John R. Sladek, Ph.D. - US grants

The goal of this project is to address the question of the role played by norepinephrine in vasopressin release by the application of a new technique, used in concert with more established methods. This new technique is the method for the simultaneous visualization of monoamines and neuropeptides as developed by our laboratory. The more established methods are radioimmunoassay of vasopressin and microspectrofluorometry of monoamine histofluorescence. We plan on examining some of the unanswered questions concerning the applicability of our technique to functional and anatomical neuro-biological problems involving neuronal interactions, particularly between aminergic afferents and peptidergic target neurons. The neural circuit of brain stem norepinephrine pathways to vasopressin neurons of the hypothalamus has been chosen as a model in part because it can be tested both anatomically and physiologically. The functional output of this system will be measured with vasopressin radioimmunoassay, while the details of the anatomical degree of interaction will be assessed with our combined immunocytochemical-histofluorescence analysis. The project also will develop the use of a newly conceived instrument, the comparator bridge microspectrofluorometer as a tool for determining neuronal activity in identified neurons during a specific functional state.

Age-related deficits in central transmitters have been discovered for several neural systems. Some groups of monoaminergic and peptidergic neurons appear to show this decline in both rodent and primate brain. Numerous human neurodegenerative disorders are characterized by similar declines in transmitters. The aged brain recently has been shown to possess a marked plasticity as measured, for example, by growth of dendrites in cortex and by growth of catecholamine fibers from the medial forebrain bundle following surgical intervention. This plasticity suggests that neural reorganization may occur during aging and further, that the aged brain may be receptive in an integrative sense, to neural transplants. Neural transplantation techniques have been shown to reverse or improve some genetic or degenerative deficits in brain function in young animals and, in one instance, in aged rats. The present proposal is designed to test the potential of neural grafts to improve lost or diminished brain function associated with vasopressin neurons of the supraoptic nucleus and noradrenergic neurons of the locus coeruleus, using a combination of endocrine, behavioral, neurochemical and morphological measures in rodent and then to extend this question to non-human primate in order to determine optimal grafting conditions in young and aged macaques as a prerequisite for potential clinical tests.

The First International Symposium of Transplantation into the mammalian CNS was held at Orenas Castle near Lund, Sweden, on June 18-22, 1984. Scientists throughout the world gathered to review the experimental basis for neural tansplantation and to discuss future directions of what has become a rapidly developing subdiscipline in neurobiology. The Conference provided numerous insights, both retrospective and prospective on the feasibility to nerve cell grafting to reverse neurological deficits in numerous model systems. It also raised provocative questions about the future. We will review new information dealing with transplantation into the primate central nervous system in animal models of Parkinson's disease, for example, and will explore the use of tissue donors alternate to fetuses as a practical approach to therapeutic grafting. New vistas in our understanding of trophic substances and exciting parameters on mechanical and neurochemical guidance of growing axons following transplantation will be explored. Numerous other topics as detailed in the application will serve to balance the meeting with respect to basic understanding of graft host interactions. The Second International Symposium maintains the organizational structure of the first meeting as well as the guiding principles of program selection. It is in part supported by philantropic efforts and a host university, but due to the expansion of the size of the meeting to meet the growth of the discipline, it will be necessary to generate financial assistance beyond that already dedicated to the Conference.

The morphology and tissue culture core will provide histological and tissue culture support for all three projects. Immunohistochemistry for tyrosine hydroxylase will be used as a primary marker for dopaminergic neurons. Quantitation of dopamine neuron numbers in grafted tissue and in the substantia nigra of MPTP treated monkeys will be assessed for each relevant experiment. Computer assisted three-dimensional reconstructions will be made, particularly for attempts at circuit reconstruction using striatal- nigral co-grafts. Additional morphological methods and techniques provided by this core and its personnel are cryopreservation, fluorescence histochemistry, riboprobe in situ hybridization for tyrosine hydroxylase, immunochemistry for a number of other transmitters. Core personnel perform all fetal brain dissections and participate in adult brain dissections and micropunches that rely upon a combined histochemical/biochemical procedure. The tissue culture facility will carry out pharmacologic studies described in Project 1.

One of the foremost difficulties with the application of neural transplantation to the treatment of neurodegenerative disorders is enhancing the survival of grafted donor tissue to ensure that adequate amounts of dopamine are produced and that transplant effect are long- lasting. One prominent laboratory reported that the survival rate of grafted, human dopamine neurons is as low as 2-5% using current techniques, tested in rodents. This laboratory and others have used this conclusion as the basis for substantially increasing the amount of tissue implanted in clinical experiments. We have been able to achieve a considerably higher survival rate in primates by studying factors which might enhance it. Improved survival of grafts may be achieved from tissue obtained from a critical stage of dopamine neurogenesis. It may be noteworthy that clinical experiments have utilized a much wider range of gestational ages than would be suggested from our new data. Moreover, we have found that chronically administered levodopa in rodents results in grafts with stunted dopamine neurons and diminished behavioral improvement. Other factors related to the preparation and storage of the donor tissue or pharmacological treatments after grafting may be equally important. Although some studies performed in rat may provide useful clues, it is understood that the greater size and complexity of the monkey brain and its similarity to the human brain makes it essential to optimize the viability of grafts in monkey models. Variables to be studied include the age of the donor tissue, graft survival over time, the method of preparation of the donor tissue such as cell suspension, solid grafts, and cryopreservation, and the effects of conventional pharmacotherapies such as levodopa, deprenyl and others on graft survival. These studies will be carried out using grafts of mesencephalic tissue into monkeys over several time periods, controlling for fetal age, matched comparisons of suspended vs. small solid grafts implanted on opposite sides of the caudate nucleus of the same monkey, fresh vs. cryopreserved tissue also implanted on opposite sides of the brain. Pharmacological studies will be done first in primate cell cultures and, if positive, subsequent experiments will test these effects on grafted monkeys. This project also will examine the effectiveness of grafts of embryonic striatum to promote growth of neurites from co-grafted mesencephalic tissue as a means of achieving greater outgrowth from grafted neurons. During the current award period we also discovered that striatal grafts have a growth enhancing effect on the host brain that can result int he growth of tyrosine hydroxylase positive fibers into grafts placed into the dorsolateral striatum. We will examine this phenomenon from the perspective of early intervention designed at protecting the residual host dopaminergic neurons.

Our proposed program will serve to enhance the infrastructure of this large multiple Institutional Review Board (IRB) serving the University of Colorado Health Sciences Center and our five affiliated institutions. Our long term goal is to establish a highly efficient and effective system of IRB review of protocols that provides accessible technical and educational support to investigators for human subject protection. Our specific aims are to 1) improve the quality of research protocols by developing an electronic protocol application that utilizes directed questions and routed follow-up questions 2) streamline administrative tracking and processing of protocols by improving the existing database 3) enhance the review process by use of electronic submission and review of protocols improving access to consultant or distant site reviewers, 4) strengthen Serious Adverse Event (SAE) review and monitoring by utilizing electronic SAE reporting which will allow for shared SAE review with Data Safety Monitoring Boards 5) enhance continuing education efforts by labelling investigators in the database with research topics in order to direct specific human subject protections education to them and 6) renovate space recently allocated in the IRB office to create an electronic IRB meeting room and additional storage space. In order to accomplish these specific aims, we will hire two additional information technology consultants to assist our database developer in making the necessary improvements to the database. LCD monitors and an LCD projector will be purchased to create the electronic meeting room. A new server and software will be purchased to support the higher web-based activity of the database proprosal. The database developer will work with the IRB director to create the electronic protocol application and laptops will be purchased for community members to participate in the electronic review of protocols. An electronic SAE form will be developed by the director, and a procedure for shared SAE review will be established with the local DSMBs. An electronic board room will be constructed in currently unrenovated space, which will include a table for the LCD monitors and an LCD projector with screen. Additional space will be rennovated to allow for storage of closed protocol files. This new comprehensive program will provide our institution with a higher level of quality, investigator participation and efficiency in review and oversight of human subject research.

Our proposed program will serve to improve the quality of education and enhance the infrastructure of this large multiple Institutional Review Board (IRB) serving the University of Colorado Health Sciences Center and our five affiliated institutions. Our long term goal is to establish a highly efficient and effective system of IRB review of protocols that provides accessible technical and educational support to investigators for human subject protection. Our specific aims are to 1) improve the quality of education in human subject protections by the creation of a comprehensive online tutorial for investigators; 2) improve the quality of continuing education for investigators by creating a series of online topical tutorials; 3) improve the quality of IRB Member education by creating educational programs available in online tutorial; 4) provide these online tutorials to affiliate IRBs within the University of Colorado system to improve consistency between IRBs; 5) Continue enhancement of the COMIRB database to support the development of electronic protocol submission. A major advantage of these new tutorials is that they include the expansion of case studies that will be tailored to individual disciplinary needs such as pediatrics since our current case studies deal with adults. Another major advantage of this program is that IRB member training, virtually unavailable in electronic format now, will be utilized widely by our large IRB and the CU System IRBs to enhance a consistent application and understanding of the federal regulations. In order to accomplish these specific aims, we will work with two co-investigators who have previously developed technology in the creation of online tutorials. Dr. Allan Prochazka who was previously a Co-chair in COMIRB and has extensive expertise in both ethics and human subject protections, will develop the educational modules for investigators, IRB members, and continuing education. Dr. Laurie Shroyer has become an expert in the technological aspects of creating these online tutorials. We also will utilize an Information Technology consultant to assist our database developer in continuing the necessary improvements to the database. A new computer will be established as an additional workstation for updating the current system. A laptop computer will be purchased for presentations of our system to other IRBs and in training investigators in the use of the electronic protocol submission process. We believe this comprehensive program will provide our institution with a higher level of quality, investigator participation and efficiency in review and oversight of human subject research.

PROJECT 3. NIGRAL TARGETING STRATEGIES FOR RESTORATION OF NEURAL FUNCTION Although intrastriatal grafts of embryonic DA neurons survive, send neurites into the host brain, elevate DA levels in the vicinity of the grafts, and restore motor function in parkinsonian MPTP-treated monkeys, the functional benefits in humans have been variable, somewhat limited and sometimes associated with dyskinesia. These limitations may be due to the ectopic placement of grafts in the striatum and the resulting dysregutated release of dopamine. Recent studies have shown the feasibility of grafting embryonic substantia nigra (SN) into the host SN and grafts of embryonic striatum into the striatum, which can be used to promote directed fiber outgrowth from DA-containing neurites towards appropriate targets. The experiments proposed build upon this knowledge. The hypothesis is that co-grafting embryonic striatum into the striatum and ventral mesencephalic tissue adjacent to the substantia nigra will improve recovery, since replacement nigral cells can be regulated in the nigra and provide appropriate release of dopamine in the normal target areas. Behavioral measures will assess functional recovery, immunohistochemical markers for DA neurons will be used as indices of graft survival in the target regions, and biochemical effects will be determined. Confocal and electron microscopy will aid the depiction of circuit reconstruction that may result from co-grafts. Prior to sacrifice, the retrograde tracer fluorogold wilt be injected in the caudate or putamen to determine if grafted dopaminergic neurons in the nigra are labeled. Using the same outcome measures encapsulated GDNF-producing cells will be implanted strategically to attract appropriate outgrowth in another experiment. Some monkeys will be studied for three years to allow for more complete restoration, determine extent of recovery, and possible side effects, such as dyskinesia. Together, these experiments will measure the degree of functional recovery attained through the placement of DA-specific grafts, subject to proper regulatory influences, into more physiological sites. Results from these experiments will be compared with behavioral, morphological, and biochemical data generated from Projects 1 & 2, as well as with prior transplants done under similar conditions in St. Kitts green monkeys.

DESCRIPTION (provided by applicant): This application seeks partial support for the 9th International conference on Neural Transplantation and Repair, INTR9, to be held June 8-11, 2005 at the Grand Hotel, Taipei, Taiwan. The conference will provide a much needed multidisciplinary forum for the presentation and critical discussion of the most recent developments in the field of neural transplantation and repair. The conference will focus on repair of neurodegenerative disease and trauma, in particular Parkinson's, Huntington's, and Alzheimer's diseases as well as spinal cord injury and stroke. It will highlight new techniques in translational medicine including stem cell biology and gene therapy. The general theme of the conference is on neural protection and neural repair, particularly as these relate to gene therapy, stem cells, and cell replacement. In particular, there is an international transplant registry and a practice committee that meets during this conference and develops guidelines for clinical practice regarding cell replacement and gene therapy in neuroscience. A broad range of speakers has been sought for this conference, chosen to represent a cross-section of leading scientists in this expanding field. The venue for the ninth international meeting was chosen by an international program committee of the American Society of Neural Transplantation and Repair, and represents the first time the international meeting will be hosted by a Pacific rim nation. Thus, it will provide easier access to a large audience of scientists who may not have been able to attend the prior meetings because of geographical limitations.

PROJECT 3. NIGRAL TARGETING STRATEGIES FOR RESTORATION OF NEURAL FUNCTION Although intrastriatal grafts of embryonic DA neurons survive, send neurites into the host brain, elevate DA levels in the vicinity of the grafts, and restore motor function in parkinsonian MPTP-treated monkeys, the functional benefits in humans have been variable, somewhat limited and sometimes associated with dyskinesia. These limitations may be due to the ectopic placement of grafts in the striatum and the resulting dysregutated release of dopamine. Recent studies have shown the feasibility of grafting embryonic substantia nigra (SN) into the host SN and grafts of embryonic striatum into the striatum, which can be used to promote directed fiber outgrowth from DA-containing neurites towards appropriate targets. The experiments proposed build upon this knowledge. The hypothesis is that co-grafting embryonic striatum into the striatum and ventral mesencephalic tissue adjacent to the substantia nigra will improve recovery, since replacement nigral cells can be regulated in the nigra and provide appropriate release of dopamine in the normal target areas. Behavioral measures will assess functional recovery, immunohistochemical markers for DA neurons will be used as indices of graft survival in the target regions, and biochemical effects will be determined. Confocal and electron microscopy will aid the depiction of circuit reconstruction that may result from co-grafts. Prior to sacrifice, the retrograde tracer fluorogold wilt be injected in the caudate or putamen to determine if grafted dopaminergic neurons in the nigra are labeled. Using the same outcome measures encapsulated GDNF-producing cells will be implanted strategically to attract appropriate outgrowth in another experiment. Some monkeys will be studied for three years to allow for more complete restoration, determine extent of recovery, and possible side effects, such as dyskinesia. Together, these experiments will measure the degree of functional recovery attained through the placement of DA-specific grafts, subject to proper regulatory influences, into more physiological sites. Results from these experiments will be compared with behavioral, morphological, and biochemical data generated from Projects 1 & 2, as well as with prior transplants done under similar conditions in St. Kitts green monkeys.

DESCRIPTION (provided by applicant): This exploratory project is designed to advance an unexpected finding that resulted from our program project grant on improving grant function in parkinsonian non-human primates. As part of the design we tested the ability of "helper" grafts to stimulate and guide the extension of neurites from grafts of ventral mesencephalon that contained dopaminergic neurons. We found that neurites from dopamine neurons could bridge the distance between the brain stem and the striatum and that they could extend from one distant helper graft to a second and ultimately to a third that was located in the striatum. This is an important finding with respect to the potential for reconstruction of elements of the nigrostriatal pathway because it places the grafted dopamine neurons in the mesencephalon where they have the opportunity to be contacted by appropriate afferent inputs. Graft therapy for Parkinsonism in both animal models and humans has utilized the striatum as the graft site for placement of dopaminergic neurons. While this convenient placement resulted in the release of dopamine directly into the region of the striatal target neurons, its shortcoming is that it places the donor neurons in a position that is removed from the normal pathway. In some clinical trials patients who initially showed some level of motor improvement eventually showed dyskinesias that are postulated to be due to uncontrolled or excessive release of dopamine by the grafts. The interesting thing about our finding is that we used striatal "helper" grafts to bridge the relatively long distance between the rostral mesencephalon and caudal striatum in the non-human primate and maintained the correct caudal to rostral position of the dissected pieces of the striatal anlagen. Thus, the dopamine neurites first reached the developing caudal portion of the striatum, and then somewhat remarkably continued to grow in a rostral direction to reach the second, later developing middle portion of the striatal donor graft. Ultimately, the fibers reached the third striatal "step" which was located in the host striatum. We hypothesize that a developmental gradient exists for the striatum from caudal to rostral that normally guides dopamine fibers that approach the striatum from the brain stem, and that a progressive wave of striatal growth factor activity exists to account for the continued extension of these fibers to reach the most distant sites within the striatum. Thus, we propose to test this in rodents prior to any further exploration in non-human primates. The program project grant is not going to be submitted for renewal which is a decision made by the executive committee of the P01 and if this exploratory proposal in rodent is awarded and proves successful then we will utilize this information to plan additional tests in the non-human primate of helper graft circuit reconstruction in behaviorally impaired animals as a possible future R01. PUBLIC HEALTH RELEVANCE: The proposed study has the potential to identify a key aid for successful circuit reconstruction of the dopaminergic pathway that is responsible for the manifestation of the signs and symptoms of Parkinson's disease. Our earlier studies in non-human primates provided a novel finding that can be tested more fully presently in a rodent model prior to moving forward with the more complex primate. We believe that more complete circuit reconstruction is crucial for optimizing graft function.