8-months old mice were treated with vehicle only (Ctr) or 30 mg/kg kainate (Kai). 24 hours after treatment, total RNA was extracted from hippocampi and subjected to real-time RT-PCR using primers specific for each of the indicated genes. Values were normalized to 18S gene expression and expressed as relative quantity (RQ) of the controls. * = p<0.05; ** = p<0.01 (two tails Student T-Test).

(A) Recruitment of endogenous HSP47 by overexpressed APP. HeLa cells were transfected with YFP –APP and HSP47 detected by a specific anti-HSP47 antibody. Arrows indicate some of the intracellular structures showing an obvious colocalization. (B) Primary rat cortical neurons (4 DIV) were exposed to DSP cross linking agent and total cell lysates were immunoprecipitated with control (MBP), with anti-HSP47 or anti-APP (6E10) antibodies. The immunoprecipitates and 40 µg of the total lysate were then immunoblotted with anti APP (C-Term) or with anti HSP47. (C) Colocalization of HSP47 and APP in 14 DIV neurons. A high magnification field of dendrites is shown in the right panel. The signals were in general justaxposed (white arrows) and in some cases were colocalized (yellow arrows).

(A) Immunohistochemical analysis of Aβ and HSP47 on wild type (WT) and APPPS1 transgenic mice (12 months old). Arrows indicate HSP47 positive plaques. (B–C) Double immunostaining of APPPS1 brain slices with HSP47 and Aβ antibodies. The diffuse plaques (B) show a marked colocalization of the two signals. In dense core plaques (C), the HSP47 antibody stained the Aβ core and a surrounding coronal area. (D–E) Dense core plaques double-stained with HSP47 and Tau (D) or APP C-terminal (E) antibodies.

(A) Immunofluorescence analysis of HSP47 distribution (green) in normal human brain (Ctr). The arrowhead indicates a positive vessel. (B) Analysis of the distribution of HSP47 (green) and Aβ (red) in one of the III–IV stage AD cases. Arrows indicate two HSP47-positive amyloid plaques. (C–D) High magnification fields of AD brain sections analyzed as in B, showing diffuse (C) and dense-core (D) HSP47-positive plaques.

(A) APPsw cells were transiently transfected with two independent siRNA oligonucleotides (H2 and H3) designed against the human HSP47 sequence or with a mismatch control (C1). After additional 36 h in culture, total cell lysates were analyzed by western blotting (upper panel) with anti HSP47, APP and Vinculin (Vinc) antibodies. In parallel, the amount of Aβ peptide species was determined in the conditioned medium by ELISA (lower panel). (B) APPsw were infected with miRNA-based RNAi lentiviral vectors directed towards HSP47 or towards an unrelated sequence (RNAi Ctr), expressing also a GFP protein. Polyclonal populations were then analyzed as in (A) 36 h after the last medium change. (C) Extracellular (E-Aβ) and intracellular (I-Aβ) Aβ peptides were measured separately in APPsw cells treated as in panel A. (D) Primary rat cortical neurons were electroporated with two indipendent plKO plasmids (R1 and R2) encoding for shRNAs directed towards HSP47 or with a plKO containing a mismatched sequence (Ctr). 48 h after electroporation medium was changed and, after additional 72 hours, RNA was exctracted to evaluate Hsp47 knockdown by a Real Time PCR assay. In parallel, the amount of Aβ40 in the conditioned medium was determined by an ELISA assay directed against rodent Aβ40 (WAKO). ** = p<0.01; *** = p<0.001 (two tails Student T-Test).

(A) Dose dependent reduction of Aβ peptides levels by increasing concentrations of HSP47 inhibitors in 293 APPsw cells. The amount of Aβ peptides species in the conditioned medium of cells treated with the indicated inhibitors was determined by ELISA 24 hours after the last medium change and expressed as ratio on the vehicle control. (B) Primary rat cortical neurons were treated 24 h after plating with Compound IV (7.5 βµM) or Vehicle (DMSO). The amount of Aβ40 in conditioned medium was then evaluated as in Fig. 5C. (C) 293 APPsw cells were transiently transfected with an unrelated control plasmid (Mock) or with an HSP47 overexpression construct. 12 hours later, cells were treated with vehicle only or with 7.5 µM Compound IV (IV). The amount of Aβ peptides in the conditioned medium was determined after additional 24 hours as in (A). ** = p<0.01 (two tails Student T-Test).