An attenuation of the electroretinogram b-wave is characteristic of Duchenne and Becker Muscular dystrophies and Muscle-Eye-Brain disease but the mechanisms underlying the abnormal retinal physiology in patients is not understood. Here we show that deletion of dystroglycan in the central nervous system causes an attenuation of the electroretinogram b-wave similar to what is observed in patients. The abnormal retinal physiology was associated with a selective loss of dystrophin and Kir4.1 clustering in glial endfeet, suggesting a critical role for dystroglycan intracellular interactions for the physiology of the retina.
In skeletal muscle, loss of dystroglycan results in the disruption of dystrophin and other components of the DGC. In the retina, β-dystroglycan anchors dystrophin in Müller glial endfeet and perivascular glial endfeet but it is not necessary for the localization of dystrophin in the outer plexiform layer, suggesting that another protein anchors dystrophin in the absence of dystroglycan. Although dystrophin does not require dystroglycan for its localization in the outer plexiform layer, the two proteins are closely associated at the synapse. DP260, a retina specific isoform of dystrophin, is localized at photoreceptor synapses (D'Souza et al., 1995
) and its disruption results in a selective loss of dystroglycan in the outer plexiform layer (Kameya et al., 1997
). Mice with impaired expression of DP260 have an electroretinogram with a prolonged b-wave implicit time but no change in b-wave amplitude (Kameya et al., 1997
). Interestingly, we observed a delay in the implicit time of electroretinograms from Nestin-Cre/DG null mice although the expression of dystrophin was preserved, suggesting that dystroglycan is important for the physiology of the ribbon synapse.
Recently, it has been shown that a novel α-dystroglycan-binding protein, pykachurin, is necessary for the opposition of pre-and post-synaptic termini in the photoreceptor ribbon synapse (Sato et al., 2008
). Deletion of pykachurin produced an abnormal electroretinogram (Sato et al., 2008
) similar to that observed in Nestin-Cre/DG null mice. Similar defects are also present in mice with mutations of laminin β2 chain (Libby et al., 1999
) but it is not known if dystroglycan binds to the laminin β2 chain. Patients with Muscle-Eye-Brain disease (Santavuori et al., 1989
) and mouse models with mutations of large
show an attenuation of the electroretinogram b-wave (Holzfeind et al., 2002
; Takeda et al., 2003
; Lee et al., 2005
), suggesting that a disruption of α-dystroglycan glycosylation and extracellular ligand binding is sufficient to disrupt its function. Hypoglycosylation of dystroglycan may also affect the intracellular interactions of dystroglycan by disrupting dystroglycan localization.
Our results suggest that dystroglycan contributes to the physiology of the retina by more than one mechanism. GFAP-Cre inactivation of dystroglycan in glial cells was sufficient to attenuate the b-wave even though expression of dystroglycan was present in the outer plexiform layer. Although it remains controversial, there is evidence that potassium channels in Müller glia may contribute to the b-wave (Wen and Oakley, 1990
; Connors and Kofuji, 2002
). In the GFAP-Cre/DG null retina the localization of dystrophin and Kir4.1 was disrupted in Müller glial endfeet, supporting the hypothesis that abnormal glial potassium currents may contribute to the abnormal retinal physiology. Mutation of the cytoplasmic domain of β-dystroglycan was sufficient to disrupt the localization of dystrophin and Kir4.1 in glial endfeet and attenuate the electroretinogram b-wave. Prior research shows that deficiency in Kir4.1 causes a defect in the amplitude of the c-wave but the b-wave is preserved (Wu et al., 2004
). One of the important roles of Müller glial potassium channels is spatial buffering (Connors et al., 2004
). In contrast to the Kir4.1 deficient mice, dystroglycan deficient mice lose Kir4.1 expression selectively at glial endfeet and expression is preserved on the Müller glial processes. Furthermore, a selective disruption of Kir4.1 expression in glial endfeet is also associated with an attenuation of electroretinogram b-wave in mice with mutations of dystrophin (Pillers et al., 1995
; Connors and Kofuji, 2002
), providing support for this hypothesis. Alternatively, other yet to be identified β-dystroglycan intracellular interactions in Müller glia may also be contribute to the b-wave amplitude.
Loss of dystroglycan expression in the neuroepithelium in Nestin-Cre/DG null mice causes an attenuation of the scotopic threshold response as well as the b-wave of the bright flash scotopic electroretinogram. Based on the electrophysiology, we would predict that the animals would have some degree of useful scotopic and photopic vision but that it would be attenuated with respect to the wild-type mice. The Nestin-Cre/DG null mice demonstrated severe visual impairment on two visual paradigms, which is likely attributed to defects in the central nervous system in addition to the abnormal retinal physiology. Structural defects, which may have been missed on examination by slit lamp photography, may have also contributed to the poor performance of the mice on visual tasks. A combination of brain and eye defects is also likely to contribute to the loss of vision in congenital muscular dystrophy patients.
Preservation of the scotopic a-wave but attenuation of the b-wave observed in both Nestin-Cre/DG null and GFAP-Cre/DG null animals is also similar to that observed in ‘typical’ juvenile X-linked retinoschisis (XLRS) (Pawar et al.; Heckenlively and Arden, 2006
). Exceptions have been observed and are termed ‘atypical’ (Sieving et al.). Sieving assesses the cause of the reduced b-wave as “defective signaling by depolarizing bipolar cells in the rod pathway” (Heckenlively and Arden, 2006
). There is also a similarity to two different types of congenital stationary night blindness (CSNB) (Heckenlively and Arden, 2006 MIT press 2006
). Incomplete CSNB has the same b-wave attenuation observed in the nestin and GFAP animals but unlike the mutant animals, incomplete CSNB typically has a severely attenuated cone response. Complete CSNB has an intact cone response, as observed in the nestin and GFAP animals, but has an absent scotopic b-wave. Thus the absent pSTR observed in these animals is a notable difference from the responses of human patients affected with XLRS or CSNB. This facet of the Nestin-Cre/DG null and GFAP-Cre/DG null mutant animals may be a valuable tool for the further dissection of retinal physiology.