Teresa Nicolson

Sensorimotor deficits can profoundly affect the life quality of patients with neurological disorders, yet our understanding of central dysfunction at the cellular or circuit level is limited. Using a forward genetic approach to generate loss of auditory function in zebrafish larvae, we identified a recessive mutation in split ends ( spen ), which encodes a transcription factor implicated in neurodevelopmental disorders in humans. We found that auditory sensorimotor deficits in spen mutants are associated with chronically elevated calcium levels in hindbrain radial astrocytes. Astrocytes have been previously implicated in the integration of neuronal inputs that alter arousal states and sensorimotor behavior. Inhibition of noradrenergic receptors, which are expressed in astrocytes, lowered calcium levels in mutant radial astrocytes and, notably, restored behavioral responses to sound in spen mutants. Our study highlights a role for noradrenergic signaling in spen -related disease and points to potential therapeutic approaches for pediatric patients.

Mutations in human VPS4A are associated with neurodevelopmental defects, including motor delays and defective muscle tone. VPS4A encodes a AAA-ATPase required for membrane scission, but how mutations in VPS4A lead to impaired control of motor function is not known. Here we identified a mutation in zebrafish vps4a , T248I, that affects sensorimotor transformation. Biochemical analyses indicate that the T248I mutation reduces the ATPase activity of Vps4a and disassembly of ESCRT filaments, which mediate membrane scission. Consistent with the role for Vps4a in exosome biogenesis, vps4a ^T248I larvae have enlarged endosomal compartments in the CNS and decreased numbers of circulating exosomes in brain ventricles. Resembling the central form of hypotonia in VPS4A patients, motor neurons and muscle cells are functional in mutant zebrafish. Both somatosensory and vestibular inputs robustly evoke tail and eye movements, respectively. In contrast, optomotor responses, vestibulospinal, and acoustic startle reflexes are absent or strongly impaired in vps4a ^T248I larvae, indicating a greater sensitivity of these circuits to the T248I mutation. ERG recordings revealed intensity-dependent deficits in the retina, and in vivo calcium imaging of the auditory pathway identified a moderate reduction in afferent neuron activity, partially accounting for the severe motor impairments in mutant larvae. Further investigation of central pathways in vps4a ^T248I mutants showed that activation of descending vestibulospinal and midbrain motor command neurons by sensory cues is strongly reduced. Our results suggest that defects in sensorimotor transformation underlie the profound yet selective effects on motor reflexes resulting from the loss of membrane scission mediated by Vps4a.

Mutations in human VPS4A are associated with neurodevelopmental defects, including motor delays and defective muscle tone. VPS4A encodes a AAA-ATPase that is required for membrane scission, but how mutations in VPS4A lead to impaired control of motor function is not known. Here we identified a mutation in zebrafish vps4a , T248I, that affects sensorimotor transformation. In biochemical experiments we show that the T248I mutation reduces the ATPase activity of Vps4a and disassembly of its substrate, ESCRT filaments, which mediate membrane scission. Consistent with the established role for Vps4a in the endocytic pathway and exosome biogenesis, vps4a ^T248I mutants have enlarged endosomal compartments in the CNS and decreased numbers of circulating exosomes. Resembling the central form of hypotonia in human VPS4A patients, motor neurons and muscle cells are unaffected in mutant zebrafish as touch sensitivity in intact. Unlike somatosensory function, optomotor responses, vestibulospinal (VS), and acoustic startle reflexes are severely impaired in vps4a ^T248I mutants, indicating a greater sensitivity of these circuits to the T248I mutation. ERG recordings indicate that visual ability is largely reduced in the mutants, however, in vivo imaging of tone-evoked responses in the inner ear and ascending auditory pathway show comparable activity. Further investigation of central pathways in vps4a ^T248I mutants revealed that sensory cues failed to fully activate neurons in the VS and medial longitudinal fasciculus (MLF) nuclei that directly innervate motor neurons. Our results suggest that a defect in sensorimotor transformation underlies the profound yet selective effects on motor reflexes resulting from the loss of membrane scission mediated by Vps4a.

Ciliated sensory cells such as photo- and olfactory receptors employ multiple types of opsins or hundreds of unique olfactory G-protein coupled receptors to respond to various wavelengths of light or odorants. With respect to hearing and balance, the mechanotransduction machinery involves fewer variants; however, emerging evidence suggests that specialization occurs at the molecular level. To address how the mechanotransduction complex varies in the inner ear, we characterized the expression of paralogous genes that encode components required for mechanotransduction in zebrafish hair cells using RNA-FISH and bioinformatic analysis. Our data indicate striking zonal differences in the expression of two components of the mechanotransduction complex which are known to physically interact, the transmembrane channel-like 1 and 2 (tmc1/2) family members and the calcium and integrin binding 2 and 3 (cib2/3) paralogues. tmc1, tmc2b, and cib3 are largely expressed in peripheral or extrastriolar hair cells, whereas tmc2a and cib2 are enriched in central or striolar hair cells. In addition, a gene implicated in deaf-blindness, ush1c, is highly enriched in a subset of extrastriolar hair cells. These results indicate that specific combinations of these components may optimize responses to mechanical stimuli in subtypes of sensory receptors within the inner ear.

Information about dynamic head motion is conveyed by a central striolar zone of vestibular hair cells and afferent neurons in the inner ear. How vestibular hair cells are tuned to transduce dynamic stimuli at the molecular level is not well understood. Here we take advantage of the differential expression pattern of tmc1 , tmc2a , and tmc2b , which encode channel subunits of the mechanotransduction complex in zebrafish vestibular hair cells. To test the role of various combinations of Tmc subunits in transducing dynamic head movements, we measured reflexive eye movements induced by high-frequency stimuli in single versus double tmc mutants. We found that Tmc2a function correlates with the broadest range of frequency sensitivity, whereas Tmc2b mainly contributes to lower-frequency responses. Tmc1 , which is largely excluded from the striolar zone, plays a minor role in sensing lower-frequency stimuli. Our study suggests that the Tmc subunits impart functional differences to the mechanotransduction of dynamic stimuli. Significance Statement Information about dynamic head movements is transmitted by sensory receptors, known as hair cells, in the labyrinth of the inner ear. The sensitivity of hair cells to fast or slow movements of the head differs according to cell type. Whether the mechanotransduction complex that converts mechanical stimuli into electrical signals in hair cells participates in conveying frequency information is not clear. Here we find that the transmembrane channel-like 1/2 genes, which encode a central component of the complex, are differentially expressed in the utricle and contribute to frequency sensitivity in zebrafish.

Inherited forms of deafness account for a sizable portion of hearing loss among children and adult populations. Many patients with sensorineural deficits have pathological manifestations in the peripheral auditory system, the inner ear. Within the hearing organ, the cochlea, most of the genetic forms of hearing loss involve defects in sensory detection and to some extent, signaling to the brain via the auditory cranial nerve. This review focuses on peripheral forms of hereditary hearing loss and how these impairments can be studied in diverse animal models or patient-derived cells with the ultimate goal of using the knowledge gained to understand the underlying biology and treat hearing loss.

The lipid phosphatase synaptojanin 1 (synj1) is required for the disassembly of clathrin coats on endocytic compartments. In neurons such activity is necessary for the recycling of endocytosed membrane into synaptic vesicles. Mutations in zebrafish synj1 have been shown to disrupt the activity of ribbon synapses in sensory hair cells. After prolonged mechanical stimulation of hair cells, both phase locking of afferent nerve activity and the recovery of spontaneous release of synaptic vesicles are diminished in synj1 mutants. Presumably as a behavioral consequence of these synaptic deficits, synj1 mutants are unable to maintain an upright posture. To probe vestibular function with respect to postural control in synj1 mutants, we developed a method for assessing the vestibulospinal reflex (VSR) in larvae. We elicited the VSR by rotating the head and recorded tail movements. As expected, the VSR is completely absent in pcdh15a and lhfpl5a mutants that lack inner ear function. Conversely, lhfpl5b mutants, which have a selective loss of function of the lateral line organ, have normal VSRs, suggesting that the hair cells of this organ do not contribute to this reflex. In contrast to mechanotransduction mutants, the synj1 mutant produces normal tail movements during the initial cycles of rotation of the head. Both the amplitude and temporal aspects of the response are unchanged. However, after several rotations, the VSR in synj1 mutants was strongly diminished or absent. Mutant synj1 larvae are able to recover, but the time required for the reappearance of the VSR after prolonged stimulation is dramatically increased in synj1 mutants. Collectively, the data demonstrate a behavioral correlate of the synaptic defects caused by the loss of synj1 function. Our results suggest that defects in synaptic vesicle recycling give rise to fatigue of ribbons synapses and possibly other synapses of the VS circuit, leading to the loss of postural control.

Detection of sound and head movement requires mechanoelectrical transduction (MET) channels at tips of hair-cell stereocilia. In vertebrates, the transmembrane channel-like (TMC) proteins TMC1 and TMC2 fulfill critical roles in MET, and substantial evidence implicates these TMCs as subunits of the MET channel. To identify developmental and functional roles of this Tmc subfamily in the zebrafish inner ear, we tested the effects of truncating mutations intmc1,tmc2a, andtmc2bonin vivomechanosensation at the onset of hearing and balance, before gender differentiation. We find thattmc1/2a/2btriple-mutant larvae cannot detect sound or orient with respect to gravity. They lack acoustic-evoked behavioral responses, vestibular-induced eye movements, and hair-cell activity as assessed with FM dye labeling and microphonic potentials. Despite complete loss of hair-cell function,tmctriple-mutant larvae retain normal gross morphology of hair bundles and proper trafficking of known MET components Protocadherin 15a (Pcdh15a), Lipoma HMGIC fusion partner-like 5 (Lhfpl5), and Transmembrane inner ear protein (Tmie). Transgenic, hair cell-specific expression of Tmc2b-mEGFP rescues the behavioral and physiological deficits intmctriple mutants. Results fromtmcsingle and double mutants evince a principle role for Tmc2a and Tmc2b in hearing and balance, respectively, whereas Tmc1 has lower overall impact. Our experiments reveal that, in developing cristae, hair cells stratify into an upper, Tmc2a-dependent layer of teardrop-shaped cells and a lower, Tmc1/2b-dependent tier of gourd-shaped cells. Collectively, our genetic evidence indicates that auditory/vestibular end organs and subsets of hair cells therein rely on distinct combinations of Tmc1/2a/2b.SIGNIFICANCE STATEMENTWe assessed the effects oftmc1/2a/2btruncation mutations on mechanoelectrical transduction (MET) in the inner-ear hair cells of larval zebrafish.tmctriple mutants lacked behavioral responses to sound and head movements, while further assays demonstrated no observable mechanosensitivity in thetmc1/2a/2btriple mutant inner ear. Examination oftmcdouble mutants revealed major contributions from Tmc2a and Tmc2b to macular function; however, Tmc1 had less overall impact. FM labeling of lateral cristae intmcdouble mutants revealed the presence of two distinct cell types, an upper layer of teardrop-shaped cells that rely on Tmc2a, and a lower layer of gourd-shaped cells that rely on Tmc1/2b.

1 Hair cells sense and transmit auditory, vestibular, and hydrodynamic information by converting mechanical stimuli into electrical signals. This process of mechano-electrical transduction (MET) requires a mechanically-gated channel localized in the apical stereocilia of hair cells. In mice, lipoma HMGIC fusion partner-like 5 (LHFPL5) acts as an auxiliary subunit of the MET channel whose primary role is to correctly localize PCDH15 and TMC1 to the mechanotransduction complex. Zebrafish have two lhfpl5 genes ( lhfpl5a and lhfpl5b ), but their individual contributions to MET channel assembly and function have not been analyzed. Here we show that the zebrafish lhfpl5 genes are expressed in discrete populations of hair cells: lhfpl5a expression is restricted to auditory and vestibular hair cells in the inner ear, while lhfpl5b expression is specific to hair cells of the lateral line organ. Consequently, lhfpl5a mutants exhibit defects in auditory and vestibular function, while disruption of lhfpl5b affects hair cells only in the lateral line neuromasts. In contrast to previous reports in mice, localization of Tmc1 does not depend upon Lhfpl5 function in either the inner ear or lateral line organ. In both lhfpl5a and lhfpl5b mutants, GFP-tagged Tmc1 and Tmc2b proteins still localize to the stereocilia of hair cells. Using a stably integrated GFP-Lhfpl5a transgene, we show that the tip link cadherins Pcdh15a and Cdh23, along with the Myo7aa motor protein, are required for correct Lhfpl5a localization at the tips of stereocilia. Our work corroborates the evolutionarily conserved co-dependence between Lhfpl5 and Pcdh15, but also reveals novel requirements for Cdh23 and Myo7aa to correctly localize Lhfpl5a. In addition, our data suggest that targeting of Tmc1 and Tmc2b proteins to stereocilia in zebrafish hair cells occurs independently of Lhfpl5 proteins.