CoChat
Sign Up
Cited by 120

The DOI matches its Crossref record; 8 of the 8 references it cites were found in a registry or an index.

Mitophagy Failure in Fibroblasts and iPSC-Derived Neurons of Alzheimer’s Disease-Associated Presenilin 1 Mutation

Martín-Maestro, Patricia, Gargini, Ricardo, A. Sproul, Andrew, García, Esther, Antón, Luis C., Noggle, Scott, Arancio, Ottavio, Avila, Jesús, García-Escudero, Vega·2017·Frontiers in Molecular Neuroscience
DOI 10.3389/fnmol.2017.00291PMC PMC5603661
Open this paper in CoChat — free account

Overview

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.3389/fnmol.2017.00291

Cites

8 of 8 references verified0 citation errors · 0 not checkable

Statistics

Figures

Figure 1 Autophagy degradation phase impairment in Familial Alzheimer’s disease associated to presenilin 1 A246E mutation (FAD1) fibroblasts. (A) Representative Western blot of Microtubule-associated protein 1 light chain 3 (LC3) expression for the study of autophagy flux as explained in “Materials and Methods” Section and Supplementary Figure S1, in control and FAD1 fibroblasts treated or not with carbonyl cyanide m-chlorophenylhydrazone (CCCP; 20 μM) in the absence or presence of NH4Cl (15 mM). (B,C) Quantification of LC3II levels (B) and LC3II/LC3I ratio (C) in FAD1 cells with respect to the control ones under basal conditions. (D,E) Quantification of LC3II synthesis (D) and degradation (E) ratios as described in “Materials and Methods” Section. (F,G) Western blot of p62 expression after the treatment as in (A) and quantification of basal levels (G). (H,I) Quantification of p62 accumulation (H) and degradation (I) ratios (n = 3 independent experiments using the control/Alzheimer’s disease (AD) fibroblast couple AG04148/AG06840; †p < 0.08; *p < 0.05; **p < 0.01).
Figure 1 Autophagy degradation phase impairment in Familial Alzheimer’s disease associated to presenilin 1 A246E mutation (FAD1) fibroblasts. (A) Representative Western blot of Microtubule-associated protein 1 light chain 3 (LC3) expression for the study of autophagy flux as explained in “Materials and Methods” Section and Supplementary Figure S1, in control and FAD1 fibroblasts treated or not with carbonyl cyanide m-chlorophenylhydrazone (CCCP; 20 μM) in the absence or presence of NH4Cl (15 mM). (B,C) Quantification of LC3II levels (B) and LC3II/LC3I ratio (C) in FAD1 cells with respect to the control ones under basal conditions. (D,E) Quantification of LC3II synthesis (D) and degradation (E) ratios as described in “Materials and Methods” Section. (F,G) Western blot of p62 expression after the treatment as in (A) and quantification of basal levels (G). (H,I) Quantification of p62 accumulation (H) and degradation (I) ratios (n = 3 independent experiments using the control/Alzheimer’s disease (AD) fibroblast couple AG04148/AG06840; †p < 0.08; *p < 0.05; **p < 0.01).CC BY · PubMed Central
Figure 2 Altered endolysosomal compartment in FAD1 fibroblasts. (A) Representative confocal microscopy immunofluorescence images showing EEA1 in red and Rab5 in green of control and FAD1 fibroblasts in basal conditions. Images on the right show enlargement of the squared area. Quantification of average size of early endosomes using each label. (B) Similar study as shown in (A) with the lysosomal markers Lysosomal-associated membrane protein 2 (LAMP2) in red and Rab7 in green and quantification of the data (Control/AD fibroblast couple used AG04148/AG06840; ***p < 0.001). Scale bar: 40 μm.
Figure 2 Altered endolysosomal compartment in FAD1 fibroblasts. (A) Representative confocal microscopy immunofluorescence images showing EEA1 in red and Rab5 in green of control and FAD1 fibroblasts in basal conditions. Images on the right show enlargement of the squared area. Quantification of average size of early endosomes using each label. (B) Similar study as shown in (A) with the lysosomal markers Lysosomal-associated membrane protein 2 (LAMP2) in red and Rab7 in green and quantification of the data (Control/AD fibroblast couple used AG04148/AG06840; ***p < 0.001). Scale bar: 40 μm.CC BY · PubMed Central
Figure 3 Dysfunctional mitochondrial clearance in FAD1 fibroblasts. (A) Representative Western blot of control and FAD1 fibroblasts in the absence or presence of CCCP (20 μM) for 24 h and quantification of Parkin RBR E3 ubiquitin protein ligase (PARK2) levels under basal conditions. (B) Representative Western Blot and quantification of full length (FL)-PTEN-induced putative kinase 1 (PINK1) and Δ1-PINK1 after the treatment with CCCP. (C,D) Quantification of the colocalization between PARK2 and Translocase of outer mitochondrial membrane 20 homolog (TOMM20) expressed as area occupied by the overlapping elements per cell data (C) from representative confocal microscopy immunofluorescence images showing PARK2 in red and TOMM20 as a mitochondrial constitutive marker in green in the same cells treated with CCCP (20 μM) for 1 h (D). Inserts represent a magnification of the same squared representative area. On the right, binary image representing the colocalization of both labels and dotted line delimits cytoplasm of each cell according to phalloidin staining (not shown). (E,F) Representative confocal images and quantification of the mitochondrial surface per cell showing TOMM20 in cyan in basal conditions (n = 3 independent experiments using the control/AD fibroblast couple AG04148/AG06840; †p < 0.08; *p < 0.05). Scale bar: 40 μm.
Figure 3 Dysfunctional mitochondrial clearance in FAD1 fibroblasts. (A) Representative Western blot of control and FAD1 fibroblasts in the absence or presence of CCCP (20 μM) for 24 h and quantification of Parkin RBR E3 ubiquitin protein ligase (PARK2) levels under basal conditions. (B) Representative Western Blot and quantification of full length (FL)-PTEN-induced putative kinase 1 (PINK1) and Δ1-PINK1 after the treatment with CCCP. (C,D) Quantification of the colocalization between PARK2 and Translocase of outer mitochondrial membrane 20 homolog (TOMM20) expressed as area occupied by the overlapping elements per cell data (C) from representative confocal microscopy immunofluorescence images showing PARK2 in red and TOMM20 as a mitochondrial constitutive marker in green in the same cells treated with CCCP (20 μM) for 1 h (D). Inserts represent a magnification of the same squared representative area. On the right, binary image representing the colocalization of both labels and dotted line delimits cytoplasm of each cell according to phalloidin staining (not shown). (E,F) Representative confocal images and quantification of the mitochondrial surface per cell showing TOMM20 in cyan in basal conditions (n = 3 independent experiments using the control/AD fibroblast couple AG04148/AG06840; †p < 0.08; *p < 0.05). Scale bar: 40 μm.CC BY · PubMed Central
Figure 4 Mitochondria accumulation in autophagic vesicles (AV) and impaired mitochondrial potential recovery in FAD1 fibroblasts. (A) Representative confocal microscopy immunofluorescence images showing LC3 in green and TOMM20 in red of control and FAD1 fibroblasts treated with CCCP (20 μM) for 24 h. On the right, binary image representing the colocalization of both labels and dotted line delimits cytoplasm of each cell according to phalloidin staining (not shown). (B) Quantification of the colocalization between LC3 and TOMM20 expressed as area occupied by the overlapping elements per cell. (C) Representative confocal immunofluorescence images of fibroblasts infected with a lentivector encoding mCherry-EGFP-LC3B and treated with CCCP for 6 h showing TOMM20 in blue. The upper and lower rows show magnifications of the squared area. Arrows point colocalization areas. (D) Quantification of the colocalization between TOMM2 and green vesicles or only-red vesicles. (E) Mitochondrial membrane potential of the same fibroblasts treated with CCCP (20 μM) for 6 h and then allowed to recover for 1 h (Reversible CCCP) or treated for 7 h with CCCP (Total CCCP). Results are expressed as percentage with respect to the mean of untreated cells (n = 3 independent samples; Control/AD fibroblasts couples used AG12988/AG06848 A,B,E and AG04148/AG06840 C,D; †p < 0.07; *p < 0.05). Scale bar: 40 μm.
Figure 4 Mitochondria accumulation in autophagic vesicles (AV) and impaired mitochondrial potential recovery in FAD1 fibroblasts. (A) Representative confocal microscopy immunofluorescence images showing LC3 in green and TOMM20 in red of control and FAD1 fibroblasts treated with CCCP (20 μM) for 24 h. On the right, binary image representing the colocalization of both labels and dotted line delimits cytoplasm of each cell according to phalloidin staining (not shown). (B) Quantification of the colocalization between LC3 and TOMM20 expressed as area occupied by the overlapping elements per cell. (C) Representative confocal immunofluorescence images of fibroblasts infected with a lentivector encoding mCherry-EGFP-LC3B and treated with CCCP for 6 h showing TOMM20 in blue. The upper and lower rows show magnifications of the squared area. Arrows point colocalization areas. (D) Quantification of the colocalization between TOMM2 and green vesicles or only-red vesicles. (E) Mitochondrial membrane potential of the same fibroblasts treated with CCCP (20 μM) for 6 h and then allowed to recover for 1 h (Reversible CCCP) or treated for 7 h with CCCP (Total CCCP). Results are expressed as percentage with respect to the mean of untreated cells (n = 3 independent samples; Control/AD fibroblasts couples used AG12988/AG06848 A,B,E and AG04148/AG06840 C,D; †p < 0.07; *p < 0.05). Scale bar: 40 μm.CC BY · PubMed Central
Figure 5 Impaired degradation phase of autophagy in FAD1-derived neurons. (A) Representative Western blot of LC3 expression in control and FAD1 neurons treated or not with CCCP (20 μM) in the absence or presence of NH4Cl (15 mM). (B,C) Quantification of LC3II levels (B) and LC3II/LC3I ratio (C) in FAD1 cells with respect to the control ones under basal conditions. (D,E) Quantification of LC3II synthesis (D) and degradation (E) ratios as described in “Materials and Methods” Section and Supplementary Figure S1. (F,G) Western blot of p62 expression after the treatment as in (A) and quantification of basal levels (G). (H,I) Quantification of p62 synthesis (H) and degradation (I) ratios as described before (n = 3 independent experiments using neurons derived from the control and AD iPSC lines 6842 A/7671 C; †p < 0.08; *p < 0.05).
Figure 5 Impaired degradation phase of autophagy in FAD1-derived neurons. (A) Representative Western blot of LC3 expression in control and FAD1 neurons treated or not with CCCP (20 μM) in the absence or presence of NH4Cl (15 mM). (B,C) Quantification of LC3II levels (B) and LC3II/LC3I ratio (C) in FAD1 cells with respect to the control ones under basal conditions. (D,E) Quantification of LC3II synthesis (D) and degradation (E) ratios as described in “Materials and Methods” Section and Supplementary Figure S1. (F,G) Western blot of p62 expression after the treatment as in (A) and quantification of basal levels (G). (H,I) Quantification of p62 synthesis (H) and degradation (I) ratios as described before (n = 3 independent experiments using neurons derived from the control and AD iPSC lines 6842 A/7671 C; †p < 0.08; *p < 0.05).CC BY · PubMed Central
Figure 6 Lysosomal anomalies in FAD neurons. (A,B) Representative Western blot and quantification of LAMP1 protein levels (B) under basal conditions FAD1 iPSC-derived neurons with respect to controls. (C) Analysis of microarray expression of indicated genes. Graphs show dot plots of the expression of the genes using n = 7 controls and n = 7 FAD brain samples retrieved from the Antonell dataset (Antonell et al., 2013). Correspondent p value was determined by Student’s t-test and it is showed for each graph. (D–G) Representative Western blot of LAMP2 and cathepsin B of iPSC-derived neurons under basal conditions and quantification of LAMP2 levels (E), pro-cathepsin B levels (F) and active-/pro-cathepsin B ratio (G) (n = 3 for A,B,E and n = 4 for F,G independent experiments using neurons derived from the control and AD iPSC lines 6842 A/7671 C; *p < 0.05; **p < 0.01).
Figure 6 Lysosomal anomalies in FAD neurons. (A,B) Representative Western blot and quantification of LAMP1 protein levels (B) under basal conditions FAD1 iPSC-derived neurons with respect to controls. (C) Analysis of microarray expression of indicated genes. Graphs show dot plots of the expression of the genes using n = 7 controls and n = 7 FAD brain samples retrieved from the Antonell dataset (Antonell et al., 2013). Correspondent p value was determined by Student’s t-test and it is showed for each graph. (D–G) Representative Western blot of LAMP2 and cathepsin B of iPSC-derived neurons under basal conditions and quantification of LAMP2 levels (E), pro-cathepsin B levels (F) and active-/pro-cathepsin B ratio (G) (n = 3 for A,B,E and n = 4 for F,G independent experiments using neurons derived from the control and AD iPSC lines 6842 A/7671 C; *p < 0.05; **p < 0.01).CC BY · PubMed Central
Figure 7 Mitophagy impairment in FAD1-derived neurons. (A) Representative Western blot of control and FAD1 neurons in the absence or presence of CCCP (20 μM) for 24 h and quantification of PARK2 levels under basal conditions. (B) Representative Western blot and quantification of FL-PINK1 and Δ1-PINK1 after the treatment with CCCP. (C) Representative confocal microscopy immunofluorescence images showing PARK2 in green, TOMM20 in red and DAPI in blue of control and FAD1 neurons treated with CCCP (20 μM) for 1 h. Quantification of the colocalization between PARK2 and TOMM20 expressed as area occupied by the overlapping elements per cell. (D) Representative confocal images of control and FAD1 neurons showing TOMM20 in cyan and DAPI in purple in basal conditions. Quantification of the mitochondrial surface per cell (n = 3 independent experiments using neurons derived from the control and AD iPSC lines 6842 A/7671 C, *p < 0.05; **p < 0.01; ***p < 0.001). Scale bar: 50 μm.
Figure 7 Mitophagy impairment in FAD1-derived neurons. (A) Representative Western blot of control and FAD1 neurons in the absence or presence of CCCP (20 μM) for 24 h and quantification of PARK2 levels under basal conditions. (B) Representative Western blot and quantification of FL-PINK1 and Δ1-PINK1 after the treatment with CCCP. (C) Representative confocal microscopy immunofluorescence images showing PARK2 in green, TOMM20 in red and DAPI in blue of control and FAD1 neurons treated with CCCP (20 μM) for 1 h. Quantification of the colocalization between PARK2 and TOMM20 expressed as area occupied by the overlapping elements per cell. (D) Representative confocal images of control and FAD1 neurons showing TOMM20 in cyan and DAPI in purple in basal conditions. Quantification of the mitochondrial surface per cell (n = 3 independent experiments using neurons derived from the control and AD iPSC lines 6842 A/7671 C, *p < 0.05; **p < 0.01; ***p < 0.001). Scale bar: 50 μm.CC BY · PubMed Central
Figure 8 Scheme of the mitophagy failure in FAD1. Dysfunctional oversized lysosomes in FAD1 cells cause the accumulation of AV containing mitochondria showing that their recycling is impaired.
Figure 8 Scheme of the mitophagy failure in FAD1. Dysfunctional oversized lysosomes in FAD1 cells cause the accumulation of AV containing mitochondria showing that their recycling is impaired.CC BY · PubMed Central

Verification

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Citations

Cites

Show all 8 references

Research works printed but not linked: 34

  1. Antonell, 2013, A preliminary study of the whole-genome expression profile of sporadic and monogenic early-onset Alzheimer’s disease, Neurobiol. Aging, 34, 1772, 10.1016/j.neurobiolaging.2012.12.026 · DOI
  2. Baxter, 2007, 2-Amino-3,4-dihydroquinazolines as inhibitors of BACE-1 (β-site APP cleaving enzyme): use of structure based design to convert a micromolar hit into a nanomolar lead, J. Med. Chem., 50, 4261, 10.1021/jm0705408 · DOI
  3. Bjørkøy, 2005, p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death, J. Cell Biol., 171, 603, 10.1083/jcb.200507002 · DOI · Paper page
  4. Borchelt, 1996, Familial Alzheimer’s disease-linked presenilin 1 variants elevate Aβ1–42/1–40 ratio in vitro and in vivo, Neuron, 17, 1005, 10.1016/s0896-6273(00)80230-5 · DOI · Paper page
  5. Coffey, 2014, Lysosomal alkalization and dysfunction in human fibroblasts with the Alzheimer’s disease-linked presenilin 1 A246E mutation can be reversed with cAMP, Neuroscience, 263, 111, 10.1016/j.neuroscience.2014.01.001 · DOI
  6. Cruts, 1998, Estimation of the genetic contribution of presenilin-1 and -2 mutations in a population-based study of presenile Alzheimer disease, Hum. Mol. Genet., 7, 43, 10.1093/hmg/7.1.43 · DOI
  7. Dagda, 2009, Loss of PINK1 function promotes mitophagy through effects on oxidative stress and mitochondrial fission, J. Biol. Chem., 284, 13843, 10.1074/jbc.M808515200 · DOI · Paper page
  8. Dinnage, 2012, Diversity of plant evolutionary lineages promotes arthropod diversity, Ecol. Lett., 15, 1308, 10.1111/j.1461-0248.2012.01854.x · DOI
  9. Dovey, 2001, Functional γ-secretase inhibitors reduce β-amyloid peptide levels in brain, J. Neurochem., 76, 173, 10.1046/j.1471-4159.2001.00012.x · DOI
  10. Fedorowicz, 2014, Cytosolic cleaved PINK1 represses Parkin translocation to mitochondria and mitophagy, EMBO Rep., 15, 86, 10.1002/embr.201337294 · DOI
  11. Gegg, 2010, Mitofusin 1 and mitofusin 2 are ubiquitinated in a PINK1/parkin-dependent manner upon induction of mitophagy, Hum. Mol. Genet., 19, 4861, 10.1093/hmg/ddq419 · DOI · Paper page
  12. Hansen, 2011, Following autophagy step by step, BMC Biol., 9, 39, 10.1186/1741-7007-9-39 · DOI
  13. Ikeda, 2000, The expression of presenilin 1 mRNA in skin fibroblasts and brains from sporadic Alzheimer’s disease, Dement. Geriatr. Cogn. Disord., 11, 245, 10.1159/000017246 · DOI
  14. Kuusisto, 2002, Early accumulation of p62 in neurofibrillary tangles in Alzheimer’s disease: possible role in tangle formation, Neuropathol. Appl. Neurobiol., 28, 228, 10.1046/j.1365-2990.2002.00394.x · DOI
  15. Kuusisto, 2001, Ubiquitin-binding protein p62 expression is induced during apoptosis and proteasomal inhibition in neuronal cells, Biochem. Biophys. Res. Commun., 280, 223, 10.1006/bbrc.2000.4107 · DOI · Paper page
  16. Lee, 2015, Presenilin 1 maintains lysosomal Ca2+ homeostasis via TRPML1 by regulating vATPase-mediated lysosome acidification, Cell Rep., 12, 1430, 10.1016/j.celrep.2015.07.050 · DOI · Paper page
  17. Lee, 1996, Expression of presenilin 1 and 2 (PS1 and PS2) in human and murine tissues, J. Neurosci., 16, 7513, 10.1523/JNEUROSCI.16-23-07513.1996 · DOI
  18. Lee, 2010, Lysosomal proteolysis and autophagy require presenilin 1 and are disrupted by Alzheimer-related PS1 mutations, Cell, 141, 1146, 10.1016/j.cell.2010.05.008 · DOI · Paper page
  19. Lemasters, 2005, Selective mitochondrial autophagy, or mitophagy, as a targeted defense against oxidative stress, mitochondrial dysfunction, and aging, Rejuvenation Res., 8, 3, 10.1089/rej.2005.8.3 · DOI
  20. Martín-Maestro, 2016, PARK2 enhancement is able to compensate mitophagy alterations found in sporadic Alzheimer’s disease, Hum. Mol. Genet., 25, 792, 10.1093/hmg/ddv616 · DOI · Paper page
  21. Moreira, Autophagocytosis of mitochondria is prominent in Alzheimer disease, J. Neuropathol. Exp. Neurol., 66, 525, 10.1097/01.jnen.0000240476.73532.b0 · DOI
  22. Moreira, Increased autophagic degradation of mitochondria in Alzheimer disease, Autophagy, 3, 614, 10.4161/auto.4872 · DOI
  23. Prè, 2014, A time course analysis of the electrophysiological properties of neurons differentiated from human induced pluripotent stem cells (iPSCs), PLoS One, 9, e103418, 10.1371/journal.pone.0103418 · DOI
  24. Rubinsztein, 2009, In search of an “autophagomometer”, Autophagy, 5, 585, 10.4161/auto.5.5.8823 · DOI · Paper page
  25. Sardiello, 2009, A gene network regulating lysosomal biogenesis and function, Science, 325, 473, 10.1126/science.1174447 · DOI · Paper page
  26. Sauvat, 2015, Quantification of cellular viability by automated microscopy and flow cytometry, Oncotarget, 6, 9467, 10.18632/oncotarget.3266 · DOI
  27. Sproul, 2015, Being human: the role of pluripotent stem cells in regenerative medicine and humanizing Alzheimer’s disease models, Mol. Aspects Med., 54, 10.1016/j.mam.2015.06.007 · DOI
  28. Sproul, 2014, Characterization and molecular profiling of PSEN1 familial Alzheimer’s disease iPSC-derived neural progenitors, PLoS One, 9, e84547, 10.1371/journal.pone.0084547 · DOI · Paper page
  29. Tanaka, 2010, Parkin-mediated selective mitochondrial autophagy, mitophagy: parkin purges damaged organelles from the vital mitochondrial network, FEBS Lett., 584, 1386, 10.1016/j.febslet.2010.02.060 · DOI
  30. Vives-Bauza, 2010, PINK1-dependent recruitment of Parkin to mitochondria in mitophagy, Proc. Natl. Acad. Sci. U S A, 107, 378, 10.1073/pnas.0911187107 · DOI · Paper page
  31. Wang, 2016, Synaptosomal mitochondrial dysfunction in 5xFAD mouse model of Alzheimer’s disease, PLoS One, 11, e0150441, 10.1371/journal.pone.0150441 · DOI
  32. Wolfe, 2013, Autophagy failure in Alzheimer’s disease and the role of defective lysosomal acidification, Eur. J. Neurosci., 37, 1949, 10.1111/ejn.12169 · DOI
  33. Yamano, 2016, The ubiquitin signal and autophagy: an orchestrated dance leading to mitochondrial degradation, EMBO Rep., 17, 300, 10.15252/embr.201541486 · DOI
  34. Ye, 2015, Parkin-mediated mitophagy in mutant hAPP neurons and Alzheimer’s disease patient brains, Hum. Mol. Genet., 24, 2938, 10.1093/hmg/ddv056 · DOI · Paper page

Cited by

Sorted by citations

Metadata

Authors
Martín-Maestro, P., Gargini, R., A. Sproul, A., García, E., Antón, L. C., Noggle, S., Arancio, O., Avila, J., & García-Escudero, V.
Venue
Frontiers in Molecular Neuroscience
Publisher
Frontiers Media SA
Published
2017-09-14
Volume
10
ISSN
1662-5099
DOI
10.3389/fnmol.2017.00291
PMC
PMC5603661
Cited by
120
References
8
Funders
Ministerio de Economà-a y Competitividad (SAF 2011 program, project 24841, SAF2016 program, project 79607-R, BES-2012-055068)

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review