Research Article 4509 Va is required for normal photoreceptor synaptic activity

Richard T. Libby1,*, Concepcion Lillo2, Junko Kitamoto2, David S. Williams2 and Karen P. Steel1,‡,§ 1MRC Institute of Hearing Research, University Park, Nottingham, NG7 2RD, UK 2Departments of Pharmacology and Neurosciences, UCSD School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093-0983, USA *Present address: The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609, USA ‡Present address: Wellcome Trust Sanger Institute, Hinxton, Cambridge, CB10 1SA, UK §Author for correspondence (e-mail: [email protected])

Accepted 14 May 2004 Journal of Cell Science 117, 4509-4515 Published by The Company of Biologists 2004 doi:10.1242/jcs.01316

Summary Myosin Va is an -based motor molecule, one of a large Va in synaptic function. Photoreceptor synapses in family of unconventional . In humans, mutations in neurologically affected myosin Va mutant mice have both MYO5A cause type 1 and Elejalde anatomical and physiological abnormalities. Thus, myosin syndrome, diseases characterized by pigmentation defects Va is required for normal photoreceptor signalling, and the prepubescent onset of severe neurological deficits suggesting that it might function in central nervous system that ultimately lead to a shortened lifespan. Mutations in synapses in general, with aberrant synaptic activity the Myo5a in mouse cause the dilute series of mouse potentially underlying the neurological defects observed in mutants, demonstrating that myosin Va is involved in dilute lethal mice and patients with Griscelli syndrome type pigmentation and neural function. Although the reason for 1 and . the pigmentation abnormalities is well understood, the role of myosin Va in neural function is not. Myosin Va has been found in synaptic terminals in the retina and brain. We Key Words: Unconventional myosin, Myosin Va, Photoreceptor, report here new physiological evidence for a role of myosin Synapse

Introduction distribution of along the dendrites of Myosin Va is an unconventional myosin (Kalhammer and melanocytes (Provance et al., 1996; Wu et al., 1997). Bahler, 2000) that functions as a processive actin-based motor Although mutations in myosin Va clearly cause severe molecule (Mehta et al., 1999). It is known to function in neurological disease in mammals, its function(s) in the central organelle and vesicle transport (Langford, 2002). In humans, nervous system (CNS) is not well understood. Myosin Va has mutations in MYO5A cause Griscelli syndrome type 1 and been found in abundance in pre- and post-synaptic terminals Elejalde syndrome, both rare autosomal recessive disorders in the retina and brain, suggesting a role in synaptic activity (Anikster et al., 2002; Bahadoran et al., 2003b; Menasche et (Bridgman, 1999; Evans et al., 1998; Prekeris and Terrian, al., 2002; Menasche et al., 2003; Pastural et al., 1997). In fact, 1997; Schlamp and Williams, 1996; Walikonis et al., 2000). Myosin Va appears to associate with synaptic vesicles, adding these two clinically defined diseases probably describe a single support to a role in synaptic transmission (Evans et al., 1998; disease (Anikster et al., 2002; Bahadoran et al., 2003b; Prekeris and Terrian, 1997). Also, an abnormal accumulation Ivanovich et al., 2001; Menasche et al., 2002). The two of presynaptic vesicles are present in cerebellar granular syndromes are characterized by hypopigmentation of the skin cells of dilute lethal mutants (Bridgman, 1999), but the and hair as well as severe neurological abnormalities. physiological consequences of these features are not known. In Neurological abnormalities are usually early onset and include an extensive analysis of hippocampal synaptic function in seizures, mental retardation and hypotonia. Griscelli syndrome dilute lethal mice, no functional defects were found in synaptic type 1 and Elejalde syndrome patients usually have a shortened transmission or plasticity (Schnell and Nicoll, 2001). Lethal life span owing to their neurological abnormalities (Bahadoran dilute mutants also have abnormal Purkinje cell smooth et al., 2003a; Duran-McKinster et al., 1999; Elejalde et al., endoplasmic reticulum and no inositol-triphosphate-receptor- 1979; Pastural et al., 1997; Pastural et al., 2000). The dilute laden reticular tubules in the postsynaptic spine of cerebellar series of mouse mutants is caused by mutations in Myo5a and, neurons (Dekker-Ohno et al., 1993; Petralia et al., 2002; as in humans, dysfunction of this gene leads to both pigment Takagishi et al., 1996). Although no abnormalities were found and neurological abnormalities. Some alleles have a diluted in either morphology or transmission at this synapse, long-term coat color only, whereas others show both diluted coat and synaptic depression was absent, suggesting an important role progressive ataxia and convulsions leading to early death for myosin Va neurophysiology (Miyata et al., 2000). (Huang et al., 1998a; Huang et al., 1998b; Mercer et al., 1991). In the retina, myosin Va is expressed predominantly in The pigmentation defect is believed to be due to abnormal photoreceptor cells. In photoreceptor cells myosin Va is 4510 Journal of Cell Science 117 (19) localized mainly to presynaptic terminals and, to a lesser Histology extent, to inner segments (Schlamp and Williams, 1996). The All eyes were harvested at least 3 hours after light onset and before subcellular localization of myosin Va suggests several roles in lights out. The eyes were fixed in 2% paraformaldehyde + 2% photoreceptor function, including in synaptic transmission. glutaraldehyde in 0.1 M cacodylate buffer, pH 7.6. Eyecups were We report here new physiological evidence for a role of processed for embedding in Epon 812 resin. Semithin sections (0.7 myosin Va in synaptic function. Electroretinography of µm) were obtained along the entire dorsoventral axis, passing dilute lethal mutants revealed prolonged implicit times through the optic nerve head, and stained with 0.25% toluidine blue and disproportionately-reduced b-wave amplitudes, and in 0.1% sodium borate. The number of photoreceptor cells was determined by counting the number of nuclei in an 85 µm length of ultrastructural analysis showed abnormal presynaptic ribbons retina at 0.5 mm intervals from the optic nerve head. Ultrathin in the photoreceptor cells. We propose that myosin Va is sections were mounted on copper grids and stained with uranyl required for normal photoreceptor synaptic morphology and acetate and lead citrate before observation in a Philips (model 208) synaptic physiology. electron microscope.

Materials and Methods Results Mice Electroretinography can be used to measure several parameters Myo5ad and Myo5ad-l mutants were originally obtained on an undefined genetic background from the MRC Mammalian Genetics of retinal physiology, including photoreceptor stimulation and Unit (Harwell, UK). The two mutations were maintained in a single synaptic activity (Peachey and Ball, 2003). The a-wave of the segregating stock. Maintenance of the stock was facilitated by the electroretinogram (ERG) is a result of photoreceptor activation visible recessive phenotype of the short ear (Bmp5se) mutation that is and the b-wave is the result of interneuron activity (Peachey very closely linked to the Myo5a and was carried on the same and Ball, 2003). ERGs were recorded from mice at postnatal as Myo5ad, allowing Myo5ad/Myo5ad-l mice to be day 19-25, when dilute lethal mice are clearly ataxic. ERG distinguished readily from Myo5ad/Myo5ad. Homozygous Myo5ad-l waveforms showed clear reductions in amplitudes of both a- mutants were recognized by their progressive ataxia, which begins and b-waves in dilute lethal homozygotes (Myo5ad-l/ about postnatal day 12 and is clearly visible by 2 weeks of age. d-l d-l Myo5a ), which have diluted coat color and neurological Myo5a mutants die between the third and fourth weeks of life from defects compared with Myo5ad/Myo5ad and Myo5ad/Myo5ad-l unknown causes. This phenotype is consistent with all known myosin Va lethal mutations (presumably, it is a null mutation) (Huang et al., mice, which have pigmentation defects alone (the two control 1998a; Huang et al., 1998b). The Myo7a4626SB mutation was genotypes were physiologically indistinguishable; Fig. 1A). maintained on a background comprising approximately 50% CBA/Ca, Measurements of a-wave amplitudes showed significant 50% BS with a small BALBc contribution (Libby and Steel, 2001), reductions of around 40% across a range of stimulus and homozygotes were distinguished by their characteristic head- intensities, whereas b-wave amplitudes were reduced by bobbing and circling behavior from about 2 weeks of age. Double around 50% (Fig. 1B). Light microscopy of retinas from these mutants were obtained by intercrossing, and the Myo7a4626SB mice revealed no obvious structural defects in the retina (Fig. genotype was established by molecular typing as described previously 1C). Photoreceptor outer segments did not appear to be any (Holme and Steel, 2002). The control groups included both d d d d-l shorter in the dilute lethal mutants, and photoreceptor cell Myo5a /Myo5a and Myo5a /Myo5a grouped together, because numbers were actually a little larger (by approximately 10%) there were no significant differences between these two genotypes for any of the parameters measured. Likewise, the control groups for the in the dilute lethal group compared with the non-lethal group cross with Myo7a4626SB included both +/Myo7a4626SB and +/+ mice, (data not shown). because no significant differences between these two genotypes were Inspection of the ERG waveform shape suggested that b- detected. All experiments were carried out in full compliance with UK waves were affected to a greater degree than a-waves in dilute Home Office regulations and in accordance with the ARVO Statement lethal mutants (Fig. 1). We measured implicit times (which for the Use of Animals in Ophthalmic and Vision Research. represent synaptic activity) for both a- and b-waves, and found significantly longer times for both waves in dilute lethal mutants, suggesting a deficiency in synaptic activity (Fig. Electroretinography 2A,B). Furthermore, b:a wave amplitude ratios were smaller in Electroretinography was performed as previously described (Libby et dilute lethal mutants than in the control group. This observation al., 2003; Libby and Steel, 2001). Mice were anaesthetized with urethane and the right pupil was then dilated with atropine and the suggests that transmission of activity from photoreceptors to mice dark adapted for 30 minutes. A series of flashes (photic bipolar and other inner retinal cells is less efficient than it stimulator PS22; Grass-Telefactor, Quincy, MA) over a 7.8 log unit should be (Fig. 2C). scale (in 0.6 log unit intervals) were presented to the mice. At the Photoreceptors have an unusual presynaptic specialization: lowest light intensities, 20 flashes were presented at three second a ribbon of electron-dense material surrounded by a cluster of intervals and the average response was recorded. As the light intensity synaptic vesicles. These ribbons are found mainly in cells increased, the number of flashes was reduced and the interval between that need to pass on information very rapidly, such as stimuli was increased so that the animal would not light adapt. At the photoreceptors and inner hair cells in the cochlea (Morgans, highest intensities, only two flashes were averaged and the inter- 2000). In photoreceptors, these ribbons are normally located at stimulus time was 1 minute. The unattenuated flash was 466 candela the presynaptic complex, immediately opposite the synaptic m–2. The a-wave amplitude was measured from the baseline to the lowest point of the initial negative deflection and the b-wave contact with bipolar and horizontal cell terminals (Fig. 3A). In amplitude was measured from the baseline or when the a-wave was dilute lethal mutants, the localization of the ribbons did not present from its trough to the highest maximum positive value. appear to be altered, but their morphology was abnormal. In Implicit times were measured from the time of the stimulus to the a- approximately half of all rod spherules, one end of the ribbon or b-wave maximum. or the entire ribbon was enlarged to give the ribbon a club-like Synaptic function of Myosin Va 4511

Fig. 1. Mutants in dilute lethal have severely disrupted electroretinographic responses. (A) Waveforms of electroretinograms from a dilute mouse (Myo5ad/Myo5ad) on the left and a dilute lethal mutant (Myo5ad-l/Myo5ad-l) on the right, at the maximum light intensity. The a-wave is the first negative (downward) deflection and the b-wave is the later positive deflection, with oscillatory potentials caused by retinal interneuron activity superimposed. The dilute lethal waveforms show reduced amplitudes of response and an anomalous shape caused by disproportionate reduction of the b-wave. Scale bar for both waveforms shown on left. (B) Response amplitudes ± standard error plotted as a function of light intensity: (left) a-waves; (right) b-waves. There is a significant difference between dilute and dilute lethal amplitudes for a-waves from 4.2 log units of attenuation (4.2-3.6, P<0.01; 3.3-0, P<0.001) and from 7.2 log units of attenuation for the b-waves (7.2-4.8, P<0.005; 4.2-0, P<0.001; see inset). At maximum flash intensity, a-wave amplitudes are reduced by 38% and b-wave amplitudes by 51% in Myo5ad-l/Myo5ad-l (n=12) compared with Myo5ad/Myo5ad or d-l (n=12; includes six Myo5ad/Myo5ad and six Myo5ad/Myo5d-l; there was no significant difference between these two genotypes for any electroretinographic parameter analysed, P>0.25 for all). All mice were between postnatal days 19 and 25 (both groups averaged 21 days of age), ages when the Myo5ad-l/Myo5ad-l are clearly afflicted with the neurological phenotype. (C) Light microscopy of retinas from a Myo5ad/Myo5ad dilute control (left) and a Myo5ad-l/Myo5ad-l dilute lethal mutant (right), at 20 days of age. No obvious differences were seen in outer segment length or any other feature of gross structure. Scale bar, 10 µm.

or completely circular profile (Fig. 3B,E; >95% of the ribbons behavior, which included head bobbing and circling as well in control retinas had a normal ribbon shape). Many of the rod as progressive ataxia and paralysis. ERG a- and b-wave spherules with normal-looking ribbons were still abnormal. In responses from the double mutants were reduced to around these spherules, clusters of presumed synaptic vesicles were 40% and 50%, respectively, of the level of responses of the ectopically located, within the spherule but not opposite the control group (Fig. 4). The same reduction in a- and b- synapse (Fig. 3C,D,F). We also saw abnormal ribbons and wave response amplitude was measured in dilute lethal ectopic clusters of vesicles in cone photoreceptors (data not homozygotes that were either homozygous mutant, shown). The ribbon synapses appeared normal in the non-lethal heterozygous or wild type at the Myo7a locus, suggesting dilute genotypes. Moreover, the ribbon synapses of bipolar that there was no additive effect. Mice that were homozygous cells, in the inner plexiform layer, appeared normal in dilute for Myo7a4626SB but carried at least one wild-type allele at lethal and non-lethal mutants (Fig. 3G). the Myo5a locus showed a 20% reduction in both a- and b- Myosin Va is one of several unconventional myosin waves (Fig. 4), similar to the reduction in Myo7a mutants molecules expressed in the retina. We have demonstrated that we have reported previously (Libby and Steel, 2001). previously that ERG amplitudes are reduced by around 20% Finally, the control group included a mixture of mice in Myo7a4626SB mutants (Libby and Steel, 2001), and that heterozygous or wild type at both the Myo5a and Myo7a loci, myosin VIIa is expressed in photoreceptor cells (Liu et al., and were thus wild type for coat color (agouti). The 1997; Wolfrum et al., 1998). We asked if myosin VIIa and relationship between dilute lethal ERG amplitudes and those myosin Va showed any signs of interactions by intercrossing of this non-dilute control group was the same as for the the two mutant stocks and deriving mice that were comparison of dilute lethal and dilute non-lethal (Myo5ad) homozygous for both dilute lethal and the Myo7a4626SB ERGs shown in Fig. 1. This observation confirms that the mutation, which is believed to be a null allele (Hasson et al., ERG abnormalities we see in dilute lethal mutants are 1997; Liu et al., 1999). These double mutants were viable associated with the neurological effects of the mutation and could be distinguished by their dilute coat color and their rather than the pigmentation effects. 4512 Journal of Cell Science 117 (19)

Fig. 2. Electroretinographic responses suggest abnormal synaptic transmission between photoreceptors and retinal interneurons. (A) Implicit times of a-wave (left) and b-wave (right) peaks, with standard errors, plotted as a function of flash intensity. Times are significantly longer in the Myo5ad-l/Myo5ad-l (n=10) group compared with the controls [Myo5ad/ Myo5ad or d-l (n=12; no difference was observed between the two control genotypes, P>0.25); a-wave P<0.01 from 3 log units of attenuation; b-wave, 7.2-2.4, P<0.05, 1.8-0 P<0.001]. (B) Ratios of b- to a-wave amplitudes are significantly smaller in dilute lethal mutants than the control dilute group (Pр0.001 from 2.4 log units of attenuation).

Discussion (consistent with its expression pattern) in photoreceptor We report here clear evidence of a role for myosin Va in synaptic function. synaptic function in the retina. In the dilute lethal mutant, we Several other mutants have been reported to show reduced observed a large reduction in ERG a-wave amplitude, a larger ERG amplitudes that disproportionately affect the b-wave, decrease in b-wave amplitude and increased implicit times for including laminin β2 (Lamb2), neurotrophic tyrosine kinase both waves. We also observed defective synaptic ultrastructure receptor type 2 (Ntrk2) and bassoon (Bsn) mouse mutants and in the photoreceptor cells, which probably underlies the the no optokinetic response c (nrc) zebrafish mutant (Allwardt aberrant electrophysiology. et al., 2001; Dick et al., 2003; Libby et al., 1999; Rohrer et al., Interestingly, the ribbon synapses of the bipolar cells were 1999). The Ntrk2-null mouse showed retarded development of normal, consistent with the retinal localization of myosin Va, the retina (Rohrer et al., 1999), whereas we saw no evidence which is concentrated in the photoreceptor but not second- of a general retardation in development in dilute lethal retinas. order neuron synapses (Schlamp and Williams, 1996). Myosin Bsn, nrc and Lamb2 mutants all have electroretinographic Va has also been detected at the photoreceptor inner segment responses – similar to those found in dilute lethal – that suggest as well as the synapse (Schlamp and Williams, 1996), so that involvement with photoreceptor synaptic function (Allwardt et a role in inner segment function, in addition to its role in al., 2001; Dick et al., 2003; Libby et al., 1999). All three of synaptic activity, might explain our observation of a reduced these mutants have floating ribbons, suggesting that these ERG a-wave in mutant mice. Alternatively, there might be are required (either directly or indirectly) for some feedback from post-synaptic activity upon photoreceptor anchoring the ribbon to the synapse and that this defect leads function. to synaptic failure. The dilute lethal mice have a different Mice with the dilute lethal mutation show deteriorating morphological defect. The ribbon in dilute lethal condition and are undersized. It is possible that the photoreceptors is properly localized but it often has an physiological and anatomical abnormalities observed are a abnormal club-like shape. Furthermore, in dilute lethal mice, secondary result of this progressive illness. However, a rat synaptic vesicles often do not associate with the ribbon, which model of desmosterolosis, a lethal disease involving they do in the Bsn, nrc and Lamb2 mutants. These data suggest cholesterol biosynthesis, has several similarities to dilute lethal that myosin Va is not necessary for proper ribbon localization mutants, including small size and death by 4 weeks of age but might be important in establishing or maintaining the (only 18% survival rate after postnatal day 28). There were no function of the ribbon, in the fast and continuous synaptic electroretinographic abnormalities found in these animals vesicle release that occurs at the photoreceptor synapse. (Fliesler et al., 2000). Therefore, it is unlikely the phenotypes Myosin Va is present in conventional presynaptic terminals described here are the result of the general health of the dilute in the CNS and localizes with markers mutants, but rather are the result of a direct role of myosin Va (Bridgman, 1999; Evans et al., 1998; Prekeris and Terrian, Synaptic function of Myosin Va 4513

Fig. 3. Dilute lethal mutants have abnormal photoreceptor synapses. Transmission electron micrographs of synaptic regions. Ribbon synapses (arrows) look normal in the control mice (A). In Myo5ad-l/Myo5ad-l mice, the ribbons of photoreceptor synapses appear club-like (B,E), although the ribbons of bipolar cell synapses appear normal (G). Photoreceptor synapses of Myo5ad-l/Myo5ad-l mutants also possess clusters of presumed synaptic vesicles that are ectopically located (C,D,F, arrows). At least three sections were examined from each of four Myo5ad-l/Myo5ad-l mice (abnormalities were consistently observed in the mutant mice) and two control mice (Myo5ad/Myo5ad or d-l; the synaptic morphology of these mice is similar to all wild-type mice we have previously examined, this dilute genotype does not affect synaptic morphology, which would be expect from their ERGs). Scale bars, 100 nm (F), 300 nm (others).

1997). It has been hypothesized that myosin Va is important in vesicle availability, it could explain why some ribbons are recycling and/or replenishing synaptic vesicles (Bridgman, found without synaptic vesicles in the dilute lethal 1999; Evans et al., 1998; Prekeris and Terrian, 1997; Schnell photoreceptors. It could also explain why so many ribbons have and Nicoll, 2001). In fact, Bridgman reported abnormal an abnormal morphology. It is now well established that the accumulation of synaptic vesicles in the presynaptic terminals length and gross morphology of the mouse photoreceptor of cerebellar granule cells of dilute lethal mice. In chromaffin ribbons can change and that this change is directly related to cells, myosin Va has been shown to be important in the light cycle (or potentially synaptic activity) (Adly et al., replenishing neurosecretory vesicles (Rose et al., 2003). These 1999; Balkema et al., 2001; Spiwoks-Becker et al., 2004). Our data, coupled with our results, support the hypothesis that results suggest that myosin Va might play a role in this dynamic myosin Va has an important role in maintaining an adequate regulation of synaptic ribbon morphology. number of vesicles available for release. This might be Although myosin Va is found in many CNS presynaptic particularly important at the photoreceptor synapse because terminals, no defects in synaptic transmission have yet been there can be long periods of sustained, continuous synaptic reported. Because of the continuous vesicle release from the vesicle release. If myosin Va has a direct role in synaptic photoreceptor synapses, requiring greater synaptic vesicle 4514 Journal of Cell Science 117 (19) cells (Miyata et al., 2000). This post-synaptic physiological phenotype might be caused by myosin Va being necessary for proper organelle localization (its more traditional role) and not directly in synaptic transmission. Importantly, Miyata et al. (Miyata et al., 2000) did not find any abnormalities in synaptic transmission or morphology. Therefore, there are at least two distinct physiological roles for myosin Va in the mammalian CNS. One involves postsynaptic processing that might or might not be a secondary consequence of myosin Va’s normal function (Miyata et al., 2000). The other, described here, is a role of myosin Va in presynaptic physiology. This is supported by the fact that myosin Va is localized to the synapse. Our work suggests the possibility that myosin Va mutations lead to synaptic dysfunction at certain CNS synapses and/or under certain conditions. This possibility should be considered as a potential cause of the more generalized neurological defects observed in the dilute lethal mouse mutant and patients with Griscelli syndrome type 1 and Elejalde syndrome.

This work was supported by the MRC, Defeating Deafness, the European Commission (BMH4-CT96-1324) and the NIH (R01 EY07042 and P30 EY12598).

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