Parkinsons Disease The Second Largest Neurodegenerative Disorder Biology Essay

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Parkinson's disease is the second largest neurodegenerative disorder. The disease is characterized by tremor, rigidity and bradykinesia (Hughes et al., 2002).

It has been proposed that Parkinson's disease is caused by the misfolding of proteins that lead to accumulation and aggregation of Lewy bodies which contain α-synuclein and ubiquitin. This lead to researchers currently proposing that there is a potential use for heat shock proteins. Heat shock proteins are known to act as protein folding machinery and assist ubiquitin-proteasome system to aid the decomposition of abnormal proteins. Transgenic animal models expressing Parkinson's disease have shown that induction or overexpression of certain heat shock proteins such as Hsp70 can alleviate α-synuclein aggregation and accumulation hence alleviate the symptoms of Parkinson's disease.

Therefore, I aim to investigate the role of further chaperones such as Hsp100, Hsp22 and Hsp60, which can potentially alleviate the pathological characteristic of Parkinson's disease in Drosophila models. The GAL4/UAS system is a technique used to study gene expression and function in animal models such as the Drosophila. I will be using GAL4/UAS system to drive the expression of the different chaperone in the transgenic Drosophila. I propose to evaluate the efficacy of different chaperones to alleviate Parkinson's disease pathology. Also, I will be examining the potential role of combining heat shock proteins to test whether it is more effective as a treatment.

Background to the project:


Parkinson's disease is the second largest neurodegenerative disorder. It affects more than 1% of 55 year olds and more than 3% of those over the age of 75 years (Bilen and Bonini, 2005). Parkinson's disease is characterized and diagnosed by symptoms of Parkinson's disease. These include tremor, rigidity and bradykinesia. Rigidity is characterised by the resistance to passive limb movement, muscle stiffness and postural instability, and bradykinesia is characterised by slow movements and a delayed stop and start. Also, patients may experience non-motor symptoms which include cognitive decline, pain, sleep disturbances, depression and visual hallucinations (Hughes et al., 2002). Moreover, imaging techniques such as Positron Emission Tomography (PET) or Single Photon Computed Emission Tomography (SPECT) support the diagnosis but this is not available for clinical purposes and usually limited to researchers. These techniques work by using radioactively labelled ligands of the pre-synaptic dopaminergic neurons.

The pathological characteristic of Parkinson's disease is the degeneration of dopaminergic neurons in the substantia nigra (Luo et al., 2006) with Lewy body accumulation and aggregation in intact dopamine neurons. Lewy bodies are intracytoplasmic inclusions composed mainly of α-synuclein and ubiquitin (Braak and Braak, 2000). It has been shown that a loss of 60% of the dopaminergic neurons are required before symptoms appear in patients, until then intact neurons compensate for the loss (Braak et al., 2004). In the past, the presence of Lewy bodies was required to confirm Parkinson's disease in patients (Bilen and Bonini, 2005). However, discovery of new subtypes of Parkinson's disease, for example, patients with PARK2 mutation have shown an absence of Lewy bodies and yet still developed Parkinson's disease. This means that presence of Lewy bodies alone cannot explain Parkinson disease but in major cases sporadic and familial Parkinson's disease the presence of Lewy bodies is the main feature.


Most cases of Parkinson's disease are sporadic with a late onset of the disease and no known cause (idiopathic). Environmental toxins have been shown to be a risk factor and increase the chances of Parkinson's disease. These toxins include agrochemicals, pesticides (Chaudhuri et al., 2007) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Braak et al., 2004). MPTP have been used in mice models to generate the pathology of the disease. It is now known that Parkinson's disease is a result of genetic mutation for about less than 5% of the cases and a number of different genes have been implicated. Currently, researchers have identified mutations in PARK1‐11 genes to be linked to Parkinson's disease (Dawson and Dawson, 2003). Moreover, these mutations have generally been linked to dysfunction of the ubiquitin-proteasome system, oxidative stress and inflammation.

PARK1 codes for a protein called α-synuclein and many researchers believe that it plays a important role in regulating synaptic vesicle relevant to dopamine storage. Moreover, it has been shown that stress can increase the α-synuclein protein aggregation and Lewy body formation (Luo et al., 2006). The misfolding of the α-synuclein can change impair proteasome mediated protein degradation, proteasome composition and alter protein synthesis (Luo et al., 2006; Chen et al., 2005).

Individuals with Parkinson's disease who have a mutation in α-synuclein have similar clinical and pathological characteristics to those with idiopathic Parkinson's disease, including the presence of Lewy bodies (Polymeropoulos et al., 1997; Celotto and Palladino, 2005).

In studies using animal models, it has been shown that over-expression of the normal α-synuclein is sufficient to cause Parkinson's disease. This suggests that the more common sporadic cases of Parkinson's disease may also be triggered by alterations in α-synuclein production. In addition, recent studies have reported that mutation in α-synuclein is a risk factor for sporadic disease (Farrer et al., 2004; Ibanez et al., 2004). Genetic studies have revealed that two mutations of A30P and A53T in α-synuclein may be a possible cause for Parkinson's disease (Polymeropoulos et al., 1997; Zourlidou et al., 2004).

However, studies which aimed to determine the mRNA expression of α-synuclein in sporadic cases have given contradictory results to the current view. A study conducted by (Neystat et al., 1999) showed that the α-synuclein was actually diminished in the substantia nigra but not in the cortex of patients with Parkinson's disease compared with a control group. Other studies suggests that over-expression of α-synuclein is actually protective (Manning-Bog et al., 2003) and that reduced expression levels are associated with Parkinson's disease (Cantuti-Castelvetri et al., 2005). However, these results are not in line with most studies currently undertaken and the current view that α-synuclein is found accumulated in the Lewy bodies in patients with Parkinson's disease is still the main view.

Another gene implicated in Parkinson's disease is PARK2 which codes for a protein called parkin. Parkin is an E3-specific ubiquitin ligase, which is required for the transfer of ubiquitin to specific target proteins for their degradation (Tsai et al., 2003). The loss of parkin potentially causes the build up of selective target proteins. Patients with parkin mutations do not have Lewy bodies, and these patients develop a syndrome that closely resembles the sporadic form of Parkinson's disease. However, the patients tend to develop symptoms at a much younger age (Bilen and Bonini, 2005). Furthermore, Kalia and her colleagues (2004) showed that the bcl-2-associated athanogene 5 (BAG5) can enhance dopaminergic neuronal death in a vivo model of Parkinson's disease by inhibiting the activity of the E3 ligase activity and hsp70. This suggests the importance of the gene parkin in humans and that mutations can cause loss of dopaminergic neurons (Kalia et al., 2004).

Another gene is PARK6 which is also known as PTEN-induced putative kinase 1 (PINK1) which codes for a protein called Serine/threonine-protein kinase. Mutations in PARK6 gene is characterised by early onset of the Parkinson's disease, and a slow progression of the disease (Park et al., 2006).

PARK7 which is another gene implicated in Parkinson's disease codes for a protein called DJ-1. DJ-1 is thought to play a role in oxidative stress and mitochondrial injury. PARK7 has been shown to cause protein aggregation and neuronal cell death in patients (Luo et al., 2006). Moreover, mutations have shown to make the protein less stable, prevent synthesis, or promote degradation through the ubiquitin-proteasome pathway, thereby reducing the amount of protein present, thus increasing oxidative stress (Bonifati et al., 2003).

Moreover, mitochondrial dysfunction has also been implicated in the cause of Parkinson's disease. Patients with a specific variant of the NADH complex I enzyme had a significantly lower risk of getting Parkinson's disease than patients who had the most common form of the enzyme (van der Walt et al., 2003). This suggests that variations in complex I proteins is an important risk factor for Parkinson's disease.


It is important to realise that Parkinson's disease is fatal and currently there is no cure for it. However, the main treatment for Parkinson's disease consist of replacing dopamine, which is most commonly done by giving patients a drug called levodopa which is a precursor of dopamine. It is given along with carbidopa which is a DOPA decarboxylase. It is given alongside since levodopa can cause side effects such as vomiting and nausea by acting on dopamine receptors in the peripheral nervous system. Combining carbidopa allows activation of levodopa only in the central nervous system so improves symptoms of Parkinson's disease while limiting the side effects. Levodopa does however, lead to motor fluctuations and dyskinesia which is uncontrollable movement especially in the upper portion of the body in patients when used over a long period of time. Other medications include anticholinergics, selegiline, and amantadine which are also currently used to replace dopamine in the brain. Dopamine agonists, inhibitors of catechol-O-methyltransferase (COMT) or monoamine oxidase-B (MAO-B) can also be used to treat Parkinson's disease (Olanow and Stocchi, 2004; Hristova and Koller, 2000). Individuals with Parkinson's disease may also benefit from physical, occupational, or speech therapy and finally surgical procedures such as deep brain stimulation of the subthalamic nucleus, pallidotomy or fetal stem cell transplant to the caudate nucleus to restore dopamine neurons is also used. However, surgical procedures are only recommended to patients as a last resort when drugs such as levodopa lose their efficacy or other therapies have shown little success (Smeding et al., 2005).

It is clear more research is required to find better treatments for patients with Parkinson's disease and possibly a cure. Although current treatments provide some relief from the disease we need better treatments especially for the sporadic cases, to decrease Lewy body accumulation in the substantia nigra. Recently, some success has come from using chaperone proteins especially Hsp70 to reduce aggregation of the Lewy bodies (Luo et al., 2006; Dedmon et al., 2005; Hartl, 1996). It would be worthwhile to examine whether other chaperones also reduce the pathology of Parkinson's disease.

Furthermore, heat shock proteins can easily be tested using Drosophila models. Animal models such as Drosophila are important to better our understanding of Parkinson's disease and discover new treatments (Betarbet and Greenamyre, 2007; Betarbet et al., 2002). Drosophila have been used to study mechanisms of a range of neurodegenerative diseases in the past including Alzheimer's disease, Huntington's disease and Parkinson's disease (Lu, 2009; Feany and Bender, 2000). Although mice and rat models offer excellent in vivo opportunities and are extensively similar to the human brain, studies based on Drosophila are less time-consuming and more cost efficient (Marsh and Thompson, 2006). Moreover Drosophila allow excellent genetic manipulation and in vivo readouts of the pathology. The pathways are considered generally highly conserved with vertebrates, with about 75% of human genes known to be associated with disease having a Drosophila homologue (Betarbet and Greenamyre, 2007; Betarbet et al., 2002; Marsh and Thompson, 2006). Therefore, I propose experiments using Drosophila models to study Parkinson's disease for potential use of chaperones as therapy.

There are four approaches that have been employed successfully to study neurodegeneration in Drosophila. The first one is the forward genetic screens, which have been carried out to identify genes that when mutated can cause degeneration of the brain. For example, the Drosophila mutants such as swisscheese, and drop-dead, the mammalian homologues of some of these genes caused neurodegeneration when mutated (Rogina et al., 1997; Akassoglou et al., 2004). The second one is transgenic over-expression, which has been used to model diseases caused by a toxic gain-of-function mechanism such as over-expressed α-synuclein in Drosophila using GAL4/UAS system (Feany and Bender, 2000). The third one is the genetic inhibition of an endogenous gene, which has been used to model the subset of familial diseases transmitted in a recessive fashion, which are likely to be caused by a loss-of-function (Pesah et al., 2004). Transposon-mediated mutagenesis, transgenic RNA interference (RNAi), and homologous recombination-based gene knockout can be used for this purpose such as co-expression of Pael-R, a substrate of human PARK2 with RNAi knockdown of endogenous Drosophila parkin has shown an enhanced loss of dopaminergic neurons (Pesah et al., 2004; Yang et al., 2003). Finally the fourth one is a pharmacological approach, which can be used to model neurodegenerative diseases and to test candidate therapeutics in animals (Chaudhuri et al., 2007; Lu, 2009). For example, Chaudhuri and his colleagues (2007) exposed Drosophila to environmental factors such as paraquat or H2O2 to see if this increased the development of Parkinson's disease (Chaudhuri et al., 2007).

Drosophila Models of Parkinson's disease:

A number of models have been developed to study Parkinson's disease on Drosophila.

Alpha-synuclein (PARK1):

Drosophila does not have a clear α-synuclein homologue yet, over-expression of the wild-type and the mutant human α-synuclein in Drosophila give rise to key features of Parkinson's disease, including Lewy body-like formation, degeneration of dopaminergic neurons, and climbing defects indicating abnormal locomotor behaviour (Feany and Bender, 2000; Auluck and Bonini, 2002; Auluck et al., 2002). The fact that both mutant and wild-type α-synuclein form aggregates in Drosophila neurons and cause Parkinson's disease supports the idea that improper disposal of aggregated abnormal proteins can result in Parkinson's disease (Lu, 2009).

Parkin (PARK2):

The key feature of Drosophila with mutated parkin is a defective mitochondrial morphology and apoptotic muscle degeneration. In addition, these mutant Drosophila exhibit sterility, reduced lifespan, reduced cell number and size, increased sensitivity to oxidative stress and loss of dopaminergic neurons (Lu, 2009). Loss-of-function of the parkin gene in Drosophila causes increased sensitivity to oxidative stress and abnormal wings (Feany and Bender, 2000). This suggests that mutations in Parkin may have toxic property (Lu, 2009).

Pink1 (PARK6):

Inhibition of Drosophila Pink1 (dPink1) results in similar characteristics to that seen in a Parkin mutant, including male sterility, apoptotic muscle degeneration, dopaminergic neuron loss, defective mitochondrial morphology, and hypersensitivity to oxidative stress (Park et al., 2006).

Moreover, over-expression of Parkin in Drosophila, rescued the Pink1 mutant characteristics, while over-expression of Pink1 had no effect on Parkin mutant characteristics. Furthermore, Pink1 Parkin double mutants showed characteristics identical to those observed in either of the single mutants. These observations suggest that Pink1 and Parkin function in the same pathway, with Parkin acting downstream of Pink1 (Park et al., 2006) Pink1 has also recently been shown to directly phosphorylate Parkin and regulate its mitochondrial localization (Park et al., 2006), providing a possible molecular explanation for their genetic relationship (Lu, 2009).

DJ-1 (PARK7):

In Drosophila, DJ-1α is the homologue of human DJ-1. DJ-1α knockout display altered sensitivity to environmental toxins such as paraquat, H2O2, and rotenone (Menzies et al., 2005). Moreover, DJ-1α Drosophila showed an accumulation of H2O2, increased sensitivity to oxidative stress, and dysfunction and degeneration of dopaminergic neurons (Lu, 2009; Menzies et al., 2005). Again this is showing that DJ-1 gene may play an important role in pathology of Parkinson's disease.

Overview of chaperones:

Heat shock proteins have recently become a focus in Parkinson's disease, because the pathology of this disease is the Lewy body formation and protein misfolding. It is suggested that heat shock proteins may serve as a protein folding system, which works together with the ubiquitin-proteasome system to help decompose abnormal proteins. Failure of ubiquitin-proteasome system is thought to play role in the Parkinson's disease. In addition, it is thought that heat shock proteins have anti-apoptotic effect and control dopamine neurons against stress conditions (Luo et al., 2006).

The heat shock proteins are a large protein family with different classes of proteins classified according to their molecular weight, these include Hsp70, Hsp40, and Hsp27. Different classes of heat shock proteins play a diverse role in protein assembly, folding and translocation. Regulation of these heat shock proteins creates a unique defence system to maintain cellular protein homeostasis and to ensure survival of our cells (Luo et al., 2006; Hartl, 1996; Hightower, 1991). Heat shock proteins also protect major pathways in the brain by reducing oxidative stress and mitochondrial dysfunction, and prevent ubiquitin-proteasome system impairment (Luo et al., 2006; Hartl, 1996; Hightower, 1991). The level of heat shock proteins is decreased with increasing age, thus it becomes more difficult to keep the cellular protein homeostasis, which may be the reason why neurodegenerative diseases such as Parkinson's disease affects more people as the age increases (Luo et al., 2006; Hartl, 1996).

In the α-synuclein model of Parkinson's disease, Drosophila over-expressing wild-type α-synuclein were found to have about 50% of dopaminergic neuronal loss compared to normal Drosophila. When hsp70 was co-expressed at the same time as α-synuclein a complete maintenance of normal numbers of dopaminergic neurons was seen. It is suggested that the expression of Hsp70 altered the neurotoxicity of α-synuclein. Immunoblot analysis confirmed that the levels of α-synuclein were not altered. Yet, Hsp70 reduced the toxicity of α-synuclein to dopaminergic neurons (Luo et al., 2006; Lu, 2009; Auluck et al., 2002).

The fact that human hsp70 protected Drosophila dopamine neurons from α-synuclein toxicity, and was present in the inclusions, raised the possibility of an interaction between endogenous chaperone activity and α-synuclein (Bilen and Bonini, 2005; Dedmon et al., 2005). Moreover, post-mortem studies carried out on patients with Parkinson's disease suggested that altered chaperone activity may be involved in progression of Parkinson's disease (Auluck et al., 2002).

Studies done in vitro and in vivo models of Parkinson's disease have shown that increasing the expression of heat shock proteins especially hsp70 by gene transfer or heat shock protein inducers such as valproic acid and radicicol (Pan et al., 2005) reduce the amount of abnormal protein misfolding. Moreover, heat shock proteins inhibit apoptotic pathway to reduce the loss of dopaminergic neurons. Thus, research on heat shock proteins provides us with a promising therapy for Parkinson's disease (Luo et al., 2006).

There has been some success with a drug called geldanamycin. It works by binding to an ATP site on hsp90 and blocks its function (Luo et al., 2006; Waza et al., 2006). Recent studies looking at the function of hsp90 indicate its potential therapy for Parkinson's disease (Luo et al., 2006). However, in case of hsp90, inhibiting the chaperone is suggested. This is because studies have shown that inhibiting hsp90 leads to an increase in hsp70 and prevents α-synuclein aggregation and toxicity in cells in cultures. Increasing the expression of any chaperones in the brain might not have a positive impact in reducing the toxicity of α-synuclein. As some chaperones such as Hsp90 need to be blocked in order to reduce the toxicity of α-synuclein (Falsone et al., 2009).

Treatment with geldanamycin resulted in complete protection against α-synuclein toxicity. It was seen that it gave similar protection just like hsp70 (Auluck and Bonini, 2002; Auluck et al., 2002). Geldanamycin and hsp70, both protect against α-synuclein toxicity despite the continued presence of Lewy body−like inclusion. Geldanamycin did not reduce the expression of α-synuclein in Drosophila, instead geldanamycin protected against the toxicity of α-synuclein in dopaminergic neurons (Auluck and Bonini, 2002; Auluck et al., 2002). Although studies suggest that geldanamycin acts by up-regulating or modulating molecular chaperone activity, the drug may also modulate other chaperones such as hsp90. Regardless, these studies revealed that geldanamycin can fully protect against the toxicity of α-synuclein to dopaminergic neurons in Drosophila models (Auluck and Bonini, 2002).

Hsp90 is now thought to be the main chaperone that has been implicated in the negative regulation of α-synuclein aggregation in dopaminergic neurons (Luo et al., 2006; Dedmon et al., 2005; Waza et al., 2006). Moreover, microglia which plays a key role in the inflammation of brain tissues have to shown to express high levels of hsp90 following excitotoxic lesion in the mouse hippocampus. The protective function of hsp90 can be very important since inflammation evoked by microglia may increase the risk of Parkinson's disease (Luo et al., 2006; Waza et al., 2006). Geldanamycin is thought to function by interfering with hsp90 activity, which normally functions as a negative regulator of the heat shock transcription factor that mediates hsp70 and hsp40 expression (Celotto and Palladino, 2005; Auluck and Bonini, 2002).

Furthermore, geldanamycin does not alter the level of hsp70 expression, suggesting that geldanamycin acts only to increase levels of chaperone in stressed cells and does not alter the activity of chaperone in neighbouring, healthier cells. It is currently believed that using geldanamycin induces specific heat shock proteins expression and this in turns reduces the pathology of Parkinson's disease (Luo et al., 2006; Waza et al., 2006).

Another heat shock protein suggested to play a role in accumulation of α-synuclein is hsp40. HDJ-1 is one of the human homologues of hsp40. Researchers using MPTP exposure in mice increased α-synuclein mRNA expression. MPP+ is the active metabolic product in MPTP (Fan et al., 2006; Gomez-Santos et al., 2002). Fan and his colleagues (2006), found that heat shock proteins were able to suppress MPP+ induced α-synuclein mRNA expression, increase protein ubiquitination and increase proteasome activity, therefore decreasing the amount of α-synuclein protein aggregation. In cells with α-synuclein, over-expression of HDJ-1 can significantly reduce α-synuclein aggregation (Fan et al., 2006).

Another chaperone is the hsp27. It has been shown that over-expression of hsp27 protects against apoptotic cell death triggered by various stimuli such as oxidative stress. Various mechanisms have been proposed to account for such an anti-apoptotic activity. Hsp27 could increase the antioxidant protection of cells by decreasing reactive oxygen species in cell and has also been shown to interfere with caspase activation upstream of the mitochondria (Parcellier et al., 2003). The precise mechanism of how hsp27 mediates its protective effect in various situations is very complex and not completely clear (Zourlidou et al., 2004).

Researchers have only recently started to for therapeutic use for heat shock proteins in Parkinson's disease. There have been contradicting studies. Some researchers suggest hsp70 to reduce aggregation of the Lewy bodies (Luo et al., 2006; Dedmon et al., 2005; Hartl, 1996). Another study suggests that hsp27 has a protective role while hsp70 does not. Therefore, it would be worthwhile to examine whether chaperones previously invested and future chaperones reduce the pathology of Parkinson's disease.

Hypothesis and objectives of the project:

The identification of certain heat shock proteins as protective agent against Parkinson's disease presents itself as a potential form of therapy. However, it is not clear which heat shock proteins confer the best protection. Hence I propose to survey further of their activity. I aim to investigate the role of different chaperones which can possibly alleviate Parkinson's disease pathology associated with several of the Drosophila Parkinson's disease models. I propose to evaluate the efficacy of different heat shock proteins in a variety of Parkinson's models. These include Hsp100, Hsp90, Hsp83, Hsp70, Hsp68, Hsp64, Hsp60, Hsp40, Hsp27, Hsp26, Hsp23 and Hsp22. Also, it would be interesting to note, what will happen if all the chaperones are expressed. The intended outcome is to identify chaperones most effective at alleviating the pathology in Parkinson's disease generated via the different causes and to inform future work in developing therapy regardless of whether the cause is sporadic or due to a mutation.

To do this I am going to use Drosophila models which express wild- type α-synuclein and use it as a control to test the chaperone expressing Drosophila models. I will note the Lewy body-like accumulation and aggregation, number of degenerated dopaminergic neurons, locomotor behaviour by looking at the climbing behaviour of the Drosophila, sterility, lifespan, and sensitivity to oxidative stress.

I will then repeat this procedure using different Parkinson's models, these include parkin, pink1, DJ-1 and the mutant forms of α-synuclein A30P and A53T. This will provide further information on which heat shock protein provides best protection against the different models. As mentioned earlier, it is currently suggested that hsp70 alleviates the pathology of Parkinson's disease by reducing the neurotoxicity of α-synuclein, but we do not know if hsp70 alleviates the pathology of Parkinson's disease in the parkin model (Auluck et al., 2002). This is what I intent to find out, whether I can generalise the effects of heat shock proteins to all Parkinson's disease cases regardless of the cause or if different heat shock proteins have different efficacy for the different Parkinson's disease models

Since it is known that heat shock proteins do not work on their own, it will be interesting to look at how combinations of heat shock proteins will affect model of Parkinson's disease. And this is what I intend to do after I have tested the individual heat shock proteins against their control model.

Plan of work and anticipated outcome:

This work will be performed by using the GAL4/UAS system to drive expression of the chaperones in differences Drosophila models of Parkinson's disease. The GAL4/UAS system is a technique used to control gene expression and function in animal models such as the Drosophila. The GAL4 gene encodes the yeast transcription activator protein Gal4, and the Upstream Activation Sequence (UAS) is a short section of the promoter region, to which Gal4 specifically binds to activate gene transcription. I will examine whether expression of the chaperones alleviates the Parkinson's disease pathology seen in the various models to identify how these chaperones compare in their activity to those previously investigated and to test which heat shock proteins are most effective against pathology of the disease.

To carry this out, I am going to cross transgenic Drosophila expressing the different chaperones, for example hsp100 with the Drosophila models modelling the Parkinson's disease. The UAS/GAL4 system will allow me to drive expression of the candidate chaperones and examine their effect on the pathology of Parkinson's disease model.

To create the transgenic Drosophila expressing each of the chaperone, the DNA encoding the coding sequence for each chaperone will be cloned into the pUAST vector (Marsh and Thompson, 2006; Brand and Dormand, 1995; Brand and Perrimon, 1993). PCR primer will be designed to allow the amplification of the chaperones sequence from cDNAs obtained from the Drosophila Genome Resource Centre ( The primers will incorporate the restriction sites EcoR1 and Xba and at their 5' and 3' ends respectively. The sticky ends of the cDNAs of the chaperones will combine itself to the pUAST sticky ends. Once the chaperones have been cloned into the pUAST the constructs will be sent to Bestgene ( to generate the transgenic Drosophila containing each heat shock proteins (Marsh and Thompson, 2006; Brand and Dormand, 1995; Brand and Perrimon, 1993).

Once the transgenic Drosophila expressing all the different heat shock proteins are made, I will cross them with the available Drosophila models. I will request wild-type human α-synuclein and both the mutant forms of α-synuclein, A30P and A53T from Mel B. Feany from Harvard Medical School, USA (Feany and Bender, 2000; Auluck and Bonini, 2002). The parkin model will be requested from Graeme Mardon from Baylor College of Medicine, USA (Pesah et al., 2004). The Pink1 models will be obtained from Jongkyeong Chung from Korea Advanced Institute of Science and Technology, Korea (Park et al., 2006) and the DJ-1 models will be obtained from Kyung-Tai Min from National Institutes of Health, Maryland (Menzies et al., 2005).

Once all the transgenic Drosophila are available to me, I will make cross the wild-type human α-synuclein with the Drosophila expressing ddc-GAL4 obtained from Bloomington Drosophila Stock Center at Indiana University ( to drive expression of α-synuclein. This procedure will be repeated for the other Parkinson's disease models. These Drosophila will form my control group and I will take note of the mean value of their Lewy body-like accumulation and aggregation, number of degenerated dopaminergic neurons, locomotor behaviour by looking at the climbing behaviour of the Drosophila, sterility, lifespan, and sensitivity to oxidative stress, (Figure 1) (Marsh and Thompson, 2006; Brand and Dormand, 1995; Brand and Perrimon, 1993).

Next, I will see the effect of each chaperone on the α-synuclein model. I will then take transgenic Drosophila containing the UAS α-synuclein and cross it with transgenic Drosophila containing the pUAST Hsp100 obtained from Bestgene. The offspring express UAS α-synuclein/pUAST Hsp100 which is then further crossed with transgenic Drosophila containing a tissue-specific promoter fused to the yeast ddc-GAL4 transcription factor. The offspring will express ddc-GAL4/UAS α-synuclein/pUAST Hsp100. This will be repeated will all the different chaperones on the-synuclein model and on each case 3 times to increase accuracy. I will compare this against the control to see if expressing any of the heat shock proteins can alleviate the phenotypes associated with Parkinson's disease (Figure 1). I will repeat this with the different Parkinson's models (Marsh and Thompson, 2006; Brand and Dormand, 1995; Brand and Perrimon, 1993).

I intend to evaluate the Drosophila models in the footsteps of previous researchers. To the measure the loss of dopamine neurons and lewy body number, I will stain the Drosophila brain with an antibody to tyrosine hydroxylase, which is an enzyme in the dopamine synthesis pathway. It will act as a marker for dopaminergic neurons and I will be able to count the number of dopamine neurons present and the number of lewy-bodies on each surviving dopamine neuron in the Drosophila (Menzies et al., 2005). To measure the locomotor behaviour, I will place 10 Drosophila per 10cm long vials and leave the Drosophila for 1 hour so they can acclimatise to the environmental, then the vial will be tapped at the bottom, and I will record the amount of time taken for each Drosophila to climb 5cm (Chaudhuri et al., 2007; Park et al., 2006). To check if the Drosophila are sterile, I will place the transgenic males with normal female Drosophila, (vice versa) and check 7 days later for the presence of larvae (Pesah et al., 2004).Life span will be check by counted the number of live Drosophila (Pesah et al., 2004). Finally to evaluate the sensitivity to oxidative stress, Drosophila will be exposed to the media which will contain paraquat, and the control Drosophila for each group will be placed under the same conditions minus paraquat (Pesah et al., 2004).

In this investigation I have limited the expression to just dopamine neurons because the loss of dopamine neurons is the main pathology of Parkinson's disease. Although, expression can be driven in all the neurons using an elav-GAL4 driver or It can also be expressed in all cells of the eye using a gmr-GAL4 driver where neurotoxicity can be monitored by measuring the loss of photoreceptor neurons, lethality of the Drosophila, or changes in their behavioural characteristics (Marsh and Thompson, 2006).

At the conclusion of the study, I will have examined the role of different heat shock proteins on Parkinson's disease by various cases. Hopefully this will identity the most effective heat shock protein or those active in Parkinson's a variety of causes. With this information I can test the role of newly identified chaperones in mouse models or maybe as therapies for humans.