is a conserved mechanism for mRNAspecific translational regulation that could be involved in regulating translation in axons.
Local protein synthesis regulates the turning of growth cones to guidance cues yet little is know... more Local protein synthesis regulates the turning of growth cones to guidance cues yet little is known about which proteins are synthesized or how they contribute to directional steering. Here we show that β-actin mRNA resides in Xenopus retinal growth cones where it binds to the RNA-binding protein, Vg1RBP. Netrin-1 induces the movement of Vg1RBP granules into filopodia suggesting that it may direct the localization and translation of mRNAs in growth cones. Indeed, a gradient of netrin-1 activates the translation initiation regulator, eIF4E-BP, asymmetrically and triggers a polarized increase in β-actin translation on the near side of the growth cone prior to growth cone turning. Inhibition of β-actin translation abolishes both the asymmetric rise in β-actin and attractive, but not repulsive, turning. Our data suggest that newly synthesized β-actin, concentrated near sites of signal reception, provides the directional bias for polymerizing actin in the direction of an attractive stimulus. stimulation with neurotrophins, such as NT-3, BDNF or NGF, increases the transport of βactin mRNA in both embryonic and adult axons13-15. Asymmetric actin polymerization is thought to play a major role in growth cone turning, since asymmetrical application of cytochalasin, an actin depolymerizing drug, causes repulsive turning16. These findings suggest that asymmetrical β-actin synthesis may be involved in growth cone turning.
It has been proposed that growth cones navigating through gradients adapt to baseline concentrati... more It has been proposed that growth cones navigating through gradients adapt to baseline concentrations of guidance cues. This adaptation process is poorly understood. Using the collapse assay, we show that adaptation in Xenopus laevis retinal growth cones to the guidance cues Sema3A or netrin-1 involves two processes: a fast, ligand-specific desensitization that occurs within 2 min of exposure and is dependent on endocytosis, and a slower, ligand-specific resensitization, which occurs within 5 min and is dependent upon protein synthesis. These two phases of adaptation allow retinal axons to adjust their range of sensitivity to specific guidance cues.
Nerve growth factor induces sensory neuron survival via retrograde signalling from the axon to th... more Nerve growth factor induces sensory neuron survival via retrograde signalling from the axon to the cell body. Local translation of the transcription factor CREB in the axon, followed by its transport to the nucleus, is involved in this process.
We have developed a compartmentalised culture model for the purification of axonal mRNA from embr... more We have developed a compartmentalised culture model for the purification of axonal mRNA from embryonic, neonatal and adult rat dorsal root ganglia. This mRNA was used un-amplified for RT-qPCR. We assayed for the presence of axonal mRNAs encoding molecules known to be involved in axon growth and guidance. mRNAs for β-actin, β-tubulin, and several molecules involved in the control of actin dynamics and signalling during axon growth were found, but mRNAs for microtubule-associated proteins, integrins and cell surface adhesion molecules were absent. Quantification of β-actin mRNA by means of qPCR showed that the transcript is present at the same level in embryonic, newborn and adult axons. Using the photoconvertible reporter Kaede we showed that there is local translation of β-actin in axons, the rate being increased by axotomy. Knock down of β-actin mRNA by RNAi inhibited the regeneration of new axon growth cones after in vitro axotomy, indicating that local translation of actin-related molecules is important for successful axon regeneration.
be investigated in vivo. In this study, we tried to build up the system to observe mesoderm cell ... more be investigated in vivo. In this study, we tried to build up the system to observe mesoderm cell migration in chicken embryo using live imaging.
Slit is a large secreted protein that provides important guidance cues in the developing nervous ... more Slit is a large secreted protein that provides important guidance cues in the developing nervous system and in other organs. Signaling by Slit requires two receptors, Robo transmembrane proteins and heparan sulfate (HS) proteoglycans. How HS controls Slit-Robo signaling is unclear.
In Vitro Cellular & Developmental Biology - Animal, 1996
... JC BURNS, L. McNEILL, C. SHIMIZU, T. MATSUBARA, J.-K. YEE, T. FRIEDMANN, B.KURDI-HAIDAR, E. M... more ... JC BURNS, L. McNEILL, C. SHIMIZU, T. MATSUBARA, J.-K. YEE, T. FRIEDMANN, B.KURDI-HAIDAR, E. MALIWAT, AND CE HOLT 1 ... transferred to 2.5-ml vials, and incubated in Xgal solution [10 mM KaFe(CN)6 , 10 mM K4Fe(CN)6 , and 0.15% x-gal in dimethylsulfoxide ...
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Papers by Christine Holt