In vivo screening for toxicity-modulating drug interactions identifies antagonism that protects against ototoxicity in zebrafish.

Introduction: Ototoxicity is a debilitating side effect of over 150 medications with diverse mechanisms of action, many of which could be taken concurrently to treat multiple conditions. Approaches for preclinical evaluation of drug-drug interactions that might impact ototoxicity would facilitate design of safer multi-drug regimens and mitigate unsafe polypharmacy by flagging combinations that potentially cause adverse interactions for monitoring. They may also identify protective agents that antagonize ototoxic injury. Methods: To address this need, we have developed a novel workflow that we call Parallelized Evaluation of Protection and Injury for Toxicity Assessment (PEPITA), which empowers high-throughput, semi-automated quantification of ototoxicity and otoprotection in zebrafish larvae via microscopy. We used PEPITA and confocal microscopy to characterize in vivo the consequences of drug-drug interactions on ototoxic drug uptake and cellular damage of zebrafish lateral line hair cells. Results and discussion: By applying PEPITA to measure ototoxic drug interaction outcomes, we discovered antagonistic interactions between macrolide and aminoglycoside antibiotics that confer protection against aminoglycoside-induced damage to lateral line hair cells in zebrafish larvae. Co-administration of either azithromycin or erythromycin in zebrafish protected against damage from a broad panel of aminoglycosides, at least in part via inhibiting drug uptake into hair cells via a mechanism independent from hair cell mechanotransduction. Conversely, combining macrolides with aminoglycosides in bacterial inhibition assays does not show antagonism of antimicrobial efficacy. The proof-of-concept otoprotective antagonism suggests that combinatorial interventions can potentially be developed to protect against other forms of toxicity without hindering on-target drug efficacy.

In vivo screening for toxicity-modulating drug interactions identifies antagonism that protects against ototoxicity in zebrafish.

Ototoxicity is a debilitating side effect of over 150 medications with diverse mechanisms of action, many of which could be taken concurrently to treat multiple conditions. Approaches for preclinical evaluation of drug interactions that might impact ototoxicity would facilitate design of safer multi-drug regimens and mitigate unsafe polypharmacy by flagging combinations that potentially cause adverse interactions for monitoring. They may also identify protective agents that antagonize ototoxic injury. To address this need, we have developed a novel workflow that we call Parallelized Evaluation of Protection and Injury for Toxicity Assessment (PEPITA), which empowers high-throughput, semi-automated quantification of ototoxicity and otoprotection in zebrafish larvae. By applying PEPITA to characterize ototoxic drug interaction outcomes, we have discovered antagonistic interactions between macrolide and aminoglycoside antibiotics that confer protection against aminoglycoside-induced damage to lateral line hair cells in zebrafish larvae. Co-administration of either azithromycin or erythromycin in zebrafish protected against damage from a broad panel of aminoglycosides, at least in part via inhibiting drug uptake into hair cells via a mechanism independent from hair cell mechanotransduction. Conversely, combining macrolides with aminoglycosides in bacterial inhibition assays does not show antagonism of antimicrobial efficacy. The proof-of-concept otoprotective antagonism suggests that combinatorial interventions can potentially be developed to protect against other forms of toxicity without hindering on-target drug efficacy.

Activity regulates a cell type-specific mitochondrial phenotype in zebrafish lateral line hair cells.

Hair cells of the inner ear are particularly sensitive to changes in mitochondria, the subcellular organelles necessary for energy production in all eukaryotic cells. There are over 30 mitochondrial deafness genes, and mitochondria are implicated in hair cell death following noise exposure, aminoglycoside antibiotic exposure, as well as in age-related hearing loss. However, little is known about the basic aspects of hair cell mitochondrial biology. Using hair cells from the zebrafish lateral line as a model and serial block-face scanning electron microscopy, we have quantifiably characterized a unique hair cell mitochondrial phenotype that includes (1) a high mitochondrial volume and (2) specific mitochondrial architecture: multiple small mitochondria apically, and a reticular mitochondrial network basally. This phenotype develops gradually over the lifetime of the hair cell. Disrupting this mitochondrial phenotype with a mutation in opa1 impacts mitochondrial health and function. While hair cell activity is not required for the high mitochondrial volume, it shapes the mitochondrial architecture, with mechanotransduction necessary for all patterning, and synaptic transmission necessary for the development of mitochondrial networks. These results demonstrate the high degree to which hair cells regulate their mitochondria for optimal physiology and provide new insights into mitochondrial deafness.

Our ability to perceive sounds relies on tiny cells deep inside our ears which can convert vibrations into the electrical signals that our brain is able to decode. These 'hair cells' sport a small tuft of short fibers on one of their ends that can move in response to pressure waves. The large amount of energy required for this activity is provided by the cells' mitochondria, the small internal compartments that act as cellular powerhouses. In fact, reducing mitochondrial function in hair cells can lead to hearing disorders. Mitochondria are often depicted as being bean-like, but they can actually adopt different shapes based on the level of energy they need to produce. Despite this link between morphology and function, little is known about what mitochondria look like in hair cells. Filling this knowledge gap is necessary to understand how these structures support hair cells and healthy hearing. To address this question, McQuate et al. turned to zebrafish, as these animals detect vibrations in water through easily accessible hair cells on their skin that work just like the ones in the mammalian ear. Obtaining and analysing series of 3D images from a high-resolution microscope revealed that hair cells are more densely populated with mitochondria than other cell types. Mitochondrial organisation was also strikingly different. The side of the cell that carries the hair-like structures featured many small mitochondria; however, on the opposite side, which is in contact with neurons, the mitochondria formed a single large network. The co-existence of different types of mitochondria within one cell is a novel concept. Further experiments investigated how these mitochondrial characteristics were connected to hair cell activity. They showed that this organisation was established gradually as the cells aged, with cellular activity shaping the architecture (but not the total volume) of the mitochondria. Overall, the work by McQuate et al. provides important information necessary to develop therapeutics for hearing disorders linked to mitochondrial dysfunction. However, by showing that various kind of mitochondria can be present within one cell, it should also inform studies beyond those that focus on hearing.

Finding the balance: The elusive mechanisms underlying auditory hair cell mitochondrial biogenesis and mitophagy.

In all cell types, mitochondrial biogenesis is balanced with mitophagy to maintain a healthy mitochondrial pool that sustains specific energetic demands. Cell types that have a higher energetic burden, such as skeletal muscle cells and cardiomyocytes, will subsequently develop high mitochondrial volumes. In these cells, calcium influx during activity triggers cascades leading to activation of the co-transcriptional regulation factor PGC-1α, a master regulator of mitochondrial biogenesis, in a well-defined pathway. Despite the advantages in ATP production, high mitochondrial volumes might prove to be perilous, as it increases exposure to reactive oxygen species produced during oxidative phosphorylation. Mechanosensory hair cells are highly metabolically active cells, with high total mitochondrial volumes to meet that demand. However, the mechanisms leading to expansion and maintenance of the hair cell mitochondrial pool are not well defined. Calcium influx during mechanotransduction and synaptic transmission regulate hair cell mitochondria, leading to a possibility that similar to skeletal muscle and cardiomyocytes, intracellular calcium underlies the expansion of the hair cell mitochondrial volume. This review briefly summarizes the potential mechanisms underlying mitochondrial biogenesis in other cell types and in hair cells. We propose that hair cell mitochondrial biogenesis is primarily product of cellular differentiation rather than calcium influx, and that the hair cell high mitochondrial volume renders them more susceptible to reactive oxygen species increased by calcium flux than other cell types.

Rapid exchange of synaptic and extrasynaptic NMDA receptors in hippocampal CA1 neurons.

N-methyl-d-aspartate receptors (NMDARs) are fundamental coincidence detectors of synaptic activity necessary for the induction of synaptic plasticity and synapse stability. Adjusting NMDAR synaptic content, whether by receptor insertion or lateral diffusion between extrasynaptic and synaptic compartments, could play a substantial role defining the characteristics of the NMDAR-mediated excitatory postsynaptic current (EPSC), which in turn would mediate the ability of the synapse to undergo plasticity. Lateral NMDAR movement has been observed in dissociated neurons; however, it is currently unclear whether NMDARs are capable of lateral surface diffusion in hippocampal slices, a more physiologically relevant environment. To test for lateral mobility in rat hippocampal slices, we rapidly blocked synaptic NMDARs using MK-801, a use-dependent and irreversible NMDAR blocker. Following a 5-min washout period, we observed a strong recovery of NMDAR-mediated responses. The degree of the observed recovery was proportional to the amount of induced blockade, independent of levels of intracellular calcium, and mediated primarily by GluN2B-containing NMDA receptors. These results indicate that lateral diffusion of NMDARs could be a mechanism by which synapses rapidly adjust parameters to fine-tune synaptic plasticity.NEW & NOTEWORTHYN-methyl-d-aspartate-type glutamate receptors (NMDARs) have always been considered stable components of synapses. We show that in rat hippocampal slices synaptic NMDARs are in constant exchange with extrasynaptic receptors. This exchange of receptors is mediated primarily by NMDA receptors containing GluN2B, a subunit necessary to undergo synaptic plasticity. Thus this lateral movement of synaptic receptors allows synapses to rapidly regulate the total number of synaptic NMDARs with potential consequences for synaptic plasticity.

A Wnt/Calcium Signaling Cascade Regulates Neuronal Excitability and Trafficking of NMDARs.

Wnt signaling controls multiple biological process, particularly the embryonic development of metazoans. Sustained expression of Wnt signaling components in the mature mammalian CNS and their apparent deregulation in certain neuropathologies suggest that it also plays a part beyond embryonic development to regulate normal brain function. We describe a noncanonical Wnt/Ca2+ signaling cascade that regulates the electrophysiological intrinsic properties of rat neurons, resulting in sustained membrane depolarization and the mobilization of Ca2+ from internal stores. These effects require tyrosine kinase-like orphan receptor 2 (RoR2), activation of PLC, and voltage-gated Ca2+ channels. Activation of this signaling cascade then promotes surface expression of N-methyl-D-aspartate receptors (NMDARs) through a SNARE-dependent mechanism. This neuronal Wnt/Ca2+ signaling pathway represents a mechanism for Wnt ligands to regulate normal brain processes in the mature animal and provides a framework for understanding how alterations in this pathway may contribute to the etiology of psychiatric disorders where NMDARs are compromised.

An epigenetic feedback regulatory loop involving microRNA-195 and MBD1 governs neural stem cell differentiation.

BACKGROUND: Epigenetic mechanisms, including DNA methylation, histone modification, and microRNAs, play pivotal roles in stem cell biology. Methyl-CpG binding protein 1 (MBD1), an important epigenetic regulator of adult neurogenesis, controls the proliferation and differentiation of adult neural stem/progenitor cells (aNSCs). We recently demonstrated that MBD1 deficiency in aNSCs leads to altered expression of several noncoding microRNAs (miRNAs). METHODOLOGY/PRINCIPAL FINDINGS: Here we show that one of these miRNAs, miR-195, and MBD1 form a negative feedback loop. While MBD1 directly represses the expression of miR-195 in aNSCs, high levels of miR-195 in turn repress the expression of MBD1. Both gain-of-function and loss-of-function investigations show that alterations of the MBD1-miR-195 feedback loop tip the balance between aNSC proliferation and differentiation. CONCLUSIONS/SIGNIFICANCE: Therefore the regulatory loop formed by MBD1 and miR-195 is an important component of the epigenetic network that controls aNSC fate.

Crosstalk among Epigenetic Pathways Regulates Neurogenesis.

The process of neurogenesis includes neural stem cell proliferation, fate specification, young neuron migration, neuronal maturation, and functional integration into existing circuits. Although neurogenesis occurs largely during embryonic development, low levels but functionally important neurogenesis persists in restricted regions of the postnatal brain, including the subgranular zone of the dentate gyrus in the hippocampus and the subventricular zone of the lateral ventricles. This review will cover both embryonic and adult neurogenesis with an emphasis on the latter. Of the many endogenous mediators of postnatal neurogenesis, epigenetic pathways, such as mediators of DNA methylation, chromatin remodeling systems, and non-coding RNA modulators, appear to play an integral role. Mounting evidence shows that such epigenetic factors form regulatory networks, which govern each step of postnatal neurogenesis. In this review, we explore the emerging roles of epigenetic mechanisms particularly microRNAs, element-1 silencing transcription factor/neuron-restrictive silencing factor (REST/NRSF), polycomb proteins, and methyl-CpG bindings proteins, in regulating the entire process of postnatal and adult neurogenesis. We further summarize recent data regarding how the crosstalk among these different epigenetic proteins forms the critical regulatory network that regulates neuronal development. We finally discuss how crosstalk between these pathways may serve to translate environmental cues into control of the neurogenic process.