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1.
Figure 4.

Figure 4. From: Centaurin-?1-Ras-Elk-1 Signaling at Mitochondria Mediates ?-Amyloid-Induced Synaptic Dysfunction.

Ras is involved in Aβ-dependent reduction of mEPSC amplitude and frequency. A, Representative traces of mEPSCs in CA1 pyramidal neurons in organotypic slices, filtered with a 1 ms window. Slices were treated with vehicle (Veh) or 1 μm Aβ together with 0.25% DMSO or 10 μm FTI-277 (FTI; DMSO as vehicle) for 7 d. B, mEPSC frequency. *p < 0.05. The number of neurons was 38, 18, 14, and 20, respectively. Mean frequency of mEPSCs was calculated over each neuron and the mean and SEM of the averaged mEPSCs were plotted. C, mEPSC amplitude. Same data as in B.

Erzsebet M. Szatmari, et al. J Neurosci. 2013 March 20;33(12):5367-5374.
2.
Figure 3.

Figure 3. From: Centaurin-?1-Ras-Elk-1 Signaling at Mitochondria Mediates ?-Amyloid-Induced Synaptic Dysfunction.

Elk-1 is involved in Aβ-mediated dendritic spine loss. A, Representative images of apical dendrites of CA1 pyramidal neurons in organotypic slices transfected with EGFP and pSuper (cloning vector for shRNA) or Elk-1-specific shRNA (sh-Elk-1) and treated with vehicle (Veh) or 1 μm Aβ for 7 d. Scale bar, 5 μm. B, Spine density in apical dendrites of CA1 pyramidal neurons treated as in A. *p < 0.01. The number of neurons was 9, 5, 6, and 8, respectively. Error bars indicate SEM. C, Spine density in apical dendrites of CA1 pyramidal neurons transfected with EGFP and one of the following combinations of constructs for 7 d: pSuper and empty cloning vector (Vec), pSuper and wild-type CentA1 (pSuper + WT-CentA1) or sh-Elk-1 and wild-type CentA1 (sh-Elk-1 + WT-CentA1). *p < 0.05. The number of neurons was 12, 8, and 8, respectively. Error bars indicate SEM. D, Spine density in apical dendrites of CA1 pyramidal neurons transfected with EGFP and cloning vector (pcDNA3) or wild-type Elk-1 (WT-Elk-1). *p < 0.01. The number of neurons was 10 and 16. Error bars indicate SEM.

Erzsebet M. Szatmari, et al. J Neurosci. 2013 March 20;33(12):5367-5374.
3.
Figure 6.

Figure 6. From: Centaurin-?1-Ras-Elk-1 Signaling at Mitochondria Mediates ?-Amyloid-Induced Synaptic Dysfunction.

Increase in CentA1 and mitochondrial Elk-1 in a mouse model of AD. A, Expression level of CentA1 in hippocampi of APP mice overexpressing a mutant human form of amyloid precursor protein (APP) and in their non-transgenic littermates (WT). The level of CentA1 in hippocampi from 3-month-old (n = 5 WT, n = 6 APP), 6-month-old (n = 6 WT, n = 6 APP), 8-month-old (n = 5 WT, n = 7 APP), and 12-month-old (n = 4 WT, n = 5 APP) male mice was analyzed by Western blotting, followed by reprobing of the membranes for β-actin. B, The amount of Elk-1 in mitochondria isolated from hippocampi of male WT and APP mice. Mitochondrial lysates from hippocampi of WT and APP 3-months-old mice (n = 5 WT, n = 6 APP), 6-month-old mice (n = 11 WT, n = 11 APP), 8-month-old mice (n = 5 WT, n = 7 APP), and 12-month-old mice (n = 5 WT, n = 6 APP) were immunoblotted for Elk-1, followed by VDAC reprobing. C, Time dependence of CentA1 upregulation and Elk-1 mitochondrial association in APP mice compared with non-transgenic littermates. D, Hypothetical signaling scheme linking Aβ and neuronal dysfunctions.

Erzsebet M. Szatmari, et al. J Neurosci. 2013 March 20;33(12):5367-5374.
4.
Figure 2.

Figure 2. From: Centaurin-?1-Ras-Elk-1 Signaling at Mitochondria Mediates ?-Amyloid-Induced Synaptic Dysfunction.

CentA1 is required for Aβ-induced impairment of spine structural plasticity. A, Time-lapsed images of spine structural plasticity induced by 2-photon glutamate uncaging in neurons transfected with sh-SC or sh-CentA1 and treated with vehicle (Veh) or Aβ for 4–6 d. The arrows indicate the stimulated spines. Structural plasticity was induced by applying a low-frequency train of two-photon uncaging pulses (6 ms, 30 pulses, 0.5 Hz) to a single dendritic spine in zero extracellular Mg2+ and 2 mm MNI-caged glutamate. B, Time course of spine volume change in stimulated spines or adjacent spines (Adj) in neurons treated as in A. The number of samples (spine/neuron) was 10/8, 9/6, 10/7, and 10/8, respectively. C, Transient spine volume change (volume change averaged over 25–30 min subtracted by volume change at 2 min (Matsuzaki et al., 2004). *p < 0.05. Error bars indicate SEM. D, Sustained spine volume change (volume change averaged over 25–30 min (Matsuzaki et al., 2004). *p < 0.05. Error bars indicate SEM.

Erzsebet M. Szatmari, et al. J Neurosci. 2013 March 20;33(12):5367-5374.
5.
Figure 1.

Figure 1. From: Centaurin-?1-Ras-Elk-1 Signaling at Mitochondria Mediates ?-Amyloid-Induced Synaptic Dysfunction.

CentA1-Ras signaling mediates Aβ-induced loss of dendritic spines. A, Expression level of CentA1 in dissociated hippocampal neurons treated with Aβ(1–42). Dissociated hippocampal neurons from P0 rats were transfected with sh-SC or sh-CentA1 using electroporation, cultured for 3 d and treated with vehicle (Veh; final 0.01% NH4OH) or 1 μm Aβ for 48 h. The level of CentA1 was analyzed by Western blotting and β-actin was used as a loading control. Numbers under blots represent normalized CentA1 expression in four independent experiments. B, Immunoblot analysis of the expression level of CentA1 in organotypic hippocampal slices treated with Aβ(1–42). Slices were prepared from postnatal day 6 rats and cultured for 1–2 weeks, then treated with Veh or 1 μm Aβ for 48 h and 7 d, respectively. The level of CentA1 was analyzed by Western blotting and β-actin was used as a loading control. Numbers under blots represent normalized CentA1 expression in 15 (48 h) and 22 (7 d) independent experiments. C, Representative images of apical dendrites from CA1 pyramidal neurons in organotypic slices. Slices were prepared from postnatal day 6 rats and cultured for 1–2 weeks. Neurons were biolistically transfected with sh-SC or sh-CentA1 together with EGFP, treated with either vehicle or 1 μm Aβ (1–42) for 7 d, and imaged with two-photon laser scanning microscopy. Scale bar, 5 μm. D, Quantification of spine density in neurons treated as in C. Error bars indicate SEM. *p < 0.01, ANOVA followed by post hoc test using least significant difference. The number of neurons was 8, 11, 8, and 8, respectively. E, Spine density in apical dendrites of CA1 pyramidal neurons transfected with sh-SC or sh-CentA1 together with an empty vector (Vec) or human amyloid precursor protein (APP). The number of neurons was 5, 6, and 5, respectively. Error bars indicate SEM. F, Images of apical dendrites of CA1 pyramidal neurons expressing EGFP and Vec or WT-CentA1. Scale bar, 5 μm. G, Images of apical dendrites of CA1 pyramidal neurons expressing EGFP and Vec or dominant-negative Ras (HRasS17N, DN-Ras). Scale bar, 5 μm. H, Quantification of spine density in neurons expressing EGFP and Vec, WT-CentA1, or DN-Ras and treated with Veh or 1 μm Aβ for 1 week. *p < 0.01. The number of neurons was 18, 9, 8, 6, and 6, respectively. I, Spine density in apical dendrites of CA1 pyramidal neurons treated with Veh or 1 μm Aβ together with DMSO or 10 μm FTI-277 for 1 week. *p < 0.01. Number of neurons was 8, 5, 6, and 11, respectively. Error bars indicate SEM.

Erzsebet M. Szatmari, et al. J Neurosci. 2013 March 20;33(12):5367-5374.
6.
Figure 5.

Figure 5. From: Centaurin-?1-Ras-Elk-1 Signaling at Mitochondria Mediates ?-Amyloid-Induced Synaptic Dysfunction.

Aβ-induced mitochondrial association of Elk-1 requires the CentA1-Ras-ERK pathway A, Nuclear Elk-1 phosphorylation/activation in organotypic slice cultures treated with vehicle (Veh) or 1 μm Aβ for 48 h followed by subcellular protein extraction and Western blotting of nuclear and cytosolic extracts for phospho-Elk-1 (Ser383). Blots were reprobed for total Elk-1 to ensure equal loading. GAPDH was used as a cytosolic marker and NeuN was used as a neuronal nuclear marker. Numbers under the blots represent relative Elk-1 phosphorylation/activation from four independent experiments. B, Elk-1 activity in dissociated hippocampal neurons quantified with the luciferase gene reporter assay. Neurons were transfected with either wild-type CentA1 (WT-CentA1) or empty vector (Vec) together with plasmids required for the assay (pFR-Luc, pFA2-Elk-1 and EF1ɑ-LacA), and treated with Veh (final 0.01% NH4OH) or 1 μm Aβ for 20 h. Error bars indicate SEM. *p < 0.05. C, Amount of Elk-1 in isolated mitochondria from hippocampal neuron cultures. Dissociated hippocampal neurons were electroporated with sh-SC or sh-CentA1, cultured for 3 d, treated with Veh or 1 μm Aβ for 48 h, followed by mitochondrial isolation. Elk-1 levels in mitochondrial fractions were determined by Western blotting. COX IV and VDAC were used as loading controls and mitochondrial markers. Numbers under blots represent COX-normalized changes in mitochondrial association of Elk-1 from seven independent experiments. D, Immunoblots of Elk-1 in whole lysates (total Elk-1; Top) and in mitochondria (Bottom) prepared from organotypic hippocampal slices treated with vehicle or 1 μm Aβ for 7 d. Numbers under the blots represent changes in expression of total Elk-1 normalized to actin (Top, n = 3) or mitochondrial Elk-1 normalized to VDAC (Bottom, n = 11). E, Ras signaling mediates mitochondrial association of Elk-1. Organotypic hippocampal slices were treated for 7 d with Veh or 1 μm Aβ together with 0.25% DMSO or 10 μm FTI-277 (DMSO as vehicle). The amount of total Elk-1 and Elk-1 from isolated mitochondria was analyzed by Western blotting. VDAC was used as the loading control and mitochondrial marker. NeuN was used to test the purity of the mitochondrial fractions. Numbers under blots represent normalized changes in Elk-1 mitochondrial association from five independent experiments. F, Organotypic hippocampal slices were treated for 7 d as in E in the presence or absence of FTI-277. Mitochondrial association and phosphorylation level of Elk-1 at Ser383 were determined by Western blotting, followed by VDAC reprobing as a loading control and mitochondrial marker. Numbers under the blots represent VDAC-normalized changes in mitochondrial Elk-1 phosphorylation from four independent experiments. G, Mitochondrial activity measured with TMRM fluorescence in neurons treated with DMSO or FTI-277. Dissociated hippocampal neurons (DIV6) were treated as in D for 24 h, followed by treatment with 100 nm TMRM (Invitrogen) for 30 min. The overall cellular fluorescence levels were visualized by fluorescence microscopy. The brightness of TMRM in each neuron was analyzed using ImageJ. Data are normalized to control condition (Veh + DMSO). Data represent average of four independent experiments. *p < 0.05. Error bars indicate SEM. H, Representative images of apical dendrites from CA1 pyramidal neurons in organotypic slices transfected with EGFP and treated with either vehicle or 1 μm Aβ (1–42) for 7 d, followed by cotreatment for additional 3 d with vehicle or 1 μm bongkrekic acid (BKA; DMSO as vehicle) or 1 μm cyclosporin A (CsA; DMSO as vehicle) and imaged with two-photon laser scanning microscopy. I, Quantification of spine density in neurons treated as in H. *p < 0.01. The number of neurons was 18, 16, 10, and 8, respectively. Error bars indicate SEM.

Erzsebet M. Szatmari, et al. J Neurosci. 2013 March 20;33(12):5367-5374.

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