Mitochondrial DNA rearrangements in aging human brain and in situ PCR of mtDNA

Neurobiology of Aging 20 (1999) 565–571

Mitochondrial DNA rearrangements in aging human brain and in situ PCR of mtDNA Simon Melova,*,1, Julie A. Schneiderb, Pinar E. Coskuna, David A. Bennettb, Douglas C. Wallacea a

Center For Molecular Medicine, Emory University, 1462 Clifton Rd, Atlanta, GA 30322, USA Rush Alzheimer’s Disease Center, Rush Institute For Healthy Aging, Rush-Presbyterian St. Lukes, 1645 West Jackson, Suite 675, Chicago, IL 60612, USA

b

Received 22 June 1999; received in revised form 25 August 1999; accepted 3 September 1999

Abstract Deletions of the mitochondrial DNA (mtDNA) have been shown to accumulate with age in a variety of species regardless of mean or maximal life span. This implies that such mutations are either a molecular biomarker of senescence or that they are more causally linked to senescence itself. One assay that can be used to detect these mtDNA mutations is the long-extension polymerase chain reaction assay. This assay amplifies ⬃16 kb of the mtDNA in mammalian mitochondria and preferentially amplifies mtDNAs that are either deleted or duplicated. We have applied this assay to the aging human brain and found a heterogeneous array of rearranged mtDNAs. In addition, we have developed in situ polymerase chain reaction to detect mtDNA within individual cells of both the mouse and the human brain as a first step in identifying and enumerating cells containing mutant mtDNAs in situ. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Aging; Oxidative stress; Superoxide; Energy; Mitochondria; Mitochondrial DNA mutations; Human brain; In situ PCR; mtDNA; Deletions; Mouse; Adenine nucleotide translocator; ANT1; Superoxide dismutase

1. Introduction Eukaryotic mitochondria contain small covalently closed, circular DNA molecules that encode 13 subunits that are vital to the production of adenosine 5⬘-triphosphate (ATP) through the process of oxidative phosphorylation (OXPHOS) [49]. Mutations in the mitochondrial DNA (mtDNA) can have profound consequences for both cellular and organismal homeostasis [49]. Accordingly, a number of severe diseases are associated with mutations of the mtDNA and can present with an extremely heterogeneous phenotype because of the ability of tissues to be mosaic for both mutant and wild-type mtDNAs [37]. There is a great diversity of mutations that cause human mitochondrial disease, including gross rearrangements of the mtDNA (encompassing both deletions and duplications) and point mutations [39, 40]. The role of mtDNA mutations in relation to aging * Corresponding author. Tel.: ⫹1-415-899-1800; fax: ⫹1-415-2092231. E-mail address: [email protected] (S. Melov) 1 Present address: Buck Center For Research in Aging, P.O. Box 638, Novato, CA 94948-0638

remains controversial because there is considerable variation in both laboratory techniques and in the evaluation of reported data [5,27,46]. However, new evidence from mouse models of mitochondrial disease may help in delineating what role mtDNA mutations play in aging and their relationship with oxidative stress [14,26,28,33]. Since the early 1990s, rearrangements of the mitochondrial genome have been correlated with senescence in a variety of species as diverse as Caenorhabditis elegans [32], Drosophila melanogaster [7], mice [9,13,31,45], rhesus monkeys [23] chimpanzees [29], and humans [10 –12,24,25, 34,41,43]. These age-related mtDNA mutations appear to occur primarily in post-mitotic tissues, and the accumulation of mtDNA rearrangements with age is a remarkably consistent feature of senescent multicellular animals regardless of the mean or maximal life span. This implies that either mtDNA rearrangements are a robust molecular biomarker of the senescent state or that they are more intimately linked to the process of senescence itself. During the course of normal metabolism, mitochondria generate reactive oxygen species (ROS) within the cell. Moreover, an increase in oxidative damage has been correlated with age in a variety of species [1,21,22,42,44,53]. The endogenous

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mitochondrial oxidative stress was hypothesized to result in mitochondrial DNA damage that causes a plethora of everincreasing, degenerative processes contributing to the overall phenotype of senescence [35]. Recently, ROS have been implicated in the formation of mtDNA rearrangements in a mouse model of mitochondrial disease—the adenine nucleotide translocator (ANT) mutant mouse [14]. This mouse lacks the heart/skeletal muscle isoform of the ANT (ANT1), a protein responsible for exchanging ATP from the mitochondrion with ADP from the cytosol for the phosphorylation of ADP to occur at complex V during normal respiration. As most ADP is imported into the mitochondria via the ANT, the ANT1 mutant mouse has a greatly reduced level of ADP for phosphorylation at complex V. Therefore, the mitochondrial electrochemical gradient becomes hyperpolarized, inhibiting electron transport and causing electrons to build up within the respiratory chain. As a result, these electrons can be donated to O2 to give O2⫺, which is further dismutated by mitochondrial superoxide dismutase (Sod2) into H2O2, which can diffuse out of the mitochondria. This increase in mitochondrial oxidative stress is observable by a four to eightfold increase in H2O2 formation from isolated mitochondria of tissues that are reliant upon ANT1 for ADP/ ATP exchange [14]. Hence, a decline in OXPHOS can give rise to an increase in oxidative stress. The observation of mtDNA rearrangements in the hearts of the ANT1 mutant mice, concurrent with an increase in oxidative stress, makes a persuasive argument that these two phenomena are inextricably linked. In the case of the ANT1 mutant mouse, it is clear that the increased oxidative stress is a direct consequence of the inactivation of the endogenous gene for ANT1 via homologous recombination [16]. Hence, one could argue that the mtDNA rearrangements identified in the hearts of the mutant animals are a secondary consequence of a synergy between reduced OXPHOS and increased oxidative stress. Given these new observations, what is the cause of mtDNA rearrangements in aging organisms, and what are their implications for senescence? First, it would appear that the steady accumulation of mitochondrial damage caused by ROS would result in a decline in OXPHOS with age, both because of the accumulation of mtDNA mutations and the inactivation of mitochondrial functions. This hypothesis would explain why increased oxidative damage and mtDNA mutations correlate with senescence in a variety of evolutionarily divergent species, regardless of the mean and maximal life span. However, to support this hypothesis, specific pathological consequences to the cell by the oxidative stress that are produced as a result of normal metabolism need to be identified. During the course of normal metabolism, between 0.4% and 4.0% of all oxygen consumed is converted into superoxide [4,6,17,47,48]. Under normal circumstances, most of the superoxide produced is dismutated into H2O2 by the mitochondrial form of Sod2. The resulting H2O2 can then

diffuse out of the mitochondrion into the cytosol where it can be converted into water by catalase or cytoplasmic glutathione peroxidase (Gpx). Alternatively, a mitochondrial form of Gpx can reduce H2O2 to water within the mitochondrion [18]. Although the level of superoxide produced within the mitochondria during normal respiration is low, such levels of endogenous oxidative stress are potentially very deleterious to a variety of mitochondrial functions [28]. Genetic inactivation of Sod2 is lethal in mice, with death generally occurring within the first week of life [26]. This is associated with a complex heterogeneous phenotype, including a dilated cardiomyopathy, lipid accumulation in the liver, organic aciduria, and catalytic decreases in the activity of several enzymes that are sensitive to endogenous mitochondrial oxidative stress [26,28]. These enzymes include the mitochondrial holoenzyme respiratory chain complexes I and II (nicotinamide adenine dinucleotide phosphateubiquinone oxidoreductase and succinate dehydrogenase, respectively), the tricarboxylic acid cycle enzyme aconitase, and the ketogenic/leucine catabolic enzyme 3-hydroxy-3methylglutaryl-coenzyme A lyase [28]. In addition, by preventing the early death of Sod2 mutant mice through pharmacological antioxidant treatment, the life span of Sod2 mutant mice can be extended. However, by the third week of life they develop severe brain defects. This manifests as a profound spongiform encephalopathy in association with gliosis and membranous profiles at the ultrastructural level that are reminiscent of Leigh’s disease [33]. Behavioral defects, including circling, tremor, ataxia, and rolling behavior, ensue [33]. Hence, in the absence of a single protective mitochondrial enzyme—Sod2—acute endogenous mitochondrial oxidative stress can lead to severe problems in many organ systems, both in the viscera and the central nervous system. The Sod2-deficient model demonstrates the acute toxicity that can result from exposure to high levels of O2⫺. The effects of lower levels of mitochondrial oxidative stress have been confirmed in the Sod2 heterozygous mouse with 50% the level of wild-type Sod2 [50] and in the ANT1 mutant mouse discussed above [14,16]. In the ANT1 mutant mouse, the increase in oxidative stress is correlated with a dramatic increase in the level of mtDNA rearrangements in the heart compared to wild-type controls [14]. The assay employed to detect such mtDNA mutations is the long-extension polymerase chain reaction assay (LX-PCR) [8,31,34]. This assay functions through the use of primers that are homologous to the mitochondrial genome with the 5⬘ end of the primers in close physical proximity to each other, but with the 3⬘ ends facing away from each other. Typically, after the use of 10-min extensions during the PCR, nearly the entire mitochondrial genome is amplified, ⬃16.3 kb in humans [8,34]. However, if mutational events that bring the two primers closer together occur, then truncated PCR products result [34]. By shortening the length of extension time, one can prevent amplification of the full-length genome, and preferentially am-

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plify mutant mtDNAs. This assay has been used to map mitochondrial DNA rearrangements from patients suffering from mitochondrial disease [15], as well as in numerous aging studies [20,34]. Sequencing of products from LXPCR has revealed breakpoints for the rearrangements, confirming that the genomes have been rearranged. By serially diluting the DNA target derived from senescent tissues [31], it can be seen that the ability to amplify mutations is not dependant on a particular concentration of target and hence artifactual, as has recently been suggested [27]. An advantage of using the LX-PCR assay to detect mtDNA mutations is that it is not biased toward any one mutational event, such as the 5 kb deletion in human tissues [11]. Its disadvantage is that it is not a quantitative assay, but rather is qualitative in nature, revealing the spectrum of mtDNA rearrangements present within a sample, and no inferences should be made about relative intensities of specific products amplified in a given sample. Using the LX-PCR assay, researchers have shown that heterogeneous mutations of the mtDNA accumulate with age in C. elegans, mice, chimpanzees, and human skeletal muscle [29 –32,34]. Previous reports indicate that the brain also accumulates mtDNA mutations with age [10,43], although these reports only assayed the levels of the 5 kb deletion.

2. LX-PCR in human brain and in situ PCR of mtDNA In human mitochondrial disease, there is a vast panoply of mtDNA mutations [36], and there is no reason a priori why a single mutation would be more prevalent with aging than others would. We therefore used LX-PCR to determine the spectrum of mtDNA mutations that accumulate with age in the human brain. Fresh autopsy tissue was obtained from various regions of the human brain to carry out LX-PCR from a number of differently aged individuals (Fig. 1). These individuals had no history of neurodegenerative disease, and all individuals were neuropathologically evaluated and showed no histopathological abnormalities. Causes of death were cancer, lymphoma, heart disease, hepatitis, and acute respiratory distress syndrome. LX-PCR analysis revealed an apparent increase in the number and variety of mutations in the brains of individuals older than 50 years relative to individuals less than 50 years old (Fig. 1a vs. b). A single mutant mtDNA PCR product was isolated from a 79-year-old woman (Fig. 1c), and the entire fragment was sequenced, including the breakpoint, by standard techniques [31]. This particular rearrangement has not been described previously [36] and was noteworthy in that it was detectable by LX-PCR from three separate extractions of the cortex, perhaps indicating a high level of this mutation within a broad region of the cortex. Although preliminary, these results suggest that older individuals accumulate a heterogeneous array of mtDNA rearrangements

Fig. 1. LX-PCR of young and old human brain. Molecular weight markers shown are in kb, and the PCR product seen at 16.3 kb is the expected sized PCR product from wild-type mtDNA by using LX-PCR. (a) Young human brain. Numbers above lines refer to the ages of the individual from whom the tissue was obtained. Lane 1, cerebellum; lane 2, cerebellum; lane 3, frontal cortex; lane 4, putamen (region 1); lane 5, putamen (region 2); lane 6, cerebellum; lane 7, frontal cortex; lane 8, putamen; lane 9, cerebellum; lane 10, entorhinal cortex; lane 11, frontal cortex; lane 12, substantia nigra; lane 13, cerebellum; lane 14, frontal cortex; lane 15, putamen. “-ve” refers to negative control for the PCR in which target was omitted. (b) Old human brain. Numbers above lines refer to the ages of the individual from whom the tissue was obtained. Lane 1: putamen; lane 3, substantia nigra; lane 4, caudate; lane 5, entorhinal cortex; lane 6, cerebellum; lane 7, caudate; lane 8, entorhinal cortex; lane 9, frontal cortex; lane 10, substantia nigra; lane 11, cerebellum; lane 12, frontal cortex; lane 13, substantia nigra; lane 14, cerebellum; lane 15, putamen; lane 16, temporal cortex. (c) LX-PCR of 79-year-old frontal cortex reveals a unique mtDNA rearrangement. Sequencing of the LX-PCR product seen at ⬃4 kb reveals the breakpoint at 1989/14366 base-pairs, implying an ⬃12-kb deletion event. This particular rearrangement was identified in LX-PCR from three separate extractions of tissue from the frontal cortex.

as well as a heterogeneity of mutations between different brain regions within the same individual. To better define the distribution of mtDNA rearrangements within tissues and individual cell types, it would be preferable to analyze mtDNAs at the cellular level. In situ PCR (ISPCR) offers an opportunity for this type of analysis, and we have begun to develop such an approach for study-

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ing the brain. Our initial experiments were to amplify a small region of mtDNA in situ within sections of brain tissue from both mice and humans. ISPCR was carried out by first permeabilizing the tissue sections of brain by using a brief protease treatment, which allows the PCR mix access to the mtDNA. At the start of the experiment, the PCR cocktail was “sealed” over the permeabilized tissue, and the PCR profile was carried out with the entire glass slide being carried through sequential denaturation, annealing, and extension steps as in ordinary PCR. The “sealing” of the PCR mix over the surface of the tissue can be done in many different ways [38], however we employed a dedicated ISPCR system, the Perkin-Elmer in situ PCR 1000 (Norwalk, CT). The PCR cocktail contained a digoxigenin-labeled nucleotide, which was incorporated in situ to the mtDNA target being amplified within the mitochondria. After the PCR profile was complete, the amplified mtDNA could then be detected through standard immunohistochemical techniques. The target we chose to amplify in mtDNA of both humans and mice was a small region of the mtDNA that contained within the D-loop, a noncoding region of the mtDNA that is responsible for regulating mtDNA transcription and replication. During the ISPCR, the reaction took place in two phases: a bound phase, in which the mtDNA molecules were retained in their original location because of the fixation of the mtDNA, and a solution phase occurring both above and within the tissue on the slide. After the PCR was complete, the reaction was “unsealed,” and the solution phase was free to be aspirated off the surface of the glass slide. Agarose gel electrophoresis of the reaction products allows the determination of the size of the PCR products from the solution phase (presumably a reflection of the bound phase) as well as the potential identification of the breakpoints of mtDNA rearrangements through subsequent nested PCR and sequencing. We had employed a rigorous set of controls within each experiment to determine that the signal we detected after immunohistochemistry corresponded to genuine mtDNA localized in situ. It has previously been reported in ISPCR protocols (“direct-PCR”) that nicked-nuclear DNA allows a nonspecific incorporation of labeled nucleotides by the Taq polymerase, thereby giving a positive nuclear signal when no hybridization/extension of the primers is occurring within the nuclear DNA (false nuclear positives) [19]. One can determine this level of artifactual background by omitting the primers from the PCR mix and carrying out ISPCR. This control allows the detection of the nonspecific nuclear background for each slide. We routinely carry this out on tissue sections within any individual ISPCR run (Fig. 2b, inset). Typically, we observe very few false nuclear positives, although this can be dependent on the fixation of the tissue. Fixation of the tissue is a critical factor in ISPCR: too long a period of fixation and many false nuclear positives are seen (presumably because of excessive nicking of nuclear DNA). Too little fixation and the tissue disintegrates

with repeated thermal cycling. We have found that in the mouse brain, up to 24-h immersion fixation in 10% neutralbuffered formalin gives very reproducible ISPCR results for the detection of mtDNA. For human brain tissue, bouin’s fixative is to be avoided, as is prolonged formalin fixation and methacarn fixation. The optimal fixation protocol for ISPCR we have found for human brain is a short paraformaldehyde fixation. A further control in evaluating new primer pairs for in situ amplification of mtDNA products is the evaluation of the capacity for a single primer to give a positive signal. Single PCR primer ISPCR controls allow the determination of whether a fortuitous annealing of a single primer can result in nonspecific products in either the nucleus or in the mitochondrial genomes. We have not found significant nonspecific background in either the human or the mouse brain for any of the primers we report here. Typical results for ISPCR of mtDNA in the mouse and in the human brain are seen in Figs. 2 and 3. Fig. 2 shows mouse cerebellum, whereas Fig. 3 shows human frontal cortex. Consistent with the location of mitochondria within the cytoplasm, a strong cytoplasmic signal is observed (Figs. 2a and 3b). In addition, not all cells stain positive, and this may reflect that mitochondrial content can vary substantially among different cell types in the brain [51,52]. Confirmation of the specificity of the ISPCR was obtained by aspirating off the solution phase from the glass slide after the PCR and electrophoresing the reaction products on an agarose gel. The expected PCR product size was seen for both mouse and human ISPCR (data not shown). Furthermore, by removing the slides from the PCR machine at various cycle numbers, it is possible to obtain a punctatecytoplasmic signal consistent with the amplification of individual clusters of mitochondria within single cells (Fig. 3a). After 35 cycles of the ISPCR in mouse brain sections, individual neuronal processes were clearly visible, as were axons from the large Purkinje cells in the cerebellum (Fig. 2a). The positive signal presumably arises from axonal and dendritic locations of mitochondria in individual neurons [51,52]. In addition, signal was detectable in the perikaryon, and it is conceivable that ISPCR may be capable of resolving individual mitochondria within individual cells (Fig. 3a, arrows). The application of LX-PCR [31] has shown that there is a complex array of mtDNA rearrangements that accumulate in the human and mouse brain with age. We have also demonstrated that it is possible to detect mtDNA in situ via the PCR in a robust and reproducible fashion. To detect mtDNA rearrangements via ISPCR, primer pairs were employed to amplify mutant mtDNA fragments ⬍1 kb in size because ISPCR is not efficient in amplifying fragments over 1 kb in length [2]. Such LX-PCR primer pairs could be employed on adjacent sections of brain tissue, with the extension phase restricted during the PCR to preferentially amplify mtDNA rearrangements. This would prevent the amplification of the wild-type mtDNA from normal cells.

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Fig. 2. In situ PCR of mtDNA in mouse cerebellum. (a) The brown color represents mtDNA amplified in situ. The large cells are Purkinje cells and stain prominently consistent with their high level of mitochondria. Various processes are visualized as well (400⫻ ). Variability in staining is seen in the cerebellar granular layer, presumably reflecting variation in mitochondrial content. (b) A lower power view of the cerebellum clearly showing neuronal processes staining for mitochondria (200⫻). The inset represents a negative control on cerebellum; ISPCR carried out without primers, demonstrating an absence of stain, this demonstrates the lack of detection of any target because of nonspecific extension of nicks in the DNA by Taq-polymerase (100⫻).

Such an approach allows the enumeration of cells containing mtDNA mutations to be resolved in a more rigorous fashion than has been possible to date. In addition, by combining ISPCR on aged tissues in parallel with immunohistochemical approaches for oxidative damage and other assays, we were able to address the potential functional consequences for cells that contain mutant mtDNAs. 3. Materials and methods 3.1. Tissues Female C57Bl/6Jnia mice were obtained from the National Institute on Aging colonies of aging rodents at

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Fig. 3. In situ PCR of mtDNA in human cortex. (a) At 25 cycles of ISPCR, a punctate brown cytoplasmic signal can be seen in neurons consistent with discrete in situ amplification of mitochondrial DNA presumably from clusters of mitochondria (arrows) (630⫻). (b) At 35 cycles of amplification, neuronal processes are visualized as is the cell body. Note the strong cytoplasmic staining and lack of nuclear staining consistent with in situ amplification of mtDNA. There is also an absence of staining in some small cells, consistent with the low copy number of mitochondria in glia.

Charles River Laboratories, Inc., Wilmington, MA. Brains were harvested after a lethal overdose of Avertin. The brain was then immersion fixed in 10% buffered formaldehyde (Fisher, Fairlawn, NJ) for 24 h. All procedures with mice were carried out under Emory University Institutional Animal Use and Care Committee ethical guidelines (Institutional Animal Use and Care Committee 255–97). Mouse brains were then processed and paraffin embedded via standard protocols [3]. Coronal mouse brain sections were then cut on an RM2155 Leica microtome at 6 ␮ thickness and placed onto in situ PCR glass slides (Perkin Elmer). For LX-PCR, human tissue was obtained after routine autopsy, 7–20 h post mortem, and snap-frozen in liquid nitrogen. For ISPCR, human cortex was obtained 7 h post mortem and placed, for 48 h, in 4% paraformaldehyde, then transferred into graded cryoprotectant (10% glycerol; 2% dimethyl sul-

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foxyde; 0.1 M phosphate buffer, pH 7.4) for 1 day at 4°C, and then stored at 4°C in 20% glycerol, 2% dimethyl sulfoxyde phosphate buffer. Approximately 5-mm cubed pieces of tissue were then dissected from the cryoprotected tissue and processed and embedded via standard protocols [3], with care being taken to not exceed 30 min in any individual step during the processing to minimize potential damage to the mtDNA. Tissue was then cut and placed onto slides in the same fashion as for the mouse brain. 3.2 LX-PCR DNA was extracted from human tissue via the use of the Puregene DNA extraction kit (Gentra, Minneapolis, MN) from 100 –200 mg of tissue that was fractured while frozen in liquid nitrogen. In some cases, repeated extractions were carried out on separate pieces of tissue (i.e., 79-year-old frontal cortex). LX-PCR was carried out on 50 ng of total DNA, as previously described [34]. The 4-kb fragment amplified from frontal cortex (Fig. 1c) was sequenced in its entirety, on both strands, to identify the breakpoint, as previously described [31]. 3.3. In situ PCR In humans, the D-loop primers correspond to nucleotides 16431–16456 for the forward primer of the mtDNA and to 280 –255 for the reverse primer. In mice, the primers correspond to 15681–15705 for the forward primer and to 16030 –16006 for the reverse primer of the mtDNA. For both human and mouse brain tissue, slides were deparaffinized and rehydrated through standard protocols [3]. Slides were then transferred into and equilibrated briefly with phosphate-buffered saline (PBS; pH 7.5). The tissue sections were then permeabilized by a brief 10-min treatment with proteinase K at 10 ␮g/mL in 100 mM Tris-HCl (pH 7.5) and 5 mM ethylenediaminetetraacetate in a 37°C humidichamber. The proteinase K was then inactivated by placing the slides into boiling PBS for 90 s, and then the slides were transferred into cold PBS for at least 2 min. The slides were then removed from the PBS and carefully dried around the tissue section with kimwipes. PCR cocktail [50 ␮L; final concentrations: 0.5 ␮M forward and reverse primers, 200 ␮M deoxy nucleotide triphosphate, 2.5 ␮M digoxigenin-11-deoxy uridine triphosphate (Boehringer Mannheim, Indianapolis, IN; # 1093– 088), 2 mM MgCl2, 3 units of Gibco Taq, and 1⫻ PCR buffer (Gibco)] was then applied to the slide, and the amplicover discs (Perkin Elmer, # N804 – 0600) under hot-start conditions were applied to seal the reaction over the tissue as recommended for the Perkin Elmer GeneAmp In situ PCR system. The PCR profile consisted of an initial 2-min denaturation at 95°C, typically followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 2 min in a Perkin Elmer GeneAmp in situ PCR system. After completion of the PCR, the amplicover discs were removed

and the solution on the surface of the tissue was removed via aspiration for later examination/re-PCR for confirmation of specificity of the PCR primers. The slides were then briefly rinsed with PBS, and 50 ␮L of antibody diluent (Dako, Carpinteria, CA) containing 0.075 of a unit of AntiDigoxigenin-POD Fab fragments (Boehringer Mannheim, Germany, # 1207 733) were applied and incubated for 1 h at room temperature in a humidichamber. The slides were then briefly rinsed again with PBS, and the in situ PCR products were visualized by developing the sections with diaminobenzidine (Zymed Laboratories, San Francisco, CA) revealing a brown color where the mtDNA was amplified in situ. Slides were then briefly washed, counterstained with nuclear fast red, dehydrated, coverslipped, and examined as per standard protocols [3].

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