Consequences of Chronic Stress on the PINE Network

Stress is a risk factor for the development and progression of a variety of disorders. At the same time stress is essential to initiate adaptation to the current situation and to promote survival of the fittest. Thus, responses to stress evolved to be fast and efficient. This is implemented by a tight networking of the psycho-immune-neuro-energy (PINE) system. Within the PINE network, glucocorticoids are the universal messengers that regulate overall physiology jointly with cytokines, neurotransmitters and energy status. While the secretion of glucocorticoids in response to stress is itself a rather unspecific reaction to any kind of stressor, complexity of the outcome is encoded by lifetime, recent and present events. Together, these individual experiences modulate the diurnal and ultradian rhythmicity of glucocorticoid levels. Given the time- and dose-dependency of glucocorticoid signaling, this rhythmicity allows for flexibility in the coping with stress. In a chronic stress setting, the interaction of PINE network components is altered. While stress-resilient individuals retain adaptive capacity, vulnerable individuals lose flexibility in their responsiveness. Gene (cid:1) environment interactions could explain individual differences. To better elucidate the molecular underpinnings of risk and resiliency, models that allow studying the consequences of chronic stress on the PINE network are required. well). On the next day, a full medium exchange was performed. On post plating day (PPD) 8 the confluent astrocyte layer was treated with AraC-medium (Cytosine Arabinoside, Sigma, #251010, 8 μ M) for 4 days. On PPD11, the medium was exchanged to LME-medium (L-leucine methyl esters, Sigma, #L1002, 50 mM) for 1 hour and astrocytes were subsequently washed three times with medium. On PPD14, the medium was exchanged to serum-free medium and the assay was performed the next day. Experimental details – qPCR: Native or stimulated cells were lysed in 250 μ l RLT buffer (Qiagen, Hilden, Germany, #79216) containing 1% beta-mercapto-ethanol (Sigma #M3148-100ML) and frozen at (cid:3) 80 ∘ C prior to RNA isolation. RNA was isolated using RNeasy Plus kit (Qiagen #74192) following the manufacturer ’ s recommendations. Integrity was confirmed to be RIN > 8 (Fragmentanalyser,Thermo Fisher Scientific, Langenselbold, Germany). For normalization, 500 ng of total mRNA were used during reverse transcription (high capacity cDNA kit, Qiagen #4368813). All TaqMan gene expression assays were labeled with FAM (Thermo Fisher #4352042; succinate dehydrogenase complex subunit A (Sdha, Mm01352366_m1), murine Fkbp5 (Mm00487403_m1), glucocorticoid receptor (Nr3c1, Mm00433832_m1)) and used in conjunction with the fast universal PCR Master Mix (Thermo Fisher #4351368). Samples were analyzed in technical triplicates on a QuantStudio 6 (Thermo Fisher). All gene expression levels were normalized within the same cell type relative to the cycle thresholds measured for Sdha and for stimulation experiments relative to DMSO-treated cells. Results: Primary murine astrocytes, microglia and neurons differ in their basal mRNA expression of the glucocorticoid receptor (Nr3c1) and its functional inhibitor FK506