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BACKGROUND: Alzheimer's disease (AD) is the most common aging-associated neurodegenerative dementia. Current epidemiological trends indicate that a rapidly aging population, in conjunction with the economic impact of AD and lack of disease-modifying agents for AD, make AD an enormous public health challenge. AD pathology has been well characterized: it consists of extracellular plaques composed of Aß protein and intraneuronal tangles of hyperphosphorylated tau protein. Genetic analyses of AD cases have identified causative mutations in the pathways of Aß protein production but these mutations are rare. Therefore environmental factors that modify AD risk are of increasing importance. One such environmental factor that has received attention recently is stress. Biomarkers of stress (i.e., plasma and urinary cortisol) are associated with increased AD risk and more precipitous AD decline. Animal models have also largely recapitulated these results: stress exacerbates the AD phenotype in several studies. One of the actions of stress hormones such as glucocorticoids, is to upregulate the activity of the 5-lipoxygenase protein (5LO). 5LO is widely expressed in the central nervous system and is responsible for producing leukotrienes from arachidonic acid. 5LO has been previously shown to positively modulate Aß production as well the phosphorylation of tau protein. Therefore, while stress is associated with increased AD vulnerability, stress hormones modulate the 5LO protein, and the 5LO protein has been shown to modulate AD pathology, but the importance of 5LO in the stress-mediated exacerbation of the AD phenotype has not yet been explored. HYPOTHESIS: The central hypothesis of this thesis is that 5LO plays a central role in the stress-mediated exacerbation of the AD phenotype. METHODS: We used the 3xTg animal system, an AD transgenic mouse model which expresses both plaques and tangles and crossed 3xTg animals with 5LO knockout mice to create 3xTg animals without 5LO (3xTg/5LO-/-). We challenged both 3xTg and 3xTg/5LO-/- animals with dexamethasone (7 d, 5mg/kg i.p.) and restraint/isolation stress (28 d, 60 min/d) in separate studies to interrogate how the stress-response to Aß, tau and fear-conditioned memory were altered by lack of 5LO in the AD context. RESULTS: In our study with dexamethasone, we found that no memory insults occurred in either 3xTg or 3xTg/5LO-/- animals as a result of a 7 d 5mg/kg dexamethasone i.p. injection challenge. We also found no elevation in brain levels of Aß after dexamethasone exposure, although 3xTg/5LO-/- animals had less Aß than 3xTg animals, a finding our group has previously published. However we found that 3xTg animals had greater phosphorylation of tau and generation of insoluble tau following dexamethasone treatment. This tau pathology was associated with elevation in GSK3ß activity. 3xTg/5LO-/- animals lacked any dexamethasone-associated advancement of tau pathology or elevation in GSK3ß activity. In our study with restraint/isolation stress, we found that 3xTg/5LO-/- animals were protected against fear-conditioned contextual and cued insult recall caused by stress found in 3xTg animals. No change in Aß was found as a function of either genotype or stress condition. As with our study with dexamethasone, we found that 3xTg animals had greater phosphorylation of tau and generation of insoluble tau following restraint/isolation stress. This tau pathology was associated with elevation in GSK3ß activity. 3xTg/5LO-/- animals lacked any restraint/isolation-associated tau pathology or GSK3ß activity. We additionally found that knockout of 5LO exerted a protective effect against restraint/isolation-mediated impairment in long-term potentiation. CONCLUSION: Our work reveals, for the first time, the importance of the 5LO protein in stress-mediated exacerbation of the AD phenotype. These data indicate that 5LO-targeted interventions could be of use in individuals vulnerable to this environmental risk factor, and more broadly, in a preventative strategy against AD. / Pharmacology

Der niedrig dosierte Dexamethason-Suppressionstest (LDDST) wird als Screeningverfahren in der Diagnostik des Cushing-Syndroms angewendet. Allerdings können Faktoren wie die variable Resorption, sowie ein gesteigerter Metabolismus von Dexamethason die Testergebnisse beeinflussen und zu falsch positiven Resultaten führen. Ein falsch positives Testresultat wird hierbei insbesondere bei adipösen Patienten häufiger beobachtet. In der vorliegenden Arbeit wurde daher der Einfluss des Körpergewichts auf das Ergebnis des Dexamethason-Suppressionstestes (DST) untersucht. Hierzu wurden hospitalisierte Patienten und ein aus gesunden Probanden bestehendes Kontrollkollektiv rekrutiert und diese einem regulären LDDST unterzogen. Es konnte gezeigt werden, dass übergewichtige Menschen im Rahmen des DST signifikant niedrigere Dexamethasonwerte erreichen als normalgewichtige Personen. Es ist daher davon auszugehen, dass Unterschiede im Body-Mass-Index (BMI) Einfluss auf die Resorptionsrate und den Metabolismus von Dexamethason nehmen und daraus resultierend die Serum-Dexamethasonspiegel wesentlich verringern können. Diese Prozesse scheinen allerdings keinen nachhaltigen Einfluss auf die Cortisolsuppression im DST zu haben, da die Cortisolwerte nach Dexamethasongabe zwischen adipösen und nicht adipösen Testpersonen nicht signifikant differierten. Diese Ergebnisse belegen, dass ein Zusammenhang zwischen BMI-Unterschieden und der Dexamethasonkinetik im LDDST besteht. Die erniedrigten Dexamethasonspiegel bei übergewichtigen Patienten scheinen hierbei insbesondere durch das wesentlich höhere Verteilungsvolumen und durch Unterschiede im hepatogenen Metabolismus bedingt zu sein. Trotz der erniedrigten Dexamethasonwerte bleibt die Feed-Back-Sensitivität der Hypothalamus-Hypophysen-Nebennieren-Achse (HHNA) auch bei Adipositas erhalten, weshalb der LDDST als zuverlässiges Screeningverfahren bei adipösen Patienten mit Verdacht auf Cushing-Syndrom einzustufen ist.

Dexamethasone and dexamethasone 21-phosphate were loaded into NDGA-polymerized collagen fibers and release rate studies were performed to calculate their diffusion coefficients. Dexamethasone loaded fibers were placed in a PBS solution for specified time intervals (1, 3, 6, 7, 12, 24, 30, and 48 hours) after which the eluant was removed and analyzed by capillary zone electrophoresis (CZE). CZE is a tool that can be utilized for quantitative analysis of chemical compounds. This data was incorporated into mathematical models to determine the diffusion coefficient. The diffusion coefficient (D) for dexamethasone in NDGA-polymerized collagen fibers is D = 1.86 x 10⁻¹⁴ m²/s. Similarly, dexamethasone 21-phosphate loaded fibers were placed into a PBS solution and analyzed using CZE at these specified intervals (15, 30, 45, 60, and 75 minutes). Applying this data to the mathematical model provided a diffusion coefficient for dexamethasone 21-phosphate in NDGA-polymerized collagen fibers of D = 2.36 x 10⁻¹³ m²/s. In an effort to control drug delivery from these fibers a polylactic-co-glycolic acid (PLGA) coating was applied to the fibers. This coating helped sustain delivery of dexamethasone 21-phosphate for over a 100 day period. CZE experiments were again conducted in conjunction with another mathematical model to characterize release. A semi steady-state diffusion coefficient was estimated to be D = 4.59 x 10⁻¹⁴ m²/s.

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