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

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.

Characterization of 100 sequential SARS-CoV-2 convalescent plasma donations.

BACKGROUND: Transfusion of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) convalescent plasma is a promising treatment for severe coronavirus disease 2019 (COVID-19) cases, with success of the intervention based on neutralizing antibody content. Measurement by serologic correlates without biocontainment needs as well as an understanding of donor characteristics that may allow for targeting of more potent donors would greatly facilitate effective collection. STUDY DESIGN AND METHODS: One hundred convalescent plasma units were characterized for functionally active SARS-CoV-2 neutralizing antibodies, as well as for SARS-CoV-2 binding antibodies, with the intention to establish a correlation between the functionally more relevant neutralization assay and the more accessible enzyme-linked immunosorbent assay (ELISA). Donor demographics such as COVID-19 severity, age, and sex were correlated with antibody titers. RESULTS: A mean neutralization titer 50% of 230 (range, <8-1765) was seen for the 100 convalescent plasma units, with highly significant (P < .0001) yet quantitatively limited (R2 = 0.2830) correlation with results of the ELISA. Exclusion of units with particularly high titers (>500) from analysis improved correlation (R2 = 0.5386). A tendency of higher-titer plasma units from donors with increased disease severity, of advanced age, and of male sex was seen, yet the functional relevance of this difference is questionable. CONCLUSION: The ELISA-based correlation to neutralization titer enabled a threshold proposal that could be used to eliminate lower-titer units from the clinical supply for COVID-19 treatment. Disease severity may be associated with the development of higher titers of neutralizing antibodies, although larger case numbers will be needed for additional confirmation.

Green-Light-Induced Inactivation of Receptor Signaling Using Cobalamin-Binding Domains.

Optogenetics and photopharmacology provide spatiotemporally precise control over protein interactions and protein function in cells and animals. Optogenetic methods that are sensitive to green light and can be used to break protein complexes are not broadly available but would enable multichromatic experiments with previously inaccessible biological targets. Herein, we repurposed cobalamin (vitamin B12) binding domains of bacterial CarH transcription factors for green-light-induced receptor dissociation. In cultured cells, we observed oligomerization-induced cell signaling for the fibroblast growth factor receptor 1 fused to cobalamin-binding domains in the dark that was rapidly eliminated upon illumination. In zebrafish embryos expressing fusion receptors, green light endowed control over aberrant fibroblast growth factor signaling during development. Green-light-induced domain dissociation and light-inactivated receptors will critically expand the optogenetic toolbox for control of biological processes.

A Phytochrome Sensory Domain Permits Receptor Activation by Red Light.

Optogenetics and photopharmacology enable the spatio-temporal control of cell and animal behavior by light. Although red light offers deep-tissue penetration and minimal phototoxicity, very few red-light-sensitive optogenetic methods are currently available. We have now developed a red-light-induced homodimerization domain. We first showed that an optimized sensory domain of the cyanobacterial phytochrome 1 can be expressed robustly and without cytotoxicity in human cells. We then applied this domain to induce the dimerization of two receptor tyrosine kinases-the fibroblast growth factor receptor 1 and the neurotrophin receptor trkB. This new optogenetic method was then used to activate the MAPK/ERK pathway non-invasively in mammalian tissue and in multicolor cell-signaling experiments. The light-controlled dimerizer and red-light-activated receptor tyrosine kinases will prove useful to regulate a variety of cellular processes with light.

Light-assisted small-molecule screening against protein kinases.

High-throughput live-cell screens are intricate elements of systems biology studies and drug discovery pipelines. Here, we demonstrate an optogenetics-assisted method that avoids the need for chemical activators and reporters, reduces the number of operational steps and increases information content in a cell-based small-molecule screen against human protein kinases, including an orphan receptor tyrosine kinase. This blueprint for all-optical screening can be adapted to many drug targets and cellular processes.

Spatio-temporally precise activation of engineered receptor tyrosine kinases by light.

Receptor tyrosine kinases (RTKs) are a large family of cell surface receptors that sense growth factors and hormones and regulate a variety of cell behaviours in health and disease. Contactless activation of RTKs with spatial and temporal precision is currently not feasible. Here, we generated RTKs that are insensitive to endogenous ligands but can be selectively activated by low-intensity blue light. We screened light-oxygen-voltage (LOV)-sensing domains for their ability to activate RTKs by light-activated dimerization. Incorporation of LOV domains found in aureochrome photoreceptors of stramenopiles resulted in robust activation of the fibroblast growth factor receptor 1 (FGFR1), epidermal growth factor receptor (EGFR) and rearranged during transfection (RET). In human cancer and endothelial cells, light induced cellular signalling with spatial and temporal precision. Furthermore, light faithfully mimicked complex mitogenic and morphogenic cell behaviour induced by growth factors. RTKs under optical control (Opto-RTKs) provide a powerful optogenetic approach to actuate cellular signals and manipulate cell behaviour.

The optogenetic promise for oncology: Episode I.

As light-based control of fundamental signaling pathways is becoming a reality, the field of optogenetics is rapidly moving beyond neuroscience. We have recently developed receptor tyrosine kinases that are activated by light and control cell proliferation, epithelial-mesenchymal transition, and angiogenic sprouting-cell behaviors central to cancer progression.

Expression of the IGF axis is decreased in local prostate cancer but enhanced after benign prostate epithelial differentiation and TGF-ß treatment.

The insulin-like growth factor (IGF) axis is a molecular pathway intensively investigated in cancer research. Clinical trials targeting the IGF1 receptor (IGF1R) in different tumors, including prostate cancer, are under way. Although studies on the IGF axis in prostate cancer have already entered into clinical trials, the expression and functional role of the IGF axis in benign prostate and in prostate cancer needs to be better defined. We determined mRNA expression levels of the IGF axis in microdissected tissue specimens of local prostate cancer using quantitative PCR. All members of the IGF axis, including IGF1, IGF2, IGF binding proteins 1 through 6, and insulin receptor, were measured in both the stromal and epithelial compartments of the prostate. IGF1, IGF2, IGF1R, and insulin receptor were down-regulated in local prostate cancer tissue compared with matched benign tissue, suggesting that the IGF axis is not induced during prostate cancer development. Using a new prostate epithelial differentiation model, we demonstrate that the expression of the IGF axis is enhanced during normal prostate epithelial differentiation and regulated by tumor growth factor (TGF)-ß. Our data reveal a functional role of the IGF axis in prostate differentiation, underscoring the importance of the IGF axis in normal development and emphasizing the importance of accurate target validation before moving to advanced clinical trials.