α-Synucleinopathy in Parkinson's Disease

What is a Parkinson's disease?
Overview of what a Parkinson's disease is. This introductory video provides basic
understanding of pathophysiology of PD that will aid in understanding role of α-Syn.

Virtually all sporadic and familial forms of Parkinson’s disease (PD) are associated with two major disease processes: selective loss of midbrain dopamine (DA) neurons in substantia nigra pars compacta (SNpC), and accumulations of intraneuronal inclusions known as Lewy bodies (LB)[1]. While the relationship between these two conditions were largely unexplored in the past, recent researche point to a strong causal relationship. More specifically, scientists believe that α-synucleins (α-Syn), a protein of primary component in LB, may be the cause of PD. Normally α-Syn function to maintain proper synaptic processes; however, certain changes in its structure initiates a cascade of pathological events commonly referred to as α-synucleinopathy. Converging lines of evidence suggest loss of DA neurons in SNpC of PD patients are the result of the α-synucleinopathy[2]. As such, it is hoped that future insights into α-Syn and their mechanism of pathology will help shed new light in developing effective therapies for PD of which we know little.

1) Normal α-synuclein properties

α-Synuclein
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Molecular image of an α-synuclein.
Adapted from Lashuel et al. (2013).

1.1 Molecular properties

α-Syn is a part of a family of synuclein proteins encoded by the SNCA gene in humans. It is a monomeric protein with a very highly conserved structure: large hydrophobic core within its N-terminal domain and large negative charge on its C-terminal domain due to high acidic (i.e. Glutamic and aspartic acids) residue contents[3]. An interesting property of an α-Syn is that it predominantly exists as a stable unfolded monomer in physiological conditions. Optical activity studies show complete lack of tertiary structures and a very weak presence of helical secondary conformation in its native conformation[4].

1.2 Tissue expression

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Schematic highlighting functional properties of α-synuclein. Of note, diagram demonstrates
α-synuclein activity in regulation of SNARE-complex assembly as well as regulation in neurotransmitter
vesicle refilling. Adapted from Lashuel et al. (2013).

Although small amounts are known to be expressed in somatic cells, α-synuclein expression is highly localized to neurons within the central nervous system[3]. Expression is especially prominent in DA neurons in the SNpC, the brain area commonly associated with PD. The majority of neuronal α-Syn are subcellularly localized around presynaptic terminals where they actively participate in a variety of synaptic processes[4].

1.3 Molecular functions and interaction with synaptic complex

α-Syn are involved in many synaptic processes and are essential for proper synaptic transmissions. The most researched role is its regulatory role in SNARE-complex assembly. A recent study by Burré et al. demonstrated that α-Syn plays a direct role in promoting SNARE-complex assembly in presynaptic terminal[1]. This occurs by a nonenzymatic mechanism that involves simultaneous binding of α-Syn to phospholipids by their hydrophobic N-terminal and to synaptobrevin-2 (a key subunit of SNARE-complex) by their negative C-terminal[1,5].

There is also evidence to suggest α-Syn in SNpC may be an important regulator in trafficking of DA vesicles from readily releasable pool of DA. The current model proposes that α-Syn may negatively regulate short-term release capacity of DA vesicles. This can allow sufficient time in between DA releases for replenishment of DA pool to take place. Overall, by preventing excessive short-term release of DA vesicles, it helps to maintain stable long-term DA transmission capacity [5,6,7]. α-Syn are also believed to partake in many other cellular functions like Golgi apparatus and ER vesicle trafficking and antioxidant activity[5].

2) Fibrillar α-synuclein pathology

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Immunohistochemical image of a substantia nigral pars
compacta neuron affected with Lewy bodies. Retrieved from:
http://www.virtualmedstudent.com/links/neurological/parkinsons.html

The essential functions of α-Syn require the protein to be maintained as an unfolded structure.[5]. Unfortunately, α-Syn may adopt different structures due to mutagenic processes and various external factors. While many conformations are observed, adopting β-sheet conformations are considered to be the most predominant and relevant to PD because of their ability to oligomerize into α-Syn fibrils[8]. β-sheet allows α-Syn to self-associate (stack) with other β-sheet α-Syn to form amyloid-like fibers commonly referred to as α-Syn fibrils. These α-Syn fibrils have the ability to accumulate and recruit other neuronal proteins (crosslinks) to produce large intracellular inclusions known as Lewy bodies – which are the neurological hallmarks of PD[5,9].

2.1 Critical sequence alterations

Based on genome wide association studies of Parkinson’s disease patients, various mutations of the SNCA gene have been proposed to cause α-Syn to adopt a β-sheet conformations that promote fibrillization. Based on the data, the most predominant mutation types were the missense mutations that convert adenine to proline: A56P and triple mutant A30P/A56P/A76P[3,10]. Unfortunately, molecular reasoning for why conversion of adenine to proline at certain sequence cause β-sheet formation is not yet understood. Gene multiplication (duplication/triplication) of SNCA gene have also been proposed to promote fibrillization of α-Syn. While the gene itself is not mutated to directly express β-sheets, overcrowding from excessive α-Syn expression can enhance a chance conformation change to β-sheets[11].

There are also evidences that certain non-mutagenic factors can cause natively unfolded α-Syn to aggregate into fibrillar forms. One such factor includes certain changes in physiological conditions within neurons that alter protein’s hydrophobicity or net charge – for instance, low pH or high temperature. Also, many studies point to roles that dysfunctional post-translational modifications and oxidative stress may play in α-Syn fibrillization[5,8,10].

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Schematic diagram explains current theories for monomeric α-Syn aggregation into α-Syn fibrils and LB (bottom half)
as well as mechanism of α-Syn fibril propagation through the synapses. Adapted from Lashuel et al. (2013).

2.2 Conversion of α-synuclein into Lewy body formation and prion-like propagation

There are three properties of α-Syn fibrils that makes it pathologically significant: 1) they aggregate into large insoluble LB; 2) they promote conversion of natively unfolded α-Syn into more fibrils; 3) they can be transmitted synaptically. As described before α-Syn fibrils tend to recruit various neuronal proteins into a large sphere of insoluble inclusions known as LB. Some of these neuronal proteins include ubiquitins, tau proteins, β-synucleins, and ϒ-synucleins; but α-Syn are typically of the highest proportion in LB[8,13]. Accumulation of these neuronal proteins into LB is believed to occur by a nucleated polymerization mechanism in which a small number of initial α-Syn fibrils act as seeds for further aggregate growth[14]. Unfortunately, the underlying molecular mechanism is not yet understood. In addition to their seeding activity, studies have demonstrated that α-Syn fibrils have the ability to actively induce endogenous non-folded α-Syn into α-Syn fibrils. It is proposed that interaction with the β-sheet structure of α-Syn fibrils induce unfolded α-Syn to adopt similar β-sheet conformations[5,15].

Furthermore, experiments in vitro and in vivo point to an ability for α-Syn fibrils to transmit themselves to interconnected neurons. Many studies have repeatedly demonstrated this phenomenon by showing that a single injection of preformed α-Syn fibrils into a small region of neural network in vivo can self-propagate and spread throughout the entire network given sufficient time[2,15]. Number of mechanisms through which α-Syn fibrils could propagate to other cells have been proposed. One model of spread is described as follows: 1) intracellular α-Syn fibrils are first released into the synapse either by exocytosis or by cell death, then 2) extracellular α-Syn fibrils can enter acceptor neurons either by receptor-mediated endocytosis or by direct penetration[16,17,18]. Unfortunately, no studies have been done to examine validities of the different models. But it is clear that once it is inside the acceptor neuron, α-Syn fibril can act as a new focal point for further intracellular aggregation[5].

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Schematic illustration of potential α-Syn fibril-mediated cellular and synaptic dysfunctions within a SNpC neuron.
It is proposed that combinations of these dysfunctions can initiate neurodegeneration. Adapted from Belluci et al. (2012).

2.3 Synaptic dysfunction and neurodegeneration

There is a general consensus that presence of LB drives deprivation of neuron’s synaptic abilities and eventual neuron death (neurodegeneration). The lethal nature of LB can be explained primarily by the pathological nature of α-Syn fibrils. When unfolded α-Syn are recruited into insoluble LB, they lose all their functional capabilities like SNARE-complex regulation[3]. A study by Volcipelli-Daley and his team examined this by inoculating small quantities of exogenous α-Syn fibrils into wild-type (WT) mice SNpC. Within two weeks after the addition of α-Syn fibrils, vesiscle-associated SNARE proteins like synaptobrevin-II began to lose their ability to assemble along presynaptic terminals as expected. Interestingly they also detected marked reductions in a subpopulation of many synaptic proteins involved in SNARE complex such as Snap 25, VAMP2, and synapsin II. The team also confirmed that decreased SNARE-complex assembly by α-Syn fibrils translates into impairment in neural network activity[15]. In comparison to control rat SNpC neurons treated with PBS, SNpC neurons treated with α-Syn fibrils showed significant reduction in both excitatory tone of the network as well as network-wide synchronous activity. Both the reduced excitatory tone and synchronicity within the neural network can be explained by an impairment in individual neuron’s ability to send synaptic signals to neighboring cells.

In addition to reduced synaptic abilities, neurons affected with LB can degenerate when intracellular accumulations of LB reach a critical level. Studies have found that neurodegeneration tends to initiate once 1) the neurons start to lose their synaptic transmissions and 2) LB accumulation occurs within the neuron body. LB formation typically initiate within the presynaptic terminal regions where α-Syn levels are at the highest, but within days they can spread towards the neuron body[6,15]. When LB start to accumulate within the neuron body they become highly toxic, affecting mitochondrial functions, ER-Golgi trafficking, and many other intracellular processes[5,19]. The combined effects of intracellular toxicity and reduced synaptic transmissions are believed to disrupt overall integrity of neurons and initiate neurodegeneration.

3) Clinical correlation with Parkinson's disorder symptoms

Neurodegeneration of DA neurons and motor dysfunction
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Results following a single intrastriatal inoculation of preformed α-Syn fibril into WT mice.
Mice displayed progressive deficit in motor performances of (A) rotarod and (B) wire hang tests;
control mice (PBS) showed no significant difference for both measures. (C) Immunoblot analysis
of striata using antibodies against DA transporters (DAT) shows similar decline in DA neuron
density within striata. Adapted from Luk et al. (2012).

Converging lines of evidences indicate that initiation of α-Syn pathology plays a central role in PD: 1) All forms of PD shows presence of LB within SNpC neurons; 2) Only SNpC and few connected cortical areas are affected with LB; 3) There is a reduction in striatal DA levels and selective loss of SNpC DA neurons[5]. While it is still unclear if PD onsets can be explained solely by α-Syn pathology, recent study by Luk and his team have demonstrated that α-Syn pathology are fully capable of inducing all of Parkinsonian symptoms in mice models.

3.1 Early motor dysfunction and late stage Parkinson's dementia

Luk et al. was the first to demonstrate that a single intrastrial inoculation of preformed α-Syn fibrils into SNpC of a wild-type mice in vivo is sufficient in inducing Parkinsonian motor dysfunctions. This was achieved by examining the relationship between the degree of SNpC DA neuron loss and motor performance at 30, 90, and 180 days post injection. Results showed that the amount of reduction in the number of SNpC DA neurons and motor performances were positively dependent on time to a very similar degree[2]. This implies that neurodegeneration of SNpC DA neurons by α-Syn fibril pathology can successfully translate into progressive motor performance deterioration that are characteristic of PD. Unfortunately, it is very difficult to determine if the motor deterioration seen in mice models are equivalent to those seen in human PD patients.

Moreover, emerging evidences suggests that α-Syn pathology may also explain the high occurence of dementia for PD patients. Many PD patients with late-onset dementia show signs of LB formation and neurodegeneration within neocortical areas that are connected to SNpC neurons. Based on this pathophysiology, it is speculated that PD dementia may be a result of transmission of α-Syn fibrils from SNpC to cortical areas followed by α-Syn mediated synaptic dysfunction and neurodegeneration[21,22].

4) Potential therapeutic targets in α-pathology

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Images of Lewy bodies in grafted DA cells stained with antibodies
against α-synuclein. The grafted neurons were initially without any traces
of Lewy bodies during implantation into brains of two patients with PD 16 (left)
and 11(right) years before this post-mortem images were taken.
Adapted from Brundin et al. (2008)

Currently, there are no known therapeutic methods that can effectively halt the progression of Parkinson’s disease because very little was understood about its etiology and pathogenesis in the past. However, emerging insights into α-Syn-mediated neurodegeneration offers opportunities for novel therapeutic interventions based on disrupting the cascade of events leading to LB formation and α-Syn fibril propagation. Already many suggestions for therapeutic targets have been made that targets different components of α-Syn pathology[5]. Lashuel et al. suggests an approach in reducing baseline level of α-Syn expression to prevent α-Syn fibrils. One way to do this may be by silencing SNCA gene with microRNA or promotor repressors[23]. Another approach could involve enhanced targetting of α-Syn for autophagy or proteosomes[5].

Novel insights into α-Syn also help to explain why past therapy attempt using grafts of neural tissue from healthy individuals were largely ineffective in treating PD. Grafted striatal tissue are not excluded from the pathological spread of α-Syn that are already present in PD patients; thus, grafted tissue will eventually degrade even if it did not normally contain α-Syn fibrils[24]. By extension, future researches on novel therapy can be suggested to stay away from the use of stem cells. Same explanation used to explain ineffectiveness of neural tissue grafts can be used to explain the rationale for disinterest in stem cell treatments[8].

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