Additional file 2: Figure S2. of Alzheimer’s disease-like APP processing in wild-type mice identifies synaptic defects as initial steps of disease progression

AAV-PS1 and AAV-APP mice do not exhibit neuronal defects in terms of PSD-95, GLT-1 and tonic glutamatergic current. C57Bl/6 J mice (all males) were injected at 8 weeks of age either with AAV-CAG-PS1M146L (AAV-PS1 mice) or AAV-CAG-APPSL (AAV-APP mice). Mice were killed three months later for analyses. (A) Western blot of PSD-95 and GLT-1 (n = 3-4 per group). (B) Densitometric analyses of the antibody immunoreactivities shown in panel A. (C) Tonic glutamatergic current recorded at a holding potential of +40 mV by the whole-cell patch-clamping of CA1 pyramidal cells. No significant difference in tonic glutamatergic current intensity was observed between the AAV-PS1 and AAV-APP groups (whole cell patch-clamp of CA1 pyramidal cells, n = 11/group from n = 10 mice per group). (PNG 447 kb

Additional file 1: Figure S1. of Alzheimer’s disease-like APP processing in wild-type mice identifies synaptic defects as initial steps of disease progression

APP is processed in sporadic AD cases. Human samples were obtained from late-onset AD cases (Braak 6, Thal 5) and age-matched controls (n = 5 per group). The hippocampus was the studied structure. (A) Western blot analysis of NeuN. (B) Densitometric analyses of western blots showing the expression of human APP in the hippocampus of human controls and AD cases (n = 5 per group). Bars represent means ± SEM, and data were normalized with respect to GAPDH. Statistical analyses were performed with Student’s t-test: **p < 0.01. (C) Densitometric analyses of A, showing the expression of NeuN in the hippocampus of human controls and AD cases (n = 5 per group). Bars represent means ± SEM, and data were normalized with respect to GAPDH. Statistical analyses were performed with Student’s t-test: *p < 0.05. (D) Representation of the APP/NeuN ratio following densitometric analyses of the corresponding western blots (n = 5 per group). Note that APP seems to be processed in sporadic AD cases. Bars represent means ± SEM and data were normalized with respect to GAPDH. Statistical analyses were performed with Student’s t-test: *p < 0.05. (PNG 325 kb

Theta-burst-induced LTP in Tg<i>hCBS</i>60.4 mice.

<p>Comparison of averaged LTP expressed as percent change in the slope of fEPSP <i>vs</i> time, induced by theta-burst stimulation (TBS, arrow) of glutamate afferents and recorded in slices from Tg<i>hCBS</i>60.4 (12 slices/9 animals) and control (10 slices/7 animals) mice. In the insert, representative traces of fEPSPs, recorded before and 60 min after TBS, are superimposed.</p

Involvement of CBS in sulfur-containing amino acid metabolism.

<p>From Stipanuk <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029056#pone.0029056-Stipanuk1" target="_blank">[54]</a>.</p

Basal synaptic transmission in Tg<i>hCBS</i>60.4 mice.

<p>(A) Superimposed sample traces of evoked AMPA-R-mediated fEPSPs induced in a control (left) and a Tg<i>hCBS</i>60.4 mouse (right) by increased intensities of electrical stimulation of glutamate afferents (arrow). Traces are averages of 3 consecutive responses. (B) Comparison of synaptic efficacy as determined by the fEPSP/PFV ratio calculated at a stimulus intensity from 500 to 900 µA in Tg<i>hCBS</i>60.4 (23 slices/5 animals) and control (22 slices/5 animals) mice.</p

CBS expression and activity in brain regions of Tg<i>hCBS</i>60.4 mice.

<p>(A, top) Immunoblots for CBS and β-actin in cerebellum (left panel), hippocampus (middle panel) and forebrain (right panel). Total CBS proteins (human CBS plus mouse CBS) were detected using the anti-hCBS polyclonal antibody that recognises both the human and mouse proteins. (A, bottom) Quantification of CBS proteins normalized to β-actin and relative to control mice in the three brain regions (<i>n</i> = 3 Tg<i>hCBS</i>60.4 and <i>n</i> = 3 control) (B) CBS activity in cerebellum (left panel), hippocampus (middle panel) and forebrain (right panel). Units are nanomoles of cystathionine formed per milligram of protein extract per hour. For cerebellum and forebrain, data represent mean ± S.E.M. of activity measurements in each group (<i>n</i> = 9 Tg<i>hCBS</i>60.4 and <i>n</i> = 7 control; <i>n</i> = 8 Tg<i>hCBS</i>60.4 and <i>n</i> = 8 control, respectively). For hippocampus, data represent mean ± S.E.M. of three experimental assays performed on same pooled hippocampi (<i>n</i> = 7 Tg<i>hCBS</i>60.4 and <i>n</i> = 8 control). *for <i>p</i><0.05. ***for <i>p</i><0.001.</p

Behavioral assessments in Tg<i>hCBS</i>60.4 mice.

<p>The analysis was conducted on 8 Tg<i>hCBS</i>60.4 and 8 control mice. Data represent mean+S.E.M. for each group. (A) Rotarod test: latencies of first fall, second fall and third fall recorded in the 10 min accelerating periods of the two rotarod sessions (S1, S2) are presented. (B) Passive avoidance test: latency to enter into the dark compartment in acquisition session and in retention session was recorded.</p

Concentrations (nmoles/mg protein) of 30 amino acids and metabolite compounds in cerebellum extracts of Tg<i>hCBS</i>60.4 and control mice.

<p>Data correspond to mean ± S.E.M. BD: below detection. ND: not determined.</p