Parkinson’s disease : Alpha synuclein’s non-amyloidal component (NAC) aids the protein’s movement through axons


Research by University at Buffalo biologists is providing new insights into alpha-synuclein, a small acidic protein associated with Parkinson’s disease.

Alpha-synuclein is known to form abnormal clumps in the brains of patients with Parkinson’s, but scientists are still trying to understand how and why this happens.

The new study explores alpha-synuclein’s basic properties, with a focus on a section of the protein known as the non-amyloidal component (NAC). The research was done on fruit fly larvae that were genetically engineered to produce both normal and mutated forms of human alpha-synuclein.

The study, led by University at Buffalo biologist Shermali Gunawardena, was published on Jan. 10 in the journal Frontiers in Cellular Neuroscience.

Some key findings:

  • The NAC region appears to aid alpha-synuclein in moving through pathways called axons that run from one area of a neuron to another. When the NAC region was missing, alpha-synuclein did not move within axons.
  • Alpha-synuclein that’s missing the NAC region may help to prevent unwanted aggregates of the protein. In experiments, Gunawardena’s team showed that it’s possible — at least in fruit flies — to prevent some key problems that occur when too much alpha-synuclein is produced: clumping of the protein; abnormalities in the structure of synapses, which form connections between neurons; and a decrease in the speed at which larvae crawl. The scientists found that when the larvae are engineered to produce both excess alpha-synuclein and a version of alpha-synuclein with the NAC region missing, the larvae crawl normally, the protein doesn’t aggregate, and the synapses are normal.

“We show that in fruit fly larvae, we’re able to prevent some problems mimicking symptoms of Parkinson’s disease, such as accumulation of alpha-synuclein in neurons,” says Gunawardena, PhD, associate professor of biological sciences in the UB College of Arts and Sciences.

This shows alpha synuclein

An epifluorescence microscope image shows clumps of human alpha-synuclein aggregating in the neurons of a fruit fly larva. Image is credited to Anderson, Hirpa, Zheng, Banerjee and Gunawardena, Frontiers in Cellular Neuroscience.

“Our work highlights a potential early treatment strategy for Parkinson’s disease that would leverage the use of deletion of the NAC region,” Gunawardena adds. “One reason this study is important is because it shows rescue of alpha-synuclein aggregates, synaptic morphological defects and locomotion defects seen in Parkinson’s disease in the context of a whole organism.”

The paper’s first author is Eric N. Anderson, a UB PhD graduate in biological sciences. In addition to Anderson and Gunawardena, co-authors include UB undergraduate students Delnessaw Hirpa and Kan Hong Zheng, who have since completed their degrees, as well as Rupkatha Banerjee, a current UB PhD candidate in biological sciences.

Funding: The research was partially funded by the National Institute of Neurological Disorders and Stroke in the National Institutes of Health, and the John R. Oishei Foundation. Anderson was supported by the Arthur A. Schomburg Fellowship Program at UB.


Parkinson’s disease (PD) is the second most common neurodegenerative disorder, after Alzheimer’s Disease, and the most common movement disorder (Mhyre et al., 2012). PD has a prevalence of approximately 0.5–1% among individuals 65–69 years of age, rising to 1–3% among persons 80 years of age and older (Nussbaum and Ellis, 2003).

PD is characterized by motor symptoms including tremor, rigidity, bradykinesia, postural instability, gait and balance impairment and non-motor symptoms such as cognitive decline, autonomic impairment, rapid eye movement behavioral sleep disorder, constipation, and other behavioral disturbances (Stacy, 2011; Obeso et al., 2017).

Additionally, its hallmark feature is the accumulation of alpha-synuclein (α-syn) that results in the formation of proteinaceous cytoplasmic inclusions, known as Lewy-bodies and Lewy neurites (LBs/LNs; Jakes et al., 1994).

Current therapeutic approaches are directed at controlling symptoms and delaying the progression of the disease for as long as possible (Poewe, 2009). Therefore, the development of disease-modifying therapeutics is extremely attractive in experimental and clinical research in PD.

Multiple lines of evidence support the critical importance of α-syn in PD pathogenesis. The intraneuronal proteinaceous cytoplasmic inclusions now known as LBs, the hallmark of PD, were first described by Lewy in 1912 (Lewy, 1912; Goedert et al., 2013). Several decades later, in 1996, the first link between α-syn and the PD phenotype was established with the identification of the A53T point mutation on chromosome 4q21-23 (Polymeropoulos et al., 1996). The gene encoding α-syn (SNCA) was identified the following year (Polymeropoulos et al., 1997). Subsequently, α-syn was identified as a major component of LBs and LNs (Spillantini et al., 1997).

While we have since achieved a greater understanding of PD pathogenesis, the exact mechanisms elucidating the nature of progressive dopaminergic cell loss in the substantia nigra (SN) pars compacta remain to be determined. In this review article, we examine advances in our current understanding of the pivotal role of α-syn in PD pathogenesis, including pathways implicated in α-syn toxicity, suggested seeding and propagation mechanisms that underlie cell-to-cell transmission between neighboring neurons, and viability of potential disease-modifying therapeutics targeted against pathological α-syn species.


Although the full extent of the physiological function for α-syn is yet to be revealed and there may be conflicting findings in need of resolution, α-syn is involved in synaptic activity through regulation of vesicle docking, fusion, and neurotransmitter release (Ghiglieri et al., 2018). α-syn is an abundant 14 kDa protein consisting of 140 amino acids and comprised of three domains: (1) an N-terminal lipid-binding alpha-helix; (2) a non-amyloid-component (NAC); and (3) an acidic C-terminal tail (Lashuel et al., 2013).

The N-terminal domain of α-syn is characterized by a series of seven 11-residue imperfect repeats, each based upon a highly conserved KTKEGV hexameric motif that is also observed in the α-helical domain of apolipoproteins (Davidson et al., 1998; Bussell and Eliezer, 2003; Bussell et al., 2005). This similar architecture allows α-syn to mediate lipid interactions in a similar way to how apolipoproteins do, inserting its amphipathic helices into lipid membranes to influence their curvature (Davidson et al., 1998).

The central region (residues 61–95), also known as the NAC domain, can form cross β-sheets and consists of a highly hydrophobic sequence underlying its high propensity for aggregation and leading to protofibril and fibril formation (Uéda et al., 1993; Giasson et al., 2001; Tuttle et al., 2016).

The predominantly unstructured conformation of α-syn makes it a target for various post-translational modifications such as phosphorylation. Indeed phosphorylation of Serine 129 in the C-terminal domain of α-syn has been associated with an increased propensity of aggregate formation (Samuel et al., 2016).

A number of studies have reported aberrant accumulation of phosphorylated α-syn at the Serine-129 residue (pS129) in the brains of PD patients, as well as in animal models of synucleinopathies (Tenreiro et al., 2014; Oueslati, 2016).

While only a small fraction of α-syn (~4%) is phosphorylated in healthy brains, substantial accumulation of pS129 (~90%) is observed in brains with Lewy pathology, implicating a potentially important association between this posttranslational modification and the accumulation of α-syn aggregates concomitant with LB formation and neurodegeneration (Fujiwara et al., 2002; Hasegawa et al., 2002).

Further insight on the significance of S129 phosphorylation on α-syn aggregation, LB formation, and neurotoxicity may provide insight into the nature of PD pathogenesis and PD and related disorders.

While numerous lines of evidence indicate that monomeric α-syn is not toxic, several studies highlight α-syn oligomers and fibrils as the species responsible for α-syn toxicity (Neumann et al., 2004; Outeiro et al., 2008; Peelaerts et al., 2015), and it has been shown that overexpression or triplication of synuclein is sufficient for α-syn aggregation to take place (Outeiro et al., 2008; Zambon et al., 2019).

There are conflicting findings on the nature of the native state of α-syn. Although the majority of studies suggest that cytosolic α-syn is present within cells as an intrinsically unfolded monomer, alternative hypotheses have proposed that it exists as a tetrameric alpha-helical oligomer that is resistant to fibrillization and thus distinct from pathological variants (Bartels et al., 2011; Wang et al., 2011; Fauvet et al., 2012; Burré et al., 2013; Smaldone et al., 2015). α-Syn can adopt an α-helical conformation in association with biological membranes or remain in an intrinsically unfolded state in the cytosol, suggesting it has different functions in different subcellular locations depending on its dynamic structure (Eliezer et al., 2001; Ramakrishnan et al., 2006; Ullman et al., 2011).

Indeed, a recent investigation lends further support to this notion using chemical crosslinking and FRET experiments to demonstrate that α-syn multimerizes into a tetrameric complex upon binding cellular membranes and that it is this tetrameric membrane-bound α-syn that mediates SNAP receptor (SNARE) complex assembly and that functions as a molecular chaperone for these complexes at the presynaptic membrane (Burré et al., 2014). These findings should be interpreted with caution as evidence for tetrameric α-syn stems from crosslinked samples.

Pathways Implicated in Toxicity of α-Syn

Disruption of several cellular pathways leads to the loss of dopaminergic neurons in PD, including synaptic vesicle recycling, mitochondrial function, oxidative stress, endoplasmic reticulum (ER) stress, and autophagy-lysosomal pathway (ALP) function (Figure 1).

The accumulation of α-syn into prefibrillar forms, and then its assembly into higher molecular weight aggregates, induces cellular toxicity and maybe the greatest contributor to pathogenesis in PD (Bengoa-Vergniory et al., 2017). The increased cellular toxic burden caused by aggregated α-syn may arise from overexpression of the protein, genetic multiplication, or impairment to normal protein clearance mechanisms such as autophagy (Alegre-Abarrategui et al., 2019).

Synaptic Vesicle Impairment

α-syn is typically localized to the presynaptic terminal where it associates with synaptic vesicles and influences membrane curvature (Maroteaux et al., 1988; Wong and Krainc, 2017). There it is known to promote synaptic vesicle fusion and other processes in synaptic-vesicle trafficking through its interactions with the synaptobrevin-2 component of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) complex. Large α-syn oligomers preferentially bind synaptobrevin-2 and may disrupt SNARE complex assembly, synaptic-vesicle motility, and dopamine release (Burré et al., 2010; Choi et al., 2013).

Additionally, loss of α-syn has been associated with an increase in dopaminergic release (Senior et al., 2008; Anwar et al., 2011), and so the aggregation of α-syn could also lead to loss of function effects at the synapse.

Mitochondrial Dysfunction

Dopaminergic neurons are uniquely susceptible to mitochondrial dysfunction due to their high energy demands and increased exposure to oxidative stress (Valente et al., 2004; Ricciardi et al., 2014). The selective vulnerability of these neurons was first recognized following the finding that the mitochondrial neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) resulted in the cell death of SN DA neurons in humans (Langston et al., 1983).

Moreover, MPTP was shown to be toxic to DA neurons in mouse and non-human primate models of PD (Przedborski et al., 2001). α-Syn oligomers can also inhibit the import of proteins, including some subunits of complex I, into the mitochondria by binding to the translocase of the outer membrane (TOM20) and inhibiting its interaction with the co-receptor TOM22 (Di Maio et al., 2016).

Overexpression of the α-syn mutants A53T or A30P has been shown to increase the aggregation of α-syn in human neuroblastoma cells (Parihar et al., 2009). In these cells, immunogold electron transmission microscopy revealed the localization of α-syn aggregates within the mitochondria of overexpressing cells, which exhibited decreased mitochondrial transmembrane potential and limited cellular respiration concomitant with increased production of reactive oxygen species (ROS).

The proximity between the mitochondrial electron transport chain (ETC) and mitochondria DNA (mtDNA) increases the vulnerability of mutations in mtDNA due to ROS, especially during mitochondrial dysfunction, such as in complex I inhibition (Davis et al., 1979). Indeed, mutations in mtDNA or impairment to the ETC cause mitochondrial dysfunction and energy depletion. Damaged mitochondria result in electron leakage, produce increased ROS, and release cytochrome C leading to activation of caspase-3, caspase-9, and other pro-apoptotic factors ultimately leading to cell death (Brustovetsky et al., 2002).

Additionally, α-syn can interfere with mitochondrial membrane fusion and fission, resulting in fragmentation of mitochondria, and it can inhibit mitophagy and complex I activity, and disrupt mitochondrial membrane potential (Cole et al., 2008; Devi et al., 2008; Chen and Chan, 2009). While this review concentrates on PD it is worth noting that oligodendrocytes, which are heavily loaded with pathological glial cytoplasmic inclusions in multiple system atrophy (MSA) patients, are also cells with high endogenous α-syn and a high metabolic profile.

It is interesting to consider the parallels between these two synucleinopathies and cellular selective vulnerability (Alegre-Abarrategui et al., 2019).

Oxidative Stress

Failure of the antioxidant proteins regulating ROS levels, like superoxide dismutase (SOD) and glutathione (GSH), results in oxidative stress, which may have deleterious effects inside cells (Indo et al., 2015). Interestingly, the SN appeared to contain twice as much oxidized proteins as compared with the caudate, putamen, and frontal cortex in the post-mortem brains of healthy individuals, suggesting that the increased susceptibility of SN to oxidative stress may contribute to the selective degeneration of nigral DA neurons (Floor and Wetzel, 2002).

Unregulated oxidation of intracellular macromolecules and organelles can cause cellular damage and may lead to cell death (Wiseman and Halliwell, 1996; Rego and Oliveira, 2003). As one of the major producers of ROS, mitochondria are particularly vulnerable to oxidative stress-induced cytotoxicity, especially considering mtDNA is not protected by histone proteins as seen in nuclear DNA (Richter et al., 1988).

As mentioned above, α-syn can interfere with translocation of mitochondrial-target proteins, thereby disrupting the proper functioning of the ETC, and leading to elevated levels of intracellular oxidative stress (Di Maio et al., 2016). Interestingly, while α-syn toxicity is implicated in increasing cellular oxidative stress, it has also been suggested that oxidative stress can induce α-syn toxicity.

Excessive exposure to oxidative stress causes lipid peroxidation of polyunsaturated fatty acids, which in turn leads to the formation of 4-hyroxy-2-nonenal, a product that has been shown to hamper fibrillization of α-syn and promote the formation of secondary beta sheets and toxic soluble oligomers in a dose-dependent manner (Dexter et al., 1989; Qin et al., 2007; Bae et al., 2013).

Incubation of α-syn with cytochrome c/H2O2 leads to the oxidative stress-induced aggregation of α-syn by crosslinking α-syn tyrosine residues through dityrosine bonding (Hashimoto et al., 1999; Ruf et al., 2008). Moreover, colocalization of cytochrome c and α-syn was reported in the LB of patients with PD (Hashimoto et al., 1999).

University at Buffalo


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