The acid sphingomyelinase inhibitor imipramine enhances the release of UV photoproduct-containing DNA in small extracellular vesicles in UVB-irradiated human skin.

Nucleic acids, lipids, and other cell components can be found within different types of extracellular vesicles (EVs), which include apoptotic bodies (ABs), large extracellular vesicles (LEVs), and small extracellular vesicles (SEVs). Release of LEVs from cells can be reduced by genetic or pharmacological inhibition of the enzyme acid sphinogomyelinase (aSMase), and indeed several studies have demonstrated a role for the clinically approved aSMase inhibitor imipramine in blocking LEV release, including in response to UVB exposure. Given that exposure of keratinocytes to UVB radiation results in the generation of UVR photoproducts in DNA that can subsequently be found in association with ABs and SEVs, we examined how imipramine impacts the release of extracellular DNA containing UVR photoproducts at an early time point after UVR exposure. Using several different model systems, including cultured keratinocytes in vitro, discarded human surgical skin ex vivo, and skin biopsies obtained from treated human subjects, these pilot studies suggest that imipramine treatment stimulates the release of CPD-containing, SEV-associated DNA. These surprising findings indicate that LEV and SEV generation pathways could be linked in UVB-irradiated cells and that imipramine may exacerbate the systemic effects of extracellular UVR-damaged DNA throughout the body.

Protocol for immunodot blot detection of UVB photoproducts in extracellular DNA.

UV radiation induces the formation of adducts in genomic DNA within cells that are later found to be present in cell-free fractions associated with extracellular vesicles (EVs) outside of cells. Here, we present a protocol for isolating UV photoproducts in extracellular DNA released from UVB-irradiated cells via differential centrifugation. We then detail steps for monitoring the DNA adducts using DNA immunoblotting. This protocol can be applied for detection of DNA adducts in EVs from cell culture and skin explant models. For complete details on the use and execution of this protocol, please refer to Carpenter et al.1.

Evidence for the involvement of keratinocyte-derived microvesicle particles in the photosensitivity associated with xeroderma pigmentosum type A deficiency.

Photosensitivity can be due to numerous causes. The photosensitivity associated with deficiency of xeroderma pigmentosum type A (XPA) has been previously shown to be associated with excess levels of the lipid mediator platelet-activating factor (PAF) generated by the keratinocyte. As PAF has been reported to trigger the production of subcellular microvesicle particles (MVP) due to the enzyme acid sphingomyelinase (aSMase), the goal of these studies was to discern if PAF and aSMase could serve as therapeutic targets for the XPA deficiency photosensitivity. HaCaT keratinocytes lacking XPA generated greater levels of MVP in comparison to control cells. Mice deficient in XPA also generated enhanced MVP levels in skin and in plasma in response to UV radiation. Use of a genetic strategy with mice deficient in both XPA and PAF receptors revealed that these mice generated less MVP release as well as decreased skin erythema and cytokine release compared to XPA knockout mice alone. Finally, the aSMase inhibitor imipramine blocked UV-induced MVP release in HaCaT keratinocytes, as well as XPA knockout mice. These studies support the concept that the photosensitivity associated with XPA involves PAF- and aSMase-mediated MVP release and provides a potential pharmacologic target in treating this form of photosensitivity.

DNA Containing Cyclobutane Pyrimidine Dimers Is Released from UVB-Irradiated Keratinocytes in a Caspase-Dependent Manner.

Solar radiation induces the formation of cyclobutane pyrimidine dimers (CPDs) and other UV photoproducts in the genomic DNA of epidermal keratinocytes. Although CPDs have been detected in urine from UV- and sun-exposed individuals, the pathway by which they arrive there and the mechanisms by which UV-induced DNA damage in the skin has systemic effects throughout the body are not clear. Consistent with previous reports that DNA associates with small extracellular vesicles that are released from a variety of cell types, we observed that a small fraction of CPDs formed in genomic DNA after UVB exposure can later be detected in the culture medium. These extracellular CPDs are found within large fragments of histone-associated DNA and are released in a time- and UVB dose‒dependent manner. Moreover, studies with both cultured cells and human skin explants revealed that CPD release into the extracellular environment is blocked by caspase inhibition, which indicates a role for apoptotic signaling in CPD release from UVB-irradiated keratinocytes. Finally, we show that this released CPD-containing DNA can be taken up by other keratinocytes. These results therefore provide possible mechanisms for the export of damaged DNA from UVB-irradiated cells and for systemic effects of UVB exposure throughout the body.

Topical Treatment of Human Skin and Cultured Keratinocytes with High-Dose Spironolactone Reduces XPB Expression and Induces Toxicity.

Spironolactone (SP) is used to treat a variety of disparate disease states ranging from heart failure to acne through antagonism of the mineralocorticoid and androgen receptors. Although normally taken as an oral medication, recent studies have explored the topical application of SP onto the skin. However, because SP induces the proteolytic degradation of the XPB protein, which plays critical roles in DNA repair and transcription, there may be safety concerns with the use of topical SP. In this study, we show that the topical application of a high concentration of either SP or its metabolite canrenone onto human skin ex vivo lowers XPB protein levels and induces toxic responses in the epidermis. Interestingly, although SP and canrenone both inhibit cell proliferation, induce replication stress responses, and stimulate apoptotic signaling at high concentrations in cultured keratinocytes in vitro, these effects were not correlated with XPB protein loss. Thus, high concentrations of SP and canrenone likely inhibit cell proliferation and induce toxicity through additional mechanisms to XPB proteolytic degradation. This work suggests that care may need to be taken when using high concentrations of SP directly on human skin.

Protein Proximity Observed Using Fluorogen Activating Protein and Dye Activated by Proximal Anchoring (FAP-DAPA) System.

The development and function of tissues, blood, and the immune system is dependent upon proximity for cellular recognition and communication. However, the detection of cell-to-cell contacts is limited due to a lack of reversible, quantitative probes that can function at these dynamic sites of irregular geometry. Described here is a novel chemo-genetic tool developed for fluorescent detection of protein-protein proximity and cell apposition that utilizes the Fluorogen Activating Protein (FAP) in combination with a Dye Activated by Proximal Anchoring (DAPA). The FAP-DAPA system has two protein components, the HaloTag and FAP, expressed on separate protein targets or in separate cells. The proteins function to bind and activate a compound that has the hexyl chloride (HexCl) ligand connected to malachite green (MG), the FAP fluorogen, via a poly(ethylene glycol) spacer spanning up to 28 nm. The dehalogenase protein, HaloTag, covalently binds the HexCl ligand, locally concentrating the attached MG. If the FAP is within range of the anchored fluorogen, it will bind and activate MG specifically when the bath concentration is too low to saturate the FAP receptor. A new FAP variant was isolated with a 1000-fold reduced KD of ∼10-100 nM so that the fluorogen activation reports proximity without artificially enhancing it. The system was characterized using purified FRB and FKBP fusion proteins and showed a doubling of fluorescence upon rapamycin induced complex formation. In cocultured HEK293 cells (HaloTag and FAP-expressing) fluorescence increased at contact sites across a broad range of labeling conditions, more reliably providing contact-specific fluorescence activation with the lower-affinity FAP variant. When combined with suitable targeting and expression constructs, this labeling system may offer significant improvements in on-demand detection of intercellular contacts, potentially applicable in neurological and immunological synapse measurements and other transient, dynamic biological appositions that can be perturbed using other labeling methods that stabilize these interactions.

Activity-evoked and spontaneous opening of synaptic fusion pores.

Synaptic release of neuropeptides packaged in dense-core vesicles (DCVs) regulates synapses, circuits, and behaviors including feeding, sleeping, and pain perception. Here, synaptic DCV fusion pore openings are imaged without interference from cotransmitting small synaptic vesicles (SSVs) with the use of a fluorogen-activating protein (FAP). Activity-evoked kiss and run exocytosis opens synaptic DCV fusion pores away from active zones that readily conduct molecules larger than most native neuropeptides (i.e., molecular weight [MW] up to, at least, 4.5 kDa). Remarkably, these synaptic fusion pores also open spontaneously in the absence of stimulation and extracellular Ca2+ SNARE perturbations demonstrate different mechanisms for activity-evoked and spontaneous fusion pore openings with the latter sharing features of spontaneous small molecule transmitter release by active zone-associated SSVs. Fusion pore opening at resting synapses provides a mechanism for activity-independent peptidergic transmission.