Biochemistry of Alpha-synuclein Aggregation and Lewy Body Formation

It’s fairly clear to see that there is a lot of uncertainty surrounding what is occurring mechanistically in Parkinson’s, and even in the healthy state of the substantia nigra and dopaminergic neurons. This is the reason why no consistently effective therapeutic has been identified in order to treat those suffering from the debilitating effects of PD. With that said, researchers have been working tirelessly to gain a better understanding of alpha-synuclein and the dopaminergic neurons that they inhabit in both the healthy and diseased states so we can inch our way closer to being able to better treat PD.

Formation of Lewy bodies

            Lewy bodies themselves are the hallmark of Parkinson’s disease pathogenesis. However, the reason for their formation and the direct cause of formation in idiopathic PD is unknown. The steps by which this occurs do seem to be more well understood. The process initiates with the misfolding of the monomeric soluble state of the protein into an oligomeric state. From here, the oligomer forms a proto fibril, and a subsequent elongation process leads to the fibrillary aggregate form of alpha-syn. This fibrillary is then what makes up the Lewy bodies. Although alpha-syn makes up a majority of the LBs, they are often also made up of other neuronal organelles such as mitochondria, golgi bodies, vesicles and endoplasmic reticulum. The Lewy bodies then are able to wreak havoc within the dopaminergic neurons, and ultimately cause cell death.2326While the cause of the initial misfolding is unknown, it has been suggested that the fibrilization process likely involves post-translational modifications that result in the alpha-syn fibers.27This is based off of the observation that around 90% of alpha-syn aggregates in Lewy bodies of PD have been found to have been phosphorylated at Ser129. In contrast, only 4% of healthy monomeric alpha-syn is phosphorylated at this site. Additionally, the inhibition of GRK6, the kinase responsible for Ser129 phosphorylation, results in a reduction of the ability for alpha-syn to exacerbate toxicity in a mouse model.28

Image of a Lewy body in vivo.
https://www.neurologyadvisor.com/conference-highlights/aan-2019-conference/long-term-efficacy-for-parkinsonism-and-safety-of-zonisamide-in-patients-with-lewy-body-dementia-examined/

Lewy body / Oligomer Effects on Neurons

            The method by which the neurons of the substantia nigra are degraded in PD pathology is somewhat disputed. On one side, some believe that it is the Lewy bodies that are directly impacting the neurons, whereas others believe that it’s actually the oligomeric state of alpha-syn that is toxic to the neurons.

            For the oligomeric argument, a 2014 study into the impacts of overexpression of alpha-synuclein measured the size of aggregations that formed, in order to determine if merely oligomers or Lewy bodies were the main cause of neurodegeneration. They found that neuronal cell loss occurred as a result of the formation of oligomers, which were comprised of less than 10 monomers. Additionally, they found evidence of mitochondrial fragmentation, which could actually be one of the main causes of the observed neuronal cell loss.29Other studies have supported this claim after observing that toxic alpha-syn oligomers actually interacted with mitochondrial membranes, and were able to impair complex I function.30Recent evidence has also indicated that the alpha-syn oligomers are capable of propagating from one neuron to another, spreading the misfolded features to other alpha-syn monomers in a prion-like manner. A 2014 study found that endogenous alpha-syn accumulated in cell culture after being exposed to exogenous alpha-syn fibrils, meaning that aggregation was able to spread throughout the culure. The prion-like nature of alpha-syn could thus provide a therapeutic opportunity in the future.31,32

            As for the Lewy body argument, a 2020 study that looked at the development of a neuronal model that actually reproduces the many key events that lead to the formation of Lewy bodies in a way that very closely mimics the same structural and biochemical features of true Lewy bodies. As a result of this model’s development, the authors found that the formation of Lewy bodies led to mitochondrial alterations such as a decrease in membrane potential, a loss of mitochondrial density, and significant reductions in membrane proteins such as OPA1 and mitofusin 2. Additionally, Lewy body formation seems to impact proper synaptic function, as during the transition from fibrils to Lewy bodies, there was a reduction in the synaptic density, as well as a dysregulation of the synaptic transcriptome.33

            In combination, it would appear that the likely reason for neuronal loss in PD is due primarily to mitochondrial, and synaptic impairment. The method by which this occurs, either by the oligomeric state of alpha-syn aggregates or by the full formation of Lewy bodies, remains unclear.21

Genetics of Parkinson’s Disease

            Although genetic cases of Parkinson’s disease make up only 10% of the total observed cases, there is still nonetheless a clear association between genetics and PD, and a need for therapeutic strategies that can treat the various mutations. There are many than 20 genes that have been linked to PD, however some are better understood than others in terms of what is occurring biochemically.

            SNCA, which was the first gene to be associated with Parkinson’s disease and is designated as the PARK1 locus, is located on chromosome 4q22. There have been five reported missense mutations at this gene, including A53T, A30P, E46K, H50Q, and G51D.7,8All of the mutations are located within the N-terminal lipid binding domain of the alpha-syn protein. Due to this location, it has been suggested that these mutations interfere with the typical lipid binding function, and instead promote aggregation. In vitro studies support this claim, as A53T and E46K mutations both caused significant increases in rates of fibril formation, similar to that the aggregated alpha-syn in Lewy bodies. More recently, duplication and triplication mutations of the SNCA gene have been identified, natural aggregation seems to occur because of the severe increase in neuronal alpha-syn concentration.34 35

Figure indicating the effects of genetic mutations of the development of PD and formation of Lewy bodies. https://www.annualreviews.org/doi/pdf/10.1146/annurev-pathol-011110-130242

            The parkin gene, designated as the PARK2 locus, is another that is associated with PD progression, and is actually the second most common known cause of PD. The parkin gene is located on the 6q chromosome and encodes a ubiquitin E3 ligase that ubiquitinates proteins for degradation (where alpha-synuclein is one of its substrates), and is also involved in mitochondrial quality control.36 Mutations in this gene that cause PD have a wide range; including single base pair substitutions, to even deletions that are hundreds of nucleotides in length. The most common understanding of the mechanism behind PD progression associated with parkin mutation is loss of parkin function. A 2003 study believes that a complete loss of parkin function, possibly due to deletion of an allele, will result in neuronal death, without even the presence of Lewy body formation. However, an incomplete loss of parkin function, due only to a point mutation, will result in typical Parkinsonism with LB formation. In both cases, improper regulation of protein aggregation will occur, alpha-syn toxicity, proteasomal dysfunction, and oxidative stress will run rampant.37

References

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2 replies on “Biochemistry of Alpha-synuclein Aggregation and Lewy Body Formation”

Hi there – I’m in grad school at UPenn, and two labs next door to mine study alpha synuclein (the labs of James Petersson and Elizabeth Rhoades). From posters and presentations I’ve seen, I’ve heard of other posttranslational modifications like acetylation and arginylation, and I know firsthand from a rotation project that there are other phosphorylation sites.

Can you comment further on the breadth of post translational modifications that synuclein may undergo? You point out that PTMs may cause more aggregation behavior, but could they affect other cellular behavior (uptake, for example)? In the case of Ser129, modification is coincident with the disease state, but is the ‘healthy’ protein modified at all?

Thanks!
Zach Zimmerman

Thank you for the question, Zach! Ser129 phosphorylation appears to be the most understood and most recognized post-translation modification of alpha-syn; however there still remains a lot of unknown with this modification. Many other sites of phosphorylation have been identified, such as Y-133 and Y-136, however the impact that these phosphorylated sites have on aggregation is unknown. Other modifications at various sites such as ubiquitination and nitration, have actually been shown to accelerate oligomerization and subsequent fibril formation. In contrast, studies have suggested that O-GlcNAcylation at various residues has shown aggregation, but with slower kinetics in comparison to the unmodified protein. Linked below is a review article that looks more in depth at the varying PTM possibilities.
https://doi.org/10.3389/fnins.2019.00381

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