Learning and memory formation in zebrafish: Protein dynamics and molecular tools.

Research on learning and memory formation at the level of neural networks, as well as at the molecular level, is challenging due to the immense complexity of the brain. The zebrafish as a genetically tractable model organism can overcome many of the current challenges of studying molecular mechanisms of learning and memory formation. Zebrafish have a translucent, smaller and more accessible brain than that of mammals, allowing imaging of the entire brain during behavioral manipulations. Recent years have seen an extensive increase in published brain research describing the use of zebrafish for the study of learning and memory. Nevertheless, due to the complexity of the brain comprising many neural cell types that are difficult to isolate, it has been difficult to elucidate neural networks and molecular mechanisms involved in memory formation in an unbiased manner, even in zebrafish larvae. Therefore, data regarding the identity, location, and intensity of nascent proteins during memory formation is still sparse and our understanding of the molecular networks remains limited, indicating a need for new techniques. Here, we review recent progress in establishing learning paradigms for zebrafish and the development of methods to elucidate neural and molecular networks of learning. We describe various types of learning and highlight directions for future studies, focusing on molecular mechanisms of long-term memory formation and promising state-of-the-art techniques such as cell-type-specific metabolic labeling.

Large-scale cell-type-specific imaging of protein synthesis in a vertebrate brain.

Despite advances in methods to detect protein synthesis, it has not been possible to measure endogenous protein synthesis levels in vivo in an entire vertebrate brain. We developed a transgenic zebrafish line that allows for cell-type-specific labeling and imaging of nascent proteins in the entire animal. By replacing leucine with glycine in the zebrafish MetRS-binding pocket (MetRS-L270G), we enabled the cell-type-specific incorporation of the azide-bearing non-canonical-amino-acid azidonorleucine (ANL) during protein synthesis. Newly synthesized proteins were then labeled via 'click chemistry'. Using a Gal4-UAS-ELAV3 line to express MetRS-L270G in neurons, we measured protein synthesis intensities across the entire nervous system. We visualized endogenous protein synthesis and demonstrated that seizure-induced neural activity results in enhanced translation levels in neurons. This method allows for robust analysis of endogenous protein synthesis in a cell-type-specific manner, in vivo at single-cell resolution.

A high-throughput chemical screen with FDA approved drugs reveals that the antihypertensive drug Spironolactone impairs cancer cell survival by inhibiting homology directed repair.

DNA double-strand breaks (DSBs) are the most severe type of DNA damage. DSBs are repaired by non-homologous end-joining or homology directed repair (HDR). Identifying novel small molecules that affect HDR is of great importance both for research use and therapy. Molecules that elevate HDR may improve gene targeting whereas inhibiting molecules can be used for chemotherapy, since some of the cancers are more sensitive to repair impairment. Here, we performed a high-throughput chemical screen for FDA approved drugs, which affect HDR in cancer cells. We found that HDR frequencies are increased by retinoic acid and Idoxuridine and reduced by the antihypertensive drug Spironolactone. We further revealed that Spironolactone impairs Rad51 foci formation, sensitizes cancer cells to DNA damaging agents, to Poly (ADP-ribose) polymerase (PARP) inhibitors and cross-linking agents and inhibits tumor growth in xenografts, in mice. This study suggests Spironolactone as a new candidate for chemotherapy.

Acetylation of lysine 382 and phosphorylation of serine 392 in p53 modulate the interaction between p53 and MDC1 in vitro.

Occurrence of DNA damage in a cell activates the DNA damage response, a survival mechanism that ensures genomics stability. Two key members of the DNA damage response are the tumor suppressor p53, which is the most frequently mutated gene in cancers, and MDC1, which is a central adaptor that recruits many proteins to sites of DNA damage. Here we characterize the in vitro interaction between p53 and MDC1 and demonstrate that p53 and MDC1 directly interact. The p53-MDC1 interaction is mediated by the tandem BRCT domain of MDC1 and the C-terminal domain of p53. We further show that both acetylation of lysine 382 and phosphorylation of serine 392 in p53 enhance the interaction between p53 and MDC1. Additionally, we demonstrate that the p53-MDC1 interaction is augmented upon the induction of DNA damage in human cells. Our data suggests a new role for acetylation of lysine 382 and phosphorylation of serine 392 in p53 in the cellular stress response and offers the first evidence for an interaction involving MDC1 that is modulated by acetylation.