A Humanin Derivative Reduces Amyloid Beta Accumulation and Ameliorates Memory Deficit in Triple Transgenic Mice

Matsuoka Y (2011) A Humanin Derivative Reduces Amyloid Beta Accumulation and Ameliorates Memory Deficit in Triple Transgenic Mice. PLoS ONE 6(1): e16259. doi:10.1371/journal.pone.0016259 A Humanin Derivative Reduces Amyloid Beta Accumulation and Ameliorates Memory Deficit in Triple Transgenic Mice Takako Niikura 0 Elkhansa Sidahmed 0 Chiho Hirata-Fukae 0 Paul S. Aisen 0 Yasuji Matsuoka 0 Tsuneya Ikezu, Boston University School of Medicine, United States of America 0 1 Faculty of Health Sciences, Simon Fraser University , Burnaby, British Columbia , Canada , 2 Department of Neurology, Georgetown University , Washington, D. C. , United States of America, 3 Department of Neurosciences, University of California San Diego , La Jolla, California , United States of America Humanin (HN), a 24-residue peptide, was identified as a novel neuroprotective factor and shows anti-cell death activity against a wide spectrum of Alzheimer's disease (AD)-related cytotoxicities, including exposure to amyloid beta (Abeta), in vitro. We previously demonstrated that the injection of S14G-HN, a highly potent HN derivative, into brain ameliorated memory loss in an Abeta-injection mouse model. To fully understand HN's functions under AD-associated pathological conditions, we examined the effect of S14G-HN on triple transgenic mice harboring APPswe, tauP310L, and PS-1M146V that show the age-dependent development of multiple pathologies relating to AD. After 3 months of intranasal treatment, behavioral analyses showed that S14G-HN ameliorated cognitive impairment in male mice. Moreover, ELISA and immunohistochemical analyses showed that Abeta levels in brains were markedly lower in S14G-HN-treated male and female mice than in vehicle control mice. We also found the expression level of neprilysin, an Abeta degrading enzyme, in the outer molecular layer of hippocampal formation was increased in S14G-HN-treated mouse brains. NEP activity was also elevated by S14G-HN treatment in vitro. These findings suggest that decreased Abeta level in these mice is at least partly attributed to S14G-HN-induced increase of neprilysin level. Although HN was identified as an anti-neuronal death factor, these results indicate that HN may also have a therapeutic effect on amyloid accumulation in AD. - Funding: This study was partly supported by the National Institutes of Health (grants AG026478 and AG022455 to YM) (www.nih.gov) and Natural Sciences and Engineering Research Council of Canada (386973-2010 to TN) (www.nserc-crsng.gc.ca). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: YM, a current employee of Glaxo Smith Kline, participated to this study when he was employed by Georgetown University. No materials have been procured or donated by Glaxo Smith Kline. No product is in development at Glaxo Smith Kline related to this study. This does not alter adherence to all the PLoS ONE policies on sharing data and materials. Humanin (HN) is a multi-functional 24-residue peptide (amino acid sequence: MAPRGFSCLLLLTSEIDLPVKRRA) whose cDNA was isolated from an Alzheimers disease (AD) patients occipital lobe of brain, a region that remains generally intact in many AD cases [1] (reviews in [2,3,4]). HN suppresses the neuronal death caused by all AD-related insults so far tested in vitro including the key cytotoxic molecule in AD, amyloid beta (Abeta) 1-42 [1,5,6]. HN suppressed cell death caused by Abeta toxicity not only in primary neurons but also in cerebrovascular smooth muscle cells, a model of amyloid angiopathy [7]. HN is also effective against cell death caused by non-AD-related insults under different experimental settings, such as serum depriviation, prion peptide118-135, IGFBP3 (insulin-like growth factor binding protein 3), staurosporine etc. [8,9,10,11]. However, HN is not effective against some insults such as etoposide, suggesting that HN is not a general anti-apoptotic agent. It has been hypothesized that HN stimulates its receptor(s) and activates signaling cascade(s) to exert its effects [2,4]. Upon HN stimulation, G protein coupled receptors, formyl peptide receptorlike (FPRL) 1 and FPRL2 [12,13], induce increase of Ca2+ flux and activation of extracellular signal-regulated kinase (ERK), while a receptor complex consisting of gp130, CNTFR, and WSX-1 [14] induces activation of a transcription factor, signal transducer and activator of transcription 3 (STAT3). In addition, three receptor-independent mechanisms have been proposed. (I) Intracellular HN bound to pro-apoptotic Bcl-2 family members, Bax, BimEL, and tBid, and blocked cytochrome c release from mitochondria, leading to inhibition of apoptosis [11,15,16]. (II) HN increased cellular ATP levels in human lymphocytes and a muscular cell line [8,17,18,19,20]. (III) Extracellularly added HN was detected in the cells and suppressed apoptosis induced by IGFBP3 [10]. Through structure-function analyses, we found that a substitution of Gly for 14th Ser (S14G-HN) increased potency 1000-fold [1]. S14G-HN ameliorated amnesia caused by muscarinic receptor antagonists [21,22,23] and Abeta in mice [23,24]. S14G-HN also ameliorated symptoms and/or pathology in rodent stroke model [25,26] and diabetes models [27,28]. These findings suggest the potential of HN for therapeutic application in AD and other diseases. To evaluate the effect of HN derivatives in vivo, Abeta injection model was used in the previous studies [23,29,30]. In this model, Abeta administration induces amnesia in rodents, decreases the number of cholinergic neurons [31], and reduces choline acetyltransferase activity [32,33,34,35] Although the Abeta injection model is a simple and convenient model, the transgenic mouse model has some advantages, for instance production of multiple Abeta species through physiological process and amyloid plaque formation. The triple transgenic mice harboring APPswe, tauP310L, and PS-1M146V (3xTg-AD) [36] have the advantage of the age-dependent development of multiple pathological events relating to AD. In these mice, memory impairment was observed by Morris water maze test at 9 months of age. Soluble Abeta was observed from 9 months of age and plaques from 14 months by immunohistochemical analyses. They also develop tau pathology: total tau protein increased age-dependently, correlating with a decrease of soluble tau and an increase of insoluble tau [37]. Therefore, they are beneficial to examine the effect of therapeutic candidates on pathological changes under the complex physiological conditions. We previously demonstrated that Abeta-induced amnesia was suppressed by intracerebroventricular injection of S14G-HN [23]. To fully understand HNs functions on complex AD-relevant pathology, in this study, we used triple transgenic mouse model. We found that S14G-HN ameliorated cognitive function of 3xTgAD mice and reduced amyloid burden accompanied by an increase (...truncated)