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The primary cilium is a ubiquitous microtubule-based organelle that senses external environment and modulates diverse signaling pathways in different cell types and tissues. The ...cilium originates from the mother centriole through a complex set of cellular events requiring hundreds of distinct components. Aberrant ciliogenesis or ciliary transport leads to a broad spectrum of clinical entities with overlapping yet highly variable phenotypes, collectively called ciliopathies, which include sensory defects and syndromic disorders with multi-organ pathologies. For efficient light detection, photoreceptors in the retina elaborate a modified cilium known as the outer segment, which is packed with membranous discs enriched for components of the phototransduction machinery. Retinopathy phenotype involves dysfunction and/or degeneration of the light sensing photoreceptors and is highly penetrant in ciliopathies. This review will discuss primary cilia biogenesis and ciliopathies, with a focus on the retina, and the role of CP110-CEP290-CC2D2A network. We will also explore how recent technologies can advance our understanding of cilia biology and discuss new paradigms for developing potential therapies of retinal ciliopathies.
Organoid models have quickly become a popular research tool to evaluate novel therapeutics on 3-D recapitulated tissue. This has enabled researchers to use physiologically relevant human tissue in ...vitro to augment the standard use of immortalized cells and animal models. Organoids can also provide a model when an engineered animal cannot recreate a specific disease phenotype. In particular, the retinal research field has taken advantage of this burgeoning technology to provide insight into inherited retinal disease(s) mechanisms and therapeutic intervention to ameliorate their effects. In this review we will discuss the use of both wild-type and patient-specific retinal organoids to further gene therapy research that could potentially prevent retinal disease(s) progression. Furthermore, we will discuss the pitfalls of current retinal organoid technology and present potential solutions that could overcome these hurdles in the near future.
Target identification (determining the correct drug targets for a disease) and target validation (demonstrating an effect of target perturbation on disease biomarkers and disease end points) are ...important steps in drug development. Clinically relevant associations of variants in genes encoding drug targets model the effect of modifying the same targets pharmacologically. To delineate drug development (including repurposing) opportunities arising from this paradigm, we connected complex disease- and biomarker-associated loci from genome-wide association studies to an updated set of genes encoding druggable human proteins, to agents with bioactivity against these targets, and, where there were licensed drugs, to clinical indications. We used this set of genes to inform the design of a new genotyping array, which will enable association studies of druggable genes for drug target selection and validation in human disease.
TopHat is a popular spliced aligner for RNA-sequence (RNA-seq) experiments. In this paper, we describe TopHat2, which incorporates many significant enhancements to TopHat. TopHat2 can align reads of ...various lengths produced by the latest sequencing technologies, while allowing for variable-length indels with respect to the reference genome. In addition to de novo spliced alignment, TopHat2 can align reads across fusion breaks, which can occur after genomic translocations. TopHat2 combines the ability to identify novel splice sites with direct mapping to known transcripts, producing sensitive and accurate alignments, even for highly repetitive genomes or in the presence of pseudogenes. TopHat2 is available at http://ccb.jhu.edu/software/tophat .
Retinal organoids (ROs) derived from human pluripotent stem cells largely recapitulate key features of in vivo retinal development, thus permitting the study of retinogenesis, disease modeling, and ...therapeutic development. However, the complexities of current protocols limit the use of this in vitro system in applications requiring large-scale production of organoids. Currently, widely used methods require the isolation of presumed optic vesicle-like structures from adherent cultures by dissection, a labor-intensive and time-consuming step that involves extensive practice and training.
We report a simple and efficient method for generating ROs by scraping the entire adherent culture and growing the resulting cell aggregates in a free-floating condition.
Within 1 to 7 days following the procedure, emerging morphologically well-defined optic vesicles can be identified and harvested with ease. The transition from two-dimensional (2D) to 3D culture condition favored the formation of ROs from areas devoid of typical optic vesicle-like structures, thus increasing the RO yield. Moreover, ROs generated by this approach were more often associated with the pigment epithelium.
This improved, robust, and efficient protocol should facilitate large-scale differentiation of pluripotent stem cells into retinal organoids in support of human disease modeling and therapy development.
Stem cell-derived retinal organoids recapitulate many landmarks of in vivo differentiation but lack functional maturation of distinct cell types, especially photoreceptors. Using comprehensive ...temporal transcriptome analyses, we show that transcriptome shift from postnatal day 6 (P6) to P10, associated with morphogenesis and synapse formation during mouse retina development, was not evident in organoids, and co-expression clusters with similar patterns included different sets of genes. Furthermore, network analysis identified divergent regulatory dynamics between developing retina in vivo and in organoids, with temporal dysregulation of specific signaling pathways and delayed or reduced expression of genes involved in photoreceptor function(s) and survival. Accordingly, addition of docosahexaenoic acid and fibroblast growth factor 1 to organoid cultures specifically promoted the maturation of photoreceptors, including cones. Our study thus identifies regulatory signals deficient in developing retinal organoids and provides experimental validation by producing a more mature retina in vitro, thereby facilitating investigations in disease modeling and therapies.
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•Temporal delay in cell-type-specific gene expression in developing retina organoids•Dysregulation of signaling pathways during retina development in vitro•Altered expression of genes associated with photoreceptor maturation in organoids•Improved biogenesis and maintenance of photoreceptors by DHA and FGF1
Swaroop and colleagues performed transcriptome analysis of developing mouse retina in vivo and in organoids. Retinal organoid transcriptomes revealed altered expression of cell-type-specific genes associated with functional maturation and dysregulation of signaling pathways. Addition of DHA and FGF1 to organoid cultures facilitated an early and improved differentiation of photoreceptors.
To elucidate cellular machinery on a global scale, we performed a multiple comparison of the recently available protein-protein interaction networks of Caenorhabditis elegans, Drosophila ...melanogaster, and Saccharomyces cerevisiae. This comparison integrated protein interaction and sequence information to reveal 71 network regions that were conserved across all three species and many exclusive to the metazoans. We used this conservation, and found statistically significant support for 4,645 previously undescribed protein functions and 2,609 previously undescribed protein interactions. We tested 60 interaction predictions for yeast by two-hybrid analysis, confirming approximately half of these. Significantly, many of the predicted functions and interactions would not have been identified from sequence similarity alone, demonstrating that network comparisons provide essential biological information beyond what is gleaned from the genome.
Background Major depressive disorder (MDD) is characterized by abnormalities in structure, function, and connectivity in several brain regions. Few studies have examined how these regions are ...organized in the brain or investigated network-level structural aberrations that might be associated with depression. Methods We used graph analysis to examine the gray matter structural networks of individuals diagnosed with MDD ( n = 93) and a demographically similar healthy comparison group ( n = 151) with no history of psychopathology. The efficiency of structural networks for processing information was determined by quantifying local interconnectivity (clustering) and global integration (path length). We also compared the groups on the contributions of high-degree nodes (i.e., hubs) and regional network measures, including degree (number of connections in a node) and betweenness (fraction of short path connections in a node). Results Depressed participants had significantly decreased clustering in their brain networks across a range of network densities. Compared with control subjects, depressed participants had fewer hubs primarily in medial frontal and medial temporal areas, had higher degree in the left supramarginal gyrus and right gyrus rectus, and had higher betweenness in the right amygdala and left medial orbitofrontal gyrus. Conclusions Networks of depressed individuals are characterized by a less efficient organization involving decreased regional connectivity compared with control subjects. Regional connections in the amygdala and medial prefrontal cortex may play a role in maintaining or adapting to depressive pathology. This is the first report of anomalous large-scale gray matter structural networks in MDD and provides new insights concerning the neurobiological mechanisms associated with this disorder.
Retinal organoids (ROs) derived from human pluripotent stem cells (hPSCs) recapitulate key features of retinogenesis and provide a promising platform to study retinal development and disease in a ...human context. Although multiple protocols are currently in use, hPSCs exhibit tremendous variability in differentiation efficiency, with some cell lines consistently yielding few or even no ROs, limiting their utility in research. We report here that early nicotinamide (NAM) treatment significantly improves RO yield across 8 hPSC lines from different donors, including some that would otherwise fail to generate a meaningful number of ROs. NAM treatment promotes neural commitment of hPSCs at the expense of non-neural ectodermal cell fate, which in turn increases eye field progenitor generation. Further analysis suggests that this effect is partially mediated through inhibition of BMP signaling. Our data encourage a broader use of human ROs for disease modeling applications that require the use of multiple patient-specific cell lines.
Objective: Previous functional magnetic resonance imaging (fMRI) studies in pediatric bipolar disorder (BD) have reported greater amygdala and less dorsolateral prefrontal cortex (DLPFC) activation ...to facial expressions compared to healthy controls. The current study investigates whether these differences are associated with the early or late phase of activation, suggesting different temporal characteristics of brain responses. Method: A total of 20 euthymic adolescents with familial BD (14 male) and 21 healthy control subjects (13 male) underwent fMRI scanning during presentation of happy, sad, and neutral facial expressions. Whole-brain voxelwise analyses were conducted in SPM5, using a three-way analysis of variance (ANOVA) with factors group (BD and healthy control HC), facial expression (happy, sad, and neutral versus scrambled), and phase (early and late, corresponding to the first and second half of each block of faces). Results: There were no significant group differences in task performance, age, gender, or IQ. Significant activation from the main effect of group included greater DLPFC activation in the HC group, and greater amygdala/hippocampal activation in the BD group. The interaction of Group x Phase identified clusters in the superior temporal sulcus/insula and visual cortex, where activation increased from the early to late phase of the block for the BD but not the HC group. Conclusions: These findings are consistent with previous studies that suggest deficient prefrontal cortex regulation of heightened amygdala response to emotional stimuli in pediatric BD. Increasing activation over time in superior temporal and visual cortices suggests difficulty processing or disengaging attention from emotional faces in BD. (Contains 3 tables and 2 figures.)
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