Figure 4.
Expression of mutant human parkin in Drosophila DA neurons causes age-dependent neurodegeneration.
A
, Schematic representation of DA neurons in the central brain of Drosophila.
a
, The projected confocal image shows anti-TH and phalloidin staining spanning optical sections 40 μm in depth from posterior to anterior. Two major DA clusters, DM (circles) and DL (rectangles), are indicated.
b
, A projected image shows anti-TH (red) and anti-GFP (green) colocalized in all DA neurons in both DM and DL (enlarged images to the right) clusters. Arrows indicate 5-HT neurons expressing GFP but not TH.
B
, Reductions in TH immunoreactivity at 5 weeks after eclosion are apparent in ddc::parkinQ311X (
b
) and ddc::parkinT240R (
c
) but not ddc control (
a
) or ddc::parkinwt (
d
) brains. Circles, DM; rectangles, DL.
C
, Quantification of DA neurons at 0, 3, and 5 weeks after eclosion in DM and DL clusters. Progressive loss of TH-immunoreactive neurons is induced in both clusters by ddc::parkinQ311X (blue bars) and ddc::parkinT240R (red bars) compared with the ddc control (black bars) and ddc::parkinwt (green bars). Values represent the mean ± SEM; n = 12. *p < 0.05, **p < 0.01, ***p < 0.001 relative to the ddc control.
D
, At 4 weeks, mCD8-GFP signal (green) is reduced in parkinT240R (
b
) compared with parkinwt (
a
) brain, which is similar to changes observed in corresponding images showing reduced TH immunoreactivity. Measurement of GFP pixel density (corresponding to the left y-axis) and GFP cell counts in the DM cluster (corresponding to the right y-axis) from 4-week-old brains of both genotypes showed a significant decrease (unpaired t test; n = 5; *p < 0.05) of GFP signal in parkinT240R brains compared with parkinwt (
c
). The GFP signal at eclosion was indistinguishable between parkinT240R and parkinwt (data not shown).
d
,
e
, Confocal images of ddc::parkinT240R (
e
) and ddc::parkinwt (
d
) brains stained with an anti-parkin antibody (green) reveals reduced parkin signals in ddc::parkinT240R compared with ddc::parkinwt brains.
f
, Analysis of brains at 4 weeks indicates that cell counts stained with anti-parkin for parkinT240R are decreased compared with parkinwt (unpaired t test; n = 6; *p < 0.05 for DM; ***p < 0.001 for DL). Parkin staining at eclosion was indistinguishable between parkinT240R and parkinwt (data not shown).
a
,
b
,
d
, and
e
are confocal images of adjacent brains imaged simultaneously in the same slide but reoriented for presentation.
E
, Anti-5-HT staining at 5 weeks after eclosion shows reduced neuritic immunoreactivity in 5-HT neurons expressing mutant parkin (
b
,
c
) compared with those expressing wild-type parkin (
d
) or controls (
a
). Scale bar, 40 μm.