Identification of the Photoreceptor Transcriptional Co-Repressor SAMD11 as Novel Cause of Autosomal Recessive Retinitis Pigmentosa.

Retinitis pigmentosa (RP), the most frequent form of inherited retinal dystrophy is characterized by progressive photoreceptor degeneration. Many genes have been implicated in RP development, but several others remain to be identified. Using a combination of homozygosity mapping, whole-exome and targeted next-generation sequencing, we found a novel homozygous nonsense mutation in SAMD11 in five individuals diagnosed with adult-onset RP from two unrelated consanguineous Spanish families. SAMD11 is ortholog to the mouse major retinal SAM domain (mr-s) protein that is implicated in CRX-mediated transcriptional regulation in the retina. Accordingly, protein-protein network analysis revealed a significant interaction of SAMD11 with CRX. Immunoblotting analysis confirmed strong expression of SAMD11 in human retina. Immunolocalization studies revealed SAMD11 was detected in the three nuclear layers of the human retina and interestingly differential expression between cone and rod photoreceptors was observed. Our study strongly implicates SAMD11 as novel cause of RP playing an important role in the pathogenesis of human degeneration of photoreceptors.

Age-related functional and structural retinal modifications in the Igf1-/- null mouse.

BACKGROUND: Mutations in the gene encoding human insulin-like growth factor-I (IGF-I) cause syndromic neurosensorial deafness. To understand the precise role of IGF-I in retinal physiology, we have studied the morphology and electrophysiology of the retina of the Igf1(-/-) mice in comparison with that of the Igf1(+/-) and Igf1(+/+) animals during aging. METHODS: Serological concentrations of IGF-I, glycemia and body weight were determined in Igf1(+/+), Igf1(+/-) and Igf1(-/-) mice at different times up to 360days of age. We have analyzed hearing by recording the auditory brainstem responses (ABR), the retinal function by electroretinographic (ERG) responses and the retinal morphology by immunohistochemical labeling on retinal preparations at different ages. RESULTS: IGF-I levels are gradually reduced with aging in the mouse. Deaf Igf1(-/-) mice had an almost flat scotopic ERG response and a photopic ERG response of very small amplitude at postnatal age 360days (P360). At the same age, Igf1(+/-) mice still showed both scotopic and photopic ERG responses, but a significant decrease in the ERG wave amplitudes was observed when compared with those of Igf1(+/+) mice. Immunohistochemical analysis showed that P360 Igf1(-/-) mice suffered important structural modifications in the first synapse of the retinal pathway, that affected mainly the postsynaptic processes from horizontal and bipolar cells. A decrease in bassoon and synaptophysin staining in both rod and cone synaptic terminals suggested a reduced photoreceptor output to the inner retina. Retinal morphology of the P360 Igf1(+/-) mice showed only small alterations in the horizontal and bipolar cell processes, when compared with Igf1(+/+) mice of matched age. CONCLUSIONS: In the mouse, IGF-I deficit causes an age-related visual loss, besides a congenital deafness. The present results support the use of the Igf1(-/-) mouse as a new model for the study of human syndromic deaf-blindness.

Retinal degeneration in two lines of transgenic S334ter rats.

Aim of this study was to examine synaptic connectivity changes in the retina and the location and rate of apoptosis in transgenic S334ter line-3 and line-5 rats with photoreceptor degeneration. Heterozygous S334ter-line-3 and line-5 at P11-13, P30, P60, P90 and several control non-dystrophic rats (Long Evans and Sprague-Dawley) at P60, were studied anatomically by immunohistochemistry for various cell and synaptic markers, and by PNA and TUNEL label.- S334ter line-3 exhibited the fastest rate of degeneration with an early loss of photoreceptors, with 1-2 layers remaining at P30, and only cones left at P60. Line-5 had 4-5 layers left at P30, and very few rods left at P60-90. In both lines, horizontal cell processes (including dendrites and axon) were diminished at P11-13, showing gaps in the outer plexiform layer (OPL) at P60, and at P90, almost no terminal tips could be seen. Bipolar cells showed a retraction of their dendrites forming clusters along the OPL. Synaptic terminals of A-II amacrine cells in the IPL lost most of their parvalbumin-immunoreactivity. The apoptosis rate was different in both lines. Line-3 rats showed many photoreceptors affected at P11, occupying the innermost part of the outer nuclear layer. Line-5 showed a lower number of apoptotic cells within the same location at P13. In summary, the S334ter line-3 rat has a faster progression of degeneration than line-5. The horizontal and bipolar terminals are already affected at P11-P13 in both models. Apoptosis is related to the mutated rhodopsin transgene; the first photoreceptor cells affected are those close to the OPL.

Intraretinal processing following photoreceptor rescue by non-retinal cells.

Royal College of Surgeon (RCS) rats undergo retinal degeneration due to the inability of retinal pigment epithelial (RPE) cells to phagocytose shed outer segments. We explored the effect of introducing Schwann cells to the subretinal space of RCS rats (before the onset of retinal degeneration), by relying on electroretinogram (ERG) recordings and correlative retinal morphology. Scotopic ERGs recorded from cell-injected eyes showed preserved amplitudes of mixed a-wave b-wave, rod b-waves, and cone b-waves over controls (sham-injected eyes); photopic b-wave amplitudes and critical flicker fusion were also improved. Normal retinal morphology was found in areas of retinas that had received cell injections. Since Schwann cells have no phagocytic properties, their therapeutic effect is best explained through a paracrine mechanism (secretion of factors that ensure photoreceptor survival).

Functional and structural modifications during retinal degeneration in the rd10 mouse.

Mouse models of retinal degeneration are useful tools to study therapeutic approaches for patients affected by hereditary retinal dystrophies. We have studied degeneration in the rd10 mice both by immunocytochemistry and TUNEL-labeling of retinal cells, and through electrophysiological recordings. The cell degeneration in the retina of rd10 mice produced appreciable morphological changes in rod and cone cells by P20. Retinal cell death is clearly observed in the central retina and it peaked at P25 when there were 800 TUNEL-positive cells per mm(2). In the central retina, only one row of photoreceptors remained in the outer nuclear layer by P40 and there was a remarkable deterioration of bipolar cell dendrites postsynaptic to photoreceptors. The axon terminals of bipolar cells also underwent atrophy and the inner retina was subject to further changes, including a reduction and disorganization of AII amacrine cell population. Glutamate sensitivity was tested in rod bipolar cells with the single cell patch-clamp technique in slice preparations, although at P60 no significant differences were observed with age-matched controls. Thus, we conclude that rod and cone degeneration in the rd10 mouse model is followed by deterioration of their postsynaptic cells and the cells in the inner retina. However, the functional preservation of receptors for photoreceptor transmission in bipolar cells may open new therapeutic possibilities.

Regressive and reactive changes in the connectivity patterns of rod and cone pathways of P23H transgenic rat retina.

We have used the P23H line 1 homozygous albino rat to study how progressive photoreceptor degeneration affects rod and cone relay pathways. We examined P23H retinas at different stages of degeneration by confocal microscopy of immunostained sections and electroretinogram (ERG) recordings. By 21 days of age in the P23H rat retina, there is already substantial loss of rods and reduction in rod bipolar dendrites along with reduction of metabotropic glutamate receptor 6 (mGluR6) and rod-associated bassoon staining. The cone pathway is relatively unaffected. By 150 days, when rods are absent from much of the retina, some rod bipolars remain and dendrites of rod and cone bipolar cells form synaptic complexes associated with cones and horizontal cell processes. These complexes include foci of mGluR6 and bassoon staining; they develop further by 270 days of age. Over the course of degeneration, beginning at 21 days, bipolar axon terminals atrophy and the inner retina undergoes further changes including a reduced and disorganized AII amacrine cell population and thinning of the inner plexiform layer. Electroretinogram (ERG) results at 23 days show reductions in a-wave amplitude, in rod and cone-associated b-waves (using a double flash paradigm) and in the amplitude of oscillatory potentials (OPs). By 38 days, rod scotopic a-wave responses and OPs are lost. B-wave amplitudes decline until 150 days, at which point they are purely cone-driven and remain stable up to 250 days. The results show that during the course of photoreceptor loss in the P23H rat, there are progressive degenerative changes, particularly in the rod relay pathway, and these are reflected in the changing ERG response patterns. Later reactive changes involving condensation of cone terminals and neurotransmitter receptors associated with rod and cone bipolar dendrites and with horizontal cell processes suggest that at this stage, there are likely to be complex changes in the relay of sensory information through the retina.

Localization of neurotransmitters and calcium binding proteins to neurons of salamander and mudpuppy retinas.

We wished to identify the different types of retinal neurons on the basis of their content of neuroactive substances in both larval tiger salamander and mudpuppy retinas, favored species for electrophysiological investigation. Sections and wholemounts of retinas were labeled by immunocytochemical methods to demonstrate three calcium binding protein species and the common neurotransmitters, glycine, GABA and acetylcholine. Double immunostained sections and single labeled wholemount retinas were examined by confocal microscopy. Immunostaining patterns appeared to be the same in salamander and mudpuppy. Double and single cones, horizontal cells, some amacrine cells and ganglion cells were strongly calbindin-immunoreactive (IR). Calbindin-IR horizontal cells colocalized GABA. Many bipolar cells, horizontal cells, some amacrine cells and ganglion cells were strongly calretinin-IR. One type of horizontal cell and an infrequently occurring amacrine cell were parvalbumin-IR. Acetylcholine as visualized by ChAT-immunoreactivity was seen in a mirror-symmetric pair of amacrine cells that colocalized GABA and glycine. Glycine and GABA colocalized with calretinin, calbindin and occasionally with parvalbumin in amacrine cells.

Morphological and neurochemical diversity of neuronal nitric oxide synthase-positive amacrine cells in the turtle retina.

The histochemistry of reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) and immunoreactivity of neuronal nitric oxide synthase (nNOS-IR) can be demonstrated in various cell types of the vertebrate retina. In this study, we have focused on characterizing the different NADPH-d-positive amacrine cell types in turtle retina. Cryostat sections were examined by confocal laser scanning microscopy for double immunofluorescence with antibodies against nNOS and either GABA or glycine, or by combining histochemistry with immunocytochemistry to obtain triple labeling with NADPH-d, GABA, and glycine. Forty-eight percent of the NADPH-d-labeled amacrine cells colocalized GABA, 52% glycine. Here we show that two morphologically different types of amacrine cell are nNOS/glycine-IR and three types are nNOS/GABA-IR. Antibodies against calretinin, parvalbumin, somatostatin, tyrosine hydroxylase, and choline acetyltransferase did not colocalize with nNOS-IR or NADPH-d-labeled amacrine cells, but 15% of the NOS-labeled amacrine cells showed immunoreactivity against calbindin. Only GABA has been seen to colocalize with NADPH-d in amacrine cells in previous reports in other species. The finding here of glycine colocalizing with NO-containing cells is novel. We suggest that NO, apart from its well known function in gap junction regulation, can also modulate the release of both GABA and glycine in the turtle retina.

Choline acetyltransferase is found in terminals of horizontal cells that label with GABA, nitric oxide synthase and calcium binding proteins in the turtle retina.

In this study, we discriminated the various types of horizontal cell in the turtle retina on their content of neuroactive substances. Double label immunocytochemistry was performed on sectioned and wholemount retina using antisera to neural- and endothelial-nitric oxide synthase (nNOS, and eNOS), calretinin (CR), calbindin (CB), gamma-aminobutyric acid (GABA) and choline acetyltransferase (ChAT). H1 cells and their axon terminals label with CR, CB and GABA. Only H1 axon terminals label with eNOS. H2 cells contain CB, CR, nNOS and GABA maybe in their dendrites. H3 cells label only with nNOS. The localization of nNOS in the H2 and H3 cells is a novel finding. None of these antibodies labels H4 cells. The photoreceptor subtypes have been differentiated by different intensity of labeling with CB. The accessory member of the double cone is less intensely labeled with CB than the principal member and rods and blue cones do not appear to label at all. ChAT-IR is located in terminal boutons of H1 and H2 horizontal cells and H1 axon terminals and these boutons contact rods and all spectral types of cones. Clearly, GABA is present in H1 horizontal cells and may be used in neurotransmission between horizontal cells and possibly for feedback pathways to photoreceptors. The evidence of nNOS immunoreactivity in H2 and H3 horizontal cells, combined with available physiological evidence, suggests that NO may be involved in electrical coupling and/or modulation of synaptic input to these types of cells. Furthermore, our results raise the possibility that cholinergic synaptic transmission may occur from horizontal cell processes to photoreceptors in the outer plexiform layer of the turtle retina.

Endothelial nitric oxide synthase (eNOS) is localized to Müller cells in all vertebrate retinas.

The distribution of endothelial nitric oxide synthase immunoreactivity (eNOS-IR) was investigated in the retinas of all phylogenetic vertebrate classes by using a monoclonal eNOS antibody. Confocal light microscopy showed immunoreactive labeling in Müller cells of fish, frog, salamander, turtle, chicken, rat, ground squirrel, and monkey retina. In vascularized retinas (rat, monkey), astrocytes and some blood vessels were also stained. Furthermore, eNOS-IR was localized to axon terminals of turtle and fish horizontal cells. These observations are the first to show the presence of eNOS-IR in Muller glia and horizontal cell structures of the vertebrate retina.

The localization of guanylyl cyclase-activating proteins in the mammalian retina.

PURPOSE: To explore the distribution of guanylyl cylase-activating proteins 1 and 2 (GCAP1 and GCAP2) in the mammalian retina. METHODS: Cryostat and vibratome vertical sections and wholemount retinas from mouse, rat, cat, bovine, monkey, and human eyes were prepared for immunocytochemistry and viewing by light and confocal microscopy. RESULTS: In all mammalian retinas investigated, intense GCAP1 immunoreactivity (GCAP1-IR) was seen in cone photoreceptor inner and outer segments, cell bodies, and synaptic regions. Intensity of the GCAP1-IR was strong in inner segments of rods in all species but weaker in outer segments-particularly so in primates and cats. GCAP2 immunoreactivity (GCAP2-IR) was weak in bovine, mouse, and rat cones but was intense in human and monkey cones. In all species except primates, GCAP2 staining was intense in rod inner and outer segments. In primates GCAP2-IR was intense in the rod inner segment but faint in the rod outer segment. A striking difference from the GCAP1 pattern of immunoreactivity was seen with GCAP2 antibodies as far as the inner retina was concerned. GCAP2-IR was evident in certain populations of bipolar, amacrine, and ganglion cells in all species. CONCLUSIONS: GCAP1 and GCAP2, which are involved in Ca2+-dependent stimulation and inhibition of photoreceptor guanylyl cyclase, can be detected in mammalian photoreceptor inner and outer segments, consistent with their physiological function. The occurrence of both GCAPs in the synaptic region of the photoreceptors indicates participation of these proteins in pathways other than regulation of phototransduction. The occurrence of GCAP2 in inner retinal neurons is indicative of second-messenger chemical transduction, possibly in metabotropic glutamate, gamma-aminobutyric acid (GABA) receptor, and nitric oxide-activated neural circuits.

Circuitry and role of substance P-immunoreactive neurons in the primate retina.

In this paper, we extend our previous light microscopic (LM) study of substance P (SP)-containing amacrine and ganglion cell types of the human retina (Cuenca et al. [1995] J. Comp. Neurol. 356:491-504) to an electron microscopic (EM) and confocal-imaging study in order to reveal synaptic circuitry and putative input and output neurons. SP-immunoreactive (-IR) amacrine cells in primate retina are typically wide-field cells with large cell bodies occurring in normal or displaced positions relative to the inner plexiform layer (IPL). Their main dendrites bear many spines and are monostratified in stratum 3 (S3) of the IPL. Axon-like processes arise from dendrites close to the cell body and run for hundreds of microns at the same level as the dendrites, thus forming a relatively dense plexus in S3 of the IPL. SP-IR axon processes also climb to S1 to surround some amacrine cell bodies, and others pass into the outer plexiform layer (OPL). Still other axons run down to the ganglion cell layer, where they encircle SP-IR ganglion cells and pass on to end in the nerve fiber layer. The SP-IR ganglion cell types have large cell bodies (20-22 microm diameter) and dendrites that costratify in S3 among the SP-IR amacrine cell processes. Double immunostaining and study by confocal microscopy reveals that SP-IR amacrine cells in the monkey colocalize gamma-aminobutyric acid (GABA). Their main plexus of dendrites in S3 of the IPL is skirted on the S2/S3 border by cone bipolar axons that stain for calbindin but intermingles primarily with glycinergic bipolar cell types of S3 and S3-S4. Strongly GABA-IR/weakly glycine-IR amacrine cell bodies, in addition to the SP-IR large-bodied ganglion cell type, are targets of encircling SP-IR axon processes. EM study of the human SP-IR amacrine cell indicates that input synapses to its dendrites are from bipolar cell axons of the S2/S3 border, S3, and the S3/S4 border of the IPL neuropil (33% of the synaptic input) and from amacrine cell processes (67% of the synaptic input). The input amacrine cells are of at least two distinct types based on cytological criteria. Synaptic output from the SP-IR amacrine cell dendrites is to bipolar cell axons as reciprocal synapses (31%), to amacrine cells (40%), and to ganglion cell profiles, primarily in S3 (29%) of the IPL. The SP-IR axons synapse upon SP-IR ganglion cell bodies and axons, upon normally placed and displaced amacrine cell bodies, and upon bipolar cell dendrites in the OPL. In addition, they appear to synapse among themselves. We shall discuss a wiring diagram and the possible role of SP-IR amacrine cells in the primate retina.

Formation and dissolution of spinules and changes in nematosome size require optic nerve integrity in black bass (Micropterus salmoides) retina.

Teleost retinas adapted to light show numerous spinules invaginated in the cone pedicles and small nematosomes in the distal horizontal cells. Darkness induces the dissolution of spinules and the presence of large and numerous nematosomes. The aim of this work is to study the influence of optic nerve integrity on spinule formation/dissolution and changes in nematosome size during light or dark adaptation of black bass (Micropterus salmoides) retinas. Eyes from fish, dark- or light-adapted, were removed and the eyecups placed in oxygenated Ringer's solution and immediately exposed to light or dark, respectively, for 1 h. The number of spinules per pedicle and the nematosome diameter were measured on electron micrographs. Isolation of eyecups in the dark, impaired both spinule formation and nematosome size reduction when they were superfused in light. In the same way, isolation of eyecups in the light, impaired both spinule dissolution and nematosome size increase when they were superfused in dark. No significant differences in spinule number and nematosome size, following dopamine superfusion, were found in comparison to retinas superfused with Ringer's solution only. Our results suggest: (1) optic nerve integrity is necessary to yield spinule formation/disruption and changes in nematosome size during light or dark adaptation. (2) dopamine does not appear to be the primary agent responsible for spinule formation.