Timothy J. Collier - US grants

Brain norepinephrine (NE) systems in rats, monkeys and humans, the locus coeruleus and its projections in particular, undergo degenerative changes during aging, as detected by morphological and biochemical methods. This probable decline in function of the NE system is exaggerated in the dementia of Alzheimers disease and Parkinsons disease. The proposed studies address the broad hypothesis that aging of the central NE system contributes to the expression of cognitive and memory deficits in some elderly individuals and in dementia. Specifically, aged Fischer-344 rats, screened for deficient performance of a spatial memory task (Morris water maze), will receive NE supplementation provided either by intracerebral implantation of fetal NE neurons, or chronic intraventricular infusion of NE via mini-pump. Control animals will receive no supplementation treatment or a graft of fetal cerebellar tissue. Efficacy of NE supplementation in memory- deficient aged rats will be assessed by re-testing behavior coupled with tests of the behavioral effects of drugs acting on the adrenergic system, and subsequent analysis of NE biochemistry and morphology in neural grafts and the host brain. These studies will provide further information on the state of brain NE systems in the rat model of behavioral senescence, the behavioral efficacy of chronic NE supplementation in these animals, the biochemical and morphological correlates of interactions between grafted NE neurons and the aged host brain, and whether the use of NE neuron grafts holds any promise as an experimental therapeutic approach for severe memory deficits in aging and dementia.

Dopamine (DA) neurons of the ventral mesencephalon which innervate the striatum show age-related deterioration in rodents, monkeys and humans. Severe degeneration of this nigrostriatal DA system yields the motor dysfunctions characteristic of Parkinson's disease in humans. Recent evidence from our laboratory indicates that exposure of embryonic rat DA neurons to diffusible products derived from an explanted peripheral nerve segment, or cultured Schwann cells, enhances survival and neurite outgrowth of these DA neurons in culture. Similar enhancement of DA neuron survival and growth is obtained when embryonic DA neurons are co- grafted with a peripheral nerve segment to the striatum of DA-depleted rats. In addition, intraventricular implantation of the peripheral nerve segment in 25 month old rats upregulates tyrosine hydroxylase (TH) staining in the entire nigrostriatal system ipsilateral to the implant. Finally, we have isolated a low molecular weigh fraction of Schwann cell conditioned medium that contains DA neuron survival and growth-promoting activity, referred to in this application as dopamine neurotrophic factor (DNTF). The current proposal seeks to extend these preliminary finding s to document whether compensatory growth and/or activity of nigrostriatal DA neurons can be induced in young adult and aging rats with variable degrees of damage to the DA system via exposure to products of grafted Schwann cells or intraventricular infusion of the conditioned medium fraction (DNTF). Since identification DNTF is ongoing and incomplete, we will also test the relative efficacy of DNTF to promote DA neuron survival and growth as compared to four known growth factors which have demonstrated positive effects on the viability of cultured DA neurons: bFGF, BDNF,IGF-1 and EGF. Both culture and animals studies will collect information on the magnitude of factor induced compensatory changes in the DA system, the relationship to the number and location of DA neurons information on cellular mechanisms of DA neuron growth promotion. Evidence from behavioral measures, immunocytochemical morphology, neurotransmitter and metabolite assays, and measurements of TH mRNA expression will combine to address these issues of interest. In addition, we will pursue our initial finding of enhanced efficacy of embryonic DA neuron grafts in the presence of co-grafted peripheral nerve, by comparing embryonic DA neuron grafts to mixed co-grafts of young and aged rats with short-term (3 weeks) or long-standing (12 months) lesions of the nigrostriatal DA pathway. Further study of Schwann cells and their products as a source of survival and growth- promoting activity for DA neurons may provide insights into the biology of aging as it influences mechanisms of central nervous system plasticity, as well as having exciting implications for experimental therapeutics in aging and Parkinson's disease.

DESCRIPTION: (Verbatim from the Applicant's Abstract) Transplantation of embryonic dopamine (DA) neurons as an experimental therapy for Parkinson's disease (PD) is currently under evaluation. The non-human primate treated with the neurotoxin MPTP has served as an important animal model for the disease and the grafting paradigm, and has had significant predictive value for results of early clinical trials. Despite some encouraging clinical findings, relative survival of grafted DA neurons is low and improvement of behavioral symptoms is incomplete. Part of the disparity between results in animals and PD patients may relate to failure of animal studies to model certain characteristics of potential recipients of graft therapy that impact significantly on the environment of grafted cells. One characteristic of this patient population that can be examined in animals is the influence of chronological age of the transplant recipient. The interactions between age-related changes inherent to this system in graft recipients, the response of the aging system to accelerated loss of nigral DA neurons, and the impact these changes have on the environment for grafted tissue only recently have received attention. We have found that the viability and function of grafted DA neurons is profoundly diminished in DA-depleted aged rats. In addition, these aged animals exhibit important deficits in compensatory responses to DA depletion including decreased striatal neurotrophic activity. Recent clinical results also suggest diminished graft efficacy in elderly patients. Advancing chronological age of the transplant recipient may represent a previously under-appreciated risk of diminished graft viability and function that may mandate study of novel grafting strategies 1 to achieve good therapeutic results. It is the goal of this proposal to evaluate the influence of chronological age of the host on graft viability and function in MM?-treated non-human primates. Tissue from single donors will be divided for implantation into pairs of young adult and aged hemiparkinsonian monkeys, with behavioral, mophological, and biochemical techniques employed to study rates of apoptosis in grafts, survivai, neurite outgrowth and release of DA from grafted cells, and receptor, metabolic and -trophic responses in the host. Additional analyses will examine aging-related changes in microvasculature and oxidative stress in the graft environment. These studies will provide valuable information on the response of the aged brain to accelerated DA neuron loss, the interaction between aging in the host and graft viability, indicate mechanisms of intervention with graft survival and function in the aged brain, and will aid in matching patients with the optimal therapeutic approach.

DESCRIPTION (provided by applicant): In vitro expansion of neural progenitor cells followed by induction of dopaminergic phenotype may provide a limitless source of cells for grafting into patients with Parkinson's disease (PD). However, the signals controlling the conversion of these cells into dopamine (DA) neurons must be identified. In an effort to accomplish this, single cells isolated from ventral mesencephalon were clonally expanded and exposed to hematopoeitic cytokines and neurotrophic molecules. Analysis of cell differentiation in response to this treatment yielded conversion of a high percentage (72 to 98 percent) of cells in some clones to a tyrosine hydroxylase (TH)-positive phenotype. Of the 24 clones generated, the best conversion to TH cells occurred with exposure to a combination of interleukin-1 (IL-1), interleukin- 11 (IL-11), leukemia inhibitory factor (LIF), and glial cell line-derived neurotrophic factor (GDNF). Positive clones expressed TH, the DA transporter, Nurr-1 and released DA in culture. Other cells in cytokine-exposed clones expressed GFAP (astrocyte marker) or MAP-2 (neuron marker) indicating that the original neurospheres were also capable of producing clones that differentiate into glial and nondopaminergic neurons. Initial neural grafting studies m the rat model of PD using a clone with the highest conversion rate to TH indicated that converted progenitor cell grafts produced complete amelioration of amphetamine-induced rotational behavior and continued to express the TH phenotype. However, the survival rate of these grafted progenitor cells was reduced (26 percent) compared to embryonic ventral mesencephalon (VM). The experiments proposed here will develop protocols for optimal survival of Wafted cytokine-converted mesencephalic progenitor cells. Once survival of grafted mesencephalic progenitor cells is optimized, direct comparisons will be made to fresh embryonic VM grafts on measures of behavior, in vivo dialysis, post-mortem DA biochemistry, DA receptors, cell survival and neurite extension. Lastly, this proposal will test the efficacy of the DA conversion cocktail on clonal progenitors derived from embryonic mesencephalon of nonhuman primate brain. If successful, cytokine-converted mesencephalic progenitor cells could potentially replace embryonic tissue as the primary source of cells for grafting in PD.

Studies in rodents and nonhuman primates suggest that up to 95% of grafted dopamine (DA) neurons die within the first week after implantation into the striatum, a possible contributor to poor clinical outcome in studies to date. In addition, poor survival of grafted cells appears to be exaggerated when cells are implanted into an elderly host. Multiple insults associated with the transplant procedure and early post-graft interval could render grafted cells susceptible to death, including mechanical trauma, hypoxia, oxidative stress, and neurotrophic factor withdrawal. Despite these problems, the identification of cell death mechanisms operating in DA grafts, and their probable triggers, provides access to cogent interventions to limit death of grafted neurons. This project aims to investigate four interventions that may limit death of grafted neurons, optimizing the potential for DA replacement and recovery of function: 1) reducing the apoptosis triggered by dissection and preparation of the tissue for implantation, termed "anoikis", via treatment with the cell adhesion factors L1 antibody and tenascin, and, 2) utilizing treatment with the lazaroid tirilazad mesylate and melatonin to reduce oxidant stress, 3) reducing hypoxia/ischemia by accelerating neovascularization of grafts using treatment with vascular endothelial growth factor (VEGF), 4) stimulating the DA phenotype of grafted cells by exposure to cyclic AMP. All interventions proposed have yielded significant graft augmentation in rodent experiments. In the present application, each intervention will be tested for functional efficacy in young adult MPTP-treated St. Kitts green monkeys. Optimally aged fetal mesencephalic tissue, with varying treatments, will be implanted into the striatum, and quantitative behavioral measurements will assess functional outcome, correlated with histological and biochemical evidence of more extensive grafts. A final experiment will combine treatments proven individually to augment graft function in young adult monkeys, and compare the functional outcome of this combination therapy in young adult and aged MPTP-treated monkeys. Successful methods for augmentation of grafted cell survival and growth may improve transplantation results and be applicable to stem cells or other cell-based therapies for Parkinson's disease.

Replacement of striatal dopamine (DA) remains the main goal of therapeutics for Parkinson's disease (PD). Many therapeutic alternatives to levodopa therapy are being tested. Among these is transplantation of immature DA neurons either derived from fetal donors or stem cells. This approach continues to be conceptually attractive, especially for late stage PD in which therapies that rely upon plasticity of remaining neurons are unlikely to be effective. The main problem associated with use of cell implants is the extremely poor survival of grafted DA neurons and/or the instability of DA phenotype after grafting. Over the past several years we have identified several molecules that augment survival and function of cultured and grafted fetal DA neurons. Many of these molecules ameliorate the negative impact of distinct threats to DA neuron viability. It is the goal of this proposal to systematically evaluate combinations of these factors to formulate a therapy to optimize survival and function of grafted DA neurons. Such an optimal approach will allow reduction in the number of cells required for therapeutic efficacy, improve standardization of graft composition and potentially enrich grafts in the DA neuron type relevant for striatal DA replacement. Cell grafts derived from fetal or stem cell sources contain a mixture of the major DA neuron types of the midbrain: A9 type and A10 type. It recently has been demonstrated that only A9 type neurons reinnervate the striatum after grafting. Thus, our analysis of augmentation effects will focus upon determining whether particular combination therapies specifically enrich the DA neuron population in the relevant A9 cell type. Our previous studies have identified the following molecules to be individually effective in promoting survival and function of cultured and grafted DA neurons: SO2A conditioned medium (neurotrophic support), vascular endothelial growth factor (VEGF)(neurotrophic, stimulates vascular supply), melatonin (anti-oxidant), creatine (cellular energy), erythropoeitin (anti-apoptotic), and minocycline (anti-inflammatory). We will use a series of experiments utilizing cell culture followed by grafting in DA-depleted rats to formulate a multi-factorial approach to promoting survival and stable DA phenotype in grafted fetal DA neurons and DA neurons derived from human embryonic stem cells. With optimization, cell replacement therapy could become a practical therapeutic option for late stage PD.

[unreadable] DESCRIPTION (provided by applicant): This R13 application seeks funding for the annual meeting of the American Society for Neural Therapy and Repair (ASNTR) 2008. Since its founding in 1994, the ASNTR has convened yearly each April/May in Clearwater, Florida. At the meeting various research advances in the fields of transplantation, neuroengineering and gene therapy applied to neurological diseases such as Parkinson's disease, Huntington's disease, Alzheimer's disease, stroke, spinal cord injury and traumatic brain injury are discussed via platform and poster presentations. ASNTR focuses on treatment strategies throughout all its sessions. Thus, students and postdocs not only learn the basic sciences, they also learn translational issues discussed by basic scientists and clinicians. Therefore, ASNTR is truly a unique gathering. Attendance of this meeting ranges between 125-200 registrants. Each year the ASNTR Education Committee encourages students/postdocs to submit their abstracts in a competition and the top rated applicants receive travel awards to cover the cost of their meeting attendance and travel. ASNTR has always placed a high priority on funding travel awards for students and post-doctoral fellows. In the past ASNTR has received donations from biotech companies and non-profit organizations to cover these travel awards, However, in recent years ASNTR has seen these contributions dwindle and although ASNTR will continue to seek funding from the private sector, we do not anticipate any increase in contributions above recent levels in the coming years. Therefore NIH funding for this activity is critical. As an incentive to encourage young scientists to seek and obtain independent peer reviewed funding, we conduct a very popular short grantsmanship course every 2-3 years surrounding the ASNTR annual meeting for the travel award winners and students. This application requests $20,000 in total direct costs to support travel awards for the top 20 graduate students/postdocs from US institutions that apply (awards of $1000 per student/postdoc) and $5000 to support the short grantsmanship course. NIH funding for this travel award and short grantsmanship course is crucial to continue the momentum created by previous funding of this activity and to encourage young investigators into this fledgling field of translational neuroscience. ASNTR and its educational committee includes and encourages participation of minorities including women and persons with disabilities in all its activities. PUBLIC HEALTH RELEVANCE: This application seeks funding to for the annual meeting of the American Society for Neural Therapy and Repair. This meeting will allow young and senior scientists and doctors working to discover new treatments for neurological disorders to come together and discuss their new research findings. This meeting will potentially allow new research collaborations to occur that could lead to improved treatments. [unreadable] [unreadable] [unreadable]

Core A. Administrative Core. The Administrative Core will serve to coordinate and support the activities of the Center in its entirety by facilitating communication between the PI, projects, cores, intemal advisors, external advisors, other Udall Centers, and the public. Salary support is requested for two individuals: The Center PI, Dr. Timothy Collier, and the Administrative Coordinator, Ms Michelle Gartland. Dr. Collier's effort will be devoted to oversight of the Core's activities, and participation in decision-making related to mechanisms for tracking progress and fiiture directions of the Center's scientific activities. This includesfrequentmeetings with Project Leaders and staff, annual meetings with intemal advisors and representatives of existing Udall Centers, bi-annual meetings with external advisors, and semi-annual revision of website content to communicate Center activities to the public. Day-to-day management of administrative activities (the real work) will be carried out by Ms. Michelle Gartland. Ms. Gartland has worked as an administrative/research assistant with Drs. Collier and Sortwell for five years. Her familiarity with both types of activities provides a significant advantage in execution of this role for the Center. In summary, the primary flmction of Core A is to enable communication. This will be achieved by 1) providing administrative support for the projects and cores, 2) scheduling and providing support for meetings between Center project leaders and intemal and external advisors, 3) facilitating travel and communication of data at annual Udall Centers meetings and meetings of national and international scientific societies, and 4) creating and maintaining a Center website. Ms. Gartland is experienced and talented in design of power point presentations and websites. These skills will enable center investigators to effectively communicate progress both to colleagues and to the public. The Center website will be the centerpiece of our group commitment to transparency in the use of Center funds as they apply to health and well-being of individuals with Parkinson's disease. RELEVANCE (See instructions): Core A provides the administrative structure to promote the educational and informational mission of Udall Center activities for patients with Parkinson's disease and the Cincinnati community at large.

Aging and Parkinson's disease: Models of therapeutics and neurologic comorbidity. This is an A2 application for a Udall Parkinson's Disease Center of Excellence from the University of Cincinnati directed by Timothy J. Collier, Ph.D. Two less studied aspects of Parkinson's disease (PD) are the neural mechanisms associated with development of adverse consequences of disease and treatment (such as depression and therapy-induced dyskinesias) and mechanisms associated with translational therapeutics (such as subthalamic nucleus DBS and progenitor ceU transplantation). In addition, it long has been appreciated that advancing age is a primary risk factor for PD, yet aging rarely is incorporated into experimental studies. Thus, the present proposal groups these topics under the rubric of adaptive and maladaptive plasticityand examines their expression in the context of advancing chronological age. The proposal consists of four projects and two cores that interconnect and serve the projects. Project 1 examines the roles of maladaptive changes in spine morphology in suboptimal recovery provided by grafted dopamine (DA) neurons and the development of therapy-induced dyskinesias. Project 2 will determine the degree and mechanism of neuroprotection for the DA system conferred by high frequency electrical stimulation of the subthalamic nucleus. In particular, stimulation effects on neurotrophic mechanisms wUl be examined. Project 3 tests the hypothesis that preservation of the structure and function of the injured nigrostriatal system following engraftment of undifferentiated neural progenitor ceUs is not a product of replacement of DA neurons by grafted cells, but is mediated by graft-induced protection and/or regeneration of mature host DA neurons. The goal of Project 4 is to gain insight into the co-mingling of PD, stress, anxiety and depression. It will test the hypothesis that comorbid depression exacerbates the behavioral deficits, neurochemical abnormalities, and neurodegeneration associated with PD via deleterious glucocorticoid mechanisms. AH projects will utilize well-established rat models and examine differences and similarities of mechanisms and outcomes in the context of advancing chronological age. To the extent that plasticity is characteristic of PD, it provides points of access to harness its therapeutic effects and curtail its negative effects.

Replacement of dopamine (DA) neurons via transplantation of fetal neurons as a therapy for Parkinson's disease has proven effective in some patients. One limitation of this approach is that therapeutic benefit relies upon transplantation of large numbers of cells to overcome poor survival of grafted neurons. One projected solution to this problem is the generation of a nearly inexhaustible supply of DA neurons for transplantation derived from a stem/progenitor cell source. While this has been achieved for cells derivedfrommouse, similar attempts with human cells have not produced significant numbers of DA neurons that retain phenotype following grafting. An altemate approach is not to coerce differentiation to the DA phenotype prior to grafting, but exploit inherent repair properties of stem/progenitor cells. We recently have demonstrated that grafted undifferentiated neural progenitor (NP) cells derived from embryonic rat rescue adult substantia nigra (SN) neuronsfromdegeneration following intrastriatal injection of 6- hydroxydopamine. The goal of this proposal is to document characteristics of the biology of this effect and begin to evaluate the clinical relevance of this approach by expanding our analysis to include study of human NP cell lines. Specifically we will 1) track the phenotypic fate of grafted NP cells and host SN neurons and glia over time after lesion and grafting. 2) Perform an initial study of potential mechanisms of mNP cell-induced neuroprotection by decreasing expression of the neurotrophic factors BDNF and PTN, using RNAi, in cells prior to grafting. 3) Determine whether neuroprotection is correlated with changes in additional factors associated with nigral cell injury: pro-inflammatory cytokines, apoptosis factors, neurotrophic factors and changes in DA metaboUsm as measured by PCR-based array and HPLC, to provide future targets for mechanistic studies. 4) Localize mRNA changes for relevant molecules to specific cell populations within the graft and host using in situ hybridization. 5) Determine whether NP cells remain effective in aged rats. The relative therapeutic efficacy and characteristics of NP cells from rat and human will be compared and contrasted in this neuroprotection paradigm. Implantation of undifferentiated NP cells may provide a simple, effective strategy for inducing neiu-al protection and repair. RELEVANCE (See Instructions): The findings of this project will provide important information relevant for the design of an optimal stem/progenitor cell therapy for treatment of Parkinson's disease.

? DESCRIPTION (provided by applicant): Parkinson's disease (PD) pathology is characterized by the formation of intraneuronal inclusions called Lewy bodies (LBs) and Lewy Neurites (LNs), that are comprised primarily of misfolded, fibrillar ?-synuclein (?-syn). One therapeutic strateg to slow disease progression is to reduce these toxic aggregates by preventing the native/monomeric form of ?-syn from aggregating. There is substantial need for new, efficacious disease-modifying therapies in PD. Despite the fact that antidepressants have already been shown to be safe and efficacious for depression in PD, the effects of these drugs on disease progression remain unknown. However, previous work from our laboratory suggests tricyclic antidepressants (TCAs) slow disease progression in both preclinical toxin models (Paumier et al., 2014) and in a retrospective analysis of data from an early cohort of patients with PD (Paumier et al., 2012). Together these findings, and others (Jeannotte et al., 2009a, Jeannotte et al., 2009b, Trushina et al., 2009, Chung et al., 2010, Chadwick et al., 2011, Zschocke et al., 2011, Valera et al., 2014), support the notion that antidepressants have disease-modifying potential within an existing framework of established safety. The objective of the proposed studies is to determine whether NOR can be a disease- modifying treatment for PD. We will test our central hypothesis that NOR attenuates the accumulation/aggregation of ?-syn that occurs in PD, resulting in nigrostriatal preservation. Our hypothesis has been formulated on the basis of our own preliminary findings that NOR is a potent inhibitor of ?-syn aggregation in vitro and in vivo. Rationale for the proposed studies is related to the inability to assess engagement of the ?-syn target in the clinic and subsequently link neurobiological changes directly to improvement. Absent a clinical biomarker for target engagement desirable for a prospective clinical trial, we propose to further develop the case for clinical use of NOR by: 1.) testing in nonhuman primates, and 2.) mining data from subjects enrolled in the ongoing Parkinson's Progressive Marker Initiative (PPMI) clinical trial. We predict that the capacity of NOR to reduce the rate of?-syn aggregation will prevent the spread and resulting dysfunction associated with LB-like pathology and this prevention of aggregation will be correlated with neurobiological benefit.