Prion infection and replication during differentiation impairs neural differentiation.

<p>Monolayer NSC derived from hippocampi and lateral ventricles were infected at the time of differentiation induction (bFGF and EGF removal) with ME7 or non infected brain homogenates as control. They were stained with anti-nestin and anti-DCX antibody 10 days after differentiation. They were also stained with anti-β-tubulin class III and anti-GFAP antibody after 10 days of differentiation. The percentage of βIII-tubulin positive cells as well as DCX positive cells were calculated with the ImageJ software. The bar graphics show higher percentages of DCX positive cells (A) as well as βIII-tubulin positive cells (B) in non-infected cells which are statistically different from percentages obtained with ME7 infected cells (p<0.0001Mann-Whitney Test). Data represent means +/− SEM from one experiment performed in triplicate. Similar results were obtained in the three independent experiments.</p

Impairment of the neuronal differentiation of NSC derived from prion infected mice.

<p>Immunofluorescence analysis of nestin, DCX, BetaIII-Tubulin and GFAP markers in differentiated NSC derived from the hippocampus (H) or Lateral ventricle (LV) of ME7 infected or non infected mice. (<b>A</b>. red: nestin; green: DCX, <b>B</b>. green: GFAP, red: BetaIII-Tubulin, blue: Hoechst nuclei coloration, scale bar 5 µm). <b>C</b>. Quantification of DCX and beta-III-Tubulin positive cells using ImageJ software. After differentiation induction, NSC gave rise to significantly more DCX positive cells in non infected conditions when compared with ME7 infected conditions (p<0.05 and p<0.01 by Mann-Whitney test). The number of neurons also differed significantly (p<0.01 by Mann-Whitney test) between non infected and infected NSC derived cells in differentiation conditions. Inversely astrocyte proportions were higher in the ME7 infected context (p<0.05 by Mann-Whitney test). Data represent means +/− SEM from one experiment performed in triplicate. Similar results were obtained in the three independent experiments.</p

NSC were infected before their isolation.

<p><b>A</b>. Schematic presentation of the experiment. Hippocampus and Lateral Ventricles were isolated from actin-GFP mice. An equivalent amount (weight) of non infected or infected tissue (in which cells have been frozen and then heat inactivated) was added in the same tube. The cells obtained were all positive for the GFP marker. <b>B</b>. PrP^Sc Western blot using Saf-Mix anti-PrP antibody: PK digested cell lysates of NSC cells derived from the lateral ventricle (1) and the hippocampus (2) of actin-GFP mice isolated in the presence of ME7, or from the lateral ventricle (3) and the hippocampus (4) of actin-GFP mice isolated in the presence of non infected brain samples after 2 subpassages. (5) PrP^Sc control from ME7 infected brain.</p

PrP^Sc is present in NSC and neuroblasts areas.

<p><b>A</b>. PrP^Sc immunostaining (brown+arrow) using Saf84 anti-PrP antibody in the lateral wall (SVZ) and the surrounding of the lateral ventricle (LV). <b>B</b>. Schematic representation of the LV localisation in the mouse brain. <b>C</b>. Nestin immunostaining (brown+arrow) in the SVZ. <b>D and G</b>. Double-immunostaining of PrP^Sc (Blue) and Nestin (Brown) in the surrounding of the LV. <b>E</b>. Doublecortin (DCX) immunostaining (brown+arrow) showing neuroblasts exiting the SVZ of the lateral ventricle. <b>F and H</b> Double-immunostaining of PrP^Sc (Blue) and DCX (Brown) in the surrounding of the LV. Scale bar 20 µm.</p

Infection of NSC during differentiation.

<p>Western blots showing the generation of PrP^Sc: (A) in hippocampus derived NSC, (B) in lateral ventricle derived NSC, (C) in KOPrP LV derived NSC, during the differentiation process and after incubation with ME7 brain homogenate at the time of the differentiation induction. All the samples were treated with PK, Saf Mix cocktail of anti-PrP antibodies was used to detect proteinase K resistant PrP^Sc. Cells were harvested at 2, 4, 6, 8, 11 and 14 dpi, after brain homogenate removal. III: Positive control ME7, MW : Molecular Weight (in kDa). Lanes without numbers were left empty on purpose.</p

Neural precursor cells isolated from infected mice accumulate PrP^Sc.

<p><b>A</b>. Immunofluorescence analysis of the nestin marker in proliferative NSC derived from the hippocampus (H) or lateral ventricle (LV) of mock or ME7 infected mice. (red: nestin, Blue: Hoechst nuclei coloration, scale bar 5 µm). Most of the cells are positive for the nestin NSC marker in proliferation conditions. <b>B</b>. PrP^Sc Immunofluorescence in NSC cells derived from the lateral ventricle and the hippocampus of mock or ME7 infected mice (red: PrP^Sc immunofluorescence using saf61 anti-PrP antibodies, Blue: Hoechst nuclei coloration, scale bar 5 µm). <b>C</b>. PrP^Sc Western blot using Saf-Mix anti-PrP antibody: PK digested cell lysates of NSC cells derived from the lateral ventricle (1) and the hippocampus (2) of non infected mice or the lateral ventricle (3) and the hippocampus (4) of ME7 infected mice after 2 subpassages. (5) PrP^Sc control from ME7 infected brain. Lanes without numbers were left empty on purpose. <b>D</b>. PrP^Sc Western blot using Saf-Mix anti-PrP antibody: PK digested cell lysates of NSC cells derived from the lateral ventricle (1) and the hippocampus (2) of non infected mice or the lateral ventricle (3) and the hippocampus (4) of ME7 infected mice after 15 subpassages. (5) PrP^Sc control from ME7 infected brain. Lanes without numbers were left empty on purpose.</p

Data_Sheet_1_Relevance of Aβ42/40 Ratio for Detection of Alzheimer Disease Pathology in Clinical Routine: The PLMR Scale.docx

<p>Background: Cerebrospinal fluid (CSF) biomarkers (Aβ peptides and tau proteins) improved the diagnosis of Alzheimer’s disease (AD) in research and clinical settings. We previously described the PLM-scale (Paris-Lille-Montpellier study), which combines Aβ42, tau, and phosphorylated ptau(181) biomarkers in an easy to use and clinically relevant way. The purpose of this work is to evaluate an optimized PLM_R-scale (PLM ratio scale) that now includes the Aβ42/Aβ40 ratio to detect AD versus non-AD (NAD) participants in clinical routine of memory centers.</p><p>Methods: Both scales were compared using 904 participants with cognitive impairment recruited from two independent cohorts (Mtp-1 and Mtp-2). The CSF Aβ42/Aβ40 ratio was measured systematically in Mtp-1, and only on biologically discordant cases in Mtp-2. Two different ELISA kit providers were also employed. The distribution of AD and NAD patients and the discrepancies of biomarker profiles were computed. Receiver Operating Characteristic curves were used to represent clinical sensitivity and specificity for AD detection. The classification of patients with the net reclassification index (NRI) was also evaluated.</p><p>Results: Nine hundred and four participants (342 AD and 562 NAD) were studied; 400 in Mtp-1 and 504 in Mtp-2. For AD patients, the mean CSF Aβ42 and CSF Aβ42/40 ratio was 553 ± 216 pg/mL and 0.069 ± 0.022 pg/mL in Mtp-1 and 702 ± 335 pg/mL and 0.045 ± 0.020 pg/mL in Mtp-2. The distribution of AD and NAD differed between the PLM and the PLM_R scales (p < 0.0001). The percentage AD well-classified (class 3) increased with PLM_R from 38 to 83% in Mpt-1 and from 33 to 53% in Mpt-2. A sharp reduction of the discordant profiles going from 34 to 16.3% and from 37.5 to 19.8%, for Mtp-1 and Mtp-2 respectively, was also observed. The AUC of the PLM_R scale was 0.94 in Mtp-1 and 0.87 in Mtp-2. In both cohorts, the PLM_R outperformed CSF Aβ42 or Aβ42/40 ratio. The diagnostic performance was improved with the PLM_R with an NRI equal to 44.3% in Mtp-1 and 28.8% in Mtp-2.</p><p>Conclusion: The integration of the Aβ42/Aβ40 ratio in the PLM_R scale resulted in an easy-to-use tool which reduced the discrepancies in biologically doubtful cases and increased the confidence in the diagnosis in memory center.</p

Impact of multidomain preventive strategies on functional brain connectivity in older adults with cognitive complaint: Subset from the Montpellier center of the ancillary MAPT-MRI study

International audienceIntroduction The impact of multi-domain preventive interventions on older adults, in particular on those with higher risk to develop Alzheimer's disease (AD), could be beneficial, as it may delay cognitive decline. However, the precise mechanism of such positive impact is not fully understood and may involve brain reserve and adaptability of brain functional connectivity (FC). Methods To determine the effect of multidomain interventions (involving physical activity, cognitive training, nutritional counseling alone or in combination with omega-3 fatty acid supplementation and vs. a placebo) on the brain, longitudinal FC changes were assessed after 36 months of intervention on 100 older adults (above 70 year-old) with subjective cognitive complaints. Results No global change in FC was detected after uni or multidomain preventive interventions. However, an effect of omega-3 fatty acid supplementation dependent on cognitive decline status was underlined for frontoparietal, salience, visual and sensorimotor networks FC. These findings were independent of the cortical thickness and vascular burden. Discussion These results emphasize the importance of patient stratification, based on risk factors, for preventive interventions

Histological and Immunohistolochemical illustration.

<p>Panel A. PrP^Sc immunostaining was observed in all the mice (arrow). Panel B. Vacuolar lesions revealed after Hematoxylin & Eosin staining were apparent in all conditions. Immunohistochemical detections of the GFAP astrocyte marker (Panel C) and the neuronal marker NeuN were also performed (Panel D).</p

Impact of siRNA on incubation and survival time.

<p>Panel A. The incubation time in scrapie infected mice based on the presence of at least three clinical signs (among: waddling gait, flattened back, rough coat, sticky eye discharge, weight loss, very jumpy, hunched, incontinence) was plotted as medians and interquartile ranges in the three groups: Aonys/PrP^C siRNA (siPrP 600 µg/kg), Aonys vehicle only and Aonys/scrambled-siRNA. The survival times of the three groups of infected mice were plotted as medians and interquartile ranges (Panel B.) and Kaplan-Meier survival curves (Panel C.). No statistical difference was observed between each group neither for the incubation period nor the survival time.</p