Small GTP‐binding protein RhoB is expressed in glial müller cells in the vertebrate retina

Santos-Bredariol, Andréa Silveira; Belmonte, M; Kihara, Alexandre Hiroaki; Santos, Marinilce Fagundes dos; Hamassaki‐Britto, Dânia Emi

doi:10.1002/cne.20861

Cited by 18 publications

(11 citation statements)

References 52 publications

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“…As previously described [21], [22], retinas were rapidly dissected, washed with phosphate buffered saline (PBS), and homogenized in RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1%Triton X-100 and protease inhibitors). Homogenates were centrifuged for 20 min at 14,000 G, 4°C to remove insoluble material.…”

Section: Methodsmentioning

confidence: 99%

Developmental and Functional Expression of miRNA-Stability Related Genes in the Nervous System

et al. 2013

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In the nervous system, control of gene expression by microRNAs (miRNAs) has been investigated in fundamental processes, such as development and adaptation to ambient demands. The action of these short nucleotide sequences on specific genes depends on intracellular concentration, which in turn reflects the balance of biosynthesis and degradation. Whereas mechanisms underlying miRNA biogenesis has been investigated in recent studies, little is known about miRNA-stability related proteins. We first detected two genes in the retina that have been associated to miRNA stability, XRN2 and PAPD4. These genes are highly expressed during retinal development, however with distinct subcellular localization. We investigated whether these proteins are regulated during specific phases of the cell cycle. Combined analyses of nuclei position in neuroblastic layer and labeling using anti-cyclin D1 revealed that both proteins do not accumulate in S or M phases of the cell cycle, being poorly expressed in progenitor cells. Indeed, XRN2 and PAPD4 were observed mainly after neuronal differentiation, since low expression was also observed in astrocytes, endothelial and microglial cells. XRN2 and PAPD4 are expressed in a wide variety of neurons, including horizontal, amacrine and ganglion cells. To evaluate the functional role of both genes, we carried out experiments addressed to the retinal adaptation in response to different ambient light conditions. PAPD4 is upregulated after 3 and 24 hours of dark- adaptation, revealing that accumulation of this protein is governed by ambient light levels. Indeed, the fast and functional regulation of PAPD4 was not related to changes in gene expression, disclosing that control of protein levels occurs by post-transcriptional mechanisms. Furthermore, we were able to quantify changes in PAPD4 in specific amacrine cells after dark -adaptation, suggesting for circuitry-related roles in visual perception. In summary, in this study we first described the ontogenesis and functional expression of these two miRNA-stability related proteins in the retina.

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Section: Methodsmentioning

confidence: 99%

Developmental and Functional Expression of miRNA-Stability Related Genes in the Nervous System

et al. 2013

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“…As previously described [8], retinas were rapidly dissected, washed with phosphate buffered saline (PBS), and homogenized in 25 mM Tris buffer (Calbiochem, La Jolla, CA, USA). Homogenates were centrifuged for 5 min at 2000 G to remove insoluble material.…”

Section: Methodsmentioning

confidence: 99%

Blocking of Connexin-Mediated Communication Promotes Neuroprotection during Acute Degeneration Induced by Mechanical Trauma

Paschon

Higa²,

Resende³

et al. 2012

PLoS ONE

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Accruing evidence indicates that connexin (Cx) channels in the gap junctions (GJ) are involved in neurodegeneration after injury. However, studies using KO animal models endowed apparently contradictory results in relation to the role of coupling in neuroprotection. We analyzed the role of Cx-mediated communication in a focal lesion induced by mechanical trauma of the retina, a model that allows spatial and temporal definition of the lesion with high reproducibility, permitting visualization of the focus, penumbra and adjacent areas. Cx36 and Cx43 exhibited distinct gene expression and protein levels throughout the neurodegeneration progress. Cx36 was observed close to TUNEL-positive nuclei, revealing the presence of this protein surrounding apoptotic cells. The functional role of cell coupling was assessed employing GJ blockers and openers combined with lactate dehydrogenase (LDH) assay, a direct method for evaluating cell death/viability. Carbenoxolone (CBX), a broad-spectrum GJ blocker, reduced LDH release after 4 hours, whereas quinine, a Cx36-channel specific blocker, decreased LDH release as early as 1 hour after lesion. Furthermore, analysis of dying cell distribution confirmed that the use of GJ blockers reduced apoptosis spread. Accordingly, blockade of GJ communication during neurodegeneration with quinine, but not CBX, caused downregulation of initial and effector caspases. To summarize, we observed specific changes in Cx gene expression and protein distribution during the progress of retinal degeneration, indicating the participation of these elements in acute neurodegeneration processes. More importantly, our results revealed that direct control of GJ channels permeability may take part in reliable neuroprotection strategies aimed to rapid, fast treatment of mechanical trauma in the retina.

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“…As previously described (Santos‐Bredariol et al, 2006), retinas were rapidly dissected, washed with PBS, and homogenized in 25 mM Tris buffer containing a cocktail of protease inhibitors (Calbiochem, La Jolla, CA, USA). Homogenates were centrifuged for 5 min at 2000 g (4 °C) to remove insoluble material.…”

Section: Methodsmentioning

confidence: 99%

Connexin‐mediated communication controls cell proliferation and is essential in retinal histogenesis

Kihara

Santos²,

Osuna-Melo³

et al. 2009

Intl J of Devlp Neuroscience

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Connexin (Cx) channels and hemichannels are involved in essential processes during nervous system development such as apoptosis, propagation of spontaneous activity and interkinetic nuclear movement. In the first part of this study, we extensively characterized Cx gene and protein expression during retinal histogenesis. We observed distinct spatio-temporal patterns among studied Cx and an overriding, ubiquitous presence of Cx45 in progenitor cells. The role of Cx-mediated communication was assessed by using broad-spectrum (carbenoxolone, CBX) and Cx36/Cx50 channel-specific (quinine) blockers. In vivo application of CBX, but not quinine, caused remarkable reduction in retinal thickness, suggesting changes in cell proliferation/apoptosis ratio. Indeed, we observed a decreased number of mitotic cells in CBX-injected retinas, with no significant changes in the expression of PCNA, a marker for cells in proliferative state. Taken together, our results pointed a pivotal role of Cx45 in the developing retina. Moreover, this study revealed that Cx-mediated communication is essential in retinal histogenesis, particularly in the control of cell proliferation.

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Small GTP‐binding protein RhoB is expressed in glial müller cells in the vertebrate retina

Cited by 18 publications

References 52 publications

Developmental and Functional Expression of miRNA-Stability Related Genes in the Nervous System

Developmental and Functional Expression of miRNA-Stability Related Genes in the Nervous System

Blocking of Connexin-Mediated Communication Promotes Neuroprotection during Acute Degeneration Induced by Mechanical Trauma

Connexin‐mediated communication controls cell proliferation and is essential in retinal histogenesis

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