Microglial cells are the primary immune cells in the central nervous system. In the mature brain, microglia perform functions that include eliminating pathogens and clearing dead/dying cells and cellular debris through phagocytosis. In the immature brain, microglia perform functions that include synapse development and the regulation of cell production through extensive contact with and phagocytosis of neural progenitor cells (NPCs). However, the functional role of microglia in the proliferation and differentiation of NPCs under hypoxic-ischemic (HI) injury is not clear. Here, we tested the hypothesis that microglia enhance NPCs proliferation following HI insult. Primary NPCs cultures were divided into four treatment groups: 1) normoxic NPCs (NN); 2) normoxic NPCs cocultured with microglia (NN+M); 3) hypoxic NPCs (HN); and 4) hypoxic NPCs cocultured with microglia (HN+M). Hypoxic-ischemic injury was induced by pretreatment of the cell cultures with 100 µM deferoxamine mesylate (DFO). NPCs treated with 100 µM DFO (HN groups) for 24 hours had significantly increased expression of hypoxia-inducible factor 1 alpha (HIF-1α), a marker of hypoxic cells. Cell number, protein expression, mitosis, and cell cycle phase were examined, and the data were compared between the four groups. We found that the number of cells expressing the NPCs marker Sox2 increased significantly in the HN+M group and that the number of PH3-positive cells increased in the HN+M group; flow cytometry analysis showed a significant increase in the percentage of cells in the G2/M phase in the HN+M group. In summary, these results support the concept that microglia enhance the survival of NPCs under HI injury by increasing NPCs proliferation, survival, and differentiation. These results further suggest that microglia may induce neuroprotective effects after hypoxic injury that can be explored to develop novel therapeutic strategies for the treatment of HI injury in the immature brain.

Background

Size and methylation mosaicism are a common phenomenon in Fragile X syndrome (FXS). Here, the authors report a study on twelve fragile X males with atypical mosaicism, seven of whom presented with autism spectrum disorder.

Methods

A combination of Southern Blot and PCR analysis was used for CGG allele sizing and methylation. FMR1 mRNA and FMRP expression were measured by qRT-PCR and by Homogeneous Time Resolved Fluorescence methodology, respectively.

Results

DNA analysis showed atypical size- or methylation-mosaicism with both, full mutation and smaller (normal to premutation) alleles, as well as a combination of methylated and unmethylated alleles. Four individuals carried a deletion of the CGG repeat and portions of the flanking regions. The extent of methylation among the participants was reflected in the lower FMR1 mRNA and FMRP expression levels detected in these subjects.

Conclusion

Decreased gene expression is likely the main contributor to the cognitive impairment observed in these subjects; although the presence of a normal allele did not appear to compensate for the presence of the full mutation, it correlated with better cognitive function in some but not all of the reported cases emphasizing the complexity of the molecular and clinical profile in FXS.

The prevalence of the fragile X premutation (55-200 CGG repeats) among the general population is relatively high, but there remains a lack of clear understanding of the links between molecular biomarkers and clinical outcomes. In this study we investigated the correlations between molecular measures (CGG repeat size, FMR1 mRNA, FMRP expression levels, and methylation status at the promoter region and in FREE2 site) and clinical phenotypes (anxiety, obsessive compulsive symptoms, depression and executive function deficits) in 36 adult premutation female carriers and compared to 24 normal control subjects. Premutation carriers reported higher levels of obsessive compulsive symptoms, depression, and anxiety, but demonstrated no significant deficits in global cognitive functions or executive function compared to the control group. Increased age in carriers was significantly associated with increased anxiety levels. As expected, FMR1 mRNA expression was significantly correlated with CGG repeat number. However, no significant correlations were observed between molecular (including epigenetic) measures and clinical phenotypes in this sample. Our study, albeit limited by the sample size, establishes the complexity of the mechanisms that link the FMR1 locus to the clinical phenotypes commonly observed in female carriers suggesting that other factors, including environment or additional genetic changes, may have an impact on the clinical phenotypes. However, it continues to emphasize the need for assessment and treatment of psychiatric problems in female premutation carriers.