Behavioural, genetic and epigenetic determinants of white matter pathology in a new mouse model of chronic cerebral hypoperfusion
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Recent clinical studies suggest that white matter pathology rather than grey matter abnormality is the major neurobiological substrate of age- related cognitive decline during “healthy” aging. According to this hypothesis, cerebrovascular (e.g. chronic cerebral hypoperfusion) and molecular (e.g. APOE, epigenetics) factors might contribute to age-related white matter pathology and cognitive decline. To test this, I used a new mouse model of chronic cerebral hypoperfusion and examined the following predictions: 1) hypoperfusion- induced white matter pathology might be associated with cognitive deficits, 2) APOE deficiency might be associated with white matter anomalies under normal physiological conditions and more severe hypoperfusion- induced white matter pathology, 3) chronic cerebral hypoperfusion might impact on hydroxymethylation (a newly discovered epigenetic marker) in white matter, via perturbations in associated epigenetic pathways, namely methylation and/ or TETs. I. Effects of chronic cerebral hypoperfusion on white matter integrity and cognitive abilities in mice To test the hypothesis suggesting that hypoperfusion- induced white matter pathology is associated with working memory and executive function impairment in mice, behavioural performance and neuropathology were systematically examined in two separate cohorts of sham and hypoperfused C57Bl6J mice. Spatial working memory, memory flexibility, learning capacity, short and long term memory recall were taxed using radial arm maze and water maze paradigms one month after surgery. At the completion of the behavioural testing white and grey matter integrity, inflammation were evaluated using standard immunohistochemistry with antibodies recognizing neuronal axons (APP), myelin sheath (MAG) and microglia (Iba1) as well as H&E histological staining to examine neuronal morphology and ischemic injury. In agreement with previous reports, the behavioral data indicated spatial working memory impairment in the absence of spatial memory flexibility, learning, short- and long- term memory recall deficits in hypoperfused mice However, in contrast to previous reports, a spectrum of white and grey matter abnormalities accompanied by an increased inflammation were observed in hypoperfused mice Although there was a significant association between hypoperfusion- induced inflammation in white matter and performance on a working memory radial arm maze task (p<0.05), the present pathological findings suggest that white matter abnormalities, neuronal ischemia and increased inflammation might be at the basis of hypoperfusioninduced cognitive impairment in mice. Further, chronic cerebral hypoperfusion might have affected alternative, non- examined brain processes (e.g. cerebral metabolism, neurotransmission) which might have contributed to the observed cognitive deficits in hypoperfused mice. II. Effects of APOE on white matter integrity under normal physiological and chronically hypoperfused conditions in mice To test the hypothesis suggesting that mouse APOE deficiency might be associated with white matter anomalies under normal physiological conditions and the development of more severe white matter pathology following chronic cerebral hypoperfusion, white and grey matter integrity, inflammation were examined in APOE deficient mice on a C57Bl6J background (APOEKO) and C57Bl6J wild- type (WT) counterparts one month after chronic cerebral hypoperfusion or sham surgery. A combined neuroimaging (MRI- DTI)/ immunochemical approach was attempted in these mice as an additional step towards translation of this research to human subjects. The ex vivo MRI- DTI findings demonstrated APOE genotype effects on the development of white matter abnormalities following chronic cerebral hypoperfusion in mice. Significant reductions in MRI metrics (FA and MTR) of white matter integrity were observed in examined white matter areas of APOEKO hypoperfused mice compared with WT hypoperfused counterparts (p<0.05). However, the neuroimaigng findings were not supported by the pathological analysis where no significant APOE differences were observed in hypoperfusion- induced axonal (APP), myelin (MAG, dMBP) pathology and inflammation (Iba1) (p>0.05). No significant differences in MRI parameters and pathological grades of white matter integrity were evidenced between APOEKO and WT sham mice (p>0.05). An absence of grey matter abnormalities was evidenced on T2- weighted scans and corresponding H&E stained brain sections in all experimental animals. However, significant reductions in MTR values and dMBP immunoreactivity (myelin pathology) (p<0.05) were observed in grey matter (the hippocampus) following chronic cerebral hypoperfusion in the absence of significant APOE genotype effect (p>0.05) suggesting the existence of both white and grey matter abnormalities in this animal model. Overall, the present neuroimaging data, but not pathological analysis, partially validated the main study hypothesis suggesting that APOE deficiency might be associated with the development of more severe white matter abnormalities in hypoperfused mice. III. Characterization of methylation and hydroxymethylation in white matter under normal physiological and chronically hypoperfused conditions in mice Lastly, I sought to test the hypothesis that chronic cerebral hypoperfusion might alter oxygen dependent DNA hydroxymethylation (5hmC) in white matter regions via perturbations in methylation (5mC) and/ or Ten- eleven translocation proteins (e.g. TET2) in mice. DNA methylation (5mC), hydroxymethylation (5hmC) and TET2 were immunochemically studied in white and grey matter of sham and chronically hypoperfused C57Bl6J mice a month after surgery. The immunochemical results demonstrated significant increases (p<0.05) in 5hmC in the hypoperfused corpus callosum (CC) in the absence of significant hypoperfusion- induced alterations in the distribution of 5mC and TET2 (p>0.05) in white matter. Significant hypoperfusion- induced increases were evident for TET2 in the cerebral cortex (Cx) (p<0.05). These data partially validated the main study hypothesis suggesting hypoperfusion- induced alterations in 5hmC in white matter. However, in contrast to the study hypothesis, the observed hypoperfusion- induced alterations in 5hmC occurred in the absence of changes in 5mC and TET2 in white matter. A subsequent correlation analysis between hydroxymethylation and 5mC, TET2 in the CC failed to show significant associations (p>0.05). In search of the cellular determinants of 5hmC in the CC, hydroxymethylation was examined in relation to some of the cell types in white matter- mature oligodendrocytes, oligodendrolial progenitors (OPC) and microglia both in vivo and in vitro. Specifically, a separate parametric correlation analysis between the proportion of 5hmC positive cells and the respective proportions of mature oligodendrocytes, OPC and microglia in the CC demonstrated that hydroxymethylation correlated significantly only with microglia in vivo (p<0.05). Following this, 5hmC immunochemical distribution was studied in vitro in oligodendroglia cells at different stages of maturation, and interferon γ/ lypopolisaccharide activated and nonactivated microglia. The in vitro analysis demonstrated that 5hmC is high in OPC, activated and nonactivated microglia, but it is low in mature oligodendrocytes. Taken together the in vivo and in vitro cellular analyses suggest that the processes of hydroxymethylation in white matter might be immunoregulated. However, it is possible that in vivo in addition to microglia, other cell types (e.g. astrocytes, OPC) contributed to the presently observed 5hmC upregulation in the hypoperfused CC. Conclusion The experimental work presented in this thesis further developed and characterized a new mouse model of chronic cerebral hypoperfusion by confirming previous behavioural findings (e.g. working memory deficits) and revealing previously undetected spectrum of white and grey matter pathology in this animal model. The thesis demonstrated for the first time by using a newly developed ex vivo MRI procedure that APOE might modulate hypoperfusion- induced white matter pathology in mice. Additional immunochemical analysis revealed important hypoperfusion- induced epigenetic alterations in white (5hmC) and grey (TET2) matter in this animal model. Future experiments on chronically hypoperfused mice would allow to get a better insight into the neurobiological determinants (e.g. white vs. grey matter) underlying the observed cognitive deficits in this animal model, the involved cellular and molecular pathways as well as the functional significance of genetic (APOE) and epigenetic (5hmC, TETs) alterations in the hypoperfused brain. Future experimental work on this animal model would potentially reveal new biological targets for the pre- clinical development of therapies for age- related cognitive decline. Further development and optimization of the newly developed ex vivo MRI procedure would allow its broader application in preclinical settings and would facilitate the translation of experimental findings to clinics.