Page:Human wild-type full-length tau accumulation disrupts mitochondrial dynamics and the functions via increasing mitofusins.pdf/3

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'''Expression of htau disrupts mitochondrial dynamics. (a,b)''' HEK293 cells with stable expression of htau (S293tau) or the vector (S293vec) were transfected with Mito-Dendra2 for 24 h. 50% power of 488 nm laser was applied for 50 s to the region of interest (ROI, square box) to allow full photo-conversion (from green to red) of all mitochondria within the ROI, then red mitochondria in ROI to turn yellow (arrowheads) in the entire cell was monitored and quantitatively analyzed using Zeiss-510 microscope. Data were presented as mean ± SD (n = 30, p = 0.000912). ***,p < 0.001. (c) HEK293 cells were transiently co-transfected with htau (T293tau) or the vector (T293vec) and Mito-Dendra2 (1 μg/ml, 1:1) for 24 h, and then red mitochondria in ROI to turn yellow was quantitatively analyzed. For quantification, the time used for a complete co-localization of red with green to form yellow was calculated. Data are presented as mean ± SD (n = 30, p = 0.00219). ***,p < 0.001. (d) Total tau levels in S293tau or S293vec cells were detected by Western blotting using antibody tau-5.

Overexpression of htau increases fusion proteins and decreases the ubiquitination of Mfn2 Mitochondrial morphology is regulated by dynamic fusion and fission; therefore, we measured the alteration of fusion proteins (Mfn1, Mfn2 and OPA1) and fission proteins (Fis1and DLP1). The results showed that fusion proteins (Mfn1, Mfn2, OPA1) significantly increased while the fission proteins (Fis1 and GLP1) were not changed much by stable expression of htau (Fig. 4a,b), suggesting that accumulation of fusion proteins may underlie the htau-enhanced mitochondrial fusion. We further measured the DLP1 level in the mitochondrial fraction, but no significant change was detected (Fig. S7). To explore the mechanisms underlying the htau-induced accumulation of the mitofusins, we measured the level of polyubiquitinated Mfn2 in the mitochondrial fraction. We found that expression of htau reduced the polyubiquitinated Mfn2 (Fig. 4c), while the ubiquitinated TOMM20 level had no significant change (Fig. S8), suggesting that an impaired ubiquitination may underlie the htau-induced Mfn accumulation.

Downregulating mitofusins rescues the htau-induced mitochondrial impairments with restoration of cell viability To further verify the role of mitofusins in mediating the toxicity of htau accumulation, we used shRNA to knockdown the fusion proteins. We found that knockdown of Mfn1 or Mfn2 to ~45% or ~52% of the control levels attenuated the mitochondrial fusion and the accumulation (Fig. 5a–h), while Scientific Reports