The results of studies in live mice and in mouse and human brain tissue suggest that one of the normal functions of alpha-synuclein, a protein found in the characteristic Lewy bodies that form in the brains of patients with Parkinson’s disease (PD), may to help repair double-stranded breaks (DSB) in DNA. The research, by a team at Oregon Health & Science University (OHSU) indicates that the formation of Lewy bodies effectively pulls alpha-synuclein out from the nerve cell nucleus into the cytoplasm. The protein is then prevented from carrying out its normal nuclear role of DNA repair, which leads to increased DSBs, and ultimately the death of brain cells.
“It may be the loss of that function that’s killing that cell,” said senior author Vivek Unni, MD, PhD, an associate professor of neurology in the OHSU School of Medicine. The researchers say their findings point to the potential for designing treatments that replace alpha-synuclein’s function in people with PD or related neurodegenerative disorders. Unni and colleagues at OHSU, and the University of Pennsylvania School of Medicine, report their findings today in Scientific Reports, in a paper titled, “Alpha-synuclein is a DNA binding protein that modulates DNA repair with implications for Lew body disorder.”
Alpha-synuclein is a small, 140-amino acid protein that is found at the presynaptic terminals of nerve cells, where it regulates the cycling of vesicles that carry neurotransmitters, the authors explained. As well as its role in healthy neurons, the protein is also found in the cytoplasmic Lewy bodies that are characteristic of neurodegenerative diseases such as PD and other Lewy body disorders. What isn’t known is how these Lewy body protein aggregates are linked with cell death. “It is currently unclear how cytoplasmic aggregation of alpha-synuclein into Lewy body pathology is related to the neuronal death that characterizes these neurodegenerative diseases,” the authors stated.
Recent studies have indicated that in addition to its cytoplasmic role in vesicle cycling, alpha-synuclein is also found in the cell nucleus. However, its function in this cellular compartment isn’t known. It has been proposed that the protein may directly or indirectly interact with DNA but, as the OHSU team notes, “… we still lack a clear picture of why alpha-synuclein is present in the nucleus of many neural and non-neural cell types, and how its nuclear function/s might be disrupted in neurodegenerative diseases where cytoplasmic alpha-synuclein aggregation occurs.”
The team had previously used a technique called multiphoton imaging in a live mouse model of PD to show that as the Lewy bodies form in neurons, soluble alpha-synuclein is lost from both the cytoplasm and the nucleus. “This suggests that cytoplasmic alpha-synuclein aggregation may decrease the amount of protein available for any nuclear or cytoplasmic role it may play, contributing to loss-of-function.” Alpha-synuclein demonstrates similarities to a known DNA repair protein, and studies have also shown that PD is linked with oxidative stress, which damages DNA, and that oxidative stress increases nuclear alpha-synuclein levels. With this evidence in mind, the team “… set out to test whether alpha-synuclein plays a role in regulating normal cellular responses to DNA damage and whether this function could be compromised in Lewy inclusion-bearing cells.”
The researchers used a combination of techniques, including immunocytochemistry, nuclear fractionation, western blotting, and neutral comet assay, to investigate alpha-synuclein function in both normal (wild-type; WT) cells and mouse neurons that had been genetically engineered to lack the SNCA gene that codes for alpha-synuclein. The tests showed that alpha-synuclein co-localizes with markers of DNA damage, and there was also direct evidence that alpha-synuclein plays a role in regulating cellular repair responses to DSBs that had been induced experimentally using the chemical bleomycin. “We found that bleomycin-induced DSBs were repaired more quickly in WT than in SNCA knock-out cells, suggesting that the presence of alpha-synuclein facilitates DSB repair,” the investigators wrote.
Further experiments showed that alpha-synuclein directly binds to DNA, and may be involved in rejoining the broken DNA ends. Importantly, mouse SNCA knockout cells exhibited increased levels of DSBs, which could be reduced by the introduction of surrogate, human alpha-synuclein. “These data suggest that removing alpha-synuclein in mouse brain increases DSB levels, and that this effect can be rescued by addition of the human alpha-synuclein protein, which compensates for the loss of mouse alpha-synuclein in a cell autonomous manner.”
The researchers developed a technique that allowed them to visualize fluorescently tagged alpha-synuclein in real time, in response to laser-induced DNA damage. “Using multiphoton microscopy through a cranial window, we generated small, subnuclear regions of DNA damage using focused, short laser pulses, and then measured the immediate response of GFP-tagged human alpha-synuclein (Syn-GFP).” These experiments confirmed that when DSBs were induced in the mouse cortex, fluorescently tagged forms of alpha-synuclein were rapidly recruited to the sites of damage.
Interestingly, tests in mouse cortical neurons and in post-mortem samples of brain amygdala tissue from patients with Lewy body dementia directly linked Lewy body aggregates with increased levels of DSBs. “This analysis showed a significant correlation between the overall Lewy pathology burden and DSB level in the amygdala, suggesting that in human disease, loss of soluble alpha-synuclein through aggregation into cytoplasmic Lewy pathology is associated with increased DSBs.”
The researchers say their collective results point to a model whereby alpha-synuclein aggregation into cytoplasmic Lewy bodies in neurons may upset the cell’s natural repair mechanisms. “We now demonstrate that part of the mechanism of cell death in Lewy inclusion-bearing neurons could be due to a loss of nuclear alpha-synuclein’s activity in the DSB repair pathway,” they concluded. “We suggest that in Lewy body forms of neurodegeneration, cytoplasmic aggregation of alpha-synuclein reduces soluble nuclear alpha-synuclein levels, potentially inducing a loss-of-function that causes increased DSBs and leading to neuronal programmed cell death.”
The investigators say their findings provide new insights into the importance of alpha-synuclein in relevant forms of neutodegeneration and could point to new therapeutic approaches. “We also postulate that this previously unknown set of functions for alpha-synuclein in DSB repair may extend across synuclein family members … This model could inform development of new treatments for Lewy body disorders by targeting alpha-synuclein-mediated DNA repair mechanisms … Further understanding of the synuclein protein family’s DNA repair functions could help facilitate the development of new therapeutic targets for several important forms of neurodegeneration and cancer.”
Unni, who sees patients in the OHSU Parkinson Center and Movement Disorders Program, said he hopes that the findings will lead to the development of methods for delivering alpha-synuclein proteins into the nucleus of cells, or designing methods to replace its function. “This is the first time that anyone has discovered one of its functions is DNA repair,” he said. “That’s critical for cell survival, and it appears to be a function that’s lost in Parkinson’s disease.”