Quantifying the stability of oxidatively damaged DNA by single-molecule DNA stretching.

One of the most common DNA lesions is created when reactive oxygen alters guanine. 8-oxo-guanine may bind in the anti-conformation with an opposing cytosine or in the syn-conformation with an opposing adenine paired by transversion, and both conformations may alter DNA stability. Here we use optical tweezers to measure the stability of DNA hairpins containing 8-oxoguanine (8oxoG) lesions, comparing the results to predictive models of base-pair energies in the absence of the lesion. Contrasted with either a canonical guanine-cytosine or adenine-thymine pair, an 8oxoG-cytosine base pair shows significant destabilization of several kBT. The magnitude of destabilization is comparable to guanine-thymine 'wobble' and cytosine-thymine mismatches. Furthermore, the measured energy of 8oxoG-adenine corresponds to theoretical predictions for guanine-adenine pairs, indicating that oxidative damage does not further destabilize this mismatch in our experiments, in contrast to some previous observations. These results support the hypothesis that oxidative damage to guanine subtly alters the direction of the guanine dipole, base stacking interactions, the local backbone conformation, and the hydration of the modified base. This localized destabilization under stress provides additional support for proposed mechanisms of enzyme repair.

INX-315, a Selective CDK2 Inhibitor, Induces Cell Cycle Arrest and Senescence in Solid Tumors.

Cyclin-dependent kinase 2 (CDK2) is thought to play an important role in driving proliferation of certain cancers, including those harboring CCNE1 amplification and breast cancers that have acquired resistance to CDK4/6 inhibitors (CDK4/6i). The precise impact of pharmacologic inhibition of CDK2 is not known due to the lack of selective CDK2 inhibitors. Here we describe INX-315, a novel and potent CDK2 inhibitor with high selectivity over other CDK family members. Using cell-based assays, patient-derived xenografts (PDX), and transgenic mouse models, we show that INX-315 (i) promotes retinoblastoma protein hypophosphorylation and therapy-induced senescence (TIS) in CCNE1-amplified tumors, leading to durable control of tumor growth; (ii) overcomes breast cancer resistance to CDK4/6i, restoring cell cycle control while reinstating the chromatin architecture of CDK4/6i-induced TIS; and (iii) delays the onset of CDK4/6i resistance in breast cancer by driving deeper suppression of E2F targets. Our results support the clinical development of selective CDK2 inhibitors. SIGNIFICANCE: INX-315 is a novel, selective inhibitor of CDK2. Our preclinical studies demonstrate activity for INX-315 in both CCNE1-amplified cancers and CDK4/6i-resistant breast cancer. In each case, CDK2 inhibition induces cell cycle arrest and a phenotype resembling cellular senescence. Our data support the development of selective CDK2 inhibitors in clinical trials. See related commentary by Watts and Spencer, p. 386. This article is featured in Selected Articles from This Issue, p. 384.

Multiomic integration reveals neuronal-extracellular vesicle coordination of gliotic responses in degeneration.

In the central nervous system (CNS), including in the retina, neuronal-to-glial communication is critical for maintaining tissue homeostasis including signal transmission, transfer of trophic factors, and in the modulation of inflammation. Extracellular vesicle (EV)-mediated transport of molecular messages to regulate these processes has been suggested as a mechanism by which bidirectional communication between neuronal and glial cells can occur. In this work we employed multiomics integration to investigate the role of EV communication pathways from neurons to glial cells within the CNS, using the mouse retina as a readily accessible representative CNS tissue. Further, using a well-established model of degeneration, we aimed to uncover how dysregulation of homeostatic messaging between neurons and glia via EV can result in retinal and neurodegenerative diseases. EV proteomics, glia microRNA (miRNA) Open Array and small RNA sequencing, and retinal single cell sequencing were performed, with datasets integrated and analysed computationally. Results demonstrated that exogenous transfer of neuronal miRNA to glial cells was mediated by EV and occurred as a targeted response during degeneration to modulate gliotic inflammation. Taken together, our results support a model of neuronal-to-glial communication via EV, which could be harnessed for therapeutic targeting to slow the progression of retinal-, and neuro-degenerations of the CNS.

Caspase-1-dependent inflammasomes mediate photoreceptor cell death in photo-oxidative damage-induced retinal degeneration.

Activation of the inflammasome is involved in the progression of retinal degenerative diseases, in particular, in the pathogenesis of Age-Related Macular Degeneration (AMD), with NLRP3 activation the focus of many investigations. In this study, we used genetic and pharmacological approaches to explore the role of the inflammasome in a mouse model of retinal degeneration. We identify that Casp1/11-/- mice have better-preserved retinal function, reduced inflammation and increased photoreceptor survivability. While Nlrp3-/- mice display some level of preservation of retinal function compared to controls, pharmacological inhibition of NLRP3 did not protect against photoreceptor cell death. Further, Aim2-/-, Nlrc4-/-, Asc-/-, and Casp11-/- mice show no substantial retinal protection. We propose that CASP-1-associated photoreceptor cell death occurs largely independently of NLRP3 and other established inflammasome sensor proteins, or that inhibition of a single sensor is not sufficient to repress the inflammatory cascade. Therapeutic targeting of CASP-1 may offer a more promising avenue to delay the progression of retinal degenerations.

MicroRNA-223 Regulates Retinal Function and Inflammation in the Healthy and Degenerating Retina.

INTRODUCTION: MicroRNAs (miRNAs) are small, non-coding RNA molecules that have powerful regulatory properties, with the ability to regulate multiple messenger RNAs (mRNAs) and biological pathways. MicroRNA-223-3p (miR-223) is known to be a critical regulator of the innate immune response, and its dysregulation is thought to play a role in inflammatory disease progression. Despite miR-223 upregulation in numerous neurodegenerative conditions, largely in cells of the myeloid lineage, the role of miR-223 in the retina is relatively unexplored. Here, we investigated miR-223 in the healthy retina and in response to retinal degeneration. METHODS: miR-223-null mice were investigated in control and photo-oxidative damage-induced degeneration conditions. Encapsulated miR-223 mimics were intravitreally and intravenously injected into C57BL/6J wild-type mice. Retinal functional responses were measured using electroretinography (ERG), while extracted retinas were investigated by retinal histology (TUNEL and immunohistochemistry) and molecular analysis (qPCR and FACS). RESULTS: Retinal function in miR-223-/- mice was adversely affected, indicating that miR-223 may be critical in regulating the retinal response. In degeneration, miR-223 was elevated in the retina, circulating serum, and retinal extracellular vesicles. Conversely, retinal microglia and macrophages displayed a downregulation of miR-223. Further, isolated CD11b+ inflammatory cells from the retinas and circulation of miR-223-null mice showed an upregulation of pro-inflammatory genes that are critically linked to retinal inflammation and progressive photoreceptor loss. Finally, both local and systemic delivery of miR-223 mimics improved retinal function in mice undergoing retinal degeneration. CONCLUSION: miR-223 is required for maintaining normal retinal function, as well as regulating inflammation in microglia and macrophages. Further investigations are required to determine the targets of miR-223 and their key biological pathways and interactions that are relevant to retinal diseases. Future studies should investigate whether sustained delivery of miR-223 into the retina is sufficient to target these pathways and protect the retina from progressive degeneration.

Photoreceptor Survival Is Regulated by GSTO1-1 in the Degenerating Retina.

Purpose: Glutathione-S-transferase omega 1-1 (GSTO1-1) is a cytosolic glutathione transferase enzyme, involved in glutathionylation, toll-like receptor signaling, and calcium channel regulation. GSTO1-1 dysregulation has been implicated in oxidative stress and inflammation, and contributes to the pathogenesis of several diseases and neurological disorders; however, its role in retinal degenerations is unknown. The aim of this study was to investigate the role of GSTO1-1 in modulating oxidative stress and consequent inflammation in the normal and degenerating retina. Methods: The role of GSTO1-1 in retinal degenerations was explored by using Gsto1-/- mice in a model of retinal degeneration. The expression and localization of GSTO1-1 were investigated with immunohistochemistry and Western blot. Changes in the expression of inflammatory (Ccl2, Il-1ß, and C3) and oxidative stress (Nox1, Sod2, Gpx3, Hmox1, Nrf2, and Nqo1) genes were investigated via quantitative real-time polymerase chain reaction. Retinal function in Gsto1-/- mice was investigated by using electroretinography. Results: GSTO1-1 was localized to the inner segment of cone photoreceptors in the retina. Gsto1-/- photo-oxidative damage (PD) mice had decreased photoreceptor cell death as well as decreased expression of inflammatory (Ccl2, Il-1ß, and C3) markers and oxidative stress marker Nqo1. Further, retinal function in the Gsto1-/- PD mice was increased as compared to wild-type PD mice. Conclusions: These results indicate that GSTO1-1 is required for inflammatory-mediated photoreceptor death in retinal degenerations. Targeting GSTO1-1 may be a useful strategy to reduce oxidative stress and inflammation and ameliorate photoreceptor loss, slowing the progression of retinal degenerations.