Strahil Iv. Pastuhov

Endocannabinoid-Goα signalling inhibits axon regeneration in Caenorhabditis elegans by antagonizing Gqα-PKC-JNK signalling

The ability of neurons to regenerate their axons after injury is determined by a balance between cellular pathways that promote and those that inhibit regeneration. In Caenorhabditis elegans, axon regeneration is positively regulated by the c-Jun N-terminal kinase mitogen activated protein kinase pathway, which is activated by growth factor-receptor tyrosine kinase signalling. Here we show that fatty acid amide hydrolase-1, an enzyme involved in the degradation of the endocannabinoid anandamide (arachidonoyl ethanolamide), regulates the axon regeneration response of γ-aminobutyric acid neurons after laser axotomy. Exogenous arachidonoyl ethanolamide inhibits axon regeneration via the Goα subunit GOA-1, which antagonizes the Gqα subunit EGL-30. We further demonstrate that protein kinase C functions downstream of Gqα and activates the MLK-1-MEK-1-KGB-1 c-Jun N-terminal kinase pathway by phosphorylating MLK-1. Our results show that arachidonoyl ethanolamide induction of a G protein signal transduction pathway has a role in the inhibition of post-development axon regeneration.

Axotomy-induced HIF-serotonin signalling axis promotes axon regeneration in C. elegans

The molecular mechanisms underlying the ability of axons to regenerate after injury remain poorly understood. Here we show that in Caenorhabditis elegans, axotomy induces ectopic expression of serotonin (5-HT) in axotomized non-serotonergic neurons via HIF-1, a hypoxia-inducible transcription factor, and that 5-HT subsequently promotes axon regeneration by autocrine signalling through the SER-7 5-HT receptor. Furthermore, we identify the rhgf-1 and rga-5 genes, encoding homologues of RhoGEF and RhoGAP, respectively, as regulators of axon regeneration. We demonstrate that one pathway initiated by SER-7 acts upstream of the C. elegans RhoA homolog RHO-1 in neuron regeneration, which functions via G12α and RHGF-1. In this pathway, RHO-1 inhibits diacylglycerol kinase, resulting in an increase in diacylglycerol. SER-7 also promotes axon regeneration by activating the cyclic AMP (cAMP) signalling pathway. Thus, HIF-1-mediated activation of 5-HT signalling promotes axon regeneration by activating both the RhoA and cAMP pathways.

LRRK1-phosphorylated CLIP-170 regulates EGFR trafficking by recruiting p150Glued to microtubule plus ends.

ABSTRACT The binding of ligand to epidermal growth factor receptor (EGFR) causes the receptor to become activated and stimulates the endocytosis of EGFR. Early endosomes containing activated EGFR migrate along microtubules as they mature into late endosomes. We have recently shown that LRRK1, which is related to the familial Parkinsonism gene product Park8 (also known as LRRK2), regulates this EGFR transport in a manner dependent on LRRK1 kinase activity. However, the downstream targets of LRRK1 that might modulate this transport function have not been identified. Here, we identify CLIP-170 (also known as CLIP1), a microtubule plus-end protein, as a substrate of LRRK1. LRRK1 phosphorylates CLIP-170 at Thr1384, located in its C-terminal zinc knuckle motif, and this promotes the association of CLIP-170 with dynein–dynactin complexes. We find that LRRK1-mediated phosphorylation of CLIP-170 causes the accumulation of p150 Glued (also known as DCTN1) a subunit of dynactin, at microtubule plus ends, thereby facilitating the migration of EGFR-containing endosomes. Thus, our findings provide new mechanistic insights into the dynein-driven transport of EGFR.

Phosphorylation of CLIP-170 by LRRK1 regulates EGFR trafficking by promoting recruitment of p150Glued to MT plus-ends

ABSTRACT The binding of ligand to epidermal growth factor receptor (EGFR) causes the receptor to become activated and stimulates the endocytosis of EGFR. Early endosomes containing activated EGFR migrate along microtubules as they mature into late endosomes. We have recently shown that LRRK1, which is related to the familial Parkinsonism gene product Park8 (also known as LRRK2), regulates this EGFR transport in a manner dependent on LRRK1 kinase activity. However, the downstream targets of LRRK1 that might modulate this transport function have not been identified. Here, we identify CLIP-170 (also known as CLIP1), a microtubule plus-end protein, as a substrate of LRRK1. LRRK1 phosphorylates CLIP-170 at Thr1384, located in its C-terminal zinc knuckle motif, and this promotes the association of CLIP-170 with dynein–dynactin complexes. We find that LRRK1-mediated phosphorylation of CLIP-170 causes the accumulation of p150Glued (also known as DCTN1) a subunit of dynactin, at microtubule plus ends, thereby facilitating the migration of EGFR-containing endosomes. Thus, our findings provide new mechanistic insights into the dynein-driven transport of EGFR.

The Core Molecular Machinery Used for Engulfment of Apoptotic Cells Regulates the JNK Pathway Mediating Axon Regeneration in Caenorhabditis elegans.

The mechanisms that govern the ability of specific neurons to regenerate their axons after injury are not well understood. In Caenorhabditis elegans, the initiation of axon regeneration is positively regulated by the JNK–MAPK pathway. In this study, we identify two components functioning upstream of the JNK pathway: the Ste20-related protein kinase MAX-2 and the Rac-type GTPase CED-10. CED-10, when bound by GTP, interacts with MAX-2 and functions as its upstream regulator in axon regeneration. CED-10, in turn, is activated by axon injury via signals initiated from the integrin α-subunit INA-1 and the nonreceptor tyrosine kinase SRC-1 and transmitted via the signaling module CED-2/CrkII–CED-5/Dock180–CED-12/ELMO. This module is also known to regulate the engulfment of apoptotic cells during development. Our findings thus reveal that the molecular machinery used for engulfment of apoptotic cells also promotes axon regeneration through activation of the JNK pathway. SIGNIFICANCE STATEMENT The molecular mechanisms of axon regeneration after injury remain poorly understood. In Caenorhabditis elegans, the initiation of axon regeneration is positively regulated by the JNK–MAPK pathway. In this study, we show that integrin, Rac-GTPase, and several other molecules, all of which are known to regulate engulfment of apoptotic cells during development, also regulate axon regeneration. This signaling module activates the JNK–MAPK cascade via MAX-2, a PAK-like protein kinase that binds Rac. Our findings thus reveal that the molecular machinery used for engulfment of apoptotic cells also promotes axon regeneration through activation of the JNK pathway.

Endocannabinoid signaling regulates regenerative axon navigation in Caenorhabditis elegans via the GPCRs NPR-19 and NPR-32

The axon regeneration ability of neurons depends on the interplay of factors that promote and inhibit regeneration. In Caenorhabditis elegans, axon regeneration is promoted by the JNK MAP kinase (MAPK) pathway. Previously, we found that the endocannabinoid anandamide (AEA) inhibits the axon regeneration response of motor neurons after laser axotomy by suppressing the JNK signaling pathway. Here, we show that the G‐protein‐coupled receptors (GPCRs) NPR‐19 and NPR‐32 inhibit axon regeneration in response to AEA. Furthermore, we show that sensory neuron expression of the nape‐1 gene, which encodes an enzyme synthesizing AEA, causes the regenerating motor axons to avoid sensory neurons and this avoidant response depends on NPR‐19 and NPR‐32. These results indicate that the navigation of regenerating axons is modulated by the action of AEA on NPR‐19/32 GPCRs.

Chaperone complex BAG2–HSC70 regulates localization of Caenorhabditis elegans leucine‐rich repeat kinase LRK‐1 to the Golgi

Mutations in LRRK2 are linked to autosomal dominant forms of Parkinsons disease. We identified two human proteins that bind to LRRK2: BAG2 and HSC70, which are known to form a chaperone complex. We characterized the role of their Caenorhabditis elegans homologues, UNC‐23 and HSP‐1, in the regulation of LRK‐1, the sole homologue of human LRRK2. In C. elegans, LRK‐1 determines the polarized sorting of synaptic vesicle (SV) proteins to the axons by excluding SV proteins from the dendrite‐specific transport machinery in the Golgi. In unc‐23 mutants, SV proteins are localized to both presynaptic and dendritic endings in neurons, a phenotype also observed in lrk‐1 deletion mutants. Furthermore, we isolated mutations in the hsp‐1 gene that can suppress the unc‐23, but not the lrk‐1 defect. We show that UNC‐23 determines LRK‐1 localization to the Golgi apparatus in cooperation with HSP‐1. These results describe a chaperone‐dependent mechanism through which LRK‐1 localization is regulated.

MAP kinase cascades regulating axon regeneration in C. elegans.

Mitogen-activated protein kinase (MAPK) signaling cascades are activated by diverse stimuli such as growth factors, cytokines, neurotransmitters and various types of cellular stress. Our evolving understanding of these signal cascades has been facilitated by genetic analyses and physiological characterization in model organisms such as the nematode Caenorhabditis elegans. Genetic and biochemical studies in C. elegans have shed light on the physiological roles of MAPK cascades in the control of cell fate decision, neuronal function and immunity. Recently it was demonstrated that MAPK signaling is also important for axon regeneration in C. elegans, and the use of C. elegans as a model system has significantly advanced our understanding of the largely conserved molecular mechanisms underlying axon regeneration. This review summarizes our current understanding of the role and regulation of MAPK signaling in C. elegans axon regeneration.

The C. elegans Discoidin Domain Receptor DDR-2 Modulates the Met-like RTK-JNK Signaling Pathway in Axon Regeneration.

The ability of specific neurons to regenerate their axons after injury is governed by cell-intrinsic regeneration pathways. However, the signaling pathways that orchestrate axon regeneration are not well understood. In Caenorhabditis elegans, initiation of axon regeneration is positively regulated by SVH-2 Met-like growth factor receptor tyrosine kinase (RTK) signaling through the JNK MAPK pathway. Here we show that SVH-4/DDR-2, an RTK containing a discoidin domain that is activated by collagen, and EMB-9 collagen type IV regulate the regeneration of neurons following axon injury. The scaffold protein SHC-1 interacts with both DDR-2 and SVH-2. Furthermore, we demonstrate that overexpression of svh-2 and shc-1 suppresses the delay in axon regeneration observed in ddr-2 mutants, suggesting that DDR-2 functions upstream of SVH-2 and SHC-1. These results suggest that DDR-2 modulates the SVH-2–JNK pathway via SHC-1. We thus identify two different RTK signaling networks that play coordinated roles in the regulation of axonal regeneration.

Survival microinjection into C. elegans with in vivo observation based on micromanipulation

This study presents the survival microinjection into Caenorhabditis elegans (C. elegans) with in vivo observation based on micromanipulation. The microinjections were achieved with micro-gel beads which are enable to encapsulate chemicals for injection. In this study, a fluorescent material was used to evaluate the injection positional precision inside the C. elegans. The fluorescent microbead was picked up at the tip of a micropipette injection tool and injected by a piezo actuated microinjector. The distance between the injected micro-gel bead and closest nerve axon was measured as 20.3 μm and 16.5 μm by in vivo observation of a confocal microscopy. The two types of pipette tools were used to evaluate the success and survival rates of microinjection, and the smaller pipette (pipette A, 0.8 μm in diameter) showed higher rates as 50 % and 67 % respectively.