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).

Rebuffing Tau. Under conditions of high cholesterol, few tau filaments gain access to the cytosol. Low-cholesterol conditions open the door for tau fibrils, ramping up seeding in the cytosol. [Courtesy of Tuck et al., Cell Reports, 2022.]

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., 2013Apr 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 newsclinicaltrials.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

Comments

  1. Tuck et al. make an important contribution to the problem of tau uptake as it transits between cells. This problem has become more interesting with the recent publication of many tau cryoEM structures that link specific disease phenotypes to tau conformations. Thus, the power of the prion hypothesis: Tau not only templates misfolding in neighboring cells, but does so with very high shape fidelity.

    The starting observation by the McEwan group in this report is that depletion of membrane cholesterol enhances tau uptake. They proceed to show that depletion of the Niemann-Pick C1 protein, which causes Niemann-Pick type C disease and is associated with both mis-sorted cholesterol and tau inclusions, increases tau entry into cultured neurons. Depletion of cholesterol also decreased the concentration of assembled tau needed to induce seeding. Furthermore, cholesterol supplementation was able to protect the cells in the seeding assay. Similar results were obtained in organotypic slice cultures, with the additional observation that the low amounts of tau found in the extracellular space were insufficient to induce seeded aggregation, but did so when membrane cholesterol was reduced even at low nanomolar concentrations.

    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. The authors point to distinct mechanisms of tau uptake in neurons versus non-neural cells which utilize canonical endocytic pathways. In contrast, tau endocytosis in neurons is clathrin- and dynamin-independent but curiously remained dependent on LRP1 and HSPGs.

    A summary of the literature suggests that multiple tau uptake pathways are operational and their relationship to the physical properties conferred by cholesterol on membranes, such as membrane rigidity and thickness, lateral diffusion within membranes, and receptor density is an important next direction. The cell biological direction forward will need 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.

  2. Growing evidence indicates that tau pathology spreads in the brains of AD patients and potentially other tauopathies. One of the proposed mechanisms through which spreading occurs is tau secretion/release followed by uptake into other neurons, where internalized tau aggregates can seed further aggregation in the cytosol of recipient neurons. The latter implies tau seeds have to gain access to the cytosol, although the precise mechanism underlying this process is unclear. In this elegant study, Tuck et al. developed a highly sensitive, split luciferase-based method to measure cytosolic entry of seeding-competent tau assemblies, a critical and yet understudied step in cell-to-cell transmission of tau pathology. This method will be a valuable tool for the field to further understand mechanisms involved in the uptake and cytosolic entry of tau seeds.

    In this new assay, luciferase is reconstituted by binding of two components, LgBiT and HiBiT (Dixon et al., 2016), which are restricted to the cell interior and coupled to exogenous recombinant tau, respectively. To prevent leakage of cytosolic LgBiT into the medium, the authors used a low-activity promoter coupled to a nuclear localization signal to maintain a very low concentration of cytosolic LgBiT. With the optimized protocol, luciferase signals obtained after adding tau-HiBiT were resistant to trypsin treatment, confirming intracellular reconstitution of luciferase activity derived from interactions between tau-HiBiT and LgBiT in the cytosol.

    Using this method, along with seeded tau aggregation as an alternative readout in many experiments, the authors confirmed some earlier findings on tau seeding, including distinct uptake mechanisms exploited by monomeric and aggregated tau, the importance of clathrin-mediated endocytosis and endosome sorting machinery in modulating cytosolic escape of tau seeds in HEK293 cells, and the critical role of HSPGs and LRP1 in the uptake of tau seeds in neurons.

    Importantly, the authors provided convincing data that cytosolic access of tau seeds occurs via different mechanisms in neurons versus HEK293 cells, highlighting the necessity to study this pathological process in the most relevant CNS cell types.

    The central finding of this study is that cholesterol levels in the membranes of mouse and human neurons inversely correlated with cytosolic entry of tau seeds. Treatments that deplete membrane content of cholesterol (e.g., methyl-beta-cyclodextrin treatment or activation of CYP46A1, an enzyme that promotes hydroxylation of cholesterol to 24(S)hydroxycholesterol) invariably promoted tau entry and thus seeding. Conversely, treatments that increase membrane cholesterol (e.g., TopFluor cholesterol loading) substantially reduced tau seeding.

    Other treatments were used and shown to modulate cytosolic entry of tau seeds, but the data interpretation is a little more challenging. For instance, the authors knocked down the endolysosomal cholesterol transporter Niemann-Pick type C 1 (NPC1), which causes an accumulation of free cholesterol in endolysosomes. While this treatment promotes tau seed entry, it is unclear whether it is a depletion of plasma membrane cholesterol or an accumulation of endolysosomal cholesterol that underlies the increased seeding. Treatment with 25-hydroxycholesterol increased tau seed entry, but the reason why treatment with the isomer 24(S)hydroxycholesterol caused the opposite phenotype is unclear, in the absence of assessments of membrane cholesterol levels. Generally, the findings obtained from cultured neurons were replicated in organotypic slice cultures.

    This intriguing mechanistic link between membrane cholesterol and tau seeding offers a potential explanation for the early development of tau pathology in NPC patients carrying loss-of-function mutations in NPC1 or NPC2, the protein that presents endolysosomal cholesterol to NPC1. Prior to this study, one may have speculated that endolysosomal accumulation of free cholesterol in NPC1 patients is the primary contributor to enhanced tau aggregation. However, new data from this study raises the interesting possibility that it may rather be the depletion of plasma membrane cholesterol resulting from the redistribution of free cholesterol into endolysosomes that promotes the cytosolic escape of tau seeds in NPC1 patients, culminating in greatly accelerated tau pathology.

    This interesting study also raises important new questions:

    1. The observation that cholesterol content in the membrane does not modulate cytosolic entry of control protein GFP argues against a general breaching of membrane with cholesterol depletion. Given this, what is the exact mechanism underlying cholesterol-sensitive tau entry? Which specific membrane compartment(s) do tau seeds penetrate to gain cytosolic access? Specifically, is it the plasma membrane or endocytic/lysosomal membranes?
    1. Would pathological tau found in AD brain utilize the same uptake/cytosolic entry mechanism(s) as recombinant tau aggregates? It was previously reported that AD brain-purified tau fibrils show structural and seeding properties that are distinct from recombinant tau fibrils (Guo et al., 2016; Zhang et al., 2019). To answer this question using the split luciferase method, one may need to generate HiBiT-tagged recombinant tau fibrils seeded by AD brain-purified tau fibrils or employ a newly published protocol to assemble recombinant tau fibrils with identical ultrastructure as AD tau fibrils (Lovestam et al., 2022). 
    1. Would membrane cholesterol affect cytosolic entry of α-synuclein aggregates, which also demonstrate prion-like transmission similar to tau? It has been shown that NPC1 patients frequently present α-synuclein pathology in addition to tau pathology and the two types of protein lesions are often found in the same neuron (Saito et al., 2004). 
    1. Rik van der Kant et al. (2019). showed a beneficial effect of CYP46A1 activation on reducing p-tau accumulation in iPSC-differentiated human neurons. AAV-mediated overexpression of CYP46A1 was also found to rescue cognitive deficits in a tauopathy mouse model without modulating hyperphosphorylation of tau (Burlot et al., 2015). In this study, however, CYP46A1 activation with Efavirenz increased cytosolic entry of tau and was thus deemed detrimental. What would be the recommended therapeutic approach on CYP46A1 modulation given the conflicting outcome of CYP46A1 activation?
    1. Several studies demonstrated that ApoE isoforms differentially mediate efflux of cholesterol and other lipids from cultured cells in the order of ApoE2 > ApoE3 >= ApoE4, which inversely correlates with ApoE isoform-dependent risk for AD (Michikawa et al., 2000; Minagawa  et al., 2009) and thus suggests that higher cellular content of cholesterol may increase the risk for AD. These findings appear to be at odds with this new study, which suggests higher cellular cholesterol, at least in the membrane, is protective against tau seeding. What could explain this discrepancy on the risk vs. benefit of cellular cholesterol? Answering this question likely requires quantitative assessments of cholesterol levels in the membrane.
    1. Membrane cholesterol levels have been identified as a key parameter affecting the processing of APP, with cholesterol-rich “raft-like” membranes enhancing the amyloidogenic pathway via BACE1 and presenilins (reviewed in Di Paolo and Kim, 2011). Therefore, membrane cholesterol reduction would facilitate the non-amyloidogenic pathway, while facilitating tau seed uptake and spreading based on this new study. If modulation of membrane cholesterol levels is considered a potential therapeutic approach to limit tau spreading, what effect would it have on APP processing? Does the fact that APP itself binds to membrane cholesterol (Barrett et al., 2012) have any impact on tau uptake and seeding?   

    We look forward to seeing these questions addressed by the field in future studies.

    References:

    . NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells. ACS Chem Biol. 2016 Feb 19;11(2):400-8. Epub 2015 Dec 10 PubMed.

    . Unique pathological tau conformers from Alzheimer's brains transmit tau pathology in nontransgenic mice. J Exp Med. 2016 Nov 14;213(12):2635-2654. Epub 2016 Oct 17 PubMed.

    . Heparin-induced tau filaments are polymorphic and differ from those in Alzheimer's and Pick's diseases. Elife. 2019 Feb 5;8 PubMed.

    . Assembly of recombinant tau into filaments identical to those of Alzheimer's disease and chronic traumatic encephalopathy. Elife. 2022 Mar 4;11 PubMed.

    . Aberrant phosphorylation of alpha-synuclein in human Niemann-Pick type C1 disease. J Neuropathol Exp Neurol. 2004 Apr;63(4):323-8. PubMed.

    . Cholesterol Metabolism Is a Druggable Axis that Independently Regulates Tau and Amyloid-β in iPSC-Derived Alzheimer's Disease Neurons. Cell Stem Cell. 2019 Mar 7;24(3):363-375.e9. Epub 2019 Jan 24 PubMed.

    . Cholesterol 24-hydroxylase defect is implicated in memory impairments associated with Alzheimer-like Tau pathology. Hum Mol Genet. 2015 Nov 1;24(21):5965-76. Epub 2015 Sep 10 PubMed.

    . Apolipoprotein E exhibits isoform-specific promotion of lipid efflux from astrocytes and neurons in culture. J Neurochem. 2000 Mar;74(3):1008-16. PubMed.

    . Mechanism underlying apolipoprotein E (ApoE) isoform-dependent lipid efflux from neural cells in culture. J Neurosci Res. 2009 Aug 15;87(11):2498-508. PubMed.

    . Linking lipids to Alzheimer's disease: cholesterol and beyond. Nat Rev Neurosci. 2011 May;12(5):284-96. Epub 2011 Mar 30 PubMed.

    . The amyloid precursor protein has a flexible transmembrane domain and binds cholesterol. Science. 2012 Jun 1;336(6085):1168-71. PubMed.

  3. I totally agree with the comments of Gilbert Di Paolo and Jing Guo from Denali Therapeutics pointing out that the findings by Tuck and colleagues (namely that the content of plasma membrane cholesterol determines the cytosolic entry and seeded aggregation of tau) do not agree well with some previous studies in the field. Specifically, Rik van der Kant and colleagues (van der Kant et al., 2019) showed that CYP46A1 activation by efavirenz reduces p-tau accumulation in iPSC-differentiated human neurons. Burlot and colleagues (Burlot et al., 2015) demonstrated that CYP46A1 overexpression by gene therapy in the THY-Tau22 mouse model of Alzheimer’s disease-like tau pathology rescues cognitive deficits without affecting tau hyperphosphorylation and associated gliosis. Also, enrichment with cholesterol of plasma membranes was shown to enhance the amyloidogenic pathway (reviewed in Maulik et al., 2013), i.e., it has an opposite effect as compared to tau pathology.

    In their studies, Tuck et al. used cell cultures, a convenient system to conduct some proof-of-principle experiments. Yet, these are isolated systems, which do not reflect the complexity of cholesterol maintenance in the brain and the strong homeostatic mechanisms that are operative in this organ to prevent significant fluctuations of cholesterol in different cell types. The cholesterol content in the cytosol and plasma membranes of cultured neurons was not measured after the treatments that modulated the neuronal cholesterol levels. Hence, it is not clear if the resulting modulations of cellular cholesterol levels were of physiological relevance and if they occur in the Alzheimer’s disease brain. In addition, it would be helpful in the future to generate dependence curves for tau entry versus the plasma membrane cholesterol (or exogenous oxysterol) concentrations, both below and above the sterol content.

    Despite the caveats, the study by Tuck et al. is important because it links plasma membrane cholesterol content with cellular tau entry followed by seeded aggregation. Accordingly, this finding should be kept in mind in future in vivo studies that use treatments affecting brain cholesterol homeostasis. The small-dose, anti-HIV drug efavirenz activates CYP46A1, the major cholesterol-eliminating enzyme in the brain, in a mouse model of Alzheimer’s disease (5XFAD mice) (Mast et al., 2017; Petrov et al., 2019), as well as in a Phase 1 clinical trial in subjects with mild cognitive impairment due to Alzheimer’s disease. In mouse brain, efavirenz was shown to increase both cholesterol elimination by CYP46A1 and brain cholesterol biosynthesis, i.e., it increased brain cholesterol turnover without affecting the whole brain cholesterol levels. Cholesterol turnover determines the rate of sterol flux through plasma membranes, and it was also shown that the brain sterol flux mediated by CYP46A1 affects membrane properties, e.g., cholesterol accessibility, ordering, osmotic resistance, and thickness, and membrane-dependent processes, including glutamate release and protein phosphorylation (Petrov et al., 2020). Accordingly, it would be interesting to study whether the rate of sterol flux affects cellular tau entry in any way.

    No doubt that the work of Tuck et al. will give impetus to many additional studies, which may ultimately enhance our understanding of Alzheimer’s disease and lead to disease-modifying treatment of this devastating brain disorder.

    References:

    . Cholesterol 24-hydroxylase defect is implicated in memory impairments associated with Alzheimer-like Tau pathology. Hum Mol Genet. 2015 Nov 1;24(21):5965-76. Epub 2015 Sep 10 PubMed.

    . Cholesterol-metabolizing enzyme cytochrome P450 46A1 as a pharmacologic target for Alzheimer's disease. Neuropharmacology. 2017 Sep 1;123:465-476. Epub 2017 Jun 24 PubMed.

    . Role of Cholesterol in APP Metabolism and Its Significance in Alzheimer's Disease Pathogenesis. Mol Neurobiol. 2012 Sep 16; PubMed.

    . CYP46A1 Activation by Efavirenz Leads to Behavioral Improvement without Significant Changes in Amyloid Plaque Load in the Brain of 5XFAD Mice. Neurotherapeutics. 2019 Jul;16(3):710-724. PubMed.

    . Brain sterol flux mediated by cytochrome P450 46A1 affects membrane properties and membrane-dependent processes. Brain Commun. 2020;2(1) Epub 2020 Apr 11 PubMed.

    . Cholesterol Metabolism Is a Druggable Axis that Independently Regulates Tau and Amyloid-β in iPSC-Derived Alzheimer's Disease Neurons. Cell Stem Cell. 2019 Mar 7;24(3):363-375.e9. Epub 2019 Jan 24 PubMed.

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References

News Citations

  1. Tau Receptor Identified on Cell Surface
  2. At Tau2022: Unknown Functions Emerge for Tau, LRRK2
  3. Tau Triple Threat: Do Trimers Make Bad Seeds?
  4. Cholesteryl Esters Hobble Proteasomes, Increase p-Tau

Paper Citations

  1. . TFEB regulates lysosomal exocytosis of tau and its loss of function exacerbates tau pathology and spreading. Mol Psychiatry. 2020 May 4; PubMed.
  2. . LRP1 is a master regulator of tau uptake and spread. Nature. 2020 Apr;580(7803):381-385. PubMed.
  3. . Heparan sulfate proteoglycans mediate internalization and propagation of specific proteopathic seeds. Proc Natl Acad Sci U S A. 2013 Aug 13;110(33):E3138-47. Epub 2013 Jul 29 PubMed.

External Citations

  1. clinicaltrials.gov

Further Reading

Primary Papers

  1. . Cholesterol determines the cytosolic entry and seeded aggregation of tau. Cell Rep. 2022 May 3;39(5):110776. PubMed.