Otoferlin Depletion Results in Abnormal Synaptic Ribbons and Altered Intracellular Calcium Levels in Zebrafish

Manchanda, Aayushi; Chatterjee, Paroma; Bonventre, Josephine A.; Haggard, Derik E.; Kindt, Katie S.; Tanguay, Robert L.; Johnson, Colin P.

doi:10.1038/s41598-019-50710-2

Cited by 8 publications

(29 citation statements)

References 38 publications

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“…Representative images of immunolabeled hair cells from both WT and mutant neuromasts at 72, 96, and 120 hpf exhibited a staining pattern consistent with previous immunofluorescence studies, albeit less intense (Figure 1C-H) (Chatterjee et al 2015). We compared anti-otoferlin levels between WT and mutant neuromasts by quantitating the average neuromast fluorescence intensity using the CTIF method (Manchanda et al 2019). To reduce variability due to natural neuromast size/shape differences, we averaged multiple neuromasts from multiple larvae (neuromast N = 4 mutant, 6 WT per larva, N = 4 mutant, 8 WT larvae each attempt).…”

Section: Differences In the Expression Of Wild Type And Mutant Otoferlinsupporting

confidence: 78%

“…To test for the effect of the otofB mutation on the synaptic ribbon, we analysed images of Ribeye immunolabeled lateral line neuromasts at 72, 96, and 120 hpf (Figure 3A-F). We found otofB hair cells displayed a similar number of Ribeye immunolabeled puncta relative to WT at all time points tested (Figure 3G) (Manchanda et al 2019). Although puncta number appeared unaffected, otofB neuromasts displayed significantly smaller and dimmer puncta relative to WT at 72 and 96 hpf (Figure 3H-I).…”

Section: Decreased Ribeye and Vglut3 Expression In Otofb Mutantsmentioning

confidence: 68%

“…Morpholino induced knockdown of otoferlin results in fewer synaptic ribbons relative to control (Manchanda et al 2019). Morphants also form larger anti-ribeye puncta compared to control, which may represent single, large synaptic ribbons, or tightly spaced clusters of ribbons.…”

Section: Decreased Ribeye and Vglut3 Expression In Otofb Mutantsmentioning

confidence: 93%

“…Targeted morpholino knockdown of otoferlin results in decreased endocytotic uptake of the fluorescent marker mCLING into neuromasts (Figure 2A,B) (Manchanda et al 2019). mCLING is a dye with selective uptake via endocytosis which has been used previously in mouse and zebrafish hair cells (Revelo et al 2014.…”

Section: Otofb Mutants Display Reduced Endocytosismentioning

confidence: 99%

“…Otoferlin morphant larvae exhibit abnormal transcriptional expression of several hair cell specific genes including myosin VIb (myo6b), parvalbumin (pvalb9), s100s, and the Nogo related proteins rtn4rl2a and rtn4rl2b (Manchanda et al 2019). We therefore sought to compare the expression of several neuromast hair cells markers between mutant otofB and WT larvae (Figure 5).…”

Section: Otofb Mutants Display Altered Expression Of Hair Cell Transcriptsmentioning

confidence: 99%

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Truncation of the otoferlin transmembrane domain alters the development of hair cells and reduces membrane docking

Manchanda

Bonventre

Bugel

et al. 2021

MBoC

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Release of neurotransmitter from sensory hair cells is regulated by otoferlin. Despite the importance of otoferlin in the auditory and vestibular pathways, the functional contributions of the domains of the protein have not been fully characterized. Using a zebrafish model, we investigated a mutant otoferlin with a stop codon at the start of the transmembrane domain. We found that both the phenotype severity and the expression level of mutant otoferlin changed with the age of the zebrafish. At the early developmental timepoint of 72 hours post-fertilization (hpf) low expression of the otoferlin mutant coincided with synaptic ribbon deficiencies, reduced endocytosis, and abnormal transcription of several hair cell genes. As development proceeded, expression of the mutant otoferlin increased, and both synaptic ribbons and hair cell transcript levels resembled wild type. However, hair cell endocytosis deficits and abnormalities in the expression of GABA receptors persisted even after upregulation of mutant otoferlin. Analysis of membrane-reconstituted otoferlin measurements suggest a function for the transmembrane domain in liposome docking. We conclude that that deletion of the transmembrane domain reduces membrane docking, attenuates endocytosis, and results in developmental delay of the hair cell.

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Section: Differences In the Expression Of Wild Type And Mutant Otoferlinsupporting

confidence: 78%

Section: Decreased Ribeye and Vglut3 Expression In Otofb Mutantsmentioning

confidence: 68%

Section: Decreased Ribeye and Vglut3 Expression In Otofb Mutantsmentioning

confidence: 93%

Section: Otofb Mutants Display Reduced Endocytosismentioning

confidence: 99%

Section: Otofb Mutants Display Altered Expression Of Hair Cell Transcriptsmentioning

confidence: 99%

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Truncation of the otoferlin transmembrane domain alters the development of hair cells and reduces membrane docking

Manchanda

Bonventre

Bugel

et al. 2021

MBoC

Self Cite

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Bilateral widefield calcium imaging reveals circuit asymmetries and lateralized functional activation of the mouse auditory cortex

Calhoun

Chen

2023

Proc. Natl. Acad. Sci. U.S.A.

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Coordinated functioning of the two cortical hemispheres is crucial for perception. The human auditory cortex (ACx) shows functional lateralization with the left hemisphere specialized for processing speech, whereas the right analyzes spectral content. In mice, virgin females demonstrate a left-hemisphere response bias to pup vocalizations that strengthens with motherhood. However, how this lateralized function is established is unclear. We developed a widefield imaging microscope to simultaneously image both hemispheres of mice to bilaterally monitor functional responses. We found that global ACx topography is symmetrical and stereotyped. In both male and virgin female mice, the secondary auditory cortex (A2) in the left hemisphere shows larger responses than right to high-frequency tones and adult vocalizations; however, only virgin female mice show a left-hemisphere bias in A2 in response to adult pain calls. These results indicate hemispheric bias with both sex-independent and -dependent aspects. Analyzing cross-hemispheric functional correlations showed that asymmetries exist in the strength of correlations between DM-AAF and A2-AAF, while other ACx areas showed smaller differences. We found that A2 showed lower cross-hemisphere correlation than other cortical areas, consistent with the lateralized functional activation of A2. Cross-hemispheric activity correlations are lower in deaf, otoferlin knockout (OTOF −/− ) mice, indicating that the development of functional cross-hemispheric connections is experience dependent. Together, our results reveal that ACx is topographically symmetric at the macroscopic scale but that higher-order A2 shows sex-dependent and independent lateralized responses due to asymmetric intercortical functional connections. Moreover, our results suggest that sensory experience is required to establish functional cross-hemispheric connectivity.

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Redefining the architecture of ferlin proteins: insights into multi-domain protein structure and function

Dominguez

McCord

Sutton

2022

Preprint

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Ferlins are complex, multidomain proteins whose functions involve membrane fusion, membrane repair, and exocytosis. Progress in studying the domain composition of ferlins has been problematic due to the large size of the ferlin proteins and the difficulty in computing accurate residue ranges for each respective ferlin protein domain. However, recent advances in in silico protein folding methods have significantly enhanced the understanding of the domain structure of these complex proteins. To compute unbiased domain boundaries, we used RoseTTAFold to assemble full-length models for each of the six human ferlin proteins (dysferlin, myoferlin, otoferlin, Fer1L4, Fer1L5, and Fer1L6). Despite the differences in amino acid sequence between the ferlin proteins, the domain ranges and the distinct subdomains in some of the ferlin domains were remarkably consistent. Further, the RoseTTAFold/AlphaFold2 in silico boundary prediction methods allowed us to describe and characterize a previously unknown C2 domain, ubiquitous in all human ferlins, which we refer to as C2-FerA. At present, the ferlin domain-domain interactions implied by the full-length in silico models are predicted to have a low accuracy; however, the use of RoseTTAFold and AlphaFold2 as a domain finder has proven to be a powerful research tool in understanding ferlin structure.

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Otoferlin Depletion Results in Abnormal Synaptic Ribbons and Altered Intracellular Calcium Levels in Zebrafish

Cited by 8 publications

References 38 publications

Truncation of the otoferlin transmembrane domain alters the development of hair cells and reduces membrane docking

Truncation of the otoferlin transmembrane domain alters the development of hair cells and reduces membrane docking

Bilateral widefield calcium imaging reveals circuit asymmetries and lateralized functional activation of the mouse auditory cortex

Redefining the architecture of ferlin proteins: insights into multi-domain protein structure and function

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