Large aggregates of misfolded α-synuclein form the characteristic Lewy bodies found in Parkinson's disease (PD) and other α-synucleinopathies. Smaller α-synuclein oligomers are suspected of being more toxic, but scientists have not been able to localize them in vivo, and their connection to neurodegeneration is still a mystery. In the March 7 Journal of Neuroscience, a pair of papers from the lab of Michael Lee, University of Minnesota, Minneapolis, addresses both problems. The researchers pinpoint the endoplasmic reticulum as an oligomer hotspot in neurons in α-synuclein transgenic mice and in humans with PD, and they finger oligomer-related cell stress in the neurons’ ultimate demise. The findings may have therapeutic implications for diseases related to α-synuclein.

Scientists have known for decades that α-synuclein plays a role in PD. Apart from the occurrence of Lewy bodies, dominant mutations in the α-synuclein gene cause a familial form of the disease. However, the protein's role in pathology is uncertain. As with amyloid-β in Alzheimer's disease (AD), recent evidence suggests that oligomers of α-synuclein are most toxic to neurons. But how and where would such oligomers act?

As outlined in the first paper, first author Emanuela Colla and colleagues went looking for α-synuclein oligomers in cell compartments of transgenic mice that express high amounts of mutant human α-synuclein (A53TαS mice; see Lee et al., 2002). They used certain antibodies—A11, which binds to many amyloidogenic oligomers but not fibrils or monomers (see Kayed et al., 2003), and FILΑ-1, which has been shown to pick out toxic α-synuclein oligomers and fibrils but not monomers (see Paleologou et al., 2009). With those, the scientists spotted α-synuclein oligomers in the lumen of the endoplasmic reticulum (ER) of mice nearing the age of onset of pathology, or already with pathology, but not in young, healthy transgenic mice. Samples of postmortem human brain from people afflicted with PD showed a similar elevation in α-synuclein in the ER fractions, relative to non-PD controls.

Tying α-synuclein to the ER is an important finding, said Wiep Scheper, Academic Medical Center, University of Amsterdam, the Netherlands. "That gives an idea of where you might interfere [therapeutically]," she said. "If you know where the protein is, it's easier to specifically target it." Scheper was not totally convinced the α-synuclein is contained completely within the ER lumen, however. She said the results could be consistent with α-synuclein being on the ER membrane as well, but she agreed that the protein is closely associated with this organelle.

Finding α-synuclein in the ER led the researchers to think that the oligomers cause the ER to function abnormally, said Lee. A backlog of misfolded proteins in the ER induces the unfolded protein response (UPR). In the UPR, the ER dials down general protein translation but ramps up production of select stress response factors to help fold and clear proteins. If stress persists, the ER can trigger programmed cell death, or apoptosis (see ARF related news story), and this has been tied to neurodegeneration (see review by Doyle et al., 2011). According to previous research, the UPR is activated in PD and tracks with the buildup of α-synuclein (see Hoozemans et al., 2007).

In their companion paper, Lee and colleagues asked whether the UPR might link α-synuclein with neural death in mouse models of PD. First author Colla and colleagues found that markers for UPR activation, ER chaperones, came up strongly in the damaged spinal cord just at the onset of neurological problems (a wobbly gait) in the A53TαS animals, compared to control mice. At later stages of disease, UPR markers rose in the mice’s brainstems as well. These chaperones predominated in neurons containing α-synuclein aggregates, and those neurons had dilated ER structures. This suggested to Lee and colleagues that α-synucleinopathy and neurodegeneration were tightly linked to the UPR.

Does the level of α-synuclein in the ER track with disease severity? To find out, the researchers fractionated neurons from A53TαS mice and examined the organelles. They found α-synuclein accumulated in the ER as the disease worsened. This was accompanied by more ER chaperones and a sensitization to extracellular ER stressors, such as tunicamycin and thapsigargin. Poly-ubiquitinated proteins abounded in the ER of these neurons and became more extreme with disease progression. This would suggest that ER-associated protein degradation, which helps the proteasome metabolize ubiquitin-tagged protein, was out of whack. These findings hinted that higher α-synuclein levels lead to ER malfunction. Interestingly, the rise of ER chaperones did not elicit phosphorylation of the eukaryotic translation initiation factor 2-α (eIF2α). Phosphorylated eIF2α is thought to protect cells from death when the UPR is activated. The absence of this safeguard suggests the UPR is triggered in these mice in a way that more readily allows cell death.

Might it be possible to prevent α-synuclein accumulation and, hence, activation of the UPR? The researchers treated A53TαS mice with salubrinal, a compound that protects cells from chronic ER stress. Salubrinal is believed to work by preventing dephosphorylation of eIF2α. Treated mice accumulated fewer α-synuclein monomers and oligomers in the ER and retained motor function for several months longer than controls, though eventually they did succumb to PD-like symptoms. Salubrinal treatment demonstrated both the importance of ER stress in α-synucleinopathy and the possibility of delaying disease onset, suggested the authors.

To see if salubrinal lessened neurodegeneration specifically in dopaminergic neurons, the researchers turned to a rat model. They injected an α-synuclein-carrying AAV virus unilaterally into the substantia nigra, then treated the animals one week later with the compound. Eleven weeks after injection, salubrinal drastically reduced the percentage of those neurons with fragmented Golgi, which is an early sign of impending neurodegeneration due to ER stress (see, e.g., Nakagomi et al., 2008). However, salubrinal did not protect dopaminergic neurons from death.

"To my knowledge, this is the first time a pharmacological agent significantly and dramatically delays disease onset in this type of animal model," said Lee. "We think that with additional work, this could be an important target for therapeutic development for Parkinson's and other diseases associated with α-synuclein pathology." Salubrinal is not approved for human use, and affects other pathways as well, so more specific inhibitors will be needed, Scheper pointed out. Lee said that, as his team uses other animal models to more rigorously establish the role of ER stress in α-synuclein neurodegeneration, they will also try additional compounds known to influence ER stress pathways.

One major question that remains is the nature of the synuclein oligomers. Lee and colleagues plan to purify and characterize them to assess their properties, including toxicity.—Gwyneth Dickey Zakaib.

References:
Colla E, Jensen PH, Pletnikova O, Troncoso JC, Glabe C, Lee MK. Accumulation of Toxic α-Synuclein Oligomer within Endoplasmic Reticulum Occurs in α-Synucleinopathy In Vivo. J Neurosci. 2012 Mar 7;32(10):3301-5. Abstract

Colla E, Coune P, Liu Y, Pletnikova O, Troncoso JC, Iwatsubo T, Schneider BL, Lee MK. Endoplasmic Reticulum Stress Is Important for the Manifestations of α-Synucleinopathy In Vivo. J Neurosci. 2012 Mar 7;32(10):3306-20. Abstract

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  1. This paper examines toxic α-synuclein in the endoplasmic reticulum (ER) of a mouse model of synucleinopathy and in brain tissue from people with Parkinson’s disease using antibodies against α-synuclein oligomers and phosphorylated forms. Determining the nature of the toxic protein species in neurodegenerative diseases and the mechanism by which they function have been a research focus over recent years. Starting with analysis of which form of amyloid-β (Aβ) is pathology-causing in Alzheimer’s disease (monomers, oligomers, or plaques), the field has now moved on to ask similar questions of other pathological proteins, including α-synuclein in Lewy body diseases. The detection of toxic α-synuclein oligomers in both a mouse model of synucleinopathy and human Parkinson’s disease brains is an important step on the pathway to new treatments and biomarker discoveries. Further, a pathological link between this mouse model and human disease validates this as a research tool for future use.

    How α-synuclein causes proteasome and lysosome/autophagy dysfunction remains unclear. Therefore, identifying the location and nature of toxic α-synuclein within cells is an important step in resolving its role in neurodegeneration. Furthermore, identifying the toxic form of the protein is important for developing targeted drugs with minimal impact on other forms of the protein, and for identifying if treatments are disease modifying or symptomatic. Recently, Santos et al. showed a correlation between oligomeric Aβ in the cerebrospinal fluid of people with Alzheimer’s disease and cognition—an important step in identifying a biomarker of disease progression. Progress in identifying the most toxic α-synuclein oligomers will allow for improved focus of ELISAs for finding biomarkers for disease progression.

    View all comments by Emma Jones
  2. This is an interesting paper, indicating a marked elevation in the levels of various truncated and multimeric α-synuclein species in the endoplasmic reticulum (ER) from α-synuclein transgenic mice and PD patients. In mice, aggregated α-synuclein species appear in the ER as the animals become symptomatic. However, the authors do not really provide any data that the toxicity is mediated by the oligomers per se. The evidence for that conclusion is only indirect, as the accumulated protein is immunoreactive with A11, an oligomer-selective antibody that was found to inhibit oligomer-induced toxicity in vitro. Another uncertainty relates to the fact that both A11 and a prefibrillar/fibril-selective antibody, FILA-1, were used solely for dot-blot analyses. Identification of soluble intermediates is likely to be less reliable in analyses on solid-phases as compared to assessments in solution, e.g., ELISA. Anyhow, the accumulation of prefibrillar/fibrillar α-synuclein species in the ER seems to be of pathogenic relevance also for human disease, as a robust increase in both A11 and FILA-1 reactivity could be demonstrated in a number of Parkinson's disease brains. Finally, the effect of orally administered salubrinal, an inhibitor of eIF2-dephosphorylation, was shown to reduce ER levels of prefibrillar/fibrillar α-synuclein in mice. Interestingly, the pharmacologically induced decrease of presumably toxic α-synuclein species was accompanied by an attenuation of motor dysfunction—as is reported in a parallel-published paper from the same group. This treatment paradigm provides additional evidence that pathological α-synuclein should be an attractive target for future pharmacological intervention in Parkinson’s disease and related disorders.

    View all comments by Martin Ingelsson

References

News Citations

  1. Move Over, Mitochondria—Make Room for ER-Induced Cell Death

Paper Citations

  1. . Human alpha-synuclein-harboring familial Parkinson's disease-linked Ala-53 --> Thr mutation causes neurodegenerative disease with alpha-synuclein aggregation in transgenic mice. Proc Natl Acad Sci U S A. 2002 Jun 25;99(13):8968-73. PubMed.
  2. . Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis. Science. 2003 Apr 18;300(5618):486-9. PubMed.
  3. . Detection of elevated levels of soluble alpha-synuclein oligomers in post-mortem brain extracts from patients with dementia with Lewy bodies. Brain. 2009 Apr;132(Pt 4):1093-101. PubMed.
  4. . Unfolded proteins and endoplasmic reticulum stress in neurodegenerative disorders. J Cell Mol Med. 2011 Oct;15(10):2025-39. PubMed.
  5. . Activation of the unfolded protein response in Parkinson's disease. Biochem Biophys Res Commun. 2007 Mar 16;354(3):707-11. PubMed.
  6. . A Golgi fragmentation pathway in neurodegeneration. Neurobiol Dis. 2008 Feb;29(2):221-31. PubMed.
  7. . Accumulation of toxic α-synuclein oligomer within endoplasmic reticulum occurs in α-synucleinopathy in vivo. J Neurosci. 2012 Mar 7;32(10):3301-5. PubMed.
  8. . Endoplasmic reticulum stress is important for the manifestations of α-synucleinopathy in vivo. J Neurosci. 2012 Mar 7;32(10):3306-20. PubMed.

Further Reading

Papers

  1. . Accumulation of toxic α-synuclein oligomer within endoplasmic reticulum occurs in α-synucleinopathy in vivo. J Neurosci. 2012 Mar 7;32(10):3301-5. PubMed.
  2. . Endoplasmic reticulum stress is important for the manifestations of α-synucleinopathy in vivo. J Neurosci. 2012 Mar 7;32(10):3306-20. PubMed.
  3. . ER stress and unfolded protein response in amyotrophic lateral sclerosis. Mol Neurobiol. 2009 Apr;39(2):81-9. PubMed.
  4. . Activation of the unfolded protein response in Parkinson's disease. Biochem Biophys Res Commun. 2007 Mar 16;354(3):707-11. PubMed.
  5. . Presynaptic alpha-synuclein aggregates, not Lewy bodies, cause neurodegeneration in dementia with Lewy bodies. J Neurosci. 2007 Feb 7;27(6):1405-10. PubMed.
  6. . The unfolded protein response in models of human mutant G93A amyotrophic lateral sclerosis. Eur J Neurosci. 2012 Mar;35(5):652-60. PubMed.
  7. . Phosphorylation of the translation initiation factor eIF2alpha increases BACE1 levels and promotes amyloidogenesis. Neuron. 2008 Dec 26;60(6):988-1009. PubMed.
  8. . The unfolded protein response is activated in Alzheimer's disease. Acta Neuropathol. 2005 Aug;110(2):165-72. PubMed.

Primary Papers

  1. . Accumulation of toxic α-synuclein oligomer within endoplasmic reticulum occurs in α-synucleinopathy in vivo. J Neurosci. 2012 Mar 7;32(10):3301-5. PubMed.
  2. . Endoplasmic reticulum stress is important for the manifestations of α-synucleinopathy in vivo. J Neurosci. 2012 Mar 7;32(10):3306-20. PubMed.