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• W3035163008 abstract "•NGF-dependent growth requires axonal protein prenylation in sympathetic neurons•NGF promotes prenylation of locally synthesized proteins in axons•Prenylation is essential for endocytic trafficking of TrkA receptors•Prenylation of axonally translated Rac1 is essential for NGF-dependent growth Compartmentalized signaling is critical for cellular organization and specificity of functional outcomes in neurons. Here, we report that post-translational lipidation of newly synthesized proteins in axonal compartments allows for short-term and autonomous responses to extrinsic cues. Using conditional mutant mice, we found that protein prenylation is essential for sympathetic axon innervation of target organs. We identify a localized requirement for prenylation in sympathetic axons to promote axonal growth in response to the neurotrophin, nerve growth factor (NGF). NGF triggers prenylation of proteins including the Rac1 GTPase in axons, counter to the canonical view of prenylation as constitutive, and strikingly, in a manner dependent on axonal protein synthesis. Newly prenylated proteins localize to TrkA-harboring endosomes in axons and promote receptor trafficking necessary for axonal growth. Thus, coupling of prenylation to local protein synthesis presents a mechanism for spatially segregated cellular functions during neuronal development. Compartmentalized signaling is critical for cellular organization and specificity of functional outcomes in neurons. Here, we report that post-translational lipidation of newly synthesized proteins in axonal compartments allows for short-term and autonomous responses to extrinsic cues. Using conditional mutant mice, we found that protein prenylation is essential for sympathetic axon innervation of target organs. We identify a localized requirement for prenylation in sympathetic axons to promote axonal growth in response to the neurotrophin, nerve growth factor (NGF). NGF triggers prenylation of proteins including the Rac1 GTPase in axons, counter to the canonical view of prenylation as constitutive, and strikingly, in a manner dependent on axonal protein synthesis. Newly prenylated proteins localize to TrkA-harboring endosomes in axons and promote receptor trafficking necessary for axonal growth. Thus, coupling of prenylation to local protein synthesis presents a mechanism for spatially segregated cellular functions during neuronal development. Spatial partitioning of biochemical processes is a fundamental principle that underlies cellular structure and specificity of functional responses in all cells but is particularly relevant in polarized nerve cells. Neurons rely on asymmetric distribution of RNA, proteins, and lipids to specialized sub-cellular domains to accomplish compartment-specific functions (Horton and Ehlers, 2003Horton A.C. Ehlers M.D. Neuronal polarity and trafficking.Neuron. 2003; 40: 277-295Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). Proteins involved in growth cone migration, axon extension, and neurotransmitter release are enriched in axons, whereas proteins involved in post-synaptic functions, including neurotransmitter receptors accumulate in dendrites and spines (Horton and Ehlers, 2003Horton A.C. Ehlers M.D. Neuronal polarity and trafficking.Neuron. 2003; 40: 277-295Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). How such segregation of cellular material is established and maintained in neuronal compartments to allow autonomous responses to extrinsic cues or neural activity remains poorly defined. Lipidation is a post-translational modification that makes proteins hydrophobic and facilitates their insertion into the plasma membrane or intracellular membranes (Jiang et al., 2018Jiang H. Zhang X. Chen X. Aramsangtienchai P. Tong Z. Lin H. Protein lipidation: occurrence, mechanisms, biological functions, and enabling technologies.Chem. Rev. 2018; 118: 919-988Crossref PubMed Scopus (194) Google Scholar). Protein prenylation is an irreversible modification that involves the transfer of farnesyl or geranylgeranyl isoprenoid lipids to conserved carboxyl terminal CaaX motifs in proteins and is predicted to affect at least 200 mammalian proteins (Wang and Casey, 2016Wang M. Casey P.J. Protein prenylation: unique fats make their mark on biology.Nat. Rev. Mol. Cell Biol. 2016; 17: 110-122Crossref PubMed Scopus (293) Google Scholar). Despite critical functions of proteins predicted to be prenylated in cellular signaling, cytoskeleton remodeling, and vesicular trafficking (Wang and Casey, 2016Wang M. Casey P.J. Protein prenylation: unique fats make their mark on biology.Nat. Rev. Mol. Cell Biol. 2016; 17: 110-122Crossref PubMed Scopus (293) Google Scholar), the functional relevance of prenyl groups for individual proteins is poorly understood. Further, protein prenylation is considered to be a constitutive process that occurs ubiquitously throughout the cytoplasm in eukaryotic cells (Sinensky, 2000Sinensky M. Recent advances in the study of prenylated proteins.Biochim. Biophys. Acta. 2000; 1484: 93-106Crossref PubMed Scopus (208) Google Scholar, Wang and Casey, 2016Wang M. Casey P.J. Protein prenylation: unique fats make their mark on biology.Nat. Rev. Mol. Cell Biol. 2016; 17: 110-122Crossref PubMed Scopus (293) Google Scholar). The highest expression of isoprenoid lipids and prenyl transferases, enzymes responsible for adding isoprenoid lipids to newly synthesized proteins, is found in the nervous system (Joly et al., 1991Joly A. Popják G. Edwards P.A. In vitro identification of a soluble protein:geranylgeranyl transferase from rat tissues.J. Biol. Chem. 1991; 266: 13495-13498PubMed Google Scholar, Tong et al., 2008Tong H. Wiemer A.J. Neighbors J.D. Hohl R.J. Quantitative determination of farnesyl and geranylgeranyl diphosphate levels in mammalian tissue.Anal. Biochem. 2008; 378: 138-143Crossref PubMed Scopus (37) Google Scholar). Given their complex morphologies and cellular polarity, prenylation could be particularly critical for spatially segregating protein functions in neurons. Here, we describe a mechanism where a neurotrophic factor couples local synthesis of protein effectors with their lipid modification in axonal compartments to allow acute and spatial responses necessary for axon development. Using compartmentalized cultures of sympathetic neurons, we identified a unique need for local protein prenylation in axons to promote growth in response to nerve growth factor (NGF), a target-derived axon growth and survival factor. NGF acutely triggers prenylation of proteins in distal axons and growth cones of sympathetic neurons. Notably, the lipid modifications occur on proteins that are locally synthesized in axons. The newly modified proteins localize to endosomes harboring TrkA receptors for NGF in axons and promote receptor trafficking, which is a critical determinant of trophic signaling. In mice, protein prenylation is essential for NGF-dependent axon innervation of targets and neuronal survival. Together, these results suggest that coupling of local protein synthesis with post-translational lipidation in axons is a mechanism for compartmentalized responses to spatial cues during neuronal development. To determine where protein prenylation occurs in polarized neurons, we investigated the expression of farnesyl and geranylgeranyl transferase I (FTase and GGTase I, respectively) in sympathetic neurons. FTase and GGTase I catalyze the addition of either a farnesyl or geranylgeranyl isoprenoid lipid to proteins. They are expressed as heterodimers that share a common α-subunit and have distinct β-subunits. Immunostaining with an antibody against the shared prenyl transferase α-subunit (Luo et al., 2003Luo Z.G. Je H.S. Wang Q. Yang F. Dobbins G.C. Yang Z.H. Xiong W.C. Lu B. Mei L. Implication of geranylgeranyltransferase I in synapse formation.Neuron. 2003; 40: 703-717Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) showed expression in sympathetic neuron cell bodies and axon fibers innervating a target, the salivary glands, in mice at post-natal day 5 (P5) (Figures S1A and S1B). This is a developmental period when sympathetic neurons rely on the neurotrophin, NGF, released from peripheral targets, for their survival and axon innervation (Glebova and Ginty, 2005Glebova N.O. Ginty D.D. Growth and survival signals controlling sympathetic nervous system development.Annu. Rev. Neurosci. 2005; 28: 191-222Crossref PubMed Scopus (211) Google Scholar). Immunostaining of cultured sympathetic neurons revealed prenyl transferase expression in cell bodies, axons, and growth cones (Figure S1C). Immunoblotting of lysates from compartmentalized neuron cultures, where a Teflon-grease diffusion barrier separates cell bodies from axons, showed a protein of the predicted size (44 kDa) in both compartments (Figure S1D). Antibody specificity was verified in PC12 cells by shRNA transfection and immunoblotting (Figure S1E). In neuron cell bodies and PC12 cells, two higher molecular weight bands at approximately 60–65 kDa were also observed that were reduced by short hairpin RNA (shRNA)-mediated knockdown, suggesting a likely post-translational modification. Together, these results indicate that the molecular machinery required for protein prenylation is present in cell bodies, axons, and even growth cones of sympathetic neurons. To visualize protein prenylation in sympathetic neurons, we developed a live-cell feeding assay in compartmentalized cultures. NGF (50 ng/mL) was added only to distal axon compartments, recapitulating the release of neurotrophins from target tissues. Prenylation was visualized by incubating cell bodies or axons with a membrane-permeable prenyl lipid analog, propargyl-farnesol (isoprenoid analog), which is metabolically incorporated into cellular proteins at native CaaX sites using endogenous prenyl transferase activity (DeGraw et al., 2010DeGraw A.J. Palsuledesai C. Ochocki J.D. Dozier J.K. Lenevich S. Rashidian M. Distefano M.D. Evaluation of alkyne-modified isoprenoids as chemical reporters of protein prenylation.Chem. Biol. Drug Des. 2010; 76: 460-471Crossref PubMed Scopus (59) Google Scholar). Newly modified proteins were visualized by conjugation to a fluorophore (biotin azide-streptavidin-Alexa-488) using click chemistry-based labeling in fixed cells. We observed prominent isoprenoid reporter labeling, indicative of newly prenylated proteins, appearing in a punctate or sometimes tubular pattern, in cell bodies, axon shafts, and distal axons (Figures 1A–1C ). The non-diffuse labeling pattern, specifically in axons, suggests that protein prenylation does not occur throughout the cytoplasm, but rather in discrete sub-cellular sites. No background fluorescence was observed when isoprenoid analog was omitted in the click reaction (Figures S2A–S2C), confirming the specificity of the prenylation signal in Figures 1A–1C. Moreover, inducible deletion of GGTase I-β subunit (encoded by Pggt1b) by adding Cre recombinase to sympathetic neurons from Pggt1bfl/fl mice (Sjogren et al., 2007Sjogren A.K. Andersson K.M. Liu M. Cutts B.A. Karlsson C. Wahlstrom A.M. Dalin M. Weinbaum C. Casey P.J. Tarkowski A. et al.GGTase-I deficiency reduces tumor formation and improves survival in mice with K-RAS-induced lung cancer.J. Clin. Invest. 2007; 117: 1294-1304Crossref PubMed Scopus (93) Google Scholar) substantially reduced labeling in cell bodies and distal axons (Figures S2D–S2I). GGTase I loss did not completely abolish the prenylation signal since the isoprenoid analog reports both geranylgeranylation and farnesylation, and FTase is still able to mediate farnesylation. NGF mediates growth of sympathetic axons by binding to TrkA receptors in axons and promoting the formation of NGF-TrkA signaling endosomes that signal locally or are retrogradely trafficked to cell bodies (Scott-Solomon and Kuruvilla, 2018Scott-Solomon E. Kuruvilla R. Mechanisms of neurotrophin trafficking via Trk receptors.Mol. Cell. Neurosci. 2018; 91: 25-33Crossref PubMed Scopus (50) Google Scholar). Several protein effectors involved in NGF-mediated signaling, trafficking, and cytoskeletal remodeling are predicted to be prenylated (Delcroix et al., 2003Delcroix J.D. Valletta J.S. Wu C. Hunt S.J. Kowal A.S. Mobley W.C. NGF signaling in sensory neurons: evidence that early endosomes carry NGF retrograde signals.Neuron. 2003; 39: 69-84Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar, Wang and Casey, 2016Wang M. Casey P.J. Protein prenylation: unique fats make their mark on biology.Nat. Rev. Mol. Cell Biol. 2016; 17: 110-122Crossref PubMed Scopus (293) Google Scholar, Wu et al., 2007Wu C. Ramirez A. Cui B. Ding J. Delcroix J.D. Valletta J.S. Liu J.J. Yang Y. Chu S. Mobley W.C. A functional dynein-microtubule network is required for NGF signaling through the Rap1/MAPK pathway.Traffic. 2007; 8: 1503-1520Crossref PubMed Scopus (62) Google Scholar, Zweifel et al., 2005Zweifel L.S. Kuruvilla R. Ginty D.D. Functions and mechanisms of retrograde neurotrophin signalling.Nat. Rev. Neurosci. 2005; 6: 615-625Crossref PubMed Scopus (346) Google Scholar), although the functional requirement for the lipid modifications is unknown. To address whether local protein prenylation is necessary for mediating axon growth, cell bodies or axons in compartmentalized cultures were treated with the membrane-permeable competitive inhibitors against FTase (FTI-277) or GGTase I (GGTI-2133) in conjunction with NGF treatment to distal axons (Figure 1D). FTI-277 and GGTI-2133 are peptidomimetics that mimic CaaX motifs specific for FTase or GGTase I, respectively (Clapp et al., 2013Clapp K.M. Ellsworth M.L. Sprague R.S. Stephenson A.H. Simvastatin and GGTI-2133, a geranylgeranyl transferase inhibitor, increase erythrocyte deformability but reduce low O(2) tension-induced ATP release.Am. J. Physiol. Heart Circ. Physiol. 2013; 304: H660-H666Crossref PubMed Scopus (10) Google Scholar, Lerner et al., 1995Lerner E.C. Qian Y. Blaskovich M.A. Fossum R.D. Vogt A. Sun J. Cox A.D. Der C.J. Hamilton A.D. Sebti S.M. Ras CAAX peptidomimetic FTI-277 selectively blocks oncogenic Ras signaling by inducing cytoplasmic accumulation of inactive Ras-Raf complexes.J. Biol. Chem. 1995; 270: 26802-26806Crossref PubMed Scopus (346) Google Scholar). NGF stimulation resulted in robust growth of sympathetic axons (Figures 1E, 1F, 1I–1K, and 1N). The FTase inhibitor added to either cell bodies or axons attenuated NGF-dependent axon growth (Figures 1G–1I). Treatment of axons with the GGTase I inhibitor also diminished axon growth in response to NGF (Figures 1L and 1N). Surprisingly, inhibition of GGTase I activity in cell bodies had no effect (Figures 1M and 1N). Prenyl transferase inhibition had no effect on neuron viability or morphology in the absence of NGF when neurons were maintained in the presence of BAF, a caspase inhibitor (Figures S2J–S2N). Together, these results indicate a specific need for proteins to undergo geranylgeranylation locally in axons to mediate NGF-dependent axon growth. The axon-specific requirement for GGTase I in NGF-directed axon growth raised the possibility it may be regulated by NGF signaling. A fluorescent assay to measure GGTase I enzymatic activity was performed after exposing distal axons of compartmentalized cultures to NGF for 30 min (Figure 2A). NGF treatment triggered a 4-fold increase in GGTase I activity in sympathetic axons but had no effect on enzyme activity in cell bodies within this time period (Figure 2B). Addition of GGTI-2133 (75 nM) significantly diminished NGF-induced GGTase I activity to levels comparable to the un-stimulated condition (Figure S3), indicating the specificity of the enzymatic assay. To visualize NGF-induced prenylation, we fed the isoprenoid analog to sympathetic neurons in the presence or absence of NGF for 4 h. Excess analog was washed from neurons and newly modified proteins visualized by biotin conjugation and streptavidin-Alexa-488 labeling. NGF increased prenylation of proteins locally in axons, particularly in axonal growth cones (Figures 2C–2G). Strikingly, treatment of neurons with GGTI-2133 abolished incorporation of isoprenoid analog in NGF-treated neurons (Figures 2F and 2G), suggesting that proteins modified by NGF treatment are GGTase I substrates. Together, these results indicate that NGF acutely regulates GGTase I activity and protein geranylgeranylation in both axons and growth cones of sympathetic neurons. NGF signaling is initiated by binding TrkA receptors in sympathetic axons, receptor dimerization and autophosphorylation, and internalization of ligand-receptor complexes in endosomes where internalized TrkA receptors continue to signal (Scott-Solomon and Kuruvilla, 2018Scott-Solomon E. Kuruvilla R. Mechanisms of neurotrophin trafficking via Trk receptors.Mol. Cell. Neurosci. 2018; 91: 25-33Crossref PubMed Scopus (50) Google Scholar). TrkA endosomes acutely regulate signaling pathways in axons and are also retrogradely transported to cell bodies to activate transcriptional programs needed for long-term axonal growth and neuron survival (Scott-Solomon and Kuruvilla, 2018Scott-Solomon E. Kuruvilla R. Mechanisms of neurotrophin trafficking via Trk receptors.Mol. Cell. Neurosci. 2018; 91: 25-33Crossref PubMed Scopus (50) Google Scholar). TrkA trafficking is known to rely on several effector proteins, especially small GTPases, that are predicted to be prenylated (Harrington et al., 2011Harrington A.W. St Hillaire C. Zweifel L.S. Glebova N.O. Philippidou P. Halegoua S. Ginty D.D. Recruitment of actin modifiers to TrkA endosomes governs retrograde NGF signaling and survival.Cell. 2011; 146: 421-434Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, Kawata et al., 1990Kawata M. Farnsworth C.C. Yoshida Y. Gelb M.H. Glomset J.A. Takai Y. Posttranslationally processed structure of the human platelet protein smg p21B: evidence for geranylgeranylation and carboxyl methylation of the C-terminal cysteine.Proc. Natl. Acad. Sci. USA. 1990; 87: 8960-8964Crossref PubMed Scopus (111) Google Scholar, Wu et al., 2001Wu C. Lai C.F. Mobley W.C. Nerve growth factor activates persistent Rap1 signaling in endosomes.J. Neurosci. 2001; 21: 5406-5416Crossref PubMed Google Scholar). Based on the appearance of newly prenylated proteins in discrete punctae or tubules (Figures 1B and 1C), we asked whether modified proteins localize to endosomal compartments in axons, and in particular, to TrkA-harboring endosomes. To monitor trafficking of surface TrkA receptors in sympathetic neurons, we utilized a chimeric Trk receptor-based live-cell antibody feeding assay (Ascaño et al., 2009Ascaño M. Richmond A. Borden P. Kuruvilla R. Axonal targeting of Trk receptors via transcytosis regulates sensitivity to neurotrophin responses.J. Neurosci. 2009; 29: 11674-11685Crossref PubMed Scopus (145) Google Scholar). Neurons were infected with an adenoviral vector for FLAG-tagged chimeric Trk receptors that have the extracellular domain of TrkB and the transmembrane and intracellular domain of TrkA (Ascaño et al., 2009Ascaño M. Richmond A. Borden P. Kuruvilla R. Axonal targeting of Trk receptors via transcytosis regulates sensitivity to neurotrophin responses.J. Neurosci. 2009; 29: 11674-11685Crossref PubMed Scopus (145) Google Scholar). Sympathetic neurons do not normally express TrkB, and chimeric receptors respond to the TrkB ligand, brain-derived neurotrophic factor (BDNF) but retain the signaling properties of TrkA. Surface chimeric FLAG-TrkB:A receptors were live labeled with anti-FLAG antibody. Neurons were stimulated with BDNF (50 ng/mL, 4 h) in the presence of the isoprenoid analog, and remaining surface-bound anti-FLAG antibody stripped with mild acid washes. Ligand stimulation increased intracellular accumulation of chimeric Trk receptors (Figures 3A–3C , top panel, and 3F) and protein prenylation in axons (Figures 3A–3C, second panel from top). Notably, 55% of Trk receptors that underwent ligand-induced endocytosis co-localized with newly prenylated proteins in axons (Figures 3C, third panel from top, and 3E). These results suggest that the proteins prenylated in response to neurotrophin stimulation accumulate, in part, on TrkA-containing endosomes in axons. GGTI-2133 treatment attenuated ligand-induced uptake of isoprenoid analog (Figure 3D), as expected. Surprisingly, GGTI-2133 treatment diminished neurotrophin-induced intracellular accumulation of Trk receptors in axons (Figures 3D, top panel, and 3F). Together, these results suggest that newly prenylated proteins are associated with TrkA-harboring endosomes in axons and that protein prenylation likely promotes NGF-dependent axonal growth by influencing receptor trafficking. The ability of NGF to promote protein geranylgeranylation in axons raises a question as to the source of these nascent proteins. Proteins are often prenylated immediately after synthesis (Wang and Casey, 2016Wang M. Casey P.J. Protein prenylation: unique fats make their mark on biology.Nat. Rev. Mol. Cell Biol. 2016; 17: 110-122Crossref PubMed Scopus (293) Google Scholar). Further, our results that local protein geranylgeranylation is essential for axon growth (Figures 1J–1N) suggests that anterograde transport of already-modified proteins from cell bodies is not sufficient. Previously, screens of axonal mRNAs have identified many transcripts that are known to encode for prenylated proteins (Gumy et al., 2011Gumy L.F. Yeo G.S. Tung Y.C. Zivraj K.H. Willis D. Coppola G. Lam B.Y. Twiss J.L. Holt C.E. Fawcett J.W. Transcriptome analysis of embryonic and adult sensory axons reveals changes in mRNA repertoire localization.Rna. 2011; 17: 85-98Crossref PubMed Scopus (264) Google Scholar). Together, these findings raised the intriguing possibility that the proteins being geranylgeranylated in axons in response to NGF are locally translated. To determine the contribution of intra-axonal protein synthesis to local geranylgeranylation in response to NGF, we used a sympathetic ganglia explant culture system that permits the mechanical removal of cell bodies leaving the axons in isolation (Figure 4A) (Andreassi et al., 2010Andreassi C. Zimmermann C. Mitter R. Fusco S. De Vita S. Saiardi A. Riccio A. An NGF-responsive element targets myo-inositol monophosphatase-1 mRNA to sympathetic neuron axons.Nat. Neurosci. 2010; 13: 291-301Crossref PubMed Scopus (149) Google Scholar). Isolated axons remain morphologically intact and respond to NGF for at least 8 h following removal of cell bodies (Andreassi et al., 2010Andreassi C. Zimmermann C. Mitter R. Fusco S. De Vita S. Saiardi A. Riccio A. An NGF-responsive element targets myo-inositol monophosphatase-1 mRNA to sympathetic neuron axons.Nat. Neurosci. 2010; 13: 291-301Crossref PubMed Scopus (149) Google Scholar). Isolated axons were incubated with the isoprenoid analog for 6 h in the presence or absence of NGF. To determine the contribution of axonal protein synthesis to NGF-induced prenylation, incorporation of isoprenoid analog in NGF-treated axons was assessed in the presence or absence of the translation inhibitor, cycloheximide (CHX). NGF promoted robust prenylation of proteins in isolated axons, as seen previously, which was abolished by CHX treatment (Figures 4B–4F). These results suggest that NGF-induced lipid modifications occur on proteins that are locally synthesized in axons. One explanation for dependence of axonal prenylation on local protein synthesis could be that the prenylation enzymes themselves are translated in axons. To test this prediction, we performed RT-PCR analysis of mRNA isolated from distal axons of compartmentalized cultures and observed that transcript encoding for GGTase I-α (Fnta), but not GGTase I-β (Pggt1b), was localized in axons (Figure 4G). However, GGTase I-α protein levels were unaffected by NGF treatment or NGF + CHX treatments for 6 h in isolated axons (Figures 4H and 4I). To directly determine if NGF-induced GGTase I enzymatic activity relied on protein synthesis, we assessed the effect of CHX on GGTase I activity in NGF-treated neurons. NGF stimulation enhanced GGTase I enzymatic activity as observed previously (Figure 2B), and this activation was not affected by CHX treatment (Figure 4J). Together, these results suggest that the reliance of axonal prenylation on local translation is due to the synthesis of GGTase I substrates and not GGTase I itself. Which proteins are locally lipid modified in sympathetic axons in response to NGF? The Rac1 GTPase is a known GGTase I substrate (Roberts et al., 2008Roberts P.J. Mitin N. Keller P.J. Chenette E.J. Madigan J.P. Currin R.O. Cox A.D. Wilson O. Kirschmeier P. Der C.J. Rho Family GTPase modification and dependence on CAAX motif-signaled posttranslational modification.J. Biol. Chem. 2008; 283: 25150-25163Crossref PubMed Scopus (237) Google Scholar). Rac1 is also a key effector of NGF trophic signaling, TrkA trafficking, and axonal morphology (Harrington et al., 2011Harrington A.W. St Hillaire C. Zweifel L.S. Glebova N.O. Philippidou P. Halegoua S. Ginty D.D. Recruitment of actin modifiers to TrkA endosomes governs retrograde NGF signaling and survival.Cell. 2011; 146: 421-434Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar) making it an attractive candidate for NGF-regulated prenylation in axons. To assess local Rac1 geranylgeranylation, isolated sympathetic axons were incubated with isoprenoid analog in the presence or absence of NGF for 6 h. Rac1 was immunoprecipitated from axon lysates, conjugated to a TAMRA azide tag using click chemistry, and prenylated Rac1 was detected by immunoblotting with an anti-TAMRA antibody. We observed a robust 2-fold increase in prenylated Rac1 in axons in response to NGF (Figures 5A and 5B ). Treatment of axons with the GGTase I inhibitor abolished NGF-induced Rac1 prenylation (Figures 5A and 5B) without affecting total Rac1 levels (Figure 5C). Thus, NGF induces axonal geranylgeranylation of Rac1. We next asked if prenylation of Rac1 in axons was dependent on local synthesis. RT-PCR analysis of mRNA isolated from distal axons of compartmentalized neurons demonstrated that Rac1 mRNA is detected in axons (Figure S4). To address the contribution of local protein synthesis to axonal Rac1 levels, distal axons of compartmentalized cultures maintained in the presence of NGF were locally treated with CHX. We found that localized inhibition of translation in distal axons significantly reduced axonal Rac1 protein levels, while Rac1 protein in cell bodies was unaffected (Figures 5D–5F). These results suggest that maintenance of axonal Rac1 protein levels relies on local protein synthesis. To determine if Rac1 geranylgeranylation was dependent on local synthesis, isolated axons were incubated with the isoprenoid analog and stimulated with NGF in the presence or absence of CHX. Treatment of isolated axons with CHX suppressed NGF-induced increase in Rac1 prenylation (Figures 5G and 5H) and also reduced axonal levels of Rac1 protein (Figure 5I). CHX-mediated reduction in Rac1 protein levels in isolated axons was comparable to that seen in distal axons of compartmentalized cultures (31% and 39%, respectively) (Figures 5F and 5I). Together, these results indicate that NGF-induced prenylation of Rac1 in axons is dependent on local translation. Of note, NGF treatment enhanced Rac1 prenylation by 2–2.5-fold even after accounting for increased axonal Rac1 levels elicited by NGF (Figures 5B and 5H). These data support that NGF-induced increase in Rac1 prenylation is not merely due to enhanced Rac1 protein synthesis but also regulation of GGTase I enzymatic activity (see Figures 2B and 4J). Thus, NGF regulates the local synthesis of Rac1 in axons and also ensures its post-translational modification. Asymmetric localization of mRNA transcripts depends on cis-elements commonly located within their 3′UTRs (Andreassi et al., 2018Andreassi C. Crerar H. Riccio A. Post-transcriptional processing of mRNA in neurons: the vestiges of the RNA world drive transcriptome diversity.Front. Mol. Neurosci. 2018; 11: 304Crossref PubMed Scopus (17) Google Scholar). To study the intra-axonal synthesis of Rac1 and to identify element(s) responsible for Rac1 mRNA trafficking to axons, we performed rapid amplification of 3′cDNA ends (3′RACE) on mRNA isolated from either cell bodies or distal axons in compartmentalized cultures. Interestingly, 3′RACE analysis revealed two isoforms of Rac1 mRNA with different 3′UTR lengths; a long 3′UTR (∼1,500 bp) and a short 3′UTR (∼250 bp) (Figures 6A, 6B, and S5A). Both isoforms have the same coding sequence. The sequence of the short isoform aligned to the first 250 bp of the long isoform upstream of a poly-adenylation site (Figure 6B), suggesting that the two isoforms likely arise from alternative polyadenylation. Although both Rac1 isoforms were found in cell bodies, only the Rac1 long 3′UTR isoform was detected in axons (Figures 6A and S5A). This finding for Rac1 is consistent with previous reports for other mRNA isoforms where a long 3′UTR biases for localization to neuronal processes (Andreassi et al., 2018Andreassi C. Crerar H. Riccio A. Post-transcriptional processing of mRNA in neurons: the vestiges of the RNA world drive transcriptome diversity.Front. Mol. Neurosci. 2018; 11: 304Crossref PubMed Scopus (17) Google Scholar). To further support the localization of the two Rac1 isoforms, we generated adenoviral vectors containing mCherry-tagged coding sequence of Rac1 fused to either long (mCherry-Rac1-L) or short (mCherry-Rac1-S) Rac1 3′UTR. Rac1 constructs were expressed in neurons for 36 h followed by fluorescent in situ hybridization using a RNAscope® probe against the mCherry sequence. Consistent with the results from the 3′RACE, both mCherry-Rac1-L and mCherry-Rac1-S mRNA were present in cell bodies (Figure S5B), whe" @default.
• W3035163008 created "2020-06-19" @default.
• W3035163008 creator A5005539758 @default.
• W3035163008 creator A5012924780 @default.
• W3035163008 date "2020-06-01" @default.
• W3035163008 modified "2023-10-05" @default.
• W3035163008 title "Prenylation of Axonally Translated Rac1 Controls NGF-Dependent Axon Growth" @default.
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• W3035163008 doi "https://doi.org/10.1016/j.devcel.2020.05.020" @default.
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