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Cell line specific modulation of extracellular aβ42 by Hsp40.

Carnini A, Scott LO, Ahrendt E, Proft J, Winkfein RJ, Kim SW, Colicos MA, Braun JE - PLoS ONE (2012)

Bottom Line: Moreover, transient transfection of the stress induced Hsp40, decreased CAD levels of Aβ(42).Mutation of the conserved HPD motif within Hsp40 significantly reduced the Hsp40-mediated Aβ(42) increase in hippocampal cultures indicating the importance of this motif in regulating cellular Aβ(42).Therefore, increasing Hsp40 therapeutically with the intention of interfering with the pathogenic cascade leading to neurodegeneration in AD should be pursued with caution.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pharmacology, Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta, Canada.

ABSTRACT
Heat shock proteins (Hsps) are a set of molecular chaperones involved in cellular repair. They provide protective mechanisms that allow cells to survive potentially lethal insults, In response to a conditioning stress their expression is increased. Here we examined the connection between Hsps and Aβ(42), the amyloid peptide involved in the pathological sequence of Alzheimer's disease (AD). Extracellular Aβ(42) associates with neuronal cells and is a major constituent of senile plaques, one of the hallmarks of AD. Although Hsps are generally thought to prevent accumulation of misfolded proteins, there is a lack of mechanistic evidence that heat shock chaperones directly modulate Aβ(42) toxicity. In this study we show that neither extracellular Aβ(42) nor Aβ(42/)PrP(C) trigger the heat shock response in neurons. To address the influence of the neuroprotective heat shock response on cellular Aβ(42), Western analysis of Aβ(42) was performed following external Aβ(42) application. Five hours after a conditioning heat shock, Aβ(42) association with CAD cells was increased compared to control neurons. However, at forty-eight hours following heat shock Aβ(42) levels were reduced compared to that found for control cells. Moreover, transient transfection of the stress induced Hsp40, decreased CAD levels of Aβ(42). In contrast to CAD cells, hippocampal neurons transfected with Hsp40 retained Aβ(42) indicating that Hsp40 modulation of Aβ(42) proteostasis is cell specific. Mutation of the conserved HPD motif within Hsp40 significantly reduced the Hsp40-mediated Aβ(42) increase in hippocampal cultures indicating the importance of this motif in regulating cellular Aβ(42). Our data reveal a biochemical link between Hsp40 expression and Aβ(42) proteostasis that is cell specific. Therefore, increasing Hsp40 therapeutically with the intention of interfering with the pathogenic cascade leading to neurodegeneration in AD should be pursued with caution.

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Transient transfection of Hsp40 reduces cellular Aβ42.(A) CAD cells were transiently transfected with Hsp40 as indicated for 24 hours prior to the addition of 3 µM Aβ42. Immediately following addition of Aβ42, indicated cells were subjected to heat shock at 43°C for 40 minutes and allowed to recover. At the indicated times cells were washed in PBS, lysed separated into soluble and insoluble fractions and cellular levels of Aβ42 were determined by Western analysis. Actin is shown as a loading control (B) Quantification of three independent experiments. *p<0.05. (C) Western analysis of CAD cells transfected with Hsp40 as indicated prior to incubation with 3 µM Aβ42 or 0.5 µM PrP106−126 or scrambled control. Data are representative of 4 separate experiments.
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pone-0037755-g007: Transient transfection of Hsp40 reduces cellular Aβ42.(A) CAD cells were transiently transfected with Hsp40 as indicated for 24 hours prior to the addition of 3 µM Aβ42. Immediately following addition of Aβ42, indicated cells were subjected to heat shock at 43°C for 40 minutes and allowed to recover. At the indicated times cells were washed in PBS, lysed separated into soluble and insoluble fractions and cellular levels of Aβ42 were determined by Western analysis. Actin is shown as a loading control (B) Quantification of three independent experiments. *p<0.05. (C) Western analysis of CAD cells transfected with Hsp40 as indicated prior to incubation with 3 µM Aβ42 or 0.5 µM PrP106−126 or scrambled control. Data are representative of 4 separate experiments.

Mentions: To further investigate the role that specific inducible chaperones play in heat shock induced reduction of Aβ42, CAD cells were transfected with the stress induced J protein Hsp40 and then challenged with the toxic Aβ42 (Figure 7). Both heat shock and Hsp40 transfection reduced soluble and insoluble Aβ42 (monomer) at 48 hours. Quantitative immunoblotting uncovered a 50% ±3 Hsp40-mediated reduction compared to a smaller heat shock-mediated decreases 88% ±6 in insoluble monomeric Aβ42. Soluble Aβ42 monomer was found to decrease to 67% ±12 following Hsp40 transfection and 48% ±15 following heat shock. Hsp40 and heat shock both caused changes in Aβ42 oligomerization, initially increasing the Aβ42 72 kDa oligomer followed by a decrease at 48 hrs (Figure 7). These experiments clearly establish Hsp40 as a chaperone that influences cellular clearance of Aβ42. Transfection does not induce the stress response (Figure 7C ). Likewise, residues encoding amino acids 106–126 of PrPC as well as a scrambled control do not induce Hsp70 or increase Hsp40 levels.


Cell line specific modulation of extracellular aβ42 by Hsp40.

Carnini A, Scott LO, Ahrendt E, Proft J, Winkfein RJ, Kim SW, Colicos MA, Braun JE - PLoS ONE (2012)

Transient transfection of Hsp40 reduces cellular Aβ42.(A) CAD cells were transiently transfected with Hsp40 as indicated for 24 hours prior to the addition of 3 µM Aβ42. Immediately following addition of Aβ42, indicated cells were subjected to heat shock at 43°C for 40 minutes and allowed to recover. At the indicated times cells were washed in PBS, lysed separated into soluble and insoluble fractions and cellular levels of Aβ42 were determined by Western analysis. Actin is shown as a loading control (B) Quantification of three independent experiments. *p<0.05. (C) Western analysis of CAD cells transfected with Hsp40 as indicated prior to incubation with 3 µM Aβ42 or 0.5 µM PrP106−126 or scrambled control. Data are representative of 4 separate experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3362613&req=5

pone-0037755-g007: Transient transfection of Hsp40 reduces cellular Aβ42.(A) CAD cells were transiently transfected with Hsp40 as indicated for 24 hours prior to the addition of 3 µM Aβ42. Immediately following addition of Aβ42, indicated cells were subjected to heat shock at 43°C for 40 minutes and allowed to recover. At the indicated times cells were washed in PBS, lysed separated into soluble and insoluble fractions and cellular levels of Aβ42 were determined by Western analysis. Actin is shown as a loading control (B) Quantification of three independent experiments. *p<0.05. (C) Western analysis of CAD cells transfected with Hsp40 as indicated prior to incubation with 3 µM Aβ42 or 0.5 µM PrP106−126 or scrambled control. Data are representative of 4 separate experiments.
Mentions: To further investigate the role that specific inducible chaperones play in heat shock induced reduction of Aβ42, CAD cells were transfected with the stress induced J protein Hsp40 and then challenged with the toxic Aβ42 (Figure 7). Both heat shock and Hsp40 transfection reduced soluble and insoluble Aβ42 (monomer) at 48 hours. Quantitative immunoblotting uncovered a 50% ±3 Hsp40-mediated reduction compared to a smaller heat shock-mediated decreases 88% ±6 in insoluble monomeric Aβ42. Soluble Aβ42 monomer was found to decrease to 67% ±12 following Hsp40 transfection and 48% ±15 following heat shock. Hsp40 and heat shock both caused changes in Aβ42 oligomerization, initially increasing the Aβ42 72 kDa oligomer followed by a decrease at 48 hrs (Figure 7). These experiments clearly establish Hsp40 as a chaperone that influences cellular clearance of Aβ42. Transfection does not induce the stress response (Figure 7C ). Likewise, residues encoding amino acids 106–126 of PrPC as well as a scrambled control do not induce Hsp70 or increase Hsp40 levels.

Bottom Line: Moreover, transient transfection of the stress induced Hsp40, decreased CAD levels of Aβ(42).Mutation of the conserved HPD motif within Hsp40 significantly reduced the Hsp40-mediated Aβ(42) increase in hippocampal cultures indicating the importance of this motif in regulating cellular Aβ(42).Therefore, increasing Hsp40 therapeutically with the intention of interfering with the pathogenic cascade leading to neurodegeneration in AD should be pursued with caution.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pharmacology, Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta, Canada.

ABSTRACT
Heat shock proteins (Hsps) are a set of molecular chaperones involved in cellular repair. They provide protective mechanisms that allow cells to survive potentially lethal insults, In response to a conditioning stress their expression is increased. Here we examined the connection between Hsps and Aβ(42), the amyloid peptide involved in the pathological sequence of Alzheimer's disease (AD). Extracellular Aβ(42) associates with neuronal cells and is a major constituent of senile plaques, one of the hallmarks of AD. Although Hsps are generally thought to prevent accumulation of misfolded proteins, there is a lack of mechanistic evidence that heat shock chaperones directly modulate Aβ(42) toxicity. In this study we show that neither extracellular Aβ(42) nor Aβ(42/)PrP(C) trigger the heat shock response in neurons. To address the influence of the neuroprotective heat shock response on cellular Aβ(42), Western analysis of Aβ(42) was performed following external Aβ(42) application. Five hours after a conditioning heat shock, Aβ(42) association with CAD cells was increased compared to control neurons. However, at forty-eight hours following heat shock Aβ(42) levels were reduced compared to that found for control cells. Moreover, transient transfection of the stress induced Hsp40, decreased CAD levels of Aβ(42). In contrast to CAD cells, hippocampal neurons transfected with Hsp40 retained Aβ(42) indicating that Hsp40 modulation of Aβ(42) proteostasis is cell specific. Mutation of the conserved HPD motif within Hsp40 significantly reduced the Hsp40-mediated Aβ(42) increase in hippocampal cultures indicating the importance of this motif in regulating cellular Aβ(42). Our data reveal a biochemical link between Hsp40 expression and Aβ(42) proteostasis that is cell specific. Therefore, increasing Hsp40 therapeutically with the intention of interfering with the pathogenic cascade leading to neurodegeneration in AD should be pursued with caution.

Show MeSH
Related in: MedlinePlus