Differential Effects of the Six Human TAU Isoforms: Somatic Retention of 2N-TAU and Increased Microtubule Number Induced by 4R-TAU

Abstract

In the adult human brain, six isoforms of the microtubule-associated protein TAU are expressed, which result from alternative splicing of exons 2, 3, and 10 of the MAPT gene. These isoforms differ in the number of N-terminal inserts (0N, 1N, 2N) and C-terminal repeat domains (3R or 4R) and are differentially expressed depending on the brain region and developmental stage. Although all TAU isoforms can aggregate and form neurofibrillary tangles, some tauopathies, such as Pick's disease and progressive supranuclear palsy, are characterized by the accumulation of specific TAU isoforms. The influence of the individual TAU isoforms in a cellular context, however, is understudied. In this report, we investigated the subcellular localization of the human-specific TAU isoforms in primary mouse neurons and analyzed TAU isoform-specific effects on cell area and microtubule dynamics in human SH-SY5Y neuroblastoma cells. Our results show that 2N-TAU isoforms are particularly retained from axonal sorting and that axonal enrichment is independent of the number of repeat domains, but that the additional repeat domain of 4R-TAU isoforms results in a general reduction of cell size and an increase of microtubule counts in cells expressing these specific isoforms. Our study points out that individual TAU isoforms may influence microtubule dynamics differentially both by different sorting patterns and by direct effects on microtubule dynamics.

Keywords: MAPT; SH-SY5Y cell line; TAU; axonal targeting; microtubule dynamics; microtubule-associated proteins; primary neuron cell culture; somatodendritic localization.

FIGURE 1

2N-containing TAU^HA isoforms are less efficiently sorted into the axon in mouse primary neurons. HA-tagged TAU isoforms (0N, 1N, 2N, and 3R or 4R) and tdTomato as a volume marker were co-transfected into mouse primary neurons (DIV4) and expressed for two days. (A) Schematic overview of the six TAU isoforms expressed in human brains and their estimated sizes in kDa. N1, N2 = N-terminal inserts; R1–R4 = C-terminal repeat domains. (B) Representative images of mouse primary neurons (DIV6) transfected with tdTomato (Ctrl.) or co-transfected with tdTomato and 0N3R-TAU, respectively. Neurons were stained with α-pan-TAU (for endogenous TAU in control) or α-HA antibody (for transfected TAU^HA) and α-MAP2 to distinguish axons and dendrites. Arrowheads indicate axons, asterisks indicate cell bodies. (C) Representative images of mouse primary neurons (DIV6) transfected with tdTomato (Ctrl.) or co-transfected with tdTomato and 2N4R-TAU^HA and 0N3R-TAU^HA, respectively. Neurons were stained with α-Tau (for control) or α-HA antibody to analyze axonal enrichment of transfected or endogenous TAU. Arrowheads indicate axons, asterisks indicate cell bodies. (D) Axonal enrichment of TAU was calculated from soma-to-axon ratio of TAU fluorescence intensity and normalized to soma-to-axon ratio of the tdTomato signal. An axonal enrichment of one is considered as a random distribution. N = 3, at least 10 cells were analyzed per condition. Error bars represent SEM. Shapiro–Wilk test was performed to test for normal distribution of data; statistical analysis was performed by one-way ANOVA with Tukey’s test for correction of multiple comparisons. Statistical significance: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. (E) Comparison of pan-TAU expression in axon and soma of untransfected neurons (endo.TAU) and TAU^HA isoform-expressing neurons. Error bars represent SEM. Shapiro–Wilk test was performed to test for normal distribution of data; statistical analysis was performed by Kruskal–Wallis test with Dunn’s correction for multiple comparisons. Statistical significance: *p ≤ 0.05; **p ≤ 0.01. (F–H) Linear regression of somatic TAU expression and axonal enrichment. Linear regression was performed for (F) untransfected neurons (endo. TAU), (G) 4R isoform-expressing neurons, and (H) 3R isoform-expressing neurons. No significant correlation of TAU expression levels and axonal enrichment was observed for all experimental groups.

FIGURE 2

4R-TAU isoforms decrease cell size and increase microtubule counts in undifferentiated SH-SY5Y neuroblastoma cells. Expression of TAU^D2 isoforms (0N, 1N, 2N, and 3R or 4R) in undifferentiated SH-SY5Y cells. (A) Western blot of SH-SY5Y cells transfected with the corresponding TAU^D2 isoforms. Longer exposure of TAU signal shows negligible endogenous TAU expression (∼55 kDa). GAPDH was used as a loading control. (B) Cell area was measured from SH-SY5Y cells, co-transfected with tdTomato-N1-EB3 and TAU^D2. Cell area was analyzed from at least 30 cells. Error bars represent SEM. Statistical analysis was performed by one-way ANOVA with Tukey’s test for correction of multiple comparisons. Asterisks indicate statistical significance: *p ≤ 0.05; **p ≤ 0.01. (C) Image processing for input into TrackMate. Minimum projection was calculated from all frames and subtracted from all. Afterward, threshold was set, and microtubule dynamics were analyzed by TrackMate. (D–G) Microtubule dynamics of SH-SY5Y cells co-transfected with Dendra2c-tagged TAU isoforms (0N, 1N, 2N, and 3R or 4R) and tdTomato-N1-EB3. Growing microtubule plus-ends were monitored in living cells for 2 min (1 fps). The following parameters were examined: (D) microtubule number (MT number/μm²) (normalized to corresponding cell area), (E) microtubule run length (μm), (F) microtubule stability (s), and (G) microtubule growth rate (μm/s). N = 3, at least five cells were analyzed per condition. Error bars represent SEM. Shapiro–Wilk test was performed to test for normal distribution of data; statistical analysis was performed by one-way ANOVA with Tukey’s test for correction of multiple comparisons for (D–F). Kruskal–Wallis test with Dunn’s correction for multiple comparisons was performed for panel (G).