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The Implications of α-synuclein in the Treatment of Parkinson's Disease
Kelly M. Wilmas
University of Texas at Austin
Abstract
This purpose of this research is to provide valuable information regarding the pathways that cause aggregation of the α-synuclein protein in brain nerve terminals, causing neurodegeneration and motor dysfunction, in order to diagnose Parkinson's disease early and provide effective treatment. Studies have shown that the idiopathic form of Parkinson's is strongly associated with changes in α-syn expression due to mutations and single nucleotide polymorphisms, which causes aggregation of α-syn protein into amyloid deposits in brain nerve terminals. Familial Parkinson's disease is due to autosomal-dominant inheritance of the mutated α-syn. Thus, α-syn is thought to play a fundamental role in the genetic etiology of Parkinson's and can possibly be targeted in order to treat the disease. In order to gather more evidence of this, I compiled primary and review research articles on the genetic mechanisms of this protein. In this review, I provide an overview of known interactions of the protein with the environment, genes, and aging process. I also include significant findings that improve our understanding of possible treatment options, targeting α-synuclein, for this debilitating disease.
Introduction
Parkinson's disease is currently the second most prevalent neurodegenerative disease in existence, affecting 6.3 million people worldwide (Baker, 2004). Presently, there is no cure for the disease, and it causes substantial morbidity. The main pathology of Parkinson's disease is degeneration of the dopaminergic substantia nigra pars compacta of the brain (McNaught & Jenner, 2001). It supplies the striatum, which is involved in modulating movement pathways and executive functions, with dopamine when functioning normally. However, neurodegeneration prevents this from occurring (McNaught & Jenner, 2001). The neurotransmitter dopamine relays messages to the brain in order to control movement and cognitive function, but the nerve cells that produce dopamine are at least 70 percent lost when Parkinson's symptoms develop (Heisters, 2011). Due to degeneration of these neurons associated with dopamine in the brain, affected individuals experience motor symptoms that include slow or rigid movement, tremors while at rest, or impaired posture and balance. Patients devastatingly lose the control over their own bodies, mentally and physically. Parkinson's does not discriminate against any nationality, gender, age, or race; many people will know someone with Parkinson's in his or her lifetime and witness that person's struggle. The disease is progressive in that the symptoms will continue to worsen until death results, although its rate of progression differs among individuals (Worth, 2013). The exact cause of neurodegeneration in the substantia nigra remains unknown, but through continuous experimentation and inquiry, it is possible to find the cause and apply new treatments.
The symptoms of Parkinson's fall into four different stages. The first stage is diagnosis after a patient may have experienced insomnia, depression, anxiety, or reduced sense of smell that was followed by the more obviously abnormal motor symptoms of the disease (Worth, 2013). The next stage is maintenance, which is largely pharmacological, although the drugs soon become inadequate at treating the symptoms (Worth, 2013). Psychiatric symptoms such as hallucinations and dementia are present in over three quarters of the patients 10 years after diagnosis. The ability to initiate or prevent an action, in addition to the capability to evaluate and change behavior to adapt to a circumstance is impaired in many Parkinson's patients (Căpuşan et al., 2011). These aspects of the disease significantly decrease the patient's quality of life. Third is the complex stage where the patient's drug regimen needs to become more involved. Doctors may recommend neurosurgery or patients may need psychological treatment (Thomas, 2006). The last stage of the disease is palliative care because 40 to 50 percent of affected individuals with Parkinson's experience severe pain due to withdrawal from anti-Parkinsonian drugs between doses or pain due to immobility and loss of muscle control (Thomas, 2006). Patients and their families must decide when the disease can no longer be treated and choose measures to treat the patient's emotional and physical pain. Because Parkinson's is such a debilitating disease, it is crucial for scientists to find the genetic causes and ways in which to inhibit it.
Parkinson's disease is a prion protein disease, which indicates that the disease can be elicited when a protein misfolds (Moreno-Gonzalez and Soto, 2011). The first protein found to be implicated in familial Parkinson's disease was α-synuclein (α-syn), which is a 140-amino-acid long protein expressed abundantly in the neurons of the brain from the SNCA gene locus. A missense mutation in the coding region of α-syn leads to the familial form of Parkinson's, while the non-inherited form's cause is less clear, but thought to be due to aging or certain mutagens, where toxin exposure alters gene sequences (Goedert, 2001). β-sheet-rich amyloid fibrils form when the proteins aggregate and it has been found that higher levels of aggregation are associated with familial Parkinson's disease. Thus, to find the route of aggregation of α-syn could provide valuable insight for novel treatment options.
The disease is thought to be caused by one of two different pathways: random onset or inherited as a familial disease. The random onset of Parkinson's is strongly associated with changes in α-syn expression due to mutations and single nucleotide polymorphisms, which causes aggregation of α-syn protein into amyloid deposits in brain nerve terminals that then cause neurodegenerative effects (Gasser, 2004). For this idiopathic form of disease, the onset of symptoms occurs around age 60 or later. On the other hand, familial Parkinson's disease is due to autosomal-dominant inheritance of the mutated α-syn, and the symptoms of the disease onset at an average age of 45.6 years (Goedert, 2001). Thus, α-syn is thought to play a fundamental role in the genetic etiology of Parkinson's and can possibly be targeted in order to treat the disease. This paper demonstrates that understanding the pathways that cause aggregation of α-synuclein protein in brain nerve terminals, causing motor dysfunction, can potentially be used to diagnose Parkinson's disease early and provide effective treatment.
This paper will examine studies done using or investigating the α-syn protein. Using these studies, I will evaluate the pathways of α-syn and explore how knowledge of these pathways has implications in finding a treatment for Parkinson's. I will describe how α-syn has been found to accumulate in cells and how mutations in the gene encoding α-syn can lead to pathogenic α-syn protein in the brain. Possible treatments for Parkinson's disease, specifically targeting α-syn accumulation, will also be described. In the concluding portion of the paper, I will describe where further research needs to lie in order to take the information we know about α-syn and create an effective treatment, leading to a cure, for Parkinson's disease.
Review of Literature
A hallmark of Parkinson's disease is the presence of the Lewy bodies, which are implicated in the sporadic, more common form of Parkinson's. The Lewy bodies are round, eosinophilic cytoplasmic inclusions made primarily of abnormal filaments of α-syn. The majority of Parkinson's cases with Lewy body presence are sporadic and possibly due to environmental toxin exposure, not a result of any clear family history. Through research, scientists found that there was a direct relationship to the construction of neuropathological lesions, such as Lewy bodies, and the neurodegeneration process (Goedert, 2001). Specifically, it was also found that Lewy bodies can be transferred from the peripheral or enteric nervous system to parts of the brain, which is implicated in the advancement of sporadic, non-inherited Parkinson's disease found in 90 percent of Parkinson's disease cases (Hansen & Li, 2012). One theory is that the cell-to-cell transfer of toxic α-syn from the outside environment, whether through inhalation or ingestion, can cause death in recipient cells. In an experiment, young 11-22 year old neurons were transplanted into Parkinson's disease patients. Four weeks later, Lewy bodies containing α-syn were found in the previously unaffected neurons, indicating cell-to-cell transfer of Lewy bodies (Hansen & Li, 2012). This finding demonstrates a possible way α-syn accumulates in different cells, by direct transfer, to form Lewy bodies. When mutation occurs in the proteins that comprise these lesions, familial Parkinson's disease develops (Goedert, 2001). It was clear that α-syn had transferred from one neuron to another, then causing harmful, prion-like protein aggregates to form in the recipient neuron (Hansen & Li, 2012). A further study showed that proliferating stem cells that were grafted into the hippocampi of the brains of mice producing α-syn caused the stem cells to accumulate α-syn through cell-to-cell transfer (Hansen & Li, 2012). If scientists can genetically disrupt the transfer of α-syn between cells through their routes of endocytosis and transfer, aggregation of the protein could possibly be halted, thus preventing any further neurodegeneration.
Finding a viable treatment to reverse the effects of neurodegneration or to prevent it is crucial because no such present treatment exists. Current treatments for the disease are mainly L-dihydroxyphenylalanine (L-DOPA) and dopaminergic agonists, both of which increase dopamine levels that are otherwise diminished due to the neurodegenerative effects of the disease (Heisters, 2011). L-DOPA is a precursor of dopamine that can cross the blood-brain barrier and initiate dopamine release. Dopaminergic agonists activate dopamine receptors in the absence of dopamine in order to give the same effect in the brain as when dopamine activates the receptors. Current treatments only target the motor symptoms of Parkinson's, but a treatment that could repair or prevent progression of neurodegeneration would be most beneficial. Because α-syn has been indirectly linked to the disease in a number of ways, I believe that targeting α-syn or α-syn pathways for treatment of Parkinson's will be key to preventing progression of the disease. Finding and understanding a connection between α-syn and Parkinson's development will be pivotal in the development of a cure for Parkinson's disease.
α-syn Pathways
The susceptibility of a person to developing the idiopathic or familial forms of Parkinson's differs depending on the type of mutation the person has. Some studies related to Parkinson's disease have shown that mutations in α-syn lead to the dominant, familial Parkinson's disease while recent, genome-wide association studies, which study the genetic variants of individuals, have shown that mutated variants of the SNCA gene, which encodes α-syn, increase the susceptibility of a person to develop non-inherited Parkinson's disease (Angot et al., 2011). It is possible that a pathogenic agent enters the body through ingestion or inhalation and eventually transfers from cell-to-cell and then to the brain stem and cortical regions. If this agent is not α-syn, α-syn could be the initial target of the pathogenic agent. This agent then causes a mutation in the coding region of the SNCA gene locus so that α-syn misfolds and accumulates. Consequently, α-syn can be transferred from cell to cell to spread disease from a pathological neuron to unaffected neurons, thus triggering misfolding.
Researchers debate over the exact route of α-syn transmission, but one source says that the pathogenic protein must exit the cell, transfer to the next cell through some route of access across the cell membrane, and then cause a stress to debilitate the cell (Angot et al., 2011). Mutations in the coding sequence are not always deleterious, but once the code is translated, the protein can misfold, causing prion formation (Moreno-Gonzalez & Soto, 2011). Therefore, certain alleles of the SNCA gene locus could be more prone to misfolding than others. One experiment using rat neurons showed that α-syn was present in cerebrospinal fluid, blood plasma, and saliva, indicative of α-syn traveling through a secretory pathway (Angot et al., 2011). In addition, it was found that cellular stressors and misfolded α-syn proteins cause more secretion of damaged α-syn proteins, as seen in Parkinson's disease. Host-to-graft transmission experiments have successfully been done by transferring α-syn from an α-syn-overexpressing mouse to a graft of neural stem cells, causing debilitating effects of Parkinson's disease in those subjects, but an experiment that causes formation of Lewy bodies has not yet been successful (Angot et al., 2011). With more research on what genetic factors trigger cell-to-cell transmission of α-syn and the formation of Lewy bodies, the fundamental cause of Parkinson's disease can be better understood to help many affected people around the world.
Techniques to detect Parkinson's disease will be pivotal in early diagnosis and treatment to prevent further neurodegeneration. A promising study revealed that auto-antibodies with specificity for self-antigens can serve to indicate the process of neurodegeneration in Parkinson's disease (Casaite et al., 2011). This was significant because the data could be used to study the auto-immune response to and treatment options for mutated, amyloidogenic proteins connected with Parkinson's disease. Scientists produced auto-antibodies for the amyloid α-syn proteins in Lewy bodies, and then recorded the blood sera levels of antibodies in early versus late Parkinson's disease patients as well as a control group using ELISA, Western blot, and Biacore surface plasmon resonance. In the Biacore surface plasmon resonance technique, amyloid fibrils of α-syn were used as antigens and placed on Biacore chips where they interacted with polyclonal antibodies from blood sera of early and late Parkinson's disease patients as well as non-affected control patients. There were significantly higher levels of antibodies for α-syn in the sera of Parkinson's disease patients when compared to control, with the highest levels of 0.884 in early stages of Parkinson's and lower levels, 0.779, in later Parkinson's. Thus, this may indicate that when amyloids form in the body, an autoimmune response may help the body remove the amyloids and try to maintain a stable environment. Antibodies have a unique specificity towards the amyloid deposits associated with Parkinson's disease, indicating a specific response to protect the body from neurodegeneration. ELISA or Western blot analyses did not show a correlation between the auto-immune response in patients with Parkinson's disease and the control group, which indicates that the auto-immune response specifically acts on Parkinson's disease proteins. 90 percent of these antibodies were correlated with familial Parkinson's disease, and 48 percent with non-inherited forms (Casaite et al., 2011). Due to these recent findings, the levels of antibodies to α-syn can be used in the diagnosis of stages of Parkinson's disease, possibly allowing a more tailored fit of therapeutic treatment in the future.
To find effective treatment, scientists must understand the mechanisms of neurodegeneration through α-syn's absence, presence, and differing concentrations. One study showed that inactivation of α-syn through homologous recombination did not cause any phenotypic neurological disorders, so neurodegeneration is most likely due to the presence of the protein instead of its absence (Goedert, 2001). However, it was also found that the absence of α-syn caused a greater release of dopamine suggesting that α-syn could serve as an activity dependent, negative regulator of neurotransmission in the striatum of the brain. Therefore, if α-syn is present, it will down-regulate dopamine release which has been associated with the development of motor abnormalities in Parkinson's. When about half of the neurons that contain dopamine in the substantia nigra and 70-80 percent of the dopamine released at the striatum are no longer present, the symptoms of Parkinson's disease become apparent. Although it has been possible in the past to alleviate some symptoms of Parkinson's through L-DOPA replacement therapy, a precursor of dopamine, this only is effective in earlier stages of Parksinson's and its effect wears off over time. Consequently, symptoms are not alleviated in individuals with later stages of the disease. Whether the affected individual's disease is in early or late stages, degeneration of brain neurons continues, even with L-DOPA treatment (Goedert, 2001). This shows that overexpression of α-syn, due to mutation of the protein, has been implicated in all forms of Parkinson's disease.
A recent study successfully found a new model organism to examine gene-by-environment interactions that could express a mutation of α-syn. Scientists created a bacterial artificial chromosome (BAC) transgenic rat model of Parkinson's disease expressing a mutation of α-syn, called the E46K mutation, which causes disease in humans (Cannon et al., 2012). These models could reproduce key features of Parkinson's that are relevant to humans, but had not been reproduced before in animal models. Rats were considered to be advantageous to find genetic implications in human disease for behavioral assessment, neuroanatomical comparisons, and the ability to analyze more endpoints due to tissue availability. Neurotoxin models replicated the behavioral and neurological features of Parkinson's, but were not relevant to neurotoxin exposure that changed the regulation of genes. Three different point mutations have been found to cause autosomal dominant Parkinson's, and the E46 mutation is the most recently discovered. It was found that the mutant protein was expressed to levels two to three times above endogenous α-syn levels, which is an indication of early onset Parkinson's disease in humans. They also reproduced preclinical Parkinson's features such as a decrease in dopamine metabolites, which means these rats can be used to examine the earliest mechanisms of dysfunction in Parkinson's and were concluded to be suitable for gene by environment interaction examinations relating to the disease. Therefore, the predictive value for testing disease-modifying genetic therapies in these models is promising.
To show the effect of environmental toxins on neurons, wild type rats and rats with a mutated E46 α-syn were experimentally treated with rotenone, a mitochondrial toxin found in pesticides linked to Parkinson's disease. Rats expressing the mutated E46 α-syn had greater sensitivity to rotenone in that their symptoms of a severe Parkinson's phenotype were induced sooner than in wild type rats. The symptoms included severe, mobility-limiting bradykinesia, postural instability, and rigidity as seen in human Parkinson's. Therefore, the E46K mutated rats were hypersensitive to rotenone, a toxin found in the environment that alters genes to cause Parkinson's. This study confers that individuals with the pathogenic, mutant form of α-syn have enhanced toxic effects of mitochondrial impairment due to mitochondrial toxins, which are found in certain pesticides. This gene-by-environment interaction can have deleterious effects on the age of onset and severity of Parkinson's disease in humans since these genetic models were well suited to be genetic models for investigating environmental factors (Cannon et al., 2012). Because a new route of induction of Parkinson's was found and able to be reproduced, genetic treatment can be more closely tailored to treat this form of disease. Affected genes can possibly be down-regulated or modified so that they would affect downstream targets.
Studies done on mutated genes, such as SNCA, in the familial and sporadic forms of Parkinson's disease, have been very beneficial to identifying cell pathways that contribute to neuronal death (Devine, 2012). Obtaining diseased neurons from living, affected patients has been difficult, so experimentally testing treatments have also been challenging. In 2012, transgenic mice were triggered to express human SCNA with a disease causing mutation known as A53T. This caused them to exhibit posture and motor abnormalities by age 1 to 2 months and lose 40 percent of their midbrain dopaminergic (mDA) neurons when compared to wild type mice. They had increased accumulation of α-syn and decreased Nurr1, which controls transcription of genes critical to mDA neurons and also helps the neurons survive and maintain themselves. It was also found through further experimentation that proteasomal inhibition stabilizes Nurr1 and ameliorates degeneration of mDA neurons caused by α-syn. It was previously thought that proteasomal inhibition contributed to neurodegeneration, but the possibility that it could be used as a treatment for neurodegernation was unforeseen. This study showed that the proteasome and proteins that cause neurodegeneration due to gene mutations have a complicated relationship that needs to be further tested. Proteasomal inhibition allowed for survival of Nurr1, which allowed the transcription and regulation of mDA neurons necessary for neurological function to continue (Devine, 2012). When mDA neurons are allowed to be transcribed and regulated properly, pathologic effects of Parkinson's can conceivably be avoided or decreased.
Possible Treatment Options
A possible target for therapeutic treatment of Parkinson's disease is RNA interference (RNAi) of α-syn mRNA to cause neuroprotection and neurorestoration in the central nervous system (CNS), but there has not been enough research done to find a technique to deliver the RNAi to the CNS (Sen & West, 2009). Other treatment methods involve chaperones such as Hsp70 reducing the aggregation of α-syn or degradation of the protein via a proteasome or lysosome. To increase the rate of degradation, E3 ubiquitin ligase can be overexpressed in model systems (Sen & West, 2009). This conflicts with the previous study mentioned where proteasomal inhibition ameliorated neurodegeneration, so more clinical trials and research must be done to determine ways to mechanistically target α-syn for inhibition and to find the proper route to do so. There are pathways that we may not understand and relationships to be determined in order to treat Parkinson's disease genetically.
One possible way in which to treat the disease is through immunotherapy to enhance the immune response in individuals. In one experiment, mice over-expressing α-syn were given a vaccine with CFA/IFA as an immunological agent to increase the antigen response (Schneeberger, 2012). This active immunotherapy method caused the mice to develop antibodies with high relative affinity to α-syn, which promote degradation of α-syn aggregates, most likely through a lysosomal pathway. Consequently, less pathogenic α-syn aggregates accumulated in neuronal cell bodies and synapses, which led to less neurodegeneration. These mice had improved function of neurons as a result of the immunotherapy. Then, researchers systemically administered an α-syn-specific monoclonal antibody that passed the blood-brain barrier and reduced α-syn accumulation in axons and synapses. This method reduced neurodegeneration and reduced behavioral deficits of affected mice also most likely through a lysosomal pathway. The AFFITOPE PD01 was the first vaccine candidate developed to target α-syn aggregates seen in Parkinson's. The vaccine elicited a humoral immune response with reactivity towards α-syn in mice. This reduced the cerebral α-syn levels and ameliorated neuronal cell loss, in addition to improving cognitive functions of the mice (Schneeberger, 2012). Further clinical tests are needed since these are pre-clinical tests, but this immunotherapy approach could be very promising for the cure of Parkinson's disease.
Studies have shown that immunization against α-syn reduced the accumulation of α-syn and lessened synaptic loss in the transgenic rat model, but the exact mechanisms that cause this reduction remain unknown (Bae et al., 2012). In one study, monoclonal antibodies against human α-syn were used to clear away α-syn into microglia through a passive immunotherapy approach. The microglia are macrophages in the brain that act as an immune defense to eliminate infectious agents in the central nervous system. Thus, uptake of α-syn by microglia can block cell-to-cell transfer of α-syn that would otherwise cause further neurodegeneration. Results showed that the antibodies against α-syn stimulated clearance of extracellular α-syn through internalization by microglia, which are then delivered to lysosomes for destruction. The study also showed that this clearance blocks cell-to-cell transfer of extracellular α-syn. Passive immunization using α-syn antibodies ameliorated neurodegeneration by reducing the accumulation in neurons and glial cells that are normally associated with pathogenic α-syn accumulation. By targeting extracellular α-syn, there is less risk of interfering with the essential functions of proteins in the neuronal cytoplasm, and targeting α-syn extracellularly could be the new approach to treating Parkinson's disease. These findings show the potential for immunotherapy, using antibodies against α-syn, to mediate the effects of abnormal α-syn deposition through either a vaccine or a molecule made to mimic the effect of these antibodies (Bae et al., 2012).
Conclusion
The ongoing search for a cure for Parkinson's will be prove to be a difficult task, but with the current research and interactions understood about α-syn, scientists are well on their way to finding successful treatment options for Parkinson's disease. In all cases, patients with neurologic pathology characteristic of Parkinson's had missense mutations in α-syn caused by familial inheritance, aging, toxic environmental agents that altered the gene that encodes α-syn, or due to yet undiscovered reasons. Therefore, treatment needs to target the prevention of α-syn accumulation in neurons of the brain when a patient has the pathologic SNCA gene or α-syn protein. Otherwise, treatment should focus on degrading the protein already present in order to prevent further neurodegeneration. Through further research, scientists can determine which of the treatments discussed is most effective, whether it be a combination of treatments or a single treatment. Because the pathologic agent of Parkinson's disease has been identified, it is a matter of understanding α-syn's interactions with other genes, the environment, and the cellular machinery to ultimately find a genetic treatment leading to the cure for Parkinson's disease.
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