Distinct Conformations, Aggregation and Cellular Internalization of Different Tau Strains

Abstract

The inter-cellular propagation of tau aggregates in several neurodegenerative diseases involves, in part, recurring cycles of extracellular tau uptake, initiation of endogenous tau aggregation, and extracellular release of at least part of this protein complex. However, human brain tau extracts from diverse tauopathies exhibit variant or “strain” specificity in inducing inter-cellular propagation in both cell and animal models. It is unclear if these distinctive properties are affected by disease-specific differences in aggregated tau conformation and structure. We have used a combined structural and cell biological approach to study if two frontotemporal dementia (FTD)-associated pathologic mutations, V337M and N279K, affect the aggregation, conformation and cellular internalization of the tau four-repeat domain (K18) fragment. In both heparin-induced and native-state aggregation experiments, each FTD variant formed soluble and fibrillar aggregates with remarkable morphological and immunological distinctions from the wild type (WT) aggregates. Exogenously applied oligomers of the FTD tau-K18 variants (V337M and N279K) were significantly more efficiently taken up by SH-SY5Y neuroblastoma cells than WT tau-K18, suggesting mutation-induced changes in cellular internalization. However, shared internalization mechanisms were observed: endocytosed oligomers were distributed in the cytoplasm and nucleus of SH-SY5Y cells and the neurites and soma of human induced pluripotent stem cell-derived neurons, where they co-localized with endogenous tau and the nuclear protein nucleolin. Altogether, evidence of conformational and aggregation differences between WT and disease-mutated tau K18 is demonstrated, which may explain their distinct cellular internalization potencies. These findings may account for critical aspects of the molecular pathogenesis of tauopathies involving WT and mutated tau.

The C291R Tau Variant Forms Different Types of Protofibrils

“…To exclude the possibility that tau-K18 C291R's aggregation into non-fibrillar structures was dependent on heparin induction, the experiment was repeated in native-state conditions by incubating both tau constructs in identical conditions as in Figure 3 except that no heparin was added. Aggregation was slower in this condition as anticipated (Karikari et al, 2019a). However, this approach allowed the fine mechanistic details of the process to be studied.…”

“…We generated the pProEx-HTa-Myc-6 × His-K18 plasmid carrying the C291R mutation (TGT > CGT, the same codon change previously reported by Marshall et al, 2015) by site-directed mutagenesis using a WT tau-K18 plasmid as a template (Karikari et al, 2017(Karikari et al, , 2019a. Protein expression and purification were achieved using our previously-characterized recombinant tau production protocols (Karikari et al, 2017(Karikari et al, , 2019aHill et al, 2019). The purified His-tagged tau-K18 constructs were used directly in functional experiments since the His-tag does not appear to affect aggregation (Huseby et al, 2019).…”

“…Tau-K18 is generally unfolded but adopts β-sheet conformation with increasing aggregation (Kumar et al, 2014;Karikari et al, 2017). Specific MAPT mutations have distinct effects on this process by either increasing or decreasing the propensity to form β-sheet structures (Barghorn et al, 2000;Combs and Gamblin, 2012;Karikari et al, 2019a). To understand the possible effects of the C291R mutation on β-sheet formation, the secondary structure content of monomeric tau-K18 C291R and the WT were studied with CD spectroscopy.…”

Effects of pharmacological modulators of α-synuclein and tau aggregation and internalization

“…Finally, we assessed whether blocking endocytosis would alter aggregation, possibly by reducing the seeding step of protein aggregation 8, 62 . Dynamins play essential roles in membrane remodeling and fission of clathrin-coated vesicles formed during endocytosis 63,64 .…”

An elongated tract of polyQ in the carboxyl‑terminus of human α1A calcium channel induces cell apoptosis by nuclear translocation

“…Although polyQ diseases are well known clinically, their molecular mechanism and cell biological behavior are still unclear, especially in the context of PME. To address this issue, in the present study, a wild-type (wt) human Cav2.1 (Cav2.1 wt) with 13 repeats of CAG as well as a mutant-type (mt) Cav2.1 (Cav2.1 mt) with 26 repeats of CAG in the C-terminus of this protein were constructed and delivered into cells of the SH-SY5Y neuroblastoma cell line, which is always used as the model cell line for investigations of human nervous system diseases (10)(11)(12). The results revealed that the forced expression of Cav2.1 mt significantly inhibited SH-SY5Y cell proliferation by inducing apoptosis.…”