<p> Recent research has associated obesity with cognitive impairments and alterations in brain-derived neurotrophic factor (BDNF), with the majority of studies examining this in older adult or aging animals. To expand upon these efforts, two experiments were performed to examine the effects of diet-induced obesity (DIO) on spatial performance and hippocampal BDNF expression in young adult female and male rats. To expand upon these efforts, we examined these effects over dietary interventions of differing durations. To investigate these issues rats (female rats in Experiment 1 and male rats in Experiment 2) were assigned to either a standard chow condition or a DIO condition and remained on these diets for 4-, 8- or 12-weeks. Compared to their chow-fed counterparts, female DIO rats consumed significantly more average weekly calories, weighed significantly more, and exhibited significant alterations in glucose metabolism. However, these obesity-related physiological changes were not associated with concurrent impairments in spatial ability as measured using the Morris water maze, and only the 4-week DIO dietary intervention demonstrated a difference in hippocampal BDNF mRNA expression. Compared to their chow-fed counterparts, male DIO rats consumed significantly more average weekly calories than their chow-fed counterparts, weighed significantly more, and exhibited significant alterations in glucose metabolism. However, obesity-related physiological alterations were not associated with concurrent impairments in spatial ability or differences in BDNF mRNA expression, with the exception of the 12-week DIO animals performing significantly better than their chow-fed counterparts during the reversal probe trial on the final day of training. These findings were unexpected and will be discussed further later in the thesis. </p><p>

Prefrontal cortex (PFC) is critical to behavioral flexibility and, hence, the top-down control over bottom-up sensory information. The mechanisms underlying this capacity have been hypothesized to involve the propagation of alpha/beta (8-30 Hz) oscillations via feedback connections to sensory regions. In contrast, gamma (30-160 Hz) oscillations are thought to arise as a function of bottom-up, feedforward stimulation. To test the hypothesis that such oscillatory phenomena embody such functional roles, we assessed the performance of nine monkeys on tasks of learning, categorization, and working memory concurrent with recording of local field potentials (LFPs) from PFC. The first set of tasks consisted of two classes of learning: one, explicit and, another, implicit. Explicit learning is a conscious process that demands top-down control, and in these tasks alpha/beta oscillations tracked learning. In contrast, implicit learning is an unconscious process that is automatic (i.e. bottom up), and in this task alpha/beta oscillations did not track learning. We next looked at dot-pattern categorization. In this task, category exemplars were generated by jittering the dot locations of a prototype. By chance, some of these exemplars were similar to the prototype (low distortion), and others were not (high distortion). Behaviorally, the monkeys performed well on both distortion levels. However, alpha/beta band oscillations carried more category information at high distortions, while gamma-band category information was greatest on low distortions. Overall, the greater the need for top-down control (i.e. high distortion), the greater the beta, and the lesser the need (i.e. low distortion), the greater the gamma. Finally, laminar electrodes were used to record from animals trained on working memory tasks. Each laminar probe was lowered so that its set of contacts sampled all cortical layers. During these tasks, gamma oscillations peaked in superficial layers, while alpha/beta peaked in deep layers. Moreover, these deep-layer alpha/beta oscillations entrained superficial alpha/beta, and modulated the amplitude of superficial-layer gamma oscillations. These laminar distinctions are consistent with anatomy: feedback neurons originate in deep layers and feedforward neurons in superficial layers. In summary, alpha/beta oscillations reflect top-down control and feedback connectivity, while gamma oscillations reflect bottom-up processes and feedforward connectivity.

Methylphenidate (trade name: Ritalin) resulted in a conditioned place preference, but in contrast to work in juveniles, there were no sex differences. In addition, methylphenidate produced a significant decrease in the dopamine transporter compared to controls that may have implications towards development and plasticity of the dopamine system.

Social defeat is an ethologically relevant stressor that utilizes the natural establishment of social rank in male rodents and has been shown to be relevant to major depressive disorder (MDD) and post-traumatic stress disorder (PTSD). In the present study, we wished to establish a social defeat stress model in combination with the chronic unpredictable stress model, which is considered a mild stressor to the rodent. In this way, we create a “double hit” model that may more accurately mimic severe stress that is common in both MDD and PTSD. In the present study, residents established dominance over the intruder for 10 consecutive days. In addition, social defeat stress was followed by another stressor given at random times during each day, i.e. chronic unpredictable stress. These unpredictable stressors included 30 min restraint, 1 h shaking/crowding, a cold water swim, a warm water swim or a tipped cage for 24 h. In one cohort of animals, brain tissue was taken 24 h after the last stressor for DNA. In a second cohort, animals were tested on a sucrose preference test in which two bottles containing 0.8% sucrose was placed on their cages for 3 consecutive days (days 8-10 of social defeat stress), and the total amount of sucrose was calculated relative to total volume consumed. Brain tissue analyses revealed significantly elevated DNA oxidation in white matter comparing stressed animals to non-stressed controls, consistent with what has been found in post-mortem white matter from MDD subjects. Further, animals given the social defeat + chronic unpredictable stress demonstrated a deficit in sucrose preference, a natural reward, revealing that these animals were anhedonic as compared to controls. Stressed animals also demonstrated fear of the intruder in a social interaction test performed one day after the social defeat/chronic unpredictable stress was complete. Therefore, it appears that social defeat plus chronic unpredictable stress produces a phenotype relevant to clinical data in humans.

Neonatal quinpirole (QUIN; dopamine D2/D3 agonist) administered from postnatal days (P)1-21 results in an increase of dopamine D2 receptor sensitivity, similar to schizophrenia and is now an established rodent model of schizophrenia. The day after birth, male and female Sprague-Dawley rats were given a daily 1 mg/kg injection of either QUIN or saline from P1-21. One subset of these animals were behaviorally tested on PPI, referred to as first generation (F0). A different subset of animals were allowed to reach adult age (P60) and female and male QUIN-treated pairs from separate litters were bred. Their offspring were also used as subjects (F1 generation). Prepulse inhibition (PPI) is a measure of sensorimotor gating reduced in individuals diagnosed with schizophrenia. Trial types were defined as prepulse trials (73, 76, 82dB), startle stimuli trials (120 dB), or no stimulus (70 dB white noise; no prepulse or pulse). Animals were tested for six consecutive days and given an ip saline injection 10 min before testing, followed by testing for another six consecutive days and given an ip nicotine (0.5 mg/kg free base) or saline injection 10 min before testing. PPI testing for F0 generation animals occurred between P35-46, and testing for F1 generation animals occurred between P44-55. In one subset of generation F1 animals, rats were ip injected with a 0.1 mg/kg dose of quinpirole and immediately observed for 60 min and the number of yawns were recorded at P60. Yawning is a behavioral event mediated by the dopamine D2 receptor. Results revealed that neonatal QUIN resulted in PPI deficits throughout the six days of testing in the F0 generation regardless of the prepulse stimulus, but females demonstrated a less robust PPI deficit as compared to males. Nicotine given during the final 6 days of testing partially alleviated the PPI deficits in both males and females. The F1 generation also demonstrated PPI deficits, but the impairment was only in males and dissipated by day 6. Nicotine did not affect PPI in these animals. Finally, F1 generation rats demonstrated a robust increase in yawning compared to controls, demonstrating an increase in D2 receptor sensitivity. Brain tissue is being analyzed for changes in the dopamine D2 receptor signaling pathway.

During neural development, axons rely on extracellular cues to reach their target regions. Although extracellular signaling is one of the principal determinants for the growth of developing axons, only a small handful of known signaling cues has been identified. The existence of some 86 billion neurons of different subtypes, which ultimately form numerous functional circuits in the human nervous system, means an enormous number of extracellular cues would be required during development. Current views hold that even if more extracellular cues were to be discovered, they would never number large enough to account for the complexity of the human nervous system. Rather, intracellular signaling pathways and other cell-intrinsic mechanisms expand the ways in which a neuron can respond to extracellular cues by tuning the degree of responsiveness to them. Cell-intrinsic signaling pathways also give axons the ability to actively control their own development. These pathways can operate independently of the extracellular environment or even independently of the cell body, where the majority of protein synthesis takes place. For example, the local translation of proteins in the axon gives it autonomous control to immediately respond to changing demands in the environment. Local translation also occurs in other cell types, but the compartmentalized control over growth is especially important for neurons since the axon can extend up to a meter away from the cell body. In addition to local translation, axonally derived transcription factors, which can be locally synthesized in or localized to the axon, provide another means to control axon development. Axonally derived transcription factors act as physiological sensors and relay information about events happening in the periphery back to the cell body in order to effectuate a global response. It has recently been shown that transcription factors belonging to the OASIS family are activated by proteolysis in axons. Following their activation by proteolytic cleavage, the transcriptionally active N-terminus of these factors is transported to the cell body to activate global transcriptional pathways. For at least one OASIS family member, CREB3L2, this cleavage event simultaneously produces the C-terminus, which is capable of undergoing secretion. The secreted C-terminus of CREB3L2 acts as an accessory ligand for the activation of Hh pathways in chondrocytes. The generation of two bioactive proteins from one transcription factor, a transcriptionally active portion and a secreted portion, raised the question of whether there was a local function for OASIS transcription factors in axons. Through my research, I identified a mechanism in which DRG axons secrete the C-terminus of CREB3L2, which promotes axon growth in a paracrine manner. CREB3L2 is a transcription factor whose translation is induced by physiological ER stress. For CREB3L2 to be active, it must be cleaved by S2P, which I found is expressed in developing axons. Following proteolysis of CREB3L2 by S2P, the secreted C-terminus of CREB3L2 promotes the formation of Shh and Ptch1 complexes along axons. I found that upon depletion of the secreted CREB3L2 C-terminus, binding of Shh to the Ptch1 receptor is diminished. Returning the CREB3L2 C-terminus to the cultures exogenously was sufficient to rescue the formation of these complexes. These results highlight an intrinsic role for Shh signaling in developing DRG axons. Moreover, these results demonstrate how ER stress machinery is recruited to axons and promotes axon outgrowth. Finally, these results illustrate a novel, neuron-intrinsic mechanism by which developing axons actively regulate their own growth.

Alzheimer’s disease (AD) is a progressive, neurodegenerative disorder and the most common form of dementia. The hallmark pathologies of AD are extracellular aggregates of amyloid-beta, intracellular aggregates of microtubule associated protein tau and increased neuroinflammation. Current therapeutics offer only symptomatic relief and clinical trials investigating therapeutic benefits of non-steroidal anti-inflammatory drugs have yielded no positive results. Therefore, recent work has focused on immunomodulators, such as CD200 and fractalkine, as potential therapeutic targets for AD. Fractalkine (CX3CL1; FKN) is expressed as a transmembrane protein with an N-terminal chemokine domain followed by a long, mucin-like stalk. FKN can signal as a membrane-bound protein or, upon cleavage, as a soluble ligand (sFKN). Upon binding its receptor, FKN reduces expression of pro-inflammatory genes in activated microglia. Disrupting FKN signaling has been shown to exacerbate neurodegeneration in a number or neurodegenerative diseases. Relevant to this study, there have been conflicting reports on how FKN signaling affects AD pathology and whether a soluble FKN is beneficial or not. Here, we examine the ability of soluble FKN over expression to impact tauopathy and the resulting cognitive deficits in the rTg4510 mouse model of tauopathy, focusing on cognitive improvement after the onset of tau deposition. Furthermore, we explore the functional activity of proteolytic fragments of FKN on activated microglia in vitro to rectify the contradictory findings in the literature. We observed that sFKN over expression can significantly reduce both soluble and insoluble phospho-tau in both a preventative and an early interventional study design. However, in animals with significant pathology and neurodegeneration we did not observe an impact of sFKN over expression on tau pathology. Interestingly, in these late stage animals we did observe an improvement in spatial learning and memory as well as a reduction in hyperactivity. This suggests that earlier intervention would likely be most beneficial in reducing tau pathology but in late stage AD FKN signaling can still have benefits on cognition, likely due to reductions in the inflammatory milieu. Current publications suggest that different proteolytic fragments of FKN may have different functional signaling. Here we demonstrate that the this may be due to differences in receptor binding. sFKN (which includes the mucin-like stalk) exhibited a lower EC50 than the ckFKN (soluble chemokine domain), which leads to reduced functional efficacy of ckFKN at low concentrations. More interestingly, we also observed that high concentrations of FKN, regardless of cleavage variant, is ineffective at reducing pro-inflammatory activation of microglial and may in fact elicit a proinflammatory response. We hypothesize that FKN may signal through an alternative receptor at high concentrations, suggesting an as yet unidentified signaling pathway for FKN. Furthermore, we show that the ckFKN does not rescue pathology in the rTg4510 mouse, as sFKN does. These data may clarify conflicts in the literature and demonstrate that care must be taken with respect to in vitro and in vivo studies using FKN.

The microtubule associated protein, tau, is involved in regulating microtubule stability and axonal transport. When tau becomes hyperphosphorylated it can disassociate from the microtubules and start to aggregate. These tau aggregates are the hallmarks of many diseases known as tauopathies. The heat shock protein 90 kDa (Hsp90) chaperone network is highly involved in modulating client proteins, including tau. However, during aging and disease the Hsp90 chaperone network becomes highly imbalanced with some Hsp90/co-chaperone complexes increasing, while others are repressed. This imbalance in Hsp90/co-chaperone complexes could result in a worsening of tau pathology in Alzheimer’s disease. Hsp90 inhibition has been of interest as a potential therapeutic for tauopathies for many years. However, issues with toxicity and bioavailability have dampened enthusiasm for Hsp90 as a viable therapeutic target. Hsp90 co-chaperones are currently being investigated for as potential therapeutic targets for tauopathies, with the hope that targeting co-chaperones will lead to more specific targeting without toxicity. One co-chaperone that has the potential to become a therapeutic target for tauopathies is the activator of Hsp90 ATPase homolog 1 (Aha1). Aha1 is the only known stimulator of the ATPase of Hsp90, so targeting this particular co-chaperone could potentially mimic the effects of Hsp90 inhibition with more specificity. In this study we found that Aha1 enhanced Hsp90-mediated tau aggregation and increased insoluble tau accumulation in vitro. Additionally, a novel Aha1 inhibitor was able to reduce the formation of insoluble tau in vitro. We also investigated the effects of Aha1 overexpression in the rTg4510 mouse model, which is a tauopathy model that stably overexpresses the P301L mutation of tau. Overexpression of Aha1 in these mice increased the accumulation of insoluble and oligomeric tau. Furthermore, Aha1 overexpression led to cognitive deficits and neurotoxicity. Due to the effect of Aha1 overexpression on tau we wanted to investigate the effects of Aha1 knock-down in the rTg4510 mice. Incredibly, Aha1 knock-down led to reductions in pathological Gallyas silver positive tau tangles and was able to rescue neuronal loss. Overall, this work highlights Aha1 as an important regulator of tau pathology through Hsp90. The Hsp90/Aha1 complex could provide a novel therapeutic target for the treatment of tauopathies.

No description available.

<p>Recently discovered neuropeptides orexins (orexin-A and -B) act as endogenous ligands for G-protein-coupled receptors called OX₁ and OX₂ receptors. Our previous studies have established model systems for investigation of the pharmacology and signaling of these receptors in recombinant CHO cells. OX₁ receptor-expressing CHO cells were mainly utilized in this thesis.</p><p>Orexin-A and -B activate both OX₁ and OX₂ receptors. However, orexin-B is less potent in activating OX₁ receptors than orexin-A, whereas the peptides are equipotent on OX₂ receptors. We have performed mutagenesis on orexin-A to investigate the basis for this selectivity. We show that OX₂ receptor is generally less affected by the mutations and thus OX₂receptor appears to have less strict requirements for ligand binding, likely explaining the lack of difference in affinity/potency between orexin-A and orexin-B on OX₂ receptor.</p><p>The other studies focus on orexin receptor signaling. OX₁ receptors are shown to regulate adenylyl cyclase both in positive and negative manner, activate different MAP-kinases (ERK1/2 and p38) and induce cell death after long-lasting stimulation. Adenylyl cyclase regulation occurs likely through three different G-protein families, Gi, Gs and Gq. For ERK1/2, several downstream pathways, such as Ras, Src, PI3-kinase and protein kinase C (PKC) are implicated. OX₁ receptor-mediated activation of ERK is suggested to be cytoprotective whereas p38 MAP-kinase induces programmed cell death. </p><p>Three particularly interesting findings were made. Firstly, novel PKC δ (delta) is suggested to regulate adenylyl cyclase, whereas conventional and atypical PKCs are involved in activation of ERK. Secondly, adenylyl cyclase and ERK activation is fully dependent on extracellular Ca²⁺. Further experiments suggest that the previously discovered receptor-operated Ca²⁺ influx is not affecting the downstream effectors of orexin receptors but that it instead enables orexin receptors to couple to several signal cascades. Thirdly, upon inhibition of caspases, classical mediators of programmed cell death, OX₁receptor-mediated cell death is not reversed, but instead the pathways to death are altered so de novo gene transcription is no longer required for cell death.</p>