Neuromol Med (2014) 16:175–190 DOI 10.1007/s12017-013-8272-8

ORIGINAL PAPER

Monitoring of Neuronal Loss in the Hippocampus of Ab-Injected Rat: Autophagy, Mitophagy, and Mitochondrial Biogenesis Stand Against Apoptosis Fatemeh Shaerzadeh • Fereshteh Motamedi • Dariush Minai-Tehrani • Fariba Khodagholi

Received: 28 June 2013 / Accepted: 23 October 2013 / Published online: 8 November 2013 Ó Springer Science+Business Media New York 2013

Abstract In the present study, we tried to answer the following questions: which kind of defense pathways are activated after Ab insult? How defense systems react against noxious effects of Ab and whether they are able to deal against apoptosis or not? So, we traced some molecular pathways including autophagy, mitophagy, and mitochondrial biogenesis before reaching to the endpoint of apoptosis. Besides, we measured the function of mitochondria after injection of Ab (1–42) in CA1 area of hippocampus as a model of Alzheimer’s disease (AD). Based on our data, autophagy markers reached to their maximum level and returned to the control level as apoptotic markers started to increase. As a specialized form of autophagy, mitophagy markers followed the trend of autophagy markers. Whereas mitochondrial dynamic processes shifted toward fission, mitochondrial biogenesis was severely affected by Ab and significantly decreased. Alongside suppression of mitochondrial biogenesis, activity of specific enzymes involved in antioxidant defense system,

F. Shaerzadeh  F. Motamedi  F. Khodagholi NeuroBiology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran F. Shaerzadeh  F. Motamedi Neurophysiology Research Center, Department of Physiology, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran F. Shaerzadeh  F. Motamedi  F. Khodagholi (&) Neuroscience Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran e-mail: [email protected] D. Minai-Tehrani Bioresearch Lab, Faculty of Biological Sciences, Shahid Beheshti University G.C., Tehran, Iran

electron transport chain, and tricarboxylic acid cycle (TCA) decreased in response to the Ab. Activity of antioxidant enzymes increased at first and then decreased significantly compared to the control. TCA enzymes aconitase and malate dehydrogenase activities reduced immediately while citrate synthase and fumarase activities did not change. Based on our finding, monitoring of the master molecules of intracellular cascades and determining their trends before the destructive function of Ab could be the target of therapeutic issues for AD. Keywords Alzheimer’s disease  Amyloid beta  Autophagy  Mitochondrial biogenesis  Apoptosis

Introduction Extracellular b-amyloid (Ab) and intracellular hyperphosphorylated tau protein depositions have been studied to be implicated in the histopathology of Alzheimer disease (AD) and the signaling of oxidative stress, inflammation, and eventually apoptosis (Braak and Braak 1991; Morishima-Kawashima and Ihara 2002; Christen 2000; Tuppoa and Arias 2009; Morishima et al. 2001). The most toxic and predominant form of Ab found in AD brains is Ab 1–42 (Glenner and Wong 1984; Haass and Selkoe 2007). It has been well documented that the presence of Ab leads to neurotoxicity even before generation of amyloid plaques (Watson et al. 2005; Haass and Selkoe 2007). Also, it has been shown that Ab directly and indirectly interacts with signaling molecules to activate and/or inactivate intracellular cascades eventually leading to neuronal death (Morishima et al. 2001; Troy et al. 2000; Yan et al. 1996; Small et al. 2009). It can be predicted that defense systems of neuronal cells, as an intelligent system, are activated

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immediately after Ab deposition or administration in order to deal with poisonous effects of Ab. Ab as an aggregated form of protein stimulates intracellular scavenging systems. Eukaryotic cells have two scavenging systems for degradation of unwanted proteins and organelles: the ubiquitin–proteasome system (UPS) and the autophagy–lysosome pathway. UPS core targets low molecular weight proteins (Goldberg 2003). In autophagy–lysosome pathway, misfolded and aggregated proteins are targeted by micro/macro- or chaperone-assisted autophagy pathways based on their structures (Ravikumar et al. 2009). Unlike micro- and chaperone-assisted autophagy that only remove dysfunctional proteins, macroautophagy additionally could digest organelles (Ravikumar et al. 2009; Li et al. 2011; Sahu et al. 2011). Macroautophagy (for simplicity after here referred to as autophagy) is an evolutionary conserved pathway existent from yeasts to mammals that acts as quality control (QC) mechanism of organelles and proteins with aberrant structures. Furthermore, products of a family of genes, known as autophagy-related genes (Atg), are involved in different processes of autophagy (Ravikumar et al. 2009; Tanida 2011; Klionsky et al. 2003). To be noticed, mitophagy is a specialized form of autophagy including processes targeting dysfunctional mitochondria directly to lysosomes (Ashrafi and Schwarz 2013; Narendra and Youle 2011). Since neurons are highly energy consuming cells and being mitochondria as the powerhouse of cells, mitochondrial biogenesis occurs in a parallel manner with mitophagy to maintain mitochondrial mass. Mitochondrial biogenesis is such a complex that is under control of both nuclear and mitochondrial genomes (Qi and Ding 2012). Moreover, specific mitochondrial enzymes have been determined in the modulatory processes of mitochondrial function. In the present study, we aimed to simulate the early stages of events occurring in the rats with Ab injection into their hippocampal CA1 area. Along with our investigations on autophagy pathway as a clearance mechanism, we determined probable alterations in mitophagy, mitochondrial biogenesis and dynamic as major mitochondrial QC mechanism.

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(CREB), p-CREB, cytochrome c, caspase-3, light chain 3 (LC3), Atg7, Atg12, dynamin-related protein 1 (Drp1), p70S6 kinase (p70S6K), and p-p70S6K were obtained from Cell Signaling Technology (Beverly, MA, USA). Nuclear respiratory factor-1(NRF-1), parkin, mitofusin (Mfn) 2, and PTENinduced putative kinase-1 (PINK-1) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1a) and mitochondrial transcription factor A (TFAM) were bought from ABCAM (Cambridge, UK) and BioVision (Palo Alto, CA, USA), respectively. Animals All procedures of animal handling were confirmed by National Institutes of Health Publication (No. 80-23, revised 1996) and Animal Care Committee of Shahid Beheshti University of Medical Sciences. Adult male Albino Wistar rats, weighing 240–280 g, were obtained from Pasteur institute (Tehran, Iran). Rats were housed under standard laboratory conditions (12 h/12 h light/dark cycle with free access to food and water). Ab Preparation and Thioflavin T (ThT)-Induced Fluorescence The solid Ab 1–42 (200 ng/ll) was dissolved in phosphatebuffered saline (PBS 0.1 M) and incubated at 37 °C for 5 days to get a fibrils form. This condense solution was diluted with PBS for injection (10 ng/ll). In order to confirm the amyloid fibril formation, ThT-induced fluorescence changes were measured spectrofluorophotometrically (Cottingham et al. 2002). Briefly, Th T reagent (10 lM) in phosphate buffer (100 mM, pH 7.4) was added to solution containing Ab 1–42 (50 mM). The fluorescent intensity was measured at the excitation/emission wavelengths of 450/490 nm. Based on the data analysis, 5-day incubation was conducted due to well-fibrilled formation of Ab. Experimental Procedures

Materials and Methods

Animals were divided into 11 groups. First group was injected intrahippocampally by 3 ll PBS and immediately killed (control group). Others received 3 ll of Ab 1–42 (2.2 nM) in CA1 area of hippocampus bilaterally.

Materials

Stereotaxic Surgery and Micro-Injection

All applied materials and reagents in the present study were from Sigma Aldrich (St. Louis, MO, USA) except those mentioned in the following text. Antibodies directed against pyruvate dehydrogenase (PDH), AMP-activated protein kinase (AMPK), p-AMPK, b-actin, c-AMP response element-binding

For Ab injection using a Hamilton syringe, the animals were anesthetized with an intramuscular injection of 0.2 ml mixture of ketamine hydrochloride (80 mg/kg) and xylazine (10 mg/kg). They were then injected into the CA1 area coordinates of hippocampus bilaterally by using stereotaxic

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apparatus [AP: 3.8 mm, L: ±3.2 (both respect to bregma), and DV: 2.8 mm (from the skull surface)] according to the rat brain atlas of Paxinos and Watson (2007). All microinjections were performed slowly over a period of 60 s. Tissue Collection All the animals were anaesthetized and decollated. Brain tissues were immediately excised and rinsed in ice-cold PBS. The hippocampus region was isolated and immediately stored at -80 °C. Mitochondrial Isolation Dissected hippocampi were minced finely in ice-cold isolation medium (0.25 M sucrose, 10 mM Tris, 0.5 mM K? EDTA, pH 7.4). Tissues were manually homogenized in isolation buffer and then centrifuged at 2,0009g for 3 min at 2 °C. The supernatant fraction was collected and centrifuged again at 12,5009g for 8 min. The resultant mitochondrial pellets were then washed and resuspended in a 3 % Ficoll medium (3 % Ficoll 0.12 M, mannitol 0.03 M, sucrose 25 lM and K? EDTA, pH 7.4) and then in a 6 % Ficoll medium. In brief, the solution was centrifuged at 11,5009g for 30 min, and then, the resultant was solved in isolation medium (Clarke and Nicklas 1970). Protein concentrations of samples were determined using colorimetric method of Bradford (Bradford 1976).

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mitochondria were added to a medium containing 100 mM Tris–HCl, pH 8.0, 0.1 % Triton X-100 at 30 °C, 10 mM acetyl-CoA, and 20 mM 5–50 -dithiobis(2-nitrobenzoic acid) (DTNB). The enzymatic reaction was started by the addition of 20 mM oxaloacetate. Changes in the absorbance were assessed at 412 nm wavelength. Determination of Aconitase Activity Aconitase activity was assayed using the method reported by Zheng et al. (1998) based on the conversion of citrate to cis-aconitate. Mitochondria of hippocampal neurons were added to a medium containing 50 mM Tris–HCl, 30 mM sodium citrate, and 0.6 mM fresh MnCl2. The reaction was initiated by the addition of mitochondria. Changes in the absorbance were monitored at 240 nm applying a UV/VIS spectrophotometer. Determination of Fumarase Activity As known, fumarase or fumarate hydratase catalyzes the reversible reaction that converts fumarate to L-malate. Then, fumarase activity was measured by following the changes in fumarate concentration (Racker 1950). Isolated mitochondria were added to the assay mixture containing 0.1 M phosphate buffer, pH 7.4, 0.3 M sodium L-malate, 0.2 and 0.25 M sucrose. Absorption was evaluated at 240 nm.

Determination of Antioxidant Superoxide Dismutase (SOD) Activity

Measurement of Malate Dehydrogenase (MDH) Activity

SOD activity was measured based on the method of Kakkar et al. (1984) by some modifications. Isolated mitochondria were added to assay mixture containing sodium pyrophosphate buffer (pH 8.3, 0.052 M), phenazine methosulphate (186 lM), and nitroblue tetrazolium (300 lM). Reaction was started by the addition of nicotinamide adenine dinucleotide (NADH) (780 lM) and stopped by adding glacial acetic acid. Color intensity was measured spectrophotometrically at 560 nm.

MDH activity was measured as described by Wang et al. (2010). Assay mixture was composed of 5.6 mM oxaloacetate in Tris–HCl (56 mM, pH 7.5) and 10 ll of NADH solution containing 0.5 mg NADH in 100 ll of Tris–HCl (50 mM, pH 9); the reaction was initiated by the addition of freshly isolated sample. Absorbance was monitored at 340 nm with a UV/VIS spectrophotometer.

Measurement of Catalase Activity

Reduced glutathione level was determined by the method of Ellman (1959). Sixty microgram of mitochondrial lysate was added to Ellman’s reagent containing 19.8 mg of DTNB in 100 ml of 0.1 % sodium nitrate and phosphate buffer (0.2 M pH 8.0). The absorbance was measured at 412 nm.

Catalase activity was determined according to the method described by Aebi (1984). Briefly, H2O2 (0.01 M) was added to 60 lg of isolated mitochondria. The break down rate of H2O2 by the activity of catalase was monitored at 240 nm, spectrophotometrically.

Measurement of Reduced Glutathione (GSH)

Spectrophotometric Analysis of Electron Transport Chain (ETC) Complexes

Measurement of Citrate Synthase (CS) Activity CS activity was measured based on the method described by Powell and Jackson (2003). Briefly, isolated

Complex I The oxidation of NADH by complex I was recorded using an electron acceptor, decylubiquinone (BirchMachin et al. 1989). The assay medium (35 mM KH2PO4,

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5 mM MgCl2, and 2 mM KCN, pH 7.2) was supplemented with 65 mM decylubiquinone and 10 mM NADH. The reaction was started by the addition of isolated mitochondria. The decrease in absorption was measured at 340 nm. Complex II–III Accumulative activity of complex II and III was assayed in medium buffer containing 50 mM Tris– HCl, 100 mM EDTA, 2 mM succinate, and 1 % dodecylmaltoside. The reaction was initiated adding 100 mM cytochrome c. Alteration in complexes activity was measured at 550 nm (Veereshwarayya et al. 2006). Complex IV The oxidation of cytochrome c was monitored at 550 nm at 37 °C according to the method originally described by Rustin et al. (1991). The reaction buffer was composed of 40 mM potassium phosphate, pH 7.0, and homogenate proteins. The reaction was initiated by the addition of reduced cytochrome c. Measurement of Cytochrome Contents Spectra of the mitochondrial cytochrome were detected using double-beam spectrophotometer (PerkinElmer lambda25). Isolated mitochondria were reduced and oxidized by adding a small quantity of sodium dithionate and hydrogen peroxide, respectively. Reduced minus oxidized spectra were obtained at 400–650 nm. The extinction coefficient for cytochrome c was 19/mM/cm at 540–550 wavelengths and for cytochrome b was 22/mM/cm at 563–575 wavelengths (Jones and Poole 1985). Western Blot Analysis The brains and consequently hippocampi were dissected out immediately and homogenized in a lysis buffer containing protease inhibitor (n = 6). Proteins (60 lg) were separated by SDS–PAGE gel and transferred to the polyvinylidene difluoride membrane (Millipore Corporation, Billerica, MA, USA). The membranes were blocked in 2 % non-fat dry milk in TBST and then incubated with primary antibodies (1:1,000 dilutions) overnight. Subsequently, the membranes were washed and incubated with HPR-conjugated secondary antibody. Different emittances of protein were detected with Electrochemiluminescence kit (Amersham Bioscience, Piscataway, NJ, USA) and then exposed to X-ray films. Relative density of the protein bands was measured by Image J software.

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Sections Preparation and Terminal-Transferase dUTP Nick End Labeling (TUNEL) Staining In each group, rats were perfused through the ascending aorta with PBS, followed by fresh 4 % paraformaldehyde in PBS (pH 7.4) (n = 4). The brains were removed rapidly and placed in 4 % paraformaldehyde in PBS for 24 h. Then, the tissues were embedded with paraffin and sectioned at a thickness of 5 lm. The TUNEL method was performed using a TUNEL assay kit (Chemicon Millipore, Temecula, CA, USA) to detect apoptotic cells in rat hippocampi. In brief, after the sections were dewaxed and hydrated, endogenous peroxidase activity was blocked by incubating with 3 % hydrogen peroxide for 3–5 min at room temperature. After washing with PBS, the sections were digested with 20 lg/ml proteinase K at room temperature for 15 min. Then, tissues were incubated with 50 ll TUNEL reaction mixture for 60 min at 37 °C and further incubation with 50 ll converter-POD for 30 min at 37 °C. After washing with PBS, sections were incubated with 50 ll substrate solution diaminobenzidine (DAB) for 10 min at 15–25 °C and rinsing with PBS was done in the next step. Counter staining was achieved by 0.5 % methyl green. For positive staining, tissues were incubated with DNase solution for 10 min at 15–25 °C. Then, sections were dehydrated and cover-slipped for analysis under light microscopy. Negative controls were performed by the omission of enzyme solution. Four images were randomly selected for each section, and the positive cells were counted (n = 4). The image for each group represented in the Fig. 1d is of four images taken. The apoptotic index was expressed as the average percentage of TUNEL-positive nerve cells. Immunofluorescence The prepared sections were blocked with goat serum and incubated with LC3-specific antibody (1: 400 dilution) overnight at 4 °C followed by fluorescent secondary antibody Alexa Fluor 488-conjugated anti-rabbit IgG (1:500, Molecular Probes, Invitrogen, CA). Data analysis was conducted using a fluorescence microscope (Olympus IX71, Japan). Data Analysis

Caspase-3 Activity Assay To measure caspase-3 activity, we used caspase-3/CPP32 Colorimetric kit (Invitrogen, Molecular Probes, CA, USA) in total hippocampus extracts and read the absorbance according to the manufacturer’s protocol.

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All data are represented as the mean ± SEM (n = 4–6). Comparison between groups was made by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. The statistical significances were achieved when P \ 0.05 (*P \ 0.05, **P \ 0.01, ***P \ 0.001).

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Day Fig. 1 Intrahippocampal injection of Ab 1–42 induces apoptotic cell death in a time-dependent manner. a Caspase-3 cleavage in the presence of Ab 1–42 in hippocampus neurons during 10 days. 60 lg proteins are separated on SDS–PAGE, transferred, probed with anticaspase-3 and reprobed with anti-b-actin, respectively (one representative Western blot is shown; n = 6). b The densities of caspase-3

bands were measured and the ratio to the b-actin was calculated. c Caspase-3 activity after Ab 1–42 injection using a colorimetric kit. d Representative microphotographs of coronal sections obtained from hippocampus CA1 area and stained by TUNEL kit (scale bar 20 lm). e Represents the quantitative analysis of the data, n = 4.*P \ 0.05; **P \ 0.01; ***P \ 0.001 different from the control group

Results

procedure of apoptosis pathway in the presence of Ab, we measured caspase-3 activity using a colorimetric kit, its cleavage level by Western blotting analysis, and morphological evaluation by TUNEL assay. As shown in Fig. 1b, the cleavage of caspase-3 started to increase insignificantly 3 days after Ab injection. This increase became significant 7 days after Ab injection. Moreover, the highest level of caspase-3 was detected on the 8th day. The same pattern

Impact of Ab on Cleavage of Caspase-3 in Hippocampal Cells Cleavage of caspase-3 occurs at the latest stages of apoptosis and known as endpoint process of this pathway (Saikumar et al. 1999). So, in order to determine the

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Fig. 2 Intrahippocampal injection of Ab 1–42 induces autophagic factors time dependently. a 60 lg proteins was separated on SDS– PAGE, Western blotted, probed with specific primary antibodies, and reprobed with anti b-actin antibody (one representative Western blot is shown; n = 6). b The densities of LC3II and I bands were measured and their ratio was calculated. Each point shows the mean ± SEM. c The densities of Atg7 bands were measured and the ratio to the b-actin bands was calculated. d The ratio of Atg12-Atg5

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complex bands to the b-actin bands densities. e The ratio of P62 bands to the b-actin bands densities. f Representative images of immunofluorescently stained brain sections from Ab-injected rat. Rats were injected by Ab1-42. The brains were dissected, fixed, sectioned, and stained with anti-LC3 antibody and secondary antibody Alexa Fluor 488-conjugated (green) and DAPI (blue), n = 4 (scale bar 50 lm). (*P \ 0.05; **P \ 0.01; ***P \ 0.001 different from the control group) (Color figure online)

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immediately after the presence of Ab (Tanida 2011). Another autophagic factor, P62 known as ubiquitinated cargo receptor, increased significantly 2 days after Ab injection (Fig. 2e). P62 delivers targets to autophagosomes (Lamark et al. 2009). Levels of autophagic factors started to fall down 6 days after Ab injection.

was observed for caspase-3 activity, as well (Fig. 1c). Histological analysis also confirmed more TUNEL-positive (apoptotic) cells in the sections of rats more than 7 days after Ab injection (Fig. 1d, e). It should be mentioned that while biochemical assays were performed on entire hippocampus, all morphological data referred to specific subdivision, i.e. CA1 area.

Impact of Ab on Parkin-Mediated Clearance of Mitochondria in Rat Brain

Influence of Ab on Autophagy Markers in Hippocampal Neurons in a Time-Dependent Manner

Depolarized mitochondrial membrane as a result of its disturbance leads to the localization of PINK-1 on cytoplasmic face of outer mitochondrial membrane. Parkin, E3 ubiquitin ligase, is recruited to mitochondria by PINK-1 (Narendra and Youle 2011). Moreover, it has been reported that parkin suppresses ceramide-mediated cell death via prevention of mitochondrial swelling (Darios et al. 2003). As shown in Fig. 3b, 3 days after Ab injection, the level of PINK-1 increased significantly and continued its trends for 5 days. Five days after Ab injection, level of PINK-1 increased 1.6-fold compared to the control group. With a delay, elevation of parkin level was observed (Fig. 3c). The interval time between the changes in PINK-1 and parkin level presumably is because of the required time for PINK-1 localization on mitochondrial membrane.

As known, LC3 has a role in the elongation and closure of the isolation membrane in autophagy processes (Fujita et al. 2009). As shown in Fig. 2b, 5 days after Ab injection, LC3II/LC3I ratio reached its highest level. At the mentioned time, LC3II/LC3I ratio increased 1.4-fold relative to the control group. Consistent with Western blot finding, our data from immunofluorescence method also revealed the highest level of LC3 5 days after Ab injection (Fig. 2f). Also, 5 days after Ab injection, the level of Atg7 and Atg12-5 complex (Atgs proteins family members) increased 1.6- and 1.5-fold, respectively, compared to the control group (Fig. 2c, d). Changes in Atg7 level were significantly higher than the control group 2 days after Ab injection (Fig. 2c). Based on the role of Atg7 in LC3 and Atg12 activation, its elevation in level seems reasonable

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Fig. 3 Time course effect of Ab 1–42 intrahippocampal injection on mitophagy factors. a 60 lg proteins was separated on SDS–PAGE, Western blotted, probed with specific primary antibodies, and reprobed with anti b-actin antibody (one representative Western blot is shown; n = 6). b The densities of PINK-1 bands were measured

and the ratio to the b-actin bands was calculated. Each point shows the mean ± SEM. c The ratio of parkin bands to the b-actin bands densities. (*P \ 0.05; **P \ 0.01; ***P \ 0.001 different from the control group)

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Influence of Ab on Mitochondrial Dynamic Markers

Fig. 5 Time course effect of Ab 1–42 intrahippocampal injection on c mitochondrial biogenesis factors. a 60 lg proteins was separated on SDS–PAGE, Western blotted, probed with specific primary antibodies, and reprobed with anti b-actin antibody (one representative Western blot is shown; n = 6). b The densities of pAMPK bands were measured and the ratio to the AMPK bands was calculated. Each point shows the mean ± SEM. c The ratio of pCREB bands to the CREB bands densities. d The ratio of pP70S6K bands to the P70S6K bands densities. e The ratio of PGC-1a bands to the b-actin bands densities. f The ratio of NRF-1 bands to the b-actin bands densities. g The ratio of TFAM bands to the b-actin bands densities. h The ratio of cytochrome c bands to the b-actin bands densities. (*P \ 0.05; **P \ 0.01; ***P \ 0.001 different from the control group)

Fission and fusion, two main processes of mitochondrial dynamic, constantly occur in mitochondrial network (Chen and Chan 2009). Fragmented and/or enlargement mitochondria are the results of disruption in fission–fusion equilibrium (Cipolat et al. 2004). To determine the mitochondrial dynamic toward fission and/or fusion, we assessed the level of Drp-1 as fission and Mfn2 as fusion markers. As shown in Fig. 4c, increasing the level of Drp-1 coincided with autophagy and mitophagy markers and reached to the maximum level 5 days after Ab injection. This increase in Drp-1 level was 1.5-fold relative to its basal level in control group, while the level of Mfn2 slightly increased and then declined significantly in the rats killed 5 days after injection of Ab compared to the control group (Fig. 4b).

master regulator of mitochondrial biogenesis (Scarpulla 2011). Based on the critical role of mitochondrial biogenesis on cell survival, we traced the alteration in PGC-1a, NRF-1, TFAM, and cytochrome c levels to determine the trend of their changes in the presence of Ab. Besides, we measured the level of phosphorylated form of AMPK and CREB as well as p70S6k functioning upstream to PGC-1a (Wenz 2013; Wang et al. 2011). As shown in Fig. 5, the levels of mitochondrial biogenesis markers decreased in the presence of Ab. The level of PGC-1a begins to decrease 2 days after Ab injection and continued by 10 days (Fig. 5e). Also, phosphorylation of AMPK, CREB, and p70S6K decreased in the presence of Ab (Fig. 5b–d). Interestingly, upstream

Effect of Ab on Mitochondrial Biogenesis Markers During 10 Days in Hippocampus Neurons Mitochondrial biogenesis is a multiplex process of cooperation between mitochondrial and nuclear transcriptional factors (Scarpulla 2002; Gleyzer et al. 2005). Mitochondrial biogenesis is governed in different levels. A considerable part of regulation is impressed on PGC-1a introduced as a

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Fig. 4 Time course effect of Ab 1–42 intrahippocampal injection on mitochondrial dynamic factors Mfn2 and Drp-1. a 60 lg proteins was separated on SDS–PAGE, Western blotted, probed with specific primary antibodies, and reprobed with anti b-actin antibody (one representative Western blot is shown; n = 6). b The densities of

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Mfn2 bands were measured and the ratio to the b-actin bands was calculated. Each point shows the mean ± SEM. c The ratio of Drp-1 bands to the b-actin bands densities. (*P \ 0.05; **P \ 0.01; ***P \ 0.001 different from the control group)

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factors of PGC-1a are affected sooner than the downstream like NRF-1, Tfam, and cytochrome c. Impact of Ab on Antioxidant Enzymes During 10 Days Following Ab Injection As the main sources of endogenous ROS production, antioxidant enzymes including SOD and catalase are abundant in mitochondria (Kohler et al. 2009). Based on the interconnection between oxidative stress and Ab, we followed the activity of antioxidant enzymes in the presence of Ab in a period of 10 days. As shown in Table 1, the activity of SOD and catalase enzymes elevated significantly compared to control group 1 day after Ab injection. Then, their activities returned to the basic level 72 h after Ab injection. From this point, activity of antioxidant enzymes started to decrease notably compared to the control group and continued up to 10 days (Table 1). Effect of Ab on GSH Content of Hippocampal Neurons in a Time-Dependent Manner Tripeptide molecule GSH is one of the most important compounds involved in detoxification of free radicals because of its antioxidant properties (Jones 2002). Based on our data, the level of GSH increased immediately after the injection of Ab, but 72 h later its level dropped down to its basal level. Then, its content decreased to 93 % compared to the control group 4 days after Ab injection, and this trend continued up to 10 days (Table 1). Influence of Ab on the Activity of ETC Complexes in the Period of 10 Days

of H? gradient across the inner membrane by transferring electron from NADH and flavin adenine dinucleotide (FADH2) to the oxygen (Adam-Vizi and Chinopoulos 2006). The complexes because of their structures and locations are targeted by Ab and their functions are destructed in the presence of Ab (Rhein et al. 2009). Based on our data, decline trend in complex IV activity was initiated 24 h after Ab injection and decreased significantly 5 days, while the activities of complex I and II–III were affected by Ab 2 and 4 days after Ab injection, respectively. This observation could be due to the sensitivity and deeper location of complex IV in the membrane (Mora´n et al. 2012). The slope of alteration in complex II–III activity was lesser than two other complexes (Table 2). Effect of Ab Injection in Hippocampus on PDH Level in 10 Days PDH enzyme generates 2 carbon units that enter the TCA cycle and react with oxaloacetate. Regulation of PDH activity is considered as one of the controlling points of TCA activity. As shown in Fig. 6b, the level of PDH decreased 3 days after Ab injection in CA1 area of hippocampus. Effect of Ab Injection on TCA Enzymes Activity in Neuronal Cells of Hippocampus Based on the majority role of TCA cycle enzymes in adenosine triphosphate (ATP) production and cell metabolism, any changes in its elements’ activity could affect cell function, widely. So, we followed the behavior of some

Mitochondrial respiratory complexes locating in the inner membrane of mitochondria are involved in the generation Table 1 Time course effect of Ab 1–42 on SOD and catalase activity and GSH content of neurons of rat hippocampus Groups

SOD (U/mg protein)

Catalase (nmol/ mg protein)

Control

78.34 ± 5.1

2.46 ± 0.078

Table 2 Time course effect of Ab 1–42 on mitochondrial complex I, II/III, and IV activity neurons of rat hippocampus Groups

Complex I (nmol/min/mg protein)

11.8 ± 0.54

Control

58.63 ± 3.7

25.81 ± 1.53

3.73 ± 0.75

57.6 ± 2.45

25.72 ± 2.07

3.16 ± 0.59

53.14 ± 2.05**

24.85 ± 1.2

2.78 ± 0.61

GSH (U/mg protein)

Complex II/III (nmol/min/mg protein)

Complex IV (nmol/min/mg protein)

Day 1

83.00 ± 4.21*

2.61 ± 0.097***

13.72 ± 0.5***

Day 1

Day 2

79.01 ± 5.37

2.79 ± 0.093***

13.140 ± 0.59***

Day 2

Day 3

75.53 ± 4.9

2.28 ± 0.082

11.61 ± 0.48

Day 3

47.68 ± 2.1***

24.16 ± 1.6

2.52 ± 0.67

Day 4

72.79 ± 4.4**

1.98 ± 0.088***

11.02 ± 0.47***

Day 4

46.26 ± 3.9***

23.32 ± 1.9*

2.65 ± 0.52

Day 5

71.02 ± 4.8***

2.04 ± 0.091***

10.67 ± 0.52***

Day 5

38.43 ± 2.55***

21.67 ± 1.5***

2.30 ± 0.59*

Day 6

40.33 ± 2.15***

21.15 ± 2.02***

2.53 ± 0.51

Day 6

69.61 ± 6.1***

1.95 ± 0.094***

10.19 ± 0.58***

Day 7

69.68 ± 4.15***

1.99 ± 0.083***

10.04 ± 0.49***

Day 7

33.45 ± 3.1***

20.66 ± 1.95***

2.15 ± 0.48*

9.79 ± 0.57***

Day 8

33.92 ± 1.8***

19.67 ± 1.83***

1.81 ± 0.53**

Day 8

67.61 ± 5.09***

1.82 ± 0.095***

Day 9

67.68 ± 4.5***

1.7 ± 0.087***

Day 10

65.88 ± 3.8***

1.5 ± 0.083***

9.68 ± ± 0.63***

Day 9

31.8 ± 1.47***

18.83 ± 1.72***

1.61 ± 0.62**

8.98 ± 0.47***

Day 10

30.6 ± 2.35***

18.01 ± 2.12***

1.39 ± 0.41***

n=4

n=4

* P \ 0.05; ** P \ 0.01; *** P \ 0.001

* P \ 0.05; ** P \ 0.01; *** P \ 0.001

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185

A

B

1.8

Day Control 1

2

3

4

5

6

7

8

9

10

PDH

43 kDa

β-actin

45 kDa

PDH/β-actin ratio (Arbitrary Unit)

1.6 1.4

***

1.2

*** ***

1

*** ***

***

***

0.8

***

0.6 0.4 0.2 0

Day

Fig. 6 Time course effect of Ab 1–42 intrahippocampal injection on PDH. a 60 lg proteins were separated on SDS–PAGE, Western blotted, probed with PDH primary antibody, and reprobed with anti b-actin antibody (one representative Western blot is shown; n = 6). Table 3 Time course effect of Ab 1–42 on CS, aconitase, fumarase, and MDH activity neurons of rat hippocampus

Groups

CS (nmol/min/mg protein)

b The densities of PDH bands were measured and the ratio to the b-actin bands was calculated. Each point shows the mean ± SEM. (*P \ 0.05; **P \ 0.01; ***P \ 0.001 different from the control group)

Aconitase (nmol/min/mg protein)

Fumarase (U/g protein)

MDH (nmol/min/ mg protein)

Control

19.5 ± 2.2

35.9 ± 2.82

98.24 ± 4.1

23.27 ± 1.4

Day 1

20.54 ± 1.9

34.29 ± 2.59

94.24 ± 3.97

21.77 ± 1.07

Day 2

20.68 ± 1.2

Day 3

21.2 ± 1.5

32.52 ± 1.05

89.80 ± 4.5

19.34 ± 1.2*

23.54 ± 1.7***

79.60 ± 4.2

17.26 ± 1.05*

Day 4

21.11 ± 1.7

20.08 ± 3.14***

71.86 ± 5.2

16.60 ± 1.7*

Day 5

19.68 ± 2.3

21.36 ± 2.03***

79.18 ± 3.7

17.01 ± 1.33*

Day 6

20.22 ± 1

21.17 ± 3.53***

81.88 ± 4.9

16.84 ± 1*

Day 7

19.97 ± 1.9

17.47 ± 3.22***

72.72 ± 4.9

16.16 ± 1.5*

Day 8

19.61 ± 1.6

14.16 ± 2***

88.63 ± 4.3

17.21 ± 1.6*

n=4

Day 9

21.25 ± 2.5

13.55 ± 1.07***

95.06 ± 4.1

16.82 ± 0.9*

* P \ 0.05; ** P \ 0.01; *** P \ 0.001

Day 10

20.17 ± 1.8

12.03 ± 3.02***

92.51 ± 3.8

15.93 ± 1.8*

of TCA cycle enzymes after Ab injection. As shown in Table 3, activity of CS did not show any changes during 10 days of Ab presence, but activity of aconitase decreased notably 72 h after Ab injection. The activities of two other enzymes, fumarase and MDH, are also monitored in the presence of Ab. As shown in Table 3, activity of MDH enzyme decreased significantly 2 days after Ab injection compared to the control group. To be noticed, our data revealed that fumarase activity was not affected in the presence of Ab.

Table 4 Time course effect of Ab 1–42 on cytochrome content of neurons of rat hippocampus Groups Control Day 1

Cytochrome c (mM/mg protein)

Cytochrome b (mM/mg protein)

4 9 10-3

3.9 9 10-3

-3

3.7 9 10-3

-3

3.9 9 10

Day 2 Day 5

2.8 9 10 ** 2.5 9 10-3***

2.7 9 10-3** 2.4 9 10-3***

Day 7

2.3 9 10-3***

2.4 9 10-3***

n=4 * P \ 0.05; ** P \ 0.01; *** P \ 0.001

Effect of Ab on Cytochrome Content of Hippocampal Neurons Cytochromes belong to the family of hemeproteins (Hui Bon Hoa et al. 2002). Because of their particular structures, cytochromes involve in electron transport in ETC eventually leading to ATP synthesis. Based on their essential role, we assessed the content of cytochrome c and b in Ab-injected hippocampi. As shown in Table 4, alterations in cytochrome c and b values took place, synchronously. Moreover, 2 days

after Ab injection, quantity of cytochrome c and b decreased significantly compared to the control group.

Discussion The main purpose of this study was to answer these questions: which kind of defensive pathways are activated after Ab insult? How defense systems react against noxious

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186

effects of Ab and whether they are able to oppose apoptosis or not? So, we first evaluated the level and activity of caspase-3 in order to determine apoptotic cell death. Based on our data, 8 days after Ab injection the cleavage of caspase-3 was in its peak and after this point decreased gradually as time passed. Cleavage of caspase-3 is known as irreversible step of apoptosis that leads to elimination of neurons (Stadelmann et al. 1999). Hence, it would be envisaged that any effort of neuronal cells to survive occurs to combat against this commitment point of apoptosis. For these purposes, cellular defense systems should be activated. Autophagy was the first signaling pathway that we tried to investigate its trend in a period of 10 days after the presence of Ab particularly before remarkable cleavage of caspase-3. There are conflicting reports about the role of autophagy in central nervous system (CNS). As de Duve and Wattiaux have used the term of autophagy for the first time, autophagy is induced under starvation conditions to provide metabolic components for cell survival (1966). Later observations have indicated that autophagy impairment involves neurodegenerative disorders like AD, Parkinson’s disease, and Huntington’s disease (Rubinsztein 2006; Levine and Kroemer 2008). Furthermore, it was also shown that inhibition of autophagy induces degeneration of neuronal cells in CNS (Hara et al. 2006; Komatsu et al. 2006). On the other hand, Yu et al. introduced autophagosomes as the new source of Ab production in neurons (2005). In addition to its beneficial effects, autophagy is introduced as type II of programmed cell death versus type I, apoptosis (Schweichel and Merker 1973). Accordingly, it seems that autophagy has a dual role in CNS. Besides these controversial reports, we monitored the autophagy markers 10 days after Ab injection in order to determinate primitive events occurred at early stages of AD. We used Ab-injected rat model of AD to resemble the pathology of AD. Elevation of LC3II level accompanied by Atg7 and Atg5–12 complexes revealed that the level of autophagy elevated since the second day of Ab administration and reached to its maximum level 5 days later. It seems that following the presence of Ab, autophagy is induced that has been confirmed by immunofluorescently detection of LC3 (Fig. 1f). However, this could not be kept up after a while and the markers then decreased in a mild manner, followed by increased caspase-3 cleavage. Consistent with some previous studies, our data show supportive evidence for the protective role of autophagy at the early stages of AD. Eisenberg-Lerner et al. (2009) have shown that cross-talk between apoptosis and autophagy depends on cell settings and conditions. They can interplay as agonistic and/or antagonistic ways. Therefore, autophagy in stress situation could determine cell fate by accompanying and/or counteracting apoptosis. Mitochondria are the major source of ROS production within the cells (Murphy 2009). In non-pathological

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situations, 1–5 % of electrons escape from complexes I and III along the ETC and produce ROS that lately are detoxified by antioxidant defense system enzymes like SOD, catalase, and glutathione peroxidase (Sazanov 2007; Muller et al. 2004; Murphy 2009). Based on our data, 4 days after Ab injection, activity of antioxidant enzymes notably decreased, whereas, during the first 2 days, activity of these enzymes increased significantly. Monitoring complexes I, II, III, and IV indicated that their activities were affected by Ab before than antioxidant enzymes. We propose that disturbance of mitochondrial ETC complexes by Ab results in excessive production of ROS. As a defensive response, antioxidant enzymes activity induces the detoxification of ROS resulted from complexes. It seems that the challenge between antioxidant enzymes and exacerbated ROS production continues until ROS defeats antioxidant system and disrupts its function or being neutralized by the system. These data are in accordance with Kim et al. (2003) report revealing that SOD and glutathione oxidant system immunoreactivity are reduced by Ab. Our data from cytochrome spectra measurement also confirmed detrimental effect of Ab on ETC complexes. Ab results in dysfunctional and depolarized mitochondria. Dysfunctional mitochondria are targeted by parkin-mediated mitochondrial clearance, known as mitophagy (Narendra and Youle 2011). Based on our data, mitophagy were induced 3 days after Ab injection and continued until the fifth day. It is predictable that mitophagy factors have the same pattern as autophagy. It seems that significant induction of mitophagy is synchronized with deactivation of antioxidant enzymes, demonstrating that over production of ROS results in mitochondria dysfunction. The cooperation between PINK-1, parkin and P62 with LC3 and Atgs leads to the elimination of mitochondria. As known, neurons are glucose-dependent cells and main part of glucose metabolism takes place in mitochondria. So, replacement of eliminated dysfunctional mitochondria is an inevitable strategy. Induction of mitochondrial dynamic process and mitochondrial biogenesis pathway are two major events for the regeneration of new mitochondria (Zhu et al. 2013). Mitochondria are highly dynamic organelles with perpetually division and fusion. Based on cells conditions, the balance tends to fission and/or fusion (Chen and Chan 2009; Cipolat et al. 2004). It has been shown that elevated level of dysfunctional mitochondria shifts mitochondrial dynamic process toward fission (Santos et al. 2010). In agreement with the previous findings, our data revealed that after Ab injection, Drp-1 level increased demonstrating shift toward fission. Moreover, decline in Mfn2 level is in parallel with the elevation of Drp-1 confirming the shift toward fission rather than fusion as in the early events of Ab injection. It seems that fission of healthy mitochondria was elevated before significant cleavage of caspase-3, although this trend did not continue.

Neuromol Med (2014) 16:175–190

However, fission of mitochondria is not sufficient and production of new mitochondrion is unavoidable. In addition to direct relation between mitophagy and mitochondrial biogenesis, decrease in mitochondria is sensed indirectly by specific products of this semi-self governed organelle. Decrease in ATP/AMP ratio is of the safety valves of cells sensed by AMPK. AMPK phosphorylation by upstream kinases leads to PGC-1a activation and consequently nuclear translocation (Yu and Yang 2010). PGC1a concomitant with NRF-1 induces transcription of mitochondrial proteins that are encoded by nuclear DNA (Scarpulla 2011). Regardless to aside genome, 99 % of mitochondrial proteins are encoded by nucleus. So, nuclear transcription of mitochondrial proteins has pivotal role in the generation of new mitochondria (Boengler et al. 2011). Based on our data, the amount of PGC-1a, NRF-1, and TFAM decreased in the presence of Ab. As the upstream of this cascade, PGC-1a is immediately affected by Ab, while NRF-1 and consequently TFAM and cytochrome c levels are influenced by Ab in the second level. Our data also revealed that phosphorylation of CREB and AMPK reduced as the activators of PGC-1a. Immediate reduction in CREB phosphorylation in the presence of Ab is in accordance with the previous investigations done by Vitolo et al. (2002). They have shown that Ab suppresses CREB phosphorylation directly by the inhibition of protein kinase A (PKA). The most well-known role of cAMP/PKA/CREB pathway is the induction of long-term potentiation (Vitolo et al. 2002). Also, we determined the amount of phosphorylated form of p70S6K. p70S6K is known as downstream factor of mammalian target of rapamycin (mTOR) complex 1 that regulates mitochondrial oxidative function by controlling transcription factor yin-yang 1 (YY1). The direct reciprocal relation between YY1 and PGC-1a has been well documented (Heinonen et al. 2008; Cunningham et al. 2007). Reduction in p70S6K phosphorylation that indicates the decreased activity of mTOR ultimately leads to decline in PGC-1a level as we indicated in our study. Reduction in glucose metabolism is reported at early stages of AD before clinical symptoms diagnosis (Minoshima et al. 1997). It has been well known that decrease in brain metabolism leads to cognition disabilities and memory deficits. Glucose metabolism initiates in cytoplasm, where throughout glycolysis pathway, pyruvate is generated. PDH is the link between glycolysis and TCA via acetyl-coA production. Decrease in PDH by decline in accessible substrates leads to reduction in TCA cycle activity (Manczak et al. 2004). TCA cycle includes biochemical reactions that are catalyzed by 8 enzymes, alternatively. The first enzyme is CS and the last one is MDH. Based on the major role of TCA cycle in glucose metabolism, we investigated the behavior of 4 enzymes of the cycle in the presence of Ab. As our data showed, the

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activity of CS was not influenced by Ab. Since the production of ATP and NADH, two endogenous inhibitors of CS, disrupted in AD brain, it can be predictable that activity of CS is unchanged (Srere 1974). Unlike CS, aconitase enzyme is the most sensitive enzyme to ROS. Flint et al. (1993) reported that upon exposure to superoxide, iron reversibly is released from aconitase cluster leading to enzyme deactivation. Based on this report, others proposed using this enzyme for the estimation of superoxide concentration in mammalian cells (Gardner et al. 1995; Patel et al. 1996). Monitoring aconitase for 10 days revealed that 2 days later, activity of aconitase decreased insignificantly. It seems that antioxidant enzymes detoxify ROS and so prevent the oxidant-dependent release of iron from aconitase. But, in the third day, when the activity of antioxidant enzymes impaired, ROS accumulated and activity of aconitase remarkably diminished as a marker of oxidative stress. Continuing TCA cycle, the next enzyme we analyzed was fumarase. Based on our data, fumarase activity was not affected by Ab which was in accordance with the previous studies (Bubber et al. 2005). Increased activity of MDH, the last enzyme of TCA cycle, has been reported in the postmortem brain of AD patient and rat brain synoptosomes, while our data revealed a decline in enzyme activity. It seems that this difference is because of opposing effect of Ab on cytosolic and mitochondrial molecules providing substrate for MDH. Increase in MDH activity is due to increase in malate production in cytosol that has been reported by Bubber et al. (2005). Alongside with disrupted TCA enzymes activity by Ab, reduction in mitochondrial biogenesis leads to less content of mitochondria as well as their enzymes. Taken together, decrease in TCA cycle enzymes activities produces less NADH and FADH2, two essential substrates of ETC. Besides, reduction in NADH production leads to decrease in the mitochondrial membrane potential and generates a good mouthful for autophagy machinery. In conclusion, it seems that in the presence of Ab, neuronal cells attempt to overcome deleterious effect of the peptide. In this way, autophagy as a clearance system is influenced by the accumulation of Ab, whereas mitochondrial biogenesis markers decrease. First, reduction in mitochondrial contents could help cells to prevent targeting of more mitochondria by Ab. Second, on the other hand, it may lead to depletion in energy production. It seems that based on neuronal requirement for energy, decline in energy levels results in cell death (Scheme 1). So, two therapeutic strategies may be suggested for targeting AD treatment: (1) manipulation of autophagy, (2) induction of mitochondrial biogenesis by targeting the main factors like PGC-1a. This study opens up a new window in future AD research as to select the appropriate targets at molecular levels. For this purpose, future studies are now in progress in our laboratory.

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Scheme 1 A schematic diagram of time course alteration of intracellular molecules and mitochondrial specific enzymes in the presence of Ab Acknowledgments This work is part of PhD student thesis of F. Shaerzadeh at the Shahid Beheshti University of Medical Sciences. Conflict of interest

The authors have no conflict of interest.

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