Overview

Pathogenicity: Frontotemporal Dementia : Uncertain Significance
Clinical Phenotype: Progressive Supranuclear Palsy
Position: (GRCh38/hg38):Chr17:45962351 G>T
Position: (GRCh37/hg19):Chr17:44039717 G>T
dbSNP ID: rs63750959
Coding/Non-Coding: Coding
DNA Change: Substitution
Expected RNA Consequence: Substitution
Expected Protein Consequence: Missense
Codon Change: CGC to CTC
Reference Isoform: Tau Isoform Tau-F (441 aa)
Genomic Region: Exon 1

Findings

This mutation was identified in the U.S. in a screen of MAPT in people with progressive supranuclear palsy (PSP). The R5L mutation was identified in one of 96 patients with PSP and in none of the 96 controls. The presenting clinical features of the female patient included falls, dysarthria (difficulty pronouncing words), and micrographia (abnormally small, cramped handwriting), with onset at age 62 (Poorkaj et al., 2002). Disease duration was 5 years. No family history was available.

This variant was reported in the gnomAD variant database at a global frequency of 0.0000034, including five heterozygotes, three of European ancestry (gnomAD v4.1.0, Apr 2024).

Neuropathology

Aggregated insoluble tau in subcortical regions was predominantly 4-repeat (4R) tau with 0 or 1 amino terminal inserts (i.e. 0N4R or 1N4R). Insoluble tau in cortical regions also contained 1N3R tau (Poorkaj et al., 2002).

Biological Effect

Microtubule dynamics

This missense mutation may alter tau protein’s regulation of microtubule dynamics, but results from different experimental systems have not always been consistent. In an early report, using an in vitro microtubule assembly assay (using the 2N4R isoform), assembly initiation was delayed and the mass of microtubules formed was lower, but the assembly rate was faster compared to wild-type tau (Poorkaj et al., 2002).

A subsequent in vitro study, using a fluorescence-based assay, indicated that R5L tau had no significant effect on maximum microtubule polymerization, the rate of microtubule polymerization, or the polymerization lag time compared to wild-type tau (Combs and Gamblin 2012). Of note, a follow-up study by the same authors, using the same methods, later showed that microtubule assembly parameters were dependent on the isoform background used (Mutreja et al., 2019). For instance, polymerization levels and kinetics, as well as microtubule assembly stabilization, did not differ from wild-type tau when on the 2N4R isoform background, but did differ on the 0N4R, and sometimes 1N4R, background.

In another study, HEK293T cells transfected with the R5L mutation were compared to cells expressing the wild-type form, and R5L-expressing cells were found to have increased microtubule binding in a cell-based assay (Xia et al., 2019).

More recently, a group of researchers examined the function of the R5L variant in an in vitro reconstituted system (Cario et al., 2022a; Cario et al., 2022b). They found that while the R5L mutation did not lead to a lower affinity of tau for microtubules, it did lead to a decreased ability of tau to form “patches”—larger-order complexes on the microtubule surface—under high tau concentration conditions (Cario et al., 2022b). In addition, the R5L mutation did not influence the interaction between tau and tubulin, nor did it affect the growth rate of microtubules or the catastrophe (a switch from microtubule growth to depolymerization) or rescue (switch from microtubule depolymerization to growth) frequencies (Cario et al., 2022a). Instead, the R5L variant was associated with a higher microtubule shrinkage rate, through a disruption of larger-order tau patches.

Tau aggregation

R5LH may also alter tau aggregation, but again, results have been mixed. In a tau seeding model using mutant HEK293T cells, the propensity of the R5L variant to form inclusions was examined (Strang et al., 2018). In R5L-expressing cells that were exposed to fibrils of wild-type K18 tau peptides, which are aggregation-prone, tau did not accumulate, indicating a lack of seeding and tau inclusion formation.

Aggregation of R5L tau has also been evaluated in vitro using electron microscopy, a thioflavin S fluorescence assay, and a laser light scattering assay (Combs and Gamblin 2012). Compared to wild-type tau (isoform 2N4R), the R5L variant had similar levels of total polymerization. However, R5L tau formed fewer oligomers and fewer, albeit longer, filaments than wild-type tau. Tau aggregation kinetics were also investigated in this study, but the conclusions were mixed. A follow-up study showed isoform-dependence, with mutant 2N4R aggregating similarly to wildtype 2N4R, while mutant 0N4R reached a lower maximum degree of aggregation with a shorter lag time than wildtype 0N4R (Mutreja et al., 2019).  

Another study also used electron microscopy to analyze aggregation parameters of the R5L variant (Chang et al., 2008). Compared to wild-type tau (isoform 2N4R), R5L tau had a significantly shorter lag time to aggregation, suggesting an acceleration of the nucleation phase. Chang et al. did not observe any differences from wild-type tau in the extension kinetics.

Additional subcellular effects

In Drosophila carrying the human R5L variant, increased phosphorylation of tau at sites linked to disease was observed, alongside heightened caspase activity—a hallmark of neurotoxic stress—and an elevation in JNK signaling, suggesting oxidative stress. (Bardai et al., 2018).  Despite these indicators of cellular dysfunction, R5L flies did not exhibit more tau inclusions than wild-type expressing flies, nor did they express increased aggregation (measured by thioflavin S fluorescence) or altered structural parameters (e.g., filament length) of polymerized tau protein. However, increased F-actin (measured by phalloidin staining) was observed in R5L flies, as was increased F-actin stabilization (measured by ELISA). Finally, cellular responses like autophagy and the unfolded protein response were increased with the R5L mutation. These data suggest that the R5L variant of tau influences various aspects of cellular function beyond aggregation.

R5L’s PHRED-scaled CADD score, which integrates diverse information in silico, is 24.2, above the commonly used threshold of 20 for predicting deleteriousness (CADD v1.7, Apr 2024).

Research Models

A Drosophila model carrying the human R5L variant (0N4R isoform) was generated using a site-directed insertion strategy to drive expression in neuronal cells (Bardai et al., 2018).

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References

Paper Citations

1. Bardai FH, Wang L, Mutreja Y, Yenjerla M, Gamblin TC, Feany MB. A Conserved Cytoskeletal Signaling Cascade Mediates Neurotoxicity of FTDP-17 Tau Mutations In Vivo. J Neurosci. 2018 Jan 3;38(1):108-119. Epub 2017 Nov 14 PubMed.
2. Poorkaj P, Muma NA, Zhukareva V, Cochran EJ, Shannon KM, Hurtig H, Koller WC, Bird TD, Trojanowski JQ, Lee VM, Schellenberg GD. An R5L tau mutation in a subject with a progressive supranuclear palsy phenotype. Ann Neurol. 2002 Oct;52(4):511-6. PubMed.
3. Combs B, Gamblin TC. FTDP-17 tau mutations induce distinct effects on aggregation and microtubule interactions. Biochemistry. 2012 Oct 30;51(43):8597-607. Epub 2012 Oct 18 PubMed.
4. Mutreja Y, Combs B, Gamblin TC. FTDP-17 Mutations Alter the Aggregation and Microtubule Stabilization Propensity of Tau in an Isoform-Specific Fashion. Biochemistry. 2019 Feb 12;58(6):742-754. Epub 2018 Dec 31 PubMed.
5. Xia Y, Sorrentino ZA, Kim JD, Strang KH, Riffe CJ, Giasson BI. Impaired tau-microtubule interactions are prevalent among pathogenic tau variants arising from missense mutations. J Biol Chem. 2019 Nov 29;294(48):18488-18503. Epub 2019 Oct 24 PubMed.
6. Cario A, Wickramasinghe SP, Rhoades E, Berger CL. The N-terminal disease-associated R5L Tau mutation increases microtubule shrinkage rate due to disruption of microtubule-bound Tau patches. J Biol Chem. 2022 Nov;298(11):102526. Epub 2022 Sep 24 PubMed.
7. Cario A, Savastano A, Wood NB, Liu Z, Previs MJ, Hendricks AG, Zweckstetter M, Berger CL. The pathogenic R5L mutation disrupts formation of Tau complexes on the microtubule by altering local N-terminal structure. Proc Natl Acad Sci U S A. 2022 Feb 15;119(7) PubMed.
8. Strang KH, Croft CL, Sorrentino ZA, Chakrabarty P, Golde TE, Giasson BI. Distinct differences in prion-like seeding and aggregation between Tau protein variants provide mechanistic insights into tauopathies. J Biol Chem. 2018 Feb 16;293(7):2408-2421. Epub 2017 Dec 19 PubMed.
9. Chang E, Kim S, Yin H, Nagaraja HN, Kuret J. Pathogenic missense MAPT mutations differentially modulate tau aggregation propensity at nucleation and extension steps. J Neurochem. 2008 Nov;107(4):1113-23. Epub 2008 Sep 18 PubMed.

Further Reading