Microarray analysis of gene expression in adult retinal ganglion cells

Ivanov, Dmitry; Dvoriantchikova, Galina; Nathanson, Lubov; McKinnon, Stuart J.; Shestopalov, Valery I.

doi:10.1016/j.febslet.2005.12.017

Cited by 57 publications

(47 citation statements)

References 31 publications

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“…These data greatly extend the single time point snapshots of gene expression we and others have published previously for RGCs (Farkas et al, 2004;Fischer et al, 2004;Ivanov et al, 2006) and allowed comparison with development surveys of gene expression in other CNS neurons, including striatal (Lobo et al, 2006) and cortical (Arlotta et al, 2005) projection neurons. Interestingly, it appears that the genes that are developmentally regulated in each of these three populations are almost entirely different.…”

Section: Discussionmentioning

confidence: 85%

Disease Gene Candidates Revealed by Expression Profiling of Retinal Ganglion Cell Development

Wang

Kunzevitzky²,

Dugas³

et al. 2007

J. Neurosci.

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To what extent do postmitotic neurons regulate gene expression during development or after injury? We took advantage of our ability to highly purify retinal ganglion cells (RGCs) to profile their pattern of gene expression at 13 ages from embryonic day 17 through postnatal day 21. We found that a large proportion of RGC genes are regulated dramatically throughout their postmitotic development, although the genes regulated through development in vivo generally are not regulated similarly by RGCs allowed to age in vitro. Interestingly, we found that genes regulated by developing RGCs are not generally correlated with genes regulated in RGCs stimulated to regenerate their axons. We unexpectedly found three genes associated with glaucoma, optineurin, cochlin, and CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide 1), previously thought to be primarily expressed in the trabecular meshwork, which are highly expressed by RGCs and regulated through their development. We also identified several other RGC genes that are encoded by loci linked to glaucoma. The expression of glaucoma-linked genes by RGCs suggests that, at least in some cases, RGCs may be directly involved in glaucoma pathogenesis rather than indirectly involved in response to increased intraocular pressure. Consistent with this hypothesis, we found that CYP1B1 overexpression potentiates RGC survival.

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Section: Discussionmentioning

confidence: 85%

Disease Gene Candidates Revealed by Expression Profiling of Retinal Ganglion Cell Development

Wang

Kunzevitzky²,

Dugas³

et al. 2007

J. Neurosci.

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“…Analysis of the FACS results showed a 2:1 stoichiometry in the yields of small (6-13 μm) vs large (15-25 μm) cells, which is consistent with the ratios reported by other investigators (Kashiwagi et al 2000). Relatively high yields of undegraded, high-quality RNA from both sub-populations were consistently obtained, for which the quality and cell type-specificity of the extracted RNA were determined by BioAnalyzer and quantitative RT-PCR (qRT-PCR) amplification of several single copy genes, including Thy1, Gfap, Cd11b, Cd68 as described earlier (Ivanov et al 2006). …”

Section: Quality Of Cell Purificationmentioning

confidence: 99%

Differential gene expression profiling of large and small retinal ganglion cells

Ivanov

Dvoriantchikova

Barakat

et al. 2008

Journal of Neuroscience Methods

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Different sub-populations of retinal ganglion cells (RGCs) vary in their sensitivity to pathological conditions such as retinal ischemia, diabetic retinopathy and glaucoma. Comparative Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. transcriptomic analysis of such groups will likely reveal molecular determinants of differential sensitivity to stress. However, gene expression profiling of primary neuronal sub-populations represent a challenge due to the cellular heterogeneity of retinal tissue. In this manuscript, we report the use of a fluorescent neural tracer to specifically label and selectively isolate RGCs with different soma sizes by fluorescence-activated cell sorting (FACS) for the purpose of differential gene expression profiling. We identified 145 genes that were more active in the large RGCs and 312 genes in the small RGCs. Differential data were validated by quantitative RT-PCR, several corresponding proteins were confirmed by immunohistochemistry. Functional characterization revealed differential activity of genes implicated in synaptic transmission, neurotransmitter secretion, axon guidance, chemotaxis, ion transport and tolerance to stress. An in silico reconstruction of cellular networks suggested that differences in pathway activity between the two sub-populations of RGCs are controlled by networks interconnected by SP-1, Erk2(MAPK1), Egr1, Egr2 and, potentially, regulated via transcription factors C/EBPbeta, HSF1, STAT1-and cMyc. The results show that FACS-aided purification of retrogradely labeled cells can be effectively utilized for transcriptional profiling of adult retinal neurons. NIH Public Access

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“…We analyzed only those genes that passed several Agilent-recommended quality control criteria, as described previously (17). To determine differential gene expression, the "naïve RGC-5/reference RNA" ratios were divided by the ratios of "silenced RGC-5/reference RNA."…”

Section: Methodsmentioning

confidence: 99%

“…RNA Amplification, Labeling, and Array HybridizationAmplification was performed using the Amino Allyl MessageAmp TM Kit (Ambion) that is configured to incorporate the modified nucleotide 5-(3-aminoallyl)-UTP (aaUTP) into the aRNA during in vitro transcription, as described previously (17). Prior to amplification, the RNA quality was tested in each sample using the NanoDrop and Agilent 2100 Bioanalyzer.…”

Section: Methodsmentioning

confidence: 99%

Effect of γ-Synuclein Silencing on Apoptotic Pathways in Retinal Ganglion Cells

Surgucheva

Shestopalov

Surguchov

2008

Journal of Biological Chemistry

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␥-Synuclein (Syn G) is highly expressed in retinal ganglion cells and the loss of these cells in glaucoma is associated with significant reduction of the intracellular Syn G level. However, a causative relationship between these two events has not been established. Here we show that the knockdown of Syn G results in a decreased viability of the immortalized retinal ganglion cells (RGC-5). The Syn G silencing reduces phosphorylation of serine 112 (Ser 112 ) in Bad protein, a member of the Bcl-2 family that plays a critical role in apoptotic cell death signaling. Our gene expression analysis data suggests that changes in Bad phosphorylation status may be caused by a coordinated shift in activities of kinases controlling Bad phosphorylation and phosphatases catalyzing its dephosphorylation. Moreover, increased phosphorylation of Bad-sequestering protein 14-3-3 detected in these cells is also pro-apoptotic. These results suggest that the homeostatic level of Syn G in RGC-5 cells is required for transcriptional regulation of protein kinases and phosphatases, controlling phosphorylation of Bad and 14-3-3. Lowering Syn G causes Bad dephosphorylation, dissociation from phosphorylated 14-3-3, and translocation to mitochondria where it initiates apoptotic death cascade.Glaucoma is a leading cause of irreversible world vision loss (1). This neuropathy is characterized by progressive damage of the optic nerve associated with a selective loss of the retinal ganglion cells (RGC) 2 (2-6). The precise mechanisms involved in glaucoma pathogenesis have yet to be determined, but a better understanding of the factors involved in ganglion cell death is central to the development of treatment of this neuropathy (7-9). It has been established that in glaucoma RGCs die by apoptosis (10) and a variety of key events in apoptosis focus on mitochondria, including the participation of pro-and antiapoptotic Bcl-2 family proteins (11, 12). These results imply that dysregulation of molecular mechanisms controlling mitochondrial apoptotic signaling may be important for the progression of glaucoma. In glaucoma progression, considerable changes in the transcriptome occur in the optic nerve (13), whole retina (14, 15), , trabecular meshwork cells (20, 21), and lymphocytes (22). However, the mechanisms controlling disease-induced changes in transcriptional regulation of mitochondrial apoptotic cascade in RGCs are not completely understood. Syn G is one of the genes that is highly expressed in RGC (16, 23, 24) and down-regulated in the course of glaucomatous alterations (25,26). The reduction of Syn G in RGC may have vital consequences for these cells, because Syn G is involved in cellular signaling and modulates the level of transcription of selected genes (27, 28). It is not clear whether the reduction of Syn G in RGC initiates the changes leading to glaucomatous alterations or it is just a result of the upstream biochemical processes that take place in glaucoma. The role of Syn G in the regulation of kinases and signaling pathways is well establi...

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Microarray analysis of gene expression in adult retinal ganglion cells

Cited by 57 publications

References 31 publications

Disease Gene Candidates Revealed by Expression Profiling of Retinal Ganglion Cell Development

Disease Gene Candidates Revealed by Expression Profiling of Retinal Ganglion Cell Development

Differential gene expression profiling of large and small retinal ganglion cells

Effect of γ-Synuclein Silencing on Apoptotic Pathways in Retinal Ganglion Cells

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