Debora B. Farber

Retinal Degeneration in Mice Lacking the γ Subunit of the Rod cGMP Phosphodiesterase

The retinal cyclic guanosine 3′,5′-monophosphate (cGMP) phosphodiesterase (PDE) is a key regulator of phototransduction in the vertebrate visual system. PDE consists of a catalytic core of α and β subunits associated with two inhibitory γ subunits. A gene-targeting approach was used to disrupt the mouse PDEγ gene. This mutation resulted in a rapid retinal degeneration resembling human retinitis pigmentosa. In homozygous mutant mice, reduced rather than increased PDE activity was apparent; the PDEαβ dimer was formed but lacked hydrolytic activity. Thus, the inhibitory γ subunit appears to be necessary for integrity of the photoreceptors and expression of PDE activity in vivo.

Opsin, G-protein and 48-kDa protein in normal and rd mouse retinas: developmental expression of mRNAs and proteins and light/dark cycling of mRNAs.

Retinal degeneration in rd mice is manifested during the most rapid period of postnatal photoreceptor differentiation and is hypothesized to be caused by a lesion in cGMP metabolism. We have studied the sequence of developmental expression of three proteins involved in the cGMP cascade and the mRNAs from which they are translated, in rd and control mouse retinas. Slot blot analysis of retinal RNAs indicates that the mRNAs coding for opsin, the alpha, beta and gamma subunits of G-protein and 48-kDa protein each has the same time for onset of expression in normal and diseased retinas. G beta and 48-kDa protein mRNAs are already detectable at birth, opsin mRNA appears by postnatal day 5 (P5), G gamma mRNA at P6 and G alpha mRNA by P8. The levels of all these mRNAs decrease in the diseased retinas after P11-P12, correlating with the reduction in photoreceptor cell number that characterizes the rd disease. Immunocytochemistry indicates that the 48-kDa protein is present at birth, G gamma and opsin are detectable at P4 and G alpha at P7. After P7, opsin and G-protein immunoreactivity are localized throughout the photoreceptor cell in the rd retinas but they are found only in the outer segment in control retinas. The 48-kDa protein immunoreactivity, which is observed in the whole photoreceptor layer both in rd and control retinas throughout development, is the only one of all immunoreactivities analysed that remains at 2 months of age in the rd retina and is probably localized in cones. However, at 6 months of age, 48-kDa protein immunoreactive cells are no longer present in the rd retina. We have also investigated whether there is a daily rhythm for the levels of mRNA present at different times during the light/dark periods in developing rd/rd and rd/+ retinas and in adult normal (+/+) retinas. We find that the levels of each mRNA analysed appear to cycle in the +/+ adult retina, with the greatest amount of opsin and the three subunits of G-protein mRNAs occurring just before light onset and the greatest amount of 48-kDa protein mRNA occurring just before lights off. Cycling in the developing diseased or control retinas (P0-P12) is not observed and may be masked by the pronounced cell growth that occurs during this period.

The β subunit of cyclic GMP phosphodiesterase mRNA is deficient in canine rod-cone dysplasia 1

Irish setter dogs affected with rod-cone dysplasia 1 have elevated levels of retinal cGMP resulting from deficient rod-specific cGMP phosphodiesterase (cGMP PDE) activity. We investigated the mRNAs coding for the three subunits of cGMP PDE and for the proteins involved in the activation/deactivation of this enzyme in the retinas of developing affected and control dogs. While the photoreceptor cells are viable in the diseased retinas, opsin, transducin alpha 1 and beta 1, 48 and 33 kd proteins, and cGMP PDE alpha and gamma mRNAs have normal transcript sizes and levels. In contrast, a different pattern of cGMP PDE beta mRNAs with lower than normal concentrations is present in the developing affected retinas prior to degeneration. Our observations suggest that an abnormality involving cGMP PDE beta expression is implicated in rod-cone dysplasia 1.

A QTL on distal chromosome 3 that influences the severity of light-induced damage to mouse photoreceptors.

Abstract. C57BL/6J-c2J (c2J) albino mice showed much less damage to their photoreceptors after exposure to prolonged light than BALB/c mice and seven other albino strains tested. There were no gender differences, and preliminary studies suggested that the c2J relative protective effect was a complex trait. A genome-wide scan using dinucleotide repeat markers was carried out for the analysis of 194 progeny of the backcross (c2J × BALB/c)F1× c2J and the thickness of the outer nuclear layer (ONL) of the retina was the quantitative trait reflecting retinal damage. Our results revealed a strong and highly significant quantitative trait locus (QTL) on mouse Chromosome (Chr) 3 that contributes almost 50% of the c2J protective effect, and three other very weak but significant QTLs on Chrs 9, 12, and 14. Interestingly, the Chrs 9 and 12 QTLs corresponded to relative susceptibility alleles in c2J (or relative protection alleles in BALB/c), the opposite of the relative protective effect of the QTLs on Chrs 3 and 14. We mapped the Rpe65 gene to the apex of the Chr 3 QTL (LOD score = 19.3). Northern analysis showed no difference in retinal expression of Rpe65 message between c2J and BALB/c mice. However, sequencing of the Rpe65 message revealed a single base change in codon 450, predicting a methionine in c2J and a leucine in BALB/c.When the retinas of aging BALB/c and c2J mice reared in normal cyclic light were compared, the BALB/c retinas showed a small but significant loss of photoreceptor cells, while the c2J retinas did not. Finding light damage-modifying genes in the mouse may open avenues of study for understanding age-related macular degeneration and other retinal degenerations, since light exposures may contribute to the course of these diseases.

Progressive photoreceptor degeneration, outer segment dysplasia, and rhodopsin mislocalization in mice with targeted disruption of the retinitis pigmentosa-1 (Rp1) gene

Retinitis pigmentosa (RP), a common group of human retinopathic diseases, is characterized by late-onset night blindness, loss of peripheral vision, and diminished or absent electroretinogram (ERG) responses. Mutations in the photoreceptor-specific gene RP1 account for 5–10% of cases of autosomal dominant RP. We generated a mouse model of the RP1 form of RP by targeted disruption of the mouse ortholog (Rp1) of human RP1. In Rp1−/− mice, the number of rod photoreceptors decreased progressively over a period of 1 year, whereas that of cone photoreceptors did not change for at least 10 months. Light and electron microscopic analysis revealed that outer segments of Rp1−/− rods and cones were morphologically abnormal and became progressively shorter in length. Before photoreceptor cell death, rhodopsin was mislocalized in inner segments and cell bodies of Rp1−/− rods. Rod ERG amplitudes of Rp1−/− mice were significantly smaller than those of Rp1+/+ mice over a period of 12 months, whereas those of Rp1+/− mice were intermediate. The decreases in cone ERG amplitudes were slower and less severe than those in rods. These findings demonstrate that Rp1 is required for normal morphogenesis of photoreceptor outer segments and also may play a role in rhodopsin transport to the outer segments. The phenotype of Rp1 mutant mice resembles the human RP1 disease. Thus, these mice provide a useful model for studies of RP1 function, disease pathology, and therapeutic interventions.

Isolation and characterization of a novel oligodendrocyte-specific protein

Myelin plays a critical role in nervous system function and alterations in myelin-specific proteins cause a variety of neurologic disorders. We isolated a novel cDNA from the CNS that shares little nucleotide sequence homology with previously reported genes but appears to encode a protein related to peripheral myelin protein-22 (PMP-22) based on its amino acid sequence, predicted structure, and cellular localization. PMP-22 is important in peripheral myelination and Schwann cell proliferation, and mutations in its gene cause diseases of peripheral nerves. The isolated cDNA is 1.8 kb in length with an open reading frame of 621 bp. Northern blot analysis detected hybridization of labeled cDNA with a single 2.1-kb transcript only in the CNS. In situ hybridization revealed expression of this cDNA in oligodendrocytes of brain and spinal cord as well as in oligodendrocyte-enriched cultures; therefore we have named it oligodendrocyte-specific protein (OSP) cDNA. An OSP-specific polyclonal antibody reacted with a single 22-kd protein present in CNS myelin and oligodendrocytes. Developmental expression of OSP mRNA in the spinal cord was similar to that of the mRNA for a major myelin protein, proteolipid protein (PLP), and similar to PMP-22 in peripheral nerves. Since OSP is localized to oligodendrocytes and myelin, has a similar structure with PMP-22, and has a developmental pattern of expression like other myelin proteins, it probably has an important role in CNS myelinogenesis. NEUROLOGY 1996;47: 772-778

Retinal degenerations in the dog III abnormal cyclic nucleotide metabolism in rod-cone dysplasia

In dogs bred to develop rod-cone dysplasia, retinal development is normal until 13 days of age.Afterwards, there is an arrest of visual cell differentiation. Rod inner segments remain diminutive and outer segments fail to elongate as in controls; the outer segments show lamellar disorientation and disorganization. Affected visual cells degenerate, but the degeneration process is more rapid and extensive for rods than cones. Cyclic GMP levels become elevated in affected retinas early during the postnatal differentiation of visual cells; this elevation precedes any morphological evidence of photoreceptor disease. Retinal protein synthesis is normal during the time that retinal cGMP levels are rising above control values. The results indicate that the biochemical abnormality which results in elevated retinal cGMP levels is the earliest recognized defect in rod-cone dysplasia.

Regulation of type-II calmodulin kinase: Functional implications

Calmodulin-kinase II (CaM kinase) is a calcium/calmodulin-dependent protein kinase which is highly enriched in the nervous system and mediates many of calciums actions. Regulation of CaM kinase activity plays an important role in modulating synaptic transmission, synaptic plasticity and in neuropathology. Primary regulation of CaM kinase occurs via changes in intracellular calcium concentrations. Increased calcium stimulates protein kinase activity and induces autophosphorylation. Autophosphorylation of CaM kinase at specific sites results in altered activity and responsiveness to subsequent changes in calcium concentrations. Intracellular translocation of CaM kinase also appears to result from autophosphorylation. These mechanisms of regulation play an important role in synaptic plasticity (e.g., Aplysia ganglia), status epilepticus and cerebral ischemia. Long-lasting alterations in the expression of CaM kinase have been demonstrated in the kindling model of epilepsy and in monocular deprivation and therefore modulation of gene expression, in addition to autophosphorylation and translocation, appears to be another important mechanism of regulating CaM kinase activity.

Kindling induces a long-lasting change in the activity of a hippocampal membrane calmodulin-dependent protein kinase system

Septal kindling has been shown to produce a long-lasting decrease in endogenous calcium/calmodulin-dependent phosphorylation of hippocampal synaptic plasma membrane proteins, including two major bands of approximately 50,000 and 60,000 Daltons. These two proteins differ from the B-50 protein and tubulin, as evidenced by differences in migration in SDS-PAGE gels and by lack of cross-immunoreactivity with specific antibodies. Identity of these two proteins with the rho and sigma subunits of purified calmodulin-dependent kinase (CaM Kinase II) is suggested by similar migration in SDS-PAGE and two-dimensional gels, by similar calmodulin binding in two-dimensional gels, and similar 125I-peptide mapping of the 50,000 Dalton protein. These results demonstrate that septal kindling is associated with changes in the activity of a major Ca2+/calmodulin-dependent kinase system in hippocampal synaptic plasma membrane. This long-lasting modulation of kinase activity may provide a molecular insight into some aspects of neuronal plasticity.

Alterations in cyclic amp metabolism associated with photoreceptor cell degeneration in the c3h mouse.

Previous studies have shown an abnormality in cyclic nucleotide phosphodiesterase activity in the retina of mice (C3H/HeJ) with an inherited degeneration of the photoreceptor layer. Adenyl cyclase activity and cyclic AMP content have been measured in C3H retina and compared with that in normal retina (DBA/1J) during postnatal maturation, to assess the influence of the mutation upon cyclic AMP metabolism. Adenyl cyclase activity increases normally for the first 7 days of age; thereafter, it becomes greater than normal. Cyclic AMP becomes obviously abnormal after 10 days of age. The elevated levels of cyclic AMP persist in the surviving cells of the inner layers of the adult C3H retina. Adenyl cyclase activity and cyclic AMP content are concentrated in the inner layers of the normal retina, while the photoreceptor layer has only a very low level of enzyme activity and cyclic AMP. The data suggest that the synthesis of cyclic AMP in the inner layers of C3H retina is significantly enhanced, during the period of postnatal development in which the photoreceptor cells have begun to degenerate.