Isolation and Molecular Profiling of Primary Mouse Retinal Ganglion Cells: Comparison of Phenotypes from Healthy and Glaucomatous Retinas

Chintalapudi, Sumana R; Djenderedjian, Levon; Stiemke, Andrew B.; Steinle, Jena J.; Jablonski, Monica M.; Morales-Tirado, Vanessa M.

doi:10.3389/fnagi.2016.00093

Cited by 29 publications

(26 citation statements)

References 44 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…RNA was extracted from Rb cell lines or PDX eye cross-sections with the Qiagen RNeasy Mini Kit (Valencia, CA, USA) according to manufacturer's instructions and our previously described protocols. 26 , 28 , 29 Rb cells were lysed and homogenized prior to the addition of chloroform. The upper, colorless phase underwent a DNase digestion and a series of alcohol precipitations prior to elution through a spin column.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Targeting the Platelet-Derived Growth Factor-beta Stimulatory Circuitry to Control Retinoblastoma Seeds

Goldsmith

Coppess

Irvine

et al. 2018

Invest. Ophthalmol. Vis. Sci.

Self Cite

View full text Add to dashboard Cite

PurposeVitreous seeding remains the primary reason for treatment failure in eyes with retinoblastoma (Rb). Systemic and intra-arterial chemotherapy, each with its own inherent set of complications, have improved salvage rates for eyes with advanced disease, but the location and biology of vitreous seeds present a fundamental challenge in developing treatments with minimal toxicity and risk. The aim of this study was to target the platelet-derived growth factor (PDGF)– PDGF-receptor β (PDGFRβ) signaling pathway and investigate its role in the growth of Rb seeds, apoptotic activity, and invasive potential.MethodsWe performed ex vivo analyses on vitreous samples from Rb patients that underwent enucleation and from patient-derived xenografts. These samples were evaluated by quantitative PCR, immunohistochemistry, and ELISA. The effects of disruption of the PDGF-PDGFRβ signaling pathway, both by pharmacologic and genomic knockdown approaches, were evaluated in vitro by cell proliferation and apoptotic assays, quantitative PCR analyses, Western blotting, flow cytometry, and imaging flow cytometry. A three-dimensional cell culture system was generated for in-depth study of Rb seeds.ResultsOur results demonstrated that PDGFRβ signaling is active in the vitreous of Rb patients and patient-derived xenografts, sustaining growth and survival in an AKT-, MDM2-, and NF-κB-dependent manner. The novel three-dimensional cell culture system mimics Rb seeds, as the in vitro generated spheroids have similar morphologic features to Rb seeds and mimicked their natural physiology.ConclusionsTargeting the PDGFRβ pathway in vitro reduces Rb cell growth, survival, and invasiveness and could augment current therapies. This represents a novel signaling pathway for potential targeted therapy to further improve ocular survival in advanced Rb.

show abstract

Section: Methodsmentioning

confidence: 99%

“…The SuperScript VILO cDNA Synthesis Kit (Life Technologies, Grand Island, NY, USA) was used to synthesize cDNA as previously described. 26 , 28 , 29 …”

Section: Methodsmentioning

confidence: 99%

Targeting the Platelet-Derived Growth Factor-beta Stimulatory Circuitry to Control Retinoblastoma Seeds

Goldsmith

Coppess

Irvine

et al. 2018

Invest. Ophthalmol. Vis. Sci.

Self Cite

View full text Add to dashboard Cite

show abstract

“…A previously reported flow cytometry-based Thy-1-labeling RGC counting method was also applied to assess RGC survival rates. RGCs were isolated using the method described by Chintalapudi [22,23], with some modifications. Suspensions of retinal cells were generated using enzymatic digestion and subsequently filtered through a sterile 70-lm nylon strainer to obtain single-cell suspensions.…”

Section: Thy-1-positive Rgc Countingmentioning

confidence: 99%

Altered Energy Metabolism During Early Optic Nerve Crush Injury: Implications of Warburg-Like Aerobic Glycolysis in Facilitating Retinal Ganglion Cell Survival

Zhu

Zhou

et al. 2020

Neurosci. Bull.

View full text Add to dashboard Cite

Neurons, especially axons, are metabolically demanding and energetically vulnerable during injury. However, the exact energy budget alterations that occur early after axon injury and the effects of these changes on neuronal survival remain unknown. Using a classic mouse model of optic nerve-crush injury, we found that traumatized optic nerves and retinas harbor the potential to mobilize two primary energetic machineries, glycolysis and oxidative phosphorylation, to satisfy the robustly increased adenosine triphosphate (ATP) demand. Further exploration of metabolic activation showed that mitochondrial oxidative phosphorylation was amplified over other pathways, which may lead to decreased retinal ganglion cell (RGC) survival despite its supplement to ATP production. Gene set enrichment analysis of a microarray (GSE32309) identified significant activation of oxidative phosphorylation in injured retinas from wild-type mice compared to those from mice with deletion of phosphatase and tensin homolog (PTEN), while PTEN-/-mice had more robust RGC survival. Therefore, we speculated that the oxidation-favoring metabolic pattern after optic nervecrush injury could be adverse for RGC survival. After redirecting metabolic flux toward glycolysis (magnifying the Warburg effect) using the drug meclizine, we successfully increased RGC survival. Thus, we provide novel insights into a potential bioenergetics-based strategy for neuroprotection.

show abstract

“…RNA was extracted from cells or tissue using RNeasy ® Mini Kit (Qiagen Inc., Valencia, CA, USA) following manufacturer's conditions and published protocols. [14][15][16] We used 100 ng of RNA material for complementary DNA (cDNA) synthesis. Resulting cDNA material was pre-amplified prior to assay set up.…”

Section: Qpcr Analysesmentioning

confidence: 99%

“…Immunofluorescence staining for p/tMDM2 and p/TP53 was performed as before. 15 Tissue sections were blocked with 10% goat serum for 30 min and permeabilized with 2.5% Triton X-100 (ThermoScientific) to identify intracellular localization patterns. Tissue sections were then separately labeled with anti-MDM2 (1:200), anti-p53 antibody (1:200), anti-pMDM2 (1:200) and anti-pP53 antibody (1:200).…”

Section: Confocal Imagingmentioning

confidence: 99%

Sirtuin Inhibitor as a Novel Cell Cycle Checkpoint and Regulator of the TP53-MDM2 Pathway in Uveal Melanoma

Goldsmith¹,

McEwen²,

Kibe³

et al. 2019

Ophthalmol Open J.

Self Cite

View full text Add to dashboard Cite

Cite this articleGoldsmith ZK, McEwen MW, Kibe MW, et al. Sirtuin inhibitor as a novel cell cycle checkpoint and regulator of the TP53-MDM2 pathway in uveal melanoma. ABSTRACT PurposeThe liver is the most common site of uveal melanoma (UM) metastasis with approximately 50% of UM patients being affected. With no proven therapies that mitigate metastases the mortality rate is 85% within the first year after detection of the liver disease. In this study, we provide a mechanistic understanding of the de-regulation of the TP53-MDM2 pathway in UM, which plays a central role in tumor biology. MethodsWe investigated the TP53-MDM2 signaling pathway in the microenvironment of liver metastases taken from both a murine orthotopic xenograft and post-mortem metastatic UM human liver. These findings were studied in-depth using both primary and metastatic UM cell lines treated with the MDM2 antagonist Nutlin-3a and the sirtuin inhibitor and transcriptional activator of TP53, Tenovin-6. ResultsDe-regulation of the TP53-MDM2 signaling pathway is specific to the liver microenvironment, providing a survival mechanism for UM metastases. Tenovin-6, not Nutlin-3a, reduced UM cell survival by increasing the percentage of cell death and reducing the percentage of proliferating cells. Tenovin-6 increased acetylation of p53, reduced ubiquitination of the protein, and acted as a cell cycle regulator. ConclusionOur findings suggest that in patients with metastatic UM de-regulation of TP53-MDM2 signaling pathway promotes growth of the liver metastases and provides pre-clinical information on the potential of targeting of the TP53-MDM2 signaling pathway via Tenovin-6.

show abstract

Isolation and Molecular Profiling of Primary Mouse Retinal Ganglion Cells: Comparison of Phenotypes from Healthy and Glaucomatous Retinas

Cited by 29 publications

References 44 publications

Targeting the Platelet-Derived Growth Factor-beta Stimulatory Circuitry to Control Retinoblastoma Seeds

Targeting the Platelet-Derived Growth Factor-beta Stimulatory Circuitry to Control Retinoblastoma Seeds

Altered Energy Metabolism During Early Optic Nerve Crush Injury: Implications of Warburg-Like Aerobic Glycolysis in Facilitating Retinal Ganglion Cell Survival

Sirtuin Inhibitor as a Novel Cell Cycle Checkpoint and Regulator of the TP53-MDM2 Pathway in Uveal Melanoma

Contact Info

Product

Resources

About