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Vol. 18, Issue 5, 1874-1886, May 2007

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Diverse Cytopathologies in Mitochondrial Disease Are Caused by AMP-activated Protein Kinase Signaling

Paul B. Bokko^*, Lisa Francione^*, Esther Bandala-Sanchez^*, Afsar U. Ahmed^*, Sarah J. Annesley^*, Xiuli Huang, Taruna Khurana, Alan R. Kimmel, and Paul R. Fisher^*

*Department of Microbiology, La Trobe University, Melbourne, Victoria 3086, Australia; and National Institutes of Health, Bethesda, MD 20892

Submitted October 2, 2006; Revised January 16, 2007; Accepted February 16, 2007
Monitoring Editor: Carole Parent

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The complex cytopathology of mitochondrial diseases is usually attributed to insufficient ATP. AMP-activated protein kinase (AMPK) is a highly sensitive cellular energy sensor that is stimulated by ATP-depleting stresses. By antisense-inhibiting chaperonin 60 expression, we produced mitochondrially diseased strains with gene dose-dependent defects in phototaxis, growth, and multicellular morphogenesis. Mitochondrial disease was phenocopied in a gene dose-dependent manner by overexpressing a constitutively active AMPK subunit (AMPKT). The aberrant phenotypes in mitochondrially diseased strains were suppressed completely by antisense-inhibiting AMPK expression. Phagocytosis and macropinocytosis, although energy consuming, were unaffected by mitochondrial disease and AMPK expression levels. Consistent with the role of AMPK in energy homeostasis, mitochondrial "mass" and ATP levels were reduced by AMPK antisense inhibition and increased by AMPKT overexpression, but they were near normal in mitochondrially diseased cells. We also found that 5-aminoimidazole-4-carboxamide-1--D-ribofuranoside, a pharmacological AMPK activator in mammalian cells, mimics mitochondrial disease in impairing Dictyostelium phototaxis and that AMPK antisense-inhibited cells were resistant to this effect. The results show that diverse cytopathologies in Dictyostelium mitochondrial disease are caused by chronic AMPK signaling not by insufficient ATP.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Mitochondrial diseases are a diverse group of degenerative disorders caused by mutations in either mitochondrial genes or nuclear genes encoding mitochondrial proteins (James and Murphy, 2002; Rossignol et al., 2003; Maassen et al., 2004; McKenzie et al., 2004). Affecting one in 4000 people, they vary in severity, age of onset, and the symptoms they cause, but they present mostly as forms of various neuromuscular disorders, heart disease, or diabetes. Although the precise nature of many of the disease-causing mutations is known, the relationship between genotype and phenotype in mitochondrial disease is complex and poorly understood. Individuals with the same genotype can exhibit very different symptoms, whereas different mutations can produce very similar clinical outcomes in different patients. This has been attributed to differences in the severity of the underlying genetic defect between individuals and to tissue-specific differences in the proportion of defective mitochondria, ATP demands, age-related accumulation of mitochondrial mutations, and the expression and function of specific isoforms of mitochondrial proteins. Despite these complexities it is accepted that mitochondrial disease pathology results primarily from a reduced capacity of the mitochondria to produce energy in the form of ATP (James and Murphy, 2002; Rossignol et al., 2003; Maassen et al., 2004; McKenzie et al., 2004). It has therefore been assumed that the associated cytopathology is caused by ATP supplies being insufficient to support the affected cellular activities. By using the Dictyostelium model for mitochondrial disease, we have been able to study the underlying mechanisms of mitochondrial disease cytopathology without the superimposed complexities of metazoan developmental biology. This has revealed that diverse mitochondrial disease phenotypes arise, not because of insufficient ATP, but because of chronic activation of an energy-sensing alarm system that shuts down or interferes with some, but not all, cellular functions. That cellular alarm is AMP-activated protein kinase (AMPK).

AMPK is a ubiquitous, highly conserved protein kinase that maintains cellular energy homeostasis in healthy cells and in a variety of pathological situations, most notably diabetes, ischemic reperfusion injury, and cancer (Hardie and Hawley, 2001; Hardie, 2004; Kahn et al., 2005; Hardie and Sakamoto, 2006). AMPK functions in vivo as a heterotrimer with a catalytic and a regulatory subunit that are assembled into the holoenzyme on a scaffold provided by the subunit. It is activated very sensitively by a reduction in ATP and concomitant increase in AMP levels resulting from stresses such as strenuous exercise, ischemia or glucose deprivation. The activated kinase phosphorylates target proteins and initiates downstream signaling pathways that shift metabolism from anabolic to catabolic pathways, stimulating glucose uptake and fatty acid oxidation, enhancing the energy-producing capacity of the cell through mitochondrial proliferation and inhibiting energy-consuming processes such as cell cycle progression and growth (Hardie and Hawley, 2001; Hardie, 2004; Kahn et al., 2005; Hardie and Sakamoto, 2006). These effects are mediated both acutely by changes in protein activities (e.g., acetyl CoA carboxylase; Witters and Kemp, 1992) or subcellular localization (e.g., the GLUT4 glucose transporter; Kurth-Kraczek et al., 1999) and by longer-term changes in gene expression (Russell et al., 1999; Woods et al., 2000). The overall effect is to restore cellular ATP/AMP ratios to normal.

In mitochondrial disease, cellular ATP generating capacity is chronically compromised so that AMPK is expected to be chronically activated. Whereas AMPK activation serves a beneficial, homeostatic role in otherwise healthy cells, we show here that AMPK is itself responsible for the diverse cytopathologies associated with mitochondrial disease in Dictyostelium—impaired signal transduction for phototaxis and thermotaxis, slow growth, and deranged multicellular morphogenesis. Thus, overexpressing the catalytic domain phenocopies mitochondrial disease in a dose-dependent manner, whereas antisense inhibition of AMPK expression in mitochondrially diseased cells suppresses all of the disease phenotypes. Major energy-consuming cellular activities such as phagocytosis and pinocytosis that are unaffected in mitochondrial disease are also impervious to AMPK signaling.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Plasmid Constructs
Chaperonin 60 antisense (pPROF128) and sense control (pPROF127) constructs were described previously (Kotsifas et al., 2002). The AMPK sense (control) and antisense constructs express a 1173-base pair AMPK sense (pPROF361) or antisense (pPROF362) RNA corresponding to base pairs 364-1536 in the snfA sequence (Figure 1b) in DictyBase (DictyBase accession no. DDB0215396). The construct (pPROF392) for overexpression of a constitutively active, truncated AMPK (Figure 1b) was created by subcloning the corresponding cDNA in the sense orientation into pA15GFP.

View larger version (31K): [in this window] [in a new window]   Figure 1. Genetic manipulation of levels of expression of the catalytic subunit of AMPK and its truncated form AMPKT. (a) Map of the Dictyostelium AMPK polypeptide showing positions of the main functional features. Numbers indicate amino acid positions. The amino acid sequence is conserved except for a short N-terminal stretch and an asparagine-rich region in the C-terminal half of the molecule. Such asparagine-rich domains are very common in Dictyostelium proteins, but their functions are unknown. The -subunit binding and autoinhibitory regions are required for assembly of the subunit into the heterotrimeric holoenzyme and its regulation by AMP/ATP-binding to the subunit. The inset contains a Northern blot showing a developmental time course for native AMPK mRNA expression. The probe was a genomic DNA fragment extending from position 2228 to 2642 base pairs in the genomic DNA sequence, numbering from the translational start codon. (b) The truncated form of AMPK used in overexpression studies. The truncated form was created by introducing a single base deletion (A1120) during RT-PCR to produce a cDNA with a reading frame shift at that position and a premature stop codon 18 nucleotides downstream. In addition, our cDNA contains the following base substitutions: T7C introduced with the 5' primer as well as T1134G and AAA1129CCC introduced with the 3' primer downstream of the frameshift. These additional changes arose because of sequence differences between GenBank record AF118151 and the since completed Dictyostelium genome sequence. S3P and VLPRGQ379 indicate amino acid substitutions, the latter resulting from the introduced frame shift at residue 374 that created a stop at codon 380. Numbers indicate amino acid positions. (c) The genomic DNA fragment used for antisense inhibition studies. The arrow indicates the direction of transcription of the fragment in the antisense RNA construct. The red section indicates the cDNA and the black bars indicate the position and size of introns in the fragment. Numbers represent nucleotide positions in the genomic sequence. (d) The amplicon used to measure antisense inhibition of expression of the native AMPK mRNA by using real-time PCR. The sequences to which the primers anneal are not present in the AMPK antisense construct. (e) Expression of the native AMPK mRNA relative to the wild-type AX2 control in cells carrying the indicated number of copies of the AMPK antisense RNA construct. Negative numbers are assigned to the copy numbers of the antisense inhibition construct, because it exerts a negative effect on expression of the native AMPK mRNA. The Southern blot in the inset made use of a chromogenic substrate to show hybridization to the 8.25-kb antisense RNA construct as a function of the number of copies per genome. (f) Overexpression of the truncated AMPK subunit (AMPKT) mRNA as a function of the number of copies of the overexpression construct per genome. Expression levels relative to expression in the transformant with the highest copy number were measured in quantitative Northern blots. Insets contain separate Southern, Northern, and Western blots using chromogenic substrates to show the AMPKT construct DNA, mRNA, and polypeptide levels at the indicated copy numbers. The antibody was not sufficiently sensitive to detect the native AMPK subunit in Western blots, suggesting that the level of expression of AMPKT is significantly higher than that of the endogenous full-length protein. This is consistent with Northern blots and quantitative RT-PCR results that showed that the levels of AMPKT mRNA were 1 to 2 orders of magnitude higher than the mRNA for the native protein (data not shown).

Double transformants were isolated by cotransformation with both required plasmids as described previously (Barth et al., 1998b). All transformants were isolated using the Ca(PO₄)₂/DNA coprecipitation method and selected as isolated, independent colonies growing on Micrococcus luteus lawns on standard medium (SM) agar supplemented with 15 or 20 µg/ml Geneticin (G-418) (Promega Corporation, Madison, WI). Cells were cultured in axenic medium (HL-5) supplemented with 100 µg/ml ampicillin and 20 µg/ml streptomycin or on bacterial (Klebsiella aerogenes) lawns on SM agar. The selective agent G-418 at 20 µg/ml was added to HL-5 medium for all transformants during routine subculture, but it was removed for phenotypic studies to exclude possible effects of the antibiotic itself.

DNA and RNA Techniques
Gene Cloning and Sequence Analysis. General cloning strategies and vectors were as described previously (Wilczynska et al., 1997; Kotsifas et al., 2002). Fragments to be cloned were amplified using gene-specific primers containing added restriction sites at the 5' end for cloning purposes. Clones were verified by restriction digestion and by sequencing at the Australian Genome Research Facility, Brisbane, Australia.

The 2.6-kb gene snfA (EMBL/GenBank accession no. AF118151) encoding the AMPK subunit was amplified and cloned in pZErO-2 (Invitrogen, Carlsbad, CA) from AX2 genomic DNA with primers PAMKF1 (5'-gcgctctagattcgaaaaaatcatgagtccatatcaacaaaatcccatt-3') and PAMKR1 (5'-gcgctctagactcgagttaaactacaaatatcaaaaatatgaatatttcacc-3'). Plasmid constructs for expression of AMPK (snfA) antisense RNA and the corresponding sense RNA control were created from the full-length genomic clone by amplifying a fragment (Figure 1c) with primers PAMPKF10 (5'-gcgcgaattccctatggatgaaaagattagaaga-3') and PAMPKR10 (5'-gcgcgaattctccatgctattgctattggtgg-3'), cloning the polymerase chain reaction (PCR) product into pZErO-2 and subcloning it into the Dictyostelium expression vector pDNeo2. A truncated cDNA encoding the catalytic domain (AMPKT; Figure 1b) was amplified with primers PACDNAF1 (5'-gcgctctagaagcttctcgagttcgaaatgagtccatatcaacaaaatcccattgg-3') and PACDNAR1A (5'-gcgctctagactcgagcccgggaattcttattggcctctggggagcactgacat-3') primers by reverse transcription (RT)-PCR by using RNA extracted from vegetative AX2 cells with TRIzol (Invitrogen). The resulting PCR product was cloned into pZErO-2 and subcloned for expression into the vector pA15GFP with replacement of the resident green fluorescent protein gene.

Sequence analyses, alignments, and database searches were conducted using Web-based software through DictyBase (http://www.dictybase.org), ExPASy (http://www.expasy.org), and the Australian Genome Research Facility (http://www.agrf.org.au).

Southern and Northern Blotting. Qualitative Southern and Northern blotting experiments were conducted using ³²P- or digoxigenin-labeled DNA probes (Roche Diagnostics Australia, Newcastle, New South Wales, Australia) in combination with the nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate color substrate for detection. For quantitative blots, fluorescein-labeled DNA probes were used in combination with anti-fluorescein alkaline peroxidase-conjugated antibody and the enhanced chemifluorescence substrate (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom). Quantitative results were obtained using the fluorescence mode of a Storm 860 FluoroImager (GE Healthcare). Copy numbers were assayed by quantitative Southern blotting and confirmed independently by our previously published Escherichia coli electroporation method (Barth et al., 1998a).

Quantitative PCR. Expression of AMPK mRNA was quantitated by real-time RT-PCR by using the iQ5 real-time PCR detection system and the iScript one-step RT-PCR Supermix (Bio-Rad, Hercules, CA) with SYBR Green I for amplicon detection. The 215-base pair target amplicon, shown in Figure 1d, was not present in the antisense RNA expression constructs or their corresponding sense RNA controls, so that the assay specifically measured the levels of the endogenous AMPK mRNA. The 50-µl reaction mixture contained 1x iScript one-step RT-PCR Supermix with SYBR Green I (50 mM KCl, 20 mM Tris-HCl, pH 8.4, 3 mM MgCl₂, 0.2 mM each dNTP, iTaq antibody-mediated hot start DNA polymerase at 50 U/ml, 500 nM SYBR Green I, and 10–50 nM fluorescein), iScript Moloney murine leukemia virus reverse transcriptase (1 µl of 50x formulation), gene-specific primers (200 nM each PAAF1 and PAAR1), and purified total RNA template (1 pg–1 µg). cDNA synthesis was performed at 42°C for 30 min. The experimental plate well factor was collected at 95°C for 90 s, and the PCR cycling and detection used 45 cycles of denaturation at 95°C for 1 min, annealing and extension at 55°C for 1 min. Expression levels for antisense RNA-expressing strains were measured relative to those of AX2 control cells.

Protein Techniques
Generation of Antipeptide Antibody. Antipeptide antibodies were raised against a mixture of two AMPK peptide sequences: KILNRTKIKNLKMDEKIRR (residues 60–78) in the catalytic domain and GSYDDEVYSPNLVSPITTPIMS (residues 352–373) in the autoinhibitory region of the D. discoideum AMPK subunit. Peptide synthesis and coupling was carried out by Mimotopes (Clayton, Victoria, Australia), and antibodies were raised in a rabbit by the Institute of Medical and Veterinary Science (Adelaide, South Australia, Australia).

Sample Preparation. The AMPKT overexpression transformants were grown to a density of 2–3 x 10⁶ cells ml^–1, harvested, washed, and resuspended in lysis buffer containing 50 mM NaCl, 120 mM Tris-Cl, 1% NP-40, and protease inhibitors (Complete EDTA-free protease inhibitor cocktail tablets; Roche Diagnostics Australia). The protein concentration was estimated using the Bradford method in the form of the Bio-Rad protein assay (Bio-Rad).

Western Blotting. Each track of 10% polyacrylamide gels was loaded with 10 µg of protein for SDS-polyacrylamide gel electrophoresis by using the Mini PROTEAN II apparatus (Bio-Rad). The proteins were electroblotted onto polyvinylidene difluoride membranes (GE Healthcare) by using the Mini Trans-Blot electrophoretic transfer cell. The AMPK primary antibody (1:500) and anti-rabbit immunoglobulin G horseradish peroxidase conjugate (1:2500; Promega, Madison, WI) as secondary antibody were used and the AMPK subunit protein was detected using the 3-amino-9-ethylcarbazole staining kit (Sigma-Aldrich, St. Louis, MO).

ATP Assays
ATP assays were conducted using the luciferase-based ATP determination kit (ENLITEN; Promega) by using cells grown axenically in HL-5 medium. Background luminescences measured before the assay were subtracted, and ATP concentrations were determined from a standard curve constructed using 10-fold serial dilutions of the ATP standard (1 x 10^–7–1 x 10^–11 M) in assay buffer.

Phenotypic Assays
Phototaxis and Thermotaxis. Semiquantitative phototaxis and thermotaxis assays were conducted as described previously (Fisher et al., 1981; Fisher and Annesley, 2006) by using a small quantity of amoebae scraped from the edges of Dictyostelium colonies on K. aerogenes lawns. After incubation, slugs and slime trails were transferred to clear polyvinyl chloride (PVC) discs. The discs were stained for 5 min with Coomassie blue (Fisher et al., 1981), and then they were rinsed gently in running water. Start and end points of the stained trails were digitized, stored as a series of x, y coordinates by using a Summagraphics 120 digitizing tablet connected to a SUN workstation (SUN Microsystems, Santa Clara, CA), and analyzed using directional statistics based on the von Mises or circular normal distribution (Fisher and Annesley, 2006).

Growth on Bacterial Lawns. Two hundred and fifty microliters of E. coli B2 cells from an overnight Luria Broth culture were harvested, resuspended in sterile saline, spread uniformly onto each normal agar (NA: 20 g/l Bacto agar [Difco, Detroit, MI]; 1 g/l Bacto peptone (Oxoid, Basingstoke, Hampshire, England), 1.1 g/l anhydrous glucose, 1.9972 g/l KH₂PO₄, and 0.356 g/l Na₂HPO₄·2H₂O, pH 6.0) plate and allowed to dry. A 10-µl droplet of a suspension containing 1 x 10⁶ Dictyostelium amoebae/ml was applied onto the center of the NA plate, allowed to dry in a laminar flow hood, and incubated at 21°C. The rate of expansion (diameter in millimeters) of the resulting D. discoideum plaque was measured at intervals of 8 or 12 h over a period of 5–7 d. The recorded values were analyzed by linear regression by using the "R" environment for statistical computing and graphics (http://www.R-project.org) to determine the growth rate from the growth curve. The 95% confidence intervals for the time taken for the plaque to expand 5 mm were determined from confidence intervals for the slope of the growth curve.

Growth in Axenic Medium. Cells from an exponentially growing Dictyostelium culture in HL-5 medium were inoculated into fresh HL-5 medium at an initial density of 1 x 10⁴ cells/ml and incubated at 21°C with shaking at 150 rpm on an orbital shaker. The cell densities were determined using a hemocytometer at 8- or 12-h intervals over a period of 5–7 d and recorded. The cell densities were then analyzed by log-linear regression using the R programming environment computer software to determine the generation time from the exponential growth curve. The 95% confidence intervals for the generation time were calculated from confidence intervals for the slope of the log-linear portion of the growth curve.

Phagocytosis
Bacterial uptake by Dictyostelium strains was determined using as prey an E. coli strain expressing a fluorescent protein termed DsRed (Maselli et al., 2002). DsRed-expressing E. coli (DsRed-Ec) cells were prepared at a density of 2–4 x 10¹⁰ bacteria/ml (equivalent to OD₆₀₀ = 10–20) in 20 mM Sorenson's buffer (2.353 mM Na₂HPO₄·2H₂O and 17.65 mM KH₂PO₄, pH 6.3). The fluorescence signal per million bacteria was determined from the density and fluorescence of the bacterial culture used in a given experiment. The relationship between OD₆₀₀ and the density of the bacterial suspension was determined in a separate calibration curve.

Dictyostelium amoebae were harvested, washed, resuspended at 1.3–23 x 10⁶ cells/ml and starved in Sorenson's buffer at 21°C for 30 min with shaking at 150 rpm. For the assay, 1 ml of the DsRed-Ec suspension added to 1 ml of starving Dictyostelium cells. At each time point in the assay (0 and 30 min), 0.5 ml of amoebae were washed free of uningested bacteria by differential centrifugation in the presence of 5 mM sodium azide (Maselli et al., 2002), and their fluorescence was measured in a Modulus fluorometer (Turner BioSystems, Sunnyvale, CA) by using a specially constructed module designed for DsRed (530-nm excitation and 580-nm emission). Measurements were performed in duplicate at each time point. The hourly rate of consumption of bacteria by a single amoeba was calculated from the increase in fluorescence over 30 min, the fluorescence signal per million bacteria and the amoebal density.

Pinocytosis
Pinocytosis assays (Klein and Satre, 1986) were performed with fluorescein isothiocyanate (FITC)-dextran (Sigma-Aldrich; average mol. mass, 70 kDa; working concentration, 2 mg/ml in HL-5 growth medium). Axenically growing Dictyostelium cells were harvested, resuspended in HL-5 at 2.5–25 x 10⁶ cells/ml, and shaken at 150 rpm for 20 min at 21°C. FITC-dextran was added to the cells, and at each time point (0 and 60 or 70 min) 200-µl aliquots were transferred to 3 ml of ice-cold phosphate buffer (2 mM Na₂HPO₄ x 2H₂O and 15 mM KH₂PO₄, pH 6.0). The cells were harvested, washed twice with ice-cold phosphate buffer, and lysed by addition of 2 ml of 0.25% (vol/vol) Triton X-100 in 100 mM Na₂HPO₄, pH 9.2. The fluorescence of the lysate was measured in duplicate at each time point in a Modulus fluorometer (Turner BioSystems) using the Green Module. The hourly rate of uptake of medium was calculated from the cell density, the increase in fluorescence over 60 or 70 min, and a separate calibration curve relating the fluorescence signal to the volume of fluorescent medium.

Mitochondrial "Mass"
MitoTracker Green fluorescence was used to estimate mitochondrial mass (Pendergrass et al., 2004). Axenically growing vegetative amoebae were harvested, washed once in Lo-Flo HL-5 (Liu et al., 2002) (3.85 g/l glucose, 1.78 g/l proteose peptone, 0.45 g/l yeast extract, 0.485 g/l KH₂PO₄, and 1.2 g/l Na₂HPO₄·12H₂O; filter sterile), incubated in Lo-Flo medium for 2 h, and then divided into two aliquots. One aliquot was resuspended in Lo-Flo HL-5 containing 200 nM MitoTracker Green FM (Invitrogen), whereas the other aliquot was resuspended in only Lo-Flo HL-5 as an unstained control. Both aliquots were incubated for an hour in the dark and then unbound MitoTracker Green was removed by washing the cells three times in Lo-Flo HL-5, with 10-min shaking on an orbital shaker (150 rpm) between washes. Finally, the cells were resuspended in Lo-Flo HL-5, and fluorescence was measured in duplicate in a Modulus fluorometer (Turner BioSystems) using the Blue Module. The MitoTracker Green fluorescence per million cells was calculated after subtraction of the background fluorescence in the unstained cells.

Fluorescence Microscopy. Fluorescence microscopy was based on our previously reported methods (Gilson et al., 2003). Vegetative amoebae were grown to log phase in HL-5 medium on sterile coverslips in six-well plates (Nalge Nunc, Naperville, IL), washed gently in Lo-Flo HL-5, and stained with 200 nM MitoTracker Red CMX-Ros (Invitrogen) in LoFlo HL-5 for 1 h in the dark. Unbound MitoTracker Red was removed by washing the cells three to four times in LoFlo HL-5 over 2 h. After two washes in phosphate buffer (12 mM Na₂HPO₄ and 12 mM NaH₂PO₄, pH 6.5), the cells were fixed and flattened at the same time by placing the coverslips upside down on a layer of 1% agarose in phosphate buffer containing 3.7% paraformaldehyde for 30 min. The fixed cells on the coverslips were washed four times (5 min each) in phosphate-buffered saline and mounted for microscopy.

Morphology
Fruiting body morphology after multicellular development was scored as described previously (Kotsifas et al., 2002) after culturing transformants on K. aerogenes lawns on SM agar plates with or without 0.5% (wt/vol) activated charcoal. The SM agar plates were prepared by spreading 0.1 ml of a dense K. aerogenes suspension in sterile saline onto the surface of the plates and allowing the inoculum to dry in the laminar flow hood before streak inoculating amoebae of the transformants onto the lawns. The plates were incubated at 21°C for 4–6 d to allow growth and multicellular development.

Statistical Techniques
Analysis of Directional Data. The directions of travel of individual slugs were analyzed using directional statistics based on the circular normal (von Mises) distribution as described previously (Fisher et al., 1981; Fisher and Annesley, 2006). The accuracy of phototaxis is the concentration parameter () of the circular normal (von Mises) distribution, which measures how concentrated individual directions are around the mean direction µ (toward the light source). The values range from 0 for no preferred direction of migration (all directions equally probable) to for perfect orientation (all directions exactly toward the light).

Regression and Correlation Analysis. Regression analysis was performed, and the coefficient of variation (R²) was calculated using standard linear regression models. R² equals the square of the Pearson product-moment r, which was used where appropriate to determine the significance probability for some correlations – the probability of the observed results occurring under the null hypothesis that there is no correlation. The significance of all correlations was also tested by calculating the nonparametric Kendall rank r and in all cases the outcomes (p > 0.1 or p < 0.01) were the same as those yielded by the Pearson coefficient. In some cases, Kolmogorov–Smirnov, Kruskal–Wallace, and Student's t two-sample tests were also performed.

Three-Dimensional Surface Regression Plots. Three-dimensional scatter plots and surface regressions were created using a local adaptation of the "scatter3d" function in the R environment for statistical computing and graphics (http://www.R-project.org). Quadratic (phototaxis and growth rate data) or planar (other data) surfaces of best fit were plotted for quantitative phenotypic data relating the phenotypes (vertical axis) to both the chaperonin 60 and AMPK expression indices (horizontal axes). The results are presented as rotating animations in Supplemental Material.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
We previously reported that mitochondrial disease can be created and studied in Dictyostelium in two ways: targeted disruption of mitochondrial genes (Wilczynska et al., 1997), or antisense inhibition of nuclear genes encoding mitochondrial proteins (e.g., chaperonin 60) essential for mitochondrial respiratory functions (Kotsifas et al., 2002). Regardless of which strategy was used, the phenotypic consequences of mitochondrial dysfunction in Dictyostelium were defects in phototaxis, thermotaxis, growth, and multicellular development. In view of the diverse regulatory roles played by AMPK in response to ATP-depleting stressors, we decided to test whether AMPK activation and signaling were responsible for these diverse phenotypic outcomes of mitochondrial disease. If this were so, then increasing cellular AMPK activities should cause the same defects as mitochondrial dysfunction, whereas depressing AMPK levels in mitochondrially diseased cells should ameliorate these defects.

Dictyostelium AMPK Is Similar to the Subunits of AMPK in Other Eukaryotes and Is Expressed throughout Development
Searches of the predicted Dictyostelium proteome (Eichinger et al., 2005; Chisholm et al., 2006) revealed a single isoform of each of the , , and subunits of the AMPK heterotrimer (DictyBase accession nos. DDB0215396, DDB0204006, and DDB0217034, respectively). The subunit gene, named snfA after the Saccharomyces cerevisiae orthologue, contains four introns and encodes a polypeptide of 727 amino acids. The predicted protein sequence was confirmed by sequencing full-length cDNAs (data not shown). The cDNA encodes a protein that is highly similar to AMPK subunits from other eukaryotes except for a short N-terminal stretch of 27 amino acids and a 241-residue asparagine-rich domain located 98 residues upstream of the C terminus, both of which are not conserved in other organisms (Figure 1a). Aside from this asparagine-rich domain, all of the features of AMPK subunits in other organisms were found to be present in the Dictyostelium protein in the expected location. These include a kinase domain near the N terminus (70% identical and 84% similar to the human 1 isoform), a phosphorylatable threonine (T₁₈₈) corresponding to the regulatory T₁₇₂ of the human AMPK2 isoform, ATP-binding and kinase catalytic site signatures, and a C-terminal region with recognizable, albeit lower similarity to the autoinhibitory and subunit-binding domain of other subunits (28% identical and 47% similar to the equivalent region of human AMPK1). In the active form of the AMPK heterotrimer, there is one molecule of AMP bound to each of two Bateman domains in the subunit (Scott et al., 2004) and the regulatory threonine on the subunit is phosphorylated (Weekes et al., 1994; Hardie et al., 2003). Although the major upstream activating kinase in mammalian cells, LKB1 (Hawley et al., 2003; Hong et al., 2003; Shaw et al., 2004), is constitutively active, phosphorylation of T₁₇₂ is normally autoinhibited in the heterotrimer, and this inhibition must be relieved by AMP binding to the subunit. ATP acts as a competitive inhibitor of AMPK at the AMP-binding sites on the subunit. Dictyostelium also possesses an LKB1 homologue (DictyBase accession no. DDB02290349).

For AMPK to be responsible for the deranged phenotypes associated with mitochondrial dysfunction in Dictyostelium, it must be present in wild-type cells at all stages of the life cycle. A Northern blot confirmed that the subunit mRNA is present throughout development and suggested that the levels may increase during the first 5 h of starvation-induced differentiation (Figure 1a, inset).

Generation of Clones with Increased or Decreased Levels of AMPK
To study the role of AMPK in mitochondrial disease in vivo in Dictyostelium, we chose a molecular genetic approach. To facilitate this, we amplified and cloned a cDNA encoding a truncated form (AMPKT) of the Dictyostelium AMPK subunit (Figure 1b). The encoded protein contains single amino acid changes at positions 3 and 374 through 379 at which point the polypeptide terminates prematurely. Although the S₃P substitution near the N terminus is not expected to affect the function of the protein, the six C-terminal substitutions and truncation of the protein mean that AMPKT will not bind to the subunit and will not be subject to the normal autoinhibitory mode of regulation of this enzyme. Truncating the human AMPK isoform 1 in this region has been shown to have both of these effects and to thereby produce an active kinase that is phosphorylated constitutively by upstream kinases (Crute et al., 1998; Iseli et al., 2005). Ectopic overexpression of AMPKT will therefore increase the total AMPK activity in the cell.

The AMPKT cDNA was subcloned into the Dictyostelium expression vector pA15GFP to create an overexpression construct. We also created antisense inhibition (and sense control) constructs by using a portion of the AMPK gene (Figure 1c). Multiple, independent, stable transformants of the wild-type Dictyostelium strain AX2 were isolated and retained in each case.

To relate the phenotypes of these transformants to their genotypes, it was necessary to verify that the level of expression of AMPK or AMPKT was correlated in the expected manner with the number of copies of the plasmids with which they had been stably transformed. Quantitative RT-PCR of the amplicon in Figure 1d showed that the reduction of the native AMPK mRNA level was correlated with the copy number of the antisense RNA-expression construct (Figure 1e). Conversely, in quantitative Northern blots the steady-state level of AMPKT mRNA was tightly correlated with the copy number of the AMPKT expression construct (Figure 1f). Western blots using an antibody directed against two specific AMPK peptides confirmed that these cells overexpressed (in a copy number-dependent manner), a novel 42-kDa protein (AMPKT) not present in wild-type cells (inset). Both constructs clearly affect expression in the expected copy number-dependent manner. Accordingly in further studies, we used the copy number of the corresponding constructs as an AMPK expression index. To simplify analysis and presentation of the data, we assigned negative values to the copy numbers for antisense constructs and positive values to the copy numbers for overexpression constructs.

Overexpression of the AMPK Catalytic Domain Phenocopies Mitochondrial Disease, whereas AMPK Antisense Inhibition Suppresses Mitochondrial Disease Phenotypes
Because mitochondrially diseased strains of Dictyostelium exhibit impaired phototactic orientation, slow growth in liquid medium and defective multicellular morphogenesis (Wilczynska et al., 1997; Kotsifas et al., 2002; Chida et al., 2004), we investigated whether AMPKT overexpression caused similar defects and whether AMPK antisense inhibition suppressed those defects (in chaperonin 60 antisense-inhibited cells). We also investigated whether the impaired growth was due to slower uptake of food by phagocytosis or macropinocytosis.

Phototaxis
In the multicellular migratory phase of its life cycle, the so-called slug, Dictyostelium exhibits extraordinarily sensitive and accurate orientation responses to light (phototaxis) and temperature gradients (thermotaxis). Although they are not well understood, the photo- and thermosensory transduction pathways in Dictyostelium are known to involve a variety of typically eukaryotic signaling molecules (Fisher, 1997, 2001; Bandala-Sanchez et al., 2006). If any of them were downstream targets of AMPK signaling, then AMPK activation would cross-talk and interfere with phototaxis and thermotaxis. Chronic AMPK activation would then account for the impaired phototaxis and thermotaxis observed in mitochondrial disease.

Figure 2 shows that slugs became increasingly disoriented in phototaxis as the copy number of either a chaperonin 60 antisense construct (Figure 2a) or an AMPKT expression construct (Figure 2b) increased. AMPKT overexpression thus phenocopies in a dose-dependent manner the phototaxis defect caused by mitochondrial dysfunction. AMPK antisense inhibition in an otherwise wild-type genetic background had little effect on slug phototaxis causing, if anything, a slight improvement in orientation. However mitochondrially diseased strains that would otherwise exhibit impaired phototaxis, behaved normally if AMPK expression was also inhibited in the same cells (Figure 2).

View larger version (20K): [in this window] [in a new window]   Figure 2. Effect of chaperonin 60 and AMPK expression levels on Dictyostelium slug phototaxis. Each circle represents a different clonal cell line (strain) carrying the indicated number of copies of either the chaperonin 60 antisense construct, the AMPK antisense construct, or the AMPKT overexpression construct. Lines are fitted only to the data represented by the circles. Each square represents a different strain carrying different numbers of copies of both the chaperonin 60 antisense construct and the AMPK antisense construct. Data for these strains therefore occurs in both panels, in each of which it is plotted according to the copy number relevant to that panel only. The accuracy of phototaxis is the concentration parameter () of the circular normal (von Mises) distribution, which measures how concentrated individual directions are around the direction toward the light source. ranges from 0 in the case of no preferred direction of migration (all directions equally probable) to in the case of perfect orientation (all directions exactly toward the light). Vertical bars are 90% confidence intervals. Negative values indicate the copy numbers of antisense inhibition constructs, whereas positive values indicate copy numbers of overexpression constructs. Copy numbers of zero include both the wild-type strain (AX2) and control strains carrying sense construct controls, but no copies of the relevant antisense or overexpression construct. (a) Phototaxis by mitochondrially diseased (chaperonin 60 antisense inhibited) Dictyostelium slugs formed and migrating on charcoal agar at 21°C. (b) Phototaxis by slugs of Dictyostelium strains with different levels of expression of the AMPK subunit (antisense inhibition, negative copy numbers) or a truncated form of the subunit containing the catalytic domain (AMPKT overexpression, positive copy numbers). An animated three-dimensional scatter plot combining data from both panels a and b is contained in the Supplemental Material video file AMPK_photo_spin.mov.

Growth Rates
Although AMPK signaling was responsible for defective phototaxis and thermotaxis in Dictyostelium mitochondrial disease, the other phenotypes could have been caused by a different mechanism. For example, the dramatically reduced cell growth and division rates reported previously in Dictyostelium mitochondrial disease (Wilczynska et al., 1997; Kotsifas et al., 2002) could have been caused by limitations in the energy available for growth. If this were the case, AMPKT overexpression would not phenocopy and AMPK antisense inhibition would not rescue the growth defects caused by mitochondrial dysfunction. Instead, the homeostatic function of AMPK in restoring the cellular energy status to normal would, if anything, produce the reverse outcomes.

Figure 3 shows that overexpression of the catalytic domain of AMPK and mitochondrial disease (antisense inhibition of chaperonin 60) dramatically impaired Dictyostelium growth both on plates on a bacterial food source (Figure 3, a and b) and in shaken culture in a nutrient broth (Figure 3, c and d). In otherwise healthy cells, AMPK antisense inhibition actually enhanced growth slightly; the generation times in liquid decreased from 9 to 7 h, whereas growth on plates also accelerated (Figure 3, b and d). Most remarkably, the impaired growth of mitochondrially diseased cells (chaperonin 60 antisense inhibition) was completely restored to normal by AMPK antisense inhibition (Figure 3, a and c). This dramatic suppression of the mitochondrial disease phenotype occurred both for growth on plates and in liquid medium. It shows that the cause of the impaired growth in Dictyostelium mitochondrial disease is chronic AMPK activation not insufficient energy.

View larger version (33K): [in this window] [in a new window]   Figure 3. Effect of chaperonin 60 and AMPK expression levels on Dictyostelium growth. Symbols used are as in Figure 2. Vertical bars are 95% confidence intervals. Negative values indicate the copy numbers of antisense inhibition constructs, whereas positive values indicate copy numbers of the overexpression construct. Copy numbers of zero include both the wild type strain (AX2) and control strains carrying sense construct controls, but no copies of the relevant antisense or overexpression construct. (a and b) Time taken for a growing Dictyostelium colony (plaque) to expand 5 mm during growth at 21°C on an E. coli B2 lawn on SM agar. The growth time was calculated from the slope of the line measured by linear regression analysis of plaque diameter versus time during 5–7 d of growth. An animated three-dimensional scatter plot combining data from both a and b is contained in the Supplemental Material video file AMPK_plate_ growth_spin.mov. (c and d) Generation time for Dictyostelium cells growing in HL-5 liquid medium at 21°C, shaken at 150 rpm. Generation times were calculated from growth curve slopes using log-linear regression analysis of cell counts during the exponential phase of growth. An animated three-dimensional scatter plot combining data from both c and d is contained in the Supplemental Material video file AMPK_HL-5_growth_spin.mov.

View larger version (23K): [in this window] [in a new window]   Figure 4. Effect of chaperonin 60 and AMPK expression levels on Dictyostelium phagocytosis and pinocytosis rates. Symbols are as in Figure 2. Negative values indicate the copy numbers of antisense inhibition constructs, whereas positive values indicate copy numbers of overexpression construct. Copy numbers of zero refer to the wild-type parental strain AX2. (a and b) Rates of phagocytosis by Dictyostelium cells engulfing E. coli cells expressing the fluorescent red protein Ds-Red. Rates were measured from duplicate fluorescence measurements immediately and 30 min after addition of fluorescent bacteria to the amoebal suspension. An animated three-dimensional scatter plot combining data from both a and b is contained in the Supplemental Material video file AMPK_phago_spin.mov. (c and d) Rates of macropinocytosis by Dictyostelium cells in HL-5 medium containing 2 mg/ml FITC-dextran. The uptake of fluorescent medium was measured in duplicate immediately and 60 or 70 min after addition of FITC-dextran to the amoebal suspension. An animated three-dimensional scatter plot combining data from both c and d is contained in the Supplemental Material video file AMPK_pino_spin.mov.

Multicellular Development
Development in Dictyostelium is initiated by starvation-induced differentiation followed by chemotactic aggregation, further differentiation into multiple cell types, and complex morphogenetic movements to form a fruiting body—a droplet of spores (sorus) atop a stalk and basal disk. Multicellularity evolved independently in the amoebozoa (Dictyostelium) and metazoa, so that some of the associated intracellular signaling molecules are different, whereas others have been conserved and recruited for use in regulation of cell type choice and differentiation in both lineages (Williams, 2006; Strmecki et al., 2005). The developmental signaling pathways thus provide a mixture of potential downstream targets of AMPK signaling in mitochondrially compromised Dictyostelium cells, some unique and others conserved.

Mitochondrial disease in Dictyostelium has been shown to result in dysmorphogenesis so that as the disease becomes more severe, fewer fruiting bodies form, and those that do are morphologically aberrant with short thick stalks (Kotsifas et al., 2002) resulting from misregulation of cell type choice in favor of the stalk differentiation pathway (Chida et al., 2004). We found that a similar phenotype results from overexpression of AMPKT in an otherwise wild-type background (Figure 5, d and f versus b). Conversely, antisense inhibition of AMPK expression restored normal development in mitochondrially diseased cells in which chaperonin 60 expression was inhibited (Figure 5, a and c versus b). Consistent with this data, prestalk gene expression is enhanced in Dictyostelium cells that accumulate AMP because of inactivation of the AMP deaminase (Chae et al., 2002). Thus, the abnormal multicellular development associated with mitochondrial disease in Dictyostelium is also mediated by AMPK signaling.

View larger version (69K): [in this window] [in a new window]   Figure 5. Effect of chaperonin 60 and AMPK expression levels on multicellular morphogenesis in Dictyostelium. Photographs were taken from above (main panels) or from the side (insets) of fruiting bodies formed during growth at 21°C on a bacterial lawn (K. aerogenes). Apart from b, the parental wild-type strain (AX2), the strains contained (a) antisense inhibition constructs for both chaperonin 60 (main panel, 73 copies; inset, 56 copies) and AMPK (main panel, 109 copies; inset, 98 copies) or (c) the chaperonin 60 antisense construct only (main panel, 48 copies; inset, top row, 67 copies; inset, other rows, 74 copies) or (e) the AMPK antisense inhibition construct (143 copies) only or (d and f) the AMPKT overexpression construct only (30 copies and 148 copies, respectively). Mitochondrial disease (chaperonin 60 antisense inhibition) and AMPKT overexpression both caused the formation of fruiting bodies with thick, short stalks (red arrows in c, d, and f). Fruiting body morphology was normal for a mitochondrially diseased strain (chaperonin 60 antisense inhibition) in which AMPK expression was antisense inhibited (cyan arrows in a). In otherwise healthy cells, AMPK antisense inhibition resulted in formation of fewer, smaller fruiting bodies that were morphologically normal (green arrows in e). For comparative purposes, the strains were selected to show the phenotypes at moderate copy numbers of the various constructs. The aberrant phenotypes in c, d, e, and f were more severe at higher copy numbers.

AMPK Stimulates Mitochondrial Biogenesis and ATP Production
In mammalian cells, particularly in muscle tissues, AMPK activity leads to mitochondrial proliferation (Bergeron et al., 2001; Zong et al., 2002). This is part of the response to strenuous physical training in athletes and is a component of roles of AMPK in energy homeostasis in healthy cells. However, mitochondrial proliferation is sometimes also a feature of mitochondrial diseases (Campos et al., 1997; Graham et al., 1997; Agostino et al., 2003). We therefore examined whether mitochondrial dysfunction and AMPK activation might cause this same cytopathology in Dictyostelium. Figure 6b shows that overexpression of the AMPK catalytic domain resulted in a stronger MitoTracker Green fluorescence signal per cell, whereas AMPK antisense inhibition reduced the fluorescence signal. However, the fluorescence signal was unaltered in the mitochondrially diseased cells whether or not AMPK expression was also antisense inhibited (Figure 6a). Fluorescence microscopy of cells stained with MitoTracker Red confirmed that mitochondrial biogenesis in Dictyostelium is stimulated by AMPK as in mammalian cells. We found no evidence for the proliferation of mitochondria in mitochondrially diseased Dictyostelium cells, presumably because in these cells any reduction in mitochondrial biogenesis caused by chaperonin 60 undersupply is counterbalanced by the effects of enhanced AMPK activity. Because mitochondrial biogenesis was stimulated in AMPKT-overexpressing cells and reduced in AMPK antisense-inhibited cells, we expected that ATP levels would be altered in a similar manner. Figure 6, c and d, shows that this is so. ATP levels were greater in AMPKT-overexpressing cells, were reduced in AMPK antisense-inhibited cells, and were not significantly altered in mitochondrially diseased cells.

View larger version (27K): [in this window] [in a new window]   Figure 6. Effect of chaperonin 60 and AMPK expression levels on mitochondrial mass and ATP levels in Dictyostelium. Symbols are as in Figure 2. Negative values indicate the copy numbers of antisense inhibition constructs, whereas positive values indicate copy numbers of the overexpression construct. Copy numbers of zero refer to the wild-type parental strain AX2. (a and b) Mitochondrial mass as measured by fluorescence with the mitochondrion-specific dye MitoTracker Green after subtraction of autofluorescence from unstained cells from the same suspension. The insets in b show MitoTracker Red fluorescence microscopy of typical cells from wild-type AX2 and from representative AMPK antisense-inhibited strains (with and without chaperonin 60 antisense inhibition) and a representative AMPKT-overexpressing strain. Compared with wild-type cells, the MitoTracker Red fluorescence seems brighter and the mitochondria more numerous in the case of AMPKT overexpression, but fainter and the mitochondria less numerous in the AMPK antisense-inhibited cells. The cells that are antisense-inhibited for both AMPK and chaperonin 60 do not seem to differ from the wild-type cells in the brightness of the MitoTracker Red fluorescence or in the numbers of mitochondria. An animated three-dimensional scatter plot combining data from both a and b is contained in the Supplemental Material video file AMPK_mtmass_spin.mov. (c and d) ATP levels in Dictyostelium cells as measured using luciferase-based luminescence in a Modulus fluorometer (Turner BioSystems) with the luminescence module. An animated three-dimensional scatter plot combining data from both c and d is contained in the Supplemental Material video file AMPK_ATP_spin.mov. The significance of the correlations in b and d was verified both by using the nonparametric Kendall rank r and by two-sample tests (t test with unequal variances, Kolmogorov–Smirnov test and Kruskal–Wallace test) of the difference between the overexpression strains and the antisense-inhibited strains.

AICAR, a Pharmacological Activator of AMPK in Mammalian Cells, Impairs Phototaxis in the Wild-Type but Not in AMPK Antisense-inhibited Cells

As a further test of whether AMPK activation could cause the defects seen in mitochondrial diseases, we examined the effect of AICAR on Dictyostelium slug phototaxis. Wild-type Dictyostelium cells were allowed to develop to the slug stage in the presence of AICAR and then to migrate in the presence of the drug. These slugs became increasingly disoriented as the AICAR concentrations increased (Figure 7a). These effects of long-term (>48 h) AICAR exposure could have been mediated directly or indirectly by some other adenosine- or AMP-binding protein. We therefore tested whether AICAR would still impair phototaxis in a strain in which the expression of the subunit of AMPK had been antisense inhibited. Figure 7b shows that the antisense-inhibited strain was resistant to the effects of AICAR on phototaxis. Although this result does not prove that AICAR impairs phototaxis by activating AMPK, it does indicate that AMPK is required for the AICAR effects and is consistent with a role for AMPK in mitochondrial disease.

View larger version (25K): [in this window] [in a new window]   Figure 7. Impairment of phototaxis by the pharmacological AMPK activator AICAR. Dictyostelium slugs of the parental strain (AX2; a) or an AMPK antisense-inhibited transformant (143 copies of the antisense construct; b) were allowed to form and migrate toward a lateral light source on water agar supplemented with the indicated concentrations of AICAR. Slug trails were blotted onto PVC discs stained with Coomassie blue protein stain, digitized, and plotted from a common origin. The light source was to the right of the Figure. In the presence of AICAR, the wild-type slugs were disoriented so that their trails wound about much more during phototaxis. This did not occur with the AMPK antisense-inhibited strain. The inset shows a separate experiment with wild-type cells in which the dose response to AICAR was clearer.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

There is every indication that this novel insight into the nature of mitochondrial disease will be generally applicable. During preparation of this article, it was reported that cell cycle progression in the developing Drosophila eye is halted specifically by AMPK signaling in response to the ATP-depleting effects of a mitochondrial mutation (Mandal et al., 2005). Mitochondrial electron transport uncouplers such as dinitrophenol have been shown, like other cellular stressors, to activate AMPK. Much of the cytopathology of mitochondrial disorders in humans (James and Murphy, 2002; Rossignol et al., 2003; Maassen et al., 2004; McKenzie et al., 2004)—mitochondrial proliferation and ragged red muscle fibers, cellular hypertrophy, and induction of apoptosis—is known also to occur in response to AMPK activation (Bergeron et al., 2001; Hardie and Hawley, 2001; Zong et al., 2002; Hardie, 2004; Kahn et al., 2005; Hardie and Sakamoto, 2006). Finally, symptoms that are strongly reminiscent of mitochondrial disease are seen in a rare human genetic disorder, AICA-ribosiduria, in which an inactive AICAR transformylase causes the AMPK activator ZMP to accumulate in cells (Marie et al., 2004). In addition to dysmorphic features, the affected patient, a female infant, suffered from severe neurological dysfunction, epilepsy, and congenital blindness.

Figure 8 shows an explanatory model for the interactions between mitochondrial function, AMPK, and the various energy-consuming cellular activities investigated in this work (flame-shaded section). Mitochondrial dysfunction was created by antisense inhibition of chaperonin 60 expression as described previously (Kotsifas et al., 2002). The model suggests that this results in a reduction in mitochondrial biogenesis and ATP-generating capacity. Rossignol et al. (2003) discussed multiple layers of biochemical "thresholds" that determine the degree to which ATP-generating capacity is actually compromised as a result of genetic defects affecting the mitochondria. When these thresholds are exceeded AMPK would become chronically activated in a homeostatic response that returns mitochondrial mass and ATP levels to near normal but chronically inhibits some of the major energy-consuming activities of the cell (e.g., growth and cell cycle progression, multicellular morphogenesis, and photo- and thermosensory signal transduction). These major symptoms of mitochondrial disease in Dictyostelium were both phenocopied by overexpression of AMPKT in otherwise healthy cells, and they were relieved by antisense inhibition of AMPK expression in mitochondrially diseased cells.

View larger version (56K): [in this window] [in a new window]   Figure 8. Model for the role of AMPK signaling in mitochondrial disease. Cpn60 is chaperonin 60 whose undersupply in antisense-inhibited cells causes mitochondrial dysfunction. Arrowheads indicate stimulation, and barred ends indicate inhibition. AMP, ADP, and ATP are interconvertible. Mitochondrial biogenesis and function favor ATP production, whereas the cellular activities in the ellipses in the flame-shaded region consume ATP and favor AMP generation. AMP activates and ATP inhibits AMPK, which in turn stimulates mitochondrial biogenesis and inhibits some energy-consuming cellular activities (e.g., growth and cell cycle progression, morphogenesis, and photosensory signal transduction), but not others (e.g., phagocytosis and macropinocytosis). In mitochondrially diseased cells, AMPK is chronically activated, which homeostatically returns mitochondrial mass and ATP levels to near normal, but it also chronically inhibits cell proliferation and impairs multicellular morphogenesis and photosensory behavior (phototaxis).

A feature of human mitochondrial disease that has been difficult to explain is the so-called threshold effect: as the underlying genetic cause becomes more severe, some cellular activities are affected before others. This is true of Dictyostelium as well. In this work, we found no significant changes to phagocytosis and pinocytosis rates in mitochondrially diseased cells. Both of these phenotypes were also unaffected by