Limits...
Transcriptome analysis using next generation sequencing reveals molecular signatures of diabetic retinopathy and efficacy of candidate drugs.

Kandpal RP, Rajasimha HK, Brooks MJ, Nellissery J, Wan J, Qian J, Kern TS, Swaroop A - Mol. Vis. (2012)

Bottom Line: These two therapies also showed dissimilar regulation of some subsets of transcripts that included alternatively spliced versions of arrestin, neutral sphingomyelinase activation associated factor (Nsmaf), SH3-domain GRB2-like interacting protein 1 (Sgip1), and axin.Diabetes alters many transcripts in the retina, and two therapies that inhibit the vascular pathology similarly inhibit a portion of these changes, pointing to possible molecular mechanisms for their beneficial effects.Our studies clearly demonstrate RNA-seq as a comprehensive strategy for identifying disease-specific transcripts, and for determining comparative profiles of molecular changes mediated by candidate drugs.

View Article: PubMed Central - PubMed

Affiliation: Neurobiology-Neurodegeneration & Repair Laboratory, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA. rkandpal@westernu.edu

ABSTRACT

Purpose: To define gene expression changes associated with diabetic retinopathy in a mouse model using next generation sequencing, and to utilize transcriptome signatures to assess molecular pathways by which pharmacological agents inhibit diabetic retinopathy.

Methods: We applied a high throughput RNA sequencing (RNA-seq) strategy using Illumina GAIIx to characterize the entire retinal transcriptome from nondiabetic and from streptozotocin-treated mice 32 weeks after induction of diabetes. Some of the diabetic mice were treated with inhibitors of receptor for advanced glycation endproducts (RAGE) and p38 mitogen activated protein (MAP) kinase, which have previously been shown to inhibit diabetic retinopathy in rodent models. The transcripts and alternatively spliced variants were determined in all experimental groups.

Results: Next generation sequencing-based RNA-seq profiles provided comprehensive signatures of transcripts that are altered in early stages of diabetic retinopathy. These transcripts encoded proteins involved in distinct yet physiologically relevant disease-associated pathways such as inflammation, microvasculature formation, apoptosis, glucose metabolism, Wnt signaling, xenobiotic metabolism, and photoreceptor biology. Significant upregulation of crystallin transcripts was observed in diabetic animals, and the diabetes-induced upregulation of these transcripts was inhibited in diabetic animals treated with inhibitors of either RAGE or p38 MAP kinase. These two therapies also showed dissimilar regulation of some subsets of transcripts that included alternatively spliced versions of arrestin, neutral sphingomyelinase activation associated factor (Nsmaf), SH3-domain GRB2-like interacting protein 1 (Sgip1), and axin.

Conclusions: Diabetes alters many transcripts in the retina, and two therapies that inhibit the vascular pathology similarly inhibit a portion of these changes, pointing to possible molecular mechanisms for their beneficial effects. These therapies also changed the abundance of various alternatively spliced versions of signaling transcripts, suggesting a possible role of alternative splicing in disease etiology. Our studies clearly demonstrate RNA-seq as a comprehensive strategy for identifying disease-specific transcripts, and for determining comparative profiles of molecular changes mediated by candidate drugs.

Show MeSH

Related in: MedlinePlus

Unique and common splice forms of transcripts are present in various groups of animals. The splice variants from various samples were compared and the numbers of transcripts unique to a specific experimental pair or common to two or more experimental pairs are indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3351417&req=5

f4: Unique and common splice forms of transcripts are present in various groups of animals. The splice variants from various samples were compared and the numbers of transcripts unique to a specific experimental pair or common to two or more experimental pairs are indicated.

Mentions: Similar analyses were also performed with the data related to alternative splicing (Figure 4). Comparison of diabetic versus nondiabetic, RAGE inhibitor-treated diabetic versus nondiabetic and p38 MAPK inhibitor-treated diabetic versus nondiabetic animals led to the identification of alternatively spliced transcripts specific to each group of animals (Table 11, Table 12, and Appendix 2). Some notable alternatively spliced transcripts in diabetic animals included doublecortin-like kinase Dclk3, death effector domain Dedd2, dynein Dync1li2, interphotoreceptor matrix protein Impg2, ankyrin 2, arrestin 3, coiled-coil domain containing Ccdc64, G-protein coupled receptor Gprasp2, Nsmaf, dynactin 6, and whirlin (Table 11). Among the transcripts responsive to RAGE inhibitor were photoreceptor specific gene arrestin, Nsmaf transcript involved in apoptosis, and endocytosis transcript Sgip1. The exacerbated levels of 76 alternatively spliced transcripts in p38 MAPK inhibitor-treated animals included the Wnt pathway transcript Axin1 and its target gene Cldn1, opsin transcript Opnm1w, Mapk8ip3, peroxisomal transcript Pex16, cilia transcript Cep164, and ocular regulator Pax6 (Table 12 and Appendix 2). A comparison of alternatively spliced transcripts revealed 12 transcripts common among nondiabetic animals, diabetic animals, diabetic animals treated with RAGE inhibitor, and diabetic animals treated with p38 MAPK inhibitor (Figure 4). Among others, these genes included G-protein coupled receptor associated sorting protein, kinesin family member 1A, nuclear receptor co-repressor, phospholipase A2, and zinc finger protein 444. Although the summary presented in Figure 4 does not allow us to decipher the mechanisms of these therapies, the results suggest that the changes in splicing patterns in response to therapy may reflect the positions of p38 MAP kinase and RAGE in the cell signaling pathways relative to each other.


Transcriptome analysis using next generation sequencing reveals molecular signatures of diabetic retinopathy and efficacy of candidate drugs.

Kandpal RP, Rajasimha HK, Brooks MJ, Nellissery J, Wan J, Qian J, Kern TS, Swaroop A - Mol. Vis. (2012)

Unique and common splice forms of transcripts are present in various groups of animals. The splice variants from various samples were compared and the numbers of transcripts unique to a specific experimental pair or common to two or more experimental pairs are indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351417&req=5

f4: Unique and common splice forms of transcripts are present in various groups of animals. The splice variants from various samples were compared and the numbers of transcripts unique to a specific experimental pair or common to two or more experimental pairs are indicated.
Mentions: Similar analyses were also performed with the data related to alternative splicing (Figure 4). Comparison of diabetic versus nondiabetic, RAGE inhibitor-treated diabetic versus nondiabetic and p38 MAPK inhibitor-treated diabetic versus nondiabetic animals led to the identification of alternatively spliced transcripts specific to each group of animals (Table 11, Table 12, and Appendix 2). Some notable alternatively spliced transcripts in diabetic animals included doublecortin-like kinase Dclk3, death effector domain Dedd2, dynein Dync1li2, interphotoreceptor matrix protein Impg2, ankyrin 2, arrestin 3, coiled-coil domain containing Ccdc64, G-protein coupled receptor Gprasp2, Nsmaf, dynactin 6, and whirlin (Table 11). Among the transcripts responsive to RAGE inhibitor were photoreceptor specific gene arrestin, Nsmaf transcript involved in apoptosis, and endocytosis transcript Sgip1. The exacerbated levels of 76 alternatively spliced transcripts in p38 MAPK inhibitor-treated animals included the Wnt pathway transcript Axin1 and its target gene Cldn1, opsin transcript Opnm1w, Mapk8ip3, peroxisomal transcript Pex16, cilia transcript Cep164, and ocular regulator Pax6 (Table 12 and Appendix 2). A comparison of alternatively spliced transcripts revealed 12 transcripts common among nondiabetic animals, diabetic animals, diabetic animals treated with RAGE inhibitor, and diabetic animals treated with p38 MAPK inhibitor (Figure 4). Among others, these genes included G-protein coupled receptor associated sorting protein, kinesin family member 1A, nuclear receptor co-repressor, phospholipase A2, and zinc finger protein 444. Although the summary presented in Figure 4 does not allow us to decipher the mechanisms of these therapies, the results suggest that the changes in splicing patterns in response to therapy may reflect the positions of p38 MAP kinase and RAGE in the cell signaling pathways relative to each other.

Bottom Line: These two therapies also showed dissimilar regulation of some subsets of transcripts that included alternatively spliced versions of arrestin, neutral sphingomyelinase activation associated factor (Nsmaf), SH3-domain GRB2-like interacting protein 1 (Sgip1), and axin.Diabetes alters many transcripts in the retina, and two therapies that inhibit the vascular pathology similarly inhibit a portion of these changes, pointing to possible molecular mechanisms for their beneficial effects.Our studies clearly demonstrate RNA-seq as a comprehensive strategy for identifying disease-specific transcripts, and for determining comparative profiles of molecular changes mediated by candidate drugs.

View Article: PubMed Central - PubMed

Affiliation: Neurobiology-Neurodegeneration & Repair Laboratory, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA. rkandpal@westernu.edu

ABSTRACT

Purpose: To define gene expression changes associated with diabetic retinopathy in a mouse model using next generation sequencing, and to utilize transcriptome signatures to assess molecular pathways by which pharmacological agents inhibit diabetic retinopathy.

Methods: We applied a high throughput RNA sequencing (RNA-seq) strategy using Illumina GAIIx to characterize the entire retinal transcriptome from nondiabetic and from streptozotocin-treated mice 32 weeks after induction of diabetes. Some of the diabetic mice were treated with inhibitors of receptor for advanced glycation endproducts (RAGE) and p38 mitogen activated protein (MAP) kinase, which have previously been shown to inhibit diabetic retinopathy in rodent models. The transcripts and alternatively spliced variants were determined in all experimental groups.

Results: Next generation sequencing-based RNA-seq profiles provided comprehensive signatures of transcripts that are altered in early stages of diabetic retinopathy. These transcripts encoded proteins involved in distinct yet physiologically relevant disease-associated pathways such as inflammation, microvasculature formation, apoptosis, glucose metabolism, Wnt signaling, xenobiotic metabolism, and photoreceptor biology. Significant upregulation of crystallin transcripts was observed in diabetic animals, and the diabetes-induced upregulation of these transcripts was inhibited in diabetic animals treated with inhibitors of either RAGE or p38 MAP kinase. These two therapies also showed dissimilar regulation of some subsets of transcripts that included alternatively spliced versions of arrestin, neutral sphingomyelinase activation associated factor (Nsmaf), SH3-domain GRB2-like interacting protein 1 (Sgip1), and axin.

Conclusions: Diabetes alters many transcripts in the retina, and two therapies that inhibit the vascular pathology similarly inhibit a portion of these changes, pointing to possible molecular mechanisms for their beneficial effects. These therapies also changed the abundance of various alternatively spliced versions of signaling transcripts, suggesting a possible role of alternative splicing in disease etiology. Our studies clearly demonstrate RNA-seq as a comprehensive strategy for identifying disease-specific transcripts, and for determining comparative profiles of molecular changes mediated by candidate drugs.

Show MeSH
Related in: MedlinePlus