Canine CNGA3 Gene Mutations Provide Novel Insights into Human Achromatopsia-Associated Channelopathies and Treatment

Cyclic nucleotide-gated (CNG) ion channels are key mediators underlying signal transduction in retinal and olfactory receptors. Genetic defects in CNGA3 and CNGB3, encoding two structurally related subunits of cone CNG channels, lead to achromatopsia (ACHM). ACHM is a congenital, autosomal recessive retinal disorder that manifests by cone photoreceptor dysfunction, severely reduced visual acuity, impaired or complete color blindness and photophobia. Here, we report the first canine models for CNGA3-associated channelopathy caused by R424W or V644del mutations in the canine CNGA3 ortholog that accurately mimic the clinical and molecular features of human CNGA3-associated ACHM. These two spontaneous mutations exposed CNGA3 residues essential for the preservation of channel function and biogenesis. The CNGA3-R424W results in complete loss of cone function in vivo and channel activity confirmed by in vitro electrophysiology. Structural modeling and molecular dynamics (MD) simulations revealed R424-E306 salt bridge formation and its disruption with the R424W mutant. Reversal of charges in a CNGA3-R424E-E306R double mutant channel rescued cGMP-activated currents uncovering new insights into channel gating. The CNGA3-V644del affects the C-terminal leucine zipper (CLZ) domain destabilizing intersubunit interactions of the coiled-coil complex in the MD simulations; the in vitro experiments showed incompetent trimeric CNGA3 subunit assembly consistent with abnormal biogenesis of in vivo channels. These newly characterized large animal models not only provide a valuable system for studying cone-specific CNG channel function in health and disease, but also represent prime candidates for proof-of-concept studies of CNGA3 gene replacement therapy for ACHM patients.     

Specific Tandem 3'UTR Patterns and Gene Expression Profiles in Mouse Thy1+ Germline Stem Cells

A recently developed strategy of sequencing alternative polyadenylation (APA) sites (SAPAS) with second-generation sequencing technology can be used to explore complete genome-wide patterns of tandem APA sites and global gene expression profiles. spermatogonial stem cells (SSCs) maintain long-term reproductive abilities in male mammals. The detailed mechanisms by which SSCs self-renew and generate mature spermatozoa are not clear. To understand the specific alternative polyadenylation pattern and global gene expression profile of male germline stem cells (GSCs, mainly referred to SSCs here), we isolated and purified mouse Thy1⁺ cells from testis by magnetic-activated cell sorting (MACS) and then used the SAPAS method for analysis, using pluripotent embryonic stem cells (ESCs) and differentiated mouse embryonic fibroblast cells (MEFs) as controls. As a result, we obtained 99,944 poly(A) sites, approximately 40% of which were newly detected in our experiments. These poly(A) sites originated from three mouse cell types and covered 17,499 genes, including 831 long non-coding RNA (lncRNA) genes. We observed that GSCs tend to have shorter 3''UTR lengths while MEFs tend towards longer 3''UTR lengths. We also identified 1337 genes that were highly expressed in GSCs, and these genes were highly consistent with the functional characteristics of GSCs. Our detailed bioinformatics analysis identified APA site-switching events at 3''UTRs and many new specifically expressed genes in GSCs, which we experimentally confirmed. Furthermore, qRT-PCR was performed to validate several events of the 334 genes with distal-to-proximal poly(A) switch in GSCs. Consistently APA reporter assay confirmed the total 3''UTR shortening in GSCs compared to MEFs. We also analyzed the cis elements around the proximal poly(A) site preferentially used in GSCs and found C-rich elements may contribute to this regulation. Overall, our results identified the expression level and polyadenylation site profiles and these data provide new insights into the processes potentially involved in the GSC life cycle and spermatogenesis.     

Silhouette Scores for Arbitrary Defined Groups in Gene Expression Data and Insights into Differential Expression Results

Background

Hierarchical Sample clustering (HSC) is widely performed to examine associations within expression data obtained from microarrays and RNA sequencing (RNA-seq). Researchers have investigated the HSC results with several possible criteria for grouping (e.g., sex, age, and disease types). However, the evaluation of arbitrary defined groups still counts in subjective visual inspection.

Results

To objectively evaluate the degree of separation between groups of interest in the HSC dendrogram, we propose to use Silhouette scores. Silhouettes was originally developed as a graphical aid for the validation of data clusters. It provides a measure of how well a sample is classified when it was assigned to a cluster by according to both the tightness of the clusters and the separation between them. It ranges from 1.0 to ??1.0, and a larger value for the average silhouette (AS) over all samples to be analyzed indicates a higher degree of cluster separation. The basic idea to use an AS is to replace the term cluster by group when calculating the scores. We investigated the validity of this score using simulated and real data designed for differential expression (DE) analysis. We found that larger (or smaller) AS values agreed well with both higher (or lower) degrees of separation between different groups and higher percentages of differentially expressed genes (P_DEG). We also found that the AS values were generally independent on the number of replicates (N_rep). Although the P_DEG values depended on N_rep, we confirmed that both AS and P_DEG values were close to zero when samples in the data showed an intermingled nature between the groups in the HSC dendrogram.

Conclusion

Silhouettes is useful for exploring data with predefined group labels. It would help provide both an objective evaluation of HSC dendrograms and insights into the DE results with regard to the compared groups.

     

Differential Effects of TPA and Pristane on Gene Expression and Transformation in Mouse Epidermal Cells

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) or 2,6,10,14-tetramethylpentadecane (pristane) on gene expression and transformation were examined using two clones (P⁺, TPA transformation sensitive and P^-, TPA resistant) of the mouse epidermal cell line JB6. Results from transformation studies indicated pristane was more efficient, i.e., lower concentrations were required to elicit an equivalent response, in transforming the P⁺, but not the P^-, clone of JB6 compared to TPA. Furthermore, results from these studies demonstrated either TPA or pristane was effective in the transactivation of the chloramphenicol acetyltransferase gene under the regulatory control of most viral promoter/enhancer elements transfected into the P⁺, but not the P^-, clone of JB6. However, if a consensus cAMP response element was linked to the simian virus 40 early promoter, pristane activation was observed in both P⁺ and P^- cells. The differential effects of these two compounds suggest that while they have similar characteristics, they may utilize different pathways to elicit their effects.     

Primary 1,25-Dihydroxyvitamin D3 Response of the Interleukin 8 Gene Cluster in Human Monocyte- and Macrophage-Like Cells

Genome-wide analysis of vitamin D receptor (VDR) binding sites in THP-1 human monocyte-like cells highlighted the interleukin 8 gene, also known as chemokine CXC motif ligand 8 (CXCL8). CXCL8 is a chemotactic cytokine with important functions during acute inflammation as well as in the context of various cancers. The nine genes of the CXCL cluster and the strong VDR binding site close to the CXCL8 gene are insulated from neighboring genes by CCCTC-binding factor (CTCF) binding sites. Only CXCL8, CXCL6 and CXCL1 are expressed in THP-1 cells, but all three are up-regulated primary 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) target genes. Formaldehyde-assisted isolation of regulatory elements sequencing analysis of the whole CXCL cluster demonstrated 1,25(OH)₂D₃-dependent chromatin opening exclusively for the VDR binding site. In differentiated THP-1 cells the CXCL8 gene showed a 33-fold higher basal expression, but is together with CXCL6 and CXCL1 still a primary 1,25(OH)₂D₃ target under the control of the same genomic VDR binding site. In summary, both in undifferentiated and differentiated THP-1 cells the genes CXCL8, CXCL6 and CXCL1 are under the primary control of 1,25(OH)₂D₃ and its receptor VDR. Our observation provides further evidence for the immune-related functions of vitamin D.     

New Insights into Cytokine Gene Expression in the Rat Hypothalamus Following Endotoxin Challenge

Peripheral injection of the endotoxin LPS in rats 3 weeks prior to a second injection of LPS derived from another bacterial strain results in elevated corticosterone and decreased pro-inflammatory cytokines in the blood. We further investigated this model by measuring cytokine expression in the hypothalamus and spleen. In LPS-pretreated rats, hypothalamic expression of a range of cytokines was attenuated in response to the second injection of LPS while splenic expression was elevated. This is the first demonstration that prior exposure to an endotoxin can differentially affect cytokine expression in the brain and peripheral tissues when a host is confronted with a second, acute, pro-inflammatory stimulus. Changes in hypothalamic cytokine expression in endotoxin pretreated rats may provide new evidence for the involvement of central cytokine pathways in modulating peripheral inflammation and mediating psychopathological alterations associated with inflammatory diseases.     

New Insights into Syndecan-2 Expression and Tumourigenic Activity in Colon Carcinoma Cells

Adhesion receptors play crucial roles in the neoplastic transformation of normal cells through induction of cancer-specific cellular behaviour and morphology. This implies that cancer cells likely express and utilize a distinct set of adhesion receptors during carcinogenesis. Colon cancer is an excellent model system for the study of this process, since both molecular genetic and morphological changes have been well established for this disease. We recently reported increased expression of the cell surface adhesion receptor, syndecan-2, in several colon carcinoma cell lines. Indeed, increased syndecan-2 expression was necessary for tumourigenic activity, suggesting that syndecan-2 might have value as both a new diagnostic marker and a possible therapeutic target. Here, we review recent advances in understanding the role of syndecan-2 in the carcinogenesis of colon cells, and discuss a leading role for this molecule in a new era for colon cancer treatment.     

Gene Expression Profiles of HLA-G1 Overexpressed in hES Cells

The goals of this study were to analyze the change in the global gene expression profile of exogenous human leukocyte antigen-G1 (HLA-G1) overexpressed in human embryonic stem (hES) cells and to explore the molecular mechanism by which the overexpression of HLA-G1 modifies immunologic pathways. Microarray and quantitative real-time PCR analyses were performed to quantify the differential expression pattern of HLA-G1?+?H1 hES cells. The results showed that HLA-G1 differentially regulated the expression of 425 genes with at least a twofold increase or decrease. These differentially expressed genes were classified into 13 functional groups, including cellular components, biological processes, and molecular functions. The pathways of focal adhesion, the TGF-β signaling pathway, and the immune response were the most predominantly affected. The synergism of these genes could explain the mechanism of the immunosuppression of HLA-G1?+?H1 hES cells. Thus, the expression pattern reflected a broad spectrum of roles of HLA-G1 in hES cells.     

Comparison of Mouse and Human Retinal Pigment Epithelium Gene Expression Profiles: Potential Implications for Age-Related Macular Degeneration

Background

The human retinal pigment epithelium (RPE) plays an important role in the pathogenesis of age related macular degeneration (AMD). AMD is the leading cause of blindness worldwide. There is currently no effective treatment available. Preclinical studies in AMD mouse models are essential to develop new therapeutics. This requires further in-depth knowledge of the similarities and differences between mouse and human RPE.

Methods

We performed a microarray study to identify and functionally annotate RPE specific gene expression in mouse and human RPE. We used a meticulous method to determine C57BL/6J mouse RPE signature genes, correcting for possible RNA contamination from its adjacent layers: the choroid and the photoreceptors. We compared the signature genes, gene expression profiles and functional annotations of the mouse and human RPE.

Results

We defined sets of mouse (64), human (171) and mouse–human interspecies (22) RPE signature genes. Not unexpectedly, our gene expression analysis and comparative functional annotation suggested that, in general, the mouse and human RPE are very similar. For example, we found similarities for general features, like “organ development” and “disorders related to neurological tissue”. However, detailed analysis of the molecular pathways and networks associated with RPE functions, suggested also multiple species-specific differences, some of which may be relevant for the development of AMD. For example, CFHR1, most likely the main complement regulator in AMD pathogenesis was highly expressed in human RPE, but almost absent in mouse RPE. Furthermore, functions assigned to mouse and human RPE expression profiles indicate (patho-) biological differences related to AMD, such as oxidative stress, Bruch’s membrane, immune-regulation and outer blood retina barrier.

Conclusion

These differences may be important for the development of new therapeutic strategies and translational studies in age-related macular degeneration.     

Cyclooxygenase-3 Gene Expression in Alzheimer Hippocampus and in Stressed Human Neural Cells

The cyclooxygenase (COX) superfamily of prostaglandin synthase genes encode a constitutively expressed COX-1, an inducible, highly regulated COX-2, and a COX-3 isoform whose RNA is derived through the retention of a highly structured, G + C-rich intron 1 of the COX-1 gene. As generators of oxygen radicals, lipid mediators, and the pharmacological targets of nonsteroidal anti-inflammatory drugs (NSAIDs), COX enzymes potentiate inflammatory neuropathology in Alzheimer's disease (AD) brain. Because COX-2 is elevated in AD and COX-3 is enriched in the mammalian CNS, these studies were undertaken to examine the expression of COX-3 in AD and in [IL-1beta + Abeta42]-triggered human neural (HN) cells in primary culture. The results indicate that while COX-2 remains a major player in propagating inflammmation in AD and in stressed HN cells, COX-3 may play ancillary roles in membrane-based COX signaling or when basal levels of COX-1 or COX-2 expression persist.