Hsp20, a novel alpha-crystallin, prevents Abeta fibril formation and toxicity

Abstract

Beta-amyloid (Abeta) is a major protein component of senile plaques in Alzheimer's disease, and is neurotoxic when aggregated. The size of aggregated Abeta responsible for the observed neurotoxicity and the mechanism of aggregation are still under investigation; however, prevention of Abeta aggregation still holds promise as a means to reduce Abeta neurotoxicity. In research presented here, we show that Hsp20, a novel alpha-crystallin isolated from the bovine erythrocyte parasite Babesia bovis, was able to prevent aggregation of denatured alcohol dehydrogenase when the two proteins are present at near equimolar levels. We then examined the ability of Hsp20 produced as two different fusion proteins to prevent Abeta amyloid formation as indicated by Congo Red binding; we found that not only was Hsp20 able to dramatically reduce Congo Red binding, but it was able to do so at molar ratios of Hsp20 to Abeta of 1 to 1000. Electron microscopy confirmed that Hsp20 does prevent Abeta fibril formation. Hsp20 was also able to significantly reduce Abeta toxicity to both SH-SY5Y and PC12 neuronal cells at similar molar ratios. At high concentrations of Hsp20, the protein no longer displays its aggregation inhibition and toxicity attenuation properties. Size exclusion chromatography indicated that Hsp20 was active at low concentrations in which dimer was present. Loss of activity at high concentrations was associated with the presence of higher oligomers of Hsp20. This work could contribute to the development of a novel aggregation inhibitor for prevention of Abeta toxicity.

Figure 1.

Light scattering of ADH, as determined by absorbance at 360 nm, at elevated temperature in the presence and absence of Hsp20-MBP. ADH at a concentration of 1.625 μM is incubated in phosphate buffer alone (open circles) and in the presence of 5.75 μM Hsp20 (closed triangles) at 58°C for 1 h.

Figure 2.

Percent reduction of light scattering at time infinity of ADH when mixed with Hsp20-MBP relative to light scattering of ADH alone. The negative of light scattering seen at 2:1 molar ratio (Hsp20:ADH) indicates that light scattering of the Hsp20:ADH mixture was 80% less than light scattering of ADH alone. The near zero value of change in light scattering at 1:1000 Hsp20:ADH mole ratio indicates that light scattering of the mixture was unchanged from light scattering of ADH seen in Figure 1 ▶. All experiments were performed at 58°C. The concentration of ADH was 1.625 μM in each experiment. Light scattering at time infinity was estimated from absorbance versus time curves for each mixture.

Figure 3.

Representative absorbance spectra of Congo Red with Aβ and Aβ-Hsp20 mixtures. One-hundred-millimolar Aβ(1–40) was used in all cases. Hsp20-MBP was added at the beginning of the 24-h aggregation at room temperature with mixing. (Bold line, Congo Red alone; fine line, Aβ alone; light gray line, 1:100 Hsp20:Aβ; dark gray line,1:1000 Hsp20:Aβ.)

Figure 4.

Relative fibril formation as estimated from Congo Red binding. Fibril formation is reported relative to fibril formation seen with pure Aβ solutions. Mean plus or minus standard deviation of at least three independent experiments are reported. (A) Relative fibril formation as a function Hsp20-MBP concentration. (B) Relative fibril formation as a function of the molar ratio of Hsp20-MBP to Aβ. Open circles, 20 μM Aβ; squares, 50μM Aβ; triangles, 100μM Aβ.

Figure 5.

Representative electron micrographs of Aβ and Hsp20-MBP after 24 h of aggregation (A) 100μM Aβ, (B) 0.1μM Hsp20-MBP, and (C) 100μM Aβ + 0.1μM Hsp20-MBP.

Figure 6.

Relative cell viability of SY5Y cells (triangles) and PC12 cells (circles) treated with 100 μM (triangles) and 2 μM (circles) Aβ as a function of Hsp20-MBP concentration. Viability is measured via the MTT reduction assay. N ≥ 6. Aβ(1–40) was incubated for 24 h in the media prior to addition to the cells. Viability as a function of molar ratio of Hsp20-MBP to Aβ; viability is reported relative to control cells.

Figure 7.

Relative Aβ fibril formation and SH-SY5Y cell viability as a function of concentration his-Hsp20. (A) Fibril formation is reported relative to fibril formation in pure 50 mM Aβ solutions and determined via Congo Red binding. (B) Cell viability was measured via MTT reduction and reported relative to cells untreated with Aβ or his-Hsp20. Aβ or his-Hsp20 mixtures were mixed, then aggregated with mixing at 37°C for 24 h prior to viability or Congo Red binding measurements.

Figure 8.

Representative size exclusion chromatograms of his-Hsp20 at (A) 100 μM his-Hsp20, (B) 29 μM his-Hsp20, and (C) 0.1 μM his-Hsp20. Separations were performed on a Pharmacia Superose 6 column at room temperature using phosphate buffered saline as an elution buffer.