An intranasally delivered peptide drug ameliorates cognitive decline in Alzheimer transgenic mice
2017; Springer Nature; Volume: 9; Issue: 5 Linguagem: Inglês
10.15252/emmm.201606666
ISSN1757-4684
AutoresYu‐Sung Cheng, Zih‐ten Chen, Tai‐Yan Liao, Chen Lin, Howard C.-H. Shen, Ya‐Han Wang, Chi‐Wei Chang, Ren‐Shyan Liu, Rita P.‐Y. Chen, Pang‐Hsien Tu,
Tópico(s)Supramolecular Self-Assembly in Materials
ResumoResearch Article29 March 2017Open Access Transparent process An intranasally delivered peptide drug ameliorates cognitive decline in Alzheimer transgenic mice Yu-Sung Cheng Yu-Sung Cheng Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan Search for more papers by this author Zih-ten Chen Zih-ten Chen Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan Search for more papers by this author Tai-Yan Liao Tai-Yan Liao Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan Search for more papers by this author Chen Lin Chen Lin orcid.org/0000-0003-1497-5899 Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan Search for more papers by this author Howard C-H Shen Howard C-H Shen Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan Search for more papers by this author Ya-Han Wang Ya-Han Wang Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan Search for more papers by this author Chi-Wei Chang Chi-Wei Chang Biomedical Imaging Research Center, Department of Nuclear Medicine, National Yang-Ming University and Taipei Veterans General Hospital, Taipei, Taiwan Search for more papers by this author Ren-Shyan Liu Ren-Shyan Liu Biomedical Imaging Research Center, Department of Nuclear Medicine, National Yang-Ming University and Taipei Veterans General Hospital, Taipei, Taiwan Molecular and Genetic Imaging Core, Taiwan Mouse Clinic, Academia Sinica, Taipei, Taiwan Search for more papers by this author Rita P-Y Chen Corresponding Author Rita P-Y Chen [email protected] orcid.org/0000-0003-3987-033X Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan Search for more papers by this author Pang-hsien Tu Corresponding Author Pang-hsien Tu [email protected] orcid.org/0000-0003-2821-7335 Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan Search for more papers by this author Yu-Sung Cheng Yu-Sung Cheng Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan Search for more papers by this author Zih-ten Chen Zih-ten Chen Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan Search for more papers by this author Tai-Yan Liao Tai-Yan Liao Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan Search for more papers by this author Chen Lin Chen Lin orcid.org/0000-0003-1497-5899 Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan Search for more papers by this author Howard C-H Shen Howard C-H Shen Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan Search for more papers by this author Ya-Han Wang Ya-Han Wang Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan Search for more papers by this author Chi-Wei Chang Chi-Wei Chang Biomedical Imaging Research Center, Department of Nuclear Medicine, National Yang-Ming University and Taipei Veterans General Hospital, Taipei, Taiwan Search for more papers by this author Ren-Shyan Liu Ren-Shyan Liu Biomedical Imaging Research Center, Department of Nuclear Medicine, National Yang-Ming University and Taipei Veterans General Hospital, Taipei, Taiwan Molecular and Genetic Imaging Core, Taiwan Mouse Clinic, Academia Sinica, Taipei, Taiwan Search for more papers by this author Rita P-Y Chen Corresponding Author Rita P-Y Chen [email protected] orcid.org/0000-0003-3987-033X Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan Search for more papers by this author Pang-hsien Tu Corresponding Author Pang-hsien Tu [email protected] orcid.org/0000-0003-2821-7335 Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan Search for more papers by this author Author Information Yu-Sung Cheng1,‡, Zih-ten Chen2,‡, Tai-Yan Liao2, Chen Lin2, Howard C-H Shen2, Ya-Han Wang2,3, Chi-Wei Chang4, Ren-Shyan Liu4,5, Rita P-Y Chen *,2,3 and Pang-hsien Tu *,1 1Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan 2Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan 3Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan 4Biomedical Imaging Research Center, Department of Nuclear Medicine, National Yang-Ming University and Taipei Veterans General Hospital, Taipei, Taiwan 5Molecular and Genetic Imaging Core, Taiwan Mouse Clinic, Academia Sinica, Taipei, Taiwan ‡These authors contributed equally to this work *Corresponding author. Tel: +886 2 27855696; Fax: +886 2 27889759; E-mail: [email protected] *Corresponding author. Tel: +886 2 26523532; Fax: +886 2 27827654; E-mail: [email protected] EMBO Mol Med (2017)9:703-715https://doi.org/10.15252/emmm.201606666 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Alzheimer's disease (AD) is the most common neurodegenerative disease. Imbalance between the production and clearance of amyloid β (Aβ) peptides is considered to be the primary mechanism of AD pathogenesis. This amyloid hypothesis is supported by the recent success of the human anti-amyloid antibody aducanumab, in clearing plaque and slowing clinical impairment in prodromal or mild patients in a phase Ib trial. Here, a peptide combining polyarginines (polyR) (for charge repulsion) and a segment derived from the core region of Aβ amyloid (for sequence recognition) was designed. The efficacy of the designed peptide, R8-Aβ(25–35), on amyloid reduction and the improvement of cognitive functions were evaluated using APP/PS1 double transgenic mice. Daily intranasal administration of PEI-conjugated R8-Aβ(25–35) peptide significantly reduced Aβ amyloid accumulation and ameliorated the memory deficits of the transgenic mice. Intranasal administration is a feasible route for peptide delivery. The modular design combining polyR and aggregate-forming segments produced a desirable therapeutic effect and could be easily adopted to design therapeutic peptides for other proteinaceous aggregate-associated diseases. Synopsis Intranasal administration of an amyloid inhibitor peptide to Alzheimer transgenic mice reduces amyloid brain deposition in the brain and ameliorates cognitive decline. A peptide, R8-Aβ(25–35), combining polyarginines (polyR) (for charge repulsion) and a segment derived from the core region of Aβ amyloid (for sequence recognition) was designed. Daily intranasal administration of the PEI-conjugated R8-Aβ(25–35) peptide significantly reduced Aβ amyloid accumulation and ameliorated the memory deficits of the transgenic mice. Intranasal administration is a feasible route for peptide delivery. The modular design combining polyR and aggregate-forming segments could be easily extended to other proteinaceous aggregate-associated diseases. Introduction Alzheimer's disease (AD) is the most common neurodegenerative disease that causes dementia across multiple cognitive domains. Its incidence increases significantly with age and doubles every 5 years among the geriatric population ≧ 65 years of age. Despite remarkable scientific advancement and the vast resources invested in drug development, no effective therapy is currently available for AD. Thus, it is listed as one of the major unmet medical needs worldwide. Although the etiology of AD remains unclear, the amyloid cascade hypothesis is the most supported explanation to date and is recently further strengthened by the finding of a protective APP mutation near the β-cleavage site against the development of late-onset dementia (Jonsson et al, 2012). Unfortunately, a series of clinical trials based on amyloid reduction therapy (ART) failed to deliver anticipated clinical improvement on mild-to-moderate patients with AD (Ross & Imbimbo, 2010; Aisen et al, 2011; Roher et al, 2011; Grundman et al, 2013; Khorassani & Hilas, 2013), raising legitimate concerns for the accuracy of amyloid cascade hypothesis and the future of ART (Extance, 2010). However, given that alternative strategies aimed at reducing neuroinflammation, cholesterol level, or oxidative stress have similarly failed to improve the clinical outcome of AD, it is fair to say that the jury is still out on finding the culprit for the failures of these clinical trials. Previous data reveal that a substantial percentage (~50%) of neurons have already disappeared in even the mild cognitive impairment or very mild AD (Gomez-Isla et al, 1996; Mufson et al, 2000; Price et al, 2001). Consistent with these, more recent findings show that alterations in amyloid biology represent the earliest detectable changes in the brain in familial AD and start in the brain more than 20 years prior to the onset of AD (Bateman et al, 2012). It is likely that the ART trials (and other alternatives) fail because they miss the most opportune "therapeutic window" of AD. Thus, ART still remains a vital and important choice when given earlier. In fact, clinical trials with very early or pre-symptomatic intervention using ART are currently being conducted or have been planned (Miller, 2012; Wadman, 2012; Moulder et al, 2013). The preliminary success of ART with aducanumab immunotherapy in decreasing cognitive decline further strengthens this hypothesis (Moreth et al, 2013; Lannfelt et al, 2014; Ratner, 2015; Underwood, 2015; Selkoe & Hardy, 2016; Sevigny et al, 2016). Peptide drugs have been used with consistent benefits for many years and have advantages over small molecules, such as higher potency and fewer off-target side effects (Craik et al, 2013). In addition, the properties of easy customization and synthesis under a well-controlled environment make peptides excellent candidates for AD drug development. Neurodegenerative diseases encompass a heterogeneous group of neurological diseases characterized by synoptic and neuronal losses caused by multiple factors. Misfolded proteinaceous aggregates which exist in a variety of these diseases besides AD, including Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, are considered one of them, and may cause or contribute to these diseases through their prionlike property (Kim & Holtzman, 2010; de Calignon et al, 2012; Luk et al, 2012; Smethurst et al, 2016). In spite of the difference in the constituent proteins and complexity of assembly mechanism, the proteinaceous aggregates across these diseases share common structural conformations such as a β-sheet conformation in the backbone (Funke & Willbold, 2012). This provides the basis for a rational design of therapeutic peptides for these misfolded aggregate-associated diseases by applying a universal principle to reverse the process of formation. In this study, we propose a novel modular approach to design an ART peptide drug and test its efficacy using the APP/PS1 transgenic mouse model. Results Design of the inhibitor peptide Amyloid in AD is an insoluble β-sheet structure formed by Aβ peptides. Thus, in order to inhibit amyloid propagation, the inhibitor should be equipped with the following characteristics: (i) the ability to interact with Aβ monomer/aggregates and (ii) the ability to prevent Aβ from association into higher order polymers and fibrils. Here, we proposed a rational approach based on the concept of modularity to design bipartite inhibitor peptides comprising two different modules, each possessing one of the aforementioned characteristics, as a prototype of potential therapeutic agents. The concept of our design was schematically presented in Fig 1A. The first module was a partial sequence derived from Aβ peptide which, because of its known propensity to self-aggregate, was anticipated to bestow on the inhibitor peptide an ability to bind to Aβ with high specificity. The second module was a charged moiety which, through the repulsion force exerted by these charges, could prevent not only self-aggregation of the inhibitor peptide, but also the propagation of amyloid after the inhibitor peptide bound to its Aβ target. Figure 1. The inhibition model of R8-Aβ(25–35) and the dose-dependent effect of R8-Aβ(25–35) on inhibition of Aβ40 fibrillizationAβ40 was mixed with R8-Aβ(25–35) in different mixing ratios (Aβ40:R8-Aβ(25–35) = 1:0.1, 1:0.2, 1:1). The Aβ40 concentration is 30 μM. The peptides were dissolved in 20 mM sodium phosphate buffer with 150 mM KCl (pH 7) and incubated at 25°C. A. Proposed working mechanism. B–E. CD spectra of Aβ40 alone (B) and three Aβ40/R8-Aβ(25–35) mixtures (C, 1:0.1; D, 1:0.2; E, 1:1) were recorded at the indicated incubation times. F–I. TEM images of the samples in (B), (C), (D), and (E) taken at the indicated incubation times are shown in (F), (G), (H), and (I), respectively. Download figure Download PowerPoint It has been reported that the sequence of residues 25–35 of Aβ is important for Aβ aggregation and toxicity (Hughes et al, 2000; Ban et al, 2004; Liu et al, 2004). In this study, we designed an L-form inhibitor peptide, R8-Aβ(25–35), and its D-form counterpart, DR8-Aβ(25–35), by combining Aβ(25–35) (the Aβ-binding module) with a segment of consecutive eight L-form or D-form Arg residues (R8 or DR8) (the charge repulsion module). The conformational properties of these two bipartite peptides and their effects on the inhibition of Aβ amyloidogenesis and toxicity were examined. Intranasal delivery has been shown to be an effective way to deliver insulin into brain to alleviate memory deficit in patients with amnestic mild cognitive impairment or AD (Craft et al, 2012; Claxton et al, 2015). It has been reported that PEI cationization facilitates protein transduction across the cell membrane and has been used in drug design to bypass blood–brain barrier into the brain parenchyma via intranasal delivery (Futami et al, 2005, 2007; Kitazoe et al, 2005, 2010; Loftus et al, 2006; Murata et al, 2008a,b; Lin et al, 2016). Thus, to facilitate entry of our peptide into the mouse brain, polyethylenimine (PEI) was conjugated with R8-Aβ(25–35), which contained the poly-R segment that could also enhance peptide penetration (Herve et al, 2008; Patel et al, 2009). We synthesized PEI-conjugated R8-Aβ(25–35) and tested its therapeutic effect in the APP/PS1 transgenic mice via intranasal administration. Conformational study of Aβ40, R8-Aβ(25–35), and DR8-Aβ(25–35) To investigate the biophysical property of our designed peptides concerning amyloid formation, Aβ40, R8-Aβ(25–35), and DR8-Aβ(25–35) dissolved in 20 mM sodium phosphate buffer with 150 mM KCl (pH 7) were individually incubated at 25°C. The circular dichroism (CD) spectra were recorded at various time points as indicated. In Aβ40 spectra, as expected, the intensity of the negative ellipticity at 218 nm increased and the intensity at 200 nm decreased with time (Fig 1B and Appendix Fig S1A), consistent with its known ability to form amyloid fibrils as shown by transmission electron microscopy (TEM) (Fig 1F and Appendix Fig S1D). In contrast, the CD spectra of R8-Aβ(25–35) showed negative ellipticity at 200 nm, indicative of random coil structure (Appendix Fig S1B). Similarly, the DR8-Aβ(25–35) also had CD spectra consistent with random coil structure, which lacked strong negative ellipticity at 200 nm due to the presence of eight D-form arginines in the peptide (Appendix Fig S1C). The CD spectra of both peptides remained largely unchanged with the incubation time. Under the same incubation condition, R8-Aβ(25–35) and DR8-Aβ(25–35) did not form amyloid fibrils at all except for small blobs of amorphous aggregates under transmission electron microscopy after incubation for 168 h (Appendix Fig S1E and F). Inhibition effect of our designed bipartite peptides on the fibrillization of Aβ40 To examine whether R8-Aβ(25–35) could interfere with the amyloidogenesis of Aβ40, the CD spectra of Aβ40 mixed with R8-Aβ(25–35) at 1:0.1, 1:0.2, or 1:1 molar ratios were measured. As shown in Fig 1C–E, the change in the CD spectrum of Aβ40 (an increase in the negative ellipticity at 218 nm and a decrease at 200 nm) clearly decreased by R8-Aβ(25–35) in a dose-dependent manner. Notably, the inhibition effect of R8-Aβ(25–35) on Aβ40 fibrillization was observed even when its concentration was ten times lower than Aβ40 (Fig 1C and E). Consistent with these CD studies, TEM revealed a clear reduction in both thickness and abundance of the amyloid fibrils at all time points we observed (Fig 1G–I). These results showed that R8-Aβ(25–35) interacted with Aβ40, interfered with its self-aggregation, and thereby significantly delayed or decreased the formation of Aβ40 amyloid fibrils. Interestingly, the inhibition effect was also observed with DR8-Aβ(25–35) (Appendix Fig S2), suggesting that it was the charge, rather than the steric structure, of the R8 moiety that prevented Aβ40 from aggregation. Attenuation of Aβ40 cytotoxicity by R8-Aβ(25–35) and DR8-Aβ(25–35) Because our designed bipartite peptides interfered with Aβ40 self-aggregation, we tested whether these peptides could decrease the toxicity of Aβ40 by measuring the viability of Neuro2a, a mouse neuroblastoma cell line with the MTT assay. Cells were treated with peptides as indicated. Aβ40 (30 μM) exerted significant cytotoxicity to Neuro2a cells; only 30% of the cells survived the treatment. In contrast, R8-Aβ(25–35) or DR8-Aβ(25–35) had no detectable toxicity to the N2a cells (Fig 2A). Interestingly, R8-Aβ(25–35) or DR8-Aβ(25–35) each decreased Aβ40 toxicity, as evidenced by an increase in cell viability from 30% to 70–75% (Fig 2B). For comparison, when Aβ40 was mixed with Aβ(25–35), very small change in cell viability was observed. Our data indicated that our designed bipartite peptides might have therapeutic potential in amyloid-induced toxicity. Figure 2. Cell viability measurement by MTT assays Neuro2a cells treated with DMSO (control), Aβ40, R8-Aβ(25–35), or DR8-Aβ(25–35). Neuro2a cells treated with DMSO (control), Aβ40, and Aβ40 with equal molar R8-Aβ(25–35), DR8-Aβ(25–35), or Aβ(25–35). Data information: Peptide concentration is 30 μM for each peptide. Standard deviations of the mean are shown as bars for each sample (N = 6 for pure peptide and N = 12 for mixture). The statistics were done by Student's t-test. Download figure Download PowerPoint Therapeutic effect of R8-Aβ(25–35)-PEI in APP/PS1 transgenic mice To test whether R8-Aβ(25–35) could prevent the deterioration of memory in vivo, PEI or PEI-coupled R8-Aβ(25–35), denoted as R8-Aβ(25–35)-PEI, was given intranasally for 4 months to APP/PS1 mice when they were 4 months of age (experimental sets 1 and 2, Appendix Fig S3). The water maze assay was performed when the mice reached 8 months of age. As shown in Fig 3A, the wild-type mice treated with PEI or R8-Aβ(25–35)-PEI showed no clear difference in the learning curve of finding the hidden platform. In contrast, the peptide-treated APP/PS1 mice exhibited a significant improvement in learning compared to the control transgenic mice treated with PEI. In addition, peptide-treated APP/PS1 mice performed better at the probe test, as evidenced by their higher crossing number (Fig 3B) and longer time spent in the quadrant where the probe used to be compared to PEI-treated control transgenics (Fig 3C). Figure 3. Effect of intranasally delivered R8-Aβ(25–35)-PEI on APP/PS1 transgenic mice after 4-month treatmentWild-type (WT) and APP/PS1 mice were treated with either PEI or R8-Aβ(25–35)-PEI from the age of 4 months to 8 months. A–C. Morris water maze. (A) The plot of the escape latency. (B) The times of the indicated mice crossing the target quadrant. (C) Percentage of time the indicated mice spent swimming in the target quadrant where the hidden platform used to be. The behavior data were expressed in mean ± SEM. The statistics of the escape trend were done with two-way ANOVA. Others were done by Student's t-test. D, E. ELISA of total Aβ40 and Aβ42 in hippocampus (D) and cortex (E) (N = 3 per group). F. Level of IL-6 and IL-1β in the cortex (N = 3 per group). Data information: The data were expressed in mean ± SD, and the statistics were done by Student's t-test for panels (D–F). Download figure Download PowerPoint We next assessed the changes in the level of Aβ peptide in the experimental set 1 animals by ELISA. As shown in Fig 3D and E, at the age of 8 months, the level of Aβ40 and Aβ42 decreased by 73% and 60%, respectively, in the hippocampus of peptide-treated APP/PS1 mice compared with those of PEI-treated transgenic mice (Fig 3D). Similarly, the level of Aβ40 and Aβ42 decreased by 86% and 32%, respectively, in the cortex of the former group compared with the latter (Fig 3E). Our data indicate that the peptide treatment effectively reduced Aβ accumulation and slowed down the clinical impairment of memory. Amyloid deposition is known to induce neuroinflammation, which contributes to disease pathogenesis in these mice. We therefore conducted cytokine assays. R8-Aβ(25–35)-PEI effectively decreased the level of pro-inflammatory cytokines interleukin (IL)-6 and IL-1β in the cortex (Fig 3F) in parallel with the changes in the level of Aβ peptides. Continuous therapeutic effect of R8-Aβ(25–35)-PEI after a suspension for 4 weeks During the water maze tests, the treatment was adjourned for about 4 weeks. To examine whether the therapeutic effect could be maintained after a suspension of treatment, administration of PEI or peptide was resumed and continued for 4 more months (experimental set 2, Appendix Fig S3). The accumulation of amyloid plaques was quantified with microPET using the tracer Pittsburg compound B (PiB). As shown in Fig 4A and B, the peptide-treated APP/PS1 mice had a much lower signal in the cortex, hippocampus, amygdala, and olfactory bulb compared with the PEI-treated mice, consistent with a beneficial therapeutic effect at this age. ELISA analyses revealed a significant decrease in SDS-insoluble Aβ40 and Aβ42 by 25–30% in the cortex or hippocampus of the peptide-treated APP/PS1 mice compared with those in PEI-treated mice (Fig 4C and D), consistent with the microPET results (18–33% reduction). Correspondingly, SDS-soluble Aβ40 and Aβ42 levels significantly increased after peptide treatment (Fig 4E and F). One important biomarker in AD diagnosis is the decrease in Aβ42 level in the cerebral spinal fluid due to Aβ aggregation; a reversion of this process is expected to increase soluble Aβ concentration. Thus, these results demonstrated that our inhibitor peptide was able to inhibit Aβ from self-associating into amyloid fibrils/plaques. Figure 4. Effect of intranasally delivered R8-Aβ(25–35)-PEI on APP/PS1 mice from 4 months to 13 months of age with a 1-month break within this period A. Representative MicroPET image of the transgenic mouse brains co-registered with mouse T2-weighted MRI brain template. CTX, cortex; HIP, hippocampus; AMY, amygdala. B. Quantitation of [11C]PiB uptake in the cortex, hippocampus, amygdala, and olfactory bulb (N = 6 per group). C–F. ELISA of SDS-insoluble Aβ40 and Aβ42 in the cortex (C) and hippocampus (D) and SDS-soluble Aβ40 and Aβ42 in the cortex (E) and hippocampus (F) (N = 5 per group). Data information: Data were expressed in mean ± SD, and the statistics were conducted with the Student's t-test. Download figure Download PowerPoint Inhibition effect of R8-Aβ(25–35)-PEI on performed amyloid plaques in older mice To test whether R8-Aβ(25–35)-PEI could prevent Aβ accumulation when amyloid plaques had already formed, we started peptide treatment with higher dosage (4 nmole/mouse/day) in another set of mice from the age of 8 months for 2 months until they were 10 months of age (experimental set 3 in Appendix Fig S3). As shown in Appendix Fig S4, treatment with R8-Aβ(25–35)-PEI did not decrease the number of amyloid plaques, but significantly decreased several parameters, including the size of individual plaques, the percentage of the cortex covered by plaques, and the total area of amyloid plaques per cortex, by 20%, 22%, and 24%, respectively. These data confirmed the therapeutic effect of this peptide even when administrated for a short time in mice with pre-existing amyloid plaques. To investigate whether the therapeutic effect of R8-Aβ(25–35)-PEI was possible with the Arg-rich segment alone, without needing Aβ(25–35), we also conducted experiments with R8-YS-PEI peptide in the set 3 mice. No therapeutic benefit was observed (Appendix Fig S4). The results confirmed that R8-Aβ(25–35)-PEI required the Aβ(25–35) segment for target recognition to reduce the amyloid accumulation. Entrance of R8-Aβ(25–35)-PEI peptide into brains To determine whether the intranasally given peptide inhibitor entered brains, we synthesized fluorescence-conjugated peptide, FITC-(Ahx)-CR8-Aβ(25–35)-PEI. A higher dosage (5 μl of 1,800 μM peptide; 9 nmole/mouse/day) was given daily to one 10-week-old female C57BL/6JNarl mouse for three consecutive days in these tests in order to enhance the success rate of detection (experimental set 4 in Appendix Fig S3). Brains were collected and processed at 0.5, 2, 6, 12, and 24 h after the completion of the 3rd peptide treatment. The amount of the intracerebral peptide was quantified by the FITC emission spectra between 500 and 600 nm of the brain filtrates excited at 446 nm (Fig 5A) against a calibration curve (Appendix Fig S5). The peptide was indeed detectable in the brains, which reached the peak (5.16 nmole) 6 h after the treatment, and then decreased at a rate about 0.086 nmole per hour (Fig 5B). In addition, the amounts of the peptide at the time points of 0.5 and 12 h were higher than that of 24 h. These data indicated that the peptide entered brain efficiently and was maintained at higher level for more than 12 h. Figure 5. Brain penetration of FITC-(Ahx)-CR8-Aβ(25–35)-PEI after the third dosing via intranasal routeThe mice were treated intranasally with FITC-(Ahx)-CR8-Aβ(25–35)-PEI (9 nmole/mouse/24 h) for three times. The mice were sacrificed, and their brains were perfused at 0.5, 2, 6, 12, and 24 h after the third treatment. Fluorescence spectra of the filtrates of mouse brain homogenates after passing through a 100-kDa filter. The unbroken, dashed, and dotted lines represent three different mice. The amount of FITC-(Ahx)-CR8-Aβ(25–35)-PEI per brain at different times after the third peptide treatment. Data were expressed in mean ± SD (n = 3). Download figure Download PowerPoint Discussion In this study, we demonstrated that the peptide R8-Aβ(25–35) reduced the formation of amyloid fibrils by Aβ40 in vitro, as well as amyloid plaques and disease manifestation in vivo. In a companion study, therapeutic peptides designed by the same modular principle also delayed disease in the R6/2 transgenic mice, a widely used mouse model for Huntington's disease (unpublished data). Thus, our data illustrated the possibility that this principle may be extended to design therapeutic peptides for other neurodegenerative diseases. A variety of therapeutic peptides to decrease the formation of amyloid fibrils has been proposed (Funke & Willbold, 2012); our bipartite design works by attaching a polyR stretch to the peptide sequence derived from the disease-specific pathogenic peptide/protein prone to aggregation. This approach possessed several unique features and advantages. First, the sequence directly taken from the pathogenic peptide/protein not only significantly reduced the labors of finding and optimizing a suitable peptide sequence, but also guaranteed high affinity with the target through its self-aggregating property. Second, the multi-charges in polyR rendered the designed therapeutic peptide (i) soluble in an aqueous environment and therefore simplifying the processes of synthesis and subsequent application, (ii) cell-penetrable (Mitchell et al, 2000), making it suitable for both extracellular and intracellular peptide/protein aggregation, and (iii) able to slow down oligomer/amyloid formation by charge repulsion after its binding to the pathogenic peptide/protein. Third, combination of the polyR with the sequence from disease-specific pathogenic protein/peptide provided great feasibility and flexibility in applying this design across different misfolded aggregate-associated diseases. Although many therapeutic peptides have been designed, only a few of them were tested in vivo (Permanne et al, 2002; van Groen et al, 2008; Frydman-Marom et al, 2009; Funke et al, 2010; Shukla et al, 2013; Lin et al, 2016). In this study, we have demonstrated the feasibility of intranasal administration of therapeutic peptidic prodrugs. When combined with technology in delivery, our study showed a proof of therapeutic principle for neurodegenerative diseases through intranasal delivery. The dose used in this study was only 2 nmoles (6 μg) per day, which was quite low compared with previous studies (Permanne et al, 2002; van Groen et al, 2008; Frydman-Marom et al, 2009; Funke et al, 2010). Using this dosage, we attempted to investigate the level of the therapeutic peptide in the brain during consecutive intranasal treatment (experimental set 5 in Appendix Figs S3 and S6). However, the peptide concentration was low and could not be reliably detected. As shown in Fig 5, after three consecutive treatments at high
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