Parkinson's Disease


Parkinson's Disease is a neurodegenerative disorder characterized by the loss of dopaminergic neurons from the substantia nigra and the presence of proteinaceous aggregates called Lewy bodies in the remaining dopaminergic neurons. These protein aggregates are a marker for cellular stress, implicating several causative sources including proteasome dysfunction, alpha-synuclein accumulation, and mitochondrial dysfunction. Due to the interconnection between these cellular stressors, we propose that each type of dysfunction can initiate and produce similar Parkinson's Disease-like features. The presence of dopamine can contribute to the selective loss of dopaminergic neurons; dopamine is a neurotransmitter that has toxic degradation products and can undergo autoxidation. We propose that the amount of dopamine and its cellular distribution can play a role in the differential susceptibility of the substantia nigra relative to other neuronal populations.

Specific Aim 1: Test the effect of dopaminergic neuron-specific proteasome knockdown.
We will use the promoter of tyrosine hydroxylase, the rate-limiting enzyme in the process of dopamine synthesis, as a tissue-specific enhancer to drive the expression of the tunable GAL4 gene, GeneSwitch. In the nervous system, tyrosine hydroxylase expression is specific to dopaminergic neurons, thus its regulatory sequence will allow us to target dopaminergic neurons with our genes of interest. Using this dopaminergic neuron-specific GeneSwitch fly line, we will express mCD8-GFP to label the cell surface and neuronal projections (Nicola?? et al. 2010), and an mRNA knockdown system (RNAi) targeting proteasomal subunit ??5. Fly survival and climbing ability will be determined and fly brains will be dissected and imaged used confocal microscopy or processed for RT-PCR. We will look for dopaminergic neuron survival, aggregate accumulation, and mitochondrial function.

Specific Aim 2: Compare the effects of proteasome knockdown, alpha-synuclein accumulation, and reduced ATP production in dopaminergic neurons.
Proteasome function, proper protein turnover, and mitochondrial activity are interconnected and due to this interconnection, their independent dysfunction can initiate Parkinson's Disease-like changes. Using the dopaminergic neuron-specific GeneSwitch driver, we will express the transmembrane mCD8-GFP with either wild-type alpha-synuclein or an mRNA knockdown system (RNAi) targeting ATP synthase subunit ??. We will look for dopaminergic neuron survival, aggregate accumulation, and mitochondrial function.

Specific Aim 3: Study the effects of manipulating dopamine levels or cellular dopamine distribution on the survival of dopaminergic neurons.
We will use our Aim 1 fly line to overexpress or knockdown the expression of the following: tyrosine hydroxylase (TH), an enzyme required to produce dopamine; vesicular monoamine transporter (VMAT), a protein required to load dopamine into vesicles; and dopamine transporter (DAT), a protein that sequesters dopamine from the synapse. Changing the level of VMAT should change the distribution of dopamine within vesicles relative to the cytoplasm. Likewise, changing DAT levels should lead to changes in intracellular and extracellular dopamine distribution. Using confocal microscopy, whole brains will be imaged to determine the state of the dopaminergic axons and soma.
Significance
Parkinson's Disease is the second-most prominent neurodegenerative disorder, directly affecting approximately one million people in the United States. In humans, there is overlap of Parkinson's Disease and the most common neurodegenerative disorder, Alzheimer Disease. Parkinson's Disease was initially described over 200 years ago, and while our understanding of its causative factors has greatly improved, our attempts at curing the disease are inadequate. Thousands of individuals die every year and thousands more are diagnosed. One therapeutic approach depends on maintaining levels of dopamine in the substantia nigra, the region of brain that degenerates during Parkinson's Disease. While some symptoms can be alleviated by providing the dopamine precursor or dopamine agonists, or by inhibiting dopamine breakdown, the disease still progresses. Surgical methods have been used to apply deep brain electrical stimulation with symptomatic success, but the underlying mechanisms are still unclear. These two types of treatments are not preventive in nature and don't address the aging-associated changes that lead to Parkinson's Disease. Through our approaches, we hope to establish a model that more accurately mimics the molecular mechanisms of Parkinson's Disease and provides possibilities for therapeutic and preventative treatments.

Innovation
The use of animal models is common in the study of Parkinson's Disease (Sang et al. 2007; Bayersdorfer et al. 2010; Pienaar et al. 2010; Mu??oz-Soriano and Paricio 2011). Most Drosophila melanogaster disease models apply pharmacological approaches (Velentzas et al. 2013) or express disease-specific proteins to reproduce features of the disease (Feany and Bender 2000). We will use the genetic, bipartite, S. cerevisiae-derived tunable GAL4-UAS system (Figure 1) to produce Parkinson's Disease models (Roman et al. 2001; Duffy 2002). The tunable GAL4 line (GeneSwitch) will be specific for dopaminergic neurons and will allow us to regulate system activity by varying RU486 concentration. This approach will avoid the non-specific effects of pharmacological approaches and due to tissue specificity the non-dopaminergic cells will not experience GeneSwitch system activation. Additionally, the Parkinson's Disease models will be developed in a living organism as opposed to dissociated cell cultures. Although the causative nature is not known, it is clear that Drosophila experience aging-associated molecular changes such as proteasome impairment and reduced ATP levels (Vernace et al. 2007). By approaching Parkinson's Disease from the aging process, we are hoping to reproduce a pathologically relevant model of Parkinson's Disease.

Introduction
The cellular proteome is in constant flux, with proteins synthesized, exported, imported, and degraded. The Ubiquitin-Proteasome Pathway (UPP) is the means by which a cell recycles the majority of its protein content (Figure 2). Proteins to be removed are covalently tagged with a chain of polyubiquitin containing at least four ubiquitin molecules, which acts as a means of localizing those proteins to the proteasome. Ubiquitin is activated by E1 ubiquitin-activating enzyme using ATP. The activated ubiquitin molecule is transferred to an E2 ubiquitin-conjugating enzyme before being bound to the target protein via the activity of an E3 ubiquitin ligase. With hundreds of E3 enzymes, specific protein substrates are selectively ubiquitinated and localized to the proteasome.

The proteasome is a protease composed of a 20S catalytic core that lyses proteins and a 19S cap that recognizes, deubiquitinates, unfolds and feeds the target protein into the 20S core. The core contains three unique catalytic activities: caspase-like (??1), trypsin-like (??2), and chymotrypsin-like (??5) (Wang and Maldonado 2006). These catalytic activities were shown to decline with age (Chondrogianni and Gonos 2005), prompting us to investigate the effects of proteasome impairment on the disease state.
The Ubiquitin-Proteasome Pathway (UPP) is required for normal protein turnover and degrades the majority of mutated, misfolded, aberrantly modified, and short-lived proteins located in the cytosol and nucleus (Inobe and Matouschek 2014). UPP genes are implicated in genetic forms of Parkinson's Disease (Davie 2008). Additionally, several UPP proteins are located within Lewy Bodies, which are proteinaceous aggregates characteristic of Parkinson's Disease (Wakabayashi et al. 2013). Pharmacological proteasome inhibition produces protein aggregates and neurodegeneration (McNaught et al. 2004; Vernon et al. 2010; Le 2014). These changes are comparable to the characteristics that define Parkinson Disease (Davie 2008). These observations lead to the conjecture that proteasome dysfunction is central to the development of Parkinson's Disease and related neurodegenerative disorders. Thus, we propose that proteasome knockdown targeted to dopaminergic neurons can initiate and reproduce key features of Parkinson's Disease.
Parkinson's Disease is a multifactor disorder; proteasome impairment, aggregate-prone protein accumulation, and reduced ATP levels are all involved in Parkinson's Disease progression (Lim and Zhang 2013; Wakabayashi et al. 2013; Celardo et al. 2014). Proteasome function is important in degrading the pool of aggregate-prone proteins, like ??-synuclein (Alvarez-Castelao et al. 2014). The UPP also has a role in mitochondrial health by recycling damaged proteins and initiating mitophagy; this process involves Parkin, an E3 ubiquitin ligase whose dysfunction accounts for some familial cases of Parkinson's Disease (Hattori et al. 2014). Mitochondrial health is important in ATP production; the UPP requires ATP (Wang and Maldonado 2006), and through proteasome inactivity, reduced ATP levels can induce aggregate formation. The accumulation of ??-synuclein can overload or block proteasome function and sequester UPP proteins (Martinez-Vicente and Vila 2013)and negatively interact with mitochondrial channel proteins and the electron transport chain (Nakamura 2013); in this manner, the degeneracy comes full circle from any point. We propose that proteasome impairment, aggregate-prone protein accumulation, and reduced ATP levels can independently initiate disorder and through their interconnectivity produce similar Parkinson's Disease-like features.
The loss of dopaminergic neurons from the substantia nigra is a key feature of Parkinson's Disease. The selective degeneration of these neurons may be in part due to their innate susceptibility to aging-associated stress. The main neurotransmitter of these neurons, dopamine, has toxic degradation derivatives and can also undergo autoxidation to produce dopaquinones (Segura-Aguilar et al. 2014). Differences in the regulation of dopamine and dopamine toxicity could explain the differential degeneration of various dopaminergic populations in the brain. Thus, we believe gaining a better understanding of dopamine, dopamine metabolism, and dopamine localization can lead to new therapeutic and preventative approaches to Parkinson Disease. We propose that preemptively reducing or redistributing dopamine can reduce dopaminergic neuron degeneration.
The common fruit fly, Drosophila melanogaster is a useful organism for transgenic studies due to the integration of the GAL4-UAS system and its tunable derivative GeneSwitch (Figure 1) (Roman et al. 2001; Duffy 2002). The system contains both a tissue-specific promoter that drives the expression of the transcriptional activator GAL4 and a GAL4-specific promoter sequence (UAS) upstream of a transgene. When both constructs are present in one organism, the transgene will be expressed in tissues that express GAL4. To avoid targeting dopaminergic neurons during larval development, we will use GeneSwitch, which introduces a steroid-binding domain to GAL4 (Roman et al. 2001), thus limiting its activity in the absence of the ligand and allowing for precise control of expression. A dopaminergic neuron-specific GAL4 line was developed (Figure 3) (Friggi-Grelin et al. 2003), allowing for targeted gene expression in dopaminergic neurons (Figure 4). We plan to apply the same approach in producing a dopaminergic neuron-specific GeneSwitch line.

The GeneSwitch system uses a chimeric transcriptional activator, comprised of the yeast
GAL4 UAS-binding domain, a mutated progesterone receptor ligand-binding domain, and the human Nf-??B p65 activation domain (Figure 1) (Burcin et al. 1999). This chimeric protein is inactive due to the presence of the mutated progesterone receptor; the mutation in this ligand-binding domain only allows binding to the antiprogestin mifepristone (RU486), a synthetic steroid used as an abortifacient and emergency contraception. Once bound by mifepristone, the mutated progesterone receptor allows for DNA-binding and transcriptional activation of target transgenes (Roman et al. 2001).
The GeneSwitch system is imperfect; its activity over time is influenced by several factors, such as genetic background, gender, mifepristone concentration, and age (Poirier et al. 2008). Aging is a two-fold concern: age-associated changes in promoter activity would influence the expression of the chimeric transcriptional activator and changes in food intake over time would influence intracellular mifepristone concentration leading to changes in the activity of the activator. Additionally, the system can experience leaky expression in a spatial and/or temporal manner (Seroude 2002). While certainly problematic, we can avoid the negative impact of these flaws by: comparing specimens of the same genetic background and gender; activating the GeneSwitch system in adulthood, minimizing differences due to early development expression leakage; setting the mifepristone concentration for minimal lifespan impact, thus synchronizing age-associated changes in transgene expression between experimental and control; and focusing most of the data collection on dopaminergic neurons, minimizing the relevance of GeneSwitch activation in undesirable tissues.
The temporal and spatial regulatory mechanisms of tyrosine hydroxylase expression in D. melanogaster are still unclear, but some properties can be inferred from studies done using the fruit fly and other organisms. The development of the successful dopaminergic neuron-specific GAL4 system required the presence of some introns in the gene sequence, in addition to the 5' promoter (Friggi-Grelin et al. 2003)(Figure 3C, Bottom Construct). Research using various mammals revealed the broad array of mechanisms that regulate the TH gene expression and enzyme activity, though differences between species do exist (Kumer and Vrana 1996). The promoter region, which can span thousands of base pairs upstream of the TH gene, contains binding sites for glucocorticoids, calcium, cAMP, and estrogen, among numerous other regulators, at least in the rat (Tekin et al. 2014). NURR1 and PITX3 are transcription factors required for the development of dopaminergic neurons and expression of TH; the TH promoter contains binding sites for these proteins. Supporting the notion of intron requirement for tissue-specific expression, primates contain regulatory elements in their first intron, as well as in certain exons (Lenartowski and Goc 2011). In humans, the sequence 3' to the TH gene is also required for correct expression. Thus, the TH promoter, the gene, and the 3' end would be relevant for proper localization of expression of our GeneSwitch system.
One of our UAS lines contains an mRNA knockdown system against the ??5 subunit of the proteasome (CH, Yeh, Unpublished). The expressed RNA contains domains complementary to the mRNA coding for ??5, a key subunit of the proteasome. Cytosolic processing of this RNA segment will inhibit translation of the ??5 mRNA (Figure 6A). The knockdown of ??5 protein leads to the knockdown of the proteasome levels and activity (Figure 6B,C). Thus a D. melanogaster line with the TH-GeneSwitch gene and the UAS-??5 RNAi gene will lead to proteasome inhibition in dopaminergic neurons in the presence of mifepristone.

Drosophila melanogaster contain less than 300 dopaminergic neurons grouped into 15 clusters, which innervate anatomically and functionally distinct parts of the brain (Mao and Davis 2009). In humans, the dopaminergic neurons of the substantia nigra regulate motor function, motivation, and learning ability (Chinta and Andersen 2005). The dopaminergic system of the fruit fly is involved in regulating motor function (Lima and Miesenbock 2005), learning (Waddell 2010) and behavior (Liu et al. 2008). Thus, the neural dopamine system of D. melanogaster has a homologous role to the dopaminergic neurons of human brains. Using our proposed models for Parkinson's Disease with dopaminergic neuron-specific manipulations, we can study the changes that occur to dopaminergic neurons and motor functionality.
Dopaminergic neurons can be imaged in dissected fly brains by using TH-GAL4 driven GFP expression or anti-TH antibodies with confocal microscopy (Friggi-Grelin et al. 2003; Mao and Davis 2009) (Figure 4 & 7). Drosophila brains can be dissociated (Egger et al. 2013), and dopaminergic neurons can be segregated from the rest of the brain using TH-GAL4 driven GFP expression and FACS (Berger et al. 2012). These dopaminergic neurons can be used for RT-PCR to confirm transgene expression. Using the natural tendency of D. melanogaster to move upwards, called negative geotaxis, the lab has established a climbing assay to determine changes in motor control (Vernace et al. 2007). Additionally, D. melanogaster can be used in alternative motion assays (White et al. 2010), learning experiments (Riemensperger et al. 2005), and behavior studies (Liu et al. 2008).

Research Design
Specific Aim 1: Test the effect of dopaminergic neuron-specific proteasome knockdown.
Friggi-Grelin, et al. (2003) developed a dopaminergic neuron-specific, continuously active GAL4 system; there was no way to control when the system is active or how much the system is activated. We want to avoid targeting dopaminergic neurons during development and we are interested in regulating the level of protein expression or knockdown as induced by our system. We will use the construct design used by Friggi-Grelin, et al. (2003) as the basis for our GeneSwitch fly line, which will allow us to vary the expression of our RNAi construct.
Parkinson's Disease is characterized in part by motor deficits, degeneration of dopaminergic neurons, Lewy bodies and Lewy neurite aggregate formation, mitochondrial dysfunction, and elevated oxidative stress, among numerous other features (Lim and Zhang 2013). We propose that proteasome knockdown in dopaminergic neurons can recapitulate several of these features.
A) In order to target the expression of GAL4 to dopaminergic neurons, Friggi-Grelin, et al. (2003) used the gene sequence for tyrosine hydroxylase. Using this approach, our final construct will contain a GeneSwitch gene flanked by two fragments of the TH gene, a Tyrosine Hydroxylase-GeneSwitch construct (TH-GS) (Figure 8).The flanking arms of our construct will be individually isolated from D. melanogaster DNA using two sets of primer pairs, resulting in a 5' and 3' fragment. The GeneSwitch sequence will be isolated from a pSwitch plasmid, and all three fragments will be amplified using a TOPO plasmid. These three DNA fragments will be ligated into a pCaSpeR vector with the GeneSwitch sequence flanked by the 5' and the 3' TH fragments. The pCaSpeR vector contains a P-element transposon that allows for the insertion of our construct into an embryonic genome. We will use an embryo injection service and the resulting fly lines (TH-GS) will be screened for tissue-specific expression using a membrane-bound UAS-mCD8-GFP line. Segregation analysis will be used to determine the chromosomal location of the GeneSwitch construct.

B) The progeny of interest will contain a UAS-mCD8-GFP gene, the TH-GS driver gene, and the internally developed UAS-??5 RNAi gene (Figure 9). The expression of the UAS genes will be absent without the activation of GeneSwitch. The inducing molecule, RU486 (mifepristone) will be administered during early adulthood to avoid development-associated defects. We will titrate different amounts of RU486 into the fly food and a survival curve will be produced to identify the two extremes: system saturation, when additional RU486 has no additional impact on survival, and the drug level that confers the smallest significant deviation from control values. Though shorter lifespan would expedite experimental cycles, the expected lifespan of D. melanogaster is short enough to allow an extended incubation period.
C) Motor defects will be detected using a climbing assay. We will place ten to twenty flies of one gender into a clear cylinder, and make note of how many make it passed a specific height within a set time limit. The values will be recorded for males and females of different ages in the induced and the control groups. Control groups have the same genetic background, but no RU486-mediated GeneSwitch system activation
D) We will dissect out the adult fly brain, fix it in paraformaldehyde, perform immunostaining using appropriate antibodies, and image them using confocal microscopy. To compare the GFP signal tissue localization, we will use an antibody against TH to mark dopaminergic neurons. The number of fluorescent dopaminergic neurons will be quantified, and their projections will be visualized to look for degeneration. To look for the accumulation of poly-ubiquitinated proteins into aggregates, which are substrates of and normally degraded by the proteasome, we will use an antibody against poly-ubiquitinated proteins.
E) We will need to confirm that our system is knocking down the expression of the ??5 subunit of the proteasome, resulting in reduced proteasome activity. In order to do so, fly brains will be dissected, individual neurons will be dissociated, dopaminergic neurons will be segregated via FACS, and resulting cellular populations will be processed to isolate RNA. Using the RNA isolate, we will perform RT-PCR, and test RNAi efficacy with primers for ??5 mRNA, as this should be reduced in the mRNA knockdown system. Additionally, dissected fly brains will be immunostained for the ??5 subunit and imaged using confocal microscopy. In order to determine proteasome activity, dissected brains will be treated with the quenched fluorogenic peptide TED (Urru et al. 2010) and imaged live using confocal microscopy (Wu and Luo 2006). The peptide is preferentially cleaved by the ??5 subunit of the proteasome.
F) Dissected brains will be plated and imaged live for general oxidative stress and for changes in mitochondrial membrane potential. Oxidative stress will be determined using the CellROX system (Liu et al. 2014), which washes cells with a membrane-permeable molecule that changes fluorescent wavelength emission in the presence of reactive oxygen species. Membrane potential will be determined using a JC-1 visual assay (Kumar et al. 2008). JC-1 is lipophilic, will make its way to the mitochondria and change its fluorescent emission wavelength depending on the mitochondrial membrane potential.
Expected Outcomes
The goal of this aim is to provide evidence in support of our hypothesis that proteasome knockdown can reproduce features of Parkinson's Disease. Thus, we fully expect these results: motor deficits; the loss of dopaminergic neuron synapses, axons, and soma; aggregate formation; enhanced oxidative stress; reduced mitochondrial membrane; and reduced ATP levels.
Anticipated Problems and Alternative Strategies
It is not guaranteed that our construct will yield flies with acceptable expression patterns of GeneSwitch with regard to dopaminergic neurons. The insertion site might play some role in the efficacy or spatiotemporal pattern of GeneSwitch expression. In order to account for that possibility, we will collect several independent fly insertion lines. Additionally, we will produce 5' extended and 3' extended versions of the construct to incorporate more enhancer sequence.
The work of this aim depends on the development of the tunable GAL4 driver, TH-GS. If this work does not come to fruition, we can switch to the constitutively active TH-GAL4 driver, as developed by Friggi-Grelin, et al. (2003). The GAL4-UAS system is active during D. melanogaster development, which would require working with larval tissue; just like adult tissue, larva brains can be dissected, imaged, dissociated, segregated, and processed for biochemical assays. Alternatively we can switch to other tissue-specific GeneSwitch lines, such as ELAV-GS, which would target all neurons; this would allow us to compare differential susceptibility of different neuronal populations in the same brain.

Specific Aim 2: Compare the effects of proteasome knockdown, alpha-synuclein accumulation, and reduced ATP production in dopaminergic neurons.
Parkinson's Disease is a multifactor disorder, and due to the communication between these factors during health and disease, we believe that the original source of dysfunction is varied for each patient and capable of producing similar disease characteristics. The three factors we will compare are proteasome dysfunction, accumulation of unfolding-prone proteins, and reduced ATP production. The first model will express wild type alpha-synuclein in dopaminergic neurons; alpha-synuclein is prone to unfolding and a common component of Lewy bodies and Lewy neurites (Yasuda et al. 2013). A dopaminergic neuron-specific mRNA knockdown system against ATP synthase subunit ?? will be used to limit the ability of mitochondria to produce ATP. This subunit is important in converting the hydrogen ion gradient into energy required to form ATP molecules.
A) Progeny containing the transgenes TH-GS, UAS-mCD8-GFP, and UAS-??-synuclein or UAS-ATP Synthase ?? RNAi (Figure 9) will be treated with varying amount of the GeneSwitch-inducing drug mifepristone (RU486). The drug concentration that produces a survival curve that is not significantly different from that of 1B flies will be used as the basis for comparison. This will allow for similar disease incubation periods. Alternatively, the drug concentration that produces the longest living flies with survival significantly different from control will be used.
B) We will use a climbing assay to determine at what age and to what extent motor dysfunction affects the flies. We will insert ten to twenty flies of one gender into a clear cylinder, and make note of how many make it passed a specific height within a set time limit.
C) Dissected fly brains will be fixed, immunostained, and imaged using confocal microscopy. An anti-TH antibody will be used to confirm a dopaminergic neuron-specific GFP signal. The state of the neurons can be viewed to determine the condition of projections and soma, as well as how many dopaminergic neurons are in each cluster. An anti-poly-ubiquitinated protein antibody will allow us to determine if these tagged proteins are accumulating and aggregating in the cell.
D) In order to confirm that our GeneSwitch systems are functional, we will use the mCD8-GFP signal and FACS to isolate dopaminergic neurons from a pool of dissociated fly brains tissue. We will process the FACS-sorted cells for RNA, perform RT-PCR, and test GeneSwitch efficacy with primers for ??-synuclein mRNA or ATP Synthase ?? mRNA. The mRNA coding for ??-synuclein should only be present in the induced sample; the mRNA coding for ATP Synthase ?? should decline due to the activity of the RNAi system. Protein levels will be determined using fixed, dissected brains and antibodies against ??-synuclein or ATP Synthase ??.
E) General oxidative stress and changes in mitochondrial membrane potential will be determined using confocal microscopy and live imaging of dissected brain samples. The CellROX system for oxidative stress and the JC-1 visual assay for mitochondrial membrane potential work on a similar basis; cells are washed with substances that change emission fluorescence wavelength when exposed to an environment that deviates from the norm.
F) In order to determine relative proteasome activity, dissected brains will be treated with the quenched fluorogenic peptide TED (Urru et al. 2010), and imaged live using confocal microscopy. The peptide is preferentially cleaved by the ??5 subunit of the proteasome.
Expected Outcomes
To obtain support for our hypothesis that proteasome impairment, ??-synuclein expression, and reduced ATP levels produce similar Parkinson's Disease-like features, we expect similar outcomes to Aim 1. We anticipate some differences between each fly line, potentially significantly different from each other, but we also expect these values to be significantly different from control samples.
Anticipated Problems and Alternative Strategies
In case of a delay in the production of the dopaminergic neuron-specific GeneSwitch driver, we can use the constitutively active TH-GAL4 driver. Alternatively, the experiments can be modified for more general neuronal analysis with the pan-neuronal driver ELAV-GS.
The purpose of the UAS responder lines is to induce different, but interconnected sources of dysfunction; for the purpose of our work, the loss of ATP can be replaced with other sources of mitochondrial dysfunction. The ATP Synthase ?? RNAi line can be swapped for any other Electron Transport Chain (ETC) RNAi. Alternatively, we could use mutant members of the ETC that mishandle electrons to produce oxidative stress (Chaturvedi and Flint Beal 2013). Superoxide dismutase, which protects the cell against oxidative stress, can be targeted to elevate levels of reactive oxygen species (Yan et al. 2013). Alternatively, proteins that promote general mitochondrial health, such as PCG1-?? (Chaturvedi and Flint Beal 2013), can be knocked down, leading to mitochondrial dysfunction. The expression of wild type ??-synuclein can be exchanged for its more aggregate-prone mutants (Yasuda et al. 2013).

Specific Aim 3: Study the effects of changing dopamine levels or localization on the survival of dopaminergic neurons.
The presence of dopamine is one of the defining features of dopaminergic neurons and due to its propensity to form toxic metabolites, dopamine might play a role in the selective degeneration of dopaminergic neurons (Park et al. 2007). The goal of this aim is to use cell survival and degeneracy as a readout for dopamine toxicity. The fly driver line will be the dopaminergic neuron-specific GeneSwitch system. To regulate the levels of dopamine, we will target the enzyme performing the rate-limiting step in the synthesis of dopamine, tyrosine hydroxylase (TH), by using overexpression (UAS-TH) and knockdown (UAS-TH RNAi) responder lines. In order to regulate the levels of dopamine in vesicles relative to the cytoplasm, we will target the vesicular monoamine transporter (VMAT) using UAS-VMAT and UAS-VMAT RNAi. To control the uptake of dopamine from the synapse via the dopamine transporter (DAT), we will use UAS-DAT to increase intracellular dopamine and UAS-DAT RNAi to reduce intracellular dopamine.
A) Each fly line will contain the dopaminergic neuron-specific driver, TH-GS, UAS-GFP, and one additional UAS responder (Figure 9). Based on the work of Aims 1 and 2, we will use a matching concentration of the inducing drug RU486 to maintain a long incubation period. Alternatively, a few broad titration points will be selected and an optimal drug concentration for all experiments will be determined by balancing results and incubation period. Fly survival will be determined with each transgene combination.
B) As a specific whole-organism approach to determining toxicity of each dopaminergic modification, we will assay motor functionality by testing climbing ability. We will insert ten to twenty flies of one gender into a clear cylinder, and make note of how many make it passed a specific height within a set time limit. This assay will allow us to determine if changes in dopaminergic neuron survival are significant on a whole-organism scale.
C) We anticipate that manipulating dopamine levels and distribution can induce neurodegeneration. In order to determine if there are any changes to total dopaminergic neurons, their soma and their axons, we will dissect fly brains, fix them in paraformaldehyde, immunostain them with an antibody against tyrosine hydroxylase and image the brains using confocal microscopy.
Expected Outcomes
The treatments that increase dopamine production, vesicle loading, and synapse reuptake might enhance climbing ability at earlier time points in comparison to same-age controls. The flies experiencing the opposite scenarios should have worse climbing ability. With regard to dopaminergic neuron survival, we expect treatments that enhance cytoplasmic dopamine would result in the degeneration of dopaminergic neurons. As such, we also expect increased vesicle loading to be least problematic on a cellular and whole organism scale. Additionally, mildly reduced dopamine production might be effective as well due to the required threshold for the appearance of motor deficits in the development of Parkinson's Disease (Meissner et al. 2003).
Anticipated Problems and Alternative Strategies
As with Aims 1 and 2, this aim hinges on the development of the dopaminergic neuron-specific GeneSwitch driver. In order to study dopamine toxicity, we will need to be able to target the neurons. Some alternatives include the constitutively active TH-GAL4 driver and the constitutively active DDC-GAL4 driver, which targets serotonergic and dopaminergic neurons. In the situation where only one or two of the responder lines produce positive results, we would apply the techniques used for Aims 1 and 2 to find a mechanism to explain those results. For example, if a fly line experienced enhanced climbing ability followed by below average ability later in life in addition to a reduced number of dopaminergic neurons, we will use live cell imaging to look for an early sign of oxidative stress, as this might implicate the autoxidation of dopamine as a causative factor in neurodegeneration.

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