(A) The PY125 antibody specifically recognizes phosphorylated Tyr125. Phenylalanine substitution at Tyr125 eliminates immunoreactivity. Lane 1, α-syn^WT; lane 2, α-syn^Y125F. The same membrane was stripped and reprobed with a phosphorylation-independent α-synuclein antibody (anti-syn). (B) The α-syn^YF mutant effectively eliminates anti-PY125 immunoreactivity. Compare lane 1 (α-syn^WT) with lane 3 (α-syn^Y125F). Lane 2 (α-syn^WT) is the same as lane 1 except for pretreatment with phosphatase. Flies were 20 days old. (C) Quantitative analysis of TH-immunoreactive dorsomedial dopamine neurons over time in transgenic flies. Values represent mean ± SEM. Accelerated loss of TH-immunoreactive neurons is observed in 10-day-old α-syn^YF transgenic flies (*P < 0.05, multivariant ANOVA with supplementary Newman-Keuls test). The driver in A–C was elav-GAL4. (D and E) Blocking tyrosine phosphorylation enhances retinal degeneration. Tangential section through the retina of an aged adult fly expressing α-syn^WT shows mild architectural distortion (D), whereas expression of α-syn^YF produced a greater loss of retinal integrity with large vacuoles (E). Original magnification in D and E, ×400. (F) Quantitative comparison demonstrating significant reduction in the percentage of normal photoreceptor clusters in α-syn^YF transgenic flies (*P < 0.01, multivariant ANOVA with supplementary Newman-Keuls test). Flies were 30 days old. The driver in D–F was GMR-GAL4. (G) Accelerated loss of climbing ability in transgenic flies expressing α-syn^S129D and α-syn^YF throughout the nervous system (elav-GAL4 driver). Asterisks indicate climbing scores that are significantly different from the control and wild-type α-synuclein transgenic scores (*P < 0.01, multivariant ANOVA with supplementary Newman-Keuls test). Control genotype: elav-GAL4/+. Error bars in F and G represent SEM.

(A) Increased tyrosine phosphorylation in flies coexpressing shark and α-synuclein in the brain compared with flies expressing α-synuclein alone. Flies were 1 day old. (B–D) Quantitative analysis of TH-immunoreactive neurons in flies coexpressing shark and α-synuclein and flies expressing α-synuclein alone. Values represent mean ± SEM. Asterisks indicate that the difference in TH-immunoreactive dopaminergic neuronal number between the 2 genotypes at the time points examined is statistically significant (*P < 0.01, **P < 0.05, multivariant ANOVA with supplementary Newman-Keuls test). The driver in A–D was elav-GAL4. (E–G) Augmented tyrosine phosphorylation suppresses retinal toxicity. Retinal degeneration in flies expressing α-syn^S129D (E) is largely prevented by coexpressing shark (F). Original magnification in E and F, ×400. (G) Quantitative comparison demonstrating significant rescue of the percentage of normal photoreceptor clusters in α-syn^S129D transgenic flies expressing shark (*P < 0.01, Student’s t test). Error bars represent SEM. The driver in E–G was GMR-GAL4. Flies were 30 days old.

(A) Representative immunoblot of soluble fractions from brain homogenates run on a 4%–12% gradient gel shows increased oligomer formation in flies expressing α-syn^S129D and decreased oligomer formation in flies expressing α-syn^S129A. Aliquots of the same protein preparations were loaded on a separate 10%–20% gel to visualize monomeric α-synuclein and tubulin. Control genotype: elav-GAL4. (B) Quantitative analysis of oligomer formation using densitometry. Values represent mean ± SEM of 3 independent experiments. Single asterisk indicates the significantly increased accumulation of high-molecular-weight species in flies expressing α-syn^S129D (*P < 0.01, multivariant ANOVA with supplementary Newman-Keuls test) at 10 days. Double asterisks indicate the significantly decreased accumulation in α-syn^S129A flies at 20 days (**P < 0.01, multivariant ANOVA with supplementary Newman-Keuls test). (C) Coexpression of the serine kinase Gprk2 with α-syn^WT increases high-molecular-weight α-synuclein species. Flies were 10 days old.

(A) Increased high-molecular-weight α-synuclein species are evident in flies expressing α-syn^YF. A lighter exposure than in Figure A is shown to demonstrate the increase in oligomeric species in α-syn^YF. Control genotype: elav-GAL4. (B) Quantitative analysis of oligomer formation. Values represent mean ± SEM of 3 independent experiments. Asterisk indicates significant difference compared with α-syn^WT (*P < 0.01, multivariant ANOVA with supplementary Newman-Keuls test). (C) Coexpression of the tyrosine kinase shark decreases high-molecular-weight α-synuclein species formation of both α-syn^WT and α-syn^S129D. (D) Quantitative analysis of shark kinase inhibition of oligomer formation. Values represent mean ± SEM of 3 independent experiments. Asterisks indicate significant decrease in flies coexpressing shark and α-synuclein (*P < 0.05, Student’s t test). Flies were 10 days old.

(A) Age-related tyrosine phosphorylation levels in humans. Shown are immunoblots from 5 young (≤28 years old) and 5 aged (≥69 years old) frontal cortical samples as detected by the antibody specific for α-synuclein phosphorylated at Tyr125. (B) The densitometry intensities of anti-PY125–positive bands pooled from both young and aged groups. Values represent mean ± SEM of 3 independent experiments (*P ≤ 0.01, Student’s t test). (C) The PY125 blot demonstrates age-related decline in tyrosine-phosphorylated wild-type α-synuclein in Drosophila brain extracts. The driver was elav-GAL4. Days after eclosion are indicated. (D) Tyr125 phosphorylation is further reduced in dementia with Lewy body (DLB) patients. Shown are immunoblots of frontal cortical samples from 5 DLB cases (≥65 years old) and 5 aged controls (≥69 years old).