RESUMEN
Apnea of prematurity (AOP) affects more than 50% of preterm infants and leads to perinatal intermittent hypoxia (IH) which is a major cause of morbimortality worldwide. At birth, the human cerebellar cortex is still immature, making it vulnerable to perinatal events. Additionally, studies have shown a correlation between cerebellar functions and the deficits observed in children who have experienced AOP. Yet, the cerebellar alterations underpinning this link remain poorly understood. To gain insight into the involvement of the cerebellum in perinatal hypoxia-related consequences, we developed a mouse model of AOP. Our previous research has revealed that IH induces oxidative stress in the developing cerebellum, as evidenced by the over-expression of genes involved in reactive oxygen species production and the under-expression of genes encoding antioxidant enzymes. These changes suggest a failure of the defense system against oxidative stress and could be responsible for neuronal death in the cerebellum. Building upon these findings, we conducted a transcriptomic study of the genes involved in the processes that occur during cerebellar development. Using real-time PCR, we analyzed the expression of these genes at different developmental stages and in various cell types. This enabled us to pinpoint a timeframe of vulnerability at P8, which represents the age with the highest number of downregulated genes in the cerebellum. Furthermore, we discovered that our IH protocol affects several molecular pathways, including proliferation, migration, and differentiation. This indicates that IH can impact the development of different cell types, potentially contributing to the histological and behavioral deficits observed in this model. Overall, our data strongly suggest that the cerebellum is highly sensitive to IH, and provide valuable insights into the cellular and molecular mechanisms underlying AOP. In the long term, these findings may contribute to the identification of novel therapeutic targets for improving the clinical management of this prevalent pathology.
RESUMEN
We have employed recently developed blind modification search techniques to generate the most comprehensive map of post-translational modifications (PTMs) in human lens constructed to date. Three aged lenses, two of which had moderate cataract, and one young control lens were analyzed using multidimensional liquid chromatography mass spectrometry. In total, 491 modification sites in lens proteins were identified. There were 155 in vivo PTM sites in crystallins: 77 previously reported sites and 78 newly detected PTM sites. Several of these sites had modifications previously undetected by mass spectrometry in lens including carboxymethyl lysine (+58 Da), carboxyethyl lysine (+72 Da), and an arginine modification of +55 Da with yet unknown chemical structure. These new modifications were observed in all three aged lenses but were not found in the young lens. Several new sites of cysteine methylation were identified indicating this modification is more extensive in lens than previously thought. The results were used to estimate the extent of modification at specific sites by spectral counting. We tested the long-standing hypothesis that PTMs contribute to age-related loss of crystallin solubility by comparing spectral counts between the water-soluble and water-insoluble fractions of the aged lenses and found that the extent of deamidation was significantly increased in the water-insoluble fractions. On the basis of spectral counting, the most abundant PTMs in aged lenses were deamidations and methylated cysteines with other PTMs present at lower levels.