Un J. Kang, Md - US grants

The ultimate purpose of this proposal is to study the fundamental molecular mechanism of aging brain and neurodegenerative disorders. The olfactory system will be used as a model system of aging because 1) the olfactory system shows changes in aging and neurodegenerative disorders such as Alzheimer's diseases; 2) it has ideal anatomic segregation of afferent inputs for detailed anatomical work; and 3) the recycling nature of its peripheral afferent receptor cells makes the study of neuronal interactions quite feasible. Aging rat and mouse brain will be studied to investigate the changes in neurotransmitter levels and transmitter synthesizing enzyme activities, correlating with previously noted morphological changes in the olfactory system. If changes in neurotransmitter phenotypes are noted, further characterization of the regulation of neurotransmitter gene expression will be undertaken by immunotitration for the amount of enzyme protein molecules, Northern and dot blot analyses for quantitation of corresponding mRNA levels, and in vitro transcription assay, if the changes are sufficiently large. Afferent regualtion of neurtransmitter phenotype in the olfactory cells will also be studied at the level of gene expression and its change with aging will be investigated. This will be the first attempt to study the gene expression in aging brain at the fundamental level by use of specific probes. The immediate focus of the proposal preceding the above project will be the basic science training period. The candidate will acquire basic metholology and understanding of biochemcial techniques, molecular biological principles, and modern anatomic methodologies through on-going laboratory projects in the sponsor's laboratory. The development of independent research program will allow the candidate to intergrate it with his clinical training in neurodegenerative disorders of aging.

DESCRIPTION: This Proposal will address mechanistic aspects of gene therapy in experimental models of Parkinson's disease (PD) and explore ways to optimize and regulate catecholamine delivery in genetically modified cells initially in vitro. Attempts to alleviate end product inhibition will use mutated tyrosine hydroxylase (TH) molecules. The stability of Th protein expressed in non-neuronal environment as well as ways to improve that stability will be explored. Once biochemical issues are resolved, the principal investigator will generate a construct using the tetracycline-regulatable promoter (tet off). Both non-neuronal (fibroblasts) and neural precursor cell lines will be utilized, the latter of which the principal investigator hyprothesizes will provide a better environment to support expression of neuronal molecules and better properties in vivo after grafting in terms of integration and dispersion into the host brain. The efficacy of these approaches will be demonstrated biochemically by in vivo microdialysis and by following a non-drug-induced spontaneous behavior that reflects the skinesia of PD better than traditional drug-induced rotational behaviors.

DESCRIPTION (provided by applicant): L-3,4-dihydroxyphenylalanine (L-DOPA) is the mainstay of therapy for Parkinson's disease (PD). Chronic L-DOPA therapy is limited, however, by the development of motor response complications, such as progressively shorter duration of improvement in akinesia (wearing-off) and the appearance of L-DOPA-induced abnormal involuntary movements. Innovative methods of sustained and localized central nervous system (CNS) dopamine delivery may further optimize L-DOPA therapy. Such methods are being explored clinically by CNS transplantation studies with fetal dopaminergic neurons and experimentally by neuronal stem cell implants and gene therapy. Our studies during the past funding cycles have defined optimal sets of genes necessary for dopamine replacement using ex vivo gene therapy using genetically modified fibroblasts. We also developed rat behavioral models that are relevant to the akinesia of PD patients. Using akinesia behaviors, we have noted that lesion severity has a major influence on the shortening of the response duration with minor contribution by the chronic intermittent L-DOPA therapy. Therefore, studies proposed in this continuing renewal application will determine the optimal parameters of gene therapy to improve akinesia and minimize and prevent motor response complications. We will use adeno-associated virus vectors to deliver tyrosine hydroxylase and guanosine triphosphate (GTP) cyclohydrolase 1 genes. The optimal combination of anatomical targets for gene therapy to improve akinesia will be defined by examining the effects of gene therapy delivered to basal ganglia structures, such as subthalamic nucleus, substantia nigra par reticulata, that receive dopaminergic inputs, in addition to the striatum. The optimal timing to initiate dopamine replacement gene therapy to forestall development of motor response complications will also be examined. These results will have significant implications beyond dopamine replacement gene therapy proposed here and guide other therapies such as fetal dopaminergic cell transplantation, neurotrophic factor therapy, stem cell therapy, and other CNS targeted delivery systems.

DESCRIPTION (provided by applicant): While multiple etiologies are likely to account for Parkinson's disease (PD), the core pathogenic feature is degeneration of dopaminergic neurons, particularly those in the substantia nigra pars compacta (SNpc), with shared common final pathways involving oxidative damage, mitochondrial dysfunction, or both. Therefore, one may hypothesize that dopaminergic neurons in the SNpc are selectively vulnerable to oxidative stresses and/or mitochondrial disruption and understanding the mechanism of this selectivity may reveal the pathogenesis. However, our data show that ventral mesencephalic dopaminergic neurons in culture have an enhanced antioxidant capacity, as they are better able to resist oxidative stresses such as glutathione depletion and peroxide treatment than nondopaminergic neurons. In addition, their enhanced antioxidant capacity is reflected in lower reactive oxygen species (ROS) and higher reduced glutathione levels than nondopaminergic neurons. We hypothesize that an enhanced antioxidant capacity is essential for the survival of dopaminergic neurons that may be subjected to increased oxidative stress exerted by dopamine and its metabolites. We postulate that disruption of this innate antioxidant capacity makes them vulnerable to additional environmental insults and thereby leads to selective degeneration. We noted that the enhanced antioxidant capacity in ventral mesencephalic dopaminergic neurons is due to tetrahydrobiopterin (BH4), which is the cofactor for tyrosine hydroxylase, the enzyme producing dopamine, but also lowers superoxide levels, partly be direct scavenging effect and modulates mitochondrial function. First, We will study the effect of BH4 on mitochondrial bioenergetics and function including initiation of death pathways. Second, we will examine the role of BH4 on NO and superoxide generation and in modulating other endogenous antioxidant systems. Third, the neuroprotective function of BH4 against PD models such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, rotenone toxicity, and glutathione depletion will be tested in vivo and in organotypic slice cultures, using hph-1 mice that are deficient in BH4, production.

DESCRIPTION (provided by applicant): Loss-of-function mutations in DJ-1 were recently identified in an autosomal recessive form of Parkinson's disease (PD). The role of DJ-1 in normal and pathological states is still unclear, and evidences for its putative functions have been quite controversial. The role of DJ-1 in protecting neurons from oxidative stress is particularly informative in understanding a potential common mechanism for cell death in this genetic form of Parkinson's disease and in sporadic forms of Parkinson's disease which is thought to result from oxidative stress generated by mitochondrial dysfunction. Although DJ-1 deficient cells are more susceptible to oxidative stress, DJ-1 does not seem to have direct scavenging effect of reactive oxygen species (ROS). Instead, DJ-1 may work on a downstream pathway from ROS. Specifically, DJ-1 may be important in ubiquitination and DJ-1-null mouse brain attenuated increase in ubiquitination in response to oxidative stress. DJ-1 has been also shown to have chaperone function. DJ-1 changes into an acidic form under oxidative stress. To investigate the pathogenesis of Parkinson's disease caused by DJ-1 deficiency, we have successfully generated DJ-1-null mice. Our preliminary studies indicate that DJ-1-null mice develop age-dependent motor deficits associated with alteration of dopamine neurotransmission, consistent with hyperactive dopamine transporter (DAT) function. Based on these observations, we hypothesize that DJ-1 facilitates ubiquitination and subsequent degradation of target proteins such as DAT and this function is activated by oxidative stress. We propose the following experiments to test these hypotheses and identify the target molecules and pathways in which DJ-1 plays a role to maintain optimal dopamine transmission and cell survival. Aim 1. To investigate whether dopamine neurons in DJ-1-null mice are more vulnerable to aging and paraquat challenge. We will examine the effect of aging and paraquat treatment in DJ-1-null mouse for its biochemical, behavioral and anatomical properties. Aim 2. To investigate whether DJ-1 influences the ubiquitin proteasomeal pathway under oxidative stress. We will examine the total ubiquitinated proteins levels and compare the relative levels of specific substrate proteins implicated in dopaminergic neuronal death in DJ-1-null and WT brains. In addition, we will examine how the effect of DJ-1 on ubiquitin-proteasome system alters DAT function by measuring its surface expression and ubiquitination. Aim 3. To investigate the chaperone function of DJ-1 and its oxidation dependent cooperation with ubiquitin pathway. Relevance Understanding the neuroprotective function of DJ-1 may lead to rational therapy for Parkinson's disease to prevent the manifestation or progression. Since Parkinson's disease leads to significant disability in a significant portion of the elderly population, such findings will have a significant impact in public health.

DESCRIPTION (provided by applicant): Parkinson's disease (PD) is a debilitating disorder resulting in severe motor dysfunction caused by progressive degeneration of the substantia nigra dopaminergic neurons. L-dihydroxyphenylalanine (L-DOPA) therapy alleviates the motor symptoms, however the utility of this agent for chronic treatment is limited due to the occurrence of abnormal involuntary movements known as dyskinesia. An understanding of how L-DOPA modulates signaling pathways in the striatum of PD is important in devising an effective treatment for L-DOPA induced dyskinesia (LID). Among the signaling molecules associated with LID is extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK) whose activation in medium spiny neurons has been associated with induction of LID. Preliminary results from our laboratory have shown that ERK is mainly activated in striatal cholinergic neurons with long-term administration of L-DOPA and is correlated with expression of LID. The objective of the proposed studies is to determine the functional significance of ERK activation in striatal cholinergic neurons with respect to the expression of LID. We will examine this issue in aphakia mice, a genetic model of nigrostriatal degeneration that expresses LID, as well as in established unilateral neurotoxin- lesion based models. The proposed experiments are focused on the following objectives: 1) Correlation of the temporal expression of dyskinesia with ERK activation in striatal cholinergic neurons; 2) Characterization of the DA receptor subtype and intracellular signaling pathways linked to L-DOPA-induced ERK activation in striatal cholinergic neurons and medium spiny neurons; and 3) Determining the functional outcome of ERK activation in cholinergic neurons with respect to neuronal excitability and cholinergic phenotypic expression. A better understanding of cell signaling mechanisms involved in LID will facilitate identification of potential targets for the treatment of PD.

? DESCRIPTION (provided by applicant): In Parkinson's disease (PD), degeneration of dopaminergic neurons leads to symptoms including slowness of movement, rigidity, tremor, and gait abnormality. The dopamine (DA) precursor levodopa (L-DOPA) dramatically alleviates motor symptoms, but can cause side effects such as L-DOPA-induced dyskinesia (LID) after prolonged treatment, thereby limiting its therapeutic use. A critical mediator of motor deficit in PD and excessive movement in LID is the striatal medium spiny neurons (MSNs), which form the sole output of the striatum and receive DA input under normal conditions. The lack of DA input in PD, and the excessive, non-physiological DA input after chronic L-DOPA leads to abnormal MSN function and contribute to slowness of movement and LID respectively. The classical model of basal ganglia (BG) proposes that MSNs can be divided into two segregated pathways: i) D1-MSNs, which express dopamine D1 receptors (D1Rs) and form the direct pathway, targeting only the output nuclei of BG; ii) D2-MSNs, which express D2 receptors (D2Rs) and form the indirect pathway, targeting the output nuclei of BG indirectly via the external globus pallidus (GPe). However, subsequent anatomical studies found that axons from D1-MSNs form collaterals that also project to the GPe. These bridging collaterals thus allow D1-MSNs to influence the targets of both direct and indirect pathway. We recently found that the terminal density of these bridging collaterals is highly plastic and changes in response to alterations in DA signaling. D-1MSN bridging collateral is reduced by chronic D2R blockade but is almost doubled by D2R overexpression. Importantly, changes in bridging collateral density dramatically alter D1-MSN's influence on motor output. While optogenetic stimulation of D1-MSNs normally increases locomotor behavior, increasing bridging collaterals by D2R overexpression caused the same stimulation to inhibit locomotor behavior. This motor inhibition was reversed back to motor activation by normalizing bridging collateral terminal density via chronic D2R blockade. Experiments proposed here will test the hypothesis that bridging collaterals are also altered by abnormal DA signaling in PD and LID. We will use transgenic mice that express green fluorescent protein (GFP) under the control of the Drd1a promoter, which allows the axonal projections of D1-MSNs to be visualized by immunohistochemistry against GFP. The terminal density of D1- MSN bridging collateral can therefore be determined by GFP staining intensity in the GPe. DA denervation by 6-hydroxydopamine will be used to induce hemi-parkinsonism. Limb-use bias will be used to assess akinesia, and LID will be induced by chronic L-DOPA treatment. We propose that DA depletion will progressively reduce bridging collateral terminal density, and that it would parallel the progressive ability of a D1R agonist to reverse akinesia. Furthermore, we propose that chronic L-DOPA treatment, which causes increases in LID severity, will be associated with further alterations in the terminal density of bridging collaterals. The identification of abnormal bridging collateral changes in PD will justify future development of therapy to reverse these changes.

? DESCRIPTION (provided by applicant): Dopaminergic therapy in Parkinson's disease (PD) is the most successful example of rationale treatment approach addressing neurotransmitter deficit in neurodegenerative disorders. However, it is limited by motor fluctuations including dyskinesia that develops over several years of treatment. It is not clear if disease progression or treatment is the major factor in producing L- DOPA-induced dyskinesia (LID), but clinical and experimental evidences point to contributions of age of onset, disease severity, and chronic dopaminergic drug exposure. We have recently reported that elevated cholinergic signaling may be a major contributor to LID. Repeated L- DOPA administration in parkinsonian mice produces LID, which is associated with hyperexcitability of striatal cholinergic interneuron (ChI) evidenced by extracellular signal- regulated kinase (ERK) activation and enhanced response of ChI to dopamine. Moreover, the expression of LID was partially attenuated by preventing ERK activation or a muscarinic receptor antagonist. Ablation of ChI dramatically reduces LID in a mouse model of PD created by 6-OHDA lesion. To define the role of ChI further, we will utilize a novel method of selectively activating or suppressing ChI by Designer Receptor Exclusively Activated by Designer Drug (DREADD) system using transgenic mice expressing Cre in ChI and adenovirus-mediated delivery of floxed construct of DREADD to the striatal ChI. We will determine the role of ChI in LID development and expression separately. We will characterize cellular mechanisms of ChI hyperactivity associated with LID. Finally, we will examine the effect of ChI on the excitability of the medium spiny neuron that is the major striatal output neuron.

Project Summary/Abstract Dopaminergic therapy in Parkinson?s disease (PD) is the most successful example of rationale treatment approach addressing neurotransmitter deficit in neurodegenerative disorders. However, it is limited by motor fluctuations including dyskinesia that develops over several years of treatment. It is not clear if disease progression or treatment is the major factor in producing L-DOPA-induced dyskinesia (LID), but clinical and experimental evidences point to contributions of age of onset, disease severity, and chronic dopaminergic drug exposure. We have recently reported that elevated cholinergic signaling may be a major contributor to LID. Repeated L-DOPA administration in parkinsonian mice produces LID, which is associated with hyperexcitability of striatal cholinergic interneuron (ChI) evidenced by extracellular signal-regulated kinase (ERK) activation and enhanced response of ChI to dopamine. Moreover, the expression of LID was partially attenuated by preventing ERK activation or a muscarinic receptor antagonist. Ablation of ChI dramatically reduces LID in a mouse model of PD created by 6-OHDA lesion. To define the role of ChI further, we will utilize a novel method of selectively activating or suppressing ChI by Designer Receptor Exclusively Activated by Designer Drug (DREADD) system using transgenic mice expressing Cre in ChI and adenovirus-mediated delivery of floxed construct of DREADD to the striatal ChI. We will determine the role of ChI in LID development and expression separately. The outcome of this experiment would indicate fundamentally different approaches, either as a prophylaxis to prevent LID development or for symptomatic control of LID expression once it has already developed. We will then characterize cellular mechanisms of ChI hyperactivity associated with LID by examining gene expression changes, morphological alterations and electrophysiological properties. Multidisciplinary approaches will provide us necessary insights and tools to devise therapeutic approaches to LID.

ABSTRACT In Parkinson's disease (PD), degeneration of dopaminergic (DA) neurons leads to profound motor impairment. Although motor symptom is initially treatable by the DA precursor levodopa (L-DOPA), patients experience dis- abling motor fluctuations, including a shortened duration of action for L-DOPA, only partially treated with pharmacological means and deep brain stimulation. Preventing L-DOPA's declining effectiveness will greatly improve patients' quality of life and reduce social cost. Emergence of disabling motor fluctuation is associated with the decline of a component of L-DOPA's antiparkinsonian response, known as the long duration response (LDR). The LDR is a long-lasting motor improvement that persists long after L-DOPA plasma level has re- turned to baseline, gradually decaying over many hours to days after discontinuation of L-DOPA. Motor fluctuation may be caused by LDR declining too rapidly, and treatments that halt LDR decay may prevent mo- tor fluctuations. However, the mechanism underlying LDR is currently unknown. Using two distinct motor tasks, we recently found that both induction and decay of LDR is task-specific, requiring the pairing of task exposure with L-DOPA (for LDR induction) or with L-DOPA withdrawal (for LDR decay). These results point to associa- tive learning and neuroplasticity as the underlying mechanism. Furthermore, indirect pathway medium spiny neurons (iMSNs) are activated by LDR decay, and D2 receptor (D2R) knockout greatly slowed LDR decay. Based on the above results and previous findings that i) iMSN activation suppresses movement and may nor- mally function to inhibit competing responses; ii) iMSNs undergo aberrant long-term potentiation (LTP) when DA depleted, we will test the hypothesis that gradual motor impairment during LDR decay results from aberrant LTP in specific ensembles of iMSNs that are normally suppressed during normal movement by D2R stimula- tion, but become pathologically active during task exposure if DA is depleted. Using Drd2-EGFP mice to label iMSNs, we will first examine whether L-DOPA-rescued motor performance vs. LDR decay activate different iMSN ensembles in the same mouse: we will tag task-activated iMSN ensemble at the 1st time point using a Fos-promoter driven, doxycycline-gated fluorophore, then tag task-activated iMSN ensemble at the 2nd time point using endogenous Fos labeling, and compare their co-localization. We will then examine whether this ?incorrect? iMSN ensemble activated during LDR decay (visualized by a Fos-driven fluorophore) has synaptic input changes that are consistent with the occurrence of LTP. Finally, we will use Fos-driven opsins to bi- directionally modulate this ?incorrect? iMSN ensemble to show its causal, pathological role: that its activation leads to task-specific motor impairment, and its inhibition recues impairment task-specifically. By demonstrat- ing the existence of, and the role of, pathological iMSN ensembles in task-specific motor impairment in PD, and by identifying the form of aberrant neuroplasticity that leads to their recruitment during movement, these experiments will form a basis for future studies to develop novel treatments to halt or reverse LDR decay and motor fluctuation.

PROJECT SUMMARY L-DOPA-induced dyskinesias (LID) are common and significant complications of dopaminergic therapy for the treatment of Parkinson's disease (PD). LID arises from complex compensatory changes throughout the basal ganglia, including to the substantia nigra pars reticulata (SNr), a primary output structure. The loss of dopamine (DA) in PD is thought to increase SNr activity, which would inhibit motor thalamus output. Additionally, DA loss causes changes in neuronal firing patterns, including increased burst firing and oscillatory and synchronous activity, in nearly all basal ganglia nuclei; whether changes in firing rate or firing pattern serve as the neurological substrate for PD symptoms or LID expression remains highly debated. To address firing rate, we directly modulated SNr activity with chemogenetics in a unilateral 6-OHDA lesion PD-LID mouse model and found that akinesia was improved by either increasing or decreasing SNr firing rate. By contrast, LID was inhibited by increasing SNr firing, while decreasing the SNr firing rate exacerbated LID from threshold doses of L-DOPA. These data indicate akinesia and LID are mediated dissociable aspects of SNr activity and lead us to propose that akinesia is improved by disrupting firing patterns in the SNr, supporting the abnormal firing pattern model. By contrast, LID is inhibited by counteracting the decrease in SNr firing rate after doses of L-DOPA, supporting the firing rate model. Using the same approach, we directly increased the firing rate of the parafascicular nucleus (Pf), a target of the GABAergic output of the SNr. This also blocked LID expression, suggesting that increasing SNr activity could block dyskinesia by paradoxically increasing Pf firing. However, the activity of SNr neurons and the effect of SNr activity on thalamic targets has not been measured in awake, behaving animals during LID. In this proposal, we will 1) characterize the activity of the SNr and its thalamic targets in awake, behaving animals during states of DA depletion and LID expression; 2) define the connectivity and effect of direct modulation of SNr firing (conditions that alter akinesia and LID) on the activity of SNr target thalamic nuclei (parafascicular, ventrolateral, and reticular thalamic nuclei); and 3) determine the thalamic nuclei at which GABA release from SNr terminals is sufficient to block LID. As LIDs are major limiting factors for symptomatic control of PD, completion of this project and the successful elucidation of the pathological processes that underlie LID expression would advance the development of rationally targeted therapies that extend the efficacy of current drugs and potentially improve the quality of life of those with PD.