The Oral History of Vic Salerno

<p>AEA, anandamide; PEA, palmitoylethanolamide; OEA, oleoylethanolamide; 2-AG, 2-arachidonoylglycerol. Values are means ± SD, n = 10.</p><p>* P< 0.05.</p><p>Visceral adipose tissue N-Acylethanolamides (NAEs), 2-AG and fatty acid concentrations in phospholipids (PL), triacylglycerols (TAG) and non esterified fatty acids (NEFA), in rats fed Lsn-2 PA or Hsn-2 PA for 5 weeks.</p

AP and endocytic activities in the retina.

<p>A–F: acid phosphatase (AP) activity is visualized with Gomori's (A, C, E) and Barka & Anderson (B and D) staining, counterstained with methyl green. 12 h after I/R, positive cells are located only in the ganglion cell layer (GCL), and are identified by their content of typical brown cytoplasmic granules (arrowheads in A) and by the red reaction (B, arrows) in the GCL and INL. 24 h after I/R, AP-positive cells are visible in both the GCL (arrows) and INL (arrowheads) (C–D). Only weak staining in the GCL is seen with the Gomori technique 48 h after I/R (E). No labeling is found in control retinas (F). Markedly positive cytoplasmic granules are visible in GCL-cells at higher magnification (G). H–I: 24h after I/R and intravitreal injection of HRP (H) or 4.4 kDa FITC-labelled dextran (I, arrows), corresponding granules are visible in neurons. GCL  =  ganglion cell layer; IPL  =  inner plexiform layer; INL  =  inner nuclear layer; OPL  =  outer plexiform layer; ONL  =  outer nuclear layer. Scale bars  =  100 µm and 10 µm (G and H).</p

Experimental plan.

1<p>For each animal, the left eye was injected, the right eye served as control.</p>2<p>IHC was performed on 2 animals of each group.</p

Effects of 3-MA treatment in I/R after 24 h.

<p>In 3-MA-treated I/R retinas treated, LC3-positivity is markedly reduced (A), whereas lysosomal activity (LAMP1) is unchanged (B) compared to I/R retinas. (C) Cleaved caspase-3-positive cells are absent in the untreated retinas (upper panel), whereas a strong increase in cleaved caspase 3-positivity in the GCL is seen at 24 h (middle panel), significantly prevented by 3-MA treatment (bottom panel). (D) TUNEL-positive neurons are found occasionally in the control retina, whereas they increase dramatically after I/R in the GCL (arrowheads) and in INL (arrow). This increase in TUNEL-positive cells is prevented by 3-MA treatment (bottom panel). (E) I/R increase GFAP immunoreactivity (red) in the retina: in the untreated retina, only the end feet of the Müller cells (arrowheads) are GFAP-positive. GFAP expression strongly increase 24 h after I/R, and is prevented by 3-MA administration. In the I/R and I/R + 3-MA retinas, GFAP positivity is detectable in the end feet (arrowheads) and radial processes (arrows) of Müller cells. Following 3-MA treatment, the immunoreactivity is decreased versus I/R retina especially in the end feet of Müller cells. ILM: inner limiting membrane. Abbreviations as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022514#pone-0022514-g001" target="_blank">Figure 1</a>. (*): vitreous. Scale bar: 50 µm (100 µm in D).</p

Additional file 3: Figure S3. of Autophagic and lysosomal defects in human tauopathies: analysis of post-mortem brain from patients with familial Alzheimer disease, corticobasal degeneration and progressive supranuclear palsy

Diffuse staining pattern of Cathepsin D (Cat D) (ab6313, Abcam) in tauopathies a-h Merged pictures show immunofluorescence staining of Cat D (green) and DAPI (nuclear marker, blue). a-c Diffuse immunoreactivity for Cat D in FAD, CBD and PSP patients (arrows) and distinct vesicle-like structures (asterisks). d and h Control samples show Cat D-positive vesicular-like structure (asterisks) and not diffuse staining. e-h High magnification of the boxed areas in a-d. Scale bars: 50 μm (20 μm high magnification). (TIF 25515 kb

Immunofluorescence against LAMP1 and LC3.

<p>All sections were counterstained with bisbenzimide (Hoechst staining) to show retinal layers. Twelve hours after I/R, LAMP1 positive cytoplasmic granules are present (A) and 24 h after I/R both LAMP1 (B) and LC3 (C) positive granules are present in the GCL (arrow) and INL (arrowheads). LC3-positive vesicles are more represented than LAMP1 vesicles. LC3-immunopositivity is absent after 48 h (D). At high magnification (E–F), clear cytoplasmic lysosomal LAMP1 positive vesicles (arrowheads) can be observed in the GCL at 24 h from the insult (E). LC3 labelling appear as numerous fluorescent dots (arrowheads) after 24 h from the I/R (F). Double immunolabeling against LAMP1 (red) and LC3 (green) at 24 h shows the relationship between autophagosomes and lysosomes (G). The increase in punctuate LC3 and LAMP1 occurs in the same neurons after 24 h (G, arrowheads). At high magnification, confocal microscopy reveals that autophagic marker and LAMP1 colocalize, suggesting that fusion of autophagosomes with lysosomes occur in dying neurons after I/R (arrowheads, H). Abbreviations as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022514#pone-0022514-g001" target="_blank">Figure 1</a>. Scale bars = 50 µm (A); 100 µm (B, C, D and G) and 5 µm (E, F and H).</p

Additional file 2: Figure S2. of Autophagic and lysosomal defects in human tauopathies: analysis of post-mortem brain from patients with familial Alzheimer disease, corticobasal degeneration and progressive supranuclear palsy

Colocalization between hyperphosphorylated tau (Tau/pS422) and LC3 in AD cases. a-d Double immunofluorescence staining against hyperphosphorylated tau (a) and LC3 (b) and merge (c) pictures in human brain samples show colocalization in neurons (NFT-like structures) in FAD samples. d Superposition of confocal stacks. Scale bar: 10 μm (TIF 25517 kb

pri-miRNA-206, mature miRNA-206, HDAC4 and FGFBP1 expression levels in quadriceps of WT and SMA mice at P5 and P10/P12.

<p>Panels A,B,D, evaluation of pri-miRNA-206 (panel A), mature miRNA-206 (panel B) and FGFBP1 (panel D) mRNA levels in WT and SMA mice, five (P5) and ten (P10/12) days after birth. #p<0.05 vs P10/12 WT, § p<0.05 vs all group by ANOVA followed by post hoc Newman-Keuls test. Panels C, representative Western blots displaying HDAC4 protein levels in WT and SMA mice, at P5 (left blot) and P10 (right blot). Graphs below each image report the quantification of HDAC4/GAPDH protein levels. *p<0.05 vs WT by t-test analysis.</p

Cell counts in the GCL.

<p>Representative transverse sections through rat retina at 24 h, Hoechst staining. Retinal thickness is markedly reduced, compared to the control (A), after injury (B), mainly due to decrease in IPL thickness and number of GCL-neurons; these effects are partially prevented by 3-MA treatment (C). No statistical significant alterations are apparent in the other layers. (D) Quantification of GCL-neurons numbers in each group: in the I/R retina, the number of the GCL-neurons significantly decrease compared to controls (** <i>P<0.01</i>); 3-MA partially prevent neuron death (* <i>P<0.05</i>). Scale bar: 50 µm.</p

Additional file 1: Figure S1. of Autophagic and lysosomal defects in human tauopathies: analysis of post-mortem brain from patients with familial Alzheimer disease, corticobasal degeneration and progressive supranuclear palsy

Tau pathology observed with the Tau/pS422 antibody. a-f Immunofluorescence staining against hyperphosphorylated tau (Tau/pS422) in human brain samples. Intense immunoreactivity in FAD, CBD and PSP. a FAD cases show immunoreactivity in neurofibrillary tangles, pretangles, neuropil threads and dystrophic neurites. b-c CBD and PSP cases show immunoreactivity in neurons and glial cells. d-f High magnification of the boxed areas in a-c. Scale bars: 50 μm (10 μm high magnification) (TIF 3632 kb