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Journal of Alzheimer’s Disease 32 (2012) 291–305 DOI 10.3233/JAD-2012-120571 IOS Press

Amyloid-␤ and Tau Pathology of Alzheimer’s Disease Induced by Diabetes in a Rabbit Animal Model Claudine L. Bitela , Chinnaswamy Kasinathanb , Rajesh H. Kaswalab , William L. Kleinc and Peter H. Frederiksea,b,∗ a Department

of Pharmacology and Physiology and Rutgers-UMDNJ Integrative Neurosciences Program, UMDNJ-New Jersey Medical School, Newark, NJ, USA b Department of Oral Biology, UMDNJ-New Jersey Medical School, Newark, NJ, USA c Department of Neurobiology and Physiology, Northwestern University, Evanston, IL, USA

Accepted 6 June 2012

Abstract. Alzheimer’s disease (AD) is the major age-dependent disease of the brain, but what instigates late-onset AD is not yet clear. Epidemiological, animal model, and cell biology findings suggest links between AD and diabetes. Although AD pathology is accelerated by diabetes in mice engineered to accumulate human-sequence amyloid-␤ (A␤) peptides, they do not adequately model non-inherited AD. We investigated AD-type pathology induced solely by diabetes in genetically unmodified rabbits which generate human-sequence A␤ peptides. After 15 weeks, alloxan-treated diabetic rabbits with expected high blood glucose showed ∼5-fold increase in A␤40 /A␤42 in cortex and hippocampus, and significantly, generated A␤-derived assemblies found in human AD. Deposits of these putative pathogenic toxins were detected by A␤/A␤ oligomer antibodies in brain parenchyma and surrounding vasculature, also co-localizing with markedly elevated levels of RAGE. Soluble brain extracts showed diabetesinduced buildup of A␤ oligomers on dot-blots. Phospho-tau also was clearly elevated, overlapping with ␤III-tubulin along neuronal tracts. Indications of retina involvement in AD led to examination of AD-type pathology in diabetic retinas and showed A␤ accumulation in ganglion and inner nuclear cell layers using A␤/oligomer antibodies, and RAGE again was elevated. Our study identifies emergence of AD pathology in brain and retina as a major consequence of diabetes; implicating dysfunctional insulin signaling in late-onset AD, and a potential relationship between A␤-derived neurotoxins and retinal degeneration in aging and diabetes, as well as AD. AD-type pathology demonstrated in genetically unmodified rabbits calls attention to the considerable potential of the model for investigation of AD pathogenesis, diagnostics, and therapeutics. Keywords: Alzheimer’s disease, amyloid-␤, brain, diabetes, oligomers, retina

INTRODUCTION Alzheimer’s disease (AD) is a progressive brain disorder constituting the major cause of dementia as well as the sixth leading cause of death [1]. Its molecular ∗ Correspondence

to: Peter H. Frederikse, PhD, Pharmacology and Physiology and Rutgers-UMDNJ Integrative Neuroscience Program, UMDNJ New Jersey Medical School, 185 South Orange Avenue MSB, H645, Newark, NJ 07103, USA. Tel.: +1 973 972 1686 lab-4785; Fax: +1 973 972 7950; E-mail: [email protected].

basis is linked to accumulation of pathogenic toxins [2, 3]. What is not yet evident, except for a very small subpopulation of patients with familial AD (FAD), is how this accumulation of toxins is instigated. In the 12% of patients with FAD, various point mutations lead to elevated production of the amyloid-␤ (A␤) peptide [4], ordinarily a physiological metabolite, but which at elevated concentrations drives its self-association into gain-of-function toxic assemblies. These toxins comprise the well-known insoluble amyloid plaques

ISSN 1387-2877/12/$27.50 © 2012 – IOS Press and the authors. All rights reserved

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and the more recently discovered toxic soluble A␤ oligomers, currently a focus of interest for their role early in dementia [3, 5]. Overproduction of A␤ in FAD has been modeled in rodents expressing human transgenes, which give rise to plaques, toxic oligomers, and ensuing behavioral dysfunction and neuropathology [6]. Transgenic animals have limited value, however, for elucidating mechanisms that generate these toxins in sporadic AD (SAD), which comprises the great majority of cases. Current models also have been illsuited for predicting whether investigational new drugs will be successful in treating SAD [7, 8]. An emerging possibility now garnering attention is that initiation and development of SAD, at least in some cases, may be related to diabetes [9]. Recent etiological data have identified type 2 diabetes as an AD risk factor [10]. Because AD brain is relatively insulin resistant [10, 11], and insulin treatment can enhance memory function in some AD patients [12, 13], it also has been suggested that AD could be regarded as a type 3 diabetes [14]. This is consistent with findings that brain insulin has a role in synaptic plasticity as well as energy metabolism [15]. At the molecular level, synaptic insulin receptors are greatly down-regulated by toxic A␤ oligomer binding [2], a loss that has been attributed to their impact on receptor trafficking [16, 17]. Reciprocally, lack of insulin signaling renders neurons more vulnerable to oligomer synaptotoxicity [18, 19], suggestive of a vicious cycle [20]. Whether deficiencies in insulin signaling could act at even earlier stages, as an instigator of AD pathogenesis, is uncertain. Because insulin plays a role in clearance of A␤ monomers and oligomers at synapses, it is feasible that failed brain insulin signaling could promote formation of A␤ synaptic insulin receptor complexes leading to the down-regulation of insulin receptors by these A␤-derived toxins. Consistent with this possibility, diabetes causes the onset of amyloid pathology to develop earlier in transgenic mouse AD models [21]. In the current study, the relationship between diabetes and the onset of AD has been examined in a non-transgenic animal to segregate the intrinsic effects of insulin deficiency from effects also requiring genetic manipulations. Our experiments used genetically unmodified rabbits which, unlike rodents, produce A␤ peptides identical in sequence to those expressed in human brain [22]. Diabetes induced due to degeneration of pancreatic beta-cells with alloxan [23, 24] was found to produce AD-type neuropathology in both brain and retina of wild-type rabbits, with significant accumulation of A␤ monomers and oligomers and

of aberrant phospho-tau. Results strongly support the hypothesis that diabetes can act as a primary factor in inducing early-stage AD phenotype. Results further suggest the potential of the wild type rabbit as a model for aspects of sporadic AD, a new resource for investigating disease initiation and treatment strategies.

MATERIALS AND METHODS Induction of diabetes in rabbits New Zealand white rabbits (Covance) were used according to NIH guidelines and University approved protocols. Seven rabbits were included per group, and males only were used to limit hormonal considerations at this stage of our studies. The study was repeated with a second group of rabbits and replicated our findings. 3-4 month-old rabbits were housed at room temperature with a 12 h light/12 h dark cycle and fed 260 gm/day of a standard pellet diet with free access to dH2 O. Rabbits were given 150 mg/kg alloxan (Sigma) in a single bolus injection through an ear vein catheter. Alloxan is structurally related to streptozotocin, and both toxic glucose analogues selectively enter pancreatic ␤-cells via GLUT2 glucose transporters [23, 25]. Alloxan predominantly produces reactive oxygen species consistent with its 1-2 min half-life and rapid clearance, with GLUT1 expressed at the blood brain barrier and GLUT3 in neurons [26, 27]. Fasting blood glucose was measured with a glucometer four times per week. Rabbits maintaining >350 mg/dl blood glucose remained in the study, and rabbits with lower glucose values were removed. Rabbits weighed ∼2.75 kg at the outset of our study. Diabetic rabbits gained ∼225 gm (s.d. 180 gm), and controls gained ∼500 gm (s.d. 50 gm), with no rabbits losing weight. Retina and brain were examined after 15 wks, similar to related studies of diabetic complications in alloxan-treated rabbits [e.g., 28]. Brains were removed for study and bisected for tissue fixation or immersed in liquid nitrogen for biochemical studies. Enucleated eyes were fixed in buffered formalin for preparation of histological sections. Serum cholesterol at the end of the 15 wk trial showed a range of increases in diabetic animals when compared with controls (Antech Diagnostics, NY): Cholesterol in normal controls 21–40 mg/dl (S.D. 11), showed increases in diabetic rabbit serum that varied from 39 to 498 mg/dl (S.D. 101), and appeared consistent with related studies in alloxan-treated rabbits [29].

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Immunochemistry and immunofluorescence Paraffin sections prepared from paraformaldehyde fixed brains and eyes were de-waxed and endogenous peroxidases were quenched in 3% H2 O2 in 10% MeOH for 5 min. Sections were blocked in PBS, 0.01% tween-20, 3% BSA for 60 min, or PBS with 10% normal serum corresponding to the 2◦ antibody for immunofluorescence. HRP conjugated 2◦ antibodies (Vector Labs) or fluor-conjugated 2o antibodies (Invitrogen) were used to visualize immune complexes. Antibodies used included: mAb anti-A␤ 6E10 and 4G8 (Covance), rabbit mAb anti-A␤42 (Invitrogen), and anti-A␤40 (Genscript). Anti-A␤ oligomer antibodies included NU1, NU4 [30, 31], A11, and OC (Invitrogen, gift of C. Glabe, UC Irvine) [32], as well as mouse anti-A␤ oligomer IgM (Agrisera). Additional antibodies included anti-RAGE (Santa Cruz, Genscript), mAb ␤III-tubulin (Sigma), and GAPDH (Abcam). Immunoblots used equal amounts of protein samples resolved by MW (NuPAGE, Invitrogen) blotted to filters and probed with antibodies using supplied protocols. Immune complexes were visualized with HRP-conjugated 2◦ antibodies (Jackson) and chemiluminescence kits (Amersham).

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4 ␮g and 1.5 ␮g of the first PBS soluble fraction and final RIPA insoluble fraction were spotted onto nitrocellulose filters, and probed with A11, OC or 4G8 antibodies, or stained for total protein with amido black. RESULTS Aβ40 and Aβ42 peptides increased in the cortex and hippocampus of diabetic rabbit brains Fifteen weeks after onset of reduced insulin production and sustained hyperglycemia, we used standard ELISAs to measure A␤40 and A␤42 peptides in tissue samples from diabetic and normal control brains. Both A␤ forms increased several fold in the cortex and hippocampus in diabetic rabbit brains in total protein samples solubilized in 5M guanidine buffer (Fig. 1),

ELISA assays A␤ was quantified in cortex and hippocampus tissues using ELISA assays and protocols specific for A␤40 or A␤42 and peptide standards provided by the supplier (Covance). Equal brain total protein samples solubilized in 5M guanidine were assayed by colorimetric quantification of HRP product with a spectrometer plate reader. ELISAs were performed 3X to measure A␤40 and A␤42 for each sample. For analysis of ELISA data, p-values were calculated using a 2-tailed ANOVA statistical test. Immuno dot-blots Small soluble A␤ oligomeric forms were assayed using nitrocellulose filter dot blot assays as described by Tomic et al. [3]. A␤ peptides and soluble oligomers were extracted from ∼60 mg brain tissue by repeated pipetting in 4 volumes of ice-cold non-denaturing PBS [A buffer: PBS, 0.02% NaN3 , pH 7.4 and protease inhibitors (Calbiochem)], and centrifuged at 100K×G for 1 h. After taking supernatants for assay, the pellets were resuspended in 4 volumes RIPA buffer (Sigma) and centrifuged again as above. This RIPA insoluble pellet was again resuspended in A buffer for assay.

Fig. 1. A␤40 and A␤42 peptides increased significantly in brain cortex and hippocampus of genetically un-modified diabetic rabbits. A␤40 and A␤42 peptides were assayed separately by ELISA in total protein samples from cortex and hippocampus in four diabetic and four control rabbits. A␤40 cortex: p < 0.03, A␤40 hippocampus: p < 0.005, A␤42 cortex: p < 0.05, A␤42 hippocampus: p < 0.03.

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Fig. 2. Pathological A␤ accumulations in the hippocampus and cortex in diabetic rabbit brain tissues, and normoglycemic control brains detected via immunohistochemistry. A␤40 and A␤42 showed similar distributions in histological sections in cortex and hippocampus from genetically unmodified diabetic rabbits using peptide-specific antibodies. Brain tissues were examined 15 weeks after induction of reduced insulin production and onset of hyperglycemia. Photographs are representative of seven diabetic and seven control rabbits in each of two experimental trials.

and more deleterious A␤42 peptides increased to a greater extent than A␤40 in both regions. A␤40 was ∼3–5-fold higher in diabetic brains, whereas A␤42 was detected at lower levels in control brain tissue and increased >5-fold in the cortex and hippocampus in diabetic animals. When we examined diabetic brains in situ for hallmark A␤ accumulations similar to what occurs in AD brains, we observed A␤ deposits in many areas in the cortex and hippocampus from wild-type (wt) diabetic rabbits. In contrast, little or no evidence of A␤ deposits was observed in control rabbit brains using mAb anti-A␤ 6E10 and 4G8 antibodies or with anti-A␤42 peptide-specific antibodies recognizing a Cterminus epitope (Fig. 2). Deposits were observed in the brain parenchyma and associated with capillary walls in the cortex and hippocampus similar to what has been described in human AD brains and transgenic (Tg) AD mouse models [33, 34]. A␤ deposits varied in size from small deposits that appeared to be contained within a cell or lysis of a single cell, to larger deposits and may suggest bystander effects on adjacent cells. A␤ was also seen in accumulations surrounding cell nuclei in apparently intact neurons in different brain regions. When we compared the distribution of specific A␤ peptide forms in diabetic brains with anti-A␤42

and A␤40 specific antibodies, we observed similar distributions of A␤ deposits with both antibodies in the cortex and hippocampus of diabetic brains (Fig. 2), and again found little or no evidence of A␤ deposits in either of these brain regions in euglycemic control 7-8 month-old rabbit brains. Spontaneous Aβ conversion to oligomeric forms in vivo in wt diabetic rabbit brains with evidence of Aβ oligomer accumulation in brain The ability of ∼4 kDa A␤ monomers to form small diffusible A␤ oligomers which can be ∼60 kDa or greater in size has been characterized by a number of laboratories [3, 30, 31]. To date, human-sequence A␤ peptides have been used to produce oligomers that elicit production of conformation-specific antibodies which recognize A␤ oligomers. Observations of substantially increased human-sequence A␤ peptides in diabetic rabbit brains indicated significant potential for oligomer formation in vivo. To examine A␤ oligomers in diabetic brains, we probed brain sections with a number of anti-A␤ oligomer antibodies produced in different laboratories. Similar to findings with anti-A␤ peptide antibodies above, few or no oligomers were

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detected in brain tissue or surrounding vessels in normoglycemic control brains using NU1 or NU4 mAb antibodies, or A11 or OC anti-A␤ oligomer rabbit antibodies [32] that have been shown capable of preferentially detecting higher order A␤ structures (Fig. 3). In contrast, a similar distribution of A␤ oligomer deposits as shown above using anti-A␤ detection was detected throughout the cortex and hippocampus of diabetic brains, and associated with vessel walls of the surrounding microvasculature using anti-A␤ oligomer antibodies (Fig. 3). Deposits which we could orient similarly in adjacent histological sections were identified with anti-A␤ and A␤ oligomer antibodies. In addition to A␤/ADDL accumulations in the cortex and hippocampus, similar deposits were also observed in other brain areas including the ventromedial hypothalamus (not shown). This brain region has also been shown to be affected in human AD brains [35, 36] and may have particular relevance in diabetes in light of its previously described roles in glucose sensing and regulating glucose homeostasis [37]. To further characterize A␤ peptide and ADDL distributions, we used in situ immunfluorescence with A␤42 specific antibodies and mouse IgM mAb anti-oligomer (OMAB) antibodies [38] to examine brain pathology (Fig. 3 C). These antibodies also demonstrated substantial overlap in detection of A␤ peptides and oligomers in the cortex and hippocampus of diabetic animals. Our results indicate significant ADDL formation occurs in diabetic rabbit brains that we could detect using mouse and rabbit IgG as well as IgM anti-A␤ oligomer antibodies. Immuno dot-blot assays of brain proteins detected increased PBS extractable oligomeric Aβ in diabetic brains Recent studies showed that in contrast to harsh guanidine denaturing buffers used in ELISAs above, oligomeric A␤ forms in brain tissue are extracted by repeated pipetting in phosphate buffered saline (PBS). A␤ oligomers obtained with these methods were shown to increase in human AD brains when compared with non-demented controls, detected on dot-blots using A␤ oligomer-specific antibodies [3]. To test these findings in the rabbit model, and to provide further evidence of A␤ conversion to oligomeric forms in diabetic brains, we used a similar dot-blot immunodetection protocol to analyze A␤ higher order assemblies eluted from diabetic and control rabbit brain tissues (Fig. 4). Aliquots of non-denatured proteins in PBS extracted brain tissue were compared with protein material in a detergent insoluble pellet

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fraction after ultracentrifugation, by spotting protein samples onto filters which were then were probed with anti-oligomer A11 and OC antibodies, and antiA␤ 4G8. After immunohistochemical detection, we observed increased levels of soluble oligomeric A␤ forms in diabetic brains using OC as well as A11 anti-A␤ oligomer antibodies. Increased RAGE expression and localized accumulation with Aβ/oligomers occurs in cortical and hippocampal regions and vessels in diabetic brains Advanced glycation end-products (AGEs) result from non-enzymatic addition of sugar units to a variety of macromolecules and increase significantly with hyperglycemia in diabetes. These increases have been shown to be matched by increased RAGE scavenger receptors [39–41]. To examine these effects in diabetic rabbit brains, we measured RAGE expression on immunoblots and observed a significant increase from low baseline expression in diabetic brains (Fig. 5), in contrast to little apparent overall change in IR␣ or GAPDH. We note the GAPDH doublet on immunoblots was also indicated by the antibody supplier. When we examined RAGE protein in situ, we observed that RAGE accumulation was also readily detected in deposits in the cortex and hippocampus in diabetic brains but not control brain tissue, as were also observed in surrounding vascular structures (Fig. 5). Increased phosphorylated tau occurs in diabetic brains and was detected predominantly along neuronal tracts co-localizing with βIII-tubulin Tau pathology is also a well-characterized fundamental neuropathological characteristic of AD. Phosphorylation at tau serine and threonine residues associated with AD and other neurodegenerative conditions also increase in response to oxidative and osmotic stress [42, 43]. In addition, tau phosphorylation was found to correlate with A␤ oligomer toxicity [44]. We examined tau phosphorylation in brain samples from diabetic and control brains, probing for phosphorylated threonine 205 and phospho-serines 396 and 404 on immunoblots (Fig. 6). We observed increased phosphorylation at residues threonine 205 and serine 404 in brain proteins from diabetic rabbits. When we next examined the localization of phosphorylated tau protein in situ in diabetic brains, we observed increased phospho-tau that corresponded with axonal tracts in the cortex of diabetic animals

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Fig. 3. Accumulation of A␤ peptides and A␤ oligomers detected in diabetic rabbit brain. A) Immunohistochemical detection of A␤ oligomers (NU4) in pathological deposits in control (Ctl, bar: 200 ␮m) and diabetic cortex (Diab, bar: 100 ␮m). B) Detection of A␤ (6E10, A␤42 ) and A␤ oligomers (NU4, A11) in diabetic cortex and hippocampus (upper bar: 50 ␮m, lower bar: 200 ␮m). C) Immunofluorescence assay of A␤ oligomers (IgM mA␤) and A␤42 in cortex and hippocampus (upper bar: 25 ␮m, lower bar: 50 ␮m). Photographs are representative of seven diabetic and seven control rabbits.

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Fig. 4. Dot-blot analysis of soluble A␤ oligomeric forms in diabetic and control brain samples identified greater A␤ oligomers in diabetic brains. Brain tissue samples from two control and two diabetic rabbits homogenized in PBS were centrifuged at 100K×g. Pellets were re-suspended in RIPA buffer and centrifuged as above. 4 ␮g and 1.5 ␮g of 1st PBS supernatant (S) or final RIPA insoluble pellet resuspended in PBS (P) were spotted onto nitrocellulose filters and probed with A11 and OC anti-A␤ oligomer antibodies, or stained with Amido Black to visualize total proteins. Data is representative of seven diabetic and seven control rabbits used in two experimental trials.

Fig. 5. Increased RAGE receptor expression in diabetic brain with local accumulation in cortex and hippocampus of diabetic rabbits. Upper right panels show immunoblot analysis of RAGE, IR␣, and GAPDH expression in control and diabetic brain tissue samples. Graph below shows relative densitometry band densities (Dark bar: control. Light bar: diabetic). The remaining panels shows IHC detection of RAGE in control hippocampus (bar = 100 ␮m), diabetic hippocampus (bar: 50 ␮m) and below: Control cortex (bar: 100 ␮m), and diabetic hippocampus (bar: 200 ␮m), Higher magnification view is shown in lower right panel (bar: 50 ␮m). Photographs are representative of two experimental trials that used seven diabetic and seven control rabbits.

using immunohistochemical and immunofluorescence methods (Fig. 7). However, we found little or no evidence of tangles in diabetic brains. To provide further evidence of intra-neuronal phosphorylated tau, we examined the distribution of neuronal ␤III-tubulin (tubb3) along neuronal tracts. We detected an overlapping distribution of phospho-tau and tubb3 in neurons

present in the cortex and hippocampus of diabetic brains (Fig. 7). In the hippocampus, phospho-tau ser404 and thr-205 was observed mainly in neurons surrounding nuclei present in the CA3 area in diabetic rabbits. The present findings of tau hyperphosphorylation with few neurofibrillary tangles formed in diabetic rabbit brains agrees with a recent report that identified

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Fig. 6. Increased tau phosphorylation is detected in diabetic brains. Immunoblots contain equal samples of total brain protein samples from control and diabetic rabbits, resolved by MW. Individual filters were probed with of anti-Tau (tau5), and antibodies recognizing tau phosphorylated at ser-404, ser-396, and thr-205. Graph below shows relative densitometry band densities (Dark bar: control. Light bar: diabetic).

links between insulin resistance and tau AD brain pathology in humans [10]. Corresponding Aβ, Aβ oligomers, and RAGE accumulation and pathology is produced in the retina of diabetic rabbits largely in outer ganglion cell and inner nuclear layers The mammalian retina contains stratified layers of several neural cell types interspersed with Muller glial cells. The present diabetes model predicts that AD-related neurodegeneration and A␤ pathology is

coordinately produced in these neural retina layers together with AD brain pathology described above in diabetic rabbits. To examine retinas in diabetic rabbits, we prepared whole eye histological sections from diabetic and control animals and probed them with anti-A␤ and anti-ADDL antibodies also used above to examine the brain. Analysis of retina morphology at 15 weeks after diabetes onset did not show obvious changes in structure at this time point (Fig. 8). Diabetic retinas probed with mAb 4G8 as well as anti-A␤42 antibodies that recognize a C-terminus epitope on 42 amino acid A␤ peptides both identified A␤ accumulation predominantly in the outer ganglion cell layer (GCL) and inner nuclear layer (INL), and to a lesser degree the inner plexiform layer of the retina (Fig. 8). This distribution of A␤ peptides in diabetic rabbit retinas showed a strikingly similarity to A␤ accumulation reported on in retinas from 14 month-old Tg2576 mice by others [45]. We next probed retinas with mouse (NU2, NU4) and rabbit (A11) anti-A␤ oligomer conformation specific antibodies and observed a similar distribution of A␤ oligomer accumulation with each of these antibodies that overlapped with our detection of A␤ in diabetic retina. ADDLs were detected in the GCL adjacent to the vitreous of the eye, as well as the INL neural layers. The distribution of A␤ and A␤ oligomers in these GCL and INL retinal layers also suggested perinuclear accumulations occurred in the cells, similar to their perinuclear detection in neurons in the brain above. We next probed eye sections with anti-RAGE antibodies, and detected RAGE accumulation in diabetic retinas that was not present in control eyes. RAGE deposits overlapped with the distribution of A␤ and A␤ oligomers in GCL and INL layers of the retina. However, we also identified increased RAGE in photoreceptor cells, and more strongly at the border with the outer nuclear layer of the retina. DISCUSSION At present, factors contributing to pathological increases of A␤ in disease and, as importantly, to the accumulation of its toxic derivatives, are not well understood. This is largely due to the absence of physiological, wild-type animal models. The present study addressed this gap by investigating AD-type neuropathology in genetically unmodified rabbits. Rabbit was chosen as it shares physiological production of human-sequence A␤ peptides throughout the body [22]. Our findings show that AD-type CNS neuropathology is instigated in rabbit by alloxan-induced diabetes. Loss of insulin production and consequent

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Fig. 7. Increased detection of phosphorylated tau protein is observed along axonal tracts in brain cortex of diabetic rabbit brains. A) Immunohistochemical detection of phospho-tau (p-ser404 and p-thr205 in control and diabetic cortex. B) Immunofluorescence detection of phopho-Tau p-ser404 (green) in diabetic cortex. For comparison, anti-␤III-tubulin (red) detected in diabetic cortex shows an overlapping distribution along axonal tracts in diabetic brains. Photographs are representative of seven diabetic and seven control rabbits.

hyperglycemia led to large increases of both A␤40 and A␤42 peptides in the CNS, in hippocampus, cortex, and retina. Phosphorylated tau characteristic of AD pathology also increased. Significantly, diabetic brains in our study manifested the spontaneous accumulation of soluble A␤ oligomers, which are widely implicated in AD-related memory loss and brain cell damage [46–48]. These oligomers, which were assayed here on dot blots of PBS extractable protein from brain tissue and observed in situ in several brain regions using mouse and rabbit IgG and IgM anti-oligomer antibodies, have not previously been detected in a

non-transgenic experimental animal. Results identify rabbit as a new resource to investigate mechanisms that cause accumulation of AD-linked oligomers and related facets of neuropathology. The new findings provide strong support for the hypothesis that systemic insulin deficiency and hyperglycemia are contributing factors to the onset of neuropathology in sporadic AD [48–52], the major form of the disease [53]. Pathological accumulation of A␤ peptides also notably manifests in diabetic rabbit retina. As in brain, there is substantial conversion to A␤ oligomers. The pathology in retina, like brain, is evident at

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Fig. 8. A␤ peptide, A␤ oligomers and RAGE accumulate in retinas of diabetic rabbits, and corresponding with brain pathology described above. Antibodies included anti-RAGE, anti-A␤42 , anti-A␤ mAb 4G8, anti-A␤ oligomer NU2, NU4, and A11. Retinal layers numbered at the upper-left as 1–5 indicate 1: ganglion cell layer, 2: inner plexiform layer, 3: inner nuclear layer, 4: outer nuclear layer, 5: photoreceptor layer.

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15 weeks after alloxan treatment, which produced hyperglycemia in the animals studied (>350 mg/dl). The distribution of A␤ peptide and oligomer accumulation in diabetic rabbits is remarkably similar to the pattern of A␤ accumulation reported in retinas in Tg2576 mice carrying the human A␤PP transgene [44]. Overlapping distributions of A␤ and oligomers with RAGE deposits were consistently detected in the ganglion cell layer adjacent to the vitreous, and inner nuclear cell layers of the retina. This distribution in rabbit agrees with analysis of human retinas chronically exposed to hyperglycemia [54, 55]. The present findings with diabetic brain and retina are consistent with our earlier study of muscle of alloxan-treated rabbits [56], which showed A␤ accumulation similar to the A␤ muscle pathology linked with aging and muscle disease in humans. Given that A␤ oligomers have been identified in human muscle degenerative disease [57], and that oligomers are firmly linked to neuronal damage in brain [58–63], the current findings suggest these toxins likely also contribute to diabetic retinopathy. Our findings may have added significance in light of studies suggesting that retinal changes may inform about AD onset and progression in humans [64, 65]. Analogous to findings reported for AD brain [33, 66], the immunohistochemistry of vessel walls in diabetic brain showed indications of oligomer deposits. Whether the deposits represent early stages of cerebral amyloid angiopathy due to diabetes is not yet known. More advanced A␤ deposition localized adjacent to vessels walls in the brain has been reported in Tg AD mouse models [34]. Together, the observations are consistent with clinical indications that a majority of AD cases in older patients represent a combination of neurodegeneration and cerebral angiopathy, which may be typical in late-onset AD [67]. We noted here a large diabetes-induced increase in RAGE expression and accumulation in vessels and brain parenchyma, along with the overlap of RAGE with A␤ distribution. These findings are consistent with the hypothesis that RAGE- A␤ binding could have a role in promoting and influencing A␤ accumulation and localization in diabetic brains [39]. Diabetic rabbit brain also showed increased tau phosphorylation but not tau tangles. It previously has been established that there is increased brain phosphotau linked to insulin resistance in human diabetes [68]. The mechanism likely involves dysfunctional control over the GSK3␤ signaling pathway, which acts as a tau kinase [69–71]. Another possibility is stimulation of tau phosphorylation by the diabetes-induced A␤ oligomers. A␤ oligomers appear to be upstream

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of tau pathology, as oligomers stimulate AD-type tau hyperphosphorylation in cultured neurons [19, 72], and oligomer-specific antibodies reduce tau pathology in transgenic mice [73]. Tau, moreover, is required to mediate aspects of A␤ oligomer-induced neuronal damage [74, 75]. Interestingly, type 1 diabetes in STZtreated wild-type mice and rats consistently manifests tau hyperphosphorylation in brain, but unlike what was found with rabbits, the diabetic rodents show little or no A␤ pathology [76–78]. As seen here, diabetes can instigate major elements of AD neuropathology. Whether there are specific distinctions between diabetes-dependent and other sporadic forms of AD neuropathology involving A␤ peptides and tau proteins remains to be determined. Significantly, the impact of diabetes is likely exacerbated by cellular interactions between neuronal insulin signaling and A␤ oligomer synaptotoxicity [79, 80]. Insulin signaling greatly reduces the binding and toxicity of oligomers [2, 81, 82], so if such signaling were to be lost, neurons would become vulnerable to oligomer exposure [16, 82]. Moreover, once attached to neurons, oligomers cause insulin receptors to be eliminated from surface membranes, and contributes to neuronal insulin resistance, also observed in some AD patients [10, 11, 83]. These phenomena have the potential to create a vicious cycle in which diabetes induces oligomer accumulation, neuron vulnerability to oligomers is elevated because of deficiencies in their insulin signaling, and oligomers exacerbate the deficiency by making neurons even more insulin resistant. Although exploratory efforts are underway to treat AD with insulin and insulin enhancers [83–86], such a vicious cycle indicates it may be difficult to affect ADrelated cognitive failures with single-modality insulin treatments. It has been proposed that minimal efficacy may depend on co-treatments in which insulin enhancement is supplemented with antibodies to help reduce oligomer levels. In conclusion, the current observations highlight the potential of the rabbit as a genetically unmodified animal model to investigate mechanisms leading to sporadic, late-onset AD. The present study provides evidence that one such AD-promoting mechanism is diabetes. Results show that untreated diabetes, in the absence of any other contributing factors, can instigate AD-type neuropathology, including tau phosphorylation and A␤ oligomer accumulation, over a relatively short period. The consequences of this pathology for cognitive function are open to future investigation, as rabbit affords experimental access to mechanisms of memory formation at the synaptic [87] and circuit

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[88] levels. The rabbit thus offers potential value for investigating mechanisms of progressive dementia as well as disease onset. Because of growing awareness that transgenic mouse may have restricted value as general AD models [89, 90], the various attributes of the rabbit model indicate it may prove to be a useful new system for use in therapeutic research and development strategies. Goals of such strategies would include treatments for milder cognitive impairments induced by diabetes as well as for the catastrophic forms of dementia that develop in sporadic, late-onset AD. ACKNOWLEDGMENTS We are grateful to Paula Pierce, Excalibur pathology for assistance in preparation of histological sections and consultation in immunohistological and immunofluorescence studies. We also thank Dr. Bruce Scharf, Director of the UMDNJ Comparative Medicine Resource Facility for advice regarding implementation of our animal protocol, and thank Vir Singh and Yicheng Feng for technical assistance. We also acknowledge NIH grant R01 EY015855 and Neuroscience Education and Research Foundation grant to PHF, and NIH grant R01 AG022547 to WLK. Authors’ disclosures available online (http://www.jalz.com/disclosures/view.php?id=1381). REFERENCES [1] [2]

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