EMBO/soda-nel huggingface dataset:
THE C99 FRAGMENT OF APP REGULATES CHOLESTEROL TRAFFICKING (2020)
Figure 1

Figure 1. C99 accumulation promotes cholesterol uptake and trafficking to MAM.

(A) Quantification of free cholesterol levels by lipidomics analysis of total homogenates (TH), ER or MAM fractions isolated from WT and PS-DKO cells. Lipid units are represented as molar mass over total moles of lipids analyzed (mol %). Graphs represent fold change over controls. Unpaired t-test (n= 6 for TH, n=3 for MAM or ER; * p<0.05).

(B) Quantification of free cholesterol levels in total homogenates of APP-DKO cells transiently expressing C99, C83 or AICD peptides, or treated with 5 µM Aβ42 oligomers for 16h. Cholesterol levels of WT cells are shown as a control. Lipid units are represented as molar mass over total moles of lipids analyzed (mol %). Graphs represent fold change over controls. One-way ANOVA (n = 4; * p<0.05).

(C) Quantification of HMGCR enzymatic activity in PS-DKO and DAPT-treated WT cells. unpaired t-test (n=3; * p< 0.05).

(D) Quantification of cholesterol uptake by incubating WT and PS-DKO cells, treated or untreated with BACE inhibitor IV (BI, 100 nM), with 2.5 µCi/mL 3H-cholesterol for the indicated times. Graph represents cholesterol radioactivity levels in total cell homogenates (TH). Two-way repeated measures ANOVA (Time, Group)(n = 3 independent experiments; * p< 0.05).

(E-F) Quantification of 3H-cholesterol delivery to MAM (E) or bulk ER (F) after the indicated times. Control and BI-treated or untreated PS-DKO cells incubated with 2.5 µCi/mL 3H-cholesterol for the indicated times, before subcellular fractionation (Western blot of isolated MAM fractions shown in Fig. EV1F). Cholesterol delivery to MAM calculated by quantification of radioactivity levels in the isolated fractions. Two-way repeated measures ANOVA (Time, Group)( n=3 independent experiments; * p< 0.05)

(G) Measurement of cholesterol uptake in the indicated cells by internalization of a fluorescent cholesterol analog [NBD-cholesterol (2 µM; upper panel, nuclei in blue)]. Endogenous free cholesterol was stained with filipin (lower panel). Graphs on the right represent fluorescence intensity (using ImageJ) and the percentage of cells with filipin punctae. Scale bar = 20 µm. One-way ANOVA (30-50 cells/condition from at least 3 independent experiments; * p<0.05, ** p<0.01, *** p<0.001).
THE C99 FRAGMENT OF APP REGULATES CHOLESTEROL TRAFFICKING (2020)
Figure 2

Figure 2. SMase inhibition prevents lipid droplet generation but fails to rescue the increase in cholesterol uptake caused by C99 accumulation.

(A-C) Representative confocal images of WT and PS-DKO cells treated with the indicated SMase inhibitors (10 µM desipramine or 5 µM GW4869) or DMSO (VEH) for 12-16h. (A) Endogenous levels of lipid droplets (LDs) were visualized by incubation with LipidTox. Graph shows the Lipidtox fluorescence intensity/cell. Endogenous levels of free cholesterol revealed by filipin staining of (B) WT or (C) PS-DKO cells under the indicated treatments. Graphs show the filipin fluorescence intensity as a percentage over VEH.

One-way ANOVA. (30-50 cells/condition from at least 3 independent experiments; * p< 0.05).
THE C99 FRAGMENT OF APP REGULATES CHOLESTEROL TRAFFICKING (2020)
Figure 3

Figure 3. The cholesterol binding domain of C99 is necessary for its localization to MAM.

(A) Crude membrane fractions from APP-DKO cells expressing C99WT or C99MUT were treated with 0.2% Triton-X-100, loaded onto continuous density sucrose gradients and centrifuged for 16h. Fractions from these gradients were analyzed by western blot to determine the migration of the indicated proteins [two parallel gels (bold vertical line)].

(B-G) Graphs represent the relative abundance of the indicated proteins in each fraction of the gradient, as measured by densitometry analysis of western blot signals (ImageJ). Note the reduced degree of co-migration of C99MUT with MAM markers (Acsl4), compared to C99WT.
THE C99 FRAGMENT OF APP REGULATES CHOLESTEROL TRAFFICKING (2020)
Figure 4

Figure 4. C99's cholesterol binding domain facilitates MAM formation and activation.

(A) Representative confocal images of APP-DKO cells expressing GFP-tagged C99WT or C99MUT (in green) and fluorescent markers of mitochondria (MitoDsRed, in red) and ER (Sec61β-BFP, in blue), and treated with DAPT to prevent C99 cleavage. Note the different distribution of C99WT or C99MUT forms. Scale bar = 10 µm. Insets show 5X amplifications of individual (C99 in green, and mitochondria in red) and merged images. Black and white bottom panels represent the areas where C99 and mitochondria signals colocalize. Upon thresholding each channel, a mask for mitochondria was generated and the C99 channel was superimposed on the mitochondria mask, so the positive pixels found in both channels are shown in black.

(B) Quantification of the degree of colocalization of C99WT or C99MUT forms with mitochondria or ER measured by ImageJ. Note the significantly decreased colocalization between C99MUT and mitochondria, when compared to that of C99WT. Unpaired t-test. (20-30 cells/condition from 3 independent experiments and 4-5 images/experiment; * p<0.05; n.s., not significant).

(C) Representative confocal images of APP-DKO cells expressing GFP-tagged C99WT or C99MUT and ER and mitochondria markers as in (A) (upper panel). Individual zoomed single channels (middle panel) as well as areas of ER-mitochondria colocalization (bottom panel) are shown for each condition. Scale bar = 10 µm.

(D) Quantification of ER-mitochondria colocalization per cell analyzed. Upon thresholding each channel, a mask for ER was generated and the mitochondria channel was superimposed on the ER mask, so the positive pixels found in both channels were quantified and referenced over the total area of the ER mask. The number of cells analyzed is indicated in white text within each column. Levels of ER-mitochondria colocalization in APP-WT cells are shown for reference as a dashed line. EV, empty vector. One-way ANOVA (n=3 independent experiments and 5-8 images/experiment; * p< 0.05).
THE C99 FRAGMENT OF APP REGULATES CHOLESTEROL TRAFFICKING (2020)
Figure 5

Figure 5. C99's cholesterol binding domain is necessary for cholesterol trafficking to MAM.

(A) Scheme of PhotoClick cholesterol methodology to detect C99 interaction with cholesterol.

(B) Representative immunoblot to reveal C99 levels in total homogenate (TH) and MAM fractions of APP-DKO cells expressing C99WT or C99MUT before (input) and after cholesterol pull-down. Acsl4 was used as a MAM marker. Streptavidin-HRP was used to detect total biotinylation (biotin conjugated to PhotoClick cholesterol).

(C) Quantification of pulled-down C99 levels versus input from the experiment in B. Two-way repeated measures ANOVA (Fraction, mutation) (n=5; * p< 0.05, ** p< 0.01).

(D) Immunoblot showing the levels of pulled-down PhotoClick Cholesterol (streptavidin-HRP) in ER and MAM fractions. Note how the levels of pulled-down C99 in the ER are negligible when compared to those from MAM. Isolated MAM and ER fractions were assessed by western blot (shown in AppendixFig S1E).

(E) Quantification of free cholesterol levels (FC) analyzed by lipidomics after subcellular fractionation to obtain MAM from the indicated cells. Lipid units are represented as molar mass over total moles of lipids analyzed (mol%). Graphs represent percentage over controls. Dashed line represents control levels. One-way ANOVA. (n=4; * p< 0.05)

(F-G) Quantification of cholesterol uptake and esterification in the indicated cells was measured by 4h incubation with 3H-cholesterol and subsequent analysis of radiolabel incorporation. The dashed line indicates control levels. Graphs represent fold change over controls. Treatment with Sandoz 58-035, a specific ACAT1 inhibitor, caused a ~95% reduction in cholesterol esterification. EV, empty vector. One-sample t-test. (n=3-4; * p< 0.05).

(H) Ratio of cholesteryl esters:free cholesterol (CE:FC) in the indicated cells.

One-way ANOVA. (n=4; * p< 0.05).
Repeat-associated non-AUG translation in C9orf72-ALS/FTD is driven by neuronal excitation and stress (2018)
Figure 1

Figure 1. C9orf72-based constructs containing the sense, (G4C2)~188, NRE undergo pathologically relevant non-AUG-dependent translation with DPR levels that vary by cell-type.

(A) Plasmid reporter constructs developed to monitor non-AUG-dependent translation using the photoconvertible fluorescent protein, Dendra2. The endogenous C9orf72 5ʹ gene region is immediately upstream and Dendra2 is immediately downstream of the NRE insertion site. Each construct is modified +1 or +2 base pairs to fluorescently monitor all individual ORFs that are in-frame with the Dendra2 with an n-terminal HA affinity tag.

(B) All ORFs for the sense non-AUG-dependent translation of the (G4C2)n~188, DPR coding for (GA), (GP), (GR) can be detected and quantified using a filter-trap binding assay probing for the c-terminal HA tag of the C9 DPR reporter construct. The (GA) ORF is the most abundantly detected DPR and GR the lowest in HEK293T cell 24 h post transient transfection of the C9 DPR reporter construct. n = 5.

(C) The C9orf72 NRE non-AUG-dependent reporter constructs recapitulate localization patterns and pathological features identified in C9orf72 NRE models. The Dendra2 (green) c-terminal fusion was used to monitor cellular localization and fluorescence intensity for each DPR translated from the G4C2 strand in HEK293T cells transiently cotransfected with an NES-mIFP-mIFP-NES (NES-mIFP) fusion protein cytoplasmic marker.

(D) C9 DPR reporter constructs show cell-type variability in the number of cells that are DPR positive and the overall DPR levels. Transient cotransfections of HEK293T cells robustly undergo G4C2 -Dendra2 non-AUG-dependent translation compared to NSC34 or rat primary cortical neurons. DPR positive cells and DPR fluorescent intensity were analyzed and calculated 24 h post-transfection only in cells expressing the co-transfected NES-mIFP construct. DPR fluorescent intensity was normalized to NES-mIFP fluorescent intensity to adjust for cell specific AUG-dependent translation levels; represented as fold-change relative to HEK293T. HEK293T: n = 5 with m > 500 cells analyzed per n. NSC34: n = 5 with m > 500 cells per n. Rat primary cortical neuron: n = 5 with m > 40 cells per n. (***P < 0.001, **P < 0.01, *P <0.05) See also Figure EV1-2.
Repeat-associated non-AUG translation in C9orf72-ALS/FTD is driven by neuronal excitation and stress (2018)
Figure 2

Figure 2. Cellular stress or neuronal excitotoxic stress increases DPR levels for all C9orf72 NRE ORFs in rat cortical neurons and in patient derived iPS spinal motor neurons.

(A) Quantitative fluorescent microscopy imaging shows a dose-dependent increase in the number of C9 DPR reporter positive cells with increasing concentrations of an apoptotic stimulator, staurosporine (SSP). The C9 DPR reporter in the GP frame was utilized for these experiments. n = 4.

(B) Filter trap binding assays using DPR-specific antibodies show all DPR levels are increased for the sense-coding C9 DPR reporter when NSC34 cells are challenged with stress-inducing compounds. These stress-inducing compounds broadly cover distinct or overlapping cellular pathways (see Table EV2) Antibodies specific for the C9orf72 NRE DPRs, poly-GA, -GP, or -GR, were used to quantify DPR levels normalized to GAPDH 24 h post transient transfection of the C9 DPR reporter constructs. n = 4.

(C) Quantitative fluorescent microscopy imaging shows that NSC34, rat primary cortical neurons have increased relative DPR fluorescent intensity levels following compound induced cellular stress. In NES-mIFP cotransfection positive NSC34 and rat primary cortical neurons there is a significant increase in C9 DPR reporter fluorescent intensity levels 24 post treatment with most stress-inducing compounds. Fluorescent intensity was not normalized to NES-mIFP fluorescent levels. NSC34: n = 10 with m > 500 cells analyzed per n. Cortical neuron: n = 6 with m > 40 cells per n.

(D) Endogenously expression of DPRs in iPS spinal motor neurons (iPS sMN) derived from C9orf72 NRE patients increases following induction of cellular stress. DPR fluorescent intensity in iPS sMN was measured using the DPR-specific antibodies described above. IPS sMN: n = 4 with m > 20 cells per n.

All changes in DPR levels are shown relative to non-stressed or DMSO-only (vehicle) treated CTRL cells unless otherwise noted.

Control (CTRL), thapsigargin (TG), Menadione (Mena), staurosporine (SSP), Diamide (Daim), Cytochalasin D (Cyto D), Etoposide (Etop), Leptomycin B (Lepto), Homocystine (HC), Sodium Arsenite (NaArs), Tunicamycin (TM), Glutamate (Glut)

Statistical comparisons in (A) were calculated using t-tests per concentration and (B) and (C) were calculated using an uncorrected Fisher LSD two-way anova for CTRL vs. treatment. (****P < 0.0001, ***P < 0.001, **P < 0.01, *P <0.05) See also Table EV2 Figure EV2-3.
Repeat-associated non-AUG translation in C9orf72-ALS/FTD is driven by neuronal excitation and stress (2018)
Figure 3

Figure 3. Excitotoxic stress-induced C9orf72 NRE-linked DPR levels can be decreased by blocking AMPA or NMDA in primary cortical neurons and in patient derived iPS spinal motor neurons.

(A) Fluorescent microscopy images shows Glutamate-induced (Glut) excitotoxic stress increases DPR levels in rat cortical neurons expressing the C9 DPR reporter and in iPS sMN derived from C9orf72 NRE patients. Glutamate-induced DPR levels are reduced by pretreating cells with the AMPA and NMDA specific antagonists NBQX and MK801, respectively.

(B) Relative fluorescent intensity quantification demonstrates there is a significant increase in DPR levels when primary cortical neurons and sMNs are challenged with the excitotoxic stressors: glutamate, AMPA, NMDA, and HC. Blocking either the AMPA or NMDA receptors reduces DPR levels in the presence of excitotoxic stressors. NES-mIFP or MAP2 were used to mark transfection positive rat cortical neurons or iPS sMNs, respectively, for quantification. DPR fluorescent intensity in iPS sMN was measured using DPR-specific antibodies. Changes in DPR levels are relative to non-stressed cells (CTRL) Cortical neuron: n = 6 with m > 40 cells per n. IPSC sMN: n = 4 with m > 25 cells per n. (****P < 0.0001, ***P < 0.001, **P < 0.01, *P <0.05).
Repeat-associated non-AUG translation in C9orf72-ALS/FTD is driven by neuronal excitation and stress (2018)
Figure 4

Figure 4. Increased neuronal activity increases non-AUG dependent translation of DPRs in primary cortical neurons.

(A) Neuronal activity was induced with Chr2 stimulation by performing trains of LED light pulses at various frequencies: No stim = 0 Hz; Low = 1 Hz; Med = 10 Hz; High = 100 Hz. Trains included 30 s of light pulses followed by 25 s of recovery for desensitized ChR2; trains were performed 20 times.

(B). Microscopy images show increased DPR fluorescent intensity levels from medium and high frequency stimulation of ChR2 in primary cortical neurons transfected with the C9 DPR reporter.

(C) Relative fluorescent intensity quantification shows a significant increase in DPR levels with increases in neuronal activity through medium and high stimulation in primary cortical neurons. No stimulation and low stimulation frequencies did not increase levels of DPR. The C9 DPR reporter, monitoring the GP DPR (ORF2), was cotransfected with ChR2. Cortical neuron: n = 4 with m > 10 cells per n. (****P < 0.0001).
Repeat-associated non-AUG translation in C9orf72-ALS/FTD is driven by neuronal excitation and stress (2018)
Figure 5

Figure 5. Increases in ISR proteins are concomitant with DPR inducing cellular or excitotoxic stressors.

(A) Filter trap binding assays show that ISR proteins are enhanced by all stressors. Significant increases following cellular stress treatments are seen in the ratio of phospho-eif2α to total eif2α as well as phospho-PERK and ATF4 levels compared to untreated controls. Changes in ISR proteins are normalized to GAPDH and shown relative to non-stressed or DMSO-only treated cells (CTRL) depending on the vehicle compounds were dissolved in. n = 4.

(B) Fluorescent intensity measurements show the levels of two ISR-related proteins, PERK and relative phospho-eif2α (phospho-eif2α / total eif2α), are increased following different excitotoxic stress treatments in cortical neurons. Representative images indicate fluorescent intensity differences for phospho-eif2α and phospho-PERK levels 24 h post treatment with different excitotoxic stressor paradigms. Blocking of AMPA and NMDA receptors using known antagonists significantly reduces ISR-related protein levels, which parallels reduced DPR levels (Figure 3B). Changes in ISR proteins are relative to non-stressed cells (CTRL). n = 4 with m > 40 cells per n. (****P < 0.0001, ***P < 0.001, **P < 0.01, *P <0.05).
Repeat-associated non-AUG translation in C9orf72-ALS/FTD is driven by neuronal excitation and stress (2018)
Figure 6

Figure 6. DPR production is inhibited through pharmacological intervention of ISR-related pathways.

(A) Relative fluorescent intensity quantification shows a significant decrease in C9 DPR reporter levels when stressor challenged NSC34 cells were treated with compounds inhibiting ISR-related or translation-targeting pathways. Dibenzoylmethane (1,3DBM; 20 µM), which inhibits eif2α-P activity, had the most significant decreases in DPR production. Trazodone (Traz; 20 µM) showed similar significant reduction in DPR levels as the PERK inhibitor, GSK2606414 (500 nM).

(B) Relative fluorescent intensity quantification shows a significant decrease in C9 DPR reporter levels when glutamate stressed primary cortical neurons were treated with the compounds Trazodone (Traz; 20 µM) or Dibenzoylmethane (1,3DBM; 20 µM). Similar to the treatments in NSC34, 1,3DBM shows the most significant decrease in DPR production.

Changes in DPR levels are relative to DMSO-only treated cells (CTRL). GSK2606414 (500 nM), CX5461 (250 nM), Salubrinal (Sal; 15 µM), Cercosporamide (Cerco; 120 nM), Metformin (Metf; 500 µM), Trazodone (Traz; 20 µM), Dibenzoylmethane (1,3DBM; 20 µM). NSC34: n = 4 with m > 100 cells per n. Cortical neuron: n = 4 with m > 40 cells per n. (****P < 0.0001, ***P < 0.001, **P < 0.01, *P <0.05).
cAMP and EPAC are key players in the regulation of the signal transduction pathway involved in the α-hemolysin autophagic response. (2012)
Figure 1

cAMP inhibits Hla-induced autophagy.

(A) GFP-LC3 CHO cells were preincubated with 1 mM dybutiryl cAMP (+dbcAMP, panels d, e, and f) for 30 min and then were treated for 4 h with 10 µg/ml of α-hemolysin (Hla) or subjected to starvation conditions (Stv) in the continuous presence of dbcAMP. Cells without any treatment were used as control (−dbcAMP, panels a, b, and c). Cells were immediately analyzed by confocal microscopy. A quantification of the percentage of cells presenting LC3 puncta (i.e., stimulated cells) upon incubation with the different conditions is shown in the right panel (n = 100 cells/condition, * p<0.05 paired Student's t-test). These data are representative of three independent experiments. (B) GFP-LC3 CHO cells were incubated with complete medium in the absence (−dbcAMP) or presence of dbcAMP (+dbcAMP) and treated for 4 h with 10 µg/ml of α-hemolysin (Hla) or subjected to starvation conditions (Stv) with (lower panel) or without (upper panel) bafilomycin A1 to block lysosomal degradation. Afterwards, cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti-LC3 and the corresponding HRP-labeled secondary antibody, and subsequently developed with an enhanced chemiluminescence detection kit. These data are representative of three independent experiments. (C) The band intensities of two independent experiments were quantificated with the Adobe Photoshop program, and normalized against tubulin. * p<0.05 (paired Student's t-test).
cAMP and EPAC are key players in the regulation of the signal transduction pathway involved in the α-hemolysin autophagic response. (2012)
Figure 2

PKA inhibition does not affect the autophagic response induced by Hla.

(A) GFP-LC3 CHO cells were preincubated for 30 min with 10 µM H89, a PKA inhibitor, in the presence (panels a, b, and c) or absence (panels d, e, and f) of 1 mM dbcAMP. Subsequently, they were incubated for 2 h in starvation medium (Stv, panels b and e) or treated for 4 h with 10 µg/ml of α-hemolysin (Hla, panels c and f) in full nutrient media. Cells without any treatment were used as control (Ctr, panels a and d). Cells were finally analyzed by confocal microscopy. Images are representative of three independent experiments. (B) Quantification of the percentage of cells presenting LC3 puncta (i.e., stimulated cells) upon incubation with the different conditions. ** p<0.01 (paired Student's t-test, n = 100 cells/condition). These data are representative of two independent experiments. (C) GFP-LC3 CHO cells were preincubated for 30 min in the presence or absence of 10 µM H89, and then they were incubated for 2 h with 1 mM dbcAMP to verify the activity of H89. Afterwards, cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti-phosphoCREB and the corresponding HRP-labeled secondary antibody, and subsequently developed with an enhanced chemiluminescence detection kit. The band intensities were quantified with the Adobe Photoshop program (lower panel). These data are representative of two independent experiments.
cAMP and EPAC are key players in the regulation of the signal transduction pathway involved in the α-hemolysin autophagic response. (2012)
Figure 3

EPAC activation inhibits the Hla-dependent autophagy.

(A) GFP-LC3 CHO cells were preincubated with 10 µM 8-pCPT-2′-O-Me-cAMP (8-pCPT) for 30 min and then they were treated for 4 h with 10 µg/ml of α-hemolysin (Hla) or subjected to starvation conditions (Stv). Cells without any treatment were used as control. Cells were immediately analyzed by confocal microscopy. Quantification of the percentage of cells presenting LC3 puncta upon incubation under the different conditions is shown in the right panel (n = 50 cells/condition). These data are representative of two independent experiments. (B) CHO cells were cotransfected with GFP-LC3 and myc-EPAC wt (left panel) or myc-ΔEPAC (right panel). Twenty-four hours after transfection they were incubated for 2 h in starvation medium (Stv) or treated for 4 h with 10 µg/ml of α-hemolysin (Hla). Cells without any treatment were used as control (Ctr). Cells were analyzed by confocal microscopy. Images are representative of two independent experiments. (C) Quantification of the percentage of cells presenting LC3 puncta upon incubation under the different conditions (n = 20 cells/condition). These data are representative of two independent experiments.