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Modulation of the Progenitor Cell and Homeostatic Capacities of Müller Glia Cells in Retina: Focus on α2-Adrenergic and Endothelin Receptor Signaling Systems
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Neuroscience. Uppsala University. (Finn Hallböök group)
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Müller cells are major glial cells in the retina and have a broad range of functions that are vital for the retinal neurons. During retinal injury gliotic response either leads to Müller cell dedifferentiation and formation of a retinal progenitor or to maintenance of mature Müller cell functions. The overall aim of this thesis was to investigate the intra- and extracellular signaling of Müller cells, to understand how Müller cells communicate during an injury and how their properties can be regulated after injury. Focus has been on the α2-adrenergic receptor (α2-ADR) and endothelin receptor (EDNR)-induced modulation of Müller cell-properties after injury.

The results show that α2-ADR stimulation by brimonidine (BMD) triggers Src-kinase mediated ligand-dependent and ligand-independent transactivation of epidermal growth factor receptor (EGFR) in both chicken and human Müller cells. The effects of this transactivation in injured retina attenuate injury-induced activation and dedifferentiation of Müller cells by attenuating injury-induced ERK signaling. The attenuation was concomitant with a synergistic up-regulation of negative ERK- and RTK-feedback regulators during injury. The data suggest that adrenergic stress-signals modulate glial responses during retinal injury and that α2-ADR pharmacology can be used to modulate glial injury-response. We studied the effects of this attenuation of Müller cell dedifferentiation on injured retina from the perspective of neuroprotection. We analyzed retinal ganglion cell (RGC) survival after α2-ADR stimulation of excitotoxically injured chicken retina and our results show that α2-ADR stimulation protects RGCs against the excitotoxic injury. We propose that α2-ADR-induced protection of RGCs in injured retina is due to enhancing the attenuation of the glial injury response and to sustaining mature glial functions. Moreover, we studied endothelin-induced intracellular signaling in Müller cells and our results show that stimulation of EDNRB transactivates EGFR in Müller cells in a similar way as seen after α2-ADR stimulation. These results outline a mechanism of how injury-induced endothelins may modulate the gliotic responses of Müller cells.

The results obtained in this thesis are pivotal and provide new insights into glial functions, thereby uncovering possibilities to target Müller cells by designing neuroprotective treatments of retinal degenerative diseases or acute retinal injury.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. , 73 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1201
Keyword [en]
Alpha2-adrenergic receptor, Brimonidine, Brn3a, Dedifferentiation, Endothelin, EGFR, ERK1/2, Neuroprotection, NMDA, MIO-M1 human Müller cell, Müller cells, Retina, Retinal ganglion cells, Src-kinase, Transactivation.
National Category
Neurosciences
Research subject
Medical Science
Identifiers
URN: urn:nbn:se:uu:diva-281569ISBN: 978-91-554-9527-5 (print)OAI: oai:DiVA.org:uu-281569DiVA: diva2:914619
Public defence
2016-05-19, B21, BMC, Husagatan 03, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2016-04-28 Created: 2016-03-24 Last updated: 2016-05-12
List of papers
1. Transactivation of EGF Receptors in Chicken Muller Cells by α2A-Adrenergic Receptors Stimulated by Brimonidine
Open this publication in new window or tab >>Transactivation of EGF Receptors in Chicken Muller Cells by α2A-Adrenergic Receptors Stimulated by Brimonidine
2014 (English)In: Investigative Ophthalmology and Visual Science, ISSN 0146-0404, E-ISSN 1552-5783, Vol. 55, no 6, 3385-3394 p.Article in journal (Refereed) Published
Abstract [en]

Purpose: α2-Adrenergic receptor agonists are used in glaucoma treatment and have been shown to have some neuroprotective effects. We performed this study to test the hypothesis that epidermal growth factor receptors on chicken Müller cells are transactivated by α2-adrenergic receptors and we focused on the extracellular signal-activated kinases 1/2 (ERK) pathway. Methods: Embryonic chicken retina and cultures of primary Müller cells were stimulated by α2-adrenergic receptor agonist brimonidine. Immunostaining, qRT-PCR and western blot techniques in combination with Src-, epidermal growth factor receptor kinase-, and matrix metalloproteinase inhibitors were used for analysis of the cellular responses. Results: Our results showed that Müller cells express α2A-adrenergic receptors in vivo and in vitro and that brimonidine triggered a robust and transient phosphorylation of ERK1/2. This ERK-response was Src-kinase dependent, associated with tyrosine phosphorylation of epidermal growth factor receptors (phospho-Y1068, Y1173) and was mediated by matrix metalloproteinase-activity on the Müller cells. Conclusions: Müller cells express the α2A-adrenergic receptor and brimonidine triggers both Src-kinase- and matrix metalloproteinase-mediated autocrine ligand-dependent activation of epidermal growth factor receptors on Müller cell. This response is consistent with transactivation of epidermal growth factor receptors by stimulation of α2-adrenergic receptors.

National Category
Ophthalmology
Identifiers
urn:nbn:se:uu:diva-224134 (URN)10.1167/iovs.13-13823 (DOI)000339485800003 ()24781942 (PubMedID)
Available from: 2014-05-05 Created: 2014-05-05 Last updated: 2017-12-05Bibliographically approved
2. Alpha2-Adrenergic Agonist Brimonidine Stimulates ERK1/2 and AKT Signaling Via Transactivation of EGF Receptors in MIO-M1 Human Müller Cells
Open this publication in new window or tab >>Alpha2-Adrenergic Agonist Brimonidine Stimulates ERK1/2 and AKT Signaling Via Transactivation of EGF Receptors in MIO-M1 Human Müller Cells
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Alpha2-adrenergic receptor (α2-ADR) agonist brimonidine is clinically used in glaucoma and ocular hypertension treatment and has been shown to protect the retina from adverse effects. The purpose of this study to test the hypothesis that brimonidine stimulates extracellular signal-regulated kinases (ERK) 1/2 and AKT signaling via transactivation of epidermal growth factor receptors in MIO-M1 human Müller cells. MIO-M1 cells were treated with brimonidine in combination with Src-kinase, epidermal growth factor receptor kinase, and matrix metalloproteinase inhibitors and analyzed by immunocytochemistry, quantitative PCR and western blot techniques. Our results show that human MIO-M1 cells express α2A-ADR and stimulation of that by brimonidine caused a robust increase of ERK1/2 and AKT (Thr-308) phosphorylation in MIO-M1 cells. The P-ERK1/2 and P-AKT (Thr-308) signaling were mediated by Src-kinase, associated with phosphorylation of tyrosine residue of epidermal growth factor receptor (P-EGFR Y1173) and activation of matrix metalloproteinase. These effects could be blocked by Src-kinase inhibitors (PP1, PP2), EGFR-kinase inhibitor (AG1478) and matrix metalloproteinases inhibitor (GM6001). These results conclude that human MIO-M1 cells express α2A-ADR and brimonidine triggers Srckinase mediated both ligand-dependent and ligand-independent EGFR transactivation. Transactivation leads to activation of ERK1/2 and AKT signaling in MIO-M1 human Müller cells.

Keyword
AKT pathway, Alpha 2-adrenergic receptors, Brimonidine, EGF receptor, ERK1/2, Matrix metalloproteinases, MIO-M1 human Müller cell, and Src-kinase
National Category
Neurosciences
Research subject
Medical Science
Identifiers
urn:nbn:se:uu:diva-281576 (URN)
Available from: 2016-03-24 Created: 2016-03-24 Last updated: 2016-05-12
3. Alpha2-Adrenergic-Agonist Brimonidine Stimulates Negative Feedback and Attenuates Injury-Induced Phospho-ERK and Dedifferentiation of Chicken Müller Cells
Open this publication in new window or tab >>Alpha2-Adrenergic-Agonist Brimonidine Stimulates Negative Feedback and Attenuates Injury-Induced Phospho-ERK and Dedifferentiation of Chicken Müller Cells
2015 (English)In: Investigative Ophthalmology and Visual Science, ISSN 0146-0404, E-ISSN 1552-5783, Vol. 56, no 10, 5933-5945 p.Article in journal (Refereed) Published
Abstract [en]

Purpose:

Retinal injury induces Müller cell dedifferentiation by activating extracellular signal-regulated kinase (ERK) signaling. Stimulation of α2-adrenergic receptors protects against injury but also activates ERK in Müller cells. The purpose of this work was to study the effect of α2-adrenergic signaling on injury-induced ERK and Müller cell dedifferentiation. We tested the hypothesis that α2-stimulation triggers negative feedback regulation of the injury-induced ERK pathway that attenuates Müller cell dedifferentiation.

Methods:

Chicken retina injured by N-methyl-D-aspartate and cultured primary Müller cells were stimulated by the α2-adrenergic agonist brimonidine. Immunostaining, quantitative RT-PCR, and Western blot techniques in combination with receptor blockers were used for analysis of the cellular responses.

Results:

Alpha2-adrenergic receptor stimulation attenuated injury-induced ERK activation and dedifferentiation of Müller cells as seen by decreased phospho-ERK, expression of transitin, and retinal progenitor cell genes. The attenuation was concomitant with a synergistic upregulation of several negative ERK-signal feedback regulators including ERK-phosphatases, Raf1-, and growth factor receptor–binding proteins. The results were also seen in cultures of primary Müller cells.

Conclusions:

Alpha2-adrenergic signaling on Müller cells elicits an intracellular attenuation of the injury response that comprises negative ERK-signaling feedback leading to attenuated Müller cell dedifferentiation. The implications of this study are that adrenergic stress signals may directly modulate glial function in retina and that α2-adrenergic receptor pharmacology may be used to control glial injury response.

National Category
Neurosciences
Identifiers
urn:nbn:se:uu:diva-262700 (URN)10.1167/iovs.15-16816 (DOI)000368426300032 ()
Funder
Swedish Research Council, M 12187
Available from: 2015-09-18 Created: 2015-09-18 Last updated: 2017-12-04Bibliographically approved
4. Neuroprotection by α2-Adrenergic Receptor Stimulation after Excitotoxic Injury of Chicken Retinal Ganglion Cells: A Population Study
Open this publication in new window or tab >>Neuroprotection by α2-Adrenergic Receptor Stimulation after Excitotoxic Injury of Chicken Retinal Ganglion Cells: A Population Study
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Retinal ganglion cells (RGCs) loss is one of the most common causes of blindness in worldwide. In this work we studied the RGC population in normal and excitotoxically injured chicken retina after pretreatment with the α2-adrenergic receptor (α2-ADR) agonist brimonidine. The main objective of this work was to study the effects of brimonidine on injured chicken RGCs. A lesion was inflicted by intraocular injection of N-methyl-D-aspartate (NMDA) at embryonic day 18 and the total population of retinal ganglion cells was studied using an automated cell counting of cells positive for the retinal ganglion cell marker Brn3a in flat-mounted retinas. Surviving Brn3a positive RGCs and their distribution in the retina were analyzed 7 and 14 days post lesion. In addition, the total population of retinal ganglion cells was analyzed in a series of normal embryonic day 8 to post-hatch day 11 retinas. The result showed the distribution of total population of RGCs both in embryonic and post-nantal chicken retina. The pretreatment with brimonidine in excitotoxic retina significantly reduced RGC death as seen both 7 and 14 days post lesion. The excitotoxic lesion was more severe in the dorsal quadrants of the retina than in the ventral ones. The regional difference was also seen in the effect of brimonidine. Thus, we conclude that α2-ADR signaling protects RGCs against the excitotoxic injury in the chicken retina.

Keyword
Alpha2-adrenergic receptor agonist, brimonidine, Brn3a, excitotoxin, flat-mount retina, NMDA, retinal ganglion cells, topographical distribution, development
National Category
Neurosciences
Research subject
Medical Science
Identifiers
urn:nbn:se:uu:diva-281583 (URN)
Available from: 2016-03-24 Created: 2016-03-24 Last updated: 2016-05-12
5. The Endothelin B Receptor Transactivates Epidermal Growth Factor Receptors in primary chicken Müller cells and in MIO-M1 Human Müller Cells
Open this publication in new window or tab >>The Endothelin B Receptor Transactivates Epidermal Growth Factor Receptors in primary chicken Müller cells and in MIO-M1 Human Müller Cells
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Injury to the nervous system elicits signals that trigger a variety of cellular responses. Injury to the retina triggers Müller cells, the major glia cell of the retina, to dedifferentiate, proliferate, attain retinal progenitor properties and in some species generate new neurons. The epidermal growth factor receptor (EGFR) system and extracellular signal-regulated kinase (ERK) signalling are key regulators of these processes in Müller cells. The complexity of the extracellular signals that modulate and control the process are not fully understood. In this work we studied whether endothelin receptor signalling can activate EGFR and ERK signalling in Müller cells. Endothelin expression is robustly up-regulated at retinal injury and endothelin receptors have been shown to transactivate EGFRs in other cell-types. We treated chicken Müller cells in vivo, cultured primary chicken Müller cells and the human Müller cell line MIO-M1 with receptor agonists and enzyme blockers, and analyzed endothelin receptor mediated transactivation of EGFRs by using western blot analysis, quantitative reverse transcriptase PCR and immunocytochemistry. The results showed that both chicken and human Müller cells express endothelin receptor B. Stimulation by using the endothelin receptor B agonist IRL1620 caused Src-kinase mediated ligand-dependent and ligand-independent EGFR transactivation. The effects could be blocked by Src-kinase inhibitors (PP1, PP2), EGFR inhibitor (AG1478) and by inhibitors to extracellular matrix metalloproteinases (GM6001). Our data outline a mechanism how injury-induced endothelins may modulate the Müller cell responses by transactivation of EGFRs. The data give support to a view in which endothelins, among several other functions, serve as an injury-signal that regulate the gliotic response of Müller cells.

Keyword
AG1478, endothelin receptor B agonist IRL1620, ERK1/2, gliosis, matrix-metalloproteinases, MAPK, N-methyl-D-aspartate, excitotoxic retinal injury, Src-kinase
National Category
Neurosciences
Research subject
Medical Science
Identifiers
urn:nbn:se:uu:diva-281586 (URN)
Available from: 2016-03-24 Created: 2016-03-24 Last updated: 2016-05-12

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