Paul M. Carvey, Ph.D. - US grants

Dopaminergic drugs used in the treatment of schizophrenia and Parkinson's disease (PD) are associated with complications which often compromise otherwise efficacious drug therapy (i.e., tardive dyskinesia (TD) and loss of efficacy, respectively). Using a novel approach for studying these drugs, we have demonstrated that cell-free striatal extracts from animals chronically treated with dopaminergic drugs alter the growth of mesencephalic cell cultures containing dopamine (DA) neurons: DA antagonist treatment enhanced, while DA agonist treatment reduced, the effect striatal extracts had on culture growth. These data suggest that drug-sensitive striatal-derived neurotrophic or neuroinhibitory factors influence the growth of DA neurons in culture. These studies have led to an operational hypothesis which states that "the growth promoting effect cell-free striatal extracts have on mesencephalic cultures is inversely related to DA tone." We intend to test this hypothesis directly using the traditional approach of varying the dose and duration of treatment with dopaminergic drugs (haloperidol and levodopa) in well established behavioral models (behavioral hypersensitivity and 6-hydroxydopamine rotation). DA biochemistry, spiroperidol receptor density, as well as stereotypical and rotational behavior alterations induced by these drug treatments will represent the dependent measures of DA tone. A highly sensitive low cell density culture system will be used to identify drug therapies which induce alterations in striatal growth promoting activity (GPA). Whether or not cultures using immunocytochemical stains and cell counts of DA and GABA neurons, as well as astrocytes. The effect this GPA has on neuron sprouting in the cultures will be evaluated by assessing DA and GABA uptake. The dependent measures of DA tone will then be used to predict GPA (defined as cell counts and/or transmitter uptake) using multiple regression analysis. Identifying a statistically significant inverse relationship using this equation will directly support the operational hypothesis. This analysis will take two years to complete. In the third year of the proposal, other dopaminergic drugs with differing pharmacological profiles and clinical activities will then be evaluated using this model system. These analyses will further support or refute the operational hypothesis as well as begin to address the potential relationships between drug treatment and side effects. If these drug sensitive growth factors were to influence DA neuron growth in vivo in a similar fashion (as our preliminary results appear to suggest), the resulting alterations in synaptic architecture could contribute to the complications which attend dopaminergic drug therapy. Thus, increased DA neuron growth associated with DA antagonist treatment could contribute to TD while reduced DA neuron growth associated with DA agonist therapy could contribute to the progression of PD. The results of this proposal would therefore be expected to influence the current therapeutic strategies employed in the treatment of schizophrenia and PD.

Dopaminergic drugs used in the treatment of schizophrenia and Parkinson's disease (PD) are associated with complications which often compromise otherwise efficacious drug therapy (i.e., tardive dyskinesia (TD) and loss of efficacy, respectively). Using a novel approach for studying these drugs, we have demonstrated that cell-free striatal extracts from animals chronically treated with dopaminergic drugs alter the growth of mesencephalic cell cultures containing dopamine (DA) neurons: DA antagonist treatment enhanced, while DA agonist treatment reduced, the effect striatal extracts had on culture growth. These data suggest that drug-sensitive striatal-derived neurotrophic or neuroinhibitory factors influence the growth of DA neurons in culture. These studies have led to an operational hypothesis which states that "the growth promoting effect cell-free striatal extracts have on mesencephalic cultures is inversely related to DA tone." We intend to test this hypothesis directly using the traditional approach of varying the dose and duration of treatment with dopaminergic drugs (haloperidol and levodopa) in well established behavioral models (behavioral hypersensitivity and 6-hydroxydopamine rotation). DA biochemistry, spiroperidol receptor density, as well as stereotypical and rotational behavior alterations induced by these drug treatments will represent the dependent measures of DA tone. A highly sensitive low cell density culture system will be used to identify drug therapies which induce alterations in striatal growth promoting activity (GPA). Whether or not cultures using immunocytochemical stains and cell counts of DA and GABA neurons, as well as astrocytes. The effect this GPA has on neuron sprouting in the cultures will be evaluated by assessing DA and GABA uptake. The dependent measures of DA tone will then be used to predict GPA (defined as cell counts and/or transmitter uptake) using multiple regression analysis. Identifying a statistically significant inverse relationship using this equation will directly support the operational hypothesis. This analysis will take two years to complete. In the third year of the proposal, other dopaminergic drugs with differing pharmacological profiles and clinical activities will then be evaluated using this model system. These analyses will further support or refute the operational hypothesis as well as begin to address the potential relationships between drug treatment and side effects. If these drug sensitive growth factors were to influence DA neuron growth in vivo in a similar fashion (as our preliminary results appear to suggest), the resulting alterations in synaptic architecture could contribute to the complications which attend dopaminergic drug therapy. Thus, increased DA neuron growth associated with DA antagonist treatment could contribute to TD while reduced DA neuron growth associated with DA agonist therapy could contribute to the progression of PD. The results of this proposal would therefore be expected to influence the current therapeutic strategies employed in the treatment of schizophrenia and PD.

Results form early clinical trials suggest that fetal nigral transplants may offer an exciting new therapeutic strategy for the treatment of Parkinson's disease (PD). However, tissue loss associated with the storage of aborted fetal tissue prior to transplantation, inadequate knowledge about the impact of patient age and/or stage of disease on graft survival, and the lack of a continuous source of tissue for transplantation are among the factors that compromises an otherwise straight forward surgical procedure. Recent work in our laboratory suggests that dopamine (DA) neuron survival can be increased by "preconditioning" DA neurons with trophic molecules during storage. We have also shown that trophic activity in the rat striatum is reduced by aging and increased in direct proportion to the degree of DA denervation. These data suggest that patient age and Parkinsonian stage may have a significant impact on the trophic environment into which grafted tissue is transplanted. In Specific Aim 1 we will therefore systematically evaluate the effect of supplementing storage media with trophic molecules (bFGF, BDNF, GDNF, EGF), antioxidants (U83836E and U74389G), and tissue extracts (striatal, mesencephalic, placental serum). Rat mesencephalon will be stored for up to 5 days under these conditions and then co-cultured with striatal cells. DA neuron survival in these cultures will be evaluated by assessing K+-evoked DA release and tyrosine hydroxylase immunoreactive (THir) cell counts. Optimal storage conditions will be further evaluated by implanting the "enhanced" cells into unilaterally lesioned (6OHDA) rats. Specific Aim 2 will focus on the effects the transplant recipients age and degree of DA denervation has on graft survival. Enhanced cells will be implanted into the striata of 2,9, 17, and 22 month old rats and THir cell counts in the implant site will be compared across groups. Using the intraventricular 6OHDA model, 17 month old rats will be bilaterally denervated 35,50,75, and 90% and transplanted with enhanced cells. THir cell counts will again be compared across groups. Although optimizing fetal tissue storage conditions and identifying the age and degree of DA denervation that maximizes DA neuron survival is likely to have an immediate, positive impact on transplant programs currently under way, these Aims do not address the issues surrounding access to a continuous source of DA neuron enriched tissue. In this regard, we have recently shown that a natural, pluripotent, progenitor cell line can be derived from fetal and adult mesencephalon. Co-culture with freshly harvested mesencephalon converted many of these cells into the DA neuron phenotype whereas known trophic factors did not. Many of these cultures were highly enriched with THir cells. Specific Aim 3 will therefore focus on the future use of the pluripotent progenitor cell as a continuous passage; 2-optimizing and identifying and identifying the crude components of the mesencephalon responsible for its conversion to the DA phenotype; and 3- grafting the progenitor cells in 6OHDA lesioned rats. The successful implementation of the Specific Aims of this proposal addresses several practical aspects of transplantation that could be readily transferred to the clinical setting.

DESCRIPTION (provided by applicant): We previously demonstrated that pups born to gravid females treated with the bacteriotoxin lipopolysaccharide (LPS) were born with fewer than normal dopamine (DA) neurons. We have extended these studies out through 16 months and showed that rats exposed to LPS prenatally exhibit progressive loss of DA neurons (46%) associated with increased DA activity, elevations in the proinflammatory cytokine tumor necrosis factor (TNFalpha), aggregates of alpha-synuclein, and Lewy-like bodies. We have recently received funding from NINDS to characterize this new animal model of Parkinson's disease (PD) through 22 months. However, the mechanism(s) responsible for this DA neuron loss during development are currently unknown. Recent preliminary data (Real-time RT-PCR) suggests that the ratio of pro-/anti-inflammatory cytokine mRNA is increased. In specific aim 1 we will therefore assess both mRNA and cytokine protein (ELISA) content for TNF-a, interleukin (IL) -113, IL-6, transforming growth factor (TGFbeta), and IL-6 in the mesencephalon, striatum, and cerebellum (control region) to determine if prenatal LPS alters transcriptional and translational control of these factors during development or early postnatal life (P 1-21). Since Nurr-1, sonic hedgehog, Ptx-3, fibroblast growth factor-8 (FGF8) and glial cell line derived neurotrophic factor (GDNF) are transcriptional and neurotrophic factors, respectively, that regulate the development of the DA neuron phenotype, we will assess mRNA and protein levels of these factors in Specific Aim 2 to determine their involvement in the LPS-induced reduction in DA neurons. Our preliminary data further suggests that LPS induces increases in TNF-alpha in microglia and we will assess the specific roles of both microglia and astrocytes in the LPS effect in Aim 3. The results of these studies will not only compliment our long-term studies, but will test the hypothesis that prenatal LPS permanently alters genes that regulate the development of the DA neuron and affect its phenotype during adult life. The results of these studies will also provide a unique opportunity to determine if prenatal neurotoxin exposure can permanently alter genes that increase the risk of neurodegenerative disease in later life.

Convention suggests that the blood brain bar ier (BBB) prevents certain drugs and neurotoxins from gaining access to dopamine (DA) neurons in patients with Parkinson's disease (PD) unless a specific transport mech nism exists. However, we showed in animal, in vitro, and autopsy studies that the BB is dysfunctional in models of PD and patients. Animal and in vitro studies suggest d that products of activated microglial were responsible for this dysfunction. Ifbarri r function is compromised, the brain will be exposed to elements in the peripheral vasc lature. We hypothesize that BBB dysfunction williead to increased exposure 0 brain parenchyma to DA neurotoxins in the blood as well as peripheral immune syste mediators (T cells and monocytes) that will contribute to disease progression. Aim l'"II evaluate progressive DA neuron loss in mice with pre-existing DA lesions (induced b MPTP and LPS) for evidence of an increased entry of a tritium labeled, systemic By administered, DA neurotoxin (MPP+), and immune mediators. Aim 2 will assess fra tions from activated microglia (cell line and microglia isolated from DA lesioned mic for effects on endothelial cell (EC) monolayers (human cell line and mouse prim ry cultures) and changes in transport of DA toxins as well as transmigration of immu e mediators. These studies will also identify protein as well as ultrastructural cha ges (EM studies) in the tight junctions that create the BBB. We anticipate these stud es demonstrating that neuroinflammatory-mediated events affect p tein structure in tight junctions disrupting the BBB that leads to entry of peri heral vascular elements that contribute to further DA neuron loss and disease progressi n. These findings will systemat ically demonstrate, for the first time, that barrier d sfunction occurs in animal models of PD and identify potential mechanisms for this dy function. These findings would provide an entirely new hypothesis for progression pa hogenesis and identify new targets for therapeutic intervention in PD designed arou d affecting BBB integrity.