Neurology Today: October 5, 2017 - Volume 17 - Issue 19 - p 45–46
doi: 10.1097/01.NT.0000526515.38695.dd
ARTICLE IN BRIEF
A new study confirms a central role for the lysosome in degrading aggregated alpha-synuclein, and strengthens the case that Parkinson's is one of the “prion-like” diseases, spread by transmission of misfolded protein from cell to cell.
A novel imaging technique has revealed new details about how alpha-synuclein is taken up and processed by neurons, and how defects in that processing may lead to the protein aggregation thought to be at the center of Parkinson's disease (PD) pathogenesis.
The study, published August 11 in the Journal of Biological Chemistry, confirms a central role for the lysosome in degrading aggregated alpha-synuclein, and strengthens the case that PD is one of the “prion-like” diseases, spread by transmission of misfolded protein from cell to cell.
A role for cell-to-cell spreading of misfolded alpha-synuclein in PD pathogenesis has been hypothesized for almost a decade, since the discovery of Lewy bodies in fetal tissue transplanted into PD patients. Strong support for this mechanism has come from both cell and animal models, in which exposure to insoluble alpha-synuclein has led to development of neuronal intracellular inclusions similar to Lewy bodies.
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| R. MICHEL GOEDERT said the case for Parkinson's disease being a prion-like disease is strong. However, he noted, despite the mounting evidence supporting it, it may remain circumstantial until aggregated alpha-synuclein can be imagedin vivo, probably with a PET ligand, and its spread from one region of the brain to another can be followed. |
But the specific processes responsible for uptake of misfolded protein from the extracellular milieu, and the fate of the protein once inside the cell, have remained unclear, said Virginia Lee, PhD, professor of pathology and laboratory medicine at the Perelman School of Medicine at the University of Pennsylvania and the principal investigator of the new study.
Alpha-synuclein can be tagged with a fluorescent marker, but a major obstacle to tracking it as it is taken within the neuron has been that the small fluorescent signal of internalized protein is swamped by the much larger amount of protein that remains outside the cell.
STUDY DESIGN
To overcome this obstacle, Dr. Lee, first author Richard Karpowicz, PhD, and colleagues exposed neurons in culture to pre-formed alpha-synuclein fibrils (PFFs) tagged with green fluorescent protein. They then let them interact long enough to begin the uptake process, adding trypan blue, a dye that “quenches” fluorescent molecules and prevents them from emitting light. Trypan blue is not membrane-soluble, so it stayed outside the neurons, quenching only the PFFs outside the cells, and allowing the fluorescent signal from the internalized PFFs to shine out without interference from any background fluorescence.
They found that PFFs were rapidly taken up by neurons, and could be seen in both neuronal processes and cell bodies. “These results represent the first time that intracellular pre-formed alpha-synuclein fibrils have been quantitatively imaged in both neuronal somata and processes with confocal resolution and no signal contribution from extracellular fibrils,” the authors noted in their report.
R. VIRGINIA LEE said the specific processes responsible for uptake of misfolded protein from the extracellular milieu, and the fate of the protein once inside the cell, have remained unclear.
Absorption of PFFs at 37 degrees Celsius was more than 200 times greater than at 4 degrees Celsius, “strongly implicating an energy-dependent mechanism of translocation across the membrane,” Dr. Lee said, indicating that fibrils were taken up by endocytosis, rather than passive diffusion. “But we still don't know the exact mechanism,” and whether there is a specific receptor for alpha-synuclein or fibrils made from it.
By using a dye that marks the acidic environment of endosomes, the team found that most of the PFFs were trafficked through the endosomal-lysosomal pathway, the principal means used by cells to degrade large protein aggregates. Lysosomal dysfunction has been implicated in PD, most strongly by the discovery that mutations in the lysosomal enzyme glucocerebrosidase (GBA) are a risk factor for the disease.
To explore the consequences of decreased lysosome function, they added a temporary and weak lysosomal inhibitor, which is known to disrupt the normally tight control of lysosome membrane chemistry. They found that the number of extra-lysosomal fluorescent “puncta,” indicating PFFs that had escaped from the endosome/lysosome vesicles, more than tripled. “We don't know the mechanism of escape,” Dr. Lee said, “but if the rate of fibril entry into the lysosome is larger than the lysosome's ability to degrade them, then some of them will escape.”
That escape led to an almost eight-fold increase in aggregation of endogenous alpha-synuclein. “What's escaping from the lysosome is a potential seed for aggregation,” Dr. Lee said. The protein normally doesn't aggregate, she said, even when highly overexpressed. “It needs a trigger,” and the pre-formed fibrils that are taken up by the neuron and then escape from the lysosome likely provide that trigger. Dr. Lee concluded, “It is entirely possible that the development of intracellular alpha-synuclein inclusions in neurons is a result of a relatively small number of seeds escaping endocytic degradation to initiate recruitment.”
EXPERT COMMENTARY
The study “takes new strides toward a mechanistic understanding” of alpha-synuclein spread, said Masato Hasegawa, PhD, chair of the neuroscience department at Tokyo Metropolitan Institute of Medical Science, who was not involved in the study.
The quenching method used by the team to reveal the internalized fibrils was “elegant,” he said, and the data “provide confirmation that endocytosis is the principal uptake mechanism, and that lysosomal processing is the predominant fate of internalized fibrils.”
Importantly, he said, the increase in intracellular aggregates as a result of disruption of lysosome function provide strong support for a model of PD pathogenesis in which defects in lysosomal activity and integrity contribute to aggregation and transmission.
The involvement of lysosomes in PD makes sense, said Michel Goedert, PhD, head of neurobiology at the Medical Research Council Laboratory of Molecular Biology in Cambridge, England, since the lysosomal degradation pathway is a principal means of removing misfolded proteins. He thought it was intriguing, though still unproved, that escape of endocytosed alpha-synuclein fibrils from lysosomes is the trigger for misfolding of endogenous protein. He added that the fact that glucocerebrosidase mutation is a risk factor for PD “fits these results nicely,” although it remains unclear whether or how the mutation contributes to alpha-synuclein aggregation.
The case for PD being a prion-like disease is strong, he said, and these results fit well within that model. However, he noted, despite the mounting evidence supporting it, it may remain circumstantial until aggregated alpha-synuclein can be imaged in vivo, probably with a PET ligand, and its spread from one region of the brain to another can be followed. Even then, he noted, it could be argued that the sequential development of aggregates in contiguous parts of the brain represents sequential development of vulnerability to a process that occurs within individual neurons.
The final word may only come with development of therapies to interrupt cell-to-cell transmission of misfolded proteins, Dr. Goedert said. By pointing out that uptake is likely through endocytosis, this new study may help point toward targets for such therapies.
http://journals.lww.com/neurotodayonline/Fulltext/2017/10050/At_the_Bench___Parkinson_s_Disease__Novel_Imaging.12.aspx
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