Faculty Bibliography

Mutations in genes encoding Isocitrate Dehydrogenase (IDH) isoforms are found in80%of low-grade gliomas (LGGs). Sequencing of LGGs has revealed branching cancer genetics; mutant IDH1 astrocytomas contain p53 and ATRX loss of function mutations, while IDH1-mutated oligodendrogliomas have a different set of mutations that includes chr 1p/19q co-deletion. The IDH mutation is a gain-of-function change at its catalytic core that results in the production of (R)-2-hydroxyglutarate, an oncometabolite, which causes characteristic DNA and histone hypermethylation changes that may contribute to tumorigenesis. Mouse models have thus far failed to demonstrate the role of IDH1 mutations in LGG formation. To test the hypothesis that mutant IDH1 is a driver of gliomagenesis, we use human embryonic stem cell (hESC)-derived neural stem cells (NSCs) to overexpress mutant IDH1 protein in combination with p53 and ATRX knockdown. We have generated twelve NSC lines that harbor combinations of mutant IDH1, wt IDH1 or an empty vector, in combination with ATRX and/or p53 knockdown. Our preliminary data indicate that mutantIDH1 does not alter the proliferative capacity of NSCs, as shown by cell cycle analysis and Ki67 staining, but paradoxically increases their apoptotic rate (15.8% vs 5.9% n = 4), a phenotype that is exacerbated by ATRX knockdown, as detected by annexin V and TUNEL staining (17.7% vs 2.6% n = 3). shRNA-mediated knockdown of p53 salvages the pro-apoptotic phenotype of mutant IDH1 and ATRX NSCs. Furthermore, initial observations suggest that mutant IDH1 biases NSCs toward glial fates, as evidenced by upregulation of the CD44 cell surface marker. We are currently testing the effects of IDH1 mutation on i) NSC differentiation to astrocytic and neuronal lineages, ii) NSC metabolism via metabolomics profiling and iii) in vivo tumorigenesis. We propose that mutant IDH1 alters the differentiation program of human NSCs toward glial rather than neuronal fates

In order to localize the neural circuits involved in generating behaviors, it is necessary to assign activity onto anatomical maps of the nervous system. Using brain registration across hundreds of larval zebrafish, we have built an expandable open-source atlas containing molecular labels and definitions of anatomical regions, the Z-Brain. Using this platform and immunohistochemical detection of phosphorylated extracellular signal-regulated kinase (ERK) as a readout of neural activity, we have developed a system to create and contextualize whole-brain maps of stimulus- and behavior-dependent neural activity. This mitogen-activated protein kinase (MAP)-mapping assay is technically simple, and data analysis is completely automated. Because MAP-mapping is performed on freely swimming fish, it is applicable to studies of nearly any stimulus or behavior. Here we demonstrate our high-throughput approach using pharmacological, visual and noxious stimuli, as well as hunting and feeding. The resultant maps outline hundreds of areas associated with behaviors.

Dysregulated endoplasmic reticulum stress and phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha) are associated with pancreatic beta-cell failure and diabetes. Here we report the first homozygous mutation in the PPP1R15B gene (also known as constitutive repressor of eIF2alpha phosphorylation, CReP), encoding the regulatory subunit of an eIF2alpha-specific phosphatase, in two siblings affected by a novel syndrome of diabetes of youth, with short stature, intellectual disability and microcephaly. The R658C mutation in PPP1R15B affects a conserved amino acid within the domain important for protein phosphatase 1 (PP1) binding. The R658C mutation decreases PP1 binding and eIF2alpha dephosphorylation, and results in beta-cell apoptosis. Our findings support the concept that dysregulated eIF2alpha phosphorylation, whether decreased by mutation of the kinase (EIF2AK3) in Wolcott-Rallison syndrome or increased by mutation of the phosphatase (PPP1R15B), is deleterious to beta-cells and other secretory tissues, resulting in diabetes associated with multi-system abnormalities.

DNA double-strand breaks (DSBs) disrupt the continuity of chromosomes and their repair by error-free mechanisms is essential to preserve genome integrity. Microhomology-mediated end joining (MMEJ) is an error-prone repair mechanism that involves alignment of microhomologous sequences internal to the broken ends before joining, and is associated with deletions and insertions that mark the original break site, as well as chromosome translocations. Whether MMEJ has a physiological role or is simply a back-up repair mechanism is a matter of debate. Here we review recent findings pertaining to the mechanism of MMEJ and discuss its role in normal and cancer cells.

Curly, described almost a century ago, is one of the most frequently used markers in Drosophila genetics. Despite this the molecular identity of Curly has remained obscure. Here we show that Curly mutations arise in the gene dual oxidase (duox), which encodes a reactive oxygen species (ROS) generating NADPH oxidase. Using Curly mutations and RNA interference (RNAi), we demonstrate that Duox autonomously stabilizes the wing on the last day of pupal development. Through genetic suppression studies, we identify a novel heme peroxidase, Curly Su (Cysu) that acts with Duox to form the wing. Ultrastructural analysis suggests that Duox and Cysu are required in the wing to bond and adhere the dorsal and ventral cuticle surfaces during its maturation. In Drosophila, Duox is best known for its role in the killing of pathogens by generating bactericidal ROS. Our work adds to a growing number of studies suggesting that Duox's primary function is more structural, helping to form extracellular and cuticle structures in conjunction with peroxidases.

The Suppressor of Cytokine Signaling (SOCS) proteins are critical, highly conserved feedback inhibitors of signal transduction cascades. The family of SOCS proteins is divided into two groups: ancestral and vertebrate-specific SOCS proteins. Vertebrate-specific SOCS proteins have been heavily studied as a result of their strong mutant phenotypes. However, the ancestral clade remains less studied, a potential result of genetic redundancies in mammals. Use of the genetically tractable organism Drosophila melanogaster enables in vivo assessment of signaling components and mechanisms with less concern about the functional redundancy observed in mammals. In this study, we investigated how the SOCS family member Suppressor of Cytokine Signaling at 36E (Socs36E) attenuates Janus Kinase/Signal Transducer and Activator of Transcription (Jak/STAT) activation during specification of motile border cells in Drosophila oogenesis. We found that Socs36E genetically interacts with the Cullin2 (Cul2) scaffolding protein. Like Socs36E, Cul2 is required to limit the number of motile cells in egg chambers. We demonstrated that loss of Cul2 in the follicle cells significantly increased nuclear STAT protein levels, which resulted in additional cells acquiring invasive properties. Further, reduction of Cul2 suppressed border cell migration defects that occur in a Stat92E-sensitized genetic background. Our data incorporated Cul2 into a previously described Jak/STAT-directed genetic regulatory network that is required to generate a discrete boundary between cell fates. We also found that Socs36E is able to attenuate STAT activity in the egg chamber when it does not have a functional SOCS box. Collectively, this work contributes mechanistic insight to a Jak/STAT regulatory genetic circuit, and suggests that Socs36E regulates Jak/STAT signaling via a Cul2-dependent mechanism, as well as by a Cullin-independent manner, in vivo.

PURPOSE: We tested what native features have been preserved with a new culture protocol for adult human RPE. METHODS: We cultured RPE from adult human eyes. Standard protocols for immunohistochemistry, electron microscopy, electrophysiology, fluid transport, and ELISA were used. RESULTS: Confluent monolayers of adult human RPE cultures exhibit characteristics of native RPE. Immunohistochemistry demonstrated polarized expression of RPE markers. Electron microscopy illustrated characteristics of native RPE. The mean transepithelial potential (TEP) was 1.19 +/- 0.24 mV (mean +/- SEM, n = 31), apical positive, and the mean transepithelial resistance (RT) was 178.7 +/- 9.9 Omega.cm2 (mean +/- SEM, n = 31). Application of 100 muM adenosine triphosphate (ATP) apically increased net fluid absorption (Jv) by 6.11 +/- 0.53 muL.cm2.h-1 (mean +/- SEM, n = 6) and TEP by 0.33 +/- 0.048 mV (mean +/- SEM, n = 25). Gene expression of cultured RPE was comparable to native adult RPE (n = 5); however, native RPE RNA was harvested between 24 and 40 hours after death and, therefore, may not accurately reflect healthy native RPE. Vascular endothelial growth factor secreted preferentially basally 2582 +/- 146 pg/mL/d, compared to an apical secretion of 1548 +/- 162 pg/mL/d (n = 14, P < 0.01), while PEDF preferentially secreted apically 1487 +/- 280 ng/mL/d compared to a basolateral secretion of 864 +/- 132 ng/mL/d (n = 14, P < 0.01). CONCLUSIONS: The new culture model preserves native RPE morphology, electrophysiology, and gene and protein expression patterns, and may be a useful model to study RPE physiology, disease, and transplantation.

To facilitate large scale functional studies in Drosophila, the Drosophila Transgenic RNAi Project (TRiP) at Harvard Medical School (HMS) was established along with several goals: developing efficient vectors for RNAi that work in all tissues, generating a genome scale collection of RNAi stocks with input from the community, distributing the lines as they are generated through existing stock centers, validating as many lines as possible using RT-qPCR and phenotypic analyses, and developing tools and web resources for identifying RNAi lines and retrieving existing information on their quality. With these goals in mind, here we describe in detail the various tools we developed and the status of the collection, which is currently comprised of 11,491 lines and covering 71% of Drosophila genes. Data on the characterization of the lines either by RT-qPCR or phenotype is available on a dedicated web site, the RNAi Stock Validation and Phenotypes Project (RSVP; www.flyrnai.org/RSVP.html), and stocks are available from three stock centers, the Bloomington Drosophila Stock Center (USA), National Institute of Genetics (Japan), and TsingHua Fly Center (China).

BACKGROUND: Diabetes and aging are known risk factors for impaired neovascularization in response to ischemic insult, resulting in chronic wounds, and poor outcomes following myocardial infarction and cerebrovascular injury. Hypoxia-inducible factor (HIF)-1alpha, has been identified as a critical regulator of the response to ischemic injury and is dysfunctional in diabetic and elderly patients. To better understand the role of this master hypoxia regulator within cutaneous tissue, the authors generated and evaluated a fibroblast-specific HIF-1alpha knockout mouse model. METHODS: The authors generated floxed HIF-1 mice (HIF-1) by introducing loxP sites around exon 1 of the HIF-1 allele in C57BL/6J mice. Fibroblast-restricted HIF-1alpha knockout (FbKO) mice were generated by breeding our HIF-1 with tamoxifen-inducible Col1a2-Cre mice (Col1a2-CreER). HIF-1alpha knockout was evaluated on a DNA, RNA, and protein level. Knockout and wild-type mice were subjected to ischemic flap and wound healing models, and CD31 immunohistochemistry was performed to assess vascularity of healed wounds. RESULTS: Quantitative real-time polymerase chain reaction of FbKO skin demonstrated significantly reduced Hif1 and Vegfa expression compared with wild-type. This finding was confirmed at the protein level (p < 0.05). HIF-1alpha knockout mice showed significantly impaired revascularization of ischemic tissue and wound closure and vascularity (p < 0.05). CONCLUSIONS: Loss of HIF-1alpha from fibroblasts results in delayed wound healing, reduced wound vascularity, and significant impairment in the ischemic neovascular response. These findings provide new insight into the importance of cell-specific responses to hypoxia during cutaneous neovascularization.

Invariant natural killer T (iNKT) cells recognize glycolipids as antigens and diversify into NKT1 (IFN-gamma), NKT2 (IL-4), and NKT17 (IL-17) functional subsets while developing in the thymus. Mechanisms that govern the balance between these functional subsets are poorly understood due, partly, to the lack of distinguishing surface markers. Here we identify the heparan sulfate proteoglycan syndecan-1 (sdc1) as a specific marker of naive thymic NKT17 cells in mice and show that sdc1 deficiency significantly increases thymic NKT17 cells at the expense of NKT1 cells, leading to impaired iNKT cell-derived IFN-gamma, both in vitro and in vivo. Using surface expression of sdc1 to identify NKT17 cells, we confirm differential tissue localization and interstrain variability of NKT17 cells, and reveal that NKT17 cells express high levels of TCR-beta, preferentially use Vbeta8, and are more highly sensitive to a-GalCer than to CD3/CD28 stimulation. These findings provide a novel, noninvasive, simple method for identification, and viable sorting of naive NKT17 cells from unmanipulated mice, and suggest that sdc1 expression negatively regulates homeostasis in iNKT cells. In addition, these findings lay the groundwork for investigating the mechanisms by which sdc1 regulates NKT17 cells.