November 2007
Volume 48, Issue 11
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Glaucoma  |   November 2007
Distribution of Amyloid Precursor Protein and Amyloid-β Immunoreactivity in DBA/2J Glaucomatous Mouse Retinas
Author Affiliations
  • David Goldblum
    From the Department of Ophthalmology, Inselspital, University of Bern, Switzerland; and the
    Department of Ophthalmology, University Hospital Basel, University of Basel, Switzerland.
  • Anna Kipfer-Kauer
    From the Department of Ophthalmology, Inselspital, University of Bern, Switzerland; and the
  • Gian-Marco Sarra
    From the Department of Ophthalmology, Inselspital, University of Bern, Switzerland; and the
  • Sebastian Wolf
    From the Department of Ophthalmology, Inselspital, University of Bern, Switzerland; and the
  • Beatrice E. Frueh
    From the Department of Ophthalmology, Inselspital, University of Bern, Switzerland; and the
Investigative Ophthalmology & Visual Science November 2007, Vol.48, 5085-5090. doi:10.1167/iovs.06-1249
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      David Goldblum, Anna Kipfer-Kauer, Gian-Marco Sarra, Sebastian Wolf, Beatrice E. Frueh; Distribution of Amyloid Precursor Protein and Amyloid-β Immunoreactivity in DBA/2J Glaucomatous Mouse Retinas. Invest. Ophthalmol. Vis. Sci. 2007;48(11):5085-5090. doi: 10.1167/iovs.06-1249.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. Evidence suggests that altered metabolism of amyloid precursor protein (APP) may play a role in the pathophysiology of retinal ganglion cell (RGC) death in the etiology of glaucoma. The authors sought to determine the distribution of APP and amyloid-β (Aβ) in DBA/2J glaucomatous mouse retinas.

methods. The retinas of 3- and 15-month-old DBA/2J mice and C57/BL-6 mice (control group) were fixed with 4% paraformaldehyde and processed for immunohistochemistry. Antibodies used included a polyclonal antibody to the C terminus of Aβ 40 and a polyclonal antibody to the APP ectodomain. Immunohistochemically stained tissue was graded using light microscopy. Distribution and semiquantitative expression of APP and Aβ in young and old glaucomatous and normal retinas were determined and compared.

results. Strong APP and Aβ immunoreactivity was found in the RGC layer, optic nerve, and pia/dura of old DBA/2J retinas, with considerably higher intensity found in the old compared with the young DBA/2J mice. In contrast to glaucomatous mice, the control group did not show any notable age-related difference.

conclusions. Disruption of the homeostatic properties of secreted APP with consecutive Aβ cytotoxicity might be a contributing factor of ganglion cell loss in glaucomatous mouse retinas.

Amyloid-β A4 precursor protein (APP) is a transmembrane neuronal protein that has several isoforms generated by alternative splicing. It is expressed throughout the brain, including the retinal ganglion cells (RGCs). APP plays a central role in neurite outgrowth, synaptogenesis, and cell survival. 1 2 APP activates cGMP and increases inward K+ currents, 3 inducing a membrane hyperpolarization, and therefore protects against excitotoxicity. Furthermore, it acts at the cellular level in several processes, such as axonal transports, cell adhesion, 4 cholesterol metabolism, and gene transcription regulation. 5 In the eye, APP is synthesized in RGCs, rapidly transported to the optic nerve (ON) in small vesicles, and finally transferred to the axonal plasma membrane and synapses. 6 When it accumulates it can be cleaved pathologically into amyloid β (Aβ)-42, a hydrophobic 42 amino acid-containing polypeptide prone to aggregation (e.g., seen in plaques of Alzheimer disease [AD]) 7 8 or into Aβ 40, which is more hydrophilic, has a lower tendency to aggregate, and is found in microvascular angiopathies in the abluminal basement membrane. 9 10 11  
Recent studies imply 12 that there is a significantly higher incidence of glaucoma among patients with AD than in the healthy population, suggesting a possible relationship between these two diseases. Additionally, the same Aβ peptide found in AD was found in 40% of the aqueous of patients with glaucoma and in 38% of patients with exfoliation syndrome (XFS), suggesting that these diseases may share common features in the biochemistry and etiologies of AD. 13  
The aim of this study was to provide immunohistochemical evidence of APP and Aβ and to determine the amount and ocular distribution of these peptides in glaucomatous mouse retinas. 
Materials and Methods
Animal Tissue
Mice were anesthetized and perfused with 4% ice-cold buffered paraformaldehyde (PFA). Each eye and its ON were immediately enucleated and postfixed in 4% PFA for 24 hours and embedded in paraffin. Female DBA/2J-mice (aged rodent colonies; National Institute on Aging, Bethesda, MD), which have been shown to spontaneously develop elevated intraocular pressure with subsequent loss of RGCs from the age of 6 months, were used. 14 Two age groups were compared: old (15 months of age; n = 6) DBA/2J with established morphologic changes caused by long-lasting ocular hypertension and young (3 months of age; n = 6) DBA/2J in which no ocular hypertension had yet developed. The nonhypertensive control group consisted of the same number of age-matched female C57/BL-6 mice (aged rodent colonies; National Institute on Aging). 
Paraffin-embedded brain sections of 24-month-old APP23 transgenic (AD) mice served as positive controls for Aβ 40 and APP immunohistochemistry (Institute for Pathology, University of Basel, Basel, Switzerland; Novartis, Basel, Switzerland). 15 All experiments adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the federal and local ethical and agricultural committees. 
Immunohistochemistry
Antibodies were initially tested in a pilot study (data not shown) before the following protocol was established. Central sections through the optic nerve were mounted on coated glass slides and deparaffinized. For all sections, a peroxidase-mediated amplification system, based on the deposition of biotinylated tyramide (BT) molecules (TSA Biotin Kit; Perkin Elmer Life Sciences, Boston, MA), was used to amplify the staining signals. After rehydration, endogenous peroxidase activity was quenched in methanol containing 0.3% H2O2. After buffering in 0.1 M Tris-HCl, 0.15 M NaCl, and 0.05% Tween 20 (TNT) and preincubation in 0.10 M Tris-HCl, 0.15 M NaCl, and 0.5% blocking reagent (TNB; TSA Biotin Kit, Perkin Elmer Life Sciences), the sections were incubated with the primary antibody diluted in TNB for 1 hour at room temperature. Affinity-purified polyclonal antibodies used were Aβ 17–40/23, a polyclonal antibody to the C terminus of Aβ 40, and APP 474, a polyclonal antibody to the APP ectodomain. These noncommercial antibodies were kindly provided by Paolo Paganetti (Novartis). The optimal concentration of the primary antibody was experimentally determined to be 1:500 (Aβ 17–40/23) and 1:200 (APP 474). 
After several washes in TNT, streptavidin-horseradish peroxidase (SA-HRP) was added for 30 minutes. The slides were rinsed before amplification with biotinyl-tyramide-reagent, which was added and incubated for 5 minutes; this was followed by several washing steps and further incubation with SA-HRP. Chromogenic visualization was achieved with diaminobenzidine tetrahydrochloride (DAB) as substrate (DAKO, Baar, Switzerland). After a last washing step, the slides were counterstained with hematoxylin and then were dehydrated through ascending alcohols and xylene. Finally the slides were mounted and coverslipped with mounting medium (Eukitt; Inselspital-Apotheke, Bern, Switzerland). 
To test the specificity of the primary antibody, control sections were stained simultaneously according to the same procedure, with the exception that the primary antibody was omitted. Additionally, brain tissue of APP 23 served as a positive control. 
Two masked observers assessed all amplified sections for localization and intensity of specific immunoreactivity on a semiquantitative scale, with grades 0 (absolutely no staining was visible), + (for staining intensities other than 0 or ++, or when the two independent observers disagreed), and ++ (intensity and color equaled those of the positive control). Different ocular structures were graded separately, such as the RGC layer (RCGl), the ON, and the pial/dural tissue around the ON. Magnifications of 100×, 200×, 400×, and 1000× were used. 
Results
Different structures within the back of the eye displayed specific APP and Aβ immunoreactivity, whereas adjacent control sections in which the primary antibody was omitted revealed no immune reaction (Figs. 1 2) . Sections of old DBA/2J mice (Figs. 3 4 5 6)more often revealed evidence of highest staining intensities for APP and Aβ than did those of young and old controls (Figs. 7 8 9 10 11 12) . High accumulation of these peptides was found in specific areas, such as the RGCl, the pia/dura, and the ON. Overall the highest staining intensity was most frequently found in the old DBA/2J mice, and no staining was found in the age-matched C57/BL-6 mice or in the omission controls (without first antibody; Figs. 1 2 ). Figures 13 and 14display the percentage distributions of APP and Aβ immunoreactivity in different anatomic locations. 
Overall, the most frequent APP and Aβ levels were found among the old DBA/2J mice, especially in the pia/dura (APP, 71%; Aβ, 71%), followed by the ON (APP, 71%; Aβ, 14%) and the RGCl (APP, 14%; Aβ, 14%; Figs. 13 14 ). 
In sections stained for APP, the old DBA/2J mice showed the highest intensities in every structure (pia/dura, 71%; ON, 71%; RGCl, 14%) compared with the young DBA/2J (pia/dura, 25%; ON, 0%; RGCl, 0%), the old C57/BL-6 (pia/dura, 22%; ON, 22%; RGCl, 0%), and the young C57/BL-6 (pia/dura, 0%; ON, 0%; RGCl, 0%) mice (Fig. 13)
In the same eyes stained for Aβ, the old DBA/2J mice had the highest levels in all ocular structures (pia/dura, 71%; ON, 14%; RGCl, 14%). The young DBA/2J mice (pia/dura, 25%; ON, 0%; RGCl, 0%) showed no relevant staining compared with the old C57/BL-6 mice (pia/dura, 0%; ON, 0%; RGCl, 0%) or the young C57/BL-6 mice (pia/dura, 0%; ON, 0%; RGCl, 0; Fig. 14 ). 
In summary, staining intensity in the APP-marked tissue was remarkably more concentrated in old than in young DBA/2J retinas and was most frequent in the ON and the pia/dura, followed by the RGCl. 
In Aβ-stained eyes, the results were less obvious, and there was a noteworthy difference between the old DBA/2J pia/dura and the young DBA/2J and old control pia/dura (C57/BL-6). 
Discussion
This study demonstrated an increased accumulation of APP and Aβ in ocular structures in a spontaneous mouse model (DBA/2J) of long-lasting, chronic glaucoma. Using an antibody detecting Aβ 40, not the classical Aβ plaque formation, as in AD, was found in these retinas. This might have occurred because Aβ 40 is more hydrophilic and has a lesser tendency to aggregate than Aβ 42. McKinnon 16 analyzed both forms of Aβ by Western blotting in an induced glaucomatous rat model and found exclusively higher Aβ 40 than Aβ 42 levels in ocular structures. 
Elevated APP and Aβ accumulation was found in distinct ocular tissues, such as the RGC layer, the ON, and abundantly in the pial/dural complex. Consistent with the results of McKinnon, 17 the accumulation of APP and Aβ 40 in the ON was distinctively adjacent to the lamina cribrosa and was highest in the area behind the lamina cribrosa, suggesting traumatic alterations to the ON probably because of elevated IOP. Recently, other groups have also reported similar APP and Aβ distributions in mouse and rat glaucoma models (Schmid P, et al. IOVS 2004;45:ARVO E-Abstract 4684; Cordeiro M, et al. IOVS 2006;47: E-Abstract 2698). 
The highest levels of Aβ 40 were found in the area of the pial/dural tissue around the optic nerve, behind the lamina cribrosa of glaucomatous mice, where the arterioles and venules were located. It has been shown that Aβ can be isolated from meningeal arterioles/venules and from capillaries within the cerebral cortex in amyloid microangiopathy found in patients with AD. 18 19 This angiopathy was ultrastructurally characterized by amyloid fibrils found in the abluminal basement membrane of the vessels, with some extension into the surrounding perivascular neuropil. The immunohistochemically identified Aβ filaments were, as in our study, primarily Aβ 40. 20  
Changes in the pia/dura might have corresponded to a failure of APP elimination through their elimination pathways in glaucoma, additionally worsened by increased age. APP overload thereafter led to cytotoxic Aβ accumulation. Martin et al. 21 showed that disruption of dynein transport in glaucoma contributes to a failure of retrograde axonal transport and thus may be a contributing factor to RGC death. The distribution pattern of dynein accumulation described by Martin et al. 21 resembled ours found for APP. APP interacts with cytoplasmic kinesin, possibly as a transport cargo adaptor, and appears to be directly involved in the disease, 22 which may explain the similar distribution pattern found. 
Interestingly, in the RGC layer, a higher proportion of APP and a lesser extent of Aβ accumulation was observed, possibly because of the specific neurofilament (NF)-triplet content in RGCs, explaining their higher vulnerability. 23 24 AD is characterized not only by plaque formation but also by neurofibrillar tangles, which consist of altered neuronal cytoskeletal proteins such as NF and tau. 25 26 The vulnerability of neurons could be delineated by their content of NF. 27 The subpopulation containing NF in the human retina likely corresponds to large ganglion cells. 24 Loss of these NF protein-containing cells were evident in a glaucoma model, in which they showed a heightened vulnerability to degeneration. 23 As mentioned, the larger human RGCs are characterized by their content of NF-triplet proteins. 24 Similar to the more vulnerable neurons of AD, such as the hippocampal neurons containing NF, 26 28 the larger RGCs are preferentially affected by increased IOP, especially those located in the periphery of the retina. 29 Some cells may be more susceptible to damage because of their specific NF content. In addition, caspase-3 activity has been colocalized with abnormal NF-triplet proteins, described not only in the hippocampus of AD brains but also in the large RGCs. 23 28 We are aware that a large number of displaced amacrine cells are present in the RGC layer of the rat retina, which can constitute up to 50% of the total cells in areas of the RGC layer. Therefore, this hypothesis must be proven further with specific stains for RGC colocalizing them with APP or Aβ. 30  
No difference could be found comparing old with young control mouse retinas (C57/BL-6). Therefore, we excluded the possibility that the high accumulation of APP and Aβ in the old glaucoma mice were attributed to the physiological aging processes. Generally, low levels of APP and Aβ were also found in the age-matched C57/BL-6 control mice. Older mice seemed to have higher levels than younger ones. This trend probably represents a physiological accumulation, in ranges normally found in aging neuronal tissue. Amyloid deposits were described to a lesser extent in the brains of elderly persons without AD. 31  
Our findings point to a probable correlation of APP/Aβ accumulation and glaucoma, suggesting that an APP altered metabolism plays a role in the pathophysiology of RGC death in glaucoma. Aβ accumulation subsequent to ischemia or mechanical trauma, such as elevated IOP, with the consequence of heat shock protein elaboration, was shown in several studies. 32 33 34 35 36 Not only could this explain the correlation between elevated IOP and optical neuropathy, it might help to better understand normal-pressure glaucoma or unresponsive primary open-angle glaucoma. 
Disruption of the homeostatic properties of secreted APP with consecutive Aβ cytotoxicity might be a contributing factor of sustaining apoptotic cell death in glaucomatous mouse retinas. Hence, new medical treatment modalities for glaucoma may warrant further study, including the known neuroprotective anti-Alzheimer drugs (cholinesterase inhibitors, memantine) and the recently developed “anti-amyloid” therapeutic strategies, which decrease Aβ production by secretase inhibitors 37 and caspase inhibitors 38 or by interfering with Aβ aggregation through Aβ vaccination. 39 40 41 42  
 
Figure 1.
 
Amplified APP staining in an old DBA/2J mouse without first antibody (negative control). Original magnification, 200×.
Figure 1.
 
Amplified APP staining in an old DBA/2J mouse without first antibody (negative control). Original magnification, 200×.
Figure 2.
 
Amplified Aβ in an old DBA/2J mouse without first antibody (negative control). Original magnification, 200×.
Figure 2.
 
Amplified Aβ in an old DBA/2J mouse without first antibody (negative control). Original magnification, 200×.
Figure 3.
 
Amplified APP staining in an old DBA/2J mouse. Original magnification, 200×.
Figure 3.
 
Amplified APP staining in an old DBA/2J mouse. Original magnification, 200×.
Figure 4.
 
Amplified APP staining in a young DBA/2J mouse. Original magnification, 200×.
Figure 4.
 
Amplified APP staining in a young DBA/2J mouse. Original magnification, 200×.
Figure 5.
 
Amplified Aβ staining in an old DBA/2J mouse. Original magnification, 200×.
Figure 5.
 
Amplified Aβ staining in an old DBA/2J mouse. Original magnification, 200×.
Figure 6.
 
Amplified Aβ staining in a young DBA/2J mouse. Original magnification, 200×.
Figure 6.
 
Amplified Aβ staining in a young DBA/2J mouse. Original magnification, 200×.
Figure 7.
 
Amplified APP staining in an old control C57/BL-6 mouse. Original magnification, 200×.
Figure 7.
 
Amplified APP staining in an old control C57/BL-6 mouse. Original magnification, 200×.
Figure 8.
 
Amplified APP staining in a young control C57/BL-6 mouse. Original magnification, 200×.
Figure 8.
 
Amplified APP staining in a young control C57/BL-6 mouse. Original magnification, 200×.
Figure 9.
 
Amplified Aβ staining in an old control C57/BL-6 mouse. Original magnification, 200×.
Figure 9.
 
Amplified Aβ staining in an old control C57/BL-6 mouse. Original magnification, 200×.
Figure 10.
 
Amplified Aβ staining in a young control C57/BL-6 mouse. Original magnification, 200×.
Figure 10.
 
Amplified Aβ staining in a young control C57/BL-6 mouse. Original magnification, 200×.
Figure 11.
 
Amplified APP staining in the brain of a transgenic human AD mouse (positive control). Original magnification, 200×.
Figure 11.
 
Amplified APP staining in the brain of a transgenic human AD mouse (positive control). Original magnification, 200×.
Figure 12.
 
Amplified Aβ staining in the brain of a transgenic human AD mouse (positive control). Original magnification, 200×.
Figure 12.
 
Amplified Aβ staining in the brain of a transgenic human AD mouse (positive control). Original magnification, 200×.
Figure 13.
 
Distribution of APP staining for ON, pia/dura matter, and RGCl in old and young glaucomatous mice (DBA/2J) and old and young control mice (C57/BL-6). (▪) Score ++; (▒) score +; (□) score 0.
Figure 13.
 
Distribution of APP staining for ON, pia/dura matter, and RGCl in old and young glaucomatous mice (DBA/2J) and old and young control mice (C57/BL-6). (▪) Score ++; (▒) score +; (□) score 0.
Figure 14.
 
Distribution of Aβ staining for ON, pia/dura matter, and RGCl in old and young glaucomatous mice (DBA/2J) and old and young control mice (C57/BL-6). (▪) Score ++; (▒) score +; (□) score 0.
Figure 14.
 
Distribution of Aβ staining for ON, pia/dura matter, and RGCl in old and young glaucomatous mice (DBA/2J) and old and young control mice (C57/BL-6). (▪) Score ++; (▒) score +; (□) score 0.
The authors thank Mathias Jucker (Neuropathology, Hertie-Institut für klinische Hirnforschung, Tuebingen, Germany) and Mathias Staufenbiel (Novartis, Basel, Switzerland) for generously providing brain tissue of APP23 transgenic mice and Paolo Paganetti (Novartis, Basel, Switzerland) for the kindly supplying the primary antibodies. They also thank Jürg Kummer, Anastasia Amoo, Anezka Chrenkowa, and Aniela Olac, members of the laboratory, for expert technical and research assistance, and Andrew Goldman, (Boulder, CO) and Istvan Vajtai (Institute of Pathology, University of Bern, Bern, Switzerland) for their helpful collaboration in the writing of this paper. 
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Figure 1.
 
Amplified APP staining in an old DBA/2J mouse without first antibody (negative control). Original magnification, 200×.
Figure 1.
 
Amplified APP staining in an old DBA/2J mouse without first antibody (negative control). Original magnification, 200×.
Figure 2.
 
Amplified Aβ in an old DBA/2J mouse without first antibody (negative control). Original magnification, 200×.
Figure 2.
 
Amplified Aβ in an old DBA/2J mouse without first antibody (negative control). Original magnification, 200×.
Figure 3.
 
Amplified APP staining in an old DBA/2J mouse. Original magnification, 200×.
Figure 3.
 
Amplified APP staining in an old DBA/2J mouse. Original magnification, 200×.
Figure 4.
 
Amplified APP staining in a young DBA/2J mouse. Original magnification, 200×.
Figure 4.
 
Amplified APP staining in a young DBA/2J mouse. Original magnification, 200×.
Figure 5.
 
Amplified Aβ staining in an old DBA/2J mouse. Original magnification, 200×.
Figure 5.
 
Amplified Aβ staining in an old DBA/2J mouse. Original magnification, 200×.
Figure 6.
 
Amplified Aβ staining in a young DBA/2J mouse. Original magnification, 200×.
Figure 6.
 
Amplified Aβ staining in a young DBA/2J mouse. Original magnification, 200×.
Figure 7.
 
Amplified APP staining in an old control C57/BL-6 mouse. Original magnification, 200×.
Figure 7.
 
Amplified APP staining in an old control C57/BL-6 mouse. Original magnification, 200×.
Figure 8.
 
Amplified APP staining in a young control C57/BL-6 mouse. Original magnification, 200×.
Figure 8.
 
Amplified APP staining in a young control C57/BL-6 mouse. Original magnification, 200×.
Figure 9.
 
Amplified Aβ staining in an old control C57/BL-6 mouse. Original magnification, 200×.
Figure 9.
 
Amplified Aβ staining in an old control C57/BL-6 mouse. Original magnification, 200×.
Figure 10.
 
Amplified Aβ staining in a young control C57/BL-6 mouse. Original magnification, 200×.
Figure 10.
 
Amplified Aβ staining in a young control C57/BL-6 mouse. Original magnification, 200×.
Figure 11.
 
Amplified APP staining in the brain of a transgenic human AD mouse (positive control). Original magnification, 200×.
Figure 11.
 
Amplified APP staining in the brain of a transgenic human AD mouse (positive control). Original magnification, 200×.
Figure 12.
 
Amplified Aβ staining in the brain of a transgenic human AD mouse (positive control). Original magnification, 200×.
Figure 12.
 
Amplified Aβ staining in the brain of a transgenic human AD mouse (positive control). Original magnification, 200×.
Figure 13.
 
Distribution of APP staining for ON, pia/dura matter, and RGCl in old and young glaucomatous mice (DBA/2J) and old and young control mice (C57/BL-6). (▪) Score ++; (▒) score +; (□) score 0.
Figure 13.
 
Distribution of APP staining for ON, pia/dura matter, and RGCl in old and young glaucomatous mice (DBA/2J) and old and young control mice (C57/BL-6). (▪) Score ++; (▒) score +; (□) score 0.
Figure 14.
 
Distribution of Aβ staining for ON, pia/dura matter, and RGCl in old and young glaucomatous mice (DBA/2J) and old and young control mice (C57/BL-6). (▪) Score ++; (▒) score +; (□) score 0.
Figure 14.
 
Distribution of Aβ staining for ON, pia/dura matter, and RGCl in old and young glaucomatous mice (DBA/2J) and old and young control mice (C57/BL-6). (▪) Score ++; (▒) score +; (□) score 0.
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