Ophelia Weeks

Loss of Asxl1 leads to myelodysplastic syndrome-like disease in mice.

ASXL1 is mutated/deleted with high frequencies in multiple forms of myeloid malignancies, and its alterations are associated with poor prognosis. De novo ASXL1 mutations cause Bohring-Opitz syndrome characterized by multiple congenital malformations. We show that Asxl1 deletion in mice led to developmental abnormalities including dwarfism, anophthalmia, and 80% embryonic lethality. Surviving Asxl1(-/-) mice lived for up to 42 days and developed features of myelodysplastic syndrome (MDS), including dysplastic neutrophils and multiple lineage cytopenia. Asxl1(-/-) mice had a reduced hematopoietic stem cell (HSC) pool, and Asxl1(-/-) HSCs exhibited decreased hematopoietic repopulating capacity, with skewed cell differentiation favoring granulocytic lineage. Asxl1(+/-) mice also developed mild MDS-like disease, which could progress to MDS/myeloproliferative neoplasm, demonstrating a haploinsufficient effect of Asxl1 in the pathogenesis of myeloid malignancies. Asxl1 loss led to an increased apoptosis and mitosis in Lineage(-)c-Kit(+) (Lin(-)c-Kit(+)) cells, consistent with human MDS. Furthermore, Asxl1(-/-) Lin(-)c-Kit(+) cells exhibited decreased global levels of H3K27me3 and H3K4me3 and altered expression of genes regulating apoptosis (Bcl2, Bcl2l12, Bcl2l13). Collectively, we report a novel ASXL1 murine model that recapitulates human myeloid malignancies, implying that Asxl1 functions as a tumor suppressor to maintain hematopoietic cell homeostasis. Future work is necessary to clarify the contribution of microenvironment to the hematopoietic phenotypes observed in the constitutional Asxl1(-/-) mice.

Alzheimer’s β‐amyloid peptide blocks vascular endothelial growth factor mediated signaling via direct interaction with VEGFR‐2

J. Neurochem. (2010) 112, 66–76.

Effects of extracellular human immunodeficiency virus type 1 Vpr protein in primary rat cortical cell cultures

Recent evidence suggests that HIV-1 Vpr exists in soluble form in the serum and cerebrospinal fluid (CSF). Further, its abundance in the bloodstream, and the CSF, and its activity on other cell types suggest that it could have an effect on brain activity. Using mixed embryonic rat brain cultures as a model to examine the effects of physiological concentrations of extracellular Vpr protein, Vpr-induced cell death was observed. We also observed similar Vpr-induced effects in enriched primary cortical rat astrocytes, as well as in the C6 glioma cell line. Vpr-induced cell death observed in the astrocytic cells appeared to be caused primarily by a necrotic mechanism, although a few apoptotic nuclei were also present. We did not observe Vpr-induced effects on any primary cortical neurons, although we did observe Vpr-induced cell death in hippocampal neurons and astrocytes. Finally, we observed no cell cycle effects due to extracellular Vpr protein. This data points out that different cell types are affected by the toxic effects of extracellular Vpr protein, and that differential toxic effects of extracellular Vpr protein are observed in similar cell types.

Combined loss of Tet1 and Tet2 promotes B-cell, but not myeloid malignancies in mice

TET1/2/3 are methylcytosine dioxygenases that regulate cytosine hydroxymethylation. Tet1/2 are abundantly expressed in HSC/HPCs and are implicated in hematological malignancies. Tet2 deletion in mice causes myeloid malignancies, while Tet1-null mice develop B cell lymphoma after an extended period of latency. Interestingly, TET1/2 are often concomitantly downregulated in acute B-lymphocytic leukemia. Here, we investigated the overlapping and non-redundant functions of Tet1/2 using Tet1/2 double-knockout (DKO) mice. DKO and Tet2(-/-) HSC/HPCs show overlapping and unique 5 hmC and 5 mC profiles. DKO mice exhibit strikingly decreased incidence and delayed onset of myeloid malignancies in comparison to Tet2(-/-) mice and in contrast develop lethal B cell malignancies. Transcriptome analysis of DKO tumors reveals expression changes in many genes dysregulated in human B cell malignancies, including LMO2, BCL6, and MYC. These results highlight the critical roles of TET1/2 individually and together in the pathogenesis of hematological malignancies.

Estradiol and lithium chloride specifically alter NMDA receptor subunit NR1 mRNA and excitotoxicity in primary cultures

Glutamate facilitates calcium influx via NMDAR, and excess calcium influx increases excitotoxicity--a pathological characteristic of neurological diseases. Both 17beta-estradiol (E2) and lithium influence NMDAR expression/signaling and excitotoxicity. This led us to hypothesize that combined E2 and lithium will alter NMDAR expression and excitotoxicity. We tested this hypothesis using primary cell cultures from the cortex and hippocampus of C57BL/6J fetal mice pretreated with E2, lithium chloride (LiCl) and combined E2/LiCl for 12, 24 or 48 h. We examined cultures for brain cell type and changes in cell type caused by experimental procedures using glia and neuron gene specific primers. These cultures expressed increased glial fibrillary acidic protein (GFAP) mRNA with low neurofilament-heavy chain (NF-H) mRNA expression. Subsequent analysis of cortical cell cultures indicated that combined E2/LiCl decreased NR1 mRNA expression after a 12 and 48 h treatment period. Combined E2/LiCl also reduced NR1 mRNA expression in hippocampal cultures but only after a 48 h treatment period. LiCl-treated hippocampal cultures also reduced NR1 mRNA expression after a 24 and 48 h treatment. We next examined the response of 48 h pretreated cultures to a toxic level of glutamate. Excitotoxicity was measured using fluorescein diacetate/propidium iodide (FDA/PI) cell viability assay. Results from FDA/PI assay revealed that LiCl pretreatment increased viability for cortical cultures while E2 and combined E2/LiCl reduced viability. All pretreatments for hippocampal cultures failed to increase viability. Our results showed combined E2/LiCl reduced NR1 mRNA and prevented protection against glutamate excitotoxicity in glial primary cultures.

Tet2 loss leads to hypermutagenicity in haematopoietic stem/progenitor cells

TET2 is a dioxygenase that catalyses multiple steps of 5-methylcytosine oxidation. Although TET2 mutations frequently occur in various types of haematological malignancies, the mechanism by which they increase risk for these cancers remains poorly understood. Here we show that Tet2−/− mice develop spontaneous myeloid, T- and B-cell malignancies after long latencies. Exome sequencing of Tet2−/− tumours reveals accumulation of numerous mutations, including Apc, Nf1, Flt3, Cbl, Notch1 and Mll2, which are recurrently deleted/mutated in human haematological malignancies. Single-cell-targeted sequencing of wild-type and premalignant Tet2−/− Lin−c-Kit+ cells shows higher mutation frequencies in Tet2−/− cells. We further show that the increased mutational burden is particularly high at genomic sites that gained 5-hydroxymethylcytosine, where TET2 normally binds. Furthermore, TET2-mutated myeloid malignancy patients have significantly more mutational events than patients with wild-type TET2. Thus, Tet2 loss leads to hypermutagenicity in haematopoietic stem/progenitor cells, suggesting a novel TET2 loss-mediated mechanism of haematological malignancy pathogenesis.

CD40/CD40L interaction induces Aβ production and increases γ-secretase activity independently of tumor necrosis factor receptor associated factor (TRAF) signaling

CD40, a member of tumor necrosis factor receptor superfamily, and its cognate ligand CD40L are both elevated in the brain of Alzheimers disease (AD) patients compared to controls. We have shown that pharmacological or genetic interruption of CD40/CD40L interaction results in mitigation of AD-like pathology in vivo in transgenic AD mouse models, and in vitro. Recently, we showed that CD40L stimulation could increase Abeta levels via NFkappaB signaling, presumably through TRAFs. In the present work, using CD40 mutants, we show that CD40L can increase levels of Abeta(1-40), Abeta(1-42), sAPPbeta, sAPPalpha and CTFbeta independently of TRAF signaling. We report an increase in mature/immature APP ratio after CD40L treatment of CD40wt and CD40-mutant cells, reflecting alterations in APP trafficking. In addition, results from CD40L treatment of a neuroblastoma cell line over-expressing the C-99 APP fragment suggest that CD40L has an effect on gamma-secretase. Furthermore, inhibition of gamma-secretase activity significantly reduces sAPPbeta levels in the CD40L treated HEK/APPsw CD40wt and the CD40-mutant cells. The latter suggests CD40/CD40L interaction primarily acts on gamma-secretase and affects beta-secretase via a positive feedback mechanism. Taken together, our data suggest that CD40/CD40L interaction modulates APP processing independently of TRAF signaling.

The TET2 interactors and their links to hematological malignancies

Ten‐eleven translocation (TET) family proteins are dioxygenases that oxidize 5‐methylcytosine to 5‐hydroxymethylcytosine, 5‐formylcytosine, and 5‐carboxylcytosine in DNA, early steps of active DNA demethylation. TET2, the second member of TET protein family, is frequently mutated in patients with hematological malignancies, leading to aberrant DNA methylation profiling and decreased 5hmC levels. Located in the nucleus and acting as a DNA‐modifying enzyme, TET2 is thought to exert its function via TET2‐containing protein complexes. Identifying the interactome network of TET2 likely holds the key to uncover the mechanisms by which TET2 exerts its function in cells. Here, we review recent literature on TET2 interactors and discuss their possible roles in TET2 loss‐mediated dysregulation of hematopoiesis and pathogenesis of hematological malignancies.

Science Café Course: An Innovative Means of Improving Communication Skills of Undergraduate Biology Majors

To help bridge the increasing gap between scientists and the public, we developed an innovative two-semester course called Science Café. In this course, undergraduate biology majors learn to develop communication skills to be better able to explain science concepts and current developments in science to non-scientists. Students develop and host outreach events on various topics relevant to the community, thereby increasing interactions between budding scientists and the public. Such a Science Café course emphasizes development of science communication skills early, at the undergraduate level, and empowers students to use their science knowledge in everyday interactions with the public to increase science literacy, get involved in the local community and engage the public in a dialogue on various pressing science issues. We believe that undergraduate science majors can be great ambassadors for science and are often overlooked since many aspire to go on to medical/veterinary/pharmacy schools. However, science communication skills are especially important for these types of students because when they become healthcare professionals, they will interact with the public as part of their everyday jobs and can thus be great representatives for the field.

Confluence: A Seminar Series as a Teaching Tool

In 2007 Florida International University (FIU) received NIH, NSF, and internal support to create a curriculum that was quantitative in nature, and that incorporated some of the most contemporary approaches to the classroom. The QBIC (Quantifying Biology In the Classroom; http://qbic. fiu.edu) Program is a specialized program in the Department of Biology specifically set up to implement ‘vision and change’ principles in the department’s overall approach to students. It is now an optional track within the Department of Biological Sciences. We discuss the major objectives of this series, the affect areas that it addresses, as well as how students incorporate the series’ lessons for their own professional development. It became apparent to us anecdotally that our undergraduates were making career choices without the awareness of the many other viable career options in biology. This is not an issue unique to our institution and other authors (1) have discussed steps to address career choice and exposure. At our institution, we created a number of career development initiatives, one of which was a seminar series called “Confluence: where life and science meet.” For this series we invite science professionals from around the country to give a seminar, mostly to undergraduates, not only on the technical specifics of their field, but also on their personal life story, and how that story informed their career choice. After the seminar, the speaker sits down with a QBIC faculty member for a half-hour interview where he or she is able to go into more specifics about the themes from the seminar. The interview is videotaped in front of a live student-only audience in a film studio on campus. The recording is published on the series’ website (http://qbic.fiu. edu/confluence). In this article we discuss using the series to address issues of identity, and how our video blog can be used in other classrooms to achieve similar objectives for science students nationally.