A light-inducible protein clustering system for in vivo analysis of α-synuclein aggregation in Parkinson disease
Fig 4
LIPA-α-syn inclusions recapitulate authentic LB features in vivo and precipitate dopaminergic neuronal loss and parkinsonian-like motor impairments.
(A) Experimental design of the experiments using the overexpression and induction of LIPA-α-syn aggregation in the midbrains of WT mice. (B) Confocal microscopy images of representative midbrain neurons with LIPA-α-syn aggregates exhibiting authentic LB markers: α-syn (BDlab), pS129, ThS, ubiquitin, HSP70, and p62 (n = 4 mice) (scale bar = 5 μm). (C) Experimental design of the long-term impact of LIPA-α-syn aggregation on DA neuronal integrity. Treatment with light stimulation (blue lines) was started 2 weeks post-AAV injection, and blue light was applied for 1 hour every other day for 8 weeks. (D) Confocal microscopy images illustrating the LIPA-α-syn or LIPA-Empty inclusions within dopaminergic midbrain neurons. The presence of pathological (pS129-positive) α-syn aggregates was observed only in dopaminergic neurons overexpressing LIPA-α-syn stimulated with blue light (n = 4 mice) (scale bar = 5 μm). (E) Representative confocal microscopy images illustrating dopaminergic neuronal loss in the midbrain of mice overexpressing LIPA constructs and exposed to blue light stimulation (n = 4 mice) (scale bar = 0.5 mm). (F) Stereological quantification of TH-positive dopaminergic neurons and (G) total neuronal markers (Nissl) in the midbrains of mice overexpressing LIPA constructs and exposed (or not exposed) to blue light stimulation. The results are expressed as percentage of the contralateral noninjected side (n = 5–8 mice per experimental condition). The data are presented as the means ± SEM. * p ≤ 0.05, ** p ≤ 0.01, and **** p ≤ 0.0001. Assessment of the behavioral impairment induced by LIPA constructs overexpression with and without blue light stimulation using (H) a grip strength test, (I) cylinder test, (J) rotarod test, and (K) gait test (n = 5–9 mice per experimental condition). The data are presented as the means ± SEM. * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.0001. The underlying data for (F) to (K) can be found in S1 Data. AAV, adeno-associated virus; α-syn, α-synuclein; LB, Lewy bodies; LIPA, light-inducible protein aggregation; pS129, phosphorylated α-syn at S129; ThS, thioflavin S; WT, wild-type.