Supplemental Data


Abstract
Full Text
Chaperone Suppression of name-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

Supplementary Material

In our analysis of Hsp70 suppression of name-synuclein toxicity, we found that the mutant forms of name-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 name-synuclein (Fig. 1, Web tables 1 and 2). Immunoblot analysis comfirmed that levels of name-synuclein protein were not altered upon co-expression of Hsp70 (Web fig. 1).

Supplemental Table 1. Hsp70 protected against name-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 name-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 name-synuclein toxicity. Serial sections from 3 to 5 fly heads were examined per data point.
ConditionMean number of dopaminergic neurons ± SEM
1 day10 days20 days
Control protein name-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 ± 0.612.3 ± 1.25.0 ± 1.4
A30P synuclein and Hsp70
(w;Ddc-GAL4,UAS-lacZ/+;UAS-A30P,UAS-HspA1L/+)16.3 ± 1.714.0 ± 2.114.0 ± 1.2
A53T synuclein
(w;Ddc-GAL4,UAS-lacZ/UAS-A53T)16.0 ± 3.57.0 ± 1.27.7 ± 0.3
A53T synuclein and Hsp70
(w;Ddc-GAL4,UAS-lacZ/UAS-A53T,UAS-HspA1L)16.7 ± 1.317.0 ± 1.216.3 ± 1.2


Supplemental Table 2. Hsp70 protected against name-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.
ConditionMean number of dopaminergic neurons ± SEM
1 day20 days
Control protein name-galactosidase
(w;Ddc-GAL4/UAS-lacZ) 16.3 ± 0.714.5 ± 2.5
Wildtype synuclein
(w;Ddc-GAL4/+;UAS-name-syn/+) 19.5 ± 0.59.7 ± 0.3
Wildtype synuclein and Hsp70
(w;Ddc-GAL4/+;UAS-name-syn,UAS-HspA1L/+) 15.3 ± 1.516.7 ± 1.7
A30P synuclein
(w;Ddc-GAL4,UAS-lacZ/+;UAS-A30P/+) 19.3 ± 0.310.0 ± 2.0
A30P synuclein and Hsp70
(w;Ddc-GAL4,UAS-lacZ/+;UAS-A30P,UAS-HspA1L/+) 17.0 ± 1.515.3 ± 1.5
A53T synuclein
(w;Ddc-GAL4,UAS-lacZ/UAS-A53T) 18.7 ± 1.214.0 ± 1.0
A53T synuclein and Hsp70
(w;Ddc-GAL4,UAS-lacZ/UAS-A53T,UAS-HspA1L) 19.3 ± 0.718.8 ± 1.3


Supplemental Figure 1. Immunoblot analysis of heads from flies expressing name-synuclein alone (lanes 1 and 3) or with Hsp70 (lanes 2 and 4). No change in the expression level of name-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-name-syn/+ (lanes 1 and 3); w;Ddc-GAL4,UAS-lacZ/+;UAS-name-syn,UAS-HspA1L/+ (lanes 2 and 4). Sixteen fly heads of appropriate genotype were analyzed. Primary antibodies used detected name-synuclein [syn204 (1)], human Hsp70 (StressGen Biotechnologies Corp., Victoria, BC, Canada), name-tubulin (Developmental Studies Hybridoma Bank, Iowa City, IA), and name-galactosidase (Promega Corp., Madison, WI).


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REFERENCES

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