but with peak CS and acuity about 50% less than those of wild-type mice (Fig. 63.3a; Bonfield 2009).

Cacna1fnob2: A spontaneous mutant having no b-wave was isolated from colonies at the Jackson Laboratory. It was first reported to have an insertion of a transposable element (ETn) into exon 2 of Cacna1f, predicted to produce an inframe premature stop codon (Chang et al. 2005). However, by northern blotting we identified not one but two mRNA species in nob2 mice; 90% of the transcripts contained the in-frame stop codon previously identified, but 10% of the transcripts lacked this stop codon because it was removed by alternative splicing within the ETn element. Thus nob2 mice were predicted to encode a complete (but altered) Cav1.4 protein with 22 novel amino acids in the N-terminal region (Doering et al. 2008). Biophysical analysis of channels in HEK cells transfected with this complete but altered cDNA revealed activation and inactivation characteristics indistinguishable from those of wild-type; but binding with filamin proteins, a property of the N-terminal portion of wild-type α1F protein, was absent from the mutant protein (Doering et al. 2008). We also have observed: reductions in amplitude of both scotopic and photopic ERG b-waves and oscillatory potentials (Fig. 63.2) (Doering et al. 2008), reduction in thickness of the outer plexiform layer, and sprouting of rod bipolar and horizontal cell dendrites into the outer nuclear layer, as in the G305X

mouse. Remarkably, the peak optokinetic CS was normal in most of the affected (Cacna1f nob2/nob2 and Cacna1f nob2/y) nob2 mice; however, their acuity was almost

50% lower than in WT mice, and CS was significantly below normal at most spatial frequencies in one of the three litters tested (Fig. 63.3b: litter 1).

63.4 Discussion

CSNB2 is a clinically variable retinal neurotransmission disorder, difficult to diagnose without detailed assessment of visual acuity, visual field, refractive error, nystagmus and strabismus, plus rigorous electroretinography (Tremblay et al. 1995; Boycott et al. 2000). Identification of the gene responsible for CSNB2 has made possible DNA-definitive diagnosis (nearly 80% of patients with the clinical features of iCSNB have mutations in CACNA1F [Bech-Hansen and Tobias, unpublished]). Signs and symptoms of CSNB2 are variable, not only in patients having different CACNA1F mutations (Lodha et al. Submitted), but also among members of families sharing the same CACNA1F founder mutation (Boycott et al. 2000). This variability may be due in part to mutations in modifier genes, thus altering interactions of their protein products with Cav1.4 protein and directly impairing Cav1.4 channel assembly, integrity, and function; this is suggested by the observation that the biophysical properties of some mutant CACNA1F-encoded channels are normal (McRory et al. 2004; Hoda et al. 2005; Peloquin et al. 2007). The functional

phenotypes of the G305X and nob2 mice are distinctive, even though both are due to mutations in the Cacna1f gene. The phenotype of Cacna1f G305X mice is more

severe than that of Cacna1f nob2 mice; the OKR, visual evoked potential, and ERG

findings indicate virtually complete loss of cone function as well as cone-to-bipolar cell synaptic transmission in Cacna1f G305X (Mansergh et al. 2005; Bonfield et al. 2007; Orton et al. 2007), whereas the OKR and ERG indicate substantial (although diminished) cone viability and cone-bipolar cell transmission in Cacna1f nob2 (Bonfield et al. 2007; Doering et al. 2008). Loss and disorganization of rod and cone ribbon synapses is accompanied by thinning of the outer plexiform layer in both mutants; however, cone ribbon synapses are completely lost in G305X mice, while some survive in nob2 mice (Chang et al. 2006; Raven et al. 2008). The presumably small fraction of complete (though abnormal) Cav1.4 protein in Cacna1fnob2 may sustain sufficient binding with critical protein partners that nob2 cones survive and function at near-normal levels, and cone synaptic transmission in nob2 retinas may be defective because of the loss of Cav1.4-protein interactions that are critical for the normal development and functioning of ribbon synapses. It is noteworthy that knockouts of genes for bassoon, CABP4, and calcium-channel β2-subunits cause similar defects in the development and function of photoreceptor ribbon synapses (Ball and Gregg 2002; Dick et al. 2003; Haeseleer et al. 2004), suggesting that the formation of functional ribbon synapses depends on the assembly of microdomains containing key proteins. Furthermore, accumulation of abnormal proteins (such as α1F) may cause endoplasmic reticulum (ER) stress, leading to an unfolded protein response and cell death (Xia and Link 2008). Cones are likely to be more susceptible to ER stress, because each cone has 20–40 times as many synaptic ribbon complexes as each rod (Haverkamp et al. 2000) and therefore is likely to be stressed by a

much larger synthetic load of any abnormal ribbon synapse-related proteins. Cones may be spared in Cacna1f nob2 mice, because even though the complete Cav1.4/α1F

protein is somewhat abnormal in structure and amount, it is less likely to remain unfolded in the ER than the truncated protein.

How do the differences in mutations account for the differences in optokinetic

behavior of the two mouse strains? Because of random inactivation of most genes on the X-chromosome, 50% of cones in Cacna1f G305X/+ females should express

the normal, and 50% the truncation-encoding, Cacna1f gene; the loss of 50% of cones is the most economical explanation for the 50% reduction in photopic contrast sensitivity and acuity of females heterozygous for the G305X mutation. Nob2 mice, on the other hand, may have enough ‘complete’ Cav1.4 channel protein to support normal assembly of ribbon synapse proteins, in all cones thus maintaining quasi-normal synaptic transmission to cone bipolar cells. Despite their superior performance under photopic conditions, however, nob2 mice should perform as poorly as G305X mice under scotopic conditions, because of the massive loss of rod ribbon synapses in both. However, the optokinetic contrast sensitivity functions of both mutants are velocity-tuned for photopic vision (when present), indicating that velocity-tuning is dependent on the unaffected circuitry of the inner plexiform layer rather than the mutation-altered circuitry of the outer plexiform layer.

Our findings in these two mutant mice highlight the importance of Cav1.4 protein in the structure and function of photoreceptor ribbon synapses and synaptic transmission. Even minor expression of a Cacna1f transcript, and presumably a reduced amount of Cav1.4 protein, are sufficient to support some visual function (OKR).

OPL = outer plexiform layer, neur = neuron, ERG = electroretinogram, RGCs = retinal ganglion cells, resp = response, OKR = optokinetic response, homozyg = homozygous, hetero = heterozygous, CS = contrast sensitivity (Manserg et al. 2005; Doering et al. 2009).

On the other hand, we also recognize that patients with the same founder mutation show extensive clinical variability, raising the suspicion that modifier genes also contribute to the phenotypic variability seen in CSNB2 patients.

63.5 Conclusion

Cav1.4 plays key roles in photoreceptor synaptogenesis and synaptic function in the mouse retina. Cacna1f G305X is a true knockout model for human CSNB2, with

prominent defects in cone function, whereas Cacna1f nob2 is not a true knockout model for CSNB2, because the synthesis of some functional Cav1.4 protein such as to maintain cone survival and photopic visual function. Differences in Cav1.4 synthesis and cone survival might explain some of the phenotypic variability seen in human CSNB2, as well as in these two murine models.

Acknowledgments Supported by: FFB-Canada, CIHR, NSERC, and AHFMR.

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