Single cell analysis of CAG repeat in brains of dentatorubral-pallidoluysian atrophy (DRPLA)
Introduction
Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disorder characterized by a combination of the clinical features of epilepsy, myoclonus, choreoathetoid movement, cerebellar ataxia, and dementia [1], [2]. DRPLA is caused by the expansion of the CAG trinucleotide repeat in the DRPLA gene located on chromosome 12p [3], [4].
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 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 [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15]. 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 [11]. 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. We here report its use to analyze CAG repeats in single neurons from DRPLA brains.
Section snippets
Brain samples
Somatic mosaicism was investigated in two autopsied DRPLA brains, stored at −80 °C, which had been used in a previous study [11]. In one case, there was early disease onset and the myoclonus epilepsy phenotype (case 2 in our previous report), and in the other late disease onset (case 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.
Single cell isolation by the excimer laser microdissection system
The
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.
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
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 [11].
The smallest expansion pattern has been associated with the anatomical features of the cerebellum and the cerebellar granular layer, in which most
Acknowledgements
This work was supported by grants-in-aid from CREST, Japan Science and Technology.
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