The HSP70 molecular chaperone is not beneficial in a mouse model of alpha-synucleinopathy

Abstract

Background: Aggregation and misfolded alpha-synuclein is thought to be central in the pathogenesis of Parkinson's disease (PD). Heat-shock proteins (HSPs) that are involved in refolding and degradation processes could lower the aggregate load of alpha-synuclein and thus be beneficial in alpha-synucleinopathies.

Methodology/principal findings: We co-overexpressed human A53T point-mutated alpha-synuclein and human HSP70 in mice, both under the control of Thy1 regulatory sequences. Behavior read-outs showed no beneficial effect of HSP70 expression in mice. In contrast, motor coordination, grip strength and weight were even worse in the alpha-synucleinopathy model in the presence of HSP70 overexpression. Biochemical analyses revealed no differences in alpha-synuclein oligomers/aggregates, truncations and phosphorylation levels and alpha-synuclein localization was unchanged in immunostainings.

Conclusion/significance: Overexpressing HSP70 in a mouse model of alpha-synucleinopathy did not lower the toxic load of alpha-synuclein species and had no beneficial effect on alpha-synuclein-related motor deficits.

Figure 1. Mice overexpressing human HSP70 under the control of Thy1 regulatory sequences.

A. Schematic diagram of the Thy1-HSP70 construct. B. Overview section of a Thy1-HSP70 mouse brain stained against HSP70. C. Details of brain sections from Thy1-HSP70 mice showing HSP70 expression in different brain regions. Scale bars: 200 µm. D. Co-localization of human HSP70 (green) and human α-synuclein (red) in different brain regions of double transgenic Thy1-HSP70/Thy1-haSN(A53T) mice. White arrow: Co-localization of HSP70 and α-synuclein; Red arrow: neurons expressing only human α-synuclein; Green arrow: neurons single positive for human HSP70. Scale bars: 50 µm.

Figure 2. Thy1-HSP70 mice appear and behave normally and are phenotypical indifferent to wildtype mice.

Measurement of weight, forelimb grip strength and motor coordination (rotating beam and rotarod) of single Thy1-HSP70 and wildtype mice. Error bars: SEM.

Figure 3. HSP70 overexpression in an α-synucleinopathy mouse model is not beneficial but even worsens the phenotype.

Measurement of weight, forelimb grip strength and motor coordination (rotating beam and rotarod) of single- and double-transgenic Thy1-HSP70/Thy1-haSN(A53T) mice. Asteriks indicate statistical significance (*: p<0.05, **: p<0.01; student's t-test, two-tailed). Error bars: SEM.

Figure 4. Biochemical analyses of soluble and pellet fraction protein extracts from spinal cord, brainstem and anterior brain reveal no difference in α-synuclein species.

A. Immunoblot of mouse spinal cord lysates detecting soluble α-synuclein monomers, oligomers/aggregates, truncations and S129-phosphorylation (P-aSN). B. Immunoblot of mouse spinal cord lysates detecting soluble HSP70. C. Immunoblot of soluble (supernatant) and pellet fractions of brainstem detecting HSP70 and α-synuclein monomers, oligomers/aggregates, truncations and S129-phosphorylation. D. Immunoblot of soluble (supernatant) and pellet fractions of anterior brain detecting HSP70 and α-synuclein monomers, truncations and S129-phosphorylation (P-aSN). β-actin was used as loading control. The size markers are indicated. E. Bar graphs representing quantitative analyses of the immunoblots shown in C and D for the α-synuclein monomers, oligomers/aggregates, truncated forms and S129-phosphorylation (P-aSN) normalized to the loading control β-actin in soluble and pellet fractions. n = 5 per genotype. Asteriks indicate statistical significance (*: p<0.05); student's t-test, two-tailed. Error bars: standard deviation.

Figure 5. Histological analyses reveal no difference in α-synuclein distribution/localization in the brain.

Brain sections of single and double transgenic Thy1-HSP70/Thy1-haSN(A53T) mice stained against human α-synuclein. Black arrow: cytoplasmic and nuclear α-synuclein in the CA1 area of the hippocampus and brainstem neurons. Scale bar: 100 µm.