Tag Archives: Bryan William Jones

Inner-Retinal Changes In AMD: Evidence, Mechanisms, and Future Perspectives

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We have a new manuscript out in Progress in Retinal Eye Research, Inner-retinal changes in AMD: Evidence, mechanisms, and future perspectives.

Authors: Matt Trinh , Michael Kalloniatis , Bryan William Jones @bwjones.bsky.social, Glenn C Yiu, Enrico Borrelli, Lisa Nivison-Smith.

Abstract: Age-related macular degeneration (AMD) has traditionally been regarded as a disorder of the outer-retina and choroid, characterised by drusen accumulation, retinal pigment epithelium (RPE) dysfunction, and photoreceptor degeneration. However, increasing evidence of inner-retinal involvement across the AMD spectrum, with structural and functional compromise evident from the early stages of disease, challenges this paradigm. Advances in spatially optimised optical coherence tomography (OCT), OCT angiography (OCTA), and high-resolution histology have revealed neuronal, vascular, and glial alterations within the inner-retina that reshape our understanding of AMD pathogenesis. This review synthesises clinical and experimental evidence on inner-retinal changes in AMD, including layer-specific thinning, microvascular rarefaction, impaired neurovascular coupling, and reactive gliosis. Such changes frequently emerge in early AMD, may precede, parallel, or exacerbate outer-retinal degeneration, and are associated with visual dysfunction not fully explained by photoreceptor loss alone. Importantly, mechanistic interactions between inner- and outer-retinal pathology support a bidirectional model of neurodegeneration, wherein region-specific vulnerability is shaped by perfusion dynamics, metabolic demands, and structural connectivity throughout the retina. Recognition of these processes expands the potential for earlier diagnosis, refined monitoring, and novel therapeutic targeting. By integrating structural, functional, and systemic insights, this review reframes AMD as a multi-layer neurovascular disease and underscores the central role of inner-retinal integrity in future AMD research and management.

Uncovering the electrical synapse proteome in retinal neurons via in vivo proximity labeling

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We have a new paper out in eLife: Uncovering the electrical synapse proteome in retinal neurons via in vivo proximity labeling.

Authors: Stephan Tetenborg, Eyad Shihabeddin, Elizebeth Olive Akansha Manoj Kumar, Crystal Sigulinsky @csigulinsky.bsky.social, Karin Dedek, Ya-Ping Lin, Fabio Echeverry, Hannah Hoff, Alberto Pereda, Bryan William Jones @bwjones.bsky.social, Christophe Ribelayga, Klaus Ebnet, Ken Matsuura, John O’Brien

Abstract: Electrical synapses containing Connexin 36 (Cx36) represent the main means for direct electrical communication among neurons in the mammalian nervous system. However, little is known about the protein complexes that constitute these synapses. In the present study, we applied different BioID strategies to screen the interactomes of Connexin 36 and its zebrafish orthologue Cx35b in retinal neurons. For in vivo proximity labeling in mice, we took advantage of the Cx36-EGFP strain and expressed a GFP-nanobody-TurboID fusion construct selectively in AII amacrine cells. For in vivo BioID in zebrafish, we generated a transgenic line expressing a Cx35b-TurboID fusion under control of the Cx35b promoter. Both strategies allowed us to capture a plethora of molecules that were associated with electrical synapses and showed a high degree of evolutionary conservation in the proteomes of both species. Besides known interactors of Cx36 such as ZO-1 and ZO-2 we have identified more than 50 new proteins, such as scaffold proteins, adhesion molecules and regulators of the cytoskeleton. Moreover, we determined the subcellular localization of these proteins in mouse retina and tested potential binding interactions with Cx36. Amongst these new interactors, we identified signal induced proliferation associated 1 like 3 (Sipa1l3), a protein that has been implicated in cell junction formation and cell polarity, as a new scaffold of electrical synapses. Interestingly, Sipa1l3 was able to interact with ZO-1, ZO-2 and Cx36, suggesting a pivotal role in electrical synapse function. In summary, our study provides the first detailed view of the electrical synapse proteome in retinal neurons, which is likely to apply to electrical synapses elsewhere.

Uncovering The Electrical Synapse Proteome In Retinal Neurons Via In Vivo Proximity Labeling

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We have a new paper out In BioRxiv, Uncovering The Electrical Synapse Proteome In Retinal Neurons Via In Vivo Proximity Labeling.

Authors: Stephan Tetenborg, Eyad Shihabeddin, Elizebeth Olive Akansha Manoj Kumar, Crystal Sigulinsky @csigulinsky.bsky.social, Karin Dedek, Ya-Ping Lin, Fabio Echeverry, Hannah Hoff, Alberto Pereda, Bryan William Jones @bwjones.bsky.social, Christophe Ribelayga, Klaus Ebnet, Ken Matsuura, John O’Brien

Abstract: Through decades of research, we have gained a comprehensive understanding of the protein complexes underlying function and regulation of chemical synapses in the nervous system. Despite the identification of key molecules such as ZO-1 or CaMKII, we currently lack a similar level of insight into the electrical synapse proteome. With the advancement of BioID as a tool for in vivo proteomics, it has become possible to identify complex interactomes of a given protein of interest by combining enzymatic biotinylation with subsequent streptavidin affinity capture. In the present study, we applied different BioID strategies to screen the interactomes of Connexin 36 (mouse) the major neuronal connexin and its zebrafish orthologue Cx35b in retinal neurons. For in vivo proximity labeling in mice, we took advantage of the Cx36-EGFP strain and expressed a GFP-nanobody-TurboID fusion construct selectively in AII amacrine cells. For in vivo BioID in
zebrafish, we generated a transgenic line expressing a Cx35b-TurboID fusion under control of the Cx35b promoter. Both two strategies allowed us to capture a plethora of molecules that were associated with electrical synapses and showed a high degree of evolutionary conservation in the proteomes of both species. Besides known interactors of Cx36 such as ZO-1 and ZO-2 we have identified more than 50 new proteins, such as scaffold proteins, adhesion molecules and regulators of the cytoskeleton. We further determined the subcellular localization of these proteins in AII amacrine and tested potential binding interactions with Cx36. Of note, we identified signal induced proliferation associated 1 like 3 (SIPA1L3), a protein that has been implicated in cell junction formation and cell polarity as a new scaffold of electrical synapses. Interestingly, SIPA1L3 was able to interact with ZO-1, ZO-2 and Cx36, suggesting a pivotal role in electrical synapse function. In summary, our study provides a first detailed view of the electrical synapse proteome in retinal neurons.

Retinal Connectomics: A Review

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We have a new manuscript out of the lab in Volume 10 of the Annual Review of Vision Science titled Retinal Connectomics: A Review by Crystal L. Sigulinsky, Rebecca L. Pfeiffer, and Bryan William Jones. A .pdf is here.

Abstract
The retina is an ideal model for understanding the fundamental rules for
how neural networks are constructed. The compact neural networks of
the retina perform all of the initial processing of visual information be-
fore transmission to higher visual centers in the brain. The field of retinal
connectomics uses high-resolution electron microscopy datasets to map
the intricate organization of these networks and further our understand-
ing of how these computations are performed by revealing the fundamental
topologies and allowable networks behind retinal computations. In this ar-
ticle, we review some of the notable advances that retinal connectomics
has provided in our understanding of the specific cells and the organi-
zation of their connectivities within the retina, as well as how these are
shaped in development and break down in disease. Using these anatomi-
cal maps to inform modeling has been, and will continue to be, instrumental
in understanding how the retina processes visual signals.

Preprint: Neural Circuit Revision in Retinal Remodeling, A Pathoconnectomics Approach

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We have a new preprint out, Neural Circuit Revision in Retinal Remodeling, A Pathoconnectomics Approach.

Authors: Rebecca L Pfeiffer, Jeebika Dahal, Crystal L Sigulinsky, James R Anderson, Isabel A Barrera, Jia-Hui Yang, Olivia Haddadin, Alexis R Houser, Jessica C Garcia, Bryan William Jones

Abstract: The Aii glycinergic amacrine cell (Aii) plays a central role in bridging rod pathways with cone pathways, enabling an increased dynamic range of vision from scotopic to photopic ranges. The Aii integrates scotopic signals via chemical synapses from rod bipolar cells (RodBCs) onto the arboreal processes of Aii ACs, injecting signals into ON-cone bipolar cells (CBbs) via gap junctions with Aiis on the arboreal processes and the waist of the Aii ACs. The CBbs then carry this information to ON and OFF ganglion cell classes. In addition, the Aii is involved in the surround inhibition of OFF cone bipolar cells (CBas) through glycinergic chemical synapses from Aii ACs onto CBas. We have previously shown changes in RodBC connectivity as a consequence of rod photoreceptor degeneration in a pathoconnectome of early retinal degeneration: RPC1. Here, we evaluated the impact of rod photoreceptor degeneration on the connectivity of the Aii to determine the impacts of photoreceptor degeneration on the downstream network of the neural retina and its suitability for integrating therapeutic interventions as rod photoreceptors are lost. Previously, we reported that in early retinal degeneration, prior to photoreceptor cell loss, Rod BCs make pathological gap junctions with Aiis. Here, we further characterize this altered connectivity and additional shifts in both the excitatory drive and gap junctional coupling of Aiis in retinal degeneration, along with discussion of the broader impact of altered connectivity networks. New findings reported here demonstrate that Aiis make additional gap junctions with CBas increasing the number of BC classes that make pathological gap junctional connectivity with Aiis in degenerating retina. In this study, we also report that the Aii, a tertiary retinal neuron alters their synaptic contacts early in photoreceptor degeneration, indicating that rewiring occurs in more distant members of the retinal network earlier in degeneration than was previously predicted. This rewiring impacts retinal processing, presumably acuity, and ultimately its ability to support therapeutics designed to restore image-forming vision. Finally, these Aii alterations may be the cellular network level finding that explains one of the first clinical complaints from human patients with retinal degenerative disease, an inability to adapt back and forth from photopic to scotopic conditions.

Mitochondrial Transfer Between Inner Retinal Neurons

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This abstract was presented today, April 26th at the 2023 Association for Research in Vision and Opthalmology (ARVO) meetings in New Orleans, Louisiana by Selena Wirthlin, Crystal Sigulinsky, James Anderson, and Bryan William Jones.

Full resolution version here.

Purpose
Intercellular mitochondrial transfer has been reported across a variety of cells and tissues under both physiological and pathological conditions. Such transfer has shown broad therapeutic potential. The effectiveness of this therapy, however, is limited by a lack of understanding of the cellular and molecular mechanisms. Here, the ultrastructural features of mitochondrial transfer between inner retinal neurons discovered through retinal connectomics analysis is shown.

Methods
Retinal Connectome 2 (RC2) was built by automated transmission electron microscopy at ultrastructural (2nm/pixel) resolution. RC2 is a 0.25mm diameter volume of retina obtained from a 5-month-old female C57BL/6J mouse. The Viking application was used to visualize and annotate inter- and intracellular features of interest in the connectome.

Results
Exploration of RC2 revealed material transfer between apposing neural processes within the OFF subliminal of the inner plexiform layer. The transferred material can be defined as a mitochondria, confirmed by the presence of crustae. At the transfer site, a short, electron-dense 140-nm diameter tube with a curved cap tightly associated with the inner mitochondrial membrane of one neuritis extends into a vacuole within the apposing neuritis formed by the plasma membranes of the two cells. Thin cytoskeletal components consistent with actin microfilaments extend into the mitochondrion. Morphology and synaptology of the acceptor cell confirm it is an Aii amacrine cell, while preliminary findings suggest the donor cell is a type of ON/OFF ganglion cell.

Conclusions
These findings demonstrate active mitochondrial transfer between different classes of endogenous inner retinal neurons and suggests it may represent an important component of tissue homeostasis in the retina. Features of this transfer differ from previously reported mitochondrial transfer between photoreceptors upon transplantation, which may indicate cell type- or context-dependent differences in the cellular or molecular mechanisms. Our findings demonstrate active mitochondrial transfer between different classes of endogenous inner retinal neurons and suggest it may represent an important component of tissue homeostasis in the retina. Features of this transfer differ from previous reports by the Wallace and Pearson groups of material transfer between photoreceptors upon transplantation through tunneling nanotubes (Ortin- Martinez et al., 2021; Kalargyrou et al., 2021), which may indicate cell type- or context-dependent differences in the cellular or molecular mechanisms. Understanding these mechanisms could serve as a catalyst for development of novel therapeutics for disease in the retina and beyond.

Structural Motifs Of Excitatory Synapses In The Mammalian Retina

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This abstract was presented today, April 24th at the 2023 Association for Research in Vision and Opthalmology (ARVO) meetings in New Orleans, Louisiana by Taylor Otterness, Crystal Sigulinsky, James Anderson, and Bryan William Jones.

Full resolution version here.

Purpose
Connectivity within the nervous system is precise and disruptions lead to degraded performance and disease, yet the rules that govern connectivity remain unknown. Recent efforts reveal that different types of cone bipolar cells in the neural retina show preferences in the selection and frequency of presynaptic structure types used for signal transmission. However, it is not yet known how these differences are related to the quantity or type of postsynaptic partner. We used Retinal Connectome 1 (RC1) to analyze the synaptic output of rabbit CBb6 cells, a type of ON cone bipolar cell that forms excitatory synapses via diverse presynaptic structure types, to identify patterns in how these cells interact with their postsynaptic partners.

Methods
RC1 is a 0.25 mm diameter volume sampled from mid-peripheral retina of a 13 month old female Dutch-Belted rabbit, serially sectioned at 70 nm, and imaged at ultrastructural resolution (2nm/px) using transmission electron microscopy. Postsynaptic partners of CBb6 cell 6156’s presynaptic structures were annotated using the Viking Viewer for Connectomics. Statistical analyses were conducted in Microsoft Excel and investigated further with 3D rendering and graph visualization of connectivity.

Results
The factors tracked for comparison included presynaptic structure type, target number, and postsynaptic partner type. Multiribbon synapses of CBb6 cell 6156 trended towards having a greater number of output partners, with a greater proportion of dyads than monads. Despite this, triads and quadrads were only found opposing single ribbon synapses. As the different presynaptic structure types may differ in the strength of neurotransmitter release (ribbonless < single ribbon < multiribbon), these findings are inconsistent with scaling of output to the number of postsynaptic targets. Both amacrine cells (AC) and ganglion cells (GC) are postsynaptic partners of 6156. However, single ribbon and ribbonless structures appear biased towards AC only targets, while multiribbon synapses appear biased toward mixed AC and GC targets.

Conclusions
Target type relationships appear more important than the number of targets in determining presynaptic structure type in CBb6. Future efforts will examine size differences of postsynaptic structures and presynaptic ribbon size, and even compare across bipolar cell classes, in order to provide further insight on the connectivity rules underlying excitatory synapses.

Current Perspective on Retinal Remodeling: Implications for Therapeutics

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We have a new paper out of the lab, a perspectives paper on Retinal Remodeling: Implications for Therapeutics. (pdf here).

Authors are Rebecca L. Pfeiffer @BeccaPfeiffer19, and Bryan W. Jones @BWJones.

Abstract: The retinal degenerative diseases retinitis pigmentosa and age-related macular degeneration are a leading cause of irreversible vision loss. Both present with progressive photoreceptor degeneration that is further complicated by processes of retinal remodeling. In this perspective, we discuss the current state of the field of retinal remodeling and its implications for vision-restoring therapeutics currently in development. Here, we discuss the challenges and pitfalls retinal remodeling poses for each therapeutic strategy under the premise that understanding the features of retinal remodeling in totality will provide a basic framework with which therapeutics can interface. Additionally, we discuss the potential for approaching therapeutics using a combined strategy of using diffusible molecules in tandem with other vision-restoring therapeutics. We end by discussing the potential of the retina and retinal remodeling as a model system for more broadly understanding the progression of neurodegeneration across the central nervous system.