Cathepsin B/L genetic depletion in MEF cells causes accumulation of cholesterol and lysosomal proteins.

<p>(A) Confocal microscopy of CtsB KO, CtsL KO and CtsB/L double KO MEFs. Cholesterol (filipin staining, white) and NPC1 (green). (B) Western blot analysis of CtsB KO, CtsL KO and CtsB/L double KO MEFs using LC3, LAMP1 and NPC1 antibody. α-Tubulin was used as a loading control. (C) Quantification of Western blot results of the 3 independent experiments was performed by ImageJ. Student t-test was used for statistical analysis. Error bars present the mean ± standard deviation (** p < 0.01, *** p < 0.001).</p

Development and evaluation of novel PET tracers for imaging cannabinoid receptor type 2 in brain

The cannabinoid receptor type 2 (CB2) has a very low expression level in brain tissue under basal conditions, but it is up-regulated in diverse pathological conditions. Two promising lead structures from the literature, N-((3S,5S,7S)-adamantan-1-yl)-8-methoxy-4-oxo-1-pentyl-1,4-dihydroquinoline-3-carboxamide and 8-butoxy-N-(2-fluoro-2-phenylethyl)-7-methoxy-2-oxo-1,2-dihydroquinoline-3-carboxamide - designated KD2 and KP23, respectively - were evaluated as potential PET ligands for imaging CB2. Both KD2 and KP23 were synthesized and labeled with carbon-11. In vitro autoradiographic studies on rodent spleen tissues showed that [(11)C]KD2 exhibits superior properties. A pilot study using [(11)C]KD2 on human post mortem ALS spinal cord slices indicated high CB2 expression level and specific binding, a very exciting finding if considering the future diagnostic application of CB2 ligands and their utility in therapy monitoring. In vivo blocking studies in rats with [(11)C]KD2 showed also high specific uptake in spleen tissue. Although the protein-bound fraction is relatively high, KD2 or KD2 derivatives could be very useful tools for the non-invasive investigation of CB2 levels under various neuroinflammatory conditions

Cathepsin B/L inhibition causes NPC disease-like cholesterol accumulation in CHO cells.

<p>(A) Confocal microscopy of CHOwt cells treated with different inhibitors. Cholesterol (filipin staining, white) and NPC1 (green). (B) Western blot of CHOwt cells treated with different inhibitors using ABCA1, NPC1 and NPC2 antibody. β-Actin was used as a loading control. (C) Quantification of Western blot results of the 3 independent experiments was performed by ImageJ. Student t-test was used for statistical analysis. Error bars present the mean ± standard deviation (** p < 0.01, *** p < 0.001).</p

Sez6L co-staining with BACE1 reveals their increased immunoreactivity in the cell body of NPC1 vs. wt mouse primary cortical neurons.

<p>Representative images of primary mouse cortical neurons stained with Sez6L antibody co-stained with BACE1. In NPC1^-/- (NPC1) cortical neurons, in contrast to NPC1^+/+ (wt) neurons, we observed an increased punctate staining of Sez6L in the cell body and in the neuronal processes. The cell body co-staining of Sez6L and BACE1 was enhanced in NPC1 vs. wt neurons (indicated by arrows).</p

The increased levels of soluble Sez6, Sez6L and APP ectodomains reveal enhanced proteolysis by BACE1 in 4-weeks old NPC1 vs. wt mouse brains.

<p>(A-C) Western blot analyses of soluble Sez6 (sSez6), soluble Sez6L (sSez6L), total soluble APP (sAPPt) and actin (Actin-DEA) in DEA fractions of the cortex (A), hippocampus (B) and cerebellum (C) collected from 4-weeks old wt (NPC1^+/+; N = 6) and NPC1 (NPC1^-/-; N = 6) mice. A standard protein lysate (S) was included twice on each gel. (D-F) Graphs representing quantified protein signals of sSez6 (D), sSez6L (E) and sAPPt (F) which were normalized against actin (Actin-DEA) in the cortex (CX), hippocampus (HP) and cerebellum (CB) of 4-weeks old wt- and NPC1-mice.</p