Elucidating and Optimizing the Photochemical Mechanism of Coumarin-Caged Tertiary Amines.

Photoactivatable or "caged" pharmacological agents combine the high spatiotemporal specificity of light application with the molecular specificity of drugs. A key factor in all optopharmacology experiments is the mechanism of uncaging, which dictates the photochemical quantum yield and determines the byproducts produced by the light-driven chemical reaction. In previous work, we demonstrated that coumarin-based photolabile groups could be used to cage tertiary amine drugs as quaternary ammonium salts. Although stable, water-soluble, and useful for experiments in brain tissue, these first-generation compounds exhibit relatively low uncaging quantum yield (Φu < 1%) and release the toxic byproduct formaldehyde upon photolysis. Here, we elucidate the photochemical mechanisms of coumarin-caged tertiary amines and then optimize the major pathway using chemical modification. We discovered that the combination of 3,3-dicarboxyazetidine and bromine substituents shift the mechanism of release to heterolysis, eliminating the formaldehyde byproduct and giving photolabile tertiary amine drugs with Φu > 20%─a 35-fold increase in uncaging efficiency. This new "ABC" cage allows synthesis of improved photoactivatable derivatives of escitalopram and nicotine along with a novel caged agonist of the oxytocin receptor.

Loss of MPC1 reprograms retinal metabolism to impair visual function.

Glucose metabolism in vertebrate retinas is dominated by aerobic glycolysis (the "Warburg Effect"), which allows only a small fraction of glucose-derived pyruvate to enter mitochondria. Here, we report evidence that the small fraction of pyruvate in photoreceptors that does get oxidized by their mitochondria is required for visual function, photoreceptor structure and viability, normal neuron-glial interaction, and homeostasis of retinal metabolism. The mitochondrial pyruvate carrier (MPC) links glycolysis and mitochondrial metabolism. Retina-specific deletion of MPC1 results in progressive retinal degeneration and decline of visual function in both rod and cone photoreceptors. Using targeted-metabolomics and 13C tracers, we found that MPC1 is required for cytosolic reducing power maintenance, glutamine/glutamate metabolism, and flexibility in fuel utilization.

ARL13B, a Joubert Syndrome-Associated Protein, Is Critical for Retinogenesis and Elaboration of Mouse Photoreceptor Outer Segments.

Mutations in the Joubert syndrome-associated small GTPase ARL13B are linked to photoreceptor impairment and vision loss. To determine the role of ARL13B in the development, function, and maintenance of ciliated photoreceptors, we generated a pan-retina knock-out (Six3-Cre) and a rod photoreceptor-specific inducible conditional knock-out (Pde6g-CreERT2) of ARL13B using murine models. Embryonic deletion of ARL13B led to defects in retinal development with reduced cell proliferation. In the absence of ARL13B, photoreceptors failed to develop outer segment (OS) membranous discs and axonemes, resulting in loss of function and rapid degeneration. Additionally, the majority of photoreceptor basal bodies did not dock properly at the apical edge of the inner segments. The removal of ARL13B in adult rod photoreceptor cells after maturation of OS resulted in loss of photoresponse and vesiculation in the OS. Before changes in photoresponse, removal of ARL13B led to mislocalization of rhodopsin, prenylated phosphodiesterase-6 (PDE6), and intraflagellar transport protein-88 (IFT88). Our findings show that ARL13B is required at multiple stages of retinogenesis, including early postnatal proliferation of retinal progenitor cells, development of photoreceptor cilia, and morphogenesis of photoreceptor OS discs regardless of sex. Last, our results establish a need for ARL13B in photoreceptor maintenance and protein trafficking.SIGNIFICANCE STATEMENT The normal development of photoreceptor cilia is essential to create functional, organized outer segments with stacked membrane discs that house the phototransduction proteins necessary for sight. Our study identifies a complex role for ARL13B, a small GTPase linked to Joubert syndrome and visual impairment, at various stages of photoreceptor development. Loss of ARL13B led to defects in retinal proliferation, altered placement of basal bodies crucial for components of the cilium (transition zone) to emanate, and absence of photoreceptor-stacked discs. These defects led to extinguished visual response and dysregulated protein trafficking. Our findings show the complex role ARL13B plays in photoreceptor development, viability, and function. Our study accounts for the severe retinal impairment observed in ARL13B-linked Joubert syndrome patients.

Bardet-Biedl syndrome-8 (BBS8) protein is crucial for the development of outer segments in photoreceptor neurons.

Bardet-Biedl syndrome (BBS) is an autosomal recessive ciliopathy characterized by developmental abnormalities and vision loss. To date, mutations in 21 genes have been linked to BBS. The products of eight of these BBS genes form a stable octameric complex termed the BBSome. Mutations in BBS8, a component of the BBSome, cause early vision loss, but the role of BBS8 in supporting vision is not known. To understand the mechanisms by which BBS8 supports rod and cone photoreceptor function, we generated animal models lacking BBS8. The loss of BBS8 protein led to concomitant decrease in the levels of BBSome subunits, BBS2 and BBS5 and increase in the levels of the BBS1 and BBS4 subunits. BBS8 ablation was associated with severe reduction of rod and cone photoreceptor function and progressive degeneration of each photoreceptor subtype. We observed disorganized and shortened photoreceptor outer segments (OS) at post-natal day 10 as the OS elaborates. Interestingly, loss of BBS8 led to changes in the distribution of photoreceptor axonemal proteins and hyper-acetylation of ciliary microtubules. In contrast to properly localized phototransduction machinery, we observed OS accumulation of syntaxin3, a protein normally found in the cytoplasm and the synaptic termini. In conclusion, our studies demonstrate the requirement for BBS8 in early development and elaboration of ciliated photoreceptor OS, explaining the need for BBS8 in normal vision. The findings from our study also imply that early targeting of both rods and cones in BBS8 patients is crucial for successful restoration of vision.

Imaging the extracellular matrix in live tissues and organisms with a glycan-binding fluorophore.

All multicellular systems produce and dynamically regulate extracellular matrices (ECM) that play important roles in both biochemical and mechanical signaling. Though the spatial arrangement of these extracellular assemblies is critical to their biological functions, visualization of ECM structure is challenging, in part because the biomolecules that compose the ECM are difficult to fluorescently label individually and collectively. Here, we present a cell-impermeable small molecule fluorophore, termed Rhobo6, that turns on and red shifts upon reversible binding to glycans. Given that most ECM components are densely glycosylated, the dye enables wash-free visualization of ECM, in systems ranging from in vitro substrates to in vivo mouse mammary tumors. Relative to existing techniques, Rhobo6 provides a broad substrate profile, superior tissue penetration, nonperturbative labeling, and negligible photobleaching. This work establishes a straightforward method for imaging the distribution of ECM in live tissues and organisms, lowering barriers for investigation of extracellular biology.

The SARS-CoV-2 accessory protein Orf3a is not an ion channel, but does interact with trafficking proteins.

The severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) and SARS-CoV-1 accessory protein Orf3a colocalizes with markers of the plasma membrane, endocytic pathway, and Golgi apparatus. Some reports have led to annotation of both Orf3a proteins as viroporins. Here, we show that neither SARS-CoV-2 nor SARS-CoV-1 Orf3a form functional ion conducting pores and that the conductances measured are common contaminants in overexpression and with high levels of protein in reconstitution studies. Cryo-EM structures of both SARS-CoV-2 and SARS-CoV-1 Orf3a display a narrow constriction and the presence of a positively charged aqueous vestibule, which would not favor cation permeation. We observe enrichment of the late endosomal marker Rab7 upon SARS-CoV-2 Orf3a overexpression, and co-immunoprecipitation with VPS39. Interestingly, SARS-CoV-1 Orf3a does not cause the same cellular phenotype as SARS-CoV-2 Orf3a and does not interact with VPS39. To explain this difference, we find that a divergent, unstructured loop of SARS-CoV-2 Orf3a facilitates its binding with VPS39, a HOPS complex tethering protein involved in late endosome and autophagosome fusion with lysosomes. We suggest that the added loop enhances SARS-CoV-2 Orf3a's ability to co-opt host cellular trafficking mechanisms for viral exit or host immune evasion.

The SARS-CoV-2 accessory protein Orf3a is not an ion channel, but does interact with trafficking proteins.

The severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) and SARS-CoV-1 accessory protein Orf3a colocalizes with markers of the plasma membrane, endocytic pathway, and Golgi apparatus. Some reports have led to annotation of both Orf3a proteins as a viroporin. Here we show that neither SARS-CoV-2 nor SARS-CoV-1 form functional ion conducting pores and that the conductances measured are common contaminants in overexpression and with high levels of protein in reconstitution studies. Cryo-EM structures of both SARS-CoV-2 and SARS-CoV-1 Orf3a display a narrow constriction and the presence of a basic aqueous vestibule, which would not favor cation permeation. We observe enrichment of the late endosomal marker Rab7 upon SARS-CoV-2 Orf3a overexpression, and co-immunoprecipitation with VPS39. Interestingly, SARS-CoV-1 Orf3a does not cause the same cellular phenotype as SARS-CoV-2 Orf3a and does not interact with VPS39. To explain this difference, we find that a divergent, unstructured loop of SARS-CoV-2 Orf3a facilitates its binding with VPS39, a HOPS complex tethering protein involved in late endosome and autophagosome fusion with lysosomes. We suggest that the added loop enhances SARS-CoV-2 Orf3a ability to co-opt host cellular trafficking mechanisms for viral exit or host immune evasion.

ARL2BP, a protein linked to retinitis pigmentosa, is needed for normal photoreceptor cilia doublets and outer segment structure.

The outer segment (OS) of photoreceptor cells is an elaboration of a primary cilium with organized stacks of membranous disks that contain the proteins needed for phototransduction and vision. Though ciliary formation and function has been well characterized, little is known about the role of cilia in the development of photoreceptor OS. Nevertheless, progress has been made by studying mutations in ciliary proteins, which often result in malformed OSs and lead to blinding diseases. To investigate how ciliary proteins contribute to OS formation, we generated a knockout (KO) mouse model for ARL2BP, a ciliary protein linked to retinitis pigmentosa. The KO mice display an early and progressive reduction in visual response. Before photoreceptor degeneration, we observed disorganization of the photoreceptor OS, with vertically aligned disks and shortened axonemes. Interestingly, ciliary doublet microtubule (MT) structure was also impaired, displaying open B-tubule doublets, paired with loss of singlet MTs. On the basis of results from this study, we conclude that ARL2BP is necessary for photoreceptor ciliary doublet formation and axoneme elongation, which is required for OS morphogenesis and vision.