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Single cell analysis of CAG repeat in brains of dentatorubral-pallidoluysian atrophy (DRPLA)
Journal of the Neurological Sciences 190 Ž2001. 87–93 www.elsevier.comrlocaterjns
Single cell analysis of CAG repeat in brains of dentatorubral-pallidoluysian atrophy žDRPLA/ Hideji Hashida a,b,) , Jun Goto a,b, Takashi Suzuki a,c , Seon-Yong Jeong a,b, Naoki Masuda a,b, Tomonori Ooie d , Yoshiaki Tachiiri d , Hiroshi Tsuchiya d , Ichiro Kanazawa a,b b
a CREST, Japan Science and Technology Corporation, Japan Department of Neurology, Graduate School of Medicine, UniÕersity of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-8655, Japan c Department of Medical Zoology, Nagoya City Medical School, Nagoya 467-8601, Japan d Hamamatsu Photonics K.K., Hamamatsu 434-8601, Japan
Received 6 February 2001; received in revised form 23 July 2001; accepted 26 July 2001
Abstract Somatic mosaicism of an expanded repeat is present in tissues of patients with triplet repeat diseases. Of the spinocerebellar ataxias associated with triplet repeat expansion, the most prominent heterogeneity of the expanded repeat is seen in dentatorubral-pallidoluysian atrophy ŽDRPLA.. The common feature of this somatic mosaicism is the difference in the repeat numbers found in the cerebellum as compared to other tissues. The expanded allele in the cerebellum shows a smaller degree of expansion. We previously showed by microdissection analysis that the expanded allele in the granular layer in DRPLA cerebellum has less expansion than expanded alleles in the molecular layer and white matter. Whether this feature of lesser expansion in granule cells is common to other types of neurons is yet to be clarified. We used a newly developed excimer laser microdissection system to analyze somatic mosaicism in the brains of two patients, one with early- and another with late-onset DRPLA, and used single cell PCR to observe the cell-to-cell differences in repeat numbers. In the late onset patient, repeat expansion was more prominent in Purkinje cells than in granule cells, but less than that in the glial cells. In the early onset patient, repeat expansion in Purkinje cells was greater than in granule cells but did not differ from that in glial cells. These findings suggest that there is a difference in repeat expansion among neuronal subgroups and that the number of cell division cycles is not the only determinant of somatic mosaicism. q 2001 Published by Elsevier Science B.V. Keywords: DRPLA; Dentatorubral-pallidoluysian atrophy; Single neuron; Microdissection; Laser; Somatic mosaicism; CAG repeat; Triplet repeat
1. Introduction Dentatorubral-pallidoluysian atrophy ŽDRPLA. is an autosomal dominant neurodegenerative disorder characterized by a combination of the clinical features of epilepsy, myoclonus, choreoathetoid movement, cerebellar ataxia, and dementia w1,2x. DRPLA is caused by the expansion of the CAG trinucleotide repeat in the DRPLA gene located on chromosome 12p w3,4x. Tissue-to-tissue variation in triplet repeat expansion was initially reported in blood cells and muscles of patients with myotonic dystrophy. Somatic mosaicism of the triplet repeat expansion in the central nervous system also has )
Corresponding author. Department of Neurology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-8655, Japan. Tel.: q81-3-5800-8672; fax: q81-3-5800-6548. E-mail address: [email protected] ŽH. Hashida..
been reported in cases of childhood-onset Huntington disease, DRPLA, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, Machado-Joseph disease, and spinal and bulbar muscular atrophy w5–15x. The common feature of the somatic mosaicism in these diseases is that the expansion is least in the cerebellum. Using microdissection analysis of patients with DRPLA, we previously showed that the expanded allele of the cerebellar granular layer is less expanded than in the molecular layer and white matter w11x. Whether this lesser degree of expansion of the triplet repeat is specific to cerebellar granule cells or is a general feature in all neuron types is yet to be clarified. The brain consists of a heterogeneous variety of cells. To analyze gene expression in neurons of patients with neurodegenerative disorders, the specific types of neurons must be isolated from postmortem brain tissues. We recently developed an excimer laser microdissection system that provides high resolution and minimal thermal damage.
0022-510Xr01r$ - see front matter q 2001 Published by Elsevier Science B.V. PII: S 0 0 2 2 - 5 1 0 X Ž 0 1 . 0 0 5 9 6 - 2
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We here report its use to analyze CAG repeats in single neurons from DRPLA brains.
2. Materials and methods 2.1. Brain samples Somatic mosaicism was investigated in two autopsied DRPLA brains, stored at y80 8C, which had been used in a previous study w11x. In one case, there was early disease onset and the myoclonus epilepsy phenotype Žcase 2 in our previous report., and in the other late disease onset Žcase 8 in our previous report. and the cerebellar ataxia phenotype. The PCR product bands in the latter brain tissue had a wider distribution range in the tissues, except for the cerebellum, than in the former.
2.4. RNA extraction and reÕerse transcription The single neurons isolated were treated with 50 ml of TRIZOLe reagent ŽGibco BRL. as the manufacturer instructed. After extraction and precipitation with 1 mg of yeast tRNA, the pellet was dissolved in 9 ml of the DNase reaction mixture, incubated at room temperature for 15 min then heat inactivated at 65 8C for 10 min with 2.5 mM EDTA. The DNase reaction mixture contained 10r9 = DNase buffer, 1 unit of DNase I ŽGibco BRL., and 1 unit of PRIME RNase Inhibitor Ž5 Prime™ 3 Prime.. DNasetreated samples were reverse transcribed with Superscript II reverse transcriptase ŽGibco BRL., according to the manufacturer’s instruction with a gene specific RT primer Ž5X-AGGGAGACAT GGCGTAA-3X ., followed by heat inactivation of the reverse transcriptase and treatment with RNase H.
2.2. Single cell isolation by the excimer laser microdissection system The microdissection system used combines a laser dissector and semiautomatic computer-assisted stage controller. This system uses a Acold laserB; an argon fluoride excimer laser with a wavelength of 193 nm, shorter than that of the nitrogen laser with a 337-nm wavelength that is used for laser pressure catapulting or of the infrared carbon dioxide laser used for laser capture microdissection w16– 21x. Because a laser with a short wavelength has a small ablation depth and high resolution, our system has the advantages of reduced damage by heat and scattered laser beams. Moreover, the diameter of the laser focus can be reduced to 2 mm w16,22x. Single cells were isolated as follows: sections of 20-mm thick were sliced from frozen cerebellar samples. These were mounted on 1-mm-thick slide glasses made of artificial quartz, which permits the excimer laser beam to pass. After being air-dried, the sections were stained with toluidine blue then dried completely in a freeze dryer. Single cells were dissected by means of the new laser microdissection system ŽHamamatsu Photonics.. The specimen was set on a stage, observed under a microscope and monitored with a CCD camera. Software for semiautomatic dissection was used to dissect the cells at their margins. After inverting the holder, single cells were released from the slide glass by a weak laser beam emitted from the reverse side and collected into a microtube cap for further processing. 2.3. Genomic DNA preparation The isolated single cell was lysed at 65 8C for 15 min in 5 ml of alkali lysis buffer containing 0.25 M KOH and 0.05 M DTT, then neutralized with 10 ml of neutralizing buffer containing 75 mM KCl, 0.125 M HCl and 225 mM Tris–HCl, pH 8.5.
Fig. 1. Single Purkinje cell isolation from a cerebellar specimen of a DRPLA brain. ŽA. Cerebellar tissue stained with toluidine blue. ŽB. The margin of the target Purkinje cell Žasterisk. has been dissected by the laser beam. ŽC. The target Purkinje cell has been released from the slide glass by a weak laser beam given from the backside. Scale bar indicates 20 mm.
H. Hashida et al.r Journal of the Neurological Sciences 190 (2001) 87–93
2.5. PCR This reaction used the total volume of sample obtained after alkali lysis or reverse transcription. The first round reaction mixture of PCR contained 1 = KlenTaq Advantage cDNA polymerase mix ŽClontech., 1.5 M betaine, 1.5 mM MgCl 2 , 200 mM of each dNTP, 20 mM of each primer, 0.7 = LA PCR buffer ŽTaKaRa., and 0.3 = reaction buffer used in reverse transcription or alkali-lysis and neutralization. The primers for the first round PCR were DRPLA ex F1583 Ž5X-GGCCGCCTCT TAGCCAACAG CAAT-3X . and DRPLA R1813 Ž5X-GTAAGGGTGT GCGTGGTGGG AGC-3X .. The conditions for the first amplification round were 40 cycles at 94 8C for 1 min, 60 8C for 1 min, and 72 8C for 2 min. The second round was done with 12.5 ml of a reaction mixture containing 1.4 ml of the first round PCR product, 10% dimethylsulfoxide, 1.0 mM MgCl 2 , 1 = LA PCR buffer, 200 mM of each dNTP, 2 = KlenTaq Advantage cDNA polymerase mix, and 20 mM of each primer. Primers in the second round PCR were DRPLA R1813 and fluorescence-labeled for-
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ward primer Ž5X FITC-CACCCACCAG TCTCAACACA TCACCATC-3X .. Conditions for the second round amplifications were 40 cycles at 94 8C for 1 min, 65 8C for 1 min, and 72 8C for 2 min. 2.6. Electrophoresis and scanning After being mixed with an equal volume of formamideloading dye, the PCR products were heat denatured, loaded on a 5% Long Ranger ŽFMC. sequencing gel and electrophoresed. Gels were scanned in a Fluoroimager SI ŽAmersham Pharmacia..
3. Results 3.1. Isolation of single neurons and glial cells Single cells were dissected individually by the laser microdissection system. Fig. 1 shows the microdissection process of a Purkinje cell.
Fig. 2. Gel image of the electrophoresis of single cell PCR of a late onset patient. The expanded allele shows a cell-to-cell difference in repeat length. ŽA. Lane 1, cerebellar cortex; lane 2, cerebral white matter; lanes 3–23, single cerebellar granule cell. ŽB. Lane 1, cerebellar cortex; lane 2, cerebral white matter; lanes 3–23, single Purkinje cell. ŽC. Lane 1, cerebellar cortex; lane 2, cerebral white matter; lanes 3–23, single glial cell in the cerebellar white matter.
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3.2. Genomic PCR Expanded alleles of 25 granule, 33 Purkinje, and 44 glial cells in the white matter were analyzed in the early onset brain. For the late onset brain, 60 granule, 67 Purkinje, and 57 glial cells in the cerebellar white matter were analyzed. Genomic PCR of individual neurons is shown in Fig. 2. The multiple bands of PCR products of single cells were artifacts of PCR but this number of the intense bands was usually defined three. We regarded the repeat number of the uppermost of the three bands as the representative one. Repeat number distribution for the expanded allele is shown in Fig. 3. The means and standard deviations for the number of expanded CAG repeat in the early onset brain were 63.3 " 1.9 Žmean " S.D.. in the granule, 67.3 " 3.3 in the Purkinje, and 67.2 " 4.1 in the
glial cells. For the late onset brain, they were 57.0 " 1.0 in the granule, 61.7 " 4.8 in the Purkinje, and 64.6 " 4.5 in the glial cells. The CAG repeat numbers in Purkinje cells tend to be larger than those in granule cells Ž p - 0.01 in both cases by the unpaired t-test.. A difference in the repeat number distribution of Purkinje and glial cells was found for the late onset but not the early onset brain Ž p - 0.01 late onset and p s 0.86 early onset by the unpaired t-test.. 3.3. RT-PCR The RT-PCR of single isolated Purkinje neurons is shown in Fig. 4. The weak bands between normal and expanded bands are postulated to be artifacts during PCR. Cell-to-cell differences in the expanded CAG repeats are
Fig. 3. Distributions of the repeat number of single isolated cells in DRPLA cerebella. Different distribution patterns are clear for repeat numbers of the expanded allele in cerebellar granule ŽA and D., Purkinje ŽB and E. and glial cells in the cerebellar white matter ŽC and F. in the early onset ŽA–C. and late onset ŽD–F. tissues. Purkinje cells have a larger repeat number and wider distribution than granule cells in each case Ž p- 0.01.. There is no difference in repeat number between Purkinje and glial cells Ž ps 0.86. in the early onset tissues. In late onset tissues, the repeat number of the Purkinje cells is smaller than that of the glial cells Ž p- 0.01..
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Fig. 4. RT-PCR products of single isolated Purkinje cells of a late onset patient. The hemi-nested PCR products show somatic mosaicism, as seen in the genomic single cell PCR. Lane 1, cerebellar cortex; lane 2, cerebral white matter; lanes 3–12, single Purkinje cell; lanes 13 and 14, no template; lanes 3–10, RT Žq.; lanes 11 and 12, RT Žy.. The weak bands between normal and expanded bands are postulated to be artifact during PCR.
clear in the gel of the RT-PCR products of single Purkinje cells.
4. Discussion Somatic mosaicism of an expanded allele has been noted in triplet repeat diseases. The feature common to this somatic mosaicism is that the lowest degree of expansion is found in the cerebellum. Analysis of microscopically dissected cerebellar tissues showed that the mosaicism in cerebellum is identical to that in the cerebellar granular layer w11x. The smallest expansion pattern has been associated with the anatomical features of the cerebellum and the cerebellar granular layer, in which most components are postmitotic neurons. Whether stability in repeat expansion is common to all neuronal types or only to specific types is yet to be clarified. To evaluate differences in the heterogeneity of repeat expansion in various neuronal types, neurons must be isolated from brain tissues made up of heterogeneous types of cells. There is only one report on somatic mosaicism analysis of neurons of triplet repeats brain, but this was of pooled neurons w23x. However, it is difficult to evaluate the heterogeneity of the repeat number precisely unless a separate PCR is done on each single cell, as the CAG repeat sequence number is unstable during PCR. Single-cell PCR provides direct evidence of somatic mosaicism. Gametic mosaicism of the triplet re-
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peat gene has been observed using single-sperm analysis w24–31x, but there has been no report on somatic mosaicism in single isolated neurons. Our study is the first to analyze somatic mosaicism in single isolated neurons and glial cells. In our analysis of single isolated cells, there was direct evidence of a difference in the repeat heterogeneity among the various somatic cell types and between two types of neuron Purkinje and cerebellar granule cells. Our results are consistent with the report of pooled cell analysis w23x. There also was a difference in the distributions of the repeat numbers of glial cells in early onset and late onset brain tissues. The heterogeneity of the expanded allele in glial cells was increased in a brain of the late onset patient. Positive correlation between somatic mosaicism and patient age is also noted in other studies on the somatic mosaicism of DRPLA patients w10,11x. This difference of mosaicism may be explained by the difference of the number of division cycles after fertilization in glial cells of the two patients. In the study reported here, the repeat number in Purkinje cells shows wider distribution than that for granule cells, indicating that there is a difference in somatic mosaicism between neuron types. The underlying mechanism of this difference in the mosaicism between Purkinje and granule cells is still to be clarified. It is unlikely that the smaller number of repeats in granule cells represent a reduction in repeat number from baseline. Repeat contraction also results in the increase of the heterogeneity as observed in repeat expansion w32x. The distribution of repeats of the expanded allele in cerebellar granule cells is more restricted compared to that of the other cell types. To understand this difference, the following points should be considered: Ž1. the number of cell division cycles, Ž2. negative selection of neurons with larger CAG repeat expansion, Ž3. factors that affect the stability of the triplet repeat, and Ž4. the relation of the gene transcription level to repeat instability. The first point is the difference in the number of mitotic divisions in the two types of neurons. The number of mitotic divisions is estimated to be 19 in rat Purkinje cells w33x. Although the number of cell division cycles in granule cells is not known, the number of cerebellar granule cells is about 3000 times that of Purkinje cells in humans w34x. Thus, it is unlikely that the mitotic division number in granule cells is lower than that in Purkinje cells. The difference in mosaicism is not explained by the difference in the number of cell division cycles alone. Similar contradictory findings have been reported in patients with myotonic dystrophy or with spinal and bulbar muscular atrophy. The triplet repeat number for lymphocytes, one of the most proliferative cell types, is reported to be less expanded than for muscle tissue in patients with myotonic dystrophy w35–37x. A greater repeat number distribution in muscle than other tissues also has been reported in spinal and bulbar muscular atrophy w38x. These findings provide
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evidence that the cell cycle number is not the sole determinant of the difference in somatic mosaicism. Concerning the second point, the negative selection of granule neurons with longer CAG repeats, programmed cell death is an important mechanism where the neuronal population is controlled by eliminating unnecessary neurons from neuronal tissues. During normal development, granule cells die by means of apoptosis if no synapse is formed with target Purkinje cells w39,40x. The Purkinje cell is a key element in the organization of the cerebellar cortex during development and its growth and differentiation do not feature the process of programmed cell death. The difference in somatic mosaicism therefore may be explained by the apoptotic mechanism that functions in granule cells, when selective survival occurs in cells with less expanded CAG repeats. Thirdly, trans-acting factors that affect triplet repeat instability may have a function in the difference in somatic mosaicism that exists in neuronal cell groups. The mismatch repair ŽMMR. system, which affects the instability of the dinucleotide repeat sequence, is thought to affect the instability of the triplet repeat sequence w32,41,42x. DNA mismatch repair genes are expressed during and after neurogenesis, and the histological distribution of MSH2 in adult rat brain has been shown w43–46x. Purkinje and cerebellar granule cells have MSH2 immunoreactivity in adult rats w46x. The developmental expression of the MMR in the nervous system has yet to be clarified. A fourth possibility is suggested by the relationship between repeat instability and the gene expression level. In spinal and bulbar muscular atrophy, the tissue-specific pattern of somatic mosaicism is correlated with the expression pattern of the androgen receptor w38x. The untranscribed CAGrCTG repeat in the ERDA1 Žexpanded repeat domain, CAGrCTG 1. region is more stable than that in other transcribed triplet repeat genes w47x. Whether transcription activity affects the instability of the repetitive sequence of the gene must be investigated. To understand pathophysiology of triplet repeat diseases, it is necessary to analyze the relationship between somatic mosaicism and the vulnerability of specific types of neurons, and to identify the repeat numbers of the affected neurons. This is difficult because these neurons have mostly disappeared by the time of death and the residual cells are not representative of the vulnerable neurons. Further investigations using tissue of non-advanced diseased brains or transgenic animal models will help in solving this problem and answer the questions about somatic mosaicism and the histopathological distribution of vulnerable neurons.
Acknowledgements This work was supported by grants-in-aid from CREST, Japan Science and Technology.
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Single cell analysis of CAG repeat in brains of dentatorubral-pallidoluysian atrophy žDRPLA/ Hideji Hashida a,b,) , Jun Goto a,b, Takashi Suzuki a,c , Seon-Yong Jeong a,b, Naoki Masuda a,b, Tomonori Ooie d , Yoshiaki Tachiiri d , Hiroshi Tsuchiya d , Ichiro Kanazawa a,b b
a CREST, Japan Science and Technology Corporation, Japan Department of Neurology, Graduate School of Medicine, UniÕersity of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-8655, Japan c Department of Medical Zoology, Nagoya City Medical School, Nagoya 467-8601, Japan d Hamamatsu Photonics K.K., Hamamatsu 434-8601, Japan
Received 6 February 2001; received in revised form 23 July 2001; accepted 26 July 2001
Abstract Somatic mosaicism of an expanded repeat is present in tissues of patients with triplet repeat diseases. Of the spinocerebellar ataxias associated with triplet repeat expansion, the most prominent heterogeneity of the expanded repeat is seen in dentatorubral-pallidoluysian atrophy ŽDRPLA.. The common feature of this somatic mosaicism is the difference in the repeat numbers found in the cerebellum as compared to other tissues. The expanded allele in the cerebellum shows a smaller degree of expansion. We previously showed by microdissection analysis that the expanded allele in the granular layer in DRPLA cerebellum has less expansion than expanded alleles in the molecular layer and white matter. Whether this feature of lesser expansion in granule cells is common to other types of neurons is yet to be clarified. We used a newly developed excimer laser microdissection system to analyze somatic mosaicism in the brains of two patients, one with early- and another with late-onset DRPLA, and used single cell PCR to observe the cell-to-cell differences in repeat numbers. In the late onset patient, repeat expansion was more prominent in Purkinje cells than in granule cells, but less than that in the glial cells. In the early onset patient, repeat expansion in Purkinje cells was greater than in granule cells but did not differ from that in glial cells. These findings suggest that there is a difference in repeat expansion among neuronal subgroups and that the number of cell division cycles is not the only determinant of somatic mosaicism. q 2001 Published by Elsevier Science B.V. Keywords: DRPLA; Dentatorubral-pallidoluysian atrophy; Single neuron; Microdissection; Laser; Somatic mosaicism; CAG repeat; Triplet repeat
1. Introduction Dentatorubral-pallidoluysian atrophy ŽDRPLA. is an autosomal dominant neurodegenerative disorder characterized by a combination of the clinical features of epilepsy, myoclonus, choreoathetoid movement, cerebellar ataxia, and dementia w1,2x. DRPLA is caused by the expansion of the CAG trinucleotide repeat in the DRPLA gene located on chromosome 12p w3,4x. Tissue-to-tissue variation in triplet repeat expansion was initially reported in blood cells and muscles of patients with myotonic dystrophy. Somatic mosaicism of the triplet repeat expansion in the central nervous system also has )
Corresponding author. Department of Neurology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-8655, Japan. Tel.: q81-3-5800-8672; fax: q81-3-5800-6548. E-mail address: [email protected] ŽH. Hashida..
been reported in cases of childhood-onset Huntington disease, DRPLA, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, Machado-Joseph disease, and spinal and bulbar muscular atrophy w5–15x. The common feature of the somatic mosaicism in these diseases is that the expansion is least in the cerebellum. Using microdissection analysis of patients with DRPLA, we previously showed that the expanded allele of the cerebellar granular layer is less expanded than in the molecular layer and white matter w11x. Whether this lesser degree of expansion of the triplet repeat is specific to cerebellar granule cells or is a general feature in all neuron types is yet to be clarified. The brain consists of a heterogeneous variety of cells. To analyze gene expression in neurons of patients with neurodegenerative disorders, the specific types of neurons must be isolated from postmortem brain tissues. We recently developed an excimer laser microdissection system that provides high resolution and minimal thermal damage.
0022-510Xr01r$ - see front matter q 2001 Published by Elsevier Science B.V. PII: S 0 0 2 2 - 5 1 0 X Ž 0 1 . 0 0 5 9 6 - 2
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We here report its use to analyze CAG repeats in single neurons from DRPLA brains.
2. Materials and methods 2.1. Brain samples Somatic mosaicism was investigated in two autopsied DRPLA brains, stored at y80 8C, which had been used in a previous study w11x. In one case, there was early disease onset and the myoclonus epilepsy phenotype Žcase 2 in our previous report., and in the other late disease onset Žcase 8 in our previous report. and the cerebellar ataxia phenotype. The PCR product bands in the latter brain tissue had a wider distribution range in the tissues, except for the cerebellum, than in the former.
2.4. RNA extraction and reÕerse transcription The single neurons isolated were treated with 50 ml of TRIZOLe reagent ŽGibco BRL. as the manufacturer instructed. After extraction and precipitation with 1 mg of yeast tRNA, the pellet was dissolved in 9 ml of the DNase reaction mixture, incubated at room temperature for 15 min then heat inactivated at 65 8C for 10 min with 2.5 mM EDTA. The DNase reaction mixture contained 10r9 = DNase buffer, 1 unit of DNase I ŽGibco BRL., and 1 unit of PRIME RNase Inhibitor Ž5 Prime™ 3 Prime.. DNasetreated samples were reverse transcribed with Superscript II reverse transcriptase ŽGibco BRL., according to the manufacturer’s instruction with a gene specific RT primer Ž5X-AGGGAGACAT GGCGTAA-3X ., followed by heat inactivation of the reverse transcriptase and treatment with RNase H.
2.2. Single cell isolation by the excimer laser microdissection system The microdissection system used combines a laser dissector and semiautomatic computer-assisted stage controller. This system uses a Acold laserB; an argon fluoride excimer laser with a wavelength of 193 nm, shorter than that of the nitrogen laser with a 337-nm wavelength that is used for laser pressure catapulting or of the infrared carbon dioxide laser used for laser capture microdissection w16– 21x. Because a laser with a short wavelength has a small ablation depth and high resolution, our system has the advantages of reduced damage by heat and scattered laser beams. Moreover, the diameter of the laser focus can be reduced to 2 mm w16,22x. Single cells were isolated as follows: sections of 20-mm thick were sliced from frozen cerebellar samples. These were mounted on 1-mm-thick slide glasses made of artificial quartz, which permits the excimer laser beam to pass. After being air-dried, the sections were stained with toluidine blue then dried completely in a freeze dryer. Single cells were dissected by means of the new laser microdissection system ŽHamamatsu Photonics.. The specimen was set on a stage, observed under a microscope and monitored with a CCD camera. Software for semiautomatic dissection was used to dissect the cells at their margins. After inverting the holder, single cells were released from the slide glass by a weak laser beam emitted from the reverse side and collected into a microtube cap for further processing. 2.3. Genomic DNA preparation The isolated single cell was lysed at 65 8C for 15 min in 5 ml of alkali lysis buffer containing 0.25 M KOH and 0.05 M DTT, then neutralized with 10 ml of neutralizing buffer containing 75 mM KCl, 0.125 M HCl and 225 mM Tris–HCl, pH 8.5.
Fig. 1. Single Purkinje cell isolation from a cerebellar specimen of a DRPLA brain. ŽA. Cerebellar tissue stained with toluidine blue. ŽB. The margin of the target Purkinje cell Žasterisk. has been dissected by the laser beam. ŽC. The target Purkinje cell has been released from the slide glass by a weak laser beam given from the backside. Scale bar indicates 20 mm.
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2.5. PCR This reaction used the total volume of sample obtained after alkali lysis or reverse transcription. The first round reaction mixture of PCR contained 1 = KlenTaq Advantage cDNA polymerase mix ŽClontech., 1.5 M betaine, 1.5 mM MgCl 2 , 200 mM of each dNTP, 20 mM of each primer, 0.7 = LA PCR buffer ŽTaKaRa., and 0.3 = reaction buffer used in reverse transcription or alkali-lysis and neutralization. The primers for the first round PCR were DRPLA ex F1583 Ž5X-GGCCGCCTCT TAGCCAACAG CAAT-3X . and DRPLA R1813 Ž5X-GTAAGGGTGT GCGTGGTGGG AGC-3X .. The conditions for the first amplification round were 40 cycles at 94 8C for 1 min, 60 8C for 1 min, and 72 8C for 2 min. The second round was done with 12.5 ml of a reaction mixture containing 1.4 ml of the first round PCR product, 10% dimethylsulfoxide, 1.0 mM MgCl 2 , 1 = LA PCR buffer, 200 mM of each dNTP, 2 = KlenTaq Advantage cDNA polymerase mix, and 20 mM of each primer. Primers in the second round PCR were DRPLA R1813 and fluorescence-labeled for-
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ward primer Ž5X FITC-CACCCACCAG TCTCAACACA TCACCATC-3X .. Conditions for the second round amplifications were 40 cycles at 94 8C for 1 min, 65 8C for 1 min, and 72 8C for 2 min. 2.6. Electrophoresis and scanning After being mixed with an equal volume of formamideloading dye, the PCR products were heat denatured, loaded on a 5% Long Ranger ŽFMC. sequencing gel and electrophoresed. Gels were scanned in a Fluoroimager SI ŽAmersham Pharmacia..
3. Results 3.1. Isolation of single neurons and glial cells Single cells were dissected individually by the laser microdissection system. Fig. 1 shows the microdissection process of a Purkinje cell.
Fig. 2. Gel image of the electrophoresis of single cell PCR of a late onset patient. The expanded allele shows a cell-to-cell difference in repeat length. ŽA. Lane 1, cerebellar cortex; lane 2, cerebral white matter; lanes 3–23, single cerebellar granule cell. ŽB. Lane 1, cerebellar cortex; lane 2, cerebral white matter; lanes 3–23, single Purkinje cell. ŽC. Lane 1, cerebellar cortex; lane 2, cerebral white matter; lanes 3–23, single glial cell in the cerebellar white matter.
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3.2. Genomic PCR Expanded alleles of 25 granule, 33 Purkinje, and 44 glial cells in the white matter were analyzed in the early onset brain. For the late onset brain, 60 granule, 67 Purkinje, and 57 glial cells in the cerebellar white matter were analyzed. Genomic PCR of individual neurons is shown in Fig. 2. The multiple bands of PCR products of single cells were artifacts of PCR but this number of the intense bands was usually defined three. We regarded the repeat number of the uppermost of the three bands as the representative one. Repeat number distribution for the expanded allele is shown in Fig. 3. The means and standard deviations for the number of expanded CAG repeat in the early onset brain were 63.3 " 1.9 Žmean " S.D.. in the granule, 67.3 " 3.3 in the Purkinje, and 67.2 " 4.1 in the
glial cells. For the late onset brain, they were 57.0 " 1.0 in the granule, 61.7 " 4.8 in the Purkinje, and 64.6 " 4.5 in the glial cells. The CAG repeat numbers in Purkinje cells tend to be larger than those in granule cells Ž p - 0.01 in both cases by the unpaired t-test.. A difference in the repeat number distribution of Purkinje and glial cells was found for the late onset but not the early onset brain Ž p - 0.01 late onset and p s 0.86 early onset by the unpaired t-test.. 3.3. RT-PCR The RT-PCR of single isolated Purkinje neurons is shown in Fig. 4. The weak bands between normal and expanded bands are postulated to be artifacts during PCR. Cell-to-cell differences in the expanded CAG repeats are
Fig. 3. Distributions of the repeat number of single isolated cells in DRPLA cerebella. Different distribution patterns are clear for repeat numbers of the expanded allele in cerebellar granule ŽA and D., Purkinje ŽB and E. and glial cells in the cerebellar white matter ŽC and F. in the early onset ŽA–C. and late onset ŽD–F. tissues. Purkinje cells have a larger repeat number and wider distribution than granule cells in each case Ž p- 0.01.. There is no difference in repeat number between Purkinje and glial cells Ž ps 0.86. in the early onset tissues. In late onset tissues, the repeat number of the Purkinje cells is smaller than that of the glial cells Ž p- 0.01..
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Fig. 4. RT-PCR products of single isolated Purkinje cells of a late onset patient. The hemi-nested PCR products show somatic mosaicism, as seen in the genomic single cell PCR. Lane 1, cerebellar cortex; lane 2, cerebral white matter; lanes 3–12, single Purkinje cell; lanes 13 and 14, no template; lanes 3–10, RT Žq.; lanes 11 and 12, RT Žy.. The weak bands between normal and expanded bands are postulated to be artifact during PCR.
clear in the gel of the RT-PCR products of single Purkinje cells.
4. Discussion Somatic mosaicism of an expanded allele has been noted in triplet repeat diseases. The feature common to this somatic mosaicism is that the lowest degree of expansion is found in the cerebellum. Analysis of microscopically dissected cerebellar tissues showed that the mosaicism in cerebellum is identical to that in the cerebellar granular layer w11x. The smallest expansion pattern has been associated with the anatomical features of the cerebellum and the cerebellar granular layer, in which most components are postmitotic neurons. Whether stability in repeat expansion is common to all neuronal types or only to specific types is yet to be clarified. To evaluate differences in the heterogeneity of repeat expansion in various neuronal types, neurons must be isolated from brain tissues made up of heterogeneous types of cells. There is only one report on somatic mosaicism analysis of neurons of triplet repeats brain, but this was of pooled neurons w23x. However, it is difficult to evaluate the heterogeneity of the repeat number precisely unless a separate PCR is done on each single cell, as the CAG repeat sequence number is unstable during PCR. Single-cell PCR provides direct evidence of somatic mosaicism. Gametic mosaicism of the triplet re-
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peat gene has been observed using single-sperm analysis w24–31x, but there has been no report on somatic mosaicism in single isolated neurons. Our study is the first to analyze somatic mosaicism in single isolated neurons and glial cells. In our analysis of single isolated cells, there was direct evidence of a difference in the repeat heterogeneity among the various somatic cell types and between two types of neuron Purkinje and cerebellar granule cells. Our results are consistent with the report of pooled cell analysis w23x. There also was a difference in the distributions of the repeat numbers of glial cells in early onset and late onset brain tissues. The heterogeneity of the expanded allele in glial cells was increased in a brain of the late onset patient. Positive correlation between somatic mosaicism and patient age is also noted in other studies on the somatic mosaicism of DRPLA patients w10,11x. This difference of mosaicism may be explained by the difference of the number of division cycles after fertilization in glial cells of the two patients. In the study reported here, the repeat number in Purkinje cells shows wider distribution than that for granule cells, indicating that there is a difference in somatic mosaicism between neuron types. The underlying mechanism of this difference in the mosaicism between Purkinje and granule cells is still to be clarified. It is unlikely that the smaller number of repeats in granule cells represent a reduction in repeat number from baseline. Repeat contraction also results in the increase of the heterogeneity as observed in repeat expansion w32x. The distribution of repeats of the expanded allele in cerebellar granule cells is more restricted compared to that of the other cell types. To understand this difference, the following points should be considered: Ž1. the number of cell division cycles, Ž2. negative selection of neurons with larger CAG repeat expansion, Ž3. factors that affect the stability of the triplet repeat, and Ž4. the relation of the gene transcription level to repeat instability. The first point is the difference in the number of mitotic divisions in the two types of neurons. The number of mitotic divisions is estimated to be 19 in rat Purkinje cells w33x. Although the number of cell division cycles in granule cells is not known, the number of cerebellar granule cells is about 3000 times that of Purkinje cells in humans w34x. Thus, it is unlikely that the mitotic division number in granule cells is lower than that in Purkinje cells. The difference in mosaicism is not explained by the difference in the number of cell division cycles alone. Similar contradictory findings have been reported in patients with myotonic dystrophy or with spinal and bulbar muscular atrophy. The triplet repeat number for lymphocytes, one of the most proliferative cell types, is reported to be less expanded than for muscle tissue in patients with myotonic dystrophy w35–37x. A greater repeat number distribution in muscle than other tissues also has been reported in spinal and bulbar muscular atrophy w38x. These findings provide
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evidence that the cell cycle number is not the sole determinant of the difference in somatic mosaicism. Concerning the second point, the negative selection of granule neurons with longer CAG repeats, programmed cell death is an important mechanism where the neuronal population is controlled by eliminating unnecessary neurons from neuronal tissues. During normal development, granule cells die by means of apoptosis if no synapse is formed with target Purkinje cells w39,40x. The Purkinje cell is a key element in the organization of the cerebellar cortex during development and its growth and differentiation do not feature the process of programmed cell death. The difference in somatic mosaicism therefore may be explained by the apoptotic mechanism that functions in granule cells, when selective survival occurs in cells with less expanded CAG repeats. Thirdly, trans-acting factors that affect triplet repeat instability may have a function in the difference in somatic mosaicism that exists in neuronal cell groups. The mismatch repair ŽMMR. system, which affects the instability of the dinucleotide repeat sequence, is thought to affect the instability of the triplet repeat sequence w32,41,42x. DNA mismatch repair genes are expressed during and after neurogenesis, and the histological distribution of MSH2 in adult rat brain has been shown w43–46x. Purkinje and cerebellar granule cells have MSH2 immunoreactivity in adult rats w46x. The developmental expression of the MMR in the nervous system has yet to be clarified. A fourth possibility is suggested by the relationship between repeat instability and the gene expression level. In spinal and bulbar muscular atrophy, the tissue-specific pattern of somatic mosaicism is correlated with the expression pattern of the androgen receptor w38x. The untranscribed CAGrCTG repeat in the ERDA1 Žexpanded repeat domain, CAGrCTG 1. region is more stable than that in other transcribed triplet repeat genes w47x. Whether transcription activity affects the instability of the repetitive sequence of the gene must be investigated. To understand pathophysiology of triplet repeat diseases, it is necessary to analyze the relationship between somatic mosaicism and the vulnerability of specific types of neurons, and to identify the repeat numbers of the affected neurons. This is difficult because these neurons have mostly disappeared by the time of death and the residual cells are not representative of the vulnerable neurons. Further investigations using tissue of non-advanced diseased brains or transgenic animal models will help in solving this problem and answer the questions about somatic mosaicism and the histopathological distribution of vulnerable neurons.
Acknowledgements This work was supported by grants-in-aid from CREST, Japan Science and Technology.
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