Biology subjects

Jones, B. W.

Publications and source records attributed to Jones, B. W..

9 recordsLinked to original sources

bioRxiv2024

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

AbstractElectrical 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.

neuroscience↗

bioRxiv2024

Neural Circuit Revision in Retinal Remodeling, A Pathoconnectomics Approach

Retinitis pigmentosa (RP), a retinal degenerative disease, is characterized by progressive photoreceptor loss and ongoing remodeling and rewiring of the inner retina. This study investigates rod network rewiring through pathoconnectomic evaluation and its impacts on signaling patterns. The glycinergic Aii amacrine cell (Aii) plays a central role in the healthy retina bridging rod and cone pathways, enabling an increased dynamic range of vision. Pathoconnectomics reveals altered connectivity in both the excitatory drive and gap junctional coupling of Aiis in retinal degeneration. A computational model of the rewired network was developed to assess the functional consequences of these structural changes by simulating light-evoked responses and changes in excitatory postsynaptic potentials (EPSPs). The model predicts significant changes in bipolar and Aii EPSPs between active and baseline conditions, driven by newly formed gap junctions in the degenerate retina. Notably, the aberrant circuitry induces rhythmic firing of up to 10 Hz in retinal ganglion cells, consistent with network depolarization relative to the healthy baseline state. These findings align with patch-clamp observations in rd1 and rd10 mouse models of RP, suggesting that Aii-mediated network alterations may underlie early clinical symptoms, including impaired adaptation between photopic and scotopic vision. More broadly, this work demonstrates that integrating computational modeling with pathoconnectomics enables predictive analysis of signaling in early-stage retinal degeneration and may help identify windows for therapeutic intervention. Such models could be further extended with multi-scale bioelectromagnetic simulations to optimize neurostimulation strategies aimed at slowing disease progression. Author summaryUnderstanding how retinal degeneration alters wiring topologies of the inner retina is important for the success of multiple therapeutic interventions, including cell replacement strategies, optogenetics, and electrode implants. Here, we continue our evaluation of retinal pathoconnectome 1 (RPC1), describing additional network-level changes occurring early in retinal degeneration. This analysis extends our previous findings on the emergence of gap junctions in rod bipolar cells in retinal degeneration to include the effects of these changes on the synaptic strength of inputs to the Aii. We then model how these network changes overall effect retinal processing through the creation of a more complete degenerate retina model. From these results we propose the emergence of aberrant gap junctions in the rod pathway as the network cause of atypical retinal ganglion cell firing and provide the field with a realistic network model for evaluating and optimizing therapeutic strategies.

neuroscience↗

bioRxiv2023

NUDC is critical for rod photoreceptor function, maintenance, and survival

NUDC (nuclear distribution protein C) is a mitotic protein involved in nuclear migration and cytokinesis across species. Considered a cytoplasmic dynein (henceforth dynein) cofactor, NUDC was shown to associate with the dynein motor complex during neuronal migration. NUDC is also expressed in postmitotic vertebrate rod photoreceptors where its function is unknown. Here, we examined the role of NUDC in postmitotic rod photoreceptors by studying the consequences of a conditional NUDC knockout in mouse rods (rNudC-/-). Loss of NUDC in rods led to complete photoreceptor cell death at six weeks of age. By 3 weeks of age, rNudC-/- function was diminished, and rhodopsin and mitochondria were mislocalized, consistent with dynein inhibition. Levels of outer segment proteins were reduced, but LIS1 (lissencephaly protein 1), a well-characterized dynein cofactor, was unaffected. Transmission electron microscopy revealed ultrastructural defects within the rods of rNudC-/- by 3 weeks of age. We investigated whether NUDC interacts with the actin modulator cofilin 1 (CFL1) and found that in rods, CFL1 is localized in close proximity to NUDC. In addition to its potential role in dynein trafficking within rods, loss of NUDC also resulted in increased levels of phosphorylated CFL1 (pCFL1), which would purportedly prevent depolymerization of actin. Absence of NUDC also induced an inflammatory response in Muller glia and microglia across the neural retina by 3 weeks of age. Taken together, our data illustrate the critical role of NUDC in actin cytoskeletal maintenance and dynein-mediated protein trafficking in a postmitotic rod photoreceptor. Significance StatementNuclear distribution protein C (NUDC) has been studied extensively as an essential protein for mitotic cell division. In this study, we discovered its expression and role in the postmitotic rod photoreceptor cell. In the absence of NUDC in mouse rods, we detected functional loss, protein mislocalization, and rapid retinal degeneration consistent with dynein inactivation. In the early phase of retinal degeneration, we observed ultrastructural defects and an upregulation of inflammatory markers suggesting additional, dynein-independent functions of NUDC.

cell biology↗

bioRxiv2023

Hypoxia regulate developmental coronary angiogenesis potentially through VEGFR2- and SOX17-mediated signaling

BackgroundCoronary vessels in embryonic mouse heart arises from multiple progenitor population including sinus venosus (SV), endocardium, and proepicardium. ELA/APJ signaling is shown to regulate coronary growth from SV pathway within the subepicardium, whereas VEGF-A/VEGF-R2 pathways is implicated to regulate coronary growth from endocardium pathway. Our previous study show hypoxia as a potential signaling cue to stimulate overall coronary growth and expansion within the myocardium. However, the role of hypoxia and its downstream signaling pathways in the regulation of coronary vessel development is not known. In this study, we investigated the role of hypoxia in coronary vessel development and have identified SOX17- and VEGF-R2-mediated signaling as a potential downstream pathway of hypoxia in the regulation of coronary vessel development. ResultsWe show that hypoxia gain-of-function in the myocardium through upregulation of HIF-1 disrupts the normal pattern of coronary angiogenesis in developing mouse hearts and displays phenotype that is reminiscent of accelerated coronary growth. We show that VEGF-R2 expression is increased in coronary endothelial cells under hypoxia gain-of-function in vivo and in vitro. Furthermore, we show that SOX17 expression is upregulated in developing mouse heart under hypoxia gain-of-function conditions, whereas SOX17 expression is repressed under hypoxia loss-of-function conditions. Furthermore, our results show that SOX17 loss-of-function disrupts normal pattern of coronary growth. ConclusionCollectively, our data provide strong phenotypic evidence to show that hypoxia might regulate coronary growth in the developing mouse heart potentially through VEGF-R2- and SOX17-mediated downstream signaling pathways.

developmental biology↗

bioRxiv2020

Revival of light-evoked neuronal signals in the post-mortem mammalian and human retina

The retina, a highly metabolic tissue in the central nervous system, consumes the most oxygen and energy stores in the body by tissue mass/volume. Consequently, it is not surprising that retinal ischemia leads to a rapid loss of retinal light responses and electrical transmission. In this study, we show that despite a swift decline of retinal light responses after circulatory death (decay time constant {tau} = [~]1-2 min), we were able to restore mouse rod and cone photoreceptor light signals from enucleated eyes up to 3 h postmortem with significantly better postmortem recovery of cone versus rod light responses. We also demonstrate that both rod and cone phototransduction are more resistant to postmortem enucleation delay than synaptic transmission to second order neurons (bipolar cells). Encouraged by these analyses and the lack of previously successful postmortem retinal light recordings from human foveal/macular photoreceptors, we attempted to restore light responses in the human macula using donor eyes harvested 0.5 - 5 hours postmortem. Here we show successful recordings of human macular cone photoresponses in samples obtained from eyes enucleated up to 5 hours postmortem. Comparing freshly enucleated non-human primate eyes with human eyes enucleated and reoxygenated at different times after death, we show the exponential decay of the light response amplitudes has a time constant of 74 min. We find that both cause of death and donor age are useful parameters for predicting the recovery of retinal light responses in postmortem human macular tissue. Moreover, we present evidence that hypoxia and secondary acidosis, two modifiable factors, are primary contributors to the rapid loss of retinal light signaling after death. Finally, we show postmortem hypoxia rather than acidosis is the major cause of irreversible decay of the light response amplitudes. The criteria and methodology that we have established here for reviving electrical photoresponses in the postmortem human eye will serve as a new starting point for studying neurophysiology and disease in the human retina.

neuroscience↗

bioRxiv2020

Altered substance P signaling underlies perivascular sensory nerve dysfunction in inflammatory bowel disease

ObjectiveInflammatory Bowel Disease (IBD) is associated with cardiovascular disease risk and impaired intestinal blood flow, but the functional role of perivascular nerves that control vasomotor function of mesenteric arteries (MAs) perfusing the intestine is unknown in IBD. Because perivascular sensory nerves and their transmitters calcitonin gene-related peptide (CGRP) and substance P (SP) are important mediators of both vasodilation and inflammatory responses, our objective was to identify IBD-related deficits in perivascular sensory nerve function and vascular neurotransmitter signaling. Approach and ResultsIn MAs from an IL-10-/- mouse model, we found that IBD significantly impairs EFS-mediated sensory vasodilation and sensory inhibition of sympathetic vasoconstriction, despite decreased sympathetic nerve density and vasoconstriction. The MA content and EFS-mediated release of both CGRP and SP are slightly decreased with IBD, but IBD has different effects on each transmitter. CGRP nerve density, receptor expression, hyperpolarization and vasodilation are preserved with IBD. In contrast, SP nerve density and receptor expression are increased, and SP hyperpolarization and vasodilation are impaired with IBD. A critical finding is that blocking neurokinin 1 (SP) receptors restored EFS-mediated sensory vasodilation and enhanced CGRP-mediated vasodilation in MAs from IBD but not Control mice. ConclusionsAn aberrant role for the perivascular sensory neurotransmitter SP and its downstream signaling in MAs underlies vascular dysfunction with IBD. With IBD, SP signaling impedes CGRP-mediated sensory vasodilation, contributing to impaired blood flow. Substance P and NK1 receptors may represent an important target for treating vascular dysfunction in IBD.

physiology↗

bioRxiv2020

A Pathoconnectome of Early Neurodegeneration

Connectomics has demonstrated that synaptic networks and their topologies are precise and directly correlate with physiology and behavior. The next extension of connectomics is pathoconnectomics: to map neural network synaptology and circuit topologies corrupted by neurological disease in order to identify robust targets for therapeutics. In this report, we characterize the first pathoconnectome, in this case, generated from a retina with photoreceptor degeneration. We observe aberrant connectivity in the rod-network pathway and novel synaptic connections deriving from neurite sprouting. These observations reveal principles of neuron responses to the loss of network components and can be extended to other neurodegenerative diseases.

neuroscience↗

bioRxiv2018

Neural crest cells regulate optic cup morphogenesis by promoting extracellular matrix assembly

The interactions between an organ and its surrounding environment are critical in regulating its development. In vertebrates, neural crest and mesodermal mesenchymal cells have been observed close to the eye during development, and mutations affecting this periocular mesenchyme can cause defects in early eye development, yet the underlying mechanism has been unknown. Here, using timelapse microscopy and four-dimensional cell tracking in zebrafish, we establish that genetic loss of neural crest impairs cell movements within the optic vesicle. At the ultrastructural level, neural crest cells are required for basement membrane formation specifically around the retinal pigment epithelium. Neural crest cells express the extracellular matrix crosslinking protein nidogen and, strikingly, ectopically expressing nidogen in the absence of neural crest partially restores optic cup morphogenesis. These results demonstrate that the neural crest is required for local establishment of ocular extracellular matrix superstructure, which in turn drives optic cup morphogenesis.

developmental biology↗

bioRxiv2018

Heterocellular coupling between amacrine cell and ganglion cells

All superclasses of retinal neurons display some form of electrical coupling including the key neurons of the inner plexiform layer: bipolar cells (BCs), amacrine or axonal cells (ACs) and ganglion cells (GCs). However, coupling varies extensively by class. For example, mammalian rod bipolar cells form no gap junctions at all, while all cone bipolar cells form class-specific coupling arrays, many of them homocellular in-superclass arrays. Ganglion cells are unique in that classes with coupling predominantly form heterocellular cross-class arrays of ganglion cell::amacrine cell (GC::AC) coupling in the mammalian retina. Ganglion cells are the least frequent superclass in the inner plexiform layer and GC::AC gap junctions are sparsely arrayed amidst massive cohorts of AC::AC, bipolar cell BC::BC, and AC::BC gap junctions. Many of these gap junctions and most ganglion cell gap junctions are suboptical, complicating analysis of specific ganglion cells. High resolution 2 nm TEM analysis of rabbit retinal connectome RC1 allows quantitative GC::AC coupling maps of identified ganglion cells. Ganglion cells classes apparently avoid direct cross-class homocellular coupling altogether even though they have opportunities via direct membrane touches, while transient OFF alpha ganglion cells and transient ON directionally selective (DS) ganglion cells are strongly coupled to distinct amacrine / axonal cell cohorts.\n\nA key feature of coupled ganglion cells is intercellular metabolite flux. Most GC::AC coupling involves GABAergic cells ({gamma}+ amacrine cells), which results in significant GABA flux into ganglion cells. Surveying GABA coupling signatures in the ganglion cell layer across species suggests that the majority of vertebrate retinas engage in GC::AC coupling.\n\nMulti-hop synaptic queries of the entire RC1 connectome clearly profiles the coupled amacrine and axonal cells. Photic drive polarities and source bipolar cell class selec-tivities are tightly matched across coupled cells. OFF alpha ganglion cells are coupled to OFF {gamma}+ amacrine cells and transient ON DS ganglion cells are coupled to ON {gamma}+ amacrine cells including a large interstitial axonal cell (IAC). Synaptic tabulations show close matches between the classes of bipolar cells sampled by the coupled amacrine and ganglion cells. Further, both ON and OFF coupling ganglion networks show a common theme: synaptic asymmetry whereby the coupled {gamma}+ neurons are also presynaptic to ganglion cell dendrites from different classes of ganglion cells outside the coupled set. In effect, these heterocellular coupling patterns enable an excited ganglion cell to directly inhibit nearby ganglion cells of different classes. Similarly, coupled {gamma}+ amacrine cells engaged in feedback networks can leverage the additional gain of bipolar cell synapses in shaping the signaling of a spectrum of downstream targets based on their own selective coupling with ganglion cells.

neuroscience↗