The prion-like propagation of aggregated tau may promote the characteristic spread of tangles from one neuron to the next in AD. For this type of propagation to work, extracellular aggregates must somehow gain access to the cytosol of recipient cells, where they seed aggregation. According to a study published May 3 in Cell Reports, membrane cholesterol may provide the first line of defense against this proteopathic invasion. Employing a cellular assay that detects tau’s entry into the cytosol of recipient cells, researchers led by William McEwan of the U.K. Dementia Research Institute at the University of Cambridge, England, reported that without sufficient cholesterol embedded in their membranes, neurons became particularly vulnerable to breach by tau filaments, as well as subsequent templated misfolding. Supplementing neurons with cholesterol bolstered their defenses against tau and blocked seeded aggregation.
- Depleting neurons of cholesterol promotes tau entry into cytosol, boosts seeded aggregation.
- Oxysterols had opposing effects on entry and seeding: 24(s)-OH promoted them, and 25-OH blocked them.
- Findings help explain link between cholesterol metabolism and neurodegenerative disease.
The findings provide support for a link between cholesterol metabolism and tau pathogenesis in neurodegenerative disease.
“The data in this paper will catalyze a more in-depth understanding of the relationship between tau uptake and tau seeding, a relationship linked through the shared effects of cholesterol depletion on both,” commented Kenneth Kosik of the University of California, Santa Barbara.
The idea that tau aggregates can propagate among cells—at least in cell culture and animal models—has become firmly established in recent years. However, exactly how cells manage, or fumble, this handoff remains unclear. Recent studies suggest that tau aggregates gain entry into neurons by binding low-density lipoprotein 1 (LRP1), and that the LRRK2 protein somehow aids and abets in this entry (Mar 2020 conference news; Mar 2022 conference news). Some studies suggest that this cellular uptake serves a beneficial purpose, whisking the internalized tau into lysosomes where it is processed into a form that does not readily aggregate (Xu et al., 2020).
Perhaps tau’s escape from the clutches of the endolysosomal system and into the cytosol unleashes its mischief, reasoned first author Benjamin Tuck and colleagues. To investigate the cellular mechanisms governing this breach, the researchers employed a split luciferase reporter system to detect tiny amounts of tau aggregates in the cytosol. The system consists of a luciferase enzyme called NanoLuc, which is split into two parts that only gain luciferase activity when they meet up in the same cellular compartment. The researchers expressed one part—LgBiT—in the cytosol of HEK293 cells, and fused the other—HiBiT—to P301S-tau, from which they formed filaments. Using this system, the researchers could detect entry of externally added HiBiT-tau filaments into the cytoplasm of HEK293 cells. Wielding different inhibitors, the researchers found that to gain access to the HEK-cell cytosol, tau fibrils entered the cell via clathrin-dependent endocytosis. Furthermore, they found that knocking down endolysosomal trafficking proteins, including VPS13D, VPS35, and Rab7, ramped up tau’s cytosolic entry, suggesting that a well-oiled endolysosomal machinery discourages tau’s cytosolic meanderings.
All seemed well and good, until the researchers went to validate their findings in primary mouse neurons. They were in for a surprise, and a load of further experiments, McEwan said. In neurons equipped with the split luciferase system, neither clathrin-dependent endocytosis nor endolysosomal trafficking appeared to play a role in the entry of tau filaments into the cytosol. The same was true with human induced pluripotent stem cell derived neurons. Instead, the researchers found that both LRP1 and heparan sulfate proteoglycans (HSPGs) were required, in agreement with prior work (Rauch et al., 2020; Holmes et al., 2013; Apr 2015 news).
Tuck also identified a pivotal defender against tau: cholesterol. Depletion of cholesterol from neuronal membranes with the cholesterol-extracting agent methyl-betacyclodextrin (MβCD) dramatically ramped up the entry of tau filaments into the cytosol. Supplementing neurons with extra cholesterol had the opposite effect. Notably, the researchers found that cholesterol depletion with MβCD did not appear cytotoxic, and did not lead to a seepage of other proteins, including HiBiT fused to GFP, into the cytoplasm. This suggested at least some selectivity to the pathway, although the researchers have not extensively checked for leakage of other proteins or aggregates into the cytosol.
In contrast to the inhibitory effect of membrane cholesterol, the oxysterol 24(s)-hydroxycholesterol (24(s)-HC) promoted tau’s entry into the cytoplasm. Secreted in the brain after its formation by the neuronal enzyme CYP46A1, 24(s)-OH rises in early dementia. Treatment with efavirenz, an anti-viral drug that boosts CYP46A1 activity, also elevated tau entry. Paradoxically, this drug was recently reported to reduce accumulation of phospho-tau in human neurons and to counteract Aβ accumulation, and is being tested in a clinical trial for MCI (Feb 2019 news; clinicaltrials.gov).
Another oxysterol, 25(s)-OH, had the opposite effect, preventing tau’s entry. This oxysterol has been reported to block the entry of viruses. Curiously, another recent study found that the endolysosomal machinery cells use to usher tau into the cell overlaps with that commandeered by viruses for infection (Mar 2022 conference news).
The mechanisms underlying the opposing roles of these different cholesterol derivatives on tau’s intracellular travels remains to be ironed out. Even so, the findings suggest that cholesterol metabolism is intertwined with tau trafficking, McEwan said. In support of this, the scientists also found that depletion of Niemann-Pick C1 protein (NPC1)—which transports cholesterol to the plasma membrane—opened the cytosolic floodgates for tau. Mutations in this protein cause Niemann-Pick type C, a neurodegenerative tauopathy caused by mis-sorted cholesterol.
How would tweaking cholesterol levels influence the seeded aggregation of tau? To find out, the researchers treated cultured neurons from P301S-tau transgenic mice with tau fibrils, and monitored intracellular tau aggregation using the AT8 antibody specific for hyperphosphorylated tau. Lining up with their findings on tau entry, tau aggregation was enhanced by cholesterol depletion, treatment with 24(s)-OH, or depletion of NPC1, while it was effectively squelched by cholesterol supplementation or treatment with 25-OH. The same was true in organotypic slice cultures, where cholesterol depletion resulted in a 1,000-fold increase in seeding of neurons.
Do these findings apply to what happens in the aging human brain? The current study doesn’t address this question, but McEwan pointed to substantial evidence that cholesterol metabolism influences neurodegeneration. For one, genes involved in cholesterol transport and homeostasis—most famously ApoE—have clear relationships with risk for AD and related diseases. Cholesterol levels wane in the brain with age, perhaps rendering neurons vulnerable to tau aggregation. McEwan said it remains to be seen how dietary cholesterol, or use of cholesterol lowering drugs, might influence the process.
Kosik commented that the study adds to mounting evidence that multiple tau uptake pathways are operational. Future work should investigate how these pathways relate to the physical properties cholesterol confers to membranes, such as their rigidity, thickness, receptor density, and lateral diffusion of proteins within membranes. “The direction forward will be to find the intracellular path that tau travels as it ultimately escapes from an endosomal compartment to the cytosol, all the while retaining its folded structure in a form capable of templating endogenous tau to misfold,” Kosik said.—Jessica Shugart
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