As if powering the cell wasn’t enough, mitochondria may also shoulder a share of its trash disposal duties. In the March 1 Nature, researchers led by Rong Li at Johns Hopkins University in Baltimore reported that in yeast, clumps of aggregated proteins congregate at the outer mitochondrial membrane, where a chaperone likely disentangles them before import channels funnel them into the mitochondrial matrix. There, the cytosolic riffraff meet their demise at the hands of matrix proteases. Dubbed MAGIC (mitochondria as guardian in cytosol), the process also functions in human cells, the authors claim. If extended to neurons, it could provide a tantalizing connection between two key pathologies in neurodegenerative disease: mitochondrial dysfunction and the accumulation of aggregated proteins.
“The beauty of this study is that it provides fundamental new information on how cells deal with intracellular protein aggregations,” commented Russell Swerdlow of the University of Kansas Medical Center in Kansas City. He added that the findings may help explain why researchers have spotted aggregation-prone proteins, including Aβ, in the mitochondria, and support the idea that their accumulation could interfere with mitochondrial function in neurodegenerative disease.
Munching Mitos. Protein aggregates congregate on the mitochondrial surface, where Hsp104 untangles them prior to their journey into the mitochondrial matrix for degradation. [Image courtesy of Chacinska, Nature N&V, 2017.]
Mitochondria’s claim to fame is the electron transport chain—the game of electron hot potato that produces adenosine triphosphate (ATP). But a few years ago, Li and colleagues observed yeast mitochondria doing something else: The organelles appeared to hoard aggregates on their surfaces. This held back the proteins from passing into budding daughter cells during asymmetric cell division (see Zhou et al., 2014). The clusters eventually dissolved, but not when the researchers disrupted the mitochondrial membrane potential using the toxin CCCP. This suggested that getting rid of the aggregates required fully functioning mitochondria.
For the current study, co-first authors Linhao Ruan and Chuankai Zhou and colleagues took a closer look into mitochondria’s relationship with protein clumps. First, they asked which proteins tend to aggregate during heat shock, a classic condition of stress in yeast. Using cells expressing FlucSM—a variant of luciferase that aggregates in response to heat shock and serves as a magnet for other misfolded miscreants— the researchers purified associated proteins and identified them via mass spectroscopy. Among them were components of the proteasome, chaperones, RNA binding proteins, stress granules, and elements of the mitochondrial import machinery—including Tomm40 and Tomm70.
Because of the involvement of the outer mitochondrial membrane transporters, the researchers hypothesized that aggregates might have to enter the organelles to be degraded. In support of this, Ruan found that FlucSM persisted in cells lacking functional Tim23, an import channel in the inner mitochondrial membrane.
To see if tangled proteins did travel into the mitochondrial matrix, the researchers employed a split green fluorescent protein (GFP) system, in which the first 10 of 11 β-strands of GFP are ushered into the mitochondrial matrix via a mitochondrial targeting sequence (MTS), while the last β-strand is attached to some other protein of interest. Fluorescence occurs only when all strands meet up. Using this system, the researchers confirmed that upon heat shock, several aggregation-prone proteins, including FlucSM and TDP-43, crossed into the mitochondrial matrix, whereas inherently stable ones did not. High-resolution microscopy and biochemical experiments confirmed the passage of FlucSM and other aggregates, including Lsg1 and Tma19, into the matrix in response to heat shock. Hsp104, a cytosolic chaperone and disaggregator, was necessary for the translocation of FlucSM into the matrix. Li hypothesized that Hsp104 detangles protein “hairballs” on the mitochondrial surface, allowing them to squeeze through import channels.
What fate awaited the cytosolic emigrants upon their arrival in the matrix? The GFP signal waned after normal temperature was restored, pointing to degradation. Strikingly, inhibiting the proteasome or vacuolar proteases had little effect on the degradation of FlucSM, whereas poisoning mitochondria with CCCP did, indicating that the mitochondrial pathway was important for ridding the cytosol of stress-induced aggregates. The researchers found that the matrix protease Pim1 was required for this degradation.
MAGIC happened not only after heat shock. GFP signals also shot up in yeast at 30 degrees if the researchers inactivated Hsp70, a crucial chaperone that keeps cytosolic inhabitants neatly folded. Under these conditions, mitochondria became fragmented and ramped up production of reactive oxygen species, indicating they were under stress. The GFP signal persisted unless researchers blocked translation, hinting at a steady stream of misfolded degenerates flooding the matrix. Furthermore, so-called “super-aggregators,” such as the RNA helicase Ded1, ended up in mitochondria regardless of heat shock, painting highly unstable cytosolic proteins as matrix regulars.
Finally, the researchers asked whether MAGIC happens in mammalian cells. Employing the split-GFP system in human retinal pigment epithelium cells, the researchers found that the amount of luciferase in the matrix correlated with the protein’s stability: a super unstable variant, Fluc-DM, flooded the matrix, while Fluc-SM and wild-type Fluc passed into the organelles at lower rates. Li told Alzforum that ongoing experiments hint the pathway may operate in neurons as well, and that proteins associated with neurodegenerative diseases may be among MAGIC’s clientele.
Li speculated that MTS-containing proteins, which made up about 20 percent of those associated with mitochondria, steer co-entangled cytosolic ones toward the organelle. In regard to neurodegeneration, Li proposed that potentially toxic proteins such as α-synuclein or TDP-43 could build up in mitochondria and tax their energy production, especially as the organelle’s function already wanes with age. This, in turn, could lead to further accumulation. The constant shuttling of mitochondria up and down neuronal axons to invigorate synapses make the organelles conveniently positioned to deal with clean-ups throughout the cell, she added.
As ongoing experiments in Li’s lab investigate MAGIC in neurons, at least one neuronal example of MAGIC may already exist. Shirley ShiDu Yan of Kansas University in Lawrence previously reported that clearance of Aβ by mitochondria in neuronal synapses reduced Aβ burden and neuroinflammation, and improved learning and memory in mAPP mice (see Fang et al., 2015). In light of the current findings, Yan said she views that Aβ degradation pathway as an example of MAGIC in neuronal cells.
Flint Beal of Weill Cornell Medical College in New York added that the findings dovetail with work in his lab suggesting Aβ mingles with mitochondria in distal synapses. Li’s finding that mitochondria undergo a stress response in the face of mounting cytosolic aggregates would also be in line with the mitochondrial damage, especially in synapses, observed in neurodegenerative disease, he said.
Leonidas Stefanis of the University of Athens Medical School in Greece was impressed by the findings. “Conceptually, this is very exciting. It challenges our view of the mitochondria as basically energy-producing organelles, and suggests that they may have a considerable and specific role in removing aggregated proteins,” he wrote. “Given the importance of protein aggregation in neurodegenerative diseases at large, and the therapeutic efforts underway to enhance endogenous clearance mechanisms to combat such protein aggregation, this discovery may provide a new therapeutic target for neurodegeneration.”
Michael Lutz of Duke University in Durham, North Carolina commented that the findings provide mechanistic support for the role of the outer membrane channel Tom40 in neurodegenerative disease, including AD. The TOMM40 and ApoE genes are neighbors in the genome, and researchers led by Lutz and the late Allen Roses previously suggested that co-inheritance of these alleles predicted disease risk and age at onset (see Nov 2009 conference news and Oct 2016 news).
Mark Cookson of the National Institutes of Health commented that certain aggregation-prone proteins known to interact with the mitochondrial import machinery, such as α-synuclein, may be prime substrates for the pathway, or potentially foul it up. “Mitochondria have had a few surprises for us over the last few years, so I certainly wouldn’t dismiss this,” he said. However, he wondered about MAGIC’s overall contribution to protein turnover in neurons, given the plethora of other disposal pathways available.
In an accompanying News & Views article, Agnieszka Chacinska of the International Institute of Molecular and Cellular Biology in Warsaw, Poland, wrote that the work is a striking example of cross-talk between the cytosol and mitochondria. “It is becoming increasingly clear that maintaining a productive dialogue between cellular compartments is a crucial task—one that we are just beginning to understand.”—Jessica Shugart
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