Paper in Medicine: Parkinson's disease. Alpha-synuclein.

Parkinson's disease (PD) a devastating neurodegenerative disorder. It is a disease of the nervous system that affects mostly older people over the age of 50 years. It can also affect young people from all races and geographical locations (Reeve, Simcox, & Turnbull, 2014). PD is a progressive condition which implies that it gets worse with time and it does not have a cure. The condition causes a heavy burden both on those affected, as well as their families. However, effective treatments that can relieve its symptoms are available

The prevalence of PD has not been accurately determined. Different studies used different diagnostic criteria, methodology, and study population. Nevertheless, it is generally accepted that the prevalence rate of 1%2% exists in the population older than 60 years, or 0.3% in the general population (Pedersen, Larsen, Alves, & Aarsland, 2009). A study by Dorsey and colleagues (2007) reported a prevalence of more than 4 million Parkinson's disease patients globally in 2005. According to Pedersen, Larsen, Alves, & Aarsland (2009), the annual incidence rates of PD range between 8.6 and 19 persons per 100,000. de Lau and Breteler (2006) reported that Parkinson's disease is the second most common neurodegenerative disorder after Alzheimers disease. PD is predominantly an old age disease (usually over 40 years of age). Schrag and Schott (2006) suggested that an early onset of PD increases the probability that genetic factors may be involved.

The cause of Parkinson's disease is still not known. However, it is thought that complex interactions between genetic and environmental factors might be responsible for the disease. Such as, the gene SNCA in humans. These genes are found and conserved only in vertebrates.

Previous studies report different risk factors for some case of Parkinson's disease such as family history, exposure to pesticides and other toxins, and oophorectomy. However, age remains the most important risk factor known to date (de Lau and Breteler 2006). Therefore, the prevalence of PD is expected to increase with the aging population, which may raise important social security questions in countries with an aging population (Dorsey et al. 2007). Recent studies claim that ceasing of smoking, drinking coffee or black tea, and administration of nonsteroidal anti-inflammatory drugs could be effective protective measures against the condition (de Lau and Breteler 2006). Other effective management remedies include stretching and flexibility exercises. Such exercises serve to relieve the stiffness of muscles in patients suffering from Parkinsons disease. In food and nutrition, proteins serve to inhibit the pharmacodynamics and pharmacokinetics of Parkinsons medications; limiting the amount of proteins ingested is advisable.

The relationship between Parkinsons disease and 61-95 Alpha-Synuclein is such that the latter contributes to the development of the prior in several ways. Alpha-synuclein is a presynaptic neuronal protein genetically linked to PD. Alpha-synuclein is also thought to be neuropathologically linked to PD because its aberrant soluble oligomeric conformations (also known as protofibrils) are toxic in nature and are responsible for the disruption of cellular homeostasis

Alpha-synuclein

Alpha-synuclein (a-syn) is a synaptic protein expressed abundantly in the central nervous system. a-syn has been linked to several neurodegenerative diseases collectively known as synucleinopathies. Alpha-synuclein is the gene encoding a 140-amino acid presynaptic protein. Rare but possible mutations in this gene cause Parkinsons disease. The same alpha-synuclein can bring about Lewy bodies when they accumulate inside the neuronal cytoplasmic inclusions causing related disorders such as dementia These diseases include Alzheimer's disease, dementia with Lewy bodies (abnormal aggregates of protein developing inside nerve cells in PD), multiple system atrophy, and Parkinson's disease. Alpha-Synuclein has been found high concentration at presynaptic terminals in both soluble and membrane-associated fractions of the brain. Iwai . (1995) suggested that this protein accounts for as much as 1% of the total protein in soluble cytosolic brain fractions. a-synuclein is thought to be involved in several functions such as fatty acid binding, neurotransmitter vesicles, neuronal survival, physiological regulation of specific enzymes, transporters, and synaptic vesicle release and trafficking (Dev., 2003). da Costa, Ancolio, and Checler (2000) demonstrated the involvement of this protein in the control of the neuronal apoptotic response and in the protection of neurons from various apoptotic stimuli. Greten-Harrison and associates (2010) conducted a study in which alpha, beta, and gamma components of synuclein were knocked out in mice. The researchers arrived at the conclusion that synucleins are important to the long-term neuronal function. Other studies have indicated that a-Synuclein interact with at least 30 proteins, which signifies its importance in cell signaling (Dev, 2003).

The structure of alpha-synuclein (a-syn) can be divided into three regions consisting of amino acid residues 160, (coding for amphipathic a-helices) with a conserved motif (KTKEGV) which are Lysine, Threonine, Lysine, Glutamic acid and Valine, and residues 6195, which contain the hydrophobic and highly amyloidogenic non-amyloid-beta component region and three additional KTKEGV repeats; and residues 96140, which contain acidic residues, such as Aspartic acid, Alanine, Phenylalanine and prolines C-terminal region (See Figure 1). Weinreb. (1996) demonstrated that alpha-synuclein has a larger Stokes radius, also known as Stokes-Einsten radius is the radius of a sphere that possesses the same diffusion rate as the original solute, and sediments more slowly in solution than globular proteins of similar molecular mass. These properties suggested that alpha-synuclein is either unfolded or elongated (Breydo, Wu, & Uversky, 2012). Over the course of time, researchers have supported the notion that this protein is natively unfolded (Uversky, Li, & Fink, 2001).

Figure 1. Alpha-Synuclein the lipid binding domain with seven highly conserved motifs (Source: Burre, 2015).

The structure the non-amyloid-beta Component of alpha-synuclein

The 140-amino acid protein is encoded by the SNCA gene which is found and conserved only in vertebrates. The protein does not have a defined structure in aqueous solutions (natively unfolded protein). However, the protein forms a-helical structures on binding to negatively charged lipids and b-sheet-rich structures on prolonged periods of incubation. As stated earlier, alpha-synuclein comprises three regions:

(a) an amino terminus of 1 60 residues, containing apolipoprotein lipid-binding motifs that form amphiphilic helices which in turn form a-helical structures on membrane binding.

(b) a central hydrophobic region (6195), known as NAC (non-amyloid-beta component) that confers the b-sheet structures.

(c) a highly negatively charged but unstructured carboxyl terminus.

The first two regions (residues 160 and residues 6195) consist of a membrane-binding domain, whereas residues 96140 is thought to comprise protein-protein and protein-small molecule interaction sites.

Figure 2: Schematic structure of a-synuclein (Source: Stefanis, 2012)

Figure 2: The structure of NAC region. The amino acid sequence of the NAC fragment, which constitutes the more hydrophobic region of a-synuclein (in bold). Positively and negatively charged residues are indicated.

Figure 3: Amino acid determinant of (61-95) a-synuclein aggregation (Source: Uversky and Fink, 2002).

Alpha-synuclein shows partial compactness typical of the intrinsically disordered proteins. It is containing preformed binding elements that are most probably involved in intramolecular interactions. Thus, a polypeptide chain of intrinsically disordered proteins misfolds to isolate the preformed elements thereby preventing these elements from interactions with non-native binding partners (Breydo, Wu, & Uversky, 2012). Zhao, Cascio, Sawaya, & Eisenberg (2011) crystallized several a-synuclein peptides fused to maltose binding protein for increased solubility. Zhao and associates reported that residues 113 and 2034 of a-synuclein formed a-helices, whereas the rest of the sequence remained unfolded. Other studies utilizing nuclear magnetic resonance techniques have demonstrated how alpha-synuclein is largely unfolded without tertiary structure. Additionally, (Breydo, Wu, & Uversky, 2012). suggest that the protein is relatively compact compared and the presence of transient contacts within the protein structure. This compactness is due to the clustering of hydrophobic residues of the protein. The protein is thus a highly dynamic assembly of preferred conformations (Bussell & Eliezer, 2001). Bussell & Eliezer (2001) observed that the regions around residues 20 and 120 had low mobility suggesting the existence of long-range contacts that possibly confer a more compact ensemble of conformations. The long-range interactions play an inhibitory role on the spontaneous (61-95) a-synuclein oligomerization and aggregation.

High-Performance Liquid Chromatography (HPLC)Liquid chromatography is a prominent separation technique used before mass spectrometric analysis of alpha-synuclein (Fekete, Schappler, Veuthey, & Guillarme 2014). The oligomer of

(61-95) alpha-synuclein is purified by HPLC. The highest efficiency of separation can be achieved by ultra-high-pressure liquid chromatography (UHPLC). It is important to use conditions that purify the protein using high-performance chromatography such as UHPLC and reversed phase-HPLC. The highest possible capabilities are achieved by addressing the efficiency of separation columns in order to minimize co-elution and hence matrix effects in Liquid Chromatography-Mass Spectroscopy. Matrix effects have become a major concern in LCMS. The consequences of matrix effects include detrimentally affecting the accuracy, specificity and sensitivity of the test result. (Jakimska, Kot-Wasik, and Namiesnik, 2014). It is important to deal with issues of backpressures when using new generation UHPLC devices to carry out the molecular separation under these demanding conditions (Cabooter, 2008; Omamogho, 2011). These challenges can be addressed by using Hydrophilic Interaction Liquid Chromatography separations that provide greater sensitivity and reduces matrix effects (Kohler, Derks, & Giera, 2016).

Another variant of the normal phase liquid chromatography is Hydrophilic Interaction Liquid Chromatography. The application of HILC has increased dramatically with the numerous improvements in stationary phase material and a better understanding of the retention principles of the Chromatographic technique (Beaudoin & Gangl, 2016). Because HILC separations require organic solvent rich mobile phases, the use of hydrophilic interaction liquid chromatography provides greater sensitivity and reduces effects. Kohler, Derks, & Giera (2016) provide an extensive review of commercially available HPLC stationary phases...