All Publications

  • CRISPR Interference-Based Platform for Multimodal Genetic Screens in Human iPSC-Derived Neurons NEURON Tian, R., Gachechiladze, M. A., Ludwig, C. H., Laurie, M. T., Hong, J. Y., Nathaniel, D., Prabhu, A. V., Fernandopulle, M. S., Patel, R., Abshari, M., Ward, M. E., Kampmann, M. 2019; 104 (2): 239-+


    CRISPR/Cas9-based functional genomics have transformed our ability to elucidate mammalian cell biology. However, most previous CRISPR-based screens were conducted in cancer cell lines rather than healthy, differentiated cells. Here, we describe a CRISPR interference (CRISPRi)-based platform for genetic screens in human neurons derived from induced pluripotent stem cells (iPSCs). We demonstrate robust and durable knockdown of endogenous genes in such neurons and present results from three complementary genetic screens. First, a survival-based screen revealed neuron-specific essential genes and genes that improved neuronal survival upon knockdown. Second, a screen with a single-cell transcriptomic readout uncovered several examples of genes whose knockdown had strikingly cell-type-specific consequences. Third, a longitudinal imaging screen detected distinct consequences of gene knockdown on neuronal morphology. Our results highlight the power of unbiased genetic screens in iPSC-derived differentiated cell types and provide a platform for systematic interrogation of normal and disease states of neurons. VIDEO ABSTRACT.

    View details for DOI 10.1016/j.neuron.2019.07.014

    View details for Web of Science ID 000492678000009

    View details for PubMedID 31422865

    View details for PubMedCentralID PMC6813890

  • Heterochromatin anomalies and double-stranded RNA accumulation underlie C9orf72 poly(PR) toxicity SCIENCE Zhang, Y., Guo, L., Gonzales, P. K., Gendron, T. F., Wu, Y., Jansen-West, K., O'Raw, A. D., Pickles, S. R., Prudencio, M., Carlomagno, Y., Gachechiladze, M. A., Ludwig, C., Tian, R., Chew, J., DeTure, M., Lin, W., Tong, J., Daughrity, L. M., Yue, M., Song, Y., Andersen, J. W., Castanedes-Casey, M., Kurti, A., Datta, A., Antognetti, G., McCampbell, A., Rademakers, R., Oskarsson, B., Dickson, D. W., Kampmann, M., Ward, M. E., Fryer, J. D., Link, C. D., Shorter, J., Petrucelli, L. 2019; 363 (6428): 707-+


    How hexanucleotide GGGGCC (G4C2) repeat expansions in C9orf72 cause frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) is not understood. We developed a mouse model engineered to express poly(PR), a proline-arginine (PR) dipeptide repeat protein synthesized from expanded G4C2 repeats. The expression of green fluorescent protein-conjugated (PR)50 (a 50-repeat PR protein) throughout the mouse brain yielded progressive brain atrophy, neuron loss, loss of poly(PR)-positive cells, and gliosis, culminating in motor and memory impairments. We found that poly(PR) bound DNA, localized to heterochromatin, and caused heterochromatin protein 1α (HP1α) liquid-phase disruptions, decreases in HP1α expression, abnormal histone methylation, and nuclear lamina invaginations. These aberrations of histone methylation, lamins, and HP1α, which regulate heterochromatin structure and gene expression, were accompanied by repetitive element expression and double-stranded RNA accumulation. Thus, we uncovered mechanisms by which poly(PR) may contribute to the pathogenesis of C9orf72-associated FTD and ALS.

    View details for DOI 10.1126/science.aav2606

    View details for Web of Science ID 000458874100023

    View details for PubMedID 30765536

  • Mapping chromatin modifications at the single cell level. Development (Cambridge, England) Ludwig, C. H., Bintu, L. n. 2019; 146 (12)


    Understanding chromatin regulation holds enormous promise for controlling gene regulation, predicting cellular identity, and developing diagnostics and cellular therapies. However, the dynamic nature of chromatin, together with cell-to-cell heterogeneity in its structure, limits our ability to extract its governing principles. Single cell mapping of chromatin modifications, in conjunction with expression measurements, could help overcome these limitations. Here, we review recent advances in single cell-based measurements of chromatin modifications, including optimization to reduce DNA loss, improved DNA sequencing, barcoding, and antibody engineering. We also highlight several applications of these techniques that have provided insights into cell-type classification, mapping modification co-occurrence and heterogeneity, and monitoring chromatin dynamics.

    View details for DOI 10.1242/dev.170217

    View details for PubMedID 31249006

  • Differential effects of partial and complete loss of TREM2 on microglial injury response and tauopathy PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sayed, F. A., Telpoukhovskaia, M., Kodama, L., Li, Y., Zhou, Y., Le, D., Hauduc, A., Ludwig, C., Gao, F., Clelland, C., Zhan, L., Cooper, Y. A., Davalos, D., Akassoglou, K., Coppola, G., Gan, L. 2018; 115 (40): 10172–77


    Alzheimer's disease (AD), the most common form of dementia, is characterized by the abnormal accumulation of amyloid plaques and hyperphosphorylated tau aggregates, as well as microgliosis. Hemizygous missense variants in Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) are associated with elevated risk for developing late-onset AD. These variants are hypothesized to result in loss of function, mimicking TREM2 haploinsufficiency. However, the consequences of TREM2 haploinsufficiency on tau pathology and microglial function remain unknown. We report the effects of partial and complete loss of TREM2 on microglial function and tau-associated deficits. In vivo imaging revealed that microglia from aged TREM2-haploinsufficient mice show a greater impairment in their injury response compared with microglia from aged TREM2-KO mice. In transgenic mice expressing mutant human tau, TREM2 haploinsufficiency, but not complete loss of TREM2, increased tau pathology. In addition, whereas complete TREM2 deficiency protected against tau-mediated microglial activation and atrophy, TREM2 haploinsufficiency elevated expression of proinflammatory markers and exacerbated atrophy at a late stage of disease. The differential effects of partial and complete loss of TREM2 on microglial function and tau pathology provide important insights into the critical role of TREM2 in AD pathogenesis.

    View details for DOI 10.1073/pnas.1811411115

    View details for Web of Science ID 000446078700091

    View details for PubMedID 30232263

    View details for PubMedCentralID PMC6176614

  • Systematic Three-Dimensional Coculture Rapidly Recapitulates Interactions between Human Neurons and Astrocytes STEM CELL REPORTS Krencik, R., Seo, K., van Asperen, J. V., Basu, N., Cvetkovic, C., Barlas, S., Chen, R., Ludwig, C., Wang, C., Ward, M. E., Gan, L., Horner, P. J., Rowitch, D. H., Ullian, E. M. 2017; 9 (6): 1745–53


    Human astrocytes network with neurons in dynamic ways that are still poorly defined. Our ability to model this relationship is hampered by the lack of relevant and convenient tools to recapitulate this complex interaction. To address this barrier, we have devised efficient coculture systems utilizing 3D organoid-like spheres, termed asteroids, containing pre-differentiated human pluripotent stem cell (hPSC)-derived astrocytes (hAstros) combined with neurons generated from hPSC-derived neural stem cells (hNeurons) or directly induced via Neurogenin 2 overexpression (iNeurons). Our systematic methods rapidly produce structurally complex hAstros and synapses in high-density coculture with iNeurons in precise numbers, allowing for improved studies of neural circuit function, disease modeling, and drug screening. We conclude that these bioengineered neural circuit model systems are reliable and scalable tools to accurately study aspects of human astrocyte-neuron functional properties while being easily accessible for cell-type-specific manipulations and observations.

    View details for DOI 10.1016/j.stemcr.2017.10.026

    View details for Web of Science ID 000417690600002

    View details for PubMedID 29198827

    View details for PubMedCentralID PMC5785708

  • Scalable Production of iPSC-Derived Human Neurons to Identify Tau-Lowering Compounds by High-Content Screening STEM CELL REPORTS Wang, C., Ward, M. E., Chen, R., Liu, K., Tracy, T. E., Chen, X., Xie, M., Sohn, P., Ludwig, C., Meyer-Franke, A., Karch, C. M., Ding, S., Gan, L. 2017; 9 (4): 1221–33


    Lowering total tau levels is an attractive therapeutic strategy for Alzheimer's disease and other tauopathies. High-throughput screening in neurons derived from human induced pluripotent stem cells (iPSCs) is a powerful tool to identify tau-targeted therapeutics. However, such screens have been hampered by heterogeneous neuronal production, high cost and low yield, and multi-step differentiation procedures. We engineered an isogenic iPSC line that harbors an inducible neurogenin 2 transgene, a transcription factor that rapidly converts iPSCs to neurons, integrated at the AAVS1 locus. Using a simplified two-step protocol, we differentiated these iPSCs into cortical glutamatergic neurons with minimal well-to-well variability. We developed a robust high-content screening assay to identify tau-lowering compounds in LOPAC and identified adrenergic receptors agonists as a class of compounds that reduce endogenous human tau. These techniques enable the use of human neurons for high-throughput screening of drugs to treat neurodegenerative disease.

    View details for DOI 10.1016/j.stemcr.2017.08.019

    View details for Web of Science ID 000412660000018

    View details for PubMedID 28966121

    View details for PubMedCentralID PMC5639430

  • Individuals with progranulin haploinsufficiency exhibit features of neuronal ceroid lipofuscinosis SCIENCE TRANSLATIONAL MEDICINE Ward, M. E., Chen, R., Huang, H., Ludwig, C., Telpoukhovskaia, M., Taubes, A., Boudin, H., Minami, S. S., Reichert, M., Albrecht, P., Gelfand, J. M., Cruz-Herranz, A., Cordano, C., Alavi, M. V., Leslie, S., Seeley, W. W., Miller, B. L., Bigio, E., Mesulam, M., Bogyo, M. S., Mackenzie, I. R., Staropoli, J. F., Cotman, S. L., Huang, E. J., Gan, L., Green, A. J. 2017; 9 (385)


    Heterozygous mutations in the GRN gene lead to progranulin (PGRN) haploinsufficiency and cause frontotemporal dementia (FTD), a neurodegenerative syndrome of older adults. Homozygous GRN mutations, on the other hand, lead to complete PGRN loss and cause neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease usually seen in children. Given that the predominant clinical and pathological features of FTD and NCL are distinct, it is controversial whether the disease mechanisms associated with complete and partial PGRN loss are similar or distinct. We show that PGRN haploinsufficiency leads to NCL-like features in humans, some occurring before dementia onset. Noninvasive retinal imaging revealed preclinical retinal lipofuscinosis in heterozygous GRN mutation carriers. Increased lipofuscinosis and intracellular NCL-like storage material also occurred in postmortem cortex of heterozygous GRN mutation carriers. Lymphoblasts from heterozygous GRN mutation carriers accumulated prominent NCL-like storage material, which could be rescued by normalizing PGRN expression. Fibroblasts from heterozygous GRN mutation carriers showed impaired lysosomal protease activity. Our findings indicate that progranulin haploinsufficiency caused accumulation of NCL-like storage material and early retinal abnormalities in humans and implicate lysosomal dysfunction as a central disease process in GRN-associated FTD and GRN-associated NCL.

    View details for DOI 10.1126/scitranslmed.aah5642

    View details for Web of Science ID 000399009200002

    View details for PubMedID 28404863

  • Memory regulatory T cells reside in human skin JOURNAL OF CLINICAL INVESTIGATION Rodriguez, R. S., Pauli, M. L., Neuhaus, I. M., Yu, S. S., Arron, S. T., Harris, H. W., Yang, S. H., Anthony, B. A., Sverdrup, F. M., Krow-Lucal, E., MacKenzie, T. C., Johnson, D. S., Meyer, E. H., Loehr, A., Hsu, A., Koo, J., Liao, W., Gupta, R., Debbaneh, M. G., Butler, D., Huynh, M., Levin, E. C., Leon, A., Hoffman, W. Y., McGrath, M. H., Alvarado, M. D., Ludwig, C. H., Truong, H., Maurano, M. M., Gratz, I. K., Abbas, A. K., Rosenblum, M. D. 2014; 124 (3): 1027-1036

    View details for DOI 10.1172/JCI72932

    View details for Web of Science ID 000332347700023