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Müller Cell Metabolic Chaos During Degeneration

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This abstract was presented today at the 2015 Association for Research in Vision and Opthalmology (ARVO) meetings in Denver, Colorado by  Rebecca L. Pfeiffer, Bryan 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.

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Retinitis Pigmentosa 2 Protein Regulates Transport Of Isoprenylated Proteins To Photoreceptor Outer Segments

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This abstract was presented today at the 2014 Association for Research in Vision and Opthalmology (ARVO) meetings in Orlando, Florida by Houbin Zhang, Li Jiang, Christin Hanke and Wolfgang Baehr.

Full size poster can be downloaded here.

Purpose: X-linked retinitis pigmentosa (XLRP) is a devastating form of retinal degeneration, manifesting early in life with symptoms of night blindness, visual field defects, and decreased visual function. In-vitro, RP2 functions as a GAP for the small GTPase ARL3, a GDI displacement factor (GDF). Mutations in the Rp2 gene account for approximately one quarter of all XLRPs. The purpose of this study was to investigate the consequences of RP2 deletion and identify mechanisms causative of XLRP.

Methods: Intracellular localization of RP2 in photoreceptors was determined by neonatal electroporation of an RP2-EGFP expression vector. An Rp2 knockout mouse was generated using a EUCOMM ES cell line containing a gene trap in intron 1. The knockout mice were characterized by Western blot, immunocytochemistry, and electroretinography (ERG).

Results: RP2-eGFP was localized to the plasma membrane of inner segments, axons and synaptic termini in photoreceptors, but not in outer segments. The Rp2 gene knockout mice were viable and developed normally. Ablation of Rp2 gene expression led to slowly progressing degeneration of cone and rod photoreceptors as indicated by ERG recordings. Scotopic a-wave and photopic b wave amplitudes were reduced as early as one month of age in the knockout mice. The Rp2Y/- ERG amplitudes were further reduced at 6 months of age. Trafficking of transmembrane phototransduction proteins, including cone opsins, to Rp2Y/- photoreceptors outer segments was normal up to 14 months of age. While targeting of transducin α and βγ to the Rp2Y/- outer segments was not affected in the knockout, transport of rod and cone PDE6 as well as GRK1 to outer segments was impeded.

Conclusions: RP2 is distributed to plasma membrane of inner segments and synaptic termini in photoreceptors. RP2 is not essential for trafficking cone opsins and transducin to photoreceptor outer segments, but regulates transport of isoprenylated proteins to photoreceptor outer segments. Our results suggest that RP2/ARL3 may allosterically release prenylated proteins from their soluble complex with PDE6D and unload them to donor membranes (e.g., TGN vesicles). ). In the Rp2 knockout, this process is impeded.

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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”

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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”

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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”

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Generation of An Inbred Miniature Pig Model of Retinitis Pigmentosa

A recent paper by Jason W. Ross, Juan P. Fernandez de Castro, Jianguo Zhao, Melissa Samuel, Eric Walters, Cecilia Rios, Patricia Bray-Ward, Bryan W. Jones, Robert E. Marc, Wei Wang, Liang Zhou, Jennifer M. Noel, Maureen A. McCall, Paul J. DeMarco, Randall S. Prather and Henry J. Kaplan describes the creation of a new model of retinal degenerative disease, specifically autosomal dominant retinitis pigmentosa in a miniature pig model, serving as an additional tool to study these diseases and therapeutic interventions.

Continue reading “Generation of An Inbred Miniature Pig Model of Retinitis Pigmentosa”

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Notable Paper: Long-term RNA interference gene therapy in a dominant retinitis pigmentosa mouse model

Hereditary retinal dystrophies (retinitis pigmentosa, Leber congenital amaurosis, cone-rod dystrophies, macular degeneration) are characterized by loss of visual function, sometimes starting during early childhood, other times in late adulthood.  About 30% of these dystrophies are inherited in an autosomal dominant fashion (RetNet), caused by gain-of-function mutant alleles which encode a malignant form of a normal protein. Continue reading “Notable Paper: Long-term RNA interference gene therapy in a dominant retinitis pigmentosa mouse model”

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Notable Paper: Retinal Remodeling in the Tg P347L Rabbit, a Large-Eye Model of Retinal Degeneration

This paper is the result of a collaborative effort between Bryan William Jones, Mineo Kondo and Hiroko Terasaki, Carl Watt, Kevin Rapp, James Anderson, Yanhua Lin, Maggie Shaw, Jia-Hui Yang and Robert Marc.

This work presents a substantial advance in models of Retinitis pigmentosa (RP), an set of inherited blinding diseases characterized by progressive loss of retinal photoreceptors.   Continue reading “Notable Paper: Retinal Remodeling in the Tg P347L Rabbit, a Large-Eye Model of Retinal Degeneration”