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Previous Article | Next Article
Volume 17, Number 20, Issue of October 15, 1997 pp. 7746-7753
Copyright ©1997 Society for NeuroscienceMyoclonic Epilepsy and Ragged Red Fibers (MERRF) Syndrome: Selective Vulnerability of CNS Neurons Does Not Correlate with the Level of Mitochondrial tRNAlys Mutation in Individual Neuronal Isolates
Li Zhou1, Anne Chomyn2, Giuseppe Attardi2, and Carol A. Miller1 1 Departments of Pathology and Neurology, University of Southern California School of Medicine, Los Angeles, California 90033, and 2 Division of Biology, California Institute of Technology, Pasadena, California 91125
ABSTRACT
Selective vulnerability of subpopulations of neurons is a striking feature of neurodegeneration. Mitochondrially transmitted diseases are no exception. In this study CNS tissues from a patient with myoclonus epilepsy and ragged red fibers (MERRF) syndrome, which results from an A to G transition of nucleotide (nt) 8344 in the mitochondrial tRNALys gene, were examined for the proportion of mutant mtDNA. Either individual neuronal somas or the adjacent neuropil and glia were microdissected from cryostat tissue sections of histologically severely affected brain regions, including dentate nuclei, Purkinje cells, and inferior olivary nuclei, and from a presumably less affected neuronal subpopulation, the anterior horn cells of the spinal cord. Mutant and normal mtDNA were quantified after PCR amplification with a mismatched primer and restriction enzyme digestion. Neurons and the surrounding neuropil and glia from all CNS regions that were analyzed exhibited high proportions of mutant mtDNA, ranging from 97.6 ± 0.7% in Purkinje cells to 80.6 ± 2.8% in the anterior horn cells. Within each neuronal group that was analyzed, neuronal soma values were similar to those in the surrounding neuropil and glia or in the regional tissue homogenate. Surprisingly, as compared with controls, neuronal loss ranged from 7% of the Purkinje cells to 46% of the neurons of the dentate nucleus in MERRF cerebellum. Thus, factors other than the high proportion of mutant mtDNA, in particular nuclear-controlled neuronal differences among various regions of the CNS, seem to contribute to the mitochondrial dysfunction and ultimate cell death.
Key words: MERRF syndrome; CNS microdissection; PCR; neurodegeneration; tRNALys; mtDNA
INTRODUCTION
Selective vulnerability of neuronal or glial subpopulations is a feature of many neurological diseases. The myoclonus epilepsy and ragged red fibers (MERRF) syndrome is characterized by myoclonic epilepsy, cerebellar ataxia, and progressive muscular weakness. In this maternally inherited disease (Wallace et al., 1988
) the symptoms of myoclonic epilepsy, ataxia, and muscular weakness may reflect severe pathological changes in neurons of the dentato-rubral and the spinocerebellar pathways and of the inferior olivary nuclei and in skeletal muscle. The myopathic changes include the generation of ragged red fibers indicative of proliferation and subsarcolemmal accumulation of mitochondria.
The MERRF syndrome is most commonly the result of a single base pair substitution at position 8344 in the mitochondrial tRNALys gene (Shoffner et al., 1990
Previous quantifications of the mutant mtDNA proportion in the CNS have been made in tissue homogenates of anatomical regions (Tanno et al., 1993
In the present study we have compared neuronal loss with the percentage of mutant mtDNA in the more severely and the less severely affected neuronal groups. Individual neuronal somas were microdissected from CNS tissue sections. Mutant and normal mtDNA were quantified after PCR amplification with a mismatched primer and restriction enzyme digestion (Zeviani et al., 1991
MATERIALS AND METHODS
CNS tissue. CNS tissues were obtained 20 hr postmortem from a 14-year-old Hispanic female with clinical and neuropathologically confirmed MERRF syndrome (Sanger and Jain, 1996
Neuronal loss was compared in MERRF and control tissues in four sections from each paraffin block. Neurons were counted at 250× magnification in five fields of either dentate nuclei or inferior olivary nuclei, or in the entire bilateral anterior horn region of the lower cervical and upper thoracic spinal cord, or Purkinje cells in five foliar loops. Counts were confirmed in all sites by two independent observers. The mean values were calculated for each.
For PCR analysis, blocks of tissue (1 cm3) were dissected at autopsy, immediately snap-frozen in liquid nitrogen-chilled isopentane, and stored at 90°C. Four neuronal subpopulations were selectively analyzed: the Purkinje neurons and dentate nuclei neurons of the cerebellum, the inferior olivary nuclei neurons of the medulla, and motor neurons from the anterior horns of the spinal cord. Two types of samples were examined from each neuronal group: pooled neuronal somas and neuropil with glia. The dissected Purkinje cell somas also were analyzed individually. Maternal and patient's leukocytes were each prepared for PCR, using the same procedure as for the CNS tissues. Snap-frozen postmortem CNS tissues from one neurologically normal patient (age 40) were used for PCR analysis.
Neuronal dissection. Cryostat tissue sections (35 µm) were stained with 1% toluidine blue in 1% Na-borate in water for 30 sec and then rinsed in water until the excess stain was removed. Stained slides were viewed with an inverted microscope. Individual neurons were dissected out and suctioned into a glass micropipette guided by a Narashige micromanipulator (Narashige Scientific Instrument Lab, Tokyo, Japan). The pipette system consisted of a siliconized, 1-mm-diameter thin glass micropipette (World Precision Instruments, Sarasota, FL). Fine tips for dissection were prepared, using a model P-77 Brown-Flaming micropipette puller (Sutter Instrument, San Francisco, CA) at a heater temperature setting of 30 for 7 sec. The tip was gently, manually broken to yield an opening of ~50 µm diameter. The blunt end was inserted into Tygon SILASTIC tubing [0.02 inches inner diameter (i.d.)], which was connected to amber latex rubber tubing (1/8 inch i.d.). A disposable syringe (30 ml) tip was inserted into the latex tubing, and gentle suction was provided to the system. Before isolation of the neuronal somas, the field was moistened with one or two drops of TE buffer (10 mM Tris-HCl, pH 7.6, and 1 mM EDTA). Neuropil plus glia samples included the residual tissue immediately surrounding the site of the neuronal soma after its dissection. After isolation of the neuronal somas or neuropil, the samples were transferred into separate microfuge tubes and centrifuged at 12,800 × g for 2 min. The supernatant fluid was removed and replaced with DNA extraction solution (50 mM Tris-HC1, pH 8.5, 1 mM EDTA, and 0.5% Tween 20, containing 200 µg/ml proteinase K). The samples were incubated at 55°C for 2 hr and then immediately heated at 95°C for 10 min to inactivate the proteinase K.
PCR amplification of mtDNA. For PCR analysis, 20-30 neuronal soma isolates from either a specific nucleus or neuronal subpopulation were pooled in 50 µl of digestion buffer. Thus, ~2.5 soma equivalents were included in each reaction sample of 10 µl. Neuropil samples were obtained from sections after the removal of somas. For tissue samples, material from ~100 µm2 areas that included neuronal somas, neuropil, and glia was isolated from a tissue section. For "single-cell" analyses two Purkinje cell soma sections (approximately one soma equivalent) were isolated in 25 µl of digestion buffer.
The PCR conditions were based on the method of Zeviani et al. (1991) -3
G transition at position 8344, a restriction site for the enzyme BglI in the amplified fragments from the mutant, but not the wild-type, mtDNA.
The extracted DNA was combined with 20 pmol of primer 1 and 40 pmol of primer 2, 1.5 mM MgCl2, and 0.4 µl (2 U) of Taq polymerase (Perkin-Elmer, Norwalk, CT). The mixture was brought to a final volume of 50 µl by the addition of distilled H20, and then it was submitted to an initial 30 cycle PCR amplification. The template DNA was added to the reaction mixture after it was heated to 90°C, before the thermal cycling was started. The PCR protocol involved an initial incubation at 95°C for 5 min before cycling. The cycling procedure then followed, with a 30 sec incubation at 94°C for denaturation, a 30 sec annealing step at 55°C, and a 30 sec elongation at 72°C. The addition of 1.5 U of Taq polymerase was followed by 10 more PCR cycles. For some experiments the PCR product was purified after the initial 30 cycles by the Gene-Clean Kit II (BIO-101, Vista, CA). The DNA purification step, however, reduced significantly the final amount of the product. In such cases we then performed an additional 30 cycles as described above, after mixing 300 ng of purified DNA and 25 µl of a PCR reaction mixture and adding distilled H20 to a final volume of 50 µl.
The PCR product plus 300 ng of pBluescript II SK (pBS II) DNA, added to each tube as an internal marker to assess completeness of digestion, were digested with 25 U of BglI (Fisher, Pittsburgh, PA) from 2 hr to overnight at 37°C. Samples of the digestion products were electrophoresed on a 1% agarose gel for detection of the pBS II and on a nondenaturing 12% polyacrylamide gel (29:1 acrylamide/bis-acrylamide) to detect the mutant mtDNA. The molecular weight marker for the agarose gels was a 1 kb DNA ladder (Life Technologies, Gaithersburg, MD) and HinfI-digested X174 (Promega, Madison, WI) for the polyacrylamide gel. The ethidium bromide-stained gels were photographed with Polaroid instant film (positive-negative), type 55.
To quantify the percentages of mutant and wild-type genomes, we used a BioImage laser densitometer. The data were corrected for the presence of heteroduplexes resistant to BglI digestion, formed in the last PCR cycle, by using a standard curve (Yoneda et al., 1994
RESULTS
Typical histological features of MERRF syndrome, characterized by neuronal cytoplasmic eosinophilia, striking shrinkage of many of the remaining neurons, and astrocytic gliosis, were seen in all sites that were examined (Fig. 1). The most severe changes were in the dentate nucleus, and the Purkinje cells were the least involved. Neuronal counts (Fig. 2) indicated a 46% reduction in the number per unit area of dentate nucleus neurons as compared with an age-matched control. Furthermore, there was a 29% loss of inferior olivary nucleus neurons and a comparable reduction (28%) of spinal motor neurons of the lower cervical and upper thoracic segments. By contrast, there was a striking preservation of Purkinje cells, with no significant loss.
Fig. 1. Tissue sections stained with hematoxylin and eosin of cerebellum (A, B), dentate nucleus (C, D), inferior olivary nucleus (E, F), and anterior horns of the thoracic spinal cord (G, H). A, C, E, and G show tissues from the neurologically normal individual; B, D, F, and H show tissues from the MERRF syndrome patient. In the diseased tissues neurons exhibit shrunken somas when compared with the normal control samples. In the control cerebellum there is an artifactual separation of the Purkinje cells from the underlying internal granule layer. Scale bar, 40 µm.
[View Larger Version of this Image (150K GIF file)]
Fig. 2. Bar graph representing the percentage of remaining neurons in different CNS regions. The number of neurons per microscopic field (at 250× magnification) was determined as described in Materials and Methods. The bars represent the ratios of the mean number in the MERRF sections to the mean number in the control sections. Error bars represent the SEM.
[View Larger Version of this Image (66K GIF file)]
For PCR analysis Purkinje cells, neurons of the dentate and inferior olivary nuclei, and motor neurons from the cervical or thoracic spinal cord were microdissected individually. As shown in Figure 3, for Purkinje cells and neurons of the inferior olivary nucleus, individual somas were isolated intact with nearly total exclusion of the surrounding neuropil. The isolated Purkinje cells either were analyzed in pairs or were pooled in samples of 20-30 somas.
Fig. 3. Microdissection procedure. The tissue sections of cerebellum are stained with 1% toluidine blue. A, The neuron to be removed (arrow) is targeted by the tip of a glass micropipette. B, The neuron is drawn into the glass micropipette. C, The same section is shown after suction of the neuronal soma. Scale bar, 70 µm. D, Neurons (arrows) in the inferior olivary nucleus. E, The same section after the neurons have been removed by syringe suction. Scale bar, 50 µm.
[View Larger Version of this Image (93K GIF file)]
The results of BglI digestion of the PCR fragments amplified from the DNA of the four different regions of the CNS are shown in Figure 4. For each site the samples included pooled, isolated neuronal somas and homogenates of nondissected tissue sections (Fig. 4A-C). A sample of neuropil minus the neuronal somas is shown in Figure 4B, and DNA from the equivalent of an individual Purkinje cell is shown in Figure 4C. 
Fig. 4. Results of BglI digestions of the PCR fragments amplified from the DNA of three different regions of the CNS. The top band is the 108 bp wild-type mtDNA fragment. The two lower bands are the 35 and 73 bp fragments derived from mutant mtDNA. A, Regional tissue compared with pooled neuronal isolates. Unmarked lane, Size marker,
[View Larger Version of this Image (28K GIF file)]
The presence of mutant mtDNA with the A
An additional band of ~60 bp was seen occasionally, especially in samples with small quantities of DNA, such as those derived from individual Purkinje cells (Fig. 4D, lane 2) and in a control sample lacking template DNA but containing the primers (Fig. 4C, lane 3). The 3 60 bp band most likely resulted from primer dimerization rather than from degradation.
Quantification of the results revealed that the mean percentage of mutant mtDNA was high in all four anatomical areas, but it was the highest in the Purkinje neurons (96.7% ± 0.7% in the individual cells and 90.5 ± 0.5% in the pooled cells; Fig. 5). The motor neurons of the anterior horn of the spinal cord had a statistically significant or nearly significant lower proportion of mutant mtDNA (84.3 ± 4.2%; p < 0.06, when compared with the pooled Purkinje cells; p < 0.01, when compared with the individual Purkinje cells). The neurons of the dentate nuclei of the cerebellum and those of the inferior olivary nuclei of the medulla contained intermediate proportions of mutant mtDNA (89.0 ± 1.5% and 86.2 ± 3.8%, respectively). 
Fig. 5. Bar graph representing the percentage of mutant mtDNA in different CNS regions. Each bar represents the mean of two to six determinations by PCR and gel analysis of the proportion of mutant DNA in the specified tissue or neuronal isolate. The gel analysis was done on at least two pools of each type of sample (some pools were sampled more than once for PCR and gel analysis), with the exception of blood. Seven determinations were made on one sample of the patient's blood. neuron, Neuronal soma; Purkinje cells, Purkinje cell soma; neuropil, the material immediately surrounding the neuronal soma collected after the soma was removed; tissue, material including somas, neuropil, and glia from a tissue section. The bar labeled individual Purkinje cells represents the mean of six pairs of Purkinje soma halves. The proportion of mutant DNA in each pair is 97.3, 97.6, 96.5, 95.8, 97.0, and 95.6%. The error bars represent 2 SEM.
[View Larger Version of this Image (69K GIF file)]
There were comparable values of the percentage of mutant mtDNA in tissue samples within each CNS region. Thus, samples of cerebellar cortical homogenates showed a mutant DNA proportion similar to that of the Purkinje cell component. Omitting the toluidine blue tissue stain caused no quantitative or qualitative changes in the PCR amplification or BglI cleavage of the PCR product (Fig. 5, lane 2). Internal granule cells exhibited slightly lower proportions of mutant DNA than Purkinje cells (88.8 ± 1.3%; Fig. 5). In the other CNS regions the mutant mtDNA proportion determined for the tissue homogenate or the neuropil was only slightly lower than that found in the isolated neurons.
DNA from individually microdissected Purkinje cells subjected in pairs to PCR amplification of the mtDNA component revealed remarkably similar proportions of mutant DNA from pair to pair, with mean values of 96.7% ± 0.7% (Fig. 5, lane 3). Such a small error suggests that the variability is minimal from cell to cell. Shrunken somas contained the same amount of the mutant form as histologically normal neighboring neurons (data not shown). There is no obvious explanation for the ~5% difference between individual and pooled Purkinje cell somas. Overall, results pointed to a relatively homogeneous distribution of mutant mtDNA in a given region regardless of cell class or whether samples were restricted to neuronal somas or neuropil only.
DISCUSSION
In the present study regional and single neuronal PCR analyses have revealed high percentages of mutant mtDNA in all of the CNS neuronal populations tested. There were, however, differences in the proportion of mutant mtDNA among the neuronal populations analyzed, with the Purkinje cells exhibiting the highest proportion (90-97%), the motor neurons of the spinal cord anterior horns exhibiting a statistically significant lower proportion (84%), and the neurons of dentate nuclei and of the inferior olivary nuclei exhibiting intermediate percentages (89 and 86%, respectively). Tanno et al. (1993)
In the present work the separation of neuronal somas from the surrounding tissue also has allowed the analysis of the distribution of the mutant mtDNA among neuronal somas and neuropil. Thus, the slight differences observed in mutant mtDNA proportion between neuronal somas and the surrounding neuropil would tend to exclude a segregation of the mutant mtDNA exclusively or predominantly in neurons or in glia.
The microdissection of neuronal somas from specific regions of the brain from the MERRF patient also has permitted a comparison of the histopathology with the percentage of mutant mtDNA in these regions. Thus, the high proportion of mutant mtDNA did not correlate directly with the histopathology, including neuronal loss. This discrepancy was especially striking for Purkinje neurons. "Individual" somas of residual Purkinje cells consistently showed the highest percentage of mutant mtDNA among the brain regions analyzed (97%), yet there was remarkably little loss of these neurons (7%). There was no significant individual variability among these neurons in their proportion of mutant DNA. Only slightly lower proportions of mutant mtDNA were found in the neurons of the dentate nucleus, as compared with Purkinje cells. However, in striking contrast to the latter cells, >45% of neurons were lost in this nucleus. This loss may be relevant to the clinical manifestation of myoclonic epilepsy.
There are several possible explanations for the discrepancy between the proportion of mutant mtDNA and the neuronal loss discussed above. As evidenced by the selective vulnerability to hypoxia (Brierly et al., 1973
Recent studies by Enriquez et al. (1995) 50-60% of the normal level, is not sufficient to support normal respiratory chain function. An addition of a minimum of 10% wild-type mtDNA is required for full respiratory competence (Yoneda et al., 1994
The cellular model of the MERRF syndrome mentioned above has been used recently to study the rules that govern complementation of the mutation (Yoneda et al., 1994
The sensitivity of PCR amplification extended to the single-cell level and the reliability of the dissection under direct visualization described in the present work provide a powerful method for analyzing, at the cellular level, the distribution of an mtDNA mutation, which is applicable to other mtDNA-linked diseases affecting the CNS. In contrast to in situ PCR, quantification of mutant mtDNA is possible in single neurons. Resolution of dissection at the light microscopic level followed by PCR is, in fact, higher than that achievable by applying PCR directly to sections (O'Leary et al., 1996
Single muscle fiber PCR analysis has been applied previously to tissue sections from patients carrying either the mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) nt3243 mutation (Moraes et al., 1992
Further analysis by the method described here of CNS tissues from this and other patients carrying the MERRF or other mtDNA mutations, in conjunction with a determination of respiratory chain activity at the single-cell level, is needed to define the relationship of mitochondrial genotype to phenotype in mutant neurons. Sensitivity at the level of the single soma isolate is within reach with the present method and would allow for the identification of any heterogeneity of mtDNA distribution as well as an examination of the possible mechanisms leading to loss of cell function and death. These probably include a variety of nuclear gene-controlled factors that contribute to the partitioning of mutant mtDNA within the mitochondria in a cell, to neuroprotection, or to selective vulnerability of neurons to oxidative stress or apoptosis.
FOOTNOTES
Received Feb. 27, 1997; revised July 15, 1997; accepted July 30, 1997.
These studies were supported by Grants from the National Institute of Aging (P50-AG05142) and the National Institute of Mental Health (5R37-MH39145) to C.A.M. and from the National Institute of General Medical Sciences (GM-11726) to G.A. We are grateful to Ms. Jeanette Espinosa and Carol Church for their excellent secretarial assistance, to Dr. Roger Duncan of the School of Pharmacy, University of Southern California, for assistance with quantitative densitometry, and to Drs. Karen Jain and Terence Sanger for helpful discussions.
Correspondence should be addressed to Dr. Carol A. Miller, Department of Pathology, University of Southern California School of Medicine, 2011 Zonal Avenue, Los Angeles, CA 90033.
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