Journal: Molecular Neurodegeneration

Article Title: AMPA-ergic regulation of amyloid-β levels in an Alzheimer’s disease mouse model

doi: 10.1186/s13024-018-0256-6

Figure Lengend Snippet: AMPA treatment decreases levels of ISF Aβ levels. a Varying doses of AMPA or vehicle (artificial CSF) were administered to 2–4 month-old APP/PS1 mice via reverse microdialysis (rev md), and changes in interstitial fluid (ISF) Aβ 40 were measured using ELISA. AMPA has a dose-dependent effect on ISF Aβ levels. Though treatment with 0.5 μM and 2 μM AMPA did not alter ISF Aβ levels significantly ( n = 3, n = 5 respectively), treatment with 5 μM AMPA decreased levels 31.7 ± 9.5% ( p = 0.015, n = 4, one-way ANOVA, Dunnet’s post hoc test), and 10 μM AMPA decreased levels by 73.8 ± 12.2% ( p < 0.0001, n = 2, one-way ANOVA, Dunnet’s post hoc test). b APP/PS1 mice ( n = 4) were treated with 5 μM AMPA for 24 h and ISF Aβ 42 levels decreased by 37.0 ± 9.4% ( p < 0.0043, two-tailed t-test). c Wild-type, littermate C3H/B6 mice were dosed with 5 μM AMPA using rev md and levels of murine ISF Aβ 40 levels decreased by 49.4 ± 8.4% ( p < 0.0001, n = 6, two-tailed t-test). d APP/PS1 mice were treated with 300 μM cyclothiazide (CTZ) for 4 h ( n = 6), after which increasing doses of AMPA (0.5, 2, and 5 μM) were added to the perfusion buffer. CTZ administered alone did not change ISF Aβ levels. Aβ levels decreased 31.9 ± 11.1% ( p = 0.030, one-way ANOVA, Dunnet’s post hoc test) by 0.5 μM AMPA, 63.6 ± 11.1% ( p < 0.0001, one-way ANOVA, Dunnet’s post hoc test) by 2 μM, and maximally decreased 83.2 ± 11.1% ( p < 0.0001, one-way ANOVA, Dunnet’s post hoc test) when treated with 5 μM AMPA. Data plotted as mean ± SEM

Article Snippet: The cannulas were securely affixed to the head with dental cement, and microdialysis probes (BR-2, 2 mm, 38 kDa MWCO, Bioanalytical Systems) were inserted into the hippocampus through the guide cannula.

Techniques: Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Molecular Neurodegeneration

Article Title: AMPA-ergic regulation of amyloid-β levels in an Alzheimer’s disease mouse model

doi: 10.1186/s13024-018-0256-6

Figure Lengend Snippet: AMPA treatment alters Aβ levels through multiple pathways. a APP/PS1 mice ( n = 6) were treated with 100 μM NBQX, an AMPA receptor antagonist, for 8 h then co-treated with either 40 μM NMDA ( n = 6), 5 μM AMPA ( n = 7), or vehicle ( n = 12). After 6 h of co-treatment with NBQX, the addition of AMPA had no effect on Aβ levels, though NMDA still reduced Aβ by 37.5 ± 3.3% ( p < 0.0001, one-way ANOVA, Bonferroni post hoc test). b Animals ( n = 6 per group) were treated with 5 μM tetrodotoxin (TTX) for 16 h then co-treated with TTX and either 5 μM AMPA, 100 μM NBQX, or vehicle for an additional 14 h. After 8 h of co-treatment, ISF Aβ levels remained unchanged in all groups. 14 h co-treatment with AMPA reduced Aβ levels by 34.6 ± 9.9% ( p = 0.0027, two-way ANOVA, Sidak post hoc test) and co-treatment with NBQX reduced levels by 32.8 ± 9.3% (p = 0.0027, two-way ANOVA, Sidak post hoc test). c APP/PS1 mice were treated with either 5 μM AMPA ( n = 7) or vehicle ( n = 5) for 14 h, leading to a decrease in ISF Aβ levels of 66.3 ± 11.8% ( p = 0.0001, two-way ANOVA, Sidak post hoc test). d 100 μM MK801 or vehicle was administered by reverse microdialysis for 6 h to APP/PS1 mice followed by co-administration with 5 μM AMPA or vehicle. After 8 h, mice treated with AMPA alone had significance decreases in ISF Aβ as compared to vehicle-treated mice, but mice receiving both MK801 and AMPA showed no change ( p = 0.996, two-way ANOVA, Sidak post hoc test). After 14 h, however, AMPA treatment significantly decreased ISF Aβ levels to the same extent regardless of the presence of MK801 ( p = 0.384, two-way ANOVA, Sidak post hoc test). Data plotted as mean ± SEM

Article Snippet: The cannulas were securely affixed to the head with dental cement, and microdialysis probes (BR-2, 2 mm, 38 kDa MWCO, Bioanalytical Systems) were inserted into the hippocampus through the guide cannula.

Techniques:

Journal: Molecular Neurodegeneration

Article Title: AMPA-ergic regulation of amyloid-β levels in an Alzheimer’s disease mouse model

doi: 10.1186/s13024-018-0256-6

Figure Lengend Snippet: AMPA treatment results in potent, long-lasting decreases in ISF Aβ levels that slowly recover. a APP/PS1 mice ( n = 5) were treated with 5 μM AMPA using reverse microdialysis for 8 h resulting in a decrease in ISF Aβ levels of 32.7 ± 3.0% from baseline. After 8 h, AMPA was removed from the microdialysis perfusion buffer. Aβ levels continued to decline for 3 h post-treatment to reach a maximum reduction of 56.7 ± 1.7% from baseline. For the next 40 h, ISF Aβ levels gradually increased. When the experiment was ended at 52 h, ISF Aβ levels had increased 23.5 ± 3.0% to reach 64.8 ± 3.0% of basal levels, which was a significant increase from the lowest Aβ levels post-treatment ( p = 0.0245, two-way ANOVA, Sidak post hoc test). b APP/PS1 mice ( n = 3) were treated with 5 μM AMPA followed by co-treatment with AMPA and 100 μM NBQX for 14 h. The addition of NBQX did not alter the decrease in Aβ levels caused by AMPA treatment (one-way ANOVA, Sidak post hoc test). c 5 μM AMPA was infused by rev md into APP/PS1 mice for a 30-min period, after which the perfusion buffer was changed to artificial CSF for 24 h. AMPA treatment caused a 41.30 ± 9.45% decrease in ISF Aβ levels in the 22–24 h after 30-min dosage ( n = 3, p = 0.035, two-tailed t-test). Data plotted as mean ± SEM

Article Snippet: The cannulas were securely affixed to the head with dental cement, and microdialysis probes (BR-2, 2 mm, 38 kDa MWCO, Bioanalytical Systems) were inserted into the hippocampus through the guide cannula.

Techniques: Two Tailed Test

Journal: Molecular Neurodegeneration

Article Title: AMPA-ergic regulation of amyloid-β levels in an Alzheimer’s disease mouse model

doi: 10.1186/s13024-018-0256-6

Figure Lengend Snippet: 8 and 14 h AMPA treatment does not alter expression of genes related to Aβ metabolism. 5 μM AMPA or vehicle was given to 2–4 month old APP/PS1 mice for 8 h ( a ) or 14 h ( b ; n = 6 per group) before the hippocampal tissue surrounding the microdialysis probe was collected and analyzed with quantitative PCR. a qPCR analysis revealed no differences in expression for major genes involved in Aβ production and clearance between mice treated with AMPA or vehicle. Expression of cFos , a marker for neuronal activity, increased 7.5 ± 3.7 fold ( p < 0.0001, two-way ANOVA, Sidak post hoc test) for the AMPA-treated group over cFos expression in controls. b After 14 h of AMPA treatment, expression of genes involved in Aβ processing was not changed as shown by qPCR analysis. AMPA-treated animals showed a 14.4 ± 1.8 fold increase in cFos expression over controls ( p < 0.0001, two-way ANOVA, Sidak post hoc test). Data plotted as mean ± SEM

Article Snippet: The cannulas were securely affixed to the head with dental cement, and microdialysis probes (BR-2, 2 mm, 38 kDa MWCO, Bioanalytical Systems) were inserted into the hippocampus through the guide cannula.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Marker, Activity Assay

Journal: Molecular Neurodegeneration

Article Title: AMPA-ergic regulation of amyloid-β levels in an Alzheimer’s disease mouse model

doi: 10.1186/s13024-018-0256-6

Figure Lengend Snippet: Extended treatment with AMPA decreases Aβ levels through clearance. a 2–4 month old APP/PS1 mice were treated with either 5 μM AMPA ( n = 6) or aCSF ( n = 8) via reverse microdialysis for 14 h. Tissue surrounding the microdialysis probe was analyzed via Western blot for full-length APP, CTF-β, sAPPα, sAPPβ, and total sAPP, and no significant change was observed between treatment groups (two-way ANOVA, Sidak post hoc test). Bands were normalized to GAPDH and displayed relative to control. Blot images are representative examples. b APP/PS1 mice were treated with 14 h of AMPA ( n = 6) or vehicle ( n = 7). With microdialysis collection ongoing, animals were administered a 4 mg/kg subcutaneous injection (s.c.) of LY411575, a γ-secretase inhibitor, or vehicle (corn oil). c ISF Aβ half-life for each treatment group was calculated by taking the slope of the semi-log plot of concentration versus time for the time points between drug delivery and the plateauing of Aβ concentrations. Mice treated with 5 μM AMPA had an Aβ half-life of 0.9 ± 0.1 h compared to a half-life of 1.5 ± 0.2 h for the mice treated with aCSF ( p = 0.0298, two-tailed t-test)

Article Snippet: The cannulas were securely affixed to the head with dental cement, and microdialysis probes (BR-2, 2 mm, 38 kDa MWCO, Bioanalytical Systems) were inserted into the hippocampus through the guide cannula.

Techniques: Western Blot, Control, Injection, Concentration Assay, Two Tailed Test

Journal: Molecular Neurodegeneration

Article Title: AMPA-ergic regulation of amyloid-β levels in an Alzheimer’s disease mouse model

doi: 10.1186/s13024-018-0256-6

Figure Lengend Snippet: AMPA-mediated decrease in Aβ not due to changes in clearance-related proteins or proteases. a 2–4 month old APP/PS1 mice were treated with either 5 μM AMPA ( n = 6) or aCSF ( n = 8) via reverse microdialysis for 14 h. Tissue surrounding the microdialysis probe was analyzed via Western blot to determine levels of proteins involved in Aβ elimination and clearance. Bands were normalized to GAPDH and displayed relative to control. Blot images are representative examples. cFos protein expression was increased 2.9 ± 0.4 fold ( p < 0.0001, two-way ANOVA, Sidak post hoc test) in the AMPA group compared to the controls. No other proteins showed a significant difference between treatment groups. b Reverse microdialysis was used to treat APP/PS1 mice ( n = 7) with 10 μM thiorphan (neprilysin inhibitor), 25 μM GM6001 (broad-spectrum MMP inhibitor), or vehicle for 6 h, followed by 14 h of co-treatment with 5 μM AMPA. The Aβ concentrations in the last 3 h of each treatment were averaged and the differences between the end of inhibitor/vehicle treatment and after the addition of AMPA were compared. Inhibiting protease activity with thiorphan or GM6001 did not alter the decrease in ISF Aβ levels observed following AMPA treatment ( p = 0.40, one-way ANOVA, Dunnet’s post hoc test). Data plotted as mean ± SEM

Article Snippet: The cannulas were securely affixed to the head with dental cement, and microdialysis probes (BR-2, 2 mm, 38 kDa MWCO, Bioanalytical Systems) were inserted into the hippocampus through the guide cannula.

Techniques: Western Blot, Control, Expressing, Activity Assay

Journal: Molecular Neurodegeneration

Article Title: AMPA-ergic regulation of amyloid-β levels in an Alzheimer’s disease mouse model

doi: 10.1186/s13024-018-0256-6

Figure Lengend Snippet: Glial recruitment unchanged and IL-6 levels enhanced following AMPA treatment. a Wild-type C3H/B6 mice (for the 8 h treatment, n = 6 per group) or APP/PS1 mice (for the 14 h treatment, n = 3 per group) were implanted with microdialysis probes and treated with either 5 μM AMPA or aCSF for 8 or 14 h. Brain sections were immunostained with DAB using anti-GFAP antibody to mark astrocytes or anti-Iba1 antibody to mark microglia. Immunoreactivity between control and AMPA-treated sections were compared, and representative images are shown. b 2–4 month old APP/PS1 mice were treated with either 5 μM AMPA ( n = 6) or aCSF ( n = 8) via reverse microdialysis for 14 h. Tissue surrounding the microdialysis probe was analyzed via Western blot for GFAP or CD45, markers of astrocytes and microglia, respectively, and no difference was observed between treatment groups (two-way ANOVA, Sidak post hoc test). Bands were normalized to GAPDH and displayed relative to control. Blot images are representative examples. c As in Fig. 7b, APP/PS1 mice were treated with either 5 μM AMPA ( n = 9) or vehicle ( n = 7) for 14 h, and hippocampal lysates were analyzed for pro-inflammatory cytokines using a MSD multiplex assay. Levels of IL-1β ( p = 0.991, two-way ANOVA, Sidak post hoc test) and TNF-α ( p = 0.999, two-way ANOVA, Sidak post hoc test) were unchanged. IL-6 levels were significantly elevated following AMPA treatment, increasing from 52.3 to 773.8 pg/mL ( p = 0.0014, two-way ANOVA, Sidak post hoc test). Data plotted as mean ± SEM

Article Snippet: The cannulas were securely affixed to the head with dental cement, and microdialysis probes (BR-2, 2 mm, 38 kDa MWCO, Bioanalytical Systems) were inserted into the hippocampus through the guide cannula.

Techniques: Control, Western Blot, Multiplex Assay

Journal: Molecular Neurodegeneration

Article Title: AMPA-ergic regulation of amyloid-β levels in an Alzheimer’s disease mouse model

doi: 10.1186/s13024-018-0256-6

Figure Lengend Snippet: IL-6 is required for AMPA-R regulation of ISF Aβ levels. Both IL-6 −/− mice ( n = 5) and C3H/B6 WT mice ( n = 6) were treated with 100 μM MK801 for 6 h via reverse microdialysis, then co-treated with MK801 and 7.5 μM AMPA for an additional 17 h. The last five hours of each treatment (MK801 alone vs MK801 + AMPA) were averaged for each treatment group and compared (two-way ANOVA, Sidak post hoc test). In WT animals, ISF Aβ levels decreased by 67.34% from MK801 alone to MK801 + AMPA ( p = 0.002). In IL-6 −/− animals, the addition of AMPA resulted in a non-significant decrease in ISF Aβ levels of 23.96% ( p = 0.652). Furthermore, ISF Aβ levels IL-6 −/− mice following extended AMPA treatment are significantly higher than observed in WT mice (80.26 and 23.0%, respectively; p = 0.027). Data plotted as mean ± SEM

Article Snippet: The cannulas were securely affixed to the head with dental cement, and microdialysis probes (BR-2, 2 mm, 38 kDa MWCO, Bioanalytical Systems) were inserted into the hippocampus through the guide cannula.

Techniques: