March 01, 2014
Characterization of Familial Mutants and Splice Variants of Parkinson’s Disease Protein α- Synuclein in Yeast Models
Natalie is a 2013 Lake Forest College alumna who double majored in neuroscience and biology, and minored in studio art. S...
Natalie Kukulka
Department of Biology
Lake Forest College
Lake Forest, Illinois 60045
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Abstract
The misfolding of the protein α-synuclein is a major contributor to Parkinson’s disease (PD). Three mutations (A53T, A30P and E46K) cause familial PD, and three newly discovered spliced variant forms of the protein (syn-126, syn-112, and syn-98) are also found in many PD patients. Little is known about whether these familial mutants can influence each other’s contributing properties and whether the spliced variants are protective or harmful. Each familial mutant distinctively affects α-synuclein’s cellular localization, aggregation, and toxicity. For my thesis, I first tested the hypothesis that all three familial mutants equally influence a-synuclein’s pathological contributions in yeast models and found unexpected support for the dominance of the A30P mutant over E46K and A53T, shedding new light on A30P’s influence of a-synuclein’s conformation. Using polymerase chain reaction-based strategies, I have also made significant progress in creating the three spliced-variants for future evaluation in yeasts to assess their contributions to PD.
INTRODUCTION
Nerve-Racking Protein Responsibilities
Understanding of the complexity of the human body is one of the most sought after abilities that doubtfully will ever be acquired. Take into consideration the human brain; it is a mass of about three pounds composed of one hundred billion nerve cells and trillions of supporting cells (Purves et al., 2012). This fundamental processing unit is responsible for everything one does, sees, smells, interacts with, has an emotional responses to or ignores altogether. Anatomically speaking, the brain is a part of the central nervous system, and along with the spine, both are responsible for integrating information and coordinating activity (Purves et al., 2012). In addition to this command system, the peripheral nervous system helps communicate with the rest of the body, thereby establishing a circuit of sensing, integrating, and acting.
As a newborn develops from an inexperienced ‘creature’ to a skilled adult, the range of processed information increases exponentially. Such overload of information is sequentially synchronized by the establishment of neuronal connections, which can either be retained with repetition or lost due to their irrelevance (Purves et al., 2012). On the molecular level, each neuron operates by an array of proteins whose functions are determined through their specific shapes. Although proteins are made up of as few as twenty amino acids, the sequence of the amino acid assortment is what leads to the diversity in protein shapes and consequent varied functions. While the protein folding machinery is quite robust, it operates best at an equilibrium state between
Figure 1. Protein folding. A) There are many factors that affect the folding process of the protein. Exhibited are just some of the factors that will influence protein conformation: environmental factors, cellular conditions, genetics, and exposure to foreign substances. If the environmental factors do not provide any harm to the organism, cellular conditions are appropriate, genetic information is not impaired, and exposure to foreign substances is minimal, then the protein will fold correctly. The proper conformation will then lead to a well-functioning protein and a healthy cell. However, if the balance between these factors is disrupted or they individually become unfavorable for the organism, then the protein may misfold. The altered shape will lead to neutral, beneficial, or harmful consequences. B) Neurodegenerative diseases are one branch of harmful consequences due to protein misfolding. Exemplified are five common neurodegenerative diseases along with a list of their pathological characteristics and the misfolded protein(s). The images for various neurodegenerative diseases were acquired from http://www.nature.com/nrn/journal/v4/n1/fig_tab/nrn1007_F1.html.
environmental factors, cellular conditions, genetic influences, and foreign substances (Shin et al., 2009; Figure 1A). Increased exposure to negative environmental factors such as pollution or UV radiation or prolonged exposure to metals or toxins (such as pesticides) could contribute to the misfolding of the protein (Alberts et al., 2011). The molecular factors negatively influencing protein conformation could arise from incorrect encoding/ transcription of the DNA via various pathways or the failure of one of the intermolecular organelles like the mitochondria that serves as the power house for the cell (Shin et al., 2009). Still, more often than not, the brain withstands the negative factor of various sources and allows us to function flawlessly on a daily basis.
Neurodegeneration Initiated by Protein Misfolding
One of the negative consequences of improper protein folding can lead to selective death of neurons consequently leading to a neurodegenerative disorder (Figure 1B). Neurodegenerative diseases are disorders that are derived from the progressive deterioration of a specific portion of the nervous system, or in other words, a specific part of the brain that specializes in a particular ability (Ross & Poirier, 2004). Some of the leading neurodegenerative diseases are: Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington disease (HD), Multiple sclerosis (MS), amyotrophic lateral sclerosis or Lou Gehrig’s disease (ALS), and Prion diseases (Figure 1B). While each of these diseases differs in the progressive death of highly specialized neurons, the common feature among the pathology of neurodegenerative disease is the formation of protein aggregates from the misfolded protein in the affected neurons (Taylor et al., 2002; Figure 1B). The vast spectrum of neurodegenerative disease can be further narrowed down by focusing on the exact protein that misfolds. Synucleinopathies are thus neurodegenerative diseases in which the α-synuclein protein misfolds. They include dementia with Lewy bodies (DLB), multiple system atrophy (MSA), Lewy body dysphagia (LBD) and Parkinson’s disease (Galvin & Trokanowscki, 2001). Despite years of extensive research, cures for any of these neurodegenerative diseases remain elusive, afflicting several regions of the brain. Due to the shared pathology , understanding the mechanisms of α-synuclein protein misfolding in one disease may unlock mysteries for the other synucleinopathies and neurodegenerative diseases. My thesis focuses on gaining insight into the molecular basis of α -synuclein that contributes to familial and sporadic PD. Nevertheless, my findings have the potential to help understand the basis of protein misfolding-linked problems in all synucleinopathies.
Understanding Parkinson’s Disease
PD is the second most common neurodegenerative disease and the first most common bradykinesia disorder, known as the slowness of movement disorder (National Institute of Neurological Disorders and Stroke, 2004). While its occurrence was first noted by James Parkinson and documented in 1817 as a short article called “An Essay on Shaking Palsy”, it continues to persist in even higher frequency two centuries later (Parkinson, 1817). PD afflicts close to ten million people worldwide and approximately 60,000 Americans annually (Parkinson’s Disease Foundation, 2012). While the search for a cure for PD has broadened worldwide, so did the cost of possible treatments. With no possible cure on the horizon, patients try their luck with administering L-Dopa or using much more invasive treatment options, such as surgical implantation of metal electrodes for deep brain stimulation (Parkinson’s Disease Foundation, 2012). While some of the symptoms may be initially suppressed, such as rigidity, resting tremors, masked facial expressions, patients ultimately die of organ failure (Olanow & Tatton, 1999; Galvin & Trokanowscki, 2001; Figure 2A).
PD Causes
PD can be classified as sporadic, which accounts for 90% of all diagnoses, or familial, which accounts for the remaining 10%. The sporadic form of PD can be initiated by various factors similar to those influencing protein misfolding (Figure 1A). For a long time, environmental factors were neglected in relation to the onset of PD, and it was not until a famous incident with the use of MPTP (1-Methyl-4-phenyl-1,2,5,6-tetrahydropyridine), a by-product of synthetic heroin, that people began pay closer attention (Sian et al., 1999). For example, exposure to chemicals like rotenone (an organic farming pesticide), (Betarbet et al ., 2000), heavy metals (Calne et al., 1994), and free radicals (Maguire-Zeiss et al., 2005) have been linked with sporadic PD (Figure 2B).
Another factor, classified as a long-term environmental influence, was linked with traumatic brain injury, such as a traumatic blow to the head or a severe concussion. In terms of cellular dysfunction, oxidative stress (Jenner & Olanow, 1996; Maguire-Zeiss et al., 2005) and mitochondrial incapability (Langston et al., 1983; Langston et al., 1984) have been tied into PD (Figure 2B). Nevertheless, the precise mechanisms behind these factors are not well known, hence researchers tend to focus on the genetic factors within the familial branch of PD. The most common gene mutations that lead to PD occur in SNCA (Polymeropoulos et al., 1997; Kruger et al., 1998; Zarranz et al., 2004), Parkin
Figure 2. The basis of PD. A) Showcased are behavioral, anatomical, and cellular consequences of PD pathology demonstrated by restricted range of movement, neuronal death and reduced neurotransmitter signaling, respectively. The behavioral panel showcases the affected brain circuitry leading to the symptoms of the disease (image borrowed from https://www.dana.org/news/brainhealth/detail.aspx?id=9860). The anatomical panel shows the midbrain region of the substantia nigra, where at the onset of the disease, 90% of dopaminergic neurons die; this demise of neural cells can be clearly identified during patient autopsy through the lack of dark pigmentation (melanin) as exemplified on the right-side panel (image borrowed from http://www.umm.edu/patiented/articles/what_parkinsons_disease_what_causes_it_000051
_1.htm). The cellular panel shows the release of the neurotransmitter, dopamine, from the specific dopaminergic neurons and how it diminishes during PD (image borrowed from http://www.webmd.com/parkinsons-disease/guide/parkinsons-causes). B) Showcased are molecular causes leading to the onset of the disease. The cellular cataclysms panel shows both the external influences on the neuron, as well as the molecular dysfunctions that lead to neurodegenration, especially in regard to PD. The genetics panel shows different genes that result in specific molecular consequences leading to the formation of Lewy bodies.
Kidata et al., 1998), UCH-L1 (Liu et al., 2002), DJ-1 (Bonifati et al., 2003), PINK1 (Valente et al., 2004) and LRRK2/PARK8 (Funayama et al., 2002; Paisan-Ruiz et al., 2004, Liu et al., 2012), PARK2, PARK7 (Neuytemans et al., 2010) autosomal-dominant PD mutations in ( UCHL-1, SNCA, LRRK2), and autosomal-recessive PD ( parkin, PINK1 and DJ-1; Figure 2B).
Parkinson’s Disease Pathology
PD is characterized by the death of dopaminergic neurons predominately concentrated within the midbrain structure known as the substantia nigra, which is Latin for “black substance” (Figure 2A). The substantia nigra is critical in the circuitry known to govern reward, addiction, and movement, which is further consistent with the exhibited symptomatic death of these neurons (Figure 2A). PD pathology reveals protein aggregates within degenerating neurons, which Frederick Lewy first named Lewy bodies in 1912. It was not until 1997 that Polymeropoulos and colleagues found a mutation in the α-synuclein gene, SNCA, on the fourth human chromosome, that caused autosomal-dominant PD through the A53T mutation in the α-synuclein protein (Polymeropoulos, 1997). The identified misfolded and aggregated α-synuclein protein was thus recognized as the major component that makes up Lewy bodies (Spillantini et al., 1998). Recently, it has been shown that Lewy bodies are formed from both full length and truncated versions of α-synuclein, indicating that the balance between these isomers plays an important role in the onset and progression of the disease (McLean et al., 2102).
Insight into α-Synuclein
The synuclein protein was first isolated from Torpedo californica, or Pacific electric ray,
but now its complete sequence has been analyzed in more than 20 species with its respective mutations, posttranscriptional modifiers, polymorphisms and truncations (Xiong et al., 2010). While much about the α-synuclein protein is still unknown, it appears to play a role in regulation of cell differentiation, synaptic plasticity, size of presynaptic vesicular pools, and dopaminergic neurotransmission (Luckin et al., 2000, Beyer et al., 2004). This is further supported by its history of binding to the phospholipid membranes in vitro and co-localization with synaptic vesicles in vivo (Luckin et al., 2000). While α-synuclein is predominantly expressed in the central nervous system, it is not a brain-specific protein because its presence is recognized in other tissues, such as the heart, muscles and pancreas (Beyer et al., 2004, Beyer et al., 2006). Further research has specified and confirmed its high expression in skin, lungs, kidney, spleen, heart, liver and muscle samples (Beyer et al., 2008). Still, its highest expression is found in the brain (Beyer et al., 2008).
Structurally speaking, α-synuclein is a small, 140-amino-acid-long acidic protein, which is encoded by the SNCA gene on chromosome 4q21 (Beyer et al., 2006). Its native unfolded form increases its predisposition to self-aggregate based on varying environmental conditions, making it a very dynamic molecule (Lucking & Brice, 2000, Beyer et al., 2006, Bisaglia et al., 2009). More specifically, in an aqueous solution, it conserves its unfolded and randomly coiled structure. However, when it comes in contact with acidic phospholipid vesicles, it folds into an α-helical structure or forms insoluble fibrils with a high beta-sheet structure leading to formation of Lewy bodies (Beyer et al., 2006, Beyer et al., 2008).
Aside from environmental conditions, there are genetic factors that may alter the characteristics of a protein. Three distinct α-synuclein single amino acid point mutations lead to the development of familial PD. The data in my thesis depends heavily upon the understanding of each individual mutant’s characteristics. Therefore, I will elaborate on them in the overview for study one. In additional genetic mutations, there are more than three hundred different posttranslational protein modifications which lead to changes in protein size, charge, structure, and conformation, affecting key protein characteristics. As part of background information for study two, I will provide insight into understanding one of these posttranslational protein modifications: the formation of splice variants.
STUDY 1
Understanding Familial PD Point Mutations
A53T:
This missense mutation, where alanine charges to threonine on the 53 rd amino acid of the α-synuclein protein, was the first familial PD point mutation identified in the Contursi family and three additional unrelated Greek families (Polymeropoukous et al., 1997; Figure 3). In in vitro studies, the A53T mutant showed that in the absence of other Lewy body-associated molecules, it was disordered in dilute solution just as WT, but at high concentrations it formed discrete spherical assemblies at the fastest rate (Conway et al., 1998; Figure 3). Further in vitro experimentation supported A53T’s ability to aggregate best at lower concentrations and showed that A53T had greater propensity to polymerize than A30P, one of the other familial mutants (Giasson et al., 1998). In 2000, Conway et al.’s research findings added that the fibrillation of A53T was relative to that of WT, and faster than that of A30P, while the consumption of the A53T monomer was the most rapid.
