Theo D. Palmer - US grants

The Route28 Summits in Neurobiology provide an outstanding training experience on topics with significant promise in the applied clinical sciences. Well-known scientists and trainees are brought together with an express goal of developing novel therapeutic strategies for disease and injury in the central nervous system. The workshop incorporates a unique and successful training strategy. Formal talks alternate with critical and candid discussions led by each speaker. To facilitate interaction, graduate students and fellows must work throughout the conference to integrate the research presented into mini-grant proposals. Working in groups of 7, participants collaborate to solve one of three assigned problems on tissue repair in the CNS. On the last day, the groups finalize and present the strategies they have developed. A study section of senior scientists score each proposal based on creativity and scientific merit. In August 23-30 of 2001, the workshop "The Id of Stem Cells" focuses on a controversial yet growing concept that stem cells from a given organ system may not be limited to the repair and regeneration of the source tissue. This idea is supported by a number of novel and entirely unexpected observations. For example, stem-like cells introduced into a recipient during bone marrow transplant appear able to generate new muscle, bone, cartilage, vascular cells and potentially even glia and neurons. Conversely, neural stem cells isolated from the adult brain seem able to reconstitute the hematopoetic system when transplanted in lieu of bone marrow stem cells. Although the identity of the cells responsible for these phenomena is not yet known, the concept that embryonic, mesenchymal, hematopoetic and neural stem cells may share a significant overlap in lineage potential has significant implications for novel approaches to CNS repair. The workshop held in August of 2003, builds on the theme of stem cell- mediated CNS repair by focusing on the use of modern genomics, proteomics and informatics to identify and manipulate the cellular and molecular cues most important for orchestrating repair and cell replacement in the CNS. In August of 2005, our focus continues in novel therapeutic strategies by introducing chip-based implantable interfaces. Although considered to be in the realm of conceptual devices, significant progress is being made in bridging the gap between computer hardware and the biological "wetware" of the mammalian brain.

DESCRIPTION (provided by applicant): The therapeutic recruitment of endogenous neural stem/progenitor cells provides a glimmer of hope for reversing the devastating loss of function that accompanies central nervous system injury. To better understand neural stem cell behavior in vivo, we have completed a detailed anatomical study of adult hippocampal neurogenesis. An unexpected finding is that angiogenesis accompanies neurogenesis and may be an essential requisite for the de novo generation of neurons from adult neural stem cells. To determine the importance of angiogenesis in adult neurogenesis, it will be necessary to block and stimulate angiogenesis in vivo, preferably without simultaneously perturbing stem cell mitogenesis. Preliminary data shows that FGF-2, EGF, and VEGF are mitogens for both endothelium and neural stem cells. This warns against the use of the readily available antagonists that target VEGF or FGF-2 receptors (or the more generic MAP kinase pathway inhibitors) since these may not be useful for our attempts to uncouple angiogenesis from neurogenesis. Fortunately, we have found that a synthetic mimetic of vitronectin is able to block integrin avb3 signaling and inhibits endothelial cell but not neural stem cell proliferation in culture. These studies will provide important insights into the microenvironments that control "adult" neural stem cell activity and fate. The primary methodologies will be IV administration of synthetic integrin antagonists to block angiogenesis. These data will be contrasted to local stimulation of angiogenesis by VEGF gene transfer. Neurogenesis in experimental animals will be monitored with systemic BrdU-labeling, post-hoc stereology and confocal analysis.

DESCRIPTION (provided by applicant): A common and debilitating side effect of cranial radiation therapy is a permanent and continually progressive decline in cognition that includes impaired learning and memory, Although previous work has implicated altered hippocampal function in this deficit, we show for the first time that cranial radiation injures adult neural stem cells, ablates hippocampal neurogenesis and permanently disrupts the hippocampal microenvironment making it unable to support neurogenesis, even following reconstitution with non-irradiated stem cells, The irradiated hippocampal subgranule zone displays two important long-term alterations that may account for its inability to support neurogenesis: a loss of normal neural stem cell activity and disruption of the "neuro-angiogenic" microenvironment. The proposed experiments further characterize the radiation sensitivity of adult neural stem cells and explore the potential utility of radio-protective as well as reconstitution strategies to reduce the impact of radiation on neurogenesis in the adult brain.

DESCRIPTION (provided by applicant): Physiological changes that affect hippocampal function in the adult are accompanied by parallel alterations in neurogenesis and it is thought that learning and memory may depend on continued neurogenesis. Cranial radiation therapy for the treatment of cancer is 1 of the most striking examples of injury that causes impaired neurogenesis and progressively severe deficits in hippocampal function. In modeling this process in rodents, we observed that chronic inflammation accompanies radiation injury, suggesting that inflammatory processes may contribute to neural stem cell dysfunction. Subsequent work has shown that neuroinflammation alone is a potent inhibitor of neurogenesis and that inflammatory blockade with indomethacin, a common non-steroidal anti-inflammatory drug, restores neurogenesis following endotoxin-induced inflammation and augments neurogenesis following cranial irradiation. In addition, animals with deficits in the chemokine MCP-1 are resistant to the long-term effects of radiation and neurogenesis returns to normal levels 1 month after irradiation. Although inflammatory blockade reverses the inhibition of neurogenesis, it is not known how inflammation influences neurogenesis or whether these perturbations in stem cell activity influence learning and memory function. This application proposes that the pro-inflammatory phase of inflammation influences neural stem cells in the hippocampus by 1) the direct action of inflammatory cells, cytokines and chemokines on stem cells and their progeny, 2) by the indirect effects of inflammatory cells, cytokines on the stem cell microenvironment and 3) by the inflammatory modulation of the hypothalamic-pituitary-adrenal axis and subsequent elevation of glucocorticoids. The use of primary neural stem cell cultures as well as animals that are genetically or surgically deficient in key inflammatory mediators will allow us to test these hypotheses with regards to neural stem cell activity, adult hippocampal neurogenesis, and hippocampal learning and memory function.

DESCRIPTION (provided by applicant): Cranial radiation therapy is associated with progressive deficits in cognitive function; particularly in children treated a very young age. Although cancer therapies can yield promisingly high cure rates, the quality of life after therapy declines and most children eventually require special education programs or even institutionalization. Hippocampal deficits in learning and memory are a predominant aspect of the cognitive decline and observations in animal models suggest that a chronic inflammatory response induced by irradiation may significantly contribute to hippocampal dysfunction. Adult neurogenesis in one aspect of normal hippocampal biology that is severely impacted by inflammation and recent work shows neurogenesis can be sheltered from these effects by oral administration of the non-steroidal anti-inflammatory drug (NSAID) indomethacin. Pilot studies show that hippocampal function is also normalized by indomethacin and the ease with which inflammation can be modulated with NSAIDs suggests that inflammatory blockade may have utility in sparing cognitive function following cranial radiotherapy. However, there may be risks associated with the use of indomethacin that might be avoided by more selective NSAIDs. The anti-inflammatory effects of Indomethacin are mediated via Cox-1, Cox-2 and PPAR(. Cox-1 inhibition also impairs platelets and the protective function of gastric mucosa. Since megakaryocytes and mucosal epithelium are also particularly sensitive to chemotherapy, concurrent Cox-1 inhibition could conceivably place a patient at higher risk of spontaneous and possibly life-threatening hemorrhage. The more selective inhibition of Cox-2 (celecoxib) and/or activation of PPAR( (rosiglitazone) may offer equally effective strategies that minimize risks associated with Cox-1 inhibition. Here we propose to evaluate these drugs in genetically manipulated mice to determine the relative importance of Cox-1, Cox-2 and PPARgamma in normalizing hippocampal function following inflammatory challenge.

DESCRIPTION (provided by applicant): This proposal requests partial funding for the 5th workshop in a series of well optimized and uniquely effective training venues for graduate students and post-graduate fellows. The 2005 Route 28 Summit focuses on the emerging links between injury, inflammation and neurodegeneration in the central nervous system dysfunction. With the help of attending faculty, trainees work in small collaborative groups to competitively produce and present research strategies addressing current issues in multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS) and spinal cord injury. The workshop involves 45 trainees and formal lectures by 19 attending faculty members. Trainees and teaching faculty are drawn from the international research and clinical communities. The current organizing committee is well experienced and has produced four prior Route 28 Summits in 1999, 2001, 2002, and 2004. The organizers of the Route 28 Summits have made a commitment to attain four Aims: Aim 1. Provide outstanding student access to leading scientists in neurobiology and related disciplines. Aim 2. Promote long-lasting and rewarding cross-disciplinary interactions on the workshop topic. Aim 3. Educate trainees in the process of collaborative thinking and group planning of a competitive research proposal. Aim 4. Provide a cost package that does not discriminate against promising students with a limited travel budget. To our knowledge, the Route 28 Summit workshops provide the only graduate and post-graduate level training venue that purposefully highlights the strengths of collaborative multi-disciplinary research in the biomedical sciences. In addition, the workshop topics and trainee assignments provide a thorough introduction to cutting edge research in disease or injury processes and explore problems facing the translation of basic science into clinical application. These strengths are clearly called out in the newly generated NIH Roadmap and are fundamental to the programmatic goals of the NINDS.

DESCRIPTION (provided by applicant): Integrins and their interactions with extracellular matrix proteins (ECM) regulate many cellular processes but their role in neural stem cell biology is poorly defined. Here we focus on a novel integrin-ECM interaction that defines a permissive environment for stem cell self-renewal and neurogenesis in the adult brain. Neural stem cells persist in the adult mammalian brain and support the continuing production of neurons and glia. Although glia are produced in all areas of the brain, native neurogenesis is restricted to the subventricular zone (SVZ) and hippocampal subgranular zone (SGZ). Neurogenesis can be triggered in other areas of the brain, for example by injury, but the production and retention of new neurons is inefficient and abortive at best. A better understanding of local environments that naturally support the maintenance of a stem cell pool as well as their efficient use in neurogenesis could lead to significant advances in neural repair/regeneration. Toward this end, we have identified several integrin-ECM interactions that are selectively localized to the native neurogenic areas of the brain. In vitro, we show that these interactions gate neural stem and progenitor cell response to mitogens and here we propose studies to define the specific mechanisms used within the adult neurogenic niche to: 1) maintain and regulate stem cell self-renewal and stem cell pool size;2) regulate the transient amplification and survival of newly generated neuroblasts;and 3), mediate physiological signaling that controls hippocampal neurogenesis in response to physical exercise or learning experiences. PUBLIC HEALTH RELEVANCE: Neural repair and regeneration in the brain is one of the most difficult yet potentially rewarding goals in stem cell research. In this application we take advantage of an area of the brain that naturally produces new neurons to more precisely define the microenvironment that maintains stem cells and regenerative activity throughout life. This unique neurogenic niche utilizes a complex interaction of cells and signals to maintain and regulate neurogenesis and we have recently found that extracellular matrix proteins are integral and essential in this cellular and biochemical environment. Here we propose to 1) further refine our understanding of the molecular interactions that promote or inhibit the stem cells in the generation, survival and integration of new neurons;2) evaluate the intracellular signaling cascades that underlie the phenomenon behind the generation of new neurons;3) develop and demonstrate the role of these cellular and molecular components in regulating adult neurogenesis in transgenic animal models. We anticipate that the outcome of the proposed studies will significantly advance our understanding of natural regenerative processes and provide insights into improving the efficacy of future stem cell therapies for a variety of neurological injuries and diseases.

DESCRIPTION (provided by applicant): The GABA(A) receptor ¿3 subunit gene GABRB3 has been strongly implicated in the pervasive developmental disorders (PDD). GABRB3 lies within chromosome 15q11-13, and anomalies in this region are associated with Rett syndrome, Prader-Willi syndrome, Angelman Syndrome, and are the single most frequent cytogenetic abnormality found in the autism spectrum disorders (ASD). It has recently been recognized that peripheral GABA(A) receptors are involved in immune cell regulation and that activation of GABA(A) receptors can attenuate tissue inflammation. Maternal inflammation during pregnancy has been an environmental factor associated with ASD and we have found that loss of one GABRB3 allele in mother and fetus is sufficient to markedly increase placental pathology in response to cytokines produced by a mild maternal innate immune response in mice. In contrast, activating GABA(A) receptors during a maternal immune event attenuates the impact to the fetus. This implicates GABA(A)-related immune alterations and placental vulnerability as a potential gene-environment interaction which is associated with increased risk of ASD and other PDDs. Importantly, the maternal inheritance pattern of 15q11-13 anomalies suggests that GABA(A)-related immune functions may be altered in mother, as well as in the placenta and fetus. The experiments proposed here test whether selective loss of GABARB3 in the mother, placenta or fetus increase the risk of developing autistic features in offspring following a prenatl maternal immune event.

DESCRIPTION (provided by applicant): Molecules involved in immune cell signaling can have independent functions in the central nervous system. Such molecules may have dual roles in cellular therapy where neural progenitor cells are transplanted into the active immune signaling environment of the diseased or injured brain. Class I major histocompatibility complex (MHC1) molecules are prominent examples with well defined function in both allograft rejection and in neurodevelopment. In addition to mediating antigen presentation by immune cells, MHC1 and cognate receptors are expressed by neurons and influence axonal growth and synapse formation and elimination. MHC expression in neurons is also pathologically up-regulated by immune cytokines present in the injured or degenerating brain. This proposal examines both immunological and neurodevelopmental roles of MHC1 in transplantation. Allogeneic cell or tissue transplants are being used in numerous clinical settings and while purified allogeneic cells can survive well in the CNS, we have found that neuron abundance in neural progenitor cell grafts is significantly reduced relative to syngeneic grafts. This may be due to the selective elimination of MHC-expressing neurons by an allo-specific T cells but classical immunosuppression does not alter outcome. In contrast, we find that attenuating innate immune signaling and cytokine production is more effective and can increase the abundance of allogeneic neurons to levels approaching syngeneic grafts. This highlights a growing awareness that T cell mediated graft rejection is only one variable in a more complex immunological equation that influences the function of graft-derived neurons in cellular therapy. The immune mechanisms may be diverse but historical data highlights the relative importance of class I MHC in both immune recognition and neurodevelopment. Experiments in this proposal focus on defining the specific roles of MHC1 in classical innate and adaptive immune recognition as well as the non-immunological roles in neuron connectivity and survival following transplant to the adult brain.

? DESCRIPTION (provided by applicant): Genetic studies indicate that Autism and Autism spectrum disorders (ASD) result from a complex genetic predisposition that acts with non-genetic factors to alter brain development. Common genetic variation may account for up to 50% of ASD risk with remaining causes attributed to other factors 1,2. Even penetrant single gene mutations are not alone sufficient to cause autism in all individuals who carry the alteration and it is clear that non-genetic or environmental factors must interact with genetics to modify risk of ASD. With diagnoses at 1 in 68 in the United States8, it has become vital to develop animal models that reflect gene-environment interactions that contribute to the development of ASDs. One of the strongest environmental associations with ASD is a maternal immune event during early pregnancy9-11. Infections of the fetus are not required for these effects and simple activation of a maternal inflammatory response is associated with increased risk9. Despite strong evidence that that both genetic and immune factors contribute to ASD, there are very few reports confirming that specific genetic alterations interact with a gestational immune events to cause or worsen the severity of ASD. Animal models confirm that activation of an innate maternal immune event is alone sufficient to cause alterations in neurodevelopment and behavior 15. It is also known that immune-induced placental damage14,16 or alterations in placental function 17 contribute to these effects. Some ASD risk genes are involved in placentation and we propose that certain types of illness or immune-activating exposures during early pregnancy will synergistically converge with genetic risk to endanger the placenta and developing fetus. Here we provide evidence that microdeletions or duplications on chromosome 16p11.2 that are associated with ASD mechanistically converges with immune-related mechanisms in both the placenta and developing brain. In this proposal, experiments in mice and with human pluripotent stem cells will determine whether environmental immune risk and 16p11.2 copy number variation synergize to more severely impact neurodevelopment and function to cause ASD.

? DESCRIPTION (provided by applicant): Genetic studies indicate that Autism and Autism spectrum disorders (ASD) result from a complex genetic predisposition that acts with non-genetic factors to alter brain development. Common genetic variation may account for up to 50% of ASD risk with remaining causes attributed to other factors 2,3. Even penetrant single gene mutations are not alone sufficient to cause autism in all individuals who carry the alteration and it is clear that non-genetic or environmental factors must interact with genetics to modify risk of ASD. With diagnoses at 1 in 68 in the United States8, it has become vital to develop animal models that reflect gene-environment interactions that contribute to the development of ASDs. One of the strongest environmental associations with ASD is a maternal immune event during early pregnancy9-11. Infections of the fetus are not required for these effects and simple activation of a maternal inflammatory response is associated with increased risk9. Despite strong evidence that that both genetic and immune factors contribute to ASD, there are very few reports confirming that specific genetic alterations interact with a gestational immune events to cause or worsen the severity of ASD. Animal models confirm that activation of an innate maternal immune event is alone sufficient to cause alterations in neurodevelopment and behavior 15. It is also known that immune-induced placental damage14,16 or alterations in placental function 17 contribute to these effects. Some ASD risk genes are involved in placentation and we propose that certain types of illness or immune-activating exposures during early pregnancy will synergistically converge with genetic risk to endanger the placenta and developing fetus. Here we provide evidence that the ASD risk gene chromodomain helicase DNA binding 8 (CHD8) mechanistically converges with immune-related mechanisms in both the placenta and developing brain. In this proposal, experiments in mice and with human pluripotent stem cells will determine whether environmental and CHD8-related genetic risks synergize to more severely impact neurodevelopment and function in ASD.

Project Summary: Human pluripotent stem cells provide an attractive experimental platform for studying the normal and abnormal development of human neural networks. In vitro, induced pluripotent stem cells (iPSC) have been used to study early developmental mechanisms and the initial stages of synaptogenesis and circuit formation. iPSC also offer the tantalizing but unrealized potential to study human neural circuits in vitro. Our efforts to model the development of human cortical circuits in vitro indicate that differentiation can be segmented into a 5-step progression involving: 1) patterning of the neuroectoderm and early neurogenesis - robustly evident after 3 weeks; 2) neuroblast migration and initial differentiation into regionally-specific neuronal subtypes - evident after 6 weeks; 3) development of spontaneous electrical activity and early synaptogenesis ? evident after 2-3 months; 4) initial appearance of astrocytes and the formation of vigorous and synchronous oscillatory bursting within large ensembles ? evident within 3-5 months; 5) resolution of large hypersynchronous neuronal ensembles into smaller, discreetly firing sub-ensembles ? occasionally observed after more than 6 months. Appearance of oligodendrocyte progenitors and myelination of axons seen in vivo has not yet been observed or reported. Although it is comforting to confirm that human-specific developmental programs are temporally intact in iPSC models, the protracted timeline makes it challenging to study late developmental mechanisms or mature circuit function. The ultimate goal of this project is to develop methods to accelerate the formation of networks of highly interconnected human neurons with mature synapses. Synchronized and oscillatory neuronal activity appears to be a fundamental and obligate transitional property of developing nervous systems. Young neurons in structures as diverse as retina, cerebral cortex and spinal cord experience periods of high connectivity and robust neuronal activity that are subsequently refined to produce specific synaptic connections and regulated action potential activity. Human neurons developing in vitro presumably require the acquisition of these same properties in order to become functional neuronal networks. Our preliminary work suggests that these obligate properties are acquired by the interactions of cell types generated at different times in development and/or from different regions of the developing brain. Experiments in this proposal focus on novel methods that recapitulate these interactions to accelerate the acquisition of synchronous oscillatory bursting and promote the further maturation of iPSC-derived human neural networks.

PROJECT SUMMARY This new T32 application seeks support for predoctoral training in the newly established doctoral degree program in Stem Cell Biology and Regenerative Medicine (SCBRM) at Stanford University. The SCBRM graduate program is the first new doctoral degree program at the School of Medicine in more than 20 years. The program faculty span multiple schools and departments at Stanford and our primary goal is to train the best scientists in this new discipline through a combination of basic stem cell biology and the role of stem cells in the development, maintenance, and disease pathogenesis of human tissues and organs. Over the last decade, fundamental discoveries in the biomedical and physical sciences have generated a strong climate of expectation that discoveries should and will lead to new medical applications and novel therapies. There is no greater expectation than that produced within the broad discipline of stem cell biology and regenerative medicine, an established area of study that rests squarely on an unprecedented intersection of disciplines. On the one hand, rapid advances in basic biology, genetics, material sciences, nanotechnology, engineering, and physics are providing extraordinary new tools for harnessing stem cells in regenerative medicine. On the other hand, rapid and continued growth of financial investment in translational medicine by national, state, and private entities targets research dollars with an increasing focus on applied science. Research in stem cell biology and regenerative medicine also raises ethical and legal concerns that are unique in all of biological research; thus, this broad intersection of disciplines is producing a new breed of scientist, one with skills that embrace biological and physical sciences, medicine, business, ethics and law. In the following application we outline a novel predoctoral curriculum and training plan that provides a strong foundation in developmental and cellular biology, genetics, and biochemistry. These intellectual strengths are partnered with clinical science, bioengineering, business, ethics and law to produce a new generation of scientist who is both motivated and capable of translating stem cell discoveries into new applications in regenerative medicine.