Abstract
DNA repair protects neurons against spontaneous or disease-associated DNA damage. Dysfunctions of this mechanism underlie a growing list of neurodegenerative disorders. The Purkinje cell (PC) degeneration mutation causes the loss of nna1 expression and is associated with the postnatal degeneration of PCs. This PC degeneration dramatically affects nuclear architecture and provides an excellent model to elucidate the nuclear mechanisms involved in a whole array of neurodegenerative disorders. We used immunocytochemistry for histone variants and components of the DNA damage response, an in situ transcription assay, and in situ hybridization for telomeres to analyze changes in chromatin architecture and function. We demonstrate that the phosphorylation of H2AX, a DNA damage signal, and the trimethylation of the histone H4K20, a repressive mark, in extensive domains of genome are epigenetic hallmarks of chromatin in degenerating PCs. These histone modifications are associated with a large scale reorganization of chromatin, telomere clustering, and heterochromatin-induced gene silencing, all of them key factors in PC degeneration. Furthermore, ataxia telangiectasia mutated and 53BP1, two components of the DNA repair pathway, fail to be concentrated in the damaged chromatin compartments, even though the expression levels of their coding genes were slightly up-regulated. Although the mechanism by which Nna1 loss of function leads to PC neurodegeneration is undefined, the progressive accumulation of DNA damage in chromosome territories irreversibly compromises global gene transcription and seems to trigger PC degeneration and death.
Highlights
The cell nucleus is highly organized in nuclear compartments mainly involved in transcription and RNA processing
In the case of the PC2 nucleus, active euchromatin is distributed in extensive nuclear domains, whereas inactive chromatin is mainly confined to the centromeric and telomeric heterochromatin masses located around the nucleolus and at the nuclear periphery [3,4,5,6]
Given that in the cerebellar cortex of pcd mice at postnatal day 20 (P20) both healthy Purkinje cell (PC) and PCs undergoing different stages of neuronal degeneration coexist, we selected this postnatal day to study the reorganization of the nuclear architecture that is associated with this type of neurodegeneration
Summary
Mouse Genotyping—C57BL/6J male mice heterozygous for the pcd1J mutated gene were purchased from The Jackson Laboratory (Bar Harbor, ME). The number of PCs per mm of the PC layer was estimated in sagittal sections of the vermis by means of confocal images of P15, P20, and P25 control and pcd mice (n ϭ 3 animals for each group). Immunoblotting—For the preparation of nuclear extracts from the cerebellar vermis of wild-type and pcd mice, tissue samples were lysed using a pellet-pestle motor (Sigma) on ice in cold extraction buffer RSB (10 mM Tris, pH 7.5, 10 mM NaCl, 3 mM MgCl2, 1% Nonidet P-40) supplemented with a protease and phosphatase inhibitor mixture (HaltTM protease and phosphatase inhibitor single use mixture, Thermo Scientific) and incubated for 20 min on ice. After centrifugation (5 min at 1,000 rpm), the pellets were washed with RSB buffer (without Nonidet P-40) and centrifuged at 2,000 rpm for 5 min at 4 °C; the supernatant was discarded. Note the significant dramatic decrease in the latency to fall of the P20 mutants
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