- Email: [email protected]
The effects of the apoE4 genotype on the developing mouse retina
Accepted Manuscript The effects of the ApoE4 genotype on the developing mouse retina Idit Maharshak, Shiran Salomon-Zimri, Ran Antes, Ori Liraz, Yael Nisgav, Tami Livnat, Dov Weinberger, Carol A. Colton, Arieh S. Solomon, Daniel M. Michaelson PII:
S0014-4835(15)30060-9
DOI:
10.1016/j.exer.2015.11.001
Reference:
YEXER 6803
To appear in:
Experimental Eye Research
Received Date: 31 May 2015 Revised Date:
27 October 2015
Accepted Date: 3 November 2015
Please cite this article as: Maharshak, I., Salomon-Zimri, S., Antes, R., Liraz, O., Nisgav, Y., Livnat, T., Weinberger, D., Colton, C.A., Solomon, A.S., Michaelson, D.M., The effects of the ApoE4 genotype on the developing mouse retina, Experimental Eye Research (2015), doi: 10.1016/j.exer.2015.11.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT The Effects of the ApoE4 Genotype on the Developing Mouse Retina By Idit Maharshaka,b, Shiran Salomon-Zimria, Ran Antesa , Ori Liraza, Yael Nisgavc, Tami Livnatc, Dov
From a
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Weinbergere,b, Carol A. Coltonf, Arieh S. Solomond, b, and Daniel M. Michaelsona
Department of Neurobiology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel
Aviv, Israel
Department of Ophthalmology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel
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b
Laboratory of Eye Research, Felsenstein Medical Research Center, Rabin Medical Center, Petach
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Tikva, Israel
Goldschleger Eye Research Institute, Tel-Aviv University, Tel-Hashomer, Israel
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Department of Ophthalmology, Rabin Medical Center, Petach Tikva, Israel
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Division of Neurology, Duke University Medical Center, Department of Medicine, Durham, NC,
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USA
Abbriviated title: The Effects of ApoE4 on the Developing Mouse Retina Address correspondence to:
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Daniel M. Michaelson Department of Neurobiology
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Faculty of Life Sciences Tel Aviv University Israel 69978
Tel 972-3-6409624
Email: [email protected]
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ACCEPTED MANUSCRIPT Abstract Apolipoprotein E4 (apoE4), the most prevalent genetic risk factor for Alzheimer's disease (AD), is associated with neuronal and vascular impairments. The retina, which is as an extension of the central nervous system (CNS), is a particularly suitable model for studying developmental and functional aspects of the neuronal and vascular systems. This study investigates the apoE4-dependent
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developmental effects on the retinal vasculature and neuronal systems and on the levels of apoE and the vascular endothelial growth factor (VEGF) in the retina. This was performed utilizing retinas of 4, 7, 12, and of 120-day-old human-apoE4-targeted replacement mice and of corresponding mice that express the AD benign isoform, apoE3. The results obtained revealed retinal vascular pathology in the
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apoE4 mice, which started on the early post-natal days. This includes transient increase in vascular branching, and vascular buds which are round vascular elements representing sprouting or retracting
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vessels. These effects peaked and ended during the neonatal period. Examination of the synaptic system utilizing the pre-synaptic marker synaptophysin revealed a significant decrease of retinal synaptic density in the apoE4 mice, which was detectable by post-natal day 12 (P12). These morphological changes are associated with neonatal age-dependent elevation in the apoE levels in both apoE3 and apoE4 retinas which is more profound in the apoE4 mice and a corresponding increase in VEGF levels, which is less profound in the apoE4 mice. Additionally, we observed lower levels of
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retinal VEGF in the apoE4 mice compared to the apoE3 mice retinas on P12. These results show that apoE4 has a transient vascular effect during retinal development that ends in the neonatal period, which is accompanied by a synaptic effect that begins at the end of the neonatal period. These findings show that the apoE4 genotype can have distinct developmental effects on both the retinal vasculature
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VEGF.
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and on neurons and suggest that the vascular effects of apoE4 may be related to reduced levels of
Keywords: Apolipoprotein E4 (apoE4), retina, targeted replacement mice, vascular endothelial growth factor (VEGF), Vasculature
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ACCEPTED MANUSCRIPT 1. Introduction Apolipoprotein E (apoE), the most prevalent brain lipoprotein, exists in 3 common human isoforms (apoE2, apoE3, and apoE4) that differ from each other by one or two amino acids (apoE2: Cys112, Cys 158; apoE3: Cys112, Arg158; apoE4: Arg112, Arg158)(Hallman et al.). The apoE4 allele was identified as the most prevalent genetic risk factor for Alzheimer's disease (AD) in genetic and
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epidemiological studies (Corder et al., 1993; Saunders et al., 1993; Roses, 1996; Strittmatter et al., 1993).
ApoE4 is also associated with pronounced vascular pathology in AD (Zlokovic, 2013), and with an
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increased risk of vascular dementia relative to apoE3 carriers (Jellinger, 2008). Furthermore, the contribution of apoE4 to the risk of vascular cognitive impairments is independent of the vascular risk factors of hypertension and dyslipidemia (Yin et al., 2012). In addition, apoE4 is also a risk factor for
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other vascular impairments and vascular diseases such as cerebrovascular disease (CVD), atherosclerosis, stroke, and coronary heart disease as well as cerebral amyloid angiopathy (CAA) (de Knijff and Havekes, 1996; McCarron et al., 1999). It has been proposed that the pathological effects of apoE4 are driven by an initial cerebrovascular insult, which in turn, leads to subsequent neurodegeneration (Zlokovic, 2013). The mechanisms underlying the neuronal and vascular effects of
be determined.
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apoE4 and the extent to which they are mediated via overlapping biochemical mechanisms remain to
AD is associated with visual dysfunction and retinal pathology. These impairments include loss of ganglion cells (Chiu et al., 2012; Koronyo et al., 2012) as well as the accumulation of Aβ-containing
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deposits termed drusen (Koronyo-Hamaoui et al., 2011). It has been suggested that apoE4 is a risk factor for macular edema in type 2 diabetes (Santos et al., 2002) and that it protects against age-related
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macular degeneration (AMD) (Fritsche et al., 2009; Bojanowski et al., 2006). Animal model studies utilizing aged apoE4-targeted replacement mice, which were maintained on a cholesterol-enriched diet, revealed pathological changes that resemble those associated with human AMD (Malek et al., 2005). Moreover, anti-VEGF treatment for neovascular AMD (Wickremasinghe et al., 2011) is more effective for humans carrying the APOE4 allele than for non-APOE4 carriers. Taken together, these observations suggest that apoE4 interacts isoform-specifically with the retina and can play a role in retinal pathology. Developmentally, the retina is an extension of the CNS. The anatomic organization of the retinal cells and vasculature render it particularly suitable for studying the interplay between the vascular and 3
ACCEPTED MANUSCRIPT neuronal systems. The main vascular plexus and the retinal astrocytes lie at the inner border of the retina at the nerve fiber layer (NFL) level. The retina is a layered structure with several layers of interconnected neurons. These include the outer nuclear layer (ONL), which contains the cell nuclei of the photoreceptor cells. These cells connect via the bipolar cells that reside in the inner nuclear layer (INL) to the ganglion cell layer (GCL) whose axons comprise the NFL, and project from the retina via
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the optic nerve to the brain. The Muller retinal glial cell bodies also reside in the INL. The synaptic connections between these neurons form two layers. Accordingly, the outer plexiform layer (OPL) contains the synapses linking the ONL to the INL, whereas the inner plexiform layer (IPL) contains the synaptic connections between INL and GCL.
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We have recently shown that apoE4 induces neuronal impairment in young adult apoE4-targeted replacement mice, which is associated with ERG abnormalities (Antes et al., 2013). In view of the
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previously observed vascular and neuronal effects of apoE4 (de Knijff and Havekes, 1996; McCarron et al., 1999; Arendt et al., 1997; Buttini et al., 2002; Ji et al., 2003; Wang et al., 2005; Blain et al., 2006; Zhong et al., 2008; Sen et al., 2012) and of recent reports that the effects of apoE4 in man can start at a very early age (Dean et al., 2014; Malkki, 2014; Mc Donald and Krainc, 2014), the experiments will focus on neonate mice and on their retinal vascular and neuronal systems. The vascular endothelial growth factor (VEGF) plays a key role in angiogenesis and vascular plasticity
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(Leung et al., 1989) and was recently also shown to be neuroprotective (Licht and Keshet, 2013). In view of these findings, the study will also focus on the effects of apoE4 on the levels and spatial distribution of VEGF in the retina and on assessing the extent to which they correlate with the effects of apoE4 on the retinal vascular and neuronal systems. This was performed immunohistochemically
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and by immunoblot measurements of the retina of neonatal-targeted replacement apoE4 neonatal mice
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and by comparing them to the results obtained with the corresponding apoE3 mice.
2. Materials and methods
2.1 Ethics statement. The experiments were approved by the Tel-Aviv University Animal Care Committee (Permit # L-11-041). Every effort was made to reduce animal stress and to minimize animal usage. 2.2 Transgenic mice. ApoE-targeted replacement mice, in which the endogenous mouse apoE was replaced by either human apoE3 or apoE4, were created by gene targeting, as previously described.(Sullivan et al., 1997) The mice used were purchased from Taconic (Germantown, NY). The 4
ACCEPTED MANUSCRIPT mice were back-crossed to C57BL/6J (Harlan 2BL/610) for ten generations and were homozygous for the apoE3 (3/3) or apoE4 (4/4) alleles; hereafter, these mice are referred to as apoE3 and apoE4 mice, respectively. The apoE genotype of the mice was confirmed by PCR analysis, as described previously (Levi et al., 2003; Belinson and Michaelson, 2009). All the experiments were performed on agematched male animals (4, 7, and 12 days and 4 months of age).
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2.3 Frozen sections preparation and staining. Mice were euthanized by cervical dislocation and their eyes were enucleated. The eyes were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 1 hr, after which the cornea was dissected and the lens was removed. The eye cups were then fixed in 4% PFA in PBS for an additional hour, washed in PBS, and then placed in 15%
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sucrose for 1 hr, followed by 30% sucrose overnight. The fixed eyes were then embedded in TissueTek OCT (Optimal Cutting Temperature) compound (Sakura Finetek, Torrance, CA, USA) for 1 hr
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and frozen on dry ice. The eye cups were serially dissected into 16 µm sagittal sections, using a cryostat at -20°C and then mounted on slides. The slides were washed X3 in PBS, after which they were blocked using PBS with 0.2% Tween and 0.2% Gelatine (PBS-TG) for 2 hrs and washed with PBS. The slides were then incubated with the indicated primary antibody overnight at 4°C, after which they were washed (X3 with PBS-TG followed by X3 with PBS), incubated with secondary antibody for 2 hrs at room temperature, and finally washed again (X3 with PBS-TG followed by X3 with PBS).
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The immunostained sections were then covered with coverslips utilizing Fluoroshield Mounting Medium that contained the nuclear stain DAPI (Abcam). The sections were immunostained with the following primary antibodies:
mouse anti-Synaptophysin 1:250 (Sigma), goat anti-human apoE
1:5000 (Calbiochem), rabbit anti-VEGF (1:500, calbiochem PC315), rabbit anti-GFAP (1:300, DAKO
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Z0334), and goat anti-Collagen IV (1:30, Millipore AB769). 2.4 Retinal flat-mount preparation and staining. The eyes of neonatal (post-natal days 4,7, and 12:
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P4, P7 and P12, respectively) and young (4 months old) human apoE4 and apoE3 mice were enucleated and fixed in 4% PFA for 48 hours. Retinas were isolated, rinsed with PBS, blocked with PBS/0.5% Triton/1% bovine serum albumin (BSA) /10% normal donkey serum (NDS), and then stained with an endothelial cell marker, FITC-labeled Isolectin B4 (Vector Laboratories) overnight at 4°C. The secondary antibody used was anti-rat-Alexa Fluor 488 (Invitrogen). The retinas were then whole-mounted onto gelatin-coated slides, according to a standard protocol (Connor et al., 2009).
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ACCEPTED MANUSCRIPT 2.5 Image analysis. The retinal sections were visualized using a confocal scanning laser microscope (Zeiss, LSM 510). Images (1024 × 1024 pixels at X25 or X40 magnification) were obtained by averaging 8 scans per slice. The intensities of immunofluorescence staining, expressed as the percentage of the area stained above a fixed threshold background and the Integrated Optical Density (IOD equals the area x the
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average density of each object), were calculated utilizing the Image-Pro Plus System (version 5.1, Media Cybernetics) as previously described (Belinson et al., 2008). In naïve mice three sections of each retina per slide were stained and analyzed together. All the images for each immunostaining were obtained under identical conditions, and their quantitative analyses were performed with no further
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handling.
In the flat-mount analysis of vascular parameters, images of the superficial vascular plexus were
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photographed with a Zeiss LSM 510 Confocal microscope. Two to four mid-peripheral fields from each retina were photographed, blindly analyzed with the Image-Pro Plus 5.0 software (Media Cybernetics, Inc.) and the Image J software (Schneider et al., 2012; Schindelin et al., 2012) and then averaged (per eye and then per group). The total retinal vascular density was measured as the percentage of stained vascular area out of the total photographed field.
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2.6 Western Blot (WB) Analysis. Mice were euthanized by cervical dislocation and their retinas were rapidly excised and frozen in liquid nitrogen. The retinas were then homogenized in 200µl of 10mM Tris HCl pH 7.6, which contained NaCl 0.15M, Triton 1%, Deoxicholic acid 0.5%, SDS 0.1% PMSF 0.3mM, DTT 0.1mM, Sodium Orto-Vanadate 0.2mM as well as Protease Inhibitor Cocktail
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(Calbiochem). The homogenates were then aliquoted and stored at –70°C. The samples were boiled for 10 min prior to gel electrophoresis, after which the electrophoresis and immunoblot assays were
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performed utilizing the following antibodies: anti-apoE 1:10,000 (goat anti-apoE, Santa Cruz, sc-6384) and anti-VEGF 1:1000 (rabbit anti-VEGF, PC315 calbiochem). Protein concentration was determined utilizing the BCA protein assay kit (Pierce). The immunoblot bands were visualized utilizing the ECL chemiluminescent substrate (Pierce), after which their intensities were quantified using EZQuantGel software (EZQuant, Tel Aviv, Israel). GAPDH levels were employed as gel loading controls and the results are presented relative to the apoE3 mice.
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ACCEPTED MANUSCRIPT 2.7 Statistical Analysis. Values are presented as the mean ± SEM. Student's T-test was performed between the apoE3 and apoE4 groups or between two different ages (Figures 1, 3, 4, 6). Bonferroni corrections were employed for multiple comparisons when needed. Further analysis of interactions between genotype and age (Figures 1, 2, 4) were performed utilizing two-way ANOVA tests using STATISTICA software (Version 8.0 StatSoft, Inc., Tulsa,USA). A significant difference between
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groups was set at P<0.05. Significant results are indicated by * for P<0.05; **for P<0.01, and ***for P<0.001.
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3. Results
3.1. The effects of apoE4 on retinal vascular density and buds in naïve apoE3 and apoE4 mice at different ages. The possibility that apoE4 affects the retinal vasculature was first evaluated
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histologically utilizing retinas of neonate mice at the ages of 4, 7, and 12 days as well as the retinas of young adults aged 120 days. The results thus obtained are depicted in Figures 1 and 2. A representative mosaic of adjacent retinal images (Figure 1A) shows a whole mount of the retina of a 120 days old apoE3 mouse containing the mid-peripheral retinal fields that were analyzed. As can be seen in the confocal images in Figure 1B, the vascular density decreased in general from day 4 to day 120 in both
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groups most prominently from P7 to P12. Analysis of the vascular density revealed lower vascular density in day 12 compared to day 7, over both genotypes (Student's T-test P<0.0001 with Bonferroni correction for multiple comparisons). Next, another morphological feature was evaluated: "bud-like" structures, which are round vascular elements 15µ x 12µ in size. These “buds” are sprouting or
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retracting vessels and seem to be related to the stress response of the retina (Stone et al., 1995; Connolly et al., 1988; Bromberg-White et al., 2009). As can be seen in the confocal images in Figure 1B, vascular buds are most prominently apparent in apoE4 retinas in P4 and decrease with time.
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Analysis of the number of buds revealed a higher number of buds in the apoE4 compared to the apoE3 mice at day 4, and lower numbers of buds in day 7 compared to day 4 only in the apoE4 mice, (P<0.05 for the effect of group x age by two-way ANOVA, P<0.0001; P<0.0001 respectively for the post hoc comparison by Fisher post hoc analysis.)
3.2. The effects of apoE4 on retinal vascular branching in naïve apoE3 and apoE4 mice at different ages. We next examined the extent to which apoE4 affects the branching of the vasculature in the different age groups. This was pursued by focusing on individual arteries starting at the optic 7
ACCEPTED MANUSCRIPT nerve head and terminating at the periphery and counting the number of branching points along the way. In Figure 1A there is a representative mosaic of adjacent retinal images showing the full course of a retinal artery in a 120 days old apoE3 mouse. As can be seen in the confocal images in Figure 2A, on day P4 there was no branching as the vasculature at this time point is still a continuous vascular network with very few major vessels and branches. However, this architecture changed as the retina
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matured such that on day 7 branching appeared, and it was markedly higher in the apoE4 group than in the apoE3 group. On day 12 the levels of branching for apoE4 mice plateaued and changed very little, whereas in the apoE3 mice it increased and almost reached the level of the apoE4 mice. Importantly, in adult mice the level of branching was lower and was similar in both groups. Thus, apoE4 affected the
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vascular branching similarly to its effect on the vascular buds, an effect that was transient and disappeared in the adult mice. Quantization of the number of branching points per total vessel length revealed an early increase in branching points on P7 for apoE4 vs. the apoE3 mice and a decrease in
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branching between P12 to P120 over both genotypes (P<0.05 for genotype x age effect by two-way ANOVA; P<0.01; P<0.01 respectively for post hoc comparison by Fisher post hoc analysis). Taken together, these results show that apoE4 is associated with transient vascular morphological changes in the retina of mice, which is apparent by the early neonatal days. However, as the retina
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matures and in adult retina, this difference disappears.
3.3. The effects of apoE4 on vascular and astrocyte outgrowth in the retina of naïve apoE3 and apoE4 mice at different ages. The effects of the apoE genotype on retinal astrocytes was next examined. These cells that spread radially during retinal development from the center to the periphery
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of the retina to form a ‘scaffold’ on which the vessels grow, play an important role in vascular development (Stone et al., 1995). Retinal sections were immunohistochemically stained for blood vessels (Collagen IV) and astrocytes (GFAP). Next, the vessel-covered and the astrocyte-covered
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internal retinal surface (facing the vitreous body) lengths were measured. The results thus obtained are depicted in Figure 3. As can be seen in the representative confocal images, there was a slight delay in vessel outgrowth for apoE4 compared with apoE3 on P4 and no difference between the astrocyte outgrowth for apoE4 versus apoE3 on P4. Both blood vessels' outgrowth and astrocyte outgrowth increased from day 4 to day 7 and to a lesser extent from day 7 to day 12 over both genotypes (Figure 3A). Quantization of both the vessel outgrowth and astrocyte outgrowth revealed increased outgrowth in day 7 compared to day 4, over both genotypes (Student's T-test P<0.001 with Bonferroni correction for multiple comparisons).
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ACCEPTED MANUSCRIPT 3.4. The effects of apoE4 on synaptic staining in the naïve apoE3 and apoE4 neonatal retina. We next examined the extent to which synapse density is affected by apoE4. As can be seen in the confocal images in figure 4A, and in accordance with our previous findings (Antes et al., 2013), both the inner plexiform layer (IPL) and the outer plexiform layer (OPL) displayed immunohistochemical staining for the pre-synaptic marker synaptophysin. Synaptic immunoreactivity increased with age in
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both genotypes in the IPL and in the OPL of apoE3 retinas, but not in the OPL of apoE4 retinas. On P12 it was much lower in the OPL of apoE4 retinas than in the corresponding apoE3 retinas. Quantization of synaptophysin immunoreactivity in the IPL reveled higher levels of synaptic immunoreactivity in the IPL on day 12 compared to day 7, over both genotypes (Student's T-test
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P<0.0001 with Bonferroni correction for multiple comparisons). Quantization of synaptophysin immunoreactivity in the OPL revealed a genotype x age effect on the synaptophysin immunoreactivity (two-way ANOVA, P<0.05) with a significant decrease in synaptophysin levels in apoE4 compare to
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apoE3 retinas on P12 (P<0.01 for the post hoc comparison of apoE3 and apoE4 on P12 by Fisher post hoc analysis).
3.5. The effects of apoE4 on ApoE levels in naïve apoE3 and apoE4 neonatal retina. In view of the
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finding that the vasculature and neuronal systems are affected by apoE4 during retinal development, and the fact that the Muller cells and astrocytes synthesize apoE during retinal development (Kurumada et al., 2007), we next assessed the extent to which these effects correlate with the effects of the apoE genotype on the levels and localization of apoE in the developing retina. The results thus
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obtained are depicted in Figure 5. As can be seen in the representative confocal images, apoE was present in several layers in the retina including the GCL, INL, OPL, and ONL (Kurumada et al., 2007; Boyles et al., 1985; Lorber et al., 2009; Hayashi et al., 2009) (Figure 5A). Quantization of these results
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is depicted in Figures 5B & 5C. There is a significant age effect by two-way ANOVA (P<0.001 and P<0.05, in the GCL and INL+OPL+ONL respectively) with a trend towards a genotype effect on P4 (apoE4 levels being lower, Student's T-test: GCL:
P=0.115, INL+OPL+ONL: P=0.086) that
disappears in later days.
3.6. The effects of apoE4 on VEGF levels in naïve apoE3 and apoE4 neonatal retina. We next examined the effect of apoE4 and age on the levels of VEGF in the retina. The results thus obtained are depicted in Figure 6. As can be seen in the confocal images in figure 6A, VEGF was present in the 9
ACCEPTED MANUSCRIPT OPL of the retina and its levels were increase over time. Quantization of VEGF levels revealed there was an increase with age in VEGF levels which tended be more pronounced in apoE3 then in apoE4.On P12 we observed a trend toward lower levels of VEGF in the apoE4 retina compare to the apoE3 retina, though this difference was not statistically significant due to high variance (Figure 6B). Complementary immunoblot measurements of the levels of VEGF in apoE4 and apoE3 retinas on P12
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were performed (Figure 6C) and statistical analysis revealed that consistent with the trend in the histological analysis, there was a significant decrease in VEGF levels in apoE4 mice compared to
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apoE3 mice (Student's T-test: P<0.0001, with Bonferroni correction for multiple comparisons).
4. Discussion
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This study investigated the extent to which apoE4 and apoE3 targeted replacement mice's retina is affected by the apoE4 genotype at different ages, mainly during the neonatal period. Immunohistochemical studies revealed that vascular density decreases with age, in both genotypes. Vascular buds were strongly associated with apoE4 on post-natal day 4 and eventually decreased with age in both genotypes. The vascular branching was significantly higher in the apoE4 than in the apoE3 retina during development. The levels of the pre-synaptic marker synaptophysin were low during the
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early days of development. As the retina matured, however, the synaptic levels increased, and this was considerably more pronounced in apoE3 compared to apoE4 retinas in the OPL. In contrast, there was no apparent effect of apoE4 on astrocyte outgrowth during retinal development, as was described in the hippocampus (Sullivan et al., 1997). These morphological changes are accompanied, in accordance
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with previous findings (Kurumada et al., 2007), by an age-dependent increase in apoE levels in both apoE3 and apoE4 retinas during post-natal development and a significant decrease in VEGF levels in
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apoE4 mice compared to apoE3 mice on post-natal day 12. These results are summarized in Table 1. The vascular buds observed on P4 are similar to previously described vascular structures. Accordingly, such buds were observed in neonatal retina after injecting anthrax toxin, which arrests the development of the deep retinal vascular plexuses, and is associated with arrested vessel sprouting (Bromberg-White et al., 2009). Connolly et al (1988) described buds that appear on P4-P6 as retracting vessels, whereas Stone et al. (Stone et al., 1995) described buds as sprouting vessels that are about to dive perpendicularly and create the deep vascular plexus. We suggest two possible mechanisms that may underlie the increased appearance of buds in apoE4 retinas, either as sprouting vessels or retracting vessels: the buds may be a sign of the direct effect of apoE4 on the retinal vasculature, causing either a 10
ACCEPTED MANUSCRIPT delay in the vasculature maturation or early maturation. Another mechanism is a biochemical one: apoE levels were shown to have a trend towards a decrease in levels in apoE4 retinas compared with apoE3 retinas, suggesting a possible relation to the increase in vascular buds in apoE4 retinas on P4, since the apoE protein is anti-angiogenic (Pencheva et al., 2012). Accordingly, the lower apoE levels in apoE4 retina on P4 may allow for the transient creation of angiogenesis and subsequently of
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vascular buds. With time, the levels of apoE in apoE4 retinas increase and thus the number of buds decreases until they disappear. Since the apoE levels at P12 are similar in both apoE genotypes, the previously observed decreased levels of apoE in retina of young adult mice (Antes et al., 2013),
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develops at a later stage.
Synaptic expression and VEGF levels increase with age in the neonate mice and at P12 are both significantly and specifically lower in the apoE4 retina. These results are consistent with the neuronal
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growth factor effects of apoE4 and suggest that the observed synaptic deficit in the apoE4 mice at P12 may be mediated by the low VEGF levels. The decrease in synaptic expression and VEGF levels in apoE4 vs. apoE3 mice is maintained throughout young adulthood (Antes et al., 2013; Antes et al., 2015), implying that a similar mechanism may play a role in both age groups.
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The fact that during development both VEGF and apoE increase with age in apoE3 and apoE4 retinas suggests that these two effects are related. This suggestion is supported by previous studies which showed that the VEGF levels correlate positively with the expression levels of apoE (Chiappelli et al., 2006; Provias and Jeynes, 2011). Further support for this coupling between apoE and VEGF is
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provided by our previous studies with young mice in which both factors were similarly decreased by the apoE4 genotype under naive conditions and rose respectively following stress related stimulation
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(e.g. laser hit) (Antes et al., 2013; Antes et al., 2015).
In conclusion, our results revealed that apoE4 has a transient vascular effect during retinal development during the neonatal period and a neuronal effect that begins at the end of the neonatal period and is sustained in young adult mice. These findings show that apoE4 affects both the retinal vasculature and neurons, with apoE and VEGF as possible mediators. The specific mechanisms underlying these effects require further investigation.
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ACCEPTED MANUSCRIPT Legends to Figures Figure 1. The effects of apoE4 on retinal vascular density and buds in naïve apoE3 and apoE4 mice at different ages. (A) A low magnification mosaic of adjacent retinal images stained for Isolectin-B4 showing the retinal vasculature from a 120 days old apoE3 mouse (scale bar, 200 microns). The mid-peripheral retinal fields were assessed for vascular density and buds. (B)
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Representative Isolectin-B4 stained high magnification confocal images of retinas of apoE3 and apoE4 mice at the ages of 4, 7, 12, and 120 days. Vascular buds are indicated by white arrows (scale bar, 50 microns). As can be seen the vascular density decreases in general with time in both groups, most prominently from P7 to P12 and vascular buds are most prominently apparent in apoE4 retinas in P4
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and decrease with time. (C) Quantization of the vascular density in apoE3 (white bars) and apoE4 (black bars) retinas at the indicated time points. Results (mean ± SEM; n = 6 retinas per group per
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age) are presented relative to the P4 apoE3 mice whose values were set as 1.0. *** indicate statistically significant decrease of vascular density in day 12 compared to day 7, over both genotypes (Student's Ttest ***P<0.0001 with Bonferroni correction for multiple comparisons). (D) Quantization of the number of buds in apoE3 (white bars) and apoE4 (black bars) retinas at the indicated time points. Results are mean ± SEM; n = 6 retinas per group per age. *** indicate statistically significant increase in the number of buds in the apoE4 mice in comparison to the apoE3 mice at day 4, and lower numbers
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of buds in day 7 compared to day 4 in the apoE4 mice, (P<0.05 for the effect of group x age by twoway ANOVA, ***P<0.0001; ***P<0.0001 respectively for the post hoc comparison by Fisher post hoc analysis).
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Figure 2. The effects of apoE4 on retinal vascular branching in naïve apoE3 and apoE4 mice at different ages. The number of arterial branching points was assessed utilizing high magnification,
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sequential, partially overlapping confocal retinal images along the whole length of an artery and counting the number of branching points (see Figure 1A). (A) Representative Isolectin-B4 stained high magnification confocal images of retinas of apoE3 and apoE4 mice at the ages of 4, 7, 12, and 120 days. Arterial branching points are indicated by white arrows (scale bar, 50 microns). As can be seen branching is more pronounced in P7 and P12 then at the earlier and later time points. (B) Quantization of the number of branching points per artery in apoE3 (white bars) and apoE4 (black bars) retinas at the indicated time points. Results are mean ± SEM; n = 3-6 retinas per group per age, 2 arteries per retina. **indicate higher number of branching points in the apoE4 mice compare to the apoE3 mice at day 7, and lower number of branching points in day 120 compared to day 12, in apoE4 mice (P<0.05 12
ACCEPTED MANUSCRIPT for genotype x age effect by two-way ANOVA; **P<0.01; **P<0.01 respectively for post hoc comparison by Fisher post hoc analysis).
Figure 3. The effects of apoE4 on vascular and astrocyte outgrowth in the retina of naïve apoE3
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and apoE4 mice at different ages. (A) Representative confocal retinal sections of apoE3 and apoE4 mice at the ages of 4, 7, and 12 days stained for Collagen IV (green, upper panel) and for the astrocytic marker GFAP (red, lower panel). The arrows denote the proximal (on the right) and distal (on the left) boundaries of the vessels' and astrocytes' outgrowth on the internal surface of the retina (scale bar, 100
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microns). Quantization of the vessel outgrowth and astrocyte outgrowth are presented in (B) and (C) respectively. Results of the apoE3 (white bars) and apoE4 (black bars) retinas at the indicated time points (mean ± SEM; n = 4-6 per group per age) are presented as the percentage of either the vascular
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or astrocytic outgrowth length out of the total internal retinal surface length. As can be seen both blood vessels' outgrowth and astrocyte outgrowth increased in day 7 compared to day 4, over both genotypes (Student's T-test ***P<0.001 with Bonferroni correction for multiple comparisons).
Figure 4. The effects of apoE4 on synaptic staining in the naïve apoE3 and apoE4 neonatal
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retina. (A) Representative sections from 4, 7, and 12 days old apoE4 and apoE3 mice were stained immunohistochemically for synaptophysin. As can be seen, synaptophysin positive immunoreactivity was present in both the IPL (inner plexiform layer) and the OPL (outer plexiform layer) and increased with time in the OPL most prominently in apoE3 retinas from P7 to P12 (scale bar, 50 microns). (B)
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Quantization of synaptophysin immunoreactivity in the IPL in apoE3 (white bars) and apoE4 (black bars) retinas at the indicated time points. (C) Quantization of synaptophysin immunoreactivity in the
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OPL in apoE3 (white bars) and apoE4 (black bars) retinas at the indicated time points. Results for both retinal areas (mean ± SEM; n = 5 per group per age) are presented relative to the P12 apoE3 mice whose values were set to 1.0. ***indicate higher levels of synaptic immunoreactivity in the IPL on day 12 compared to day 7, over both genotypes (Student's T-test ***P<0.0001 with Bonferroni correction for multiple comparisons). For the OPL, P<0.05 for the effect of genotype x age on the synaptophysin immunoreactivity by two-way ANOVA; **P<0.01 for the post hoc comparison of apoE3 and apoE4 on P12 by Fisher post hoc analysis).
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Figure 5. The effects of apoE4 on ApoE levels in naïve apoE3 and apoE4 neonatal retinas. Retinal sections from apoE3 and apoE4 mice at the ages of 4, 7, and 12 days were stained for panapoE immunoreactivity. (A) Representative sections of apoE3 and apoE4 mice at the ages of 4, 7, and
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12 days. As can be seen apoE staining was present in several layers in the retina, including the GCL, INL, OPL, and ONL (scale bar, 50 microns). (B) Quantization of apoE immunoreactivity in the GCL in apoE3 (white bars) and apoE4 (black bars) retinas at the indicated time points. Results (mean ± SEM; n = 5 per group per age) are presented relative to the P12 apoE3 mice whose values were set to
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1.0 (C) Quantization of apoE immunoreactivity in the INL+OPL+ONL layers in apoE3 (white bars) and apoE4 (black bars) retinas at the indicated time points. Results (mean ± SEM; n = 5 per group per age) are presented relative to the P12 apoE3 mice whose values were set to 1.0. In both areas there was
P=0.115, INL+OPL+ONL: P=0.086).
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a transient tendency towards a decrease in apoE4 compared to apoE3 on P4 (Student's T-test: GCL:
Figure 6. The effects of apoE4 on VEGF levels in naïve apoE3 and apoE4 neonatal retina. Retinal sections and corresponding homogenates from apoE3 and apoE4 neonatal mice were stained
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immunohistochemically and immunoblotted for VEGF. (A). Representative immunohistochemical sections of apoE3 and apoE4 mice at the ages of 4, 7, and 12 days. As can be seen VEGF was present in the OPL of the retina and increased over time (scale bar, 50 microns). (B) Quantization of VEGF immunoreactivity in the OPL in apoE3 (white bars) and apoE4 (black bars) retinas at the indicated
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time points. Results (mean ± SEM; n = 5 per group per age) are presented relative to the P12 apoE3 mice whose values were set to 1.0. As can be seen, there was an increase with age in VEGF levels
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which tended to be more pronounced in apoE3 then in apoE4. (C) Complementary immunoblot measurements of the levels of VEGF in apoE3 and apoE4 retinas on P12. Representative blots are depicted in the upper panel with GAPDH as the loading control. Quantization of the results (mean ± SEM a; n=5 per group, normalized to the apoE3 mice) of apoE3 (white bar) and apoE4 (black bar) retinas at P12 is presented in the lower panel. Statistical analysis revealed, consistently with the immunohistochemical results, a significant decrease in VEGF levels in apoE4 mice compared to apoE3 mice (Student's T-test ***P<0.0001with Bonferroni correction for multiple comparisons).
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ACCEPTED MANUSCRIPT Acknowledgements This research was supported in part by grants from the "Legacy heritage biomedical program of the Israel Science Foundation" grant No. 1575/14); from the Esterson Trust; from the Joseph K. and Inez Eichenbaum Foundation, and from the Harold and Eleanore Foonberg Foundation. DMM is the
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technical assistance and for maintaining the mouse colonies.
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incumbent of the Myriam Lebach Chair in Molecular Neurodegeneration. We thank Alex Smolar for
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ACCEPTED MANUSCRIPT Highlights Maharshak et al
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• ApoE4 induces retinal vascular pathology transiently in P4 neonate mice. • ApoE4 induces retinal synaptic pathology starting at P12 neonate mice. • ApoE4 driven retinal vascular pathology correlate temporally with low levels of apoE4. • ApoE4 driven retinal pathology is associated with decrease levels of VEGF.
S0014-4835(15)30060-9
DOI:
10.1016/j.exer.2015.11.001
Reference:
YEXER 6803
To appear in:
Experimental Eye Research
Received Date: 31 May 2015 Revised Date:
27 October 2015
Accepted Date: 3 November 2015
Please cite this article as: Maharshak, I., Salomon-Zimri, S., Antes, R., Liraz, O., Nisgav, Y., Livnat, T., Weinberger, D., Colton, C.A., Solomon, A.S., Michaelson, D.M., The effects of the ApoE4 genotype on the developing mouse retina, Experimental Eye Research (2015), doi: 10.1016/j.exer.2015.11.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT The Effects of the ApoE4 Genotype on the Developing Mouse Retina By Idit Maharshaka,b, Shiran Salomon-Zimria, Ran Antesa , Ori Liraza, Yael Nisgavc, Tami Livnatc, Dov
From a
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Weinbergere,b, Carol A. Coltonf, Arieh S. Solomond, b, and Daniel M. Michaelsona
Department of Neurobiology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel
Aviv, Israel
Department of Ophthalmology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel
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b
Laboratory of Eye Research, Felsenstein Medical Research Center, Rabin Medical Center, Petach
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Tikva, Israel
Goldschleger Eye Research Institute, Tel-Aviv University, Tel-Hashomer, Israel
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Department of Ophthalmology, Rabin Medical Center, Petach Tikva, Israel
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Division of Neurology, Duke University Medical Center, Department of Medicine, Durham, NC,
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USA
Abbriviated title: The Effects of ApoE4 on the Developing Mouse Retina Address correspondence to:
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Daniel M. Michaelson Department of Neurobiology
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Faculty of Life Sciences Tel Aviv University Israel 69978
Tel 972-3-6409624
Email: [email protected]
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ACCEPTED MANUSCRIPT Abstract Apolipoprotein E4 (apoE4), the most prevalent genetic risk factor for Alzheimer's disease (AD), is associated with neuronal and vascular impairments. The retina, which is as an extension of the central nervous system (CNS), is a particularly suitable model for studying developmental and functional aspects of the neuronal and vascular systems. This study investigates the apoE4-dependent
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developmental effects on the retinal vasculature and neuronal systems and on the levels of apoE and the vascular endothelial growth factor (VEGF) in the retina. This was performed utilizing retinas of 4, 7, 12, and of 120-day-old human-apoE4-targeted replacement mice and of corresponding mice that express the AD benign isoform, apoE3. The results obtained revealed retinal vascular pathology in the
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apoE4 mice, which started on the early post-natal days. This includes transient increase in vascular branching, and vascular buds which are round vascular elements representing sprouting or retracting
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vessels. These effects peaked and ended during the neonatal period. Examination of the synaptic system utilizing the pre-synaptic marker synaptophysin revealed a significant decrease of retinal synaptic density in the apoE4 mice, which was detectable by post-natal day 12 (P12). These morphological changes are associated with neonatal age-dependent elevation in the apoE levels in both apoE3 and apoE4 retinas which is more profound in the apoE4 mice and a corresponding increase in VEGF levels, which is less profound in the apoE4 mice. Additionally, we observed lower levels of
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retinal VEGF in the apoE4 mice compared to the apoE3 mice retinas on P12. These results show that apoE4 has a transient vascular effect during retinal development that ends in the neonatal period, which is accompanied by a synaptic effect that begins at the end of the neonatal period. These findings show that the apoE4 genotype can have distinct developmental effects on both the retinal vasculature
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VEGF.
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and on neurons and suggest that the vascular effects of apoE4 may be related to reduced levels of
Keywords: Apolipoprotein E4 (apoE4), retina, targeted replacement mice, vascular endothelial growth factor (VEGF), Vasculature
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ACCEPTED MANUSCRIPT 1. Introduction Apolipoprotein E (apoE), the most prevalent brain lipoprotein, exists in 3 common human isoforms (apoE2, apoE3, and apoE4) that differ from each other by one or two amino acids (apoE2: Cys112, Cys 158; apoE3: Cys112, Arg158; apoE4: Arg112, Arg158)(Hallman et al.). The apoE4 allele was identified as the most prevalent genetic risk factor for Alzheimer's disease (AD) in genetic and
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epidemiological studies (Corder et al., 1993; Saunders et al., 1993; Roses, 1996; Strittmatter et al., 1993).
ApoE4 is also associated with pronounced vascular pathology in AD (Zlokovic, 2013), and with an
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increased risk of vascular dementia relative to apoE3 carriers (Jellinger, 2008). Furthermore, the contribution of apoE4 to the risk of vascular cognitive impairments is independent of the vascular risk factors of hypertension and dyslipidemia (Yin et al., 2012). In addition, apoE4 is also a risk factor for
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other vascular impairments and vascular diseases such as cerebrovascular disease (CVD), atherosclerosis, stroke, and coronary heart disease as well as cerebral amyloid angiopathy (CAA) (de Knijff and Havekes, 1996; McCarron et al., 1999). It has been proposed that the pathological effects of apoE4 are driven by an initial cerebrovascular insult, which in turn, leads to subsequent neurodegeneration (Zlokovic, 2013). The mechanisms underlying the neuronal and vascular effects of
be determined.
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apoE4 and the extent to which they are mediated via overlapping biochemical mechanisms remain to
AD is associated with visual dysfunction and retinal pathology. These impairments include loss of ganglion cells (Chiu et al., 2012; Koronyo et al., 2012) as well as the accumulation of Aβ-containing
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deposits termed drusen (Koronyo-Hamaoui et al., 2011). It has been suggested that apoE4 is a risk factor for macular edema in type 2 diabetes (Santos et al., 2002) and that it protects against age-related
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macular degeneration (AMD) (Fritsche et al., 2009; Bojanowski et al., 2006). Animal model studies utilizing aged apoE4-targeted replacement mice, which were maintained on a cholesterol-enriched diet, revealed pathological changes that resemble those associated with human AMD (Malek et al., 2005). Moreover, anti-VEGF treatment for neovascular AMD (Wickremasinghe et al., 2011) is more effective for humans carrying the APOE4 allele than for non-APOE4 carriers. Taken together, these observations suggest that apoE4 interacts isoform-specifically with the retina and can play a role in retinal pathology. Developmentally, the retina is an extension of the CNS. The anatomic organization of the retinal cells and vasculature render it particularly suitable for studying the interplay between the vascular and 3
ACCEPTED MANUSCRIPT neuronal systems. The main vascular plexus and the retinal astrocytes lie at the inner border of the retina at the nerve fiber layer (NFL) level. The retina is a layered structure with several layers of interconnected neurons. These include the outer nuclear layer (ONL), which contains the cell nuclei of the photoreceptor cells. These cells connect via the bipolar cells that reside in the inner nuclear layer (INL) to the ganglion cell layer (GCL) whose axons comprise the NFL, and project from the retina via
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the optic nerve to the brain. The Muller retinal glial cell bodies also reside in the INL. The synaptic connections between these neurons form two layers. Accordingly, the outer plexiform layer (OPL) contains the synapses linking the ONL to the INL, whereas the inner plexiform layer (IPL) contains the synaptic connections between INL and GCL.
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We have recently shown that apoE4 induces neuronal impairment in young adult apoE4-targeted replacement mice, which is associated with ERG abnormalities (Antes et al., 2013). In view of the
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previously observed vascular and neuronal effects of apoE4 (de Knijff and Havekes, 1996; McCarron et al., 1999; Arendt et al., 1997; Buttini et al., 2002; Ji et al., 2003; Wang et al., 2005; Blain et al., 2006; Zhong et al., 2008; Sen et al., 2012) and of recent reports that the effects of apoE4 in man can start at a very early age (Dean et al., 2014; Malkki, 2014; Mc Donald and Krainc, 2014), the experiments will focus on neonate mice and on their retinal vascular and neuronal systems. The vascular endothelial growth factor (VEGF) plays a key role in angiogenesis and vascular plasticity
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(Leung et al., 1989) and was recently also shown to be neuroprotective (Licht and Keshet, 2013). In view of these findings, the study will also focus on the effects of apoE4 on the levels and spatial distribution of VEGF in the retina and on assessing the extent to which they correlate with the effects of apoE4 on the retinal vascular and neuronal systems. This was performed immunohistochemically
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and by immunoblot measurements of the retina of neonatal-targeted replacement apoE4 neonatal mice
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and by comparing them to the results obtained with the corresponding apoE3 mice.
2. Materials and methods
2.1 Ethics statement. The experiments were approved by the Tel-Aviv University Animal Care Committee (Permit # L-11-041). Every effort was made to reduce animal stress and to minimize animal usage. 2.2 Transgenic mice. ApoE-targeted replacement mice, in which the endogenous mouse apoE was replaced by either human apoE3 or apoE4, were created by gene targeting, as previously described.(Sullivan et al., 1997) The mice used were purchased from Taconic (Germantown, NY). The 4
ACCEPTED MANUSCRIPT mice were back-crossed to C57BL/6J (Harlan 2BL/610) for ten generations and were homozygous for the apoE3 (3/3) or apoE4 (4/4) alleles; hereafter, these mice are referred to as apoE3 and apoE4 mice, respectively. The apoE genotype of the mice was confirmed by PCR analysis, as described previously (Levi et al., 2003; Belinson and Michaelson, 2009). All the experiments were performed on agematched male animals (4, 7, and 12 days and 4 months of age).
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2.3 Frozen sections preparation and staining. Mice were euthanized by cervical dislocation and their eyes were enucleated. The eyes were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 1 hr, after which the cornea was dissected and the lens was removed. The eye cups were then fixed in 4% PFA in PBS for an additional hour, washed in PBS, and then placed in 15%
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sucrose for 1 hr, followed by 30% sucrose overnight. The fixed eyes were then embedded in TissueTek OCT (Optimal Cutting Temperature) compound (Sakura Finetek, Torrance, CA, USA) for 1 hr
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and frozen on dry ice. The eye cups were serially dissected into 16 µm sagittal sections, using a cryostat at -20°C and then mounted on slides. The slides were washed X3 in PBS, after which they were blocked using PBS with 0.2% Tween and 0.2% Gelatine (PBS-TG) for 2 hrs and washed with PBS. The slides were then incubated with the indicated primary antibody overnight at 4°C, after which they were washed (X3 with PBS-TG followed by X3 with PBS), incubated with secondary antibody for 2 hrs at room temperature, and finally washed again (X3 with PBS-TG followed by X3 with PBS).
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The immunostained sections were then covered with coverslips utilizing Fluoroshield Mounting Medium that contained the nuclear stain DAPI (Abcam). The sections were immunostained with the following primary antibodies:
mouse anti-Synaptophysin 1:250 (Sigma), goat anti-human apoE
1:5000 (Calbiochem), rabbit anti-VEGF (1:500, calbiochem PC315), rabbit anti-GFAP (1:300, DAKO
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Z0334), and goat anti-Collagen IV (1:30, Millipore AB769). 2.4 Retinal flat-mount preparation and staining. The eyes of neonatal (post-natal days 4,7, and 12:
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P4, P7 and P12, respectively) and young (4 months old) human apoE4 and apoE3 mice were enucleated and fixed in 4% PFA for 48 hours. Retinas were isolated, rinsed with PBS, blocked with PBS/0.5% Triton/1% bovine serum albumin (BSA) /10% normal donkey serum (NDS), and then stained with an endothelial cell marker, FITC-labeled Isolectin B4 (Vector Laboratories) overnight at 4°C. The secondary antibody used was anti-rat-Alexa Fluor 488 (Invitrogen). The retinas were then whole-mounted onto gelatin-coated slides, according to a standard protocol (Connor et al., 2009).
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ACCEPTED MANUSCRIPT 2.5 Image analysis. The retinal sections were visualized using a confocal scanning laser microscope (Zeiss, LSM 510). Images (1024 × 1024 pixels at X25 or X40 magnification) were obtained by averaging 8 scans per slice. The intensities of immunofluorescence staining, expressed as the percentage of the area stained above a fixed threshold background and the Integrated Optical Density (IOD equals the area x the
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average density of each object), were calculated utilizing the Image-Pro Plus System (version 5.1, Media Cybernetics) as previously described (Belinson et al., 2008). In naïve mice three sections of each retina per slide were stained and analyzed together. All the images for each immunostaining were obtained under identical conditions, and their quantitative analyses were performed with no further
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handling.
In the flat-mount analysis of vascular parameters, images of the superficial vascular plexus were
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photographed with a Zeiss LSM 510 Confocal microscope. Two to four mid-peripheral fields from each retina were photographed, blindly analyzed with the Image-Pro Plus 5.0 software (Media Cybernetics, Inc.) and the Image J software (Schneider et al., 2012; Schindelin et al., 2012) and then averaged (per eye and then per group). The total retinal vascular density was measured as the percentage of stained vascular area out of the total photographed field.
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2.6 Western Blot (WB) Analysis. Mice were euthanized by cervical dislocation and their retinas were rapidly excised and frozen in liquid nitrogen. The retinas were then homogenized in 200µl of 10mM Tris HCl pH 7.6, which contained NaCl 0.15M, Triton 1%, Deoxicholic acid 0.5%, SDS 0.1% PMSF 0.3mM, DTT 0.1mM, Sodium Orto-Vanadate 0.2mM as well as Protease Inhibitor Cocktail
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(Calbiochem). The homogenates were then aliquoted and stored at –70°C. The samples were boiled for 10 min prior to gel electrophoresis, after which the electrophoresis and immunoblot assays were
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performed utilizing the following antibodies: anti-apoE 1:10,000 (goat anti-apoE, Santa Cruz, sc-6384) and anti-VEGF 1:1000 (rabbit anti-VEGF, PC315 calbiochem). Protein concentration was determined utilizing the BCA protein assay kit (Pierce). The immunoblot bands were visualized utilizing the ECL chemiluminescent substrate (Pierce), after which their intensities were quantified using EZQuantGel software (EZQuant, Tel Aviv, Israel). GAPDH levels were employed as gel loading controls and the results are presented relative to the apoE3 mice.
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ACCEPTED MANUSCRIPT 2.7 Statistical Analysis. Values are presented as the mean ± SEM. Student's T-test was performed between the apoE3 and apoE4 groups or between two different ages (Figures 1, 3, 4, 6). Bonferroni corrections were employed for multiple comparisons when needed. Further analysis of interactions between genotype and age (Figures 1, 2, 4) were performed utilizing two-way ANOVA tests using STATISTICA software (Version 8.0 StatSoft, Inc., Tulsa,USA). A significant difference between
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groups was set at P<0.05. Significant results are indicated by * for P<0.05; **for P<0.01, and ***for P<0.001.
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3. Results
3.1. The effects of apoE4 on retinal vascular density and buds in naïve apoE3 and apoE4 mice at different ages. The possibility that apoE4 affects the retinal vasculature was first evaluated
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histologically utilizing retinas of neonate mice at the ages of 4, 7, and 12 days as well as the retinas of young adults aged 120 days. The results thus obtained are depicted in Figures 1 and 2. A representative mosaic of adjacent retinal images (Figure 1A) shows a whole mount of the retina of a 120 days old apoE3 mouse containing the mid-peripheral retinal fields that were analyzed. As can be seen in the confocal images in Figure 1B, the vascular density decreased in general from day 4 to day 120 in both
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groups most prominently from P7 to P12. Analysis of the vascular density revealed lower vascular density in day 12 compared to day 7, over both genotypes (Student's T-test P<0.0001 with Bonferroni correction for multiple comparisons). Next, another morphological feature was evaluated: "bud-like" structures, which are round vascular elements 15µ x 12µ in size. These “buds” are sprouting or
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retracting vessels and seem to be related to the stress response of the retina (Stone et al., 1995; Connolly et al., 1988; Bromberg-White et al., 2009). As can be seen in the confocal images in Figure 1B, vascular buds are most prominently apparent in apoE4 retinas in P4 and decrease with time.
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Analysis of the number of buds revealed a higher number of buds in the apoE4 compared to the apoE3 mice at day 4, and lower numbers of buds in day 7 compared to day 4 only in the apoE4 mice, (P<0.05 for the effect of group x age by two-way ANOVA, P<0.0001; P<0.0001 respectively for the post hoc comparison by Fisher post hoc analysis.)
3.2. The effects of apoE4 on retinal vascular branching in naïve apoE3 and apoE4 mice at different ages. We next examined the extent to which apoE4 affects the branching of the vasculature in the different age groups. This was pursued by focusing on individual arteries starting at the optic 7
ACCEPTED MANUSCRIPT nerve head and terminating at the periphery and counting the number of branching points along the way. In Figure 1A there is a representative mosaic of adjacent retinal images showing the full course of a retinal artery in a 120 days old apoE3 mouse. As can be seen in the confocal images in Figure 2A, on day P4 there was no branching as the vasculature at this time point is still a continuous vascular network with very few major vessels and branches. However, this architecture changed as the retina
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matured such that on day 7 branching appeared, and it was markedly higher in the apoE4 group than in the apoE3 group. On day 12 the levels of branching for apoE4 mice plateaued and changed very little, whereas in the apoE3 mice it increased and almost reached the level of the apoE4 mice. Importantly, in adult mice the level of branching was lower and was similar in both groups. Thus, apoE4 affected the
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vascular branching similarly to its effect on the vascular buds, an effect that was transient and disappeared in the adult mice. Quantization of the number of branching points per total vessel length revealed an early increase in branching points on P7 for apoE4 vs. the apoE3 mice and a decrease in
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branching between P12 to P120 over both genotypes (P<0.05 for genotype x age effect by two-way ANOVA; P<0.01; P<0.01 respectively for post hoc comparison by Fisher post hoc analysis). Taken together, these results show that apoE4 is associated with transient vascular morphological changes in the retina of mice, which is apparent by the early neonatal days. However, as the retina
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matures and in adult retina, this difference disappears.
3.3. The effects of apoE4 on vascular and astrocyte outgrowth in the retina of naïve apoE3 and apoE4 mice at different ages. The effects of the apoE genotype on retinal astrocytes was next examined. These cells that spread radially during retinal development from the center to the periphery
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of the retina to form a ‘scaffold’ on which the vessels grow, play an important role in vascular development (Stone et al., 1995). Retinal sections were immunohistochemically stained for blood vessels (Collagen IV) and astrocytes (GFAP). Next, the vessel-covered and the astrocyte-covered
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internal retinal surface (facing the vitreous body) lengths were measured. The results thus obtained are depicted in Figure 3. As can be seen in the representative confocal images, there was a slight delay in vessel outgrowth for apoE4 compared with apoE3 on P4 and no difference between the astrocyte outgrowth for apoE4 versus apoE3 on P4. Both blood vessels' outgrowth and astrocyte outgrowth increased from day 4 to day 7 and to a lesser extent from day 7 to day 12 over both genotypes (Figure 3A). Quantization of both the vessel outgrowth and astrocyte outgrowth revealed increased outgrowth in day 7 compared to day 4, over both genotypes (Student's T-test P<0.001 with Bonferroni correction for multiple comparisons).
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ACCEPTED MANUSCRIPT 3.4. The effects of apoE4 on synaptic staining in the naïve apoE3 and apoE4 neonatal retina. We next examined the extent to which synapse density is affected by apoE4. As can be seen in the confocal images in figure 4A, and in accordance with our previous findings (Antes et al., 2013), both the inner plexiform layer (IPL) and the outer plexiform layer (OPL) displayed immunohistochemical staining for the pre-synaptic marker synaptophysin. Synaptic immunoreactivity increased with age in
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both genotypes in the IPL and in the OPL of apoE3 retinas, but not in the OPL of apoE4 retinas. On P12 it was much lower in the OPL of apoE4 retinas than in the corresponding apoE3 retinas. Quantization of synaptophysin immunoreactivity in the IPL reveled higher levels of synaptic immunoreactivity in the IPL on day 12 compared to day 7, over both genotypes (Student's T-test
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P<0.0001 with Bonferroni correction for multiple comparisons). Quantization of synaptophysin immunoreactivity in the OPL revealed a genotype x age effect on the synaptophysin immunoreactivity (two-way ANOVA, P<0.05) with a significant decrease in synaptophysin levels in apoE4 compare to
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apoE3 retinas on P12 (P<0.01 for the post hoc comparison of apoE3 and apoE4 on P12 by Fisher post hoc analysis).
3.5. The effects of apoE4 on ApoE levels in naïve apoE3 and apoE4 neonatal retina. In view of the
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finding that the vasculature and neuronal systems are affected by apoE4 during retinal development, and the fact that the Muller cells and astrocytes synthesize apoE during retinal development (Kurumada et al., 2007), we next assessed the extent to which these effects correlate with the effects of the apoE genotype on the levels and localization of apoE in the developing retina. The results thus
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obtained are depicted in Figure 5. As can be seen in the representative confocal images, apoE was present in several layers in the retina including the GCL, INL, OPL, and ONL (Kurumada et al., 2007; Boyles et al., 1985; Lorber et al., 2009; Hayashi et al., 2009) (Figure 5A). Quantization of these results
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is depicted in Figures 5B & 5C. There is a significant age effect by two-way ANOVA (P<0.001 and P<0.05, in the GCL and INL+OPL+ONL respectively) with a trend towards a genotype effect on P4 (apoE4 levels being lower, Student's T-test: GCL:
P=0.115, INL+OPL+ONL: P=0.086) that
disappears in later days.
3.6. The effects of apoE4 on VEGF levels in naïve apoE3 and apoE4 neonatal retina. We next examined the effect of apoE4 and age on the levels of VEGF in the retina. The results thus obtained are depicted in Figure 6. As can be seen in the confocal images in figure 6A, VEGF was present in the 9
ACCEPTED MANUSCRIPT OPL of the retina and its levels were increase over time. Quantization of VEGF levels revealed there was an increase with age in VEGF levels which tended be more pronounced in apoE3 then in apoE4.On P12 we observed a trend toward lower levels of VEGF in the apoE4 retina compare to the apoE3 retina, though this difference was not statistically significant due to high variance (Figure 6B). Complementary immunoblot measurements of the levels of VEGF in apoE4 and apoE3 retinas on P12
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were performed (Figure 6C) and statistical analysis revealed that consistent with the trend in the histological analysis, there was a significant decrease in VEGF levels in apoE4 mice compared to
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apoE3 mice (Student's T-test: P<0.0001, with Bonferroni correction for multiple comparisons).
4. Discussion
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This study investigated the extent to which apoE4 and apoE3 targeted replacement mice's retina is affected by the apoE4 genotype at different ages, mainly during the neonatal period. Immunohistochemical studies revealed that vascular density decreases with age, in both genotypes. Vascular buds were strongly associated with apoE4 on post-natal day 4 and eventually decreased with age in both genotypes. The vascular branching was significantly higher in the apoE4 than in the apoE3 retina during development. The levels of the pre-synaptic marker synaptophysin were low during the
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early days of development. As the retina matured, however, the synaptic levels increased, and this was considerably more pronounced in apoE3 compared to apoE4 retinas in the OPL. In contrast, there was no apparent effect of apoE4 on astrocyte outgrowth during retinal development, as was described in the hippocampus (Sullivan et al., 1997). These morphological changes are accompanied, in accordance
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with previous findings (Kurumada et al., 2007), by an age-dependent increase in apoE levels in both apoE3 and apoE4 retinas during post-natal development and a significant decrease in VEGF levels in
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apoE4 mice compared to apoE3 mice on post-natal day 12. These results are summarized in Table 1. The vascular buds observed on P4 are similar to previously described vascular structures. Accordingly, such buds were observed in neonatal retina after injecting anthrax toxin, which arrests the development of the deep retinal vascular plexuses, and is associated with arrested vessel sprouting (Bromberg-White et al., 2009). Connolly et al (1988) described buds that appear on P4-P6 as retracting vessels, whereas Stone et al. (Stone et al., 1995) described buds as sprouting vessels that are about to dive perpendicularly and create the deep vascular plexus. We suggest two possible mechanisms that may underlie the increased appearance of buds in apoE4 retinas, either as sprouting vessels or retracting vessels: the buds may be a sign of the direct effect of apoE4 on the retinal vasculature, causing either a 10
ACCEPTED MANUSCRIPT delay in the vasculature maturation or early maturation. Another mechanism is a biochemical one: apoE levels were shown to have a trend towards a decrease in levels in apoE4 retinas compared with apoE3 retinas, suggesting a possible relation to the increase in vascular buds in apoE4 retinas on P4, since the apoE protein is anti-angiogenic (Pencheva et al., 2012). Accordingly, the lower apoE levels in apoE4 retina on P4 may allow for the transient creation of angiogenesis and subsequently of
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vascular buds. With time, the levels of apoE in apoE4 retinas increase and thus the number of buds decreases until they disappear. Since the apoE levels at P12 are similar in both apoE genotypes, the previously observed decreased levels of apoE in retina of young adult mice (Antes et al., 2013),
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develops at a later stage.
Synaptic expression and VEGF levels increase with age in the neonate mice and at P12 are both significantly and specifically lower in the apoE4 retina. These results are consistent with the neuronal
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growth factor effects of apoE4 and suggest that the observed synaptic deficit in the apoE4 mice at P12 may be mediated by the low VEGF levels. The decrease in synaptic expression and VEGF levels in apoE4 vs. apoE3 mice is maintained throughout young adulthood (Antes et al., 2013; Antes et al., 2015), implying that a similar mechanism may play a role in both age groups.
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The fact that during development both VEGF and apoE increase with age in apoE3 and apoE4 retinas suggests that these two effects are related. This suggestion is supported by previous studies which showed that the VEGF levels correlate positively with the expression levels of apoE (Chiappelli et al., 2006; Provias and Jeynes, 2011). Further support for this coupling between apoE and VEGF is
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provided by our previous studies with young mice in which both factors were similarly decreased by the apoE4 genotype under naive conditions and rose respectively following stress related stimulation
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(e.g. laser hit) (Antes et al., 2013; Antes et al., 2015).
In conclusion, our results revealed that apoE4 has a transient vascular effect during retinal development during the neonatal period and a neuronal effect that begins at the end of the neonatal period and is sustained in young adult mice. These findings show that apoE4 affects both the retinal vasculature and neurons, with apoE and VEGF as possible mediators. The specific mechanisms underlying these effects require further investigation.
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ACCEPTED MANUSCRIPT Legends to Figures Figure 1. The effects of apoE4 on retinal vascular density and buds in naïve apoE3 and apoE4 mice at different ages. (A) A low magnification mosaic of adjacent retinal images stained for Isolectin-B4 showing the retinal vasculature from a 120 days old apoE3 mouse (scale bar, 200 microns). The mid-peripheral retinal fields were assessed for vascular density and buds. (B)
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Representative Isolectin-B4 stained high magnification confocal images of retinas of apoE3 and apoE4 mice at the ages of 4, 7, 12, and 120 days. Vascular buds are indicated by white arrows (scale bar, 50 microns). As can be seen the vascular density decreases in general with time in both groups, most prominently from P7 to P12 and vascular buds are most prominently apparent in apoE4 retinas in P4
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and decrease with time. (C) Quantization of the vascular density in apoE3 (white bars) and apoE4 (black bars) retinas at the indicated time points. Results (mean ± SEM; n = 6 retinas per group per
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age) are presented relative to the P4 apoE3 mice whose values were set as 1.0. *** indicate statistically significant decrease of vascular density in day 12 compared to day 7, over both genotypes (Student's Ttest ***P<0.0001 with Bonferroni correction for multiple comparisons). (D) Quantization of the number of buds in apoE3 (white bars) and apoE4 (black bars) retinas at the indicated time points. Results are mean ± SEM; n = 6 retinas per group per age. *** indicate statistically significant increase in the number of buds in the apoE4 mice in comparison to the apoE3 mice at day 4, and lower numbers
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of buds in day 7 compared to day 4 in the apoE4 mice, (P<0.05 for the effect of group x age by twoway ANOVA, ***P<0.0001; ***P<0.0001 respectively for the post hoc comparison by Fisher post hoc analysis).
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Figure 2. The effects of apoE4 on retinal vascular branching in naïve apoE3 and apoE4 mice at different ages. The number of arterial branching points was assessed utilizing high magnification,
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sequential, partially overlapping confocal retinal images along the whole length of an artery and counting the number of branching points (see Figure 1A). (A) Representative Isolectin-B4 stained high magnification confocal images of retinas of apoE3 and apoE4 mice at the ages of 4, 7, 12, and 120 days. Arterial branching points are indicated by white arrows (scale bar, 50 microns). As can be seen branching is more pronounced in P7 and P12 then at the earlier and later time points. (B) Quantization of the number of branching points per artery in apoE3 (white bars) and apoE4 (black bars) retinas at the indicated time points. Results are mean ± SEM; n = 3-6 retinas per group per age, 2 arteries per retina. **indicate higher number of branching points in the apoE4 mice compare to the apoE3 mice at day 7, and lower number of branching points in day 120 compared to day 12, in apoE4 mice (P<0.05 12
ACCEPTED MANUSCRIPT for genotype x age effect by two-way ANOVA; **P<0.01; **P<0.01 respectively for post hoc comparison by Fisher post hoc analysis).
Figure 3. The effects of apoE4 on vascular and astrocyte outgrowth in the retina of naïve apoE3
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and apoE4 mice at different ages. (A) Representative confocal retinal sections of apoE3 and apoE4 mice at the ages of 4, 7, and 12 days stained for Collagen IV (green, upper panel) and for the astrocytic marker GFAP (red, lower panel). The arrows denote the proximal (on the right) and distal (on the left) boundaries of the vessels' and astrocytes' outgrowth on the internal surface of the retina (scale bar, 100
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microns). Quantization of the vessel outgrowth and astrocyte outgrowth are presented in (B) and (C) respectively. Results of the apoE3 (white bars) and apoE4 (black bars) retinas at the indicated time points (mean ± SEM; n = 4-6 per group per age) are presented as the percentage of either the vascular
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or astrocytic outgrowth length out of the total internal retinal surface length. As can be seen both blood vessels' outgrowth and astrocyte outgrowth increased in day 7 compared to day 4, over both genotypes (Student's T-test ***P<0.001 with Bonferroni correction for multiple comparisons).
Figure 4. The effects of apoE4 on synaptic staining in the naïve apoE3 and apoE4 neonatal
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retina. (A) Representative sections from 4, 7, and 12 days old apoE4 and apoE3 mice were stained immunohistochemically for synaptophysin. As can be seen, synaptophysin positive immunoreactivity was present in both the IPL (inner plexiform layer) and the OPL (outer plexiform layer) and increased with time in the OPL most prominently in apoE3 retinas from P7 to P12 (scale bar, 50 microns). (B)
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Quantization of synaptophysin immunoreactivity in the IPL in apoE3 (white bars) and apoE4 (black bars) retinas at the indicated time points. (C) Quantization of synaptophysin immunoreactivity in the
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OPL in apoE3 (white bars) and apoE4 (black bars) retinas at the indicated time points. Results for both retinal areas (mean ± SEM; n = 5 per group per age) are presented relative to the P12 apoE3 mice whose values were set to 1.0. ***indicate higher levels of synaptic immunoreactivity in the IPL on day 12 compared to day 7, over both genotypes (Student's T-test ***P<0.0001 with Bonferroni correction for multiple comparisons). For the OPL, P<0.05 for the effect of genotype x age on the synaptophysin immunoreactivity by two-way ANOVA; **P<0.01 for the post hoc comparison of apoE3 and apoE4 on P12 by Fisher post hoc analysis).
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Figure 5. The effects of apoE4 on ApoE levels in naïve apoE3 and apoE4 neonatal retinas. Retinal sections from apoE3 and apoE4 mice at the ages of 4, 7, and 12 days were stained for panapoE immunoreactivity. (A) Representative sections of apoE3 and apoE4 mice at the ages of 4, 7, and
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12 days. As can be seen apoE staining was present in several layers in the retina, including the GCL, INL, OPL, and ONL (scale bar, 50 microns). (B) Quantization of apoE immunoreactivity in the GCL in apoE3 (white bars) and apoE4 (black bars) retinas at the indicated time points. Results (mean ± SEM; n = 5 per group per age) are presented relative to the P12 apoE3 mice whose values were set to
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1.0 (C) Quantization of apoE immunoreactivity in the INL+OPL+ONL layers in apoE3 (white bars) and apoE4 (black bars) retinas at the indicated time points. Results (mean ± SEM; n = 5 per group per age) are presented relative to the P12 apoE3 mice whose values were set to 1.0. In both areas there was
P=0.115, INL+OPL+ONL: P=0.086).
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a transient tendency towards a decrease in apoE4 compared to apoE3 on P4 (Student's T-test: GCL:
Figure 6. The effects of apoE4 on VEGF levels in naïve apoE3 and apoE4 neonatal retina. Retinal sections and corresponding homogenates from apoE3 and apoE4 neonatal mice were stained
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immunohistochemically and immunoblotted for VEGF. (A). Representative immunohistochemical sections of apoE3 and apoE4 mice at the ages of 4, 7, and 12 days. As can be seen VEGF was present in the OPL of the retina and increased over time (scale bar, 50 microns). (B) Quantization of VEGF immunoreactivity in the OPL in apoE3 (white bars) and apoE4 (black bars) retinas at the indicated
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time points. Results (mean ± SEM; n = 5 per group per age) are presented relative to the P12 apoE3 mice whose values were set to 1.0. As can be seen, there was an increase with age in VEGF levels
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which tended to be more pronounced in apoE3 then in apoE4. (C) Complementary immunoblot measurements of the levels of VEGF in apoE3 and apoE4 retinas on P12. Representative blots are depicted in the upper panel with GAPDH as the loading control. Quantization of the results (mean ± SEM a; n=5 per group, normalized to the apoE3 mice) of apoE3 (white bar) and apoE4 (black bar) retinas at P12 is presented in the lower panel. Statistical analysis revealed, consistently with the immunohistochemical results, a significant decrease in VEGF levels in apoE4 mice compared to apoE3 mice (Student's T-test ***P<0.0001with Bonferroni correction for multiple comparisons).
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ACCEPTED MANUSCRIPT Acknowledgements This research was supported in part by grants from the "Legacy heritage biomedical program of the Israel Science Foundation" grant No. 1575/14); from the Esterson Trust; from the Joseph K. and Inez Eichenbaum Foundation, and from the Harold and Eleanore Foonberg Foundation. DMM is the
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technical assistance and for maintaining the mouse colonies.
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incumbent of the Myriam Lebach Chair in Molecular Neurodegeneration. We thank Alex Smolar for
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ACCEPTED MANUSCRIPT Highlights Maharshak et al
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• ApoE4 induces retinal vascular pathology transiently in P4 neonate mice. • ApoE4 induces retinal synaptic pathology starting at P12 neonate mice. • ApoE4 driven retinal vascular pathology correlate temporally with low levels of apoE4. • ApoE4 driven retinal pathology is associated with decrease levels of VEGF.