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Targeting Tau Mitigates Mitochondrial Fragmentation and Oxidative Stress in Amyotrophic Lateral Sclerosis

Abstract

Understanding the mechanisms underlying amyotrophic lateral sclerosis (ALS) is crucial for the development of new therapies. Previous studies have demonstrated that mitochondrial dysfunction is a key pathogenetic event in ALS. Interestingly, studies in Alzheimer’s disease (AD) post-mortem brain and animal models link alterations in mitochondrial function to interactions between hyperphosphorylated tau and dynamin-related protein 1 (DRP1), the GTPase involved in mitochondrial fission. Recent evidence suggest that tau may be involved in ALS pathogenesis, therefore, we sought to determine whether hyperphosphorylated tau may lead to mitochondrial fragmentation and dysfunction in ALS and whether reducing tau may provide a novel therapeutic approach. Our findings demonstrated that pTau-S396 is mis-localized to synapses in post-mortem motor cortex (mCTX) across ALS subtypes. Additionally, the treatment with ALS synaptoneurosomes (SNs), enriched in pTau-S396, increased oxidative stress, induced mitochondrial fragmentation, and altered mitochondrial connectivity without affecting cell survival in vitro. Furthermore, pTau-S396 interacted with DRP1, and similar to pTau-S396, DRP1 accumulated in SNs across ALS subtypes, suggesting increases in mitochondrial fragmentation in ALS. As previously reported, electron microscopy revealed a significant decrease in mitochondria density and length in ALS mCTX. Lastly, reducing tau levels with QC-01-175, a selective tau degrader, prevented ALS SNs-induced mitochondrial fragmentation and oxidative stress in vitro. Collectively, our findings suggest that increases in pTau-S396 may lead to mitochondrial fragmentation and oxidative stress in ALS and decreasing tau may provide a novel strategy to mitigate mitochondrial dysfunction in ALS.

Graphical abstract

pTau-S396 mis-localizes to synapses in ALS. ALS synaptoneurosomes (SNs), enriched in pTau-S396, increase oxidative stress and induce mitochondrial fragmentation in vitro. pTau-S396 interacts with the pro-fission GTPase DRP1 in ALS. Reducing tau with a selective degrader, QC-01-175, mitigates ALS SNs-induced mitochondrial fragmentation and increases in oxidative stress in vitro.

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Data Availability

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Code Availability

Not applicable.

Abbreviations

ALS:

Amyotrophic lateral sclerosis

AD:

Alzheimer’s disease

DRP1:

Dynamin-related protein 1

SNs:

Synaptoneurosomes

mCTX:

Motor cortex

pTau-S396:

Phosphorylated tau at S396

ROS:

Reactive oxygen species

SOD1:

Superoxide dismutase 1

TARDBP:

TAR DNA binding protein

FUS:

Fused in sarcoma

MAP:

Microtubule-associated protein

OXPHOS:

Oxidative phosphorylation

ALS/FTD:

Amyotrophic lateral sclerosis/frontotemporal dementia

DTT:

Dithiothreitol

SDS:

Sodium dodecyl sulphate

TBST:

Tris-buffered saline with Tween 20

DMEM:

Dulbecco’s modified Eagle medium

FBS:

Fetal bovine serum

ELISA:

Enzyme-linked immunosorbent assay

coIP:

Co-immunoprecipitation

EDTA:

Ethylenediaminetetraacetic acid

PSD-95:

Postsynaptic density protein 95

siRNA :

Small Interference RNA

Mfn1:

Mitofusin 1

Mfn2:

Mitofusin 2

OPA1:

Dominant optic atrophy 1

FDA:

Food and Drug Administration

RNAi:

RNA interference

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Acknowledgments

The authors would like to thank the patients and their families for sample donations.

Funding

T.P. was supported by an award from the Judith and Jean Pape Adams Charitable Foundation and Byrne Family Endowed Fellowship in ALS Research. S.M.K.F. was supported by the ALS Canada Tim E. Noël Postdoctoral Fellowship. S.D. was supported by the Alzheimer’s association (2018-AARF-591935) and the Jack Satter Foundation. D.H.O. is a recipient of an Alzheimer’s Association Clinician Scientist Fellowship (2018-AASCF-592307) and a Jack Satter Foundation Award; he is partially supported by the Dr. and Mrs. E. P. Richardson, Jr Fund for Neuropathology at MGH. S.J.H. was supported by the Alzheimer’s Association/Rainwater Foundation Tau Pipeline Enabling Program and the Stuart & Suzanne Steele MGH Research Scholars Program. The Massachusetts Alzheimer’s Disease Research Center is supported by the National Institute on Aging NIA (Grant P30AG062421). The Philly Dake Electron Microscopy Facility was supported by the Dake Family Foundation and by the NIH grant (1S10RR023594S10) to M.D.

Author information

Authors and Affiliations

Authors

Contributions

T.P. and E.A.B. contributed to the study design, data collection, data analysis, and drafting of the manuscript. A.N.M., S.E.K., E.S., B.A.D., A.A.O., S.M.K.F., A.C.A., S.D., and P.M.D. contributed to the data collection, data analysis, and editing of the manuscript. C.H., D.H.O., A.N., B.T.H., T.S.J., S.D.B., K.V., M.E.C., J.D.B., M.D., M.C.S., and S.J.H. contributed to the study design and editing of the manuscript. G.S.V. contributed to the study design, data analysis, and drafting of the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Ghazaleh Sadri-Vakili.

Ethics declarations

Ethics Approval

The study was approved by the Mass General Brigham Healthcare Institutional Review Board (IRB).

Consent to Participate

Written informed consent was obtained from all participants prior to study enrollment. Post-mortem consent was obtained from the appropriate representative (next of kin or health care proxy) prior to autopsy.

Consent for Publication

Not applicable.

Conflict of Interest

B.T.H. is a member of Novartis, Dewpoint, and Cell Signaling Scientific Advisory Board (SAB), and of Biogen DMC, and acts as consultant for US DoJ, Takeda, Virgil, W20, and Seer; he receives grants from Abbvie, F prime, NIH, Tau consortium, Cure Alzheimer’s fund, Brightfocus, and JPB foundations. T.S.J. is on the scientific advisory board of Cognition Therapeutics and receives grant funding from European Research Council (grant 681181), UK Dementia Research Institute, MND Scotland, and Autifony. M.E.C. acts as consultant for Aclipse, Mt Pharma, Immunity Pharma Ltd., Orion, Anelixis, Cytokinetics, Biohaven, Wave, Takeda, Avexis, Revelasio, Pontifax, Biogen, Denali, Helixsmith, Sunovian, Disarm, ALS Pharma, RRD, Transposon, and Quralis, and as DSBM Chair for Lilly. J.D.B. has received personal fees from Biogen, Clene Nanomedicine and MT Pharma Holdings of America, and grant support from Alexion, Biogen, MT Pharma of America, Anelixis Therapeutics, Brainstorm Cell Therapeutics, Genentech, nQ Medical, NINDS, Muscular Dystrophy Association, ALS One, Amylyx Therapeutics, ALS Association, and ALS Finding a Cure. S.J.H. is or/has been a member of the SAB and equity holder in Rodin Therapeutics, Psy Therapeutics, Frequency Therapeutics, and Souvien Therapeutics, and has received consulting or speaking fees from Sunovion, Biogen, AstraZeneca, Amgen, Merck, Juvenescence, Regenacy Pharmaceuticals, and Syros Pharmaceuticals, and funding from F-Prime, Tau Consortium, Alzheimer’s Association/Rainwater Foundation Tau Pipeline Enabling Program and the Stuart & Suzanne Steele MGH Research Scholars Program. None of these had any influence over the current paper.

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Supplementary Information

Supplementary Fig. 1

Antimycin A decrease c- and m-aconitase activity. Antimycin A reduced (a) c-aconitase and (b) m-aconitase activity in SH-SY5Y cells (Mann-Whitney U test=0, p=0.0286, and Mann-Whitney U test=0, p=0.0286, respectively). Data in a-b are represented as bra graph indicating mean ± SD. *p<0.05. (PNG 64 kb)

High resolution image (TIF 479 kb)

Supplementary Fig. 2

ALS SNs induce mitochondrial fragmentation. (a) Two-way ANOVA revealed an effect of length ([F(4,234)=1148], p<0.0001) and length X treatment interaction ([F(12,234)=9.629], p<0.0001) in SH-SY5Y cells. Tukey’s test revealed an increase in the frequency of smaller mitochondria (<2μm) in recombinant tau- and ALS SNs-treated cells compared to vehicle- (p<0.0001, and p<0.0001, respectively) and control SNs-treated cells (p<0.0001, and p<0.0001, respectively) as well as a decrease in the frequency of larger mitochondria (>8μm) in recombinant tau- and ALS SNs-treated cells compared to control SNs-treated cells (p=0.0041, and p=0.0003, respectively). Data are represented as scatter plots with bar indicating mean±SD. (b) Two-way ANOVA demonstrated an effect of volume ([F(5,266)=1878], p<0.0001) and volume X treatment interaction ([F(15,266)=5.943], p<0.0001) in SH-SY5Y cells. Tukey’s test revealed an increase in the frequency of smaller mitochondria (<2μm3) in recombinant tau- and ALS SNs- compared to vehicle- (p<0.0001, and p<0.0001, respectively) and control SNs-treated cells (p=0.0028, and p<0.0001, respectively) as well as a decrease in the frequency of larger mitochondria (>10μm3) in ALS SNs- compared to vehicle-treated cells (p=0.0178) and in recombinant tau- and ALS SNs- compared to control SNs-treated cells (p=0.0418, and p<0.0001, respectively). Data are represented as scatter plots with bar indicating mean±SD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. (PNG 254 kb)

High resolution image (TIF 19785 kb)

Supplementary Figure 3.

Pro-fusion proteins are not altered in ALS SNs. (a) Representative western blot images of Mfn1, Mfn2 and OPA1 in control and ALS SNs. (b) There was no significant change in Mfn1 (Mann-Whitney U test=144, p=0.8597) (c) Mfn2 (Mann-Whitney U test=61, p=0.9321) or (d) OPA1 levels (Mann-Whitney U test=135, p=0.3994) in ALS SNs (n=32) compared to controls (n=12). Data are represented as individual value plots with the central line representing the median and the whiskers representing the interquartile range. (PNG 264 kb)

High resolution image (TIF 1029 kb)

Supplementary Figure 4.

siDRP1 decreases DRP1 levels in ALS SNs-treated cells. (a) Representative western blot images of DRP1 in SH-SY5Y cells following siDRP1. DRP1 levels were reduced following transfection with 150nM siDRP1 for 24h and following 48h treatment. (b) There was a significant effect of treatment on DRP1 levels in SH-SY5Y cells (one-way ANOVA, [F(3,12)=18.52], p<0.0001) with a significant decrease in DRP1 levels in siDRP1- and siDRP1+ALS SNs-treated cells compared to siControl- (Tukey’s test, p=0.0023, and p=0.0033, respectively) and siControl+ALS SNs-treated cells (Tukey’s test, p=0.00004, and p=0.0005, respectively). Data are represented as a bar graph with individual values with bar indicating mean ± SD. **p<0.01; ***p<0.001. (PNG 394 kb)

High resolution image (TIF 1824 kb)

Supplementary Figure 5.

siDRP1 mitigates ALS SNs-induced alteration in mitochondrial connectivity. (a) Representative images of siControl- and siDRP1-transfected SH-SY5Y cells treated with vehicle, recombinant tau, control and ALS SNs stained with Hoechst (blue), CellMask (white), and Tomm20 (red). (b) Cumulative frequency graph indicated an effect of treatment on length (Kruskal-Wallis, H=33.14, p<0.0001) with smaller mitochondria in siControl+tau- and siControl+ALS SNs- (n=3) compared to siControl- (p=0.0023, and p=0.0477, respectively) and siControl+control SNs-treated (n=3) cells (p=0.0008, and p=0.0377, respectively). (c) Cumulative frequency graph indicated an effect of treatment on volume (Kruskal-Wallis, H=40.04, p<0.0001) with smaller mitochondria in siControl+tau- and siControl+ALS SNs- compared to siControl+control SNs-treated cells (p<0.00001, and p<0.0001, respectively). (d) There was no effect of treatment on networks/cell following treatments (two-way ANOVA, [F(3,38)=0.9047], p=0.4479). (e) Two-way ANOVA revealed an effect of treatment ([F(3,39)=3.953], p=0.0149), siDRP1 ([F(1,39)=12.80], p=0.0009), and treatment X siDRP1 interaction ([F(3,39)=5.659], p=0.0026) on large networks/cell in SH-SY5Y cells. Tukey’s test revealed a decrease in tau- and ALS SNs- compared to vehicle- (p=0.0056, and p=0.0374, respectively) or control SNs-treated cells (p=0.0064, and p=0.0416, respectively). siDRP1 prevented tau- and ALS SNs-induced decrease in large networks/cell (Tukey’s test, p=0.0137, and p=0.0082, respectively). (f) Two-way ANOVA demonstrated an effect of treatment ([F(3,39)=4.034], p=0.0136) and treatment X siDRP1 interaction ([F(3,39)=6.527], p=0.0011) on mean branch length. Tukey’s test demonstrated a decrease in siControl+tau and siControl+ALS SNs- compared to siControl- (p=0.0068, and p=0.0195, respectively) or siControl+control SNs-treated cells (p=0.0153, and p=0.0398, respectively). siDRP1 prevented ALS SNs-mediated branch length alterations (p=0.0398). Data in d-f are represented as bar graphs with individual values indicating mean ± SD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. (PNG 2520 kb)

High resolution image (TIF 37137 kb)

Supplementary Figure 6.

siDRP1 prevents ALS SNs-induced mitochondrial fragmentation. (a) There was a significant effect of length (two-way ANOVA, [F(4,200)=1502], p<0.0001) and length X treatment interaction (two-way ANOVA, [F(28,200)=3.621], p<0.0001) in SH-SY5Y cells. Tukey’s test revealed a significant increase in the frequency of smaller mitochondria (<2μm) in siControl+tau- and siControl+ALS SNs- (n=3) compared to siControl (p<0.0001, and p=0.0002, respectively) and siControl+control SNs-treated (n=3) cells (p<0.0001 and p=0.0002, respectively). Similarly, Tukey’s test demonstrated a significant decrease in the frequency of larger mitochondria (>8 μm) in recombinant tau- and ALS SNs- compared to siControl-treated cells (p=0.0032 and p=0.0024, respectively) as well as in ALS SNs- compared to control SNs-treated cells (p=0.0433). siDRP1 prevented ALS SNs-induced increases in smaller mitochondria (p=0.0015) and reductions in larger mitochondria (p=0.0433). (b) There was a significant effect of both mitochondrial volume (two-way ANOVA, [F(5,237)=3239], p<0.0001) and volume X treatment interaction (two-way ANOVA, [F(35,237)=3.702], p<0.0001) in SH-SY5Y cells. Tukey’s test revealed a significant increase in the frequency of smaller mitochondria (<2μm3) in siControl+tau- and siControl+ALS SNs compared to siControl (p<0.0001, and p<0.0001, respectively) and control SNs-treated cells (p<0.0001 and p=0.0007, respectively). Similarly, Tukey’s test demonstrated a significant decrease in the frequency of larger mitochondria (>10 μm3) in recombinant tau- and ALS SNs-treated cells compared to siControl (p<0.0001) as well as compared to siControl+control SNs-treated cells (p=0.0203 and p<0.0001, respectively). siDRP1 prevented ALS SNs-induced increases in smaller mitochondria (p=0.0289) and reductions in larger mitochondria (p<0.0001). Data in a-b are represented as bar graphs with individual values indicating mean ± SD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. (PNG 266 kb)

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Supplementary Figure 7.

QC-01-175 prevents ALS SNs-induced mitochondrial fragmentation. (a) There was a significant effect of length (two-way ANOVA, [F(4,215)=955.7], p<0.0001), and length X treatment interaction (two-way ANOVA, [F(28,215)=4.756], p<0.0001). Tukey’s test revealed an increase in the frequency of smaller mitochondria (<2 μm) in recombinant tau- or ALS SNs- (n=3) compared to vehicle- (p=0.0021, and 0.0235, respectively) and control SNs-treated (n=3) cells (p=0.0005, and p=0.0068, respectively). QC-01-175 prevented recombinant tau- and ALS SNs-induced increase in the frequency of smaller mitochondria (p=0.0014 and p<0.0001, respectively). (b) There was a significant effect of volume (two-way ANOVA, [F(5,264)=1510], p<0.0001), and volume X treatment interaction (two-way ANOVA, [F(35,264)=2.864], p<0.0001) in SH-SY5Y cells. Tukey’s test revealed an increase in the frequency of smaller mitochondria (<2 μm3) in recombinant tau- and ALS SNs- compared to vehicle- (p=0.0034, p=0.0027, respectively) and control SNs-treated cells (p=0.0449, and p=0.0373, respectively). QC-01-175 prevented recombinant tau- and ALS SNs-induced increase in the frequency of smaller mitochondria (p=0.0179 and p<0.0001, respectively) as well as ALS SNs-induced decrease in the frequency of larger mitochondria (p=0.0470). Data in a-b are represented as bar graphs with individual values indicating mean ± SD. *p<0.05; **p<0.01; ****p<0.0001. (PNG 262 kb)

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Supplementary Figure 8.

Antimycin A decreases ROS levels. Antimycin A decreased ROS levels in SH-SY5Y cells (Mann-Whitney U test=7, p=0.0037). Data are represented as a bar graph indicating mean ± SD. **p<0.01. (PNG 35 kb)

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Petrozziello, T., Bordt, E.A., Mills, A.N. et al. Targeting Tau Mitigates Mitochondrial Fragmentation and Oxidative Stress in Amyotrophic Lateral Sclerosis. Mol Neurobiol 59, 683–702 (2022). https://doi.org/10.1007/s12035-021-02557-w

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  • DOI: https://doi.org/10.1007/s12035-021-02557-w

Keywords

  • Hyperphosphorylated tau
  • Mitochondrial dynamics
  • Mitochondrial dysfunction
  • Tau degrader
  • Amyotrophic lateral sclerosis