Mitochondria may be the universal powerhouses of eukaryotic cells, but there is more to these organelles than just ATP production. According to a paper in the September 9 Nature Neuroscience, mouse mitochondria express distinct subsets of proteins that imbue them with cell-specific functions. Researchers led by Thomas Misgeld of the German Center for Neurodegenerative Diseases in Munich reported that the mitochondrial proteome differed between astrocytes and neurons, and even between different types of neuron. Using these distinctive proteomes to identify cell-type-specific mitochondrial markers, the researchers spotted a dearth of neuronal mitochondria around amyloid plaques and in the spinal cord in mouse models of Alzheimer’s disease and amyotrophic lateral sclerosis (ALS), respectively. They saw the same in postmortem tissue from people with those diseases.
- A new tagging system plucks intact mitochondria from the brain.
- Their proteomes vary depending on cellular origin.
- Around amyloid plaques, neuronal mitochondria are scarcer than astrocytic ones.
“We’ve known for over a decade that the mitochondrial proteome varies across organs. This paper is now reporting differences across even cell types,” commented Vamsi Mootha of Massachusetts General Hospital in Boston. “This is an exciting advance, one that will hopefully help the community unravel the role of mitochondrial dysfunction in neurodegeneration.”
Russell Swerdlow, University of Kansas Medical Center in Kansas City, agreed. “This is a very creative methods-development paper that will likely fill a much-needed niche in the mitochondrial research field,” he wrote to Alzforum. “As an investigator who labors over separating neurons, astrocytes, microglia, and endothelial cells from mouse brains in order to get a better grasp of what their mitochondria are up to, the potential of this approach to facilitate studies of brain mitochondria is obvious.”
Mitochondria Vacate Premises. In an amyloid plaque (left, dashed line), astrocyte mitochondria bearing the marker Sfxn5 (green, middle) and neuronal mitochondria expressing Ociad2 (purple, right) are relatively depleted from its core (solid white line). [Courtesy of Fecher et al., Nature Neuroscience, 2019.]
Mitochondrial function deteriorates in neurodegenerative diseases, including Alzheimer’s, Parkinson’s, and ALS. Still, it has been difficult to nail down exactly when malfunction begins, and if it is limited to specific cell types. For one thing, mitochondria often get damaged when they are purified from isolated cells; for another, their abundance makes it difficult to determine their origin.
To address these problems, co-first authors Caroline Fecher, Laura Trovò, and colleagues generated MitoTag mice. These animals harbor a Cre-activated gene that expresses a green fluorescent protein (GFP) targeted to the outer mitochondrial membrane. By crossing MitoTag mice with a strain that expresses Cre recombinase under the control of cell-specific promoters, the researchers were able to limit the green tag to those particular cells. This allowed them to isolate the tagged mitochondria using GFP antibodies, a far gentler approach than using mechanical homogenization and centrifugation to separate mitochondria from cell preparations.
In the resent study, the researchers used MitoTag to label mitochondria from three cell types in the cerebellum: Purkinje cells, a major inhibitory neuron; granule cells, a major excitatory neuron in this region; and astrocytes. Mitochondrial proteomes from all three cell types were 85 percent identical, and yet nearly 200 proteins were differentially expressed.
The proteome of astrocyte mitochondria most clearly diverged from those of neuronal ones, and pointed to functional differences. For example, astrocytic mitochondria abundantly expressed proteins involved in lipid metabolism that were absent or scarce in neuronal mitochondria, and they expressed higher levels of enzymes involved in fatty-acid metabolism. Though the brain primarily relies on glucose and lactate for energy, some studies have suggested it can metabolize fatty acids in a pinch. The researchers went on to confirm their proteomic results with functional assays, finding that astrocytic mitochondria more efficiently metabolized a long-chain fatty acid than did their neuronal counterparts.
The proteomes also laid bare differences between the two subtypes of neuronal mitochondria. Granule cell mitochondria were flush with the calcium channel Mcu, while Purkinje cell and astrocyte mitochondria had little of it. Functional assays confirmed that the granule cell organelles robustly took up calcium in a Mcu-dependent manner, while calcium uptake in Purkinje cell mitochondria was more sluggish. Maria Ankarcrona of Karolinska Institute in Stockholm noted that these findings mesh with granule cells being excitatory and using glutamate receptors and calcium influx for their signaling.
Moreover, the proteomes pointed to cell-type-specific mitochondrial markers. Sfxn5 marked astrocytic mitochondria, while Ociad2 and Nipsnap1 were neuronal. The German study used these markers to track different types of mitochondria in mouse models of disease and in human tissue, sans MitoTag.
In preliminary experiments using three APP/PS1 mice and three wild-type, the researchers found that the density of both Ociad2-positive neuronal mitochondria and Sfxn5-positive astrocytic mitochondria were less around Aβ plaques than in the normal parenchyma (see image above). In the spinal cords of three SOD-G93A mice, a model of motor neuron disease, neuronal mitochondria were morphologically altered, while astrocytic mitochondria appeared normal. Antibodies against these same markers revealed similar changes in distribution and shape of mitochondria in postmortem brain and spinal cord samples from four AD and four ALS patients, respectively. The researchers did not quantify any of these changes. Misgeld said these experiments merely served as a proof of concept that the markers could tag mitochondria from different cell types in disease models. Fecher said this initial study did not distinguish between loss of cells and loss of mitochondria, but that future studies could clarify how neuronal, astrocytic, and even microglial mitochondria are affected by pathology.
Mark Cookson of the National Institutes of Health thinks the models could help nail down in which cells mitochondria malfunction in Parkinson’s—where mutations in mitochondrial proteins PINK1 and parkin lead to early onset disease. “It would be of huge interest to express the MitoTag in different cells in PINK1 and parkin-deficient mice and see how the [mitochondrial] proteome reshapes under different conditions,” he wrote.
Fecher told Alzforum that MitoTag mice may also prove useful in examining differences in mitochondria that reside in axons and dendrites, versus those in the cell body. She pointed out that mitochondria located far from the soma rely heavily on local translation from nearby ribosomes, and may therefore have a distinct set of proteins and functions.
MitoTag mice are now available for purchase at The Jackson Laboratory.—Jessica Shugart
Research Models Citations
- Studies Suggest Mitochondria Changes Precede Aging, Alzheimer’s
- Aβ and Mitochondria—When It Reigns, They Pore
- Mitochondrial Damage in Alzheimer's Disease
- C9ORF72 Toxicity Tied to Mitochondria, Transcriptional Machinery
- Mitochondria Sag Early in AD Mouse Model
- Could Disposing of Damaged Mitochondria Treat Alzheimer’s Disease?
- Pink Mutations Link Parkinson’s Disease to Mitochondria
- Fecher C, Trovò L, Müller SA, Snaidero N, Wettmarshausen J, Heink S, Ortiz O, Wagner I, Kühn R, Hartmann J, Karl RM, Konnerth A, Korn T, Wurst W, Merkler D, Lichtenthaler SF, Perocchi F, Misgeld T. Cell-type-specific profiling of brain mitochondria reveals functional and molecular diversity. Nat Neurosci. 2019 Sep 9; PubMed.