Disease-relevant Phenotypes Research Articles

Smoking-Induced CXCL14 Expression in the Human Airway Epithelium Links Chronic Obstructive Pulmonary Disease to Lung Cancer

CXCL14, a recently described epithelial cytokine, plays putative multiple roles in inflammation and carcinogenesis. In the context that chronic obstructive pulmonary disease (COPD) and lung cancer are both smoking-related disorders associated with airway epithelial disorder and inflammation, we hypothesized that the airway epithelium responds to cigarette smoking with altered CXCL14 gene expression, contributing to the disease-relevant phenotype. Using genome-wide microarrays with subsequent immunohistochemical analysis, the data demonstrate that the expression of CXCL14 is up-regulated in the airway epithelium of healthy smokers and further increased in COPD smokers, especially within hyperplastic/metaplastic lesions, in association with multiple genes relevant to epithelial structural integrity and cancer. In vitro experiments revealed that the expression of CXCL14 is induced in the differentiated airway epithelium by cigarette smoke extract, and that epidermal growth factor mediates CXCL14 up-regulation in the airway epithelium through its effects on the basal stem/progenitor cell population. Analyses of two independent lung cancer cohorts revealed a dramatic up-regulation of CXCL14 expression in adenocarcinoma and squamous-cell carcinoma. High expression of the COPD-associated CXCL14-correlating cluster of genes was linked in lung adenocarcinoma with poor survival. These data suggest that the smoking-induced expression of CXCL14 in the airway epithelium represents a novel potential molecular link between smoking-associated airway epithelial injury, COPD, and lung cancer.

Modeling key pathological features of frontotemporal dementia with C9ORF72 repeat expansion in iPSC-derived human neurons

The recently identified GGGGCC repeat expansion in the noncoding region of C9ORF72 is the most common pathogenic mutation in patients with frontotemporal dementia (FTD) or amyotrophic lateral sclerosis (ALS). We generated a human neuronal model and investigated the pathological phenotypes of human neurons containing GGGGCC repeat expansions. Skin biopsies were obtained from two subjects who had >1,000 GGGGCC repeats in C9ORF72 and their respective fibroblasts were used to generate multiple induced pluripotent stem cell (iPSC) lines. After extensive characterization, two iPSC lines from each subject were selected, differentiated into postmitotic neurons, and compared with control neurons to identify disease-relevant phenotypes. Expanded GGGGCC repeats exhibit instability during reprogramming and neuronal differentiation of iPSCs. RNA foci containing GGGGCC repeats were present in some iPSCs, iPSC-derived human neurons and primary fibroblasts. The percentage of cells with foci and the number of foci per cell appeared to be determined not simply by repeat length but also by other factors. These RNA foci do not seem to sequester several major RNA-binding proteins. Moreover, repeat-associated non-ATG (RAN) translation products were detected in human neurons with GGGGCC repeat expansions and these neurons showed significantly elevated p62 levels and increased sensitivity to cellular stress induced by autophagy inhibitors. Our findings demonstrate that key neuropathological features of FTD/ALS with GGGGCC repeat expansions can be recapitulated in iPSC-derived human neurons and also suggest that compromised autophagy function may represent a novel underlying pathogenic mechanism.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-013-1149-y) contains supplementary material, which is available to authorized users.

Human iPSC-derived neurons reveal novel effects of the APP London mutation and show cell fate-specific Alzheimer’s disease relevant phenotypes

Human iPSC-derived neurons reveal novel effects of the APP London mutation and show cell fate-specific Alzheimer’s disease relevant phenotypes

Structural basis of kynurenine 3-monooxygenase inhibition

Inhibition of kynurenine 3-monooxygenase (KMO), an enzyme in the eukaryotic tryptophan catabolic pathway (i.e. kynurenine pathway), leads to amelioration of Huntington’s disease-relevant phenotypes in yeast, fruit fly, and mouse models1–5, as well as a mouse model of Alzheimer’s disease3. KMO is a FAD-dependent monooxygenase, and is located in the outer mitochondrial membrane where it converts L-kynurenine to 3-hydroxykynurenine. Perturbations in the levels of kynurenine pathway metabolites have been linked to the pathogenesis of a spectrum of brain disorders6, as well as cancer7,8, and several peripheral inflammatory conditions9. Despite the importance of KMO as a target for neurodegenerative disease, the molecular basis of KMO inhibition by available lead compounds has remained hitherto unknown. Here we report the first crystal structure of KMO, in the free form and in complex with the tight-binding inhibitor UPF 648. UPF 648 binds close to the FAD cofactor and perturbs the local active site structure, preventing productive binding of the substrate kynurenine. Functional assays and targeted mutagenesis revealed that the active site architecture and UPF 648 binding are essentially identical in human KMO, validating the yeast KMO:UPF 648 structure as a template for structure-based drug design. This will inform the search for new KMO inhibitors that are able to cross the blood-brain barrier in targeted therapies against neurodegenerative diseases such as Huntington’s, Alzheimer’s, and Parkinson’s diseases.

MicroRNA-31 Sensitizes Human Breast Cells to Apoptosis by Direct Targeting of Protein Kinase C ϵ (PKCϵ)

MicroRNAs post-transcriptionally regulate gene expression and thereby contribute to the modulation of numerous complex and disease-relevant cellular phenotypes, including cell proliferation, cell motility, apoptosis, and stress response. In breast cancer cell systems, miR-31 has been shown to inhibit cell migration, invasion, and metastasis. Here, we link enhanced expression of miR-31 to the inhibition of the oncogenic NF-κB pathway, thus supporting the tumor-suppressive function of this microRNA. We identified protein kinase C epsilon (PKCε encoded by the PRKCE gene) as a novel direct target of miR-31 and show that down-regulation of PKCε results in impaired NF-κB signaling, enhanced apoptosis, and increased sensitivity of MCF10A breast epithelial and MDA-MB-231 triple-negative breast cancer cells toward ionizing radiation as well as treatment with chemotherapeutics. Mechanistically, we attribute this sensitization to anti-cancer treatments to the PRKCE-mediated down-regulation of the anti-apoptotic factor BCL2. In clinical breast cancer samples, high BCL2 expression was associated with poor prognosis. Furthermore, we found an inverse correlation between miR-31 and BCL2 expression, highlighting the functional relevance of the indirect down-regulation of BCL2 via direct targeting of PRKCE by miR-31.

A novel human embryonic stem cell‐derived Huntington's disease neuronal model exhibits mutant huntingtin (mHTT) aggregates and soluble mHTT‐dependent neurodegeneration

Most neurodegenerative diseases are linked to aberrant accumulation of aggregation-prone proteins. Among them, Huntington's disease (HD) is caused by an expanded polyglutamine repeat stretch in the N terminus of the mutant huntingtin protein (mHTT), which gets cleaved and aggregates in the brain. Recently established human induced pluripotent stem cell-derived HD neurons exhibit some disease-relevant phenotypes and provide tools for HD research. However, they have limitations such as genetic heterogeneity and an absence of mHTT aggregates and lack a robust neurodegeneration phenotype. In addition, the relationship between the phenotype and mHTT levels has not been elucidated. Herein, we present a human embryonic stem cell (hESC)-derived HD neuronal model expressing HTTexon1 fragments, which addresses the deficiencies enumerated above. The wild-type and HD lines are derived from an isogenic background and exhibit insoluble mHTT aggregates and neurodegeneration. We also demonstrate a quantitative relationship between neurodegeneration and soluble monomeric (but not oligomeric or aggregated) mHTT levels. Reduction of ~10% of mHTT is sufficient to prevent toxicity, whereas ~90% reduction of wild-type HTT is safe and well-tolerated in these cells. A known HD toxicity modifier (Rhes) showed expected rescue of neurodegeneration. Therefore, the hESC-derived neuronal models complement existing induced pluripotent stem cell-derived neuronal models and provide valuable tools for HD research.

Disease Modeling and Drug Discovery Using Induced Pluripotent Stem Cells From CINCA Syndrome Patients

AbstractAbstract 4680Chronic infantile neurologic cutaneous and articular syndrome (CINCA syndrome; MIM #607715) is a dominantly inherited autoinflammatory disease characterized by systemic inflammation with an urticaria-like rash, neurological manifestations, and arthropathy. NLRP3 mutation is the first and so far the only identified mutation that is responsible for CINCA syndrome. NLRP3 is expressed mainly in myelomonocytic lineage cells and chondrocytes, and acts as an intracellular sensor of danger signals from various cellular insults. In normal macrophages, a first stimulus, such as lipopolysaccharide (LPS), induces the synthesis of NLRP3 and the biologically inactive proIL-1β. A second stimulus, such as ATP, enhances the assembly of a protein complex called the NLRP3-inflammasome. The inflammasome contains caspase1, which executes the proteolytic maturation and secretion of IL-1β. While normal monocytes/macrophages show no or limited IL-1β secretion in response to LPS stimulation alone, CINCA patients’ cells exhibit robust IL-1β secretion, because the mutant NLRP3-inflammasome is auto-activated without the need for any second stimulus.While approximately half of CINCA patients carry heterozygous gain-of-function mutations of the NLRP3 gene, 30 to 40% of all patients have mutations in NLRP3 in only a small number of somatic cells. Since the population of mutant cells is relatively small (4.2–35.8% in blood cells), it remains controversial whether the small fraction of NLRP3-mutated cells actually causes the strong autoinflammation observed in CINCA patients, or whether the NLRP3 mutations found in mosaic patients are just a bystander, with all cells carrying an unknown mutation of another gene that causes the disease. The pathogenesis of CINCA syndrome patients who carry NLRP3mutations as somatic mosaicism has not been precisely described because of the difficulty in separating live cells based on the presence or absence of the mutation.It is believed that most of the manifestations of CINCA syndrome are caused by the excessive secretion of the proinflammatory cytokine, IL-1β, and this concept is supported by the efficacy of an IL-1 receptor antagonist (IL-1Ra) for decreasing most of the symptoms. However, an NLRP3-targeted therapeutic approach would be attractive because 1) the progressive arthropathy despite anti-IL-1 therapy indicates that the presence of additional proteins processed by the inflammasome is also involved in the pathogenesis of CINCA syndrome 2) specific inhibition of the NLRP3-inflammasome can avoid unfavorable suppression of other IL-1β processing pathways in response to various triggers, and 3) these drugs may be also effective for various other NLRP3-related chronic inflammatory conditions, such as Alzheimer’s disease, diabetes, severe gout and atherosclerosis.Here, we report the generation of NLRP3-mutant and non-mutant induced pluripotent stem cell (iPSC) lines from two CINCA syndrome patients with somatic mosaicism, and describe their differentiation into macrophages (iPS-MPs). We found that mutant cells are predominantly responsible for the pathogenesis in these mosaic patients because only mutant iPS-MPs showed the disease relevant phenotype of abnormal IL-1β secretion.Next, after confirming that the existing anti-inflammatory compounds inhibited the abnormal IL-1β secretion from iPS-MPs, we started drug screening for CINCA syndrome and other NLRP3-related inflammatory conditions using a recently-established feeder-free differentiation protocol. Among the 3,939 chemically-potent low molecule compounds, 127 candidates inhibited IL-1β secretion by more than 30%. We then excluded compounds that also inhibited IL-6 secretion and selected 23 candidate compounds. We are now validating the potency of these compounds.Our results illustrate that patient-derived iPSCs are useful for dissecting somatic mosaicism, and that NLRP3-mutant iPSCs can provide a valuable platform for drug discovery for multiple NLRP3-related disorders. Disclosures:Yamanaka:iPierian: Membership on an entity's Board of Directors or advisory committees.

The population genetics of the Jewish people

Adherents to the Jewish faith have resided in numerous geographic locations over the course of three millennia. Progressively more detailed population genetic analysis carried out independently by multiple research groups over the past two decades has revealed a pattern for the population genetic architecture of contemporary Jews descendant from globally dispersed Diaspora communities. This pattern is consistent with a major, but variable component of shared Near East ancestry, together with variable degrees of admixture and introgression from the corresponding host Diaspora populations. By combining analysis of monoallelic markers with recent genome-wide variation analysis of simple tandem repeats, copy number variations, and single-nucleotide polymorphisms at high density, it has been possible to determine the relative contribution of sex-specific migration and introgression to map founder events and to suggest demographic histories corresponding to western and eastern Diaspora migrations, as well as subsequent microevolutionary events. These patterns have been congruous with the inferences of many, but not of all historians using more traditional tools such as archeology, archival records, linguistics, comparative analysis of religious narrative, liturgy and practices. Importantly, the population genetic architecture of Jews helps to explain the observed patterns of health and disease-relevant mutations and phenotypes which continue to be carefully studied and catalogued, and represent an important resource for human medical genetics research. The current review attempts to provide a succinct update of the more recent developments in a historical and human health context.Electronic supplementary materialThe online version of this article (doi:10.1007/s00439-012-1235-6) contains supplementary material, which is available to authorized users.

Induced pluripotent stem cells from CINCA syndrome patients as a model for dissecting somatic mosaicism and drug discovery

AbstractChronic infantile neurologic cutaneous and articular (CINCA) syndrome is an IL-1–driven autoinflammatory disorder caused mainly by NLRP3 mutations. The pathogenesis of CINCA syndrome patients who carry NLRP3 mutations as somatic mosaicism has not been precisely described because of the difficulty in separating individual cells based on the presence or absence of the mutation. Here we report the generation of NLRP3-mutant and nonmutant-induced pluripotent stem cell (iPSC) lines from 2 CINCA syndrome patients with somatic mosaicism, and describe their differentiation into macrophages (iPS-MPs). We found that mutant cells are predominantly responsible for the pathogenesis in these mosaic patients because only mutant iPS-MPs showed the disease relevant phenotype of abnormal IL-1β secretion. We also confirmed that the existing anti-inflammatory compounds inhibited the abnormal IL-1β secretion, indicating that mutant iPS-MPs are applicable for drug screening for CINCA syndrome and other NLRP3-related inflammatory conditions. Our results illustrate that patient-derived iPSCs are useful for dissecting somatic mosaicism and that NLRP3-mutant iPSCs can provide a valuable platform for drug discovery for multiple NLRP3-related disorders.

Modeling Brain Disease in a Dish: Really?

Cellular programming and reprogramming technology (CPART) presents a novel approach for understanding disease progression and mechanism. In addition, CPART provides an innovative opportunity for developing diagnostic tools and novel drug candidates for therapy. In this Forum, we will discuss obstacles and solutions for modeling brain disease using CPART.

Z’ Factor including siRNA design quality parameter in RNAi screening experiments

RNA interference (RNAi) high-content screening (HCS) enables massive parallel gene silencing and is increasingly being used to reveal novel connections between genes and disease-relevant phenotypes. The application of genome-scale RNAi relies on the development of high quality HCS assays. The Z’ factor statistic provides a way to evaluate whether or not screening run conditions (reagents, protocols, instrumentation, kinetics, and other conditions not directly related to the test compounds) are optimized. Z’ factor, introduced by Zhang et al.,1 is a dimensionless value that represents both the variability and the dynamic range between two sets of sample control data. This paper describe a new extension of the Z' factor, which integrates bioinformatics RNAi non-target compounds for screening quality assessment. Currently presented Z’ factor is based on positive and negative control, which may not be sufficient for RNAi experiments including oligonucleotides (oligo) with lack of knock-down. This paper proposes an algorithm which extends existing algorithm by using additional controls generetaed from on-target analysis.

Abnormal Mitochondrial Trafficking and Cytoskeletal Organization in a Human Induced Pluripotent Stem Cell and a Mouse Model of Charcot-Marie-Tooth Disease Type 2E (IN7-2.001)

Objective: To characterize human neuronal cells derived from induced pluripotent stem cells of a patient with CMT2E and a mouse model of the same mutation. Background Induced pluripotent stem cells (iPS) offer an alternative human model for the study of neurogenetic conditions. Previous studies have demonstrated the feasibility of generating and differentiating iPS cells from patients with neurological diseases into functional neurons and glia. This approach could be particularly useful in the pathophysiological study of distinct forms of inherited neuropathies. Charcot-Marie-Tooth disease type 2E (CMT2E) is caused by heterozygous point mutations in the NEFL gene, which encodes the intermediate filament neurofilament light chain, an essential component of the neuronal cytoskeleton. Design/Methods: We developed iPS cell lines from skin fibroblasts of a patient with a N98S NFL point mutation. Knockin mice carrying the same N98S mutation were also generated. Following a differentiation protocol for generating spinal cord motor neurons, we obtained human CMT2E neuronal cultures consisting of at least 50% ISLET1 immunoreactive cells. These neuronal cultures were characterized by time-lapse microscopy and axonal mitochondrial kinetics was analyzed. Results: Immunostaining for intermediate filaments revealed an increased content of these proteins in the neuronal body of CMT2E spinal cord motor neurons. Immunostaining of both the spinal cord and brain of a N98S mouse shows abundant NFL positive aggregates in the cell bodies and axons of the motor neurons, as well as in the cerebellum. Multiple filamentous spheroid-like structures were also observed in the brain stem of the mice. An increased proportion of paused mitochondria was found in axons from human CMT2E neurons, compared to an age-matched control. Conclusions: This study demonstrates disease relevant phenotypes in human CMT2E motor neurons that were supported by a mouse model of the same mutation, demonstrating the potential of iPS cell technology to model neurogenetic diseases. Supported by: M.S. was funded by a Rare Disease Clinical Research Network Fellowship from the National Institutes of Health. This study was also supported by iPierian Inc. Disclosure: Dr. Saporta has nothing to disclose. Dr. Volfson has received personal compensation for activities with iPierian Inc. Dr. Volfson has received research support from iPierian Inc. Dr. Liem has nothing to disclose. Dr. Shy has nothing to disclose. Dr. Liem has nothing to disclose. Dr. Dimos has received personal compensation for activities with iPierian Inc. as an employee.Dr. Dimos has received research support from iPierian Inc.

Induced pluripotent stem cell models from X‐linked adrenoleukodystrophy patients

Because of a lack of an appropriate animal model system and the inaccessibility of human oligodendrocytes in vivo, X-linked adrenoleukodystrophy (X-ALD)-induced pluripotent stem cells (iPSCs) would provide a unique cellular model for studying etiopathophysiology and development of therapeutics for X-ALD. We generated and characterized iPSCs of the 2 major types of X-ALD, childhood cerebral ALD (CCALD) and adrenomyeloneuropathy (AMN), and differentiated them into oligodendrocytes and neurons. We evaluated disease-relevant phenotypes by pharmacological and genetic approaches. We established iPSCs from the patients with CCALD and AMN. Both CCALD and AMN iPSCs normally differentiated into oligodendrocytes, the cell type primarily affected in the X-ALD brain, indicating no developmental defect due to the ABCD1 mutations. Although low in X-ALD iPSCs, very long chain fatty acid (VLCFA) level was significantly increased after oligodendrocyte differentiation. VLCFA accumulation was much higher in CCALD oligodendrocytes than AMN oligodendrocytes but was not significantly different between CCALD and AMN neurons, indicating that the severe clinical manifestations in CCALD might be associated with abnormal VLCFA accumulation in oligodendrocytes. Furthermore, the abnormal accumulation of VLCFA in the X-ALD oligodendrocytes can be reduced by the upregulated ABCD2 gene expression after treatment with lovastatin or 4-phenylbutyrate. X-ALD iPSC model recapitulates the key events of disease development (ie, VLCFA accumulation in oligodendrocytes), provides new clues for better understanding of the disease, and allows for early and accurate diagnosis of the disease subtypes. X-ALD oligodendrocytes can be a useful cell model system to develop new therapeutics for treating X-ALD.

The Kynurenine Pathway Modulates Neurodegeneration in a Drosophila Model of Huntington's Disease

Neuroactive metabolites of the kynurenine pathway (KP) of tryptophan degradation have been implicated in the pathophysiology of neurodegenerative disorders, including Huntington's disease (HD) [1]. A central hallmark of HD is neurodegeneration caused by a polyglutamine expansion in the huntingtin (htt) protein [2]. Here we exploit a transgenic Drosophila melanogaster model of HD to interrogate the therapeutic potential of KP manipulation. We observe that genetic and pharmacological inhibition of kynurenine 3-monooxygenase (KMO) increases levels of the neuroprotective metabolite kynurenic acid (KYNA) relative to the neurotoxic metabolite 3-hydroxykynurenine (3-HK) and ameliorates neurodegeneration. We also find that genetic inhibition of tryptophan 2,3-dioxygenase (TDO), the first and rate-limiting step in the pathway, leads to a similar neuroprotective shift toward KYNA synthesis. Importantly, we demonstrate that the feeding of KYNA and 3-HK to HD model flies directly modulates neurodegeneration, underscoring the causative nature of these metabolites. This study provides the first genetic evidence that inhibition of KMO and TDO activity protects against neurodegenerative disease in an animal model, indicating that strategies targetedattwo key points within the KP may have therapeutic relevance in HD, and possibly other neurodegenerative disorders.

Induced pluripotent stem cells and neurodegenerative diseases

Neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease and Amyotrophic Lateral Sclerosis, are characterized by idiopathic neuron loss in different regions of the central nervous system, which contributes to the relevant dysfunctions in the patients. The application of cell replacement therapy using human embryonic stem (hES) cells, though having attracted much attention, has been hampered by the intrinsic ethical problems. It has been demonstrated that adult somatic cells can be reprogrammed into the embryonic state, called induced pluripotent stem (iPS) cells. It is soon realized that iPS cells may be an alternative source for cell replacement therapy, because it raises no ethical problems and using patient-specific iPS cells for autologous transplantation will not lead to immunological rejection. What's more, certain types of neurons derived from patient-specific iPS cells may display disease-relevant phenotypes. Thus, patient-specific iPS cells can provide a unique opportunity to directly investigate the pathological properties of relevant neural cells in individual patient, and to study the vulnerability of neural cells to pathogenic factors in vitro, which may help reveal the pathogenesis of many neurodegenerative diseases. In this review, the recent development in cellular treatment of neurodegenerative diseases using iPS cells was summarized, and the potential value of iPS cells in the modeling of neurodegenerative disease was discussed.

Induced Pluripotent Stem Cells from Individuals with Recessive Dystrophic Epidermolysis Bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited blistering skin disorder caused by mutations in the COL7A1 gene-encoding type VII collagen (Col7), the major component of anchoring fibrils at the dermal-epidermal junction. Individuals with RDEB develop painful blisters and mucosal erosions, and currently, there are no effective forms of therapy. Nevertheless, some advances in patient therapy are being made, and cell-based therapies with mesenchymal and hematopoietic cells have shown promise in early clinical trials. To establish a foundation for personalized, gene-corrected, patient-specific cell transfer, we generated induced pluripotent stem (iPS) cells from three subjects with RDEB (RDEB iPS cells). We found that Col7 was not required for stem cell renewal and that RDEB iPS cells could be differentiated into both hematopoietic and nonhematopoietic lineages. The specific epigenetic profile associated with de-differentiation of RDEB fibroblasts and keratinocytes into RDEB iPS cells was similar to that observed in wild-type (WT) iPS cells. Importantly, human WT and RDEB iPS cells differentiated in vivo into structures resembling the skin. Gene-corrected RDEB iPS cells expressed Col7. These data identify the potential of RDEB iPS cells to generate autologous hematopoietic grafts and skin cells with the inherent capacity to treat skin and mucosal erosions that typify this genodermatosis.

Sensitized phenotypic screening identifies gene dosage sensitive region on chromosome 11 that predisposes to disease in mice

The identification of susceptibility genes for human disease is a major goal of current biomedical research. Both sequence and structural variation have emerged as major genetic sources of phenotypic variability and growing evidence points to copy number variation as a particularly important source of susceptibility for disease. Here we propose and validate a strategy to identify genes in which changes in dosage alter susceptibility to disease-relevant phenotypes in the mouse. Our approach relies on sensitized phenotypic screening of megabase-sized chromosomal deletion and deficiency lines carrying altered copy numbers of ∼30 linked genes. This approach offers several advantages as a method to systematically identify genes involved in disease susceptibility. To examine the feasibility of such a screen, we performed sensitized phenotyping in five therapeutic areas (metabolic syndrome, immune dysfunction, atherosclerosis, cancer and behaviour) of a 0.8 Mb reciprocal chromosomal duplication and deficiency on chromosome 11 containing 27 genes. Gene dosage in the region significantly affected risk for high-fat diet-induced metabolic syndrome, antigen-induced immune hypersensitivity, ApoE-induced atherosclerosis, and home cage activity. Follow up studies on individual gene knockouts for two candidates in the region showed that copy number variation in Stat5 was responsible for the phenotypic variation in antigen-induced immune hypersensitivity and metabolic syndrome. These data demonstrate the power of sensitized phenotypic screening of segmental aneuploidy lines to identify disease susceptibility genes.

Targeting Kynurenine 3-Monooxygenase (KMO): Implications for Therapy in Huntingtons Disease

Huntington's disease (HD) is an adult onset neurodegenerative disease caused by a polyglutamine expansion in the huntingtin protein. Recent work has shown that perturbation of kynurenine pathway (KP) metabolism is a hallmark of HD pathology, and that changes in brain levels of KP metabolites may play a causative role in this disease. The KP contains three neuroactive metabolites, the neurotoxins 3-hydroxykynurenine (3-HK) and quinolinic acid (QUIN), and the neuroprotectant kynurenic acid (KYNA). In model systems in vitro and in vivo, 3-HK and QUIN have been shown to cause neurodegeneration via a combination of excitotoxic mechanisms and oxidative stress. Recent studies with HD patient samples and in HD model systems have supported the idea that a shift away from the synthesis of KYNA and towards the formation of 3-HK and QUIN may trigger the neuropathological features observed in HD. The enzyme kynurenine 3-monooxygenase (KMO) is located at a critical branching point in the KP such that inhibition of this enzyme by either pharmacological or genetic means shifts the flux in the pathway towards the formation of KYNA. This intervention ameliorates disease-relevant phenotypes in HD models. Here we review the work implicating the KP in HD pathology and discuss the potential of KMO as a therapeutic target for this disorder. As several neurodegenerative diseases exhibit alterations in KP metabolism, this concept has broader implications for the treatment of brain diseases.

Kernelized Z’ factor in multiparametric screening technology

RNA interference (RNAi) high-content screening (HCS) enables massive parallel gene silencing and is increasingly being used to reveal novel connections between genes and disease-relevant phenotypes. The application of genome-scale RNAi relies on the development of high quality HCS assays. The Z’ factor statistic provides a way to evaluate whether or not screening run conditions (reagents, protocols, instrumentation, kinetics, and other conditions not directly related to the test compounds) are optimized. Z’ factor, introduced by Zhang et al.1, is a dimensionless value that represents both the variability and the dynamic range between two sets of sample control data. This paper describes a new extension of the Z' factor, which integrates multiple readouts for screening quality assessment. Currently presented multivariate Z’ factor is based on linear projection, which may not be suitable for data with nonlinear structure. This paper proposes an algorithm which extends existing algorithm to deal with nonlinear data by using the kernel function. Using kernel methods for projections, multiple readouts are condensed to a single parameter, based on which the screening run quality is monitored.

B02 Validation of suppressors of mutant huntingtin toxicity in a fly model of Huntington's disease

Studies using yeast models of Huntington's disease (HD) have provided insight into mechanisms underlying mutant huntingtin (htt) toxicity and have also identified several promising therapeutic targets for this disorder. Of...