Disarranged neuroplastin environment upon aging and chronic stress recovery in female Sprague Dawley rats.

Chronic stress produces long-term metabolic changes throughout the superfamily of nuclear receptors, potentially causing various pathologies. Sex hormones modulate the stress response and generate a sex-specific age-dependent metabolic imprint, especially distinct in the reproductive senescence of females. We monitored chronic stress recovery in two age groups of female Sprague Dawley rats to determine whether stress and/or aging structurally changed the glycolipid microenvironment, a milieu playing an important role in cognitive functions. Old females experienced memory impairment even at basal conditions, which was additionally amplified by stress. On the other hand, the memory of young females was not disrupted. Stress recovery was followed by a microglial decrease and an increase in astrocyte count in the hippocampal immune system. Since dysfunction of the brain immune system could contribute to disturbed synaptogenesis, we analyzed neuroplastin expression and the lipid environment. Neuroplastin microenvironments were explored by analyzing immunofluorescent stainings using a newly developed Python script method. Stress reorganized glycolipid microenvironment in the Cornu Ammonis 1 (CA1) and dentate gyrus (DG) hippocampal regions of old females but in a very different fashion, thus affecting neuroplasticity. The postulation of four possible neuroplastin environments pointed to the GD1a ganglioside enrichment during reproductive senescence of stressed females, as well as its high dispersion in both regions and to GD1a and GM1 loss in the CA1 region. A specific lipid environment might influence neuroplastin functionality and underlie synaptic dysfunction triggered by a combination of aging and chronic stress.

High-throughput rat immunoglobulin G N-glycosylation profiling revealed subclass-specific changes associated with chronic stress.

Immunoglobulin G (IgG) glycosylation corresponds well with immune system changes, so it can potentially be used as a biomarker for the consequences of chronic stress such as low-grade inflammation and enhanced immunosenescence in older animals. Here we present a high-throughput glycoproteomic workflow, including IgG enrichment, HILIC glycopeptide purification, and nano-LC-MS analysis of tryptic glycopeptides applied for the analysis of rat IgG. A cohort of 80 animals was exposed to seven stressors in a customized chronic stress protocol with blood and tissue sampling in three timepoints. Young female rats experienced an increase in agalactosylated glycoforms on IgG2a and IgG2c accompanied by a decrease in monogalactosylation. Among old females, increased galactosylation was observed in the IgG2b subclass, pointing to an anti-inflammatory activity of IgG. Additionally, IgG Fc N-glycosylation patterns in Sprague Dawley rats were analyzed, quantified, and reported for the first time. Our findings emphasize age-, sex- and subclass-dependent differences in IgG glycosylation related to chronic stress exposure, confirming the relevance of newly developed methods for further research in glycobiology of rodent immune response. SIGNIFICANCE: In this study, we showed that a high-throughput streamlined methodology based on protein L 96-well monolithic plates for efficient rat IgG immunoaffinity enrichment from blood plasma, paired with appropriate tryptic glycopeptide preparation, HILIC-SPE enrichment, and nano-LC-MS methods was suitable for quick processing of large sample sets. We report a subclass-specific profiling and changes in rat IgG Fc galactosylation and adrenal gland immunohistochemistry of male and female animals exposed to a customized chronic stress protocol.