Marius Wernig - US grants

DESCRIPTION (provided by applicant): The recent identification of transcription factors (TFs) that can induce conversion of fibroblasts into pluripotent stem (iPS) cells makes it potentially possible to generate patient-specific neurons from fibroblasts. However, the neurons thus produced are difficult to obtain. The present project builds on preliminary results in which we demonstrate direct conversion of adult fibroblasts cells into neurons, referred to as 'induced neuronal cells'(iN cells), without an iPS intermediate. The resulting iN cells have the functional properties of neurons, including the ability to form functional synapses as assayed by electrophysiology. Thus, the iN cell technology provides a novel, more facile approach to generating and studying human neurons, and opens up a new avenue to investigating human disease processes. However, at this point the iN cell technology has only been developed for mouse fibroblasts, fundamental questions regarding the conversion process and the molecular identity of iN cells were not determined, the generation of iN cells from human fibroblasts has not yet been established, and most importantly, the feasibility of the iN cell technology to study diseases affecting neuronal function has not been demonstrated. In this project, we propose to address these important challenges in an interdisciplinary approach capitalizing from the combined expertises of the Wernig and S|dhof laboratories. We propose experiments that will systematically investigate the cellular and molecular identity of iN cells, and develop protocols to induce specific neuronal subpopulations from fibroblasts. These protocols will then be employed to model genetic diseases in mouse iN cells. Furthermore, we will extend our findings to human fibroblasts, with the long term goal to establish a cell model for neuropsychiatric diseases. Our goals will be pursued by a combination of tissue culture experiments with cells cultured from mice and humans, cell biology, molecular biology, and electrophysiology. We believe our proposed experiments have the potential to fundamentally change existing paradigms of cellular differentiation and epigenetic gene regulation, and could provide a novel platform to study human neurons from patients suffering from a variety of brain diseases. PUBLIC HEALTH RELEVANCE: This application will develop methods to generate neurons directly from non-neuronal cells, allowing the production of neurons from skin fibroblasts of human patients. These methods will then be used to test the effects of mutations associated with neuropsychiatric disorders on neuronal biology, with the long-term goal of establishing a better understanding of the pathomechanism of these diseases in human neurons.

DESCRIPTION (provided by applicant): This project covers 3 thematic areas: Applying Genomics and Other High Throughput Technologies, Translating Basic Science Discoveries into New and Better Treatments and Reinvigorating the Biomedical Research Community. Somatic cells are highly stable in adult animals due to robust gene expression patterns, which are stabilized by epigenetic mechanisms. The seminal invention of induced pluripotent stem (iPS) cells, however, provided the surprising conclusion that the differentiated state can be reversed by simple expression of four transcription factors (TFs). This finding proved that even supposedly stable epigenetic modifications of genes are essentially controlled by TFs. We asked whether this concept can be extended to trans-differentiation of one cell type into another, and recently succeeded in converting mouse fibroblasts directly into functional neurons, referred to as induced neuronal (iN) cells, by overexpression of only three lineage-specific TFs. Our findings indicate that TFs suffice to not only reverse a particular pathway of differentiation, but also to redirect the transcriptional regulatory network in a cell into a completely different pathway. This fundamental result answered one of the key open questions in the field, and is the basis of the current proposal. Apart from documenting the dominance of TFs over epigenetic modifications, iN cells could represent an attractive way to derive patient- specific neurons from skin fibroblasts. This may be used to model various neurological diseases or for cell transplantation therapy. This proposal aims to characterize the process of iN cell generation on the molecular level, with the expectation to gain fundamental insights into the biology of the underlying trans-differentiation process. In addition to identifying the molecular events underlying the fibroblast-to-neuron conversion, this study will in particular assess the epigenetic stability of the iN cell state as well as their safety with respect to their potential tumorigenicity, key prerequisites for clinical application of iN cell technologies. Our multidisciplinary approach entails state-of-the art high-throughput sequencing technologies for genome-level interrogation of epigenetic states and transcription, newly developed microfluidic devices enabling genome- wide analyses of small cell populations as well as multiplex gene expression on the single cell level allowing the determination of cellular heterogeneity, electrophysiology, and neurodevelopmental techniques. PUBLIC HEALTH RELEVANCE: This application will develop methods to generate neurons directly from non-neuronal cells, allowing the production of neurons from skin fibroblasts of human patients. Patient-derived neurons could be used for modeling neurological diseases or as cell grafts to treat neurodegenerative diseases like Parkinson's disease.

DESCRIPTION (provided by applicant): The recent identification of transcription factors (TFs) that can induce conversion of fibroblasts into pluripotent stem (iPS) cells makes it potentially possible to generate patient-specific neurons from fibroblasts. However, the neurons thus produced are difficult to obtain. The present project builds on preliminary results in which we demonstrate direct conversion of adult fibroblasts cells into neurons, referred to as 'induced neuronal cells' (iN cells), without an iPS intermediate. The resulting iN cells have the functional properties of neurons, including the ability to form functional synapses as assayed by electrophysiology. Thus, the iN cell technology provides a novel, more facile approach to generating and studying human neurons, and opens up a new avenue to investigating human disease processes. However, at this point the iN cell technology has only been developed for mouse fibroblasts, fundamental questions regarding the conversion process and the molecular identity of iN cells were not determined, the generation of iN cells from human fibroblasts has not yet been established, and most importantly, the feasibility of the iN cell technology to study diseases affecting neuronal function has not been demonstrated. In this project, we propose to address these important challenges in an interdisciplinary approach capitalizing from the combined expertises of the Wernig and Sidhof laboratories. We propose experiments that will systematically investigate the cellular and molecular identity of iN cells, and develop protocols to induce specific neuronal subpopulations from fibroblasts. These protocols will then be employed to model genetic diseases in mouse iN cells. Furthermore, we will extend our findings to human fibroblasts, with the long term goal to establish a cell model for neuropsychiatric diseases. Our goals will be pursued by a combination of tissue culture experiments with cells cultured from mice and humans, cell biology, molecular biology, and electrophysiology. We believe our proposed experiments have the potential to fundamentally change existing paradigms of cellular differentiation and epigenetic gene regulation, and could provide a novel platform to study human neurons from patients suffering from a variety of brain diseases.

Schizophrenia is a frequent psychotic disorder leading to severe human suffering and increased suicide rates. Quality and severity of disease phenotypes vary dramatically between patients and the pathogenetic mechanisms on the molecular, cell biological, or neuronal circuit level are poorly understood. However, human genetics studies have demonstrated that genetic factors contribute to the highest risk factors to develop disease. The importance of genetic factors for disease development is widely accepted in the field. Because of our ability to manipulate and define genetic backgrounds and specific factors (as opposed to potential environmental factors) in cultured cells, it is possible that critical disease traits of the human brain can be recapitulated in cultured cell-based models. Recent advances by us and others in the field of epigenetic reprogramming and stem cell biology has made it possible to generate fully functional human neurons from pluripotent stem cells. We are therefore very close to interrogate human disease neurons for abnormal cell- biological traits in a meaningful way. Projects 1 and 2 will begin to analyze disease-specific traits using existing methods. However, current technology has several shortcomings limiting the full phenotypic characterization. The goal of Project 3 (this project) is to further develop and optimize existing stem cell methods that will allow to substantially increase the spectrum of assays to be analyzed. As the protocols are being developed and become available in Project 3, they will be immediately implemented and utilized in Projects 1 and 2. In particular, Project 3 will develop two critical components for the overall consortium grant: (1) It will provide the tools to genetically engineer conditional and/or definitive single gene and large CNV mutations. (2) The project will develop methods to develop defined inhibitory neuronal subtypes. Together with our already existing method to generate pure excitatory neuronal subtypes this will allow us to generate mixed cultures with defined excitatory/ inhibitory neuronal components which will allow the characterization of inhibitory synaptic transmission. In addition, the project will work on two non-essential but highly desired protocol developments: (1) Methods will be devised for industrial generation of neurons in large scale to improve consistent phenotypic analyses and enable the establishment of human cell models for pharmaceutical drug development. (2) We will develop ways to eliminate the currently required co-culture of mouse glia to be replaced with defined substances or human cells because use of cell models containing supportive mouse cells may complicated therapeutic drug development for human use in vivo. We believe the proposed technology developments will be critical contributions to the field and ultimately enable the generation of authentic human disease cell models.

DESCRIPTION (provided by applicant): The goal of the NCRCRG program is to create multidisciplinary research groups, in partnership with academia and industry, to use patient-derived reprogrammed cells to develop validated platforms for identifying novel targets and developing new therapeutics ... to reduce the burden of mental illness. Here we propose to use induced neuronal (iN) cells, derived from induced pluripotent stem (iPS) cells, to model defects in synaptic function due to three engineered or naturally-occurring mutations known to substantially increase the risk of schizophrenia (SCZ): NRXN1 exonic deletions, 22q11.2 deletions, and 16p11.2 duplications. The study includes three projects. Project 1 will analyze the functional characteristics of human iN cells. For each mutation, one component will study cells with engineered mutations vs. non-mutated cells from the same control individual, while a second component will study iN cells derived from 5 SCZ patients with the mutation of interest vs. 5 control individuals. Project 2 will analyze the same mutations in mouse models, providing both a cross-species validation of human findings, and an novel attempt to determine whether the synaptic phenotypes of these mutations are the same in iN cells, cultured primary neurons, and brain slices (medical pre-frontal cortex). Project 3 will develop and optimize stem cell methods that are required for this project (large-scale implementation of iN cell protocols; new targeted mutation strategies for large CNVs; derivation of pure inhibitory iN cells; development of a mouse-free iN cell protocol), and which will also be needed to develop future high-throughput screening assays based on the pathophysiological models developed in this study. These experiments will provide new insights into the characteristics of neurons derived by reprogramming method, into synaptic phenotypes produced by each of these mutations, and ultimately into the pathophysiology susceptibility to the risk of psychotic disorders including SCZ. We will determine whether these mutations produce distinct or at least partially overlapping synaptic phenotypes. Either observation has profound implications for future SCZ research. To accomplish this work, we have assembled an outstanding scientific team from Stanford University (Drs. Sudhof in molecular neuroscience, Wernig in stem cell biology and Levinson in genetics of schizophrenia); Rutgers University (Dr. Pang in neuroscience and stem cell biology); Cincinnati Children's Hospital Medical Center (Dr. Aronow in bioinformatics and high-content imaging); Eli Lilly and Company (Drs. Isaac and Ursu in electrophysiology, Merchant in drug development, Dage in high-content imaging and assay development, Collier in genomics and systems biology, and Eastwood in biostatistics); and Cellular Dynamics, Inc. (Dr. Swanson, representing a leading biotechnology company in stem cell biology). These projects represent a multidisciplinary effort of academic and industrial institutions to gain insight into te pathophysiology of psychotic disorders by studying three mutations that are associated with SCZ.

DESCRIPTION (provided by applicant): In this Project, we will characterize synaptic, cellular and biochemical phenotypes of high-risk Alzheimer Disease (AD) mutations in human induced neuronal (iN) cells derived from iPS cells. We believe that the new advances in pluripotent stem cell biology and epigenetic reprogramming will provide an important breakthrough as they allow the genetic modification and functional evaluation of human neurons. Therefore, it is now possible to functionally interrogate risk mutations and study their cell biological effects in huma neurons. In particular, recent advances of gene targeting tools in human induced pluripotent stem (iPS) cells and our recent development of rapid methods that generate fully functional induced neuronal (iN) cells from iPS cells provide ideal conditions to begin to apply this technology to disease modeling for brain diseases such as AD. We will introduce into control iPS cell line derived from a well-characterized healthy normal subject conditional mutations that confer high risk for AD. Mutations will be introduced using homologous recombination in a protocol that we have developed in preliminary studies, and the conditionally mutant iPS cells will then be converted into precisely matched wild-type and mutant iN cells. Mutant and control cells will be characterized for Ab and Tau biochemistry and importantly for detailed synaptic properties. We believe the focus on the precise synaptic characterization represents a key innovative factor of our proposal as synaptic dysfunction may be much more sensitive than other cell biological assays such as cell death. Finally, we have confirmed in our iN cell/ astrocyte co-culture system that ApoE is primarily produced by the glia and that ApoE is a critical mediator of the glia-induced synaptic maturation of primary neurons and human iN cells, with possibly different effects of ApoE3 and ApoE4. Building on these results, we propose to evaluate in this specific aim the precise effects of ApoE3 and ApoE4 on synaptic maturation in wild type and APP-mutant iN cells generated in Aim 1, with the goal of gaining insight into the role of ApoE4 in AD pathology. Applied together, these specific aims will allow us to perform a well-controlled assessment of the effect of AD-associated APP mutations on the properties of human neurons and their synapses.

Abstract The goal of this project is to describe the function of synaptic adhesion molecules of the Neuroligin family (Nlgns) in the mouse brain and in human neurons. Recent single cell expression studies have highlighted the obversation that Nlgns are expressed also in non-neuronal cells, in particular oligodendrocyte precursors cells (OPCs) and astrocytes who express Nlgns to even higher levels than neurons. Since little is known about the function of Nlgns in glia and their effect on neurons and neural circuits, we propose to specifically delete Nlgns in OPCs and astrocytes using our triple conditional Nlgn1-3 knock-out strain. Brains will be characterized morphologically, electrophysiologically on the cellular and circuit level, and mutant mice will be characterized by behavior. Next, we will perform an in-depth molecular characterization of the Neuroligin proteins by characterizing the molecular mechanisms underlying the surprising functional diversity of Nlgns. We will map their functional domains in mouse neurons by expressing various domain-mutant proteins in Nlgn1-4 quadruple knock-out cells. We will explore whether Nlgn sequence relates to functional specificity and investigate the notion of a synaptic Neurexin ?code? that may determine Nlgn specificity. To complement our mouse studies and explore human-specific Neuroligin function as well as human disease-associated mutations, we will capitalize on our previous human stem cell and reprogramming work in which we have developed human induced neuronal (iN) cells that exhibit all principal functional properties of primary mouse neurons including robust synapse formation. We propose to utilize this system to investigate the so far obscure function of NLGN4Y, a Y chromosomal gene closely related to NLGN4 on the X- chromosome and a member of the family not present in mouse. We will assess subcellular targeting by tagging the endogenous locus and assess the functional consequences of genetic deletion. Another frequently mutated Nlgn gene is NLGN3. Unlike NLGN4 it is better conserved in mice, but almost nothing is known about its function in human cells. In addition to generate loss-of-function alleles, we will study the functional consequences of distinct ASD-associated mutations introduced into the human NLGN3 gene. We will use a conditional mutagenesis approach as we have successfully done in the past, as it allows the generation of a perfect control conidition derived from the identical cell line as the experimental condition. Mutant human neurons and controls will be characterized biochemically, morphologically, by gene expression, and electrophysiologically. Finally, we propose to investigate the role of the proposed Nlgns-modulators MDGAs which are also found mutated in ASD and other neurodevelopmental disorders. We will assess their requirement for proper synapse formation and function by generating loss-of-function alleles in human neurons. We will further probe their function as Neuroligin modulators as competitive Nlgn binding molecules.

We found that the three transcription factors Ascl1, Myt1-like (Myt1l), and Brn2 can reprogram fibroblasts directly into functional neurons and are thus powerful neuronal lineage determination factors. Ascl1 and Brn2 are well studied genes. Myt1l on the other hand is a fairly uncharacterized zinc finger domain containting protein predicted to be a transcription factor. It has a remarkably unique expression pattern: it is expressed in virtually all neurons, but at the same time is also specific for neurons, to our knowledge the only transcription factor known to be specific and pan-neuronal at the same time. Independent of the reprogramming work, recent sequencing studies showed that MYT1L is frequently mutated in neuropsychiatric disease including autism and schizophrenia. Nevertheless, very little is known about this gene. Not even a mouse knock-out has been reported yet. We have therefore begun to investigate Myt1l's role in reprogramming and during normal development. Our first insights about its molecular function suggest that Myt1l is important for neuronal reprogramming and normal embryonic neurogenesis acting predominantly by transcriptional repression of non- neuronal lineage programs. The goal of this research project is to better understand the role of Myt1l in neurons after neurogenesis is completed. We propose to investigate its role on the molecular, cellular circuit, and behavioral level using the mouse as model system. We have intriguing preliminary data that about a third of high confidence autism- causing chromatin factors are also candidate binding partners of Myt1l. This suggests that all these mutations might converge on a hypothetical Myt1l-associated ?chromatin pathway? which is dysfunctional in at least subset of autism. This project will test this hypothesis and evaluate whether interference with the members of this chromatin ?pathway? might rectify molecular, cellular or behavioral phenotypes caused by Myt1l deletion. Since chromatin modifying enzymes are in principle pharmacologically tractable the hope would be that a functional intervention of such chromatin factors may be of therapeutic value for autistic children carrying MYT1L mutations. We will therefore test throughout all our three aims whether manipulation of these chromatin factors can rescue the molecular, cellular, or behavioral Myt1l phenotypes.