Though tau goes rogue in many neurodegenerative conditions, exactly how it contributes to disease still puzzles scientists. To gain a better idea, researchers led by Li Gan, now at Weill Cornell Medicine in New York City, used a relatively new technology to tag proteins that bumped up against wild-type or mutant tau in human neurons. In the January 14 Cell, they presented the first comprehensive tau interactome from human iPSC-derived neurons. The data reinforced previous findings in the field, but added some surprises. For example, the scientists found numerous interactions between mitochondrial proteins and wild-type, but not mutant, tau. Moreover, in the presence of mutant tau, mitochondria became sluggish. The finding hints at a role for tau in bolstering cellular energy production.
- Comprehensive survey of tau’s “interactome” hints at multifarious roles.
- In response to neuronal activity, tau binds synaptic proteins responsible for exocytosis.
- Mutant tau poorly binds mitochondrial proteins, correlating with energy deficits.
The interactome data also bulked up tau’s synaptic portfolio. Not only did tau snuggle up to many proteins at the surface of synaptic vesicles, but when neurons were stimulated, tau dallied with additional proteins that regulated exocytosis. These data may help explain the activity-dependent release of tau that is thought to spread it among neurons.
Amy Pooler at Sangamo Therapeutics in California’s San Francisco Bay area called the findings exciting. “This is a wonderfully comprehensive analysis of interactions between neuronal proteins and tau,” she wrote. Giuseppina Amadoro at the Institute of Translational Pharmacology-National Resource Council, Rome, said it would be a valuable resource. “This interesting report not only provides a mechanistic explanation of the direct action of tau on mitochondrial bioenergetics and presynaptic function, both in health and in disease, but also reveals exhaustive mapping of potential microtubule-independent interactors of the protein that can be therapeutically targeted to slow down the progression of human tauopathies,” she wrote to Alzforum (full comments below).
Most previous attempts to delineate the tau interactome have focused on mice (Liu et al., 2016; Wang et al., 2017; Maziuk et al., 2018). To extend these findings to people, Gan and colleagues used a human iPSC line modified to generate glutamatergic neurons (Wang et al., 2017). The APEX enzyme modifies tyrosine residues on any protein that comes within 10-20 nanometers of it, allowing these residues to be subsequently biotinylated. Biotinylated proteins can then be identified by mass spectrometry (Rhee et al., 2013).
Joint first authors Tara Tracy, Jesus Madero-Pérez, and Danielle Swaney conjugated APEX to the 2N4R isoform of human wild-type tau, and expressed the hybrid protein in the iPSC-derived neurons. Mass spectrometry identified 246 proteins that had contacted tau. The list included known interactors, such as microtubule and cytoskeletal proteins, ribonucleoproteins, heat shock proteins, and α-synuclein. Nuclear and RNA-binding proteins also turned up, as did lysosomal and proteasome proteins involved in waste removal. There were also a large number of synaptic proteins responsible for vesicle docking and fusion, including SNARE complex proteins, dynamin, and syntaxin (see image above). Because the synaptic vesicle proteins were biotinylated only on their cytosolic side, never the luminal, the researchers concluded that tau bound to the surface of synaptic vesicles.
“I was excited to find the study corroborated several earlier findings, including, from our own work, the prominent association of tau with the ribonucleo-proteome. I look forward to digging deeper into the data files to see what else they may tell us about candidate interactors,” Gerold Schmitt-Ulms at the University of Toronto wrote to Alzforum. However, he cautioned that proteins that come close to each other do not necessarily interact, highlighting the importance of validating these findings by other methods (comment below).
One unique feature of APEX is that it detects transient and even unstable interactions, providing a snapshot of cellular activity. Gan exploited this feature to examine the effects of neuronal activity on the tau interactome. When the scientists stimulated the cultures with potassium chloride, to depolarize them and trigger synaptic vesicle release, new proteins interacted with tau. Many were synaptic proteins that regulated exocytosis, such as synaptotagmin-1, Mint1, SV2C, and synapsin-1. Gan was intrigued by these data. She thinks the neuronal hyperexcitability seen in early AD could help drive the spread of tau between cells.
Mutant Tau Ignores Mitochondria. Some mitochondrial proteins (green) interact with wild-type but not V337M tau; others (purple) interact with wild-type but not P301L tau, and some (blue) interact with wild-type but neither mutant variety. The data hint at a role for wild-type tau in energy production. [Courtesy of Tracy et al., Cell.]
Although APEX can capture fleeting interactions, it has limitations. For one, it is less sensitive than other methods, and can miss protein partners that bind infrequently. To complement the APEX data, the authors expressed tagged wild-type or mutant tau in neurons, then lysed the cells and isolated the tau isoforms using beads that recognized the tag. Mass spectrometry identified any bound proteins. This technique largely replicated the APEX findings. However, when the authors compared interactomes, they were surprised to find that P301L and V337M mutant tau had far fewer contacts with mitochondrial proteins than did wild-type (see image above). Wild-type tau bound to a large variety of mitochondrial proteins, including cytochrome C, ATP transporters, amino acid transporters, fatty acid oxidizers, the protein importer TOMM40, chaperone TIMM13, and ATPase inhibitor ATP5IF1.
There are previous reports in the literature of mutant tau entering mitochondria, Gan noted, but this was usually assumed to be aberrant (Dec 2017 news). She believes the new data suggest tau has a physiological function at mitochondria.
However, Ben Wolozin at Boston University questioned if these interactions occur inside or even on mitochondria. Instead, he wondered if they reflect tau helping to traffic these proteins from the nucleus or endoplasmic reticulum to their final destination. “Tau is not a mitochondrial protein,” he noted (comment below).
Whatever explains the interactions, the authors found that mitochondrial function was impaired in neurons expressing mutant tau. More protons leaked from mitochondria, requiring the cells to use more oxygen to generate the same amount of energy as did wild-type cells. Because this occurred in the absence of any tau filaments or inclusions, the finding hints that weakened bioenergetics could be an early event in tauopathy, Gan said.
Does this happen in human brain? Analyzing mRNA and protein data from AMP-AD, the authors found that in the brains of people who had Alzheimer’s disease at the time of death, many mitochondrial tau partners were in short supply compared to levels in healthy age-matched controls. Intriguingly, the lower the protein level, the more amyloid and tau pathology that brain contained, as measured by CERAD and Braak scores, respectively. The authors found a similar pattern in postmortem brains from the University of Pennsylvania Brain Bank. Notably, levels of these mitochondrial interlocutors were low in tauopathies besides AD, as well, including frontotemporal dementia and progressive supranuclear palsy.
“Taken together, these findings suggest that defects in mitochondrial bioenergetics may represent a converging disease mechanism across primary tauopathies and AD,” noted Wilfried Rossoll at the Mayo Clinic in Jacksonville, Florida. Nicholas Seyfried at Emory University, Atlanta, called the human data a strength of the paper (full comments below).
Commenters believe the methods described here could be extended to gather more data. Pooler suggested looking at additional neuronal subpopulations besides glutamatergic, while Wolozin noted the importance of studying neurons containing oligomeric and fibrillized tau. Overall, they agreed the techniques will be broadly useful. “One can envision how this discovery platform can be used to map and compare interactomes under a variety of different conditions in neuronal health and disease,” Rossoll wrote.—Madolyn Bowman Rogers
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