Tau assemblies enter the cytosol of neurons in a cholesterol sensitive manner

Introduction:

Alzheimer’s disease (AD) is the most common neurodegenerative disease in the world, contributing to approximately 50 million dementia cases worldwide. In AD, Tau, a major microtubule associated protein which promotes the assembly of tubulin into microtubules, becomes hyper-phosphorylated and aggregated into bundles¹. This is also true for other related neurodegenerative diseases called tauopathies.

There are two main ways in which tau becomes aggregated within the diseased brain.

1. Endogenous tau in each individual cell aggregates without endogenous tau.
2. Tau can move in a prion-like fashion propagating from one cell to the next².

It has been shown that the spread between cells may occur by uptake of naked tau, or through membrane-bound vesicles during endocytosis or micropinocytosis. However, how tau is released from intracellular vesicles into the cytosol is relatively unknown. It has been shown that low-density lipoprotein receptor Lrp1 and Heparan sulphate proteoglycans (HSPGs) are involved with tau seeding^3,4 but how these may effect tau entry into the cytosol is yet to be known. In this preprint, Tuck et al. designed a novel assay to study entry of tau into the cytosol and established a role for the endocytic proteins clathrin and dynamin, as well as cholesterol, in the tau entry process.

Tau entry assay

The authors developed a highly sensitive method for the detection of tau entry into the cytosol.  This assay can be conducted in live cells and relies on a split luciferase system. This system is composed of 11 amino acid fragments of luciferase, HiBiT, which is fused to tau, and a cytosolic LgBiT fragment.

When tau-HiBiT enters a cell through endocytosis it is unable to fuse with cytosolic LgBiT fragments until it is released from the intracellular vesicles. Upon release, the LgBiT can form a complete luciferase (NLuc), and with the addition of a substrate, luminescence can be measured. Thus, uptake of tau can be detected via measuring luminescence signal.

The authors found that the NLuc signal was concentration-dependent and increased proportionally to the amount of tau-HiBiT present in the cytosol.  Thus, the authors expressed LgBiT by lentiviral transduction under the mammalian housekeeping promoter and with a nuclear localisation signal. This kept the cytosolic levels low to allow the authors to establish an assay that reports solely on cytosolic intracellular tau.

Key points of the paper

Tau uptake and entry into the cytosol of HEK cells is clathrin and dynamin dependent. However, this is cell-type dependent and does not happen in primary mouse neurons. 

First, the authors transduced HEK293 cells with tau-HiBiT and inhibited key components of endocytic pathway, coat protein clathrin and GTPase dynamin, using inhibitors PitStop2 and Dyngo4a. The inhibition of these key endocytic protein resulted in reduced uptake of the fluorescently targeted transferrin, a key clathrin mediated endocytic cargo, and reduced entry of tau into the cytosol. This indicates that tau uptake and entry into the cytosol are clathrin and dynamin dependent.

Vacuole Protein Sorting 13 (VPS13) is a ubiquitin binding protein which has been shown to promote the seeded aggregation of tau through endolysosomal escape of tau seeds⁵. The authors depleted VPS13D in HEK293 cells, which resulted in an increase in tau entry into the cytosol. Similarly, a reduction in VPS35, known to be a genetic risk factor for Parkinson’s Disease and also linked to increased tau burden in late onset AD^6,7, was correlated with a decrease in tau entry into the cytosol. These data suggest a role for endosome sorting machinery in preventing tau entry. Together, these data suggest that the endocytic and endosomal pathways are key regulators of tau uptake, entry to the cytosol and therefore tau seeding.

Next, the authors wished to study the mechanisms of tau entry in a more physiologically relevant system and so adapted their tau seeding assay to primary mouse neurons. LgBiT with nuclear localisation sequence and eGFP was expressed in primary mouse neurons using the human Synapsin promoter, a neuronal maker. Results showed that there was no change in entry of tau with the addition of clathrin and dynamin inhibitors, no difference to micropinocytosis, and no difference with knock-down of RAB GTPases in both Day 7 and Day 14 primary mouse neurons. This suggests that the entry of tau into the cytosol in primary mice neurons is mediated by a mechanism which is not clathrin or dynamin dependent.

Lrp1 and HSPGs facilitate the cholesterol dependent entry of tau into the cytosol of neurons

Lrp1 has recently been identified as a receptor for tau uptake⁴. The authors therefore wanted to investigate if Lrp1 has an effect on tau entry into cytosol in HEK293 cells. The depletion of Lrp1 corresponded to a decrease in tau entry into the cell 4h after the addition of LgBiT tau. In addition, Heparin, a ligand for HSPGs, cell surface receptors involved in many developmental signalling process and extracellular matrix protein, also reduced tau entry in a dose dependent manner suggesting a role of cell-surface HSPGs in promoting tau attachment.

Cholesterol has been associated with the accumulation of tau filaments in Neimann-Pick disease,⁸ and the Apolipoprotein (APOE) gene which encodes a cholesterol transporting protein, is also a genetic risk factor for AD⁹. Knowing this, the authors tested if cholesterol was needed for tau import by using both the cholesterol extracting agent methyl-beta-cyclodextin (MßCD) and cholesterol accumulating agent 25-HC. The extraction of cholesterol from the cell resulted in a significant increase in tau entry into the cytosol, while the accumulation of cholesterol in intracellular membrane bound organelles saw a significant reduction in cytosolic tau. This was also found to be reproducible in Organotypic hippocampal slice cultures¹⁰, where MßCD increased seeded aggregation while 25-HC reduced it. Together, this suggests that cholesterol plays a role in the entry of tau into the cytosol. It is thought that cholesterol alters the properties of the lipid bilayers, thus, decreasing cholesterol makes the membrane more prone to rupture and likely increases the entry of tau into the cytosol.

Why I chose this preprint

I chose this preprint because the authors have developed an extremely interesting new tool to study the prion-like movement of aggregated proteins found in neurodegenerative diseases. This has the potential to help investigate protein aggregation and seeding in many other neurodegenerative diseases linked to the accumulation of other proteins, such as alpha-synuclein in PD.

Questions to the author.

1. Has this been tried with IPSC’s such as iNeurons which are a model of cortical neurons? Would you expect tau uptake to be similar to the human cell line or the mouse neurons?
2. Could this assay be adapted for alpha-synuclein? Do you think it would have a similar mechanism?
3. Why did you use split luciferase system rather than a split fluorescence protein system?
4. Did you check uptake of transferrin or other cargo for each of the manipulations you did? Was there a decrease in uptake which could result in a decrease in entry into the cytosol when you knockdown VPS35?
5. Are you planning to investigate uptake into the cell vs entry into the cytosol of tau?
6. Are you interested in investigating early endosome genes BIN1, PICALM and CD2AP which are the most common polymorphisms associated with late onset AD? Would it be interesting to see their impact on the seeding assay?

References

1. Goedert, M. Tau filaments in neurodegenerative diseases. FEBS Letters (2018). doi:10.1002/1873-3468.13108
2. Sanders, D. W. et al. Distinct tau prion strains propagate in cells and mice and define different tauopathies. Neuron (2014). doi:10.1016/j.neuron.2014.04.047
3. Holmes, B. B. et al. Heparan sulfate proteoglycans mediate internalization and propagation of specific proteopathic seeds. Proc. Natl. Acad. Sci. U. S. A. (2013). doi:10.1073/pnas.1301440110
4. Rauch, J. N. et al. LRP1 is a master regulator of tau uptake and spread. Nature (2020). doi:10.1038/s41586-020-2156-5
5. Anding, A. L. et al. Vps13D Encodes a Ubiquitin-Binding Protein that Is Required for the Regulation of Mitochondrial Size and Clearance. Curr. Biol. (2018). doi:10.1016/j.cub.2017.11.064
6. Vagnozzi, A. N. et al. VPS35 regulates tau phosphorylation and neuropathology in tauopathy. Mol. Psychiatry (2019). doi:10.1038/s41380-019-0453-x
7. Williams, E. T., Chen, X. & Moore, D. J. VPS35, the retromer complex and Parkinson’s disease. Journal of Parkinson’s Disease (2017). doi:10.3233/JPD-161020
8. Auer, I. A. et al. Paired helical filament tau (PHFtau) in Niemann-Pick type C disease is similar to PHFtau in Alzheimer’s disease. Acta Neuropathol. (1995). doi:10.1007/BF00318566
9. William Rebeck, G., Reiter, J. S., Strickland, D. K. & Hyman, B. T. Apolipoprotein E in sporadic Alzheimer’s disease: Allelic variation and receptor interactions. Neuron (1993). doi:10.1016/0896-6273(93)90070-8
10. Croft, C. L., Futch, H. S., Moore, B. D. & Golde, T. E. Organotypic brain slice cultures to model neurodegenerative proteinopathies. Molecular Neurodegeneration (2019). doi:10.1186/s13024-019-0346-0

Tags: ad, cholesterol, neurodegeneration, neuron, tau

doi: https://doi.org/10.1242/prelights.29943

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