Faculty Bibliography

Adenosine A2a receptor (A2aR) stimulation promotes the synthesis of collagens I and III, and we have recently demonstrated that there is crosstalk between the A2aR and WNT/beta-catenin signaling pathway. In in vitro studies, A2aR signaling for collagen III expression was mediated by WNT/beta-catenin signaling in human dermal fibroblasts; we further verified whether the crosstalk between A2aR and Wnt/beta-catenin signaling was involved in diffuse dermal fibrosis in vivo. Wnt-signaling reporter mice (Tcf/Lef:H2B-GFP) were challenged with bleomycin and treated with the selective A2aR antagonist istradefylline (KW6002) or vehicle. Dermal fibrosis was quantitated and nuclear translocation of beta-catenin in fibroblasts was assessed by double-staining for Green fluorescent protein or dephosphorylated beta-catenin or beta-catenin phosphorylated at Ser552, and vimentin. KW6002 significantly reduced skin thickness, skinfold thickness, breaking tension, dermal hydroxyproline content, myofibroblast accumulation, and collagen alignment in bleomycin-induced dermal fibrosis. Also, there was increased expression of Tcf/Lef:H2B-GFP reporter in bleomycin-induced dermal fibrosis, an effect that was diminished by treatment with KW6002. Moreover, KW6002 significantly inhibited nuclear translocation of Tcf/Lef:H2B-GFP reporter, as well as dephosphorylated beta-catenin and beta-catenin phosphorylated at Ser552. Our work supports the hypothesis that pharmacologic blockade of A2aR inhibits the WNT/beta-catenin signaling pathway, contributing to its capacity to inhibit bleomycin-induced dermal fibrosis.

The mechanisms that initiate vitiligo are poorly understood. Vitiligo triggers, such as monobenzone (MB) exposure, induce stress. Understanding the survival responses that combat this stress is key to determining why melanocytes become immune targets. MB induces oxidative and endoplasmic reticulum (ER) stress, which activates the unfolded protein response (UPR). PERK, a UPR initiator, phosphorylates eIF2alpha and master antioxidant regulator, NRF2. Here, we investigated the impact of PERK-eIF2alpha/-NRF2 activation on sensitivity to MB. Basal phospho-eIF2alpha and NRF2 levels are higher in melanocytes compared to fibroblasts or keratinocytes. PERK downregulation significantly reduced melanocyte viability (implicated in several autoimmune disorders) may link exposure to vitiligoinducing triggers with onset of autoimmunity

Current clinical and pre-clinical data suggest that both cannabinoid agents and blockage of CRF through corticotrophin releasing factor receptor type 1 (CRFr1) may offer therapeutic benefits for post-traumatic stress disorder (PTSD). Here we aim to determine whether they are more effective when combined when microinjected into the basolateral amygdala (BLA) or CA1 area of the hippocampus after exposure to a stressful event in the shock/reminders rat model for PTSD. Injection of the fatty acid amide hydrolase (FAAH) inhibitor URB597 after the shock into either the BLA or CA1 facilitated extinction, and attenuated startle response and anxiety-like behavior. These preventive effects of URB597 were found to be mediated by the CB1 receptor. Intra-BLA and intra-CA1 microinjection of the CRFr1 antagonist, CP-154,526 attenuated startle response. When microinjected into the BLA, CP-154,526 also attenuated freezing behavior during exposure to the first reminder and decreased anxiety-like behavior. The combined treatment of URB597 and CP-154,526 was not more effective than the separate treatments. Finally, mRNA levels of CRF, CRFr1 and CB1r were significantly higher in the BLA of rats exposed to shock and reminders compared to non-shocked rats almost one month after the shock. Taken together, the results show that enhancing endocannabinoid signaling in the amygdala and hippocampus produced a more favorable spectrum of effects than those caused by the CRFr1 antagonist. The findings suggest that FAAH inhibitors may be used as a novel treatment for stress-related anxiety disorders.

This paper provides an overview of the discussion and presentations from the Workshop on the Management of Large CryoEM Facilities held at the New York Structural Biology Center, New York, NY on February 6-7, 2017. A major objective of the workshop was to discuss best practices for managing cryoEM facilities. The discussions were largely focused on supporting single-particle methods for cryoEM and topics included: user access, assessing projects, workflow, sample handling, microscopy, data management and processing, and user training.

Wharton's Jelly mesenchymal stem cells (WJ-MSCs) are an attractive potential source of multipotent stem cells for bone tissue replacement therapies. However, the molecular mechanisms involved in their osteogenic conversion are poorly understood. Particularly, epigenetic control operating at the promoter regions of the two master regulators of the osteogenic program, RUNX2/P57 and SP7 has not yet been described in WJ-MSCs. Via quantitative PCR profiling and chromatin immunoprecipitation (ChIP) studies, here we analyze the ability of WJ-MSCs to engage osteoblast lineage. In undifferentiated WJ-MSCs, RUNX2/P57 P1, and SP7 promoters are found deprived of significant levels of the histone post-translational marks that are normally associated with transcriptionally active genes (H3ac, H3K27ac, and H3K4me3). Moreover, the RUNX2 P1 promoter lacks two relevant histone repressive marks (H3K9me3 and H3K27me3). Importantly, RUNX2 P1 promoter is found highly enriched in the H3K4me1 mark, which has been shown recently to mediate gene repression of key regulatory genes. Upon induction of WJ-MSCs osteogenic differentiation, we found that RUNX2/P57, but not SP7 gene expression is strongly activated, in a process that is accompanied by enrichment of activating histone marks (H3K4me3, H3ac, and H3K27ac) at the P1 promoter region. Histone mark analysis showed that SP7 gene promoter is robustly enriched in epigenetic repressive marks that may explain its poor transcriptional response to osteoblast differentiating media. Together, these results point to critical regulatory steps during epigenetic control of WJ-MSCs osteogenic lineage commitment that are relevant for future applications in regenerative medicine. J. Cell. Physiol. 9999: 1-9, 2016. (c) 2016 Wiley Periodicals, Inc.

A dysfunctional endosomal pathway and abnormally enlarged early endosomes in neurons are an early characteristic of Down syndrome (DS) and Alzheimer's disease (AD). We have hypothesized that endosomal material can be released by endosomal multivesicular bodies (MVBs) into the extracellular space via exosomes to relieve neurons of accumulated endosomal contents when endosomal pathway function is compromised. Supporting this, we found that exosome secretion is enhanced in the brains of DS patients and a mouse model of the disease, and by DS fibroblasts. Furthermore, increased levels of the tetraspanin CD63, a regulator of exosome biogenesis, were observed in DS brains. Importantly, CD63 knockdown diminished exosome release and worsened endosomal pathology in DS fibroblasts. Taken together, these data suggest that increased CD63 expression enhances exosome release as an endogenous mechanism mitigating endosomal abnormalities in DS. Thus, the upregulation of exosome release represents a potential therapeutic goal for neurodegenerative disorders with endosomal pathology.

Apela (also known as Elabela, Ende, and Toddler) is a small signaling peptide that activates the G-protein-coupled receptor Aplnr to stimulate cell migration during zebrafish gastrulation. Here, using CRISPR/Cas9 to generate a null, reporter-expressing allele, we study the role of Apela in the developing mouse embryo. We found that loss of Apela results in low-penetrance cardiovascular defects that manifest after the onset of circulation. Three-dimensional micro-computed tomography revealed a higher penetrance of vascular remodeling defects, from which some mutants recover, and identified extraembryonic anomalies as the earliest morphological distinction in Apela mutant embryos. Transcriptomics at late gastrulation identified aberrant upregulation of erythroid and myeloid markers in mutant embryos prior to the appearance of physical malformations. Double-mutant analyses showed that loss of Apela signaling impacts early Aplnr-expressing mesodermal populations independently of the alternative ligand Apelin, leading to lethal cardiac defects in some Apela null embryos.

A major hallmark of Parkinson's disease (PD) is the presence of Lewy bodies (LBs) in certain neuronal tissues. LBs are protein-rich inclusions, in which α-synuclein (α-syn) is the most abundant protein. Since these inclusions are not present in healthy individuals, despite the high concentration of α-syn in neurons, it is important to investigate whether natural control mechanisms are present to efficiently suppress α-syn aggregation. Here, we demonstrate that a CRISPR/Cas9-mediated knockout (KO) of a DnaJ protein, DNAJB6, in HEK293T cells expressing α-syn, causes a massive increase in α-syn aggregation. Upon DNAJB6 re-introduction into these DNAJB6-KO HEK293T-α-syn cells, aggregation is reduced to the level of the parental cells. We then show that the suppression of α-syn aggregation is dependent on the J-domain of DNAJB6, as the catalytically inactive protein, which carries the H31Q mutation, does not suppress aggregation, when re-introduced into DNAJB6-KO cells. We further demonstrate, that the suppression of α-syn aggregation is dependent on the molecular chaperone Hsp70, which is consistent with the well-known function of J-domains of transferring unfolded and misfolded proteins to Hsp70. These data identify a natural control strategy to suppress α-syn aggregation and suggest potential therapeutic approaches to prevent or treat PD and related disorders.

Cultured cells are useful for studying the subcellular distribution of proteins, including peripheral membrane proteins. Genetically encoded fluorescently tagged proteins have revolutionized the study of subcellular protein distribution. However, it is difficult to quantify the distribution with fluorescent microscopy, especially when proteins are partially cytosolic. Moreover, it is often important to study endogenous proteins. Biochemical assays such as immunoblots remain the gold standard for quantification of protein distribution after subcellular fractionation. Although there are commercial kits that aim to isolate cytosolic or certain membrane fractions, most of these kits are based on extraction with detergents, which may be unsuitable for studying peripheral membrane proteins that are easily extracted from membranes. Here we present a detergent-free protocol for cellular homogenization by nitrogen cavitation and subsequent separation of cytosolic and membrane-bound proteins by ultracentrifugation. We confirm the separation of subcellular organelles in soluble and pellet fractions across different cell types, and compare protein extraction among several common non-detergent-based mechanical homogenization methods. Among several advantages of nitrogen cavitation is the superior efficiency of cellular disruption with minimal physical and chemical damage to delicate organelles. Combined with ultracentrifugation, nitrogen cavitation is an excellent method to examine the shift of peripheral membrane proteins between cytosolic and membrane fractions.

By celebrating the 100th anniversary of John Kendrew's birth in 1917, the Journal of Molecular Biology recognizes his seminal contributions to science in general and structural biology in particular. John was first to use X-ray diffraction to solve the 3-dimensional structure of a protein, sperm-whale myoglobin, worthy of a Nobel Prize in Chemistry in 1962. John was the Founder and first Editor-in-Chief of the Journal of Molecular Biology, Deputy Chairman of the Laboratory of Molecular Biology and Head of its Division of Structural Studies, a Founder of the European Molecular Biology Organization, first Director-General of the European Molecular Biology Laboratory, and 33rd President of St. John's College, Oxford. In this personal perspective I relate how I came to know John as his postdoctoral fellow at the Laboratory of Molecular Biology in 1967 and as his biographer 45 years later.