Dodd DA, LaCroix M, Valdez C, Knox GM, Vega AR, Kumar A, Xing C, White CL, Diamond MI. Tau seeds translocate across the cell membrane to initiate aggregation. medRxiv, May 12, 2022
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Dodd DA, LaCroix M, Valdez C, Knox GM, Vega AR, Kumar A, Xing C, White CL, Diamond MI. Tau seeds translocate across the cell membrane to initiate aggregation. medRxiv, May 12, 2022
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University of Massachusetts Amherst
The Diamond lab has done a lot of work studying mechanisms of tau uptake and seeding. I think there are very likely many ways that tau can get into cells, and it will likely be dependent on the characteristics of the tau species itself (monomer, oligomer, fibril, etc.) as well as, importantly, the identity of the cell type (HEK293, neuron, astrocyte, etc.). There is interesting work from the Livesey group on how different endocytic pathways can be exploited by different forms of tau (Evans et al., 2018; Evans et al., 2020).
I’m not sure if LRP1 would be involved with direct translocation, as LRP1 has a very established role in endocytosis. LRP1 expression varies drastically across different cell types, and LRP1 expression in HEK293T, for example, is very low (Journal of Biological Chemistry news). Therefore, it will be important to see how these findings translate into additional cell types or different model systems.
References:
Evans LD, Wassmer T, Fraser G, Smith J, Perkinton M, Billinton A, Livesey FJ. Extracellular Monomeric and Aggregated Tau Efficiently Enter Human Neurons through Overlapping but Distinct Pathways. Cell Rep. 2018 Mar 27;22(13):3612-3624. PubMed.
Evans LD, Strano A, Campbell A, Karakoc E, Iorio I, Bassett AR, Livesey FJ. Whole genome CRISPR screens identify LRRK2-regulated endocytosis as a major mechanism for extracellular tau uptake by human neurons. bioRxiv, August 11, 2020 bioRxiv
New York University School of Medicine/Nathan Kline Institute
The alternative non-endocytic pathway for extracellular tau seeds to directly cross plasma membranes, proposed by Dodd et al. from the Marc Diamond group, is provocative and exciting, especially when considered in the context of endosomal-lysosomal dysfunction in Alzheimer’s disease. Although minor in normal, non-neural cells compared to the conventional endocytic routes, the alternative pathway is accelerated when vATPase function is downregulated, as it is in AD and AD cell/mouse models. As such, the mechanism is expected to accelerate extracellular aggregate translocation in vulnerable neuronal populations in AD. The process might not only apply to AD but also to other proteinopathies, such as Parkinson’s disease, where vATPase is reported to be impaired, and mutations within the vATPase complex even occurs in rare forms of familial PD.
Given that the mechanism by which translocation of tau seeds across cell membrane remains obscure, could the same observations possibly be explained by a conventional mechanism involving macropinocytosis and lysis of macropinosomes? The cold temperature block of endocytosis argues against this, although if blockade of endocytic routes is not complete, could a slower endocytosis compounded by accelerated lysis induce internal seed growth?
In a disease context where vesicle acidification is dysregulated, as in AD for example, lysosomal membrane permeability is increased and rupture of a subpopulation of these substrate-engorged vesicles (e.g., amphisomes, autolysosomes, late endosomes) has been documented, releasing substrate cargoes into the cytosol (Lee et al., 2022). In addition, investigators interested in delivering an extracellular (therapeutic) agent into cell cytoplasm have tagged the agent with a moiety that promotes pH-dependent lysis of the macropinosome.
Could membrane-disruptive properties of certain modified, or oligomeric, tau species achieve a similar release, particularly if pH is dysregulated in the disease? It would be interesting to follow up these intriguing studies with video and high-resolution microscope analyses on the state of tau fibrils during the translocation process. In any event, the report nicely illuminates novel adaptations that cells can make to vesicular trafficking challenges arising in disease-related states.
References:
Lee JH, Yang DS, Goulbourne CN, Im E, Stavrides P, Pensalfini A, Chan H, Bouchet-Marquis C, Bleiwas C, Berg MJ, Huo C, Peddy J, Pawlik M, Levy E, Rao M, Staufenbiel M, Nixon RA. Faulty autolysosome acidification in Alzheimer's disease mouse models induces autophagic build-up of Aβ in neurons, yielding senile plaques. Nat Neurosci. 2022 Jun;25(6):688-701. Epub 2022 Jun 2 PubMed.
University of Cambridge
MRC-LMB
Assemblies of tau have been shown to seed aggregation of native cellular tau pools, potentially explaining the spread of tau pathology during neurodegenerative disease. A critical step in this process is the crossing of cell-limiting membranes by seeding-competent tau species. This study by Dodd and colleagues provides important insight into how tau gains access to the cell. The authors demonstrate that uptake and seeding are separable events: Some treatments that inhibit tau uptake, seemingly paradoxically, promote seeded aggregation. The authors further show that tau can penetrate cell-derived lipid bilayers, crossing membranes with comparable efficiency to the cell-penetrating TAT peptide isolated from HIV-1, and with a dependence on heparin sulphate glycoproteins (HSPGs). As membrane-crossing behavior was observed for both tau monomer and tau assemblies, these observations are consistent with seed-competent tau species gaining entry to the cell via direct translocation across the plasma membrane.
The findings are in good agreement with our recent study (Tuck et al., 2022) where we developed tools for measuring tau entry to the cytosol. We found that HSGPs are required for entry and that inhibition of endocytic pathways was unable to prevent entry of tau to the cytosol of neurons. Instead, we found that the cholesterol content of membranes was important. Experimental removal of cholesterol, or its missorting by knockdown of cholesterol transporter NPC1, promoted tau entry to the cytosol and seeded aggregation. In contrast, supplying the plasma membrane with exogenous cholesterol protected against tau entry and seeding. This also led us to conclude that the plasma membrane is a likely site of tau entry.
Some interesting questions arise from this study and when compared with our study. First: Why does inhibition of endocytosis by Dodd et al. increase seeded aggregation of tau rather than leave it unchanged? One explanation is that endocytosis is required to clear membrane-associated tau from the cell surface to a degradative pathway. Arguing against this, however, is the observation that not all treatments that are expected to inhibit endocytosis lead to this increase in seeding. For instance, dominant-negative Rab7a and dynamin 2 did not have the same aggregation-enhancing effect as did dominant-negative Rab5a. Would other endocytosis inhibitors also have this effect?
An alternative explanation is that treatments that inhibit endocytosis generally (including bafilomycin and dominant-negative Rab5a mutants), lead to destabilization of membrane-bound compartments. This would speak to a model wherein the endocytic pathway is normally degradative, but becomes competent for tau cytosolic entry when certain perturbations are made.
Second, our study showed major differences in the cytosolic entry pathway between HEK293 and neurons. We found that, unlike neurons, cytosolic entry to the cytosol of HEK293 cells was principally via endocytosis. In the study by Dodd et al., seed entry was not directly measured but was inferred from seeding experiments to occur independently of endocytosis in both cell types. Is this simply an artifact of different HEK293 strains between the labs or are there missing steps between entry and seeding that we are not seeing? Could there be, for instance, treatments that differentially inhibit entry of bulk aggregates versus seed-competent aggregates that may be smaller and contribute less to an entry signal?
One of the main insights gained from the two papers is that measuring tau uptake is poorly indicative of seeded aggregation. Entry to the cytosol does not necessarily occur by the same means as tau is taken up. The use of direct measures of cytosolic entry and seeded aggregation are therefore important in order to determine how tau gains access to the cell, and ultimately to devise strategies that prevent this process.
References:
Tuck BJ, Miller LV, Katsinelos T, Smith AE, Wilson EL, Keeling S, Cheng S, Vaysburd MJ, Knox C, Tredgett L, Metzakopian E, James LC, McEwan WA. Cholesterol determines the cytosolic entry and seeded aggregation of tau. Cell Rep. 2022 May 3;39(5):110776. PubMed.
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