Shunbin Xu

MicroRNA (miRNA) Transcriptome of Mouse Retina and Identification of a Sensory Organ-specific miRNA Cluster

Although microRNAs (miRNAs) provide a newly recognized level of regulation of gene expression, the miRNA transcriptome of the retina and the contributions of miRNAs to retinal development and function are largely unknown. To begin to understand the functions of miRNAs in retina, we compared miRNA expression profiles in adult mouse retina, brain, and heart by microarray analysis. Our results show that at least 78 miRNAs are expressed in adult mouse retina, 21 of which are potentially retina-specific. Among these, we identified a polycistronic, sensory organ-specific paralogous miRNA cluster that includes miR-96, miR-182, and miR-183 on mouse chromosome 6qA3 with conservation of synteny to human chromosome 7q32.2. In situ hybridization showed that members of this cluster are expressed in photoreceptors, retinal bipolar and amacrine cells. Consistent with their genomic organization, these miRNAs have a similar expression pattern during development with abundance increasing postnatally and peaking in adult retina. Target prediction and in vitro functional studies showed that MITF, a transcription factor required for the establishment and maintenance of retinal pigmented epithelium, is a direct target of miR-96 and miR-182. Additionally, to identify miRNAs potentially involved in circadian rhythm regulation of the retina, we performed miRNA expression profiling with retinal RNA harvested at noon (Zeitgeber time 5) and midnight (Zeitgeber time 17) and identified a subgroup of 12 miRNAs, including members of the miR-183/96/182 cluster with diurnal variation in expression pattern. Our results suggest that miR-96 and miR-182 are involved in circadian rhythm regulation, perhaps by modulating the expression of adenylyl cyclase VI (ADCY6).

Embryonic stem cells assume a primitive neural stem cell fate in the absence of extrinsic influences

The mechanisms governing the emergence of the earliest mammalian neural cells during development remain incompletely characterized. A default mechanism has been suggested to underlie neural fate acquisition; however, an instructive process has also been proposed. We used mouse embryonic stem (ES) cells to explore the fundamental issue of how an uncommitted, pluripotent mammalian cell will self-organize in the absence of extrinsic signals and what cellular fate will result. To assess this default state, ES cells were placed in conditions that minimize external influences. Individual ES cells were found to rapidly transition directly into neural cells, a process shown to be independent of suggested instructive factors (e.g., fibroblast growth factors). Further, we provide evidence that the default neural identity is that of a primitive neural stem cell (NSC). The exiguous conditions used to reveal the default state were found to present primitive NSCs with a survival challenge (limiting their persistence and proliferation), which could be mitigated by survival factors or genetic interference with apoptosis.

microRNAs in Early Diabetic Retinopathy in Streptozotocin-Induced Diabetic Rats

PURPOSE Diabetic retinopathy (DR) is one of the leading causes of blindness. However, the roles of microRNAs (miRNAs) in DR are still unknown. The aims of this study were to identify miRNAs involved in early DR and to characterize their roles in the pathogenesis of DR. METHODS miRNA-expression profiling was performed in the retina and retinal endothelial cells (RECs) of streptozotocin (STZ)-induced diabetic rats 3 months after the onset of diabetes and miRNAs differentially expressed in diabetic rats were identified and compared with controls. Subsequently, functional annotation analysis was conducted to identify miRNA signatures of pathologic pathways of DR. In addition, in vitro functional assays were used to dissect interactions of miR-146 and NF-κB activation in a conditionally immortalized retinal capillary endothelial cell line, Tr-iBRB. RESULTS Approximately 350 and 220 miRNAs were detected in the retinas and RECs, respectively, in both control and diabetic rats. At least 86 and 120 miRNAs were differentially expressed (P < 0.01) in the retinas and RECs of diabetic rats and controls, respectively. Upregulation of NF-κB-, VEGF-, and p53-responsive miRNAs constituted key miRNA signatures, reflecting ongoing pathologic changes of early DR. In addition, it was demonstrated that the negative feedback regulation of miR-146 on NF-κB activation may function in Tr-iBRB endothelial cells, suggesting that miR-146 is a potential therapeutic target for the treatment of DR through its inhibition on NF-κB activation in RECs. CONCLUSIONS miRNAs are involved in the pathogenesis of DR through the modulation of multiple pathogenetic pathways and may be novel therapeutic targets for the treatment of DR.

β-Amyloid Deposition and Functional Impairment in the Retina of the APPswe/PS1ΔE9 Transgenic Mouse Model of Alzheimer’s Disease

PURPOSE To determine whether beta-amyloid (Abeta) deposition affects the structure and function of the retina of the APPswe/PS1DeltaE9 transgenic (tg) mouse model of Alzheimers disease. METHODS Retinas from 12- to 19-month old APPswe/PS1DeltaE9 tg and age-matched non-transgenic (ntg) littermates were single or double stained with thioflavine-S and antibodies against Abeta, glial fibrillar acidic protein (GFAP), microglial marker F4/80, choline acetyltransferase (ChAT), and syntaxin 1. Quantification of thioflavine-S positive plaques and retinal layer thickness was analyzed semi-quantitatively, whereas microglial cell size and levels of F4/80 immunoreactivity were evaluated using a densitometry program. Scotopic electroretinogram (ERG) recording was used to investigate retinal physiology in these mice. RESULTS Thioflavine-S positive plaques appeared at 12 months in the retinas of APPswe/PS1DeltaE9 tg mice with the majority of plaques in the outer and inner plexiform layers. Plaques were embedded in the inner plexiform layer strata displaying syntaxin 1 and ChAT. The number and size of the plaques in the retina increased with age. Plaques appeared earlier and in greater numbers in females than in male tg littermate mice. Microglial activity was significantly increased in the retinas of APPswe/PS1DeltaE9 tg mice. Although we did not detect neuronal degeneration in the retina, ERG recordings revealed a significant reduction in the amplitudes of a- and b-waves in aged APPswe/PS1DeltaE9 tg compared to ntg littermates. CONCLUSIONS The present findings suggest that Abeta deposition disrupts retinal structure and may contribute to the visual deficits seen in aged APPswe/PS1DeltaE9 tg mice. Whether Abeta is involved in other forms of age-related retinal dysfunction is unclear.

Inactivation of the microRNA-183/96/182 cluster results in syndromic retinal degeneration

The microRNA-183/96/182 cluster is highly expressed in the retina and other sensory organs. To uncover its in vivo functions in the retina, we generated a knockout mouse model, designated “miR-183CGT/GT,” using a gene-trap embryonic stem cell clone. We provide evidence that inactivation of the cluster results in early-onset and progressive synaptic defects of the photoreceptors, leading to abnormalities of scotopic and photopic electroretinograms with decreased b-wave amplitude as the primary defect and progressive retinal degeneration. In addition, inactivation of the miR-183/96/182 cluster resulted in global changes in retinal gene expression, with enrichment of genes important for synaptogenesis, synaptic transmission, photoreceptor morphogenesis, and phototransduction, suggesting that the miR-183/96/182 cluster plays important roles in postnatal functional differentiation and synaptic connectivity of photoreceptors.

MicroRNA-146 inhibits thrombin-induced NF-κB activation and subsequent inflammatory responses in human retinal endothelial cells.

PURPOSE Nuclear factor-κB (NF-κB), a key regulator of immune and inflammatory responses, plays important roles in diabetes-induced microvascular complications including diabetic retinopathy (DR). Thrombin activates NF-κB through protease-activated receptor (PAR)-1, a member of the G-protein-coupled receptor (GPCR) superfamily, and contributes to DR. The current study is to uncover the roles of microRNA (miRNA) in thrombin-induced NF-κB activation and retinal endothelial functions. METHODS Target prediction was performed using the TargetScan algorithm. Predicted target was experimentally validated by luciferase reporter assays. Human retinal endothelial cells (HRECs) were transfected with miRNA mimics or antimiRs and treated with thrombin. Expression levels of miR-146 and related protein-coding genes were analyzed by quantitative (q)RT-PCR. Functional changes of HRECs were analyzed by leukocyte adhesion assays. RESULTS We identified that caspase-recruitment domain (CARD)-containing protein 10 (CARD10), an essential scaffold/adaptor protein of GPCR-mediated NF-κB activation pathway, is a direct target of miR-146. Thrombin treatment resulted in NF-κB-dependent upregulation of miR-146 in HRECs; while transfection of miR-146 mimics resulted in significant downregulation of CARD10 and prevented thrombin-induced NF-κB activation, suggest that a negative feedback regulation of miR-146 on thrombin-induced NF-κB through targeting CARD10. Furthermore, overexpression of miR-146 prevented thrombin-induced increased leukocyte adhesion to HRECs. CONCLUSIONS We uncovered a novel negative feedback regulatory mechanism on thrombin-induced GPCR-mediated NF-κB activation by miR-146. In combination with the negative feedback regulation of miR-146 on the IL-1R/toll-like receptor (TLR)-mediated NF-κB activation in RECs that we reported previously, our results underscore a pivotal, negative regulatory role of miR-146 on multiple NF-κB activation pathways and related inflammatory processes in DR.

PHR1 Encodes an Abundant, Pleckstrin Homology Domain-containing Integral Membrane Protein in the Photoreceptor Outer Segments

We cloned human and murine cDNAs of a gene (designated PHR1), expressed preferentially in retina and brain. In both species, PHR1 utilizes two promoters and alternative splicing to produce four PHR1 transcripts, encoding isoforms of 243, 224, 208, and 189 amino acids, each with a pleckstrin homology domain at their N terminus and a transmembrane domain at their C terminus. Transcript 1 originates from a 5′-photoreceptor-specific promoter with at least three Crx elements ((C/T)TAATCC). Transcript 2 originates from the same promoter but lacks exon 7, which encodes 35 amino acids immediately C-terminal to the pleckstrin homology domain. Transcripts 3 and 4 originate from an internal promoter in intron 2 and either include or lack exon 7, respectively. In situ hybridization shows thatPHR1 is highly expressed in photoreceptors, with lower expression in retinal ganglion cells. Immunohistochemistry localizes the PHR1 protein to photoreceptor outer segments where chemical extraction studies confirm it is an integral membrane protein. Using a series of PHR1 glutathione S-transferase fusion proteins to perform in vitro binding assays, we found PHR1 binds transducin βγ subunits but not inositol phosphates. This activity and subcellular location suggests that PHR1 may function as a previously unrecognized modulator of the phototransduction pathway.

PHR1, a PH Domain-Containing Protein Expressed in Primary Sensory Neurons

ABSTRACT Previously, we identified PHR1 as an abundantly expressed gene in photoreceptors and showed that it encodes four isoforms, each with N-terminal pleckstrin homology (PH) and C-terminal transmembrane domains. To better understand PHR1 function and expression, we made a Phr1 null mouse by inserting a β-galactosidase/neor cassette into exon 3. In addition to photoreceptors, we found abundant expression of specific Phr1 splice forms in olfactory receptor neurons and vestibular and cochlear hair cells. We also found Phr1 expression in cells with a possible sensory function, including peripheral retinal ganglion cells, cochlear interdental cells, and neurons of the circumventricular organ. Despite this discrete expression in known and putative sensory neurons, mice lacking PHR1 do not have overt sensory deficits.

miRNA mutations are not a common cause of deafness.

Mutations in miRNA genes have been implicated in hearing loss in human families and mice. It is also possible that mutations in miRNA binding sites of inner ear targets alter gene expression levels and lead to hearing loss. To investigate these possibilities we screened predicted target genes of the miR‐183 miRNA cluster known to be expressed in the inner ear sensory epithelium. In one Iranian family segregating autosomal recessive non‐syndromic hearing loss (ARNSHL), we identified a homozygous variant in a predicted miR‐96/182 binding site in the 3′UTR of the RDX (DFNB24) gene. However, in vitro functional studies showed that this site is not a functional target for miR‐96/182. We extended our study to include the miR‐183 genes themselves and 24 additional predicted target genes of the miRNA‐183 cluster. Screening these miRNAs and target sequences in numerous families segregating either autosomal dominant non‐syndromic deafness (ADNSHL) or ARNSHL did not identify any potential deafness‐causing mutations. These results suggest that mutations disrupting gene regulation by the miR‐183 cluster are not a common cause of human hearing loss.

PHR1 is a vesicle-bound protein abundantly expressed in mature olfactory neurons

To characterize the role of Phr1, a gene highly expressed in primary sensory neurons where it encodes an integral membrane protein with an N‐terminal pleckstrin homology domain and a C‐terminal transmembrane domain, in the olfactory system.