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Chaperone Suppression of  -Synuclein Toxicity in a Drosophila Model for
Parkinson’s Disease
Pavan K. Auluck, H. Y. Edwin Chan, John Q. Trojanowski, Virginia M.-Y. Lee, Nancy M. Bonini
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Supplementary Material
In our analysis of Hsp70 suppression of
-synuclein toxicity, we found that the mutant forms of
-synuclein (A30P and A53T) had similar toxicity to that of the wildtype protein. This toxicity resulted in ~50% neuron loss in the dorsomedial (DM) clusters of dopaminergic neurons in flies aged to 20 days (Web table 1). Dopaminergic neuron loss in the dorsolateral-1 (DL-1) clusters was more variable. In some experiements, no cell loss was evident whereas in others, as many as 50% of the neurons were lost in 20-day-old flies (Web table 2). Hsp70 suppressed neurodegeneration due to both mutant and wildtype
-synuclein (Fig. 1, Web tables 1 and 2). Immunoblot analysis comfirmed that levels of
-synuclein protein were not altered upon co-expression of Hsp70 (Web fig. 1).
Supplemental Table 1. Hsp70 protected against -synuclein-induced loss of dopaminergic neurons in the DM clusters. Dopaminergic neurons were visualized by immunostaining for tyrosine hydroxlyase. In this experiment, UAS-lacZ, which encodes the  -galactosidase protein, was included as a control transgene, which confirmed that expression of a control protein resulted in neither loss of dopaminergic neurons, nor protection against -synuclein toxicity. Serial sections from 3 to 5 fly heads were examined per data point.
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| Condition | Mean number of dopaminergic neurons ± SEM |
| 1 day | 10 days | 20 days |
| Control protein -galactosidase |
| (w;Ddc-GAL4/UAS-lacZ) | 17.0 ± 2.0 | - | 14.0 ± 1.0 |
| A30P synuclein |
| (w;Ddc-GAL4,UAS-lacZ/+;UAS-A30P/+) | 17.0 ± 0.6 | 12.3 ± 1.2 | 5.0 ± 1.4 |
| A30P synuclein and Hsp70 |
| (w;Ddc-GAL4,UAS-lacZ/+;UAS-A30P,UAS-HspA1L/+) | 16.3 ± 1.7 | 14.0 ± 2.1 | 14.0 ± 1.2 |
| A53T synuclein |
| (w;Ddc-GAL4,UAS-lacZ/UAS-A53T) | 16.0 ± 3.5 | 7.0 ± 1.2 | 7.7 ± 0.3 |
| A53T synuclein and Hsp70 |
| (w;Ddc-GAL4,UAS-lacZ/UAS-A53T,UAS-HspA1L) | 16.7 ± 1.3 | 17.0 ± 1.2 | 16.3 ± 1.2 |
| Supplemental Table 2. Hsp70 protected against -synuclein-induced loss of dopaminergic neurons in the DL-1 clusters. Dopaminergic neurons were visualized by immunostaining for tyrosine hydroxylase. Cell loss was variable in the DL-1 clusters between experiments. Serial sections from 3 to 5 fly heads were examined for each data point.
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| Condition | Mean number of dopaminergic neurons ± SEM |
| 1 day | 20 days |
| Control protein -galactosidase |
| (w;Ddc-GAL4/UAS-lacZ) | 16.3 ± 0.7 | 14.5 ± 2.5 |
| Wildtype synuclein |
| (w;Ddc-GAL4/+;UAS--syn/+) | 19.5 ± 0.5 | 9.7 ± 0.3 |
| Wildtype synuclein and Hsp70 |
| (w;Ddc-GAL4/+;UAS--syn,UAS-HspA1L/+) | 15.3 ± 1.5 | 16.7 ± 1.7 |
| A30P synuclein |
| (w;Ddc-GAL4,UAS-lacZ/+;UAS-A30P/+) | 19.3 ± 0.3 | 10.0 ± 2.0 |
| A30P synuclein and Hsp70 |
| (w;Ddc-GAL4,UAS-lacZ/+;UAS-A30P,UAS-HspA1L/+) | 17.0 ± 1.5 | 15.3 ± 1.5 |
| A53T synuclein |
| (w;Ddc-GAL4,UAS-lacZ/UAS-A53T) | 18.7 ± 1.2 | 14.0 ± 1.0 |
| A53T synuclein and Hsp70 |
| (w;Ddc-GAL4,UAS-lacZ/UAS-A53T,UAS-HspA1L) | 19.3 ± 0.7 | 18.8 ± 1.3 |
Supplemental Figure 1. Immunoblot analysis of heads from flies expressing -synuclein alone (lanes 1 and 3) or with Hsp70 (lanes 2 and 4). No change in the expression level of -synuclein was observed in the presence of Hsp70. A co-expressed marker transgene (UAS-lacZ) also confirmed that all conditions expressed the same levels of transgene-driven protein. Genotypes: w;Ddc-GAL4,UAS-lacZ/+;UAS--syn/+ (lanes 1 and 3); w;Ddc-GAL4,UAS-lacZ/+;UAS--syn,UAS-HspA1L/+ (lanes 2 and 4). Sixteen fly heads of appropriate genotype were analyzed. Primary antibodies used detected -synuclein [syn204 (1)], human Hsp70 (StressGen Biotechnologies Corp., Victoria, BC, Canada), -tubulin (Developmental Studies Hybridoma Bank, Iowa City, IA), and -galactosidase (Promega Corp., Madison, WI).
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REFERENCES
1. B. I. Giasson et al., J. Neurosci. Res. 59, 528 (2000).
