Abstract
Phosphorylation of the histone H2AX (γH2AX form) is an early response to DNA damage and a marker of aging and disease in several cells and tissues outside the nervous system. Little is known about in vivo phosphorylation of H2AX in neurons, although it was suggested that γH2AX is an early marker of neuronal endangerment thus opening the possibility to target it as a neuroprotective strategy. After experimental labeling of DNA-synthesizing cells with 5-bromo-2-deoxyuridine (BrdU), we studied the brain occurrence of γH2AX in developing, postnatal, adult and senescent (2 years) mice by light and electron microscopic immunocytochemistry and Western blotting. Focal and/or diffuse γH2AX immunostaining appears in interkinetic nuclei, mitotic chromosomes, and apoptotic nuclei. Immunoreactivity is mainly associated with neurogenetic areas, i.e., the subventricular zone (SVZ) of telencephalon, the cerebellar cortex, and, albeit to a much lesser extent, the subgranular zone of the hippocampal dentate gyrus. In addition, γH2AX is highly expressed in the adult and senescent cerebral cortex, particularly the piriform cortex. Double labeling experiments demonstrate that γH2AX in neurogenetic brain areas is temporally and functionally related to proliferation and apoptosis of neuronal precursors, i.e., the type C transit amplifying cells (SVZ) and the granule cell precursors (cerebellum). Conversely, γH2AX-immunoreactive cortical neurons incorporating the S phase-label BrdU do not express the proliferation marker phosphorylated histone H3, indicating that these postmitotic cells undergo a significant DNA damage response. Our study paves the way for a better comprehension of the role of H2AX phosphorylation in the normal brain, and offers additional data to design novel strategies for the protection of neuronal precursors and mature neurons in central nervous system (CNS) degenerative diseases.
Highlights
First identified as an isoform of the core histone H2A, H2AX constitutes a major H2A species [1].The role of H2AX γ-phosphorylation in DNA damage was first suggested by showing that phosphorylated H2AX appeared rapidly following cell exposure to ionizing radiations [1].The amount of γH2AX was estimated to be directly related to the number of double strand breaks (DSBs) and, to the extent of DNA damage [2]
Apoptotic cells in the subventricular zone (SVZ)/rostral migratory stream (RMS)/olfactory bulb (OB) were so rare in our material, and we found no direct evidence for an increase of apoptotic cells in the senescent mouse
The semi-quantitative analysis of the distribution of γH2AX reported in Table 1 was carried out by counting, for each animal, the number of γH2AX-IR nuclei in individual areas of the brain from five coronal sections cut through the forebrain (P60/OLD: bregma −2.18, −1.22, 0.98, 1.34, and 3.56; see Figure 1), one section cut through the midbrain (P60/OLD: bregma between −4.04 and −4.60), and one section cut through the hindbrain (P60/OLD: bregma between −6.00 and −6.64)
Summary
First identified as an isoform of the core histone H2A, H2AX constitutes a major H2A species [1]. Neurons are among the most metabolically active cells, their gene expression levels being two to three fold higher compared with other cell types [16] This renders the brain one of the most vulnerable organs in terms of the damage to cellular DNA. Insults not necessarily resulting in neuronal death can induce γH2AX, and a role for H2AX alterations in determining neuronal vulnerability following damage was hypothesized Did these observations suggest a different sensitivity to irradiation between neuronal precursors and mature neurons [18], but γH2AX was proposed as an early marker of neuronal endangerment after ionotropic glutamate receptor activation and seizures in the adult brain [19,20]. We were convinced that an answer to the above question could be of help in setting a baseline for further exploitation of neuroprotective strategies tagging H2AX
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