Faculty Bibliography

Cardiovascular disease, with atherosclerosis as the major underlying factor, remains the leading cause of death worldwide. It is well established that cholesterol ester-enriched foam cells are the hallmark of atherosclerotic plaques. Multiple lines of evidence support that enhancing foam cell cholesterol efflux by HDL (high-density lipoprotein) particles, the first step of reverse cholesterol transport (RCT), is a promising antiatherogenic strategy. Yet, excitement towards the therapeutic potential of manipulating RCT for the treatment of cardiovascular disease has faded because of the lack of the association between cardiovascular disease risk and what was typically measured in intervention trials, namely HDL cholesterol, which has an inconsistent relationship to HDL function and RCT. In this review, we will summarize some of the potential reasons for this inconsistency, update the mechanisms of RCT, and highlight conditions in which impaired HDL function or RCT contributes to vascular disease. On balance, the evidence still argues for further research to better understand how HDL functionality contributes to RCT to develop prevention and treatment strategies to reduce the risk of cardiovascular disease.

A fundamental aim in developmental biology is to understand how the various cell types of the body are specified by differential gene regulation. Caenorhabditis elegans nervous system development provides a powerful system for studying this, as exemplified by a new Development paper reporting on how the BAG neurons that help the worm sense oxygen and carbon dioxide are specified. We caught up with first authors Julia Brandt and Mary Rossillo and their supervisor Niels Ringstad (Associate Professor at the Skirball Institute of Biomolecular Medicine and Department of Cell Biology at New York University) to find out more about the story.

Introduction: Noncoding small RNAs in the retina regulate gene expression by targeting and repressing mRNA. Small RNAs are secreted in extracellular vesicles (EVs). Analysis of EVs released from developing retinal tissue is an essential step in elucidating the role of EV molecular cargo and signalling during retinogenesis. A number of canonical genes are associated with retinal cell fate determination during development, but EV-mediated gene regulation in the retinal microenvironment remains undefined. In this study, we characterize the microRNA, tRNA, and piRNA composition of EVs secreted from human induced pluripotent stem cell (hiPSCs) - derived 3D retinas at three developmental time points that correlate with hallmarks of retinal cell differentiation and lamination in vivo.
Method(s): Retinal organoids were generated from hiPSCs. We selected three developmental time points (day 42, 63 and 90) that represent distinctive stages during normal retinal cell fate specification and lamination. We analysed the release rate, concentration, morphology and content (miRNA, tRNA and pi- RNA) of EVs released from human hiPSCs-derived 3D retinas.
Result(s): The genetic signalling, developmental time course and morphogenesis of these retinal organoids were comparable to those of developing human retinas in vivo. According to Gene Ontology analysis, miRNA targets at the earliest stage of development were more relevant to early differentiation and cell morphogenesis, whereas miRNA targets at the later stages were more relevant to cell proliferation, cell differentiation, and cell migration. Summary/Conclusion: For the first time, this work demonstrates the rate of release and concentration of EVs from developing hiPSC-derived 3D retinal tissue. We report a large variety of small RNAs in EVs from hiPSC-derived 3D retinas, including miRNAs, tRNAs and piRNAs. The full range of small RNAs detected in our EVs may act as regulatory elements to modulate gene activity and may serve as biomarkers of normal development. This work represents the first sequencing analysis of small RNA species contained in hiPSCderived 3D retinas and their released EVs

Developmental programs that generate the astonishing neuronal diversity of the nervous system are not completely understood and thus present a major challenge for clinical applications of guided cell differentiation strategies. Using direct neuronal programming of embryonic stem cells, we found that two main vertebrate proneural factors, Ascl1 and neurogenin 2 (Neurog2), induce different neuronal fates by binding to largely different sets of genomic sites. Their divergent binding patterns are not determined by the previous chromatin state, but are distinguished by enrichment of specific E-box sequences that reflect the binding preferences of the DNA-binding domains. The divergent Ascl1 and Neurog2 binding patterns result in distinct chromatin accessibility and enhancer activity profiles that differentially shape the binding of downstream transcription factors during neuronal differentiation. This study provides a mechanistic understanding of how transcription factors constrain terminal cell fates, and it delineates the importance of choosing the right proneural factor in neuronal reprogramming strategies.

Genomic studies have identified hundreds of candidate genes near loci associated with risk for schizophrenia. To define candidates and their functions, we mutated zebrafish orthologs of 132 human schizophrenia-associated genes. We created a phenotype atlas consisting of whole-brain activity maps, brain structural differences, and profiles of behavioral abnormalities. Phenotypes were diverse but specific, including altered forebrain development and decreased prepulse inhibition. Exploration of these datasets identified promising candidates in more than 10 gene-rich regions, including the magnesium transporter cnnm2 and the translational repressor gigyf2, and revealed shared anatomical sites of activity differences, including the pallium, hypothalamus, and tectum. Single-cell RNA sequencing uncovered an essential role for the understudied transcription factor znf536 in the development of forebrain neurons implicated in social behavior and stress. This phenotypic landscape of schizophrenia-associated genes prioritizes more than 30 candidates for further study and provides hypotheses to bridge the divide between genetic association and biological mechanism.

Habituation is a simple form of learning where animals learn to reduce their responses to repeated innocuous stimuli [1]. Habituation is thought to occur via at least two temporally and molecularly distinct mechanisms, which lead to short-term memories that last for seconds to minutes and long-term memories that last for hours or longer [1, 2]. Here, we focus on long-term habituation, which, due to the extended time course, necessitates stable alterations to circuit properties [2-4]. In its simplest form, long-term habituation could result from a plasticity event at a single point in a circuit, and many studies have focused on identifying the site and underlying mechanism of plasticity [5-10]. However, it is possible that these individual sites are only one of many points in the circuit where plasticity is occurring. Indeed, studies of short-term habituation in C. elegans indicate that in this paradigm, multiple genetically separable mechanisms operate to adapt specific aspects of behavior [11-13]. Here, we use a visual assay in which larval zebrafish habituate their response to sudden reductions in illumination (dark flashes) [14, 15]. Through behavioral analyses, we find that multiple components of the dark-flash response habituate independently of one another using different molecular mechanisms. This is consistent with a modular model in which habituation originates from multiple independent processes, each adapting specific components of behavior. This may allow animals to more specifically or flexibly habituate based on stimulus context or internal states.

Lysosomal failure underlies pathogenesis of numerous congenital neurodegenerative disorders and is an early and progressive feature of Alzheimer's disease (AD) pathogenesis. Here, we report that lysosomal dysfunction in Down Syndrome (Trisomy 21), a neurodevelopmental disorder and form of early onset AD, requires the extra gene copy of amyloid precursor protein (APP) and is specifically mediated by the beta cleaved carboxy terminal fragment of APP (APP-βCTF, C99). In primary fibroblasts from individuals with Down Syndrome (DS), lysosomal degradation of autophagic and endocytic substrates is selectively impaired causing them to accumulate in enlarged autolysosomes/lysosomes. Direct measurements of lysosomal pH uncovered a significant elevation (0.6 units) as a basis for slowed LC3 turnover and the inactivation of cathepsin D (CTSD) and other lysosomal hydrolases known to be unstable or less active when lysosomal pH is persistently elevated. Normalizing lysosome pH by delivering acidic nanoparticles to lysosomes ameliorated lysosomal deficits, while RNA sequencing analysis excluded a transcriptional contribution to hydrolase declines. Cortical neurons cultured from the Ts2 mouse model of DS exhibited lysosomal deficits similar to those in DS cells. Lowering APP expression with siRNA or BACE1 inhibition reversed cathepsin deficits in both fibroblasts and neurons. Deleting one BACE1 allele from adult Ts2 mice had similar rescue effects in vivo The modest elevation of endogenous APP-βCTF needed to disrupt lysosomal function in DS is relevant to sporadic AD where APP-βCTF, but not APP, is also elevated. Our results extend evidence that impaired lysosomal acidification drives progressive lysosomal failure in multiple forms of AD.SIGNIFICANCE STATEMENTDown Syndrome (trisomy 21) (DS) is a neurodevelopmental disorder invariably leading to early-onset Alzheimer's Disease (AD). We showed in cells from DS individuals and neurons of DS models that one extra copy of a normal amyloid precursor protein (APP) gene impairs lysosomal acidification, thereby depressing lysosomal hydrolytic activities and turnover of autophagic and endocytic substrates - processes vital to neuronal survival. These deficits, which were reversible by correcting lysosomal pH, are mediated by elevated levels of endogenous β-cleaved carboxy-terminal fragment of APP (APP-βCTF). Notably, similar endosomal-lysosomal pathobiology emerges early in sporadic AD, where neuronal APP-βCTF is also elevated, underscoring its importance as a therapeutic target and underscoring the functional and pathogenic interrelationships between the endosomal-lysosomal pathway and genes causing AD.

Thoracic aortic aneurysm (TAA) is a complex life-threatening disease characterized by extensive extracellular matrix (ECM) fragmentation and persistent inflammation, culminating in a weakened aorta. Although evidence suggests defective canonical signaling pathways in TAA, the full spectrum of mechanisms contributing to TAA is poorly understood, therefore limiting the scope of drug-based treatment. Here, we used a sensitive RNA sequencing approach to profile the transcriptomic atlas of human TAA. Pathway analysis revealed upregulation of key matrix-degrading enzymes and inflammation coincident with the axonal guidance pathway. We uncovered their novel association with TAA and focused on the expression of Semaphorins and Netrins. Comprehensive analysis of this pathway showed that several members were differentially expressed in TAA compared to controls. Immunohistochemistry revealed that Semaphorin4D and its receptor PlexinB1, similar to Netrin-1 proteins were highly expressed in damaged areas of TAA tissues but faintly detected in the vessel wall of non-diseased sections. It should be considered that the current study is limited by its sample size and the use of internal thoracic artery as control for TAA for the sequencing dataset. Our data determines important neuronal regulators of vascular inflammatory events and suggest Netrins and Semaphorins as potential key contributors of ECM degradation in TAA.

Organoids derived from stem cells or tissues in culture can develop into structures that resemble the in vivo anatomy and physiology of intact organs. Human organoid cultures provide the potential to study human development and model disease processes with the same scrutiny and depth of analysis customary for research with nonhuman model organisms. Resembling the complexity of the actual tissue or organ, patient-derived human organoid studies may accelerate medical research, creating new opportunities for tissue engineering and regenerative medicine, generating knowledge and tools for preclinical studies, including drug development and testing. Biologists are drawn to this system as a new "model organism" to study complex disease phenotypes and genetic variability among individuals using patient-derived tissues. The American Society for Cell Biology convened a task force to report on the potential, challenges, and limitations for human organoid research. The task force suggests ways to ease the entry for new researchers into the field and how to facilitate broader use of this new model organism within the research community. This includes guidelines for reproducibility, culturing, sharing of patient materials, patient consent, training, and communication with the public.

Semaphorins (SEMAs) and their Plexin (PLXN) receptors are central regulators of metazoan cellular communication. SEMA-PLXND1 signaling plays important roles in cardiovascular, nervous, and immune system development, and cancer biology. However, little is known about the molecular mechanisms that modulate SEMA-PLXND1 signaling. As PLXND1 associates with GIPC family endocytic adaptors, we evaluated the requirement for the molecular determinants of their association and PLXND1's vascular role. Zebrafish that endogenously express a Plxnd1 receptor with a predicted impairment in GIPC binding exhibit low penetrance angiogenesis deficits and antiangiogenic drug hypersensitivity. Moreover, gipc mutant fish show angiogenic impairments that are ameliorated by reducing Plxnd1 signaling. Finally, GIPC depletion potentiates SEMA-PLXND1 signaling in cultured endothelial cells. These findings expand the vascular roles of GIPCs beyond those of the Vascular Endothelial Growth Factor (VEGF)-dependent, proangiogenic GIPC1-Neuropilin 1 complex, recasting GIPCs as negative modulators of antiangiogenic PLXND1 signaling and suggest that PLXND1 trafficking shapes vascular development.