A cellular model for sporadic ALS using patient-derived induced pluripotent stem cells.

Development of therapeutics for genetically complex neurodegenerative diseases such as sporadic amyotrophic lateral sclerosis (ALS) has largely been hampered by lack of relevant disease models. Reprogramming of sporadic ALS patients' fibroblasts into induced pluripotent stem cells (iPSC) and differentiation into affected neurons that show a disease phenotype could provide a cellular model for disease mechanism studies and drug discovery. Here we report the reprogramming to pluripotency of fibroblasts from a large cohort of healthy controls and ALS patients and their differentiation into motor neurons. We demonstrate that motor neurons derived from three sALS patients show de novo TDP-43 aggregation and that the aggregates recapitulate pathology in postmortem tissue from one of the same patients from which the iPSC were derived. We configured a high-content chemical screen using the TDP-43 aggregate endpoint both in lower motor neurons and upper motor neuron like cells and identified FDA-approved small molecule modulators including Digoxin demonstrating the feasibility of patient-derived iPSC-based disease modeling for drug screening.

Compound classification using image-based cellular phenotypes.

Compounds with similar target specificities and modes of inhibition cause similar cellular phenotypes. Based on this observation, we hypothesized that we could quantitatively classify compounds with diverse mechanisms of action using cellular phenotypes and identify compounds with unintended cellular activities within a chemical series. We have developed Cytometrix technologies, a highly automated image-based system capable of quantifying, clustering, and classifying changes in cellular phenotypes for this purpose. Using this system, 45 out of 51 known compounds were accurately classified into 12 distinct mechanisms of action. We also demonstrate microtubule-binding activity in one of seven related cytochalasin actin poisons. This technology can be used for a variety of drug discovery applications, including high-throughput primary screening of chemical and siRNA libraries and as a secondary assay to detect unintended activities and toxicities.

An infrastructure for high-throughput microscopy: instrumentation, informatics, and integration.

High-throughput, image-based cell assays are rapidly emerging as valuable tools for the pharmaceutical industry and academic laboratories for use in both drug discovery and basic cell biology research. Access to commercially available assay reagents and automated microscope systems has made it relatively straightforward for a laboratory to begin running assays and collecting image-based cell assay data, but doing so on a large scale can be more challenging. Challenges include process bottlenecks with sample preparation, image acquisition, and data analysis as well as day-to-day assay consistency, managing unprecedented quantities of image data, and fully extracting useful information from the primary assay data. This chapter considers many of the decisions needed to build a robust infrastructure that addresses these challenges. Infrastructure components described include integrated laboratory automation systems for sample preparation and imaging, as well as an informatics infrastructure for multilevel image and data analysis. Throughout the chapter we describe a variety of strategies that emphasize building processes that are scaleable, highly efficient, and rigorously quality controlled.

An unbiased cell morphology-based screen for new, biologically active small molecules.

We have implemented an unbiased cell morphology-based screen to identify small-molecule modulators of cellular processes using the Cytometrix (TM) automated imaging and analysis system. This assay format provides unbiased analysis of morphological effects induced by small molecules by capturing phenotypic readouts of most known classes of pharmacological agents and has the potential to read out pathways for which little is known. Four human-cancer cell lines and one noncancerous primary cell type were treated with 107 small molecules comprising four different protein kinase-inhibitor scaffolds. Cellular phenotypes induced by each compound were quantified by multivariate statistical analysis of the morphology, staining intensity, and spatial attributes of the cellular nuclei, microtubules, and Golgi compartments. Principal component analysis was used to identify inhibitors of cellular components not targeted by known protein kinase inhibitors. Here we focus on a hydroxyl-substituted analog (hydroxy-PP) of the known Src-family kinase inhibitor PP2 because it induced cell-specific morphological features distinct from all known kinase inhibitors in the collection. We used affinity purification to identify a target of hydroxy-PP, carbonyl reductase 1 (CBR1), a short-chain dehydrogenase-reductase. We solved the X-ray crystal structure of the CBR1/hydroxy-PP complex to 1.24 A resolution. Structure-based design of more potent and selective CBR1 inhibitors provided probes for analyzing the biological function of CBR1 in A549 cells. These studies revealed a previously unknown function for CBR1 in serum-withdrawal-induced apoptosis. Further studies indicate CBR1 inhibitors may enhance the effectiveness of anticancer anthracyclines. Morphology-based screening of diverse cancer cell types has provided a method for discovering potent new small-molecule probes for cell biological studies and anticancer drug candidates.

Identification of a novel light intermediate chain (D2LIC) for mammalian cytoplasmic dynein 2.

The diversity of dynein's functions in mammalian cells is a manifestation of both the existence of multiple dynein heavy chain isoforms and an extensive set of associated protein subunits. In this study, we have identified and characterized a novel subunit of the mammalian cytoplasmic dynein 2 complex. The sequence similarity between this 33-kDa subunit and the light intermediate chains (LICs) of cytoplasmic dynein 1 suggests that this protein is a dynein 2 LIC (D2LIC). D2LIC contains a P-loop motif near its NH(2) terminus, and it shares a short region of similarity to the yeast GTPases Spg1p and Tem1p. The D2LIC subunit interacts specifically with DHC2 (or cDhc1b) in both reciprocal immunoprecipitations and sedimentation assays. The expression of D2LIC also mirrors that of DHC2 in a variety of tissues. D2LIC colocalizes with DHC2 at the Golgi apparatus throughout the cell cycle. On brefeldin A-induced Golgi fragmentation, a fraction of D2LIC redistributes to the cytoplasm, leaving behind a subset of D2LIC that is localized around the centrosome. Our results suggest that D2LIC is a bona fide subunit of cytoplasmic dynein 2 that may play a role in maintaining Golgi organization by binding cytoplasmic dynein 2 to its Golgi-associated cargo.