Barry J. Hoffer - US grants

Inducible Cre recombinase systems have been developed to bypass initial lethal phenotypes and to provide access to later embryonic or adult phenotypes. We have generated a recombinant mouse that combines a tetracycline dependent switch with generalized Cre recombinase expression by targeting the ubiquitously expressed ROSA26 locus. This transgenic strain was developed using a simplified gene delivery system integrating both elements, the reverse tetracycline controlled trans-activator (rtTA) and rtTA inducible promoter into a single vector. In this transgenic strain, the endogenous ROSA26 promoter drives rtTA expression through a splice acceptor site. The tetracycline inducible promoter, cloned in opposite orientation to the ROSA26 locus and separated from the rtTA element by a 5 kb human p53 intron, drives Cre recombinase expression. Crossing these mice with a Cre reporter strain showed that Cre DNA-mediated recombination was ubiquitously and effectively induced during various prenatal developmental windows. Background Cre recombinase expression levels were observed in some tissues in the absence of the inducer, mostly during late embryonic developmental stages and in adult animals. Background recombination levels were low during development and most prominent in nervous tissue. Cre recombinase expression could not be effectively induced in adult animals. While rtTA mRNA levels were high in developmental and adult tissues, Cre recombinase mRNA levels remained low after doxycycline treatment. The mouse strain described here provides a valuable tool to further analyze the function of genes during specific developmental windows, by allowing the effective inactivation of their function throughout defined stages of embryonic development.

We study effects of CDNF protein in the rat unilateral maximal lesion PD 6-OHDA model. CDNF is administered prior to, and two weeks after, intracranial injection of 6-OHDA into the ascending DA bundle.We study effects of CDNF protein in the mouse bilateral PD model induced by parental injection of MPTP. We study effects of CDNF protein in the MitoPark mouse model. We are developing a mouse CDNF null mutation and will study development of the midbrain DA system. We will generate AAV-CDNF and in order to test effects in the 6-OHDA rat model and in the MPTP and MitoPark mouse models. In vivo tests of such a vector, if successfully generated, will be carried out in future research.

We have used a reverse genetic approach to investigate this question and created conditional knockout mice with reduced mtDNA expression in midbrain DA neurons. These mice have adult onset of slowly progressive, typical parkinsonian symptoms accompanied by progressive formation of intracellular inclusions and characteristic neuropathology. The inclusions are present in most DA neurons and contain both mitochondrial membranes and proteins. Our findings thus support an etiological role for respiratory chain dysfunction Parkinsons disease and suggest a novel mechanism for the formation of Lewy bodies.

We are characterizing the CNS phenotypes caused by elevated mtDNA mutation levels and relating these findings to age-related functional changes. Furthermore, we will investigate the affects of ischemic brain injury in these same mice. We have already documented change in cortical thickness and lamination patterns. There are also changes in oxidative energy production and mitochondrial function. These changes parallel cognitive and motor deficiencies in the mtDNA mutators. Drugs that upregulate mitochondrial function are being evaluated.

The Office of the Clinical Director (OCD), Intramural Research Program (IRP), National Institute on Drug Abuse (NIDA), NIH provides research support to two clinical research branches at the IRP. The scope of the research program is broad and is strongly represented in neuroimaging, while also incorporating both behavioral and pharmacological treatments for substance abuse disorders. [unreadable] The Office of the Clinical Director has a staff of two Administrative Assistants who provides primary support to the Director, Deputy Director, and Medical Officer. These Administrative Assistants also coordinate with the IRP Pharmacy, Matthews Media Group, Inc. (MMG), Medical Records Department, the Mid-Level Providers and Nursing. The four fulltime Mid-Level Providers (Federal) medically screen potential participants as well as assist in the day to day running of various protocols. Additional Clinical staff supporting the NDA/IRP programs include 5 full time RNs, 5 full time Research Associates, 1 part time Unit Secretary and 1 full time Patient Care Manager. These positions are part of the clinical services provided through the Johns Hopkins Bayview Medical Contract with NIDA/IRP. This contract also provides essential infrastructure and services including professional physician consultations, laboratory medicine and staff support. There are collaborative relationships with Behavioral Pharmacological Research Unit (BPRU) located on the Bayview campus and the Maryland Psychiatric Research Center (MRPC) located on the campus of Spring Grove State Hospital. The changes that have come about as a result of the move to the BRC have brought into focus the need to expand the numbers of nursing staff. [unreadable] The NIDA IRB office is under the direction of the OCD and is staffed by an IRB Administrator (Federal) and a Protocol Specialist (Contract). The office handles approximately 500 IRB related actions a year, from 65 active protocols. This year the IRB office has joined the NIH CNS IRB, and is in the process of merging with their electronic Protocol Tracking and Management System (PTMS), as well as integrating with NIHs Office of Protocol Services (OPS).[unreadable] The OCD oversees a fully funded contract with MMG, an outside organization. MMG represents NIDA/IRP, recruiting research participants in the Baltimore Washington area. At this time MMG employs 13 screening specialists, 2 patient counselors and a 3 member management team. There is a yearly budget of $225,000 for print, web, radio and television advertising and $50,000 for community outreach.[unreadable] [unreadable] The Medical Records Department at NIDA/IRP is designed to maintain department compliance of Policy and Procedures while safekeeping the Privacy of over 6300 electronic (HuRIS System) medical records annually. Both microfilm and/or DVD as well as hardcopy versions of the medical records are accessed for both in-house and outside sources. This is done according to the National Institute of Health, Federal and State Rules and Regulations (Including the Privacy Act of 1974 and HIPPA).[unreadable] [unreadable] The IRP Pharmacy employs two fulltime pharmacists. Along with the main pharmacy at the IRP, they manage a satellite location at MPRC on the Spring Grove Hospital Campus. This is part of the collaborative research agreement. The IRP pharmacy currently supports about 20 clinical studies including 5 Archway studies and 70 researchers/labs for nonclinical studies. One pharmacist devotes about 80% of her time supporting the Archway studies, including IND support. The other pharmacist spends about 60 % effort on clinical and 40 % effort on nonclinical studies. Clinical research support includes reviewing, preparing, compounding, and dispensing the study medications. Nonclinical support includes ordering, compounding, dispensing, and laboratory auditing. In addition, the pharmacy monitors drug inventories, and meets all DEA and FDA regulatory requirements including licensing and IND reporting.

The Biomedical Informatics Section:[unreadable] Interacts with NIDA/IRP investigators to develop bioinformatics applications and databases that can access, manage, disseminate, and analyze large quantities of high quality data.[unreadable] Assists in developing, researching, and/or applying computational tools to assist NIDA/IRP investigators in the acquisition and analysis of biological, medical behavioral or health data, within a specific time frame determined to be appropriate by the NIDA. [unreadable] Helps facilitate new system initiatives and changes to existing systems to meet legislative, regulatory, and departmental requirements within the specific time frames designated by each requirement. [unreadable] Conducts routine system analysis of automatic data processing resources and techniques of existing projects.[unreadable] Recommends, as needed, the implementation of new technologies that are efficient and timely.[unreadable] Provides technical and professional support to help integrate computer systems, design or acquire computer programs, configure and support networks and streamline automated data processing within the NIDAs specified timeframe.[unreadable] Integrates scientific data systems with network services and security services to foster safe NIDA/IRP laboratory collaboration and for collaboration with extramural entities, when possible integrate scientific data systems with one another and ensure design for interoperable data.[unreadable] Reports experimental findings and conclusions (in oral or written format) routinely to members of the BIS and others at the NIDA/IRP as well as presenting our findings at various scientific conferences or journals.[unreadable] Automates various aspects of clinical program at the NIDA/IRP. These include but are not limited to functions related to research participant recruiting, pharmacy, nurses, physicians, and other investigators including those at the Maryland Psychiatric Research Center.

Veterinary resources branch provides veterinary care for laboratory animals. This includes not only veterinary medical care but also basal husbandry tasks such as animal feeding and the cleaning of the animal facility. [unreadable] We also provide regulatory guidance to the PIs and SD by supporting the Animal Care and Use Committee, providing consultations to the PIs and training animal users. [unreadable] The branch also facilitates the procurement of animals, breeds transgenic lines of rodents and performs gross necropsies as necessary.

DESCRIPTION (provided by applicant): Parkinson's disease (PD) affects at least 1 million individuals in the US alone. Although much is known about its pathophysiology and information is emerging about its cause, there are no pharmacological treatments shown to have a significant, sustained impact on the prevention of PD or on attenuation of its progress. However, clinical evidence suggests that physical exercise is such a treatment, and this is supported by studies of animal models of the dopamine (DA) deficiency associated with the motor symptoms of PD. Moreover, exercise is a practical and sustainable therapeutic intervention likely to act simultaneously on most if not all of the cellular events capable of protecting DA neurons and restoring DA function. This proposal is designed test the hypothesis that exercise increases DA function and protects DA neurons against toxic insult due in part to increased neurotrophic factor (NTF) signaling, protection of mitochondrial respiration, and stimulation of angiogenesis. An MPTP mouse model will be used to test this hypothesis. Aim 1. To determine the effects of exercise on the impact of MPTP on dopaminergic function (1a) the optimal temporal relationship between exercise and toxin exposure will first be established, thus exploring both protection (exercise before toxin) and rescue (exercise after toxin). (1b) The impact of the optimal exercise paradigm on DA cell loss and on changes in pre- and post-synaptic DA receptors will then be assessed. (1c) Levels of DA and metabolites in striatal tissue and extracellular fluid will be measured in MPTP animals treated with exercise as dictated by Aim 1a. (1d) Mitochondrial respiration will be assessed after MPTP exercise. Aim 2. To assess the role of NTFs in exercise-induced protection members of four distinct NTF families known to protect DA neurons will be examined: GDNF, BDNF, MANF, and VEGF. (2a) The temporal and anatomical profile of NTF changes will be determined after exercise, MPTP, and the optimal combination of MPTP and exercise. (b) The ability of exogenous NTF to mimic the effects of exercise identified in Aim 1 will then be assessed. (2c) The NTFs and/or their receptors suggested by Aim 2a and 2b will be conditionally knocked out to determine if this increases the toxin impact of MPTP and reduces the protective impact of exercise, thereby strengthening the hypothesis of a causal relationship between NTF and exercise-induced protection. Aim 3. To examine the role of angiogenesis in exercise- and NTF-induced neuroprotection, (a) vasculature be assessed in substantia nigra, striatum, and cortex using BrdU to measure new cells and PECAM-1 (CD31), to mark blood vessels. (3b) FITC-conjugated tomato lectin will be used to detect patent blood vessels. (3c) A 3-dimensional analysis of vascular density will be performed. (3d) Finally, expression of angiogenesis-promoting proteins, including angiopoetins 1 and 2, will be measured. These results will provide the basis for the next iteration of a research program focusing on additional mechanisms underlying exercise-induced protection, provide stronger evidence for exercise-induced protection in PD, and establish targets for pharmacological treatment of the condition. PUBLIC HEALTH RELEVANCE: Parkinson's disease affects well over a million individuals in the US alone. Although we know a good deal about how the brain is affected by the disease, there are no pharmacological treatments shown to have a significant and sustained impact on the prevention of the disease or on the attenuation of its progress. Such treatments are urgently needed. Clinical evidence suggests that physical exercise is just such a treatment, and studies of animal models of the dopamine loss associated with the motor symptoms of the condition further support this hypothesis. This study is designed to test that hypothesis. Such a demonstration would not only strengthen the case for advising individuals to engage in exercise, but may also provide insights into drug development for the condition.

The resulting mutant mice showed neuronal hypertrophy, and an increased number of dopaminergic neurons and fibers in the ventral mesencephalon. Interestingly, quantitative microdialysis studies in Pten KO mice revealed no alterations in basal DA extracellular levels or evoked DA release in the dorsal striatum, despite a significant increase in total DA tissue levels. Striatal dopamine receptor D1 (DRD1) and prodynorphin (PDyn) mRNA levels were significantly elevated in KO animals, suggesting an enhancement in neuronal activity associated with the striatonigral projection pathway, while dopamine receptor D2 (DRD2) and preproenkephalin (PPE) mRNA levels remained unchanged. In addition, PTEN inactivation protected DA neurons and significantly enhanced DA-dependent behavioral functions in KO mice after a progressive 6OHDA lesion. These results provide further evidence about the role of PTEN in the brain and suggest that manipulation of the PTEN/Akt signaling pathway during development may alter the basal state of dopaminergic neurotransmission and could provide a therapeutic strategy for the treatment of Parkinsons disease, and other neurodegenerative disorders. PTEN deletion or Akt/PKB activation in dopamine neurons of the ventral midbrain results in remarkable hypertrophy of the substantia nigra and VTA. Our initial characterization of a DAT-PTEN KO strain has provided a clear definition of some of the neuroadaptations in the mesolimbic and nigrostriatal systems, and clearly show dopamine neurotransmission is permanently altered in PTEN KO mice. However, while DAT-PTEN KO animals are viable and appear behaviorally competent, an in depth study of behavioral parameters will clarify if the lack of PTEN interferes with essential functions related to the dopamine system in young, adult and aged animals. Studies performed over the past few years have clearly shown that phenotypes caused by specific genetic modifications are strongly influenced by genes unlinked to the target locus. This problem is exacerbated through the use of Cre-LoxP models as two strains, often containing their own (obscure) genetic backgrounds, are crossed through very specific breeding schedules to generate control and experimental animals. Clearly, it becomes important to avoid the use of a mixed genetic background so complex as to preclude any reasonable use of controls and prevent replication by other investigators. To perform complex behavioral studies, we will use a c57bl/6 congenic DAT-PTEN KO mouse line, generated in our lab. A new mechanism in the brain of rats that may mediate the rewarding and reinforcing properties of drugs of abuse has recently been discovered. This mechanism involves the physical interaction between two proteins in midbrain dopamine neurons, the tumor suppressor PTEN and the brain specific receptor for serotonin, the 5-HT2c receptor (5-HT2cR). Blocking the interaction of PTEN with 5-HT2cR prevents the development of conditioned place preference to nicotine and marijuana. In addition, PTEN has been shown to physically interact with the NR1 subunit of NMDAR in hippocampus, and PTEN downregulation decreases NMDAR surface expression. These studies suggest PTEN in dopamine neurons may directly modulate functions intimately linked to the development of addiction, and dopamine mediated cognition, such as responses to reward and motivation. We will use a congenic PTEN-KO line to analyze in detail the behavioral profile of KO animals in relationship to drug abuse, overall locomotor performance, as well as other dopamine related cognitive functions. We have shown Pten deletion in differentiated DA neurons causes a significant increase in the number and size of surviving neurons in both the mesolimbic and nigrostriatal projecting pathways (see above). Because at the time of Pten deletion DA neurons have already completed mitosis and phenotypic determination, it is unlikely the reported increase in DA neurons is due to an increase in newly formed neurons. It is thus likely PTEN ablation preserves DA neurons that normally would undergo apoptosis due to the lack of target support, by repressing the initiation of apoptotic pathways. We are now intrigued about several aspects induced by PTEN deletion in dopamine cells: Do all dopamine neurons preserved in DAT-PTEN KO animals project to target areas and form functional connections? Can exposure to an enriched environment enhance dopaminergic function in young and aged KO animals? Do the mesolimbic and nigrostriatal dopamine projections remain functional into aging? Is the PTENless aging dopaminergic system more or less resistant to neurotoxic insults applied during different stages of the mouse life span? Are PTENless dopamine neurons prone to form tumors? Obviously, the tremendous adaptations observed in the PTENless dopaminergic system during development, may pose problems for interpretation of results;however, as previous studies have shown Akt/PKB activation in the adult dopaminergic system can also result in remarkable hypertrophy and plasticity of the nigra and VTA, the results obtained in this study may provide valuable insights into how PTEN ablation changes dopamine function at the molecular and behavioral levels, and the long-term consequences of such adaptations. We believe these studies are important, as manipulations of the PTEN pathway are being considered as a possible venue for therapeutic strategies involving the brain.

DESCRIPTION (provided by applicant): Traditional Chinese Medicine (TCM) may provide some novel approaches to Parkinson's Disease treatment that deserve further attention by Western medicine. A prime example is electroacupuncture, a well-known type of TCM that has been used as an alternative therapy in patients with PD with some success. We propose to establish a collaboration among three institutions, Case Western Reserve University (CWRU), the University of Pittsburgh (Pitt), and China Medical University (CMU), to initiate a research program on acupuncture as an intervention for PD. General hypothesis: EA reduces the vulnerability of DA neurons to both environmental and genetic factors by activating gene expression for neurotrophic factors (NTFs) including GDNF, which then reduces the susceptibility of these neurons to ER stress and mitochondrial dysfunction. This R21 has three specific aims. Aim 1: To establish in our U.S. labs a well-characterized model of EA neuroprotection in MPTP-treated mice. In the first experiment we will use C57/b16 mice and a progressive MPTP model. Six groups will be involved: (1) acute MPTP plus 100 Hz EA, (2) MPTP plus 0 Hz acupuncture, (3) MPTP plus EA at a non-effective site, (4) MPTP treatment alone, (5) vehicle-treated mice given EA, and (6) vehicle treated mice plus sham acupuncture. Behavioral, histological and biochemical analysis will be used to assess neuroprotection after 2.5 and 5.5 weeks. Aim 2: To determine the influence of EA on indices of ER stress and mitochondrial function. Using tissue generated in Experiments 1 and 2, we will assess activation of downstream targets of ER stress, protein upregulation of UPR target genes and mitochondrial parameters using biochemical and immunohistochemical methods. Aim 3: To determine if there is an interaction between GDNF availability on the response to EA. Two different groups of GDNF null mutants will be treated acutely with MPTP, subjected to EA or sham treatments, and then EA-induced neuroprotection assessed.

? DESCRIPTION (provided by applicant): Dipeptidyl protease-4 (DPP-4) inhibitors - also known as gliptins - are widely used in the effective treatment of type 2 diabetes to safely regulate bloo glucose levels. DPP-4 is the key enzyme responsible for the metabolism of the endogenous incretins, glucagon-like peptide-like-peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) whose elevated levels in brain, we hypothesized, would provide neurotrophic/neuroprotective actions in cellular and in vivo rodent models of Parkinson's disease (PD). On evaluating several DPP-4 inhibitors, brain and plasma incretin levels were, indeed, substantially elevated in rodents, and this resulted in amelioration of Parkinsonism and elevations in brain dopamine levels in a well- characterized acute rodent PD model as well as reducing toxicity in vitro cellular model. Our proposed studies will extend our evaluation of dipeptidyl protease-4 (DPP-4) inhibitors as a new treatment strategy for Parkinson's disease by assessing the DPP-4 inhibitor sitagliptin in chronic toxin and genetics rodent PD models. In these chronic rodent PD models, we will evaluate neurorestorative activity of sitagliptin by measuring behavioral, biochemical, and immunocytochemical parameters. In addition, mechanistic studies will be carried out to correlate sitagliptin efficacy with analysis of ER stress/unfolded protein responses, mitochondrial function and neuroinflammation.