research article ap39, a mitochondria-targeted hydrogen...

Research ArticleAP39 a Mitochondria-Targeted Hydrogen Sulfide DonorSupports Cellular Bioenergetics and Protects againstAlzheimerrsquos Disease by Preserving Mitochondrial Function inAPPPS1 Mice and Neurons

Feng-li Zhao1 Fang Fang1 Pei-feng Qiao1 Ning Yan2 Dan Gao1 and Yong Yan1

1Experimental Research Center Department of Neurology The First Affiliated Hospital of Chongqing Medical UniversityChongqing Medical University Chongqing 400016 China2Department of Neurology The University-Town Hospital of Chongqing Medical University Chongqing 400016 China

Correspondence should be addressed to Yong Yan yanyongzflh163com

Received 10 November 2015 Revised 11 December 2015 Accepted 15 December 2015

Academic Editor David Sebastian

Copyright copy 2016 Feng-li Zhao et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Increasing evidence suggests that mitochondrial functions are altered in AD and play an important role in AD pathogenesis Ithas been established that H

2S homeostasis is balanced in AD The emerging mitochondrial roles of H

2S include antioxidation

antiapoptosis and the modulation of cellular bioenergetics Here using primary neurons from the well-characterized APPPS1transgenic mouse model we studied the effects of AP39 (a newly synthesized mitochondrially targeted H

2S donor) on

mitochondrial function AP39 increased intracellular H2S levels mainly in mitochondrial regions AP39 exerted dose-dependent

effects on mitochondrial activity in APPPS1 neurons including increased cellular bioenergy metabolism and cell viability at lowconcentrations (25ndash100 nM) and decreased energy production and cell viability at a high concentration (250 nM) FurthermoreAP39 (100 nM) increasedATP levels protectedmitochondrial DNA and decreased ROS generation AP39 regulatedmitochondrialdynamics shifting from fission toward fusion After 6 weeks AP39 administration to APPPS1 mice significantly ameliorated theirspatial memory deficits in the Morris water maze and NORT and reduced A120573 deposition in their brains Additionally AP39inhibited brain atrophy in APPPS1 mice Based on these results AP39 was proposed as a promising drug candidate for ADtreatment and its anti-AD mechanism may involve protection against mitochondrial damage

1 Introduction

Alzheimerrsquos disease (AD) is the most universal age-relatedneurodegenerative disease and form of dementia affectingapproximately 53 million American people Of those 51million are over the age of 65 years [1] AD initially causesmemory impairment and as the disease progresses patientsexhibit motor aberrancies personality changes languagedeficiencies and other neuropsychiatric symptoms BecauseAD is a complex and multifaceted disease involving variousmechanisms including energy metabolism [2] inflamma-tion and abnormal cell cycle control [3] the currently avail-able treatments include a combination treatment regimen tar-geting two ormore aspects of AD pathology [4] For example

drug treatment forAD is primarily based on the acetylcholinehypothesis or the amyloid-120573 (A120573) accumulation hypothesisbut none of these drugs can stop or reverse the progression ofAD

Swerdlow first proposed the ldquoAD mitochondrial cascadehypothesisrdquo in 2004 [5] Recently increasing evidence hasshown that mitochondrial dysfunction is a prominent factorin AD pathogenesis [6] as either a cause or a consequence ofA120573 toxicity Severe structural and functional abnormalities ofthe mitochondria were observed in the immediate vicinity ofA120573 plaques [7] Moreover A120573 gradually accumulates withinthe mitochondria of both living mouse models of AD andhuman AD brain sections and A120573 has been found to directlyinteract with several mitochondrial proteins for example

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2016 Article ID 8360738 19 pageshttpdxdoiorg10115520168360738

2 Oxidative Medicine and Cellular Longevity

cyclophilin D (CypD) amyloid-120573 binding alcohol (ABAD)dehydrogenase and dynamin-related protein 1 (Drp1) [8]These interactions impair the physiological functions ofmitochondria resulting in limited electron transfer abnor-mal adenosine 51015840-triphosphate (ATP) production increasedproduction of reactive oxygen species (ROS) and alteredmitochondrial morphology and mobility in AD transgenicmodels [9 10] Furthermore a growing body of research sug-gests that mitochondrial biogenesis is considerably decreasedin the brains of both AD patients and AD model mice [911ndash13] Impaired mitochondrial biogenesis is conducive tomitochondrial dysfunction in AD [13]

Hydrogen sulfide (H2S) has recently been recognized as

an endogenous gaseous mediator that plays multiple regula-tory roles in humans and mammals [14] Increasing evidencehas demonstrated that the dysregulation of H

2S homeostasis

is implicated in the pathological processes of AD Recentlyour experimental research has revealed that the level of H

2S is

decreased in AD patients and that this change in the H2S lev-

els may be related to the severity of AD The emerging mito-chondrial roles ofH

2S include antioxidant antiapoptotic and

anti-inflammatory effects [15] Tang revealed that H2S mod-

ulates A120573-induced damage to PC12 cells by ameliorating thedecrease in the mitochondrial membrane potential (MMPΔΨm) and attenuating the increase in intracellular ROS levels[16] Moreover Xuan et al found that H

2S may attenuate

spatial memory impairment and neuroinflammation withinthe hippocampus of AD model mice [17] With regard tothe regulatory role of H

2S in cellular bioenergy metabolism

recent studies have shown that H2S can serve as a physiologi-

cal electron donor and as an inorganic energy source inmam-malian cells Recently Modis et al reported that an endoge-nous intramitochondrial H

2S-producing pathway which is

governed by 3-mercaptopyruvate sulfurtransferase (3-MST)complements and balances the bioenergetic activity of Krebscycle-derived electron donors in the Hepa1c1c7 cell line [18]

Taken together these findings indicate that the potentialeffects of H

2S on mitochondrial dysfunction in AD models

are significant Therefore to clarify whether the new H2S

donor AP39 which contains the mitochondria-targetingcompound triphenylphosphonium (TPP+) coupled to anH2S-donating moiety (dithiolethione) via an aliphatic linker

regulates mitochondrial function and cellular bioenergeticsis of great interest Using APPPS1 neurons and mice we firstdetected the overall viability of the neurons and evaluated theeffects of AP39 on cellular bioenergetics and mitochondrialfunction In addition we examined the effects of AP39 onspatial memory deficits and on magnetic resonance imaging(MRI) characteristics of the mouse brains

2 Materials and Methods

21 Materials AP39 a novel mitochondria-targeted H2S

donor was designed and synthesized by Medicilon Inc asdescribed previously [19 20] Antimycin A 025 trypsin2-deoxyglucose oligomycin carbonyl cyanide-4-(trifluoro-methoxy)phenylhydrazone (FCCP) trichloroacetic acidrotenone 7-azido-4-methylcoumarin (AzMC) monobasicpotassium phosphate dibasic sodium succinate hexahydrate

adenosine 51015840-triphosphate (ATP) disodium salt hydrate fattyacid-free BSA sodium hydrosulfide hydrate (NaSHsdot119909H

2O)

NN-diethyl-p-phenylenediamine sulfate magnesium chlo-ride and iron(III) chloride (FeCl

3) were purchased from

Sigma-Aldrich Company The monoclonal anti-A120573 antibody6E10 was obtained from Covance Inc (Princeton NJ USA)Drp1 was purchased from Novus Biologicals Inc Fis1 waspurchased from Proteintech Group Mfn1 and Mfn2 anti-bodies were purchased from Santa Cruz Biotech andOPA-1 antibody was purchased from BD Transduction Lab-oratories The secondary antibodies goat anti-mouse-HRPand donkey anti-rabbit-HRP antibodies were purchasedfrom GE Healthcare The Pierce BCA protein assay kit waspurchased from Thermo Fisher Scientific (Waltham MAUSA) Dulbeccorsquos modified Eaglersquos medium (DMEM) 1glutamine and 1 penicillin were obtained from InvitrogenAll other chemicals were purchased from Amresco (SolonOH USA)

22 Animals and Treatments This experiment was per-formed as described previously [21] All animal protocolswere approved by the Animal Research Committee of ChongQing Medical University Heterozygous APPPS1 double-transgenic mice (APPswe-PS1dE9) were crossed with non-transgenic female mice (the Model Animal Research Centerof Nanjing University Jiangsu China) to produce hemizy-gous transgenic mice and nontransgenic littermates In thedouble-transgenic mice that coexpressed the Swedish muta-tion of APP (APPswe) and two FAD-PS1 variants A120573 plaquesaccumulated in the brain and learning and memory abilitydeclined over time [22] The mice were housed at 23ndash25∘Cwith 60 humidity under 12 12 h light-dark cycles and wereprovided with free access to food and water throughout theexperiment Pregnant females were sacrificed at gestationaldays 15-16 (E15-16) and the embryos were removed for thepreparation of brain neuronal cultures The genotyping forAPP and PS1 was performed according to the literatureto assign cultures to transgenic and nontransgenic groupsaccording to the PCR results with genomic DNA For theMorris water maze test and the novel object recognition task(NORT) these 12-month-old mice were divided into fourgroups of fifteen mice in each group wild-type (WT) micetreated with deionized water or 100 nMkg AP39 and ADmodel mice treated with deionized water or 100 nMkg AP39Themice were treated once daily via intraperitoneal injectionfor 6 weeks prior to the experiments After the behavioraltests the mice were scanned by MRI and then sacrificedfor the collection of their blood and brains The bloodwas collected into heparinized tubes and then centrifugedat 1000 g for 10min at 4∘C The plasma (supernatant) wascollected to detect A120573

40and A120573

42by ELISA The brains

were removed and collected to examine A120573 deposition byimmunohistochemistry

23 Primary Neuron Culture Primary cultures of corti-cal neurons were generated from the brains of individualembryos at E15-16 Briefly the brains were removed andthen placed in Hankrsquos balanced salt solution at 4∘C Thecortex was dissected from the brain chopped and digested

Oxidative Medicine and Cellular Longevity 3

in 025 trypsin for 16min at 37∘C with gentle shakingFor the mixed neuronal cultures after cell counting thedissociated cells were plated at a density of 2times 105 cellscm2 ina 35mm dish on poly-d-lysine-coated cover slips in DMEMcontaining 10 F-12 and 10 fetal bovine serum (FBS) andwere maintained at 37∘C in a humidified atmosphere of95 air and 5 CO

2 After culturing for 24 h in vitro the

medium was replaced with serum-free neurobasal mediumcontaining 2 B27 serum-free supplement 1 glutamineand 1penicillinThereafter half of themediumwas replacedwith neurobasal medium every three days Pure neuronalcultures were supplemented with glia-conditioned medium(GCM) These neurons were prepared in a similar mannerexcept that cytosine arabinoside (5 120583M) was added to theculture media at DIV 4 to block the proliferation of gliaand the cultures were maintained in GCM After 16 days theneurons were treated with different concentrations of AP39diluted in the appropriate medium for 24 h

24 Measurement of H2S Production

241 H2S Production in Neurons and Brain Tissue (Methy-lene Blue Assay) This experiment was performed based onprevious literature [23 24] Neurons were scraped collectedand then washed 3 times with phosphate-buffered saline(PBS) The neurons were suspended in ice-cold 50mM Tris-HCl buffer and homogenized by sonication Homogenizedsamples were centrifuged at 10000 g for 10min at 4∘C andthe supernatants were collected as cell lysates The proteinconcentrations of the supernatants were determined usingthe Quick Start Protein Assay Kit

We used spectrophotometric measurements based onthe formation of methylene blue by H

2S to detect H

2S

production We performed all reactions in duplicate Celllysates (310 120583L 1mgmL) in 15-mL tubes were mixedwith trichloroacetic acid (20wv 60 120583L) zinc acetate(2wv 30 120583L) and N N-dimethyl-p-phenylenediaminesulfate (NNDPD) (20mM 40 120583L) in 72M HCl and FeCl

3

(30mM 30 120583L) in 12M HCl The optical absorbance of theresulting solution (670 nm)wasmeasured after 15min using a96-well microplate reader (Tecan Systems Inc Switzerland)H2S was calculated against a calibration curve of NaHSTo explore the effect of AP39 on the generation of H

2S

in mice these 12-month-old WT or APPPS1 mice weretreated with different concentrations of AP39 (25 nMkgndash250 nMkg) The mice were treated once daily via intraperi-toneal injection for 6 weeks prior to determining H

2S con-

centration In brief mice from each group were anesthetizedwith chloral hydrate and euthanized by decapitation Tissuesfrom the hippocampus and cerebral cortex were immedi-ately removed and then homogenized in ice-cold 50mMpotassium phosphate buffer (12 wtvol pH 80) with aPolytron homogenizerThe homogenates were centrifuged at47000timesg for 10min at 4∘C and supernatants were collectedThen the H

2S production was measured as above described

via methylene blue assay

242 H2S Detection in Mitochondria Neurons were seededat a density of 5 times 104 cellswell in a Lab-Tek II chamber

coverglass system and were maintained at 37∘C in a humidi-fied atmosphere of 95 air and 5 CO

2 The H

2S-sensitive

fluorescent dye AzMC was incorporated into a cell-basedassay to detect H

2S production [19 25] The neurons were

loaded with the fluorogenic dyes 10 120583M AzMC and 200 nMMitoTracker Red CMXRos (Invitrogen) at 37∘C for 30minDifferent concentrations of AP39 were added to fresh mediaand the neurons were further incubated for 2 h after whichH2S imaging was performed After washing 3 times with PBS

the specific fluorescence from the various dyes was examinedusing a confocal laser scanning microscope

25 Bioenergetic Analysis in Neurons We used the XF24Extracellular Flux Analyzer (Seahorse Bioscience BillericaMA USA) to measure cellular bioenergetic function aspreviously described [26] Neurons were treated with dif-ferent concentrations of AP39 in medium Neurons wereseeded at the optimal density of 35 times 104 cells per welland were maintained at 37∘C in a humidified atmosphereof 95 air and 5 CO

2 Next the indices of mitochondrial

function were measured according to the following protocolAfter recording the basal OCR and PPR levels oligomycin(15 120583gmL) was used to assess the mitochondrial ATP pro-duction rate and FCCP (05 120583M) was used to assess themaximal mitochondrial respiratory capacity (a parameterthat characterizes overall mitochondrial function in neurons)via the measurement of the oxygen consumption rate (OCR)Finally antimycin A (2120583gmL) and rotenone (2120583M) wereused to inhibit the flux of electrons through complexes IIIand I and to detect the residual nonmitochondrial OCRwhich is considered to be mediated by cytosolic oxidaseenzymes Bioenergetic parameters were normalized to theneuron count as the same number of neurons was seeded ineach well

26 MTT Assay Cell viability was determined by the MTTreduction assay method as follows The neurons were cul-tured and treated with different concentration of AP39 for24 h in 96-well platesMTTwas added to eachwell with a finalconcentration of 05mgmL for 4 h After a 4-h incubationat 37∘C the MTT solution was removed and the insolubleformazan crystal was dissolved in DMSO The absorbanceof the colored solution was measured at 570 nm using amicroplate reader (Tecan USA)

27 LDH Assay Lactate dehydrogenase (LDH) release anindirect measurement of cell death was determined using acytotoxicity assay according to the manufacturerrsquos instruc-tions Briefly 30 120583L of supernatant was saved before theaddition of MTT and mixed with 100 120583L of freshly preparedLDH assay reagent LDH release into the culturemediumwasdetected using a colorimetric reaction reading of absorbanceat 490 nm according to manufacturerrsquos protocol for the LDHassay kit (Beyotime Jiangsu China) LDH activity values areshown as 119881max for kinetic assays in mODmin

28 Cellular ATP Measurements Neurons were plated at adensity of 05 times 104 cellswell in 96-well plates before theexperiment WT and APPPS1 neurons were treated with


water or 100 nMAP39 and the samplesweremeasured in trip-licate After incubation for 24 h the ATP levels were assessedusing the ATP Bioluminescent Assay Kit (Sigma-Aldrich)The fluorescence intensity which was linearly related tothe ATP concentration was measured using a microplateluminometer [27]

29 Measurement of Mitochondrial and Nuclear DNAIntegrity DNA integrity was assessed using gene-specificsemiquantitative PCR assays as described previously [28]Briefly total DNA from experimental neurons was isolatedusing the DNase Blood and Tissue Kit Quantification of aPCR-amplified 9-kb nuclear-specific DNA fragment usingPicoGreen fluorescent dye to measure double-stranded DNAwas used to estimate damage to nuclear DNA Quantificationof a PCR-amplified 10-kb mitochondrial-specific DNAfragment using PicoGreen dye was used to detect damage tomitochondrial DNA The obtained data were normalized bythe PCR amplification of a 117-bp mitochondrial genome-specific fragment to correct for multiple copies of themitochondrial genome Preliminary assays were performedto ensure the linearity of PCR amplification with respect tothe number of cycles and DNA concentration

210 Measurement of Intracellular ROS The fluorescent dye21015840 71015840-dichlorofluorescein diacetate (DCFH-DA) was usedto assess the intracellular production of ROS accordingto the manufacturerrsquos instructions Following the indicatedtreatment the neurons were harvested washed twice withPBS and centrifuged at 1000 g for 10minThen the cells wereresuspended in 10 120583MDCFH-DA (Beyotime Jiangsu China)solution and incubated in a CO

2incubator at 37∘C for 30min

in the dark followed by three washes with PBS Finally thecells were resuspended in 05mL of PBS and the fluorescenceintensity of the samples was analyzed by flow cytometry

211 Western Blot Approximately 1 times 107 neurons werecollected for each experiment The cell samples were lysedwith Western and IP lysis buffer (Beyotime Jiangsu China)and centrifuged at 12000 g for 15min The protein con-centrations were determined using the BCA protein assaykit A total of 20120583g protein was loaded per lane andsubjected to electrophoretic separation in a 10 SDS-PAGEgel After separation the proteinswere electrically transferredto a nitrocellulose transfer membrane (PerkinElmer LifeSciences) for 1 h After blocking with 5 nonfat milk in Tris-buffered saline containing Tween-20 (TBST) themembraneswere incubated in primary antibodies against Mitofusin-1(Mfn-1 1 200 rabbit polyclonal)Mitofusin-1 (Mfn-2 1 200rabbit polyclonal) Optic Atrophy-1 (OPA-1 1 400 mousemonoclonal) Fission-1 (Fis-1 1 1500 rabbit polyclonal)Drp-1 (1 200 rabbit polyclonal) or VDAC (1 500 to 1 1000)overnight at 4∘C The membranes were subsequently incu-bated in donkey anti-rabbit or goat anti-mouse antibodiesfor 1 h at 37∘C Chemiluminescent detection was performedusing the Pierce ECL Western blotting substrate and thesignals were analyzed using Quantity One software

212 Behavioral Testing

2121 Morris Water Maze Test The Morris water maze testwas performed according to the protocols of a previousstudy [29] Animals were randomly assigned to differenttreatment groups and then were pretreated with once-dailyadministration of either water or AP39 for 6 weeks prior totesting Briefly the mice were put into each quadrant in arandom order to locate a hidden platform within 1min andthe experimenter would guide them to the platform if themice failed to find the platform Then the mice were left onthe platform for 30 s to help them remember the platformlocation The mice were trained to locate the platform usinga two-trial-per-day regimen and the training was dividedinto two blocks of four trials The first block of four trialswas performed in the morning and the second block of fourtrials was performed in the afternoon In the probe trial theplatformwas removed and the mice were placed in the waterfor a single 60-s period The latency to reach the platformwas recorded for both the training and probe trials and theparameters were analyzed using an EthoVision 31 analysissystem by an observer who was blinded to the treatmentstatus of the mice

2122 Novel Object Recognition Task (NORT) The novelobject recognition task (NORT) which is based on thenatural instinct of rodents to interactmorewith a novel objectthan a familiar object was performed to assess the memoryof the experimental mice as described in the literature[30] Briefly on the first day of acclimation each mousewas provided with 10min to interact with either object forcombined 30 s The total distance traveled was recorded andanalyzed to evaluate the motor ability of the mice On thefollowing day the mice were exposed to two identical objects(A1 and A2) for 10min for familiarization Both objects A1and A2 had identical textures colors and sizes and werepositioned in two adjacent corners from the walls After a 24-h delay in the mice cages the mice were again placed into thesame arena used before for 10min and then exposed to thefield for 5min in the presence of the old familiar (A1) and anew different novel (B) object We determined the amountof time spent at both familiar and novel objects throughvideo analysis using a ldquowithin object areardquo scoring methodA mouse was scored as interacting with the object when itsnose was in contact with the object or directed at the objectwithin le 2 cm Time spent standing sitting or leaning onthe secured object was not scoredThe exploratory preferencewas defined as the percentage of total time that the animalspent investigating the novel object and calculated for eachanimal by the following ratio TB(TA + TB) times 100 [TAtime spent exploring the familiar object A TB time spentexploring the novel object B]

213 Mouse Brain MRI The mice were scanned by MRIafter treatment with water or AP39 for 6 weeks as describedpreviously in the literature [31] The MRI experiment wasconducted in Da Ping Hospital Research Institute of SurgeryThird Military Medical University China The mice wereanesthetized with 10 chloral hydrate-saline and then placed


in the prone position Their chests were connected to a lifesignal detector to monitor their physiological status Theheads of the mice were scanned using a 70 T ultrahigh fieldanimal MRI scanner (70 T ClinScan system Bruker BioSpinEttlingenGermany) T2-weighted images of themouse headswere acquired from the sagittal plane axial direction andcoronal plane according to the following conditions TR =3200ms TE = 45ms field of view (FOV) = 22 times 22mmslice thickness = 05mm a scan matrix = 384 times 384 andrepetitions (NEX) = 5

214 ELISA for A12057340 and A12057342 A commercially availableELISA kit for A120573

40and A120573

42was purchased from Invitrogen

The plasma from samples was collected by centrifugation(1000 g at 4∘C for 10min) and 100 120583L of plasma was used forA12057340and A120573

42measurementThe secreted levels of A120573

40and

A12057342in blood were quantitatively measured according to the

manufacturerrsquos instructions

215 Immunohistochemistry After the behavioral teststhe mice were deeply anaesthetized with chloral hydrate(400mgkg body weight) and sacrificed for the preparationof brain slices [32] The brains were removed from theskull postfixed overnight in 4 paraformaldehyde at 4∘Cand transferred to 30 sucrose solution for dehydrationSubsequently the brains were frozen and sliced into 30-mmthick sections using a Leica CM3050 S cryostat Aftermultiple washes in PBS the slices were incubated with a6E10 primary antibody (1 1000 diluted) in 10 goat serumovernight at 4∘CThe tissue sections were washed 3 times for10min in PBS and then incubated with a TRITC-conjugatedgoat anti-mouse IgG antibody at room temperature for 1 hfollowed by PBS washing The slides were subsequentlystained with diaminobenzidine and counterstained withhematoxylin The images were acquired using a 10x objectiveon a Carl Zeiss LSM780 confocal microscope (OlympusBX60 Tokyo Japan) The A120573 plaque areas were quantifiedusing Image-Pro Plus 60 software

216 Statistical Analysis All statistical analyses were per-formed using SPSS 220 software All data were presented asthe means plusmn SEM The data in the Morris water maze wereanalyzed using two-way ANOVA coupled with a post hocFisherrsquos LSD test usingGraphPadPrism60 softwareThedatafrom other experiments were analyzed by one-way ANOVAcoupled with Dunnettrsquos posttest using GraphPad Prism 60software and 119875 le 005 was considered to be statisticallysignificant

3 Results

31 AP39 Increased the Generation of H2S in Neurons andMitochondria H

2S levels were comparable between WT

neurons treated with different concentrations of AP39 for2 h AP39 (25ndash250 nM) induced a concentration-dependentincrease in H

2S generation and in the fluorescence of the

H2S-detecting dye AzMC and the signal was significantly

colocalized with the mitochondria (Figures 1(a) and 1(b))Notably baseline fluorescent signal was detectable in the

neurons In this study the low basal levels of H2S did not

indicate a mitochondrial preference (Figure 1(b)) In conclu-sion the data in Figure 1 indicate that AP39 contributes to thesynthesis of H

2S in neurons especially in the mitochondria

32 Biphasic Effects of AP39 on Cellular Bioenergetics Similarto the responses previously noted for authentic H

2S [25 33

34] exposure of WT neurons to AP39 induced a significantincrease in the basal OCR at 100 nM but induced a decreasein the basal OCR at 250 nM (Figure 2(a)) Additionally AP39caused a dose-dependent increase in the FCCP-stimulatedOCR at 25 and 100 nM but caused a decrease in the FCCP-stimulated OCR at 250 nM (Figure 2(b))The FCCP-inducedincrease in the OCR represents the ldquomaximal respirationrdquoThus AP39 acts as a supplemental bioenergetic stimulatorat low concentrations The pattern of the effects of AP39is consistent with the previously established bell-shapedpharmacological properties of H

2S [19 35] These results

demonstrated that this inhibitory effect is only conspicuousat higher concentrations (Figure 2(b))

33 AP39 Exerted Cytoprotective Effects on APPPS1 NeuronsWe cultured cortical neurons from the brains of embryonicAPPPS1 transgenic mice as an experimental model to studythe effects of AP39 We detected the levels and time course ofthe release of A120573

42from these neurons into the culture media

by ELISA Figure 3(a) shows that at 3 DIV a small amountof A120573

42was released into the media of neurons cultured

fromAPPPS1mice but significant differences were observedbetween neurons cultured from APPPS1 mice and their WTlittermates (161plusmn69 and 59plusmn27 pgmL resp) By 7DIV thelevel of A120573

42produced in the cultures from theAPPPS1mice

was significantly higher than that produced in the culturesfrom the WT mice (2857 plusmn 386 versus 62 plusmn 07 pgmL 119875 lt001) and this level was further increased at 16 DIV (8983 plusmn273 versus 68 plusmn 05 pgmL 119875 lt 001) The total proteinfrom WT and APPPS1 neurons was extracted separatelyand analyzed by Western blot We observed that A120573 wasoverexpressed in the APPPS1 primary neurons compared tothe WT neurons (Figures 3(b) and 3(c))

Exposure of WT neurons to AP39 for 24 h had no effecton cell viability but treating the APPPS1 neurons with AP39(25100 nM) for 24 h resulted in increase in cell viability(Figures 3(d) and 3(e)) To confirm this result we measuredthe release of LDH in the supernatant of the primary neuronsAP39 had no effect on basal LDH release inWT neurons butthere was a significant increase in the amount of LDH in theAPPPS1 neuron culture mediumThis result indicated a lossof cell membrane integrity and an increase in cell necrosis Incontrast after treatment with AP39 particularly at 100 nMthe amount of LDH in the APPPS1 neuron culture mediumwas decreased (Figures 3(f) and 3(g))

34 AP39 Attenuated the Loss of Cellular Bioenergetics andProtected Mitochondrial Function in APPPS1 Neurons Toinvestigate the effects of AP39 on mitochondrial dysfunc-tion we measured cellular bioenergetics using the XF24Extracellular Flux Analyzer Cellular bioenergetic parameterswere significantly decreased in the mitochondria of APPPS1


0

5

10

15

20

AP39 minus 10025 50

H2S

prod

uctio

n (n

mol

mg

min

)

250(nM)

lowastlowast

lowastlowastlowast

lowast

(a)

AP39 Nuclei Mitochondria AzMC Merge

minus

50nM

100nM

(b)

Figure 1 AP39 generates H2S in WT neurons primarily in the mitochondria (a) The contribution of AP39 to H

2S production in neurons

Neurons fromWTmice were treated with various concentrations of AP39 for 2 h and H2S production was detected by methylene blue assay

(b) Neurons were treated with different concentrations of AP39 for 2 h and intracellular H2S was detected using the fluorescent probe AzMC

DAPI was used to stain nuclei and MitoTracker was used to stain mitochondria The colocalization of H2S with mitochondria was indicated

by the overlapping of red (mitochondria) and green (H2S) fluorescence in the merged image Note the concentration-dependent increase in

the H2S signal in response to AP39 treatment lowast119875 lt 005 lowastlowast119875 lt 001 compared with control treatment (no AP39)


0

20

40

60

80

100

120

Control

Oligo FCCP AA

Basalrespiration

Proton leak

ATPproduction

Maximalrespiration

capacity

ADP

(min)0 10 20 30 40 50

OCR

(pm

olm

in120583

g pr

otei

n)

250nM AP39100nM AP39

25nM AP39(a)

0

30

60

90

120

Control

Basalrespiration

Proton leakATPproduction

Maximalrespiration

OCR

(pm

olm

in120583

g pr

otei

n)


25nM AP39

lowastlowast

lowast

(b)

Figure 2 Biphasic effects of AP39 on the cellular bioenergetics of the WT neurons Neurons from WT mice were incubated in AP39 (25-250 nM) for 2 h and bioenergetic parameters were measured using an Extracellular Flux Analyzer (a) Representative tracings (b) Thecalculated bioenergetic parameters and indicate a significant enhancement in the bioenergetic parameter compared to the controlgroup (no AP39) (119875 lt 005 and 119875 lt 001 resp) lowast indicates a significant reduction in the bioenergetic parameter compared to the controlgroup (119875 lt 005)

neurons compared with those of WT neurons ImportantlyAP39 (100 nM) significantly increased the basal respiratoryrate and the OCR-linked maximal respiratory capacity of theAPPPS1 neurons (Figure 4) These data indicated that AP39may attenuate the loss of cellular bioenergetics in APPPS1neurons

ATP production mitochondrial DNA (mtDNA)integrity and ROS production were measured to evaluatemitochondrial function AP39 significantly increased theATP production in WT and APPPS1 neurons (Figure 5(a))Next mtDNA and nuclear genomic DNA integrity wasassessed via PCR of long DNA fragments We found thatmtDNA but not nuclear DNA integrity was clearly reducedin APPPS1 neurons compared to WT neurons HoweverAP39 significantly protected against mtDNA damage inAPPPS1 neurons by partially restoring mtDNA integrity(Figure 5(b))

Intracellular ROS levels are a basic indicator of oxidativestress A fluorescence assay using a DCFH-DA probe wasperformed to quantify the generation of ROS AP39 (100 nM)effectively decreased ROS levels in APPPS1 neurons (Figures5(c) and 5(d)) In conclusion these results suggested thatAP39 protects againstmitochondrial dysfunction in APPPS1neurons

35 AP39 Shifted the Mitochondrial Dynamics toward Fissionin APPPS1 Neurons The changes in the levels of pro-teins involved in mitochondrial dynamics (Drp1 Fis1 Mfn1Mfn2 and OPA1) were determined via Western blot analysis(Figure 6) The levels of these mitochondrial dynamics-related proteins were shifted toward fission in APPPS1neuronsThe levels of themitochondrial fusion proteinsMfn1

and OPA1 were significantly reduced and the level of themitochondrial fission protein Fis1 was markedly increased inAPPPS1 neurons compared with WT neurons The proteinlevels of Mfn2 and Drp1 were not significantly differentbetween APPPS1 and WT neurons OPA1 Mfn1 and Mfn2catalyze mitochondrial fusion AP39 increased the levels ofOPA1 andMfn1 but notMfn2 Moreover AP39 decreased thelevels of Fis1 but notDrp1These findings suggested thatAP39may shift mitochondrial dynamics in APPPS1 neurons fromfission towards fusion

36 AP39 Increased the Generation of H2S in WT andAPPPS1 Mice AP39 (25ndash250 nM) induced a dose-dependent increase in H

2S generation in the cortex and

hippocampus of WT and APPPS1 mice (Figures 7(a) and7(b)) We also observed that H

2S levels in the cortex were

lower than those of the hippocampus However the level ofH2S was significantly decreased in APPPS1 mice compared

to WT mice In conclusion the data indicate that AP39contributes to the synthesis of H

2S in WT and APPPS1

mice

37 AP39 Reversed theMemory Deficits of APPPS1 TransgenicMice TheAPPPS1 transgenicmice developed a pronouncedspatial learning and memory deficit and produced A120573plaques by 12months of age [22] To test the ability of AP39 toameliorate the spatial learning deficits of the ADmodel micewe treated 12-month-old AD model mice and WT controlswith 100 nMkgAP39 Then behavioral testing was initiated6 weeks after the initiation of AP39 therapy using the Morriswater maze and the NORT


0

200

400

600

800

1000

WT neurons

3 DIV 7 DIV 16 DIV

APPPS1 neuronslowast

lowast

A120573

1minus42

rele

ase (

pgm

L m

ediu

m)

(a)

WT neurons APPPS1 neurons

A120573

Actin

(b)

WT APPPS10

1

2

3

4

5

6

7

Rela

tive A

120573ex

pres

sion

( o

f

cont

rol l

evel)

(c)

0

50

100

150

MTT

redu

ctio

n (

of c

ontro

l)

AP39 minus 10025 250(d)

0

50

100

150

MTT

redu

ctio

n (

of c

ontro

l)

AP39 minus minus

APPPS1WT

lowast

lowastlowast

10025 250

(e)

0

50

100

150

LDH

rele

ase (

o

f con

trol)

AP39 minus 10025 250

lowast lowast

(f)

0

50

100

150

200

LDH

rele

ase (

o

f con

trol)

APPPS1WTAP39 minus minus 10025 250

lowastlowast

(g)

Figure 3 Cytoprotective effects of AP39 in APPPS1 neurons (a) A time course shows that increasing levels of A12057342are released into the

culture media of neurons from transgenic mice but not their WT littermates (b) A120573 was overexpressed in neurons from APPPS1 micecompared to A120573 expression in neurons from theirWT littermates (c) Representative of A120573 blots is shown with quantification (d)The effectsof AP39 (25ndash250 nM) treatment for 24 h on cell viability in WT neurons AP39 alone did not affect MTT conversion (e) The effects of AP39(25ndash250 nM) onMTT conversion inAPPPS1 neuronsTherewas a decrease inMTT conversion in theAPPPS1 neurons compared to theWTneurons these effects were attenuated by AP39 (f) After treatment ofWT neurons with AP39 for 24 h AP39 alone did not affect LDH releasein the cellular medium (g) The effects of AP39 on LDH release from APPPS1 neurons lowast119875 lt 005 versus the APPPS1 group 119875 lt 005 theAPPPS1 group versus the WT group


OCR

(pm

olm

in120583

g pr

otei

n)

0 10 20 30 40 500

30

60

90

120

Oligo FCCP AA

Basalrespiration

Proton leak

ATPproduction

Maximalrespiration

capacity

ADP

AD + 100nM AP39AD + H2O

WT + 100nM AP39WT + H2O

(a)O

CR (p

mol

min

120583g

prot

ein)

Basalrespiration


Maximalrespiration

0

50

100

150



lowast

lowast

lowast

lowast

lowast

lowast

(b)

Figure 4 Protective effects of AP39 on the cellular bioenergetics of APPPS1 neurons APPPS1 neurons were incubated in AP39 (100 nM)for 24 h and bioenergetic parameters were measured using the Extracellular Flux Analyzer (a) Representative tracings are shown (b) Thecalculated bioenergetic parameters are shown lowast indicates a significant enhancement of the bioenergetic parameter compared to the control(H2O) (119875 lt 005) indicates a significant reduction in the bioenergetic parameter compared to the control (WT + H

2O) (119875 lt 005)

In the training trials treatment with AP39 for 6 weekseliminated the preexisting deficits of the transgenic miceas demonstrated by a reduced latency to locate the hiddenplatform compared to the latency of the WT control withAP39 (Figure 8(a)) The WT mice with or without AP39treatment preferred the target quadrant in the probe trialIn contrast the AD model mice treated with H

2O spent

much less time in the target quadrant near the chance levelof 15 s The AD model mice treated with AP39 showed thesame strong preference for the target quadrant as the WTmiceThus after 6weeks AP39 treatment reversed the spatiallearning and memory deficits of the aged AD model mice(Figure 8(b)) This benefit was concentration-dependent as25 nMkg AP39 treatment did not improve the water mazeperformance of the AD model mice (Figure 8(c)) Next thesame mice with 100 nMkg AP39 or H

2O treatment were

subjected to the NORT test (Figure 8(d)) The NORT testdoes not put stress on the animal and does not requirespatial orientation It has been used to measure deficits inlearning and memory in various AD mouse models [36 37]Importantly AD mice treated with water performed poorlyand presented an impairment compared to the WT miceindicated by a significant reduction in the percentage of timeexploring the novel object this behavior was consistent withan impairment in memory function for familiarization onthe previous day In contrast the AP39-treated AD miceshowed significantly improved performance levels comparedto the water-treated AD mice level Thus treatment of agedAPPPS1 mice with 100 nMkg AP39 for 6 weeks reversedthe age-dependent memory impairment All of these resultsindicated that appropriate doses of AP39 improved thelearning and working memory ability of AD model mice

38 AP39 Inhibited the Brain Atrophy of APPPS1 TransgenicMice In addition to behavioral assessments mice wererandomly selected for intravital scanning to noninvasivelystudy brain structure usingMRI dataWe studied the coronaland axial sections of the mouse brains (Figures 9(a)ndash9(f)) Asexpected there were no apparent structural abnormalities inthe WT mouse brains For example the sizes of the ventriclewere symmetric between the two brain hemispheres and theedges of the ventricle were clear and sharp (Figures 9(a) and9(b)) However we observed visible atrophy in the brains ofthe 12-month-old ADmodelmice treated with water (Figures9(c) and 9(d)) Their ventricles were asymmetric with amuch larger size on the right than the left side and hadunclear edges The MRI imaging data revealed that AP39might alleviate brain atrophy and ventricle asymmetry in ADmodel mice (Figures 9(e) and 9(f))

The apparent diffusion coefficient (ADC) value in parietalcortex and hippocampus of each group of mice was com-pared with diffusion weighted imaging (DWI) sequencesFigure 9(g) showed the ADC value in parietal cortex and hip-pocampus of APPPS1 mice significantly declined comparedto those in WT mice whereas AP39 significantly increasedthe ADC values in parietal cortex and hippocampus ofAPPPS1 mice (119875 lt 005)

39 AP39 Reduced the Levels of A120573 and A120573 Deposition inAPPPS1 Transgenic Mice After different treatments for 6weeks A120573 levels and plaque development were examinedin vivo The A120573

40and A120573

42levels in the 12-month-old AD

transgenic model mouse brains were 925 and 532 pgmLrespectively Importantly treatment with 100 nMkg AP39decreased the A120573

40and A120573

42levels in AD mice compared


0

1

2

3Cellular ATP level

ATP

conc

entr

atio

n (n

mol

)

AP39 100WT APPPS1

H2O 100H2O

lowastlowastlowastlowast

(a)

00

05

10

15 DNA integrity

DN

A in

tegr

ity (

)

Mitochondrial DNANuclear DNA

AP39 100WT APPPS1

H2O 100H2O

lowastlowast

(b)

150

100

50

0

Cou

nt

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

50

40

30

20

10

0

100

50

0

100

50

0

WT + H2O WT + AP39 APPPS1 + H2O + AP39APPPS1

(c)

0

1

2

3

4

5Cellular ROS level

DCF

fluo

resc

ence

(rel

ativ

e uni

ts)

AP39 100WT APPPS1

H2O 100H2O

lowastlowast

(d)


(e)

Figure 5 Continued


0

1

2

3Mitochondria membrane potential

JC-1

fluo

resc

ence

(rel

ativ

e uni

ts)

AP39 100WT APPPS1

H2O 100H2O

lowastlowast

(f)

Figure 5 AP39 protectedmitochondrial function inAPPPS1 neurons (a)AP39 increased the cellularATP levels (b)AP39 protectedmtDNAintegrity (c) ROS levels were detected by flow cytometry (d) AP39 reduced ROS levels 119875 lt 001 compared with theWT group lowastlowast119875 lt 001compared with the control group (H

2O)

to water treatment (Figures 10(a) and 10(b)) As expectedamyloid plaques were not observed in WT mice Howeverintracellular amyloid in the hippocampal and cortical tissuesof the AD model mice treated with water was remarkablyincreased compared with the WT control mice This obser-vation is consistent with those from a previous study [31]Importantly treatment with AP39 significantly reduced boththe number and the size of intracellular amyloid plaques inthe cortical and hippocampal tissues compared to treatmentwith water in the ADmice (Figure 10(c))The amyloid plaqueburden in the brains ofADmodelmice treatedwithAP39wasdecreased compared with the brains of ADmice treated withwater (Figure 10(c)) These results demonstrated the abilityof AP39 to inhibit A120573 plaque deposition in the brains of ADmice

4 Discussion

Our findings demonstrate that AP39 a newly synthesizedmitochondrially targeted H

2S donor maintains cellular

bioenergetics and exerts mitochondrial protective effects inAD neurons and mice First AP39 exerted concentration-dependent modulatory effects on cell viability and cellularbioenergetic function At 25 or 100 nM AP39 enhanced cellviability and bioenergetics but the highest AP39 concentra-tion (250 nM) reduced these parameters in APPPS1 neuronsIn addition treatment with an appropriately selected con-centration of AP39 (100 nM) increased ATP levels protectedmtDNA integrity decreased intracellular ROS levels andregulated mitochondrial dynamics Moreover AP39 signifi-cantly inhibited brain atrophy and ameliorated the memorydeficits and A120573 deposition in APPPS1 mice

H2S which has recently been determined to be the third

most abundant gasotransmitter plays a variety of physiolog-ical and pathological roles in the central nervous system andother systems [38 39] An increasing amount of evidencesuggests that H

2S is a potential therapeutic drug for AD

Recently it was reported that H2S-modulating agents such as

S-ally-l-cysteine (SAC) and Tabiano spa waters (enriched inH2S) protected against impairments in learning andmemory

in AD transgenic mice by modulating inflammation andapoptosis [40ndash42] In addition our group reported thatNaHScan promote the nonamyloidogenic processing of APP andimprove spatial learning andmemory acquisition in APPPS1mice [43] However due to technical limitations for H

2S

detection at present its biological roles and metabolitesin vivo have remained elusive resulting in considerablecontroversy [44] Moreover the use of sulfide salts such asNaHS is limited although the solutions to these problemshave recently become apparent [36] Importantly recentstudies indicated that AP39 acts as an endogenous H

2S donor

and exerts antioxidative and cytoprotective effects on bEnd3cells [19] AP39 decreased blood pressure heart rate andpulse wave velocity in rats [37] Based on these limitationsand progress we synthesized AP39 to evaluate its protectiveeffects on mitochondrial function in APPPS1 mice

We observed that AP39 increased H2S levels in neurons

and mitochondria Given that AP39 has the TPP+ groupwhich has been used to selectively target various molecules[45ndash47] this resulted in AP39 successfully and selectivelydelivering H

2S to mitochondriaThis result is consistent with

previous literature [19]Previous research demonstrated that H

2S can act as an

electron donor and as a potential inorganic source of energyin mammalian cells resulting in an increase in cellular oxy-gen utilization and ATP production [48 49] Subsequentlystudies reported opposing (stimulatory at lower concentra-tions inhibitory at higher concentrations) effects of H

2S

on cellular bioenergetics in HT-29 Glc(minus+) cells and someimmune cells [50 51] However owing to the various mea-surement methods used in experiment the exact concentra-tions of H

2S reported to induce stimulation versus inhibition

were different Recently Modis et al reported that 3-MST-derived H

2S functions as an endogenous bioenergetic factor

that donates electrons to SQR the subsequent mitochondrialelectron transport is coupled to aerobic ATP generation In


Fis1

Drp1

VDAC

Mfn1

Mfn2

OPA-1

AP39 100WT APPPS1

H2O 100H2O

(a)

0

50

100

150 Mfn1

WT APPPS1100nM AP39H2O 100nM AP39H2O

lowastlowast

WT

(H2O

) (de

nsito

met

ry) (

)

(b)

0

50

100

150 Mfn2

WT

(H2O

) (de

nsito

met

ry) (

)


(c)

OPA-1

lowastlowast

0

50

100

150W

T (H

2O

) (de

nsito

met

ry) (

)


(d)

0

50

100

150

200 Fis1

WT

(H2O

) (de

nsito

met

ry) (

)


lowastlowast

(e)

0

50

100

150 Drp1

WT

(H2O

) (de

nsito

met

ry) (

)


(f)

Figure 6 AP39 reduced the expression of proteins involved in mitochondrial fusion but increased the expression of a mitochondrial fissionprotein (a) Protein samples were probed for Drp1 Fis1 Mfn1 Mfn2 OPA-1 and VDAC expression (bndashf) Representative blots are shownwith quantification 119875 lt 001 AD mice receiving water compared to WT mice receiving water lowastlowast119875 lt 001mice receiving AP39 comparedwith mice receiving water


0

20

40

60

80

CortexHippocampus

AP39 minus

(nM)25 100 250

H2S

prod

uctio

n (n

mol

mg

prot

ein)

50

(a)

0

20

40

60

80

100

CortexHippocampus

AP39APPPS1WT

H2S

prod

uctio

n (n

mol

mg

prot

ein)

minus minus 100 25025

lowastlowast

lowastlowast

lowastlowast

(b)

Figure 7 AP39 generates H2S inWTmice and AP39 increased H

2S levels in APPPS1 mice (a)The contribution of AP39 to H

2S production

in the cortex and hippocampus of WT mice (b) AP39 increased H2S levels in APPPS1 mice 119875 lt 001 compared with the WT group

lowastlowast119875 lt 001 compared with the control group (H

2O)

Hepa1c1c7 low concentrations of H2S (01ndash1120583M) elicited a

significant increase in mitochondrial electron transport andcellular bioenergetics and higher concentrations of H

2S (3ndash

30 120583M) were inhibitory [18] In our experiments AP39 (25ndash100 nM) induced the stimulation of cellular bioenergetics andAP39 (250 nM) induced marked inhibition in WT neuronsMoreover APPPS1 neurons exhibited higher ldquobaselinerdquo ratesof oxygen consumption electron transport and cellularbioenergetic status than WT neurons However it is notedthat AP39 (100 nM) increased the basal respiratory rate andexerted a protective effect on the cellular bioenergetics of theAPPPS1 neuronsThese results are consistent with a previousstudy showing a stimulatory role forH

2S on bioenergetics but

are opposed to the inhibitory effects of H2S onmitochondrial

electron transport at complex IV [52ndash54] which occurs atsupraphysiological concentrations Interestingly the concen-trations of AP39 (nM) that resulted in either stimulation orinhibition were lower than those previously reported usingH2SWe considered that AP39 accumulated inmitochondria

in which the AP39 concentration is likely substantially higherthan the ldquonominalrdquo concentration that was applied to theculture medium resulting in this discrepancy Overall thesedata indicated that the stimulatory and inhibitory effects ofH2S occur in a narrow concentration range (in a specific

concentration range to exert positive bioenergetic effectsexceeding this range produces inhibitory effects) Howeverthe exact molecular mechanism underlying the protectiveeffect of AP39 remains to be investigated in further experi-ments

It has been reported that transgenic mouse strains har-boring a single (APP) or double (APP and PSEN1) geneticmutation generate amyloid plaques [55] In our experimentsA12057342release was significantly decreased in neurons cultured

from APPPS1 mice compared to neurons from WT miceand this result may be attributed to these two (APP and

PSEN1) gene mutations Previous reports have commonlyused A120573PP cultured primary neurons to evaluate A120573PPprocessing and A120573 secretion [56 57] and have shown that A120573accumulates intracellularly in A120573PP cultured neurons [58]Our data are consistent with the reports of these previousstudies

We first evaluated the effects of AP39 on APPPS1neurons In APPPS1 neurons cell viability was decreasedand LDH release was improved compared to WT neuronsThe results may be attributable to the fact that mediafrom the A120573PP primary neuronal cultures contained toxiccomponents that led to neuritic degeneration [59] HoweverAP39 improved the situation in a dose-dependent mannerThese findings demonstrate the beneficial effects of AP39

RecentlymtDNAdamage and oxidative stresswere foundto play important roles in the pathogenesis of AD [60]A120573 can decrease mitochondrial energy production increaseROS levels within the mitochondria and injure sensitivemtDNA [61] In our study treatment of APPPS1 neuronswith AP39 was shown to increase neuronal ATP productionand reduce ROS levels Interestingly the APPPS1 neuronsshowed significant damage to their mitochondrial DNAand this damage may be due to A120573 plaque deposition inthese neurons More importantly we found that AP39 clearlyincreased mitochondrial but not nuclear DNA integrityOur results suggest that AP39 may partially contribute tothe enhancement of mitochondrial biogenesis the repairof oxidative damage and the attenuation of mitochondrialdysfunction in AD However we can not exclude the pos-sibility that H

2S may also affect the integrity or activity

of various mitochondrial DNA repair proteins in APPPS1neurons This potential relationship remains to be exploredin further experiments Recently accumulating evidence hassuggested that there is an imbalance in mitochondrial fissionand fusion in AD progression [62 63] Our study showed


0 1 2 3 4 5 60

10

20

30

40

50

60La

tenc

y (s

)



(a)

0

20

40

60

80

Qua

dran

t tim

e (

)


lowastlowast

(b)

0 1 2 3 4 5 60

10

20

30

40

50

60

Late

ncy

(s)

AD + 100nM AP39AD + 25nM AP39WT + H2O

(c)

0

10

20

30

40

Obj

ect i

nter

actio

n (s

)

NovelOld


lowastlowast lowastlowastlowastlowast

(d)

Figure 8 AP39 ameliorated the learning andmemory deficits of APPPS1miceWTorADmicewere treatedwithwater or AP39 (100 nMkg)for 6 weeks followed by assessment using the Morris water maze and novel object recognition task tests (a) Spatial learning and memory inADmice are scored as the latency to locate a hidden platform (b) After 24 h a 60-s probe trial was performed (c) Spatial learning was testedas the latency to locate a hidden platform for the 25 nMkg AP39-treated mice and scored (d) Effects of AP39 on the memory performanceof 12-month WT or AD mice treated with water or AP39 were tested in the NORT test

that the levels of the fission proteins Mfn1 and OPA1 weresignificantly decreased but that the levels of the fusion proteinFis1 were markedly increased in APPPS1 neurons Theseresults suggested that mitochondrial fission and fusion werealtered in APPPS1 neurons However no apparent changesin the levels of the mitochondrial fusion protein Mfn2 orthe mitochondrial fission protein Drp1 were observed in theAPPPS1 neurons This result was consistent with the obser-vation of mitochondria with increased fission and decreasedfusion based on the related mRNA and protein levels inA120573PP primary neurons [9] Importantly AP39 treatmentreversed these changes in APPPS1 neurons The molecularbasis for the extensive changes in mitochondrial fusion andfission observed in neurons in response to the pathogenesisof mitochondrial-related neurodegenerative diseases has notbeen described [64] Interestingly our data are not consistent

with the findings of all previous reportsThese inconsistenciesmay be due to the use of a different experimental modeland different environments in vivo and in vitro ImportantlyAP39 treatment reversed these changes in APPPS1 neuronsWhether the three genes exhibiting altered expression medi-ate the effects of AP39 onAD is currently under investigation

To test whether AP39 protects AD model mice againstimpaired cognitive function we examined both spatial mem-ory and object recognition memory in APPPS1 mice Aftersix weeks of AP39 treatment the age-dependent memorydeficit in the APPPS1 mice was fully rescued on bothmemory tests AP39 ameliorated their spatial learning andmemory impairments as shown by faster movement to thetarget quadrant of the pool The reduced swimming timenear the periphery of the pool indicated that AP39 can helpto relieve anxiety a common symptom of AD in mice In


(a) (b)

(c) (d)

(e) (f)

0

2

4

6

8

CortexHippocampus

100minus

APPPS1WTminus

lowast

lowast

(AD

C10

minus4

mm

2s

)

(g)

Figure 9 AP39 alleviated the brain atrophy of APPPS1 mice as observed by brain magnetic resonance imaging (MRI) Brain MRI datawere collected at different coronal and axial sections in 12-month-old mice receiving different treatments Notes panels (a) (c) and (e)show coronal sections and panels (b) (d) and (f) show axial sections The highlighted white regions indicate cerebrospinal fluid (CSF)in the ventricle and the hippocampus is adjacent to the ventricle Representative images from brain MRI depict slices of the T2-weightedmorphologic images of 12-month-old WT mice treated with water (a)-(b) or AD mice treated with water (c)-(d) or AP39 (e)-(f) (g) ADCvalue determination of the mouse brains

the present study AP39 (100 nMkg) was a more effectivetherapeutic concentration than AP39 (25 nMkg) The dose-dependence of the observed memory improvement impliesthat an appropriate dose of AP39 is required for its beneficialeffects on learning and memory It remains possible thathigher doses of AP39 are deleterious to memory function

Due to its advantages such as noninvasiveness and theability to perform repeated in vivo measurements MRI hasbeen widely used in AD research [65] and in the auxiliaryclinical diagnosis of AD [66] It was used to directly visualizethe inner structure of the intravital cerebrum and to measurethe in vivo changes in brain volume [67] amyloid burden

[68] and white matter The hippocampus is a brain areacritical for learning and memory and is especially vulnerableto damage in the early stages of AD cases [69] In our studythe changes in the ventricle including the deepening andwidening of the sulci and the gyri were clearly observedby MRI In the water-treated AD mice we observed asym-metrical ventricles which indicated the atrophy of regionssurrounding the ventricle However in the AP39-treated ADmice we observed that the ventricles were symmetrical andexhibited clear edges Because the hippocampus is adjacentto the ventricle we hypothesized that AP39 may partiallyprevent hippocampal atrophy However we must further


0

500

1000

1500(p

gm

L)

100APPPS1WT

AP39 minus H2O

lowastlowast

A12057340

(a)

(pg

mL)

0

200

400

600

800

100APPPS1WT

AP39 minus H2O

lowastlowast

A12057342

(b)

WT APPPS1 + H2O APPPS1 + AP39

(c)

Figure 10 AP39 reduced A120573 production and A120573 deposition in APPPS1 mice (a)-(b) After intraperitoneal injection of AP39 for 6 weeksA12057340and A120573

42levels in the mouse brains were decreased from 925 pgmL and 532 pgmL to 531 pgmL and 365 pgmL respectively (c) A120573

plaqueswere detected in the cortex and the hippocampus of theWTandAPPPS1mouse brains based on 6E10 immunostainingThe statisticalresults show the area of the A120573 plaques Original magnification ndash40x scale bars = 100 120583m

investigate this hypothesis Our data suggest that MRI is auseful tool to observe the dynamic neuropathology changesin AD mouse brains and those changes in aged AD miceobserved by MRI correlated with their cognitive dysfunctionin the current study

Interestingly recent research has indicated that the mito-chondrial translocation of H

2S-producing enzymes produces

an endogenous protective response to various insults [70]In our study AP39 served as a pharmacological tool or

potential therapeutic agent that ldquomimicsrdquo this endogenousprotective mechanism However some limitations of thisstudy should be noted First we have not precisely deter-mined the exact cellular metabolism of AP39 It will be neces-sary to conduct additional studies to establish the dynamicsof the cellular metabolism of AP39 in the future Second thebell-shaped dose-response properties of AP39 are similar tothose observed for authentic H

2S or other H

2S donors and

we must admit that there may be additional pharmacologicalmechanisms that may be conducive to the detected decreasein cellular bioenergetics at higher concentrations of AP39The above limitations must be studied in future experiments

5 Conclusions

Taken together the results of the current study revealedthat the main target of protection by AP39 was mito-chondria based on the APPPS1 model We showed thatAP39 exerts concentration-dependent modulatory effects oncellular bioenergetics in APPPS1 neurons In addition AP39increasedATP levels decreased oxidative damage tomtDNAand reduced ROS levels in APPPS1 neurons MoreoverAP39 regulated the balance between mitochondrial fissionand fusion All of these events contribute to an attenuationof mitochondrial dysfunction in APPPS1 neurons Basedon these data AP39 exerts multiple protective effects inthe APPPS1 model including the amelioration of memorydeficits the prevention of brain atrophy and the decreaseof A120573 deposition Therefore AP39 represents a good can-didate disease-modifying therapy for AD Despite recentadvancements in our understanding of the role of AP39 inmitochondrial bioenergetics and function whether AP39 isactually beneficial for AD patients remains to be determinedand warrants further study


Conflict of Interests

The authors confirm that this paper content has no conflict ofinterests

Acknowledgments

The authors thank Professor Zhang (Associate Profes-sor Neuroscience Center the First Affiliated Hospital ofChongqingMedicalUniversity) for significant comments andscientific support and Professor Tang for technical assistanceThis study was supported by the National Natural ScienceFoundation of China (Grant no 81271222)

References

[1] Alzheimerrsquos Association ldquo2015 Alzheimerrsquos disease facts andfiguresrdquo Alzheimerrsquos amp Dementia vol 11 no 3 pp 332ndash3842015

[2] W S Liang E M Reiman J Valla et al ldquoAlzheimerrsquos disease isassociated with reduced expression of energymetabolism genesin posterior cingulate neuronsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 105 no11 pp 4441ndash4446 2008

[3] R Bowser and M A Smith ldquoCell cycle proteins in Alzheimerrsquosdisease plenty of wheels but no cyclerdquo Journal of AlzheimerrsquosDisease vol 4 no 3 pp 249ndash254 2002

[4] K Herrup M C Carrillo D Schenk et al ldquoBeyond amyloidgetting real about nonamyloid targets in Alzheimerrsquos diseaserdquoAlzheimerrsquos amp Dementia vol 9 no 4 pp 452e1ndash458e1 2013

[5] R H Swerdlow and S M Khan ldquoA ldquomitochondrial cascadehypothesisrdquo for sporadic Alzheimerrsquos diseaserdquoMedical Hypothe-ses vol 63 no 1 pp 8ndash20 2004

[6] X Wang W Wang L Li G Perry H-G Lee and X ZhuldquoOxidative stress andmitochondrial dysfunction in Alzheimerrsquosdiseaserdquo Biochimica et Biophysica ActamdashMolecular Basis ofDisease vol 1842 no 8 pp 1240ndash1247 2014

[7] H Xie J Guan L A Borrelli J Xu A Serrano-Pozo and B JBacskai ldquoMitochondrial alterations near amyloid plaques in anAlzheimerrsquoS diseasemousemodelrdquo Journal of Neuroscience vol33 no 43 pp 17042ndash17051 2013

[8] O Benek L Aitken L Hroch et al ldquoA direct interactionbetween mitochondrial proteins and amyloid-beta peptide andits significance for the progression and treatment of Alzheimerrsquosdiseaserdquo Current Medicinal Chemistry In press

[9] M J Calkins M Manczak P Mao U Shirendeb and P HReddy ldquoImpaired mitochondrial biogenesis defective axonaltransport of mitochondria abnormal mitochondrial dynamicsand synaptic degeneration in a mouse model of Alzheimerrsquosdiseaserdquo Human Molecular Genetics vol 20 no 23 Article IDddr381 pp 4515ndash4529 2011

[10] S Rosales-Corral D Acuna-Castroviejo D X Tan et al ldquoAccu-mulation of exogenous amyloid-beta peptide in hippocampalmitochondria causes their dysfunction a protective role formelatoninrdquo Oxidative Medicine and Cellular Longevity vol2012 Article ID 843649 15 pages 2012

[11] I Pedros D Petrov M Allgaier et al ldquoEarly alterations inenergy metabolism in the hippocampus of APPswePS1dE9mouse model of Alzheimerrsquos diseaserdquo Biochimica et BiophysicaActa vol 1842 no 9 pp 1556ndash1566 2014

[12] A C Rice P M Keeney N K Algarzae A C Ladd R RThomas and J P Bennett Jr ldquoMitochondrial DNA copy num-bers in pyramidal neurons are decreased and mitochondrialbiogenesis transcriptome signaling is disrupted in Alzheimerrsquosdisease hippocampirdquo Journal of Alzheimerrsquos Disease vol 40 no2 pp 319ndash330 2014

[13] B Sheng XWang B Su et al ldquoImpairedmitochondrial biogen-esis contributes to mitochondrial dysfunction in Alzheimerrsquosdiseaserdquo Journal of Neurochemistry vol 120 no 3 pp 419ndash4292012

[14] C Szabo ldquoHydrogen sulphide and its therapeutic potentialrdquoNature Reviews Drug Discovery vol 6 no 11 pp 917ndash935 2007

[15] H-J Wei X Li and X-Q Tang ldquoTherapeutic benefits of H2S

in Alzheimerrsquos diseaserdquo Journal of Clinical Neuroscience vol 21no 10 pp 1665ndash1669 2014

[16] X Q Tang C T Yang J Chen et al ldquoEffect of hydrogensulphide on 120573-amyloid-induced damage in PC12 cellsrdquo Clinicaland Experimental Pharmacology and Physiology vol 35 no 2pp 180ndash186 2008

[17] A Xuan D Long J Li et al ldquoHydrogen sulfide attenuates spa-tial memory impairment and hippocampal neuroinflammationin beta-amyloid rat model of Alzheimerrsquos diseaserdquo Journal ofNeuroinflammation vol 9 article 202 2012

[18] K Modis C Coletta K Erdelyi A Papapetropoulos and CSzabo ldquoIntramitochondrial hydrogen sulfide production by 3-mercaptopyruvate sulfurtransferase maintains mitochondrialelectron flow and supports cellular bioenergeticsrdquo The FASEBJournal vol 27 no 2 pp 601ndash611 2013

[19] B Szczesny K Modis K Yanagi et al ldquoAP39 a novelmitochondria-targeted hydrogen sulfide donor stimulates cel-lular bioenergetics exerts cytoprotective effects and protectsagainst the loss of mitochondrial DNA integrity in oxidativelystressed endothelial cells in vitrordquo Nitric Oxide vol 41 pp 120ndash130 2014

[20] S Le Trionnaire A Perry B Szczesny et al ldquoThe synthesisand functional evaluation of a mitochondria-targeted hydro-gen sulfide donor (10-oxo-10-(4-(3-thioxo-3H-12-dithiol-5-yl)phenoxy)decyl)triphenylphosphonium bromide (AP39)rdquoMedChemComm vol 5 no 6 pp 728ndash736 2014

[21] S Albers F Inthathirath S K Gill et al ldquoNuclear 82-kDacholine acetyltransferase decreases amyloidogenic APP meta-bolism in neurons from APPPS1 transgenic micerdquo Neurobiol-ogy of Disease vol 69 pp 32ndash42 2014

[22] J L Jankowsky D J Fadale J Anderson et al ldquoMutantpresenilins specifically elevate the levels of the 42 residue 120573-amyloid peptide in vivo evidence for augmentation of a 42-specific 120574 secretaserdquo Human Molecular Genetics vol 13 no 2pp 159ndash170 2004

[23] R Miyamoto K-I Otsuguro S Yamaguchi and S Ito ldquoCon-tribution of cysteine aminotransferase and mercaptopyruvatesulfurtransferase to hydrogen sulfide production in peripheralneuronsrdquo Journal of Neurochemistry vol 130 no 1 pp 29ndash402014

[24] X-Q Tang H-R Fang C-F Zhou et al ldquoA novel mechanismof formaldehyde neurotoxicity inhibition of hydrogen sulfidegeneration by promoting overproduction of nitric oxiderdquo PLoSONE vol 8 no 1 Article ID e54829 2013

[25] K Modis C Coletta A Asimakopoulou et al ldquoEffect ofS-adenosyl-l-methionine (SAM) an allosteric activator ofcystathionine-120573-synthase (CBS) on colorectal cancer cell pro-liferation and bioenergetics in vitrordquo Nitric Oxide vol 41 pp146ndash156 2014


[26] D A Ferrick A Neilson and C Beeson ldquoAdvances in mea-suring cellular bioenergetics using extracellular fluxrdquo DrugDiscovery Today vol 13 no 5-6 pp 268ndash274 2008

[27] U Keil A Bonert C A Marques et al ldquoAmyloid beta-inducedchanges in nitric oxide production and mitochondrial activitylead to apoptosisrdquoThe Journal of Biological Chemistry vol 279no 48 pp 50310ndash50320 2004

[28] B Szczesny G Olah D K Walker et al ldquoDeficiency in repairof the mitochondrial genome sensitizes proliferating myoblaststo oxidative damagerdquo PLoS ONE vol 8 no 9 Article ID e752012013

[29] A C Kaufman S V Salazar L T Haas et al ldquoFyn inhibitionrescues established memory and synapse loss in Alzheimermicerdquo Annals of Neurology vol 77 no 6 pp 953ndash971 2015

[30] R A Bevins and J Besheer ldquoObject recognition in rats andmice a one-trial non-matching-to-sample learning task tostudy lsquorecognition memoryrsquordquo Nature Protocols vol 1 no 3 pp1306ndash1311 2006

[31] J Zhang Q Cao S Li et al ldquo3-Hydroxybutyrate methyl esteras a potential drug against Alzheimerrsquos disease viamitochondriaprotection mechanismrdquo Biomaterials vol 34 no 30 pp 7552ndash7562 2013

[32] D-Y Choi JW Lee J Peng et al ldquoObovatol improves cognitivefunctions in animal models for Alzheimerrsquos diseaserdquo Journal ofNeurochemistry vol 120 no 6 pp 1048ndash1059 2012

[33] R Wang ldquoPhysiological implications of hydrogen sulfide awhiff exploration that blossomedrdquo Physiological Reviews vol92 no 2 pp 791ndash896 2012

[34] C Szabo C Coletta C Chao et al ldquoTumor-derived hydro-gen sulfide produced by cystathionine-120573-synthase stimulatesbioenergetics cell proliferation and angiogenesis in coloncancerrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 110 no 30 pp 12474ndash12479 2013

[35] K Modis E M Bos E Calzia et al ldquoRegulation of mito-chondrial bioenergetic function by hydrogen sulfide Part IIPathophysiological and therapeutic aspectsrdquo British Journal ofPharmacology vol 171 no 8 pp 2123ndash2146 2014

[36] M Whiteman S Le Trionnaire M Chopra B Fox and JWhatmore ldquoEmerging role of hydrogen sulfide in health anddisease critical appraisal of biomarkers and pharmacologicaltoolsrdquo Clinical Science vol 121 no 11 pp 459ndash488 2011

[37] L Tomasova M Pavlovicova L Malekova et al ldquoEffectsof AP39 a novel triphenylphosphonium derivatised anetholedithiolethione hydrogen sulfide donor on rat haemodynamicparameters and chloride and calciumCav3 and RyR2 channelsrdquoNitric Oxide vol 46 pp 131ndash144 2015

[38] K Umemura and H Kimura ldquoHydrogen sulfide enhancesreducing activity in neurons neurotrophic role of H

2S in the

brainrdquo Antioxidants and Redox Signaling vol 9 no 11 pp2035ndash2041 2007

[39] H Kimura ldquoPhysiological roles of hydrogen sulfide andpolysulfidesrdquo in Chemistry Biochemistry and Pharmacology ofHydrogen Sulfide vol 230 of Handbook of Experimental Phar-macology pp 61ndash81 Springer Basel Switzerland 2015

[40] N B Chauhan ldquoEffect of aged garlic extract on APP processingand tau phosphorylation in Alzheimerrsquos transgenic modelTg2576rdquo Journal of Ethnopharmacology vol 108 no 3 pp 385ndash394 2006

[41] Q-H Gong L-L Pan X-H Liu Q Wang H Huang and Y-Z Zhu ldquoS-propargyl-cysteine (ZYZ-802) a sulphur-containingamino acid attenuates beta-amyloid-induced cognitive deficits

and pro-inflammatory response involvement of ERK12 andNF-kappaB pathway in ratsrdquo Amino Acids vol 40 no 2 pp601ndash610 2011

[42] D Giuliani A Ottani D Zaffe et al ldquoHydrogen sulfide slowsdown progression of experimental Alzheimerrsquos disease by tar-geting multiple pathophysiological mechanismsrdquo Neurobiologyof Learning and Memory vol 104 pp 82ndash91 2013

[43] X-L He N Yan H Zhang et al ldquoHydrogen sulfide improvesspatial memory impairment and decreases production of A120573in APPPS1 transgenic micerdquoNeurochemistry International vol67 no 1 pp 1ndash8 2014

[44] L Li P Rose and P K Moore ldquoHydrogen sulfide and cellsignalingrdquo Annual Review of Pharmacology and Toxicology vol51 no 1 pp 169ndash187 2011

[45] C Constant-Urban M Charif E Goffin et al ldquoTriphenylphos-phonium salts of 124-benzothiadiazine 11-dioxides relatedto diazoxide targeting mitochondrial ATP-sensitive potassiumchannelsrdquo Bioorganic and Medicinal Chemistry Letters vol 23no 21 pp 5878ndash5881 2013

[46] M Millard J D Gallagher B Z Olenyuk and N Neamati ldquoAselectivemitochondrial-targeted chlorambucil with remarkablecytotoxicity in breast and pancreatic cancersrdquo Journal of Medic-inal Chemistry vol 56 no 22 pp 9170ndash9179 2013

[47] H Yuan H Cho H H Chen M Panagia D E Sosnovik andL Josephson ldquoFluorescent and radiolabeled triphenylphospho-nium probes for imaging mitochondriardquo Chemical Communi-cations vol 49 no 88 pp 10361ndash10363 2013

[48] M Goubern M Andriamihaja T Nubel F Blachier and FBouillaud ldquoSulfide the first inorganic substrate for humancellsrdquoThe FASEB Journal vol 21 no 8 pp 1699ndash1706 2007

[49] E Lagoutte S Mimoun M Andriamihaja C ChaumontetF Blachier and F Bouillaud ldquoOxidation of hydrogen sulfideremains a priority in mammalian cells and causes reverseelectron transfer in colonocytesrdquo Biochimica et Biophysica Acta(BBA)mdashBioenergetics vol 1797 no 8 pp 1500ndash1511 2010

[50] S Mimoun M Andriamihaja C Chaumontet et al ldquoDetoxi-fication of H

2S by differentiated colonic epithelial cells impli-

cation of the sulfide oxidizing unit and of the cell respiratorycapacityrdquo Antioxidants and Redox Signaling vol 17 no 1 pp 1ndash10 2012

[51] MGroeger JMatallo OMcCook et al ldquoTemperature and cell-type dependency of sulfide effects on mitochondrial respira-tionrdquo Shock vol 38 no 4 pp 367ndash374 2012

[52] H Aslami M J Schultz and N P Juffermans ldquoPotential appli-cations of hydrogen sulfide-induced suspended animationrdquoCurrent Medicinal Chemistry vol 16 no 10 pp 1295ndash13032009

[53] P Nicholls ldquoInhibition of cytochrome c oxidase by sulphiderdquoBiochemical Society Transactions vol 3 no 2 pp 316ndash319 1975

[54] A A Khan M M Schuler M G Prior et al ldquoEffects ofhydrogen sulfide exposure on lung mitochondrial respiratorychain enzymes in ratsrdquo Toxicology and Applied Pharmacologyvol 103 no 3 pp 482ndash490 1990

[55] C Sturchler-Pierrat D Abramowski M Duke et al ldquoTwoamyloid precursor protein transgenic mouse models withAlzheimer disease-like pathologyrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 94 no24 pp 13287ndash13292 1997

[56] Z Qiu D L Naten J C Liston J Yess and G W Rebeck ldquoAnovel approach for studying endogenous abeta processing usingcultured primary neurons isolated from APP transgenic micerdquoExperimental Neurology vol 170 no 1 pp 186ndash194 2001


[57] S Burger Y Yafai M Bigl P Wiedemann and R SchliebsldquoEffect of VEGF and its receptor antagonist SU-5416 aninhibitor of angiogenesis on processing of the 120573-amyloidprecursor protein in primary neuronal cells derived from braintissue of Tg2576 micerdquo International Journal of DevelopmentalNeuroscience vol 28 no 7 pp 597ndash604 2010

[58] R H Takahashi C G Almeida P F Kearney et al ldquoOligomer-ization of Alzheimerrsquos beta-amyloid within processes andsynapses of cultured neurons and brainrdquo Journal of Neuro-science vol 24 no 14 pp 3592ndash3599 2004

[59] M-H Wu and C-Y Kuo ldquoApplication of high throughput per-fusion micro 3-D cell culture platform for the precise study ofcellular responses to extracellular conditionsmdasheffect of serumconcentrations on the physiology of articular chondrocytesrdquoBiomedical Microdevices vol 13 no 1 pp 131ndash141 2011

[60] G J Tranah J S Yokoyama S M Katzman et al ldquoMitochon-drial DNA sequence associations with dementia and amyloid-beta in elderly African Americansrdquo Neurobiology of Aging vol35 no 2 pp 442e1ndash442e8 2014

[61] M PMattson ldquoMotherrsquos legacymitochondrial DNAmutationsand Alzheimerrsquos diseaserdquo Trends in Neurosciences vol 20 no 9pp 373ndash375 1997

[62] XWang B Su H-G Lee et al ldquoImpaired balance ofmitochon-drial fission and fusion in Alzheimerrsquos diseaserdquo The Journal ofNeuroscience vol 29 no 28 pp 9090ndash9103 2009

[63] P H Reddy ldquoAmyloid beta mitochondrial structural andfunctional dynamics inAlzheimerrsquos diseaserdquo Experimental Neu-rology vol 218 no 2 pp 286ndash292 2009

[64] M Cagalinec D SafiulinaM Liiv et al ldquoPrinciples of themito-chondrial fusion and fission cycle in neuronsrdquo Journal of CellScience vol 126 no 10 pp 2187ndash2197 2013

[65] M R Brier J B Thomas A Z Snyder et al ldquoLoss of intranet-work and internetwork resting state functional connectionswith Alzheimerrsquos disease progressionrdquo Journal of Neurosciencevol 32 no 26 pp 8890ndash8899 2012

[66] M J de Leon S Desanti R Zinkowski et al ldquoMRI and CSFstudies in the early diagnosis of Alzheimerrsquos diseaserdquo Journal ofInternal Medicine vol 256 no 3 pp 205ndash223 2004

[67] J Yin G H Turner S W Coons M Maalouf E M Reimanand J Shi ldquoAssociation of amyloid burden brain atrophyand memory deficits in aged apolipoprotein 1205764 micerdquo CurrentAlzheimer Research vol 11 no 3 pp 283ndash290 2014

[68] T M Wengenack C R Jack Jr M Garwood and J F PodusloldquoMR Microimaging of amyloid plaques in Alzheimerrsquos diseasetransgenic micerdquo European Journal of Nuclear Medicine andMolecular Imaging vol 35 supplement 1 pp S82ndashS88 2008

[69] Y Mu and F H Gage ldquoAdult hippocampal neurogenesis and itsrole in Alzheimerrsquos diseaserdquo Molecular Neurodegeneration vol6 article 85 2011

[70] H Teng B Wu K Zhao G Yang L Wu and R WangldquoOxygen-sensitive mitochondrial accumulation of cystathion-ine 120573-synthase mediated by Lon proteaserdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 110 no 31 pp 12679ndash12684 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


cyclophilin D (CypD) amyloid-120573 binding alcohol (ABAD)dehydrogenase and dynamin-related protein 1 (Drp1) [8]These interactions impair the physiological functions ofmitochondria resulting in limited electron transfer abnor-mal adenosine 51015840-triphosphate (ATP) production increasedproduction of reactive oxygen species (ROS) and alteredmitochondrial morphology and mobility in AD transgenicmodels [9 10] Furthermore a growing body of research sug-gests that mitochondrial biogenesis is considerably decreasedin the brains of both AD patients and AD model mice [911ndash13] Impaired mitochondrial biogenesis is conducive tomitochondrial dysfunction in AD [13]

Hydrogen sulfide (H2S) has recently been recognized as

an endogenous gaseous mediator that plays multiple regula-tory roles in humans and mammals [14] Increasing evidencehas demonstrated that the dysregulation of H

2S homeostasis

is implicated in the pathological processes of AD Recentlyour experimental research has revealed that the level of H

2S is

decreased in AD patients and that this change in the H2S lev-

els may be related to the severity of AD The emerging mito-chondrial roles ofH

2S include antioxidant antiapoptotic and

anti-inflammatory effects [15] Tang revealed that H2S mod-

ulates A120573-induced damage to PC12 cells by ameliorating thedecrease in the mitochondrial membrane potential (MMPΔΨm) and attenuating the increase in intracellular ROS levels[16] Moreover Xuan et al found that H

2S may attenuate

spatial memory impairment and neuroinflammation withinthe hippocampus of AD model mice [17] With regard tothe regulatory role of H

2S in cellular bioenergy metabolism

recent studies have shown that H2S can serve as a physiologi-

cal electron donor and as an inorganic energy source inmam-malian cells Recently Modis et al reported that an endoge-nous intramitochondrial H

2S-producing pathway which is

governed by 3-mercaptopyruvate sulfurtransferase (3-MST)complements and balances the bioenergetic activity of Krebscycle-derived electron donors in the Hepa1c1c7 cell line [18]

Taken together these findings indicate that the potentialeffects of H

2S on mitochondrial dysfunction in AD models

are significant Therefore to clarify whether the new H2S

donor AP39 which contains the mitochondria-targetingcompound triphenylphosphonium (TPP+) coupled to anH2S-donating moiety (dithiolethione) via an aliphatic linker

regulates mitochondrial function and cellular bioenergeticsis of great interest Using APPPS1 neurons and mice we firstdetected the overall viability of the neurons and evaluated theeffects of AP39 on cellular bioenergetics and mitochondrialfunction In addition we examined the effects of AP39 onspatial memory deficits and on magnetic resonance imaging(MRI) characteristics of the mouse brains

2 Materials and Methods

21 Materials AP39 a novel mitochondria-targeted H2S

donor was designed and synthesized by Medicilon Inc asdescribed previously [19 20] Antimycin A 025 trypsin2-deoxyglucose oligomycin carbonyl cyanide-4-(trifluoro-methoxy)phenylhydrazone (FCCP) trichloroacetic acidrotenone 7-azido-4-methylcoumarin (AzMC) monobasicpotassium phosphate dibasic sodium succinate hexahydrate

adenosine 51015840-triphosphate (ATP) disodium salt hydrate fattyacid-free BSA sodium hydrosulfide hydrate (NaSHsdot119909H

2O)

NN-diethyl-p-phenylenediamine sulfate magnesium chlo-ride and iron(III) chloride (FeCl

3) were purchased from

Sigma-Aldrich Company The monoclonal anti-A120573 antibody6E10 was obtained from Covance Inc (Princeton NJ USA)Drp1 was purchased from Novus Biologicals Inc Fis1 waspurchased from Proteintech Group Mfn1 and Mfn2 anti-bodies were purchased from Santa Cruz Biotech andOPA-1 antibody was purchased from BD Transduction Lab-oratories The secondary antibodies goat anti-mouse-HRPand donkey anti-rabbit-HRP antibodies were purchasedfrom GE Healthcare The Pierce BCA protein assay kit waspurchased from Thermo Fisher Scientific (Waltham MAUSA) Dulbeccorsquos modified Eaglersquos medium (DMEM) 1glutamine and 1 penicillin were obtained from InvitrogenAll other chemicals were purchased from Amresco (SolonOH USA)

22 Animals and Treatments This experiment was per-formed as described previously [21] All animal protocolswere approved by the Animal Research Committee of ChongQing Medical University Heterozygous APPPS1 double-transgenic mice (APPswe-PS1dE9) were crossed with non-transgenic female mice (the Model Animal Research Centerof Nanjing University Jiangsu China) to produce hemizy-gous transgenic mice and nontransgenic littermates In thedouble-transgenic mice that coexpressed the Swedish muta-tion of APP (APPswe) and two FAD-PS1 variants A120573 plaquesaccumulated in the brain and learning and memory abilitydeclined over time [22] The mice were housed at 23ndash25∘Cwith 60 humidity under 12 12 h light-dark cycles and wereprovided with free access to food and water throughout theexperiment Pregnant females were sacrificed at gestationaldays 15-16 (E15-16) and the embryos were removed for thepreparation of brain neuronal cultures The genotyping forAPP and PS1 was performed according to the literatureto assign cultures to transgenic and nontransgenic groupsaccording to the PCR results with genomic DNA For theMorris water maze test and the novel object recognition task(NORT) these 12-month-old mice were divided into fourgroups of fifteen mice in each group wild-type (WT) micetreated with deionized water or 100 nMkg AP39 and ADmodel mice treated with deionized water or 100 nMkg AP39Themice were treated once daily via intraperitoneal injectionfor 6 weeks prior to the experiments After the behavioraltests the mice were scanned by MRI and then sacrificedfor the collection of their blood and brains The bloodwas collected into heparinized tubes and then centrifugedat 1000 g for 10min at 4∘C The plasma (supernatant) wascollected to detect A120573

40and A120573

42by ELISA The brains

were removed and collected to examine A120573 deposition byimmunohistochemistry

23 Primary Neuron Culture Primary cultures of corti-cal neurons were generated from the brains of individualembryos at E15-16 Briefly the brains were removed andthen placed in Hankrsquos balanced salt solution at 4∘C Thecortex was dissected from the brain chopped and digested








2S to detect H

2S


3


2S


2S con-






2 The H

2S-sensitive

























40and A120573




40and





3 Results











2S [25 33














0

5

10

15

20

AP39 minus 10025 50

H2S

prod

uctio

n (n

mol

mg

min

)

250(nM)

lowastlowast

lowastlowastlowast

lowast

(a)


minus

50nM

100nM

(b)









0

20

40

60

80

100

120

Control

Oligo FCCP AA

Basalrespiration

Proton leak

ATPproduction

Maximalrespiration

capacity

ADP

(min)0 10 20 30 40 50

OCR

(pm

olm

in120583

g pr

otei

n)


25nM AP39(a)

0

30

60

90

120

Control

Basalrespiration


Maximalrespiration

OCR

(pm

olm

in120583

g pr

otei

n)


25nM AP39

lowastlowast

lowast

(b)













2S in WT and APPPS1

mice



0

200

400

600

800

1000

WT neurons

3 DIV 7 DIV 16 DIV


lowast

A120573

1minus42

rele

ase (

pgm

L m

ediu

m)

(a)


A120573

Actin

(b)

WT APPPS10

1

2

3

4

5

6

7

Rela

tive A

120573ex

pres

sion

( o

f

cont

rol l

evel)

(c)

0

50

100

150

MTT

redu

ctio

n (

of c

ontro

l)

AP39 minus 10025 250(d)

0

50

100

150

MTT

redu

ctio

n (

of c

ontro

l)

AP39 minus minus

APPPS1WT

lowast

lowastlowast

10025 250

(e)

0

50

100

150

LDH

rele

ase (

o

f con

trol)

AP39 minus 10025 250

lowast lowast

(f)

0

50

100

150

200

LDH

rele

ase (

o

f con

trol)


lowastlowast

(g)




OCR

(pm

olm

in120583

g pr

otei

n)

0 10 20 30 40 500

30

60

90

120

Oligo FCCP AA

Basalrespiration

Proton leak

ATPproduction

Maximalrespiration

capacity

ADP



(a)O

CR (p

mol

min

120583g

prot

ein)

Basalrespiration


Maximalrespiration

0

50

100

150



lowast

lowast

lowast

lowast

lowast

lowast

(b)


2O) (119875 lt 005)


2O spent


2O treatment were





40and A120573



40and A120573



0

1

2

3Cellular ATP level

ATP

conc

entr

atio

n (n

mol

)

AP39 100WT APPPS1

H2O 100H2O


(a)

00

05

10

15 DNA integrity

DN

A in

tegr

ity (

)


AP39 100WT APPPS1

H2O 100H2O

lowastlowast

(b)

150

100

50

0

Cou

nt

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

50

40

30

20

10

0

100

50

0

100

50

0


(c)

0

1

2

3

4

5Cellular ROS level

DCF

fluo

resc

ence

(rel

ativ

e uni

ts)

AP39 100WT APPPS1

H2O 100H2O

lowastlowast

(d)


(e)

Figure 5 Continued


0

1

2


JC-1

fluo

resc

ence

(rel

ativ

e uni

ts)

AP39 100WT APPPS1

H2O 100H2O

lowastlowast

(f)


2O)


4 Discussion










2S


2S donor






2S can act as an


2S







Fis1

Drp1

VDAC

Mfn1

Mfn2

OPA-1

AP39 100WT APPPS1

H2O 100H2O

(a)

0

50

100

150 Mfn1


lowastlowast

WT

(H2O

) (de

nsito

met

ry) (

)

(b)

0

50

100

150 Mfn2

WT

(H2O

) (de

nsito

met

ry) (

)


(c)

OPA-1

lowastlowast

0

50

100

150W

T (H

2O

) (de

nsito

met

ry) (

)


(d)

0

50

100

150

200 Fis1

WT

(H2O

) (de

nsito

met

ry) (

)


lowastlowast

(e)

0

50

100

150 Drp1

WT

(H2O

) (de

nsito

met

ry) (

)


(f)



0

20

40

60

80

CortexHippocampus

AP39 minus

(nM)25 100 250

H2S

prod

uctio

n (n

mol

mg

prot

ein)

50

(a)

0

20

40

60

80

100

CortexHippocampus

AP39APPPS1WT

H2S

prod

uctio

n (n

mol

mg

prot

ein)


lowastlowast

lowastlowast

lowastlowast

(b)



2S production



2O)



2S (3ndash















0 1 2 3 4 5 60

10

20

30

40

50

60La

tenc

y (s

)



(a)

0

20

40

60

80

Qua

dran

t tim

e (

)


lowastlowast

(b)

0 1 2 3 4 5 60

10

20

30

40

50

60

Late

ncy

(s)


(c)

0

10

20

30

40

Obj

ect i

nter

actio

n (s

)

NovelOld



(d)






(a) (b)

(c) (d)

(e) (f)

0

2

4

6

8

CortexHippocampus

100minus

APPPS1WTminus

lowast

lowast

(AD

C10

minus4

mm

2s

)

(g)






0

500

1000

1500(p

gm

L)

100APPPS1WT

AP39 minus H2O

lowastlowast

A12057340

(a)

(pg

mL)

0

200

400

600

800

100APPPS1WT

AP39 minus H2O

lowastlowast

A12057342

(b)


(c)









2S or other H

2S donors and


5 Conclusions





Acknowledgments


References









































2S in the











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























2S to detect H

2S


3


2S


2S con-






2 The H

2S-sensitive

























40and A120573




40and





3 Results











2S [25 33














0

5

10

15

20

AP39 minus 10025 50

H2S

prod

uctio

n (n

mol

mg

min

)

250(nM)

lowastlowast

lowastlowastlowast

lowast

(a)


minus

50nM

100nM

(b)









0

20

40

60

80

100

120

Control

Oligo FCCP AA

Basalrespiration

Proton leak

ATPproduction

Maximalrespiration

capacity

ADP

(min)0 10 20 30 40 50

OCR

(pm

olm

in120583

g pr

otei

n)


25nM AP39(a)

0

30

60

90

120

Control

Basalrespiration


Maximalrespiration

OCR

(pm

olm

in120583

g pr

otei

n)


25nM AP39

lowastlowast

lowast

(b)













2S in WT and APPPS1

mice



0

200

400

600

800

1000

WT neurons

3 DIV 7 DIV 16 DIV


lowast

A120573

1minus42

rele

ase (

pgm

L m

ediu

m)

(a)


A120573

Actin

(b)

WT APPPS10

1

2

3

4

5

6

7

Rela

tive A

120573ex

pres

sion

( o

f

cont

rol l

evel)

(c)

0

50

100

150

MTT

redu

ctio

n (

of c

ontro

l)

AP39 minus 10025 250(d)

0

50

100

150

MTT

redu

ctio

n (

of c

ontro

l)

AP39 minus minus

APPPS1WT

lowast

lowastlowast

10025 250

(e)

0

50

100

150

LDH

rele

ase (

o

f con

trol)

AP39 minus 10025 250

lowast lowast

(f)

0

50

100

150

200

LDH

rele

ase (

o

f con

trol)


lowastlowast

(g)




OCR

(pm

olm

in120583

g pr

otei

n)

0 10 20 30 40 500

30

60

90

120

Oligo FCCP AA

Basalrespiration

Proton leak

ATPproduction

Maximalrespiration

capacity

ADP



(a)O

CR (p

mol

min

120583g

prot

ein)

Basalrespiration


Maximalrespiration

0

50

100

150



lowast

lowast

lowast

lowast

lowast

lowast

(b)


2O) (119875 lt 005)


2O spent


2O treatment were





40and A120573



40and A120573



0

1

2

3Cellular ATP level

ATP

conc

entr

atio

n (n

mol

)

AP39 100WT APPPS1

H2O 100H2O


(a)

00

05

10

15 DNA integrity

DN

A in

tegr

ity (

)


AP39 100WT APPPS1

H2O 100H2O

lowastlowast

(b)

150

100

50

0

Cou

nt

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

50

40

30

20

10

0

100

50

0

100

50

0


(c)

0

1

2

3

4

5Cellular ROS level

DCF

fluo

resc

ence

(rel

ativ

e uni

ts)

AP39 100WT APPPS1

H2O 100H2O

lowastlowast

(d)


(e)

Figure 5 Continued


0

1

2


JC-1

fluo

resc

ence

(rel

ativ

e uni

ts)

AP39 100WT APPPS1

H2O 100H2O

lowastlowast

(f)


2O)


4 Discussion










2S


2S donor






2S can act as an


2S







Fis1

Drp1

VDAC

Mfn1

Mfn2

OPA-1

AP39 100WT APPPS1

H2O 100H2O

(a)

0

50

100

150 Mfn1


lowastlowast

WT

(H2O

) (de

nsito

met

ry) (

)

(b)

0

50

100

150 Mfn2

WT

(H2O

) (de

nsito

met

ry) (

)


(c)

OPA-1

lowastlowast

0

50

100

150W

T (H

2O

) (de

nsito

met

ry) (

)


(d)

0

50

100

150

200 Fis1

WT

(H2O

) (de

nsito

met

ry) (

)


lowastlowast

(e)

0

50

100

150 Drp1

WT

(H2O

) (de

nsito

met

ry) (

)


(f)



0

20

40

60

80

CortexHippocampus

AP39 minus

(nM)25 100 250

H2S

prod

uctio

n (n

mol

mg

prot

ein)

50

(a)

0

20

40

60

80

100

CortexHippocampus

AP39APPPS1WT

H2S

prod

uctio

n (n

mol

mg

prot

ein)


lowastlowast

lowastlowast

lowastlowast

(b)



2S production



2O)



2S (3ndash















0 1 2 3 4 5 60

10

20

30

40

50

60La

tenc

y (s

)



(a)

0

20

40

60

80

Qua

dran

t tim

e (

)


lowastlowast

(b)

0 1 2 3 4 5 60

10

20

30

40

50

60

Late

ncy

(s)


(c)

0

10

20

30

40

Obj

ect i

nter

actio

n (s

)

NovelOld



(d)






(a) (b)

(c) (d)

(e) (f)

0

2

4

6

8

CortexHippocampus

100minus

APPPS1WTminus

lowast

lowast

(AD

C10

minus4

mm

2s

)

(g)






0

500

1000

1500(p

gm

L)

100APPPS1WT

AP39 minus H2O

lowastlowast

A12057340

(a)

(pg

mL)

0

200

400

600

800

100APPPS1WT

AP39 minus H2O

lowastlowast

A12057342

(b)


(c)









2S or other H

2S donors and


5 Conclusions





Acknowledgments


References









































2S in the











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























40and A120573




40and





3 Results











2S [25 33














0

5

10

15

20

AP39 minus 10025 50

H2S

prod

uctio

n (n

mol

mg

min

)

250(nM)

lowastlowast

lowastlowastlowast

lowast

(a)


minus

50nM

100nM

(b)









0

20

40

60

80

100

120

Control

Oligo FCCP AA

Basalrespiration

Proton leak

ATPproduction

Maximalrespiration

capacity

ADP

(min)0 10 20 30 40 50

OCR

(pm

olm

in120583

g pr

otei

n)


25nM AP39(a)

0

30

60

90

120

Control

Basalrespiration


Maximalrespiration

OCR

(pm

olm

in120583

g pr

otei

n)


25nM AP39

lowastlowast

lowast

(b)













2S in WT and APPPS1

mice



0

200

400

600

800

1000

WT neurons

3 DIV 7 DIV 16 DIV


lowast

A120573

1minus42

rele

ase (

pgm

L m

ediu

m)

(a)


A120573

Actin

(b)

WT APPPS10

1

2

3

4

5

6

7

Rela

tive A

120573ex

pres

sion

( o

f

cont

rol l

evel)

(c)

0

50

100

150

MTT

redu

ctio

n (

of c

ontro

l)

AP39 minus 10025 250(d)

0

50

100

150

MTT

redu

ctio

n (

of c

ontro

l)

AP39 minus minus

APPPS1WT

lowast

lowastlowast

10025 250

(e)

0

50

100

150

LDH

rele

ase (

o

f con

trol)

AP39 minus 10025 250

lowast lowast

(f)

0

50

100

150

200

LDH

rele

ase (

o

f con

trol)


lowastlowast

(g)




OCR

(pm

olm

in120583

g pr

otei

n)

0 10 20 30 40 500

30

60

90

120

Oligo FCCP AA

Basalrespiration

Proton leak

ATPproduction

Maximalrespiration

capacity

ADP



(a)O

CR (p

mol

min

120583g

prot

ein)

Basalrespiration


Maximalrespiration

0

50

100

150



lowast

lowast

lowast

lowast

lowast

lowast

(b)


2O) (119875 lt 005)


2O spent


2O treatment were





40and A120573



40and A120573



0

1

2

3Cellular ATP level

ATP

conc

entr

atio

n (n

mol

)

AP39 100WT APPPS1

H2O 100H2O


(a)

00

05

10

15 DNA integrity

DN

A in

tegr

ity (

)


AP39 100WT APPPS1

H2O 100H2O

lowastlowast

(b)

150

100

50

0

Cou

nt

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

50

40

30

20

10

0

100

50

0

100

50

0


(c)

0

1

2

3

4

5Cellular ROS level

DCF

fluo

resc

ence

(rel

ativ

e uni

ts)

AP39 100WT APPPS1

H2O 100H2O

lowastlowast

(d)


(e)

Figure 5 Continued


0

1

2


JC-1

fluo

resc

ence

(rel

ativ

e uni

ts)

AP39 100WT APPPS1

H2O 100H2O

lowastlowast

(f)


2O)


4 Discussion










2S


2S donor






2S can act as an


2S







Fis1

Drp1

VDAC

Mfn1

Mfn2

OPA-1

AP39 100WT APPPS1

H2O 100H2O

(a)

0

50

100

150 Mfn1


lowastlowast

WT

(H2O

) (de

nsito

met

ry) (

)

(b)

0

50

100

150 Mfn2

WT

(H2O

) (de

nsito

met

ry) (

)


(c)

OPA-1

lowastlowast

0

50

100

150W

T (H

2O

) (de

nsito

met

ry) (

)


(d)

0

50

100

150

200 Fis1

WT

(H2O

) (de

nsito

met

ry) (

)


lowastlowast

(e)

0

50

100

150 Drp1

WT

(H2O

) (de

nsito

met

ry) (

)


(f)



0

20

40

60

80

CortexHippocampus

AP39 minus

(nM)25 100 250

H2S

prod

uctio

n (n

mol

mg

prot

ein)

50

(a)

0

20

40

60

80

100

CortexHippocampus

AP39APPPS1WT

H2S

prod

uctio

n (n

mol

mg

prot

ein)


lowastlowast

lowastlowast

lowastlowast

(b)



2S production



2O)



2S (3ndash















0 1 2 3 4 5 60

10

20

30

40

50

60La

tenc

y (s

)



(a)

0

20

40

60

80

Qua

dran

t tim

e (

)


lowastlowast

(b)

0 1 2 3 4 5 60

10

20

30

40

50

60

Late

ncy

(s)


(c)

0

10

20

30

40

Obj

ect i

nter

actio

n (s

)

NovelOld



(d)






(a) (b)

(c) (d)

(e) (f)

0

2

4

6

8

CortexHippocampus

100minus

APPPS1WTminus

lowast

lowast

(AD

C10

minus4

mm

2s

)

(g)






0

500

1000

1500(p

gm

L)

100APPPS1WT

AP39 minus H2O

lowastlowast

A12057340

(a)

(pg

mL)

0

200

400

600

800

100APPPS1WT

AP39 minus H2O

lowastlowast

A12057342

(b)


(c)









2S or other H

2S donors and


5 Conclusions





Acknowledgments


References









































2S in the











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

5

10

15

20

AP39 minus 10025 50

H2S

prod

uctio

n (n

mol

mg

min

)

250(nM)

lowastlowast

lowastlowastlowast

lowast

(a)


minus

50nM

100nM

(b)









0

20

40

60

80

100

120

Control

Oligo FCCP AA

Basalrespiration

Proton leak

ATPproduction

Maximalrespiration

capacity

ADP

(min)0 10 20 30 40 50

OCR

(pm

olm

in120583

g pr

otei

n)


25nM AP39(a)

0

30

60

90

120

Control

Basalrespiration


Maximalrespiration

OCR

(pm

olm

in120583

g pr

otei

n)


25nM AP39

lowastlowast

lowast

(b)













2S in WT and APPPS1

mice



0

200

400

600

800

1000

WT neurons

3 DIV 7 DIV 16 DIV


lowast

A120573

1minus42

rele

ase (

pgm

L m

ediu

m)

(a)


A120573

Actin

(b)

WT APPPS10

1

2

3

4

5

6

7

Rela

tive A

120573ex

pres

sion

( o

f

cont

rol l

evel)

(c)

0

50

100

150

MTT

redu

ctio

n (

of c

ontro

l)

AP39 minus 10025 250(d)

0

50

100

150

MTT

redu

ctio

n (

of c

ontro

l)

AP39 minus minus

APPPS1WT

lowast

lowastlowast

10025 250

(e)

0

50

100

150

LDH

rele

ase (

o

f con

trol)

AP39 minus 10025 250

lowast lowast

(f)

0

50

100

150

200

LDH

rele

ase (

o

f con

trol)


lowastlowast

(g)




OCR

(pm

olm

in120583

g pr

otei

n)

0 10 20 30 40 500

30

60

90

120

Oligo FCCP AA

Basalrespiration

Proton leak

ATPproduction

Maximalrespiration

capacity

ADP



(a)O

CR (p

mol

min

120583g

prot

ein)

Basalrespiration


Maximalrespiration

0

50

100

150



lowast

lowast

lowast

lowast

lowast

lowast

(b)


2O) (119875 lt 005)


2O spent


2O treatment were





40and A120573



40and A120573



0

1

2

3Cellular ATP level

ATP

conc

entr

atio

n (n

mol

)

AP39 100WT APPPS1

H2O 100H2O


(a)

00

05

10

15 DNA integrity

DN

A in

tegr

ity (

)


AP39 100WT APPPS1

H2O 100H2O

lowastlowast

(b)

150

100

50

0

Cou

nt

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

50

40

30

20

10

0

100

50

0

100

50

0


(c)

0

1

2

3

4

5Cellular ROS level

DCF

fluo

resc

ence

(rel

ativ

e uni

ts)

AP39 100WT APPPS1

H2O 100H2O

lowastlowast

(d)


(e)

Figure 5 Continued


0

1

2


JC-1

fluo

resc

ence

(rel

ativ

e uni

ts)

AP39 100WT APPPS1

H2O 100H2O

lowastlowast

(f)


2O)


4 Discussion










2S


2S donor






2S can act as an


2S







Fis1

Drp1

VDAC

Mfn1

Mfn2

OPA-1

AP39 100WT APPPS1

H2O 100H2O

(a)

0

50

100

150 Mfn1


lowastlowast

WT

(H2O

) (de

nsito

met

ry) (

)

(b)

0

50

100

150 Mfn2

WT

(H2O

) (de

nsito

met

ry) (

)


(c)

OPA-1

lowastlowast

0

50

100

150W

T (H

2O

) (de

nsito

met

ry) (

)


(d)

0

50

100

150

200 Fis1

WT

(H2O

) (de

nsito

met

ry) (

)


lowastlowast

(e)

0

50

100

150 Drp1

WT

(H2O

) (de

nsito

met

ry) (

)


(f)



0

20

40

60

80

CortexHippocampus

AP39 minus

(nM)25 100 250

H2S

prod

uctio

n (n

mol

mg

prot

ein)

50

(a)

0

20

40

60

80

100

CortexHippocampus

AP39APPPS1WT

H2S

prod

uctio

n (n

mol

mg

prot

ein)


lowastlowast

lowastlowast

lowastlowast

(b)



2S production



2O)



2S (3ndash















0 1 2 3 4 5 60

10

20

30

40

50

60La

tenc

y (s

)



(a)

0

20

40

60

80

Qua

dran

t tim

e (

)


lowastlowast

(b)

0 1 2 3 4 5 60

10

20

30

40

50

60

Late

ncy

(s)


(c)

0

10

20

30

40

Obj

ect i

nter

actio

n (s

)

NovelOld



(d)






(a) (b)

(c) (d)

(e) (f)

0

2

4

6

8

CortexHippocampus

100minus

APPPS1WTminus

lowast

lowast

(AD

C10

minus4

mm

2s

)

(g)






0

500

1000

1500(p

gm

L)

100APPPS1WT

AP39 minus H2O

lowastlowast

A12057340

(a)

(pg

mL)

0

200

400

600

800

100APPPS1WT

AP39 minus H2O

lowastlowast

A12057342

(b)


(c)









2S or other H

2S donors and


5 Conclusions





Acknowledgments


References









































2S in the











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

20

40

60

80

100

120

Control

Oligo FCCP AA

Basalrespiration

Proton leak

ATPproduction

Maximalrespiration

capacity

ADP

(min)0 10 20 30 40 50

OCR

(pm

olm

in120583

g pr

otei

n)


25nM AP39(a)

0

30

60

90

120

Control

Basalrespiration


Maximalrespiration

OCR

(pm

olm

in120583

g pr

otei

n)


25nM AP39

lowastlowast

lowast

(b)













2S in WT and APPPS1

mice



0

200

400

600

800

1000

WT neurons

3 DIV 7 DIV 16 DIV


lowast

A120573

1minus42

rele

ase (

pgm

L m

ediu

m)

(a)


A120573

Actin

(b)

WT APPPS10

1

2

3

4

5

6

7

Rela

tive A

120573ex

pres

sion

( o

f

cont

rol l

evel)

(c)

0

50

100

150

MTT

redu

ctio

n (

of c

ontro

l)

AP39 minus 10025 250(d)

0

50

100

150

MTT

redu

ctio

n (

of c

ontro

l)

AP39 minus minus

APPPS1WT

lowast

lowastlowast

10025 250

(e)

0

50

100

150

LDH

rele

ase (

o

f con

trol)

AP39 minus 10025 250

lowast lowast

(f)

0

50

100

150

200

LDH

rele

ase (

o

f con

trol)


lowastlowast

(g)




OCR

(pm

olm

in120583

g pr

otei

n)

0 10 20 30 40 500

30

60

90

120

Oligo FCCP AA

Basalrespiration

Proton leak

ATPproduction

Maximalrespiration

capacity

ADP



(a)O

CR (p

mol

min

120583g

prot

ein)

Basalrespiration


Maximalrespiration

0

50

100

150



lowast

lowast

lowast

lowast

lowast

lowast

(b)


2O) (119875 lt 005)


2O spent


2O treatment were





40and A120573



40and A120573



0

1

2

3Cellular ATP level

ATP

conc

entr

atio

n (n

mol

)

AP39 100WT APPPS1

H2O 100H2O


(a)

00

05

10

15 DNA integrity

DN

A in

tegr

ity (

)


AP39 100WT APPPS1

H2O 100H2O

lowastlowast

(b)

150

100

50

0

Cou

nt

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

50

40

30

20

10

0

100

50

0

100

50

0


(c)

0

1

2

3

4

5Cellular ROS level

DCF

fluo

resc

ence

(rel

ativ

e uni

ts)

AP39 100WT APPPS1

H2O 100H2O

lowastlowast

(d)


(e)

Figure 5 Continued


0

1

2


JC-1

fluo

resc

ence

(rel

ativ

e uni

ts)

AP39 100WT APPPS1

H2O 100H2O

lowastlowast

(f)


2O)


4 Discussion










2S


2S donor






2S can act as an


2S







Fis1

Drp1

VDAC

Mfn1

Mfn2

OPA-1

AP39 100WT APPPS1

H2O 100H2O

(a)

0

50

100

150 Mfn1


lowastlowast

WT

(H2O

) (de

nsito

met

ry) (

)

(b)

0

50

100

150 Mfn2

WT

(H2O

) (de

nsito

met

ry) (

)


(c)

OPA-1

lowastlowast

0

50

100

150W

T (H

2O

) (de

nsito

met

ry) (

)


(d)

0

50

100

150

200 Fis1

WT

(H2O

) (de

nsito

met

ry) (

)


lowastlowast

(e)

0

50

100

150 Drp1

WT

(H2O

) (de

nsito

met

ry) (

)


(f)



0

20

40

60

80

CortexHippocampus

AP39 minus

(nM)25 100 250

H2S

prod

uctio

n (n

mol

mg

prot

ein)

50

(a)

0

20

40

60

80

100

CortexHippocampus

AP39APPPS1WT

H2S

prod

uctio

n (n

mol

mg

prot

ein)


lowastlowast

lowastlowast

lowastlowast

(b)



2S production



2O)



2S (3ndash















0 1 2 3 4 5 60

10

20

30

40

50

60La

tenc

y (s

)



(a)

0

20

40

60

80

Qua

dran

t tim

e (

)


lowastlowast

(b)

0 1 2 3 4 5 60

10

20

30

40

50

60

Late

ncy

(s)


(c)

0

10

20

30

40

Obj

ect i

nter

actio

n (s

)

NovelOld



(d)






(a) (b)

(c) (d)

(e) (f)

0

2

4

6

8

CortexHippocampus

100minus

APPPS1WTminus

lowast

lowast

(AD

C10

minus4

mm

2s

)

(g)






0

500

1000

1500(p

gm

L)

100APPPS1WT

AP39 minus H2O

lowastlowast

A12057340

(a)

(pg

mL)

0

200

400

600

800

100APPPS1WT

AP39 minus H2O

lowastlowast

A12057342

(b)


(c)









2S or other H

2S donors and


5 Conclusions





Acknowledgments


References









































2S in the











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

200

400

600

800

1000

WT neurons

3 DIV 7 DIV 16 DIV


lowast

A120573

1minus42

rele

ase (

pgm

L m

ediu

m)

(a)


A120573

Actin

(b)

WT APPPS10

1

2

3

4

5

6

7

Rela

tive A

120573ex

pres

sion

( o

f

cont

rol l

evel)

(c)

0

50

100

150

MTT

redu

ctio

n (

of c

ontro

l)

AP39 minus 10025 250(d)

0

50

100

150

MTT

redu

ctio

n (

of c

ontro

l)

AP39 minus minus

APPPS1WT

lowast

lowastlowast

10025 250

(e)

0

50

100

150

LDH

rele

ase (

o

f con

trol)

AP39 minus 10025 250

lowast lowast

(f)

0

50

100

150

200

LDH

rele

ase (

o

f con

trol)


lowastlowast

(g)




OCR

(pm

olm

in120583

g pr

otei

n)

0 10 20 30 40 500

30

60

90

120

Oligo FCCP AA

Basalrespiration

Proton leak

ATPproduction

Maximalrespiration

capacity

ADP



(a)O

CR (p

mol

min

120583g

prot

ein)

Basalrespiration


Maximalrespiration

0

50

100

150



lowast

lowast

lowast

lowast

lowast

lowast

(b)


2O) (119875 lt 005)


2O spent


2O treatment were





40and A120573



40and A120573



0

1

2

3Cellular ATP level

ATP

conc

entr

atio

n (n

mol

)

AP39 100WT APPPS1

H2O 100H2O


(a)

00

05

10

15 DNA integrity

DN

A in

tegr

ity (

)


AP39 100WT APPPS1

H2O 100H2O

lowastlowast

(b)

150

100

50

0

Cou

nt

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

50

40

30

20

10

0

100

50

0

100

50

0


(c)

0

1

2

3

4

5Cellular ROS level

DCF

fluo

resc

ence

(rel

ativ

e uni

ts)

AP39 100WT APPPS1

H2O 100H2O

lowastlowast

(d)


(e)

Figure 5 Continued


0

1

2


JC-1

fluo

resc

ence

(rel

ativ

e uni

ts)

AP39 100WT APPPS1

H2O 100H2O

lowastlowast

(f)


2O)


4 Discussion










2S


2S donor






2S can act as an


2S







Fis1

Drp1

VDAC

Mfn1

Mfn2

OPA-1

AP39 100WT APPPS1

H2O 100H2O

(a)

0

50

100

150 Mfn1


lowastlowast

WT

(H2O

) (de

nsito

met

ry) (

)

(b)

0

50

100

150 Mfn2

WT

(H2O

) (de

nsito

met

ry) (

)


(c)

OPA-1

lowastlowast

0

50

100

150W

T (H

2O

) (de

nsito

met

ry) (

)


(d)

0

50

100

150

200 Fis1

WT

(H2O

) (de

nsito

met

ry) (

)


lowastlowast

(e)

0

50

100

150 Drp1

WT

(H2O

) (de

nsito

met

ry) (

)


(f)



0

20

40

60

80

CortexHippocampus

AP39 minus

(nM)25 100 250

H2S

prod

uctio

n (n

mol

mg

prot

ein)

50

(a)

0

20

40

60

80

100

CortexHippocampus

AP39APPPS1WT

H2S

prod

uctio

n (n

mol

mg

prot

ein)


lowastlowast

lowastlowast

lowastlowast

(b)



2S production



2O)



2S (3ndash















0 1 2 3 4 5 60

10

20

30

40

50

60La

tenc

y (s

)



(a)

0

20

40

60

80

Qua

dran

t tim

e (

)


lowastlowast

(b)

0 1 2 3 4 5 60

10

20

30

40

50

60

Late

ncy

(s)


(c)

0

10

20

30

40

Obj

ect i

nter

actio

n (s

)

NovelOld



(d)






(a) (b)

(c) (d)

(e) (f)

0

2

4

6

8

CortexHippocampus

100minus

APPPS1WTminus

lowast

lowast

(AD

C10

minus4

mm

2s

)

(g)






0

500

1000

1500(p

gm

L)

100APPPS1WT

AP39 minus H2O

lowastlowast

A12057340

(a)

(pg

mL)

0

200

400

600

800

100APPPS1WT

AP39 minus H2O

lowastlowast

A12057342

(b)


(c)









2S or other H

2S donors and


5 Conclusions





Acknowledgments


References









































2S in the











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















OCR

(pm

olm

in120583

g pr

otei

n)

0 10 20 30 40 500

30

60

90

120

Oligo FCCP AA

Basalrespiration

Proton leak

ATPproduction

Maximalrespiration

capacity

ADP



(a)O

CR (p

mol

min

120583g

prot

ein)

Basalrespiration


Maximalrespiration

0

50

100

150



lowast

lowast

lowast

lowast

lowast

lowast

(b)


2O) (119875 lt 005)


2O spent


2O treatment were





40and A120573



40and A120573



0

1

2

3Cellular ATP level

ATP

conc

entr

atio

n (n

mol

)

AP39 100WT APPPS1

H2O 100H2O


(a)

00

05

10

15 DNA integrity

DN

A in

tegr

ity (

)


AP39 100WT APPPS1

H2O 100H2O

lowastlowast

(b)

150

100

50

0

Cou

nt

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

50

40

30

20

10

0

100

50

0

100

50

0


(c)

0

1

2

3

4

5Cellular ROS level

DCF

fluo

resc

ence

(rel

ativ

e uni

ts)

AP39 100WT APPPS1

H2O 100H2O

lowastlowast

(d)


(e)

Figure 5 Continued


0

1

2


JC-1

fluo

resc

ence

(rel

ativ

e uni

ts)

AP39 100WT APPPS1

H2O 100H2O

lowastlowast

(f)


2O)


4 Discussion










2S


2S donor






2S can act as an


2S







Fis1

Drp1

VDAC

Mfn1

Mfn2

OPA-1

AP39 100WT APPPS1

H2O 100H2O

(a)

0

50

100

150 Mfn1


lowastlowast

WT

(H2O

) (de

nsito

met

ry) (

)

(b)

0

50

100

150 Mfn2

WT

(H2O

) (de

nsito

met

ry) (

)


(c)

OPA-1

lowastlowast

0

50

100

150W

T (H

2O

) (de

nsito

met

ry) (

)


(d)

0

50

100

150

200 Fis1

WT

(H2O

) (de

nsito

met

ry) (

)


lowastlowast

(e)

0

50

100

150 Drp1

WT

(H2O

) (de

nsito

met

ry) (

)


(f)



0

20

40

60

80

CortexHippocampus

AP39 minus

(nM)25 100 250

H2S

prod

uctio

n (n

mol

mg

prot

ein)

50

(a)

0

20

40

60

80

100

CortexHippocampus

AP39APPPS1WT

H2S

prod

uctio

n (n

mol

mg

prot

ein)


lowastlowast

lowastlowast

lowastlowast

(b)



2S production



2O)



2S (3ndash















0 1 2 3 4 5 60

10

20

30

40

50

60La

tenc

y (s

)



(a)

0

20

40

60

80

Qua

dran

t tim

e (

)


lowastlowast

(b)

0 1 2 3 4 5 60

10

20

30

40

50

60

Late

ncy

(s)


(c)

0

10

20

30

40

Obj

ect i

nter

actio

n (s

)

NovelOld



(d)






(a) (b)

(c) (d)

(e) (f)

0

2

4

6

8

CortexHippocampus

100minus

APPPS1WTminus

lowast

lowast

(AD

C10

minus4

mm

2s

)

(g)






0

500

1000

1500(p

gm

L)

100APPPS1WT

AP39 minus H2O

lowastlowast

A12057340

(a)

(pg

mL)

0

200

400

600

800

100APPPS1WT

AP39 minus H2O

lowastlowast

A12057342

(b)


(c)









2S or other H

2S donors and


5 Conclusions





Acknowledgments


References









































2S in the











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

1

2

3Cellular ATP level

ATP

conc

entr

atio

n (n

mol

)

AP39 100WT APPPS1

H2O 100H2O


(a)

00

05

10

15 DNA integrity

DN

A in

tegr

ity (

)


AP39 100WT APPPS1

H2O 100H2O

lowastlowast

(b)

150

100

50

0

Cou

nt

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

50

40

30

20

10

0

100

50

0

100

50

0


(c)

0

1

2

3

4

5Cellular ROS level

DCF

fluo

resc

ence

(rel

ativ

e uni

ts)

AP39 100WT APPPS1

H2O 100H2O

lowastlowast

(d)


(e)

Figure 5 Continued


0

1

2


JC-1

fluo

resc

ence

(rel

ativ

e uni

ts)

AP39 100WT APPPS1

H2O 100H2O

lowastlowast

(f)


2O)


4 Discussion










2S


2S donor






2S can act as an


2S







Fis1

Drp1

VDAC

Mfn1

Mfn2

OPA-1

AP39 100WT APPPS1

H2O 100H2O

(a)

0

50

100

150 Mfn1


lowastlowast

WT

(H2O

) (de

nsito

met

ry) (

)

(b)

0

50

100

150 Mfn2

WT

(H2O

) (de

nsito

met

ry) (

)


(c)

OPA-1

lowastlowast

0

50

100

150W

T (H

2O

) (de

nsito

met

ry) (

)


(d)

0

50

100

150

200 Fis1

WT

(H2O

) (de

nsito

met

ry) (

)


lowastlowast

(e)

0

50

100

150 Drp1

WT

(H2O

) (de

nsito

met

ry) (

)


(f)



0

20

40

60

80

CortexHippocampus

AP39 minus

(nM)25 100 250

H2S

prod

uctio

n (n

mol

mg

prot

ein)

50

(a)

0

20

40

60

80

100

CortexHippocampus

AP39APPPS1WT

H2S

prod

uctio

n (n

mol

mg

prot

ein)


lowastlowast

lowastlowast

lowastlowast

(b)



2S production



2O)



2S (3ndash















0 1 2 3 4 5 60

10

20

30

40

50

60La

tenc

y (s

)



(a)

0

20

40

60

80

Qua

dran

t tim

e (

)


lowastlowast

(b)

0 1 2 3 4 5 60

10

20

30

40

50

60

Late

ncy

(s)


(c)

0

10

20

30

40

Obj

ect i

nter

actio

n (s

)

NovelOld



(d)






(a) (b)

(c) (d)

(e) (f)

0

2

4

6

8

CortexHippocampus

100minus

APPPS1WTminus

lowast

lowast

(AD

C10

minus4

mm

2s

)

(g)






0

500

1000

1500(p

gm

L)

100APPPS1WT

AP39 minus H2O

lowastlowast

A12057340

(a)

(pg

mL)

0

200

400

600

800

100APPPS1WT

AP39 minus H2O

lowastlowast

A12057342

(b)


(c)









2S or other H

2S donors and


5 Conclusions





Acknowledgments


References









































2S in the











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

1

2


JC-1

fluo

resc

ence

(rel

ativ

e uni

ts)

AP39 100WT APPPS1

H2O 100H2O

lowastlowast

(f)


2O)


4 Discussion










2S


2S donor






2S can act as an


2S







Fis1

Drp1

VDAC

Mfn1

Mfn2

OPA-1

AP39 100WT APPPS1

H2O 100H2O

(a)

0

50

100

150 Mfn1


lowastlowast

WT

(H2O

) (de

nsito

met

ry) (

)

(b)

0

50

100

150 Mfn2

WT

(H2O

) (de

nsito

met

ry) (

)


(c)

OPA-1

lowastlowast

0

50

100

150W

T (H

2O

) (de

nsito

met

ry) (

)


(d)

0

50

100

150

200 Fis1

WT

(H2O

) (de

nsito

met

ry) (

)


lowastlowast

(e)

0

50

100

150 Drp1

WT

(H2O

) (de

nsito

met

ry) (

)


(f)



0

20

40

60

80

CortexHippocampus

AP39 minus

(nM)25 100 250

H2S

prod

uctio

n (n

mol

mg

prot

ein)

50

(a)

0

20

40

60

80

100

CortexHippocampus

AP39APPPS1WT

H2S

prod

uctio

n (n

mol

mg

prot

ein)


lowastlowast

lowastlowast

lowastlowast

(b)



2S production



2O)



2S (3ndash















0 1 2 3 4 5 60

10

20

30

40

50

60La

tenc

y (s

)



(a)

0

20

40

60

80

Qua

dran

t tim

e (

)


lowastlowast

(b)

0 1 2 3 4 5 60

10

20

30

40

50

60

Late

ncy

(s)


(c)

0

10

20

30

40

Obj

ect i

nter

actio

n (s

)

NovelOld



(d)






(a) (b)

(c) (d)

(e) (f)

0

2

4

6

8

CortexHippocampus

100minus

APPPS1WTminus

lowast

lowast

(AD

C10

minus4

mm

2s

)

(g)






0

500

1000

1500(p

gm

L)

100APPPS1WT

AP39 minus H2O

lowastlowast

A12057340

(a)

(pg

mL)

0

200

400

600

800

100APPPS1WT

AP39 minus H2O

lowastlowast

A12057342

(b)


(c)









2S or other H

2S donors and


5 Conclusions





Acknowledgments


References









































2S in the











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Fis1

Drp1

VDAC

Mfn1

Mfn2

OPA-1

AP39 100WT APPPS1

H2O 100H2O

(a)

0

50

100

150 Mfn1


lowastlowast

WT

(H2O

) (de

nsito

met

ry) (

)

(b)

0

50

100

150 Mfn2

WT

(H2O

) (de

nsito

met

ry) (

)


(c)

OPA-1

lowastlowast

0

50

100

150W

T (H

2O

) (de

nsito

met

ry) (

)


(d)

0

50

100

150

200 Fis1

WT

(H2O

) (de

nsito

met

ry) (

)


lowastlowast

(e)

0

50

100

150 Drp1

WT

(H2O

) (de

nsito

met

ry) (

)


(f)



0

20

40

60

80

CortexHippocampus

AP39 minus

(nM)25 100 250

H2S

prod

uctio

n (n

mol

mg

prot

ein)

50

(a)

0

20

40

60

80

100

CortexHippocampus

AP39APPPS1WT

H2S

prod

uctio

n (n

mol

mg

prot

ein)


lowastlowast

lowastlowast

lowastlowast

(b)



2S production



2O)



2S (3ndash















0 1 2 3 4 5 60

10

20

30

40

50

60La

tenc

y (s

)



(a)

0

20

40

60

80

Qua

dran

t tim

e (

)


lowastlowast

(b)

0 1 2 3 4 5 60

10

20

30

40

50

60

Late

ncy

(s)


(c)

0

10

20

30

40

Obj

ect i

nter

actio

n (s

)

NovelOld



(d)






(a) (b)

(c) (d)

(e) (f)

0

2

4

6

8

CortexHippocampus

100minus

APPPS1WTminus

lowast

lowast

(AD

C10

minus4

mm

2s

)

(g)






0

500

1000

1500(p

gm

L)

100APPPS1WT

AP39 minus H2O

lowastlowast

A12057340

(a)

(pg

mL)

0

200

400

600

800

100APPPS1WT

AP39 minus H2O

lowastlowast

A12057342

(b)


(c)









2S or other H

2S donors and


5 Conclusions





Acknowledgments


References









































2S in the











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

20

40

60

80

CortexHippocampus

AP39 minus

(nM)25 100 250

H2S

prod

uctio

n (n

mol

mg

prot

ein)

50

(a)

0

20

40

60

80

100

CortexHippocampus

AP39APPPS1WT

H2S

prod

uctio

n (n

mol

mg

prot

ein)


lowastlowast

lowastlowast

lowastlowast

(b)



2S production



2O)



2S (3ndash















0 1 2 3 4 5 60

10

20

30

40

50

60La

tenc

y (s

)



(a)

0

20

40

60

80

Qua

dran

t tim

e (

)


lowastlowast

(b)

0 1 2 3 4 5 60

10

20

30

40

50

60

Late

ncy

(s)


(c)

0

10

20

30

40

Obj

ect i

nter

actio

n (s

)

NovelOld



(d)






(a) (b)

(c) (d)

(e) (f)

0

2

4

6

8

CortexHippocampus

100minus

APPPS1WTminus

lowast

lowast

(AD

C10

minus4

mm

2s

)

(g)






0

500

1000

1500(p

gm

L)

100APPPS1WT

AP39 minus H2O

lowastlowast

A12057340

(a)

(pg

mL)

0

200

400

600

800

100APPPS1WT

AP39 minus H2O

lowastlowast

A12057342

(b)


(c)









2S or other H

2S donors and


5 Conclusions





Acknowledgments


References









































2S in the











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 1 2 3 4 5 60

10

20

30

40

50

60La

tenc

y (s

)



(a)

0

20

40

60

80

Qua

dran

t tim

e (

)


lowastlowast

(b)

0 1 2 3 4 5 60

10

20

30

40

50

60

Late

ncy

(s)


(c)

0

10

20

30

40

Obj

ect i

nter

actio

n (s

)

NovelOld



(d)






(a) (b)

(c) (d)

(e) (f)

0

2

4

6

8

CortexHippocampus

100minus

APPPS1WTminus

lowast

lowast

(AD

C10

minus4

mm

2s

)

(g)






0

500

1000

1500(p

gm

L)

100APPPS1WT

AP39 minus H2O

lowastlowast

A12057340

(a)

(pg

mL)

0

200

400

600

800

100APPPS1WT

AP39 minus H2O

lowastlowast

A12057342

(b)


(c)









2S or other H

2S donors and


5 Conclusions





Acknowledgments


References









































2S in the











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c) (d)

(e) (f)

0

2

4

6

8

CortexHippocampus

100minus

APPPS1WTminus

lowast

lowast

(AD

C10

minus4

mm

2s

)

(g)






0

500

1000

1500(p

gm

L)

100APPPS1WT

AP39 minus H2O

lowastlowast

A12057340

(a)

(pg

mL)

0

200

400

600

800

100APPPS1WT

AP39 minus H2O

lowastlowast

A12057342

(b)


(c)









2S or other H

2S donors and


5 Conclusions





Acknowledgments


References









































2S in the











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

500

1000

1500(p

gm

L)

100APPPS1WT

AP39 minus H2O

lowastlowast

A12057340

(a)

(pg

mL)

0

200

400

600

800

100APPPS1WT

AP39 minus H2O

lowastlowast

A12057342

(b)


(c)









2S or other H

2S donors and


5 Conclusions





Acknowledgments


References









































2S in the











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Acknowledgments


References









































2S in the











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























2S in the











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article ap39, a mitochondria-targeted hydrogen...

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