Hair cell toxicology: With the help of a little fish

Hearing or balance loss are disabling conditions that have a serious impact in those suffering them, especially when they appear in children. Their ultimate cause is frequently the loss of function of mechanosensory hair cells in the inner ear. Hair cells can be damaged by environmental insults, like noise or chemical agents, known as ototoxins. Two of the most common ototoxins are life-saving medications: cisplatin against solid tumors, and aminoglycoside antibiotics to treat infections. However, due to their localization inside the temporal bone, hair cells are difficult to study in mammals. As an alternative animal model, zebrafish larvae have hair cells similar to those in mammals, some of which are located in a fish specific organ on the surface of the skin, the lateral line. This makes them easy to observe in vivo and readily accessible for ototoxins or otoprotective substances. These features have made possible advances in the study of the mechanisms mediating ototoxicity or identifying new potential ototoxins. Most importantly, the small size of the zebrafish larvae has allowed screening thousands of molecules searching for otoprotective agents in a scale that would be highly impractical in rodent models. The positive hits found can then start the long road to reach clinical settings to prevent hearing or balance loss

The Snail genes as inducers of cell movement and survival: implications in development and cancer

11 pages, 5 figures.-- PMID: 15983400 [PubMed].-- Printed version published Jul 2005.Full-text version available Open Access at the journal site.The functions of the Snail family of zinc-finger transcription factors are essential during embryonic development. One of their best-known functions is to induce epithelial to mesenchymal transitions (EMTs), which convert epithelial cells into migratory mesenchymal cells. In recent years, many orthologues of the Snail family have been identified throughout the animal kingdom, and their study is providing new clues about the EMT-dependent and -independent functions of Snail proteins. Here, we discuss these functions and how they influence cell behaviour during development and during diseases such as metastatic cancer. From these findings, we propose that Snail genes act primarily as survival factors and inducers of cell movement, rather than as inducers of EMT or cell fate.We are grateful to all members of M. A. Nieto’s laboratory for encouraging discussions. Work in the laboratory is mainly supported by grants from the Spanish Ministry of Education and Science. A.B.-G. is a researcher of the Ramón y Cajal Programme (MEC).Peer reviewe

Relationship between vestibular hair cell loss and deficits in two anti-gravity reflexes in the rat.

The tail-lift reflex and the air-righting reflex in rats are anti-gravity reflexes that depend on vestibular function. To begin identifying their cellular basis, this study examined the relationship between reflex loss and the graded lesions caused in the vestibular sensory epithelia by varying doses of an ototoxic compound. After ototoxic exposure, we recorded these reflexes using high speed video. The movies were used to obtain objective measures of the reflexes: the minimum angle formed by the nose, the back of the neck and the base of the tail during the tail-lift maneuver and the time to right in the air-righting test. The vestibular sensory epithelia were then collected from the rats and used to estimate the loss of type I (HCI), type II (HCII) and all hair cells (HC) in both central and peripheral parts of the crista, utricle, and saccule. As expected, tail-lift angles decreased, and air-righting times increased, while the numbers of HCs remaining in the epithelia decreased in a dose-dependent manner. The results demonstrated greater sensitivity of HCI compared to HCII to the IDPN ototoxicity, as well as a relative resiliency of the saccule compared to the crista and utricle. Comparing the functional measures with the cell counts, we observed that loss of the tail-lift reflex associates better with HCI than with HCII loss. In contrast, most HCI in the crista and utricle were lost before air-righting times increased. These data suggest that these reflexes depend on the function of non-identical populations of vestibular HCs

Scratch2 prevents cell cycle re-entry by repressing miR-25 in postmitotic primary neurons

During the development of the nervous system the regulation of cell cycle, differentiation, and survival is tightly interlinked. Newly generated neurons must keep cell cycle components under strict control, as cell cycle re-entry leads to neuronal degeneration and death. However, despite their relevance, the mechanisms controlling this process remain largely unexplored. Here we show that Scratch2 is involved in the control of the cell cycle in neurons in the developing spinal cord of the zebrafish embryo. scratch2 knockdown induces postmitotic neurons to re-enter mitosis. Scratch2 prevents cell cycle re-entry by maintaining high levels of the cycle inhibitor p57 through the downregulation of miR-25. Thus, Scratch2 appears to safeguard the homeostasis of postmitotic primary neurons by preventing cell cycle re-entry

Identification and characterization of the zebrafish ClC-2 chloride channel orthologs

ClC-2 is a Cl− channel that belongs to the CLC family of chloride channel/transport proteins. ClC-2 molecular role is not clear, and Clcn2 knockout mice develop blindness, sterility, and leukodystrophy by unknown reasons. ClC-2 is associated in the brain with the adhesion molecule GlialCAM, which is defective in a type of leukodystrophy, involving ClC-2 in the homeostasis of myelin. To get more insight into the functions of ClC-2, we have identified in this work the three ClC-2 orthologs in zebrafish. clcn2a and clcn2b resulted from the teleost-specific whole genome duplication, while clcn2c arose from a gene duplication from clcn2b. The expression patterns in adult tissues and embryos of zebrafish clcn2 paralogs support their subfunctionalization after the duplications, with clcn2a being enriched in excitable tissues and clcn2c in ionocytes. All three zebrafish clc-2 proteins interact with human GLIALCAM, that is able to target them to cell junctions, as it does with mammalian ClC-2. We could detect clc-2a and clc-2b inward rectified chloride currents with different voltage-dependence and kinetics in Xenopus oocytes, while clc-2c remained inactive. Interestingly, GlialCAM proteins did not modify clc-2b inward rectification. Then, our work extends the repertoire of ClC-2 proteins and provides new tools for structure-function and physiology studies

Sec24D-Dependent Transport of Extracellular Matrix Proteins Is Required for Zebrafish Skeletal Morphogenesis

Protein transport from endoplasmic reticulum (ER) to Golgi is primarily conducted by coated vesicular carriers such as COPII. Here, we describe zebrafish bulldog mutations that disrupt the function of the cargo adaptor Sec24D, an integral component of the COPII complex. We show that Sec24D is essential for secretion of cartilage matrix proteins, whereas the preceding development of craniofacial primordia and pre-chondrogenic condensations does not depend on this isoform. Bulldog chondrocytes fail to secrete type II collagen and matrilin to extracellular matrix (ECM), but membrane bound receptor β1-Integrin and Cadherins appear to leave ER in Sec24D-independent fashion. Consequently, Sec24D-deficient cells accumulate proteins in the distended ER, although a subset of ER compartments and Golgi complexes as visualized by electron microscopy and NBD C6-ceramide staining appear functional. Consistent with the backlog of proteins in the ER, chondrocytes activate the ER stress response machinery and significantly upregulate BiP transcription. Failure of ECM secretion hinders chondroblast intercalations thus resulting in small and malformed cartilages and severe craniofacial dysmorphology. This defect is specific to Sec24D mutants since knockdown of Sec24C, a close paralog of Sec24D, does not result in craniofacial cartilage dysmorphology. However, craniofacial development in double Sec24C/Sec24D-deficient animals is arrested earlier than in bulldog/sec24d, suggesting that Sec24C can compensate for loss of Sec24D at initial stages of chondrogenesis, but Sec24D is indispensable for chondrocyte maturation. Our study presents the first developmental perspective on Sec24D function and establishes Sec24D as a strong candidate for cartilage maintenance diseases and craniofacial birth defects

Comparison of zebrafish and mice knockouts for Megalencephalic Leukoencephalopathy proteins indicates that GlialCAM/MLC1 forms a functional unit

[Abstract] Background: Megalencephalic Leukoencephalopathy with subcortical Cysts (MLC) is a rare type of leukodystrophy characterized by astrocyte and myelin vacuolization, epilepsy and early-onset macrocephaly. MLC is caused by mutations in MLC1 or GLIALCAM, coding for two membrane proteins with an unknown function that form a complex specifically expressed in astrocytes at cell-cell junctions. Recent studies in Mlc1−/− or Glialcam−/− mice and mlc1−/− zebrafish have shown that MLC1 regulates glial surface levels of GlialCAM in vivo and that GlialCAM is also required for MLC1 expression and localization at cell-cell junctions. Methods: We have generated and analysed glialcama−/− zebrafish. We also generated zebrafish glialcama−/− mlc1−/− and mice double KO for both genes and performed magnetic resonance imaging, histological studies and biochemical analyses. Results: glialcama−/− shows megalencephaly and increased fluid accumulation. In both zebrafish and mice, this phenotype is not aggravated by additional elimination of mlc1. Unlike mice, mlc1 protein expression and localization are unaltered in glialcama−/− zebrafish, possibly because there is an up-regulation of mlc1 mRNA. In line with these results, MLC1 overexpressed in Glialcam−/− mouse primary astrocytes is located at cell-cell junctions. Conclusions: This work indicates that the two proteins involved in the pathogenesis of MLC, GlialCAM and MLC1, form a functional unit, and thus, that loss-of-function mutations in these genes cause leukodystrophy through a common pathway.Ministerio de Ciencia e Innovación; SAF2015–70377Ministerio de Ciencia e Innovación; RTI2018–093493-B-I00Generalitat de Catalunya; SGR2014–1178Generalitat de Catalunya; SGR014–2016Instituto de Salud Carlos III; PI16/00267-R-Fede

The vestibular calyceal junction is dismantled following subchronic streptomycin in rats and sensory epithelium stress in humans

Hair cell (HC) loss by epithelial extrusion has been described to occur in the rodent vestibular system during chronic 3,3'-iminodipropionitrile (IDPN) ototoxicity. This is preceded by dismantlement of the calyceal junction in the contact between type I HC (HCI) and calyx afferent terminals. Here, we evaluated whether these phenomena have wider significance. First, we studied rats receiving seven different doses of streptomycin, ranging from 100 to 800 mg/kg/day, for 3 to 8 weeks. Streptomycin caused loss of vestibular function associated with partial loss of HCI and decreased expression of contactin-associated protein (CASPR1), denoting calyceal junction dismantlement, in the calyces encasing the surviving HCI. Additional molecular and ultrastructural data supported the conclusion that HC-calyx detachment precede HCI loss by extrusion. Animals allowed to survive after the treatment showed functional recuperation and rebuilding of the calyceal junction. Second, we evaluated human sensory epithelia obtained during therapeutic labyrinthectomies and trans-labyrinthine tumour excisions. Some samples showed abnormal CASPR1 label strongly suggestive of calyceal junction dismantlement. Therefore, reversible dismantlement of the vestibular calyceal junction may be a common response triggered by chronic stress, including ototoxic stress, before HCI loss. This may partly explain clinical observations of reversion in function loss after aminoglycoside exposure

Comparison of zebrafish and mice knockouts for Megalencephalic Leukoencephalopathy proteins indicates that GlialCAM/MLC1 forms a functional unit

Background: Megalencephalic Leukoencephalopathy with subcortical Cysts (MLC) is a rare type of leukodystrophy characterized by astrocyte and myelin vacuolization, epilepsy and early-onset macrocephaly. MLC is caused by mutations in MLC1 or GLIALCAM, coding for two membrane proteins with an unknown function that form a complex specifically expressed in astrocytes at cell-cell junctions. Recent studies in Mlc1-/- or Glialcam-/- mice and mlc1-/- zebrafish have shown that MLC1 regulates glial surface levels of GlialCAM in vivo and that GlialCAM is also required for MLC1 expression and localization at cell-cell junctions. Methods: we have generated and analysed glialcama-/- zebrafish. We also generated zebrafish glialcama-/- mlc1-/- and mice double KO for both genes and performed magnetic resonance imaging, histological studies and biochemical analyses. Results: glialcama-/- shows megalencephaly and increased fluid accumulation. In both zebrafish and mice, this phenotype is not aggravated by additional elimination of mlc1. Unlike mice, mlc1 protein expression and localization are unaltered in glialcama-/- zebrafish, possibly because there is an up-regulation of mlc1 mRNA. In line with these results, MLC1 overexpressed in Glialcam-/- mouse primary astrocytes is located at cell-cell junctions. Conclusions: this work indicates that the two proteins involved in the pathogenesis of MLC, GlialCAM and MLC1, form a functional unit, and thus, that loss-of-function mutations in these genes cause leukodystrophy through a common pathway