Mitochondria in Sporadic Amyotrophic Lateral Sclerosis

EXPERIMENTAL NEUROLOGY ARTICLE NO.

153, 135–142 (1998)

EN986866

Mitochondria in Sporadic Amyotrophic Lateral Sclerosis Russell H. Swerdlow,* Janice K. Parks,* David S. Cassarino,* Patricia A. Trimmer,* Scott W. Miller,† David J. Maguire,‡ Jason P. Sheehan,* Robyn S. Maguire,* Gary Pattee,§ Vern C. Juel,* Lawrence H. Phillips,* Jeremy B. Tuttle,* James P. Bennett, Jr.,* Robert E. Davis,† W. Davis Parker, Jr.* *Center for the Study of Neurodegenerative Diseases and Department of Neurology, University of Virginia Health Sciences Center, 1 Hospital Drive, Charlottesville, Virginia 22908; †MitoKor, San Diego, California; ‡Department of Science and Technology, Griffith University, Brisbane, Australia; and §Lincoln, Nebraska Received January 21, 1998; accepted May 29, 1998

MATERIALS AND METHODS Mitochondria are abnormal in persons with amyotrophic lateral sclerosis (ALS) for unknown reasons. We explored whether aberration of mitochondrial DNA (mtDNA) could play a role in this by transferring mitochondrial DNA (mtDNA) from ALS subjects to mtDNA-depleted human neuroblastoma cells. Resulting ALS cytoplasmic hybrids (cybrids) exhibited abnormal electron transport chain functioning, increases in free radical scavenging enzyme activities, perturbed calcium homeostasis, and altered mitochondrial ultrastructure. Recapitulation of defects previously observed in ALS subjects and ALS transgenic mice by expression of ALS mtDNA support a pathophysiologic role for mtDNA mutation in some persons with this disease. r 1998 Academic Press Key Words: ALS; amyotrophic lateral sclerosis; cybrids; DNA; mitochondria; oxidative; calcium.

Subjects Enrollment of human subjects at the University of Virginia followed IRB approval and informed consent. ALS subjects diagnosed by a neurologist specializing in neuromuscular diseases met World Federation of Neurology criteria for definite or probable ALS (43). Patients with a family history of ALS were excluded. Determination of control status was made on the basis of history and clinical examination. No ALS subjects (n 5 11) required ventilatory support, 55% were male, and mean age was 56.4 6 3.3 (SEM). For the agematched control group 17% were male and mean age was 62.4 6 3.2.

Generation of Cybrid Cell Lines INTRODUCTION

Mitochondria are abnormal in persons with amyotrophic lateral sclerosis (ALS) (14, 15, 26). The reasons for this are unknown. Other late-onset, ‘‘sporadic’’ neurodegenerative diseases that rarely exhibit Mendelian inheritance also manifest mitochondrial pathology (25, 30, 31, 34). Recent data obtained using cytoplasmic hybrid (cybrid) systems suggest that in two of these diseases, Alzheimer’s disease (AD) and Parkinson’s disease (PD), mitochondrial dysfunction is associated with mutation of mitochondrial DNA (mtDNA) (38, 39). In this work, mitochondrial DNA (mtDNA) was transferred from AD, PD, or control patients into mtDNA-depleted immortalized cells (r0 cells) to form unique cell lines that differ only in the source of their mtDNA; any phenotypic differences between ‘‘control’’ and ‘‘disease’’ cybrid lines most likely arise from differences in mtDNA and imply the presence of mtDNA mutation (19). We used a cybrid system to investigate whether mtDNA mutation could play a role in ALS.

Six milliliters of blood was obtained from each subject using test tubes containing acid-citrate-dextrose. Platelets were isolated and fused with human SHSY5Y neuroblastoma cells containing no mtDNA (r0 cells, MitoKor, San Diego, CA) as previously described (24). Cells successfully incorporating platelet contents were selected for and grown over 5 to 6 weeks. Formation of multinucleated hybrids during the fusion step was ruled out by chromosome counts of the resultant cybrid lines. Confocal microscopy of cybrids was performed as previously described to confirm stable transfer of exogenous platelet mitochondria to r0 cells (38).

Electron Transport Chain Biochemistry Electron transport chain (ETC) analysis followed digitonin-induced lysis of cell membranes (38). Spectrophotometric assays of NADH:ubiquinone oxidoreductase (complex I), cytochrome reductase (complex III), and cytochrome c oxidase (complex IV) were performed as previously described (32,38). Each cybrid line was

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0014-4886/98 $25.00 Copyright r 1998 by Academic Press All rights of reproduction in any form reserved.

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FIG. 1.

Complex I activity is significantly reduced in ALS cybrids.

assayed in duplicate and the average of these two independent assays served as the final enzyme activity for a given cell line. Mean complex I, III, and IV activities from ALS and control cell lines were compared using Student’s t test (unpaired). Calcium Measurements ALS (n 5 7) and control (n 5 4) cybrid cell line cytosolic calcium before and after exposure to 10 µM carbonyl cyanide m-chlorophenylhydrazone (CCCP) was determined using the fluorescent dye fura-2 as previously described (36). Mean basal and post-CCCP cytosolic calcium levels were determined for the ALS and control groups and these means were compared using Student’s t test (unpaired).

Free Radical Scavenging Enzyme Activities Glutathione reductase, glutathione peroxidase, total superoxide dismutase (SOD), Mn SOD, Cu/Zn SOD, and catalase activities were determined as previously described (3). The mean of each free radical scavenging enzyme assayed was determined for ALS cybrids (n 5 6) and control cybrids (n 5 8), and these means were compared using Student’s t test. MPP1 Exposure To assess susceptibility of cybrid lines to a mitochondrial toxin, ALS (n 5 8) and control cybrid lines (n 5 8) were exposed to 80 or 160 µM 1-methyl-4-phenyl pyridinium (MPP1) for 24 h. Cells were plated, transferred

FIG. 2. Mitochondrial calcium handling is impaired in ALS cybrids. Basal cytosolic calcium is increased (A). After CCCP administration, the control cybrid cytosolic calcium concentration rises by more than twice the amount that it does in the ALS cybrids, indicating that the ALS mitochondria have an impaired ability to sequester calcium.

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FIG. 3. Free radical scavenging enzyme activities are increased in ALS cybrids. Data are expressed as the mean of a particular enzyme for the control or ALS cybrid lines 6 SEM.

to serum-free media, exposed to MPP1, fixed, stained, and visualized as previously described (38). Ratios of condensed, damaged nuclei to normal nuclei were determined. The means of the percentage of cells killed for ALS and control lines were compared using Student’s t test (unpaired). Electron Microscopy For electron microscopy studies of cybrid ultrastructure, ALS (n 5 6) and control (n 5 6) cybrid lines were grown for at least 3 weeks in media containing sodium pyruvate as previously described (24). Cybrid processing and sectioning were performed by the Central Electron Microscope Facility at the University of Virginia. Cells in culture were fixed with 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.2). Fixed monolayers were washed, scraped from the flask surface, and transferred to

microfuge tubes. The tubes were centrifuged at 5000 rpm for 4 min after each solution change. Cell pellets were postfixed with 2% osmium tetroxide for 1 h, rinsed with distilled water, and dehydrated using a graded series of acetone. Cell pellets were embedded in epoxy resin. Thin sections were cut on a Reichert Ultracut E (Reichert, Austria), stained with uranyl acetate and lead citrate, and photographed at 10,0003 on a JEOL 100CX transmission electron microscope (JEOL, Japan). The perinuclear regions from 10 individual cells per line were imaged. Features considered typical for ALS or of mitochondrial pathology in general (16, 20) were counted for these 10 cells and included membranous whorls, lysosomal bodies, swollen mitochondria, and intermediate filament aggregates. The mean number of pathologic features for the control and ALS cybrid groups were compared using Student’s t test (unpaired). RESULTS

TABLE 1 Cybrid Cell Killing Following MPP1 Exposure

ALS (n 5 8) Control (n 5 8)

Percentage of cells killed by 80 µM MPP1 (mean percentage killed 6 SEM)

Percentage of cells killed by 160 µM MPP1 (mean percentage killed 6 SEM)

ns 5.7 6 1.0

ns 23.5 6 5.0

4.9 6 1.0

18.9 6 4.6

Note. ns, not significant.

Generation of Cybrids Following execution of the r0 cell-platelet fusion procedure, cells were placed in selection media which was not supplemented with uridine or pyruvate. This resulted in the removal of r0 cells not incorporating donor mitochondria/mtDNA. Mitochondrially transformed cells grew initially as individual colonies, which became confluent over time to form unique ‘‘mixed population’’ cybrid cell lines. ‘‘Mock fusions’’ in which r0 cells experienced all stages of the fusion procedure except coincubation with platelets selected completely

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and indicated reversion of potential residual endogenous mtDNA molecules did not occur. To further demonstrate the integrity of the fusion strategy used (5, 24), platelet mitochondria were independently stained with the dye Mito Tracker CMXRos-H2 (Molecular Probes, Eugene, OR) and following fusion were observed residing within newly created cybrid cells (data not shown). This confirmed that the source of mtDNA repopulation within cybrid lines originated from the mtDNA of the platelet donor individual. Electron Transport Chain Activities Activities of the ETC enzymes complex I, III, and IV were determined since the physical generation of these three enzymes requires input from 11 of the 13 structural genes and the 24 tRNA/rRNA genes of mtDNA (Fig. 1). After 5–6 weeks of growth in culture the mean complex I activity of the ALS cybrid cell lines (n 5 11) was 26.4 6 1.2 nmol/min/mg (SEM) and for the control cybrid cell lines (n 5 12) it was 32.7 6 1.2 nmol/min/mg (Fig. 1). This represented a 19% defect of complex I activity in the ALS cybrid lines (P , 0.001). Mean COX activity in the ALS cybrid lines (n 5 11) was 0.048 6 0.004 s21/mg, and in the control cybrid lines (n 5 12) it was 0.057 6 0.005 s21/mg. Complex IV activity was 16% lower in the ALS cybrids, but this difference was not significant. Mean complex III activity in the ALS cybrid cell lines (n 5 10) was 0.015 6 0.002 s21/mg and in the control cybrid lines (n 5 10) it was 0.019 6 0.002 s21/mg. While complex III activity was 21% lower in the ALS cybrids, this difference was not significant. Within the ALS cybrid lines, degrees of ETC impairment did not correlate with the subjective clinical severity of the disease. Calcium Measurements ALS and control cybrid mean cytosolic calcium concentrations before and after CCCP are shown in Fig. 2. In the ALS cybrids basal cytosolic calcium was 170.26 6 5.65 nM (n 5 7, 45 total cells analyzed) and in the control cybrids it was 142.56 6 7.40 nM (n 5 4, 30 total cells analyzed) (P , 0.01). Following CCCP, an electron transport uncoupler which triggers mitochondrial calcium efflux, the cytosolic calcium concentration in the same ALS cybrid cells was 269.85 6 4.87 nM and in the controls it was 360.65 6 16.21 nM (P , 0.0001). This difference is consistent with decreased calcium sequestration by ALS mitochondria. The control cybrid cytosolic calcium concentration increased by 154% after

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CCCP administration, whereas the ALS cybrid cytosolic calcium increased by only 58%. Free Radical Scavenging Enzyme Activities Free radical scavenging enzyme activities were assayed in cybrid lines as an index of oxidative stress. Glutathione reductase, glutathione peroxidase, total SOD, Mn SOD, and catalase activities were increased in ALS cybrids (Fig. 3). This is most consistent with an absolute increase in ALS cybrid reactive oxygen species (ROS) production. MPP1 Exposure Following both 80 and 160 µM MPP1 treatment, the mean percentage of cells killed for the ALS and control cybrid lines was not statistically different. Therefore, in contrast to what was previously observed in PD cybrids (38), ALS cybrids exposed to the complex I inhibitor MPP1 were not more likely to undergo demise than control cybrids (Table 1). Electron Microscopy Although electron microscopy revealed the presence of normal mitochondria in all ALS cybrid lines analyzed, in each ALS cybrid line there were also cells which contained swollen mitochondria whose few remaining cristae were peripherally distributed (Fig. 4). This type of pathology was virtually absent from control cybrid line cells. Other pathological changes present in all ALS cybrid lines included increased numbers of lysosomes and membranous bodies, which represent degenerating mitochondria and cellular components (20). Four of six ALS cybrid lines contained cells that possessed intermediate filament aggregates, which were observed in only one of the control lines (Fig. 4). The mean number of membranous bodies, lysosomes, swollen mitochondria, and intermediate filament aggregates counted from the perinuclear regions of 10 cells of a cybrid line was 10.5 6 1.6 for the ALS lines (n 5 6) and 5.2 6 0.8 for the control lines (n 5 6) (P , 0.05) (Fig. 5). The increased frequency of enlarged mitochondria, lysosomes, membranous bodies, and neurofilament aggregates in ALS (compared to control) cybrid cell lines demonstrates that in tissue culture, ALS mtDNA can give rise to ultrastructural pathology observed in persons with the disease and in ALS transgenic mice (6, 8, 9, 14, 15, 16, 28, 44).

FIG. 4. Altered mitochondrial and neurofilament ultrastructure in ALS cybrids. Mitochondria from a representative control cybrid line are shown in (A). Pathological changes characteristic of ALS cybrid cells included mitochondrial swelling, loss, and peripheral distribution of cristae (C), as well as increased numbers of lysosomes and membranous bodies (arrowheads in B, D, and E). Approximately 5% of the ALS cybrid cells also contained intermediate filament aggregates (right side of E). The scale bar in (A) also applies in (B, C); scale bars in A–E represent 1 µm.

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FIG. 5. The sum total of lysosomes, membranous bodies, enlarged mitochondria, and intermediate filament aggregates from the perinuclear regions of ten individual cells was determined for ALS (n 5 6) and control (n 5 6) cybrid lines. This figure shows the ‘‘summed pathology score’’ from each of the cybrid lines analyzed by electron microscopy. More pathologic features were present in ALS cybrid lines than in control cybrid lines (P , 0.05).

DISCUSSION

Mitochondria containing mtDNA from sporadic ALS subjects were abnormal by several parameters. Defective electron transport chain function, perturbed mitochondrial calcium buffering, oxidative stress, and altered mitochondrial ultrastructure were observed and recapitulate pathology already described in ALS patients and animal models of the disease. Demonstration of such phenomena in a cybrid system supports the presence of mtDNA aberration in sporadic ALS, since in cybrids nuclear genetic and environmental input is controlled for. Nuclear genes are the same in ALS and control cybrid lines and should not account for phenotypic differences. Cell lines are grown and assayed under identical conditions and environmental factors should not contribute to phenotypic differences. Transferred platelet mRNA and nongenetic factors such as platelet proteins degrade and dilute over the many cycles of cell replication and are therefore irrelevant. Biochemical differences between ALS and control cybrids are therefore most consistent with differences in ALS and control mtDNA. Complex I activity was reduced in ALS cybrids and trends toward low complex III and IV activities were observed. These data are potentially consistent with a point mutation of an mtDNA gene coding for a complex

I subunit, although the trend toward depression of multiple mtDNA encoded complexes could be consistent with a tRNA or rRNA mutation, point mutations in multiple ETC subunit-encoding mtDNA genes, deletion(s), or varying patterns of mtDNA aberration between ALS patients. Our data also do not indicate whether the mtDNA lesion(s) is somatic or inherited, but the possible presence of a maternal link in ALS suggest inheritance may represent the origin of mtDNA mutation in this disease (21). Furthermore, although we recently used cybrid methodology to demonstrate the complex I defect of PD arises from mtDNA mutation, PD cybrids exhibit selective susceptibility to MPP1 (38). The absence of increased MPP1 susceptibility in our ALS cybrids suggests the mtDNA defects of PD and ALS are not identical and argues against the possibility that mtDNA aberration in these diseases results from random degradation of the mitochondrial genome. Still, this latter finding does not prove that mitochondrial dysfunction in these experiments is specific for ALS, nor does it address whether or not putative mtDNA aberration in our ALS cybrids is a primary or secondary event. Defects of calcium handling exist in multiple tissues in ALS (7, 18, 37, 42). In our cybrid experiments mutated mtDNA from the platelets of ALS patients was sufficient to cause increased basal cytosolic calcium and decreased mitochondrial calcium sequestration. Mitochondria are important for buffering increases in cell cytosolic calcium. When this buffering capacity is reduced, alterations in calcium can act as an apoptotic trigger and cause neurodegeneration (13, 27, 29, 40, 41). Studies of cell oxidative markers in ALS suggest that in this disease there is either increased production or decreased scavenging of ROS (2, 35). Our demonstration of increased ROS scavenging enzymes suggests the former mechanism is operant in sporadic ALS. A compensatory increase in free radical scavenging enzymes occurs in PD and AD (1, 4, 10, 11, 17, 23, 33, 45) and we have observed similar changes in PD and AD cybrids, indicating these changes are ultimately triggered by mtDNA defects (3, 39). In ALS cybrids the ETC also appears to act as a genetically determined ROS generator. Observed ALS cybrid mitochondrial ultrastructure abnormalities are similar to those described both in persons with ALS (14, 15, 26) and in transgenic mouse models of the disease (8, 44). Neurofilament aggregations observed in ALS subjects and ALS transgenic mice were also seen in our ALS cybrid lines (6, 9, 16, 28). In ALS transgenic mice, abnormal SOD is primarily responsible for giving rise to ultrastructural abnormalities. There is, however, no demonstrable superoxide dismutase abnormality in the majority of ALS patients. Our data suggests that mtDNA mutation may

MITOCHONDRIA IN ALS

play a role in driving structural mitochondrial/neurofilament pathology in sporadic ALS. Recapitulation of ALS pathology by expression of ALS mtDNA in a cybrid system suggests mtDNA aberration is present in this disease. Abnormal ETC functioning, oxidative stress, perturbed calcium homeostasis, and altered mitochondrial/neurofilament ultrastructure are present in ALS cybrids and reinforce previous hypotheses proposing these mechanisms are relevant to ALS neurodegeneration. Coexpression of dementia, parkinsonism, and motor neuron disease as well as epidemiologic observations of increased AD and PD risk for families of ALS subjects (12, 22) argue that a ‘‘common genetic susceptibility factor’’ exists for these diseases. This ALS data, combined with recent data implicating mtDNA mutation in AD and PD (38, 39) indicates aberration of mtDNA could represent this genetic factor. ACKNOWLEDGMENTS We are grateful to Judy Warder, Cindy Barnhill, George Olcott, and Bonita Eppard of the University of Virginia Neuromuscular Clinic for providing blood samples from ALS patients. Dr. Wendy Golden performed chromosome counts. This work was supported by AG00800 (R.H.S.), a Commonwealth of Virginia Alzheimer’s Disease and Related Research Award (R.H.S.), AG104667 (W.D.P), and NS35325 (J.P.B.).

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