February 28, 2012

9:05 AM

Systems Approaches to Neurodegenerative Diseases and the Emergence of Transforming Techniques

Lee Hood, MD, PhD – Institute for Systems Biology

The challenge for biology in the 21st century is the need to deal with the incredible complexity of biological systems and disease. The challenge in understanding disease complexity is that of deciphering the operations of dynamic biological networks and molecular machines. One approach to deciphering this complexity is to generate enormous amounts of information about the systems of interest—across the multiple scales of biological information (DNA, RNA, protein, interactions, etc.) and then integrate these data into predictive models of health and disease.

I will focus on our efforts at taking a systems approach to disease—looking at a neurodegenerative (prion) disease, We published a few years ago a study on prion disease that has taken more than 6 years to integrates 6 different types of data and lay out the principles of a systems approach to disease. From these studies and the others come a clear understanding of some of the principal opportunities systems biology brings to medicine and the study of disease. These include: powerful new approaches to delineate disease mechanisms, making blood a window into health and disease, the stratification of diseases into different subtypes so as to be able create an impedance match with effective drugs, a new approach to the identification of drug targets and the ability to analyze multiple organ responses to disease.

I will also discuss the emerging technologies that will transform biology and medicine over the next 10 years—e.g., next generation DNA sequencing and its applications to human genome sequencing, targeted mass spectrometry, microfluidic protein chips, new approaches to proteincapture agents, single-cell analyses and the use of induced pluripotential cells to understand development, disease mechanisms and stratify disease.

It appears that systems medicine, together with emerging technologies and the development of powerful new computational and mathematical tools will transform medicine over the next 5-20 years from its currently reactive state to a mode that is predictive, personalized, preventive and participatory (P4). I will describe the nature of P4 medicine and its societal implications for healthcare.

9:50 AM

The Application of Systems Biology to the Discovery and Development of HD Therapeutics

Keith Elliston, PhD – CHDI

Systems biology aims to describe and understand the operation of complex biological systems to give insight into the fundamental governing principles of the disease and, ultimately, to apply this knowledge to guide drug discovery and improve health. While the proximal cause of Huntington’s disease has been well known for some time - the expanded triplet repeat in the huntingtin gene - the molecular mechanisms that manifest in the disease are still unclear. The nascent systems biology effort at CHDI is applying state-of-the-art techniques and tools to define the underlying molecular mechanisms of disease in HD patients to inform drug discovery, and to identify sensitive and appropriate animal- and cellbased models, new targets for therapeutic intervention, and biomarkers to assess mechanisms of disease and therapeutic response in the clinic. Systems approaches to biology rely upon coherent large-scale gene, protein and metabolite datasets (‘omics’) to develop computational models of disease that enable efficient hypothesis generation and testing, expediting discovery cycles. This marriage of large-scale empirical biology with advanced computational analysis and modeling promises to streamline and accelerate the discovery of therapeutics for HD. The goals, strategy, organization, and progress of the CHDI systems biology effort will be presented and discussed.

11:00 AM

A Genetic Foundation for Integrating Large Datasets to Define HD Pathogenesis

James F. Gusella, PhD – Massachusetts General Hospital

Huntington’s disease is triggered by the presence of an expanded CAG repeat that is expressed, as polyglutamine in the huntingtin protein, with biochemical consequences throughout the lifespan though its effects usually become apparent clinically only in mid-life. Human genetic studies involving thousands of individuals have established that the rate of HD pathogenesis leading to neurologic onset is largely determined by the length of a single HD gene CAG repeat expansion but is also modified by other, as yet unidentified genetic factors. This finding provides the foundation for two broad types of investigations in which the integration of large datasets can support unbiased approaches for defining the pathogenic process and identifying critical factors that can alter it. In ongoing collaborative efforts, genome-wide association and DNA sequencing strategies are being pursued to allow integration of DNA variation in large numbers of HD subjects with a large set of phenotypic measures, beginning with age at motor onset. These studies aim to identify additional genetic factors that impact on the pathogenic process and result in alteration of the ultimate outcome measure. However, they also “book-end” in both function and time the intervening dynamic of biochemical and physiological processes that connect the cause of pathogenesis with its clinical consequence. This offers the opportunity to fill the intervening gap with molecular measures on the same sets of individuals to define the pathogenic process by integrated analysis of varied data types, guided by knowledge of the genetic underpinnings of disease in each individual. In particular, the strong mathematical relationship between CAG repeat length and rate of pathogenesis offers a reliable filter with which to extract relevant alterations in gene expression, microRNA expression, chromatin modification, protein expression, protein modification, metabolomics, clinical phenotype, etc., from large but inherently noisy datasets. The goal of this “system genetics’ strategy is to better define and model in an integrated manner the network of changes that lead from the initial mutation to the clinical disease, providing both clues to interventions that could alter this network and measures with which to test them.

11:40 AM

3D Brain Atlases of Model Animals: Synapses, Neurons and Brain Compartments

Hanchuan Peng, PhD – Howard Hughes Medical Institute

Presentation not available.

We built 3D digital neuron atlases for model animals including C. elegans, fruit fly, dragon fly, and mouse. In these models we quantitatively describe the wiring of neurite structures, projection and potential connectivity patterns of neurons, and distribution of synapses. For instance, we build a 3D map of the spatially stereotyped neurite tracts throughout a fruit fly’s brain, and reconstruct an initial fly brain wiring diagram how neurites connect and project across a complete set of 64 brain compartments. To produce this informative atlases, we developed a pipeline of image analysis and informatics tools to register high-resolution 3D laser scanning microscopic images accurately, followed by 3D image-visualization assisted neuron reconstruction methods (Vaa3D-Neuron) to digitize the morphology and location of neurite structures. We also demonstrate the usefulness of our approach for complicated mammalian (mouse) brains by quantitatively measuring the synapses visualized using the newly developed mouse-GRASP labeling technique. Our methods can be applied to a variety of brain structure-function studies.

2:10 PM

RNA Quality Control Pathways as Potential Therapeutics Targets in HD

Melissa J. Moore, PhD – University of Massachusetts Medical School

Although research in the HD field has traditionally focused on expanded poly-Gln toxicity, recent work has indicated that expanded CAG repeats can also exert neuronal toxicity at the RNA level. Further, it is now known that CAG repeats can be translated in vivo in all three reading frames to generate poly-Ser and poly-Ala in addition to poly-Gln. While the extent to which RNA and poly-Ser/poly- Ala toxicity contribute to HD in humans is not yet established, specific removal of mutant HD RNA would have the advantage of eliminating both RNA and protein toxicities. One auspicious approach that recently entered large animal trials is specific knock down of mutant HD RNA by allele-specific RNAi. Yet because allele-specific RNAi relies on SNP heterozygosities between mutant and wild type HD alleles, it may not be applicable in all cases. Therefore, other means of silencing the mutant allele need to be explored. One idea is to harness one of the endogenous RNA quality control (QC) pathways. In particular, mRNA QC pathways that detect and eliminate stalled translation complexes may hold promise as targets whose upregulation could specifically down regulate mRNAs containing expanded CAG repeats.

2:45 PM

A Role for Hunting in Post-Transcriptional Gene Expression

Naoko Tanese, PhD – New York University

We recently reported a new role for the HD protein huntingtin (Htt) in post-transcriptional gene regulation and maintenance of processing bodies / neuronal RNA granules (PNAS 2008:105,10820; JBC 2010:285,13142). Endogenous Htt was found to co-localize and co-traffic with mRNA in dendrites. An emerging body of evidence suggests regulated transport and local translation of mRNAs in neurons play a critical role in establishing their connectivity. Our findings implicate normal Htt in these important dynamic processes in neurons; it is possible that mutant Htt perturbs them in some way, contributing to the HD pathogenesis. We hypothesize that Htt is associated with a subset of mRNAs in neuronal granules and regulate transport and local translation of these mRNAs in response to synaptic activity.

Our previous study used non-neural cell lines to purify and identify Htt-associated proteins. To better understand the functions of Htt in the brain, we immunopurified Htt from mice expressing endogenous FLAG-Htt (provided by Dr. Scott Zeitlin), and identified proteins and RNA selectively associated with wild type and mutant Htt. Previously unreported Htt interactions with Myo5a, Prkra, Gnb2l1, Rps6, G3bp2, and Syt2 were confirmed by immunoblotting. Gene ontology analysis of Htt-associated proteins revealed a statistically significant enrichment for proteins involved in RNA processing and translation among other categories. Wild type and mutant Htt co-localized with the stress granule protein component Tia1 in metabolically stressed cultured cells. We also found cosedimentation of Htt with polysomes in cytoplasmic mouse brain extracts dependent on the presence of intact ribosomes. Together, these data support a role for Htt in protein translation. We think our new findings on Htt will lead to its previously undiscovered role in pathways that regulate gene expression at the post-transcriptional level.

3:35 PM

Defining Posttranslational Modifications in Huntingtin

Lisa M. Ellerby, PhD – The Buck Institute for Age Research

Presentation not available.

Polyglutamine expansion in the huntingtin protein is the cause of Huntington’s disease. A potential disease-modifying therapy would be one that causes the mutant Htt protein itself to no longer be toxic to cells. One approach is to determine which post-translational modifications in the context of mutant Htt that abolish toxicity. Post-translational modifications (PTMs) are rapid, effective and reversible ways to regulate the stability, proteolysis, localization, function, toxicity and protein interactions of Htt. A number of laboratories have explored how specific PTMs affect some of these events. We have developed methods to identify and quantify PTMs in huntingtin using an unbiased approach with mass spectrometry. This approach has identified novel sites of phosphorylation, acetylation and oxidation. The role of post-translational modifications in the biology and pathogenesis of Huntington’s disease will be discussed as well as potential approaches to target these PTMs for drug discovery.

4:10 PM

The Role of Posttranslational Modifications in Clearance of Mutant Huntingtin

Dimitri Krainc, MD, PhD - Massachusetts General Hospital

In eukaryotic cells, acetylation is among the most common covalent modifications and ranks similar to the important master switch phosphorylation. The correlation between histone acetylation and increased transcription has been known for many years, but acetylases are now being identified to modify a number of non-histone proteins. Acetylation can affect many cellular functions including protein-protein interactions, microtubule dynamics, splicing, mRNA function, protein localization, metabolism, protein stability and aging. We have previously shown that acetylation of mutant huntingtin facilitates clearance of the mutant protein via macroautophagy. Here we examined the mechanisms of acetylation-mediated clearance of mutant Htt in more detail and tested whether inhibition of specific HDACs plays a role in the process. Specifically, we found that genetic or pharmacological inhibition of a subset of HDACs leads to increased acetylation and clearance of mutant huntingtin. This clearance occurs primarily via acetylation-dependent ubiqutination suggesting across talk between these posttranslational modifications. Together, our results suggest that isoform-specific HDAC inhibitors would be potentially useful to increase acetylation and clearance of mutant huntingtin.

4:45 PM

Genetics Based Discovery of Huntingtin in Post-Translational Modification

Marcy MacDonald, PhD – Massachusetts General Hospital

Expanded HD (now HTT) CAG trinucleotide repeat alleles trigger the HD pathogenic process, in a dominant CAG length dependent fashion, and predict the eventual onset of HD symptoms. Furthermore, the CAG repeat is polymorphic in the population and there is now considerable molecular and biochemical evidence that the repeat acts as a quantitative functional polymorphism even through the normal allele range. A parsimonious explanation is an impact of the CAG-encoded polyglutamine tract on huntingtin that affects its structural properties, functional activity, subcellular location or other parameter. To uncover the pathogenic trigger mechanism and its immediate consequences, we employ a strategy that couples global phenotyping methods to the HD genetic criteria (true dominance, progressivity with CAG size, striatal specificity). An observation that fully conforms to these criteria was evidence, from Hdh CAG knock-in mice, for distinct conformational isoforms of full-length huntingtin, displaying distinct subsets of antibody epitopes, which appeared to be altered in location within striatal neurons early in the disease process. Now, application of the genetic approach to the discovery of the structural properties and posttranslational modifications of human huntingtin, using biophysical and mass spectrometry analysis of a panel of recombinant full-length proteins, has revealed that increasing polyglutamine tract length yields alternate patterns of phosphorylation encompassing more than a dozen sites across the entire protein, consistent with the progressive impact of the polyglutamine tract on huntingtin’s continuous α-helical structure. Indeed, these patterns were not found with huntingtin sub-domains. Moreover, global de-phosphorylation, like increased polyglutamine tract length, enhanced recombinant huntingtin activity in an in vitro assay system. Thus, structural conformers marked by alternate phosphorylation patterns may offer a posttranslational mechanism for polyglutamine tract modulation of huntingtin function, subcellular location or other property that may trigger pathogenesis. This view strongly urges PTM-based therapeutic approaches that target conformational isoforms of huntingtin, marked by distinct sets of coordinately altered phosphor-epitopes, in a manner designed to alleviate or circumvent the impact of the polyglutamine tract on huntingtin’s continuous α-helical structure.