Replication Fork Remodeling and Therapy Escape in DNA Damage Response-Deficient Cancers.

Most cancers have lost a critical DNA damage response (DDR) pathway during tumor evolution. These alterations provide a useful explanation for the initial sensitivity of tumors to DNA-targeting chemotherapy. A striking example is dysfunctional homology-directed repair (HDR), e.g., due to inactivating mutations in BRCA1 and BRCA2 genes. Extensive efforts are being made to develop novel targeted therapies exploiting such an HDR defect. Inhibitors of poly(ADP-ribose) polymerase (PARP) are an instructive example of this approach. Despite the success of PARP inhibitors, the presence of primary or acquired therapy resistance remains a major challenge in clinical oncology. To move the field of precision medicine forward, we need to understand the precise mechanisms causing therapy resistance. Using preclinical models, various mechanisms underlying chemotherapy resistance have been identified. Restoration of HDR seems to be a prevalent mechanism but this does not explain resistance in all cases. Interestingly, some factors involved in DNA damage response (DDR) have independent functions in replication fork (RF) biology and their loss causes RF instability and therapy sensitivity. However, in BRCA-deficient tumors, loss of these factors leads to restored stability of RFs and acquired drug resistance. In this review we discuss the recent advances in the field of RF biology and its potential implications for chemotherapy response in DDR-defective cancers. Additionally, we review the role of DNA damage tolerance (DDT) pathways in maintenance of genome integrity and their alterations in cancer. Furthermore, we refer to novel tools that, combined with a better understanding of drug resistance mechanisms, may constitute a great advance in personalized diagnosis and therapeutic strategies for patients with HDR-deficient tumors.

The Aryl Hydrocarbon Receptor Pathway Defines the Time Frame for Restorative Neurogenesis.

Zebrafish have a high capacity to replace lost neurons after brain injury. New neurons involved in repair are generated by a specific set of glial cells, known as ependymoglial cells. We analyze changes in the transcriptome of ependymoglial cells and their progeny after injury to infer the molecular pathways governing restorative neurogenesis. We identify the aryl hydrocarbon receptor (AhR) as a regulator of ependymoglia differentiation toward post-mitotic neurons. In vivo imaging shows that high AhR signaling promotes the direct conversion of a specific subset of ependymoglia into post-mitotic neurons, while low AhR signaling promotes ependymoglial proliferation. Interestingly, we observe the inactivation of AhR signaling shortly after injury followed by a return to the basal levels 7 days post injury. Interference with timely AhR regulation after injury leads to aberrant restorative neurogenesis. Taken together, we identify AhR signaling as a crucial regulator of restorative neurogenesis timing in the zebrafish brain.

Adult neural stem cell behavior underlying constitutive and restorative neurogenesis in zebrafish.

Adult Neural Stem Cells (aNSCs) generate new neurons that integrate into the pre-existing networks in specific locations of the Vertebrate brain. Moreover, aNSCs contribute with new neurons to brain regeneration in some non-mammalian Vertebrates. The similarities and the differences in the cellular and molecular processes governing neurogenesis in the intact and regenerating brain are still to be assessed. Toward this end, we recently established a protocol for non-invasive imaging of aNSC behavior in their niche in vivo in the adult intact and regenerating zebrafish telencephalon. We observed different modes of aNSC division in the intact brain and a novel mode of neurogenesis by direct conversion, which contributes to stem cell depletion with age. After injury, the generation of neurons is increased both by the activation of additional aNSCs and a shift in the division mode of aNSCs, thereby contributing to the successful neuronal regeneration. The cellular behavior we observed opens new questions regarding long-term aNSC maintenance in homeostasis and in regeneration. In this commentary we discuss our data and new questions arising in the context of aNSC behavior, not only in zebrafish but also in other species, including mammals.

Single-cell in vivo imaging of adult neural stem cells in the zebrafish telencephalon.

Adult neural stem cells (aNSCs) in zebrafish produce mature neurons throughout their entire life span in both the intact and regenerating brain. An understanding of the behavior of aNSCs in their intact niche and during regeneration in vivo should facilitate the identification of the molecular mechanisms controlling regeneration-specific cellular events. A greater understanding of the process in regeneration-competent species may enable regeneration to be achieved in regeneration-incompetent species, including humans. Here we describe a protocol for labeling and repetitive imaging of aNSCs in vivo. We label single aNSCs, allowing nonambiguous re-identification of single cells in repetitive imaging sessions using electroporation of a red-reporter plasmid in Tg(gfap:GFP)mi2001 transgenic fish expressing GFP in aNSCs. We image using two-photon microscopy through the thinned skull of anesthetized and immobilized fish. Our protocol allows imaging every 2 d for a period of up to 1 month. This methodology allowed the visualization of aNSC behavior in vivo in their natural niche, in contrast to previously available technologies, which rely on the imaging of either dissociated cells or tissue slices. We used this protocol to follow the mode of aNSC division, fate changes and cell death in both the intact and injured zebrafish telencephalon. This experimental setup can be widely used, with minimal prior experience, to assess key factors for processes that modulate aNSC behavior. A typical experiment with data analysis takes up to 1.5 months.

Neurodevelopment. Live imaging of adult neural stem cell behavior in the intact and injured zebrafish brain.

Adult neural stem cells are the source for restoring injured brain tissue. We used repetitive imaging to follow single stem cells in the intact and injured adult zebrafish telencephalon in vivo and found that neurons are generated by both direct conversions of stem cells into postmitotic neurons and via intermediate progenitors amplifying the neuronal output. We observed an imbalance of direct conversion consuming the stem cells and asymmetric and symmetric self-renewing divisions, leading to depletion of stem cells over time. After brain injury, neuronal progenitors are recruited to the injury site. These progenitors are generated by symmetric divisions that deplete the pool of stem cells, a mode of neurogenesis absent in the intact telencephalon. Our analysis revealed changes in the behavior of stem cells underlying generation of additional neurons during regeneration.

Stab wound injury of the zebrafish telencephalon: a model for comparative analysis of reactive gliosis.

Reactive glia, including astroglia and oligodendrocyte progenitors (OPCs) are at the core of the reaction to injury in the mammalian brain with initially beneficial and later partially adverse functions such as scar formation. Given the different glial composition in the adult zebrafish brain with radial ependymoglia but no parenchymal astrocytes, we examined the glial response to an invasive stab wound injury model in the adult zebrafish telencephalon. Strikingly, already a few days after injury the wound was closed without any scar tissue. Similar to mammals, microglia cells reacted first and accumulated close to the injury site, while neither GFAP+ radial ependymoglia nor adult OPCs were recruited to the injury site. Moreover, OPCs failed to increase their proliferation after this injury, while the number of proliferating GFAP+ glia was increased until 7 days after injury. Importantly, neurogenesis was also increased after injury, generating additional neurons recruited to the parenchyma which survived for several months. Thus, these data suggest that the specific glial environment in the adult zebrafish telencephalon is not only permissive for long-term neuronal survival, but avoids scar formation. Invasive injury in the adult zebrafish telencephalon may therefore provide a useful model to untangle the molecular mechanisms involved in these beneficial glial reactions.

MicroRNA 18 and 124a down-regulate the glucocorticoid receptor: implications for glucocorticoid responsiveness in the brain.

Glucocorticoids (GCs) exert profound effects on a variety of physiological processes, including adaptation to stress, metabolism, immunity, and neuronal development. Cellular responsiveness to GCs depends on numerous factors, including the amount of the glucocorticoid receptor (GR) protein. We tested the hypothesis that micro-RNAs (miRs), a recently discovered group of noncoding RNAs involved in mRNA translation, might control GR activity by reducing GR protein levels in neuronal tissues. We tested a panel of five miRs consisting of 124aa, 328, 524, 22, and 18. We found that miRs 18 and 124a reduced GR-mediated events in addition to decreasing GR protein levels. miR reporter assays revealed binding of miR-124a to the 3' untranslated region of GR. In correspondence, the activation of the GR-responsive gene glucocorticoid-induced leucine zipper was strongly impaired by miR-124a and -18 overexpression. Although miR-18 is expressed widely throughout the body, expression of miR-124a is restricted to the brain. Endogenous miR-124a up-regulation during neuronal differentiation of P19 cells was associated with a decreasing amount of GR protein levels and reduced activity of luciferase reporter constructs bearing GR 3' untranslated regions. Furthermore, we show that miR-124a expression varies over time during the stress hyporesponsive period, a neonatal period when GC signaling is modulated. Our findings demonstrate a potential role for miRs in the regulation of cell type-specific responsiveness to GCs, as may occur during critical periods of neuronal development. Ultimately, our results may provide a better understanding of the etiology of stress-related diseases as well as the efficacy of GC therapy.