Müller Cell Metabolic Chaos During Degeneration

Muller-cell-chaos1 copy

This abstract was presented today at the 2015 Association for Research in Vision and Opthalmology (ARVO) meetings in Denver, Colorado by  Rebecca L. PfeifferBryan W. Jones and Robert E. Marc.

Purpose: Müller cells (MCs) play a critical role in glutamate (E) metabolism and carbon skeleton cycling in retina. MCs demonstrate changes in metabolism and morphology during retinal degeneration. The timing, extent, regulation, and impacts of these changes are not yet known. We evaluated metabolic phenotypes of MCs and evaluated their capacity to transport glutamate during degeneration.

Methods: Retinas were harvested from wild-type (WT) and rhodopsin Tg P347L rabbits, divided into chips mounted on filters, and incubated in Ames medium with 5 mM D-aspartate (D-Asp), D-glutamate (D-Glu), or D-glutamine (D-Gln) for 10 min at 35 deg to explore transport and metabolism. Chips were fixed in mixed aldehydes and resin embedded for computational molecular phenotyping (CMP) of a range of L- and D-amino acid markers and selected proteins including glutamine synthetase (GS) (J Comp Neurol. 464:1, 2003).

Results: CMP revealed wide variations in metabolite levels across individual MCs from Tg P347L retinas, generating chaotic patterns. GS decreased significantly while glutamine levels (Q) increased, although to varying degrees. Remarkably, E levels were variable and much higher in some MCs than normal, but did not correlate (inversely) with GS levels. Transport experiments using D-Glu, D-Asp, and D-Gln showed that alterations in MC metabolites are not the product of defective transporters, in contrast to previous reports. These results are also inconsistent with conventional models of GS-based E-Q metabolism and microenvironmental regulation of MC phenotypes.

Conclusions: These observations suggest three conclusions. (1) Although degeneration of the retina is certainly the trigger, MC phenotype changes are not a coherent response to the surrounding microenvironment but are, rather, uncoordinated individual MC responses. (2) Although GS is accepted as the primary enzyme responsible for the conversion of E to Q in the normal retina, alternative pathways appear unmasked in the degenerate state. (3) It has been previously hypothesized that MCs in retinal degenerations exhibit deficient E transport. Our experiments show no transport deficiency. This indicates that chaotic metabolite levels emerge from changes in individual MC metabolic processing.

Seminar: Cellular Response Associated to Retinal Diseases and Therapeutic Approaches

Nico Cuenca Seminar

Nicolás Cuenca, professor Department of Physiology,Genetics and Microbiology University of Alicante Spain will be delivering a seminar, Cellular Response Associated to Retinal Diseases and Therapeutic Approaches on Monday, May 11thth at Noon in the the Moran Eye Center auditorium.

 

Abstract: At the cellular and molecular level, the response to retinal injury is similar in diseases like AMD, glaucoma, diabetic retinopathy and retinitis pigmentosa. All exhibit a set of cell signals that lead to controlled cell death and retinal remodeling, inflammatory responses, oxidative stress and activation of apoptotic pathways. In our hands anti-apoptotic compounds such as TUDCA and proinsulin and an3oxidant like safranal prevent retinal degeneration in animal models. In addition using Human CNS stem cells (HuCNS-SCs) we have been able to protect photoreceptors and preserve retinal integrity in a rat model of AMD. Such approaches could potentially delay retinal degeneration in human retinal diseases.

 

Seminar: How Inhibiting The Dimerization Of Vitamin A and/or Catalyzing The Reduction of Quinones May Prevent Retinal Degeneration

Ilyas W Flyer

Ilyas Washington from Columbia University Medical Center will be delivering a seminar on How Inhibiting The Dimerization Of Vitamin A and/or Catalyzing The Reduction of Quinones May Prevent Retinal Degeneration on Wednesday, February 11th, 2015 in the Moran Eye Center auditorium.

Abstract:  Many human conditions, originating from multiple genetic and environmental pressures, converge to overproduce superoxide radical anions. The bulk synthesis of cellular superoxides is believed to result from redox cycling of the quinone, coenzyme q. We have designed small molecules to control quinone redox cycling and correct superoxide fluxes as a means to prevent retinal degeneration.

Metabolic Changes Associated With Müller Cells In A Transgenic Rabbit Model Of Retinal Degeneration

Retina RLP

This abstract was presented today at the 2014 Association for Research in Vision and Opthalmology (ARVO) meetings in Orlando, Florida by  Rebecca L. Pfeiffer, Bryan W. Jones and Robert E. Marc.

Purpose: Müller cells play a central role in retinal metabolism via the glutamate cycle. During retinal degeneration Müller cells are among the first to demonstrate changes, reflected in alterations of metabolic signatures and morphology. The timing, extent and regulation of these changes is not fully characterized. To address this issue, we evaluated Müller cell metabolic phenotypes at multiple stages of retinal remodeling.

Methods: Samples were collected post-mortem from both WT and P347L rabbits. The retinas were then divided into fragments, fixed in buffered aldehydes, and embedded in epoxy resins. Tissues were sectioned at 200nm followed by classification with computational molecular phenotyping (CMP) using an array of small and macromolecular signatures (aspartate (D), glutamate (E), glycine (G), glutamine (Q), glutathione (J), GABA (yy), taurine (T), CRALBP, Glutamine Synthetase (GS), and GFAP). Levels of amino acid or protein were quantified by selecting a region of interest either within the Müller cell population or surrounding neurons and evaluating the intensity of the signal within that region.

Results: CMP reveals overall decreases in GS levels over the course of degeneration. Of notable importance, we saw that in regions of near complete photoreceptor loss neighboring Müller cells may express independent variation in metabolic signatures of E, Q, and GS. Also observed in these Müller cells, ratios of GS:E and GS:Q are not consistent with the ratios seen in WT retina. These results are inconsistent with the current models of both E to Q metabolism and microenvironment regulation of Müller cell phenotypes.

Conclusions: These observations indicate two conclusions. First, although the degenerate state of the retina is the likely trigger inducing Müller cells to express altered metabolic signatures, the rate at which the metabolic state changes is not purely a product of the surrounding environment, but also a stochastic change within individual Müller cells. Second, although it is commonly accepted that GS is the primary enzyme which converts Q to E as part of the glutamate cycle, in degenerate retina alternative pathways may be utilized following decrease in GS.

Support:  NIH EY02576 (RM), NIH EY015128 (RM), NSF 0941717 (RM), NIH EY014800 Vision Core (RM), RPB CDA (BWJ), Thome AMD Grant (BWJ).

Seminar: Genetics of Inherited Retinal Degenerations: Genetic Diagnostic Testing and Novel Disease Gene Discovery

 

Eric Pierce from the Massachusetts Eye and Ear Infirmary and Harvard Medical School Department of Ophthalmology will be delivering a talk on Genetics of Inherited Retinal Degenerations: Genetic Diagnostic Testing and Novel Disease Gene Discovery Monday, December 10th at 12:00pm in the John A. Moran Eye Center Auditorium.

Abstract:

Inherited retinal degenerations (IRDs) are important causes of vision loss. Over 200 different genetics types of IRDs have been identified to date. Despite the notable progress made in identifying the genetic causes for IRDs, the specific genetic cause remains elusive in half of IRD patients. Identifying the genetic cause of patients’ IRD has become especially important with the recent success of clinical trials of gene therapy for RPE65 Leber’s congenital amaurosis (LCA). We are using a two-tiered next- generation sequencing (NGS) approach for disease gene identification. In the first tier, we use selective exon capture and NGS for all known IRD disease genes perform diagnostic genetic testing. Using this approach, we identified disease-causing mutations in 54 of 117 (46%) families tested to date. In tier 2, we are using whole exome sequencing to search for new IRD disease genes in patients and families who do not have mutations in known IRD disease genes. This approach has already been fruitful, and lead to the identification of NMNAT1 as a novel LCA disease gene. Our exome sequencing studies have also shown that sequencing does not provide a genetic solution for many of families that we have analyzed to date, indicating that improved approaches are needed to identify novel disease genes in patients with IRDs.

Interesting Paper: Gene Expression Changes Within Müller Glial Cells in Retinitis Pigmentosa

Retinal degenerations are accompanied by retinal remodeling events.  These events alter the structure and function of the retina and involve to a large extent, Müller cells which seem to serve as pathways for neuronal migration.  This paper by Karin Roesch, Michael B. Stadler and Constance L. Cepko looks at gene expression changes in the Müller cells, one of the glial cells of the retina as the rd1 mouse retina degenerates.

While the paper is not terribly conclusive in its definition of genes or pathways involved, (partially I suspect because of the limited time points examined and the late point in the examinations), this paper does however point in a direction that is useful to the retinal degeneration community.  Specifically, Müller cells are fundamentally involved in the remodeling process.  Intervening there is an opportunity to arrest or slow down the retinal remodeling process to allow for interventions and understanding which genes are involved is a good first step.

Notable Paper: Receptor interacting protein kinase mediates necrotic cone but not rod cell death in a mouse model of inherited degeneration

How photoreceptor cells go through the process of cell death has been an outstanding question.  The authors of this paper by Yusuke Murakami, Hidetaka Matsumoto, Miin Roh, Jun Suzuki, Toshio Hisatomi, Yasuhiro Ikeda, Joan W. Miller, and Demetrios G. Vavvas have further defined the process and identified the receptor interacting protein kinase (RIP) pathway as a possible target for intervention in patients with retinitis pigmentosa (RP).  The authors used the rd10 mouse model, a mouse model of retinitis pigmentosa to examine the cell death process.  They defined RIP kinase as a mediator of necrotic cell death in cones.  RIP3, has been defined as they key regulator of programmed necrosis and its expression was elevated in rd10 retinas during cone photoreceptor death and not rod photoreceptor death.  Furthermore, the cone photoreceptor cell death was rescued by RIP3 deficiency and by pharmacological treatment with RIPkinase inhibitors. Continue reading “Notable Paper: Receptor interacting protein kinase mediates necrotic cone but not rod cell death in a mouse model of inherited degeneration”

The Rd8 mutation of the Crb1 gene is present in vendor lines of C57BL/6N mice and embryonic stem cells

This is an important issue for anyone involved in using murine models of retinal degeneration.  It turns out that contamination of Rd8 mutation in the B6 mice is more wide spread than the C57BL/6N mice.  Labs worldwide are going to have to reassess their data due to this mutation and all reviewers will ask about this in the immediate future.  The genotyping analysis of a variety of vendor lines is described in this paper by Mary J. Mattapallil, Eric F. Wawrousek, Chi-Chao Chan, Hui Zhao, Jayeeta Roychoudhury, Thomas A. Ferguson, and Rachel R. Caspi.  The take home message is that the rd8 mutation is in the C57BL/6N strain which is used worldwide to produce transgenic and knockout models.  The implications for non-vision labs are not as clear, but for vision labs, substantial disease can be present unrelated to another specific disease gene and will need to be accounted for.

Undersized Dendritic Arborizations in Retinal Ganglion Cells of the rd1 Mutant Mouse: A Paradigm of Early Onset Photoreceptor Degeneration

This paper by Devid Damiani, Elena Novelli, Francesca Mazzoni and Enrica Strettoi documents continued negative plasticity in retina by examining ganglion cells in the rd1 mouse.  The rd1 mouse is one of many models of retinal degenerative disease, in this case as an autosomal recessive retinal degenerative disease.  This work gets at the remodeling issue in retinal degenerative diseaseby examining the last cells in the chain of retinal cells that process information before sending it out to the brain and other CNS centers for further processing.   Continue reading “Undersized Dendritic Arborizations in Retinal Ganglion Cells of the rd1 Mutant Mouse: A Paradigm of Early Onset Photoreceptor Degeneration”

Review: Defective Trafficking of Rhodopsin and Its Role In Retinal Degenerations

This is a great review paper on the role of rhodopsin trafficking and its influence on retinal degenerative disease by TJ Hollingsworth and Alecia Gross.  Rhodopsin delocalization in rod photoreceptors has been recognized for some time as one of the first indications of retinal photoreceptor cell stress in retinal degenerative diseases, so I was intrigued when seeing this paper come up in PubMed.   Continue reading “Review: Defective Trafficking of Rhodopsin and Its Role In Retinal Degenerations”

Dysmorphic Photoreceptors in a P23H Mutant Rhodopsin Model of Retinitis Pigmentosa Are Metabolically Active and Capable of Regenerating to Reverse Retinal Degeneration

This paper (and the cover article) is the result of a collaborative effort between Damian C. Lee, Felix R. Vazquez-Chona, W. Drew Ferrell, Beatrice M. Tam, Bryan W. JonesRobert E. Marc, and Orson L. Moritz.

Continue reading “Dysmorphic Photoreceptors in a P23H Mutant Rhodopsin Model of Retinitis Pigmentosa Are Metabolically Active and Capable of Regenerating to Reverse Retinal Degeneration”

Notable Paper: Gene Therapy Rescues Photoreceptor Blindness in Dogs and Paves The Way for Treating Human X-linked Retinitis Pigmentosa

This paper in PNAS by William A. Beltran, Artur V. Cideciyan, Alfred S. Lewin, Simone Iwabe, Hemant Khanna, Alexander Sumaroka, Vince A. Chiodo, Diego S. Fajardo, Alejandro J. Román, Wen-Tao Deng, Malgorzata Swider, Tomas S. Alemán, Sanford L. Boye, Sem Genini, Anand Swaroop, William W. Hauswirth, Samuel G. Jacobson and Gustavo D. Aguirre is a continuation of their work in retinal degeneration, this form of retinal degeneration, X-linked retinitis pigmentosa (RP).

Continue reading “Notable Paper: Gene Therapy Rescues Photoreceptor Blindness in Dogs and Paves The Way for Treating Human X-linked Retinitis Pigmentosa”