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Studies headed by researchers at the Gladstone Institutes have generated new insights into the role played by genes known as PINK1 and Parkin, which are associated with Parkinson’s disease, in the recycling of energy-generating mitochondria in brain cells. The team, headed by Gladstone associate investigator Ken Nakamura, MD, PhD, suggested that the results of their reported studies, backed by future investigations, could point to new therapeutic avenues.

“This work gives us unprecedented insight into mitochondria’s life cycle and how they are recycled by key proteins that, when mutated, cause Parkinson’s disease,” Nakamura commented. “It suggests that mitochondrial recycling is critical to maintaining healthy mitochondria, and disruptions to this process can contribute to neurodegeneration … Our future studies will investigate how these pathways contribute to disease and how they can be targeted therapeutically.”

Nakamura is senior author of the researchers’ published paper in Science Advances, which is titled, “Longitudinal tracking of neuronal mitochondria delineates PINK1/Parkin-dependent mechanisms of mitochondrial recycling and degradation.”

Scientists have long known that living cells are master recyclers, constantly breaking down old parts and building them back up into new molecular machines. In most cells, damaged mitochondria are decomposed in a process known as mitophagy, which is initiated by two proteins, PINK1 and Parkin. Mutations in these same proteins also cause hereditary forms of Parkinson’s disease. While the role of PINK1 and Parkin in mitophagy has been heavily studied in many cell types, it has been unclear whether these proteins act the same way in neurons, which are also the cells that die in Parkinson’s disease. “Altered mitochondrial quality control and dynamics may contribute to neurodegenerative diseases, including Parkinson’s disease, but we understand little about these processes in neurons,” the investigators noted. Indeed, neurons have unusually high energy needs and their mitochondria are much more resistant to degradation by Parkin than those in other cell types.

For their newly reported study, Nakamura’s group followed mitochondria inside living neurons and examined how PINK1 and Parkin affected their fate. Mitochondria are small and they move inside cells, frequently fusing with each other or splitting in two, which makes them difficult to track. “We had to develop a new way of tracking individual mitochondria over long periods of time, almost a full day,” explained Zak Doric, a graduate student at Gladstone and the University of California, San Francisco (UCSF), and is co-first author of the new study. “Getting that technique up and running was quite a challenge.”

The scientists also used a method that allowed them to generate larger-than-normal mitochondria, making them easier to see under a microscope. “We combined time-lapse microscopy and correlative light and electron microscopy to track individual mitochondria in neurons lacking the fission-promoting protein dynamin-related protein 1 (Drp1) and delineate the kinetics of PINK1-dependent pathways of mitochondrial quality control,” they pointed out. The high resolution of the approach should help researchers gain a more detailed understanding of how Parkin and PINK1 affect mitochondrial degradation in Parkinson’s disease.

Gladstone researchers, including Ken Nakamura (left), Zak Doric (center), and Huihui Li (right), tracked mitochondria inside neurons and uncovered a new recycling pathway that may be linked to Parkinson’s disease. [Photo: Michael Short/Gladstone Institutes]
Through the newly reported studies, the team found that Parkin proteins encircled damaged mitochondria and targeted them for degradation, demonstrating that mitophagy starts in neurons in the same way as it does in other cell types. “Depolarized mitochondria recruit Parkin to the outer mitochondrial membrane, triggering autophagosome formation, rapid lysosomal fusion, and Parkin redistribution,” the team noted.

But, through use of the new technology, the scientists could also watch the process unfold in great detail. They documented the key initial steps in which damaged, Parkin-coated mitochondria fuse with other components inside the cell to form mitochondria-degrading structures called mitolysosomes. “We were able to visualize these steps at a level that hasn’t been done before in any cell type,” noted Nakamura, who is also an associate professor of neurology at UCSF.

The researchers then examined the later phases of mitophagy, monitoring what happens to mitochondria in the mitolysosomes. “Until now, nobody has known what happens next to these mitolysosomes,” said Nakamura. Scientists had generally assumed that they rapidly break down into molecules that the cell can reuse to build new mitochondria from scratch. But Nakamura and his team found that, instead, the mitolysosomes survived for hours inside cells.

Remarkably, and unexpectedly, some mitolysosomes were engulfed by healthy mitochondria, while other times, they suddenly burst, releasing their contents into the interior of the cell, including some proteins that were still functional. “We found that following fusion of mitophagosomes with lysosomes, the resulting acidic mitolysosomes continue to actively interact with other healthy mitochondria for many hours and are sometimes engulfed by functional mitochondria,” the investigators stated. “Moreover, a subset eventually normalize their pH, burst, and release their contents into the cytosol.”

“This appears to be a new mitochondrial quality control, recycling system,” said Huihui Li, PhD, a Gladstone postdoctoral scholar and co-first author of the paper. “We think we’ve uncovered a pathway of mitochondrial recycling—which is like salvaging valuable furniture in a house before demolishing it.”

Importantly, the study shows that the recycling pathway identified by the scientists requires PINK1 and Parkin, supporting the concept that mitochondrial recycling may also be critical in protecting against neurodegeneration in Parkinson’s disease. “Dopamine neurons that die in Parkinson’s disease are particularly susceptible to mutations in PINK1 and Parkin,” commented Nakamura. “Our study advances our understanding of how these two key Parkinson’s disease proteins degrade and recycle mitochondria.”

The authors pointed out that “critical questions” are raised by the new findings. Scientists need to understand how and why mitolysosomes fuse with and are engulfed by mitochondria, including the extent to which mitochondrial contents are recycled through these processes. “It will also be important to better define the process by which mitolysosomes burst and whether this facilitates recycling of degraded mitochondrial contents and degradation of nonrecyclable components,” they wrote, and also to understand how and when these processes are required for neuronal health and survival. “Further research is required to determine whether the mechanisms of mitochondrial quality control we delineate here, including the release of mitochondrial contents into the cytosol, may initiate immune and other signaling pathways and contribute to the selective vulnerability of neurons to PINK1 and Parkin mutations.”


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