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THE EFFECTS OF OXIDATIVE STRESS AND ALTERED INTRACELLULAR CALCIUM LEVELS ON VESICULAR TRANSPORT OF APOE-EGFP
Cell Biology International 2002, Vol. 26, No. 5, 407–420 doi:10.1006/cbir.2002.0868, available online at http://www.idealibrary.com on
THE EFFECTS OF OXIDATIVE STRESS AND ALTERED INTRACELLULAR CALCIUM LEVELS ON VESICULAR TRANSPORT OF APOE-EGFP ROBERT M. DEKROON and PATRICIA J. ARMATI1 Neuroscience Unit, School of Biological Sciences A08, University of Sydney, NSW 2006, Australia Received 16 November 2001; accepted 5 January 2002
Apolipoprotein E (apoE) plays a role in the distribution of lipid within many organs and cell types in the human body, including the central nervous system (CNS). The apoE4 isoform is also an established risk factor for late-onset Alzheimer’s disease (AD), however its role in the aetiology of the disease remains largely unknown. Therefore, as AD is a late-onset disease, we sort to investigate how conditions hypothesised to model ageing affect apoE metabolism, such as the transport of apoE along the secretory pathway. Two of these models include oxidative stress and calcium deregulation. Using apoE-EGFP-expressing astrocytoma cell lines we established that vesicle number and velocity are up-regulated under oxidative stress conditions, and slowed under KCl induced calcium deregulation. Although these findings apply to cells in general under these two stress conditions, the up-regulation of apoE in particular may be a response to cell injury with implications for neurodegeneration such as that found with 2002 Elsevier Science Ltd. All rights reserved. late-onset AD. K: apolipoprotein E; oxidative stress; vesicle trafficking; exocytosis; ageing. A: apoE, Apolipoprotein E; CNS, central nervous system; AD, Alzheimer’s disease; EGFP, modified form of the green fluorescent protein; ER, endoplasmic reticulum.
INTRODUCTION Apolipoprotein E (apoE) is a 34 kDa protein that appears to play a role in the redistribution of cholesterol amongst the cells of the central nervous system (CNS) and in the maintenance of cholesterol homeostasis (Boyles et al., 1985; Pitas et al., 1987). There are 3 common isoforms of apoE, E2, E3 and E4, which are the products of a single gene locus on chromosome 19. The apoE3 isoform contains cysteine and arginine at positions 112 and 158, respectively, while apoE2 has cysteine at both positions and apoE4 has arginine at both positions (Mahley, 1988). The association of the E4 isoform of apoE as a genetic risk factor for late-onset AD has been well established (Corder et al., 1993; Roses et al., 1995) The E4 isoform also lowers the age of onset of AD. Individuals homozygous for E4 are 95% more 1
Corresponding author: Patricia Armati: Neuroscience Unit, School of Biological Sciences A08, University of Sydney, NSW 2006, Australia. Tel: 612-9351-2062; Fax: 612-9351-4119; E-mail: [email protected] 1065–6995/02/$-see front matter
likely to acquire the disease at an average age of 68.4 years, compared to individuals heterozygous for the E4 isoform who are 65% more likely to acquire the disease at an average age of 75.5 years (Corder et al., 1993). However, even though these susceptibility data strongly suggest that apoE is involved in disease onset (Roses et al., 1995), the role of apoE in the aetiology of late-onset AD remains largely unknown. An important way of addressing this problem is to understand how the process of ageing affects apoE metabolism in the CNS, such as the transport of synthesised apoE along the secretory pathway from Golgi to the cell membrane. There are a number of conditions hypothesised to contribute to the ageing process, two of which are oxidative stress and deregulation of intracellular calcium concentrations (Butterfield et al., 1999; Gutteridge and Halliwell, 2000; Khachaturian, 1990). There is some evidence that apoE is resistant to oxidative damage and is involved in reducing neuronal death induced by oxidative stress (Miyata 2002 Elsevier Science Ltd. All rights reserved.
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and Smith, 1996). The effectiveness of this resistance was isoform dependent with E2 being more effective than E3, which, in turn, was more effective than E4. It is interesting that not only has oxidative stress been associated with the process of ageing, but the E4 isoform has been associated with a poorer prognosis for other conditions which are likely to result in the production of oxidative stress conditions in the CNS including cognitive recovery from head injury, cardiac bypass surgery and stroke (Sabo et al., 2000; Sheng et al., 1998; Tardiff et al., 1997). There is also evidence that apoE increases intracellular calcium concentrations in both neurons and astrocytes in the order E4>E3>E2 (Muller et al., 1998). As well as oxidative stress, calcium deregulation leads to changes in Ca2+ homeostasis which contributes to the production of free radicals, membrane damage and the deregulation of synaptic plasticity (Malenka, 1991; Neveu and Zucker, 1996). While calciumdependent exocytosis from the plasma membrane is well characterised in neurons and other cell types (Holz et al., 1991; Kasai, 1999; Knight et al., 1989), its effect on secretory vesicle trafficking is not well defined. The effects of oxidative stress on vesicle trafficking have also not been investigated. Oxidative stress causes numerous cellular changes including disruption of microtubules, along which secretory vesicles are normally transported to the cell surface from the Golgi-complex and endoplasmic reticulum (ER) (Bellomo and Mirabelli, 1992; Mirabelli et al., 1989; Rogers et al., 1989). Therefore we have examined the effects of oxidative stress and calcium deregulation on vesicle trafficking and on spatial changes to the Golgi and ER distribution. As we are specifically interested in how these models of ageing effect the CNS, we employed a human astrocyte cell line transfected with human apoE isoforms, E2, E3 or E4, to examine the effects of oxidative stress and calcium deregulation on vesicle trafficking. These cell lines were particularly appropriate as astrocytes are the major producers of CNS apoE (Pitas et al., 1987), the extracellular form of apoE which is more highly sialylated than that found in serum (Boyles et al., 1985), and the CNS form of apoE does not cross the blood brain barrier (Martel et al., 1997). MATERIALS AND METHODS Astrocytoma cultures The human glioblastoma astrocytoma cell line U-87 MG (European Collection of Cell Cultures), was a gift from Dr Ron Weinberger, Westmead
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hospital, Sydney, and was maintained in ‘Dmedium’ which consisted of DMEM (Sigma) supplemented with 10% FBS, 50 U/ml penicillin, 50 g/ml streptomycin, 2.5 g/ml amphotericin B and 200 m L-glutamine. Cultures were maintained at 37C in 5% CO2 with a change of medium every 72 h. For all experiments, cells were plated onto matrigel-coated polished glass coverslips (Lomb Scientific) in 24 well plates (Falcon) at a density of 1105 cells/well. Cells were cultured for a minimum of 48 h before use. Construction of expression vectors and transfections Human apoE cDNA clones for the E2; E3 and E4 isoforms were a gift from Dr Pu-Ting Xu and Dr Ann Saunders, Department of Medicine, Duke University Medical Center, Durham, NC. The coding region was amplified by PCR using the primers pApoE-F (5 -AAGCTTAAGATGAAGGTTCT GTGGGGC-3 ) and pApoE-R (5 -AGGATCCT CGTGATTGTCGCTGGGCACA-3 ) (Cybersyn), and were designed to introduce Hind III and Bam HI restriction sites to the 5 and 3 ends of the PCR product, respectively. The resulting PCR products were subcloned into a pGEM-T vector (Promega), digested with Hind III and Bam HI (Boehringer Mannheim), and ligated into the pEGFP-N1 expression vector (Clontech) (Dr Nicki Thompson, Glaxo Smith Kline, U.K.). U-87 MG astrocytoma cells were transfected with LipofectAMINE and PLUS reagents (Gibco BRL) according to the manufacturer’s recommendations. ‘L-medium’ was used during transfections and consisted of DMEM supplemented with 4% monomed and 200 m L-glutamine. U-87 MG astrocytes were plated onto 12 well plates at a density of 6–8106 cells per well. They were grown to approximately 80% confluence, then washed and re-fed with 500 l L-medium before transfections were performed. For each well 2 g of DNA was diluted to 50 l in L-medium and mixed by vortex. 5 l of PLUS reagent was then added, mixed again by vortex, and incubated at RT for 15 mins to form DNAPLUS reagent complexes. During this incubation 4 l LipofectAMINE was diluted to 50 l in L-medium and mixed. The diluted LipofectAMINE was added to the DNA-PLUS reagent mixture, mixed and incubated for a further 15 mins at RT. The final DNA-PLUS reagentLipofectAMINE complex was mixed with the L-medium on the cells (50 l/well) and incubated at 37C at 5% CO2 for 5 h. A further 500 l of
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D-medium, containing 20% FBS, was then added and the cultures incubated overnight at 37C at 5% CO2. The medium was then changed to G418-Dmedium (D-medium containing 400 g/ml G418) to select for stably transfected cells. Immunohistochemistry Mouse anti--tubulin antisera (Sigma), diluted (1/1000) was a gift from Dr John Harper, Dr Nim Weerakoon, and John Gardiner, School of Biological Sciences, University of Sydney, NSW, Australia. Secondary antibody, Alexa-594 conjugated rabbit anti-mouse (Molecular Probes), was diluted 1/500. All antisera were diluted in Tris buffered saline (TBS) consisting of 50 m Tris, 150 m NaCl, pH 7.5. All treatments in all experiments had at least two replicates (n=2). Controls consisted of untreated duplicate cultures incubated in (i) normal mouse serum, and (ii) minus primary antibody. No staining was apparent with minus primary antibody or normal serum controls. Cultures were fixed and permeablised by incubating in cold 5% acetic acid/95% ethanol for 20 mins at RT, washed three times in TBS and blocked in 1% BSA/1% FBS/TBS for 1 h at RT. After washing in TBS, cultures were incubated at 4C overnight in primary antibody, washed again in TBS and incubated in secondary antibody for 1 h at RT. After washing as before, duplicate coverslips were inverted on microscope slides in 3.5 l fluorescence anti-fade solution (Vectashield) and sealed with clear nail varnish. Stress conditions and the disruption of microtubules (i) Oxidative stress conditions included the addition of 1 m or 0.01 m H2O2 to D-medium. (ii) Deregulation of intracellular calcium was performed with either 50 m KCl or 10 m CaCl2 in calcium and magnesium free Hanks solution. Cultures for each of these conditions were incubated for at least 2 h at 37C. Microtubule depolymerisation was induced by incubating cells for 3 h at 37C in D-medium supplemented with 2.5 g/ml nocodazole (kindly provided by Dr. Monica Miranda, Westmead Millennium Institute, Westmead hospital, NSW, Australia). More stable polymerisation of microtubules was induced by incubating cells for 1 h at 37C in D-medium supplemented with 20 taxol (kindly provided by John Gardiner, Dr John Harper, and Dr Nim Weerakoon, School of Biological Sciences, University of Sydney, NSW, Australia).
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Confocal microscopy Immunostained cultures were examined by confocal microscopy (Nikon E800 with a Bio-Rad Radiance 2000 confocal unit) at the Electron Microscope Unit, University of Sydney, NSW, Australia. Single or time series images from each source (EGFP or Alexa-594, and transmission) were collected from the same field of view. For time series files an image was collected every 4 s for up to 180 min. Image analysis Time series files were assessed and merged, or ‘projected’, using Confocal Assistant version 4.02. Individual images from a time series were assessed for the total area of EGFP fluorescence, the number of particles, and particle size using Scion Image beta 4.0.2 for Windows. At least five consecutive images were assessed to avoid any effect of photo-damage, and each measurement was performed for at least six replicate time series. Particle speed and distance travelled were measured on the projected image using the ‘segmented line’ tool in Scion Image, while playing the time series file in parallel using Confocal Assistant to confirm the pathway of individual vesicles. This meant that the total travel distance was measured rather than the direct distance between starting and end points, and thus the average velocity was calculated for these vesicles. Online supplemental material Time-series files associated with Figure 4, from which vesicle number, size and speed were measured, are available as Quicktime files. An example of a control time-series file is provided in Figure 4A. The ER and Golgi are clearly discernible throughout the cell and vesicle traffic is predominantly found at the edges of the ER. In comparison, vesicle traffic appears faster and more prevalent under H2O2 conditions, Figure 4B, while those under KCl conditions were slower when compared to controls, Figure 4C. Under CaCl2 conditions vesicle traffic was similar to controls, Figure 4D. RESULTS ApoE-EGFP expression localises to vesicles, ER and Golgi The stably transfected U-87 MG astrocytoma cell lines, expressing apoE-EGFP for each of the
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common apoE isoforms (E2, E3 or E4), were established by transfecting the cell line with a cDNA encoding the recombinant protein and selected for neomycin-resistance. The expression of apoE-EGFP fusion protein in the cell lines was confirmed by SDS-PAGE and western transfer of cell lysates developed with an antiserum to apoE. Western transfers of all the cell lines showed a fusion protein of the expected size of approximately 63 kDa (data not shown). Examination of the cell lines expressing apoE2 and E3 by confocal microscopy revealed apoEEGFP present in vesicles and in a latticework structure, resembling endoplasmic reticulum (ER) and Golgi cisternae, which filled the majority of the cell body (Fig. 1A and B). Conversely, the E4 cell line produced no visible intracellular apoE4-EGFP, even though its synthesis was confirmed by western-transfer. To enhance the expression of apoE4-EGFP we incubated this cell line with the histone-deacetylation inhibitor sodium butyrate (5 m) for at least 24 h (Collas et al., 1998; Wacker et al., 1997), after which apoE-EGFP was visible in the same pattern as the other cell lines. The effects of H2O2 induced stress on apoE-EGFP localisation Oxidative stress in the apoE-EGFP expressing cell lines was induced by the addition of 1 m H2O2 and assessed by confocal microscopy. Assessment included the location of apoE-EGFP positive vesicles and determination of the structure of apoEEGFP positive Golgi and ER. In comparison to untreated control cells, apoE-EGFP fluorescence was seen within a condensed Golgi complex which localised to the perinuclear region, and was barely discernible in the ER (Fig. 1D). ApoE-EGFP positive vesicles were concentrated near the plasma membrane. The effect of H2O2 on apoE-EGFP localisation was not completely synchronised between different cells in the same culture. However, in the majority of cells, the changes in apoEEGFP localisation were apparent after 2 hours at 37C. Also, as expected, the addition of 0.01 m H2O2 resulted in a compacted Golgi structure but this was to a lesser degree than that found with 1 m H2O2 used in the previous experiments. The ER structure was also more apparent with apoEEGFP positive vesicles found throughout the cell body (Fig. 1C). With H2O2 treatment there also appeared to be an isoform specific difference in the number of cells affected by this treatment after 2 h, with 34% of E2, 41% of E3 and 58% of E4 cells affected.
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The effects of KCl and CaCl2 induced stress on apoE-EGFP localisation To investigate the effects of deregulated intracellular calcium levels, cells were incubated in calcium and magnesium free Hanks containing either 50 m KCl or 10 m CaCl2 for at least 2 h at 37C. KCl treatment had no discernible effect on the structure of the Golgi or the ER (Fig. 1E). However, in contrast to control cultures, cells treated with KCl had reduced vesicle traffic with the majority of vesicular movement resembling Brownian motion. By 30 min of KCl treatment, astrocytes also decreased in volume, resulting in a more stellate appearance. CaCl2 treatment of cells did not appear to alter vesicle movement or Golgi and ER structure (Fig. 1F).
The effects of stress on apoE-EGFP intracellular area To further characterise the spatial changes to the apoE-EFGP secretory pathway after treatment with H2O2, KCl or CaCl2, a series of photomicrographs, taken every 4 s, were collected by confocal microscopy. The average total area of apoE-EGFP fluorescence was determined from the first five consecutive images to avoid any effect of photo-damage. The area of apoE-EGFP fluorescence was also determined for five randomly selected fields-of-view per coverslip. The average total area of apoE-EGFP fluorescence was determined as an indication of the compaction or disruption of the Golgi and ER. After treatment with H2O2 the area of apoE-EGFP fluorescence in cells was significantly less (P<0.001) than controls (Fig. 2A) (Table 1). Conversely, KCl and CaCl2 treated cells showed no significant difference in the total area of apoE-EGFP fluorescence when compared with controls.
The effects of stress on the number of apoE-EGFP particles To measure the changes in vesicle formation, the total number of apoE-EGFP particles per cell was determined from the same images. H2O2 treated cells contained significantly more particles per cell (P<0.05), but with some overlap with control cells, while KCl and CaCl2 had no significant effect on the number of apoE-EGFP particles (Fig. 2B) (Table 1).
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Fig. 1. Representative confocal micrographs of apoE-EGFP localisation within astrocytoma cells under conditions modelling ageing. Untreated cells are represented at two different magnification in (A) and (B). At the higher magnification in (B) apoE-EGFP is present in vesicles and a latticework structure, resembling ER and Golgi cisternae. (C) and (D) are representative images of cells treated with 0.01 and 1 m H2O2 respectively. Both concentrations resulted in a condensed Golgi structure that remained perinuclear. A less discernible ER and the concentration of vesicles near the plasma membrane are also apparent in (D) with 1 m H2O2. With the deregulation of intracellular calcium with either (E) 50 m KCl or (F) 10 m CaCl2, the structure of the ER and Golgi do not appear to be effected, although cells appeared more stellate. Bar, 20 m.
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The overall effect of stress conditions on the spatial parameters of the apoE-EGFP secretory pathway
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To gain an overall view of the degree to which the apoE-EGFP containing structures were fragmented, the total area of fluorescence in each cell was divided by the number of particles it contained, thus accounting for any variation between cell size and shape. The area per particle in H2O2 treated cells was significantly lower (P<0.01) than that found for controls, while, in comparison, neither KCl nor CaCl2 treated cells were significantly different from controls (Fig. 2C) (Table 1). To further investigate the size of particles (or vesicles) in treated and control cultures we categorised particles on the basis of size. The size categories used were 0–0.025, 0.025–0.05, 0.05– 0.075, 0.075–0.1, 0.1–0.15, 0.15–0.2, 0.2–0.3, 0.3– 0.4, 0.4–0.5, 0.5–1.0, 1.0–1.5 and 1.5–2.0. Particles larger than 2 m2, normally less than 5% of all particles, were excluded as they were likely to be part of the ER or Golgi and not vesicles. 35–48% of vesicles, were between 0.025 and 0.05 m2 with no significant difference between any of the treatments and control cells (Fig. 3).
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Fig. 2. The effects of ageing-related stress on apoE-EGFP intracellular spatial parameters. (A) The average total area of apoE-EGFP fluorescence under H2O2 (), control (), KCl (), and CaCl2 ( ) conditions, showing significantly lower values under H2O2 conditions. (B) The average number of apoE-EGFP particles per cell under stress conditions, with significantly more particles under H2O2 conditions. (C) The average area per apoE-EGFP particle under stress conditions giving an overall indication of fragmentation and accounting for any variation in cell size and shape. Again, H2O2 conditions resulted in a significant reduction in area per particle.
In vivo analysis of movement of individual apoEEGFP vesicles was carried out by confocal microscopy. Time-series micrographs were collected, with an image taken every 4 seconds for up to 180 minutes. Vesicles in both treated and control cells moved in several different ways: rapid movement with no or one change in direction, movement with variations of speed, movement but with several reversals of direction, and small movements about a fixed position in several directions or Brownian movement. To determine if there were any significant changes in vesicle traffic under H2O2 induced stress, the total distance travelled was measured for at least 20 vesicles selected at random. The exact pathway of vesicles was determined time-series images by merging, or ‘projecting’, using Confocal Assistant v4.02 and the pathway of individual vesicles confirmed by playing the time-series file in parallel. Examples of time-series and projected images for treated and control cells are shown in Figure 4, and time-series files are available online (see Online Supplemental Material under Materials and Methods). The distance travelled by vesicles was measured on the projected image using the
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Fig. 3. The percentage of particles in each size category. There was no significant difference between treatments and controls, and the majority of particles were between 0.025 and 0.05 m2.
‘segmented line’ tool in Scion Image. This meant that the total travel distance was measured rather than the direct distance between starting and end points, and thus the average velocity was calculated for these vesicles. The distance travelled by vesicles in H2O2 treated cells was significantly longer than those in controls (P<0.0001), even when the two greatest values in the treated group were excluded from the t-test (Fig. 5A) (Table 1). Surprisingly, the distance travelled by vesicles in CaCl2 treated cells was also significantly longer than in controls (P<0.0001), while, in contrast, the distance travelled by vesicles in KCl treated cells was not significantly different from controls. The average velocity of vesicles was calculated from the distance travelled and the total time in which the vesicle was moving (ie the number of frames multiplied by 4 s). Vesicles in H2O2 treated cells travelled at velocities significantly faster than those found in controls (P<0.0001), even though 30% of velocities in the H2O2 treated group were within the same range as controls (Fig. 5B) (Table 1). In contrast, vesicles in KCl treated cells were significantly slower (P<0.0001) than controls. However, in spite of travelling a significantly greater distance than control vesicles, CaCl2 treated cells travelled at a similar velocity.
ApoE isoforms under stress conditions There was no significant difference in effect on vesicle trafficking between apoE isoforms under stress conditions (Fig. 6). However, there was an isoform specific difference in the number of cells in each of the transfected cell types being affected by H2O2 treatment, with 34% of E2, 41% of E3 and 58% of E4 cells being affected. The effects of nocodazole and taxol on apoE-EGFP vesicle movement as well as Golgi and ER structure To further investigate the changes in vesicle speeds under oxidative stress and KCl conditions, cells were treated with nocodazol to completely depolymerise microtubules, and taxol to promote stable microtubule polymerisation. After 3 h in nocodazole the Golgi and ER were no longer discernible and apoE-EGFP positive vesicles were distributed throughout the cell (Fig. 7A). Only Brownian movement of apoE vesicles was observed, with directional movement no longer apparent (not shown). In contrast, in taxol treated cells showed directional movement of apoE-EGFP vesicles but this was in the range of velocities seen with the KCl treated cells. The Golgi and ER structure remained unchanged.
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Fig. 4. The effects of ageing-related stress conditions on vesicle movement. Representative frames from confocal time-series micrographs for each treatment are shown, with the first, middle and last frame from which a vesicle path was measured. The vesicle location is circled in consecutive frames and indicated by the arrows in the projected, or merged, image. Vesicles in (A) control and (D) CaCl2 treated cells show similar movements. Vesicles in (B) H2O2 treated cells were faster, and those in (C) KCl treated cells slower, when compared to controls. Time-series files are also available online (see Online Supplemental Material). Bar, 10 m.
The effects of H2O2 and KCl stress conditions on microtubule organisation As the majority of vesicle traffic is along microtubule tracks, cells were immunostained for -tubulin after incubation for 2 h in the same H2O2 and KCl stress conditions used to study Golgi/ER
structure and vesicle traffic. Nocodazole and taxol treated cells were also immunostained as controls for microtubule depolymerisation and polymerisation respectively. A comparison between the -tubulin staining of H2O2 treated cells and the controls suggests that there is only a partial depolymerisation of microtubules (Fig. 7). In
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Fig. 6. ApoE isoforms under ageing-related stress conditions. The average area per particle is shown for each apoE isoform as a representative aspect of apoE-EGFP spatial changes. No isoform-specific difference was apparent under any of the conditions after analysis of the data using a Student’s t-test.
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structure, while the apoE-EGFP was distributed throughout the cytoplasm as vesicular structures (Fig. 7G). However, no directional vesicle movement was detected. The -tubulin polymerisation was also found to be completely disrupted (Fig. 7H), appearing similar to that found after nocodazole treatment.
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Fig. 5. Distance travelled (A) and velocity (B) of vesicles under ageing-related stress conditions. Under H2O2 and CaCl2 conditions vesicles travelled significantly further that in KCl or control cells. Vesicles under H2O2 conditions were also significantly faster than controls and vesicles under KCl conditions were significantly slower.
contrast, KCl treated cells showed an increase in the polymerisation of microtubules corresponding to a more activated form of astrocyte. The longer-term exposure of astrocytes to H2O2 stress conditions As a 2 h treatment of astrocytes with H2O2 resulted in only partial microtubule disruption, an increase in vesicle speed and partial disruption of Golgi and ER structure we investigated the effects of a 4 h exposure to H2O2 stress conditions. After 4 h there appeared to be no perinuclear Golgi or ER
We report here that oxidative stress conditions increased the speed and number of apoE positive secretory vesicles in our transfected astrocytoma cells, with no difference detected between the three isoforms. We also found that the apoE-positive Golgi and ER appeared disrupted but remained perinuclear. There was, however, an apoE isoformspecific difference in the number of cells resistant to oxidative stress with the E2 cell line being most resistant and the E4 cell line the least resistant. As the Golgi-complex and ER are well established as the major sites of protein synthesis, posttranslational modification and sorting in eucaryotic cells (Alberts et al., 1994), the association of apoE with these structures was not unexpected. What is unusual, however, is that vesicle speed increased after 2 h under oxidative stress conditions. Although the effect of oxidative stress on vesicular trafficking has not been previously reported such stress is known to disrupt microtubule proteins (Banan et al., 2000; Neely et al., 1999). Therefore, we expected oxidative stress to progressively slow
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Fig. 7. The effects of nocodazole, taxol, and H2O2 and KCl stress conditions on microtubule organisation. Nocodazole disruption of apoE-EGFP localisation in astrocytoma cells is apparent in (A) where the Golgi and ER are no longer visible. (B) Shows the corresponding disruption of -tubulin. Taxol, on the other hand, increased -tubulin polymerisation (C) when compared to untreated control cells (D). In comparison, (E) -tubulin appeared partially disrupted after 2 h under H2O2 conditions, while under KCl conditions (F) -tubulin was more polymerised and cell volume decreased. After 4 h under H2O2 conditions apoE-EGFP localisation (G) was entirely vesicular and was not concentrated in a perinuclear region. The apoE-EGFP localisation was also similar to that found in cells treated with nocodazole (A). The -tubulin staining of cells after 4 h under H2O2 was also similar to nocodazole treatment, showing a greater disruption of microtubule organisation. Bar, 20 m (H).
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apoE vesicle movement and that this slowing would correspond to a disruption of microtubules. Instead while the velocity and number of apoE vesicles increased after 2 h, the microtubules appeared only partially disrupted. One explanation for this increase in vesicle speed is an increase in protein phosphorylation, mediated by the activation of protein kinases or inhibition of protein phosphatases, as an increase in phosphorylation is known to affect both vesicle speed and microtubule stability (Hammalvarez et al., 1993; Lee, 1993; Olmsted, 1986). Microtubule stability is governed in part by the binding of microtubuleassociated-proteins (MAPs), which in a phosphorylated state dissociate from microtubules. The binding of MAPs to tubulin is also inhibited by the molecule mapmodulin, which in its phosphorylated state binds to the tubulin-binding domain of MAPs (Itin et al., 1999; Ulitzur et al., 1997). This binding of mapmodulin also occurs under normal conditions allowing the transport of organelles along microtubule tracks, although it is not essential for vesicle movement to occur. The inhibition of protein phosphatases by okadaic acid has also been shown to disrupt microtubules, as well as increase the speed of vesicles in CV-1 cells (Hammalvarez et al., 1993). In addition, the phosphorylation of kinesin motor proteins induces their binding to microtubules and correlates with an increase in organelle transport to the plus ends of microtubules (Lee and Hollenbeck, 1995). Such changes in protein phosphorylation could therefore account for the increased rate of transport of the 70% of vesicles seen in our experiments with oxidative stress. Further, the incomplete disruption of microtubules, as seen with a comparison of -tubulin staining of controls, at 2 h with H2O2 and in nocodazol treated cells, accounts for the 30% of vesicles under oxidative stress conditions travelling at speeds within the lower range observed in our control cells. Oxidative stress conditions however also lead to lipid peroxidation, a major product of which is 4-hydroxy-2 (E)-nonenal (4-HNE) (Vanwinkle et al., 1994). Interestingly, 4-HNE also disrupts microtubules by modifying tubulin (Neely et al., 1999) and binding to the MAP, tau, preventing its dephosphorylation and moderately promoting its phosphorylation (Mattson et al., 1997). Therefore, this combination of events resulting from oxidative stress could result in disruption of microtubules independently of an increase in vesicle speed. As microtubule disruption also causes Golgi and ER fragmentation, the above scenario offers a further explanation for the increase in the number
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of apoE vesicles in our experiments. The perinuclear location of Golgi and ER after 2 h of oxidative stress, is also consistent with our immunohistochemical evidence that not all microtubules are disrupted. However, by 4 h of oxidative stress disruption appeared complete. Complete disruption of microtubules was also observed in our experiments with nocodazole-treated cells, and consistent with studies of (Turner and Tartakoff, 1989) using Madin-Darby bovine kidney cells. Complete microtubule disruption indicates that cell damage increases significantly after 4 h of oxidative stress. Interestingly, the phenomenon of Golgi dispersal and reassembly also occurs during mitosis (Lucocq et al., 1987; Robbins and Gonatas, 1964). Thus our results may have more general implications for vesicle trafficking during this stage of the cell cycle. More specifically, fragmented Golgi are known to be specifically dispersed through the cytoplasm to sites corresponding to the location of disrupted ER (Cole et al., 1996). With oxidative stress, this may also be a fortuitous response to injury, aiding cell recovery in concert with the local synthesis of protein and/or lipid. While the vesicle speed of Golgi redistribution and a dependence on microtubules has not previously been reported, Golgi reassembly is known to be microtubule dependent (Ho et al., 1989). The reported vesicle speed (0.1 to 0.4 m/s) during microtubule and Golgi reassembly in Vero fibroblast cells is comparable to our findings in control cultures, but in contrast to the speeds in our astrocytes after 2 h of oxidative stress. Nocodazole treatment and the resulting complete disruption of microtubules, however, is known to dramatically slow the movement of secretory vesicles (Wacker et al., 1997), which was also seen in our experiments with nocodazole. Interestingly, 4 h of oxidative stress slowed vesicle movement in our cell lines with a resulting loss of directional vesicle traffic. In addition, the increase in vesicle number and velocity may reflect a direct apoE response to membrane damage, which would be important considering the role of apoE in cholesterol redistribution (Mahley, 1988). It has been reported that apoE is retained without degradation in lysosomal vesicles (DeKroon and Armati, 2001; Heeren et al., 1999; Ji et al., 1998; Rensen et al., 2000), a point where the endocytic and secretory pathways meet (Mellman, 1996; Mukherjee et al., 1997). From this point in the endocytic/secretory pathway, apoE may be recycled or secreted. Further, apoE is synthesised and secreted in response to extracellular signals, such as nerve growth factor, as well
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as cellular damage (Futerman and Banker, 1996; Soulie et al., 1999). Therefore the activation of the secretory pathway in response to cell growth and repair (Kozu et al., 1997; Miyata et al., 1988; Zhang et al., 1998 and our results), as indicated by the increase in vesicle number and velocity, may offer a purpose for this unique retention of apoE in cells under standard culture conditions. In our second stress model, utilising KCl and CaCl2, the lack of changes observed in both the number of apoE vesicles and the structure of the ER, Golgi and microtubules in cells, were not unexpected. In contrast to the oxidative stress findings, the increase in extracellular KCl concentration slowed the movement of intracellular apoE vesicles after 2 h and induced the transformation of some cells into so-called ‘activated astrocytes’ (Gehrmann et al., 1995; Landis, 1994), with a corresponding decrease in cell volume and increase in -tubulin staining, a marker of microtubule polymerisation. Interestingly, extracellular K + concentrations which rise after ischemic damage and induce a wave of intracellular Ca2+ , also results in activated astrocytes (Verkhratsky et al., 1998). In our experiments, cells were exposed to a high extracellular concentration of K + for 2 h, with an initial wave of Ca2+ into the cells and later lowering of the [Ca2+ ]i, as confirmed by Fluo-4 AM (not shown). Under these conditions the speed of vesicle movement was significantly slower, suggesting that intracellular Ca2+ may play a role in vesicle movement. Of note is that an increased [Ca2+ ]i in HT4 and cortical neurons has been reported to activate protein phosphatase 2B, leading to dephosphorylation of actin depolymerising factor/cofilin and the polymerisation of actin (Meberg et al., 1998). In addition, the stabilisation of microtubules with taxol in CV-1 cells (Hammalvarez et al., 1993), as well as our astrocyte cells, slows vesicle speed after 2 h. In contrast, an increase in [Ca2+ ]i induced by A23187 increases vesicle speed in CV-1 cells. These results together suggest that an initial wave of Ca2+ influx, resulting in further polymerisation of -tubulin, could be responsible for the slowing of vesicle traffic in our second stress model. Furthermore, the lower extracellular Ca2+ levels in these experiments could contribute to the slower vesicle speed, as reported by Lin et al. in cultured fibroblasts (Lin and Collins, 1993). However, the KClstress conditions are likely to have a number of other effects, including changes in the concentration of other ions such as sodium and the depletion or inactivation of ATPases. Therefore, although our results with the KCl-stress conditions
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are interesting, the influence of other KCl-affected factors on vesicle movement cannot be discounted in these experiments. It is not surprising that we found no isoformspecific differences in the apoE synthesis or secretory pathways under either oxidative or calcium stress conditions, as apoE is not directly involved in vesicle trafficking and the structural differences between the apoE isoforms are single amino acid substitutions (Mahley, 1988). It was instead the number of astrocyte cells resistant to oxidative stress that presented apoE isoformspecific differences, with E2>E3>E4. This finding is directly comparable to the protective antioxidant effects of apoE isoforms added exogenously to neurons as reported by (Miyata and Smith, 1996) with again, E2>E3>E4. These results also suggest that the antioxidant effects of apoE isoforms are mediated extracellularly as it is only after apoE secretion that the isoform difference is apparent. While our results are of importance to basic knowledge of vesicle transport under conditions of oxidative stress and deregulated intracellular Ca2+ , they also have implications for the role of apoE in the CNS. Our results with oxidative stress conditions suggest that the up-regulation of apoE vesicle number and transport, linked to incomplete microtubule depolymerisation, may be an early response to oxidative stress. The deregulation of [Ca2+ ]i may also contribute to a deregulation of the apoE response to injury. Therefore, our report of the regulation of the apoE secretory pathway under both oxidative and calcium stress conditions is important information of relevance to our understanding of the ageing process in the brain as well as in recovery from head injury and ischemic damage.
ACKNOWLEDGEMENTS We thank Ellie Kable and Dr Guy Cox, Electron Microscope Unit, University of Sydney, for valuable advice with confocal image analysis. This work was supported by Glaxo Smith Kline Ltd and The Nerve Research Foundation of the University of Sydney. REFERENCES A B, B D, L J, R M, R K, W JD, 1994. Molecular Biology of the Cell. 3rd ed. New York, Garland.
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THE EFFECTS OF OXIDATIVE STRESS AND ALTERED INTRACELLULAR CALCIUM LEVELS ON VESICULAR TRANSPORT OF APOE-EGFP ROBERT M. DEKROON and PATRICIA J. ARMATI1 Neuroscience Unit, School of Biological Sciences A08, University of Sydney, NSW 2006, Australia Received 16 November 2001; accepted 5 January 2002
Apolipoprotein E (apoE) plays a role in the distribution of lipid within many organs and cell types in the human body, including the central nervous system (CNS). The apoE4 isoform is also an established risk factor for late-onset Alzheimer’s disease (AD), however its role in the aetiology of the disease remains largely unknown. Therefore, as AD is a late-onset disease, we sort to investigate how conditions hypothesised to model ageing affect apoE metabolism, such as the transport of apoE along the secretory pathway. Two of these models include oxidative stress and calcium deregulation. Using apoE-EGFP-expressing astrocytoma cell lines we established that vesicle number and velocity are up-regulated under oxidative stress conditions, and slowed under KCl induced calcium deregulation. Although these findings apply to cells in general under these two stress conditions, the up-regulation of apoE in particular may be a response to cell injury with implications for neurodegeneration such as that found with 2002 Elsevier Science Ltd. All rights reserved. late-onset AD. K: apolipoprotein E; oxidative stress; vesicle trafficking; exocytosis; ageing. A: apoE, Apolipoprotein E; CNS, central nervous system; AD, Alzheimer’s disease; EGFP, modified form of the green fluorescent protein; ER, endoplasmic reticulum.
INTRODUCTION Apolipoprotein E (apoE) is a 34 kDa protein that appears to play a role in the redistribution of cholesterol amongst the cells of the central nervous system (CNS) and in the maintenance of cholesterol homeostasis (Boyles et al., 1985; Pitas et al., 1987). There are 3 common isoforms of apoE, E2, E3 and E4, which are the products of a single gene locus on chromosome 19. The apoE3 isoform contains cysteine and arginine at positions 112 and 158, respectively, while apoE2 has cysteine at both positions and apoE4 has arginine at both positions (Mahley, 1988). The association of the E4 isoform of apoE as a genetic risk factor for late-onset AD has been well established (Corder et al., 1993; Roses et al., 1995) The E4 isoform also lowers the age of onset of AD. Individuals homozygous for E4 are 95% more 1
Corresponding author: Patricia Armati: Neuroscience Unit, School of Biological Sciences A08, University of Sydney, NSW 2006, Australia. Tel: 612-9351-2062; Fax: 612-9351-4119; E-mail: [email protected] 1065–6995/02/$-see front matter
likely to acquire the disease at an average age of 68.4 years, compared to individuals heterozygous for the E4 isoform who are 65% more likely to acquire the disease at an average age of 75.5 years (Corder et al., 1993). However, even though these susceptibility data strongly suggest that apoE is involved in disease onset (Roses et al., 1995), the role of apoE in the aetiology of late-onset AD remains largely unknown. An important way of addressing this problem is to understand how the process of ageing affects apoE metabolism in the CNS, such as the transport of synthesised apoE along the secretory pathway from Golgi to the cell membrane. There are a number of conditions hypothesised to contribute to the ageing process, two of which are oxidative stress and deregulation of intracellular calcium concentrations (Butterfield et al., 1999; Gutteridge and Halliwell, 2000; Khachaturian, 1990). There is some evidence that apoE is resistant to oxidative damage and is involved in reducing neuronal death induced by oxidative stress (Miyata 2002 Elsevier Science Ltd. All rights reserved.
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and Smith, 1996). The effectiveness of this resistance was isoform dependent with E2 being more effective than E3, which, in turn, was more effective than E4. It is interesting that not only has oxidative stress been associated with the process of ageing, but the E4 isoform has been associated with a poorer prognosis for other conditions which are likely to result in the production of oxidative stress conditions in the CNS including cognitive recovery from head injury, cardiac bypass surgery and stroke (Sabo et al., 2000; Sheng et al., 1998; Tardiff et al., 1997). There is also evidence that apoE increases intracellular calcium concentrations in both neurons and astrocytes in the order E4>E3>E2 (Muller et al., 1998). As well as oxidative stress, calcium deregulation leads to changes in Ca2+ homeostasis which contributes to the production of free radicals, membrane damage and the deregulation of synaptic plasticity (Malenka, 1991; Neveu and Zucker, 1996). While calciumdependent exocytosis from the plasma membrane is well characterised in neurons and other cell types (Holz et al., 1991; Kasai, 1999; Knight et al., 1989), its effect on secretory vesicle trafficking is not well defined. The effects of oxidative stress on vesicle trafficking have also not been investigated. Oxidative stress causes numerous cellular changes including disruption of microtubules, along which secretory vesicles are normally transported to the cell surface from the Golgi-complex and endoplasmic reticulum (ER) (Bellomo and Mirabelli, 1992; Mirabelli et al., 1989; Rogers et al., 1989). Therefore we have examined the effects of oxidative stress and calcium deregulation on vesicle trafficking and on spatial changes to the Golgi and ER distribution. As we are specifically interested in how these models of ageing effect the CNS, we employed a human astrocyte cell line transfected with human apoE isoforms, E2, E3 or E4, to examine the effects of oxidative stress and calcium deregulation on vesicle trafficking. These cell lines were particularly appropriate as astrocytes are the major producers of CNS apoE (Pitas et al., 1987), the extracellular form of apoE which is more highly sialylated than that found in serum (Boyles et al., 1985), and the CNS form of apoE does not cross the blood brain barrier (Martel et al., 1997). MATERIALS AND METHODS Astrocytoma cultures The human glioblastoma astrocytoma cell line U-87 MG (European Collection of Cell Cultures), was a gift from Dr Ron Weinberger, Westmead
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hospital, Sydney, and was maintained in ‘Dmedium’ which consisted of DMEM (Sigma) supplemented with 10% FBS, 50 U/ml penicillin, 50 g/ml streptomycin, 2.5 g/ml amphotericin B and 200 m L-glutamine. Cultures were maintained at 37C in 5% CO2 with a change of medium every 72 h. For all experiments, cells were plated onto matrigel-coated polished glass coverslips (Lomb Scientific) in 24 well plates (Falcon) at a density of 1105 cells/well. Cells were cultured for a minimum of 48 h before use. Construction of expression vectors and transfections Human apoE cDNA clones for the E2; E3 and E4 isoforms were a gift from Dr Pu-Ting Xu and Dr Ann Saunders, Department of Medicine, Duke University Medical Center, Durham, NC. The coding region was amplified by PCR using the primers pApoE-F (5 -AAGCTTAAGATGAAGGTTCT GTGGGGC-3 ) and pApoE-R (5 -AGGATCCT CGTGATTGTCGCTGGGCACA-3 ) (Cybersyn), and were designed to introduce Hind III and Bam HI restriction sites to the 5 and 3 ends of the PCR product, respectively. The resulting PCR products were subcloned into a pGEM-T vector (Promega), digested with Hind III and Bam HI (Boehringer Mannheim), and ligated into the pEGFP-N1 expression vector (Clontech) (Dr Nicki Thompson, Glaxo Smith Kline, U.K.). U-87 MG astrocytoma cells were transfected with LipofectAMINE and PLUS reagents (Gibco BRL) according to the manufacturer’s recommendations. ‘L-medium’ was used during transfections and consisted of DMEM supplemented with 4% monomed and 200 m L-glutamine. U-87 MG astrocytes were plated onto 12 well plates at a density of 6–8106 cells per well. They were grown to approximately 80% confluence, then washed and re-fed with 500 l L-medium before transfections were performed. For each well 2 g of DNA was diluted to 50 l in L-medium and mixed by vortex. 5 l of PLUS reagent was then added, mixed again by vortex, and incubated at RT for 15 mins to form DNAPLUS reagent complexes. During this incubation 4 l LipofectAMINE was diluted to 50 l in L-medium and mixed. The diluted LipofectAMINE was added to the DNA-PLUS reagent mixture, mixed and incubated for a further 15 mins at RT. The final DNA-PLUS reagentLipofectAMINE complex was mixed with the L-medium on the cells (50 l/well) and incubated at 37C at 5% CO2 for 5 h. A further 500 l of
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D-medium, containing 20% FBS, was then added and the cultures incubated overnight at 37C at 5% CO2. The medium was then changed to G418-Dmedium (D-medium containing 400 g/ml G418) to select for stably transfected cells. Immunohistochemistry Mouse anti--tubulin antisera (Sigma), diluted (1/1000) was a gift from Dr John Harper, Dr Nim Weerakoon, and John Gardiner, School of Biological Sciences, University of Sydney, NSW, Australia. Secondary antibody, Alexa-594 conjugated rabbit anti-mouse (Molecular Probes), was diluted 1/500. All antisera were diluted in Tris buffered saline (TBS) consisting of 50 m Tris, 150 m NaCl, pH 7.5. All treatments in all experiments had at least two replicates (n=2). Controls consisted of untreated duplicate cultures incubated in (i) normal mouse serum, and (ii) minus primary antibody. No staining was apparent with minus primary antibody or normal serum controls. Cultures were fixed and permeablised by incubating in cold 5% acetic acid/95% ethanol for 20 mins at RT, washed three times in TBS and blocked in 1% BSA/1% FBS/TBS for 1 h at RT. After washing in TBS, cultures were incubated at 4C overnight in primary antibody, washed again in TBS and incubated in secondary antibody for 1 h at RT. After washing as before, duplicate coverslips were inverted on microscope slides in 3.5 l fluorescence anti-fade solution (Vectashield) and sealed with clear nail varnish. Stress conditions and the disruption of microtubules (i) Oxidative stress conditions included the addition of 1 m or 0.01 m H2O2 to D-medium. (ii) Deregulation of intracellular calcium was performed with either 50 m KCl or 10 m CaCl2 in calcium and magnesium free Hanks solution. Cultures for each of these conditions were incubated for at least 2 h at 37C. Microtubule depolymerisation was induced by incubating cells for 3 h at 37C in D-medium supplemented with 2.5 g/ml nocodazole (kindly provided by Dr. Monica Miranda, Westmead Millennium Institute, Westmead hospital, NSW, Australia). More stable polymerisation of microtubules was induced by incubating cells for 1 h at 37C in D-medium supplemented with 20 taxol (kindly provided by John Gardiner, Dr John Harper, and Dr Nim Weerakoon, School of Biological Sciences, University of Sydney, NSW, Australia).
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Confocal microscopy Immunostained cultures were examined by confocal microscopy (Nikon E800 with a Bio-Rad Radiance 2000 confocal unit) at the Electron Microscope Unit, University of Sydney, NSW, Australia. Single or time series images from each source (EGFP or Alexa-594, and transmission) were collected from the same field of view. For time series files an image was collected every 4 s for up to 180 min. Image analysis Time series files were assessed and merged, or ‘projected’, using Confocal Assistant version 4.02. Individual images from a time series were assessed for the total area of EGFP fluorescence, the number of particles, and particle size using Scion Image beta 4.0.2 for Windows. At least five consecutive images were assessed to avoid any effect of photo-damage, and each measurement was performed for at least six replicate time series. Particle speed and distance travelled were measured on the projected image using the ‘segmented line’ tool in Scion Image, while playing the time series file in parallel using Confocal Assistant to confirm the pathway of individual vesicles. This meant that the total travel distance was measured rather than the direct distance between starting and end points, and thus the average velocity was calculated for these vesicles. Online supplemental material Time-series files associated with Figure 4, from which vesicle number, size and speed were measured, are available as Quicktime files. An example of a control time-series file is provided in Figure 4A. The ER and Golgi are clearly discernible throughout the cell and vesicle traffic is predominantly found at the edges of the ER. In comparison, vesicle traffic appears faster and more prevalent under H2O2 conditions, Figure 4B, while those under KCl conditions were slower when compared to controls, Figure 4C. Under CaCl2 conditions vesicle traffic was similar to controls, Figure 4D. RESULTS ApoE-EGFP expression localises to vesicles, ER and Golgi The stably transfected U-87 MG astrocytoma cell lines, expressing apoE-EGFP for each of the
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common apoE isoforms (E2, E3 or E4), were established by transfecting the cell line with a cDNA encoding the recombinant protein and selected for neomycin-resistance. The expression of apoE-EGFP fusion protein in the cell lines was confirmed by SDS-PAGE and western transfer of cell lysates developed with an antiserum to apoE. Western transfers of all the cell lines showed a fusion protein of the expected size of approximately 63 kDa (data not shown). Examination of the cell lines expressing apoE2 and E3 by confocal microscopy revealed apoEEGFP present in vesicles and in a latticework structure, resembling endoplasmic reticulum (ER) and Golgi cisternae, which filled the majority of the cell body (Fig. 1A and B). Conversely, the E4 cell line produced no visible intracellular apoE4-EGFP, even though its synthesis was confirmed by western-transfer. To enhance the expression of apoE4-EGFP we incubated this cell line with the histone-deacetylation inhibitor sodium butyrate (5 m) for at least 24 h (Collas et al., 1998; Wacker et al., 1997), after which apoE-EGFP was visible in the same pattern as the other cell lines. The effects of H2O2 induced stress on apoE-EGFP localisation Oxidative stress in the apoE-EGFP expressing cell lines was induced by the addition of 1 m H2O2 and assessed by confocal microscopy. Assessment included the location of apoE-EGFP positive vesicles and determination of the structure of apoEEGFP positive Golgi and ER. In comparison to untreated control cells, apoE-EGFP fluorescence was seen within a condensed Golgi complex which localised to the perinuclear region, and was barely discernible in the ER (Fig. 1D). ApoE-EGFP positive vesicles were concentrated near the plasma membrane. The effect of H2O2 on apoE-EGFP localisation was not completely synchronised between different cells in the same culture. However, in the majority of cells, the changes in apoEEGFP localisation were apparent after 2 hours at 37C. Also, as expected, the addition of 0.01 m H2O2 resulted in a compacted Golgi structure but this was to a lesser degree than that found with 1 m H2O2 used in the previous experiments. The ER structure was also more apparent with apoEEGFP positive vesicles found throughout the cell body (Fig. 1C). With H2O2 treatment there also appeared to be an isoform specific difference in the number of cells affected by this treatment after 2 h, with 34% of E2, 41% of E3 and 58% of E4 cells affected.
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The effects of KCl and CaCl2 induced stress on apoE-EGFP localisation To investigate the effects of deregulated intracellular calcium levels, cells were incubated in calcium and magnesium free Hanks containing either 50 m KCl or 10 m CaCl2 for at least 2 h at 37C. KCl treatment had no discernible effect on the structure of the Golgi or the ER (Fig. 1E). However, in contrast to control cultures, cells treated with KCl had reduced vesicle traffic with the majority of vesicular movement resembling Brownian motion. By 30 min of KCl treatment, astrocytes also decreased in volume, resulting in a more stellate appearance. CaCl2 treatment of cells did not appear to alter vesicle movement or Golgi and ER structure (Fig. 1F).
The effects of stress on apoE-EGFP intracellular area To further characterise the spatial changes to the apoE-EFGP secretory pathway after treatment with H2O2, KCl or CaCl2, a series of photomicrographs, taken every 4 s, were collected by confocal microscopy. The average total area of apoE-EGFP fluorescence was determined from the first five consecutive images to avoid any effect of photo-damage. The area of apoE-EGFP fluorescence was also determined for five randomly selected fields-of-view per coverslip. The average total area of apoE-EGFP fluorescence was determined as an indication of the compaction or disruption of the Golgi and ER. After treatment with H2O2 the area of apoE-EGFP fluorescence in cells was significantly less (P<0.001) than controls (Fig. 2A) (Table 1). Conversely, KCl and CaCl2 treated cells showed no significant difference in the total area of apoE-EGFP fluorescence when compared with controls.
The effects of stress on the number of apoE-EGFP particles To measure the changes in vesicle formation, the total number of apoE-EGFP particles per cell was determined from the same images. H2O2 treated cells contained significantly more particles per cell (P<0.05), but with some overlap with control cells, while KCl and CaCl2 had no significant effect on the number of apoE-EGFP particles (Fig. 2B) (Table 1).
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Fig. 1. Representative confocal micrographs of apoE-EGFP localisation within astrocytoma cells under conditions modelling ageing. Untreated cells are represented at two different magnification in (A) and (B). At the higher magnification in (B) apoE-EGFP is present in vesicles and a latticework structure, resembling ER and Golgi cisternae. (C) and (D) are representative images of cells treated with 0.01 and 1 m H2O2 respectively. Both concentrations resulted in a condensed Golgi structure that remained perinuclear. A less discernible ER and the concentration of vesicles near the plasma membrane are also apparent in (D) with 1 m H2O2. With the deregulation of intracellular calcium with either (E) 50 m KCl or (F) 10 m CaCl2, the structure of the ER and Golgi do not appear to be effected, although cells appeared more stellate. Bar, 20 m.
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The overall effect of stress conditions on the spatial parameters of the apoE-EGFP secretory pathway
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To gain an overall view of the degree to which the apoE-EGFP containing structures were fragmented, the total area of fluorescence in each cell was divided by the number of particles it contained, thus accounting for any variation between cell size and shape. The area per particle in H2O2 treated cells was significantly lower (P<0.01) than that found for controls, while, in comparison, neither KCl nor CaCl2 treated cells were significantly different from controls (Fig. 2C) (Table 1). To further investigate the size of particles (or vesicles) in treated and control cultures we categorised particles on the basis of size. The size categories used were 0–0.025, 0.025–0.05, 0.05– 0.075, 0.075–0.1, 0.1–0.15, 0.15–0.2, 0.2–0.3, 0.3– 0.4, 0.4–0.5, 0.5–1.0, 1.0–1.5 and 1.5–2.0. Particles larger than 2 m2, normally less than 5% of all particles, were excluded as they were likely to be part of the ER or Golgi and not vesicles. 35–48% of vesicles, were between 0.025 and 0.05 m2 with no significant difference between any of the treatments and control cells (Fig. 3).
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Fig. 2. The effects of ageing-related stress on apoE-EGFP intracellular spatial parameters. (A) The average total area of apoE-EGFP fluorescence under H2O2 (), control (), KCl (), and CaCl2 ( ) conditions, showing significantly lower values under H2O2 conditions. (B) The average number of apoE-EGFP particles per cell under stress conditions, with significantly more particles under H2O2 conditions. (C) The average area per apoE-EGFP particle under stress conditions giving an overall indication of fragmentation and accounting for any variation in cell size and shape. Again, H2O2 conditions resulted in a significant reduction in area per particle.
In vivo analysis of movement of individual apoEEGFP vesicles was carried out by confocal microscopy. Time-series micrographs were collected, with an image taken every 4 seconds for up to 180 minutes. Vesicles in both treated and control cells moved in several different ways: rapid movement with no or one change in direction, movement with variations of speed, movement but with several reversals of direction, and small movements about a fixed position in several directions or Brownian movement. To determine if there were any significant changes in vesicle traffic under H2O2 induced stress, the total distance travelled was measured for at least 20 vesicles selected at random. The exact pathway of vesicles was determined time-series images by merging, or ‘projecting’, using Confocal Assistant v4.02 and the pathway of individual vesicles confirmed by playing the time-series file in parallel. Examples of time-series and projected images for treated and control cells are shown in Figure 4, and time-series files are available online (see Online Supplemental Material under Materials and Methods). The distance travelled by vesicles was measured on the projected image using the
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Fig. 3. The percentage of particles in each size category. There was no significant difference between treatments and controls, and the majority of particles were between 0.025 and 0.05 m2.
‘segmented line’ tool in Scion Image. This meant that the total travel distance was measured rather than the direct distance between starting and end points, and thus the average velocity was calculated for these vesicles. The distance travelled by vesicles in H2O2 treated cells was significantly longer than those in controls (P<0.0001), even when the two greatest values in the treated group were excluded from the t-test (Fig. 5A) (Table 1). Surprisingly, the distance travelled by vesicles in CaCl2 treated cells was also significantly longer than in controls (P<0.0001), while, in contrast, the distance travelled by vesicles in KCl treated cells was not significantly different from controls. The average velocity of vesicles was calculated from the distance travelled and the total time in which the vesicle was moving (ie the number of frames multiplied by 4 s). Vesicles in H2O2 treated cells travelled at velocities significantly faster than those found in controls (P<0.0001), even though 30% of velocities in the H2O2 treated group were within the same range as controls (Fig. 5B) (Table 1). In contrast, vesicles in KCl treated cells were significantly slower (P<0.0001) than controls. However, in spite of travelling a significantly greater distance than control vesicles, CaCl2 treated cells travelled at a similar velocity.
ApoE isoforms under stress conditions There was no significant difference in effect on vesicle trafficking between apoE isoforms under stress conditions (Fig. 6). However, there was an isoform specific difference in the number of cells in each of the transfected cell types being affected by H2O2 treatment, with 34% of E2, 41% of E3 and 58% of E4 cells being affected. The effects of nocodazole and taxol on apoE-EGFP vesicle movement as well as Golgi and ER structure To further investigate the changes in vesicle speeds under oxidative stress and KCl conditions, cells were treated with nocodazol to completely depolymerise microtubules, and taxol to promote stable microtubule polymerisation. After 3 h in nocodazole the Golgi and ER were no longer discernible and apoE-EGFP positive vesicles were distributed throughout the cell (Fig. 7A). Only Brownian movement of apoE vesicles was observed, with directional movement no longer apparent (not shown). In contrast, in taxol treated cells showed directional movement of apoE-EGFP vesicles but this was in the range of velocities seen with the KCl treated cells. The Golgi and ER structure remained unchanged.
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Fig. 4. The effects of ageing-related stress conditions on vesicle movement. Representative frames from confocal time-series micrographs for each treatment are shown, with the first, middle and last frame from which a vesicle path was measured. The vesicle location is circled in consecutive frames and indicated by the arrows in the projected, or merged, image. Vesicles in (A) control and (D) CaCl2 treated cells show similar movements. Vesicles in (B) H2O2 treated cells were faster, and those in (C) KCl treated cells slower, when compared to controls. Time-series files are also available online (see Online Supplemental Material). Bar, 10 m.
The effects of H2O2 and KCl stress conditions on microtubule organisation As the majority of vesicle traffic is along microtubule tracks, cells were immunostained for -tubulin after incubation for 2 h in the same H2O2 and KCl stress conditions used to study Golgi/ER
structure and vesicle traffic. Nocodazole and taxol treated cells were also immunostained as controls for microtubule depolymerisation and polymerisation respectively. A comparison between the -tubulin staining of H2O2 treated cells and the controls suggests that there is only a partial depolymerisation of microtubules (Fig. 7). In
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Fig. 6. ApoE isoforms under ageing-related stress conditions. The average area per particle is shown for each apoE isoform as a representative aspect of apoE-EGFP spatial changes. No isoform-specific difference was apparent under any of the conditions after analysis of the data using a Student’s t-test.
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structure, while the apoE-EGFP was distributed throughout the cytoplasm as vesicular structures (Fig. 7G). However, no directional vesicle movement was detected. The -tubulin polymerisation was also found to be completely disrupted (Fig. 7H), appearing similar to that found after nocodazole treatment.
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Fig. 5. Distance travelled (A) and velocity (B) of vesicles under ageing-related stress conditions. Under H2O2 and CaCl2 conditions vesicles travelled significantly further that in KCl or control cells. Vesicles under H2O2 conditions were also significantly faster than controls and vesicles under KCl conditions were significantly slower.
contrast, KCl treated cells showed an increase in the polymerisation of microtubules corresponding to a more activated form of astrocyte. The longer-term exposure of astrocytes to H2O2 stress conditions As a 2 h treatment of astrocytes with H2O2 resulted in only partial microtubule disruption, an increase in vesicle speed and partial disruption of Golgi and ER structure we investigated the effects of a 4 h exposure to H2O2 stress conditions. After 4 h there appeared to be no perinuclear Golgi or ER
We report here that oxidative stress conditions increased the speed and number of apoE positive secretory vesicles in our transfected astrocytoma cells, with no difference detected between the three isoforms. We also found that the apoE-positive Golgi and ER appeared disrupted but remained perinuclear. There was, however, an apoE isoformspecific difference in the number of cells resistant to oxidative stress with the E2 cell line being most resistant and the E4 cell line the least resistant. As the Golgi-complex and ER are well established as the major sites of protein synthesis, posttranslational modification and sorting in eucaryotic cells (Alberts et al., 1994), the association of apoE with these structures was not unexpected. What is unusual, however, is that vesicle speed increased after 2 h under oxidative stress conditions. Although the effect of oxidative stress on vesicular trafficking has not been previously reported such stress is known to disrupt microtubule proteins (Banan et al., 2000; Neely et al., 1999). Therefore, we expected oxidative stress to progressively slow
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Fig. 7. The effects of nocodazole, taxol, and H2O2 and KCl stress conditions on microtubule organisation. Nocodazole disruption of apoE-EGFP localisation in astrocytoma cells is apparent in (A) where the Golgi and ER are no longer visible. (B) Shows the corresponding disruption of -tubulin. Taxol, on the other hand, increased -tubulin polymerisation (C) when compared to untreated control cells (D). In comparison, (E) -tubulin appeared partially disrupted after 2 h under H2O2 conditions, while under KCl conditions (F) -tubulin was more polymerised and cell volume decreased. After 4 h under H2O2 conditions apoE-EGFP localisation (G) was entirely vesicular and was not concentrated in a perinuclear region. The apoE-EGFP localisation was also similar to that found in cells treated with nocodazole (A). The -tubulin staining of cells after 4 h under H2O2 was also similar to nocodazole treatment, showing a greater disruption of microtubule organisation. Bar, 20 m (H).
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apoE vesicle movement and that this slowing would correspond to a disruption of microtubules. Instead while the velocity and number of apoE vesicles increased after 2 h, the microtubules appeared only partially disrupted. One explanation for this increase in vesicle speed is an increase in protein phosphorylation, mediated by the activation of protein kinases or inhibition of protein phosphatases, as an increase in phosphorylation is known to affect both vesicle speed and microtubule stability (Hammalvarez et al., 1993; Lee, 1993; Olmsted, 1986). Microtubule stability is governed in part by the binding of microtubuleassociated-proteins (MAPs), which in a phosphorylated state dissociate from microtubules. The binding of MAPs to tubulin is also inhibited by the molecule mapmodulin, which in its phosphorylated state binds to the tubulin-binding domain of MAPs (Itin et al., 1999; Ulitzur et al., 1997). This binding of mapmodulin also occurs under normal conditions allowing the transport of organelles along microtubule tracks, although it is not essential for vesicle movement to occur. The inhibition of protein phosphatases by okadaic acid has also been shown to disrupt microtubules, as well as increase the speed of vesicles in CV-1 cells (Hammalvarez et al., 1993). In addition, the phosphorylation of kinesin motor proteins induces their binding to microtubules and correlates with an increase in organelle transport to the plus ends of microtubules (Lee and Hollenbeck, 1995). Such changes in protein phosphorylation could therefore account for the increased rate of transport of the 70% of vesicles seen in our experiments with oxidative stress. Further, the incomplete disruption of microtubules, as seen with a comparison of -tubulin staining of controls, at 2 h with H2O2 and in nocodazol treated cells, accounts for the 30% of vesicles under oxidative stress conditions travelling at speeds within the lower range observed in our control cells. Oxidative stress conditions however also lead to lipid peroxidation, a major product of which is 4-hydroxy-2 (E)-nonenal (4-HNE) (Vanwinkle et al., 1994). Interestingly, 4-HNE also disrupts microtubules by modifying tubulin (Neely et al., 1999) and binding to the MAP, tau, preventing its dephosphorylation and moderately promoting its phosphorylation (Mattson et al., 1997). Therefore, this combination of events resulting from oxidative stress could result in disruption of microtubules independently of an increase in vesicle speed. As microtubule disruption also causes Golgi and ER fragmentation, the above scenario offers a further explanation for the increase in the number
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of apoE vesicles in our experiments. The perinuclear location of Golgi and ER after 2 h of oxidative stress, is also consistent with our immunohistochemical evidence that not all microtubules are disrupted. However, by 4 h of oxidative stress disruption appeared complete. Complete disruption of microtubules was also observed in our experiments with nocodazole-treated cells, and consistent with studies of (Turner and Tartakoff, 1989) using Madin-Darby bovine kidney cells. Complete microtubule disruption indicates that cell damage increases significantly after 4 h of oxidative stress. Interestingly, the phenomenon of Golgi dispersal and reassembly also occurs during mitosis (Lucocq et al., 1987; Robbins and Gonatas, 1964). Thus our results may have more general implications for vesicle trafficking during this stage of the cell cycle. More specifically, fragmented Golgi are known to be specifically dispersed through the cytoplasm to sites corresponding to the location of disrupted ER (Cole et al., 1996). With oxidative stress, this may also be a fortuitous response to injury, aiding cell recovery in concert with the local synthesis of protein and/or lipid. While the vesicle speed of Golgi redistribution and a dependence on microtubules has not previously been reported, Golgi reassembly is known to be microtubule dependent (Ho et al., 1989). The reported vesicle speed (0.1 to 0.4 m/s) during microtubule and Golgi reassembly in Vero fibroblast cells is comparable to our findings in control cultures, but in contrast to the speeds in our astrocytes after 2 h of oxidative stress. Nocodazole treatment and the resulting complete disruption of microtubules, however, is known to dramatically slow the movement of secretory vesicles (Wacker et al., 1997), which was also seen in our experiments with nocodazole. Interestingly, 4 h of oxidative stress slowed vesicle movement in our cell lines with a resulting loss of directional vesicle traffic. In addition, the increase in vesicle number and velocity may reflect a direct apoE response to membrane damage, which would be important considering the role of apoE in cholesterol redistribution (Mahley, 1988). It has been reported that apoE is retained without degradation in lysosomal vesicles (DeKroon and Armati, 2001; Heeren et al., 1999; Ji et al., 1998; Rensen et al., 2000), a point where the endocytic and secretory pathways meet (Mellman, 1996; Mukherjee et al., 1997). From this point in the endocytic/secretory pathway, apoE may be recycled or secreted. Further, apoE is synthesised and secreted in response to extracellular signals, such as nerve growth factor, as well
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as cellular damage (Futerman and Banker, 1996; Soulie et al., 1999). Therefore the activation of the secretory pathway in response to cell growth and repair (Kozu et al., 1997; Miyata et al., 1988; Zhang et al., 1998 and our results), as indicated by the increase in vesicle number and velocity, may offer a purpose for this unique retention of apoE in cells under standard culture conditions. In our second stress model, utilising KCl and CaCl2, the lack of changes observed in both the number of apoE vesicles and the structure of the ER, Golgi and microtubules in cells, were not unexpected. In contrast to the oxidative stress findings, the increase in extracellular KCl concentration slowed the movement of intracellular apoE vesicles after 2 h and induced the transformation of some cells into so-called ‘activated astrocytes’ (Gehrmann et al., 1995; Landis, 1994), with a corresponding decrease in cell volume and increase in -tubulin staining, a marker of microtubule polymerisation. Interestingly, extracellular K + concentrations which rise after ischemic damage and induce a wave of intracellular Ca2+ , also results in activated astrocytes (Verkhratsky et al., 1998). In our experiments, cells were exposed to a high extracellular concentration of K + for 2 h, with an initial wave of Ca2+ into the cells and later lowering of the [Ca2+ ]i, as confirmed by Fluo-4 AM (not shown). Under these conditions the speed of vesicle movement was significantly slower, suggesting that intracellular Ca2+ may play a role in vesicle movement. Of note is that an increased [Ca2+ ]i in HT4 and cortical neurons has been reported to activate protein phosphatase 2B, leading to dephosphorylation of actin depolymerising factor/cofilin and the polymerisation of actin (Meberg et al., 1998). In addition, the stabilisation of microtubules with taxol in CV-1 cells (Hammalvarez et al., 1993), as well as our astrocyte cells, slows vesicle speed after 2 h. In contrast, an increase in [Ca2+ ]i induced by A23187 increases vesicle speed in CV-1 cells. These results together suggest that an initial wave of Ca2+ influx, resulting in further polymerisation of -tubulin, could be responsible for the slowing of vesicle traffic in our second stress model. Furthermore, the lower extracellular Ca2+ levels in these experiments could contribute to the slower vesicle speed, as reported by Lin et al. in cultured fibroblasts (Lin and Collins, 1993). However, the KClstress conditions are likely to have a number of other effects, including changes in the concentration of other ions such as sodium and the depletion or inactivation of ATPases. Therefore, although our results with the KCl-stress conditions
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are interesting, the influence of other KCl-affected factors on vesicle movement cannot be discounted in these experiments. It is not surprising that we found no isoformspecific differences in the apoE synthesis or secretory pathways under either oxidative or calcium stress conditions, as apoE is not directly involved in vesicle trafficking and the structural differences between the apoE isoforms are single amino acid substitutions (Mahley, 1988). It was instead the number of astrocyte cells resistant to oxidative stress that presented apoE isoformspecific differences, with E2>E3>E4. This finding is directly comparable to the protective antioxidant effects of apoE isoforms added exogenously to neurons as reported by (Miyata and Smith, 1996) with again, E2>E3>E4. These results also suggest that the antioxidant effects of apoE isoforms are mediated extracellularly as it is only after apoE secretion that the isoform difference is apparent. While our results are of importance to basic knowledge of vesicle transport under conditions of oxidative stress and deregulated intracellular Ca2+ , they also have implications for the role of apoE in the CNS. Our results with oxidative stress conditions suggest that the up-regulation of apoE vesicle number and transport, linked to incomplete microtubule depolymerisation, may be an early response to oxidative stress. The deregulation of [Ca2+ ]i may also contribute to a deregulation of the apoE response to injury. Therefore, our report of the regulation of the apoE secretory pathway under both oxidative and calcium stress conditions is important information of relevance to our understanding of the ageing process in the brain as well as in recovery from head injury and ischemic damage.
ACKNOWLEDGEMENTS We thank Ellie Kable and Dr Guy Cox, Electron Microscope Unit, University of Sydney, for valuable advice with confocal image analysis. This work was supported by Glaxo Smith Kline Ltd and The Nerve Research Foundation of the University of Sydney. REFERENCES A B, B D, L J, R M, R K, W JD, 1994. Molecular Biology of the Cell. 3rd ed. New York, Garland.
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