Andrew D. Strand

Promoter-specific regulation of MyoD binding and signal transduction cooperate to pattern gene expression.

We used expression arrays and chromatin immunoprecipitation assays to demonstrate that myogenesis consists of discrete subprograms of gene expression regulated by MyoD. Approximately 5% of assayed genes alter expression in a specific temporal sequence, and more than 1% are regulated by MyoD without the synthesis of additional transcription factors. MyoD regulates genes expressed at different times during myogenesis, and promoter-specific regulation of MyoD binding is a major mechanism of patterning gene expression. In addition, p38 kinase activity is necessary for the expression of a restricted subset of genes regulated by MyoD, but not for MyoD binding. The identification of distinct molecular mechanisms that regulate discrete subprograms of myogenesis should facilitate analyses of differentiation in normal development and disease.

Dysfunction of the cholesterol biosynthetic pathway in Huntington's disease.

The expansion of a polyglutamine tract in the ubiquitously expressed huntingtin protein causes Huntingtons disease (HD), a dominantly inherited neurodegenerative disease. We show that the activity of the cholesterol biosynthetic pathway is altered in HD. In particular, the transcription of key genes of the cholesterol biosynthetic pathway is severely affected in vivo in brain tissue from HD mice and in human postmortem striatal and cortical tissue; this molecular dysfunction is biologically relevant because cholesterol biosynthesis is reduced in cultured human HD cells, and total cholesterol mass is significantly decreased in the CNS of HD mice and in brain-derived ST14A cells in which the expression of mutant huntingtin has been turned on. The transcription of the genes of the cholesterol biosynthetic pathway is regulated via the activity of sterol regulatory element-binding proteins (SREBPs), and we found an ∼50% reduction in the amount of the active nuclear form of SREBP in HD cells and mouse brain tissue. As a consequence, mutant huntingtin reduces the transactivation of an SRE-luciferase construct even under conditions of SREBP overexpression or in the presence of an exogenous N-terminal active form of SREBP. Finally, the addition of exogenous cholesterol to striatal neurons expressing mutant huntingtin prevents their death in a dose-dependent manner. We conclude that the cholesterol biosynthetic pathway is impaired in HD cells, mice, and human subjects, and that the search for HD therapies should also consider cholesterol levels as both a potential target and disease biomarker.

Expression profiling of Huntington's disease models suggests that brain-derived neurotrophic factor depletion plays a major role in striatal degeneration.

Many pathways have been proposed as contributing to Huntingtons disease (HD) pathogenesis, but generally the in vivo effects of their perturbation have not been compared with reference data from human patients. Here we examine how accurately mechanistically motivated and genetic HD models recapitulate the striatal gene expression phenotype of human HD. The representative genetic model was the R6/2 transgenic mouse, which expresses a fragment of the huntingtin protein containing a long CAG repeat. Pathogenic mechanisms examined include mitochondrial dysfunction; profiled in 3-nitropropionic acid-treated rats, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice, and PGC-1α knock-out mice; and depletion of brain-derived neurotrophic factor (BDNF) using heterozygous and forebrain-specific BDNF-knock-out mice (BDNF HET , Emx-BDNF KO ). Based on striatal gene expression, we find the BDNF models, both heterozygous and homozygous knock-outs, to be more like human HD than the other HD models. This implicates reduced trophic support as a major pathway contributing to striatal degeneration in HD. Because the majority of striatal BDNF is synthesized by cortical neurons, the data also imply that cortical dysfunction contributes to HDs hallmark effects on the basal ganglia. Finally, the results suggest that striatal lesions caused by mitochondrial toxins may arise via pathways different from those that drive neurodegeneration in HD. Based on these findings, we present a testable model of HD pathogenesis that, unlike most models, begins to account for regional specificity in human HD and the absence of such specificity in genetic mouse models of HD.

BMP-2 mediates retinoid-induced apoptosis in medulloblastoma cells through a paracrine effect

The mechanisms of retinoid activity in tumors remain largely unknown. Here we establish that retinoids cause extensive apoptosis of medulloblastoma cells. In a xenograft model, retinoids largely abrogated tumor growth. Using receptor-specific retinoid agonists, we defined a subset of mRNAs that were induced by all active retinoids in retinoid-sensitive cell lines. We also identified bone morphogenetic protein-2 (BMP-2) as a candidate mediator of retinoid activity. BMP-2 protein induced medulloblastoma cell apoptosis, whereas the BMP-2 antagonist noggin blocked both retinoid and BMP-2-induced apoptosis. BMP-2 also induced p38 mitogen-activated protein kinase (MAPK), which is necessary for BMP-2- and retinoid-induced apoptosis. Retinoid-resistant medulloblastoma cells underwent apoptosis when treated with BMP-2 or when cultured with retinoid-sensitive medulloblastoma cells. Retinoid-induced expression of BMP-2 is thus necessary and sufficient for apoptosis of retinoid-responsive cells, and expression of BMP-2 by retinoid-sensitive cells is sufficient to induce apoptosis in surrounding retinoid-resistant cells.

Altered transcription in yeast expressing expanded polyglutamine

Expanded polyglutamine tracts are responsible for at least eight fatal neurodegenerative diseases. In mouse models, proteins with expanded polyglutamine cause transcriptional dysregulation before onset of symptoms, suggesting that this dysregulation may be an early event in polyglutamine pathogenesis. Transcriptional dysregulation and cellular toxicity may be due to interaction between expanded polyglutamine and the histone acetyltransferase CREB-binding protein. To determine whether polyglutamine-mediated transcriptional dysregulation occurs in yeast, we expressed polyglutamine tracts in Saccharomyces cerevisiae. Gene expression profiles were determined for strains expressing either a cytoplasmic or nuclear protein with 23 or 75 glutamines, and these profiles were compared to existing profiles of mutant yeast strains. Transcriptional induction of genes encoding chaperones and heat-shock factors was caused by expression of expanded polyglutamine in either the nucleus or cytoplasm. Transcriptional repression was most prominent in yeast expressing nuclear expanded polyglutamine and was similar to profiles of yeast strains deleted for components of the histone acetyltransferase complex Spt/Ada/Gcn5 acetyltransferase (SAGA). The promoter from one affected gene (PHO84) was repressed by expanded polyglutamine in a reporter gene assay, and this effect was mitigated by the histone deacetylase inhibitor, Trichostatin A. Consistent with an effect on SAGA, nuclear expanded polyglutamine enhanced the toxicity of a deletion in the SAGA component SPT3. Thus, an early component of polyglutamine toxicity, transcriptional dysregulation, is conserved in yeast and is pharmacologically antagonized by a histone deacetylase inhibitor. These results suggest a therapeutic approach for treatment of polyglutamine diseases and provide the potential for yeast-based screens for agents that reverse polyglutamine toxicity.

Distinct neuroinflammatory profile in post-mortem human Huntington's disease

Neuroinflammation is a prominent feature of many neurodegenerative diseases, however, little is known about neuroinflammation in Huntingtons disease. We used quantitative real time-PCR to compare the expression level of neuroinflammation-associated mediators in the striatum, cortex, and cerebellum from post-mortem Huntingtons disease patient samples with controls. We found increased expression of several key inflammatory mediators, including CCL2 and IL-10, specifically in the striatum of Huntingtons disease patients, the main area affected by this pathology. Remarkably, we also found upregulation of IL-6, IL-8, and MMP9, in the cortex and notably the cerebellum, a brain area commonly thought to be spared by Huntingtons disease. Our data suggest that neuroinflammation is a prominent feature associated with Huntingtons disease and may constitute a novel target for therapeutic intervention.

A Large Number of Protein Expression Changes Occur Early in Life and Precede Phenotype Onset in a Mouse Model for Huntington Disease

Huntington disease (HD) is fatal in humans within 15–20 years of symptomatic disease. Although late stage HD has been studied extensively, protein expression changes that occur at the early stages of disease and during disease progression have not been reported. In this study, we used a large two-dimensional gel/mass spectrometry-based proteomics approach to investigate HD-induced protein expression alterations and their kinetics at very early stages and during the course of disease. The murine HD model R6/2 was investigated at 2, 4, 6, 8, and 12 weeks of age, corresponding to absence of disease and early, intermediate, and late stage HD. Unexpectedly the most HD stage-specific protein changes (71–100%) as well as a drastic alteration (almost 6% of the proteome) in protein expression occurred already as early as 2 weeks of age. Early changes included mainly the up-regulation of proteins involved in glycolysis/gluconeogenesis and the down-regulation of the actin cytoskeleton. This suggests a period of highly variable protein expression that precedes the onset of HD phenotypes. Although an up-regulation of glycolysis/gluconeogenesis-related protein alterations remained dominant during HD progression, late stage alterations at 12 weeks showed an up-regulation of proteins involved in proteasomal function. The early changes in HD coincide with a peak in protein alteration during normal mouse development at 2 weeks of age that may be responsible for these massive changes. Protein and mRNA data sets showed a large overlap on the level of affected pathways but not single proteins/mRNAs. Our observations suggest that HD is characterized by a highly dynamic disease pathology not represented by linear protein concentration alterations over the course of disease.

Brain-specific Proteins Decline in the Cerebrospinal Fluid of Humans with Huntington Disease

We integrated five sets of proteomics data profiling the constituents of cerebrospinal fluid (CSF) derived from Huntington disease (HD)-affected and -unaffected individuals with genomics data profiling various human and mouse tissues, including the human HD brain. Based on an integrated analysis, we found that brain-specific proteins are 1.8 times more likely to be observed in CSF than in plasma, that brain-specific proteins tend to decrease in HD CSF compared with unaffected CSF, and that 81% of brain-specific proteins have quantitative changes concordant with transcriptional changes identified in different regions of HD brain. The proteins found to increase in HD CSF tend to be liver-associated. These protein changes are consistent with neurodegeneration, microgliosis, and astrocytosis known to occur in HD. We also discuss concordance between laboratories and find that ratios of individual proteins can vary greatly, but the overall trends with respect to brain or liver specificity were consistent. Concordance is highest between the two laboratories observing the largest numbers of proteins.

A technology platform to assess multiple cancer agents simultaneously within a patient’s tumor

Simultaneous in vivo assessment of multiple cancer drugs and drug combinations using microinjection technology predicts systemic response in model tumors and has shown feasibility for assessment of drug efficacy in a pilot study in cancer patients. There’s no place like the human Animal models of human tumors and dish cultures of cancer cells are not sufficient to predict an individual patient’s response to therapy. In the emerging era of personalized medicine, why limit ourselves to rodent models and engineered in vitro tumor models when we can study a drug directly in the patient’s tumor? This question was answered by Klinghoffer et al. by creating a microinjection system called CIVO that delivers small doses of up to eight different drugs simultaneously, directly into the tumor. The tumors could then be removed and evaluated for various markers of cancer response; in short, the authors looked for markers of cell death and drug-related mechanisms of action. By using an injection-tracking dye, Klinghoffer and colleagues could see where the drug was deposited and then use an automated analyzer for quantitative image processing along the 6-mm injection tract. In mouse models of human lymphoma, the authors were able to correctly predict systemic responsiveness to doxorubicin or vincristine—or not, in the case of resistant lymphomas. They also uncovered unexpected drug sensitivities, which were not picked up by traditional cell culture, including to novel anticancer agents, and confirmed these in vivo. The authors pilot-tested the device in dog and human patients, demonstrating the ability of CIVO to inject and track local tumor response to chemotherapies. Ultimately, such a personalized approach to drug sensitivity testing will allow for rational selection of therapeutics while sparing patients the pain—and time—associated with ineffective treatments. A fundamental problem in cancer drug development is that antitumor efficacy in preclinical cancer models does not translate faithfully to patient outcomes. Much of early cancer drug discovery is performed under in vitro conditions in cell-based models that poorly represent actual malignancies. To address this inconsistency, we have developed a technology platform called CIVO, which enables simultaneous assessment of up to eight drugs or drug combinations within a single solid tumor in vivo. The platform is currently designed for use in animal models of cancer and patients with superficial tumors but can be modified for investigation of deeper-seated malignancies. In xenograft lymphoma models, CIVO microinjection of well-characterized anticancer agents (vincristine, doxorubicin, mafosfamide, and prednisolone) induced spatially defined cellular changes around sites of drug exposure, specific to the known mechanisms of action of each drug. The observed localized responses predicted responses to systemically delivered drugs in animals. In pair-matched lymphoma models, CIVO correctly demonstrated tumor resistance to doxorubicin and vincristine and an unexpected enhanced sensitivity to mafosfamide in multidrug-resistant lymphomas compared with chemotherapy-naïve lymphomas. A CIVO-enabled in vivo screen of 97 approved oncology agents revealed a novel mTOR (mammalian target of rapamycin) pathway inhibitor that exhibits significantly increased tumor-killing activity in the drug-resistant setting compared with chemotherapy-naïve tumors. Finally, feasibility studies to assess the use of CIVO in human and canine patients demonstrated that microinjection of drugs is toxicity-sparing while inducing robust, easily tracked, drug-specific responses in autochthonous tumors, setting the stage for further application of this technology in clinical trials.

SA85-1 proteins of Trypanosoma cruzi lack sialidase activity

Trypanosoma cruzi infects a wide range of mammalian species, and replicates within many different cell types [1]. Numerous reports indicate that trypomastigote surface proteins facilitate adhesion and invasion of mammalian cells [1]. Recently, several genes that are expressed specifically by mammalianstage parasites have been shown to encode surface proteins with homology to sialidases [2]. Two of these, genes have been shown to encode functional sialidases when expressed in Escherichia coli [3,4]. Other proteins, with homology to sialidase, apparently lack intrinsic sialidase activity, and their function remains unclear [4]. In addition, sialidase and trans-sialidase activities appear to be catalyzed by the same protein, and to represent coupled steps of the same reaction; the former representing sialic acid transfer to water, and the latter transfer to another carbohydrate [4]. We have described the SA85-1 family of surface proteins expressed by mammalianstage parasites that have homology to sialidases [5,6]. Hybridization to a cDNA named 1.1 (cl.1), or reactivity to antibodies purified with a recombinant protein generated by expression of cl. 1 (anti-cl. 1 antibodies) define