Branched KLVFF tetramers strongly potentiate inhibition of beta-amyloid aggregation

The key pathogenic event in the onset of Alzheimer's disease (AD) is the aggregation of beta-amyloid (Abeta) peptides into toxic aggregates. Molecules that interfere with this process might act as therapeutic agents for the treatment of AD. The amino acid residues 16-20 (KLVFF) are known to be essential for the aggregation of Abeta. In this study, we have used a first-generation dendrimer as a scaffold for the multivalent display of the KLVFF peptide. The effect of four KLVFF peptides attached to the dendrimer (K(4)) on Abeta aggregation was compared to the effect of monomeric KLVFF (K(1)). Our data show that K(4) very effectively inhibits the aggregation of low-molecular-weight and protofibrillar Abeta(1-42) into fibrils, in a concentration-dependent manner, and much more potently than K(1). Moreover, we show that K(4) can lead to the disassembly of existing aggregates. Our data lead us to propose that conjugates that bear multiple copies of KLVFF might be useful as therapeutic agents for the treatment of Alzheimer's disease

Impaired Heat Shock Response in Cells Expressing Full-Length Polyglutamine-Expanded Huntingtin

The molecular mechanisms by which polyglutamine (polyQ)-expanded huntingtin (Htt) causes neurodegeneration in Huntington's disease (HD) remain unclear. The malfunction of cellular proteostasis has been suggested as central in HD pathogenesis and also as a target of therapeutic interventions for the treatment of HD. We present results that offer a previously unexplored perspective regarding impaired proteostasis in HD. We find that, under non-stress conditions, the proteostatic capacity of cells expressing full length polyQ-expanded Htt is adequate. Yet, under stress conditions, the presence of polyQ-expanded Htt impairs the heat shock response, a key component of cellular proteostasis. This impaired heat shock response results in a reduced capacity to withstand the damage caused by cellular stress. We demonstrate that in cells expressing polyQ-expanded Htt the levels of heat shock transcription factor 1 (HSF1) are reduced, and, as a consequence, these cells have an impaired a heat shock response. Also, we found reduced HSF1 and HSP70 levels in the striata of HD knock-in mice when compared to wild-type mice. Our results suggests that full length, non-aggregated polyQ-expanded Htt blocks the effective induction of the heat shock response under stress conditions and may thus trigger the accumulation of cellular damage during the course of HD pathogenesis

Oligomer-specific Abeta toxicity in cell models is mediated by selective uptake

Alzheimer's disease (AD) is characterized by the aggregation and subsequent deposition of misfolded beta-amyloid (Abeta) peptide. Previous studies show that aggregated Abeta is more toxic in oligomeric than in fibrillar form, and that each aggregation form activates specific molecular pathways in the cell. We hypothesize that these differences between oligomers and fibrils are related to their different accessibility to the intracellular space. To this end we used fluorescently labelled Abeta1-42 and demonstrate that Abeta1-42 oligomers readily enter both HeLa and differentiated SKNSH cells whereas fibrillar Abeta1-42 is not internalized. Oligomeric Abeta1-42 is internalized by an endocytic process and is transported to the lysosomes. Inhibition of uptake specifically inhibits oligomer but not fibril toxicity. Our study indicates that selective uptake of oligomers is a determinant of oligomer specific Abeta toxicit

Reduced HSF1 levels in cells expressing polyQ-expanded Htt.

<p><b>A</b>) Western blots prepared with protein lysates from STHdh(Q7) and STHdh(Q111) cells that were grown at 33°C or heat shocked (HS, 42°C for three hours). The blots were probed with anti-HSF1 antibodies and anti-tubulin antibodies as a loading control. <b>B</b>) Upper panel: quantification of Western blots as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037929#pone-0037929-g005" target="_blank">Figure 5 A</a>. Signals of total HSF1 were normalized to the corresponding tubulin signal in each experiment. Lower panel: quantification of Western blots as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037929#pone-0037929-g005" target="_blank">Figure 5 A</a>. Signals of the top, i.e. the heat shock-induced part of HSF1 were normalized to the corresponding tubulin signal in each experiment. The signal of the top part of HSF1 at 33°C was set as 1 for each condition. Error bars represent SDs. Results from three independent experiments were analyzed. *p<0.07 (two-tailed t-test). <b>C</b>) Cross-linking of HSF1. Protein lysates were prepared as in A) followed by cross-linking with EGS (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037929#s4" target="_blank">Materials and Methods</a> for details). The ensuing Western blots were probed with and anti-HSF1 antibody. <b>D</b>) Quantification of three independent experiments as shown in C). The error bars represent SDs. * p<0.01 (two-tailed t-test). <b>E</b>) Protein lysates were prepared as in A) and then separated into cytosolic and nuclear fractions. The blots were probed with anti-HSF1 antibodies and anti-lamin A/C antibodies and tubulin antibody as a loading control and as a control for the purity of the fractions (nucleus and cytosol respectively). <b>F</b>) Viability assay (luciferase activity, Promega) of STHdh(Q7) and STHdh(Q111) cells that were grown at 33°C and heat shocked cells (three hours at 42°C) in the absence or presence of triptolide. The signal from STHdh(Q7) cells grown at 33°C without triptolide was set as 100% and the other signals were calculated accordingly. The error bars represent SDs. Results from four independent experiments were analyzed. * p<0.001 (two-tailed t-test).</p

Decreased resistance to a heat shock in cells expressing polyQ-expanded Htt.

<p><b>A</b>) Caspase assay of cells that were grown at 33°C and of heat shocked cells (six hours at 42°C). In either medium containing high concentrations of glucose and serum (HH) or lower concentration of glucose and serum (LL). The error bars represent SDs. Results from four independent experiments were analyzed. * p<0.1 (two tailed t-test). <b>B</b>) Relative caspase activities (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037929#pone-0037929-g001" target="_blank">Figure 1</a> A). The values for non heat shocked cells were set as 1 and the other signals were calculated accordingly. <b>C</b>) Viability assay (luciferase activity, Promega) of STHdh(Q7) and STHdh(Q111) cells that were grown at 33°C and of heat shocked cells (six hours at 42°C). The signals from STHdh(Q7) cells grown at 33°C was set as 100% and the other signals were calculated accordingly. The error bars represent SDs. Results from four independent experiments were analyzed. * p<0.05 (two tailed t-test). <b>D</b> MTT assay of STHdh(Q7) and STHdh(Q111) cells that were grown at 33°C and of heat shocked cells (six hours at 42°C). The signals from STHdh(Q7) cells grown at 33°C was set as 100% and the other signals were calculated accordingly. The error bars represent SDs. Results from four independent experiments were analyzed. * p<0.05 (two tailed t-test). Experiments shown under B), C), and D) were carried out using LL medium.</p

Impaired heat shock response in cells expressing polyQ-expanded Htt.

<p><b>A</b>) Western blots were prepared with protein lysates from STHdh(Q7) (St7Q) and STHdh(Q111) (St111Q) cells that were grown at 33°C or heat shocked (HS), i.e. grown at 33°C and then incubated at 42°C for three hours. The blots were probed with the indicated anti-Hsp antibodies and anti-tubulin antibodies as a loading control. The anti-Hsp70 antibody (3A3) detects both constitutive (const.) and inducible (ind.) Hsp70s. <b>B</b>) Quantification of Western blots as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037929#pone-0037929-g001" target="_blank">Figure 1 A</a>. All signals were normalized to the corresponding tubulin signal in each experiment. For Hsp70, both the constitutive and the inducible Hsp70s were quantified together. The error bars present SDs. * p<0.01 (two-tailed t-test). <b>C</b>) Immunofluorescence microscopy of fixed cells that were grown at 33°C and heat shocked cells (HS, three hours at 42°C) using the indicated anti-Hsp antibodies. The scale bars represent 75 µm <b>D</b>) Western blot probed with an anti-ubiquitin antibody of protein lysates derived from STHdh(Q7) and STHdh(Q111) cells that were grown at 33°C or exposed to a heat shock (42°C) for either three or six hours. A western blot using an anti-tubulin antibody (bottom) served as a loading control. E) Western blot probed with an anti-ubiquitin antibody of protein lysates derived from STHdh(Q7) and STHdh(Q111) cells that were grown at 33°C in the absence or presence of 10 µM MG132. A western blot using an anti-tubulin antibody (bottom) served as a loading control.</p

Decreased resistance to inhibition of the proteasome or Hsp90 in cells expressing polyQ-expanded Htt.

<p><b>A</b>) Immunofluorescence microscopy of STHdh(Q7) and STHdh(Q111) cells that were treated for six hours with the Hsp90 inhibitor radicicol (5 µM) or the proteasome inhibitor MG132 (10 µM) using anti-Hsp70 and anti-Hsp27 antibodies. The scale bars represent 75 µm <b>B</b>) Western blots prepared from protein lysates treated as in A. <b>C</b>) Viability assay (luciferase activity, Promega) of STHdh(Q7) and STHdh(Q111) cells that were treated with DMSO (control), radicicol (5 µM), or MG132 (10 µM) for six hours. The error bars represent SD. Results from four independent experiments were analyzed. * p<0.001 and **p<0.005 (two tailed t-test).</p

Reduced levels of HSF1 and Hsp70s in striata of HD knock-in mice.

<p><b>A</b>) Western blots were prepared with protein lysates from the cerebella (Cer) or striata (St) from wild-type (WT) or STHdh(Q111) knock-in mice and probed with anti-HSF1 antibodies and anti-tubulin antibodies as a loading control (left panel). Quantification of three independent Western blots. Error bars represent SDs. *p<0.07 (two-tailed t-test). <b>B</b>) Western blots were prepared with protein lysates from the cerebella (Cer) or striata (St) from wild-type (WT) or STHdh(Q111) knock-in mice and probed with anti-Hsp70 antibodies (3A3) and anti-tubulin antibodies as a loading control. Quantification of three independent Western blots. Error bars represent SDs. *p<0.07 (two-tailed t-test).</p

Aberrant Hsp70 localization upon heat shock in cells expressing polyQ-expanded Htt.

<p><b>A</b>) Immunofluorescence microscopy of fixed cells that were grown at 33°C and heat shocked cells (HS, 42°C) for either one, three, or six hours probed with anti-Hsp70 antibodies. The scale bars represent 75 µm. The 33°C samples were exposed longer to visualize Hsp70 localization. <b>B</b>) Immunofluorescence microscopy of fixed cells as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037929#pone-0037929-g004" target="_blank">Figure 4 A</a>. Pictures were taken at a higher magnification (scale bars represent 15 µm) to demonstrate the difference between diffuse and granular Hsp70 staining in the nucleus.</p