Neurobiology of Aging xxx (2014) 1e8

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Pituitary adenylate cyclase-activating polypeptide protects against b-amyloid toxicity Pengcheng Han a, *, Zhiwei Tang a, b, Junxiang Yin a, Marwan Maalouf a, Thomas G. Beach c, Eric M. Reiman d, Jiong Shi a, * a

Department of Neurology, Barrow Neurological Institute, St. Joseph Hospital and Medical Center, Dignity Health Organization, Phoenix, AZ, USA Department of Neurosurgery, The First Hospital of Kunming Medical University, Kunmming, China Civin Laboratory for Neuropathology, Banner Sun Health Research Institute, Sun City, AZ, USA d Banner Alzheimer’s Institute, Phoenix, AZ, USA b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 4 December 2013 Received in revised form 28 February 2014 Accepted 15 March 2014

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neurotrophin. However, its role in human Alzheimer’s disease (AD) is largely unknown. We examined PACAP expression in postmortem human AD and triple transgenic mouse (3xTG, Psen1/APPSwe/TauP301L) brains. We established an in vitro model of primary neuronal cell culture to study the protective effects of PACAP against b-amyloid (Ab) toxicity. We further studied the PACAP-Sirtuin 3 (Sirt3) pathway on mitochondrial function. PACAP expression was reduced in AD and 3xTG mouse brains. This reduction was inversely correlated with Ab and tau protein levels. Treatment with PACAP effectively protected neurons against Ab toxicity. PACAP stimulated mitochondrial Sirt3 production. Similar to PACAP, Sirt3 was reduced in AD and 3xTG brains. Knocking down Sirt3 compromised the neuroprotective effects of PACAP, and this was reversed by over-expressing Sirt3. PACAP is reduced in AD and may represent a novel therapeutic strategy. Ó 2014 Elsevier Inc. All rights reserved.

Keywords: Alzheimer’s disease PACAP SIRT3 Mitochondrial respiration

1. Introduction Alzheimer’s disease (AD) is the most common cause of dementia in elderly adults. With the growing number of people living to an older age, there is an urgency to better understand elements of the pathogenic pathway, discover agents that target these elements, and establish their roles in the treatment and prevention of AD. Pituitary adenylate cyclase activating polypeptide (PACAP) is intrinsically expressed in mammals and is considered to be a potent neurotrophic and neuroprotective peptide (Vaudry et al., 2009). PACAP exists in 2 forms. The 38-amino acid form (PACAP-38) is the major form in tissues, while the shorter 27-amino acids form corresponds to the N-terminal of PACAP-38 (PACAP-27). Both forms of PACAP bind to and activate G protein-coupled receptors (PAC1, VPAC1, and VPAC2). Several studies proposed to use PACAP to treat neurodegenerative diseases including AD (Reglodi et al., 2011). Cell culture studies strongly suggest that PACAP protect against b-amyloid (Ab) toxicity in PC-12 (Onoue et al., 2002) and astrocyte cell * Corresponding authors at: Department of Neurology, NRC428, Barrow Neurological Institute, St. Joseph Hospital and Medical Center, Dignity Health Organization, 350 W. Thomas Road, Phoenix, AZ 85013, USA. Tel.: þ1 602 406 4032; fax: þ1 602 798 0899. E-mail addresses: [email protected] (P. Han), Jiong.Shi@ dignityhealth.org (J. Shi). 0197-4580/$ e see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2014.03.022

lines (Shieh et al., 2008). In a gene expression survey study, 3 mouse models of AD were used. One was APP(NLh/NLh)/PS-1(P264L/ P264L) and the other 2 were amyloid precursor protein (APP) over-expression of FAD mutations (Tg2576) with the PS-1 knock-in mutation at either 1 or 2 alleles. PACAP was one of the 3 genes that were downregulated. The other 2 were brain-derived neurotrophic factor and insulin-like growth factor I receptor (Wu et al., 2006). PACAP has been shown to stimulate the nonamyloidogenic processing of APP by reducing the Ab transporter receptor for advanced glycation end products messenger RNA level and by increasing the Ab-degrading enzyme neprilysin in the APP[V717I]-transgenic mouse brain (Rat et al., 2011). It also increases the levels of brainderived neurotrophic factor and anti-apoptotic Bcl-2 protein (Rat et al., 2011). However, there is no report on the expression of PACAP in normal and AD human brains. PACAP modulates multiple apoptosis-related mechanisms including several mitochondrial targets. PACAP protects rotenoneinduced cytotoxicity, suggesting it helps to restore complex I function (Wang et al., 2005). PACAP modulates adenosine monophosphate-activated protein kinase (AMPK) (AcquaahMensah et al., 2012), which in turn stimulates the production of Sirtuin 3 (Sirt3) (Buler et al., 2012). Sirt3 is a member of the conserved deacetylases family named as silencing information regulators (Sirtuin). Among the 7 members of Sirtuin family

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(SIRT1-7), Sirt3, 4, and 5 are located in mitochondria (Albani et al., 2013; Sundaresan et al., 2009), and Sirt3 is the only one that modulates the respiratory chain. Sirt3 null mouse showed low baseline adenosine triphosphate and complex I hyperacetylation (Sundaresan et al., 2009). Sirt3 enhances mitochondrial respiration by deacetylate complex I and complex II in a NADþ-dependent fashion (Jacobs et al., 2008). The dependence on NADþ level constitutes a self-modulatory mechanism to optimize energy supply in the hunger state (Yu and Auwerx, 2009). In this study, we hypothesized that PACAP would be reduced in AD cerebral cortex and treatment with PACAP would improve mitochondrial function and protect against Ab toxicity by modulating Sirt3 expression. 2. Methods 2.1. Human and animal tissue Postmortem human cerebral cortex was obtained from the Banner Sun Health Research Institute Brain and Body Donation Program (Beach et al., 2008). The operations of the Brain and Body Donation Program have been approved by the Western Institutional Review Board. All experimental animal procedures were approved by the Institutional Animal Care and Use Committee of the Barrow Neurological Institute and performed according to the Revised Guide for the Care and Use of Laboratory Animals. Frozen cerebral cortex postmortem brain tissues were obtained from 16 subjects with a clinicopathological diagnosis of AD and 12 nondemented elderly subjects. All AD cases were selected as being “intermediate” or “high” probability for AD according to NIA-Reagan criteria (National Institute on Aging-Alzheimer’s Association criteria, 1997). In addition, they were free of other neurodegenerative disorders such as vascular dementia, Parkinson’s disease, dementia with Lewy bodies, frontotemporal dementia, hippocampal sclerosis, progressive supranuclear palsy, dementia lacking distinctive histology, multiple system atrophy, motor neuron disease with dementia, and corticobasal degeneration. Nondemented control subjects were of a similar age but did not meet clinicopathological criteria for AD or dementia. 3xTG mice (with 3 mutant alleles: Psen1 mutation, APPSwe, and TauP301L) and controls (129/sv  C57BL6 background strain) were produced in colony. This was a kind gift from Dr Jon Valla who received these mice directly from University of California, Irvine. Transgene status of mice in each cohort was blinded until the analysis was complete and was confirmed by polymerase chain reaction genotyping. The animals were kept in groups on a 12:12 hours light and/or dark cycle with food and water ad libitum. Mice were euthanized by injection of a ketamine and/or xylamine cocktail at 24-month-old. They were then perfused transcardially with 0.1 M phosphate-buffered saline (PBS). The fresh cortical tissue were dissected quickly on ice and frozen in liquid nitrogen for subsequent protein extraction and immunoblot or ELISA assay. The human or mouse brain samples were homogenized in PBS or RIPA buffer containing 1 proteinase inhibitor cocktails (SigmaAldrich, #P8340, St. Louis, MO, USA) and sonicated at 4  C. The total amount of protein was quantified using Pierce BCA protein assay kit (Thermo Scientific, #23227, Rockford, IL, USA). 2.2. Cell culture and the treatment with Ab42, PACAP, and PAC1 antagonist Mouse pups were sacrificed at the postnatal age of 0e3 days. The brain was harvested immediately into the Neurobasal media (Life Technology Inc, Grand Island, NY, USA). The brain cortex was carefully dissected out under a microscopy and minced to minimal

pieces. The tissue was then digested in diluted papain solution (Worthington Biochemical Corp, Lakewood, NJ, USA) at a final concentration of 5 mg/mL, and it was constantly shaking at 150 RPM in 37  C for 20 minutes. After stopping digestion with 10% fetal bovine serum, the cells were filtered through a 70-mm cell strainer and washed 3 times using the Neurobasal media. The cells were then plated into either 96-well plates or poly-lysine coated culture dishes at a concentration of 104 cells/mL in Neurobasal media supplemented with 0.5% L-glutamine and 2% B27 serum free supplement. To prepare soluble Ab42 oligomers, human synthetic Ab42 (#A1163-1, rPeptide, Athens, GA, USA) was dissolved in 20-mM ammonium acetate aqueous solution (pH ¼ 8, ionic strength ¼ 0.25 M) at a concentration of 1 mg/mL, lyophilized and stored at 80  C, as recommended by the manufacturer. The pretreated Ab42 was dissolved in culture media at room temperature immediately before adding into the culture. Unless stated otherwise, Ab42 was incubated with the cells overnight (24 hours) before the subsequent assays. Full length PACAP-38 (#1186, Tocris, St Louis, MO) was prepared as a stock aqueous solution at 0.1 mg/ml and diluted freshly in culture media. PACAP 6-38 (Tocris, #3236), the PAC1 receptor antagonist was applied to cultured cell at a concentration of 1 mM. 2.3. Mitochondria isolation and/or enrichment The brain tissue or cultured cells were harvested and homogenized at 4  C in mitochondrial isolation buffer (320 mM sucrose, 1 mM EDTA, and 10 mM Tris-HCl, pH ¼ 7.4). The homogenate was centrifuged 1000 g at 4  C for 10 minutes and the supernatant was collected. The supernatant was then centrifuged 13,000 g at 4  C for 20 minutes. The resulting pellet was resuspended in mitochondrial respiration buffer (110 mM sucrose, 0.5 mM EGTA, 3 mM MgCl2, 40 mM KCl, 10 mM KH2PO4, 20 mM HEPES, 1 g/l BSA) to make mitochondrial sample solution for respiration assay, or resuspended in RIPA buffer and sonicated for protein assay and Western blot. 2.4. ELISA assay PACAP was quantified with the ELISA kit (Cat# MBS160511, MyBioSource Inc, San Diego, CA, USA) according to the manufacture protocol. Briefly, protein samples were loaded and incubated with biotin-labeled PACAP antibody at 37  C for 60 minutes. The plate was washed with the washing buffer for 5 times, followed by incubation with chromogen solution at 37  C for 10 minutes. The reaction was terminated by adding the stop solution. The absorption was measured at Optical Density 450 nm. A standard PACAP-38 peptide sample provided by the manufacturer was diluted in series and quantified in parallel with the human or animal samples as a standard curve. Sample Optical Density value was converted to concentration (ng/mL) based on the standard curve calibration. The concentration was then normalized by total protein as ng PACAP/ mg total protein (ng/mg). 2.5. Immunoblot The antibodies used in these experiments include the rabbit anti-PACAP primary antibody (#sc-25439, Santa Cruz Biotechnology, Dallas, TX, USA), the rabbit anti-Sirt3 primary antibody (#sc-99143, Santa Cruz Biotechnology), mouse anti-voltagedependent anion channel (VDAC) antibody (Ab61273, Abcam Inc, Cambridge, MA, USA), goat anti-VDAC antibody (#sc-32064, Santa Cruz Biotechnology), donkey anti-mouse, anti-rabbit, and anti-goat secondary antibodies (Santa Cruz Biotechnology). Protein sample

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solution was heated 95  C for 5 minutes. Laemmli sample buffer (Biorad, Hercules, CA, USA) containing 5% b-mercaptoethanol (Sigma-Aldrich) was mixed with protein samples with 1:1 (vol/vol) ratio. The samples were loaded at 50-mg total protein per well for electrophoresis on a 10% Mini-Protean precast gel or 4%e12% gradient gel (Biorad) and transferred to a nitrocellular film at 4  C. The film was then incubated with 5% milk. The primary antibody was incubated at 4  C overnight with gentle shaking. The film was washed 3 times the next day and incubated with secondary antibody for 60 minutes, incubated with chemiluminescent substrate (ThermoScientific, Rockford, IL, USA) and analyzed with a UVP imaging system (UVP Inc, Upland, CA, USA).

sealed with mineral oil from the top to create a definite chamber for oxygen consumption. The probe fluorescence was recorded using a TECAN Spectra Fluor Detector immediately. The reaction system was excited at the wavelength of 340 nm to convert triplet oxygen into high-energy singlet oxygen. As the oxygen molecules relax from singlet to triplet, the energy was released and captured by Pdporphyrin. The fluorescence of Pd-porphyrin at 650 nm was quenched. Thus, the fluorescence signal intensity correlates with oxygen consumption. The mitochondrial function kinetics was represented as MitoXpress fluorescence intensity time series, which was fitted with Bolzman logit equation (Equation 1) with 4 parameters.

2.6. Cell viability assay

F ¼ Fmin þ

MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) crystal (Sigma-Aldrich) was prepared in PBS at 5 mg/mL and filtered through sterile filter before use. The cells were incubated with MTT-PBS solution at 37  C for 3.5 hours. The live cells convert MTT into formazan and are stained pink. After taking microphotograph, isopropanol solvent containing 4 mM HCl and 0.1% Nondet P-40 (NP40) was added to dissolve formazan and the plate was gently agitated on a shaker for 15 minutes. Absorption was measured at the wavelength of 590 nm with a reference wavelength of 620 nm. The values were averaged among repeated experiments. The MTT value of control cells (no Ab added) were normalized to 100% and all other MTT values of cells incubated with Ab were presented as a relative percentage of survival. We analyzed the results using 1-way analysis of variance (ANOVA) followed by Tukey post hoc analysis for group-to-group comparison. 2.7. Vector construction and transfection A short sequence of 19 nucleotides targeting Sirt3 location 764 was constructed into OmicsLink short hairpin RNA (shRNA) expression clone (Cat# MSH032833, Genecopoeia, Rockville, MD, USA) to knock down the expression of Sirt3. Exogenous mouse Sirt3 complementary DNA sequence with a mitochondrial targeting sequence was gift from Drs Jin and Westphal (Jin et al., 2009). We subcloned them into Lenti-CMV-GFP vector (Applied Biological Materials Inc, Vancouver, Canada) to over-express the level of Sirt3 and named as Lenti-Sirt3 construct, which was sequenced to verify the right sequence and location. The shRNA expression vector, Lenti-Sirt3 vector, and a control vector were packaged into third generation Lenti-Virus transfection system (UCLA core facility, Los Angeles, CA, USA). After the cortical neurons mature in petri dish for 10e15 days, we add in the transfecting viral particles at a multiplicity of infection of 250. All vectors contain sequence of enhanced Green fluorescence Protein so the effectiveness of transfection can be visualized after 2e5 days. The levels of knocking down and overexpression were confirmed by Western blot. 2.8. Mitochondrial respiration assay We use MitoXpress fluorescence oxygen probe (Luxcel Biosciences, Ireland) to measure the kinetics of mitochondrial respiration. The probe solution was prepared according to the manufacturer’s instruction. Mitochondrial sample solution and MitoXpress probe solution were mixed by 1:1 ratio (vol/vol) and were supplemented by either 10% (vol/vol) mitochondrial complex I substrates (100 mM glutamate and 100 mM malate) or 10% (vol/vol) mitochondrial complex II substrates (100 mM succinate supplemented by 10 mM rotenone). ADP (2 mg) was added to each 0.5 mL substrate solution. After mixing substrates with mitochondrial sample solution and probe solution, the reaction chamber was

Fmax  Fmin T  T0:5 s

1 þ e

A ¼ Fmax  Fmin

(1)

(2)

Fmin is the minimal fluorescence, Fmax is the maximal fluorescence. Amplitude (A) is defined as the difference between Fmax and Fmin, representing the capacity of mitochondrial function (Equation 2). T0.5 is defined as the time to reach 50% amplitude, representing the mitochondrial respiratory threshold or sensitivity. Slope Width (S) reciprocally represents mitochondrial respiratory speed. 2.9. Data analysis For comparing values of 2 groups, we used unpaired t test. For comparing value of multiple groups, we used 1-way ANOVA with post hoc Tukey test, or 2-way ANOVA where we applied 2 independent treatments (used in Fig. 2). For correlation analysis, we used Pearson correlation assay. For mitochondrial assay, we used time series fitting as explained previously. All the results were reported as mean value  standard error. p < 0.05 was accepted as significant. All experiments were repeated at least 3 times. 3. Results 3.1. PACAP expression is reduced in AD brain We examined a total of 28 cases that had been clinicopathologically diagnosed with AD (n ¼ 16) or cognitively normal control (non-AD, n ¼ 12), based on National Institute on Aging-Alzheimer’s Association criteria (1997). Cortical Ab neuritic plaque density (CERAD score) (Mirra et al., 1991) and Braak tangle stage (Braak and Braak, 1991) were determined by a neuropathologist (T.G.B). The average age in the AD group was 83.2  1.6, slightly younger but not significantly different from the non-AD group (88.1  1.8, p ¼ 0.06). PACAP expression was reduced in AD patients compared with nonAD subjects, as determined by an ELISA assay. The amount of PACAP was 0.057  0.003 ng/mg in non-AD group (N ¼ 12) and was reduced to 0.028  0.002 ng/mg in AD group (N ¼ 16, p < 0.001, Fig. 1A). In AD cases, the amount of PACAP was inversely correlated with amyloid plaque burden (CERAD score, Fig. 1B). The Pearson correlation coefficient (r) was 0.5788 (p < 0.05). The amount of PACAP was also inversely related to neurofibrillary tangle (Braak Stage, Fig. 1C). The Braak tangle stages ranged from IV to VI in AD cases. The average PACAP was 0.041  0.003 ng/mg (N ¼ 6) in Braak stage IV AD patients, which was significantly higher than 0.024  0.005 (N ¼ 4) in stage V patients (p < 0.05), and 0.022  0.003 (N ¼ 6) in stage VI patients (p < 0.01, Fig. 1C). This inverse correlation of PACAP with pathologic hallmarks of AD suggests that PACAP is not only reduced in AD but also represents the severity of AD pathology.

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Fig. 1. PACAP expression is reduced in Alzheimer’s disease (AD). (A) ELISA quantification of PACAP in non-AD (n ¼ 12) and AD (n ¼ 16) human postmortem cortex (superior frontal cortex). *** indicate p < 0.001 nonpaired t test. (B) PACAP quantity in AD group was negatively correlated with CERAD plaque score using Pearson correlation assay, r ¼ 0.5788, p < 0.05. (C) AD samples was sub-grouped according to Braak stage scores, PACAP level was compared among the different Braak stage (1-way ANOVA and post hoc Tukey test. * indicates p < 0.05, ** indicate p < 0.01). (D) PACAP immunoblots were demonstrated from 12 non-AD and 16 AD samples. The samples were prepared from frozen brain tissue homogenate. VDAC is a housekeeping protein as an internal reference. (E) ELISA quantification of PACAP from 3TgAD mice and wild type control (n ¼ 4 each group). * indicates p < 0.05 nonpaired t test. Abbreviations: ANOVA, analysis of variance; ELISA, enzyme-linked immunosorbent assay; PACAP, pituitary adenylate cyclase-activating polypeptide; VDAC, voltage-dependent anion channel.

We confirmed the ELISA assay with Western immunoblot (Fig. 1D). The immunoblot was normalized with a conservative housekeeping protein VDAC, to semi-quantify the relative levels of PACAP. Although a small peptide such as PACAP may be

deteriorated by the heat-denaturing process during immunoblotting and hence may compromise the accuracy of the measurement, the immunoblot semi-quantification of PACAP still reasonably represents the ELISA data, where no denaturing procedure was involved (Supplementary Fig. S1B). 3xTG mice showed apparent amyloid plaques and neurofibrillary tangles that mimic human AD pathology (Supplementary Fig. S2) (Billings et al., 2005; Oddo et al., 2003, 2008). In 3xTG mice, PACAP expression in the brain cortex was reduced compared with their wild type controls (Fig. 1E). The amount of PACAP was 0.072  0.004 ng/mg in wild type mice and were reduced to 0.055  0.009 ng/mg in 3xTG mice (N ¼ 4 for each group, p < 0.05, Fig. 1E). PACAP is reduced in both human and murine AD. We tested the possibility that Ab, a key player in AD pathogenesis, may interfere with PACAP expression. In cultured primary neurons, we added Ab42 at a dose range of 0.05e0.5 mM but did not observe a reduction of PACAP (Supplementary Fig. S3). Nontoxic APPa (0.5 mM) did not affect PACAP expression either. Thus, it is more likely that PACAP deficits may cause neuronal vulnerability to toxic milieu in AD. 3.2. PACAP protects against Ab toxicity

Fig. 2. PACAP is protective against Ab42 toxicity in cultured mouse cortical neurons. Ab42 and PACAP were incubated in the cell culture dish for 18 hours before the MTT assay. The MTT assay was performed on a standard 96-well plate. Each treatment condition was average from 6 wells. The MTT assay of untreated cultured cells is normalized to 1 unit (100%). Note that Ab42 (1 mM) significantly killed 60% cells and PACAP (50 nM) effective protects the toxicity. * indicates significant difference (p < 0.05) with 2-way ANOVA. Abbreviations: ANOVA, analysis of variance; PACAP, pituitary adenylate cyclase-activating polypeptide.

Does exogenous PACAP protect against Ab toxicity? We first applied MTT chemiluminescent assay to examine cell survival when primary cortical neurons were exposed to different doses of Ab42 (Fig. 2). Low dose Ab (0.1 mM) did not cause cell death, whereas median (0.5 mM) and high doses (1 mM) Ab reduced cell survival to 78%  5% (p < 0.05, n ¼ 6) and 38%  6% (p < 0.01, n ¼ 6), respectively. We then treated these neurons with PACAP at the time when Ab was added. PACAP protected against Ab induced toxicity in a dose-dependent manner. At 50 nM and 100 nM, PACAP rescued the cells that were incubated with Ab (1 mM) to 79%  5% and 87%  4% (p < 0.05, n ¼ 6), respectively. Based on these data, we used

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1 mM Ab to induce cell toxicity and treated cells with 50 nM PACAP (the minimal effective dose) in all the subsequent experiments unless indicated otherwise. 3.3. Sirt3 pathway mediates neuroprotective effects of PACAP PACAP activates AMPK (Acquaah-Mensah et al., 2012), which in turn enhances Sirt3 (Buler et al., 2012). Thus, PACAP is likely to enhance the expression of Sirt3. First, we investigated if Sirt3 was relevant in AD. We examined the expression of Sirt3 in postmortem human brains by Western blot. The normalized immunoblot intensity was 0.829  0.05 in non-AD (N ¼ 12) and 0.6042  0.04 in AD samples (N ¼ 15, p < 0.01, Fig. 3A). In the 3TG mouse model, Sirt3 expression was reduced to 0.09  0.004, compared with wild type (0.12  0.007, N ¼ 3 in each group, p < 0.05, Fig. 3B). Second, we investigated if there was a direct link between PACAP and Sirt3. Mitochondrial Sirt3 was increased by 40% in cells treated with PACAP (Fig. 4A1 and A4). This upregulation of Sirt3 was blocked by PACAP receptor antagonist PACAP 6-38 (Fig. 4A1 and A4) or AMPK blocker dorsomorphine (DMP) at 10 mM (Fig. 4A3 and A4). Third, we constructed a shRNA sequence to knock down the expression of intrinsic Sirt3 in neuronal cells (Fig. 4B and C). The knock down efficiency was 68% (Fig. 4A2 and A4). In the presence of shRNA, PACAP failed to enhance Sirt3 expression (Fig. 4A1 and A4). The downstream effect of PACAP in enhancing mitochondrial function could be effectively blocked by knocking down Sirt3. Noteworthy, without adding b-amyloid to cultured neurons, knocking down Sirt3 (shRNA) alone did not reduce the total capacity of mitochondrial respiration indicated by its amplitude (Fig. 5A and B), or the sensitivity of the mitochondrial respiration indicated by its threshold T0.5 (Fig. 5C), or the speed of respiration indicated by its slope width (Fig. 5D), suggesting some other mechanism may compensate for the loss of Sirt3 and maintain mitochondrial function compatible with the physiological demand. Fourth, we investigated if mitochondrial function could be enhanced by increasing Sirt3. We effectively introduced exogenous mouse Sirt3 complementary DNA into neuronal cells culture (Fig. 4D and E). When cultured neurons were incubated with Ab (1 mM) overnight, the mitochondrial respiratory capacity (amplitude) was uniformly compromised (Fig. 5EeF). Ab reduced mitochondrial capacity by 77% (Fig. 5F). Over-expressing Sirt3 alone improved the

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mitochondrial capacity by almost 2-folds (Fig. 5F). It was less effective than treatment with PACAP (50 nM) which reversed detrimental effects of Ab and improved mitochondrial capacity close to its baseline (Fig. 5F). PACAP showed no effect on T0.5 or slope width (Fig. 5GeH) despite the change of amplitude, suggesting that PACAP enhance the mitochondrial capacity without hastening the respiratory speed. Finally, knocking down Sirt3 compromised the neuroprotective effect of PACAP against Ab induced cell death (Fig. 5I) but to a similar degree as the PACAP receptor antagonist PACAP 6-38. Comparable to the effects on mitochondrial function, over-expressing Sirt3 increased cell survival to 70% (p < 0.05). 4. Discussion This is the first study showing that PACAP expression is reduced in human AD and it is inversely correlated with Ab plaques and neurofibrillary tangles, the 2 pathologic hallmarks of human AD. PACAP expression is also reduced in a transgenic mouse model of AD. This reduction of PACAP expression in AD is in agreement with the previous report of a down regulation of PACAP messenger RNA in human and animal AD (Wu et al., 2006). Is PACAP reduction a coincidence with AD, a consequence of AD or a contributing factor to AD? One explanation is that the pathogenesis of AD may reduce the expression of PACAP. The reduced PACAP fails to protect the neurons, so that a vicious cycle irreversibly exacerbates AD pathology. However, when we treated primary neuronal cells with a range of different doses of Ab, PACAP levels remained stable (Supplementary Fig. S3). A relative high dose of Ab (1 mM) reduced PACAP expression slightly in a cultured astrocyte cell line (RBA1) (Shieh et al., 2008). This difference between primary neuronal cells and astrocyte cell line may result from their unique sensitivity to Ab toxicity and individual capacity to produce PACAP. Neurons are more vulnerable to toxic damage than immortalized cell lines or glial cells. The second explanation is that PACAP deficit predisposes neurons to AD pathology. It would be ideal to evaluate the role of PACAP in AD pathogenesis by using PACAP knockout mice, but the PACAP knockout mice do not survive long enough to an advanced age to develop AD pathology (Gaszner et al., 2011; Szakaly et al., 2011). Nevertheless, PACAP stimulates nonamyloidogenic processing of APP by enhancing a secretase to increase sAPPa production

Fig. 3. Sirt3 expression is reduced in Alzheimer’s disease (AD). (A) Western blot detection of Sirt3 expression in non-AD (n ¼ 12) and AD (n ¼ 15) cortex tissue. VDAC is internal standard reference protein. Western blot relative intensity of Sirt3 was calculated by normalization with VDAC protein. ** indicate p < 0.01 nonpaired t test. (B) Protein homogenate from enriched mitochondrial preparation from WT (n ¼ 3) and 3TgAD (n ¼ 3) mice were analyzed for Sirt3 using immunoblot assay. The ratio of Sirt3/VDAC is expressed as relative intensity. * indicates significant difference (p < 0.05) with nonpaired t test. Abbreviations: VDAC, voltage-dependent anion channel; WT, wild type.

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Fig. 4. PACAP enhances the expression of mitochondrial Sirt3 in cultured neurons. (A) The cultured neurons were harvested to isolate mitochondria. The mitochondrial fraction was detected with immunoblot. PACAP increase the immunoblot density of mitochondrial Sirt3. (A1) PACAP 6-38 (abbreviated as PAC6-38), an antagonist for PACAP receptor (PAC1 receptor) blocks the effect of PACAP. Cells pre-infected with shRNA knocking down Sirt3 are incapable of upregulating Sirt3 expression by PACAP. (A2) shRNA knocked down Sirt3 expression and over-expressing Sirt3 (Lenti-Sirt3) increase Sirt3 product. The control vector contains scrambled target sequence which did not change the expression level of Sirt3. (A3) The upregulation of Sirt3 was blocked by DMP, a AMPK antagonist. (A4) Sirt3 to VDAC intensity ratio in untreated conditions was normalized to one unit. The intensity ratios from other conditions were compared with the untreated condition. Data were presented as mean  standard error. For untreated condition, n ¼ 13; for vector, shRNA and LentiSirt3, n ¼ 6; for PACAP, n ¼ 7; for PACAP þ PAC6-38, PACAP þ DMP, and PACAP þ shRNA, n ¼ 4. ** indicate p < 0.01, *** indicate p < 0.001 (1-way ANOVA). (B) shRNA targeting sequence. (C) shRNA transfected into cultured neurons via lentiviruses. (D) Construct to overexpress Sirt3 (Lenti-Sirt3). (E) Lenti-Sirt3 transfected into cultured neurons via lentiviruses. Abbreviations: AMPK, adenosine monophosphate-activated protein kinase; ANOVA, analysis of variance; DMP, dorsomorphine; PACAP, pituitary adenylate cyclaseactivating polypeptide; shRNA, short hairpin RNA.

and to reduce sAPPb secretion (Rat et al., 2011). PACAP enhances ChAT expression and maintains cholinergic function (Bourgault et al., 2011; Yuhara et al., 2003). Our experiment has demonstrated that PACAP has a direct protective effect against Ab toxicity in cultured neurons. This is consistent with a previous study using PC12 cell line (Onoue et al., 2002). As an immortalized embryonic

pre-neuronal cell line, PC12 cells seem more acceptable to PACAP protection as a relative low dose (1e10 nM) PACAP effectively protects the cells (Onoue et al., 2002). All these evidence suggests that PACAP deficit is an important contributing factor in AD pathogenesis and exacerbation. Vasoactive intestine peptide, which is highly similar to PACAP in N-terminal sequence, also protects Ab

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toxicity in PC12 cell line but requires a much higher concentration (w104 to 105-fold) to reach the same effects as PACAP (Onoue et al., 2002). Although vasoactive intestine peptide is less protective than PACAP in vitro, it is more effective than PACAP to improve cognitive retardation in ApoE-deficient mice (Gozes et al., 1997a, 1997b). We have shown that PACAP in vitro protects against Ab induced cell death by enhancing Sirt3 expression and improving mitochondrial function. AD is associated with a characteristic and progressive pattern of diminished cerebral glucose metabolism as measured by flourodeoxyglucose positron emission tomography (Reiman, 2011). Diminished cerebral glucose metabolism may begin years before the onset of cognitive symptoms and is correlated with clinical severity (Chen et al., 2010). Evidence supports the notion that mitochondrial dysfunction may be an initiating factor leading to apoptosis which is a common pathologic mechanism for neurodegeneration. Insufficient energy metabolism because of complex I malfunction has been proposed to contribute to tau phosphorylation, a crucial pathologic step in AD (EscobarKhondiker et al., 2007). PACAP has a consistent effect on increasing insulin release and sensitivity (Harmar et al., 2012). In addition, PACAP has a direct protective effect on neurons, likely targeting on intrinsic apoptotic pathway (Vaudry et al., 2004). PACAP inhibits the intrinsic apoptotic pathway, which is initiated by mitochondrial metabolic dysfunction. Thus, enzymes that modulate mitochondrial respiration could be a candidate target, such as Sirt3. Similar to PACAP, Sirt3 expression was decreased in AD. The expression of Sirt3 is enhanced by AMPK and PACAP activates AMPK function. We have shown that exogenous PACAP enhances the expression of Sirt3 in primary neurons. The PACAP-Sirt3 pathway not only improved mitochondrial function but also protected neurons from Ab induced cell death. Knocking down Sirt3 abolished the beneficial effects of PACAP, whereas over-expressing Sirt3 reversed it. Interestingly, knocking down Sirt3 alone is not sufficient to induce cell death or compromise mitochondrial respiration. This is probably because of other compensatory mechanisms that keep the integrity of mitochondria. On the other hand, over-expression of Lenti-Sirt3 could improve mitochondrial respiration to a certain degree but not as efficient as PACAP does. This is likely because of other Sirt3-independent mechanisms of PACAP. To name a few, PACAP blocks extrinsic apoptotic pathway as well, maintain the integrity of synaptic structure, modulate calcium influx, and various ion channel functions (Botia et al., 2007; Bourgault et al., 2009; Dejda et al., 2008; Falluel-Morel et al., 2007; Ravni et al., 2006; Vaudry et al., 2009). In addition, PACAP protects against Ab-induced neurotoxicity in PC12 cells, and this effect was attributed to the inhibition of caspase-3 (Onoue et al., 2002). Taken together, Sirt3 is a crucial downstream step as reducing Sirt3 diminishes the neuroprotective and neurotrophic effects of PACAP. In conclusion, PACAP deficit is associated with AD pathology. Therapeutic strategies to compensate this signaling molecule may improve mitochondrial function and protect neurons from Ab-induced cell death. Disclosure statement The authors have no conflicts of interest to disclose.

Fig. 5. PACAP enhances mitochondrial respiratory function and protects against Ab toxicity through Sirt3. (A) Mitochondrial respiratory kinetics without Ab treatment. (BeD) Mitochondrial respiratory function kinetic (A) was fitted with sigmoid equation (see Section 2). All conditions compared with control. The amplitude represents

mitochondrial function capacity (B), T0.5 represents functional sensitivity (C), and slope width inversely related to respiratory speed (D). (E) Mitochondrial respiratory kinetics with Ab treatment. (FeH) Statistical analysis of curves in (E). All conditions compared with Ab only. (I) The relative survivability of cultured neurons in multiple conditions. The MTT assay value from no-treatment cells was normalized to one unit (100%). * indicates p < 0.05, ** indicate p < 0.01 (1-way ANOVA with post hoc Tukey analysis). Abbreviations: Ab, b-amyloid; ANOVA, analysis of variance; PACAP, pituitary adenylate cyclase-activating polypeptide.

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