Oxidative stress and inflammation are often linked to the prognosis of diseases caused by chronic alcohol consumption. Chronic alcohol consumption plays a key role in brain tissue damage, often leading to the development of cognitive disorders and loss of brain function. In addition to the direct effects of alcohol on brain function, consumption of alcohol can lead to psychosocial stressors such as legal, financial, and interpersonal problems. It has been found that mice that overexpress C1q/Tumor Necrosis Factor-related protein-3 (CTRP3) and exposed to ethanol daily do not die like the mice who did not overexpress CTRP3 and fed the same diet. Although the specific physiological functions regulated by the CTRP family are largely unknown, there is evidence showing that they have diverse biological effects on inflammation, metabolism, and survival signaling in several different types of tissue. Postmortem brain tissue samples were collected from mice that were exposed to ethanol with transgenic overexpression of CTRP3 and from wild type mice that were only exposed to ethanol. Interestingly, previous immunoblotting of the cerebellum and the hippocampus using collected tissue demonstrated that glia activation was present in the CTRP3 overexpressing mice but not in the wild-type ethanol fed mice. This finding suggests that glia cells are either dying in the ethanol fed wild type mice or that CTRP3 protects and prolongs activated glia cells. The current study will determine if markers of oxidative stress and cell viability are altered in the CTRP3 overexpressing mice when compared to wild-type mice at the molecular level. RNA isolation using the Directzol system and cDNA synthesis using punch dissected homogenate tissue collected from the hippocampus was used for this investigation. Gene expression of BDNF, SOD1 and PARP1 in mouse tissue was determined using quantitative PCR. Immunoblotting of a small number of hippocampal tissue using PARP1 was performed. The mice that were CTRP3 overexpressed and fed ethanol will likely exhibit altered gene expression of cytokines and increased oxidative stress gene expression in postmortem hippocampal brain tissue when compared to wild-type ethanol fed mice. The current studies could contribute to the body of knowledge for the development of novel therapies that may alleviate the neuro-inflammatory effects of alcohol use.

Autism spectrum disorder (ASD) is a neurodevelopmental disability affecting communication and social behaviors. Research is needed because the percentage of children affected by ASD is 1 in 59, and it is diagnosed in males at a rate of 1 in 42. Animal models must be used, because the neurological changes that lead to ASD occur during prenatal development. In this study, three mouse models were used to represent possible causes of ASD. The BTBR model is a genetically engineered model that displays social behavior deficits and has neuroanatomical findings similar to ASD. The other models include the Poly-IC and valproic acid injected mice which exposes the pregnant mother to a virus activating her immune system or a drug thought to affect brain development, respectively. In all three models the effects of brain-derived neurotrophic factor or BDNF, which is an important cytokine in the brain responsible for synaptic plasticity, maintenance and recognition, are being studied via expression levels of NTRK2. BDNF activates cell signaling cascades in glutamatergic neurons via the TrkB receptor which is encoded by the NTRK2 gene. It was previously found that NTRK2 expression was reduced in glutamatergic cells in people affected by ASD. The first outcome of the study is to determine gene expression differences in glutamatergic neurons captured from the cingulate cortex in all three models as well as in wild type control mice. Additionally, a second outcome of the study is to optimize a new protocol for single cell gene expression using a nested PCR method. This was done by comparing the previously used method for relative end-point PCR with the nested method to identify gene expression alterations. To prepare for the two PCR methods, samples were dehydrated and laser capture microdissection was performed on mouse brain tissue to obtain pyramidal neurons from the cingulate area. This area is highly connected to the limbic system and plays a role in personality and communication. All animal procedures were approved by the ETSU animal care committee. RNA isolation was performed on 1000 cells after which RNA was reverse transcribed into cDNA using the Superscript III cDNA synthesis system. Initial optimization experiments included using various amounts of starting cDNA and determining expression differences using relative end-point PCR and Agilent tape station. The same starting cDNA was used and initially 20 cycles of PCR were performed using Prime5 HotStart Master Mix followed by a quantitative PCR reaction using Powerup on the BioRad CFX96 RT detection system. Gene expression was performed using NTRK2 as the target gene and GAPDH as the reference gene for each method. Both methods will allow the detection of changes in the expression levels of NTRK2 and GAPDH when different sample concentrations are used. This data could help establish a link between maternal immune system activation or exposure to certain drugs during pregnancy with the occurrence of ASD.

Exercise enhances aspects of human cognition, but its intensity may matter. Recent research in animal models suggests that vigorous exercising may be optimal as it releases greater amounts of lactate, which in turn, activates brain-derived neurotrophic factor (BDNF) in the hippocampus to support cognitive function. Furthermore, the effects of exercise on cognition may be augmented when exercise is combined with cognitive training. The sport of orienteering simultaneously combines exercise with spatial navigation and therefore may result in greater cognitive benefits than exercising only, especially when performed at a vigorous intensity. Therefore, the aim of the present study was to examine the effects of an acute bout of orienteering at different intensities compared to exercising only on cognition and BDNF. We hypothesized that vigorous-intensity orienteering would increase lactate and BDNF and improve cognition more than moderate-intensity orienteering, or vigorous exercise alone. To test this, we recruited 63 recreationally active, healthy young adults (Mage = 21.10±2.75 years) with no orienteering experience. The orienteering groups navigated a 1.3km orienteering course while exercising at either a vigorous (80-85% of HRR) or moderate (40-50% of HRR) intensity. The control group exercised at a vigorous intensity on the same course without navigation. Peak lactate, heart rate and rating of perceived exertion during the intervention were used to verify exercise intensity. Immediately before and after the intervention, serum BDNF was extracted, and cognitive function was assessed using the Mnemonic Similarity Task for high-interference memory and the Groton Maze Learning Test for spatial learning and memory. The results show that exercising (M = 5.35±2.52 mmol/L) and orienteering (M = 5.94±2.49) at a vigorous intensity elicited greater peak lactate levels than orienteering at a moderate intensity (M = 2.01±1.20). Vigorous exercise (p = .003) and orienteering (p = .043) elicited greater increases in BDNF, and individuals with higher peak lactate had greater increases in BDNF (rs (56) = .28, p = .037). Vigorous exercise also benefited high-interference memory compared to moderate orienteering (p = .019). All groups increased in spatial learning (p’s < .05), but only the vigorous orienteering group improved in delayed spatial memory performance (p = .007). Overall, the results provide evidence for the beneficial effects of combined exercise plus navigation training interventions for spatial cognitions that are closely related to the process engaged during cognitive training. / Thesis / Master of Science (MSc)

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This dissertation is composed of five major chapters. The first is a comprehensiveliterature review, followed by three chapters of research findings, and a final concludingchapter. Major Depressive Disorder (MDD) is a phenotypically complex andheterogeneous syndrome. This challenge and others faced when investigating the geneticbasis for the susceptibility to MDD are discussed, as are tools used to address andovercome these challenges. Included in this review of the literature is a discussion onfindings of genome-wide analyses of MDD, as well as candidate genes that may play arole in the susceptibility to major depression. Following the literature review, threechapters of studies are presented. The first one demonstrates that in humans, the actualnumber of risk genotypes in the serotonin system accounts for over half of the variance inmood response to tryptophan depletion. There was no association between the dopaminesystem and mood response. The main conclusion from that study is that using a pathwayanalysis, rather than a single gene approach, may lead to more informative results whenstudying the genetics of a complex behavior. The next study demonstrates a similarconclusion, however, is not pathway specific. It is shown that in a group of de pressedsubjects not capable of treatment response, the mean number of risk genotypes is greaterthan in a group without depression. This supports the thought that treatment resistancemay be a more severe form of MDD. This study also presents data on single gene resultswhich demonstrate that the genetic basis for susceptibility to major depression may bedifferent and independent from the genetic basis for the capacity to respond to treatment.Several individual polymorphisms are implicated in each case. The final investigation is apublished manuscript refuting the findings of a previously published article onpolymorphisms in the TPH2 gene and association with treatment resistance. Many otherresearch groups have also been able to replicate the results demonstrated here. A finalchapter discusses the overall conclusions about the three research studies, as well as thefield of psychiatric genetics with a focus on the continuing search for the genetic basis ofsusceptibility to major depressive disorder.

Interstitial cystitis is a chronic debilitating disease that causes pain and increased frequency of micturition, amongst other symptoms, without any identifiable cause. This disease affects a large number of the population, yet the etiology is still unknown. The present study aimed to characterize BDNF and CGRP—two neuropeptides that have both been proven to play an important role in the transmission of pain as well as in hypersensitivity. The signaling pathways regulating the expression of the two neuropeptides were also examined. Results revealed that BDNF protein expression levels increased in both L1 and L6 DRG following 48 hours post CYP-induced cystitis. CGRP protein expression levels decreased in L1 DRG, but increased in L6 DRG following 48 hours post CYP-induced cystitis. Examination of mRNA levels revealed an increase in the mRNA levels of both BDNF and CGRP in L6 DRG. NGF, a member of the neurotrophin family, mRNA levels also increased following 48 hour CYP-induced cystitis in the urinary bladder. Retrograde analysis revealed NGF possibly retrograde signaled to the DRG to increase BDNF and CGRP expression. Co-localization immunohistochemistry results revealed phospho-Akt co-localized with BDNF, but not with CGRP. Thus NGF retrograde signaling may activate the PI3-K/Akt cascade which may be involved in BDNF expression. CGRP expression may be via another signaling cascade.

In der vorliegenden Dissertation wurde die subzelluläre Lokalisation der Rezeptoren für die neurotrophen Faktoren BDNF, CNTF und GDNF in primären embryonalen und adulten Motoneuronen erstmalig genau charakterisiert. Die Rezeptoruntereinheiten des BDNF und CNTF Rezeptors, TrkB, p-TrkB, gp130 und p-Stat3, sind im Perikaryon, in Dendriten, im Axon und an den Axonterminalen bzw. Wachstumskegeln von Motoneuronen lokalisiert. Dabei sind die nativen Formen (TrkB, gp130) im Axon überwiegend membranständig, die aktivierten Formen (p-TrkB, p-Stat3) überwiegend im Inneren des Axons lokalisiert. Demgegenüber sind die Rezeptoruntereinheiten des GDNF Rezeptors, Ret und p-Ret, besonders stark in den Dendriten exprimiert. Auch im Perikaryon und an der neuromuskulären Endplatte sind Ret und p-Ret lokalisiert, nicht jedoch im Axon. Im zweiten Teil der Arbeit wurde das durch neurotrophe Faktoren bedingte Neuritenwachstum genau quantifiziert. Dabei wurde zwischen einer Stimulation des Axon- bzw. des Dendritenwachstums differenziert. Die mit GDNF behandelten Dendriten werden etwa doppelt so lang wie die Dendriten, der mit BDNF oder CNTF behandelten Motoneurone. GDNF ist somit ein potenter Stimulator des Dendritenwachstums bei isolierten primären Motoneuronen. Dieser Befund korreliert gut mit der starken Expression von Ret und p-Ret in den Dendriten. Des Weiteren wurde eine Analyse der Interaktion der neurotrophen Faktoren mit dem glutamatergen AMPA Rezeptor in Hinblick auf das Neuritenwachstum durchgeführt. Dabei zeigte sich, dass die Interaktion zwischen neurotrophen Faktoren und dem AMPA Rezeptor besonders für das Dendritenwachstum von Bedeutung ist. Die klinische Bedeutung neurotropher Faktoren und deren Rezeptoren wird im dritten Teil der Arbeit dargestellt. Die pmn Maus ist ein Mausmodell für humane degenerative Erkrankungen des Motoneurons, wie der ALS und der SMA. Pmn Motoneurone, die mit BDNF oder GDNF kultiviert werden, weisen den charakteristischen axonalen Wachstumsdefekt der pmn Motoneurone auf und werden nur etwa halb so lang wie gesunde Kontrollmotoneurone. Bemerkenswerterweise führt die Behandlung der pmn Motoneurone mit CNTF zu einer kompletten Remission des axonalen Wachstumsdefekts, so dass die Axone eine normale Axonlänge erreichen. Auch die Anzahl der pathologischen axonalen Schwellungen werden in vitro durch CNTF stark reduziert. CNTF scheint demnach der interessanteste neurotrophe Faktor für eine Behandlung degenerativer Motoneuronerkrankungen zu sein. / Neurotrophins are important factors for many different functions of motoneurons, such as survival, neurite growth, as well as neuromuscular signalling. Neurotrophin receptors are therefore thought to be differently distributed in dendrites and axons. However, their precise localization and regulation in motoneurons were not well defined. This thesis characterized the exact subcellular localisation of the BDNF, CNTF and GDNF receptor subunits on adult and embryonic motoneurons. The BDNF und CNTF receptor subunits, gp130 and p-Stat3, are located in the perikaryon, in dendrites, in the axon as well as the growth cones and neuromuscular junctions of motoneurons. Immunofluorescent staining for the native forms (TrkB, gp130) is mainly found close to the membrane of the axon. In contrast, the activated forms (p-TrkB, p-Stat3) are mainly located inside the axon. GDNF receptor subunits Ret and p-Ret are highly expressed in the dendrites of motoneurons. In addition, Ret and p-Ret are also located in the perikaryon as well as the neuromuscular junction. Moreover, neurite outgrowth stimulated by neurotrophic factors was analyzed, differentiating axonal and dendritic growth. Primary motoneurons treated with GDNF grew dendrites which were twice as long as dendrites treated with BDNF or CNTF. Thus, GDNF is an important and potent stimulator of dendrite outgrowth in isolated primary motoneurons. This finding correlates well with the high expression of Ret and p-Ret in dendrites. On the other hand BDNF, CNTF and GDNF had equally potent effects on stimulating axonal growth. This thesis also characterized the interactions of neurotrophic factors with AMPA receptors regarding effects on neurite outgrowth. Interestingly, this interaction seems to be of greater importance for dendritic growth rather than axonal growth. The pmn mouse is a mouse model for neurodegenerative diseases of motoneurons, such as amyotrophic lateral sclerosis and spinal muscular atrophy. Pmn Motoneurons, which were cultured in presence of BDNF or GDNF, displayed the characteristic axonal growth deficiency as well as typical axonal swellings. The axon of these motoneurons reached only half the length of healthy control motoneurons. Surprisingly, treatment with CNTF rescued the pmn phenotype as the axons grew to the lengths of healthy control motoneurons. CNTF treatment also significantly reduced the number of pathological axonal swellings in vitro. Therefore CNTF seems to be the most promising therapeutic neurotrophic factor for treatment of neurodegenerative diseases of the motoneuron.

The Cache County Study on Memory in Aging (CCSMA) is a longitudinal population-based study which took place in Cache County, Utah. The study followed 5092 older-adult residents (aged 65+) for approximately 12 years to examine risk and protective factors for dementia. Participants completed dementia screening and follow-up assessments across four triennial visits. Additionally, researchers gathered information regarding demographics, reproductive history (e.g. age of menopause; hormone replacement therapy [HRT]) and other health-related factors, such as physical activity. Genotyping of DNA was completed for a genetic variation of genes for brain-derived neurotrophic factor (BDNF), a protein found in the brain associated with neuronal health and survival. Estrogen has been associated with cognitive health and has been shown to interact with BDNF in the brain to promote neuronal survival. The current research investigated the associations between estrogen, BDNF, and cognitive decline in older adult women from the CCSMA. An examination of how reproductive history, including the reproductive window (age of menarche to menopause) and use of HRT, affects the cognitive health of women in older adulthood can provide a clearer understanding of how estrogen exposure across the lifespan contributes to cognition in late life. This research can be helpful in determining the implications of events such as pregnancy, breastfeeding, surgical menopause and use of HRT on cognitive decline. Additionally, an investigation of how these reproductive factors interact with BDNF genetics is important to understand gene-by-environment interactions. The results of the current project demonstrated that increased lifelong estrogen exposure, both in the form of the reproductive window and HRT use, had small cognitive benefits for women in late life. Additionally, it was shown that women who initiated HRT use closer to menopause had increased cognitive status compared to those who initiated later. The specific BDNF gene under investigation was not associated with cognitive status in late life, neither was the interaction between BDNF and lifetime estrogen exposure. This research contributes to the discussion of sex-dependent factors of cognitive health and can help provide a better understanding late life cognitive decline.

The combination of bone morphogenetic protein 7 (BMP7) and neurotrophins (e.g. brain-derived neurotrophic factor, BDNF) protects septal neurons during hypoglycemic stress. I investigated the signaling mechanisms underlying this synergistic protection. BMP7 (5 nM) increased phosphorylation and nuclear translocation of BMP-responsive Smads 1/5/8 within 30 min in cultures of rat embryonic septal neurons. BDNF (100 ng/ml) enhanced the BMP7-induced increase in phospho-Smad levels in both nucleus and cytoplasm; this effect was more pronounced after a hypoglycemic stress. BDNF increased both Akt and Erk phosphorylation, but pharmacological blockade of these kinase pathways (with wortmannin and U0126, respectively) did not reduce the Smad phosphorylation produced by the BMP7+BDNF combination. Inhibitors of casein kinase II (CK2) activity reduced the (BMP7 + BDNF)-induced Smad phosphorylation, and this trophic factor combination increased CK2 activity in hypoglycemic cultures. These findings suggest that BDNF can increase BMP-dependent Smad phosphorylation via a mechanism requiring CK2. Preliminary results indicate that a cytoplasmic component robustly inhibits CK2. Protection of septal cholinergic neurons during a hypoglycemic stress is inhibited by a CK2 inhibitor and by a Phosphatidylinositol 3-kinase inhibitor, indicating that increases in CK2 activity and in Smad phosphorylation are only part on the protective mechanisms.

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