The diverse roles of collapsin response mediator protein 4 in mitosis and nerve regeneration

Microtubule-actin interactions underlie a diverse number of biological processes including cell motility, neuronal outgrowth, cellular wound healing, cell division and cortical flow. CRMPs (Collapsin Response Mediator Proteins) are a family of cytosolic phosphoproteins that play roles in regulating both actin and microtubule dynamics. The roles of the CRMP family of proteins in regulating these cellular processes have only been partially described. Our lab has been particularly interested in the function of the CRMP4 isoform because of its unique ability to complex with RhoA, a master regulator of the actin cytoskeleton. In this thesis we explore the function of CRMP4 in two biological processes that are dependent on actin and microtubule dynamics: mitosis (Chapter 2) and axon regeneration (Chapter 3 and 4). In Chapter 2, we identify CRMP4 as an important regulator of mitotic chromosomal alignment. We show that CRMP4 localizes to spindle microtubules during mitosis and that loss of CRMP4 disrupts chromosomal alignment, mitotic progression and spindle morphology. Furthermore, we demonstrate that these processes are dependent on CRMP4 phosphorylation, which may be important for recruitment of additional proteins to the mitotic machinery. In Chapter 3, we investigate the ability of an adeno-associated virus (AAV) encoding a CRMP4 antagonist C4RIP (CRMP4-RhoA inhibitory peptide) to enhance adult retinal ganglion cell (RGC) axon regeneration in an in vivo preclinical optic nerve injury model. We describe the inability of AAV-C4RIP to promote RGC regeneration and discuss the likelihood that AAV-mediated expression levels of C4RIP may be insufficient to promote regeneration. In Chapter 4, we describe the development and validation of cell permeable recombinant C4RIP (TAT-C4RIP) and discuss our data testing the effects of TAT-C4RIP on regeneration in vitro and in vivo. Together, these studies identify CRMP4 as an important regulator of mitosis, and describe our ongoing studies testing the effects of a CRMP4 antagonist on nerve regeneration.Les interactions entre l'actine et les microtubules sont sous-jacentes à divers processus biologiques incluant la motilité cellulaire, le guidage neuronal, la cicatrisation cellulaire, la division cellulaire et la circulation corticale. Les protéines CRMPs (Collapsin Response Mediator Protein) sont une famille de phosphoprotéines cytosoliques jouant un rôle dans la régulation de la dynamique de l'actine et des microtubules. Cependant, cette régulation du cytosquelette par les CRMPs n'a été que partiellement décrite. Notre laboratoire s'intéresse à la fonction de l'isoforme CRMP4 en raison de sa capacité unique d'interagir avec RhoA, un régulateur important du cytosquelette d'actine. Dans cette thèse, nous explorons la fonction de CRMP4 dans deux processus biologiques qui dépendent de la dynamique de l'actine et des microtubules: la mitose (chapitre 2) et la régénération des axones (chapitre 3 et 4). Dans le chapitre 2 sera présentée notre identification de CRMP4 en tant que régulateur important de l'alignement chromosomique durant la mitose. Nous démontrons que, pendant la mitose, CRMP4 se situe sur les fuseaux mitotiques formés de microtubules et que la perte de CRMP4 perturbe l'alignement chromosomique, la progression de la mitose et la morphologie des fuseaux. En outre, nous démontrons que ces processus sont dépendants de la phosphorylation de CRMP4. Ceci pourrait être crucial pour le recrutement de protéines supplémentaires nécessaire pour la mitose. Dans le chapitre 3, nous étudions la capacité d'un virus adéno-associé (AAV) codant pour l'antagoniste de CRMP4, nommé C4RIP (CRMP4-RhoA inhibitory peptide), de favoriser la régénération de l'axone de cellules ganglionnaires de la rétine (RGC) chez l'adulte. Pour cela, nous utilisons un modèle in vivo de traumatismes du nerf optique chez le rat adulte. Nous décrivons l'incapacité des virus AAV-C4RIP de favoriser la régénération des RGCs et discutons de la probabilité que les niveaux de AAV-C4RIP exprimés puissent être insuffisants afin de favoriser la régénération. Le chapitre 4, quant à lui, est consacré à la description du développement et de la validation de la protéine recombinante TAT-C4RIP qui a le potentiel de traverser la membrane cellulaire. Nous y discutons les données concernant les effets de TAT-C4RIP sur la régénération in vitro et in vivo. Dans l'ensemble, ces études caractérisent CRMP4 comme important régulateur de la mitose et décrivent une nouvelle méthode de purification pour des protéines perméables à la membrane cellulaire

Extrinsic and intrinsic regulation of axon regeneration at a crossroads

Repair of the injured spinal cord is a major challenge in medicine. The limited intrinsic regenerative response mounted by adult central nervous system (CNS) neurons is further hampered by astrogliosis, myelin debris and scar tissue that characterize the damaged CNS. Improved axon regeneration and recovery can be elicited by targeting extrinsic factors as well as by boosting neuron-intrinsic growth regulators. Our knowledge of the molecular basis of intrinsic and extrinsic regulators of regeneration has expanded rapidly, resulting in promising new targets to promote repair. Intriguingly certain neuron-intrinsic growth regulators are emerging as promising targets to both stimulate growth and relieve extrinsic inhibition of regeneration. This crossroads between the intrinsic and extrinsic aspects of spinal cord injury is a promising target for effective therapies for this unmet need

Cultivated Autologous Limbal Epithelial Cell (CALEC) Transplantation: Development of Manufacturing Process and Clinical Evaluation of Feasibility and Safety

To treat unilateral limbal stem cell (LSC) deficiency, we developed cultivated autologous limbal epithelial cells (CALEC) using an innovative xenobiotic-free, serum-free, antibiotic-free, two-step manufacturing process for LSC isolation and expansion onto human amniotic membrane with rigorous quality control in a good manufacturing practices facility. Limbal biopsies were used to generate CALEC constructs, and final grafts were evaluated by noninvasive scanning microscopy and tested for viability and sterility. Cultivated cells maintained epithelial cell phenotype with colony-forming and proliferative capacities. Analysis of LSC biomarkers showed preservation of stemness. After preclinical development, a phase 1 clinical trial enrolled five patients with unilateral LSC deficiency. Four of these patients received CALEC transplants, establishing preliminary feasibility. Clinical case histories are reported, with no primary safety events. On the basis of these results, a second recruitment phase of the trial was opened to provide longer term safety and efficacy data on more patients

Establishing a Trainee Section in the CIM

The Clinician Investigator Trainee Association of Canada – Association des cliniciens-chercheurs en formation du Canada (CITAC-ACCFC) is a national organization composed of MD+ trainees enrolled in MD/MSc, MD/PhD, and Clinician Investigator Programs (CIP) across Canada. In less than three years since its conception, CITAC-ACCFC has become an established organization with over 200 members from fifteen academic institutions. The mission of the CITAC-ACCFC is to organize and promote activities that support clinician investigator trainees in Canada, with the intention to improve academic and post-graduate career opportunities, expand institutional and public awareness of clinician investigator programs, and develop a nationally accessible information database of student and program development. The CITAC-ACCFC aims to improve the early-career conditions of clinician investigators in order to expand and advance innovative research initiatives within Canada. Throughout its development, CITAC-ACCFC has relied on the Canadian Society for Clinical Investigation (CSCI) for mentorship and guidance. In an exciting development, the relationship between CITAC-ACCFC and CSCI has enabled an innovative opportunity for early career development through a new collaboration- the creation of a Trainee Section in the Clinical and Investigative Medicine (CIM) journal. As a forum through which work on diverse topics can be shared with the global community, the CIM Trainee Section will create unique opportunities for professional development. Trainees in MD+ programs represent the next generation of clinical-investigators, who will integrate cutting edge research and compassionate patient care throughout their careers. Both professions require excellent communication skills, and through the Trainee Section, MD+ trainees, their supervisors, and MD+ Program Directors, will be able to focus on mentorship, academic training, financial planning, and career development, among other topics devoted to MD+ trainees. In addition to articles highlighting the research activities of trainees, the Trainee Section will feature clinical work, review articles, first chapter of theses, book reviews, and opinion pieces. This focus of the Trainee Section will complement CIM’s current focus on original research and issues of interest to the CSCI. It is therefore fitting to establish a Trainee Section that is dedicated to addressing issues arising at an earlier stage of training. As MD+ organizations are being established globally, the Trainee Section will strive to become an international forum that focuses on MD+ training. By increasing the international readership of CIM through the CITAC website, we will aim to engage MD+ trainees, program directors, university administration, and other leaders in education, to advocate for issues of national and global relevance. As trainees will be responsible for overseeing the editorial and peer-review process of the Trainee Section, we anticipate that this will provide an avenue by which to foster stronger interactions and collaborations among trainees on our Editorial Board, junior investigators, and senior faculty, while providing critical editorial experience for board members. Our team of Editors will ensure that articles are reviewed in a fair and timely manner, respecting the need to promptly publish articles of immediate relevance. We encourage all MD+ trainees to participate in the Trainee Section editorial and/or peer-review process. Additional details can be found on our website at: http://www.citac-accfc.org/portal/ As MD+ trainees, we are optimistic about the future of both basic science and clinical research. This is an exciting time to be an MD+ trainee, and we are confident that the creation of a Trainee Section will only augment this positive experience. We strongly encourage you to publish your work in the Trainee Section and become part of an emerging global community of MD+ trainees, who are keen on tackling health issues that affect us irrespective of our geographic, cultural, or racial borders. We look forward to receiving your articles

La création d'une Section étudiante dans le CIM

L’Association des cliniciens-chercheurs en formation du Canada – the Clinician Investigator Trainee Association of Canada (ACCFC-CITAC) est une organisation nationale composée de MD+ en formation, inscrits dans un programme MD/MSc, MD/PhD ou clinicien-chercheur (PCC) à travers le Canada. Depuis sa création il y a moins de trois ans, l’ACCFC-CITAC est devenue une organisation bien établie comptant plus de 200 membres appartenant à quinze institutions académiques. La mission de l’ACCFC-CITAC est d’organiser et de promouvoir des activités qui soutiennent les cliniciens-chercheurs en formation au Canada, dans l’intention d’améliorer les opportunités de carrières académiques et post-graduées, d’augmenter la visibilité institutionnelle et publique des programmes cliniciens-chercheurs et de développer une base de donnée accessible à l’échelle nationale, des étudiants et des programmes en développement. L’ACCFC-CITAC vise également à améliorer les conditions des cliniciens-chercheurs en début de carrière, dans le but d’élargir et de faire progresser les initiatives de recherche novatrices à l’intérieur du Canada. Au cours de son développement, l’ACCFC-CITAC a pu compter sur le mentorat et les conseils de la Société canadienne de recherche clinique (SCRC). Les relations en pleine croissance entre l’ACCFC-CITAC et la SCRC ont permis, à travers une nouvelle collaboration, la création d’une opportunité innovatrice pour les cliniciens-chercheurs en formation, soit la mise sur pied d’une section dédiée (Section étudiante) dans le journal officiel de la SCRC, le « Clinical and Investigative Medicine (CIM) Journal ». En tant que forum par lequel des travaux portant sur divers sujets seront partagés, la Section étudiante du CIM constituera une belle opportunité en rapport au développement professionnel. Les étudiants en formation au sein des programmes MD+ représentent la prochaine génération de cliniciens-chercheurs qui seront appelés à intégrer la recherche dans des domaines de pointe au sein de leur pratique ainsi que dans les soins aux patients. Ces deux professions exigent d’excellentes habiletés de communication et c’est donc par le biais de la Section étudiante du CIM que les étudiants MD+, leurs superviseurs ainsi que les responsables de programmes MD+ seront en mesure de mettre en valeur des sujets touchant les étudiants MD+ tels que le mentorat, l’enseignement académique, la planification financière et le développement professionnel. En plus d’articles soulignant les activités de recherche des étudiants, la Section étudiante mettra en valeur des travaux cliniques, des articles de synthèse, des résumés de thèses, des comptes-rendus de livres ainsi que des lettres d’opinion. L’orientation de cette section complémentera l’orientation actuelle de la revue CIM qui met à l’avant-plan les articles originaux et les sujets d’intérêts à la SCRC. Il est donc tout à fait pertinent de mettre en place une Section étudiante dédiée à des sujets qui émergent rapidement au cours de la formation d’un clinicien-chercheur. Étant donné que les programmes MD+ se répandent à travers le monde, la Section étudiante favorisera la mise en place d’un forum international encadrant la formation des MD+. En augmentant la portée internationale de la revue CIM par le biais du site web de l’ACCFC-CITAC, nous souhaitons inciter les étudiants MD+, les responsables des programmes, les instances universitaires et autres leaders de l’éducation à s’impliquer et prendre position sur des questions importantes sur le plan national et international. Comme les cliniciens-chercheurs en formation seront responsables de surveiller le processus d’édition et de révision par les pairs de la Section étudiante, nous prévoyons que cela ouvre la voie vers la consolidation des collaborations entre les cliniciens-chercheurs en formation sur notre comité de rédaction, les chercheurs juniors et les professeurs séniors, tout en apportant une expérience éditoriale critique aux membres du comité. Notre équipe d’éditeurs s’assurera que les articles soient révisés de façon équitable et convenable, en respectant la nécessité de publier rapidement les articles de pertinence immédiate. Nous encourageons tous les MD+ en formation à participer au processus d’édition et/ou de révision par les pairs. Des détails additionnels sont disponibles sur notre site internet à l’adresse suivante: http://www.citac-accfc.org/portal/ En tant qu’étudiants MD+, nous sommes optimistes quant à l’avenir des sciences fondamentales et de la recherche clinique. Les temps actuels sont stimulants pour les étudiants MD+ et nous avons bon espoir que la création de la Section étudiante promouvra de surcroît cette expérience. Nous vous encourageons fortement à publier vos travaux de recherche dans la Section étudiante et ainsi prendre part à une communauté émergente d’étudiants MD+ intéressés par les enjeux du domaine de la santé qui nous affectent, peu importe notre lieu géographique, notre culture ou notre origine. Nous attendons donc vos articles

Toxic Keratoconjunctivitis from Coral Reef

The final, published version of this article is available at https://doi.org/10.1159/000517350A 25-year-old woman presented with right eye pain, lid edema, conjunctival injection and chemosis, and mild corneal epitheliopathy after exposure to fluid content from an aquarium coral reef. Topical moxifloxacin and prednisolone were started 4 times daily, with full clinical resolution after 2 weeks. Toxin-mediated keratoconjunctivitis may occur after exposure to zoanthid coral reef, particularly in aquarium enthusiasts. Topical corticosteroids in tandem with topical antibiotics appear to be effective in mild disease. However, in severe cases that exhibit corneal infiltrates and stromal thinning, close observation is warranted in case of possible keratolysis

The role of CRMP4 in nerve regenerations

The inability of CNS neurons to regenerate and reform functional connections following spinal cord injury has devastating clinical consequences. The failure of CNS neurons to spontaneously regenerate following injury can be partially attributed to the expression of neurite outgrowth inhibitory myelin associated inhibitors (MAIs). MAIs signal through a tripartite receptor complex to activate the cytosolic protein RhoA and influence cytoskeletal dynamics. RhoA antagonists promote neuronal survival and regeneration in animal models of nerve injury. However, RhoA's potential as a therapeutic target may be limited by its widespread roles in multiples cellular processes and cell types. In an attempt to discover more specific therapeutic targets to promote nerve regeneration, our lab identified the cytosolic phosphoprotein CRMP4b (Collapsin Response Mediator Protein 4b) as a protein that functionally interacts with RhoA to mediate neurite outgrowth inhibition. siRNA-mediated knockdown of CRMP4 and blockade of the RhoA-CRMP4b interaction with a competitive peptide (C4RIP) attenuates myelin-dependent neurite outgrowth inhibition. Analysis of the proximal tip of extending axons (growth cones) by time lapse video microscopy reveals that C4RIP regulates filopodial dynamics indicating that C4RIP modulates the actin cytoskeleton. We are currently investigating the in vivo roles of CRMP4 in regeneration in an optic nerve injury model by developing readily deliverable C4RIP and a CRMP4 knockout mouse. Elucidating the role of CRMP4 in nerve regeneration may provide insight into the molecular mechanisms following nervous system injury

GSK3 Regulates Mitotic Chromosomal Alignment through

Background: Glycogen Synthase Kinase 3 (GSK3) has been implicated in regulating chromosomal alignment and mitotic progression but the physiological substrates mediating these GSK3-dependent effects have not been identified. Collapsin Response Mediator Protein 4 (CRMP4) is a cytosolic phosphoprotein known to regulate cytoskeletal dynamics and is a known physiological substrate of GSK3. In this study, we investigate the role of CRMP4 during mitosis. Methodology and Principal Findings: Here we demonstrate that during mitosis CRMP4 phosphorylation is regulated in a GSK3-dependent manner. We show that CRMP4 localizes to spindle microtubules during mitosis and loss of CRMP4 disrupts chromosomal alignment and mitotic progression. The effect of CRMP4 on chromosomal alignment is dependent on phosphorylation by GSK3 identifying CRMP4 as a critical GSK3 substrate during mitotic progression. We also provide mechanistic data demonstrating that CRMP4 regulates spindle microtubules consistent with its known role in the regulation of the microtubule cytoskeleton. Conclusion and Significance: Our findings identify CRMP4 as a key physiological substrate of GSK3 in regulatin

GSK3β Regulates Myelin-Dependent Axon Outgrowth Inhibition through CRMP4

Myelin-associated inhibitors (MAIs) contribute to failed regeneration in the CNS. The intracellular signaling pathways through which MAIs block axonal repair remain largely unknown. Here, we report that the kinase GSK3β is directly phosphorylated and inactivated by MAIs, consequently regulating protein–protein interactions that are critical for myelin-dependent inhibition. Inhibition of GSK3β mimics the neurite outgrowth inhibitory effect of myelin. The inhibitory effects of GSK3β inhibitors and myelin are not additive indicating that GSK3β is a major effector of MAIs. Consistent with this, overexpression of GSK3β attenuates myelin inhibition. MAI-dependent phosphorylation and inactivation of GSK3β regulate phosphorylation of CRMP4, a cytosolic regulator of myelin inhibition, and its ability to complex with RhoA. Introduction of a CRMP4 antagonist attenuates the neurite outgrowth inhibitory properties of GSK3β inhibitors. We describe the first example of GSK3β inactivation in response to inhibitory ligands and link the neurite outgrowth inhibitory effects of GSK3β inhibition directly to CRMP4. These findings raise the possibility that GSK3β inhibition will not effectively promote long-distance CNS regeneration following trauma such as spinal cord injury