Enrica Strettoi

The Major Cell Populations of the Mouse Retina

We report a quantitative analysis of the major populations of cells present in the retina of the C57 mouse. Rod and cone photoreceptors were counted using differential interference contrast microscopy in retinal whole mounts. Horizontal, bipolar, amacrine, and Müller cells were identified in serial section electron micrographs assembled into serial montages. Ganglion cells and displaced amacrine cells were counted by subtracting the number of axons in the optic nerve, learned from electron microscopy, from the total neurons of the ganglion cell layer. The results provide a base of reference for future work on genetically altered animals and put into perspective certain recent studies. Comparable data are now available for the retinas of the rabbit and the monkey. With the exception of the monkey fovea, the inner nuclear layers of the three species contain populations of cells that are, overall, quite similar. This contradicts the previous belief that the retinas of lower mammals are “amacrine-dominated”, and therefore more complex, than those of higher mammals.

Retinal organization in the retinal degeneration 10 (rd10) mutant mouse: a morphological and ERG study.

Retinal degeneration 10 (rd10) mice are a model of autosomal recessive retinitis pigmentosa (RP), identified by Chang et al. in 2002 (Vision Res. 42:517–525). These mice carry a spontaneous mutation of the rod‐phosphodiesterase (PDE) gene, leading to a rod degeneration that starts around P18. Later, cones are also lost. Because photoreceptor degeneration does not overlap with retinal development, and light responses can be recorded for about a month after birth, rd10 mice mimic typical human RP more closely than the well‐known rd1 mutants. The aim of this study is to provide a comprehensive analysis of the morphology and function of the rd10 mouse retina during the period of maximum photoreceptor degeneration, thus contributing useful data for exploiting this novel model to study RP. We analyzed the morphology and survival of retinal cells in rd10 mice of various ages with quantitative immunocytochemistry and confocal microscopy; we also studied retinal function with the electroretinogram (ERG), recorded between P18 and P30. We found that photoreceptor death (peaking around P25) is accompanied and followed by dendritic retraction in bipolar and horizontal cells, which eventually undergo secondary degeneration. ERG reveals alterations in the physiology of the inner retina as early as P18 (before any obvious morphological change of inner neurons) and yet consistently with a reduced band amplification by bipolar cells. Thus, changes in the rd10 retina are very similar to what was previously found in rd1 mutants. However, an overall slower decay of retinal structure and function predicts that rd10 mice might become excellent models for rescue approaches. J. Comp. Neurol. 500:222–238, 2007.

Protection of retinal ganglion cells from natural and axotomy-induced cell death in neonatal transgenic mice overexpressing bcl-2.

Approximately half of the retinal ganglion cells (RGCs) present in the rodent retina at birth normally die during early development. Overexpression of the proto-oncogene bcl-2 recently has been shown to rescue some neuronal populations from natural cell death and from degeneration induced by axotomy of nerves within the peripheral nervous system. Here we study in vivo the role of the overexpression of bcl-2 in the natural cell death of RGCs and in the degenerative process induced in these cells by transection of the optic nerve. We find that in newborn bcl-2 transgenic mice, the number of RGCs undergoing natural cell death is considerably lower than in wild-type pups. Consistently, a vast majority (90%) of the ganglion cells found in the retina of neonatal transgenics are maintained in adulthood, whereas only 40% survive in wild-type mice. After transection of the optic nerve, the number of degenerating ganglion cells, determined by counting pyknotic nuclei or nuclei with fragmented DNA, is substantially reduced in transgenic mice. In wild-type animals, almost 50% of ganglion cells degenerate in the 24 hr after the lesion, whereas almost the entire ganglion cell population survives axotomy in transgenic mice. Therefore, overexpression of bcl-2 is effective in preventing degeneration of this neuronal population, raising the possibility that ganglion cells are dependent on the endogenous expression of bcl-2 for survival. The remarkable rescue capacity of bcl-2 overexpression in these neurons makes it an interesting model for studying natural cell death and responses to injury in the CNS.

Dicer Inactivation Leads to Progressive Functional and Structural Degeneration of the Mouse Retina

MicroRNAs (miRNAs) are small, highly conserved molecules that have been shown to regulate the expression of genes by binding to specific target mRNAs. Dicer, an RNase III endonuclease, is essential for the production and function of mature miRNAs, and removal of Dicer has been shown to disrupt many developmental processes. In this study, Dicer was removed specifically from the retina using a floxed Dicer conditional allele and the retinal Chx10Cre transgene. Retinal Dicer knock-out mice displayed a reproducible inability to respond to light. In addition, morphological defects were observed with the formation of photoreceptor rosettes at postnatal day 16, which progressed to more general cellular disorganization and widespread degeneration of retinal cell types as the animals aged. This was accompanied by concomitant decrease in both scotopic and photopic electroretinogram (ERG) responses. Interestingly, removing a single allele of Dicer resulted in ERG deficits throughout life but not to morphological abnormalities. Northern blot analysis of Dicer-depleted retinas showed a decrease in several miRNAs. The observation that progressive retinal degeneration occurred after removal of Dicer raises the possibility that miRNAs are involved in retinal neurodegenerative disorders.

Remodeling of second-order neurons in the retina of rd/rd mutant mice

This is a brief review of data obtained by analyzing the morphology and the physiology of the retinas in rd/rd and normal, wt mice, aged 10-90 days. Second-order neurons of the rd/rd show abnormalities that start with the anomalous development of rod bipolar cells around P10 and culminate with the atrophy of dendrites in cone bipolar cells, mostly evident at P90. Horizontal cells remodel considerably. Cone-mediated ERGs, (recorded between 13 and 16 days of age) have reduced a-wave and b-wave amplitudes and longer b-wave latency and duration. B-wave abnormalities indicate specific postreceptoral dysfunction. Morphological and ERG changes in rd/rd retinas are consistent with substantial inner retinal remodeling associated to photoreceptor degeneration.

Retinal Ganglion Cells Survive and Maintain Normal Dendritic Morphology in a Mouse Model of Inherited Photoreceptor Degeneration

Retinitis pigmentosa (RP), a family of inherited disorders characterized by progressive photoreceptor death, is a leading cause of blindness with no available cure. Despite the genetic heterogeneity underlying the disease, recent data on animal models show that the degeneration of photoreceptors triggers stereotyped remodeling among their postsynaptic partners. In particular, bipolar and horizontal cells might undergo dendritic atrophy and secondary death. The aim of this study was to investigate whether or not concomitant changes also occur in retinal ganglion cells (RGCs), the only retinal projection neurons to the brain and the proposed substrate for various therapeutic approaches for RP. We assessed the retention of morphology, overall architecture, and survival of RGCs in a mouse model of RP at various stages of the disease. To study the morphology of single RGCs, we generated a new mouse line by crossing Thy1-GFP-M mice (Feng et al., 2000), which express GFP (green fluorescent protein) in a small number of heterogeneous RGCs types, and rd10 mutants, a model of autosomal recessive RP, which exhibit a typical rod–cone degeneration (Chang et al., 2002). We show remarkable preservation of RGC structure, survival, and projections to higher visual centers in the time span from 3 to 9 months of life, well beyond the death of photoreceptors. Thus, unlike second-order neurons, RGCs appear as a considerably stable population of cells, potentially constituting a favorable substrate for restoring vision in RP individuals by means of electronic prostheses or direct expression of photosensitive proteins.

Long‐term Survival of Retina Optic Nerve Section in Adult Ganglion Cells Following bcl‐2 Transgenic Mice

The bcl‐2 gene codes for a protein that acts as a powerful inhibitor of active cell death. Since the transection of the optic nerve in adult mammalians starts a massive process of degeneration in retinal ganglion cells, we investigated whether the overexpression of bcl‐2 in adult transgenic mice can protect the axotomited ganglion cells. We performed intracranial optic nerve transection on both wild type and transgenic adult mice, and we tested cell survival 2 or 3.5 months after axotomy. The percentage of surviving ganglion cells after optic nerve section was computed by combining the counts of the optic nerve fibres in intact nerves with the cell density measures of the ganglion cell layer of axotomized retinae. From these data we found that in transgenic mice˜65% of ganglion cells survived 3.5 months after axotomy. In contrast, 2 months after surgery, <10% of ganglion cells were left in wild type retinae. We have also examined the morphology and fine structure of the proximal stump of the sectioned optic nerves by light and electron microscopy. In the transgenic mice a very large number of axons survived after surgery and they still exhibited fairly normal morphology and ultrastructure. On the other hand the wild type transected nerves had only a few visible axons that displayed clear signs of degeneration. We conclude that the overexpression of Bcl‐2 protein in central neurons is a very effective strategy to ensure long‐term survival in axotomized cells.

Transformation of cone precursors to functional rod photoreceptors by bZIP transcription factor NRL

Networks of transcriptional regulatory proteins dictate specification of neural lineages from multipotent retinal progenitors. Rod photoreceptor differentiation requires the basic motif–leucine zipper (bZIP) transcription factor NRL, because loss of Nrl in mice (Nrl−/−) results in complete transformation of rods to functional cones. To examine the role of NRL in cell fate determination, we generated transgenic mice that express Nrl under the control of Crx promoter in postmitotic photoreceptor precursors of WT and Nrl−/− retina. We show that NRL expression, in both genetic backgrounds, leads to a functional retina with only rod photoreceptors. The absence of cones does not alter retinal lamination, although cone synaptic circuitry is now recruited by rods. Ectopic expression of NRL in developing cones can also induce rod-like characteristics and partially suppress cone-specific gene expression. We show that NRL is associated with specific promoter sequences in Thrb (encoding TRβ2 transcription factor required for M-cone differentiation) and S-opsin and may, therefore, directly participate in transcriptional suppression of cone development. Our studies establish that NRL is not only essential but is sufficient for rod differentiation and that postmitotic photoreceptor precursors are competent to make binary decisions during early retinogenesis.

Long-term retinal function and structure rescue using capsid mutant AAV8 vector in the rd10 mouse, a model of recessive retinitis pigmentosa.

The retinal degeneration 10 (rd10) mouse is a well-characterized model of autosomal recessive retinitis pigmentosa (RP), which carries a spontaneous mutation in the β subunit of rod cGMP-phosphodiesterase (PDEβ). Rd10 mouse exhibits photoreceptor dysfunction and rapid rod photoreceptor degeneration followed by cone degeneration and remodeling of the inner retina. Here, we evaluate whether gene replacement using the fast-acting tyrosine-capsid mutant AAV8 (Y733F) can provide long-term therapy in this model. AAV8 (Y733F)-smCBA-PDEβ was subretinally delivered to postnatal day 14 (P14) rd10 mice in one eye only. Six months after injection, spectral domain optical coherence tomography (SD-OCT), electroretinogram (ERG), optomotor behavior tests, and immunohistochemistry showed that AAV8 (Y733F)-mediated PDEβ expression restored retinal function and visual behavior and preserved retinal structure in treated rd10 eyes for at least 6 months. This is the first demonstration of long-term phenotypic rescue by gene therapy in an animal model of PDEβ-RP. It is also the first example of tyrosine-capsid mutant AAV8 (Y733F)-mediated correction of a retinal phenotype. These results lay the groundwork for the development of PDEβ-RP gene therapy trial and suggest that tyrosine-capsid mutant AAV vectors may be effective for treating other rapidly degenerating models of retinal degeneration.

Remodeling of cone photoreceptor cells after rod degeneration in rd mice

We studied the survival of cone photoreceptors following the degeneration of rods in the rd mouse. Cones were visualized by selective expression of green fluorescent protein (GFP) following transduction with an adeno-associated virus (AAV) vector. As previously reported, many cones survive after the initial degeneration of the rods. Soon after the initial degeneration, they lose their outer segments and all but a vestigial inner segment; and they partially retract or lose their axon and synaptic pedicle. However, they retain many fundamental features of the cone phenotype, and for many weeks show a polarized morphology indicative of substantial regrowth of processes. The cells retain their laminar position, forming a cell row just distal to a much thinned outer plexiform layer. The somata subsequently enlarge. Most of the cells extend bipolar processes, recreating the original bipolar morphology of a photoreceptor cell--though now turned on its side relative to the native position. The cells express short- or middle-wavelength opsins, recoverin and connexin36. One or more of the polarized processes could often be shown to contain synaptic ribbons, as visualized by antibodies against RIBEYE. The cones do not express protein kinase C alpha, Go alpha, ChX10 or calbindin, markers of bipolar or horizontal cells. The partially differentiated cone morphology persists for at least several months, after which the processes begin to retract and there is slow loss of the cells. Thus, during the time following the loss of their rod-dominated microenvironment, the cones achieve a semi-stable state in which much of their normal phenotype is preserved. Cone photoreceptors in retinas of human RP donors appear from their morphology to undergo a similar progression. The therapeutic window for rescue of cone photoreceptors may be longer than would have been thought.