Shaker-1 Mice Research Articles

Third-generation lentiviral gene therapy rescues function in a mouse model of Usher 1B

Usher syndrome 1B (USH1B) is a devastating genetic disorder with congenital deafness, loss of balance, and blindness caused by mutations in the myosin-VIIa (MYO7A) gene, for which there is currently no cure. We developed a gene therapy approach addressing the vestibulo-cochlear deficits of USH1B using a third-generation, high-capacity lentiviral vector system capable of delivering the large 6,645-bp MYO7A cDNA. Lentivirally delivered MYO7A and co-encoded dTomato were successfully expressed in the cochlear cell line HEI-OC1. In normal-hearing mice, both cochlea and the vestibular organ were efficiently transduced, and ectopic MYO7A overexpression did not show any adverse effects. In Shaker-1 mice, an USH1B disease model based on Myo7a mutation, cochlear and vestibular hair cells, the main inner ear cell types affected in USH1B, were successfully transduced. In homozygous mutant mice, delivery of MYO7A at postnatal day 16 resulted in a trend for partial recovery of auditory function and in strongly reduced balance deficits. Heterozygous mutant mice were found to develop severe hearing loss at 6months of age without balance deficits, and lentiviral MYO7A gene therapy completely rescued hearing to wild-type hearing thresholds. In summary, this study demonstrates improved hearing and balance function through lentiviral gene therapy in the inner ear.

Dual-AAV vector-mediated expression of MYO7A improves vestibular function in a mouse model of Usher syndrome 1B

Usher syndrome is the most common cause of deafness-blindness in the world. Usher syndrome type 1B (USH1B) is associated with mutations in MYO7A. Patients with USH1B experience deafness, blindness, and vestibular dysfunction. In this study, we applied adeno-associated virus (AAV)-mediated gene therapy to the shaker-1 (Myo7a4626SB/4626SB) mouse, a model of USH1B. The shaker-1 mouse has a nonsense mutation in Myo7a, is profoundly deaf throughout life, and has significant vestibular dysfunction. Because of the ∼6.7-kb size of the MYO7A cDNA, a dual-AAV approach was used for gene delivery,which involves splitting human MYO7A cDNA into 5' and 3' halves and cloning them into two separate AAV8(Y733F) vectors. When MYO7A cDNA was delivered to shaker-1 inner ears using the dual-AAV approach, cochlear hair cell survival was improved. However, stereocilium organization and auditory function were not improved. In contrast, in the vestibular system, dual-AAV-mediated MYO7A delivery significantly rescued hair cell stereocilium morphology and improved vestibular function, as reflected in a reduction of circling behavior and improved vestibular sensory-evoked potential (VsEP) thresholds. Our data indicate that dual-AAV-mediated MYO7A expression improves vestibular function in shaker-1 mice and supports further development of this approach for the treatment of disabling dizziness from vestibular dysfunction in USH1B patients.

Myosin-XVa Controls Both Staircase Architecture and Diameter Gradation of Stereocilia Rows in the Auditory Hair Cell Bundles.

Mammalian hair cells develop their mechanosensory bundles through consecutive phases of stereocilia elongation, thickening, and retraction of supernumerary stereocilia. Many molecules involved in stereocilia elongation have been identified, including myosin-XVa. Significantly less is known about molecular mechanisms of stereocilia thickening and retraction. Here, we used scanning electron microscopy (SEM) to quantify postnatal changes in number and diameters of the auditory hair cell stereocilia in shaker-2 mice (Myo15sh2) that lack both "long" and "short" isoforms of myosin-XVa, and in mice lacking only the "long" myosin-XVa isoform (Myo15∆N). Previously, we observed large mechanotransduction current in young postnatal inner (IHC) and outer (OHC) hair cells of both these strains. Stereocilia counts showed nearly identical developmental retraction of supernumerary stereocilia in control heterozygous, Myo15sh2/sh2, and Myo15∆N/∆N mice, suggesting that this retraction is largely unaffected by myosin-XVa deficiency. However, myosin-XVa deficiency does affect stereocilia diameters. In control, the first (tallest) and second row stereocilia grow in diameter simultaneously. However, the third row stereocilia in IHCs grow only until postnatal day 1-2 and then become thinner.In OHCs, they also grow slower than taller stereocilia, forming a stereocilia diameter gradation within a hair bundle. The sh2 mutation disrupts this gradation and makes all stereocilia nearly identical in thickness in both IHCs and OHCs, with only subtle residual diameter differences. All Myo15sh2/sh2 stereocilia grow postnatally including the third row, which is not a part of normal development. Serial sections with focused ion beam (FIB)-SEM confirmed that diameter changes of Myo15sh2/sh2 IHC and OHC stereocilia resulted from corresponding changes of their actin cores. In contrast to Myo15sh2/sh2, Myo15∆N/∆N hair cells develop prominent stereocilia diameter gradation. Thus, besides building the staircase, the short isoform of myosin-XVa is essential for controlling the diameter of the third row stereocilia and formation of the stereocilia diameter gradation in a hair bundle.

A Novel Mouse Model of MYO7A USH1B Reveals Auditory and Visual System Haploinsufficiencies.

Usher’s syndrome is the most common combined blindness–deafness disorder with USH1B, caused by mutations in MYO7A, resulting in the most severe phenotype. The existence of numerous, naturally occurring shaker1 mice harboring variable MYO7A mutations on different genetic backgrounds has complicated the characterization of MYO7A knockout (KO) and heterozygote mice. We generated a novel MYO7A KO mouse (Myo7a–/–) that is easily genotyped, maintained, and confirmed to be null for MYO7A in both the eye and inner ear. Like USH1B patients, Myo7a–/– mice are profoundly deaf, and display near complete loss of inner and outer cochlear hair cells (HCs). No gross structural changes were observed in vestibular HCs. Myo7a–/– mice exhibited modest declines in retinal function but, unlike patients, no loss of retinal structure. We attribute the latter to differential expression of MYO7A in mouse vs. primate retina. Interestingly, heterozygous Myo7a+/– mice had reduced numbers of cochlear HCs and concomitant reductions in auditory function relative to Myo7a+/+ controls. Notably, this is the first report that loss of a single Myo7a allele significantly alters auditory structure and function and suggests that audiological characterization of USH1B carriers is warranted. Maintenance of vestibular HCs in Myo7a–/– mice suggests that gene replacement could be used to correct the vestibular dysfunction in USH1B patients. While Myo7a–/– mice do not exhibit sufficiently robust retinal phenotypes to be used as a therapeutic outcome measure, they can be used to assess expression of vectored MYO7A on a null background and generate valuable pre-clinical data toward the treatment of USH1B.

The shaker-1 mouse myosin VIIa deafness mutation results in a severely reduced rate of the ATP hydrolysis step

Mutations in the MYO7A gene, encoding the motor protein myosin VIIa, can cause Usher 1B, a deafness/blindness syndrome in humans, and the shaker-1 phenotype, characterized by deafness, head tossing, and circling behavior, in mice. Myosin VIIa is responsible for tension bearing and the transduction mechanism in the stereocilia and for melanosome transport in the retina, in line with the phenotypic outcomes observed in mice. However, the effect of the shaker-1 mutation, a R502P amino acid substitution, on the motor function is unclear. To explore this question, we determined the kinetic properties and the effect on the filopodial tip localization of the recombinant mouse myosin VIIa-5IQ-SAH R502P (myoVIIa-sh1) construct. Interestingly, although residue 502 is localized to a region thought to be involved in interacting with actin, the kinetic parameters for actin binding changed only slightly for the mutant construct. However, the rate constant for ATP hydrolysis (k+H + k-H) was reduced by ∼200-fold from 12 s-1 to 0.05 s-1, making the hydrolysis step the rate-limiting step of the ATPase cycle in the presence and absence of actin. Given that wild-type mouse myosin VIIa is a slow, high-duty ratio, monomeric motor, this altered hydrolysis rate would reduce activity to extremely low levels. Indeed, the translocation to the filopodial tips was hampered by the diminished motor function of a dimeric construct of the shaker-1 mutant. We conclude that the diminished motor activity of this mutant is most likely responsible for impaired hearing in the shaker-1 mice.

Descending projections from the inferior colliculus to medial olivocochlear efferents: Mice with normal hearing, early onset hearing loss, and congenital deafness

Auditory efferent neurons reside in the brain and innervate the sensory hair cells of the cochlea to modulate incoming acoustic signals. Two groups of efferents have been described in mouse and this report will focus on the medial olivocochlear (MOC) system. Electrophysiological data suggest the MOC efferents function in selective listening by differentially attenuating auditory nerve fiber activity in quiet and noisy conditions. Because speech understanding in noise is impaired in age-related hearing loss, we asked whether pathologic changes in input to MOC neurons from higher centers could be involved. The present study investigated the anatomical nature of descending projections from the inferior colliculus (IC) to MOCs in 3-month old mice with normal hearing, and in 6-month old mice with normal hearing (CBA/CaH), early onset progressive hearing loss (DBA/2), and congenital deafness (homozygous Shaker-2). Anterograde tracers were injected into the IC and retrograde tracers into the cochlea. Electron microscopic analysis of double-labelled tissue confirmed direct synaptic contact from the IC onto MOCs in all cohorts. These labelled terminals are indicative of excitatory neurotransmission because they contain round synaptic vesicles, exhibit asymmetric membrane specializations, and are co-labelled with antibodies against VGlut2, a glutamate transporter. 3D reconstructions of the terminal fields indicate that in normal hearing mice, descending projections from the IC are arranged tonotopically with low frequencies projecting laterally and progressively higher frequencies projecting more medially. Along the mediolateral axis, the projections of DBA/2 mice with acquired high frequency hearing loss were shifted medially towards expected higher frequency projecting regions. Shaker-2 mice with congenital deafness had a much broader spatial projection, revealing abnormalities in the topography of connections. These data suggest that loss in precision of IC directed MOC activation could contribute to impaired signal detection in noise.

EIAV-Based Retinal Gene Therapy in the shaker1 Mouse Model for Usher Syndrome Type 1B: Development of UshStat

Usher syndrome type 1B is a combined deaf-blindness condition caused by mutations in the MYO7A gene. Loss of functional myosin VIIa in the retinal pigment epithelia (RPE) and/or photoreceptors leads to blindness. We evaluated the impact of subretinally delivered UshStat, a recombinant EIAV-based lentiviral vector expressing human MYO7A, on photoreceptor function in the shaker1 mouse model for Usher type 1B that lacks a functional Myo7A gene. Subretinal injections of EIAV-CMV-GFP, EIAV-RK-GFP (photoreceptor specific), EIAV-CMV-MYO7A (UshStat) or EIAV-CMV-Null (control) vectors were performed in shaker1 mice. GFP and myosin VIIa expression was evaluated histologically. Photoreceptor function in EIAV-CMV-MYO7A treated eyes was determined by evaluating α-transducin translocation in photoreceptors in response to low light intensity levels, and protection from light induced photoreceptor degeneration was measured. The safety and tolerability of subretinally delivered UshStat was evaluated in macaques. Expression of GFP and myosin VIIa was confirmed in the RPE and photoreceptors in shaker1 mice following subretinal delivery of the EIAV-CMV-GFP/MYO7A vectors. The EIAV-CMV-MYO7A vector protected the shaker1 mouse photoreceptors from acute and chronic intensity light damage, indicated by a significant reduction in photoreceptor cell loss, and restoration of the α-transducin translocation threshold in the photoreceptors. Safety studies in the macaques demonstrated that subretinal delivery of UshStat is safe and well-tolerated. Subretinal delivery of EIAV-CMV-MYO7A (UshStat) rescues photoreceptor phenotypes in the shaker1 mouse. In addition, subretinally delivered UshStat is safe and well-tolerated in macaque safety studies These data support the clinical development of UshStat to treat Usher type 1B syndrome.

Myosin 7aa−/− mutant zebrafish show mild photoreceptor degeneration and reduced electroretinographic responses

Mutations in myosin VIIa (MYO7A) cause Usher Syndrome 1B (USH1B), a disease characterized by the combination of sensorineural hearing loss and visual impairment termed retinitis pigmentosa (RP). Although the shaker-1 mouse model of USH1B exists, only minor defects in the retina have been observed during its lifespan. Previous studies of the zebrafish mariner mutant, which also carries a mutation in myo7aa, revealed balance and hearing defects in the mutants but the retinal phenotype has not been described. We found elevated cell death in the outer nuclear layer (ONL) of myo7aa−/− mutants. While myo7aa−/− mutants retained visual behaviors in the optokinetic reflex (OKR) assay, electroretinogram (ERG) recordings revealed a significant decrease in both a- and b-wave amplitudes in mutant animals, but not a change in ERG threshold sensitivity. Immunohistochemistry showed mislocalization of rod and blue cone opsins and reduced expression of rod-specific markers in the myo7aa−/− ONL, providing further evidence that the photoreceptor degeneration observed represents the initial stages of the RP. Further, constant light exposure resulted in widespread photoreceptor degeneration and the appearance of large holes in the retinal pigment epithelium (RPE). No differences were observed in the retinomotor movements of the photoreceptors or in melanosome migration within the RPE, suggesting that myo7aa−/− does not function in these processes in teleosts. These results indicate that the zebrafish myo7aa−/− mutant is a useful animal model for the RP seen in humans with USH1B.

High-Speed Hopping Probe Scanning Ion Conductance Microscopy

Vertically protruding stereocilia on the apical surface of the inner ear hair cells represent the ultimate challenge for scanning probe imaging. We have previously imaged these structures, which are largely unresolvable by optical microscopy, using hopping probe scanning ion conductance microscopy (HPSICM) in glutaraldehyde-fixed mammalian auditory hair cells (Novak et al. Nat Methods, 2009). However, it required a considerable amount of time (∼44 min per bundle of ∼8x8 μm). To study live cells, we needed to significantly increase the speed of imaging.For Z-movement we have now used a faster piezo assembly with a resonant frequency of ∼18 kHz, similar to the ones we used for hair bundle deflection. Despite having a less sensitive strain gauge sensor, the vertical resolution of the system remained the same (∼5nm). After adjusting the proportional-integral-derivative controller of the Z-scanner (∼50μs delay) and increasing the speed of approach, we were able to obtain high-resolution images of live hair cell bundles at a frame rate of 12 min/bundle or less.We tested the performance of the improved HPSICM system in live rat inner hair cells (IHC) and showed, for the first time in live cells, the presence of characteristic stereocilia features at an X-Y resolution of ∼11nm. We also imaged IHC bundles from the Shaker2 and Whirler mice due to their short stereocilia with abundant stereocilia links (typically ∼5nm in diameter and ∼100-300nm in length). We confirmed the reproducibility of links in continuous time-lapse scanning and also their absence after chemical disruption with BAPTA-buffered Ca2+-free medium.Our results demonstrate that the improved HPSICM technique successfully visualizes the extremely convoluted surface of stereocilia in live auditory hair cells at a high resolution and a faster speed.Supported by NIDCD/NIH (R01DC008861).

Photoreceptors in whirler mice show defective transducin translocation and are susceptible to short-term light/dark changes-induced degeneration

Usher syndrome combines congenital hearing loss and retinitis pigmentosa (RP). Mutations in the whirlin gene (DFNB31/WHRN) cause a subtype of Usher syndrome (USH2D). Whirler mice have a defective whirlin gene. They have inner ear defects but usually do not develop retinal degeneration. Here we report that, in whirler mouse photoreceptors, the light-activated rod transducin translocation is delayed and its activation threshold is shifted to a higher level. Rhodopsin mis-localization is observed in rod inner segments. Continuous moderate light exposure can induce significant rod photoreceptor degeneration. Whirler mice reared under a 1500 lux light/dark cycle also develop severe photoreceptor degeneration. Previously, we have reported that shaker1 mice, a USH1B model, show moderate light-induced photoreceptor degeneration with delayed transducin translocation. Here, we further show that, in both whirler and shaker1 mice, short-term moderate light/dark changes can induce rod degeneration as severe as that induced by continuous light exposure. The results from shaker1 and whirler mice suggest that defective transducin translocation may be functionally related to light-induced degeneration, and these two symptoms may be caused by defects in Usher protein function in rods. Furthermore, these results indicate that both Usher syndrome mouse models possess a light-induced retinal phenotype and may share a closely related pathobiological mechanism.

The Shaker-1 Mouse Myosin VIIa has a Drastically Compromised ATP Hydrolysis Mechanism

The shaker-1 mice carry a missense mutation in the myosin VIIa gene (R502P) and exhibit deafness, circling behavior and mild retinal degeneration. Developmental and electrophysiological studies have shown that the cochlear function is altered in these mice. We carried out a detailed kinetic analysis of the baculoviral expressed truncated shaker-1 construct to assess whether the ATP hydrolysis mechanism is altered in these mice. The basal steady-state rate (0.03 s−1) is activated by actin only 1.5 -fold. ATP binding is relatively fast (1.22 uM−1s−1) and the dissociation of the myosin from the actomyosin complex is the same as for the wild type myosin VIIa (400 s−1). ADP binding to (1.8 uM−1s−1) and the dissociation (1.5 s−1) from the actomyosin complex are also similar to the wild type. Phosphate release from the M-ADP-Pi complex is slow (0.07 s−1) and is not activated by actin. Quenched flow experiments show that there is no Pi burst and the rate of the hydrolysis is 0.12 s−1. The R502 residue is in a small loop that is in the middle of an α-helical region which aligns with the switch-2 helix and is the continuation of the relay-loop. The sequence in the loop is changed from NRPM to NPPM which is likely to alter the conformation of the helix-loop-helix structure that is relatively straight in all myosins.Consequently the rate constants of the steps leading into the power stroke (hydrolysis and phosphate dissociation) are reduced greater than 10-fold whereas the post-power stoke steps are essentially unchanged. As a result the shaker-1 mutant protein has a severely compromised ATP hydrolysis mechanism in which < 10% of the normal fraction of the force-producing crossbridges are formed.Supported by NIH/RO3DC009335.

Subretinal Delivery of EIAV-based Lentiviral Vectors in the Shaker1 mouse model for Usher Syndrome Type 1B : Development of UshStat

Subretinal Delivery of EIAV-based Lentiviral Vectors in the Shaker1 mouse model for Usher Syndrome Type 1B : Development of UshStat

Moderate Light-Induced Degeneration of Rod Photoreceptors with Delayed Transducin Translocation inshaker1Mice

PURPOSE. Usher syndrome is characterized by congenital deafness associated with retinitis pigmentosa (RP). Mutations in the myosin VIIa gene (MYO7A) cause a common and severe subtype of Usher syndrome (USH1B). Shaker1 mice have mutant MYO7A. They are deaf and have vestibular dysfunction but do not develop photoreceptor degeneration. The goal of this study was to investigate abnormalities of photoreceptors in shaker1 mice. METHODS. Immunocytochemistry and hydroethidine-based detection of intracellular superoxide production were used. Photoreceptor cell densities under various conditions of light/dark exposures were evaluated. RESULTS. In shaker1 mice, the rod transducin translocation is delayed because of a shift of its light activation threshold to a higher level. Even moderate light exposure can induce oxidative damage and significant rod degeneration in shaker1 mice. Shaker1 mice reared under a moderate light/dark cycle develop severe retinal degeneration in less than 6 months. CONCLUSIONS. These findings show that, contrary to earlier studies, shaker1 mice possess a robust retinal phenotype that may link to defective rod protein translocation. Importantly, USH1B animal models are likely vulnerable to light-induced photoreceptor damage, even under moderate light.

Function of MYO7A in the Human RPE and the Validity of Shaker1 Mice as a Model for Usher Syndrome 1B

To investigate the function of MYO7A in human RPE cells and to test the validity of using shaker1 RPE in preclinical studies on therapies for Usher syndrome 1B by comparing human and mouse cells. MYO7A was localized by immunofluorescence. Primary cultures of human and mouse RPE cells were used to measure melanosome motility and rod outer segment (ROS) phagocytosis and digestion. MYO7A was knocked down in the human RPE cells by RNAi to test for a mutant phenotype in melanosome motility. The distribution of MYO7A in the RPE of human and mouse was found to be comparable, both in vivo and in primary cultures. Primary cultures of human RPE cells phagocytosed and digested ROSs with kinetics comparable to that of primary cultures of mouse RPE cells. Melanosome motility was also comparable, and, after RNAi knockdown, consisted of longer-range fast movements characteristic of melanosomes in shaker1 RPE. The localization and function of MYO7A in human RPE cells is comparable to that in mouse RPE cells. Although shaker1 retinas do not undergo degeneration, correction of mutant phenotypes in the shaker1 RPE represents a valid preclinical test for potential therapeutic treatments.

Usher syndromes due to MYO7A, PCDH15, USH2A or GPR98 mutations share retinal disease mechanism

Usher syndrome (USH) is a genetically heterogeneous group of autosomal recessive deaf-blinding disorders. Pathophysiology leading to the blinding retinal degeneration in USH is uncertain. There is evidence for involvement of the photoreceptor cilium, photoreceptor synapse, the adjacent retinal pigment epithelium (RPE) cells, and the Crumbs protein complex, the latter implying developmental abnormalities in the retina. Testing hypotheses has been difficult in murine USH models because most do not show a retinal degeneration phenotype. We defined the retinal disease expression in vivo in human USH using optical imaging of the retina and visual function. In MYO7A (USH1B), results from young individuals or those at early stages indicated the photoreceptor was the first detectable site of disease. Later stages showed photoreceptor and RPE cell pathology. Mosaic retinas in Myo7a-deficient shaker1 mice supported the notion that the mutant photoreceptor phenotype was cell autonomous and not secondary to mutant RPE. Humans with PCDH15 (USH1F), USH2A or GPR98 (USH2C) had a similar retinal phenotype to MYO7A (USH1B). There was no evidence of photoreceptor synaptic dysfunction and no dysplastic phenotype as in CRB1 (Crumbs homologue1) retinopathy. The results point to the photoreceptor cell as the therapeutic target for USH treatment trials, such as MYO7A somatic gene replacement therapy.

Assessment of myosin II, Va, VI and VIIa loss of function on endocytosis and endocytic vesicle motility in bone marrow‐derived dendritic cells

An essential feature of dendritic cell immune surveillance is endocytic sampling of the environment for non-self antigens primarily via macropinocytosis and phagocytosis. The role of several members of the myosin family of actin based molecular motors in dendritic cell endocytosis and endocytic vesicle movement was assessed through analysis of dendritic cells derived from mice with functionally null myosin mutations. These include the dilute (myosin Va), Snell's waltzer (myosin VI) and shaker-1 (myosin VIIa) mouse lines. Non muscle myosin II function was assessed by treatment with the inhibitor, blebbistatin. Flow cytometric analysis of dextran uptake by dendritic cells revealed that macropinocytosis was enhanced in Snell's waltzer dendritic cells while shaker-1 and blebbistatin-treated cells were comparable to controls. Comparison of fluid phase uptake using pH insensitive versus pH sensitive fluorescent dextrans revealed that in dilute cells rates of uptake were normal but endosomal acidification was accelerated. Phagocytosis, as quantified by uptake of E. coli, was normal in dilute while dendritic cells from Snell's waltzer, shaker-1 and blebbistatin treated cells exhibited decreased uptake. Microtubule mediated movements of dextran-or transferrin-tagged endocytic vesicles were significantly faster in dendritic cells lacking myosin Va. Loss of myosin II, VI or VIIa function had no significant effects on rates of endocytic vesicle movement.

Functional Analysis of Slac2-c/MyRIP as a Linker Protein between Melanosomes and Myosin VIIa*[boxs

Slac2-c/MyRIP, an in vitro Rab27A- and myosin Va/VIIa-binding protein, has recently been proposed to regulate retinal melanosome transport in retinal pigment epithelium cells by directly linking melanosome-bound Rab27A and myosin VIIa; however, the exact function of Slac2-c in melanosome transport has never been clarified. In this study, we used melanosome transport in skin melanocytes as a model for retinal melanosome transport and analyzed the in vivo function of Slac2-c in melanosome transport by the ectopic expression of Slac2-c, together with myosin VIIa, in Slac2-a-depleted melanocytes. In vitro binding experiments revealed that myosin VIIa had a greater affinity for Slac2-c, compared with the binding affinity of myosin Va, and that the myosin VIIa-binding domain of Slac2-c is different from the previously characterized myosin Va-binding domain that is conserved between Slac2-a/melanophilin and Slac2-c. Consistent with this result, cyan fluorescent protein-tagged Slac2-c expressed in melanocytes was localized on melanosomes via the specific interaction with Rab27A and recruited co-expressed yellow fluorescent protein-tagged myosin VIIa to the melanosomes without interfering with the normal peripheral melanosome distribution, whereas when myosin VIIa alone was expressed in melanocytes, it was not localized on the melanosomes. Moreover, Slac2-c ectopically expressed in melanocytes did not rescue the perinuclear aggregation phenotype induced by the knockdown of endogenous Slac2-a with a specific small interfering RNA, whereas the expression of the Slac2-c x myosin VIIa complex supported the normal melanosome distribution in Slac2-a-depleted melanocytes, indicating that Slac2-c functions as a myosin VIIa receptor rather than a myosin Va receptor in melanosome transport. Based on these findings, we propose that Slac2-c acts as a functional myosin VIIa receptor and that the Rab27A.Slac2-c x myosin VIIa tripartite protein complex regulates the transport of retinal melanosomes in pigment epithelium cells.

Genetic heterogeneity in Usher syndrome.

Mutations in seven different genes have been associated with Usher syndrome, and an additional four loci have been mapped. The identified genes encode myosin VIIa, harmonin (a PDZ-domain protein), cadherin 23, protocadherin 15, sans (a scaffold-like protein), usherin and clarin. Three clinical types of Usher syndrome have been described: USH1 patients have severe to profound congenital hearing loss, vestibular dysfunction, and retinal degeneration beginning in childhood, those with USH2 have moderate to severe congenital hearing loss, normal vestibular function, and later onset of retinitis pigmentosa, and USH3 patients have progressive hearing loss, which distinguishes them from the other two types. The shaker-1, waltzer, Ames waltzer, and Jackson shaker mice provide murine models for four of the genetic forms of Usher syndrome. Ongoing studies are enabling early diagnosis of Usher syndrome in children who present with hearing loss, thus providing time to prepare for the onset of visual loss.

The role of Rab27a in the regulation of melanosome distribution within retinal pigment epithelial cells.

Melanosomes within the retinal pigment epithelium (RPE) of mammals have long been thought to exhibit no movement in response to light, unlike fish and amphibian RPE. Here we show that the distribution of melanosomes within the mouse RPE undergoes modest but significant changes with the light cycle. Two hours after light onset, there is a threefold increase in the number of melanosomes in the apical processes that surround adjacent photoreceptors. In skin melanocytes, melanosomes are motile and evenly distributed throughout the cell periphery. This distribution is due to the interaction with the cortical actin cytoskeleton mediated by a tripartite complex of Rab27a, melanophilin, and myosin Va. In ashen (Rab27a null) mice RPE, melanosomes are unable to move beyond the adherens junction axis and do not enter apical processes, suggesting that Rab27a regulates melanosome distribution in the RPE. Unlike skin melanocytes, the effects of Rab27a are mediated through myosin VIIa in the RPE, as evidenced by the similar melanosome distribution phenotype observed in shaker-1 mice, defective in myosin VIIa. Rab27a and myosin VIIa are likely to be required for association with and movement through the apical actin cytoskeleton, which is a prerequisite for entry into the apical processes.

遺伝性内耳障害モデルにおける前庭形態異常と遺伝子導入の試み―ｓｈａｋｅｒ‐２マウス，ｗａｌｔｚｉｎｇ ｇｕｉｎｅａ Ｐｉｇの検討から―

We reviewed the hair cell pathology of genetic hearing loss and circling behavior and discussed possibility of gene therapy in human genetic inner ear disease through genetic inner ear disease animal models. First, we focused on the cytocauds containing pathological actin filaments in the vestibular type 1 hair cell of waltzing guinea pigs (WGP) and in auditory and vestibular hair cells of shaker-2 mice. The cytocaud, which seems like to be an actin filaments ladder, penetrates the basement membrane and runs along nerve fiber. One possible explanation is that hair cells detach from the basement membrane during development but do not in WGP and shaker-2 mice. However, the molecular signals that are required for detachment are unknown.Furthermore, we tested the possibility of gene transudation in shaker-2 mice, the model of DFNB3, i.e. human deafness, in order to treat the hair cell pathology including cytocaud. Vestibular sensory epithelia of explant organ culture obtained from mouse pups at P1, P3, and P5 were transfected by adenovirus vector containing the bacterial lacZ in vitro and processed to detect the reporter gene expression. Vestibular hair cells can be transduced at all stages in shaker-2 mice. Our data demonstrate that differentiating hair cells in mouse explants can be virally transduced, suggesting that wild-type genes can be inserted into these cells to correct the phenotype. We need further investigations of gene therapy using animal in vivo data to discuss the possibility of clinical application for the genetic inner ear diseases.