Journal: bioRxiv

Article Title: Partial Inhibition of Mitochondrial Complex I Reduces Tau Pathology and Improves Energy Homeostasis and Synaptic Function in 3xTg-AD Male and Female Mice

doi: 10.1101/2020.08.19.258236

Figure Lengend Snippet: Mechanisms of CP2-dependent restoration of pTau-mediated synaptic dysfunction. Under physiological conditions, tau transports Fyn to the dendritic spines where Fyn phosphorylates the NR2B subunit of NMDARs at Y-1472 leading to the stabilization of the NMDAR:PSD95 complex. Increased tau phosphorylation leads to the destabilization of the NMDAR:PSD95 and AMPAR:PSD95 complexes with increased AMPAR endocytosis resulting in reduced synaptic strength and LTP. Partial inhibition of MCI with CP2 increases AMP/ATP ratio that directly activates AMPK with concomitant effect on the activity of PP2Ac, CDK5 and GSK3β resulting in a significant reduction in pTau levels. Reduction in pTau and Fyn results in the stabilization of the NMDAR and AMPAR complexes and improved LTP leading to enhanced cognitive function in 3xTg-AD mice.

Article Snippet: The following primary antibodies were used: phospho-AMPK (Thr 172) (1:1000, Cell Signaling Technology, cat. # 2535), AMPK (1:1000, Cell Signaling Technology, cat. #2532), Synaptophysin (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, cat. # 17750), PSD95 (1:1000, Cell Signaling Technology, cat. # 2507), GluA1 (1:1000, Cell Signaling Technology, cat. # 13185), phospho-AMPAR 1 (Ser845) (1:1000, Cell Signaling Technology, cat. # 8084), GluN2B (1:1000, Cell Signaling Technology, cat. # 14544), phospho-NMDAR 2B (Tyr1472) (1:1000, Cell Signaling Technology, cat. # 4208), AT8 (1:1000, Thermo Fisher, cat. # MN1020), AT180 (1:1000, Thermo Fisher, cat. # MN1040), HT7 (1:1000, Thermo Fisher, cat. # MN1000), Fyn (1:1000, Cell Signaling Technology, cat. # 4023), phosphor-Src (Tyr416) (1:1000, Cell Signaling Technology, cat. # 6943), CDK5 (1:1000, Cell Signaling Technology, cat. #2506), p35/p25 (1:1000, Cell Signaling Technology, cat. # 2680), GSK3β (1:1000, Cell Signaling Technology, cat. #9832), phospho-GSK3β (Ser9) (1:1000, Cell Signaling Technology, cat. #9322), PP2Ac (1:1000, Cell Signaling Technology, cat. #2038), phospho-PP2Ac (Tyr 307) (1:1000, Santa Cruz, cat. #sc-12615), Tubulin (1:5000, Biovision, cat. #3708), β-Actin (1:5000, Sigma-Aldrich, cat. # A5316).

Techniques: Phospho-proteomics, Inhibition, Activity Assay

Journal: bioRxiv

Article Title: Partial Inhibition of Mitochondrial Complex I Reduces Tau Pathology and Improves Energy Homeostasis and Synaptic Function in 3xTg-AD Male and Female Mice

doi: 10.1101/2020.08.19.258236

Figure Lengend Snippet: A) Experimental setup for stimulation-evoked local field potential (LFP) measurement in the hippocampal acute slice. The stimulation (Stim.) electrode was placed at the Schaffer collaterals and the recording (Rec.) electrode was placed at the striatum radiatum in the CA1 region. To induce LTP, three tetanic stimulations (100 Hz, 60 µsec-pulse width for 1 sec) were applied with 3 sec intervals. In the slice, LTP was induced and maintained over 30 - 50 min. Slope of fEPSPs was measured and results normalized to the average value established during the 30 min baseline period. Recording continued for at least 60 min following a tetanic stimulation and the first 30 min was used to calculate the LTP. B) CP2 treatment improves LTP formation in 3xTg-AD female mice. Traces represent mean ± SEM per time. N = 2-3 slices from 3–5 mice per group. C) LTP intensity in CP2-treated female 3xTg-AD mice compared to vehicle-treated counterparts at 30 min after the stimulation. N = 2 - 3 slices from 3–5 mice per group. D) Representative western blot analysis of protein markers involved in synaptic function of dendritic spines in the brain tissue of CP2- and vehicle-treated female 3xTg-AD mice. N = 4 mice per group. E) Quantification of western blot data from (D) showing a percent of changes in protein expression after CP2 treatment. Significant increase was observed in levels of synaptophysin, PSD95, and GluA1 and GluN2B phosphorylation. No changes were detected in total levels of GluA1 and GluN2B. Differences between individual groups were analyzed by Student t -test. Data are presented as mean ± SEM. *P<0.05, **P<0.01.

Article Snippet: The following primary antibodies were used: phospho-AMPK (Thr 172) (1:1000, Cell Signaling Technology, cat. # 2535), AMPK (1:1000, Cell Signaling Technology, cat. #2532), Synaptophysin (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, cat. # 17750), PSD95 (1:1000, Cell Signaling Technology, cat. # 2507), GluA1 (1:1000, Cell Signaling Technology, cat. # 13185), phospho-AMPAR 1 (Ser845) (1:1000, Cell Signaling Technology, cat. # 8084), GluN2B (1:1000, Cell Signaling Technology, cat. # 14544), phospho-NMDAR 2B (Tyr1472) (1:1000, Cell Signaling Technology, cat. # 4208), AT8 (1:1000, Thermo Fisher, cat. # MN1020), AT180 (1:1000, Thermo Fisher, cat. # MN1040), HT7 (1:1000, Thermo Fisher, cat. # MN1000), Fyn (1:1000, Cell Signaling Technology, cat. # 4023), phosphor-Src (Tyr416) (1:1000, Cell Signaling Technology, cat. # 6943), CDK5 (1:1000, Cell Signaling Technology, cat. #2506), p35/p25 (1:1000, Cell Signaling Technology, cat. # 2680), GSK3β (1:1000, Cell Signaling Technology, cat. #9832), phospho-GSK3β (Ser9) (1:1000, Cell Signaling Technology, cat. #9322), PP2Ac (1:1000, Cell Signaling Technology, cat. #2038), phospho-PP2Ac (Tyr 307) (1:1000, Santa Cruz, cat. #sc-12615), Tubulin (1:5000, Biovision, cat. #3708), β-Actin (1:5000, Sigma-Aldrich, cat. # A5316).

Techniques: Western Blot, Expressing, Phospho-proteomics

Journal: bioRxiv

Article Title: Partial Inhibition of Mitochondrial Complex I Reduces Tau Pathology and Improves Energy Homeostasis and Synaptic Function in 3xTg-AD Male and Female Mice

doi: 10.1101/2020.08.19.258236

Figure Lengend Snippet: Mechanisms of CP2-dependent restoration of pTau-mediated synaptic dysfunction. Under physiological conditions, tau transports Fyn to the dendritic spines where Fyn phosphorylates the NR2B subunit of NMDARs at Y-1472 leading to the stabilization of the NMDAR:PSD95 complex. Increased tau phosphorylation leads to the destabilization of the NMDAR:PSD95 and AMPAR:PSD95 complexes with increased AMPAR endocytosis resulting in reduced synaptic strength and LTP. Partial inhibition of MCI with CP2 increases AMP/ATP ratio that directly activates AMPK with concomitant effect on the activity of PP2Ac, CDK5 and GSK3β resulting in a significant reduction in pTau levels. Reduction in pTau and Fyn results in the stabilization of the NMDAR and AMPAR complexes and improved LTP leading to enhanced cognitive function in 3xTg-AD mice.

Article Snippet: The following primary antibodies were used: phospho-AMPK (Thr 172) (1:1000, Cell Signaling Technology, cat. # 2535), AMPK (1:1000, Cell Signaling Technology, cat. #2532), Synaptophysin (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, cat. # 17750), PSD95 (1:1000, Cell Signaling Technology, cat. # 2507), GluA1 (1:1000, Cell Signaling Technology, cat. # 13185), phospho-AMPAR 1 (Ser845) (1:1000, Cell Signaling Technology, cat. # 8084), GluN2B (1:1000, Cell Signaling Technology, cat. # 14544), phospho-NMDAR 2B (Tyr1472) (1:1000, Cell Signaling Technology, cat. # 4208), AT8 (1:1000, Thermo Fisher, cat. # MN1020), AT180 (1:1000, Thermo Fisher, cat. # MN1040), HT7 (1:1000, Thermo Fisher, cat. # MN1000), Fyn (1:1000, Cell Signaling Technology, cat. # 4023), phosphor-Src (Tyr416) (1:1000, Cell Signaling Technology, cat. # 6943), CDK5 (1:1000, Cell Signaling Technology, cat. #2506), p35/p25 (1:1000, Cell Signaling Technology, cat. # 2680), GSK3β (1:1000, Cell Signaling Technology, cat. #9832), phospho-GSK3β (Ser9) (1:1000, Cell Signaling Technology, cat. #9322), PP2Ac (1:1000, Cell Signaling Technology, cat. #2038), phospho-PP2Ac (Tyr 307) (1:1000, Santa Cruz, cat. #sc-12615), Tubulin (1:5000, Biovision, cat. #3708), β-Actin (1:5000, Sigma-Aldrich, cat. # A5316).

Techniques: Phospho-proteomics, Inhibition, Activity Assay

Journal: bioRxiv

Article Title: Partial Inhibition of Mitochondrial Complex I Reduces Tau Pathology and Improves Energy Homeostasis and Synaptic Function in 3xTg-AD Male and Female Mice

doi: 10.1101/2020.08.19.258236

Figure Lengend Snippet: A) Experimental setup for stimulation-evoked local field potential (LFP) measurement in the hippocampal acute slice. The stimulation (Stim.) electrode was placed at the Schaffer collaterals and the recording (Rec.) electrode was placed at the striatum radiatum in the CA1 region. To induce LTP, three tetanic stimulations (100 Hz, 60 µsec-pulse width for 1 sec) were applied with 3 sec intervals. In the slice, LTP was induced and maintained over 30 - 50 min. Slope of fEPSPs was measured and results normalized to the average value established during the 30 min baseline period. Recording continued for at least 60 min following a tetanic stimulation and the first 30 min was used to calculate the LTP. B) CP2 treatment improves LTP formation in 3xTg-AD female mice. Traces represent mean ± SEM per time. N = 2-3 slices from 3–5 mice per group. C) LTP intensity in CP2-treated female 3xTg-AD mice compared to vehicle-treated counterparts at 30 min after the stimulation. N = 2 - 3 slices from 3–5 mice per group. D) Representative western blot analysis of protein markers involved in synaptic function of dendritic spines in the brain tissue of CP2- and vehicle-treated female 3xTg-AD mice. N = 4 mice per group. E) Quantification of western blot data from (D) showing a percent of changes in protein expression after CP2 treatment. Significant increase was observed in levels of synaptophysin, PSD95, and GluA1 and GluN2B phosphorylation. No changes were detected in total levels of GluA1 and GluN2B. Differences between individual groups were analyzed by Student t -test. Data are presented as mean ± SEM. *P<0.05, **P<0.01.

Article Snippet: The following primary antibodies were used: phospho-AMPK (Thr 172) (1:1000, Cell Signaling Technology, cat. # 2535), AMPK (1:1000, Cell Signaling Technology, cat. #2532), Synaptophysin (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, cat. # 17750), PSD95 (1:1000, Cell Signaling Technology, cat. # 2507), GluA1 (1:1000, Cell Signaling Technology, cat. # 13185), phospho-AMPAR 1 (Ser845) (1:1000, Cell Signaling Technology, cat. # 8084), GluN2B (1:1000, Cell Signaling Technology, cat. # 14544), phospho-NMDAR 2B (Tyr1472) (1:1000, Cell Signaling Technology, cat. # 4208), AT8 (1:1000, Thermo Fisher, cat. # MN1020), AT180 (1:1000, Thermo Fisher, cat. # MN1040), HT7 (1:1000, Thermo Fisher, cat. # MN1000), Fyn (1:1000, Cell Signaling Technology, cat. # 4023), phosphor-Src (Tyr416) (1:1000, Cell Signaling Technology, cat. # 6943), CDK5 (1:1000, Cell Signaling Technology, cat. #2506), p35/p25 (1:1000, Cell Signaling Technology, cat. # 2680), GSK3β (1:1000, Cell Signaling Technology, cat. #9832), phospho-GSK3β (Ser9) (1:1000, Cell Signaling Technology, cat. #9322), PP2Ac (1:1000, Cell Signaling Technology, cat. #2038), phospho-PP2Ac (Tyr 307) (1:1000, Santa Cruz, cat. #sc-12615), Tubulin (1:5000, Biovision, cat. #3708), β-Actin (1:5000, Sigma-Aldrich, cat. # A5316).

Techniques: Western Blot, Expressing, Phospho-proteomics