Dataset Information

Quantitative and Qualitative Evaluation of Photoreceptor Synapses in Developing, Degenerating and Regenerating Retinas.

ABSTRACT: Quantitative and qualitative evaluation of synapses is crucial to understand neural connectivity. This is particularly relevant now, in view of the recent advances in regenerative biology and medicine. There is an urgent need to evaluate synapses to access the extent and functionality of reconstructed neural network. Most of the currently used synapse evaluation methods provide only all-or-none assessments. However, very often synapses appear in a wide spectrum of transient states such as during synaptogenesis or neural degeneration. Robust evaluation of synapse quantity and quality is therefore highly sought after. In this paper we introduce QUANTOS, a new method that can evaluate the number, likelihood, and maturity of photoreceptor ribbon synapses based on graphical properties of immunohistochemistry images. QUANTOS is composed of ImageJ Fiji macros, and R scripts which are both open-source and free software. We used QUANTOS to evaluate synaptogenesis in developing and degenerating retinas, as well as de novo synaptogenesis of mouse iPSC-retinas after transplantation to a retinal degeneration mouse model. Our analysis shows that while mouse iPSC-retinas are largely incapable of forming synapses in vitro, they can form extensive synapses following transplantation. The de novo synapses detected after transplantation seem to be in an intermediate state between mature and immature compared to wildtype retina. Furthermore, using QUANTOS we tested whether environmental light can affect photoreceptor synaptogenesis. We found that the onset of synaptogenesis was earlier under cyclic light (LD) condition when compared to constant dark (DD), resulting in more synapses at earlier developmental stages. The effect of light was also supported by micro electroretinography showing larger responses under LD condition. The number of synapses was also increased after transplantation of mouse iPSC-retinas to rd1 mice under LD condition. Our new probabilistic assessment of synapses may prove to be a valuable tool to gain critical insights into neural-network reconstruction and help develop treatments for neurodegenerative disorders.

SUBMITTER: Akiba R

PROVIDER: S-EPMC6378395 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Quantitative and Qualitative Evaluation of Photoreceptor Synapses in Developing, Degenerating and Regenerating Retinas.

Akiba Ryutaro R Matsuyama Take T Tu Hung-Ya HY Hashiguchi Tomoyo T Sho Junki J Yamamoto Shuichi S Takahashi Masayo M Mandai Michiko M

Frontiers in cellular neuroscience 20190211

Quantitative and qualitative evaluation of synapses is crucial to understand neural connectivity. This is particularly relevant now, in view of the recent advances in regenerative biology and medicine. There is an urgent need to evaluate synapses to access the extent and functionality of reconstructed neural network. Most of the currently used synapse evaluation methods provide only all-or-none assessments. However, very often synapses appear in a wide spectrum of transient states such as during ...[more]

PMID: 30804754

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Project description:BACKGROUND:Retinal degeneration is a leading cause of blindness in the world; its etiology is complex and involves genetic defects and stress-associated aging. In addition to gene therapies for known genetically defective retinal degeneration, cellular therapies have been widely explored for restoring vision in both preclinical animal models and clinical trials. Stem cells of distinct tissue sources and their derived lineages have been tested for treating retinal degeneration; most of them were reported to be effective to some extent in restoring/improving deteriorated vision. Whether this visual improvement is due to a functional integration of grafted cells to substitute for lost retinal neurons in recipients or due to their neuroprotective and neurotrophic effects to retain recipient functional neurons, or both, is still under debate. METHODS:We compared the results of subretinal transplantation of various somatic cell types, such as stem cells and differentiated cells, into RhoP23H/+ mice, a retinal degeneration model for human retinitis pigmentosa (RP) by evaluating their optokinetic response (OKR) and retinal histology. We identified some paracrine factors in the media that cultured cells secreted by western blotting (WB) and functionally evaluated the vascular endothelial growth factor Vegfa for its potential neurotrophic and neuroprotective effects on the neuroretina of model animals by intravitreal injection of VEGF antibody. RESULTS:We found that live cells, regardless of whether they were stem cells or differentiated cell types, had a positive effect on improving degenerating retinas after subretinal transplantation; the efficacy depended on their survival duration in the host tissue. A few paracrine factors were identified in cell culture media; Vegfa was the most relevant neurotrophic and neuroprotective factor identified by our experiments to extend neuron survival duration in vivo. CONCLUSIONS:Cellular therapy-produced benefits for remediating retinal degeneration are mostly, if not completely, due to a paracrine effect of implanted cells on the remaining host retinal neurons.

Project description:Biotin (vitamin H) is a key enzyme cofactor required in all three domains of life. Although this cofactor was discovered over 70 years ago and has long been recognized as an essential nutrient for animals, our knowledge of the strategies bacteria use to sense biotin demand is very limited. The paradigm mechanism is that of Escherichia coli in which BirA protein, the prototypical bi-functional biotin protein ligase, both covalently attaches biotin to the acceptor proteins of central metabolism and represses transcription of the biotin biosynthetic pathway in response to biotin demand. However, in other bacteria the biotin protein ligase lacks a DNA-binding domain which raises the question of how these bacteria regulate the synthesis of biotin, an energetically expensive molecule. A bioinformatic study by Rodionov and Gelfand identified a protein termed BioR in ?-proteobacteria and predicted that BioR would have the biotin operon regulatory role that in most other bacteria is fulfilled by the BirA DNA-binding domain. We have now tested this prediction in the plant pathogen Agrobacterium tumefaciens. As predicted the A.?tumefaciens biotin protein ligase is a fully functional ligase that has no role in regulation of biotin synthesis whereas BioR represses transcription of the biotin synthesis genes. Moreover, as determined by electrophoretic mobility shift assays, BioR binds the predicted operator site, which is located downstream of the mapped transcription start site. qPCR measurements indicated that deletion of BioR resulted in a c. 15-fold increase of bio operon transcription in the presence of high biotin levels. Effective repression of a plasmid-borne bioB-lacZ reporter was seen only upon the overproduction of BioR. In contrast to E.?coli and Bacillus subtilis where biotin synthesis is tightly controlled, A.?tumefaciens synthesizes much more biotin than needed for modification of the biotin-requiring enzymes. Protein-bound biotin constitutes only about 0.5% of the total biotin, most of which is found in the culture medium. To the best of our knowledge, A.?tumefaciens represents the first example of profligate biotin synthesis by a wild type bacterium.

Dataset Information

Quantitative and Qualitative Evaluation of Photoreceptor Synapses in Developing, Degenerating and Regenerating Retinas.

Publications

Quantitative and Qualitative Evaluation of Photoreceptor Synapses in Developing, Degenerating and Regenerating Retinas.

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