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First published online 17 July 2007
doi: 10.1242/jcs.004598

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Cellular prion protein (PrP^C) protects neuronal cells from the effect of huntingtin aggregation

Laboratory of Cell Biology, NHBLI, NIH, Bethesda, MD 20892-0301, USA

View larger version (36K): [in this window] [in a new window]   Fig. 1. Depletion of PrPC by RNAi. (A) Western blot of PrPC levels in control (Mock) SN56 cells and cells 48 hours after being transfected with the following oligonucleotides: scramble sequence (Scram), sequence 1, or sequence 2. In each lane, 100 µg of total protein was loaded. (B) Quantification of the western blot shown in A. The different intensities of the PrPC bands were normalized to the mock-depleted value which was set at 100%. Quantification of the western blot showed that it was linear from 10 to 100 µg of cell lysate. (C) Immunoprecipitation of PrPC from 500 µg cell lysates obtained from control cells and cells transfected with the scramble sequence, sequence 1, or sequence 2. The control cells were treated with Lipofectamine, the same as the PrPC-depleted cells.

View larger version (61K): [in this window] [in a new window]   Fig. 2. Effect of PrPC depletion on Htt aggregation in neuronal cells. (A) Immunofluorescence images of SN56 cells following 48 hours after transfection with GFP-HttQ25 (a,b) and HttQ103 (c-f) constructs. The cells were transfected with (a,c) GFP-Htt constructs only, (b,e) oligonucleotide 2 and GFP-Htt constructs, (d) scramble oligonucleotide and GFP-HttQ103 construct, (f) oligonucleotide 2, GFP-HttQ103 construct and mouse PrPC expressing vector. (B) The number of aggregates was measured 48 hours after GFP-HttQ103 transfection in control neuronal cells (lane 1 and 7), and cells treated with scramble oligonucleotide (lane 2), oligonucleotide 1 (lanes 3 and 8), oligonucleotide 2 (lanes 4 and 9), oligonucleotide 2 and co-transfected with mouse PrPC-expressing vector (lanes 5 and 10), and in transfected cells treated overnight with 10 µM lactacystin (lane 6). The open and gray bars are data obtained from SN56 and N2a cells, respectively. *P<0.05 and **P<0.01 compared with transfected control cells. (C) Filtration assay to measure aggregated protein lysates from SN56 cells transfected with HttQ25, HttQ103, oligonucleotides 2 followed by transfection of HttQ103 expressing vector, and oligonucleotides 2 followed by transfection of HttQ103 and mouse PrPC expressing vectors. The intensity of the Htt retained on the membrane is quantified beneath the dot blot for each experimental condition. (D) The level of HttQ103 expression in cells transfected under varying conditions. Cell lysates (100 µg) from SN56 cells transfected with HttQ103, scrambled oligonucleotide followed by HttQ103, oligonucleotides 2 followed by HttQ103, and oligonucleotides 2 followed by HttQ103 and mouse PrPC. The western blot was probed using anti-GFP and anti-actin antibodies. (E) The level of Hsp70 expression in SN56 cells under varying conditions in the presence and absence of HttQ103. Cells were transfected with either HttQ25 or HttQ103. Cells were either mock transfected or transfected with scramble sequence, sequence 1 and sequence 2 oligonucleotides.

View larger version (19K): [in this window] [in a new window]   Fig. 3. Effects of PrPC depletion on HttQ103 induced cytotoxicity and apoptosis in SN56 cells. The extent of cytotoxicity and apoptosis were measured in mock- and PrPC-depleted cells 48 hours after transfection with either HttQ25 or HttQ103. (A) Cell viability was measured by MTT assay as described in Materials and Methods. (B) Apoptosis was measured by immunostaining for cleaved caspase-3 using Rhodamine-conjugated secondary antibody. The data represent an average of three experiments in which 500 transfected cells were analyzed to determine caspase-3 staining. *P<0.05 and **P<0.01 compared to control cultures.

View larger version (24K): [in this window] [in a new window]   Fig. 4. Effects of PrPC depletion on proteasome activity in SN56 cells. The activity of proteasome was measured in mock- and PrPC-depleted cells 48 hours following transfection with either HttQ25 or HttQ103. *P<0.05 and **P<0.01 compared with control cultures.

View larger version (24K): [in this window] [in a new window]   Fig. 5. Effects of PrPC depletion on ROS levels in SN56 cells. (A) Immunofluorescence images of fixed cells that were transfected with either scramble (a,b) or sequence 1 (c,d) oligonucleotides and conjugated to Dye547-3' as an indicator of transfection (a,c) or stained with DFA to show ROS levels (b,d). The asterisks indicate cells not transfected with oligonucleotides. The images were produced using identical scan-settings that allowed direct comparison of fluorescence intensities. Bar, 20 µm. (B) The level of ROS was measured 72 hours after siRNA oligomer transfection in the presence and absence of Htt. *P<0.05 and **P<0.01 compared with control transfected cells.

View larger version (21K): [in this window] [in a new window]   Fig. 6. Effects of PrPC depletion on proteasome activity and cellular ROS in Caco-2 and HT-29 cells. (A) Western blot of PrPC levels in siRNA-treated Caco-2 and HT-29 cells and quantification of this blot. (B) The activity of proteasome measured 72 hours after transfection with oligonucleotides in Caco-2 and HT-29 cells. (C) ROS levels measured 72 hours after transfection with oligonucleotides in Caco-2 and HT-29 cells.

View larger version (22K): [in this window] [in a new window]   Fig. 7. Effects of expressing PrPC on HttQ103 aggregation and proteasome activity in neuronal (SN56) and non-neuronal cells (HeLa and CHO). (A) Effect of overexpression of PrPC on the extent of HttQ103 aggregation in different cell types. (B) Effect of overexpression of PrPC on proteasome activity in different cell types. In addition to HttQ103, cells were co-transfected with the same concentration of either pcDNA4 or PrPC expressing vector, as indicated. *P<0.01 compared with cells transfected with only HttQ103.

View larger version (25K): [in this window] [in a new window]   Fig. 8. Effects of scrapie on HttQ103 aggregation, viability, proteasome activity, and ROS level in SN56 cells. The extent of HttQ103 aggregation (A), neurotoxicity (B), ROS level (C) and proteasome activity (D) were measured 48 hours after SN56 and ScSN56 cells were transfected with Htt Q103.

View larger version (8K): [in this window] [in a new window]   Fig. 9. Diagram showing the interrelationship between ROS, proteasome activity, huntingtin aggregation and PrPC expression. At any given time in the nerve cell, there are competing pathways in which PrPC expression reduces ROS and in turn increases proteasome activity, whereas Htt aggregation has the opposite effect.

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