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1. Alex, Katherine D. 5-HT2C SEROTONIN RECEPTORS: CELLULAR LOCALIZATION AND CONTROL OF DOPAMINERGIC PATHWAYS IN THE RAT BRAIN.

Degree: PhD, Neurosciences, 2007, Case Western Reserve University

► Dopamine (DA) is known to play a role in the pathology and/or… (more)

▼ Dopamine (DA) is known to play a role in the pathology and/or treatment of schizophrenia, drug abuse and Parkinson’s disease. Serotonin (5-HT) is capable of modulating dopamine release through actions at 5-HT receptors. In particular, 5-HT2 receptor binding, and subsequent effects on DA release, may be involved in the efficacy of atypical antipsychotic drugs and recent work suggests that they may be promising targets for the treatment of depression, anxiety, obesity, and drug abuse as well. 5-HT2C receptors have been shown to tonically inhibit DA release in the striatum and the prefrontal cortex (PFC). The localization of the receptors that mediate these effects has not been studied. These data show that 5-HT2C receptors in the terminal region of the nigrostriatal pathway are, at least in part, responsible for the tonic inhibition of DA release in the striatum. In addition, data is presented that suggests that the mesocortical pathway is not modulated by 5-HT2C receptors in the terminal region. The cellular localization of 5-HT2C receptors has not been extensively studied, in part due to a lack of specific antibodies. Here, a selective 5-HT2C receptor antibody was used in immunofluorescence studies to examine the cellular localization of the 5-HT2C receptors that mediate the tonic inhibition of DA release in the brain. These studies show that 5-HT2C receptors do not colocalize with markers for parvalbumin-containing GABAergic cells in the cortex and hippocampus. Importantly 5-HT2C receptors show a high degree of colocalization with 5-HT2A receptors in these regions. 5-HT2A and 5-HT2C receptors are similar in structure and couple to the same intracellular signaling pathways upon activation. Differences in their levels of constitutive activity and desensitization in response to chronic ligand exposure have, however, been shown. Thus, by these mechanisms 5-HT2A and 5-HT2C receptors expressed in the same cortical and hippocampal pyramidal neurons may finely tune cortical efferents. The results presented here have implications for the development of new therapeutics for the treatment of diseases and disorders in which a 5-HT2 receptor-mediated manipulation of DA is beneficial. Advisors/Committee Members: Pehek, Elizabeth A.

Subjects: Biology, Neuroscience

Keywords: Schizophrenia; dopamine; striatum; hippocampus; 5-HT2A; depression

2. Balu, Ramani. Intrinsic and Synaptic Properties of Olfactory Bulb Neurons and Their Relation to Olfactory Sensory Processing.

Degree: PhD, Neurosciences, 2007, Case Western Reserve University

► The elucidation of how sensory information is represented in the brain is… (more)

▼ The elucidation of how sensory information is represented in the brain is a fundamental challenge in neuroscience. Numerous theories exist to explain the encoding of sensory perceptions by brain activity; however, the cellular mechanisms of sensory perception remain a mystery. Because of its stereotyped and relatively simple anatomy, the olfactory bulb represents an ideal system to study sensory coding. This project used cellular electrophysiological and optical imaging methods to investigate how local circuits within the olfactory bulb process information from olfactory sensory afferents to produce a coded representation of smell that is relayed to higher centers. First, I studied the intrinsic membrane currents in mitral cells—the principal output neurons of the olfactory bulb—that shape their response properties. Mitral cells have unique intrinsic electrophysiological properties that actively sculpt their responses to depolarizing and hyperpolarizing stimuli. One class of slowly inactivating voltage gated potassium currents (D-type) controls the generation of action potential clusters in response to depolarizing stimuli and ensures precise spiking in response to phasic depolarizations that mimic trains of olfactory sensory nerve mediated excitatory postsynaptic potentials (EPSPs). A different class of inactivating voltage gated potassium currents (A-type) regulates the ability of mitral cells to fire rebound action potentials in response to inhibitory postsynaptic potentials (IPSPs) and hyperpolarizing stimuli. These results suggest that the intrinsic electrophysiological properties of mitral cells actively regulate the temporal pattern of mitral cell action potentials during odor processing I next investigated the properties of excitatory glutamatergic inputs onto granule cells. Granule cells are the most common interneuron in the olfactory bulb and make reciprocal dendrodendritic synapses with mitral cells. These interneurons possess two functionally distinct classes of synapses that differ in their short term plasticity properties: dendrodendritic inputs from mitral cells that show prominent depression in response to trains of stimuli, and feedback inputs from the piriform cortex that strongly facilitate. These results suggest that oscillatory activity in the piriform cortex—a prominent feature of odor processing—may gate the activity of dendrodendritic inputs, and has important consequences for how feedback inhibition onto mitral cells is regulated. Advisors/Committee Members: Strowbridge, Ben W.

Keywords: Potassium Channel; Neuron; Synapse; Olfaction; Neural Computation; Synaptic Plasticity; Optical Imaging; Electrophysiology; Patch Clamp

3. Busch, Sarah Ann. Cellular and molecular strategies to overcome macrophage-mediated axonal dieback after spinal cord injury.

Degree: PhD, Neurosciences, 2009, Case Western Reserve University

► Spinal cord injury initiates the processes of inflammation and glial scar formation… (more)

▼ Spinal cord injury initiates the processes of inflammation and glial scar formation that ultimately lead to regeneration failure. Interestingly, injured axons not only fail to regenerate, they retract back from the original site of axotomy over time. This phenomenon, also known as axonal dieback, coincides with the infiltration of activated macrophages into the lesion center. Our laboratory has previously shown that activated macrophages directly induce retraction of dystrophic axons in an in vitro model of the glial scar. Here we utilized this model to investigate the basic biology underlying this phenomenon and discovered that several treatments which have been primarily thought to increase neuronal regeneration actually derive a portion of their positive effects from prevention of macrophage-mediated axonal retraction. Treatment with an inhibitor of matrix metalloproteinase-9 ameliorated retraction and identified a critical protease involved in macrophage-mediated axonal dieback in vitro. Digestion of the inhibitory proteoglycan substrate with chondroitinase ABC also prevented macrophages from inducing axonal retraction. Augmentation of the intrinsic growth potential of the neurons with a conditioning lesion prevented macrophage-mediated axonal dieback both in vitro and in vivo. The regenerating front of axons is typically found in areas containing large numbers of activated macrophages, despite the fact that these cells directly induce axonal retraction. In order to investigate this paradox, we analyzed the cellular components of the glial scar and found that in these areas devoid of reactive astrocytes, there is an association of injured axons with a population of cells expressing the precursor markers nestin, vimentin, and NG2 proteoglycan. The influence of NG2+ cells on regeneration is highly controversial, but our in vivo analysis suggests that these cells are permissive to injured axons. We observed the interactions between dystrophic adult sensory neurons and primary NG2+ cells derived from the adult mouse spinal cord and found that NG2+ cells expressed high levels of growth-promoting molecules and were able to stabilize axons undergoing macrophage-mediated axonal dieback. Overall, we have identified several cellular and molecular components of the glial scar that may be critical targets for preventing the phenomenon of macrophage-mediated axonal dieback in the injured CNS. Advisors/Committee Members: Silver, Jerry.

Subjects: Biology; Neurology

Keywords: spinal cord injury, axonal injury, macrophage, growth cone, dystrophy, conditioning lesion, axonal dieback, NG2, proteoglycan

4. Cai, Xiao-Dan. Altered Production of Aβ by Mutations of the Amyloid Protein Precursor Linked to Familial Alzheimer’s Disease.

Degree: PhD, Neurosciences, 1994, Case Western Reserve University

► Alzheimer's disease (AD) is a neurodegenerative disease characterized by neuronal loss, neurofibrillary… (more)

▼ Alzheimer's disease (AD) is a neurodegenerative disease characterized by neuronal loss, neurofibrillary tangles, and amyloid plaques. The principal component of the amyloid plaques is Aβ, a peptide of 39-43 amino acid residue, which is derived from an integral membrane protein, amyloid β protein precursor (βAPP). The discoveries of mutations in the βAPP gene that cosegregate with the AD phenotype (FAD) have provided strong evidence that amyloid deposition plays a critical role in the development of AD, though how these mutations cause AD remains to be answered. I have focused my study on the pathogenic mechanism of those mutations of βAPP gene linked to FAD. By showing that the βAPPΔNL mutation increases Aβ production six-fold, I provided strong evidence for the first time that this mutation caused AD because its processing was altered in a way that increased the production of Aβ, thereby promoting amyloid deposition. However, my initial study of βAPPΔIle showed that it did not increase the total amount of Aβ production. Based on published in vivo and in vitro data suggesting that the C-termini of Aβ are heterogeneous and that longer Aβ forms aggregate more rapidly, we hypothesized that the C-terminal mutations might promote amyloid deposition by favoring the genesis of longer Aβ. In collaboration with Toby Cheung, and later, Dr. Suzuki, we tested the hypothesis and provided evidence that βAPPΔIle and βAPPΔPhe increased the proportion of Aβ1-42 when compared to βAPP wild type. To study the mechanism by which βAPPΔNL promotes Aβ production, I further characterized the proteolytic processing of βAPPWT and βAPPΔNL both in full length and shortened constructs. My results indicate that βAPPΔNL promotes Aβ production by favoring the cleavage at the N- terminal of Aβ over the one within Aβ sequence. In order for cells to produce Aβ, a cleavage C-terminal to Aβ, a site located within the transmembrane domain of βAPP, must occur. Until now, no model of βAPP processing can explain how this can happen. Based on the principles of cell biology, I will try to integrate the process of autophagocytosis into βAPP processing, and to propose a hypothesis which emphasizes topological transposition and degradation of membrane as prerequisites for Aβ genesis. I will also discuss possible strategies to test the hypothesis. Advisors/Committee Members: Younkin, Steven G.

Subjects: Biology, Neuroscience

Keywords: Alzeimer's disease; Amyloid-protein precursor; Mutation

5. Cheng, Ling. MOLECULAR MECHANISM OF L1CAM FUNCTION: AXON GROWTH AND GUIDANCE.

Degree: PhD, Neurosciences, 2004, Case Western Reserve University

► The neural cell adhesion molecule L1 is critical for human neural development,… (more)

▼ The neural cell adhesion molecule L1 is critical for human neural development, so defining the molecular basis for different aspects of L1 function in normal and abnormal development was the goal of my thesis. L1-L1 and L1-integrin binding are abolished in a new mutant mouse strain, L1-6D mice, where the 6th Ig domain of L1 is deleted. The loss of L1-integrin and L1-L1 binding doesn’t affect the formation of axon pathways in the CNS but causes hydrocephalus. L1 interactions with neuropilin and semaIIIa signaling were preserved in L1-6D mice. It is possible that some axon guidance defects in the L1-KO mice are due to loss of L1-neuropilin interactions and disruption of semaIIIa signaling, rather than due to a loss of L1-L1 homophilic binding. A major goal of my thesis is to investigate how L1-L1 homophilic interactions stimulate neurite outgrowth. By growing transfected L1 knockout cerebellar neurons on an L1 substrate, we are able to compare neurite length and branching number between full-length L1-transfected neurons and the L1 mutant-transfected neurons. Using extracellular pathological missense mutations, we found several mutations disrupt neurite branching and the results can’t be simply explained by homophilic binding or expression level. Our data suggests that a coreceptor is required for L1-mediated neurite outgrowth. Using L1 intracellular mutations, we demonstrate that the L1CD is not required for neurite outgrowth but it plays an important role in neurite branching. The juxtamembrane region of L1CD is especially important for the regulation of branching, presumably through the L1-ERM binding site. We propose that L1 on the cell surface binds to the substrate L1 in a homophilic fashion and then recruits a coreceptor by cis binding. The coreceptor can transduce signals independent of the L1CD. The L1CD, however, plays a significant regulatory role in axon branching. My thesis work has identified a novel L1-binding protein, RanBPM by yeast two-hybrid screening. We demonstrate that the L1-RanBPM interacts in vitro and in vivo and the N-terminal of RanBPM is sufficient for the interaction with L1. By directly binding to Sos, RanBPM seems to act as the adaptor protein for L1 to activate MAPK pathway. Advisors/Committee Members: Lemmon, Vance.

Subjects: Biology, Neuroscience

Keywords: L1; NEURITE; RANBPM; NEURITE OUTGROWTH; OUTGROWTH; L1CD; ADHESION MOLECULE

6. Chiang, Elizabeth C. INVESTIGATION OF THE ELECTROPHYSIOLOGICAL PROPERTIES OF THE MAJOR CELL TYPES IN THE RAT OLFACTORY TUBERCLE.

Degree: PhD, Neurosciences, 2008, Case Western Reserve University

► Olfactory information is processed by a diverse group of interconnected forebrain regions.… (more)

▼ Olfactory information is processed by a diverse group of interconnected forebrain regions. Most efforts to define the cellular mechanisms involved in processing olfactory information have been focused on understanding the function of the olfactory bulb, the primary second-order olfactory region, and its principal target, the piriform cortex. However, the olfactory bulb also projects to other targets, including the rarely-studied olfactory tubercle, a ventral brain region recently implicated in regulating cocaine-related reward behavior. We used whole-cell patch clamp recordings from rat tubercle slices to define the intrinsic properties of neurons in the dense and multiform cell layers. We find three common firing modes of tubercle neurons: regular-spiking, intermittent-discharging and bursting. Regular-spiking neurons are typically spiny-dense-cell-layer cells with pyramidal-shaped, dendritic arborizations. Intermittently-discharging and bursting neurons comprise the majority of the deeper multiform layer and share a common morphology: multipolar, sparsely-spiny cells. Rather than generating all-or-none stereotyped discharges, as observed in many brain areas, bursting cells in the tubercle generate depolarizing plateau potentials that trigger graded but time-limited intermittent discharges. We find two distinct subclasses of bursting cells that respond similarly to xv step stimuli but differ in the role transmembrane Ca currents play in their intrinsic behavior. We also created a numerical model of the bursting cell to examine the currents that allow the unique bursting pattern of firing. We varied the currents until we found a response that was similar to that of recordings. Then we ran simulations to test what the response of the model neuron in environments mimicking Cs, low Ca, and TTX. We also looked at the response to brief stimuli and two pulse stimuli. Experiments and the mathematical model both lead us to assert that the role of calcium in nonregenerative bursting tubercle neurons appears to be to decrease excitability by triggering Ca-activated K currents. Nonregenerative bursting cells exhibit a prolonged refractory period following even short discharges suggesting that they may function to detect transient events. Advisors/Committee Members: Strowbridge, Ben W.

Keywords: rat olfactory tubercle; electrophysiology; patch-clamp; major cell types; cocaine; bursting; regular-spiking; intermittent-discharging

7. Cramer, Paige E. Nuclear Receptor Activation and Alzheimer's Disease Pathogenesis.

Degree: PhD, Neurosciences, 2012, Case Western Reserve University

► Alzheimer’s disease (AD) is a progressive neurodegenerative disorder, characterized by the presence… (more)

▼ Alzheimer’s disease (AD) is a progressive neurodegenerative disorder, characterized by the presence of β-amyloid (Aβ) plaques and neurofibrillary tangles, and is accompanied by a robust inflammatory response that clinically presents as the progressive loss of cognition and memory, ultimately leading to death. The disease is associated with a disruption of Aβ homeostasis and it’s deposition in the brain, which initiates a microglial-mediated immune response that produces pro-inflammatory cytokines and reactive nitrogen and oxygen species. Using ibuprofen, a peroxisome proliferator activated receptor γ (PPARγ) agonist and non-steroidal anti-inflammatory (NSAID), we demonstrate that chronic ibuprofen treatment reduces Aβ burden by 90% in the parenchyma and prevents microglia, the primary producers of reactive oxygen and nitrogen species, from forming the NADPH oxidase. Ibuprofen prevents the phosphorylation of Vav and consequently RAC1 translocation to the plasma membrane, preventing NADPH oxidase assembly, thus inhibiting the release of superoxide radicals. Consequently, we report less oxidative damage in the brains of mice treated with ibuprofen. We further extend our studies to assess the mechanism of Aβ removal from the brain and the behavioral outcomes in mouse models of AD, by using a retinoid X receptor (RXR) selective, FDA approved agonist, bexarotene. We show that bexarotene utilizes its ability and obligation to heterodimerize with Liver X Receptor (LXR) to induce the expression of LXR response element genes, apolipoprotein E, ABCA1 and ABCG1, and elevating HDL levels in the brains of treated AD mice. We show that following 1 dose of bexarotene, soluble Aβ levels are reduced for up to 84 hours. We show that after just 72 hours of bexarotene treatment, the plaque burden is reduced by 50%. The consequence of reducing soluble Aβ levels is correlative to ameliorating the AD-related behavioral deficits in three different AD mouse models and reversing a neural circuit deficit. Together our data demonstrate the important roles for nuclear receptors in preventing the inflammation associated with AD and facilitating clearance of Aβ species. These data may provide a novel therapeutic strategy for the treatment of the neurodegenerative disease and its prodromal states. Advisors/Committee Members: Landreth, Gary.

Subjects: Neurosciences

Keywords: Retinoid X Receptor, Alzheimer's disease, amyloid beta, nuclear receptor, Liver X Receptor, Peroxisome Proliferator Activated Receptor gamma

8. Cruise, Bethany Ann. A NOVEL ROLE FOR ACTIVIN IN WOUND HEALING AND PSORIASIS: INDUCTION OF A SENSORY NEUROPEPTIDE.

Degree: PhD, Neurosciences, 2004, Case Western Reserve University

► Sensory neurons of the dorsal root ganglia (DRG) are essential for successful… (more)

Subjects: Biology, Neuroscience

Keywords: activin; sensory neurons; calcitonin gene-related peptide; skin wound; dorsal root ganglion; psoriasis; nerve growth factor

9. Ensslen, Sonya Emily Lesya. The role of signaling via the receptor tyrosine phosphatase PTPmu in retinal development and axon guidance.

Degree: PhD, Neurosciences, 2004, Case Western Reserve University

► Development of the vertebrate visual system requires the formation of a precisely… (more)

Keywords: PTPÂµ; neurite; RGC; growth cone; retina; PKCÎ´; nasal

10. Fan, Guoping. Developmental regulation of catecholaminergic phenotypic expression in primary sensory neurons.

Degree: PhD, Neurosciences, 1995, Case Western Reserve University

► One important aspect of neural development is the differentiation of neurotransmitter phenotypes… (more)

▼ One important aspect of neural development is the differentiation of neurotransmitter phenotypes in developing neurons. The studies described in this dissertation explore mechanisms of transmitter differentiation using catecholaminergic (CA) phenotypic expression in sensory neurons as a model system. The results show that both environmental factors as well as intrinsic neuronal properties play key roles in regulating sensory CA phenotypic expression. Specifically, ganglionic non-neuronal cells (NNC) inhibit expression of the CA-biosynthetic enzyme tyrosine hydroxylase (TH) in cultured sensory neurons. The effect of NNC is partially mediated by a NNC-derived cytokine, Leukemia Inhibitory Factor (LIF). The detection of LIF and LIF receptor (LIFR) mRNAs in vivo suggests that LIF may be one factor that modulates CA potential in sensory neurons in vivo. In contrast to the inhibitory effect of LIF, depolarizing conditions up-regulate TH expression in cultured embryonic and newborn sensory neurons. These results raise the possibility that membrane depolarization during this critical period of development plays a role in stimulating CA differentiation in subsets of sensory neurons. Moreover, the effects of depolarization and LIF are antagonistic, suggesting that mature levels of CA expre ssion are regulated by a balance of positive and negative regulatory signals. Regulation of intrinsic neuronal properties, such as responsiveness to environmental influences, could be another general mechanism of neurotransmitter regulation. For example, nodose ganglion (NG) neurons lose their LIF responsiveness between embryonic and newborn stages, coincident with the development of a CA phenotype in a subset of NG neurons in vivo. This result raises the possibility that modulation of LIF responsiveness may contribute to stable, phenotypic TH expression in a subset of nodose neurons during late fetal development. To examine whether LIF inhibits sensory TH expression in vivo, I compared sensory TH expression in LIFR and LIF knockout mice with wildtype mice. TH cell numbers appeared unchanged in LIFR and LIF knockout mice compared to controls, indicating that the LIF system is dispensable for the normal patterning of TH expression in sensory neurons in vivo. These data suggest either (1) the loss of LIF function can be compensated for by other TH inhibitory factor(s), or (2) LIF may only be involved under culture conditions. In conclusion, my data indicate that spatial restriction of CA phenotypic expression in subsets of adult sensory neurons arises through modulation, by positive and negative extrinsic regulatory factors, of a widespread CA potential, as well as from the regulation of intrinsic neuronal responses to these environmental factors during development Advisors/Committee Members: Katz, David M.

Subjects: Biology, Neuroscience

Keywords: Catecholamines; Phenotype expression; Sensory neurons, primary

11. Fox, Stephanie R. Engrailed is Required in Maturing Serotonin Neurons to Regulate the Cytoarchitecture and Survival of the Dorsal Raphe Nucleus.

Degree: PhD, Neurosciences, 2012, Case Western Reserve University

► Serotonin (5-HT) neuron development and signaling output is driven by a regulatory… (more)

▼ Serotonin (5-HT) neuron development and signaling output is driven by a regulatory transcriptional network. Alterations in the development and function of the 5-HT system via the transcription factor network can alter mouse behavior and physiology. While there is increasing understanding of the factors involved, there are still many important factors to identify. Analysis of constitutive Engrailed (En) null mice implicated the two homeobox paralogs in the development of 5-HT neurons; however, it is unclear whether En play intrinsic roles. En1 and En2 are expressed in maturing 5-HT neurons that will form the dorsal raphe nucleus (DRN) and dorsal part of the median raphe nucleus. En1 expression in 5-HT neurons continues in adulthood, while En2 expression diminishes by E17.5. Conditional targeting of En1/2 in 5-HT neurons after neurogenesis identified intrinsic roles in 5-HT neuron maturation. En1/2 are necessary for the formation of the DRN cytoarchitecture, apparently through regulation of 5-HT neuron secondary migration. Furthermore, En1/2 are necessary for perinatal maintenance of 5-HT neuron identity and postnatally for maintenance of 5-HT levels and 5-HT neuron survival. Analysis of an En allelic series revealed that En1 is the dormant functional paralog in maturing 5-HT neurons, while En2 contributes prior to its downregulation. Therefore, there are important intrinsic roles for En1/2 in 5-HT neuron development. The Pet-1 transcription factor is an important determinant for 5-HT neuron development and maintenance. The Pet-1-/- model has been used to study 5-HT system development and function using combinations of C57BL/6, 129/Sv and SJL backgrounds. Variations in the 5-HT system or in the interaction between 5-HT system dysfunction due to Pet-1-targeting and genetic modifiers in the background could cause inconsistencies in phenotype and confusion over 5-HT system or Pet-1 function. Indeed, conflicting anxiety behavioral data has arisen, potentially due to genetic background. Congeneric C57BL/6- and SJL-Pet-1 lines were generated and compared to the original 129/Sv*C57BL/6-Pet-1 line. Genetic background did not obviously alter the distribution of 5-HT neurons or brain 5-HT. However, differences in newborn 5-HT metabolism and adult blood 5-HT levels suggest differences in 5-HT synthesis, release, reuptake and/or degradation, which could alter brain development and 5-HT function. Advisors/Committee Members: Deneris, Evan.

Subjects: Biomedical Research; Developmental Biology; Genetics; Molecular Biology; Neurosciences

Keywords: Engrailed1; Engrailed 2; Pet-1; transcription factor; serotonin; development

12. Fuller, Molly Lynn. The Role of Bone Morphogenetic Proteins in Reactive Gliosis after Demyelinating Spinal Cord Lesions.

Degree: PhD, Neurosciences, 2007, Case Western Reserve University

► The response of glial cells to injury of the adult central nervous… (more)

▼ The response of glial cells to injury of the adult central nervous system (CNS) leads to formation of a glial scar, a dense network of astrocytes and extracellular matrix molecules. While the scar plays a protective role by creating a barrier between the CNS parenchyma and external environment, it is also one of the major factors leading to incomplete repair of tissue architecture and function. In this thesis, I examine the importance of bone morphogenetic proteins in glial cell reactivity after demyelinating injury. Using the model system of the dorsal columns of the rat thoracic spinal cord, I examined the acute behaviors of astrocytes and NG2+ glial precursors after LPC-induced demyelinating lesions. At three days after injection, levels of BMP4 and BMP7 are increased in the lesion, and the majority of cells contained phosphorylated Smad 1/5/8 in their nuclei. Expression of glial fibrillary acidic protein (GFAP) is increased in astrocytes at the lesion edge and expression of NG2 is increased on cells filling the core of the lesion. Vimentin is up-regulated in both of these populations. Cultures of mature astrocytes or NG2+ glial cells were treated with BMP4 or BMP7 and the quantity of chondroitin sulphate proteoglycans (CSPGs) were found to be increased at both the protein and RNA levels. Astrocytes doubled the mRNA levels for CSPG core proteins aggrecan and neurocan after one day of BMP treatment and displayed a maximum of 4-fold increase in CSPG protein after 4 days of treatment. NG2+ cells increased their aggrecan mRNA levels 14-fold and neurocan and versican levels 2-fold after 3 days of BMP4 treatment. BMP7 caused similar but smaller changes. These data link BMP signaling to glial scar formation after demyelination. The increase of CSPGs by BMPs is an event that is likely to inhibit migration of oligodendrocyte precursors and extension of regenerating neurites through the area of damage. Additionally, remyelination requires the recruitment of glial precursors and differentiation of those precursors into oligodendrocytes capable of myelination. Evidence of active BMP signaling in these cells, as shown here, would likely lead to the differentiation of precursors into astrocytes rather than oligodendrocytes. This work reveals BMPs as a possible therapeutic target for limiting glial scarring and optimizing the area of damage for remyelination. Advisors/Committee Members: Miller, Robert H.

Subjects: Biology, Neuroscience

Keywords: BMP; CSPG; glial scar; demyelination; CNS

13. Gao, Yuan. Mechanisms of Synaptic Plasticity in the Rat Olfactory Bulb.

Degree: PhD, Neurosciences, 2010, Case Western Reserve University

► Understanding the general principles of information coding that are used by the… (more)

▼ Understanding the general principles of information coding that are used by the brain to examine the external world is a central challenge for neuroscientists. The olfactory bulb represents an ideal model for understanding the information coding in sensory systems because of its highly organized and stereotyped anatomy. In addition, the olfactory bulb merits the study of the long-term plasticity, since its cellular roles in the olfactory learning and memory behavior remains a mystery. This thesis project used electrophysiological and novel optical imaging methods to investigate the cellular mechanisms underlying the olfactory learning and memory. Using two-photon guided focal stimulation spike timing-dependent plasticity (STDP) could be induced at the granule cell proximal synapse, which was presumably due to the feedback projection from the piriform cortex. The positive STDP protocol potentiated proximal inputs, but depressed distal inputs to granule cells. Granule cell excitatory postsynaptic potential and mitral cell inhibition were also potentiated by theta-burst stimulation in the granule cell layer. The above results provided a potential cellular basis for olfactory learning and memory.Then I investigated the source of excitatory input on mitral cells in the main olfactory bulb. Although most previous studies in the olfactory system are based on the premise that the sensory afferent in the glomeruli is the only source of excitatory input for mitral cells, this has not been rigorously tested. In my thesis work, I found that depolarization in mitral cells could be evoked by extracellular stimulation in the granule cell layer in dissected olfactory bulb slices that have no glomerular layer. This depolarization evoked in these mini slices was sensitive to AMPA receptor antagonist NBQX, TTX, and gap junction blocker carbenoxolone. Intracellular fills of mitral cells with lucifer yellow often showed small processes apparently connected to somata or secondary dendrites. These results are consistent with the presence of gap junctions between mitral cell lateral dendrites and processes of the tufted cells. The possible tufted-mitral cell excitatory pathway may enable mitral cells to respond to sensory input into “off-beam” glomeruli, in addition to the receptor neurons that drive EPSPs on the glomerulus innervated by their apical dendrites. Advisors/Committee Members: Strowbridge, Ben.

Subjects: Biomedical research

Keywords: olfactory bulb, granule cell, mitral cell, inhibition, excitation

14. Garner, Andrew Seyfarth. The regulation of avian TrkB and TrkC receptor function by alternative splicing.

Degree: PhD, Neurosciences, 1996, Case Western Reserve University

► The neurotrophins are a family of structurally and functionally related growth factors… (more)

▼ The neurotrophins are a family of structurally and functionally related growth factors that activate the Trk family of receptor tyrosine kinases. Models of neurotrophin function have been primarily based on the role of nerve growth factor in peripheral nervous system development, where it acts as a target-derived survival factor. Although neurotrophins are also expressed in the central nervous system (CNS), their role in the survival of CNS neurons is unclear. However, recent work in vitro has demonstrated that the neurotrophins elicit diverse cellular responses, suggesting that they may regulate multiple events in the development and function of the CNS. To explore the possible roles and regulation of neurotrophin signaling during development, the expression patterns an functional capabilities of TrkB and TrkC receptor isoforms were characterized. RT/PCR demonstrated that a variety of TrkB receptor isoforms are expressed in the avian visual system, a widely studied model of CNS development. The expression pattern of TrkB in the retina is consistent with brain-derived neurotrophic factor (BDNF) being a tectal-derived survival factor retinal ganglion cells (RGCs). However, the expression of kinase-containing transcripts in the tectum suggests the principle target of RGCs is also BDNF responsive. Since BDNF is synthesized in both the retina and tectum and is anterogradely transported by RGCs, BDNF may also act in an anterograde, retinotectal manner or locally as a paracrine/autocrine factor within the retina or tectum. TrkB and TrkC receptor isoforms were expressed in non-neuronal and neuron-like cells to assess receptor function. A deletion in the extracellular domain of TrkB eliminated neurotrophin-3 (NT3) induced receptor phosphorylation in fibroblasts, indicating that alternative splicing can regulate ligand specificity. The insertion of 25 amino acids in the kinase domain of TrkC eliminated NT3-induced survival, but had no apparent effect on transformation or differentiation, demonstrating that alternative splicing can regulate neurotrophin-induced cellular responses as well. This dissertation identifies alternative splicing as an important regulator of receptor function, and provides a molecular basis for the pleiotropic actions of the neurotrophins Advisors/Committee Members: Large, Thomas H.

Subjects: Biology, Neuroscience

Keywords: Alternative splicing; TrkB, TrkC receptors

15. Gutierrez, Davina V. REVERSIBLE MODIFICATIONS IN MOTOR OUTPUT FOLLOWING PURKINJE NEURON PHOTOSTIMULATION.

Degree: PhD, Neurosciences, 2011, Case Western Reserve University

► A central goal in biology has focused on the ability to manipulate… (more)

▼ A central goal in biology has focused on the ability to manipulate a defined population of cells within a model to determine the correlation between modulation and functional output. Ultimately, the technique utilized will ensure that the level of control will be precise, temporally realistic, genetically tangible and reversible. Devising a procedure that can modify a defined group of functionally related neurons both reversibly and rapidly creates a scenario where one can examine the effects of suppression or enhancement of neuro- and synaptic transmission and could establish a correlation between an electrophysiological outcome to a behavioral phenotype. While numerous innovative approaches have attempted to establish a link between cellular activity and development, plasticity and behavior by modulating a specific group of neurons, studies were often confronted with a multitude of limitations that were intrinsic to the experimental approach. Classical methods faced obstacles in either kinetic, construct or pharmacological design that often rendered the targeted cellular population irreversibly or incompletely modified. The discovery and utilization of light-sensitive opsins, coupled with traditional genetic approaches and innovation within the optogenetic field have revealed that the previously encountered spatial, temporal and reversibility restraints are essentially eliminated. Taking this into consideration, we hypothesized that the targeted expression and light activation of vertebrate rhodopsin (vRh) within the cerebellum would not only alter the characteristic, cellular firing pattern but would also exert a visible and distinct change in motor behavior. In order to examine the physiological and behavioral effects of vRh expression and photoactivation in vivo, we had to first establish a transgenic mouse line whereby the expression of vRh was exclusively driven to Purkinje neurons. Subsequent steps included the development of a surgical procedure that would allow for the targeted delivery of light to the cerebellar vermis via an optrode as well as the in vivo electrophysiological and behavioral outcomes of light application in both transgenic and wild type littermates. Our results revealed that vRh expression was restricted to the soma and proximal dendrites of Purkinje neurons in a punctate pattern. The in vivo data revealed that light activation of vRh in vermal Purkinje neurons significantly reduced the firing frequency in a manner similar to baclofen application. Furthermore, we also discovered that a brief pulse of light (26 sec) sufficiently induced a loss of motor coordination and balance in positive transgenic mice. Advisors/Committee Members: Herlitze, Stefan.

Subjects: Neurosciences

16. Han, Jing. ROLE OF THE REGULATOR OF G PROTEIN SIGNALING 2 (RGS2) FOR NEURONAL AND SYSTEM FUNCTION.

Degree: PhD, Neurosciences, 2007, Case Western Reserve University

► RGS2, one of the small members of the regulator of G protein… (more)

Subjects: Biology, Neuroscience

Keywords: RGS2; serotonergic; neurons; Gi/o; serotonergic neurons; RGS; PROTEIN

17. Hanson, Martin Gartz Jr. THE EMBRYONIC NEURAL CIRCUIT: MECHANISM AND INFLUENCE OF SPONTANEOUS RHYTHMIC ACTIVITY IN EARLY SPINAL CORD DEVELOPMENT.

Degree: PhD, Neurosciences, 2004, Case Western Reserve University

► Over the last several decades, a general consensus has emerged that molecular… (more)

Subjects: Biology, Neuroscience

Keywords: motoneurons; locomotion; guidance; mouse; chick; spontaneous; circuits; cholinergic; glycine; GABA; PSA; EphA4

18. Hawthorne, Alicia Lynn. The Development and Regeneration of the Serotonergic System.

Degree: PhD, Neurosciences, 2010, Case Western Reserve University

► Serotonergic fibers project throughout the central nervous system (CNS). Since the expression… (more)

▼ Serotonergic fibers project throughout the central nervous system (CNS). Since the expression of 5-hydroxytryptamine (5-HT) beings after the completion of migration from the germinal zone, the early migration of serotonergic neurons has not previously been studied. Two main modes of radial neuronal migration are radial glial-guided locomotion and somal translocation. Radial glial-guided locomotion has dominated as the major mode of migration within the CNS, by which radial glia act as a scaffold for migrating neurons. In somal translocation, the neurons themselves are radial and migrate by moving their cell body through their pial process. We studied the migration of serotonergic neurons by utilizing mice that have serotonergic neurons specifically labeled with enhanced yellow fluorescent protein (EYFP) under control of the Pet-1 enhancer/promoter region (ePet-EYFP), which turns on after a serotonergic neuron has differentiated but before the neuron expresses 5-HT. Both histologically and in living slice cultures, we show that E10.5-E11.5 serotonergic neurons migrate by somal translocation through areas of high chondroitin sulfate proteoglycan (CSPG) and toward regions of laminin. The intracellular motors non-muscle myosin II, kinesin, and dynamin were inhibited pharmacologically, but only dynamin slowed translocation, demonstrating for the first time that neurons use dynamin during migration. In the adult, serotonergic neurons have an enhanced capacity for sprouting after injury, but have never been investigated in an ischemic brain lesion. We used a focal cortical thermocoagulatory lesion to study the response of serotonergic and callosal projection fibers after injury. Callosal fibers became dystrophic and died back and after injury, whereas serotonergic fibers persisted and sprouted in the lesion among high levels of CSPG and laminin. Using P3-4 ePet-EYFP serotonergic neurons or cortical neurons from EYFP-negative littermates, growth cones were compared in vitro on substrates of low laminin with or without high CSPG. Serotonergic growth cones maintained a more active growth state when challenged with CSPG, while cortical growth cones collapsed. Serotonergic neurites were also longer when grown on CSPG alone. Higher levels of GAP-43 and beta1 integrin expression may explain this robust growth. The mechanisms responsible for serotonergic sprouting may lead to therapies for both brain and spinal cord injury. Advisors/Committee Members: Deneris, Evan.

Subjects: Biology; Biomedical research; Cellular biology; Neurology; Scientific imaging; Surgery

Keywords: serotonin; 5-HT; migration; somal translocation; chondroitin sulfate proteoglycan; ischemia; sprouting; glia; GAP-43; beta1 integrin; laminin; dynamin; non-muscle myosin II; kinesin; slice culture; Pet-1; growth cone

19. Henion, Paul Dean. Developmental regulation of neuropeptide expression in sympathoadrenal derivatives of the neural crest.

Degree: PhD, Neurosciences, 1991, Case Western Reserve University

► The choice and regulation of neurotransmitter phenotype is of critical importance for… (more)

Subjects: Biology, Neuroscience

Keywords: Neuropeptides; Neural crest, sympathoadrenal derivatives

20. Horn, Kevin Paul. An Investigation of the Role of Macrophages in Spinal Cord Injury and Repair.

Degree: PhD, Neurosciences, 2010, Case Western Reserve University

► The role of macrophage infiltration following spinal cord injury is a highly… (more)

▼ The role of macrophage infiltration following spinal cord injury is a highly controversial topic as research has demonstrated both beneficial and detrimental effects of these cells on regenerating neurons. Cajal observed that injured axons of the adult CNS retract extensively from the initial site of axotomy. Macrophage infiltration correlates spatio-temporally with this deleterious phenomenon, but the direct involvement of these inflammatory cells has not been demonstrated. Here, we examined the role of macrophages in axonal retraction within the dorsal columns following spinal cord injury in vivo and found that retraction occurred concomitantly with macrophage infiltration and that the ends of injured axons were associated with these ED-1+ cells. Interestingly, depletion of infiltrating macrophages resulted in a significant reduction in axonal retraction. We used time-lapse imaging to observe the interactions of adult dorsal root ganglion neurons with macrophages in an in vitro model of the glial scar. We found that adhesive contacts and considerable physical interplay between macrophages and dystrophic axons led to extensive axonal retraction. These data are the first to indicate that activated macrophages can directly induce axonal retraction by physical cell-cell interactions. Other work in the field shows that the presence of activated macrophages induces axonal sprouting and growth into the lesion penumbra. Therefore, we also examined the effects of soluble macrophage-derived factors on neurons in the absence of cell-cell contact using the same in vitro spot gradient model. Macrophage-conditioned media significantly increased neuronal adhesion and growth. Surprisingly, conditioned media from activated macrophages had a similar effect on neuronal adhesion, but did not increase the number of crossing neurites over control. Together, these data show that soluble macrophage-derived factors in the absence of cell-cell contact have beneficial effects on neuronal growth and substrate modification. However, this work also identifies physical macrophage attack as another barrier to axonal regeneration following spinal cord injury. In the end, this work further illustrates the duality of the immune response following trauma to the CNS. Modulation of neuroinflammation, rather than its complete elimination, may be best strategy to maximize tissue repair and functional recovery. Advisors/Committee Members: Silver, Jerry.

Subjects: Biology; Biomedical research; Immunology; Molecular biology; Neurology; Pathology; Surgery

Keywords: spinal cord injury; neuroinflammation; axonal retraction (dieback); macrophage; regeneration; CNS

21. Jiang, Qingguang. The Role of ApoE and Liver X Receptors in Alzheimer's Disease.

Degree: PhD, Neurosciences, 2008, Case Western Reserve University

► Apolipoprotein E (ApoE) is associated with age-related risk for Alzheimer's disease and… (more)

▼ Apolipoprotein E (ApoE) is associated with age-related risk for Alzheimer's disease and plays critical roles in Aβ metabolism. ApoE is the major apolipoprotein present in high-density lipoproteins (HDL) and plays an essential role in cholesterol homeostasis in the brain. The ATP-binding cassette A1 (ABCA1) mediates the efflux of cholesterol and phospholipids into ApoE HDL. Liver X receptors regulate the expression of ApoE and ABCA1. ApoE plays a previously unappreciated role in facilitating the proteolytic clearance of soluble Aβ from the brain. The endolytic degradation of Aβ peptides within microglia by neprilysin and related enzymes is dramatically enhanced by ApoE. Similarly, Aβ degradation extracellularly by insulin degrading enzyme is facilitated by ApoE. The capacity of ApoE to promote Aβ degradation is dependent upon the ApoE isoform and its lipidation status. The enhanced expression of lipidated ApoE, through the activation of liver X receptors, stimulates Aβ degradation. In vivo, aged Tg2576 mice treated with the LXR agonist GW3965 exhibited a dramatic reduction in brain Aβ load. Significantly, GW3965 treatment also reversed contextual memory deficits. In N2a.Swe cells, LXR activation induced ABCA1 expression and reduced steady state Aβ levels. However, it did not affect the levels of holo-APP, the generation of secreted α-APP or C-terminal fragment. LXR did not affect the distribution of APP or its processing machinery, supporting the conclusion that LXR-mediated reduction of Aβ is due to enhanced clearance but not altered APP processing. Amyloid plaques are associated with a chronic inflammatory environment. LXR activation inhibited microglia-mediated inflammation both in vitro and in vivo. This inhibition is due to its regulation of NFκB transcriptional activity by actively stimulating the nuclear export of p65. The effect of the LXR agonist on p65 nuclear export correlates with its effect on the dynamic exchange of the SMRT/HDAC3 corepressor complex for the p300/pCAF coactivator complex upon LXR activation. Furthermore, LXR activation reverses the inhibition of fibrillar Aβ-stimulated phagocytosis by pro-inflammatory stimuli in microglia and ameliorates plaque-associated microglial inflammation in aged Tg2576 mice. Altogether, our results suggest that LXR agonists may represent a novel therapy for AD through its effects on both Aβ homeostasis and inflammation. Advisors/Committee Members: Landreth, Gary.

Subjects: Biomedical research

Keywords: ApoE; LXR; ABCA1; Aβ; APP; Alzheimer's disease; NFκB; inflammation; microglia; astrocyte; IDE; neprilysin; HDL; GW3965; p65; acetylation; HDAC3; SMRT; p300; pCAF; lipidation

22. Kastanenka, Ksenia V. IN VIVO ACTIVATION OF CHANNELRHODOPSIN-2 USED TO DETERMINE THE ROLE OF SPONTANEOUS NEURAL ACTIVITY IN AXONAL GUIDANCE.

Degree: PhD, Neurosciences, 2011, Case Western Reserve University

► Rhythmic spontaneous neural activity is widespread in the developing central nervous system.… (more)

▼ Rhythmic spontaneous neural activity is widespread in the developing central nervous system. In the developing chick spinal cord this bursting activity has recently been shown to regulate motoneuron pathfinding in the limb (Hanson and Landmesser, 2004; Hanson and Landmesser, 2006). Moderate decreases in the frequency of bursting activity by inhibiting GABAA receptor signaling via in ovo picrotoxin application resulted in dorsal-ventral (D-V) pathfinding errors and a decrease in the expression of the guidance molecules involved in this pathfinding choice. In contrast, moderate increases in the frequency of bursting activity by application of the glycine reuptake inhibitor, sarcosine, resulted in major pool-specific pathfinding errors in the limb. However, it was still unclear whether the disruption in the frequency of rhythmic depolarizations or altered neurotransmitter signaling led to the pathfinding errors, since altering the frequency of bursting activity also perturbed GABAergic or glycinergic transmission. To distinguish between these possibilities, I conducted the experiments described in this thesis. Herein I show that driving bursting activity at normal frequencies by light activation of Channelrhodopsin-2 in the presence of the GABAA receptor blocker, picrotoxin, prevents the D-V pathfinding errors and maintains the expression of the guidance molecules involved in this pathfinding choice. I have also determined that increasing the frequency by light activation of Channelrhodopsin-2 without alteration of glycine signaling results in pool-specific projection errors. This body of work shows that normal patterns of spontaneous neural activity and not neurotransmitter signaling are necessary for proper pathfinding of motoneurons in the limb. Thus, these experiments clearly identify what aspect of bursting activity motoneurons are sensitive to and establish the foundation for further determination of the downstream signaling pathways through which bursting activity regulates motor axon pathfinding. Advisors/Committee Members: Landmesser, Lynn.

Subjects: Neurobiology; Neurosciences

Keywords: spinal cord, motoneuron, busting activity, EphA4, EphB1, PSA, development, axonal pathfinding

23. Kerstetter Fogle, Amber E. ROLE OF CHEMOKINES IN REGULATING OLIGODENDROCYTE DEVELOPMENT, ASTROGLIOSIS, AND DEMYELINATING DISEASES.

Degree: PhD, Neurosciences, 2010, Case Western Reserve University

► Role of Chemokines in Regulating Oligodendrocyte Development, Astrogliosis and Demyelinating DisordersAbstract by… (more)

Subjects: Biomedical research

Keywords: demyelination, chemokines, Multiple sclerosis, astrogliosis

24. Krueger, Katherine C. Transcriptional Regulation of FEV, a Human Serotonin Neuron Developmental Control Gene.

Degree: PhD, Neurosciences, 2009, Case Western Reserve University

► The ETS domain transcription factor Pet1 is critical for the differentiation and… (more)

▼ The ETS domain transcription factor Pet1 is critical for the differentiation and development of serotonin (5HT) neurons in rodents. The human homolog of Pet1, FEV, is expressed in human raphe and can rescue the Pet1 loss-of-function phenotype. However, the levels of 5HT and serotonergic genes, as well as certain 5HT-modulated behaviors, depended on the level of FEV expression. These findings suggest variation in the program that directs FEV expression to developing 5HT neurons and determines the expression level of FEV may be relevant to psychiatric and neurological disorders. Thus, an important goal is to define the cis-regulatory mechanisms for serotonergic expression of FEV, and identify polymorphisms that might affect FEV expression. Here, the transcriptional control of FEV was explored in transgenic mice. These studies predict separate spatial and temporal modules direct certain aspects of serotonergic expression, while a conserved FEV-proximal region is necessary and sufficient to direct expression to developing 5HT neurons. Both in vitro and in vivo experiments support the conclusion that Gata2 directly regulates FEV/Pet1 via binding sites within this proximal region. Using this new FEV transcriptional control map as a guide, FEV upstream regions were examined for variants in pediatric mental illness patients. These studies revealed several new, rare SNPs that can now be tested for function and disease association. Finally, FEV-directed transgenes enabled determination of 5HT neuron precursor fate in the adult Pet1 knockout midbrain, demonstrating that the majority of mutant cells are not fated to apoptosis but rather survive and retain serotonergic character, although they are aberrantly distributed. Overall, these findings suggest a conserved genetic program governs human and mouse 5HT neuron development, and identify conserved, GATA factor-dependent transcriptional regulation of FEV/Pet1. Moreover, these studies provide novel SNPs and genetic tools for further studies of the role of FEV/Pet1 in 5HT neuron development as well as psychiatric disease. Advisors/Committee Members: Deneris, Evan.

Subjects: Biology; Biomedical research

Keywords: serotonin; FEV; Pet1; transgenic; Gata2; cis-regulatory

25. Larimer, Phillip. Local Circuit and Intrinsic Mechanisms of Persistent Activity in the Dentate Hilus of the Hippocampus.

Degree: PhD, Neurosciences, 2009, Case Western Reserve University

► The ability to recall information over a short period of time (working… (more)

▼ The ability to recall information over a short period of time (working memory) is critically important for human cognition. The dominant model of working memory is persistent activity in subsets of neurons, but the cellular and circuit mechanisms underlying this activity are not known. Persistent activity in several cortical regions, including the dentate gyrus of the hippocampus (a region important for long-term memory formation) is apparent during working memory tasks in vivo. Using patch-clamp recordings in rat hippocampal slices, I found that dentate gyrus hilar cells become persistently active in vitro following brief perforant path inputs. Furthermore, when stimulating dentate gyrus inputs in multiple locations, activity recorded in small populations of hilar neurons is sufficiently unique to allow identification of the stimulus. In this thesis, I used electrophysiological and imaging methods to investigate the cellular basis of this persistent activity.I first examined whether persistent firing in mossy cells (the excitatory cells of the hilus) reflects reverberant activity among those neurons. I found that both mossy cells and hilar interneurons demonstrate marked target selectivity, with mossy cells almost never synapsing on other mossy cells and interneurons preferential contacting mossy cells over other interneurons by a ratio of 3:1. This connectivity is unlikely to support reverberance-based models of hilar persistent activity. Together with Phil Williams and Yuan Gao, I identified a new cell type (semilunar granule cells, SGCs) in the inner molecular layer of the dentate gyrus that have broad dendritic arbors and axon collaterals in the granule cell layer. SGCs enter states of prolonged depolarization following brief perforant path input while the nearby granule cells are strongly inhibited. Blockade of T-type calcium currents, or NMDA receptors, did not block synaptic transmission to the hilus but eliminated SGC plateau potentials and hilar persistent activity, consistent with the hypothesis that SGCs mediate hilar persistent activity. These results suggest an important role for SGCs in both normal memory processes and also in the pathological hyperexcitability of the dentate gyrus seen in temporal lobe epilepsy. Advisors/Committee Members: Strowbridge, Ben.

Subjects: Biomedical research; Neurology

Keywords: hippocampus; persistent activity; neurophysiology

26. Lee, Chung-Ying Daniel. The Mechanism of Apolipoprotein E in the Proteolytic Degradation of Aβ.

Degree: PhD, Neurosciences, 2012, Case Western Reserve University

► Alzheimer’s disease is a neurodegenerative disease, which results from the imbalance of… (more)

▼ Alzheimer’s disease is a neurodegenerative disease, which results from the imbalance of the generation and the clearance of β-amyloid (Aβ) peptides. Apolipoprotein E (apoE) facilitates Aβ degradation in a dose- and isoform-dependent manner. However, the mechanisms underlying these activities remain unclear. In microglia, apoE promotes Aβ degradation through eliciting cholesterol efflux. Inhibition of cholesterol efflux blocks the apoE-facilitated Aβ degradation, while eliciting cholesterol efflux by a mimetic peptide enhances it. These findings indicate that the cholesterol efflux property of apoE is both sufficient and necessary for promoting Aβ degradation. Regulation of Aβ degradation could be achieved by solely manipulating cellular cholesterol levels, suggesting that apoE-enhanced Aβ degradation is a consequence of reduced cellular cholesterol levels through cholesterol efflux. Although the transcription and activity of all known Aβ degrading enzymes were not changed, the trafficking of Aβ is regulated by cholesterol. We further showed that cholesterol regulates the recycling of Rab7, which recruits the dynein motor complex onto endosomes. Cholesterol efflux elicited by apoE lowers cellular cholesterol levels and consequently promotes efficient recycling of Rab7, which leads to faster Aβ trafficking to lysosomes. As a result, the degradation of Aβ is enhanced. These findings highlight a direct role of cholesterol in regulating microglial degradation of Aβ. The extracellular degradation of Aβ is carried out principally by insulin degrading enzyme (IDE). In contrast to the intracellular degradation, apoE facilitates IDE-mediated Aβ degradation through its direct interaction with Aβ. Enzyme kinetic analysis shows that the Km of IDE for Aβ degradation in the presence of apoE was reduced, while the Vmax remained unchanged. These results suggest that apoE promotes the interaction/recognition of IDE and Aβ. Manipulating apoE’s affinity for Aβ regulates its ability to promote Aβ degradation. ApoE2 shows the highest affinity for Aβ and greatest efficiency in facilitating Aβ degradation, while apoE4 has low Aβ-binding affinity and is insufficient in promoting Aβ degradation. Together, these data support the chaperone function of apoE in facilitating Aβ degradation by IDE. Our study advances the knowledge of the mechanisms of apoE in facilitating Aβ degradation, which provides insight for developing effective therapies for AD. Advisors/Committee Members: Landreth, Gary.

Subjects: Neurosciences

Keywords: Apolipoprotein E; Aβ; Cholesterol; Alzheimer's disease; microglia; endocytosis

27. Lerch-Haner, Jessica Katrina. Pet-1/FEV Transcriptional Regulation of Central and Peripheral Serotonergic Traits and Offspring Survival.

Degree: PhD, Neurosciences, 2008, Case Western Reserve University

► Serotonin is a critical modulator of many physiological, emotional, and behavioral processes… (more)

▼ Serotonin is a critical modulator of many physiological, emotional, and behavioral processes both in the Central Nervous System (CNS) and in the periphery. Here we demonstrate that central serotonergic neuron development is critical for establishing maternal behavior and offspring survival; that the expression level of the upstream transcriptional cascade directing serotonin neuron differentiation impacts expression of serotonergic traits and behavior; and that Pet-1 controls both central and peripheral serotonin production. In Pet- 1 null mice, which have an arrest of serotonin neuron differentiation and an almost complete loss of brain serotonin, there is a severe maternal behavior deficit resulting in offspring death. Pet-1 null mice which express FEV, the human orthologue of Pet-1, at levels comparable to or above that found in wild type, have wild type levels of serotonergic trait expression, brain serotonin level, and maternal behavior. In Pet-1 null mice with reduced expression of FEV, and therefore reduced activity of the serotonergic transcriptional cascade, serotonergic trait expression, brain serotonin level, and offspring survival are all reduced. Finally, we provide evidence that Pet-1 is a common transcriptional determinant for both central and peripheral serotonin production. We found that vi Pet-1 is expressed in the serotonin producing cells of the intestine, enterochromaffin cells. In Pet-1 null mice, TPH1 expression is reduced by ~50% along with a ~50% reduction in intestinal serotonin production. The reduction in the Pet-1 null’s intestinal serotonin level is reflected in the blood serotonin level, which is also reduced compared to wild type. Overall these findings show that serotonin is important in establishing maternal behavior and offspring survival; that the activity of the upstream transcriptional cascade directing serotonergic neuron differentiation can affect the level of serotonergic neuron trait expression and impact serotonin modulated-behaviors; and that central and peripheral serotonin production are controlled by one molecule, Pet- 1/FEV. Advisors/Committee Members: Deneris, Evan.

Subjects: Biology

Keywords: serotonin; maternal behavior; enteroendocrine cell; intestine

28. Liu, Chen. Pet-1 Is Required Across Different Stages of Life to Regulate Serotonergic Function.

Degree: PhD, Neurosciences, 2010, Case Western Reserve University

► An intrinsic transcriptional cascade directs the generation of 5-HT synthesizing neurons in… (more)

Subjects: Biology; Biomedical research; Genetics; Molecular biology; Neurology

Keywords: Pet-1; transcription factor; serotonin; development; maintenance; emotional behavior; anxiety; adult; psychiatric disorder

29. Li, Xiang. G-Protein Modulation of Ion Channels and Control of Neuronal Excitability by Light.

Degree: PhD, Neurosciences, 2007, Case Western Reserve University

► Many neurotransmitters, hormones and sensory stimuli exert their biological functions through transmembrane… (more)

▼ Many neurotransmitters, hormones and sensory stimuli exert their biological functions through transmembrane receptors which couple to heterotrimeric guanine nucleotide binding protein (G protein) and normally have 7 transmembrane domains called G-protein coupled receptor (GPCR). G proteins work as transducers which convert extracellular signals into intracellular events. Voltage-gated calcium channels (VGCC) (P,Q and N types) and G-protein inwardly rectifying potassium channels (GIRK) are important for modulation of neuronal excitability and synaptic communication. They also belong to the end-targets of GPCRs. These modulations are mediated by G protein âã subunits. Combining electrophysiological recordings and fluorescence resonance energy transfer (FRET), I characterized different regions derived from Gâ2 subunit for interaction with P/Q type calcium channels and GIRK channels. Interestingly, different parts of the Gâ2 subunit can either induce or inhibit G protein modulation of the examined ion channels. In particular, peptides derived from the Gâ2 N-terminus inhibit G protein modulation, whereas peptides derived from Gâ2 C-terminus induced channel modulation. In a second series of studies, I developed light activated probes which could control neuronal excitability. First, I demonstrated that vertebrate rhodopsin can couples to GIRK channels and voltage gated Ca2+ channels through the Gi/o pathway. When expressed in hippocampal neurons, light activation of rhodopsin reduces neuronal firing and modulates synaptic transmission. In contrast, to excite neurons, I applied the green algae channelrhodospin 2 (ChR2), which could generate inward sodium current in HEK293 cells in response to light. Moreover, when ChR2 was expressed in hippocampal neurons, light activation ChR2 can depolarizes neurons to induce action potentials and presynaptic neuronal transmitter release. To further characterize the use of these two light switches in neuronal regulation, I used embryonic chick spinal cords as a model. Electroporated with vertebrate rhodopsin, turning on light could reduce the spontaneous firing and synchronize the bursting activity of spinal cord; on the other hand, light could increase spontaneous activity of spinal cord which was electroporated with ChR2. Thus, with these two probes, I could either increase or decrease neuronal activity with light in a fast and non-invasive way. Advisors/Committee Members: Herlitze, Stefan.

Subjects: Biology, Neuroscience

Keywords: Light, neuron, G protein, rhodopsin

30. Mandrekar-Colucci, Shweta Dilip. PPARɣ Activation Rapidly Ameliorates Amyloid Pathology and Restores Cognition in a Mouse Model of Alzheimer’s Disease.

Degree: PhD, Neurosciences, 2011, Case Western Reserve University

► Alzheimer’s disease (AD) is a chronic neurodegenerative disease characterized by the progressive… (more)

▼ Alzheimer’s disease (AD) is a chronic neurodegenerative disease characterized by the progressive loss of cognition and memory. The pathological hallmarks of AD include extracellular amyloid deposits and intra-neuronal neurofibrillary tangles. Progression of the disease is associated with a disruption of Aβ homeostasis and accumulation and deposition of Aβ in the brain parenchyma, initiating a robust microglial-mediated immune response that leads to the production of pro-inflammatory cytokines, chemokines and reactive nitrogen and oxygen species which are deleterious to the CNS. Despite the abundance of activated microglia surrounding plaques, they are inefficient in clearing fibrillar Aβ deposits. It is thought that activating mechanisms that facilitate Aβ clearance will alleviate disease related pathophysiology and be of great therapeutic utility for the treatment of Alzheimer’s disease. The peroxisome proliferator activated receptor-ɣ (PPARɣ), is a ligand activated nuclear receptor, which regulates lipid and glucose homeostasis in the body and exhibits potent anti-inflammatory actions. Activation of PPARγ has been shown to reduce brain Aβ levels and ameliorate AD related cognitive deficits in animal models of AD. However, the mechanisms through which PPARγ attenuates AD related pathophysiology have yet to be elucidated. We have shown that activation of another nuclear receptor, LXRα, facilitates the proteolytic degradation of Aβ in an ApoE-dependent mechanism through induction of target genes, ABCA1 and ApoE. ABCA1 regulates cholesterol efflux via lipidation of ApoE and this process augments the degradation of soluble Aβ species. Importantly, PPARγ activation induces the expression of LXRα and its targets ApoE and ABCA1, metabolically linking these pathways. We demonstrate that PPARɣ activation using the synthetic agonist, Pioglitazone (Actos™) enhances the proteolytic degradation of Aβ in microglia and astrocytes by utilizing the PPAR-LXR-ApoE linked pathway in the brain. PPARɣ mediated intracellular degradation of Aβ is dependent on expression of ApoE and LXR. Furthermore, an acute (9 day) treatment of APP/PS1 mice with pioglitazone, rapidly increased brain levels of ABCA1 and ApoE, decreased amyloid deposition and ameliorated AD-related cognitive deficits. The reduction in plaque deposition was paralleled with an increase in amyloid-laden microglia and astrocytes in the parenchyma of treated animals. Significantly, pioglitazone treatment of APP/PS1 animals polarized CNS microglia from a “classical” to an “alternative” activation state, reducing glial activation, re-engaging the phagocytic machinery and facilitating the clearance of fibrillar Aβ deposits. Together our data demonstrates an important role for PPARɣ activation in facilitating the clearance of both soluble and fibrillar species of Aβ and provides a mechanistic explanation for how PPARɣ agonists reduce AD-related pathophysiology. Advisors/Committee Members: Landreth, Gary.

Subjects: Neurosciences

Keywords: Alzheimer's Disease; Microglia; PPARgamma

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