Evan Y. Snyder - US grants

To elucidate mechanisms which direct development of the mammalian nervous system, retroviral vectors are used to insert heritable genetic markers and modifiers into primordial neural tissue. In the course of these studies, phenomena were observed which hint at an extraordinary degree of plasticity late in development of the immature nervous system. The studies proposed endeavor to determine the pervasiveness of these phenomena and to understan the variables directing this plasticity in order to exploit its potential i the prevention, compensation, and repair of the damaged developing nervous system. Such an understanding may not only lend insight into strategies of normal neural development, but also into development "gone awry"--i.e., unchecked plasticity--which may prove to be a molecular mechanism contributing to neural oncogenesis. Work will continue IN VITRO and IN VIV in the mouse. IN VITRO, "immortalizing" genes are inserted into individual neural stem cells--both of neural tube (cerebellum) and neural crest origin--allowing, through the creation of neural cell lines, a study of the r subsequent differentiation and commitment. In cerebellum, lines from ostensibly different neural cell types appear not only to be clonally-related, but to display plasticity in the expression of their phenotype. Work in this system will seek to determine the factors which direct differentiation down a given phenotypic path or allow a selected phenotype to change, and to use these lines for neural transplantation. Lines from neural crest will be similarly characterized, searched for late vs early commitment, assessed for degrees of plasticity, and serving as transplantation material. IN VIVO, through microinjection of vectors containing "marker" genes into neonatal and embryonic mouse CEREBELLUM and embryonic mouse RETINA, individual progenitor cells have been labeled in si u allowing lineage mapping. Two conclusions are emerging: (a) multiple neura cell types are present in a given clone, suggesting that they share a commo progenitor with divergence as late as the last cell division (retina); (b) multipotent progenitors in the CNS may migrate with commitment to cell type occurring only later, following interaction with its microenvironment (postnatal cerebellum). Lineage patterns in pre- and postnatal cerebellum will be analyzed to validate these impressions and provide a basis for transplantation experiments.

In the field of neural transplantation, alternatives to primary fetal tissue are being sought as graft material. Previously, through retrovirus- mediated gene transfer, we generated immortalized, clonal, multipotent neural progenitor lines from mouse. Examination of their differentiation potential in vitro suggested enormous plasticity at the level of the individual progenitor. Upon transplantation, these immortalized progenitors engrafted in a cytoarchitecturally and functionally appropriate manner, recapitulating their multipotency in vivo & expressing retrovirally-transduced exogenous genes in a robust, stable fashion throughout brain parenchyma for prolonged periods. These progenitors could engraft & participate normally in the development of multiple structures along the neuraxis & at multiple stages spanning from embryonic to the adult. They differentiated into multiple cell types, presumably responding to signals of the respective region at the particular developmental stage. The progenitors, though most engraftable when initially mitotic, migratory, & plastic, quickly differentiated, down-regulated their immortalizing gene product, & never formed tumors. CNS cytoarchitecture was never disrupted, the blood-brain barrier remained intact, & recipient animals behaved normally. When transplanted into various animal models of neural degeneration, the progenitors integrated into cytoarchitecture often assuming the phenotype of the missing cell type. Furthermore, progenitors expressing gene products missing in the host successfully engrafted into the CNS of various animal models in which defects in those genes had been defined (e.g., certain neurovisceral storage diseases). These data suggest the feasibility of using immortalized progenitors to provide exogenous factors of therapeutic or developmental interest (constitutiveiy or through genetic engineering), or to effect repair as integral members of CNS cytoarchitecture, may be feasible. The generation of progenitor lines with similar potential from human fetal neural tissue is the next step. Starting with primary human fetal neural tissue, the proposed studies will attempt to establish a paradigm of neural progenitor transplantation as a therapy for developmental, degenerative, & acquired injury to CNS through the following 4 aims: (1) Determine conditions for maintaining human fetal neural tissue in vitro which would optimize the presence of proper progenitors for immortalization. (2) Generate & characterize in vitro clonal, immortalized, multipotent human neural progenitor lines. (3) Transplant candidate lines into mouse hosts (cyclosporin-treated or SClD mice) to assess ability to engraft, differentiate in vivo, & express a reporter transgene. (4) Attempt transplantation of engraftable progenitor lines into mouse models of neural degeneration.

As in other organs, 2 areas of inquiry into CNS dysfunction (particularly that due to inherited metabolic or neurogenetic disease) have recently converged: neural progenitor cell biology & CNS via transplantation. Through retrovirus-mediated gene transfer, we previously generated immortalized, clonal, multipotent murine neural progenitor lines. Examination of their differentiation potential in vitro suggested enormous plasticity at the level of the individual progenitor. Upon transplantation, these immortalized progenitors engrafted in a non- tumorigenic, cytoarchitecturally & functionally appropriate manner, recapitulating their multipotency in vivo & expressing retrovirally- transduced genes in a robust fashion within brain parenchyma for prolonged periods. Because the blood-brain barrier imposes restrictions to entry of enzyme supplied peripherally (either directly or through genetically-engineered somatic cells) & because bone marrow transplantation entails irradiation which is inimical to developing CNS, peripheral treatment of CNS manifestations of genetic disease has been disappointing. Delivery of gene products directLy to CNS would circumvent such problems. (Grafting primary fetal tissue for such purposes poses numerous biologic & ethical concerns). Our data suggest the feasibility of transplanting immortalized neural progenitors constituitively expressing missing gene products, or genetically engineered to do so, as a strategy for sustained therapy of CNS manifestations of neurovisceral disease, &/or to effect repair as integral members of CNS cytoarchitecture. This proposal attempts to establish a paradigm of neural precursor transplantation as a therapy for CNS insult through 4 aims: (1) Confirm preliminary findings that a given clonal progenitor line can engraft (with technical ease) & participate normally in the development of multiple structures along the neuraxis & at multiple stages spanning from embryo to adult, that the progenitors can differentiate into multiple cell types in response to prevailing spatial & temporal cues, & express their retrovirally-transduced reporter gene. (This would broaden enormously the applications of such an immortalized precursor.) (2) Insure consistently efficient engraftment & safety of recipient animals by identifying the variables which direct engraftment, & determining the properties & fate of cells in situ that do engraft & express transgenes. Grafted cells can be traced & characterized in vivo; they can be retrieved & re-examined in vitro; their efficiency of engraftment vs gene expression can be quantified; their impact on host brain can be assessed. (Such characterization is crucial prior to consideration of similar strategies in humans.) (3) Transfer an exogenous gene or factor, via transplantation of an expressing precursor, into the CNS of a prototypical mouse model of neurovisceral disease in which a defect in that gene has been defined. (4) Transplant progenitors into a lesioned or mutant mouse model of a cell type-specific neurodegeneration to determine if they will integrate into the CNS & assume the phenotype of the deficient cell-type. Pilot data attest to the feasibility of each aim.

In the field of neural transplantation, alternatives to primary fetal tissue are being sought as graft material. Previously, through retrovirus- mediated gene transfer, we generated immortalized, clonal, multipotent neural progenitor lines from mouse. Examination of their differentiation potential in vitro suggested enormous plasticity at the level of the individual progenitor. Upon transplantation, these immortalized progenitors engrafted in a cytoarchitecturally and functionally appropriate manner, recapitulating their multipotency in vivo & expressing retrovirally-transduced exogenous genes in a robust, stable fashion throughout brain parenchyma for prolonged periods. These progenitors could engraft & participate normally in the development of multiple structures along the neuraxis & at multiple stages spanning from embryonic to the adult. They differentiated into multiple cell types, presumably responding to signals of the respective region at the particular developmental stage. The progenitors, though most engraftable when initially mitotic, migratory, & plastic, quickly differentiated, down-regulated their immortalizing gene product, & never formed tumors. CNS cytoarchitecture was never disrupted, the blood-brain barrier remained intact, & recipient animals behaved normally. When transplanted into various animal models of neural degeneration, the progenitors integrated into cytoarchitecture often assuming the phenotype of the missing cell type. Furthermore, progenitors expressing gene products missing in the host successfully engrafted into the CNS of various animal models in which defects in those genes had been defined (e.g., certain neurovisceral storage diseases). These data suggest the feasibility of using immortalized progenitors to provide exogenous factors of therapeutic or developmental interest (constitutiveiy or through genetic engineering), or to effect repair as integral members of CNS cytoarchitecture, may be feasible. The generation of progenitor lines with similar potential from human fetal neural tissue is the next step. Starting with primary human fetal neural tissue, the proposed studies will attempt to establish a paradigm of neural progenitor transplantation as a therapy for developmental, degenerative, & acquired injury to CNS through the following 4 aims: (1) Determine conditions for maintaining human fetal neural tissue in vitro which would optimize the presence of proper progenitors for immortalization. (2) Generate & characterize in vitro clonal, immortalized, multipotent human neural progenitor lines. (3) Transplant candidate lines into mouse hosts (cyclosporin-treated or SClD mice) to assess ability to engraft, differentiate in vivo, & express a reporter transgene. (4) Attempt transplantation of engraftable progenitor lines into mouse models of neural degeneration.

[unreadable] DESCRIPTION (provided by applicant): Human Embryonic Stem Cells (hESC) have remarkable potential for studies of fundamental human developmental biology. The principal goal of this project is to establish the Burnham Stem Cell Center to facilitate collaborative research in the basic biology of hESC and encourage new researchers to enter the field. The Center will enable new and established investigators to develop the utility of hESC as a model system for a diverse range of biological and medical problems. The proposal for the Center has two specific aims. For Aim 1, we will establish a shared core laboratory that provides state-of-art equipment and expert support for area investigators and training in hESC methods. Core programs will be tightly focused on specific goals to provide technical support and expertise, develop new technologies, and train new investigators. The Core Programs are (1) Cell Culture and Maintenance, (2) Cell Characterization, (3) High Throughput Analysis Technology Development, and (4) Data Sharing and Training. These programs will use NIH Registry Cell lines WA01, WA09, and WA14. To encourage new investigators and collaborations, the Center will share responsibility for an annual ten-day NIH-sponsored T15 training course in hESC technology, provide 2-3 day training courses for visiting scientists, sponsor a website for sharing information about hESC, host scientific and ethics symposia twice a year, and support the monthly meetings of the Southern California Stem Cell Consortium. Aim 2 is to sponsor competitive pilot projects that take advantage of the unique potential of hESC. We will support four early-phase, hypothesis-driven pilot projects that address fundamental questions of hESC biology. These pilot projects include investigations of chemical inducers of differentiation, epigenetic controls of differentiation, development of novel high-resolution real time imaging technology to study hESCs in vitro, and molecular and developmental controls of hESC self-renewal. [unreadable] [unreadable]

Current insights into the onset of dopaminergic (DA) neuronal dysfunction and/or death (hence, the etiology of Parkinson's Disease [PD]) implicate abnormalities in the unbiquitin-proteasome system (UPS) in response to oxidative and nitrosative stress, leading to protein misfolding. Protein misfolding appears to be mediated, at least in part, by S-nitrosylation of parkin or protein-disulfide isomerase (PDI). Hence, these molecules may provide mechanism-based biomarkers for impending neuronal demise or, conversely, if levels go down, their recovery. Dysfunctional mitochondria can lead to the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). In particular, there is growing evidence that mitochondrial complex 1 dysfunction results in an increase in ROS, eventually leading to the aggregation of a-synuclein. Oligomers/protofibrils of a-synuclein appear to play a central role in neurodegeneration - and, particularly, PD pathology -- likely through proteasome inhibition. Dysfunction of the UPS is likely the basis for familial PD characterized by mutations in Parkin, PINK1 and DJ-1. Recently, the Lipton group (Project 3) has demonstrated that S-nitrosylation of parkin or PDI, a key stress-induced chaperone in the endoplasmic reticulum (ER), has been linked to protein misfolding and neurodegeneration in PD models and in brains of PD patients. In addition, in preliminary studies, we have observed that mice carrying mutant a-synuclein (asyn) show dramatically increased S-nitrosylation of PDI;i.e., increased nitrosative/oxidative stress appears to be present in the context of such a mutation. There appears to be a developmental component to PD onset. For example, although, mutant a-syn is present in the earliest CNS progenitors of patients with some familial forms of PD, the disease does not typically manifest itself until adulthood. Progressive DA dysfunction also appears to be a component of the aging process. Immature neural progenitor cells appear to be resistant to oxidative stress in a manner not observed when those same cells become mature. Although human stem cells are typically studied for their therapeutic potential, they also provide (perhaps even more compellingly) models of human cellular development and offer the prospect for modeling human disease (from which novel therapies may, in turn, be derived). We have established defined culture conditions for modeling the iterative steps of DA neuronal development from an undifferentiated human embryonic stem cell (hESC) to a differentiated DA neuron in vitro. Cells at each developmental stage can be engineered to express mutant a-syn and/or "lesioned" pharmacologically with mitochondrial complex inhibitors. In DA neurons, such manipulations produce features emulating PD. Therefore, we propose to use a human stem cell-based system to model the developmental susceptibility of neural precursors to oxidative/nitrosative stress relevant to PD in order to understand mechanisms by which endangered or dysfunctional DA neurons may ultimately be protected. A study of developmental susceptibility may help to develop drugs that will prevent oxidative/nitrosative stress in both endogenous and transplanted neural progenitors. Preserving mesostriatal circuitry is more tractable and safer than attempting to reconstruct proper new connections. However, if, in the future, transplantation into PD patients is required, protecting these exogenous stem cells will also be crucial. It is possible that different protective drugs will be necessary depending on the developmental stage of the neural progenitors used.

DESCRIPTION (provided by applicant): Somatic stem cells appear to have their greatest impact on stroke therapeutics not by literally replacing cells but by protecting host neural cells &circuitry from progressive damage as well as catalyzing endogenous host regenerative responses. When such stem cell-mediated actions have been invoked, behavioral improvements have been seen. Neuroectoderm-derived stem cells ("neural stem cells [NSCs]") have been the "gold-standard" for such therapeutics against which any alternative stem cell type must be compared. However, a number of labs have found efficacy from the intravascular administration of human umbilical cord blood-derived stem cells (hUCSCs). The intravascular administration of stem cells allows one to circumvent more invasive neurosurgical implantation, assuming that the cells can efficiently home to the region of injury. An additional advantage of cell-mediated protective therapies (as opposed to pharmacological-mediated interventions) is that the window of opportunity can be as wide as 24-72 hrs, making them well-suited to the realities of when patients with stroke present to health care facilities. Although UCSCs have been promising, the ability to extend their use to actual patients has practical &immunological limitations. In particular, based on extrapolating from animal studies, adult patients would require combining multiple cord blood units in order to obtain sufficient numbers of stem cells to achieve efficacy. Furthermore, combining units places stress on identifying compatible units with no more than 1-2 HLA mismatches to minimize an immune reaction. Also, efficient delivery of cells to where they are most needed ¬ to areas where they are not required or could even create problems - i.e., homing - is pivotal to the success of all cell-based therapeutics for any pathological condition. UCSCs must have optimal access to injured host cells &their milieu if efficacy &safety are to be maximized. When considering systemic administration of either UCSCs or NSCs for mediating brain repair following stroke, rolling &adhesion on endothelial cells is the critical 1st step in the homing cascade that is necessary for targeting these cells to the injured brain. Most hUCSCs exhibit deficient rolling &adhesion on endothelial cells, severely limiting their therapeutic potential. NSCs have a similar limitation. Recent studies have shown that this deficit can be corrected by pretreating hUCSCs with fucosyltransferase-VI which fucosylates sLeX &completes the P-selectin glycoprotein ligand (PSGL) on the CD34+ cells. The net effect is to increase recognition of the PSGL for selectins on endothelial cells. It is unknown whether this works, however, for brain vascular endothelium. It is also unknown whether this works for NSCs -- again, the "gold standard" -- although they, too, have an incomplete unfucosylated selectin ligand which compromises their homing. We propose first [Aim 1] to determine in vitro the optimal parameters for fucosylating &thereby enhancing the binding of hUCSCs &human NSCs (hNSCs) to inflamed human brain-derived endothelial cells under physiological shear stress (employing a specialized flow chamber). Subsequently [Aim 2], we will use these conditions to administer fucosylated (vs. control) hUCSCs &hNSCs to a rat model of stroke, comparing the 2 stem cell types (for the 1st time) head-to-head. Rats will be injected iv with the stem cells at 24-72 hrs post-middle cerebral artery occlusion. Outcome measures will include behavior;infarct volume (by MRI);spectroscopy (by MRS);migration (by MRI &bioluminescence imaging);sparing of host neurons &their connections (by immunohistochemistry &tract tracing). These data will be correlated with histology. These studies could help provide a strategy for enhancing the homing of stem cells to the brain in a minimally-invasive manner, using cell numbers &a time frame that is practical for providing clinical benefit in acute/subacute stroke, particularly if the stem cells serve to protect extant neural tissue &connections;diminish inflammation, scarring &secondary injury processes;detoxify the milieu;&promote neovascularization. Such studies could also provide insights that extend to other types of CNS pathology &other types of stem cells. PUBLIC HEALTH RELEVANCE: Peripheral intravascular administration of either human umbilical cord-derived stem cells (hUCSCs) or neural stem cells (hNSCs) for acute stroke therapeutics in adults requires a sufficient number of cells with fully expressed homing mechanisms for optimal targeting to the area of compromise. This proposal will assess a new technology that could expand the use of available supplies of either source of stem cells. The process of placing a sugar group - a fucosyl group - on stem cell membranes through a simple enzymatic pre-treatment process has shown in preclinical studies to increase homing of hUCSCs and likely will extend to hNSCs as well. Aim 1 will identify the optimal conditions for preparing the stem cells while Aim 2 will comparatively assess in a rat stroke model the efficacy of hUCSCs vs. hNSCs following peripheral administration 24 hrs post-stroke, particularly under conditions where their homing has been optimized by fucosylation.

DESCRIPTION (provided by applicant): ): Bipolar disorder (BPD) is a neuropsychiatric condition defined by a lifetime of relapsing &remitting manic &depressive episodes. This mood disorder has been shown to have strong genetic linkage with familial predisposition. A major impediment to proper diagnosis has been such confounders as the prevalence of substance &alcohol abuse/dependence among those meeting criteria for BPD. Substance abuse, intoxication &withdrawal may elicit mood episodes that recapitulate bipolar phenotypes. Typically, at least 6-12 months of abstinence from substances is required to diagnose a mood episode as underlying BPD. Clinical practice often resorts to early psychotropic medication because of potentially extreme irritability &impulsive suicidal actions. Lithium has been the standard of treatment for BPD, but, because of its side effects, anti-convulsants (including valproic acid, lamotrigine, topiramate &carbamazepine) &atypical anti-psychotics have also been prescribed. The underlying etiology &mechanisms of disease therapy are poorly understood {Rosenberg, 2007 #160}. Adequate laboratory (including animal) models have been difficult to establish {Fornito, 2009 #161}. Microarray analyses have been performed on post-mortem brain samples &compared with controls &patients with schizophrenia, but no clear distinction between neural subtypes, glia, &surrounding vascular cells has been evident (Kim &Webster, 2008). There is no consensus on gene expression patterns linked to BPD &, hence, very little insight into underlying molecular mechanisms. Protein kinase pathways may be altered. Lithium alters MEK &ERK phosphorylation {Pardo, 2003 #162}, decreases CREB phosphorylation &activity of CaM kinase IV {Tardito, 2007 #163} in rat hippocampal neurons. Lithium &valproate may reduce phosphorylation of rat GluR1 {Du, 2008 #164}. Lithium correlates with reduced phosphorylation of the NMDA receptor subunit NR2B in rat cortical neurons {Hashimoto, 2002 #159}. Studies in rat brains have also suggested that lithium administration reduces translocation of Protein Kinase C from the cytosol to the cell membrane {Hahn, 1999 #158}, &inhibits GSK3 activity in mice (Catapano &Manji, 2008). Given the diversity of implicated kinase pathways in BPD and limited by obstacles in studying human neuropsychiatric diseases, we sought to develop a representative, predictive model system to explore regulation of protein phosphorylation in neural cells that most faithfully recapitulates underlying defects of actual human BPD. Recent advances have allowed the conversion of patient-specific human fibroblasts to human induced pluripotent stem cells (hIPSCs), cells which become sufficiently dedifferentiated to a primordial developmental stage that they now emulate human embryonic stem cells (hESCs) in terms of their ability to give rise to the 3 primitive embryonic germ layers and following various differentiation protocols, to more mature, tissue- and organ-specific cell types, including those in the neural lineage. Our group not only has the ability to generate hIPSCs from fibroblasts from normal individuals &from those carrying difficult-to-model diseases, but has actually done so for some neurogenetic/neuropsychiatric entities, e.g., Rett Syndrome, &has differentiated them to neural lineages. We propose to generate hIPSCs from a well-defined subset of BPD patients (i.e., the lithium-responsive subpopulation) in order to begin more faithfully modeling BPD from a rigorous molecular mechanistic perspective using material derived from actual patients. We will include control cell lines from unaffected patients as well as patients with neurologic or psychiatric disorders that are not BPD. At the core of beginning to understand the molecular basis of BPD - and an aspect for which hIPSCs would be particularly well-suited &informative - is a better understanding of changes in the expression of key proteins, particularly their phosphorylation state, at the level of the proteome &phosphoproteome. Our team has been particularly adept at using phosphoproteomic analysis of hESCs (the 1st such analysis in the field) to identify key (including novel) drugable signal transduction pathways that influence neural differentiation. We believe we can apply a similar approach to hIPSCs from BPD patients. In other words, we hypothesize that identification of proteomic &phosphoproteomic differences between hIPSC-derived neurons from BPD vs. unaffected controls or controls with other neuropsychiatric disorders will help elucidate pivotal, diagnostic, and potentially drugable molecular mechanisms underlying BPD. Multidimensional liquid chromatography (MDLC) tandem mass spectrometry (MS/MS) is a powerful tool for analysis of proteomes, phosphoproteomes, and is a strength of the Burnham Institute. MDLC-MS/MS will be used to analyze the proteomes and phosphoproteomes of normal and patient-derived hIPSCs and their neural derivatives. We will use refined bioinformatic tools (another Burnham strength) to glean critical differences among the cell types. We will quantify total cellular proteins, with a particular focus on site- specific phophorylation. This large-scale analysis will likely yield a number of candidate molecular differences between control &BPD cells, any of which might suggest improved methods of diagnosis &treatment. While we will be able to perform follow-up analyses on only a few proteins predicted to be key to control of pluripotency, neural differentiation and neural function, this valuable dataset will be made available to the broader research community so that complementary parallel studies may be launched on the basis of these proteomic &phosphoproteomic results. PUBLIC HEALTH RELEVANCE: Bipolar disorder (BPD) is a severe and prominent societal malady with poorly understood etiology. Proteomic and phosphoproteomic technology is a powerful analytical platform allowing unbiased identification of molecular profiles of healthy and diseased cells, e.g. those from normal and BPD patients. We propose to merge the application of induced pluripotent cells (iPSCs) from BPD patients with comprehensive proteomic/phosphoproteomic analyses of these iPSCs and their neural derivatives, to discover molecular underpinnings of the abnormalities in BPD patients, as well as potential targets for improved diagnosis and treatment.