Myoclonic Epilepsy and Ragged Red Fibers (MERRF) Syndrome
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The Journal of Neuroscience, October 15, 1997, 17(20):7746–7753 Myoclonic 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 Zhou,1 Anne Chomyn,2 Giuseppe Attardi,2 and Carol A. Miller1 1Departments of Pathology and Neurology, University of Southern California School of Medicine, Los Angeles, California 90033, and 2Division of Biology, California Institute of Technology, Pasadena, California 91125 Selective vulnerability of subpopulations of neurons is a striking that were analyzed exhibited high proportions of mutant feature of neurodegeneration. Mitochondrially transmitted dis- mtDNA, ranging from 97.6 6 0.7% in Purkinje cells to 80.6 6 eases are no exception. In this study CNS tissues from a patient 2.8% in the anterior horn cells. Within each neuronal group that with myoclonus epilepsy and ragged red fibers (MERRF) syn- was analyzed, neuronal soma values were similar to those in the drome, which results from an A to G transition of nucleotide (nt) surrounding neuropil and glia or in the regional tissue homog- 8344 in the mitochondrial tRNA Lys gene, were examined for the enate. Surprisingly, as compared with controls, neuronal loss proportion of mutant mtDNA. Either individual neuronal somas ranged from 7% of the Purkinje cells to 46% of the neurons of or the adjacent neuropil and glia were microdissected from the dentate nucleus in MERRF cerebellum. Thus, factors other cryostat tissue sections of histologically severely affected brain than the high proportion of mutant mtDNA, in particular nuclear- regions, including dentate nuclei, Purkinje cells, and inferior controlled neuronal differences among various regions of the olivary nuclei, and from a presumably less affected neuronal CNS, seem to contribute to the mitochondrial dysfunction and subpopulation, the anterior horn cells of the spinal cord. Mutant ultimate cell death. and normal mtDNA were quantified after PCR amplification with a mismatched primer and restriction enzyme digestion. Neu- Key words: MERRF syndrome; CNS microdissection; PCR; rons and the surrounding neuropil and glia from all CNS regions neurodegeneration; tRNA Lys; mtDNA Selective vulnerability of neuronal or glial subpopulations is a mtDNA, either in their somas or in their processes within the feature of many neurological diseases. The myoclonus epilepsy surrounding neuropil, than neurons in the relatively spared re- and ragged red fibers (MERRF) syndrome is characterized by gions, or, alternatively, whether differences in nuclear gene activ- myoclonic epilepsy, cerebellar ataxia, and progressive muscular ity play a crucial role in determining the degree of damage to weakness. In this maternally inherited disease (Wallace et al., various neuronal populations. In particular, important variables 1988) the symptoms of myoclonic epilepsy, ataxia, and muscular differentially affecting cell survival may include demand for oxi- weakness may reflect severe pathological changes in neurons of dative phosphorylation of different cell types and the terminally the dentato-rubral and the spinocerebellar pathways and of the differentiated state of the neuron. inferior olivary nuclei and in skeletal muscle. The myopathic Previous quantifications of the mutant mtDNA proportion in changes include the generation of ragged red fibers indicative of the CNS have been made in tissue homogenates of anatomical proliferation and subsarcolemmal accumulation of mitochondria. regions (Tanno et al., 1993; Sanger and Jain, 1996). However, The MERRF syndrome is most commonly the result of a single these samples contained not only neurons but the surrounding base pair substitution at position 8344 in the mitochondrial glia, including oligodendrocytes, astrocytes, and microglia as well Lys tRNA gene (Shoffner et al., 1990, 1991; Yoneda et al., 1990; as blood vessels. Furthermore, these analyses could not distin- Chomyn et al., 1991; Hammans et al., 1993; Silvestri et al., 1993). guish neuronal somas from dendrites, axons, and synaptic termi- The relationship between mtDNA genotype and phenotype of nals that form the surrounding neuropil. selective neuronal vulnerability has not been defined. It is not In the present study we have compared neuronal loss with the known whether the affected neurons contain more mutant percentage of mutant mtDNA in the more severely and the less severely affected neuronal groups. Individual neuronal somas Received Feb. 27, 1997; revised July 15, 1997; accepted July 30, 1997. were microdissected from CNS tissue sections. Mutant and nor- These studies were supported by Grants from the National Institute of Aging mal mtDNA were quantified after PCR amplification with a (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 mismatched primer and restriction enzyme digestion (Zeviani et G.A. We are grateful to Ms. Jeanette Espinosa and Carol Church for their excellent al., 1991; Yoneda et al., 1991). Four neuronal subpopulations secretarial assistance, to Dr. Roger Duncan of the School of Pharmacy, University of Southern California, for assistance with quantitative densitometry, and to Drs. were compared: Purkinje cells and neurons of the dentate nuclei Karen Jain and Terence Sanger for helpful discussions. of the cerebellum and neurons of the inferior olivary nuclei of the Correspondence should be addressed to Dr. Carol A. Miller, Department of medulla, all heavily vulnerable, and motor neurons of the spinal Pathology, University of Southern California School of Medicine, 2011 Zonal Avenue, Los Angeles, CA 90033. cord anterior horns, a presumably less-affected neuronal sub- Copyright © 1997 Society for Neuroscience 0270-6474/97/177746-08$05.00/0 population. Three types of samples from each neuronal group Zhou et al. • Mitochondrial tRNALys Mutation in Neuronal Isolates J. Neurosci., October 15, 1997, 17(20):7746–7753 7747 were analyzed: neuronal somas only, neuropil and glia only, and pmol of primer 2, 1.5 mM MgCl2 , and 0.4 ml(2U)ofTaq polymerase homogenates of regions, including entire neuronal groups (neu- (Perkin-Elmer, Norwalk, CT). The mixture was brought to a final volume of 50 ml by the addition of distilled H20, and then it was submitted ronal somas plus neuropil and glia). 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 MATERIALS AND METHODS cycling was started. The PCR protocol involved an initial incubation at 95°C for 5 min before cycling. The cycling procedure then followed, with CNS tissue. CNS tissues were obtained 20 hr postmortem from a 14-year- a 30 sec incubation at 94°C for denaturation, a 30 sec annealing step at old Hispanic female with clinical and neuropathologically confirmed 55°C, and a 30 sec elongation at 72°C. The addition of 1.5 U of Taq MERRF syndrome (Sanger and Jain, 1996). Paraffin-embedded CNS polymerase was followed by 10 more PCR cycles. For some experiments tissue sections (7 mm) were stained with hematoxylin and eosin. Control the PCR product was purified after the initial 30 cycles by the Gene- tissues for histology were obtained postmortem from an age-matched Clean Kit II (BIO-101, Vista, CA). The DNA purification step, however, patient who died from non-neurological causes. reduced significantly the final amount of the product. In such cases we Neuronal loss was compared in MERRF and control tissues in four then performed an additional 30 cycles as described above, after mixing sections from each paraffin block. Neurons were counted at 2503 mag- 300 ng of purified DNA and 25 ml of a PCR reaction mixture and adding nification in five fields of either dentate nuclei or inferior olivary nuclei, distilled H20 to a final volume of 50 ml. or in the entire bilateral anterior horn region of the lower cervical and The PCR product plus 300 ng of pBluescript II SK (pBS II) DNA, upper thoracic spinal cord, or Purkinje cells in five foliar loops. Counts added to each tube as an internal marker to assess completeness of were confirmed in all sites by two independent observers. The mean digestion, were digested with 25 U of BglI (Fisher, Pittsburgh, PA) from values were calculated for each. 3 2 hr to overnight at 37°C. Samples of the digestion products were For PCR analysis, blocks of tissue (1 cm ) were dissected at autopsy, electrophoresed on a 1% agarose gel for detection of the pBS II and on immediately snap-frozen in liquid nitrogen-chilled isopentane, and a nondenaturing 12% polyacrylamide gel (29:1 acrylamide/bis- stored at 290°C. Four neuronal subpopulations were selectively ana- acrylamide) to detect the mutant mtDNA. The molecular weight marker lyzed: the Purkinje neurons and dentate nuclei neurons of the cerebel- for the agarose gels wasa1kbDNAladder (Life Technologies, Gaith- lum, the inferior olivary nuclei neurons of the medulla, and motor ersburg, MD) and HinfI-digested fX174 (Promega, Madison, WI) for neurons from the anterior horns of the spinal cord. Two types of samples the polyacrylamide gel. The ethidium bromide-stained gels were photo- were examined from each neuronal group: pooled neuronal somas and graphed with Polaroid instant film (positive–negative), type 55. neuropil with glia. The dissected Purkinje cell somas also were analyzed To quantify the percentages of mutant and wild-type genomes, we individually. Maternal and patient’s leukocytes were each prepared for used a BioImage laser densitometer. The data were corrected for the PCR, using the same procedure as for the CNS tissues. Snap-frozen presence of heteroduplexes resistant to BglI digestion, formed in the last postmortem CNS tissues from one neurologically normal patient (age 40) PCR cycle, by using a standard curve (Yoneda et al., 1994). were used for PCR analysis. Neuronal dissection. Cryostat tissue sections (35 mm) were stained with 1% toluidine blue in 1% Na-borate in water for 30 sec and then rinsed in RESULTS water until the excess stain was removed.