Neurotrophins, such as nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF), are key factors in neuronal survival and function. In Alzheimer's disease (AD), there is a change in the normal level of these neurotrophins and their precursors (proNGF and proBDNF). The mechanism/s underlying changes in the levels of these neurotrophins in AD is not fully understood. According to the amyloid cascade hypothesis, amyloid-β is the original insult in AD and tau pathology is a downstream event. Amyloid-β interferes with axonal transport and reduces BDNF levels. However, it is not clear if amyloid-β affects neurotrophin levels directly or through tau hyperphosphorylation. If tau is responsible for changes in the level of neurotrophins in AD, we expect to observe the same alteration in neurotrophin levels in other diseases with tau dysfunction such as tauopathies. We tested the levels of BDNF mRNA and proNGF protein in subjects with tauopathies. We observed significant decrease in BDNF mRNA levels in subjects with Corticobasal degeneration. Our result suggests that BDNF may be down-regulated by tau hyperphosphorylation. Moreover, we showed that there was a significant increase in the level of proNGF in Pick's disease (PiD). Interestingly, AD and PiD share common tau modifications. Our result demonstrates a role for tau dysfunction in changes in the level of proNGF. Therefore, study of the levels of NGF and BDNF in non-AD tauopathies has shed light on the mechanisms underlying neurotrophin dysregulation in AD. How do increased levels of proNGF impact the brain in AD or PiD? Is neuronal degeneration in AD or PiD due to the lack of neurotrophic support of proNGF or do increased levels of apoptotic proNGF cause neurodegeneration? Lee et al. (2001b) and Fahnestock et al. (2004a) produced two different cleavage-resistant proNGFs with opposite activities (apoptotic versus neurotrophic). Structural and procedural differences between the two cleavage resistant proNGFs and different bioassays can cause opposite activities. We showed that proNGF from Lee's lab was neurotrophic when it was expressed in the expression system used by Fahnestock et al. or when it was purified. ProNGF expressed in a different expression system was also neurotrophic. ProNGF was neurotrophic in all bioassays except the serum withdrawal assay. We conclude that proNGF is normally neurotrophic but may be apoptotic when cell survival is already compromised. We propose that in AD, cells undergo degeneration due to the lack of neurotrophic support of proNGF (impaired transport). Moreover, TrkA is downregulated in AD which compromises cell survival and may lead to apoptosis induced by increased levels of proNGF. / Thesis / Doctor of Philosophy (PhD)

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The practice of ranking objects, events, and people to determine relevance, importance, or competitive edge is ancient. Recently, the use of rankings has permeated into daily usage, especially in the fields of business and education. When determining the association among those creating the ranks (herein called sources), the traditional assumption is that all sources compare a list of the same items (herein called elements). In the twenty-first century, it is rare that any two sources choose identical elements to rank. Adding to this difficulty, the number of credible sources creating and releasing rankings is increasing. In statistical literature, there is no current methodology that adequately assesses the association among multiple sources. We introduce rank-adapted singular value decomposition (R-A SVD), a new method that uses Kendall's tau as the underlying correlation method. We begin with (P), a matrix of data ranks. The first step is to factor the covariance matrix (K) as follows: K = cov(P) = V D^2 V Here, (V) is an orthonormal basis for the rows that is useful in identifying when sources agree as to the rank order and specifically which sources. D is a diagonal of eigenvalues. By analogy with singular value decomposition (SVD), we define U^* as U^* = PVD^(-1) The diagonal matrix, D, provides the factored eigenvalues in decreasing order. The largest eigenvalue is used to assess the overall association among the sources and is a conservative unbiased method comparable to Kendall's W. Anderson's test determines whether this association is significant and also identifies other significant eigenvalues produced by the covariance matrix.. Using Anderson's test (1963) we identify the a significantly large eigenvalues from D. When one or more eigenvalues is significant, there is evidence that the association among the sources is significant. Focusing on the a corresponding vectors of V specifically identifies which sources agree. In cases where more than one eigenvalue is significant, the $a$ significant vectors of V provide insight into factions. When more than one set of sources is in agreement, each group of agreeing sources is considered a faction. In many cases, more than one set of sources will be in agreement with one another but not necessarily with another set of sources; each group that is in agreement would be considered a faction. Using the a significant vectors of U^* provides different but equally important results. In many cases, the elements that are being ranked can be subdivided into equivalence classes. An equivalence class is defined as subpopulations of ranked elements that are similar to one another but dissimilar from other classes. When these classes exist, U^* provides insight as to how many classes and which elements belong in each class. In summary, the R-A SVD method gives the user the ability to assess whether there is any underlying association among multiple rank sources. It then identifies when sources agree and allows for more useful and careful interpretation when analyzing rank data. / Statistics

5-Lipoxygenase (5LO) is a lipid-peroxidizing enzyme which inserts molecular oxygen into fatty acids leading to the biosynthesis of leukotrienes. This protein is widely expressed in the brain including the cortex and hippocampus regions, where its levels and activity increase in an age-dependent manner. Previous work has shown that 5LO modulates both amyloid beta (A) and tau pathology in Alzheimer's disease (AD) models. However, whether the effect of 5LO on tau is direct or indirect still remains unclear. Tau is a microtubule-associated protein usually found in the axons of neurons where it promotes assembly and stabilization of microtubules. In post-mortem brains of AD patients, tau is hyperphosphorylated and altered conformationally, followed by the formation of intracellular aggregates known as neurofibrillary tangles (NFTs), which are also the major pathological hallmark of another group of neurodegenerative diseases collectively referred to as tauopathies such as Pick's Disease, Progressive Supranuclear Palsy (PSP), Frontotemporal Dementia (FTD) and Parkinsonism linked to chromosome 17. The central hypothesis of the thesis is that 5LO directly influences tau metabolism, the development of related neuropathology and behavioral phenotype. To prove this hypothesis, a comprehensive genetic and pharmacologic experimental approach, combining both in vivo and in vitro experiments, was implemented. We initially showed that human brains from patients with a confirmed diagnosis of PSP, had significantly higher levels of 5LO when compared with brains form healthy controls. Next, we assayed the levels of 5LO in brains from htau (transgenic tau mice) mice at 4 different age time-points and two regions (cortex and cerebellum). Interestingly, compared with wild type controls, cortices from htau mice had a non-significant increase in 5LO protein levels as early as 6 months of age, which became significant by 10 months of age in the cortex only. Taken together, the age-dependent and region-specificity of the 5LO up-regulation supports the hypothesis that this pathway may have a functional role in the development of the tauopathy phenotype. To prove it, we treated tau mice with a selective 5LO inhibitor, zileuton, and explored the effect on learning and memory. Treatment of the htau mice with zileuton restored their short term working memory and spatial memory deficits. Shortly after completion of the behavioral tests, mice were euthanized and brains harvested for biochemistry and immunohistochemistry analyses. In association with the changes in behavior, we observed that pharmacologic inhibition of 5LO had an influence on tau metabolism, more specifically a significant decrease in tau phosphorylation. In search for the molecular mechanism involved in this biological effect, we assayed different kinases and phosphatase which have been implicated in tau metabolism and showed the specific involvement of the cdk5 pathway. This observation was further confirmed by in vitro studies, in which by using primary neuronal cells we showed that zileuton decreased tau phosphorylation via a cdk-5-dependent mechanism. Since the development of tau pathology results in biochemical and functional manifestation of synaptic deficits, next we assessed levels of pre- and post-synaptic protein markers. Compared with wild type, htau mice had significant reduction in the levels of three distinct markers of synaptic integrity (that is synaptophysin, post-synaptic density protein-95 and microtubule associated protein-2). By contrast, the decrease was completely restored to wild type levels by zileuton treatment. To further support the involvement of this pathway in the improvement of the behavioral and cognitive deficits, we explored the effects of its pharmacological blockade on synaptic function by performing electrophysiological studies. As reported previously, there was a significant difference in Long Term Potentiation (LTP) between the wild type and htau mice, with the latter showing significant deficits. However, pharmacologic blockade of 5LO in the htau mice was adequate to restore the LTP responses to a level comparable to those measured in the wild type mice. In the genetic portion of the study, WT and htau pups were intracranially injected with both AAV2/1 control vector and AAV2/1 5LO vector. Compared with the htau control group, the htau mice injected with AAV2/1 5LO displayed a significant deficits in cognition and memory associated with a decline in their synaptic integrity. Also, genetic upregulation of 5LO yielded significant increases in tau phosphorylation associated with an increase in cdk-5 kinase activation both in vivo and in vitro. Taken together these results describe a pluripotent role for 5LO in the context of tauopathy by representing its direct functional role in modulating behavior along with tau phosphorylation, neuroinflammation and synaptic function in a relevant mouse model of the human disease. The demonstration of the pleiotropic role 5LO in tauopathy pathogenesis makes it not only a valid pharmacological target, as 5LO inhibitors are already FDA approved but, more importantly represents a unique therapeutic opportunity with true disease modifying potential for the treatment of these dementing disorders for which there is no cure. / Pharmacology

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