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. 2008 Dec;15(4):625-40.
doi: 10.3233/jad-2008-15409.

Use of copper and insulin-resistance to accelerate cognitive deficits and synaptic protein loss in a rat Abeta-infusion Alzheimer's disease model

Aynun N Begum  1 , Fusheng Yang, Edmond Teng, Shuxin Hu, Mychica R Jones, Emily R Rosario, Walter Beech, Beverly Hudspeth, Oliver J Ubeda, Greg M Cole, Sally A Frautschy
Affiliations

Use of copper and insulin-resistance to accelerate cognitive deficits and synaptic protein loss in a rat Abeta-infusion Alzheimer's disease model

Aynun N Begum et al. J Alzheimers Dis. 2008 Dec.

Abstract

The rat amyloid-beta (Abeta) intracerebroventricular infusion can model aspects of Alzheimer's disease (AD) and has predicted efficacy of therapies such as ibuprofen and curcumin in transgenic mouse models. High density lipoprotein (HDL), a normal plasma carrier of Abeta, is used to attenuate Abeta aggregation within the pump, causing Abeta-dependent toxicity and cognitive deficits within 3 months. Our goal was to identify factors that might accelerate onset of Abeta-dependent deficits to improve efficiency and cost-effectiveness of model. We focused on: 1) optimizing HDL-Abeta preparation for maximal toxicity; 2) evaluating the role of copper, a factor typically in water that can impact oligomer stability; and 3) determining impact of insulin resistance (type II diabetes), a risk factor for AD. In vitro studies were performed to determine doses of copper and methods of Abeta-HDL preparation that maximized toxicity. These preparations when infused resulted in earlier onset of cognitive deficits within 6 weeks post-infusion. Induction of insulin resistance did not exacerbate Abeta-dependent cognitive deficits, but did exacerbate synaptic protein loss. In summary, the newly described in vivo infusion model may be useful cost-effective method for screening for new therapeutic drugs for AD.

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Conflict of interest statement

The authors state that there are no financial conflicts of interests.

Figures

Fig. 1
Fig. 1. Impact of HDL dose on Aβ deposition and neurotoxicity
Aβ42 was icv infused at 25 μg/200 μl (0.2% DMSO) with different mass ratios of HDL. Rats were sacrificed at 4 weeks post-infusion and evaluated for Aβ deposits using monoclonal antibodies 6E10 (A) or 10G4 (B). Neuron nuclei staining in layer 2 of the entorhinal cortex was evaluated using NeuN (C). Values are shown as the mean ± SD. * p < 0.05 represent a significant difference between vehicle (HDL) and treatment (HDL + Aβ).
Fig. 2
Fig. 2. Differential effect of HDL on Aβ toxicity in AβPP SwN2A neuroblastoma cells, depending on whether Aβ42 is pre-aggregated
N2A neuroblastoma cells were incubated for 48h with different mass ratios of HDL:Aβ and toxicity determined by maximum LDH release and morphological assessment. A. HDL caused dose-dependent protection from toxicity caused by pre-aggregated Aβ. B. But if unaggregated Aβ was co-stirred (co-aggregated) with different concentrations of HDL for 48 hours and then applied to N2A cells, HDL exacerbated Aβ toxicity. C. Toxicity of freshly made preparations of unaggregated Aβ (co-stirred with HDL) was compared to that of a preparation incubated for 2 weeks at 37°C. D. Aβ alone (not shown) and co-stirred Aβ-HDL preparations (fresh and incubated for 2 weeks) were spun down, and the soluble fraction was immunblotted with 6E10. E–H. Neuroblastoma cells were stained non-specifically with diaminobenzidine (not quenched with H202) to increase contrast and visualization of cytoplasm to reveal morphological or pyknotic changes. Magnification bar = 100 μm. Values are shown as the mean ± SD. * p < 0.05, ** p < 0.01 and *** p < 0.001 represents a significant difference between vehicle (HDL) and treatment with Aβ + HDL (co-aggregated) or with preaggregated Aβ +HDL.
Fig. 3
Fig. 3. Delayed post-synaptic protein loss in the hippocampus after pre-aggregating Aβ is co-infused with HDL
Rats were infused with pre-aggregated Aβ which was shown to be A11 positive on dot blot and neurotoxic at 100 nM in N2A cells. HDL was added in the pump to further attenuate aggregation and rats as previously described. Rats were sacrificed at 5 (A) and 15 weeks post infusion (B). Hippocampal lysates were immunobloted for post- synaptic proteins, normalized to β-actin and quantified. Values are shown as the mean ± SD. * p < 0.05 represent a significant difference between vehicle (HDL) and treatment (HDL +pre-aggregated Aβ).
Fig. 4
Fig. 4. Low dose copper enhances toxicity in N2A neuroblastoma cell without destabilizing soluble oligomers
To determine the copper effect on Aβ, N2A neuroblastoma cells were incubated for 48h with different doses of copper with and without HDL. A. At lower concentrations (0.5–30 μM) copper exacerbated toxicity caused by pre-aggregated Aβ or Aβ co-stirred with HDL. B. These preparations were electrophoresed on Western blot and determined oligomeric band by conformationally specific anti-oligomer antibody A11. Values are shown as the mean ± SD. * p < 0.05, ** p < 0.01 and *** p < 0.001 represents a significant difference between vehicle (HDL+ copper) and treatment (co-aggregated Aβ with HDL or co-aggregated Aβ with HDL and copper).
Fig. 5
Fig. 5. Behavioral effect of co-aggregated copper- Aβ-HDL by Morris Water Maze
Co-aggregated Aβ-HDL-Copper or vehicle (copper-HDL) was icv infused into Sprague Dawley rats and behavior conducted between 5–7 weeks post infusion. There were no differences in performance to find a visible platform or in swim speeds (not shown), but Aβ-HDL-Copper-infused animals showed acquisition deficits to find a hidden platform (A) and an increase in thigmotaxis (B). Values are shown as the mean ± SEM. * p < 0.05 and ** p < 0.01 represent a significant differences between vehicle (HDL+copper) and treatment (Aβ co-aggregated with HDL and copper).
Fig. 6
Fig. 6. Impact of insulin resistance on Aβ infusion-induced loss of post-synaptic protein NR2B
Male Sprague Dawley rats were fed a high fructose diet to induce insulin resistance or a regular diet for 4 months, and then at 15 months of age infused with vehicle or Aβ co-aggregated with HDL and copper. Animals were sacrificed 6 weeks later at completion of behavioral analysis and representative lanes from a Western of hippocampal lysate is shown for the post-synaptic protein NR2B and β-actin (A). Western quantification of diet dependent effect on NR2B normalized to β-actin (B). 2×2 ANOVA (Treatment x Diet) revealed significant treatment-diet interaction (p < 0.01). RD; Regular Diet, HFD; High Fructose Diet. Values are shown as the mean ± SD. * p < 0.05 represent a significant difference between vehicle (HDL +copper) and treatment (Aβ co-aggregated with HDL and copper).
Fig. 7
Fig. 7. Impact of insulin resistance on Aβ infusion-induced Aβ deposition and microgliosis (Iba-1)
Rats were fed a high fructose diet to induce insulin resistance or a regular diet, and then icv- infused with Aβ co-aggregated with HDL and copper or vehicle (HDL+copper). Sections were immunostained with monoclonal antibody against Aβ (10G4). Representative micrographs of hippocampus are shown, and Aβ burden quantified using image analysis (A). Sections were also stained for antibody to microglia (Iba-1). Representative sections are shown and quantified by image analysis (B). Magnification bar = 100 μm. Values are shown as the mean ± SD. * p < 0.05, ** p < 0.01 and *** p < 0.001 represents a significant difference between vehicle (HDL +copper) and treatment (Aβ co-aggregated with HDL and copper) within diet.
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References

    1. Atwood CS, Perry G, Zeng H, Kato Y, Jones WD, Ling KQ, Huang X, Moir RD, Wang D, Sayre LM, Smith MA, Chen SG, Bush AI. Copper mediates dityrosine cross-linking of Alzheimer’s amyloid-beta. Biochemistry. 2004;43:560–568. - PubMed
    1. Calero M, Tokuda T, Rostagno A, Kumar A, Zlokovic B, Frangione B, Ghiso J. Functional and structural properties of lipid-associated apolipoprotein J (clusterin) Biochem J. 1999;344(Pt 2):375–383. - PMC - PubMed
    1. Cheng IH, Scearce-Levie K, Legleiter J, Palop JJ, Gerstein H, Bien-Ly N, Puolivali J, Lesne S, Ashe KH, Muchowski PJ, Mucke L. Accelerating amyloid-beta fibrillization reduces oligomer levels and functional deficits in Alzheimer disease mouse models. J Biol Chem. 2007;282:23818–23828. - PubMed
    1. Cherny RA, Atwood CS, Xilinas ME, Gray DN, Jones WD, McLean CA, Barnham KJ, Volitakis I, Fraser FW, Kim Y, Huang X, Goldstein LE, Moir RD, Lim JT, Beyreuther K, Zheng H, Tanzi RE, Masters CL, Bush AI. Treatment with a copper-zinc chelator markedly and rapidly inhibits beta-amyloid accumulation in Alzheimer’s disease transgenic mice. Neuron. 2001;30:665–676. - PubMed
    1. Cleary J, Hittner JM, Semotuk M, Mantyh P, O’Hare E. b-amyloid (1-40) effects on behavior and memory. Brain Res. 1995;682:69–74. - PubMed

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