Developmental genome-wide occupancy analysis of bZIP transcription factor NRL uncovers the role of c-Jun in early differentiation of rod photoreceptors in the mammalian retina.

The basic motif-leucine zipper (bZIP) transcription factor neural retina leucine zipper (NRL) determines rod photoreceptor cell fate during retinal development, and its loss leads to cone-only retina in mice. NRL works synergistically with homeodomain protein Cone-Rod Homeobox and other regulatory factors to control the transcription of most genes associated with rod morphogenesis and functional maturation, which span over a period of several weeks in the mammalian retina. We predicted that NRL gradually establishes rod cell identity and function by temporal and dynamic regulation of stage-specific transcriptional targets. Therefore, we mapped the genomic occupancy of NRL at four stages of mouse photoreceptor differentiation by CUT&RUN analysis. Dynamics of NRL binding revealed concordance with the corresponding changes in transcriptome of the developing rods. Notably, we identified c-Jun proto-oncogene as one of the targets of NRL, which could bind to specific cis-elements in the c-Jun promoter and modulate its activity in HEK293 cells. Coimmunoprecipitation studies showed the association of NRL with c-Jun, also a bZIP protein, in transfected cells as well as in developing mouse retina. Additionally, shRNA-mediated knockdown of c-Jun in the mouse retina in vivo resulted in altered expression of almost 1000 genes, with reduced expression of phototransduction genes and many direct targets of NRL in rod photoreceptors. We propose that c-Jun-NRL heterodimers prime the NRL-directed transcriptional program in neonatal rod photoreceptors before high NRL expression suppresses c-Jun at later stages. Our study highlights a broader cooperation among cell-type restricted and widely expressed bZIP proteins, such as c-Jun, in specific spatiotemporal contexts during cellular differentiation.

Multiomics analyses reveal early metabolic imbalance and mitochondrial stress in neonatal photoreceptors leading to cell death in Pde6brd1/rd1 mouse model of retinal degeneration.

Retinal diseases exhibit extensive genetic heterogeneity and complex etiology with varying onset and severity. Mutations in over 200 genes can lead to photoreceptor dysfunction and/or cell death in retinal neurodegeneration. To deduce molecular pathways that initiate and/or drive cell death, we adopted a temporal multiomics approach and examined molecular and cellular events in newborn and developing photoreceptors before the onset of degeneration in a widely-used Pde6brd1/rd1 (rd1) mouse, a model of autosomal recessive retinitis pigmentosa caused by PDE6B mutations. Transcriptome profiling of neonatal and developing rods from the rd1 retina revealed early downregulation of genes associated with anabolic pathways and energy metabolism. Quantitative proteomics of rd1 retina showed early changes in calcium signaling and oxidative phosphorylation, with specific partial bypass of complex I electron transfer, which precede the onset of cell death. Concurrently, we detected alterations in central carbon metabolism, including dysregulation of components associated with glycolysis, pentose phosphate and purine biosynthesis. Ex vivo assays of oxygen consumption and transmission electron microscopy validated early and progressive mitochondrial stress and abnormalities in mitochondrial structure and function of rd1 rods. These data uncover mitochondrial overactivation and related metabolic alterations as determinants of early pathology and implicate aberrant calcium signaling as an initiator of higher mitochondrial stress. Our studies thus provide a mechanistic framework with mitochondrial damage and metabolic disruptions as early drivers of photoreceptor cell death in retinal degeneration.

Patient iPSC-derived neural stem cells exhibit phenotypes in concordance with the clinical severity of mucopolysaccharidosis I.

Mucopolysaccharidosis type I (MPS I) is caused by deficiency of α-l-iduronidase (IDUA), a lysosomal enzyme involved in the breakdown and recycling of glycosaminoglycans (GAGs). Although enzyme replacement therapy is available, the efficacy of the treatment for neuropathic manifestations is limited. To facilitate drug discovery and model disease pathophysiology, we generated neural stem cells (NSCs) from MPS I patient-derived induced pluripotent stem cells (iPSCs). The NSCs exhibited characteristic disease phenotypes with deficiency of IDUA, accumulation of GAGs and enlargement of lysosomes, in agreement with the severity of clinical subgroups of MPS I. Transcriptome profiling of NSCs revealed 429 genes that demonstrated a more extensive change in expression in the most severe Hurler syndrome subgroup compared to the intermediate Hurler-Scheie or the least severe Scheie syndrome subgroups. Clustering and pathway analysis revealed high concordance of the severity of neurological defects with marked dysregulation of GAG biosynthesis, GAG degradation, lysosomal function and autophagy. Gene ontology (GO) analysis identified a dramatic upregulation of the autophagy pathway, especially in the Hurler syndrome subgroup. We conclude that GAG accumulation in the patient-derived cells disrupts lysosomal homeostasis, affecting multiple related cellular pathways in response to IDUA deficiency. These dysregulated processes likely lead to enhanced autophagy and progressively severe disease states. Our study provides potentially useful targets for clinical biomarker development, disease diagnosis and prognosis, and drug discovery.

REEP6 mediates trafficking of a subset of Clathrin-coated vesicles and is critical for rod photoreceptor function and survival.

In retinal photoreceptors, vectorial transport of cargo is critical for transduction of visual signals, and defects in intracellular trafficking can lead to photoreceptor degeneration and vision impairment. Molecular signatures associated with routing of transport vesicles in photoreceptors are poorly understood. We previously reported the identification of a novel rod photoreceptor specific isoform of Receptor Expression Enhancing Protein (REEP) 6, which belongs to a family of proteins involved in intracellular transport of receptors to the plasma membrane. Here we show that loss of REEP6 in mice (Reep6-/-) results in progressive retinal degeneration. Rod photoreceptor dysfunction is observed in Reep6-/- mice as early as one month of age and associated with aberrant accumulation of vacuole-like structures at the apical inner segment and reduction in selected rod phototransduction proteins. We demonstrate that REEP6 is detected in a subset of Clathrin-coated vesicles and interacts with the t-SNARE, Syntaxin3. In concordance with the rod degeneration phenotype in Reep6-/- mice, whole exome sequencing identified homozygous REEP6-E75K mutation in two retinitis pigmentosa families of different ethnicities. Our studies suggest a critical function of REEP6 in trafficking of cargo via a subset of Clathrin-coated vesicles to selected membrane sites in retinal rod photoreceptors.

Expression of deubiquitinating enzyme genes in the developing mammal retina.

Purpose: Genes involved in the development and differentiation of the mammalian retina are also associated with inherited retinal dystrophies (IRDs) and age-related macular degeneration. Transcriptional regulation of retinal cell differentiation has been addressed by genetic and transcriptomic studies. Much less is known about the posttranslational regulation of key regulatory proteins, although mutations in some genes involved in ubiquitination and proteostasis-E3 ligases and deubiquitinating enzymes (DUBs)-cause IRDs. This study intends to provide new data on DUB gene expression during different developmental stages of mouse and human fetal retinas. Methods: We performed a comprehensive transcriptomic analysis of all the annotated human and mouse DUBs (87) in the developing mouse retina at several embryonic and postnatal time points compared with the transcriptome of the fetal human retina. An integrated comparison of data from transcriptomics, reported chromatin immunoprecipitation sequencing (ChIP-seq) of CRX and NRL transcription factors, and the phenotypic retinal alterations in different animal models is presented. Results: Several DUB genes are differentially expressed during the development of the mouse and human retinas in relation to proliferation or differentiation stages. Some DUB genes appear to be distinctly expressed during the differentiation stages of rod and cone photoreceptor cells, and their expression is altered in mouse knockout models of relevant photoreceptor transcription factors. We complemented this RNA-sequencing (RNA-seq) analysis with other reported expression and phenotypic data to underscore the involvement of DUBs in cell fate decision and photoreceptor differentiation. Conclusions: The present results highlight a short list of potential DUB candidates for retinal disorders, which require further study.

Transcriptome-based molecular staging of human stem cell-derived retinal organoids uncovers accelerated photoreceptor differentiation by 9-cis retinal.

PURPOSE: Retinal organoids generated from human pluripotent stem cells exhibit considerable variability during differentiation. Our goals are to assess developmental maturity of the neural retina in vitro and design improved protocols based on objective criteria. METHODS: We performed transcriptome analyses of developing retinal organoids from human embryonic and induced pluripotent stem cell lines and utilized multiple bioinformatic tools for comparative analysis. Immunohistochemistry, immunoblotting and electron microscopy were employed for validation. RESULTS: We show that the developmental variability in organoids was reflected in gene expression profiles and could be evaluated by molecular staging with the human fetal and adult retinal transcriptome data. We also demonstrate that the addition of 9-cis retinal, instead of the widely used all-trans retinoic acid, accelerated rod photoreceptor differentiation in organoid cultures, with higher rhodopsin expression and more mature mitochondrial morphology evident by day 120. CONCLUSION: Our studies provide an objective transcriptome-based modality for determining the differentiation state of retinal organoids and for comparisons across different stem cell lines and platforms, which should facilitate disease modeling and evaluation of therapies in vitro.

Transcriptome profiling of NIH3T3 cell lines expressing opsin and the P23H opsin mutant identifies candidate drugs for the treatment of retinitis pigmentosa.

Mammalian cells are commonly employed in screening assays to identify active compounds that could potentially affect the progression of different human diseases including retinitis pigmentosa (RP), a class of inherited diseases causing retinal degeneration with compromised vision. Using transcriptome analysis, we compared NIH3T3 cells expressing wildtype (WT) rod opsin with a retinal disease-causing single P23H mutation. Surprisingly, heterologous expression of WT opsin in NIH3T3 cells caused more than a 2-fold change in 783 out of 16,888 protein coding transcripts. The perturbed genes encoded extracellular matrix proteins, growth factors, cytoskeleton proteins, glycoproteins and metalloproteases involved in cell adhesion, morphology and migration. A different set of 347 transcripts was either up- or down-regulated when the P23H mutant opsin was expressed suggesting an altered molecular perturbation compared to WT opsin. Transcriptome perturbations elicited by drug candidates aimed at mitigating the effects of the mutant protein revealed that different drugs targeted distinct molecular pathways that resulted in a similar phenotype selected by a cell-based high-throughput screen. Thus, transcriptome profiling can provide essential information about the therapeutic potential of a candidate drug to restore normal gene expression in pathological conditions.

The cellular and compartmental profile of mouse retinal glycolysis, tricarboxylic acid cycle, oxidative phosphorylation, and ~P transferring kinases.

PURPOSE: The homeostatic regulation of cellular ATP is achieved by the coordinated activity of ATP utilization, synthesis, and buffering. Glucose is the major substrate for ATP synthesis through glycolysis and oxidative phosphorylation (OXPHOS), whereas intermediary metabolism through the tricarboxylic acid (TCA) cycle utilizes non-glucose-derived monocarboxylates, amino acids, and alpha ketoacids to support mitochondrial ATP and GTP synthesis. Cellular ATP is buffered by specialized equilibrium-driven high-energy phosphate (~P) transferring kinases. Our goals were twofold: 1) to characterize the gene expression, protein expression, and activity of key synthesizing and regulating enzymes of energy metabolism in the whole mouse retina, retinal compartments, and/or cells and 2) to provide an integrative analysis of the results related to function. METHODS: mRNA expression data of energy-related genes were extracted from our whole retinal Affymetrix microarray data. Fixed-frozen retinas from adult C57BL/6N mice were used for immunohistochemistry, laser scanning confocal microscopy, and enzymatic histochemistry. The immunoreactivity levels of well-characterized antibodies, for all major retinal cells and their compartments, were obtained using our established semiquantitative confocal and imaging techniques. Quantitative cytochrome oxidase (COX) and lactate dehydrogenase (LDH) activity was determined histochemically. RESULTS: The Affymetrix data revealed varied gene expression patterns of the ATP synthesizing and regulating enzymes found in the muscle, liver, and brain. Confocal studies showed differential cellular and compartmental distribution of isozymes involved in glucose, glutamate, glutamine, lactate, and creatine metabolism. The pattern and intensity of the antibodies and of the COX and LDH activity showed the high capacity of photoreceptors for aerobic glycolysis and OXPHOS. Competition assays with pyruvate revealed that LDH-5 was localized in the photoreceptor inner segments. The combined results indicate that glycolysis is regulated by the compartmental expression of hexokinase 2, pyruvate kinase M1, and pyruvate kinase M2 in photoreceptors, whereas the inner retinal neurons exhibit a lower capacity for glycolysis and aerobic glycolysis. Expression of nucleoside diphosphate kinase, mitochondria-associated adenylate kinase, and several mitochondria-associated creatine kinase isozymes was highest in the outer retina, whereas expression of cytosolic adenylate kinase and brain creatine kinase was higher in the cones, horizontal cells, and amacrine cells indicating the diversity of ATP-buffering strategies among retinal neurons. Based on the antibody intensities and the COX and LDH activity, Müller glial cells (MGCs) had the lowest capacity for glycolysis, aerobic glycolysis, and OXPHOS. However, they showed high expression of glutamate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate thiokinase, GABA transaminase, and ~P transferring kinases. This suggests that MGCs utilize TCA cycle anaplerosis and cataplerosis to generate GTP and ~P transferring kinases to produce ATP that supports MGC energy requirements. CONCLUSIONS: Our comprehensive and integrated results reveal that the adult mouse retina expresses numerous isoforms of ATP synthesizing, regulating, and buffering genes; expresses differential cellular and compartmental levels of glycolytic, OXPHOS, TCA cycle, and ~P transferring kinase proteins; and exhibits differential layer-by-layer LDH and COX activity. New insights into cell-specific and compartmental ATP and GTP production, as well as utilization and buffering strategies and their relationship with known retinal and cellular functions, are discussed. Developing therapeutic strategies for neuroprotection and treating retinal deficits and degeneration in a cell-specific manner will require such knowledge. This work provides a platform for future research directed at identifying the molecular targets and proteins that regulate these processes.

Loss of lysophosphatidylcholine acyltransferase 1 leads to photoreceptor degeneration in rd11 mice.

Retinal degenerative diseases, such as retinitis pigmentosa and Leber congenital amaurosis, are a leading cause of untreatable blindness with substantive impact on the quality of life of affected individuals and their families. Mouse mutants with retinal dystrophies have provided a valuable resource to discover human disease genes and helped uncover pathways critical for photoreceptor function. Here we show that the rd11 mouse mutant and its allelic strain, B6-JR2845, exhibit rapid photoreceptor dysfunction, followed by degeneration of both rods and cones. Using linkage analysis, we mapped the rd11 locus to mouse chromosome 13. We then identified a one-nucleotide insertion (c.420-421insG) in exon 3 of the Lpcat1 gene. Subsequent screening of this gene in the B6-JR2845 strain revealed a seven-nucleotide deletion (c.14-20delGCCGCGG) in exon 1. Both sequence changes are predicted to result in a frame-shift, leading to premature truncation of the lysophosphatidylcholine acyltransferase-1 (LPCAT1) protein. LPCAT1 (also called AYTL2) is a phospholipid biosynthesis/remodeling enzyme that facilitates the conversion of palmitoyl-lysophosphatidylcholine to dipalmitoylphosphatidylcholine (DPPC). The analysis of retinal lipids from rd11 and B6-JR2845 mice showed substantially reduced DPPC levels compared with C57BL/6J control mice, suggesting a causal link to photoreceptor dysfunction. A follow-up screening of LPCAT1 in retinitis pigmentosa and Leber congenital amaurosis patients did not reveal any obvious disease-causing mutations. Previously, LPCAT1 has been suggested to be critical for the production of lung surfactant phospholipids and biosynthesis of platelet-activating factor in noninflammatory remodeling pathway. Our studies add another dimension to an essential role for LPCAT1 in retinal photoreceptor homeostasis.

Rapid RGR-dependent visual pigment recycling is mediated by the RPE and specialized Müller glia.

In daylight, demand for visual chromophore (11-cis-retinal) exceeds supply by the classical visual cycle. This shortfall is compensated, in part, by the retinal G-protein-coupled receptor (RGR) photoisomerase, which is expressed in both the retinal pigment epithelium (RPE) and in Müller cells. The relative contributions of these two cellular pools of RGR to the maintenance of photoreceptor light responses are not known. Here, we use a cell-specific gene reactivation approach to elucidate the kinetics of RGR-mediated recovery of photoreceptor responses following light exposure. Electroretinographic measurements in mice with RGR expression limited to either cell type reveal that the RPE and a specialized subset of Müller glia contribute both to scotopic and photopic function. We demonstrate that 11-cis-retinal formed through photoisomerization is rapidly hydrolyzed, consistent with its role in a rapid visual pigment regeneration process. Our study shows that RGR provides a pan-retinal sink for all-trans-retinal released under sustained light conditions and supports rapid chromophore regeneration through the photic visual cycle.

Mutations in a BTB-Kelch protein, KLHL7, cause autosomal-dominant retinitis pigmentosa.

Retinitis pigmentosa (RP) refers to a genetically heterogeneous group of progressive neurodegenerative diseases that result in dysfunction and/or death of rod and cone photoreceptors in the retina. So far, 18 genes have been identified for autosomal-dominant (ad) RP. Here, we describe an adRP locus (RP42) at chromosome 7p15 through linkage analysis in a six-generation Scandinavian family and identify a disease-causing mutation, c.449G-->A (p.S150N), in exon 6 of the KLHL7 gene. Mutation screening of KLHL7 in 502 retinopathy probands has revealed three different missense mutations in six independent families. KLHL7 is widely expressed, including expression in rod photoreceptors, and encodes a 75 kDa protein of the BTB-Kelch subfamily within the BTB superfamily. BTB-Kelch proteins have been implicated in ubiquitination through Cullin E3 ligases. Notably, all three putative disease-causing KLHL7 mutations are within a conserved BACK domain; homology modeling suggests that mutant amino acid side chains can potentially fill the cleft between two helices, thereby affecting the ubiquitination complexes. Mutations in an identical region of another BTB-Kelch protein, gigaxonin, have previously been associated with giant axonal neuropathy. Our studies suggest an additional role of the ubiquitin-proteasome protein-degradation pathway in maintaining neuronal health and in disease.

A role for prenylated rab acceptor 1 in vertebrate photoreceptor development.

BACKGROUND: The rd1 mouse retina is a well-studied model of retinal degeneration where rod photoreceptors undergo cell death beginning at postnatal day (P) 10 until P21. This period coincides with photoreceptor terminal differentiation in a normal retina. We have used the rd1 retina as a model to investigate early molecular defects in developing rod photoreceptors prior to the onset of degeneration. RESULTS: Using a microarray approach, we performed gene profiling comparing rd1 and wild type (wt) retinas at four time points starting at P2, prior to any obvious biochemical or morphological differences, and concluding at P8, prior to the initiation of cell death. Of the 143 identified differentially expressed genes, we focused on Rab acceptor 1 (Rabac1), which codes for the protein Prenylated rab acceptor 1 (PRA1) and plays an important role in vesicular trafficking. Quantitative RT-PCR analysis confirmed reduced expression of PRA1 in rd1 retina at all time points examined. Immunohistochemical observation showed that PRA1-like immunoreactivity (LIR) co-localized with the cis-Golgi marker GM-130 in the photoreceptor as the Golgi translocated from the perikarya to the inner segment during photoreceptor differentiation in wt retinas. Diffuse PRA1-LIR, distinct from the Golgi marker, was seen in the distal inner segment of wt photoreceptors starting at P8. Both plexiform layers contained PRA1 positive punctae independent of GM-130 staining during postnatal development. In the inner retina, PRA1-LIR also colocalized with the Golgi marker in the perinuclear region of most cells. A similar pattern was seen in the rd1 mouse inner retina. However, punctate and significantly reduced PRA1-LIR was present throughout the developing rd1 inner segment, consistent with delayed photoreceptor development and abnormalities in Golgi sorting and vesicular trafficking. CONCLUSIONS: We have identified genes that are differentially regulated in the rd1 retina at early time points, which may give insights into developmental defects that precede photoreceptor cell death. This is the first report of PRA1 expression in the retina. Our data support the hypothesis that PRA1 plays an important role in vesicular trafficking between the Golgi and cilia in differentiating and mature rod photoreceptors.

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

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.

Wellness Through the Lens of a Medical Orchestra.

Stress and burnout afflict medical students and professionals at alarming rates, which has led institutions to invest in counseling services and other traditional wellness programming. However, the stigma of utilizing these services permeates the medical community. This narrative explores the founding of the Nebraska Medical Orchestra-an orchestra created as a nontraditional antidote to reduce stress and burnout among health care students and professionals-and also examines the concept of wellness through interactions between the orchestra's director and health care-related musicians.

How a Medical Orchestra Cultivates Creativity, Joy, Empathy, and Connection.

Background: Inspired by research indicating that exposure to humanities correlates with reduced burnout, the Nebraska Medical Orchestra was founded in 2018 as a collaboration between the University of Nebraska Medical Center and the University of Nebraska at Omaha School of Music. Methods: Semistructured interviews about orchestra participants' experiences were conducted with 9 musicians and recorded and transcribed. Transcripts were analyzed using the constant comparative method. Results: The interviews suggested that participants are drawn to the orchestra to pursue a love of music, to be part of an ensemble, and to connect with others in an environment that provides a lighthearted, nonjudgmental, noncompetitive forum in which to create and enjoy music for its own sake. Conclusions: This study has implications for designing arts-based wellness activities for clinicians and scaling them nationwide.

Next-generation sequencing facilitates quantitative analysis of wild-type and Nrl(-/-) retinal transcriptomes.

PURPOSE: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived retinal transcriptome profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRT-PCR) methods and to evaluate protocols for optimal high-throughput data analysis. METHODS: Retinal mRNA profiles of 21-day-old wild-type (WT) and neural retina leucine zipper knockout (Nrl(-/-)) mice were generated by deep sequencing, in triplicate, using Illumina GAIIx. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows-Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. qRT-PCR validation was performed using TaqMan and SYBR Green assays. RESULTS: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm9) and identified 16,014 transcripts in the retinas of WT and Nrl(-/-) mice with BWA workflow and 34,115 transcripts with TopHat workflow. RNA-seq data confirmed stable expression of 25 known housekeeping genes, and 12 of these were validated with qRT-PCR. RNA-seq data had a linear relationship with qRT-PCR for more than four orders of magnitude and a goodness of fit (R(2)) of 0.8798. Approximately 10% of the transcripts showed differential expression between the WT and Nrl(-/-) retina, with a fold change ≥1.5 and p value <0.05. Altered expression of 25 genes was confirmed with qRT-PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Hierarchical clustering of differentially expressed genes uncovered several as yet uncharacterized genes that may contribute to retinal function. Data analysis with BWA and TopHat workflows revealed a significant overlap yet provided complementary insights in transcriptome profiling. CONCLUSIONS: Our study represents the first detailed analysis of retinal transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.

Genetic Rescue of X-Linked Retinoschisis Mouse (Rs1-/y) Retina Induces Quiescence of the Retinal Microglial Inflammatory State Following AAV8-RS1 Gene Transfer and Identifies Gene Networks Underlying Retinal Recovery.

To understand RS1 gene interaction networks in the X-linked retinoschisis (XLRS) mouse retina (Rs1-/y), we analyzed the transcriptome by RNA sequencing before and after in vivo expression of exogenous retinoschisin (RS1) gene delivered by AAV8. RS1 is a secreted cell adhesion protein that is critical for maintaining structural lamination and synaptic integrity of the neural retina. RS1 loss-of-function mutations cause XLRS disease in young boys and men, with splitting ("schisis") of retinal layers and synaptic dysfunction that cause progressive vision loss with age. Analysis of differential gene expression profiles and pathway enrichment analysis of Rs1-KO (Rs1-/y) retina identified cell surface receptor signaling and positive regulation of cell adhesion as potential RS1 gene interaction networks. Most importantly, it also showed massive dysregulation of immune response genes at early age, with characteristics of a microglia-driven proinflammatory state. Delivery of AAV8-RS1 primed the Rs1-KO retina toward structural and functional recovery. The disease transcriptome transitioned toward a recovery phase with upregulation of genes implicated in wound healing, anatomical structure (camera type eye) development, metabolic pathways, and collagen IV networks that provide mechanical stability to basement membrane. AAV8-RS1 expression also attenuated the microglia gene signatures to low levels toward immune quiescence. This study is among the first to identify RS1 gene interaction networks that underlie retinal structural and functional recovery after RS1 gene therapy. Significantly, it also shows that providing wild-type RS1 gene function caused the retina immune status to transition from a degenerative inflammatory phenotype toward immune quiescence, even though the transgene is not directly linked to microglia function. This study indicates that inhibition of microglial proinflammatory responses is an integral part of therapeutic rescue in XLRS gene therapy, and gene therapy might realize its full potential if delivered before microglia activation and photoreceptor cell death. Clinical Trials. gov Identifier NTC 02317887.

Proneural genes define ground-state rules to regulate neurogenic patterning and cortical folding.

Asymmetric neuronal expansion is thought to drive evolutionary transitions between lissencephalic and gyrencephalic cerebral cortices. We report that Neurog2 and Ascl1 proneural genes together sustain neurogenic continuity and lissencephaly in rodent cortices. Using transgenic reporter mice and human cerebral organoids, we found that Neurog2 and Ascl1 expression defines a continuum of four lineage-biased neural progenitor cell (NPC) pools. Double+ NPCs, at the hierarchical apex, are least lineage restricted due to Neurog2-Ascl1 cross-repression and display unique features of multipotency (more open chromatin, complex gene regulatory network, G2 pausing). Strikingly, selectively eliminating double+ NPCs by crossing Neurog2-Ascl1 split-Cre mice with diphtheria toxin-dependent "deleter" strains locally disrupts Notch signaling, perturbs neurogenic symmetry, and triggers cortical folding. In support of our discovery that double+ NPCs are Notch-ligand-expressing "niche" cells that control neurogenic periodicity and cortical folding, NEUROG2, ASCL1, and HES1 transcript distribution is modular (adjacent high/low zones) in gyrencephalic macaque cortices, prefiguring future folds.

Retinal pigment epithelium transcriptome analysis in chronic smoking reveals a suppressed innate immune response and activation of differentiation pathways.

Cigarette smoking, a powerful mixture of chemical oxidants, is the strongest environmental risk factor for developing age-related macular degeneration (AMD), the most common cause of blindness among the elderly in western societies. Despite intensive study, the full impact of smoking on the retinal pigment epithelium (RPE), a central cell type involved in AMD pathobiology, remains unknown. The relative contribution of the known dysfunctional pathways to AMD, at what stage they are most pathogenic, or whether other processes are relevant, is poorly understood, and furthermore, whether smoking activates them, is unknown. We performed global RNA-sequencing of the RPE from C57BL/6J mice exposed to chronic cigarette smoke for 6 months to identify potential pathogenic and cytoprotective pathways. The RPE transcriptome induced by chronic cigarette smoking exhibited a mixed response of marked suppression of the innate immune response including type I and II interferons and upregulation of cell differentiation and morphogenic gene clusters, suggesting an attempt by the RPE to maintain its differentiated state despite smoke-induced injury. Given that mice exposed to chronic smoke develop early features of AMD, these novel findings are potentially relevant to the transition from aging to AMD.