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. 2021 Apr 15;17(4):e1009479.
doi: 10.1371/journal.pgen.1009479. eCollection 2021 Apr.

Optogenetic delivery of trophic signals in a genetic model of Parkinson's disease

Alvaro Ingles-Prieto  1 , Nikolas Furthmann  2 , Samuel H Crossman  3   4 , Alexandra-Madelaine Tichy  3   4 , Nina Hoyer  5 , Meike Petersen  5 , Vanessa Zheden  1 , Julia Biebl  1 , Eva Reichhart  1   3   4 , Attila Gyoergy  1 , Daria E Siekhaus  1 , Peter Soba  5 , Konstanze F Winklhofer  2 , Harald Janovjak  1   3   4
Affiliations

Optogenetic delivery of trophic signals in a genetic model of Parkinson's disease

Alvaro Ingles-Prieto et al. PLoS Genet. .

Abstract

Optogenetics has been harnessed to shed new mechanistic light on current and future therapeutic strategies. This has been to date achieved by the regulation of ion flow and electrical signals in neuronal cells and neural circuits that are known to be affected by disease. In contrast, the optogenetic delivery of trophic biochemical signals, which support cell survival and are implicated in degenerative disorders, has never been demonstrated in an animal model of disease. Here, we reengineered the human and Drosophila melanogaster REarranged during Transfection (hRET and dRET) receptors to be activated by light, creating one-component optogenetic tools termed Opto-hRET and Opto-dRET. Upon blue light stimulation, these receptors robustly induced the MAPK/ERK proliferative signaling pathway in cultured cells. In PINK1B9 flies that exhibit loss of PTEN-induced putative kinase 1 (PINK1), a kinase associated with familial Parkinson's disease (PD), light activation of Opto-dRET suppressed mitochondrial defects, tissue degeneration and behavioral deficits. In human cells with PINK1 loss-of-function, mitochondrial fragmentation was rescued using Opto-dRET via the PI3K/NF-кB pathway. Our results demonstrate that a light-activated receptor can ameliorate disease hallmarks in a genetic model of PD. The optogenetic delivery of trophic signals is cell type-specific and reversible and thus has the potential to inspire novel strategies towards a spatio-temporal regulation of tissue repair.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig 1
Fig 1. Engineering of light-activated RET receptors.
(A) hRET and dRET consist of an extracellular ligand-binding domain (LBD), single-span transmembrane domain (TMD) and intracellular domain (KD: kinase domain, CTD: C-terminal tail domain). Activation by a GFL and GFRα was shown to result in the formation of a human ternary complex (binding model of Schlee et al. [40]). (B) In light-activated Opto-h/dRET, the LOV domain of the AUREOCHROME1 photoreceptor of V. frigida is incorporated at the receptor C-terminus. (C) MAPK/ERK pathway activation in response to blue light (I = 250 μW/cm2, λ = 470 nm, 8h continuous) for HEK293 cells transfected with Opto-hRET, Opto-dRET or Opto-dRETMEN2B. The MAPK/ERK reporter utilizes a pathway-specific Elk1 trans-activator to induce transcription of luciferase (LUC; see Materials and Methods for details). Light units (LU; mean ± SD) for dark treated cells and illuminated cells are given (n = 6 to 12, three independent experiments; t-test, *: p < .0001).
Fig 2
Fig 2. Induction of retina roughening and phenotype quantification.
(A) Developmental time window targeted by light in retina experiments. (B-G) Representative retina SEM images. Scale bar: 0.1 mm. (H and I) Quantification of rough retina phenotypes of one-day old flies as fused area and the number of structures identified. “M2B” denotes Opto-dRETMEN2B. The number of analyzed flies is given (at least three independent experiments) and bars sharing the same label are not significantly different (ANOVA/Bonferroni corrected t-tests of means, p>.04). Continuous light intensity was 385 μW/cm2 for the duration shown in A.
Fig 3
Fig 3. Suppression of thorax defects and locomotion deficits.
(A) Time window targeted by light in experiments with PINK1B9 flies. Illumination of pupal and adult stages prevented lethality observed upon Opto-dRET signaling in earlier stages (e.g., Opto-dRETMEN2B flies were grown at 18°C to prevent lethality during development; see Main Text). (B) Percentage of flies with a degenerate thorax phenotype. Representative bright field thorax images shown on the right. Hollow thorax is highlighted by the red arrow. (C) Climbing ability of flies. “M2B” denotes Opto-dRETMEN2B. PINK1 “+” denotes the WT gene. For B and C, counts ± SE for the indicated number of flies (n) is given. Percentages sharing the same label are not significantly different (Fisher’s exact test, p>.04). Continuous light intensity was 320 μW/cm2 for the duration shown in A.
Fig 4
Fig 4. Improved mitochondrial structure and function.
(A) ATP content in fly thoraces from PINK1B9 flies at the indicated conditions (normalized to the mean for control flies shown as bar 1). (B-E) Representative TEM images of thoracic indirect flight muscles. Arrow heads indicate mitochondria that are either electron dense (B: controls, E: illuminated PINK1B9 Opto-dRET flies) or malformed with disintegrated cristae (C: PINK1B9 flies, D: PINK1B9 Opto-dRET flies in the absence of light). Scale bar: 2 μm. (F) Analysis of mitochondrial density in TEM images. “M2B” denotes Opto-dRETMEN2B. PINK1 “+” denotes the WT gene. In A, the number of analyzed flies is given (at least three independent experiments) and bars sharing the same label are not significantly different (ANOVA/Bonferroni corrected t-tests of means, p>.04). In F, the number of analyzed micrographs is given (at least three independent experiments) and bars sharing the same label are not significantly different (ANOVA/Bonferroni corrected t-tests of means, p>.04). Continuous light intensity was 320 μW/cm2 for the duration shown in Fig 3A.
Fig 5
Fig 5. Rescue of mitochondrial fragmentation in human cells.
(A and B) WB analysis of PINK1 knock-down by siRNA and Opto-dRET expression. (C) Representative images for fragmentation of mitochondria induced by PINK1 silencing. Magenta: MitoTracker. Green: GFP marker. Scale bar: 200 (columns 1, 2) or 20 μm (columns 3, 4). (D) Quantification of mitochondrial fragmentation upon light stimulation of RET, Opto-dRET, Opto-dRETMEN2B or Opto-dRETKD (I = 232 μW/cm2, λ = 470 nm, 4 h continuous). (E) Quantification analysis of mitochondrial fragmentation upon light activation of Opto-dRET and inhibition of NF-кB signaling (by IκB-2S/A, label “N”), PI3K (by LY294002, label “P”) or MEK1 (by PD98059, label “M”). For D and E, mean ± SD for five independent experiments is given (at least 150 cells per condition in each experiment). Means sharing the same label are not significantly different (ANOVA/Bonferroni corrected t-tests, p>.04).
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