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Dendrite regeneration in C. elegans is controlled by the RAC GTPase CED-10 and the RhoGEF TIAM-1

['Harjot Kaur Brar', 'Department Of Cellular', 'Molecular Neuroscience', 'National Brain Research Centre', 'Manesar', 'Haryana', 'Swagata Dey', 'Smriti Bhardwaj', 'Devashish Pande', 'Pallavi Singh']

Date: 2022-05

Neurons are vulnerable to physical insults, which compromise the integrity of both dendrites and axons. Although several molecular pathways of axon regeneration are identified, our knowledge of dendrite regeneration is limited. To understand the mechanisms of dendrite regeneration, we used the PVD neurons in C. elegans with stereotyped branched dendrites. Using femtosecond laser, we severed the primary dendrites and axon of this neuron. After severing the primary dendrites near the cell body, we observed sprouting of new branches from the proximal site within 6 hours, which regrew further with time in an unstereotyped manner. This was accompanied by reconnection between the proximal and distal dendrites, and fusion among the higher-order branches as reported before. We quantified the regeneration pattern into three aspects–territory length, number of branches, and fusion phenomena. Axonal injury causes a retraction of the severed end followed by a Dual leucine zipper kinase-1 (DLK-1) dependent regrowth from the severed end. We tested the roles of the major axon regeneration signalling hubs such as DLK-1-RPM-1, cAMP elevation, let-7 miRNA, AKT-1, Phosphatidylserine (PS) exposure/PS in dendrite regeneration. We found that neither dendrite regrowth nor fusion was affected by the axon injury pathway molecules. Surprisingly, we found that the RAC GTPase, CED-10 and its upstream GEF, TIAM-1 play a cell-autonomous role in dendrite regeneration. Additionally, the function of CED-10 in epidermal cell is critical for post-dendrotomy fusion phenomena. This work describes a novel regulatory mechanism of dendrite regeneration and provides a framework for understanding the cellular mechanism of dendrite regeneration using PVD neuron as a model system.

The knowledge of the repair of injured neural circuits comes from the study of the regeneration of injured axons. The information receiving neurites, namely dendrites, are also vulnerable to physical insult during stroke and trauma. However, little knowledge is available on the mechanism of dendrite regeneration since the study of Cajal. In order to get insight into this process, we severed both axon and dendrites of PVD neuron in C. elegans using laser. By comparing the roles of axon regeneration pathways in both dendrite and axon regeneration in this neuron, we found that dendrite regeneration is independent of molecular mechanisms involving axon regrowth. We discovered that dendrite regeneration is dependent on the RAC GTPase CED-10 and GEF TIAM-1. Moreover, we found that CED-10 plays roles within both neuron and in the surrounding epithelia for mounting regeneration response to dendrite injury. This work provides mechanistic insight into the process of dendrite repair after physical injury.

Funding: The Department of Biotechnology, ministry of science and technology, DBT/Wellcome Trust India Alliance (Grant # IA/I/13/1/500874) to AGR, and DBT/Wellcome Trust India Alliance (Grant # IA/E/18/1/504331) to SD.Caenorhabditis Genetics Center (CGC) is supported by the NIH Office of Research Infrastructure Programs (P40 OD010440). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2022 Brar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

In this report, by combining 2-photon laser neurosurgery and quantitative imaging, we have established both axon and dendrite injury paradigms using the PVD neurons in worms. Using both dendrite and axon regeneration assays in the same neuron, we assessed the roles of axon regeneration pathways in dendrite regeneration. Our results showed that the dendrite regeneration involves multiple cellular processes comprising regrowth, branching, and fusion events independent of conventional axon regeneration pathways, including DLK-1/MLK-1. Our results highlight the neuronal and epidermal roles of Rac GTPase, CED-10 in the initiation of dendrite regrowth and self-fusion processes. We also showed that TIAM-1, a Rho Guanine Exchange Factor (Rho GEF) acts upstream to CED-10 for dendrite regrowth and branching.

PVD neurons in C. elegans, which is responsible for proprioception and harsh touch sensation, have an elaborate dendritic branching pattern [ 28 , 29 ]. Laser-induced small damage to the dendrites of PVD neurons triggers a regenerative self-fusion process [ 30 , 31 ]. The Fusogen AFF-1 plays a crucial role in promoting fusion between the proximal and distal dendrites after injury [ 30 ]. However, the early signalling mechanisms initiating dendrite regrowth remain elusive.

The knowledge about neurite regeneration has been attained mostly from axonal injury models. An injury to the axons elicits a local calcium increase [ 11 , 12 ] that triggers elevation in Cyclic Adenosine monophosphate (cAMP) levels, activation of downstream Protein Kinase A (PKA), and mitogen-activated protein kinase kinase kinase (MAPKKK) Dual Leucine Zipper Kinase (DLK-1) [ 13 – 15 ]. DLK-1 initiates local microtubule remodeling [ 16 ] and activates Ets-C/EBP-1 transcription complex promoting axon regeneration [ 17 ]. The Dendritic arborization (da) neurons in Drosophila have been recently established as an efficient model for studying dendrite regeneration [ 18 , 19 ]. Both intrinsic and extrinsic mechanisms of neurons can regulate the efficiency of dendrite regeneration [ 20 ]. The dendrite regeneration is independent of Dual Leucine zipper Kinase (DLK) MAPK pathway [ 21 ], which is an essential factor for the initiation of axon regeneration [ 13 ]. However, other kinases like AKT, and Ror have been implicated in the process [ 18 , 22 ]. Also, Wnt effectors, which regulate the dendritic morphology and branching, can also regulate dendrite regeneration process [ 22 , 23 ]. Although some of the cytoskeleton-based mechanisms controlling the axon regrowth do not affect dendrite regeneration [ 24 ], microtubule minus-end binding protein, Patronin-1 controls both axon and dendrite regeneration [ 25 – 27 ]. The roles of the axon regeneration machineries have not been extensively tested for dendrite regeneration.

The functional nervous system of an organism requires intact neuronal processes and synaptic connections for proper transmission of electrical signals. A deficit in the structural integrity in the cognitive areas of brain leads to manifestation of neuropathologies[ 1 – 4 ]. Due to their sensitivity towards excitatory and inhibitory inputs, dendrites are often the sites of neurotoxic damage leading to severe dendritic dystrophy such as formation of dendritic varicosities, loss of dendritic spines, mitochondrial swelling and dysfunction and disruption of microtubules[ 5 – 7 ]. One or more of these hallmarks of dendrite damage have also been observed in focal stroke or anoxic depolarization[ 8 ], mild Traumatic Brain Injury (mTBI)[ 9 ], and epilepsy[ 10 ]. Though these features may appear neuroprotective and reversible in favorable conditions, their frequent or chronic occurrence may be devastating or fatal. Unlike axonal damage and regeneration, dendrite regeneration has not been comprehensively explored.

Results

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[1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1010127

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