Deficiency of SHP-1 Protein-Tyrosine Phosphatase in “Viable Motheaten” Mice Results in Retinal Degeneration

Abstract

purpose. Viable motheaten mutant mice (abbreviated allele symbol me ^v ) are deficient in Src-homology 2-domain phosphatase (SHP)-1, a critical negative regulator of signal transduction in hematopoietic cells. These mice exhibit immune dysfunction, hyperproliferation of myeloid cells, and regenerative anemia. This study focused on the role of SHP-1 in retinal homeostasis.

methods. Ophthalmoscopy, histology, transmission electron microscopy (TEM), electroretinography (ERG), immunohistochemistry, Western blot, bone marrow transplantation, and genetic crosses were performed for phenotypic characterization and functional studies of retinal degeneration (RD) in me ^v /me ^v mice.

results. Fundus examinations of me ^v /me ^v mice revealed numerous, small white spots. Histologic examination demonstrated photoreceptor loss beginning at 3 weeks of age, and TEM revealed disorganization and reduction in the number of outer segments, as well as the presence of phagocytic cells in the subretinal space. Rod- and cone-mediated ERGs were abnormal. SHP-1 protein was expressed in mouse and human retinal lysates and was localized to the outer nuclear layer of the retina in me ^v /me ^v and control mice. Autoantibodies are not necessary for RD, as B-cell-deficient me ^v /me ^v Igh-6 ^tm1Cgn mice had no attenuation of photoreceptor cell loss compared with age-matched me ^v /me ^v mice. Histologic examination of lungs and retinas from normal recipients of me ^v /me ^v marrow revealed the classic acidophilic macrophage pneumonia of me ^v /me ^v mice, but no retinal degeneration.

conclusions. me ^v /me ^v mice exhibit normal retinal development with the onset of RD at 3 weeks of age and a rapidly progressive loss of photoreceptors. These findings support the hypothesis that SHP-1 plays a critical role in retinal homeostasis.

The identification and characterization of mutations in the mouse that cause retinal degeneration are critical in identifying molecular components of the retina and the phototransduction cascade. Of great interest is the identification of molecules involved in retinal cell-signaling pathways. These pathways, through the use of enzymes or adaptor proteins, recruit and regulate downstream molecules that affect intracellular functions and regulate gene transcription. To understand the molecular mechanisms of postmitotic retinal homeostasis, cell-signaling pathways in the retina must be elucidated. 

We have identified retinal degeneration in viable motheaten mice, a previously undetected phenotype of this spontaneous mutant mouse (Lyons BL, et al. IOVS 2004;45:ARVO E-Abstract 3620). The autosomal recessive motheaten (Ptpn6 ^me ) and viable motheaten (Ptpn6 ^me-v ) mutations disrupt the structural gene for protein-tyrosine phosphatase nonreceptor 6 (Ptpn6), commonly referred to as Src-homology 2-domain phosphatase-1 (SHP-1). This gene, located on mouse chromosome 6, is primarily expressed in hematopoietic cells. ¹ The motheaten (me) mutation is a null mutation, and the allelic viable motheaten (me ^v ) mutation results in abnormal SHP-1 protein. This catalytically defective protein retains approximately 25% of wild-type activity. ² The SHP-1 protein has been well studied in the immune system and primarily functions as a negative regulator of signal transduction. ^{2 3} In the central nervous system SHP-1 has been shown to have a regulatory role in astrocyte activation and proliferation. ⁴ In our present study, SHP-1 protein was expressed in the mouse retina, and deficiency of this protein resulted in a rapidly progressive retinal degeneration. 

The purpose of the present study was to (1) characterize the temporal progression of retinal degeneration in the me ^v /me ^v mouse; (2) determine whether the retinal degeneration was due to a cell-autonomous effect of SHP-1 deficiency in the retina or was a secondary consequence of SHP-1 deficiency in the hematopoietic system; and (3) examine the manner of and possible mechanism of photoreceptor cell death. We found that me ^v /me ^v mice exhibit normal retinal development with the onset of retinal degeneration at 3 weeks of age. Progressive loss of photoreceptors is due, at least in part, to a caspase-dependent mechanism. The SHP-1 protein is expressed in the outer nuclear layer (ONL) and is upregulated in the dark-adapted retina. These findings support the hypothesis that SHP-1 is a critical signaling molecule necessary for normal retinal homeostasis, which may modulate the phototransduction cascade. 

Methods

Mice

C57BL/6J motheaten and viable motheaten mice were raised by mating C57BL/6J-Ptpn6 ^me /J (+/me) or Ptpn6 ^me-v /J (+/me ^v ) heterozygotes. Homozygous (me/me or me ^v /me ^v ) offspring were identified at 4 to 5 days of age by the presence of multifocal alopecia. Heterozygotes (+/me or +/me ^v ) were identified by polymerase chain reaction (PCR) as previously described. ^{5 6} The mean lifespan of C57BL/6J me/me and me ^v /me ^v mice in our colony is 22 days ⁷ and 96 days (Lyons BL, unpublished data, 2003), respectively. BALB/cByJ me ^v /me ^v mice were produced after 10 generations of backcrossing the me ^v mutation onto the BALB/cByJ strain. me ^v /me ^v mice on the albino C57BL/6J-Tyr ^c-2J /J (c2J) strain were generated by breeding histocompatible ovariectomized recipients of C57BL/6J-me ^v /me ^v ovaries with C57BL/6J-c2J/c2J males. F1 offspring were intercrossed. c2J/c2J homozygotes were identified by the albino coat color and +/me ^v heterozygotes were identified by PCR. Mice of the desired genotypes were produced by sib mating of +/me ^v c2J/c2J mice. B6.Cg-Ptpn6 ^me-v Itgb2 ^tm1Bay (me ^v /me ^v Mac-1 ^null ) mice were generated by breeding histocompatible ovariectomized recipients of C57BL/6J-me ^v /me ^v ovaries with B6.129S7-Itgb2 ^tm1Bay /J homozygotes. Mice doubly heterozygous for me ^v and Mac-1 ^null were identified by PCR. Mice of the desired genotypes were produced by sib mating of mice heterozygous for me ^v and homozygous for Mac-1 ^null . The same breeding scheme was used to generate B6.Cg-Ptpn6 ^me-v Igh-6 ^tm1Cgn (me ^v /me ^v Igh-6 ^null ) mice. 

All mice were bred and housed at The Jackson Laboratory under standard SPF conditions with a 14-hour light–10-hour dark cycle and were provided pasteurized food and acidified water ad libitum. The experimental protocols were in accord with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by our Institutional Animal Use Committee. 

Ocular Examination

Indirect ophthalmic examination of the fundus was performed as previously described. ⁸ Five pairs of C57BL/6J (B6) me ^v /me ^v and +/? littermate control mice at 3, 4, 8, and 12-weeks of age were examined. 

Angiography

After indirect ophthalmic examination, the integrity of the retinal vasculature was assessed by fluorescein angiography, as previously described. ⁸ Three pairs of 4-month-old B6-me ^v /me ^v and littermate control mice were examined. Multiple, sequential, timed photographs of the retinal vasculature were taken. 

Histology

B6-me ^v /me ^v and littermate control mice were euthanatized by CO₂ asphyxiation, and eyes were immediately enucleated. For temporal studies, the eyes were immersed in 16% paraformaldehyde, 25% glutaraldehyde in 0.2 M phosphate buffer at 4°C for 24 hours. They were then embedded in methacrylate historesin and 2-μm sections were cut in a horizontal plane at the level of the optic nerve. For all other studies, tissues were paraffin embedded after fixation, and 5-μm sections were cut as just described. Tissues were immersed in a modified methacarn fixative (67% [vol/vol] absolute methanol and 37% glacial acetic acid) at 4°C for 24 hours, and sections were stained with hematoxylin and eosin for routine histology. For immunohistochemical studies, eyes were fixed in 90% ethanol for SHP-1 detection or 10% neutral-buffered formalin for F4/80 and activated caspase-3 detection. 

Electron Microscopy

For ultrastructural analysis, eyes from B6-me ^v /me ^v and littermate control mice were immersed in cold fixative (2% formaldehyde and 2.5% glutaraldehyde in 100 nM cacodylate buffer [pH 7.4] containing 0.025% CaCl₂) for a minimum of 3 hours and hemisected along the ora serrata. The posterior eyecups were sectioned into four quadrants and each quadrant further sectioned into small wedges (∼1 × 2 mm). The tissue wedges were washed in cold PBS, postfixed in 1% osmium tetroxide, dehydrated through a series of increasing concentrations of ethanol, and embedded in Epon-Araldite resin (Electron Microscopy Sciences, Fort Washington, PA). 

Immunohistochemistry

Retinas from B6-me/me, B6-me ^v /me ^v c ^2J /c ^2J , and littermate control mice were assayed for SHP-1 and F4/80 protein expression by immunohistochemistry. Tissue sections were incubated with rabbit anti-human SH-PTP1 polyclonal antibody (1:100; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or rat anti-mouse F4/80 monoclonal antibody (1:2; Serotec, Raleigh, NC). For F4/80 detection, antigen retrieval was performed by incubating sections for 10 minutes with proteinase K (200 μg/mL) before labeling with the primary antibody. Antibody binding was detected for SHP-1 with EnVision+ (DakoCytomation, Carpinteria, CA) and for F4/80 with an avidin biotin complex (ABC) kit (Vectastain; Vector Laboratories, Burlingame, CA) and the peroxidase substrate 3,3′-diaminobenzidine tetrahydrochloride (DAB; Sigma-Aldrich). Eyes from B6-me ^v /me ^v and littermate control mice at 1, 2, and 3 months of age were assayed for activated caspase-3 by immunohistochemistry. Three eyes from three different mice in each age group were assayed. Tissue sections from the small intestine of lethally γ-irradiated and nonirradiated mice were used for the positive and negative controls, respectively. For antigen retrieval, the sections were heated in a microwave oven for 10 minutes in 10 mM sodium citrate (pH 6.0). Tissue sections were then incubated with rabbit anti-human caspase-3 antibody (1:200; Cell Signaling Technology, Beverly, MA) overnight at 4°C. This antibody detects the large fragment of activated caspase-3. Antibody binding was detected with a goat anti-rabbit IgG ABC kit (Vectastain; Vector Laboratories) and DAB. Antibody-treated sections were examined by light microscope and images captured (SPOT camera; Diagnostics Instruments, Sterling Heights, MI). The outer nuclear layer area was measured on computer (MetaMorph; Universal Imaging, West Chester, PA). 

For all immunohistochemistry experiments, multiple retinal sections on a slide were incubated with the appropriate primary and secondary antibodies, and, as a negative control, the primary antibody was omitted for a single retinal section on each slide. No staining was detected on negative control retinal sections. 

Electroretinography and Light Calibrations

Retinal function was assessed by ERG, as previously described. ⁹ Because B6-me ^v /me ^v mice had consistently smaller pupil sizes compared with littermate controls, we expressed stimulus intensity in retinal illuminance (RI) where RI = [corneal luminance (cd/m²)][pupil area (mm²)]. ¹⁰ All comparisons are made in terms of retinal illuminance. 

Bone Marrow Transplantation

B6 wild-type mice were irradiated with a lethal dose of 10 Gy of γ-irradiation with a ¹³⁷Cs irradiator (Shepard Mark I; J. L. Shepard and Associates, San Fernando, CA), separated into two groups, and reconstituted with 5 × 10⁶ donor bone marrow cells injected into the lateral tail vein 3 hours after irradiation. Group 1 received bone marrow cells from B6 me ^v /me ^v donors, and group 2 received bone marrow cells from B6 wild-type donors. Histopathologic examination of the retina and lung was conducted 5 to 7 weeks after transplantation (n = 3). Bone marrow transplantation was repeated using BALB/cByJ mice. Wild-type mice were irradiated with 8 Gy of γ-radiation and treated as described earlier. Group 1 received bone marrow cells from BALB/cByJ-me ^v /me ^v donors, and group 2 received bone marrow cells from BALB/cByJ wild-type donors. Histopathologic examination of the retina and lung was conducted 6 weeks after transplantation (n = 5). 

Western Blot Analysis

The neural retina was separated from the RPE and adherent choroid by microdissection, 4 to 18 retinas from mice of each genotype were pooled, and total cellular protein lysates of the neural retinas were prepared in Tris-buffered saline with 1% Igepal (Sigma-Aldrich) and protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). Protein content was determined by the Bradford method (D_c Protein Assay; Bio-Rad Laboratories, Hercules, CA) with a bovine serum albumin standard. For detection of SHP-1 protein expression, mice were dark adapted overnight and retinas collected under red light. Light-adapted retinas were collected 3 hours after light onset. Human retina normal tissue lysate was commercially obtained (Abcam Inc., Cambridge, MA). Western blot analysis was performed as previously described. ¹ The blots were probed with affinity-purified rabbit anti-SHP-1 (1:4000; Research Genetics Inc., Huntsville, AL), polyclonal IgG fraction of rabbit anti-glial fibrillary acidic protein (GFAP; 1:2000; Abcam), or monoclonal IgG1 mouse anti-cellular retinaldehyde-binding protein (CRALBP; 1:5000; Affinity Bioreagents, Golden, CO). As an internal control for protein loading, blots were washed in stripping buffer (62.5 mM Tris-HCl [pH 6.7], 100 mM 2-mercaptoethanol, and 2% sodium dodecyl sulfate) at 50°C and reprobed with either polyclonal IgG fraction of goat anti-actin or goat anti-heat shock protein 70 (HSP; 1:500 and 1:200, respectively; Santa Cruz Biotechnology). Antibodies were detected with peroxidase-conjugated anti-rabbit and anti-goat IgG (1:25,000; Research Diagnostics Inc., Flanders, NJ) or peroxidase-conjugated anti-mouse immunoglobulins (IgG, IgA, and IgM; 1:2000; Sigma-Aldrich). Blots were developed with chemiluminescence (ECL plus; GE Healthcare, Piscataway, NJ) and specifically bound protein was detected by exposure to autoradiograph film (Eastman Kodak Co., Rochester, NY). Band analysis was performed on a Macintosh computer (Apple Computer, Cupertino, CA) with the public-domain NIH Image program (developed at the US National Institutes of Health by Wayne Rasband and available at http://rsb.info.nih.gov/nih-image/). Band optical density ratios were calculated and normalized to the wild-type protein band. 

Results

Ocular Examination

Examination of me ^v /me ^v mice by indirect ophthalmoscopy demonstrated the presence of numerous, small, white foci, giving a mottled appearance to the retina (Fig. 1B) . The white spots were variable in size, randomly distributed throughout the retina and visible as early as 1 month of age. These grossly visible lesions probably reflect the atrophy of the outer nuclear layer (ONL), seen microscopically. The distribution and caliber of retinal vessels appeared normal in me ^v /me ^v mice. Normal vascular integrity and a normal retinal capillary bed were demonstrated by fluorescein angiography in me ^v /me ^v mice (data not shown). 

Histopathology

Histologic examination of retinal sections from me ^v /me ^v mice revealed a reduction in the length of the outer segments that was first evident at postnatal day 16 (Fig. 1D) . These widely scattered focal areas of outer segment shortening were frequently associated with disorganization of the outer segments and occasionally with pyknotic photoreceptor nuclei. Random, multifocal areas of photoreceptor cell loss, as evidenced by a reduction in the thickness of the ONL, were observed beginning at 1 month of age (Fig. 2A) . With increasing age, there was a diffuse, progressive thinning of the ONL and loss of outer segments. By 3 months of age, the ONL was multifocally reduced from a normal thickness of 10 to 12 rows of nuclei to two to four rows. Mice heterozygous at the me ^v locus examined at 9 months of age had no histologic evidence of retinal degeneration (data not shown). 

The principle lesion in me ^v /me ^v mice is the over proliferation of myeloid cells. To study the role of peripheral macrophages in the retinal degeneration of me ^v /me ^v mice, we generated me ^v /me ^v mice that were also homozygous for a targeted mutation in the integrin beta 2 chain (Itgb2 ^tm1Bay , common gene symbol Mac-1). Mac-1 ^null mice are macrophage deficient. Examination of the retina of these doubly homozygous mutant mice (me ^v /me ^v Mac-1 ^null ) showed photoreceptor cell loss comparable to that in age-matched me ^v /me ^v mice (data not shown). 

Electron Microscopy

Ultrastructural evaluation of retinas from me ^v /me ^v and control mice was performed using TEM. In the outer segments, there was a reduction in the length, disorganization, and, in some sections, complete absence in the me ^v /me ^v retina (Fig. 3B) . Numerous vacuoles were evident within the inner and outer segments of the photoreceptor cells, and individual cells were frequently present in the subretinal space (Fig. 3C) . These subretinal cells lacked the cytoplasmic pigment granules characteristic of retinal pigment epithelial (RPE) cells and frequently contained electron-dense material similar to packets of outer segment discs. The detection by immunohistochemistry of F4/80, a cell surface antigen expressed by macrophages, on these cells (data not shown) is consistent with the subretinal cells’ being of macrophage lineage and not RPE cells. RPE cells in me ^v /me ^v mice appeared structurally normal with apical microvilli enveloping adjacent outer segments. The number and morphology of the mitochondria present in the inner segments of the me ^v /me ^v retina appear normal (Fig. 3D) . 

Electroretinography

Rod- and cone-mediated ERGs were recorded to evaluate retinal function in me ^v /me ^v mice. Representative ERGs are presented in Figure 2 . Rod ERGs for the me ^v /me ^v mouse are comparable to those obtained from a littermate control at 1 month of age (Fig. 2B) . 

At 2 months of age, rod ERG responses in the me ^v /me ^v mouse are clearly attenuated relative to the control mouse. Rod responses progressively decline but are still detectable at 3 months of age, the oldest age at which me ^v /me ^v mice could be tested. In contrast, cone ERG responses were better preserved at 1 month, but attenuation of cone wave amplitudes was also evident in the me ^v /me ^v mouse by 3 months of age (Fig. 2C) . 

Rod mediated intensity–response functions of individual me ^v /me ^v mice and littermate controls at 1, 2, and 3 months of age are illustrated in Figure 4 . At higher retinal illuminances the a-wave amplitudes were mildly attenuated in me ^v /me ^v mice at 1 month of age but progressively diminished with age (Fig. 4A) . A similar attenuation with age was observed in the analysis of me ^v /me ^v b-wave amplitudes (Fig. 4B) . 

To quantify rod retinal responsivity, retinal illuminance versus b-wave amplitude data was fitted with a Naka-Ruston equation to estimate V _max, the maximum saturated retinal response (Fig. 4C) . There was a statistically significant reduction of V _max for me ^v /me ^v mice beginning at 2 months of age. Analysis of the cone b-wave amplitudes, at maximum stimulation, in the me ^v /me ^v mice, was not significantly different from those of the control subjects at any age, although there was a trend toward reduced amplitudes at the oldest age (Fig. 4D) . 

Together, these data demonstrate a progressive loss of rod photoreceptor function in me ^v /me ^v mice as they age. Although not statistically significant, there was a marked reduction in me ^v /me ^v cone function, compared with littermate controls, by 3 months of age. The differences in rod and cone function in me ^v /me ^v mice are consistent with an early loss of rod photoreceptors, with cone photoreceptors being more resistant to the deleterious effects of SHP-1 deficiency. 

Apoptosis of Photoreceptors

To determine whether the loss of photoreceptors in the me ^v /me ^v retina was through an apoptotic mechanism, we assayed for activated caspase-3 by immunohistochemistry. Caspase-3 is a key executor of apoptosis and has been associated with photoreceptor cell death in other mouse models of retinal degeneration. ¹¹ Activated caspase-3 was detected in a small number of photoreceptor cells from me ^v /me ^v retinas at all ages examined, and there was acceleration in the rate of photoreceptor apoptosis with increasing age (Fig. 5A) . At 2 months of age, there was a twofold increase in caspase-positive cells in the retina of me ^v /me ^v mice compared with 1-month-old me ^v /me ^v mice, and by 3 month of age there was a threefold increase. Caspase-3 expression was cytoplasmic and mostly perinuclear, and photoreceptor cells had either normal or pyknotic nuclear morphology (Fig. 5B) . A few photoreceptors in retinal sections from me ^v /me ^v mice had pyknotic nuclei, a hallmark of apoptosis, but were not caspase-3 positive. This finding may reflect loss of caspase-3 activity before nuclear condensation or apoptosis in some photoreceptors of me ^v /me ^v mice occurs via a caspase-independent pathway. There were no caspase-3 positive cells observed in the retina of control mice at any of the ages examined. 

Bone Marrow Transfer

Previous work has shown that the transfer of B6-me ^v /me ^v bone marrow into irradiated syngeneic wild-type hosts can transfer the autoimmune disease 2 to 3 weeks after transplantation, ¹² demonstrating that the immunologic dysfunction caused by the me ^v /me ^v mutation is determined at the level of bone marrow progenitor cells. To determine whether the retinal degeneration of me ^v /me ^v mice is also determined at the level of bone marrow progenitor cells, bone marrow from B6-me ^v /me ^v mice was injected intravenously into irradiated syngeneic wild-type hosts. Histopathologic examination of recipients of B6-me ^v /me ^v marrow, 5 to 7 weeks after transplantation, revealed acidophilic macrophage pneumonia, the hallmark lesion of me ^v /me ^v mice, but no retinal degeneration (Fig. 6C 6D) . Because recipients of B6-me ^v /me ^v bone marrow do not live beyond 7 weeks after transplantation, it is possible that the retinal degeneration develops more slowly and is not morphologically evident at the time of death. Bone marrow transplantation experiments were repeated in BALB/cBy mice. me ^v /me ^v mice on the BALB/cBy background show a more severe and earlier onset of retinal degeneration, with complete loss of photoreceptors by 5 weeks of age (Lyons BL, unpublished data, 2002). As in recipients of B6-me ^v /me ^v marrow, retinal degeneration was not evident in recipients of BALB/cByJ-me ^v /me ^v bone marrow (data not shown). These results suggest that there is a retina-specific defect in me ^v /me ^v mice and retinal degeneration is not determined at the level of bone marrow–derived progenitor cells. 

Protein Expression in the Retina

Recipients of me ^v /me ^v bone marrow failed to develop retinal degeneration, suggesting a local affect of SHP-1 deficiency in the retina. To determine if SHP-1 protein is expressed in the retina, Western blot analysis was performed. SHP-1 expression in retinal homogenates from dark- (D) and light-adapted (L) retinas is demonstrated in Figure 7A . SHP-1 protein is expressed in the retina with enrichment of SHP-1 protein in me ^v /me ^v compared with wild-type retinas. Densitometric semiquantification of the protein bands in light- versus dark-adapted retinas demonstrated a 112.1% and 77.8% increase of SHP-1 expression in the dark-adapted retinas of me ^v /me ^v and wild-type mice, respectively. As an internal control for the specificity of the anti-SHP-1 antibody, retinal homogenates from SHP-1 null me/me mice were included (Figs. 7A 7B) . A faint immunoreactive band was detected in all retinal lysates from me/me mice. 

Genetic mutations identified in the mouse that result in retinal degeneration have led to the identification of mutations in the homologous human gene in patients with similar disease. Therefore, it was critical to determine whether SHP-1 protein was also expressed in the human retina. Using a commercially available human normal retina cell lysate, we demonstrated SHP-1 protein expression in the human retina (Fig. 7B) . 

The differential expression of SHP-1 in light- versus dark-adapted retinas suggests that SHP-1 may have a role in visual pigment regeneration, potentially in dark adaptation. Therefore, we examined the expression level of cellular retinaldehyde binding protein (CRALBP) in me ^v /me ^v and control retinas. CRALBP is present in Müller and RPE cells and is involved in the regeneration of rod visual pigment. ¹³ Expression levels of CRALBP were increased 34.3% in retinal cell lysates from me ^v /me ^v mice over wild-type protein expression levels (Fig. 7C) . Numerous mammalian inherited retinal degenerations exhibit a reactive hypertrophy and activation of Müller cells that is reflected by an increase in glial fibrillary acidic protein (GFAP) in the retina. ¹⁴ Retinal cell lysates from me ^v /me ^v mice had a 74.7% increase in GFAP expression compared with the wild-type control lysate (Fig. 7D) . The functional significance of Müller cell activation in retinal degeneration and other retinal insults is currently unknown. 

Localization of SHP-1 in the Retina

Immunohistochemistry was used to localize SHP-1 within the retina (Fig. 8) . There is intense cytoplasmic staining for SHP-1 in the ONL that is sharply demarcated by the outer limiting membrane, with no staining of the outer segments (Fig. 8A 8B) . There also appears to be weak irregular cytoplasmic staining in the inner nuclear layer. To prevent melanin pigment granules from obscuring the visualization of the DAB chromogen in the RPE, retinas from albino C57BL/6J mice (C57BL/6J-Tyr ^c-2J ) were used (Figs. 8A 8B) . There was no staining in the RPE of C57BL/6J-Tyr ^c-2J albino mice. Retina from the SHP-1 null me/me mouse was used as an internal control for the specificity of the anti-SHP-1 antibody. There was no SHP-1 staining in the me/me retina (Fig. 8C) . 

Discussion

We describe a new mouse model of retinal degeneration that results from a deficiency in a known intracellular signaling protein. SHP-1-deficient me ^v /me ^v mice have early-onset and rapidly progressive retinal degeneration, with almost complete loss of photoreceptor cell layers by 3 months of age. 

The earliest lesions in the retina of me ^v /me ^v mice were detected at postnatal day 16. Examination of histologic sections showed a low number of pyknotic photoreceptor nuclei and focal areas with shortening and disorganization of the outer segments. Abnormalities of the outer segments are a common phenotype in many animal models of retinal degeneration and in most of these models, the outer segment defects are secondary to photoreceptor cell loss. ^{9 15 16 17 18} With the completion of outer segment development at postnatal day 21, the mouse retina is fully mature. ¹⁹ Although outer segment abnormalities are present in me ^v /me ^v mice before retinal maturity, we believe this is a degenerative process and not a developmental defect in disc morphogenesis, as the outer segments are not diffusely affected and most are morphologically normal. The failure of some areas to maintain normal outer segment length is probably due to a disruption in the localized production of outer segments secondary to photoreceptor cell death, as evidenced by the concurrent presence of pyknotic photoreceptor nuclei. 

The primary morphologic feature of me ^v /me ^v mice is the presence of a large number of myeloid cells in the lung, skin, and other tissues. ⁷ The relative paucity of phagocytic cells in the me ^v /me ^v retina and the absence of them in any other part of the eye suggest that unlike in other tissues, infiltration by peripheral myeloid cells is not the primary cause of photoreceptor cell death in these mutant mice. Examination of the retina of macrophage-deficient me ^v /me ^v Mac-1 ^null mice compared with retinas from age matched me ^v /me ^v mice revealed no attenuation of photoreceptor cell loss. In addition, evaluation of the retinal morphology in me ^v /me ^v bone marrow chimeras also supports the hypothesis that the deleterious effect of the me ^v mutation in the retina is cell specific and not dependent on bone marrow-derived cells. Recipients of me ^v /me ^v bone marrow developed acidophilic macrophage pneumonia, the hallmark lesion of me ^v /me ^v mice; however, these recipients did not undergo retinal degeneration. The presence of phagocytic cells in the subretinal space of me ^v /me ^v mice is most likely a consequence of photoreceptors cell death and the subsequent activation of resident microglial cells. Investigations in the Royal College of Surgeon rat have demonstrated that phagocytic cells found in this model of retinal degeneration are not blood-borne macrophages, but are derived from resident microglial cells that have become activated ²⁰ and that the microglia are not the initiators of, but are responding to, photoreceptor cell death. ²¹ Our data in the retinal degeneration of me ^v /me ^v mice is consistent with a similar temporal relationship between photoreceptor apoptosis and the presence of phagocytes in the subretinal space. 

Autoantibodies have been implicated in cancer-associated retinopathy and other acquired retinal diseases ^{22 23 24} and anti-recoverin antibodies have been shown to induce apoptosis of photoreceptor cells. ²⁵ me ^v /me ^v mice develop hyperimmunoglobulinemia and high levels of circulating autoantibodies. ² We have shown that autoantibodies are not essential for the development of anemia in me ^v /me ^v mice, ²⁶ and it appears that autoantibodies do not contribute in a significant way to the development of retinal degeneration in these mice. We generated me ^v /me ^v mice that were also homozygous for a targeted disruption of the membrane exon of the immunoglobulin μ gene (Igh-6 ^tm1Cgn , abbreviated gene symbol Igh-6 ^null ). Disruption of this gene results in B-cell deficiency, ²⁷ with Igh-6 ^null mice expressing little or no serum immunoglobulin except for small amounts of IgA. ²⁸ Mice doubly homozygous for the me ^v /me ^v and the Igh-6 ^null allele had no attenuation of photoreceptor cell loss compared with age-matched me ^v /me ^v mice (Lyons BL, unpublished data, 2000). Therefore, we believe it is unlikely that either autoantibodies or an increased number of peripheral macrophages present in me ^v /me ^v mice are directly responsible for the retinal degeneration in these mice. We believe that the photoreceptor cell death in these mutant mice is due to a disruption of the intrinsic SHP-1 function in the retina. 

Apoptosis of photoreceptor cells occurs in many human and animal retinal dystrophies and degenerations. ²⁹ Caspase-3 activation has been demonstrated in the photoreceptor cells of some mouse models of retinal degeneration. However, it has also been shown in the retinal degeneration 1 (Pde6b ^rd1 ) mouse that caspase-3 activation is not necessary for photoreceptor cell death. ¹¹ In me ^v /me ^v mice, photoreceptor cell loss is wholly or in part due to caspase-dependent apoptosis, as there is an increasing number of caspase-positive photoreceptor cells corresponding with the increasing loss of photoreceptors in the outer nuclear layer. The role of SHP-1 in cell survival is complex and appears contradictory. In the hematopoietic system, SHP-1 has been shown to be a negative regulator of cell proliferation, and, in the absence of SHP-1, there is amplified proliferation and activation of myeloid cells. ² In addition, splenocytes from SHP-1-deficient motheaten mice are resistant to γ-irradiation-induced apoptosis and cell cycle arrest. ³⁰ However, SHP-1 has also been identified as an important inhibitory signaling molecule in B-lymphocyte apoptosis and in SHP-1-deficient B cells there is enhanced apoptosis. ^{2 31} These disparate outcomes in cell survival are less incongruous when the absence of SHP-1 is viewed as a loss of inhibition and not active induction of apoptosis. Our work on the temporal characterization of retinal degeneration in me ^v /me ^v mice also fits this paradigm for the role of SHP-1 in photoreceptor cell survival. SHP-1 null, me/me mice, have a less severe retinal degeneration at their maximum lifespan then comparably aged me ^v /me ^v mice (Lyons BL, unpublished data, 2004). If reduction in SHP-1 protein directly induces photoreceptor cell apoptosis, then me/me mice would be expected to have a more severe retinal degeneration than age-matched me ^v /me ^v mice. 

The presence of faint immunoreactive bands on the Western blot analysis of retinal homogenates from SHP-1 null me/me mice was unexpected. Previous works in hematopoietic cells have shown Ptpn6 RNA levels are normal in me ^v /me ^v mice but undetectable in me/me mice and SHP-1 protein expression has not been detected in hematopoietic cells from me/me mice. ^{1 32 33} We cannot exclude the possibility that some SHP-1 protein is expressed in me/me photoreceptors; however, it is likely that the faint bands represent nonspecific immunoreactivity. 

SHP-1 protein expression, in both light- and dark-adapted me ^v /me ^v retinal lysates, was increased above the levels found in the wild-type retina. We speculate that this upregulation of basal SHP-1 protein levels in the me ^v /me ^v retina may be a compensatory effect of reduced SHP-1 enzymatic activity in me ^v /me ^v mice. Previous work has shown that bone marrow macrophages in me ^v /me ^v mice retain approximately 25% of the phosphatase activity found in macrophages from wild-type mice ¹ and that, in the resting state, SHP-1 is catalytically inactive as a consequence of Src homology 2 domain–mediated autoinhibition. ³⁴ Potentially, a reduction in the enzymatic activity of SHP-1 in the me ^v /me ^v retina may fail to reach the threshold necessary for autoinhibition, and consequently SHP-1 protein transcription is not terminated. 

There was an increase in CRALBP in the retina of me ^v /me ^v mice. Mutations in the human CRALBP gene (RLBP1) are responsible for autosomal recessive retinitis pigmentosa, retinitis punctata albescens, and other retinopathies that results in retinal degeneration and delayed dark adaptation. ³⁵ The mechanism by which SHP-1 deficiency may impact components of the visual cycle remains to be elucidated. 

The human homologue (PTPN6) of the mouse Ptpn6 gene is located on chromosome 12 at p13 (www.informatics.jax.org/searches/homology_report.cgi? _Marker_key=9686). No human retinal diseases have yet been identified with mutations in the PTPN6 gene (www.sph.uth.tmc.edu/retnet/disease.htm). Comparative sequence analysis of the coding segments of PTPN6 with the mouse Ptpn6 gene shows 89.2% and 96.1% nucleotide and amino acid similarity, respectively. ³⁶ This high homology and our demonstration of SHP-1 protein expression in the human retina warrant the examination of the PTPN6 gene for mutations in humans with similar retinal degenerations. 

 Supported by National Eye Institute Grants EY13742 (BLL), EY01475 (BLL, RSS, BC), and EY07758 (BC); National Cancer Institute Grant CA34196 (RSS); the Foundation Fighting Blindness (SN); and National Institute of Heart, Lung, and Blood Grant HL077642 (LDS).

 Submitted for publication August 30, 2005; revised November 9, 2005; accepted January 5, 2006.

 Disclosure: B.L. Lyons, None; R.S. Smith, None; R.E. Hurd, None; N.L. Hawes, None; L.M. Burzenski, None; S. Nusinowitz, None; M.G. Hasham, None; B. Chang, None; L.D. Shultz, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Fundus photographs of 4-month-old B6-me ^v /me ^v and littermate control mice (A, B). Normal fundus of a littermate control mouse (A). There were numerous, disseminated, small, white foci on the retina of the me ^v /me ^v mouse (B). Light micrographs of retinal sections from me ^v /me ^v and littermate control mice at 16 days of age (C, D). Normal retina of littermate control mouse (C). There was shortening of the outer segments (OS) and pyknotic photoreceptor nuclei (arrows) in the me ^v /me ^v retina (D). Scale bar, 50 μm.

Figure 2.

 

Temporal progression of retinal degeneration in B6-me ^v /me ^v mice. Representative photomicrographs of retinal sections (A) and ERGs (B, C) from a littermate control mouse at 3 months of age and from B6-me ^v /me ^v mice at 1, 2, and 3 months of age. me ^v /me ^v mice had a progressive loss of photoreceptors and outer segments, as evidenced by a reduction in the thickness and density of the ONL and outer segments (A). GC, ganglion cell layer; INL, inner nuclear layer; IS, OS, inner and outer segments, respectively; PE, pigment epithelium. Scale bar, 100 μm. The dark-adapted rod ERGs in me ^v /me ^v mice were similar in waveform to those in control mice; however, the amplitude of both the a- and b-waves were progressively attenuated with age (B). The a- and b-wave amplitudes were increased over control mice in the cone ERGs of the 1- and 2-month-old me ^v /me ^v mice. By 3 months of age, the wave amplitudes of cone ERGs in me ^v /me ^v mice were decreased compared with age-matched control mice (C).

Figure 3.

 

Ultrastructure of the retina in 1-month-old littermate control and B6-me ^v /me ^v mice (A, B). (A) Photoreceptor outer segments in the control mouse retina were normal in length and orientation. (B) In the me ^v /me ^v retina, there was almost complete loss of outer segments, and remaining outer segments were disorganized. There are vacuoles in both the inner and outer segments (arrows) and amorphous material ( ) in the subretinal space. (C) Subretinal cell in the retina of a 2-month-old me ^v /me ^v mouse. The cell adjacent to the microvilli of the RPE contained cytoplasmic inclusions and whorls of membranous material (arrows). (D) Morphologically normal mitochondria (arrows) in the inner segments of the retina from a 1-month-old me ^v /me ^v mouse. Scale bar, 2 μm.

Figure 4.

 

Plots of rod ERG amplitude versus retinal illuminance at 1, 2, and 3 months of age for individual me ^v /me ^v and control mice (A, B). Both a-wave (A) and b-wave (B) amplitudes progressively diminish with age in B6-me ^v /me ^v mice compared with the littermate control subjects. Analysis of (C) rod- and (D) cone-mediated function. Rod responsivity (V _max) was significantly reduced in me ^v /me ^v mice beginning at 2 months of age (C). There was no significant difference in the cone response of me ^v /me ^v mice and littermate control mice, although there was a trend toward reduced amplitudes in me ^v /me ^v mice at 3-months of age (D). Data are expressed as the mean ± SEM; n = 5; *P < 0.005.

Figure 5.

 

Kinetics of apoptosis and photomicrograph of an apoptotic photoreceptor in the me ^v /me ^v retina (A, B). Activated caspase-3 expression in photoreceptor cells of B6-me ^v /me ^v and littermate control mice at 1, 2, and 3 months of age. The number of caspase-3-positive photoreceptor cells in the me ^v /me ^v retina increased with age (A). No caspase-3 expression was detected in the photoreceptors of the littermate control at all ages tested. Perinuclear caspase-3 expression (arrow) and pyknotic nucleus in a photoreceptor cell from a 2-month-old me ^v /me ^v mouse (B).

Figure 6.

 

Light micrographs of tissue sections from B6+/+ bone marrow recipients 5 weeks after transplantation. Normal lung (A) and normal retina (B) from recipient of B6 wild-type bone marrow. Acidophilic macrophage pneumonia (C), the hallmark lesion of me ^v /me ^v mice, in a recipient of B6-me ^v /me ^v bone marrow. Normal retina (D) from recipient of B6-me ^v /me ^v bone marrow. Scale bar, 100 μm.

Figure 7.

 

Western blot analysis of retinal cell lysates. (A) SHP-1, a 68-kDa immunoreactive band was detected in wild-type and me ^v /me ^v light (L) and dark (D) adapted retinas. Faint immunoreactive bands were detected in the SHP-1 null, me/me, retina. SHP-1 content was increased in dark-adapted retinas (20 μg protein loaded in each lane). (B) SHP-1 protein expression was detected in human normal retina cell lysate (40 μg protein in each lane). (C) There was a mild increase in levels of CRALBP expression, a 37-kDa immunoreactive band, in me ^v /me ^v compared with littermate control lysate (20 μg protein in each lane). (D) GFAP protein expression, a 55-kDa immunoreactive band, was enhanced in me ^v /me ^v compared with littermate control lysate (50 μg protein in each lane). Blots were reprobed with anti-actin or anti-HSP70 antibodies as protein loading controls. Band optical density ratios are given below each lane.

Figure 8.

 

Immunohistochemistry of SHP-1 in the retina (A–C). SHP-1 was localized in the ONL and INL of (A) B6^+/+ Tyr ^c-2J and (B) me ^v /me ^v Tyr ^c-2J retinas. (C) There was no SHP-1 detected in the SHP-1-null, B6-me/me retina. Scale bar, 50 μm.

The authors thank Bruce Gott, Douglas Howell, and Barbara Mortimer for technical assistance and the scientific services of The Jackson Laboratory.