UNIVERSITA’ DEGLI STUDI DI TORINO
DIPARTIMENTO DI MORFOFISIOLOGIA VETERINARIA
DOTTORATO DI RICERCA IN NEUROSCIENZE
CICLO XX
DNA MODIFICATIONS LINKED TO NEURONAL PROLIFERATION AND APOPTOSIS IN BRAIN
PhD student: Serena Barral.
Supervisor: Prof. Adalberto Merighi
Abstract
The relationship between functional DNA modifications and neuronal birth, differentiation, maintenance and
death, in normal and pathological status, is still poorly understood. This thesis aims to shed some light on such
a relationship from the level of fine molecular tuning of certain histone function, to gross chromosomal
anomalies in the normal and diseased brain.
Histone H2AX is part of the nucleosome core, and carries a unique COOH-terminal tail that contains the
consensus phosphatidyl inositol 3-kinase (PI-3 kinase) motif that is activated by double strand DNA breaks
(DSBs). Phosphorylation of H2AX (H2AX form) is induced in response to DSBs arising from different origins
including external damage, oxidative damage, replication errors, V(D)J recombination, replication fork
collision, apoptosis, dysfunctional telomeres, chromosomal stability, and senescence. H2AX seems to function
both as an anchor to hold broken chromosomal DNA ends in close proximity, and as recruiter of repair factors
to damaged DNA. Therefore, the construction of a protein activation map for this histone can be useful to gain
an understanding about the apoptotic and proliferative processes in the developing, postnatal and adult nervous
system.
A map of γH2AX distribution was obtained in developing, postnatal and adult mice (E14.5, P0, P5, P10, P15,
P20, P60) as well as in very old mice (18 or more months). To do so an immunohistochemical analysis was
carried out along the entire rostro-caudal axis of the brain via coronal serial sectioning. At cellular level,
γH2AX-immunoreactivity was restricted to the nucleus, displaying three different labeling pattern: (i) a typical
distribution in foci of intense staining. (ii) a very intense staining of chromosomes with a typical anaphase
and/or metaphase morphology; (iii) strong immunoreactive nuclei displaying the characteristic features of
apoptosis. All these three patterns of immunostaining were observed throughout the brain: (i) focal γH2AXimmunoreactivity was seen in different areas and layers of the cerebral cortex in mice older than P15; (ii)
γH2AX-immunoreactivity was observed in subgranular zone (SGZ) of the dentate gyrus in P10, P15, P20 and
P60 mice, where nuclei displayed essentially the foci and metaphase/anaphase pattern of staining, while
apoptotic labeling was never seen; (iii) γH2AX-immunoreactivity was found in embryonic ventricular zone
(VZ) and subventricular zone (SVZ) and in postnatal SVZ and rostral migratory stream (RMS), albeit with very
different intensities; (iiii) a high intensity of γH2AX-immunoreactivity was observed in the external granular
layer (EGL) at P0, P5 and P10. Moreover we confirmed the presence of γH2AX in the mouse brain by Western
blot analysis on total proteins extract from brains of P0, P5, P10, P20 and P60 mice.
To analyze the relationship between γH2AX expression and mitosis, a series of double labeling experiments for
γH2AX and phosphorylated histone H3 (P-H3) were performed. A positive reaction for P-H3 was seen in the
SVZ and EGL, two of the brain areas of postnatal proliferation well documented in the literature. A number of
cells, ranging from 66 to 76% in the SVZ and from 78 to 90% in cerebellum were double stained, with no
apparent differences between the different ages studied. Double-immunostained nuclei were also seen in SVZ
of old mice.
To clarify the relationship between γH2AX and DNA synthesis during the S phase of the cell cycle, we carried
out experiments with 5-bromo-3'-deoxyuridine (BrdU) on P10 injected mice and we found a 82% of cells in the
EGL double stained for γH2AX and BrdU. We also characterized γH2AX immunostaining during different cell
cycle phases and apoptosis by post-embedding electron microscopy experiments on the P10 cerebellar cortex of
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control and BrdU-injected mice, confirming the activation of γH2AX from S phase to M phase of the cell cycle
and during first phases of apoptosis.
Positive nuclei for γH2AX can be either Neuronal nuclei (NeuN) or glial fibrillary acidic protein (GFAP)
positive, in particular in cerebral cortex where cells are differentiated post-mitotic neurons or glia, while in SVZ
only GFAP+γH2AX positive cells were found.
This study demonstrate the existence of high levels of γH2AX immunoreactivity at different stages of mouse
brain development. In rapidly dividing cells, such those of the embryonic and juvenile SVZ and postnatal
cerebellar EGL, phosphorylation of H2AX is linked to proliferation and apoptosis. In particular, we
demonstrate for the first time the activation of H2AX during S-, G2-, and M phase of cell cycle in vivo, and
confirmed the activation of H2AX in the first phases of apoptosis. The existence of high levels of γH2AX
immunoreactivity with the typical foci staining in the cerebral cortex is puzzling and can be linked to the well
known role of H2AX in DNA repair, particularly in DSBs.
In most progressive neurological diseases, the degenerative cell death of neurons lies at the vanguard of any
type of detectable cognitive pathology. In Alzheimer's disease (AD), the early loss of neurons in the basal
nucleus, hippocampus and frontal cortical associative areas foretells the decline in cognitive ability that cripples
the majority of AD patients. One recent hypothesis for AD pathogenesis poses that neuronal death is intimately
linked to aberrant cell-cycle re-entry, evidenced both by the re-expression of several key cell cycle regulators in
post-mitotic neurons, and by the demonstration of cells with tetrasomic DNA content for selected
chromosomes, both observations suggesting neuronal progression through S phase.
Human brain nuclei from pathologist-verified AD and non-demented control frontal cortex and hippocampus
were sorted by fluorescence activated cell sorting (FACS) into pools of ‘putative S phase’ hyperdiploid nuclei
based on propidium iodide fluorescence. Fluorescence in situ hybridization (FISH) analysis for chromosomal
copy number status, revealed nuclei with four FISH signals for chromosomes 4, 6 and 21 in all samples
analyzed. We employed sequential hybridizations for these chromosomes on cortical or hippocampal nuclei to
determine if these nuclei were tetraploid, or tetrasomic for the chromosomes targeted, as tetrasomy for
chromosome 21 has been reported in the normal human brain.
After the analysis of over 150 tetrasomic nuclei from each brain sample, we found that in every instance,
individual nuclei showed four signals each for chromosomes 4, 6 and 21, indicating that these nuclei are most
likely tetraploid, and had completed DNA synthesis.
Several studies have indicated that neuronal progression into S phase is a harbinger of neuronal cell death,
however the neuronal identification methods used in these studies were limited to location and morphology. We
carried out FISH analysis of NeuN+ nuclei from the flow sorted ‘putative S phase’ population, allowing for the
simultaneous determination of neuronal identity and autosomal copy number status in the same nucleus. After
analyzing over 1000 neuronal (by NeuN or HuC/D) nuclei from each sample, we failed to detect any neuronal
nuclei showing greater than 2 discrete centromeric FISH signals in the cerebral cortex, and found only one in a
single control hippocampal sample.
Our results strongly suggest that vulnerable neurons in AD arrest at the G1/S checkpoint rather than progress
through DNA replication and serve to underscore the importance of fully understanding the mechanisms of cell
cycle re-entry in AD prior to the implementation of novel therapeutic interventions.
Neural progenitor cells (NPCs) in several distinct proliferative regions of the mammalian brain, including the
developing cerebral cortex and adult SVZ, display cell division defects that result in aneuploid adult progeny.
Here, we identify the developing cerebellum as a novel proliferative region of the vertebrate central nervous
system (CNS) which exhibits this phenomenon. Metaphase chromosome analysis revealed that 15.3 and 20.8%
of cerebellar NPCs are aneuploid at P0 and P7, respectively, compared to 5.7% of cultured lymphocytes. In
addition, immunohistochemical analysis of EGL precursor cells at both developmental time points reveal the
presence of abnormal cell divisions, including both lagging chromosomes during mitosis and supernumerary
centrosomes. Finally, FACS coupled with FISH, we show that both neuronal and non neuronal aneuploid cells
populate the adult mouse and human cerebellum, indicating that at least some of the aneuploid cells generated
during development are maintained into adulthood. Taken together, these results underscore the pervasive
prevalence of mosaic aneuploidy as a salient feature of the mammalian brain and suggest that the generation
and maintenance of aneuploid cells is a fundamental part of CNS development and organization.
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