Category Archives: Retinal Disease

A Pathoconnectome of Early Retinal Remodeling

This abstract was presented today, Monday, April 30th at the 2018 Association for Research in Vision and Opthalmology (ARVO) meetings in Honolulu, Hawaii by Rebecca Pfeiffer, Robert E. Marc, James R. Anderson, Daniel P. Emrich, Carl B. Watt, Jia-Hui Yang, Kevin D. Rapp, Jeebika Dahal, Mineo Kondo, Hiroko Terasaki, and Bryan W. Jones.

Purpose:
Retinal remodeling is a consequence of retinal degenerative disease, during which neurons sprout new neurites whose synaptic structures and partners are not yet defined. Simultaneously during remodeling, Müller cells (MCs) undergo structural and metabolic changes, whose impact on surrounding neurons is an active area of research. Retinal connectomes have elucidated and validated fundamental networks. These data provide further classification of neuronal types and subtypes and a precise framework for modeling of retinal function, based on ground truth networks. The creation of the first pathoconnectome (RPC1), a connectome from pathological retinal tissue, provides the opportunity to determine connectivites between neurons, while simultaneously evaluating glial remodeling. Computational Molecular Phenotyping (CMP) embedded within the ultrastructure provides metabolic factors of pathologies.

Methods:
RPC1 was collected post-mortem from a 10mo TgP347L rabbit model of adRP, fixed in 1% FA, 2.5% GA, 3% sucrose, and 1mM MgSO4 in cacodylate buffer (pH 7.4). The tissue was osmicated, dehydrated, resin embedded, and sectioned at 70nm. Sections were placed on formvar grids, stained, and imaged on a JEOL JEM-1400 TEM using SerialEM. 1 section was reserved from every 30 section for CMP, where it was probed for small molecules: glutamate, glutamine, glycine, GABA, taurine, glutathione; or proteins GFAP and GS. RPC1 was evaluated using the Viking software suite.

Results:
RPC1 was chosen based on early features of retinal degeneration/remodeling: degeneration of rod OS, MC hypertrophy, and neuronal sprouting. RPC1 consists of 948 serial sections spanning the ONL to the vitreous, with a diameter of 90µm. We find dendrites extending from rod bipolar cells to cone pedicles, originally described in light microscopy, and active synaptic contacts. We also see alterations of synaptic structure in the IPL, and MC morphological changes affecting surface to volume and neuron/glial relationships. Network motifs are being actively investigated.

Conclusions:
We observe many features of remodeling previously described using light microscopy, and confirm active synaptic contact. We also find synaptic structural features, not previously described. In addition, early evaluation of MC morphology demonstrates marked changes in MC shape and associations with nearby neurons and glia, which, combined with CMP, will be instrumental in understanding how MCs affect retinal remodeling.

Impact of Glaucoma On Retinal Ganglion Cell Subtypes: A Single-Cell RNA-seq Analysis of the DBA/2J Mouse

This abstract was presented today, May 1st at the 2018 Association for Research in Vision and Opthalmology (ARVO) meetings in Honolulu, Hawaii by Siamak Yousefi, Hao Chen, Jesse Ingels, Sumana R. Chintalapudi, Megan Mulligan, Bryan W. Jones, Vanessa Marie Morales-Tirado, Pete Williams, Simon W. John, Felix Struebing, Eldon E. Geisert, Monica Jablonski, Lu Lu, Robert Williams

Purpose
We are developing methods to define molecular signatures of cellular stress during early stages of glaucoma for major subtypes of retinal ganglion cells (RGCs). Our first aim is to develop reliable mRNA biomarkers for RGC subtypes in the DBA/2J (D2) mouse model prior to disease onset. Our second objective is to quantify cellular stress in RGC subtypes at early stages of disease using known sets of stress-responsive transcripts (e.g. Struebing et al, 2016 PMID:27733864; Williams et al. 2017, PMID:28209901; Lu et al, ARVO 2018).

Methods
Whole retinas from D2 or D2.Cg-Tg(Thy1-CFP)23Jrs/SjJ at 130 to 150 days-of-age were dissociated gently and size selected (>10 µm). RGCs were enriched using THY1 antibody-coated beads. Fluidigm HT microfluidics plates were used to isolate and generate scRNA-seq libraries of full length polyA-positive mRNAs using SMART-Seq v4. Libraries were sequenced using HiSeq3000, PE151. Following alignment using STAR, expression was normalized to log2(FPKM+1) across ~25,000 unique transcript models. Cells with fewer than 1000 detected genes and genes expressed in fewer than 1% of RGCs were excluded. Sets of genes with high variance and/or high expression were used for principal component analysis (PCA). Twenty PCs were used for graph-based unsupervised clustering and visualized using t-distributed stochastic neighbor embedding (tSNE). Gene specificity was computed for all transcripts across all clusters. The top transcripts per cluster with expression >1 in 1% or more of cells, were used to diagnose cellular identify of clusters. The top 30 genes per cluster were searched in PubMed against a panel of cell and tissue specific terms using Chilibot.

Results
The scRNA-seq protocol generates 150,000 – 200,000 uniquely mapped mRNA reads/cell and ~5000 genes/cells. We currently have 1600 cells, of which over half are RGCs. Around 75% of cells are positive for two or more of the following RGC markers: Thy1, Rbpms, Rbpms2, Jam2, G3bp1, and Ywhaz. This set of cells and different subsets of genes are now being used for RGC clustering. We have identified at least 17 clusters in initial datasets using these protocols and are now linking clusters to major classes of RGCs.

Conclusions
Molecular signatures of cellular stress and RGC subtypes in early stage of glaucoma should now be identifiable using unsupervised learning techniques.

Circuit Remodeling In Retinal Degeneration

This abstract was presented yesterday, April 29th at the 2018 Association for Research in Vision and Opthalmology (ARVO) meetings in Honolulu, Hawaii by Bryan W. Jones.

Abstract:

The retina is a complex, heterocellular tissue with most/all retinal cell classes becoming impacted or altered in retinitis pigmentosa (RP) and age-related macular degeneration (AMD) in a process called retinal remodeling. Defining disease and the stage-specific cytoarchitectural and metabolic responses in RP and AMD is critical for highlighting targets for intervention. We now know that negative plasticity and neural retinal remodeling occurs regardless of retinal insult in models of retinal degeneration as well as in human RP and in human AMD, revealing that no retinal disease fails to trigger remodeling and reprogramming.

Evidence in the literature over the past decade has improved our understanding into mechanisms of initial retinal degeneration and informed our understanding of the subsequent remodeling events in the neural retina that occur post-photoreceptor degeneration. Remodeling associated with retinal degeneration is intimately linked with insults that cause photoreceptor stress and eventually photoreceptor cell death. These phenomena result in reprogramming of cell types in retina followed by progressive neural degeneration akin to CNS neural degenerations involving both neuronal and glial classes. No cell class in the retina is spared from the effects of remodeling. The earliest cell classes involved in remodeling are horizontal, bipolar and Müller cells and the Müller glia are the last cell class left in the remodeling retina.

Our efforts are now focused on elucidating the precise wiring changes in retina, through the creation of pathological connectomes, or “patho-connectomes” to study precisely what the circuit topologies are, compared to normal topologies derived from Retinal Connectome 1 (RC1).  Also, because temporal windows are critical to understanding when interventions may be possible, we are exploring when circuit topology revisions occur to understand their impact on information flow in the retina and their impact on rescues of vision loss.  Precise circuit topologies in early retinal degenerative events is our first area of exploration with ultrastructural reconstructions of outer retinal neurons, bipolar cells and horizontal cells.  Müller glia are also of intense interest as we are tracking the earliest metabolic and morphological changes in glia in response to retinal degenerations.

Editorial: Special Issue on Retinal Remodeling

Geoff Lewis and I worked over the last year or so to edit a special issue of Experimental Eye Research with a focus on Retinal Remodeling.  Our editorial for the issue is here. Pubmed link is here.

This special issue of Experimental Eye Research represents laboratories around the world that are involved in retinal degeneration research and was developed with two motivations: 1) to solicit manuscripts from our colleagues that would give broad, yet substantial insight into the various disorders associated with retinal degeneration and 2) to focus discussion in the vision research community into the negative plasticity now known as retinal remodeling that is associated with blinding diseases…

We selected an image for the cover from the Calkins lab (the center image in the above montage) that we felt was beautiful and represented the quality of work that went into every article in this issue and look forward to the dialogue that this special issue will foster.  Many thanks to: The Lewis/Fisher lab, the Calkins lab, the Vetter lab, the Lutty lab, the Gross lab, the Sagdullaev lab, the Merriman lab and Wei Li, Michael Kalloniatis and the Fletcher lab, the Cuenca lab, the Acosta lab, and everyone in our lab.

Retinal Remodeling And Metabolic Alterations in Human AMD

We have a new publication out (direct link, open access), Müller Cell Metabolic Chaos During Retinal Degeneration authored by Bryan W. JonesRebecca Pfeiffer, William Ferrell, Carl Watt, James Tucker, and Robert Marc.

Abstract:

Age-related macular degeneration (AMD) is a progressive retinal degeneration resulting in central visual field loss, ultimately causing debilitating blindness. AMD affects 18% of Americans from 65 to 74, 30% older than 74 years of age and is the leading cause of severe vision loss and blindness in Western populations. While many genetic and environmental risk factors are known for AMD, we currently know less about the mechanisms mediating disease progression. The pathways and mechanisms through which genetic and non-genetic risk factors modulate development of AMD pathogenesis remain largely unexplored. Moreover, current treatment for AMD is palliative and limited to wet/exudative forms. Retina is a complex, heterocellular tissue and most retinal cell classes are impacted or altered in AMD. Defining disease and stage-specific cytoarchitectural and metabolic responses in AMD is critical for highlighting targets for intervention. The goal of this article is to illustrate cell types impacted in AMD and demonstrate the implications of those changes, likely beginning in the retinal pigment epithelium (RPE), for remodeling of the the neural retina. Tracking heterocellular responses in disease progression is best achieved with computational molecular phenotyping (CMP), a tool that enables acquisition of a small molecule fingerprint for every cell in the retina. CMP uncovered critical cellular and molecular pathologies (remodeling and reprogramming) in progressive retinal degenerations such as retinitis pigmentosa (RP). We now applied these approaches to normal human and AMD tissues mapping progression of cellular and molecular changes in AMD retinas, including late-stage forms of the disease.

Müller Cell Metabolic Chaos During Retinal Degeneration

We have a new publication out (direct link, open access), Müller Cell Metabolic Chaos During Retinal Degeneration authored by Rebecca PfeifferRobert Marc, Mineo Kondo, Hiroko Terasaki and Bryan W. Jones.

Abstract:

Müller cells play a critical role in retinal metabolism and are among the first cells to demonstrate metabolic changes in retinal stress or disease. The timing, extent, regulation, and impacts of these changes are not yet known. We evaluated metabolic phenotypes of Müller cells in the degenerating retina.

Retinas harvested from wild-type (WT) and rhodopsin Tg P347L rabbits were fixed in mixed aldehydes and resin embedded for computational molecular phenotyping (CMP). CMP facilitates small molecule fingerprinting of every cell in the retina, allowing evaluation of metabolite levels in single cells.

CMP revealed signature variations in metabolite levels across Müller cells from TgP347L retina. In brief, neighboring Müller cells demonstrated variability in taurine, glutamate, glutamine, glutathione, glutamine synthetase (GS), and CRALBP. This variability showed no correlation across metabolites, implying the changes are functionally chaotic rather than simply heterogeneous. The inability of any clustering algorithm to classify Müller cell as a single class in the TgP347L retina is a formal proof of metabolic variability in the present in degenerating retina.

Although retinal degeneration is certainly the trigger, Müller cell metabolic alterations are not a coherent response to the microenvironment. And while GS is believed to be the primary enzyme responsible for the conversion of glutamate to glutamine in the retina, alternative pathways appear to be unmasked in degenerating retina. Somehow, long term remodeling involves loss of Müller cell coordination and identity, which has negative implications for therapeutic interventions that target neurons alone.

Progressive Retinal Remodeling In A Transgenic Rabbit Model Of Retinitis Pigmentosa

This poster was presented today, May 2th at the 2016 Association for Research in Vision and Opthalmology (ARVO) meetings in Seattle, Washington by Rebecca L. Pfeiffer, Bryan W. Jones, and Robert E. Marc.

Posterboard #: D0246

Abstract Number: 2256 – D0246

Author Block: Rebecca L. Pfeiffer1,2 , Bryan W. Jones1,2 , Robert E. Marc1,2 
1 Ophthalmology, University of Utah, Salt Lake City, Utah, United States; 2 Interdepartmental Program in Neuroscience, University of Utah, Salt Lake City, Utah, United States

Purpose:Retinal degenerations are a collection of neural disorders, usually precipitated by photoreceptor degeneration. All display progressive metabolic alterations and neural loss following the death of the photoreceptors. Although it has been demonstrated that the metabolism of Müller cells (MCs) is drastically altered in degeneration, the full impact of these changes on surrounding neurons and long-term characterization of remodeling was previously unavailable, due to short lifespans of model organisms.

Methods:Retinal samples were collected from WT and Tg P347L rabbits at ages ranging from 3 months to 6 years. Following enucleation, retinas were divided into fragments and incubated for 10 minutes at 35 degrees C in D-isomers of Glutamate (dE), Glutamine (dQ), or Aspartate (dD) and Ames-bicarbonate medium to explore retinal transport capabilities at each stage of degeneration. Retinas were then fixed in mixed aldehyde buffer and processed for transmission electron microscope connectomics, immunocytochemistry for a range of macromolecules, and computational molecular phenotyping for small molecules (CMP) (J Comp Neurol. 464:1, 2003).

Results:CMP reveals that single metabolic phenotype of MCs splits and diverges into many phenotypes continuously throughout degeneration. Further, all neuronal classes continue to die throughout degeneration. By 6 years, over 90% of neurons are lost, and the remaining glutamatergic neurons have altered metabolic signatures with a large increase in aspartate levels, at times exceeding glutamate. Transport of the D-isomers indicates that glutamate transport capabilities remain intact until the latest stages of degeneration. This may not be true of their GABA transporters.

Conclusions:These results suggest three main conclusions. First, retinal remodeling in degeneration is relentlessly progressive long after all photoreceptors have disappeared. Second, cell types previously thought to remain after degeneration onset, such as ganglion cells, will also ultimately die and the cells not lost often will change their metabolism. The consequence of this metabolic change in neurons is not yet fully explored. Third, the persistent robust glutamate transport capabilities of Müller cells indicate Müller cells can metabolize glutamate despite the massive loss of glutamine synthetase activity, likely unmasking alternate metabolic pathways.

Metabolic Changes During Late Stage Retinal Degeneration In Heterozygous Crx Mutant Cats (CrxRdy/+)

This abstract was presented today, May 2th at the 2016 Association for Research in Vision and Opthalmology (ARVO) meetings in Seattle, Washington by Laurence Occelli, Bryan W. Jones, and Simon M. Petersen-Jones.

Posterboard #: D0250

Abstract Number: 2260 – D0250

Author Block: Laurence M. Occelli, Bryan W. Jones, Simon M. Petersen-Jones
1 Small Animal Clinical Sciences, Michigan State University, East Lansing, Michigan, United States; 2 Ophthalmology, Moran Eye Center, University of Utah, Salt Lake City, Utah, United States

Disclosure Block:Laurence M. Occelli, None; Bryan W. Jones, None; Simon M. Petersen-Jones, None

Purpose: CRX is a transcription factor essential for normal photoreceptor development and survival. The CrxRdy cat has a spontaneous mutation in Crx. Early disease stages in heterozygous cats (CrxRdy/+) mimics severe Leber’s congenital amaurosis. This study investigated the timing and extent of retinal remodeling in the late stages of retinal degeneration. This will help optimizing the best time for therapies such as retinal prosthesis or optogenetics before retinal rewiring and glial scar become too extensive.

Methods: CrxRdy/+ cats from 6 weeks to 10 years of age were investigated. Eyes were fixed in mixed aldehyde buffer and processed for immunocytochemistry for computational molecular phenotyping for macromolecules and small molecules (CMP) including GABA, glycine, glutamate, taurine, glutamine, aspartate, rhodopsin and red green opsin (J Comp Neurol. 464:1 2003). Samples from 5 retinal areas were collected: area centralis, mid- and far-superior as well as mid- and far-inferior regions.

Results: CMP revealed an absence of red green opsin and a decrease in rhodopsin expression with mislocalization to the photoreceptor inner segments (IS) and cell bodies as early as 6 weeks of age. Inner and outer photoreceptor segments (IS/OS) were present but short at 6 weeks of age. By 12 weeks of age, very few of the stunted OS remained and IS were very short. At that age, Müller cells had become activated initiating hypertrophy, and indicating cell stress. By 5 years of age, a Müller cell seal was clearly present disrupting the retinal lamination via glial columns. Migration of inner nuclear layer cells with inverted and everted cells was also observed from an early age as well as horizontal and amacrine cell sprouting. By 5 years of age, microneuromas formations had developed (Fig.1). Extreme thinning and remodeling was observed in the peripheral retina of older animals and retinal pigment epithelium was lost from the area centralis.

Conclusions: This study indicates that retinal degeneration in the CrxRdy/+ cat retina follows the 3 proposed phases of retinal remodeling. As early as 12 weeks of age, some glial reaction to photoreceptor death was observed followed by formation of a glial seal, rewiring and inner nuclear layer cells migration. Finally, microneuroma formation, severe retinal thinning and remodeling was developed.

A Targeted Inhibitor Of The Alternative Complement Pathway Accelerates Recovery From Smoke-Induced Ocular Injury

We have a new publication out, A Targeted Inhibitor Of The Alternative Complement Pathway Accelerates Recovery From Smoke-Induced Ocular Injury authored by Alex Woodell, Bryan W. Jones, Tucker Williamson, Gloriane Schnabolk, Stephen Tomlinson, Carl Atkinson and Bärbel Rohrer.

Abstract:
PURPOSE. Morphological and genetic evidence exists that an overactive complement system driven by the complement alternative pathway (CAP) is involved in pathogenesis of age- related macular degeneration (AMD). Smoking is the only modifiable risk factor for AMD. As we have shown that smoke-related ocular pathology can be prevented in mice that lack an essential activator of CAP, we ask here whether this pathology can be reversed by increasing inhibition in CAP.

METHODS. Mice were exposed to either cigarette smoke (CS) or filtered air (6 hours/day, 5 days/week, 6 months). Smoke-exposed animals were then treated with the CAP inhibitor (CR2-fH) or vehicle control (PBS) for 3 months. Spatial frequency and contrast sensitivity were assessed by optokinetic response paradigms at 6 and 9 months; additional readouts included assessment of retinal morphology by electron microscopy (EM) and gene expression analysis by quantitative PCR.

RESULTS. The CS mice treated with CR2-fH showed significant improvement in contrast threshold compared to PBS-treated mice, whereas spatial frequency was unaffected by CS or pharmacological intervention. Treatment with CR2-fH in CS animals reversed thinning of the retina observed in PBS-treated mice as analyzed by spectral-domain optical coherence tomography, and reversed most morphological changes in RPE and Bruch’s membrane seen in CS animals by EM.

CONCLUSIONS. Taken together, these findings suggest that CAP inhibitors not only prevent, but have the potential to accelerate, the clearance of complement-mediated ocular injury. Improving our understanding of the regulation of the CAP pathway is paramount to developing novel treatment approaches for AMD.

Retinal Remodeling in Human Retinitis Pigmentosa

We have a new publication out (Direct Link, Free Open Access), Retinal Remodeling in Human Retinitis Pigmentosa authored by Bryan W. Jones, Rebecca Pfeiffer, Drew Ferrell, Carl Watt, Michael Marmor and Robert Marc.

Abstract: Retinitis Pigmentosa (RP) in the human is a progressive, currently irreversible neural degenerative disease usually caused by gene defects that disrupt the function or architecture of the photoreceptors. While RP can initially be a disease of photoreceptors, there is increasing evidence that the inner retina becomes progressively disorganized as the outer retina degenerates. These alterations have been extensively described in animal models, but remodeling in humans has not been as well characterized. This study, using computational molecular phenotyping (CMP) seeks to advance our understanding of the retinal remodeling process in humans. We describe cone mediated preservation of overall topology, retinal reprogramming in the earliest stages of the disease in retinal bipolar cells, and alterations in both small molecule and protein signatures of neurons and glia. Furthermore, while Müller glia appear to be some of the last cells left in the degenerate retina, they are also one of the first cell classes in the neural retina to respond to stress which may reveal mechanisms related to remodeling and cell death in other retinal cell classes. Also fundamentally important is the finding that retinal network topologies are altered. Our results suggest interventions that presume substantial preservation of the neural retina will likely fail in late stages of the disease. Even early intervention offers no guarantee that the interventions will be immune to progressive remodeling. Fundamental work in the biology and mechanisms of disease progression are needed to support vision rescue strategies.