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. 2012 Aug 23;75(4):618-32.
doi: 10.1016/j.neuron.2012.06.026.

Tau promotes neurodegeneration via DRP1 mislocalization in vivo

Brian DuBoff  1 , Jürgen Götz, Mel B Feany
Affiliations

Tau promotes neurodegeneration via DRP1 mislocalization in vivo

Brian DuBoff et al. Neuron. .

Abstract

Mitochondrial abnormalities have been documented in Alzheimer's disease and related neurodegenerative disorders, but the causal relationship between mitochondrial changes and neurodegeneration, and the specific mechanisms promoting mitochondrial dysfunction, are unclear. Here, we find that expression of human tau results in elongation of mitochondria in both Drosophila and mouse neurons. Elongation is accompanied by mitochondrial dysfunction and cell cycle-mediated cell death, which can be rescued in vivo by genetically restoring the proper balance of mitochondrial fission and fusion. We have previously demonstrated that stabilization of actin by tau is critical for neurotoxicity of the protein. Here, we demonstrate a conserved role for actin and myosin in regulating mitochondrial fission and show that excess actin stabilization inhibits association of the fission protein DRP1 with mitochondria, leading to mitochondrial elongation and subsequent neurotoxicity. Our results thus identify actin-mediated disruption of mitochondrial dynamics as a direct mechanism of tau toxicity in neurons in vivo.

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Figures

Figure 1
Figure 1. Tau expression promotes mitochondrial elongation in neurons in vivo
(A) Immunofluorescent staining for GFP in neurons of flies expressing mitochondrially-targeted GFP (mitoGFP) reveals normal round to modestly tubular mitochondria in control flies of the genotype elav-GAL4/+; UAS-mitoGFP/+ (control, arrowheads). Mitochondria in tau transgenic flies are elongated (tau, arrowheads). Reduction of miro (miro RNAi) increases the number of mitochondria in neuronal soma, but does not alter mitochondrial morphology (arrowheads). Nuclei are stained with DAPI. Scale bar is 2 μm. Quantification of mean mitochondrial length for control and tau transgenic neurons is displayed in the graph. Asterisk indicates P < 0.01, unpaired t-test, n = 6. Control (ctrl) is elav-GAL4/+. (B) Immunofluorescent staining for ATP synthase in CA1 hippocampal neurons of control mice demonstrates normal modestly tubular mitochondria (control, arrowheads). Mitochondria in hippocampal neurons from human tau P301L transgenic mice are elongated (tau, arrowheads). Nuclei are stained with DAPI. Scale bar is 5 μm. Quantification of mean mitochondrial length for control and tau transgenic neurons is displayed in the graph. Asterisk indicates P < 0.01, unpaired t-test, n = 3. See also Fig. S1.
Figure 2
Figure 2. Genetic modification of mitochondrial dynamics influences tau toxicity
MARF is overexpressed using a UAS-MARF transgene (MARF OE) or reduced with transgenic RNAi (MARF RNAi). DRP1 is overexpressed using a UAS-DRP1 transgene (DRP1 OE), or reduced with transgenic RNAi (DRP1 RNAi). Tau is expressed when indicated (tau). (A) Mitochondrial length in neurons is altered by DRP1 or MARF manipulation in transgenic flies. n = 6 per genotype. Control (ctrl) is elav-GAL4/+;UAS-mitoGFP/+. (B) Neuronal degeneration as assayed by TUNEL staining is modified by DRP1 or MARF manipulation. n = 6 per genotype. (C) Oxidative stress is evaluated by quantification of dihydroethidium (DHE) fluorescence in whole mount brains, and shows elevated levels in tau transgenic flies compared to control. Quantification of DHE fluorescence in brain shows modification following DRP1 or MARF manipulation. n = 3 per genotype. (D) Cell cycle activation as measured by PCNA immunostaining is modified by DRP1 and MARF manipulation. n = 6 per genotype. Asterisks indicate P < 0.01, one-way ANOVA with Student-Neuman-Keuls test. Control (ctrl) in (B–D) is elav-GAL4/+. See also Fig. S2.
Figure 3
Figure 3. Tau expression blocks mitochondrial localization of DRP1
(A) Mitochondria (mitoGFP, green) and HA-tagged DRP1, detected with an antibody to HA (red), colocalize in control neurons (control, arrowheads). Mitochondria in tau transgenic neurons show markedly reduced colocalization with DRP1 (tau, arrowheads). Elongated mitochondria produced by overexpression of MARF retain the ability to recruit DRP1 (MARF OE, arrowheads). Scale bar is 2 μm. Control is elav-GAL4/+; UAS-mitoGFP/+; HA-DRP1/+. (B) Subcellular fractionation of fly head homogenates shows depletion of HA-DRP1, detected with an antibody to HA, from the mitochondrial fraction of tau flies (Mito, M) with equivalent levels of DRP1 in the cytoplasmic fraction (Cyto, C) or total homogenate (Total, T). Control is elav-GAL4/+; HA-DRP1/+, negative control (neg ctrl) is elav-GAL4/+. (C) Subcellular fractionation of mouse hippocampal homogenates shows reduction of mitochondrially-localized DRP1 in tau animals (Mito, M), while total (Total, T) and cytoplasmic (Cyto, C) DRP1 levels are not significantly affected. The blots in (B,C) are reprobed for porin and GAPDH to demonstrate enrichment of mitochondrial and cytoplasmic proteins in the appropriate fractions. The graphs in (B,C) show DRP1 levels as percent of control in each fraction from tau transgenic flies or mice. Asterisks indicate P < 0.01, one-way ANOVA with Student-Neuman-Keuls test, n = 3. See also Fig. S3.
Figure 4
Figure 4. Actin stabilization blocks mitochondrial localization of DRP1
(A) Mitochondria (mitoGFP, green) are round to modestly tubular in control neurons and colocalize with HA-DRP1 (red, arrowheads). In flies overexpressing WASP using a UAS-WASP transgene (WASP OE) or forked using a genomic rescue construct (forked+) mitochondria are frequently elongated (arrows). DRP1 fails to localize to elongated mitochondria, but DRP1 colocalization is preserved in some mitochondria of normal length (arrowheads). Control is elav-GAL4/+; UAS-mitoGFP/+; HA-DRP1/+. Scale bar is 2 μm. Quantification of mean mitochondrial length for control, WASP OE, and forked+ is displayed in the graph. Asterisk indicates P < 0.01, one-way ANOVA with Student-Neuman-Keuls test. n = 6 per genotype. (B) Subcellular fractionation shows reduced HA-DRP1 in mitochondrial fractions of head homogenates from forked+ flies (Mito, M). DRP1 levels are equivalent in cytoplasmic fractions (Cyto, C) and total fractions (Total, T). The blot is reprobed for porin and GAPDH to demonstrate enrichment of mitochondrial and cytoplasmic proteins in the appropriate fractions. DRP1 level as percent of control is calculated for each fraction from forked flies (graph). Control is elav-GAL4/+; HA-DRP1/+, negative control (neg ctrl) is elav-GAL4/+. Asterisk indicates P < 0.01, one-way ANOVA with Student-Neuman-Keuls test, n = 3. (C) Isolation of F-actin from control and forked+ flies by precipitation with biotinylated phalloidin (BioPh) followed by immunoblotting for actin or HA-DRP1 demonstrates a physical interaction between F-actin and DRP1, which is regulated by the stabilization state of actin. Precipitation with non-coupled biotin controls for association specificity. Control is elav- GAL4/+; HA-DRP1/+. n = 3. See also Fig. S4.
Figure 5
Figure 5. Reversing actin stabilization rescues tau-induced mitochondrial defects
(A) Overexpression of gelsolin using a UAS-gelsolin transgene (gelsolin OE) rescues mitochondrial morphology (mitochondrial length, left), neurotoxicity (TUNEL, center), and oxidative stress (DHE, right) in tau transgenic animals. Control (ctrl) is elav-GAL4/+; UAS-mitoGFP/+ for (left) and elav-GAL4/+ for (center, right). Asterisk indicates P < 0.01, one-way ANOVA with Student-Neuman-Keuls test. n = 6 per genotype for mitochondrial length and TUNEL. n = 3 for DHE. (B) Normal mitochondria in control neurons colocalize with HA-DRP1 (control, arrowheads). Expression of tau reduces the frequency of DRP1-associated mitochondria (tau, arrowheads) in favor of elongated mitochondria lacking DRP1 (tau, arrows). Coexpression of the actin severing protein gelsolin with tau reduces mitochondrial length and restores DRP1 localization to mitochondria (tau; gelsolin OE, arrowheads). Control is elav-GAL4/+; UAS-mitoGFP/+; HA-DRP1/+. Scale bar is 2 μm. See also Fig. S5.
Figure 6
Figure 6. F-actin interacts with DRP1 to regulate mitochondrial morphology
(A) Immunofluorescence of DRP1 (green) and visualization of mitochondria with transfected mitochondrially-localized RFP (mitoRFP, red) in empty vector control shows small, round mitochondria colocalized with DRP1 (inset). Transgelin transfection inhibits association of DRP1 with mitochondria (inset), causing elongation of mitochondria and diffuse DRP1 localization. Scale bar is 10 μm. Mean mitochondrial length is quantified in the graph. (B) Mitochondrial membrane potential measured as percent change in TMRM fluorescence compared with empty vector control shows reduced membrane potential in response to CCCP depolarizing control and transgelin transfection. Asterisk indicates P < 0.01, unpaired t-test. n = 6. See also Fig. S6.
Figure 7
Figure 7. Myosin II facilitates colocalization of DRP1 with mitochondria
(A) In control Drosophila neurons mitochondria (green) are short and colocalize with HA-DRP1 (red, arrowheads). Neurons from flies heterozygous for zip1 or sqhAX3, loss-of-function alleles of the Drosophila homologs of mammalian myosin II heavy chain and regulatory light chain, respectively, have elongated neuronal mitochondria that do not associate with DRP1 (zip1 and sqhAX3, arrows). Normal round to modestly tubular mitochondria often retain DRP1 (zip1 and sqhAX3, arrowheads). Control is elav-GAL4/+; UAS-mitoGFP/+; HA-DRP1/+. Scale bar is 2 μm. Mean mitochondrial length is quantified in the graph. Asterisk indicates P < 0.01, one-way ANOVA with Student-Neuman-Keuls test. n = 6. (B–C) Subcellular fractionation shows reduced HA-DRP1 in mitochondrial fractions of head homogenates from flies with reduced zip (B) or sqh (C) levels (zip1 and sqhAX3, Mito, M). DRP1 levels are equivalent in cytoplasmic fractions (Cyto, C) and total fractions (Total, T). The blot is reprobed for porin and GAPDH to demonstrate enrichment of mitochondrial and cytoplasmic proteins in the appropriate fractions. DRP1 level as percent of control is calculated for each fraction (graph). Control is elav-GAL4/+; HA-DRP1/+, negative control (neg ctrl) is elav-GAL4/+, HA-DRP1/+, zip1 is elav-GAL4/+; zip1/+; HA-DRP1/+, sqhAX3 is elav-GAL4/sqhAX3; HA-DRP1/+. Asterisk indicates P < 0.01, one-way ANOVA with Student-Neuman-Keuls test, n = 3. See also Fig. S7.
Figure 8
Figure 8. Myosin II links mitochonria with F-actin
(A–B) Precipitation of F-actin with biotinylated phalloidin from isolated mitochondrial fractions of heterozygous zip1 (A, Mitochondria) and sqhAX3 (B, Mitochondria) fly heads followed by immunoblotting for actin reveals a depletion in mitochondria-associated F-actin compared to control (BiotPh Precip). The depletion is also apparent in total actin (Input). Cytoplasmic F- actin and total actin levels are equivalent between control and both zip1 and sqhAX3 flies (Cytoplasm). The blots are reprobed for Complex Vα and GAPDH to demonstrate enrichment of mitochondrial and cytoplasmic proteins in the appropriate fractions. n = 3. (C–D) Coprecipitation of DRP1 and F-actin using biotinylated phalloidin in total head homogenate from heterozygous zip1 (C) and sqhAX3 (D) flies reveals no change in total actin (Input) or F-actin (BiotPh Precip) levels compared to control. However, both zip1 and sqhAX3 increase the level of DRP1 coprecipitating with F-actin. n = 3. In (A–D) control is elav-GAL4/+; HA-DRP1/+, zip1 is elav-GAL4/+; zip1/+; HA-DRP1/+, sqhAX3 is elav-GAL4/sqhAX3; HA-DRP1/+. (E) In control Cos-1 cells transfected with mitoRFP and control siRNA mitochondria (red) are round or modestly tubular and associated with endogenous DRP1 (green, inset). Transfection of either of two siRNAs targeting non-overlapping sequences in MLC2 results in mitochondrial elongation and inhibits localization of DRP1 to mitochondria (inset). Mean mitochondrial length is quantified in the graph. Asterisk indicates P < 0.01, one-way ANOVA with Student-Neuman-Keuls test, n = 6. See also Fig. S8.
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