Sergiu P. Pasca

All Publications

• Chromatin accessibility dynamics in a model of human forebrain development. Science (New York, N.Y.) Trevino, A. E., Sinnott-Armstrong, N., Andersen, J., Yoon, S. J., Huber, N., Pritchard, J. K., Chang, H. Y., Greenleaf, W. J., Pașca, S. P. 2020; 367 (6476)

  Abstract

  Forebrain development is characterized by highly synchronized cellular processes, which, if perturbed, can cause disease. To chart the regulatory activity underlying these events, we generated a map of accessible chromatin in human three-dimensional forebrain organoids. To capture corticogenesis, we sampled glial and neuronal lineages from dorsal or ventral forebrain organoids over 20 months in vitro. Active chromatin regions identified in human primary brain tissue were observed in organoids at different developmental stages. We used this resource to map genetic risk for disease and to explore evolutionary conservation. Moreover, we integrated chromatin accessibility with transcriptomics to identify putative enhancer-gene linkages and transcription factors that regulate human corticogenesis. Overall, this platform brings insights into gene-regulatory dynamics at previously inaccessible stages of human forebrain development, including signatures of neuropsychiatric disorders.

  View details for DOI 10.1126/science.aay1645

  View details for PubMedID 31974223

• Neuronal defects in a human cellular model of 22q11.2 deletion syndrome. Nature medicine Khan, T. A., Revah, O., Gordon, A., Yoon, S. J., Krawisz, A. K., Goold, C., Sun, Y., Kim, C. H., Tian, Y., Li, M. Y., Schaepe, J. M., Ikeda, K., Amin, N. D., Sakai, N., Yazawa, M., Kushan, L., Nishino, S., Porteus, M. H., Rapoport, J. L., Bernstein, J. A., O'Hara, R., Bearden, C. E., Hallmayer, J. F., Huguenard, J. R., Geschwind, D. H., Dolmetsch, R. E., Paşca, S. P. 2020

  Abstract

  22q11.2 deletion syndrome (22q11DS) is a highly penetrant and common genetic cause of neuropsychiatric disease. Here we generated induced pluripotent stem cells from 15 individuals with 22q11DS and 15 control individuals and differentiated them into three-dimensional (3D) cerebral cortical organoids. Transcriptional profiling across 100 days showed high reliability of differentiation and revealed changes in neuronal excitability-related genes. Using electrophysiology and live imaging, we identified defects in spontaneous neuronal activity and calcium signaling in both organoid- and 2D-derived cortical neurons. The calcium deficit was related to resting membrane potential changes that led to abnormal inactivation of voltage-gated calcium channels. Heterozygous loss of DGCR8 recapitulated the excitability and calcium phenotypes and its overexpression rescued these defects. Moreover, the 22q11DS calcium abnormality could also be restored by application of antipsychotics. Taken together, our study illustrates how stem cell derived models can be used to uncover and rescue cellular phenotypes associated with genetic forms of neuropsychiatric disease.

  View details for DOI 10.1038/s41591-020-1043-9

  View details for PubMedID 32989314

• Assembling human brain organoids. Science (New York, N.Y.) Pasca, S. P. 2019; 363 (6423): 126–27

  View details for PubMedID 30630918

• Reliability of human cortical organoid generation. Nature methods Yoon, S. J., Elahi, L. S., Pașca, A. M., Marton, R. M., Gordon, A., Revah, O., Miura, Y., Walczak, E. M., Holdgate, G. M., Fan, H. C., Huguenard, J. R., Geschwind, D. H., Pașca, S. P. 2019; 16 (1): 75–78

  Abstract

  The differentiation of pluripotent stem cells in three-dimensional cultures can recapitulate key aspects of brain development, but protocols are prone to variable results. Here we differentiated multiple human pluripotent stem cell lines for over 100 d using our previously developed approach to generate brain-region-specific organoids called cortical spheroids and, using several assays, found that spheroid generation was highly reliable and consistent. We anticipate the use of this approach for large-scale differentiation experiments and disease modeling.

  View details for PubMedID 30573846

• The hidden biology of the human brain. Nature medicine Pașca, S. P. 2019

  View details for DOI 10.1038/s41591-019-0621-1

  View details for PubMedID 31700168

• Human 3D cellular model of hypoxic brain injury of prematurity. Nature medicine Pașca, A. M., Park, J. Y., Shin, H. W., Qi, Q., Revah, O., Krasnoff, R., O'Hara, R., Willsey, A. J., Palmer, T. D., Pașca, S. P. 2019

  Abstract

  Owing to recent medical and technological advances in neonatal care, infants born extremely premature have increased survival rates1,2. After birth, these infants are at high risk of hypoxic episodes because of lung immaturity, hypotension and lack of cerebral-flow regulation, and can develop a severe condition called encephalopathy of prematurity3. Over 80% of infants born before post-conception week 25 have moderate-to-severe long-term neurodevelopmental impairments4. The susceptible cell types in the cerebral cortex and the molecular mechanisms underlying associated gray-matter defects in premature infants remain unknown. Here we used human three-dimensional brain-region-specific organoids to study the effect of oxygen deprivation on corticogenesis. We identified specific defects in intermediate progenitors, a cortical cell type associated with the expansion of the human cerebral cortex, and showed that these are related to the unfolded protein response and changes. Moreover, we verified these findings in human primary cortical tissue and demonstrated that a small-molecule modulator of the unfolded protein response pathway can prevent the reduction in intermediate progenitors following hypoxia. We anticipate that this human cellular platform will be valuable for studying the environmental and genetic factors underlying injury in the developing human brain.

  View details for PubMedID 31061540

• Differentiation and maturation of oligodendrocytes in human three-dimensional neural cultures. Nature neuroscience Marton, R. M., Miura, Y., Sloan, S. A., Li, Q., Revah, O., Levy, R. J., Huguenard, J. R., Pașca, S. P. 2019

  Abstract

  Investigating human oligodendrogenesis and the interaction of oligodendrocytes with neurons and astrocytes would accelerate our understanding of the mechanisms underlying white matter disorders. However, this is challenging because of the limited accessibility of functional human brain tissue. Here, we developed a new differentiation method of human induced pluripotent stem cells to generate three-dimensional brain organoids that contain oligodendrocytes as well as neurons and astrocytes, called human oligodendrocyte spheroids. We found that oligodendrocyte lineage cells derived in human oligodendrocyte spheroids transitioned through developmental stages similar to primary human oligodendrocytes and that the migration of oligodendrocyte lineage cells and their susceptibility to lysolecithin exposure could be captured by live imaging. Moreover, their morphology changed as they matured over time in vitro and started myelinating neurons. We anticipate that this method can be used to study oligodendrocyte development, myelination, and interactions with other major cell types in the CNS.

  View details for PubMedID 30692691

• Generation and assembly of human brain region-specific three-dimensional cultures. Nature protocols Sloan, S. A., Andersen, J., Pașca, A. M., Birey, F., Pașca, S. P. 2018

  Abstract

  The ability to generate region-specific three-dimensional (3D) models to study human brain development offers great promise for understanding the nervous system in both healthy individuals and patients. In this protocol, we describe how to generate and assemble subdomain-specific forebrain spheroids, also known as brain region-specific organoids, from human pluripotent stem cells (hPSCs). We describe how to pattern the neural spheroids toward either a dorsal forebrain or a ventral forebrain fate, establishing human cortical spheroids (hCSs) and human subpallial spheroids (hSSs), respectively. We also describe how to combine the neural spheroids in vitro to assemble forebrain assembloids that recapitulate the interactions of glutamatergic and GABAergic neurons seen in vivo. Astrocytes are also present in the human forebrain-specific spheroids, and these undergo maturation when the forebrain spheroids are cultured long term. The initial generation of neural spheroids from hPSCs occurs in <1 week, with regional patterning occurring over the subsequent 5 weeks. After the maturation stage, brain region-specific spheroids are amenable to a variety of assays, including live-cell imaging, calcium dynamics, electrophysiology, cell purification, single-cell transcriptomics, and immunohistochemistry studies. Once generated, forebrain spheroids can also be matured for >24 months in culture.

  View details for PubMedID 30202107

• The rise of three-dimensional human brain cultures. NATURE Paşca, S. P. 2018; 553: 437-445

  Abstract

  Pluripotent stem cells show a remarkable ability to self-organize and differentiate in vitro in three-dimensional aggregates, known as organoids or organ spheroids, and to recapitulate aspects of human brain development and function. Region-specific 3D brain cultures can be derived from any individual and assembled to model complex cell-cell interactions and to generate circuits in human brain assembloids. Here I discuss how this approach can be used to understand unique features of the human brain and to gain insights into neuropsychiatric disorders. In addition, I consider the challenges faced by researchers in further improving and developing methods to probe and manipulate patient-derived 3D brain cultures.

  View details for DOI 10.1038/nature25032

• Building Models of Brain Disorders with Three-Dimensional Organoids. Neuron Amin, N. D., Paşca, S. P. 2018; 100 (2): 389–405

  Abstract

  Disorders of the nervous system are challenging to study and treat due to the relative inaccessibility of functional human brain tissue for research. Stem cell-derived 3D human brain organoids have the potential to recapitulate features of the human brain with greater complexity than 2D models and are increasingly being applied to model diseases affecting the central nervous system. Here, we review the use of human brain organoids to investigate neurological and psychiatric (neuropsychiatric) disorders and how this technology may ultimately advance our biological understanding of these conditions.

  View details for PubMedID 30359604

• Assembly of functionally integrated human forebrain spheroids NATURE Birey, F., Andersen, J., Makinson, C. D., Islam, S., Wei, W., Huber, N., Fan, H. C., Metzler, K. R., Panagiotakos, G., Thom, N., O'Rourke, N. A., Steinmetz, L. M., Bernstein, J. A., Hallmayer, J., Huguenard, J. R., Pasca, S. P. 2017; 545 (7652): 54-?

  Abstract

  The development of the nervous system involves a coordinated succession of events including the migration of GABAergic (γ-aminobutyric-acid-releasing) neurons from ventral to dorsal forebrain and their integration into cortical circuits. However, these interregional interactions have not yet been modelled with human cells. Here we generate three-dimensional spheroids from human pluripotent stem cells that resemble either the dorsal or ventral forebrain and contain cortical glutamatergic or GABAergic neurons. These subdomain-specific forebrain spheroids can be assembled in vitro to recapitulate the saltatory migration of interneurons observed in the fetal forebrain. Using this system, we find that in Timothy syndrome-a neurodevelopmental disorder that is caused by mutations in the CaV1.2 calcium channel-interneurons display abnormal migratory saltations. We also show that after migration, interneurons functionally integrate with glutamatergic neurons to form a microphysiological system. We anticipate that this approach will be useful for studying neural development and disease, and for deriving spheroids that resemble other brain regions to assemble circuits in vitro.

  View details for DOI 10.1038/nature22330

  View details for PubMedID 28445465

• Human Astrocyte Maturation Captured in 3D Cerebral Cortical Spheroids Derived from Pluripotent Stem Cells. Neuron Sloan, S. A., Darmanis, S., Huber, N., Khan, T. A., Birey, F., Caneda, C., Reimer, R., Quake, S. R., Barres, B. A., Paşca, S. P. 2017; 95 (4): 779–90.e6

  Abstract

  There is significant need to develop physiologically relevant models for investigating human astrocytes in health and disease. Here, we present an approach for generating astrocyte lineage cells in a three-dimensional (3D) cytoarchitecture using human cerebral cortical spheroids (hCSs) derived from pluripotent stem cells. We acutely purified astrocyte-lineage cells from hCSs at varying stages up to 20 months in vitro using immunopanning and cell sorting and performed high-depth bulk and single-cell RNA sequencing to directly compare them to purified primary human brain cells. We found that hCS-derived glia closely resemble primary human fetal astrocytes and that, over time in vitro, they transition from a predominantly fetal to an increasingly mature astrocyte state. Transcriptional changes in astrocytes are accompanied by alterations in phagocytic capacity and effects on neuronal calcium signaling. These findings suggest that hCS-derived astrocytes closely resemble primary human astrocytes and can be used for studying development and modeling disease.

  View details for PubMedID 28817799

• Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture. Nature methods Pasca, A. M., Sloan, S. A., Clarke, L. E., Tian, Y., Makinson, C. D., Huber, N., Kim, C. H., Park, J., O'Rourke, N. A., Nguyen, K. D., Smith, S. J., Huguenard, J. R., Geschwind, D. H., Barres, B. A., Pasca, S. P. 2015; 12 (7): 671-678

  Abstract

  The human cerebral cortex develops through an elaborate succession of cellular events that, when disrupted, can lead to neuropsychiatric disease. The ability to reprogram somatic cells into pluripotent cells that can be differentiated in vitro provides a unique opportunity to study normal and abnormal corticogenesis. Here, we present a simple and reproducible 3D culture approach for generating a laminated cerebral cortex-like structure, named human cortical spheroids (hCSs), from pluripotent stem cells. hCSs contain neurons from both deep and superficial cortical layers and map transcriptionally to in vivo fetal development. These neurons are electrophysiologically mature, display spontaneous activity, are surrounded by nonreactive astrocytes and form functional synapses. Experiments in acute hCS slices demonstrate that cortical neurons participate in network activity and produce complex synaptic events. These 3D cultures should allow a detailed interrogation of human cortical development, function and disease, and may prove a versatile platform for generating other neuronal and glial subtypes in vitro.

  View details for DOI 10.1038/nmeth.3415

  View details for PubMedID 26005811

• Research and training in autism spectrum disorder to catalyze the next genomic and neuroscience revolutions. Molecular psychiatry Pasca, S. P., Veenstra-VanderWeele, J., McPartland, J. C. 2020

  View details for DOI 10.1038/s41380-020-0830-5

  View details for PubMedID 32601454

• Genetically targeted chemical assembly of functional materials in living cells, tissues, and animals. Science (New York, N.Y.) Liu, J., Kim, Y. S., Richardson, C. E., Tom, A., Ramakrishnan, C., Birey, F., Katsumata, T., Chen, S., Wang, C., Wang, X., Joubert, L. M., Jiang, Y., Wang, H., Fenno, L. E., Tok, J. B., Pașca, S. P., Shen, K., Bao, Z., Deisseroth, K. 2020; 367 (6484): 1372–76

  Abstract

  The structural and functional complexity of multicellular biological systems, such as the brain, are beyond the reach of human design or assembly capabilities. Cells in living organisms may be recruited to construct synthetic materials or structures if treated as anatomically defined compartments for specific chemistry, harnessing biology for the assembly of complex functional structures. By integrating engineered-enzyme targeting and polymer chemistry, we genetically instructed specific living neurons to guide chemical synthesis of electrically functional (conductive or insulating) polymers at the plasma membrane. Electrophysiological and behavioral analyses confirmed that rationally designed, genetically targeted assembly of functional polymers not only preserved neuronal viability but also achieved remodeling of membrane properties and modulated cell type-specific behaviors in freely moving animals. This approach may enable the creation of diverse, complex, and functional structures and materials within living systems.

  View details for DOI 10.1126/science.aay4866

  View details for PubMedID 32193327

• Aberrant calcium channel splicing drives defects in cortical differentiation in Timothy Syndrome. eLife Panagiotakos, G., Haveles, C., Arjun, A., Petrova, R., Rana, A., Portmann, T., Pasca, S. P., Palmer, T. D., Dolmetsch, R. E. 2019; 8

  Abstract

  The syndromic autism spectrum disorder (ASD) Timothy Syndrome (TS) is caused by a point mutation in the alternatively spliced exon 8A of the calcium channel Cav1.2. Using mouse brain and human induced pluripotent stem cells (iPSCs), we provide evidence that the TS mutation prevents a normal developmental switch in Cav1.2 exon utilization, resulting in persistent expression of gain-of-function mutant channels during neuronal differentiation. In iPSC models, the TS mutation reduces the abundance of SATB2-expressing cortical projection neurons, leading to excess CTIP2+ neurons. We show that expression of TS-Cav1.2 channels in the embryonic mouse cortex recapitulates these differentiation defects in a calcium-dependent manner and that in utero Cav1.2 gain-and-loss of function reciprocally regulates the abundance of these neuronal populations. Our findings support the idea that disruption of developmentally-regulated calcium channel splicing patterns instructively alters differentiation in the developing cortex, providing important in vivo insights into the pathophysiology of a syndromic ASD.

  View details for DOI 10.7554/eLife.51037

  View details for PubMedID 31868578

• Engineering a Genetically Encoded Magnetic Protein Crystal. Nano letters Li, T. L., Wang, Z., You, H., Ong, Q., Varanasi, V. J., Dong, M., Lu, B., Pasca, S. P., Cui, B. 2019

  Abstract

  Magnetogenetics is a new field that leverages genetically encoded proteins and protein assemblies that are sensitive to magnetic fields to study and manipulate cell behavior. Theoretical studies show that many proposed magnetogenetic proteins do not contain enough iron to generate substantial magnetic forces. Here, we have engineered a genetically encoded ferritin-containing protein crystal that grows inside mammalian cells. Each of these crystals contains more than 10 million ferritin subunits and is capable of mineralizing substantial amounts of iron. When isolated from cells and loaded with iron in vitro, these crystals generate magnetic forces that are 9 orders of magnitude larger than the forces fromthe single ferritin cages used in previous studies. These protein crystals are attracted to an applied magnetic field and move toward magnets even when internalized into cells. While additional studies are needed to realize the full potential of magnetogenetics, these results demonstrate the feasibility of engineering protein assemblies for magnetic sensing.

  View details for DOI 10.1021/acs.nanolett.9b02266

  View details for PubMedID 31552740

• Engineered materials for organoid systems NATURE REVIEWS MATERIALS Kratochvil, M. J., Seymour, A. J., Li, T. L., Pasca, S. P., Kuo, C. J., Heilshorn, S. C. 2019; 4 (9): 606–22

  View details for DOI 10.1038/s41578-019-0129-9

  View details for Web of Science ID 000484683300006

• Cell diversity in the human cerebral cortex: from the embryo to brain organoids CURRENT OPINION IN NEUROBIOLOGY Arlotta, P., Pasca, S. 2019; 56: 194–98

  View details for DOI 10.1016/j.conb.2019.03.001

  View details for Web of Science ID 000472704500024

• Cell diversity in the human cerebral cortex: from the embryo to brain organoids. Current opinion in neurobiology Arlotta, P., Paşca, S. P. 2019; 56: 194–98

  Abstract

  The development and wiring of the central nervous system is a remarkable biological process that starts with the generation of and interaction between a large diversity of cell types. Our understanding of the developmental logic that drives cellular diversification in the mammalian brain comes, to a large extent, from studies in rodents. However, identifying the unique cellular processes underlying primate corticogenesis has been slow, due to the challenges associated with directly observing and manipulating brain tissue from these species. Recent technological advances in two areas hold promise to accelerate discovery of the mechanisms that govern human brain development, evolution, and pathophysiology of disease. Molecular profiling of large numbers of single cells can now capture cell identity and cell states within a complex tissue. Furthermore, modeling aspects of human organogenesis in vitro, even for tissues as complex as the brain, has been advanced by the use of three-dimensional organoid systems. Here, we describe how these approaches have been applied to date and how they promise to uncover the principles of cell diversification in the developing human brain.

  View details for PubMedID 31051421

• Organoid and Assembloid Technologies for Investigating Cellular Crosstalk in Human Brain Development and Disease. Trends in cell biology Marton, R. M., Pașca, S. P. 2019

  Abstract

  The biology of the human brain, and in particular the dynamic interactions between the numerous cell types and regions of the central nervous system, has been difficult to study due to limited access to functional brain tissue. Technologies to derive brain organoids and assembloids from human pluripotent stem cells are increasingly utilized to model, in progressively complex preparations, the crosstalk between cell types in development and disease. Here, we review the use of these human cellular models to study cell-cell interactions among progenitors, neurons, astrocytes, oligodendrocytes, cancer cells, and non-central nervous system cell types, as well as efforts to study connectivity between brain regions following controlled assembly of organoids. Ultimately, the promise of these patient-derived preparations is to uncover previously inaccessible features of brain function that emerge from complex cell-cell interactions and to improve our mechanistic understanding of neuropsychiatric disorders.

  View details for DOI 10.1016/j.tcb.2019.11.004

  View details for PubMedID 31879153

• A framework for the investigation of rare genetic disorders in neuropsychiatry. Nature medicine Sanders, S. J., Sahin, M., Hostyk, J., Thurm, A., Jacquemont, S., Avillach, P., Douard, E., Martin, C. L., Modi, M. E., Moreno-De-Luca, A., Raznahan, A., Anticevic, A., Dolmetsch, R., Feng, G., Geschwind, D. H., Glahn, D. C., Goldstein, D. B., Ledbetter, D. H., Mulle, J. G., Pasca, S. P., Samaco, R., Sebat, J., Pariser, A., Lehner, T., Gur, R. E., Bearden, C. E. 2019

  Abstract

  De novo and inherited rare genetic disorders (RGDs) are a major cause of human morbidity, frequently involving neuropsychiatric symptoms. Recent advances in genomic technologies and data sharing have revolutionized the identification and diagnosis of RGDs, presenting an opportunity to elucidate the mechanisms underlying neuropsychiatric disorders by investigating the pathophysiology of high-penetrance genetic risk factors. Here we seek out the best path forward for achieving these goals. We think future research will require consistent approaches across multiple RGDs and developmental stages, involving both the characterization of shared neuropsychiatric dimensions in humans and the identification of neurobiological commonalities in model systems. A coordinated and concerted effort across patients, families, researchers, clinicians and institutions, including rapid and broad sharing of data, is now needed to translate these discoveries into urgently needed therapies.

  View details for DOI 10.1038/s41591-019-0581-5

  View details for PubMedID 31548702

• Polarizing brain organoids. Nature biotechnology Miura, Y., Pașca, S. P. 2019

  View details for PubMedID 30936565

• Loss of Adaptive Myelination Contributes to Methotrexate Chemotherapy-Related Cognitive Impairment. Neuron Geraghty, A. C., Gibson, E. M., Ghanem, R. A., Greene, J. J., Ocampo, A., Goldstein, A. K., Ni, L., Yang, T., Marton, R. M., Paşca, S. P., Greenberg, M. E., Longo, F. M., Monje, M. 2019

  Abstract

  Activity-dependent myelination is thought to contribute to adaptive neurological function. However, the mechanisms by which activity regulates myelination and the extent to which myelin plasticity contributes to non-motor cognitive functions remain incompletely understood. Using a mouse model of chemotherapy-related cognitive impairment (CRCI), we recently demonstrated that methotrexate (MTX) chemotherapy induces complex glial dysfunction for which microglial activation is central. Here, we demonstrate that remote MTX exposure blocks activity-regulated myelination. MTX decreases cortical Bdnf expression, which is restored by microglial depletion. Bdnf-TrkB signaling is a required component of activity-dependent myelination. Oligodendrocyte precursor cell (OPC)-specific TrkB deletion in chemotherapy-naive mice results in impaired cognitive behavioral performance. A small-molecule TrkB agonist rescues both myelination and cognitive impairment after MTX chemotherapy. This rescue after MTX depends on intact TrkB expression in OPCs. Taken together, these findings demonstrate a molecular mechanism required for adaptive myelination that is aberrant in CRCI due to microglial activation.

  View details for DOI 10.1016/j.neuron.2019.04.032

  View details for PubMedID 31122677

• Inhibitory Interneurons in Hemimegalencephaly: A Survey of 9 Cases Lummus, S., Andersen, J., Pasca, S., Kleinschmidt-DeMasters, B., Vogel, H. OXFORD UNIV PRESS INC. 2018: 501

  View details for Web of Science ID 000434064400092

• A human cellular model of amyotrophic lateral sclerosis NATURE MEDICINE Marton, R. M., Pasca, S. P. 2018; 24 (3): 256–57

  View details for PubMedID 29509753

• The ethics of experimenting with human brain tissue. Nature Farahany, N. A., Greely, H. T., Hyman, S., Koch, C., Grady, C., Pașca, S. P., Sestan, N., Arlotta, P., Bernat, J. L., Ting, J., Lunshof, J. E., Iyer, E. P., Hyun, I., Capestany, B. H., Church, G. M., Huang, H., Song, H. 2018; 556 (7702): 429–32

  View details for DOI 10.1038/d41586-018-04813-x

  View details for PubMedID 29691509

• Absent forebrain replaced by embryonic stem cells. Nature Andersen, J., Pașca, S. P. 2018; 563 (7729): 44–45

  View details for DOI 10.1038/d41586-018-06933-w

  View details for PubMedID 30375499

• Building three-dimensional human brain organoids. Nature neuroscience 2018

  View details for PubMedID 29593345

• Nondestructive nanostraw intracellular sampling for longitudinal cell monitoring. Proceedings of the National Academy of Sciences of the United States of America Cao, Y., Hjort, M., Chen, H., Birey, F., Leal-Ortiz, S. A., Han, C. M., Santiago, J. G., Pasca, S. P., Wu, J. C., Melosh, N. A. 2017

  Abstract

  Here, we report a method for time-resolved, longitudinal extraction and quantitative measurement of intracellular proteins and mRNA from a variety of cell types. Cytosolic contents were repeatedly sampled from the same cell or population of cells for more than 5 d through a cell-culture substrate, incorporating hollow 150-nm-diameter nanostraws (NS) within a defined sampling region. Once extracted, the cellular contents were analyzed with conventional methods, including fluorescence, enzymatic assays (ELISA), and quantitative real-time PCR. This process was nondestructive with >95% cell viability after sampling, enabling long-term analysis. It is important to note that the measured quantities from the cell extract were found to constitute a statistically significant representation of the actual contents within the cells. Of 48 mRNA sequences analyzed from a population of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs), 41 were accurately quantified. The NS platform samples from a select subpopulation of cells within a larger culture, allowing native cell-to-cell contact and communication even during vigorous activity such as cardiomyocyte beating. This platform was applied both to cell lines and to primary cells, including CHO cells, hiPSC-CMs, and human astrocytes derived in 3D cortical spheroids. By tracking the same cell or group of cells over time, this method offers an avenue to understand dynamic cell behavior, including processes such as induced pluripotency and differentiation.

  View details for DOI 10.1073/pnas.1615375114

  View details for PubMedID 28223521

• The Zika threat to the periphery. Nature neuroscience Khan, T. A., Paşca, S. P. 2017; 20 (9): 1191–92

  View details for PubMedID 28849792

• MicroRNA-9 Couples Brain Neurogenesis and Angiogenesis. Cell reports Madelaine, R., Sloan, S. A., Huber, N., Notwell, J. H., Leung, L. C., Skariah, G., Halluin, C., Paşca, S. P., Bejerano, G., Krasnow, M. A., Barres, B. A., Mourrain, P. 2017; 20 (7): 1533–42

  Abstract

  In the developing brain, neurons expressing VEGF-A and blood vessels grow in close apposition, but many of the molecular pathways regulating neuronal VEGF-A and neurovascular system development remain to be deciphered. Here, we show that miR-9 links neurogenesis and angiogenesis through the formation of neurons expressing VEGF-A. We found that miR-9 directly targets the transcription factors TLX and ONECUTs to regulate VEGF-A expression. miR-9 inhibition leads to increased TLX and ONECUT expression, resulting in VEGF-A overexpression. This untimely increase of neuronal VEGF-A signal leads to the thickening of blood vessels at the expense of the normal formation of the neurovascular network in the brain and retina. Thus, this conserved transcriptional cascade is critical for proper brain development in vertebrates. Because of this dual role on neural stem cell proliferation and angiogenesis, miR-9 and its downstream targets are promising factors for cellular regenerative therapy following stroke and for brain tumor treatment.

  View details for PubMedID 28813666

• Neural Differentiation in the Third Dimension: Generating a Human Midbrain. Cell stem cell Marton, R. M., Pasca, S. P. 2016; 19 (2): 145-146

  Abstract

  In recent years, technological improvements in three-dimensional (3D) culture systems have enabled the generation of organoids or spheroids representing a variety of tissues, including the brain. In this issue of Cell Stem Cell, Jo et al. (2016) describe a 3D culture model of the human midbrain containing dopaminergic neurons and neuromelanin.

  View details for DOI 10.1016/j.stem.2016.07.017

  View details for PubMedID 27494668

• Personalized Human Cortical Spheroids. American journal of psychiatry Pasca, S. P. 2016; 173 (4): 332-333

  View details for DOI 10.1176/appi.ajp.2016.16020133

  View details for PubMedID 27035533

• A deleterious Nav1.1 mutation selectively impairs telencephalic inhibitory neurons derived from Dravet Syndrome patients. eLife Sun, Y., Pasca, S. P., Portmann, T., Goold, C., Worringer, K. A., Guan, W., Chan, K. C., Gai, H., Vogt, D., Chen, Y. J., Mao, R., Chan, K., Rubenstein, J. L., Madison, D. V., Hallmayer, J., Froehlich-Santino, W. M., Bernstein, J. A., Dolmetsch, R. E. 2016; 5

  Abstract

  Dravet Syndrome is an intractable form of childhood epilepsy associated with deleterious mutations in SCN1A, the gene encoding neuronal sodium channel Nav1.1. Earlier studies using human induced pluripotent stem cells (iPSCs) have produced mixed results regarding the importance of Nav1.1 in human inhibitory versus excitatory neurons. We studied a Nav1.1 mutation (p.S1328P) identified in a pair of twins with Dravet Syndrome and generated iPSC-derived neurons from these patients. Characterization of the mutant channel revealed a decrease in current amplitude and hypersensitivity to steady-state inactivation. We then differentiated Dravet-Syndrome and control iPSCs into telencephalic excitatory neurons or medial ganglionic eminence (MGE)-like inhibitory neurons. Dravet inhibitory neurons showed deficits in sodium currents and action potential firing, which were rescued by a Nav1.1 transgene, whereas Dravet excitatory neurons were normal. Our study identifies biophysical impairments underlying a deleterious Nav1.1 mutation and supports the hypothesis that Dravet Syndrome arises from defective inhibitory neurons.

  View details for DOI 10.7554/eLife.13073

  View details for PubMedID 27458797

• Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain. Nature Biotechnology Deverman, B. E., Pravdo, P. L., Simpson, B. P., Kumar, S. R., Chan, K. Y., Banerjee, A., Wu, W., Yang, B., Huber, N., Pasca, S., Gradinaru, V. 2016; 34 (2): 204-9

  View details for DOI 10.1038/nbt.3440

• Alteration in basal and depolarization induced transcriptional network in iPSC derived neurons from Timothy syndrome GENOME MEDICINE Tian, Y., Voineagu, I., Pasca, S. P., Won, H., Chandran, V., Horvath, S., Dolmetsch, R. E., Geschwind, D. H. 2014; 6

  Abstract

  Common genetic variation and rare mutations in genes encoding calcium channel subunits have pleiotropic effects on risk for multiple neuropsychiatric disorders, including autism spectrum disorder (ASD) and schizophrenia. To gain further mechanistic insights by extending previous gene expression data, we constructed co-expression networks in Timothy syndrome (TS), a monogenic condition with high penetrance for ASD, caused by mutations in the L-type calcium channel, Cav1.2.To identify patient-specific alterations in transcriptome organization, we conducted a genome-wide weighted co-expression network analysis (WGCNA) on neural progenitors and neurons from multiple lines of induced pluripotent stem cells (iPSC) derived from normal and TS (G406R in CACNA1C) individuals. We employed transcription factor binding site enrichment analysis to assess whether TS associated co-expression changes reflect calcium-dependent co-regulation.We identified reproducible developmental and activity-dependent gene co-expression modules conserved in patient and control cell lines. By comparing cell lines from case and control subjects, we also identified co-expression modules reflecting distinct aspects of TS, including intellectual disability and ASD-related phenotypes. Moreover, by integrating co-expression with transcription factor binding analysis, we showed the TS-associated transcriptional changes were predicted to be co-regulated by calcium-dependent transcriptional regulators, including NFAT, MEF2, CREB, and FOXO, thus providing a mechanism by which altered Ca(2+) signaling in TS patients leads to the observed molecular dysregulation.We applied WGCNA to construct co-expression networks related to neural development and depolarization in iPSC-derived neural cells from TS and control individuals for the first time. These analyses illustrate how a systems biology approach based on gene networks can yield insights into the molecular mechanisms of neural development and function, and provide clues as to the functional impact of the downstream effects of Ca(2+) signaling dysregulation on transcription.

  View details for DOI 10.1186/s13073-014-0075-5

  View details for Web of Science ID 000344567900001

  View details for PubMedCentralID PMC4213483

• Generating human neurons in vitro and using them to understand neuropsychiatric disease. Annual review of neuroscience Pasca, S. P., Panagiotakos, G., Dolmetsch, R. E. 2014; 37: 479-501

  Abstract

  Recent advances in cell reprogramming enable investigators to generate pluripotent stem cells from somatic cells. These induced pluripotent cells can subsequently be differentiated into any cell type, making it possible for the first time to obtain functional human neurons in the lab from control subjects and patients with psychiatric disorders. In this review, we survey the progress made in generating various neuronal subtypes in vitro, with special emphasis on the characterization of these neurons and the identification of unique features of human brain development in a dish. We also discuss efforts to uncover neuronal phenotypes from patients with psychiatric disease and prospects for the use of this platform for drug development.

  View details for DOI 10.1146/annurev-neuro-062012-170328

  View details for PubMedID 25002278

• Timothy syndrome is associated with activity-dependent dendritic retraction in rodent and human neurons NATURE NEUROSCIENCE Krey, J. F., Pasca, S. P., Shcheglovitov, A., Yazawa, M., Schwemberger, R., Rasmusson, R., Dolmetsch, R. E. 2013; 16 (2): 201-209

  Abstract

  L-type voltage gated calcium channels have an important role in neuronal development by promoting dendritic growth and arborization. A point mutation in the gene encoding Ca(V)1.2 causes Timothy syndrome, a neurodevelopmental disorder associated with autism spectrum disorders (ASDs). We report that channels with the Timothy syndrome alteration cause activity-dependent dendrite retraction in rat and mouse neurons and in induced pluripotent stem cell (iPSC)-derived neurons from individuals with Timothy syndrome. Dendrite retraction was independent of calcium permeation through the mutant channel, was associated with ectopic activation of RhoA and was inhibited by overexpression of the channel-associated GTPase Gem. These results suggest that Ca(V)1.2 can activate RhoA signaling independently of Ca(2+) and provide insights into the cellular basis of Timothy syndrome and other ASDs.

  View details for DOI 10.1038/nn.3307

  View details for Web of Science ID 000314260200017

  View details for PubMedCentralID PMC3568452

• A promoter in the coding region of the calcium channel gene CACNA1C generates the transcription factor CCAT. PloS one Gomez-Ospina, N., Panagiotakos, G., Portmann, T., Pasca, S. P., Rabah, D., Budzillo, A., Kinet, J. P., Dolmetsch, R. E. 2013; 8 (4)

  Abstract

  The C-terminus of the voltage-gated calcium channel Cav1.2 encodes a transcription factor, the calcium channel associated transcriptional regulator (CCAT), that regulates neurite extension and inhibits Cav1.2 expression. The mechanisms by which CCAT is generated in neurons and myocytes are poorly understood. Here we show that CCAT is produced by activation of a cryptic promoter in exon 46 of CACNA1C, the gene that encodes CaV1.2. Expression of CCAT is independent of Cav1.2 expression in neuroblastoma cells, in mice, and in human neurons derived from induced pluripotent stem cells (iPSCs), providing strong evidence that CCAT is not generated by cleavage of CaV1.2. Analysis of the transcriptional start sites in CACNA1C and immune-blotting for channel proteins indicate that multiple proteins are generated from the 3' end of the CACNA1C gene. This study provides new insights into the regulation of CACNA1C, and provides an example of how exonic promoters contribute to the complexity of mammalian genomes.

  View details for DOI 10.1371/journal.pone.0060526

  View details for PubMedID 23613729

  View details for PubMedCentralID PMC3628902

• Motor abnormalities as a putative endophenotype for Autism Spectrum Disorders. Frontiers in integrative neuroscience Esposito, G., Pasca, S. P. 2013; 7: 43-?

  Abstract

  Autism Spectrum Disorders (ASDs) represent a complex group of behaviorally defined conditions with core deficits in social communication and the presence of repetitive and restrictive behaviors. To date, neuropathological studies have failed to identify pathognomonic cellular features for ASDs and there remains a fundamental disconnection between the complex clinical aspects of ASDs and the underlying neurobiology. Although not listed among the core diagnostic domains of impairment in ASDs, motor abnormalities have been consistently reported across the spectrum. In this perspective article, we summarize the evidence that supports the use of motor abnormalities as a putative endophenotype for ASDs. We argue that because these motor abnormalities do not directly depend on social or linguistic development, they may serve as an early disease indicator. Furthermore, we propose that stratifying patients based on motor development could be useful not only as an outcome predictor and in identifying more specific treatments for different ASDs categories, but also in exposing neurobiological mechanisms.

  View details for DOI 10.3389/fnint.2013.00043

  View details for PubMedID 23781177

• Using iPSC-derived neurons to uncover cellular phenotypes associated with Timothy syndrome NATURE MEDICINE Pasca, S. P., Portmann, T., Voineagu, I., Yazawa, M., Shcheglovitov, A., Pasca, A. M., Cord, B., Palmer, T. D., Chikahisa, S., Nishino, S., Bernstein, J. A., Hallmayer, J., Geschwind, D. H., Dolmetsch, R. E. 2011; 17 (12): 1657-U176

  Abstract

  Monogenic neurodevelopmental disorders provide key insights into the pathogenesis of disease and help us understand how specific genes control the development of the human brain. Timothy syndrome is caused by a missense mutation in the L-type calcium channel Ca(v)1.2 that is associated with developmental delay and autism. We generated cortical neuronal precursor cells and neurons from induced pluripotent stem cells derived from individuals with Timothy syndrome. Cells from these individuals have defects in calcium (Ca(2+)) signaling and activity-dependent gene expression. They also show abnormalities in differentiation, including decreased expression of genes that are expressed in lower cortical layers and in callosal projection neurons. In addition, neurons derived from individuals with Timothy syndrome show abnormal expression of tyrosine hydroxylase and increased production of norepinephrine and dopamine. This phenotype can be reversed by treatment with roscovitine, a cyclin-dependent kinase inhibitor and atypical L-type-channel blocker. These findings provide strong evidence that Ca(v)1.2 regulates the differentiation of cortical neurons in humans and offer new insights into the causes of autism in individuals with Timothy syndrome.

  View details for DOI 10.1038/nm.2576

  View details for PubMedID 22120178

• Surround modulation of neuronal responses in V1 is as stable over time as responses to direct stimulation of receptive fields CORTEX Pasca, S. P., Singer, W., Nikolic, D. 2010; 46 (9): 1199-1203

  Abstract

  In the primary visual cortex (V1) the modulation of neuronal responses by surround stimuli displays considerable variability. At present, it is not known whether this variability across neurons is due to temporal instability or to neuron-specific differences. We explored this question in the cat visual cortex by making multi-channel recordings while repeatedly presenting surround gratings of collinear and orthogonal orientation to the centre stimulus for a period of 96 h. Our results indicate that surround modulation is temporally stable to about the same degree as the responses evoked by the centre stimuli. The results support the notion that the mechanisms of surround modulation exhibit a high degree of stability and play an important role in the modulation of cortical responses.

  View details for DOI 10.1016/j.cortex.2010.05.003

  View details for Web of Science ID 000282561600014

  View details for PubMedID 20557882

• Paraoxonase 1 activities and polymorphisms in autism spectrum disorders JOURNAL OF CELLULAR AND MOLECULAR MEDICINE Pasca, S. P., Dronca, E., Nemes, B., Kaucsar, T., Endreffy, E., Iftene, F., Benga, I., Cornean, R., Dronca, M. 2010; 14 (3): 600-607

  Abstract

  Autism spectrum disorders (ASD) comprise a complex and heterogeneous group of conditions of unknown aetiology, characterized by significant disturbances in social, communicative and behavioural functioning. Recent studies suggested a possible implication of the high-density lipoprotein associated esterase/lactonase paraoxonase 1 (PON1) in ASD. In the present study, we aimed at investigating the PON1 status in a group of 50 children with ASD as compared to healthy age and sex matched control participants. We evaluated PON1 bioavailability (i.e. arylesterase activity) and catalytic activity (i.e. paraoxonase activity) in plasma using spectrophotometric methods and the two common polymorphisms in the PON1 coding region (Q192R, L55M) by employing Light Cycler real-time PCR. We found that both PON1 arylesterase and PON1 paraoxonase activities were decreased in autistic patients (respectively, P < 0.001, P < 0.05), but no association with less active variants of the PON1 gene was found. The PON1 phenotype, inferred from the two-dimensional enzyme analysis, had a similar distribution in the ASD group and the control group. In conclusion, both the bioavailability and the catalytic activity of PON1 are impaired in ASD, despite no association with the Q192R and L55M polymorphisms in the PON1 gene and a normal distribution of the PON1 phenotype.

  View details for DOI 10.1111/j.1582-4934.2008.00414.x

  View details for Web of Science ID 000276950100011

  View details for PubMedID 18624774

• One carbon metabolism disturbances and the C677T MTHFR gene polymorphism in children with autism spectrum disorders JOURNAL OF CELLULAR AND MOLECULAR MEDICINE Pasca, S. P., Dronca, E., Kaucsar, T., Craciun, E. C., Endreffy, E., Ferencz, B. K., Iftene, F., Benga, I., Cornean, R., Banerjee, R., Dronca, M. 2009; 13 (10): 4229-4238

  Abstract

  Autism spectrum disorders (ASDs), which include the prototypic autistic disorder (AD), Asperger's syndrome (AS) and pervasive developmental disorders not otherwise specified (PDD-NOS), are complex neurodevelopmental conditions of unknown aetiology. The current study investigated the metabolites in the methionine cycle, the transsulphuration pathway, folate, vitamin B(12) and the C677T polymorphism of the MTHFR gene in three groups of children diagnosed with AD (n= 15), AS (n= 5) and PDD-NOS (n= 19) and their age- and sex-matched controls (n= 25). No metabolic disturbances were seen in the AS patients, while in the AD and PDD-NOS groups, lower plasma levels of methionine (P= 0.01 and P= 0.03, respectively) and alpha-aminobutyrate were observed (P= 0.01 and P= 0.001, respectively). Only in the AD group, plasma cysteine (P= 0.02) and total blood glutathione (P= 0.02) were found to be reduced. Although there was a trend towards lower levels of serine, glycine, N, N-dimethylglycine in AD patients, the plasma levels of these metabolites as well as the levels of homocysteine and cystathionine were not statistically different in any of the ASDs groups. The serum levels of vitamin B(12) and folate were in the normal range. The results of the MTHFR gene analysis showed a normal distribution of the C677T polymorphism in children with ASDs, but the frequency of the 677T allele was slightly more prevalent in AD patients. Our study indicates a possible role for the alterations in one carbon metabolism in the pathophysiology of ASDs and provides, for the first time, preliminary evidence for metabolic and genetic differences between clinical subtypes of ASDs.

  View details for DOI 10.1111/j.1582-4934.2008.00463.x

  View details for Web of Science ID 000274181900015

  View details for PubMedID 19267885

• Vomiting is not an adaption for glaucoma (and Darwinian medicine is difficult) MEDICAL HYPOTHESES Pasca, S. P., Nesse, R. M. 2008; 71 (3): 472-473

  View details for DOI 10.1016/j.mehy.2008.04.009

  View details for Web of Science ID 000258641800040

  View details for PubMedID 18513879

• Serum paraoxonase 1 activities and homocysteinemia in hemodialysis patients CLINICAL CHEMISTRY AND LABORATORY MEDICINE Dronca, M., Pasca, S. P., Nemes, B., Vlase, L., Vladutiu, D. 2008; 46 (6): 880-881

  View details for DOI 10.1515/CCLM.2008.164

  View details for Web of Science ID 000257542800023

  View details for PubMedID 18601616

• Behavioral effects of corpus callosum transection and environmental enrichment in adult rats BEHAVIOURAL BRAIN RESEARCH Miu, A. C., Heilman, R. M., Pasca, S. P., Stefan, C. A., Spanu, F., Vasiu, R., Olteanu, A. I., Miclea, M. 2006; 172 (1): 135-144

  Abstract

  A common assumption about the corpus callosum transection (CCX) is that it only affects behaviors heavily relying on interhemispheric communication. However, cerebral laterality is ubiquitous across motor and perceptual, cognitive and emotional domains, and the corpus callosum is important for its establishment. Several recent studies showed that the partial denervation of the sensorimotor isocortex through CCX derepressed neural growth processes that were sensitive to motor demand (experience-dependent neural plasticity). We investigated whether the facilitatory effects of CCX on cortical neural plasticity, shaped by differential housing, extended beyond the motor domain. Adult rats were housed in enriched (EE), standard (SE) or impoverished environments (IE) for 10 weeks, that is, 2 weeks before they underwent CCX or sham surgery, and, then, 8 weeks throughout the experiments. After they recovered from surgery, the behavioral performance of rats was tested using open-field, spontaneous alternation in the T-maze, paw preference, Morris water maze, and tone fear conditioning. The results indicated that the effects of CCX and housing on open-field behavior were independent, with CCX increasing the time spent in the center of the field at the beginning of the observation (i.e., emotionality), and EE and IE increasing rearing (emotionality) and reducing teeth-chattering (habituation), respectively. CCX reduced the frequency of spontaneous alternation, denoting spatial working memory deficits, while housing did not influence this performance. Neither CCX, nor housing significantly affected paw preference lateralization, although CCX was associated with a leftward bias in paw preference. In the Morris water maze, housing had effects on spatial acquisition, while CCX reduced activity, without interfering with spatial memory. CCX did not influence tone fear conditioning, but context fear conditioning seemed to benefit from EE. We conclude that CCX in adult rats has subtle, but specific behavioral effects pertaining to emotionality, spatial working memory, and, possibly, aversively motivated exploration, and these effects are either independent or only peripherally interact with the effects of housing.

  View details for DOI 10.1016/j.bbr.2006.05.007

  View details for Web of Science ID 000239683800016

  View details for PubMedID 16764947

• High levels of homocysteine and low serum paraoxonase 1 arylesterase activity in children with autism LIFE SCIENCES Pasca, S. P., Nemes, B., Vlase, L., Gagyi, C. E., Dronca, E., Miu, A. C., Dronca, M. 2006; 78 (19): 2244-2248

  Abstract

  Autism is a behaviorally defined disorder of unknown etiology that is thought to be influenced by genetic and environmental factors. High levels of homocysteine and oxidative stress are generally associated with neuropsychiatric disorders. The purpose of this study was to compare the level of homocysteine and other biomarkers in children with autism to corresponding values in age-matched healthy children. We measured total homocysteine (tHcy), vitamin B(12), paraoxonase and arylesterase activities of human paraoxonase 1 (PON1) in plasma and glutathione peroxidase (GPx) activity in erythrocytes from 21 children: 12 with autism (age: 8.29 +/- 2.76 years) and 9 controls (age: 8.33 +/- 1.82 years). We found statistically significant differences in tHcy levels and in arylesterase activity of PON1 in children with autism compared to the control group: 9.83 +/- 2.75 vs. 7.51 +/- 0.93 micromol/L (P < or =0.01) and 72.57 +/- 11.73 vs. 81.83 +/- 7.39 kU/L (P < or =0.005). In the autistic group there was a strong negative correlation between tHcy and GPx activity and the vitamin B(12) level was low or suboptimal. In conclusion, our study shows that in children with autism there are higher levels of tHcy, which is negatively correlated with GPx activity, low PON1 arylesterase activity and suboptimal levels of vitamin B(12).

  View details for DOI 10.1016/j.lfs.2005.09.040

  View details for Web of Science ID 000236718800012

  View details for PubMedID 16297937