Fig 1.
The LIPA system allows for long-term induction and real-time monitoring of α-syn aggregation.
(A) Schematic representation of the LIPA constructs. Under blue light stimulation, the LIPA system induces robust and reversible protein clustering. (B) Representative confocal images of HEK-293T cells overexpressing the LIPA constructs exposed to continuous blue light stimulation (0.8 mW/mm2) for different time points up to 24 hours (scale bar = 10 μm). (C) Estimation of the percentage of mCherry-positive HEK-293T cells exhibiting LIPA aggregates at different time points during continuous blue light stimulation (220–250 cells/condition/time point; n = 3). The data are presented as the means ± SEM. (D and E) Filter retardation assay showing the time course of LIPA-Empty and LIPA-α-syn aggregate formation as examined using mCherry and α-syn antibodies, respectively. The histograms present the aggregation signal intensity expressed in arbitrary units (n = 3). The data are presented as the means ± SEM. (F) Prior to the addition of SDS, the samples were subjected to filtration through a nitrocellulose membrane, allowing the retention of monomeric and aggregate proteins and the verification of the total protein levels under each experimental condition. Histograms representing the levels of mCherry expression in arbitrary units (n = 3). The data are presented as the means ± SEM. (G) Time-lapse live imaging illustration of representative HEK-293T cells overexpressing the LIPA constructs (scale bar = 10 μm). (H) Estimation of the total number of mCherry-positive aggregates and (I) the mean aggregate volume (μm3) after repetitive 5-second 488-nm light pulses (15.5 μW/mm2). (J) Time-lapse live imaging illustration of representative single-cell LIPA induction using 5-second 488-nm light pulses (15.5 μW/mm2) in an ROI (represented by the yellow circle) in HEK-293T cells overexpressing LIPA-α-syn (scale bar = 10 μm). The underlying data for (C), (D), (E), (F), (H), and (I) can be found in S1 Data. α-syn, α-synuclein; LIPA, light-inducible protein aggregation; ROI, region of interest.
Fig 2.
LIPA-α-syn inclusions recapitulate authentic LB features in cell culture.
(A) Confocal images of HEK-293T cells overexpressing LIPA-α-syn exposed to blue light for 12 hours (0.8 mW/mm2) and stained with LB markers: α-syn (BDlab), phosphorylated α-syn at S129 (pS129), ThS, ubiquitin, HSP70, and p62 (n = 5) (scale bar = 10 μm). (B) Confocal images of hiPSC-derived human neurons overexpressing LIPA-α-syn exposed to blue light for 6 hours (0.4 mW/mm2) and stained with LB markers: α-syn (BDlab), phosphorylated α-syn at S129 (pS129), ThS, ubiquitin, HSP70, p62, and MAP2 (n = 3) (scale bar = 5 μm). (C) Electron micrograph of a cell overexpressing LIPA-α-syn and not exposed to the blue light (scale bar = 2 μm). (D) Electron micrograph of a cell overexpressing LIPA-α-syn and exposed to the blue light for 12 hours. The image illustrates the presence of circular structures (dashed line) corresponding to a LIPA-α-syn inclusion (scale bar = 2 μm). (E) Electron micrograph of a cell overexpressing LIPA-Empty and exposed to the blue light. The arrowhead points to the needle-like LIPA-Empty inclusion (scale bar = 2 μm). (F) At low magnification, LIPA-α-syn inclusion appear to be surrounded by mitochondria and endoplasmic reticulum (yellow arrowhead) and are mainly composed of abundant vesicular structures and dysmorphic organelles (scale bar = 1 μm). (G) At a high magnification, the images reveal the presence of dense core vesicles (yellow asterisk) and dysmorphic mitochondria (blue asterisk) (scale bar = 500 nm). (H) EM images of filamentous structure of α-syn from purified LIPA-α-syn aggregates from HEK-293T cells exposed to blue light for 0, 6, 12 and 24 hours (scale bar 0 hour = 500 nm; scale bar 6, 12, and 24 hours = 200 nm). α-syn, α-synuclein; hiPSC, human-induced pluripotent stem cell; LB, Lewy bodies; LIPA, light-inducible protein aggregation; Nu, nucleus; ThS, thioflavin S.
Fig 3.
The LIPA system initiates stable and self-perpetuating α-syn aggregates through the seeding effect.
(A) Time course of LIPA-Empty and LIPA-α-syn aggregate dissociation after 12 hours of preexposure to blue light. The data are presented as the percentage of mCherry-positive cells exhibiting inclusions over the total number of mCherry-positive cells normalized to the number of mCherry-positive cells exhibiting inclusions at time zero, which is set as 100% (n = 5). (B) Impact of treatment with baicalein and myricetin, small-molecule inhibitors of α-syn aggregation, on LIPA-α-syn aggregate stability (n = 4). (C) Effect of β-syn and mα-syn overexpression on LIPA-α-syn aggregate stability (n = 3). The data are presented as percentage of mCherry-positive cells with inclusions over the total number of mCherry-expressing cells, which was then normalized as 100% at time zero. The data are presented as the means ± SEM. *** p ≤ 0.001. (D) Confocal microscopy images of HEK-293T cells illustrating the seeding capacity of LIPA-α-syn inclusions in recruiting α-syn-GFP after 12 hours of blue light stimulation (n = 3) (scale bar = 10 μm). (E) Co-IP of LIPA-α-syn aggregates and α-syn-GFP showing that LIPA-α-syn aggregates interact with and recruit their soluble monomeric counterparts (n = 3). (F) Confocal representative images and (G) orthogonal view showing the seeding capacity of purified LIPA-α-syn added to the culture medium and internalized by recipient N2a cells (n = 3) (scale bar = 5 μm). (H) RT-QuIC analysis illustrating the kinetics of recombinant α-syn aggregation in the presence of purified LIPA-α-syn aggregates (+light), monomeric LIPA-α-syn (−light), and recombinant α-syn Pffs. The average ThT fluorescence intensity was plotted against time (n = 3). The data are presented as the means ± SEM. (I) ThT binding kinetics of recombinant α-syn in the presence of purified LIPA-α-syn aggregates (+light), monomeric LIPA-α-syn (−light), and recombinant α-syn Pffs after 32 cycles of PMCA (n = 3). (J) Filter retardation assay and quantification of α-syn protein levels showing the accumulation of α-syn aggregates in the presence of LIPA-α-syn aggregates and recombinant α-syn Pffs after 32 cycles of PMCA (n = 3). The data are presented as the means ± SEM. * p ≤ 0.05, ** p ≤ 0.01, and **** p ≤ 0.0001. The underlying data for (A), (B), (C), (H), (I), and (J) can be found in S1 Data. α-syn, α-synuclein; β-syn, β-synuclein; IP, immunoprecipitation; LIPA, light-inducible protein aggregation; mα-syn, mouse α-syn; PMCA, protein misfolding cyclic amplification; RT-QuIC, real-time quaking-induced conversion; ThT, thioflavin T.
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.
Fig 5.
LIPA-α-syn aggregation disrupts nigrostriatal neuronal transmission.
(A) Schematic representation of the in vivo experimental paradigm of light-induced α-syn aggregation in the SNc and Ca2+ imaging in the striatum. (B) Confocal microscopy image reconstitution of a sagittal brain slice illustrating the expression of LIPA-α-syn in the SNc and the Ca2+ indicator GCaMP6s in the striatum (n = 5 mice)(scale bar = 500μm). (C) Representative time-lapse live imaging illustration of mini-endoscopic Ca2+ in the striatum. Arrowheads indicate striatal neurons showing Ca2+ activity (scale bar = 20μm). (D) Confocal images illustrating the presence of LIPA-α-syn expression in the majority of dopaminergic TH-positive neurons (n = 5 mice) (scale bar = 20 μm). (E) High magnification of confocal images showing that pathological pS129-positive LIPA-α-syn aggregates persisted in dopaminergic neurons 10 days post-optogenetic stimulation (n = 4–6 mice) (scale bar = 10 μm). (F) Temporal trajectories of synchronicity, frequency, and amplitude of Ca2+ transients before, during, and after light-induced α-syn aggregation (n = 4–5 mice). (G) Temporal trajectories of synchronicity, frequency, and amplitude of Ca2+ transients in the absence of light-induced LIPA-α-syn aggregation (n = 3 mice). (H) Temporal trajectories of synchronicity, frequency, and amplitude of Ca2+ transients before, during, and after light-induction in LIPA-α-synΔNAC (n = 4–5 mice). The data are presented as the means ± SEM. * p ≤ 0.05 and ** p ≤ 0.01. The underlying data for (F) to (H) can be found in S1 Data. α-syn, α-synuclein; LIPA, light-inducible protein aggregation; pS129, phosphorylated α-syn at S129; SNc, substantia nigra pars compacta.
Table 1.
List of antibodies used in the present study.