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Published in final edited form as: J Neurochem. 2010 Aug 12;115(1):68–78. doi: 10.1111/j.1471-4159.2010.06905.x

Autophagy and protein aggregation after brain ischemia

Chunli Liu 1,*, Yanqin Gao 2, John Barrett 1, Bingren Hu 1,2,3
PMCID: PMC3518272  NIHMSID: NIHMS224332  PMID: 20633207

Abstract

Autophagy is the main degradation pathway responsible for eliminating abnormal protein aggregates and damaged organelles prevalent in neurons after transient cerebral ischemia. This study investigated whether accumulation of protein aggregate-associated organelles in postischemic neurons is a consequence of changes in autophagy. Electron microscopic (EM) analysis indicated that both autophagosomes (AP) and autolysosomes (AL) are significantlly upregulated in hippocampal CA1 and DG neurons after ischemia. The LC3-II conjugate, a marker for APs assessed by Western blotting, was upregulated in postischemic brain tissues. Confocal microscopy showed that LC3 isoforms were located in living postischemic neurons. Treatment with chloriquine (CQ) resulted in accumulation of LC3-II in sham-operated rats, but did not further change the LC3-II levels in postischemic brain tissues. The results indicate that at least part of the accumulation of protein aggregate-associated organelles seen following ischemia is likely to be due to failure of the autophagy pathway. The resulting protein aggregation on subcellular organelle membranes could lead to multiple organelle damage and to delayed neuronal death after transient cerebral ischemia.

Keywords: delayed neuronal death, ubiquitin, proteosome, protein misfolding, protein aggregation, Brain ischemia, autophagy, ATG12-ATG5 and LC3II, and electron microscopy

There are two major routes currently known for clearance of aberrant cellular components: (i) the ubiquitin-proteasomal pathway; and (ii) the autophagy-lysosomal pathway. The ubiquitin-proteasomal pathway is responsible for degradation of short-lived proteins and has been studied intensively (Ciechanover, 2006), including following brain ischemia (Hu et al., 2000; Ge et al., 2007). The autophagy pathway, originally described as a stress response to nutrient deprivation, is now emerging as the chief route for bulk degradation of aberrant organelles, protein aggregates and invading foreign agents (Nixon et al., 2005). There are three basic types of autophagy: macroautophagy, microautophagy, and chaperone-mediated autophagy. Bulk degradation of cytoplasmic organelles is thought to be mediated largely by macroautophagy, which is commonly referred to as autophagy (hereafter). Autophagy is a nonstop life-sustaining renewal process, which is active under normal conditions and is further enhanced in response to tissue injury (Klionsky, 2005 and 2006).

The autophagy pathway is mediated by a group of autophagy-related genes (atgs) and their encoded proteins (ATGs). Autophagy starts with formation of double-membraned cisternae that subsequently engulf cytoplasmic organelles to form double-membrane vacuoles, known as autophagosomes (APs). After maturation, APs merge with lysosomes for bulk degradation of the cargo contents (Yorimitsu and Klionsky, 2005). Hence, the appearance of APs under transmission EM is a morphological hallmark unique to autophagy. Two biochemical markers unique to autophagy are the covalent conjugates of: (i) ATG12-ATG5; and (ii) microtubule-associated protein light chain 3 (LC3)-phosphatidylethanolamine (PE). LC3 is a mammalian homologue of yeast ATG8, and it is synthesized as a pro-LC3. After synthesis, pro-LC3 is cleaved by ATG4 protease and becomes the 16-18 kDa LC3-I. Upon activation of autophagy, LC3-I is conjugated with PE (lipidated). The lipidated form is referred to as LC3-II (Kabeya et al., 2000 and 2004).

The conjugation to create ATG12-ATG5 or LC3-II is carried out by two consecutive ubiquitination-like enzyme systems in an ATP-dependent manner, involving ATG7 and ATG10 for ATG12-ATG5 conjugation, and ATG7 and ATG3 for LC3-II conjugation (Klionsky, 2005). After conjugation, both ATG12–ATG5 and LC3-II become structural components of the double-membraned cisterns or APs, and are redistributed to the membrane fractions. Therefore, the protein levels and redistribution of ATG5-ATG12 and/or LC3-II conjugates in the membrane fractions are often employed as a measure to determine autophagic activity (Kabeya et al., 2004).

We have previously reported that ubi-protein aggregates and aggregate-associated organelles are drastically accumulated only in neurons destined to undergo delayed neuronal death after transient period of ischemia (Hu et al., 2000 and 2001). In this study, we investigated whether protein aggregation after brain ischemia is a consequence of insufficiency of autophagy. This study suggests that the autophagic pathway is upregulated moderately by ischemia, but fails to operate at the full capacity, probably because of its impairment. As a result, protein aggregates accumulate following ischemia. Protein aggregation on subcellular organelle membranes is likely to cause multiple organelle failure and to delayed neuronal death after transient cerebral ischemia.

MATERIALS AND METHODS

Ischemia Model

Brain ischemia was produced using the 2-vessel occlusion (2VO) model in rats (Smith et al., 1984). All experimental procedures were approved by the Animal Care and Use Committee at the University of Miami and were performed in compliance with the National Institutes of Health guidelines on the ethical use of animals. Measures were taken to reduce animal suffering and the number of animals used. Male Wistar rats (250-300g) were fasted overnight. Anesthesia was induced with 4% and maintained with 1.5% halothane in an oxygen/nitrous oxide (30/70%) gas mixture. Catheters were inserted into a femal artery and tail artery to allow blood sampling, arterial blood pressure recording and drug infusion. Both common carotid arteries were encircled by loose ligatures. Fifteen minutes prior to ischemia induction and 15 min postischemia, blood gases were measured and adjusted to PaO2 > 90 mmHg, PaCO2 35 - 45 mmHg, and pH 7.35 - 7.45 by changing the tidal volume of the respirator. Brain ischemia was induced by withdrawing blood via the femal artery catheter to produce a mean arterial blood pressure (MABP) of 50 mmHg, followed by clamping both carotid arteries for 20 min. MABP was maintained at 50 mmHg during the ischemic period by withdrawing or infusing blood through the femoral artery catheter. At the end of ischemia, the clamps were removed, halothane was discontinued and all wounds were sutured. Brain temperature was maintained at 37°C before, during and after ischemia (15 min of reperfusion). Brains were collected at the ends of 0.5, 4, 24 and 72 h of reperfusion. Each experimental group consisted of at least 4 rats. For biochemical studies, brains were frozen in situ with liquid nitrogen (Pontén, 1973). For light, electron and confocal microscopy, rats were perfusion-fixed either with 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer for light microscopy (LM) and electron microscopy (EM), or with 4% of paraformaldehyde in PBS for confocal microscopy.

Treatment with lysosomal inhibitor

Rats were subjected to either sham-surgery or 15 min of ischemia followed by 24 h of reperfusion. Treatment with chloriquine (25 mg/kg), or the vehicle was carried out by single intra-cerebroventricular injection with a needle (26-gauge) at the stereotaxic coordinates: 0.7 mm anterior to the bregma, 1.6 mm lateral to the midline, and 5.0 mm ventral to the skull surface, at 5 h before animals were sacrificed (Noble et al., 1967). Each experimental animal group consisted of at least 3 rats. Rat brains were collected by the methods described above and analyzed by light microscopy (LM), confocal microscopy and Western blot analysis (see below).

LM, EM and confocal microscopy

LM, EM and confocal microscopy were conducted with brain sections from sham-operated control rats and rats subjected to transient cerebral ischemia followed by various periods of reperfusion. Brain sections were stained with the conventional osmium-uranyl-lead method for EM, and with celestine blue and acid fuchsin for LM, respectively, as described previously (Martone et al., 1999; Hu et al., 2000; Liu et al., 2005b). Briefly, coronal brain sections were cut consecutively at the hippocampal level at a thickness of 150 μm for EM and then at 30 μm for LM with a vibratome. The EM brain sections were postfixed with 4% glutaraldehyde in 0.1 mM cacodylate buffer (pH 7.4) for 1 h, and then with 1% osmium tetroxide in 0.1 M cacodylate buffer for 2 h. After rinsing with distilled water, EM brain sections were stained with 1% aqueous uranyl acetate overnight, dehydrated in an ascending series of ethanols to 100% followed by dry acetone, and then embedded in Durcopan ACM. Ultrathin sections (0.1 μm) were cut and stained with 3% lead citrate, and examined with a Zeiss EM (Germany). The vibratome brain sections (30 μm) for LM were stained with celestine blue and acid fuchsin. Neuronal histopathology was determined according to the method of Smith et al. (1984).

Quantitative analyses of autophagosomes (APs) and autolysosomes (ALs) were conducted in CA1 and DG tissue sections from sham-opertaed control rats (n = 5) and rats subjected to 15 min of ischemia followed by 24 h of reperfusion (n = 5). Specimens were analyzed from the soma region of CA1 and the upper granule cell layer of the DG. Specimens were cut at a thickness of 100 nm, and photographed at a magnification of 6300×. Micrographs were taken from the cell layer areas between the nuclei and apical dendrites of the CA1 pyramidal neurons or the DG granule cells. For each animal, 15 micrographs were taken from both CA1 and the DG as described above. Micrographs were digitized and the numbers of APs and ALs in micrographs (see Fig. 2) were counted by two investigators who were blinded to the experimental conditions. The APs and ALs in 10 micrographs from the same rat were averaged and counted as one sample. Mean ± SD from 5 samples (5 rats) per condition (n = 5) was employed for statistical analysis. One-way ANOVA, followed by Dunnett's test was utilized to assess statistical significance (P<0.05).

Fig. 2.

Fig. 2

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A: EM micrographs of CA1 and DG neurons. CA1 and DG neurons from sham control rats contained normal polyribosomes (arrows), nucleus (N), rough endoplasmic reticulum (ER), Golgi apparatus (G), mitochondria (M), multivesicular bodies (MB), and pre-existing APs, ALs, and lysosomes (L). CA1 neurons from brains subjected to 15 min of ischemia followed by 4 and 24 h reperfusion displayed dissociation of polyribosomes, fragmentation of the neuronal Golgi apparatus and dramatic accumulation of intracellular vesicles and protein aggregates (arrows), as well as a moderate increase in autophagosomes (APs) and autolysosomes (ALs). Most abnormal morphological changes such as accumulation of protein aggregates and dissociation of polyribosomes were absent in DG neurons, whereas there was a moderate increase in APs and ALs similar to that seen in CA1 neurons at 4 and 24 h of reperfusion after ischemia. Scale bar = 0.5 μm. B: EM micrographs of autophagic ultrastructure in hippocampal neurons from rats subjected to 15 min of ischemia followed by 4 h of reperfusion. a. a double-membraned cistern (arrow) and protein aggregates (arrowhead); b. an AP containing dilated Golgi cistern (G) and its surrounding vesicles; c. APs containing a damaged mitochondrion, a protein aggregate (arrows) and undegisted membranous structures; d. an AP containing dilated ER with ribosome; e. an AP containing a shrunken mitochondrion (M) and a protein aggregate (arrow); f. two autolysosomes (AL) with partially digested organelles, and bulble-like structures, as well as a lysosome (L); g. an AL with partially digested membranous structures and protein aggregates (dark masses), as well as a lysosome; and h. two APs containing clusters of membrane whorls. Bar = 0.2 μm.

Fluorescence confocal microscopy was performed on coronal brain sections (50 μm) at the dorsal hippocampal level. Antibody to LC3 was obtained from Cell Signaling Tech (Danvers, MA). Briefly, brain sections were transferred into a 24 well microtiter plates filled with 1 ml of 0.01M citric acid/sodium citrate buffer (pH 6.0) and heated for 10 seconds in microwave set to 30% power. The sections were then washed twice with 0.2% Triton X100 (TX100)/phosphate-buffered saline (PBS) for 10 min. Non-specific binding sites were blocked in 3% BSA in PBS for 1 h. Primary antibodies were diluted at 1:250 in PBS containing 0.1% TX100 and 3% bovine serum albumin (BSA). After incubation overnight at 4°C, the sections were washed 3 times for 10 min at room temperature in PBS containing 0.1% TX100. The sections were then incubated in fluorescein-labeled anti-rabbit diluted 1:200 in PBS containing 1% BSA for 1 h at room temperature. The sections were washed, mounted on glass slides and coverslipped. The slides were analyzed on a Zeiss laser-scanning confocal microscope.

Subcellular fractionation

Frozen hippocampal CA1, DG, and dorsal neocortical tissues was microdissected in a -12°C glove box according to the method described previously (Hu et al., 1994). Brain tissues were homogenized with a Dounce homogenizer (50 strokes) in 10 times the tissue volume (1 μg tissue + 10 μl buffer) of ice-cold homogenization buffer containing 15 mM Tris base/HCl pH 7.6, 1 mM DTT, 0.25 M sucrose, 1 mM MgCL2, 1 μg/ml pepstain A, 5 μg/ml leupeptin, 2.5 μg/ml aproptonin, 0.5 mM PMSF, 2.5 mM EDTA, 1 mM EGTA, 0.25 M Na3VO4, 25 mM NaF and 2 mM sodium pyrophosphate. The homogenate was centrifuged at 10,000 g for 10 min at 4°C to obtain a pellet fraction that was washed with the homogenization buffer plus 1% Triton X100, and designated as P2 ( = P1 + P2 fractions, Hu et al., 1994). The 10,000 g supernatant was further centrifuged at 165,000 g for 60 min at 4°C to get pellet 3 (P3 or microsomal fraction) and the supernatant fraction (S3 or cytosol). All pellet fractions were suspended in the homogenization buffer containing 0.1% TX100. Protein concentration in subcellular fractions was determined by the micro-bicinchonic acid (BCA) method (Pierce Biochemicals, Rockford, USA).

Western blot analysis

Western blot analysis was carried out with 10-12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Each subcellular fraction containing 20 μg of protein was loaded on the gels for western blot analysis. Following electrophoresis, proteins were transferred to an Immobilon-P membrane. The membranes were probed with antibodies against ATG5 and LC3 (each at dilution of 1:5,000, Cell Signaling Tech, Danvers, MA). The immunoblot membranes were then incubated with horseradishperoxidase conjugated anti-mouse or anti-rabbit secondary antibody for 1 h at room temperature (Cell Signaling Tech, Danvers, MA). The blots were developed with an ECL detection method (Cell Signaling, Danvers, MA), and then exposed to Kodak films to such a degree that protein bands on the films were not saturated. The optical densities of protein bands on the films were quantified using Kodak 1D gel analysis software. Each immunolabeled protein band was quantified as the mean intensity value subtracted by the background value, and then presented as mean ± SD. One-way ANOVA, followed by Dunnett's test was used to assess statistical significance (P<0.05).

RESULTS

Histopathology

Fifteen min of cerebral ischemia did not lead to neuronal death assayed by light microscopy (acid fuscine and celestin blue staining of 30 um sections, see methods) until 72 h of reperfusion (delayed neuronal death) when more than 90% of CA1 neurons were dead (Fig. 1A, upper panel). This brief ischemia leaves DG neurons largely intact (Fig. 1A, lower panel) but induced delayed neuronal death in about 5-10% of dorsolateral neocortical neurons after 72 h of reperfusion (data not shown). These results are consistent with previous studies (Smith et al., 1984; Hu et al., 2000; Liu et al., 2005). Normal neurons are pyramidal-like or round in shape (Fig. 1A, arrows), whereas ischemic dead neurons have shrunken and acidophilic cytoplasm, as well as dark polygonal nuclei (Fig. 1A, arrowheads).

Fig. 1.

Fig. 1

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Histopathology of hippocampal CA1 and DG neurons stained with acid fuchsin and celestine blue. No neuronal death was found in brain sections from sham-operated control rats and rats subjected to 15 min of ischemia followed by 24 h of reperfusion. Normal neurons are indicated by arrows. Most CA1 neurons were dead at 72 h of reperfusion (arrowheads), but almost all DG neurons had survived the same period of ischemia. Scale bar = 50 μm.

Ultrastructual features of autophagic maturation after brain ischemia

To determine whether autophagic activity is altered in neurons after brain ischemia, we examined brain sections by transmission EM. The EM manifestation of bubblelike vacuoles enclosing recognizable cytoplasmic structures still represents the gold standard for identifying autophagosomes (APs) (Brunk et al., 2002). CA1 and DG neurons from sham-operated control rats contained normal polyribosomes (arrows), nucleus (N), rough endoplasmic reticulum (ER), Golgi apparatus (G), mitochondria (M), multivesicular bodies (MB), and pre-existing APs, ALs, and lysosomes (L) (Fig. 2A, Sham). Neurons from brains subjected to 15 min of transient cerebral ischemia followed by reperfusion displayed an increase in the numbers of APs and ALs (also see Fig. 4). In CA1 neurons after 4 - 24 h of reperfusion post-ischemically (Fig. 2A, 4h and 24 h, upper panels), the dominant ultrastructural changes were: (i) dissociation of polyribosomes, fragmentation of the neuronal Golgi apparatus and dramatic accumulation of intracellular vesicles, (ii) accumulation of a large quantities of protein aggregates (arrows), and (iii) a moderate increase in autophagosomes (APs) and autolysosomes (ALs) (see Fig. 4, below). In comparison with CA1 neurons, most abnormal morphological changes such as accumulation of protein aggregates and dissociation of polyribosome were absent in DG neurons, whereas CA1 and DG neuron showed similar moderate increases in APs and ALs after 4 - 24 h of reperfusion following ischemia (Fig. 2A, 4h and 24 h, lower panels, see below).

Fig. 4.

Fig. 4

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Immunoblots and quantification of ATG5-ATG12 conjugates and β-actin in brain samples of sham-operated rats and rats subjected to 15 min of ischemia followed by 4, 24 and 72 h of reperfusion. The tissue homogenate (H), as well as P2 (heavy membranes), P3 (intracellular membranes) and S3 (cytosol) fractions were immunoblotted with ATG5 antibody and β-actin antibody respectively. Upper panels: ATG5 antibody labeled mainly ATG12-ATG5 conjugates (>50 kDa). Lower panels: quantification of ATG5-ATG12 conjugates and β-actin. ATG12-ATG5 protein level and β-actin, on immunoblots were evaluated with Kodak 1D image software. ATG12-ATG5 protein level was calculated by ATG12-ATG5/β-actin ratios using 4 different individual rat samples. Data are expressed as mean ± SD (n = 4). *denotes p<0.05 between control and experimental conditions, one-way ANOVA followed by Dunnett's tests.

Several morphological features of AP maturation were observed in hippocampal CA1 or DG neurons at 24 h of reperfusion after ischemia (Fig. 2B). AP formation began with isolation membranes, i.e., the formation of double-membraned cisterns in the cytoplasm (Fig. 2B-a, arrows). The double-membraned cisterns enveloped cytoplasmic contents or whole organelles to form APs such as: (i) AP containing Golgi stacks and surrounding vesicles (Fig. 2B-b); (ii) APs containing a mitochondrial structures (Fig. 2B-c, M) and a protein aggregate (Fig. 2B-c, arrow, see below); (iii) AP containing the dilated ER associated with ribosomes (Fig. 2B-d, arrow); (iv) AP containing a mitochondrial structure (Fig. 2B-e, M); and (v) AP clusters containing membrane whorls, probably representing damaged mitochondria (Fig. 2B-f). APs eventually merged with lysosomes to become autolysosomes (ALs) that contained partially degisted cellular structures (Figs. 2B-f and -g). Partially degraded cargo contents within ALs were then manifested as unevenly distributed dense (dark) masses (Fig. 2B-g, AL), and eventually as evenly distributed materials (Fig. 2B-g, L). No obvious morphological changes in CA1 and DG neurons were seen by the histopathological light microscopy by 24 h of reperfusion (see Fig. 1, 24 h, CA1 and DG), suggesting that the ischemia-induced accumulation of protein aggregates, APs and ALs described above were not visible by the histopathological light microscopy.

We then measured quantitiatively the changes in autophagic activity in response to transient cerebral ischemia. A classical way to measure autophagy is quantitative electron microscopy. Although labor-intensive, EM quantitation remains the most sensitive and accurate way to detect the induction of autophagy (Tallóczy et al., 2002). Quantitative EM of autophagy estimates the accumulation of APs and ALs, which can be caused by either increased formation or decreased degradation of APs and ALs. In other words, it is possible to have increased cellular numbers of APs and ALs, but decreased autopahgy activity in the cells, (see below). We counted the numbers of recognizable APs and ALs within the defined CA1 and DG regions at 24 h of reperfusion by EM (see Methods). Relative those in sham-operated controls, the numbers of recognizable APs and ALs were significantly increased in both CA1 and DG neurons postischemically (Fig. 3).

Fig. 3.

Fig. 3

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Quantitative EM data showing that the number of recognizable AVs is significantly increased in CA1 and DG neurons after ischemia. *p<0.01 indicates significant difference compared with sham-operated control value (n=9, ANOVA followed by Dunnett's test). The unit area is a field of specimen that was cut at a thickness of 100 nm, and photographed at a magnification of 6300× as described in the method.

ATG12-ATG5 and LC3-II conjugation after brain ischemia

The biochemical hallmark of autophagic initiation is the consecutive formation and subcellular redistribution of two key ATG conjugates: first ATG12-ATG5, followed by LC3-II (LC3-II is a mammalian homologue of yeast ATG8-PE). We therefore prepared brain tissue homogenates (H) as well as standard 10,000 g pellets (P2), P3 (containing intracellular membranes) and S3 (cytosol) fractions from neocortical, as well as CA1 and DG tissues of rats subjected either to sham-surgery or 15 min of ischemia followed by 0.5, 4, 24 and 72 h of reperfusion. To ensure equal loading of protein samples on immunoblots, β-actin immunoreactivity was used as an endogenous control. As demonstrated in Fig. 4, ATG5 antibody labeled primarily the ATG12-ATG5 conjugated form (~53 kDa) in all subcellular fractions (Fig. 4), whereas the unconjugated (free form) ATG5 (~32 kDa) was barely detected (data not shown), consistent with many previous studies including our own (Liu et al., 2008). Relative to sham-operated controls, the ATG12-ATG5 conjugate was not significantly altered in tissue homogenates (H), whereas it was upregulated in the P2 and S3 fractions, and concomitantly dowregulated in P3 fractions at 24 and 72 h of reperfusion after ischemia in CA1 and DG, as well as, to a lesser degree, in neocortical (Cx) tissue samples (Fig. 4, lower panels). β-actin immunoreactivity on immunoblots was not significantly altered in brain tissue homogenates and any subcellular fraction in the neocortex samples (Fig. 4, upper panel, Anti-actin, Cx) as well as in CA1 and DG tissue samples (data not shown) after brain ischemia.

LC3-I conjugates covalently with phosphatidylethanolamine (PE) to form lipidated LC3-II which has a faster mobility on immunoblots. Thus, the N-terminal LC3 antibody was able to recognize both the upper LC3-I and lower LC3-II band in homogenate (H) as well as in subcellular P2, P3 and S3 fractions (Fig. 5). After conjugation, LC3-II becames a component of the AP and AL membranes, and thus redistributes mainly to the P2 fraction and to a lesser degree also to the P3 fraction after ischemia (Fig. 5). Only LC3-I, but not LC3-II, was detected in the S3 fraction (Fig. 5, upper panel), consistent with previous reports including our own (Liu et al., 2008). The LC3-I level was not significantly altered by the ischemia, where as LC3-II protein level was significantly upregulated in homogenates and all subcellular fractions (Fig. 5, upper panels). Quantitative analysis further demonstrated that the LC3-II level was upregulated about 15-fold of the control in P2 membrane fraction of the CA1 region, and about 10-fold and 5- fold of the control levels in DG and neocortical samples, respectively (Fig. 5, lower panels). These data suggest that increase in LC3-II levels was more pronounced in the CA1 region relative to those in the DG and neocortical (Cx) tissue samples after ischemia (Fig. 5, lower panels).

Fig. 5.

Fig. 5

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Immunoblots (upper panels) and quantification (lower panels) of LC3 in brain samples of sham-operated rats and rats subjected to 15 min of ischemia followed by 4, 24 and 72 h of reperfusion. The same brain samples as those in Fig. 5 were analyzed by immunoblotting with LC3 antibody. LC3 antibody recognizes both LC3-I and LC3-II. LC3-I is not obviously altered, whereas LC3-II is significantly increased in whole homogenate and P2 and P3 fractions of CA1 samples after ischemia. However, relative to those in CA1 samples, accumulation of LC3-II was less in dentate gyrus (DG) and neocortical (Cx) samples. *P<0.01 significant compared with sham value (n=5, ANOVA followed by Dunnett's test).

Autophagy-lysosome flux after brain ischemia

Quantitative EM and biochemical studies show that APs and their marker protein LC3-II are significantly upregulated in neurons after brain ischemia (Figs. 2, 3 and 5). However, accumulation of AP or LC3-II can be either because of increased AP formation, or decreased AP flow to lysosomes where APs, together with LC3-II, are degraded. The AP-to-lysosome flow can be blocked with lysosome inhibitors, such as chloroquine (CQ). An increase in the AP marker protein LC3-II after CQ inhibition of lysosomes indicates that there is AP flow to lysosomes for AP degradation (Hamacher-Brady et al., 2006 and 2009; Yitzhaki et al., 2009).

To test whether accumulation of LC3-II without corresponding changes in LC3-I (see Fig. 5) is attributable to decrease in the autophagy activity (degradation of AP marker protein LC3-II by lysosomes) after brain ischemia, we carried out a CQ (a lysosome inhibitor) challenge study. LC3 immunostaining as viewed by confocal microscopy is upregulated, probably owing to increases in LC3-II levels of CA1 and neocortical (Cx) neurons after brain ischemia. Intra-cerebral ventricular injection of 25 mg/kg CQ at 5 h before sacrificing animals leads to upregulation of LC3-II in sham control CA1, but has little such effect in postischemic CA1 neurons (Fig. 6A). In comparison, CQ treatment leads to upregulation of LC3-II in both sham control and postischemic neocortical neurons (Fig. 6A). Consistently, CQ treatment leads to increases in LC3-II levels on immunoblots of both sham control and postischemic neocortical (Cx) samples, but has little such effect in postischemic hippocampal (Hipp) samples after ischemia (Fig. 6B and 6C). This study provides a clue that the autophagy pathway may be impaired selectively in CA1 neurons (that undergo delayed neuronal death) at or before the step of lysosomal degradation of LC3-II after ischemia. This pathway appears to be less affected in neocortical neurons (that mostly survive the same episode of ischemia).

Fig. 6.

Fig. 6

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Effects of CQ treatment on LC3-II levels. Brain sections or samples were obtained from sham-operated control rats (Ctr) and rats subjected to 15 min of ischemia followed by 24 h of reperfusion, treated either with or without the lysosomal inhibitor, chloroquine (CQ). A: Confocal images of CA1 and Cx neurons labeled with anti-LC3 antibody. LC3 immunostaining is markedly increased in CA1 and Cx neurons at 24 h of reperfusion. Intraventricular injection of CQ leads to upregulation of LC3 level in sham Ctr neurons including CA1 neurons, but has little such effect in postischemic 24h CA1 neurons. Scale bar = 50 μm. B and C: immunoblots of LC3 and quantitative analysis of LC3-II level on immunoblots. LC3-I is not obviously altered, whereas LC3-II is upregulated in CQ-treated sham control (Ctr) hippocampal (Hipp) and neocortical (Cx) samples. CQ treatment also leads to upregulation of LC3-II in postischemic 24 h Cx but has little such effect in postischemic 24 h Hipp samples. Data are expressed as mean ± SD (n = 4). *denotes p<0.05 between control and experimental conditions, one-way ANOVA followed by Dunnett's tests.

Accumulation of protein aggregate-associated organelles after brain ischemia

As shown in Fig. 7, massive accumulation of aberrant organelles and intracellular vesicles was the most prominent early ultrastructural feature in neurons that were undergoing delayed neuronal death after brain ischemia (Hu et al., 2000; Liu et al., 2004). Aberrant aggregates were not seen in sham control neurons (Fig. 7A), but following ischemia they were associated with mitochondria (Fig. 7B), fragmented Golgi apparatus (Fig. 7C), and ER (Fig. 7D). This massive accumulation of aberrant organelles further supports postischemic impairment of the autophagy pathway that is the main route for their degradation (Klionsky, 2005).

Fig. 7.

Fig. 7

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Accumulatiom of protein aggregate-associated organelles in CA1 neurons after brain ischemia. CA1 tissue sections were obtained from rats subjected either to sham surgery or to 15 min of ischemia followed by 24 h of reperfusion. A. A sham neuron shows normal nucleus (N), Golgi (G), ER, and mitochondria (M); B. a 24 h-reperfused neuron shows abnormally aggregated ribosomes or translational complex (arrows) and a protein aggregate-associated mitochondrion (M) and accumulated APs; C. a 24 hreperfused neuron shows that bubble-like APs and abnormal protein aggregates (arrows) after ischemia; D. APs are located close to aggregate-associated ER (arrows) after ischemia. Scale bar = 1 μm.

DISCUSSION

This study provides evidence that the autophagy pathway is altered and likely impaired after brain ischemia: (i) Quantitative EM shows that AP number is moderately increased in neurons after brain ischemia; (ii) aberrant organelles, which are normally degraded by the autophagy pathway, are massively accumulated in postischemic neurons; (iii) LC3-II, an AP marker protein, increases by as much as 15-fold in the CA1 membrane fraction after brain ischemia; (iv) accumulation of LC3-II is likely caused by disruption of a late step of the pathway, because blocking the pathway at the lysosomal step with CQ leads to increase in neuronal LC3-II levels in sham-operated rats, but has little such effect in postischemic CA1 neurons, suggesting that the autophagy pathway has normal flow in CA1 neurons of sham operated animals, but has little flow after brain ischemia; and (v) the increase in LC3-II without corresponding changes in LC3-I and accumulation of aberrant organelles in postischemic neurons futher support the hypothesis that the autophagy pathway is impaired in vulnerable CA1 neurons after ischemia (see below).

Recent studies report changes in LC3 after brain hypoxia-ischemia (HI). Zhu et al. (2005 and 2006) reported that the LC3-II protein level was upregulated mainly in adult, rather than in neonatal brains after HI. Adhami et al. (2006 and 2007) found a reduction of LC3-I after HI, but did not detect upregulation of LC3-II on immunoblots in a HI model in mature mice. The present study suggests that, relative to mild changes in LC3-II after HI in the previous reports, alterations in LC3-II after transient cerebral ischemia are robust. The mature brains with the 2VO ischemia model used in the present study may produce more severe protein aggregation and metabolic failure than those of the HI model used in the previous studies (Zhu et al., 2005 and 2006; Chu, 2006). Upregulation of LC3-II was also reported after traumatic brain injury (Liu et al., 2008; Lai et al., 2008).

Ultrastructural features of autophagy after ischemia

Although labor intensive, transmission EM remains an indispensable technique for evaluation of autophagy in situ and remains the most accurate method to detect induction of autophagy in tissue (Eskelinen et al., 2005a, b and 2006; Ciechomska and Tolkovsky, 2007). The ultrastructural hallmarks for induction of autophagy are the manifestation of: (i) double-membraned cistern structures; (ii) autophagosomes containing cytoplasmic materials or aberrant organelles such as ribosomes, the ER, Golgi structures and mitochondria (see Fig. 2); and (iii) autolysosomes which contain partially digested subcellular structures such as ER or mitochondrial remnants. Ultimately, AL contents are digested and become homogeneous dense material. All these ultrastructual features of autophagy were clearly observed by transmission EM in postischemic neurons (see Fig. 2). Furthermore, the quatitative EM studies provide solid evidence that APs and ALs are more abundant after ischemia in both vulnerable CA1 as well as resistant DG, and some neocortical neurons, suggesting that upregulation of APs and ALs alone may not lead to neuronal death.

Biochemical changes in autophagy after ischemia

ATG12-ATG5 and LC3-II are biochemical hallmarks of autophagy initiation. The present study shows that ATG5 antibody predominantly labels the ATG12-ATG5 conjugated form (~53 kDa) (see Fig. 4), whereas the free or unconjugated ATG5 is hardly detected in brain tissue samples (Liu et al., 2008). This result is consistent with many previously reports showing that almost all ATG5 is present as an ATG12-ATG5 conjugated form in cells (Mizushima, et al., 2001). The present study further demonstrates that the ATG12-ATG5 conjugate is significantly reduced in P3 fractions while commitantly increased in the P2 and S3 fractions during the late periods of recovery after brain ischemia. The changes are probably caused by its redistribution among P2, P3 and S3 fractions, because ATG12-ATG5 in the total homogenate is not significantly altered after brain ischemia. The late increases in ATG12-ATG5 in P2 and S3 fractions indicate that APs may contain “heavy” denatured organelles during the late period of recovery after ischemia. These results are consistent with the increases in APs and ALs observed by EM (see Fig. 2).

LC3-II is also recruited into double-membraned cisterns in an ATG12-ATG5-dependent manner (Mizushima et al., 2001). However, unlike the ATG12-ATG5 conjugate that dissociates from the membrane immediately after AP formation, a portion of the LC3-II conjugate remains on the AP membrane throughout the AP life cycle even after the APs merge with lysosomes (Kabeya et al., 2000). Hence, the LC3-II protein level has been utilized as a reliable molecular marker to assess cellular AP numbers (Koike et al., 2005). The present study clearly demonstrates that LC3-II is robustly upregulated in neurons at 4, 24 and 72 h of reperfusion after ischemia, thus providing solid evidence for dynamic increase in APs after brain ischemia. In addition, increase in LC3-II is found mainly in P1 and P2 fractions, but, to a much lesser degree, in P3 fractions, after brain ischemia. This is consistent with the fact that LC3-II is located both in AP and AL membranes (Kabeya et al., 2000; Tanida et al., 2004). Taken together, these results are consistent with the fact that ATG12-ATG5 is associated with double-membrane cisterns (pre-autophagosomes) located mianly in the microsomal P3 fraction, whereas LC3-II in AP and AL membranes is distributed mainly to P1 and P2 fractions.

Confocal microscopy of LC3 after brain ischemia

Currently, there are no reliable biomarker proteins for unambiguous detection of autophagy in tissue sections using standard immunohistochemical techniques (Ciechomska and Tolkovsky, 2007). The punctate LC3 immunostaining of APs was not obvious in brain sections after ischemia (Fig. 7). This result is consistent with previous studies showing that although AP number increases 10-fold during 2 h amino acid starvation, the prevalence of visible punctate LC3 structures does not change (Eskelinen et al., 2005a and b, Martinet et al., 2006). The reason for not detecting punctate LC3 is likely because the LC3-I level is much higher than the LC3-II level in postischemic neurons, as indicated by immunoblotting shown in Fig. 5. This is in constrast with LC3 levels in some cultured cells in which almost all LC3-I is converted to LC3-II after CQ treatment, and thus can be labeled as punctuate LC3-II positive structures (Martinet et al., 2006). Unfortunately, there is no method currently available specifically recognizing LC3-II isoform in tissue sections. Nevertheless, confocal microscopic immunolabeling of LC3 in brain sections is still useful to identify LC3 positive living neurons.

Impairment of the autophagy pathway after brain ischemia

Quantitative EM has been used to quantify autophagic structures (Eskelinen et al., 2005a and b). The data from both quantitative EM and immunoblots of LC3-II in the present study demonstrate that the number of APs and ALs are increased in neurons after brain ischemia. However, because APs fuse with and then are degraded by lysosomes, the accumulation of APs or LC3-II protein after brain ischemia can be either due to increased formation, or decreased lysosomal degradation of APs. In pathological conditions, there are often increased APs and ALs, but decreased autophagic activity (Hamacher-Brady et al., 2006; Kiselyov et al., 2007; Gustafsson and Gottlieb, 2008). This is also exemplified by many lysosome deficiency disorders, such as Danon's disease, which show accumulation of APs and ALs, but decreased autophagic activity as a result of lysosome deficiency (Nishino, 2006; Kiselyov et al., 2007). Lysosome dysfunction creates an “AP/AL traffic jam or accmulation” mainly due to reduction of AP degradation. For that reason, we used a lysosomal inhibitor CQ to test whether accumulation of APs and ALs after brain ischemia is due to increased formation, or decreased degradation of APs by assaying the level of the AP marker protein LC3-II in the presence of a lysosomal inhibitor CQ. This study suggests that the autophagy pathway is impaired in ischemic vulnerable neurons, but still functional in ischemia resistant neurons after brain ischemia. Similar results have also been observed in several studies of cardiac ischemia, suggesting that ischemia/reperfusion may generally damage the degradation of AP in various types of cells, thus blocking or impairing the autophagy pathway (Gustafsson and Gottlieb, 2008; Huang et al., 2009; Yitzhaki et al., 2009).

Significance of the autophagy pathway in neuronal death after brain ischemia

The study of the role of autophagy in pathological conditions has drawn significant scientific interest (Liu et al., 2008). This is underscored by two studies demonstrating that conditional knockout of key autophagy genes, ATG7 or ATG5, in mice leads to neuronal death (Komatsu et al. 2006; Hara et al., 2006). These studies demonstrated that autophagy is essential for maintaining neuronal homeostasis, since its absence leads to accumulation of intracellular protein aggregates and damaged organelles, and ultimately delayed neuronal death.

Whereas normal autophagy is cell protective, overactivation of autophagy might also carry potential risks in a number of ways. For example, overcrowded lysosomes might leak hydrolytic enzymes, causing secondary cell injury. By destroying partly damaged mitochondria, autophagy might also reduce the number of mitochondria and so produce a secondary energy failure in postischemic neurons. However, the present study showed that lysosomes were only moderately increased in both ischemic vulnerable and resistant neurons after brain ischemia. The number of damaged mitochondria was not reduced, rather increased after brain ischemia. Accumulation of damaged mitochondria is likely a result of imperfect autophagy (Brunk and Terman, 2002).

It has been proposed that autophagy may take part in cell death, the so-called autophagic cell death (ACD) (Swanson, 2006). Currently, it remains controversial whether ACD is a tissue injury epiphenomenon caused by an unsuccessful autophagic attempt to rescue dying cells or is a programmed cell death response (Swanson, 2006). Several studies using chemical inhibitors of autophagy have been inconclusive, because of non-specific properties of these inhibitors (Nixon, 2005; Klionsky, 2006). On the other hand, growing evidence supports the view that autophagy is essential for maintaining neuronal homeostasis: (i) deficiency in this pathway leads to neurodegeneration and human genetic diseases (Eskelinen, 2006; Nishino, 2006); and (ii) conditional knockout of key autophagic genes, ATG5 or ATG7, leads to accumulation of intracellular protein aggregates and neuronal death (Komatsu et al. 2006; Hara et al., 2006). A protective role of autophagy was also observed in neonatal hypoxia-ischemia induced brain injury (Carloni et al., 2008). Autophagy is required for preconditioning-induced cell protection (Yitzhaki et al., 2009). Therefore, induction of the autophagy pathway may be a cell protective response, and it may be inadequate autophagy, rather than excessive autophagy, that contributes to neuronal death after brain ischemia.

Acknowledgments

This work was supported by National Institutes of Health grants NS040407 and NS36810.

Abbreviations

ubi-proteins

ubiquitin-conjugated proteins

AP

autophagosome

AL

autolysosome

atg

autophagy gene

3-MA

3-methyladenine

SDS-PAGE

sodium dodecyl sulfate polyacrylamide gel electrophoresis

EM

electron microscopy

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