1983 — 1987 |
Teitelman, Gladys [⬀] Iacovitti, Lorraine |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Induction of Adrenergic Traits in Autonomic Cholinergic Neurons @ Joan and Sanford I. Weill Medical College of Cornell University |
0.931 |
1988 — 1990 |
Iacovitti, Lorraine |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Transmitter Phenotypic Plasticity in Central Neurons @ Weill Medical College of Cornell Univ
We have recently demonstrated that the catecholamine (CA) synthesizine enzyme tyrosine hydroxylase (TH) is expressed in a large and previously undescribed population of adult neurons located in layer I of the cerebral cortex. These neurons have not yet been observed in the developing cortex. However, when the cortex of E13 rats is dissociated and placed into culture, we find several thousand TH immunoreactive neurons. Expression of the enzyme is restricted to neurons which have completed cell replication, occurs only in cells at a critical stage in their development (E13-E16) and gradually declines in culture as some neurons acquire GABAergic traits. Our findings in culture, combined with the anatomical localization of TH cells in the adult cortex, suggest, but do not prove, that TH is being expressed in a group of neurons known as Cajal-Retzuis (C-R) cells. To date, the fate and function of C-R cells remains a mystery. The fact that many of their processes project to the pial membrane raises the possibility that blood vessels, acting as targets, may in some way regulate the phenotypic expression of transmitter traits in these cells. Indeed, in our preliminary experiments, we found that the porportion of TH immunoreactive cortical neurons was dramatically increased (20 fold) by growing the cells with a soluble factor(s) derived from muscle cells of vascular origin as well as from other muscle sources. The present study seeks to determine 1) Whether TH cells are indeed C-R neurons; 2) Whether C-R cells die or transdifferentiate into cells of another neurotransmitter phenotype; 3) Whether other CA traits are expressed by these cells in vivo and in vitro; 4) Whether, during the critical period, TH expression is modified as a result of changes at the transcription or translation level; 5) The mechanism and nature of the epigenetic signal regulating the expression of TH in cortex; and 6) Whether these target-derived factors found in muscle also play a role in signally CA differentiation in vivo. These issues will be analyzed using a multidisciplinary approach which includes biochemical, autoradiographic, immunocytochemical and molecular biological techniques. In so doing, we expect to identify and characterize a new population of neurons expressing the CA enzyme TH and to further our understanding of the basic genetic and epigenetic principles which govern the choice of transmitter phenotype in brain neurons. Our hope is that these studies may provide some clues as to the processes operating during normal development of the CNS as well as in certain pathological conditions.
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0.951 |
1991 — 1993 |
Iacovitti, Lorraine |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Studies On Muscle-Derived Differentiation Factor @ Allegheny University of Health Sciences
DESCRIPTION (Investigator's Abstract): Regulation of neurotransmitter synthesis is crucial to the development and normal functioning of neurons. Much progress has been made in identifying factors which regulate catecholamine (CA) synthesis in the peripheral nervous system. In contrast, very little is known about this process in brain. Recent experiments (Iacovitti et al., 1989) have now demonstrated that synthesis of tyrosine hydroxylase (TH), the rate limiting enzyme in CA synthesis, is profoundly increased in certain embryonic brain neurons when they are exposed to a muscle cell extract in tissue culture. The induction of TH is dose dependent and results from a diffusible molecule(s) termed the Muscle-Derived Differentiation Factor (MDF). The identify of MDF remains unknown. In an exhaustive survey, its effects could not be reproduced by other factors known to regulate TH. MDF dramatically induces TH in catecholamine neurons (substantia nigra) as well as in neurons that do not normally express a CA phenotype (cerebral cortex and striatum). MDF exerts its influence at the level of gene expression, up-regulating TH mRNA transcription at least 10-fold. As such, MDF is the first factor described which regulates the TH gene in brain neurons. The proposed project aims to purify and identify MDF. Refinement of the MDF assay, based upon the immunocytochemical detection of TH, has now made purification an obtainable goal. Preliminary experiments indicate that MDF is a fairly stable macromolecule that will be amenable to a variety of fractionation methods. Concurrent with purification, experiments are proposed which can be carried out with partially pure MDF. The signal transduction pathway by which the TH gene is regulated will be investigated by artificially inducing second messenger systems in an attempt to reproduce the effects of MDF. Finally, MDF's effects will be studied in situ in animal models of disease involving compromise of brain catecholamine systems. We will assess anatomical, biochemical and behavioral recovery in lesioned/grafted brain following MDF treatment. The long range goal of this work is to elucidate the mechanisms controlling the expression of catecholamines in the brain. These studies are of clinical relevance since a number of neurological and psychiatric disorders are associated with imbalances in brain CA's; most notable, is Parkinson's Disease, but also Alzheimers Disease, clinical depression and schizophrenia.
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0.937 |
1992 |
Iacovitti, Lorraine |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Muscle-Derived Differentiation Factor @ Allegheny University of Health Sciences
DESCRIPTION (Investigator's Abstract): Regulation of neurotransmitter synthesis is crucial to the development and normal functioning of neurons. Much progress has been made in identifying factors which regulate catecholamine (CA) synthesis in the peripheral nervous system. In contrast, very little is known about this process in brain. Recent experiments (Iacovitti et al., 1989) have now demonstrated that synthesis of tyrosine hydroxylase (TH), the rate limiting enzyme in CA synthesis, is profoundly increased in certain embryonic brain neurons when they are exposed to a muscle cell extract in tissue culture. The induction of TH is dose dependent and results from a diffusible molecule(s) termed the Muscle-Derived Differentiation Factor (MDF). The identify of MDF remains unknown. In an exhaustive survey, its effects could not be reproduced by other factors known to regulate TH. MDF dramatically induces TH in catecholamine neurons (substantia nigra) as well as in neurons that do not normally express a CA phenotype (cerebral cortex and striatum). MDF exerts its influence at the level of gene expression, up-regulating TH mRNA transcription at least 10-fold. As such, MDF is the first factor described which regulates the TH gene in brain neurons. The proposed project aims to purify and identify MDF. Refinement of the MDF assay, based upon the immunocytochemical detection of TH, has now made purification an obtainable goal. Preliminary experiments indicate that MDF is a fairly stable macromolecule that will be amenable to a variety of fractionation methods. Concurrent with purification, experiments are proposed which can be carried out with partially pure MDF. The signal transduction pathway by which the TH gene is regulated will be investigated by artificially inducing second messenger systems in an attempt to reproduce the effects of MDF. Finally, MDF's effects will be studied in situ in animal models of disease involving compromise of brain catecholamine systems. We will assess anatomical, biochemical and behavioral recovery in lesioned/grafted brain following MDF treatment. The long range goal of this work is to elucidate the mechanisms controlling the expression of catecholamines in the brain. These studies are of clinical relevance since a number of neurological and psychiatric disorders are associated with imbalances in brain CA's; most notable, is Parkinson's Disease, but also Alzheimers Disease, clinical depression and schizophrenia.
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0.937 |
1994 — 1998 |
Iacovitti, Lorraine |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R55Activity Code Description: Undocumented code - click on the grant title for more information. |
Purified Dopamine Neurons @ Thomas Jefferson University
DESCRIPTION: One of the-central issues in neurobiology revolves around the importance of trophic molecules in the survival and-differentiation of dopamine neurons during development and after injury. Moreover, the absence or loss of growth factors has been implicated as a possible cause for a) the degeneration of neurons that occurs in a number of neurodegenerative diseases like Parkinson's and b) the failure of implanted brain cells to survive in transplant therapy. The research proposed in this application examines the role of growth substances (EGF, aFGF, bFGF, TGFB, CNTF, LIF, IL1, NGF, IGF, GMI) in regulating the survival and biochemical differentiation of dopamine neurons deriving development, after damage, and following transplantation. The primary model to be used in these studies was recently developed wherein populations of nearly pure dopamine neurons are isolated for study. This is accomplished by flow cytometry of neurons previously labeled with retrogradely transported fluorescent dyes (diL). Until now, dopamine neurons have comprised only a small (<1%) fraction of midbrain cells used in these studies. With this new found ability to isolate nearly purified (>90%) dopamine neurons, the principal investigator is now in an ideal position to examine the relative contributions made directly by dopamine neurons or indirectly by other cell types and/or their molecular products (ie. trophic factors).
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1 |
1995 — 2002 |
Iacovitti, Lorraine |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Factors Regulating Tyrosine Hydroxylase Gene Expression @ Thomas Jefferson University
DESCRIPTION: (Adapted from Applicant's Abstract) Imbalances in brain catecholamine (CA) neurotransmitter systems are associated with aging as well as a whole array of debilitating neurological ad psychiatric disorders; including Parkinson's disease, Alzheimer's disease, Tourette's syndrome, manic-depressive illness and schizophrenia. These ailments serve to underscore the critical importance that Cas play in the proper functioning of the brain. The availability of CA neurotransmitters is largely determined by regulation of the rate-limiting enzyme, tyrosine hydroxylase (TH). Over the last nine years, studies from this laboratory have focused on identifying the genetic and epigenetic agents which regulate the first expression of the TH gene in the hope of discovering ways in which to manipulate its expression. We have taken advantage of the fact that the GABergic neurons of the striatum are phenotypically plastic for a time in their development. We discovered that, during this brief window, neurons can trans-differentiate into TH-expressing and DA-producing cells if exposed to the synergistic interaction of specific growth factors (aFGF, bFGF, BDNF) and an obligatory co-activating molecule (CA neurotransmitters, protein kinase A and C pathway activators). Our most recent studies have begun to explore precisely how these substances trigger expression of the TH gene. Thus far, one key pathway followed by these signals is through MAP kinase to the AP-1 site on the 5' flanking region of the TH gene. Our proposed project aims are: 1) identification of known and new cis-and trans-acint factors; 2) the signal transudction pathways that lead to their activation of the TH gene; 3) the celluar/molecular changes which repress TH induction later in life and 4)finally, whether the differentiation of other neurotransmitter phenotypes can be similarly regulated. The long range goal of this work is to elucidate the cellular and molecular processes regulating neurotransmitter synthesis so that brain cells can someday be engineering or stem cells can be stimulated to produce more or less of the appropriate neurotransmitters in the damaged, diseased or aging brain.
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1 |
1998 — 2000 |
Iacovitti, Lorraine |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Melatonin Effects On Damaged Dopamine Neurons @ Thomas Jefferson University
DESCRIPTION Dopamine (DA) neurons are uniquely vulnerable to damage and cell death, and their loss in humans is associated with diseases including Parkinson's disease (PD). There is a large body of evidence suggesting that the destruction of DA neurons in PD involves the accumulation of free radicals. The antioxidant hormone, melatonin, is one of the most potent endogenous scavengers of oxyradicals yet its ability to rescue, prevent or repair damage to DA neurons has not been directly studied. This proposal seeks to test melatonin's effects on DA neurons in several laboratory models associated with oxidative stress, including growth factor deprivation or neurotoxic injury from MPP+ or 6-OHDA. Preliminary studies reveal a remarkable ability of melatonin to rescue developing and neurotoxin- damaged DA neurons from impending cell death in culture and in vivo and to further promote the growth and regeneration of DA fibers. The ultimate goal of this research is to provide a fuller understanding of the role that melatonin might play in increasing the survival of neurons in the injured DA system and lay a foundation for development of treatments for PD and other diseases with compromised DA systems.
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1 |
1999 — 2007 |
Iacovitti, Lorraine |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Studies of Purified Dopamine Neurons @ Thomas Jefferson University
DESCRIPTION (provided by applicant): Historically, there has been no good way to isolate DA neurons from other cells of the midbrain. Thus, missing DA neurons have been replaced by mixed cell populations following transplantation of embryonic midbrain tissue in animal models of disease and in Parkinson's patients. Although, in many cases, these transplants have provided long-term benefit, the presence of unwanted cells, such as glia, non-DAergic neurons, or even excessive numbers of DA neurons, has produced serious side effects, and in rare cases, even death. Discovering ways in which to segregate DA neurons from other cell types poses a significant challenge, but a necessary next step. In the present proposal, our plan is to take advantage of several new advances in the laboratory; including the recent cloning of 11kb human tyrosine hydroxylase gene promoter (hTH). This sequence accurately targets the expression of the reporter, green fluorescent protein (GFP) to DA neurons of the mammalian CNS. Because GFP can be directly visualized in live fetal DA neurons, this approach allows enrichment via flourescent activated cell sorting (FACS) for study in vivo and in vitro. Moreover, it is possible to adapt these purification methods to mouse stem and human progenitor cells using a lentiviral vector to transduce cells with the hTH-GFP transgene. Following their DA differentiation and FACS sorting, our goal is to study purified populations of engineered stem/progenitor-derived DA neurons in culture or after transplantation into the Parkinsonian rat. These models offer us a unique opportunity to determine the ideal number of DA neurons needed as well as the optimal conditions which contribute to their survival and growth following transplantation. Graft function will be assessed in live animals via behavioral testing and in vivo microdialysis which will be correlated with biochemical and anatomical (at the light and electron microscopic levels) changes following sacrifice. This work will hopefully lay the foundation for the development of therapeutic treatments for Parkinson's and other diseases involving compromised DA systems.
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1 |
2002 — 2004 |
Iacovitti, Lorraine |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Neural Stem Cells Grafts in Primate Models of Parkinsons @ Thomas Jefferson University
DESCRIPTION (provided by applicant) One promising new therapy for Parkinson's Disease (PD) involves the replacement of degenerated nigrostriatal neurons with those derived from transplanted fetal mesencephalic tissue. Although this approach has often yielded remarkable recovery of function in rats and monkeys, results in clinical trials with PD patients have been less consistent. At issue, is the relative inability to standardize a number of critical factors in human fetal transplants, including the age, type, number and integrity of cells being grafted. Consequently, finding more reliable sources of dopaminergic (DA) tissue for transplantation has become increasingly important. One direction has been to search for a line of readily available, well-characterized, continually self-renewing stem or precursor cells that possess the capacity to differentiate, ideally spontaneously and without need of genetic manipulation, into DA neurons, thus providing an inexhaustible and uniform source of replacement tissue. Towards this end, our preliminary findings demonstrate that grafts of embryonic mouse neural stem cells of the C17.2 cell can differentiate exclusively into neurons, which in a majority of cases, can express DA traits when transplanted into the brain of a Parkinsonian rat. In addition, we now have available two human NSC lines (HNSC.100 and HSP-2) which are being studied in vitro and in rat models of PD. Therefore, the goal of the present proposal is to move a step closer to the clinic by testing the utility of both mouse and human stem cells in a non-human primate model of PD. Our specific goals for this proposal are twofold: 1) Determine whether mouse NSCs behave in a primate model as they do in a rat model of PD; and 2) Determine whether human NSCs also differentiate, integrate and function as DA neurons when transplanted into the MPTP-treated monkey. The ultimate goal of this research program is a fuller understanding of the cellular and molecular processes regulating the differentiation of DA traits in stem cells and application of that knowledge to transplantation strategies for the treatment of Parkinson's Disease.
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1 |
2002 — 2006 |
Iacovitti, Lorraine |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Using Stem Cells in Animal Models of Parkinson's Disease @ Thomas Jefferson University
One promising new therapy for Parkinson's Disease (PD) involves the replacement of degenerated nigrostriatal neurons with those derived from transplanted fetal mesencephalic tissue. Although this approach has often yielded remarkable recovery of function in rats and monkeys, results in clinical trials with PD patients have been less consistent. At issue, is the relative inability to standardize a number of critical factors in human fetal transplants, including the age, type, number and integrity of cells being grafted. Consequently, finding more reliable sources of dopaminergic (DA) tissue for transplantation has become increasingly important. One direction has been to search for a line of readily available, well-characterized continually self-renewing stem or precursor cells that possess the capacity to differentiate, ideally spontaneously and with the need for little manipulation, into DA neurons, thus providing an inexhaustible and uniform source of replacement tissue. Towards this end, our preliminary findings demonstrate that grafts of embryonic mouse neural stem cells (NSCs) of the C17.2 cell can differentiate exclusively into neurons, which in a majority of cases, can express DA traits when cells are transplanted into the brain of a Parkinsonian rat. In addition, in preliminary studies using stem cells from adult human bone marrow (MSCs), we have found that nearly 100 percent of MSCs will convert into process- bearing, beta-tubulin III+ neuronal-like cells after only 1-2 hours of incubation with specific differentiation factors. If these cells also exhibit the same capacity as NSCs to respond to appropriate DA differentiation cues in vivo, patients could provide their own source of stem cells for autologous grafts in PD. Using NSC and MSC stem cell models and a multidisciplinary approach, our specific goals for this proposal are threefold: 1) Identify the conditions that promote the stable appearance of a postmitotic differentiated DA phenotype in stem cells grown in culture; 2) Identify those factors which promote the differentiation of a DA phenotype in transplanted stem cells and 3) Determine whether the DA phenotype in transplanted stem cells is stable and long lasting, and whether, it can produce functional recovery of motor deficits in a rat model of PD. The ultimate goal of this research program is a fuller understanding of the cellular and molecular processes regulating the differentiation of DA traits in stem cells and apply that knowledge to transplantation strategies for the treatment of Parkinson's Disease.
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1 |
2006 — 2007 |
Iacovitti, Lorraine |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Imaging Stem Cell Implants in Neurodegenerative Disease @ Thomas Jefferson University
DESCRIPTION (provided by applicant): Despite considerable effort, there is still no reliable way to either prevent or rescue dopamine (DA) neurons from the progressive degeneration that occurs during aging or in Parkinson's Disease (PD). Since clinical diagnosis almost always occurs after the vast majority of DA neurons have been destroyed, researchers continue to work to develop ways in which to replace lost tissue with transplanted cells capable of dopaminergic function. Our goal is l to study purified populations of engineered stem, progenitor or fetal DA neurons after transplantation into the Parkinsonian rat. A major limitation of these approaches is the inability to monitor the progression, if any, of the grafted cells without highly invasive tissue biopsy, which invariably results in the death of the animal. The vast numbers of animals required for these studies could be reduced significantly (with the concomitant reduction in costs) if each animal could be studied non-invasively and repeatedly. In addition, measurements made of the DA neuronal regeneration in a rat model ex vivo are not translatable to humans. Molecular imaging, using PET and SPECT, of animal models of PD, and other neurodegenerative diseases, enables the study of the in vivo neurochemical basis of the disorder. In this proposal we aim to perform quantitative imaging of dopaminergic neurons in vivo in longitudinal studies of the same animals over an extended period of time after stem cell implantation. We aim to validate quantitative models of dopaminergic function in rats, using ultra-high resolution] PET and SPECT. [18F]DOPA imaging will be used with PET to monitor striatal dopa decarboxylase activity, while [99mTc]TRODAT-1 and SPECT will be used to measure dopamine transporter (DAT) availability directly. These will be validated against established post mortem methods, such as GFP reporter gene expression and immunocytochemistry. Longitudinal imaging studies of rats, following stem cell implantation, will enable the visualization of the regeneration of DA neurons over an extended period of time. Once the imaging techniques have been fully validated, they will be applied to a variety of stem cell implant models, and correlated with behavioral studies. This non-invasive approach will enable the best combination of cell types and growth factors to be established without sacrificing the animals. The ultimate goal of this study is to develop methods which will enable the monitoring in vivo of DA neuron replacement treatments in Parkinson's and other neurodegenerative diseases. This will provide vital information in the animal model of PD, allow us to longitudinally follow DA neuron regeneration, and, most importantly, will be translatable to clinical human studies using similar techniques.
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1 |
2010 — 2014 |
Iacovitti, Lorraine |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Cvos as Putative Sites of Adult Stem Cells @ Thomas Jefferson University
Achieving tissue homeostasis in the adult organism requires the continual replacement of cells which have died due to aging, damage or disease. While it was once thought that the adult brain was an exception to this aspect of tissue regulation, it is now known that the brain contains several regions where neurogenesis occurs throughout life. This laboratory recently described a series of new sites in the brain, found in the adult circumventricular organs (CVOs), that contains cells expressing stem cell-like traits. Traditionally, CVOs have been referred to as the "windows of the brain" because of their unique ability to sense physiological stimuli in blood and secrete factors into cerebrospinal fluid. These properties of the CVOs, and their cells with stem cell-like properties, raise the intriguing hypothesis that CVOs represent novel niches for cell genesis important for homeostasis in the adult brain. This project is designed to characterize further the putative stem cells residing in the CVOs of mice. The PI's laboratory has generated genetically engineered CVO stem cells that are tagged and can be tracked allowing their survival, migration and differerentiation to be followed in the adult brain. In addition, this project will explore whether the fate of tagged CVO stem cells can be re-directed following their transplantation to distinct CVO niches. The overall goal of these studies is the discovery and characterization of new sites for cell genesis in the adult brain that are important for maintaining brain homeostasis in response to changing physiological, environmental, and/or pathological stimuli. Moreover, by training both undergraduate and graduate students, this project will foster the development of the next generation of scientists in stem cell biology, an important new area of scientific inquiry.
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1 |
2012 — 2016 |
Iacovitti, Lorraine |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Using Reporter Human Ips Cells to Study Fate, Function and Parkinson's Disease @ Thomas Jefferson University
DESCRIPTION (provided by applicant): Understanding the principles and processes governing the differentiation of a midbrain dopamine (mDA) phenotype in developing neurons is important not only for brain ontogeny but also for the study and treatment of diseases such as Parkinson's disease (PD). In the last decade, a great deal of insight has been gained into the transcriptional machinery regulating mDA differentiation in the embryonic mouse brain. Importantly, many of those same processes appear to be shared by human induced pluripotent stem (hiPS) cells as they differentiate into mDA neurons in the dish. Thus, when human neural progenitors (hNPs) derived either from human embryonic stem (hES) cells or adult induced pluripotent stem (hiPS) cells commit to the mDA differentiation pathway, they express many of the same mDA-specific genes/proteins (Lmx1a, Aldh1a1, Nurr1, Pitx3, TH, etc.). Importantly, regardless of the differentiation protocol used, the maximum yield of mDA neurons rarely exceeds 20% of total cells. This heterogeneity of cell types in mDA-differentiated stem cell cultures combined with the current lack of suitable cell surface markers for the selection of mDA cells, has significantly impacted the field, hampering our ability to study the mechanisms underlying mDA differentiation or to develop stem cells as a model for the study of PD in vitro or as a treatment modality in vivo. Thus, in this proposal, our goal is to create novel reporter hiPS stem cell lines using zinc finger nucleases to insert GFP-tagged mDA transgenes into the adeno-associated virus (AAVS1) safe harbor genomic integration site. These fluorescently labeled cell lines will allow us to purify cells to homogeneity at distinct stages during the mDA differentiation process and proceed with important proof-of-concept studies on the genetic and epigenetic factors governing mDA specification, midbrain regionalization and physiological function. In addition, we will use these reporter lines and DA-specific neurotoxins and PD-related genetic mutations to develop a stem cell model of PD for future studies in culture on PD pathogenesis and potential PD treatments.
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1 |
2017 — 2021 |
Iacovitti, Lorraine Mazo, Alexander M Sykulev, Yuri |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
The Role of the De-Condensed Structure of Nascent Chromatin During T Cell Differentiation @ Thomas Jefferson University
Project Summary: During cell differentiation, transcriptional programs are changed, and then must be maintained in turn. Chromatin-based epigenetic mechanisms are at the core of maintenance and switching of transcriptional programs. The fundamental issues of the nature of epigenetic marking and of the mechanisms that switch this marking during differentiation remain unclear due to lack of relevant experimental approaches. We developed new experimental paradigms that allow investigating the structure of chromatin during DNA replication at a single-cell and at a gene-specific levels. Using our new techniques, we found striking differences in the structure of chromatin during differentiation of the pluripotent human embryonic stem cells (hESC) and the antigen-inexperienced (naïve) T cells. During the first several hours after induction of differentiation of hESCs to dopamine neuron lineage, or T cells to different T cell subsets, accumulation of H3K27me3 is significantly delayed on nascent DNA. Since the occurrence of H3K27me3 in the genome coincides with the dense structure of nucleosomes, this suggests the existence of a temporarily de-condensed structure of nucleosomes on nascent DNA shortly after induction of cell differentiation. Our preliminary data indicate that the de-condensed, `open' structure of chromatin may be essential for recruitment to DNA of the lineage-specific transcription factors (TFs) that are essential to induce changes in transcriptional programs during cell differentiation. Thus, our results present a molecular explanation of how the vast areas of the repressed genome can be activated during cell differentiation. The goals of this proposal are to test two unique hypotheses using different models of differentiation to various lineages for pluripotent hESCs and for specialized T cells: 1) To examine whether the period of `open' post-replicative chromatin in early differentiating cells is a result of complex interplay of activities of several histone-modifying proteins; and 2) To examine whether this open post-replicative chromatin creates a `window of opportunity' for high accessibility of lineage- specifying TFs that are required to change the transcriptional program during cell differentiation. Examining these unique hypotheses may provide a universal chromatin-based molecular mechanism for biological plasticity of the cell.
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1 |
2017 — 2021 |
Iacovitti, Lorraine |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Using Human Ips Cells to Study Fate, Function and Neurodegenerative Disease @ Thomas Jefferson University
Human induced pluripotent stem cells (iPSCs), with their potential to generate autologous patient-derived cells, hold great promise for the study and treatment of a host of devastating diseases, including Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS), to name a few. One of the major obstacles slowing the translation of this powerful technology to the clinic is the heterogeneity of both desired and unwanted cell types generated in grafts of iPSCs, even after cells have been directed down specific differentiation pathways. Similarly in culture, a multitude of cell types are generated after treatment of iPSCs with lineage-specifying cocktails. These realities, combined with the current lack of suitable cell surface markers for the selection of specific desired cell types, has significantly impacted the field, hampering our ability to develop cell replacement therapies or to accurately model diseases in the dish. One plausible explanation for the observed cell heterogeneity is that presumptive undifferentiated pluripotent cells sometimes spontaneously initiate the process of differentiation after encountering lineage- specifying cues in culture, thereby precluding their subsequent directed differentiation by exogenously added differentiation cocktails. Currently, there are no assured ways to know if iPSCs have begun to spontaneously differentiate. However, an exciting new discovery made during our previous grant cycle suggests that the epigenetic state of chromatin shortly after DNA replication serves as a reliable and very early indicator of the state of differentiation of a stem cell. Our results suggest that it may be possible to uniformly direct the differentiation of all iPSCs toward a specific cell fate if chromatin can be kept closed until incubation with exogenous fate-specifying differentiation factors. If these insights are indicative of a more generalized principle, then it should be possible to generate pure populations of neural progenitors (NPs) of different subtypes which can give rise to homogeneous populations of various neurons for studies in culture and in animal models of multiple diseases. With these goals in mind, our Specific Aims for this proposal are: 1) to assess chromatin status during commitment to a motor neuron phenotype; 2) to generate pure populations of midbrain dopamine (mDA) and motor NPs and neurons which will be characterized for phenotype and synaptic function in culture and 3) to further determine whether homogeneous mDA-committed NPs/neurons can accurately model PD in the dish and be used in transplants to therapeutically treat PD rat models.
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1 |
2018 — 2019 |
Hou, Ya-Ming Iacovitti, Lorraine |
R03Activity Code Description: To provide research support specifically limited in time and amount for studies in categorical program areas. Small grants provide flexibility for initiating studies which are generally for preliminary short-term projects and are non-renewable. |
A Patient-Derived Cmt Cell Model @ Thomas Jefferson University
Charcot-Marie-Tooth (CMT) disease is the most common inherited peripheral neuropathy, affecting ~1 in 2,500 individuals worldwide. Patients with CMT disease display motor and sensory loss in the distal limbs, which can lead to ambulatory loss, limb amputation, and morbidity, incurring significant public health costs. The CMT2 subtype is difficult to understand at the mechanism level. Not only does it manifest impaired neuronal axon function, it is also often associated with mutations in ubiquitously expressed aminoacyl-tRNA synthetases (ARSs), the enzymes that charge tRNAs with an amino acid to generate aminoacyl-tRNAs for protein synthesis. While CMT2 pathogenic mutations are known, whether these mutations drive the disease progression by a loss- or gain-of-function effect is inconclusive. Without this knowledge, our efforts to combat CMT2 are limited. Most studies of CMT2 use vertebrate models, which while useful, differ considerably from human patients in genotypes. We hypothesize that the best approach to elucidate the pathogenic mechanism of CMT2 is to use induced pluripotent stem cells (iPSC)-derived motor neurons as a cell model. With the ability to differentiate into peripheral motor neurons, iPSC models represent a unique opportunity to study CMT2 in a platform of the human disease. We will focus on the R329H mutation in AARS (alanyl aminoacyl-tRNA synthetase), which reduces tRNA charging and is expected to cause defective protein synthesis. While we already have an iPSC model for the mutation generated from the human embryonic H9 line, we propose here to generate a patient- derived model for comparison, which will provide critical insight into whether the disease mechanism is impacted by other factors associated with the genetic background of the patient. In Aim 1, we will generate a control line based on the fibroblasts of the patient to restore the wild-type (wt) sequence at the AARS-R329H locus. We will use CRISPR/Cas9 for this restoration and will validate it by assays for the AARS enzyme. We expect that the restored wt sequence will confer full enzyme activity. In Aim 2, we will establish pluripotent iPSC lines from the control and patient fibroblasts and validate each by assays for AARS. We will then differentiate each line to motor neurons and identify phenotypes specific to the mutation. Combined, this work will produce an isogenic pair of wt and AARS-R329H iPSC lines that maintain the genetic background of the patient. The phenotype of this patient-derived pair, relative to the H9-derived pair, will provide new insight into the disease within the genetic context of the patient. Both pairs will be shared with the research community of CMT2 and related peripheral neuropathies.
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1 |