CONFORMATIONAL CHANGES IN THE EXTRACELLULAR DOMAIN OF GLUTAMATE RECEPTORS

The family of membrane protein called glutamate receptors play an important role in the central nervous system in mediating signaling between neurons. Glutamate receptors are involved in the elaborate game that nerve cells play with each other in order to control movement, memory, and learning. Neurons achieve this communication by rapidly converting electrical signals into chemical signals and then converting them back into electrical signals. To propagate an electrical impulse, neurons in the brain launch bursts of neurotransmitter molecules like glutamate at the junction between neurons, called the synapse. Glutamate receptors are found lodged in the membranes of the post-synaptic neuron. They receive the burst of neurotransmitters and respond by fielding the neurotransmitters and opening ion channels. Glutamate receptors have been implicated in a number of neuropathologies like ischemia, stroke and amyotrophic lateral sclerosis. Specifically, the NMDA subtype of glutamate receptors has been linked to the onset of Alzheimer’s disease and the subsequent degeneration of neuronal cells. While crystal structures of AMPA and kainate subtypes of glutamate receptors have provided valuable information regarding the assembly and mechanism of activation; little is known about the NMDA receptors. Even the basic question of receptor assembly still remains unanswered. Therefore, to gain a clear understanding of how the receptors are assembled and how agonist binding gets translated to channel opening, I have used a technique called Luminescence Resonance Energy Transfer (LRET). LRET offers the unique advantage of tracking large scale conformational changes associated with receptor activation and desensitization. In this dissertation, LRET, in combination with biochemical and electrophysiological studies, were performed on the NMDA receptors to draw a correlation between structure and function. NMDA receptor subtypes GluN1 and GluN2A were modified such that fluorophores could be introduced at specific sites to determine their pattern of assembly. The results indicated that the GluN1 subunits assembled across each other in a diagonal manner to form a functional receptor. Once the subunit arrangement was established, this was used as a model to further examine the mechanism of activation in this subtype of glutamate receptor. Using LRET, the correlation between cleft closure and activation was tested for both the GluN1 and GluN2A subunit of the NMDA receptor in response to agonists of varying efficacies. These investigations revealed that cleft closure plays a major role in the mechanism of activation in the NMDA receptor, similar to the AMPA and kainate subtypes. Therefore, suggesting that the mechanism of activation is conserved across the different subtypes of glutamate receptors.

The unstable (CTG)$\sb{\rm n}$ trinucleotide repeat associated with myotonic dystrophy: What it does and where it came from

Myotonic dystrophy (DM), an autosomal dominant disorder mapping to human chromosome 19q13.3, is the most common neuromuscular disease in human adults.^ Following the identification of the mutation underlying the DM phenotype, an unstable (CTG)$\sb{n}$ trinucleotide repeat in the 3$\prime$ untranslated region (UTR) of a gene encoding a ser/thr protein kinase named DM protein kinase (DMPK), the study was targeted at two questions: (1) the identification of the disease-causing mechanism(s) of the unstable repeat, and at a more basic level, (2) the identification of the origin and the mechanism(s) involved in repeat instability. The first goal was to identify the pathophysiological mechanisms of the (CTG)$\sb{n}$ repeat.^ The normal repeat is transcribed but not translated; therefore, initial studies centered on the effect on RNA transcript levels. The vast majority of DM affecteds are heterozygous for the mutant expansion, so that the normal allele interferes with the analysis of the mutant allele. A quantitative allele-specific RT-PCR procedure was developed and applied to a spectrum of patient tissue samples and cell lines. Equal levels of unprocessed pre-mRNA were determined for the wild type (+) and disease (DM) alleles in skeletal muscle and cell lines of heterozygous DM patients, indicating that any nucleosome binding has no effect at the level of transcriptional initiation and transcription of the mutant DMPK locus. In contrast, processed mRNA levels from the DM allele were reduced relative to the + allele as the size of the expansion increased. The unstable repeat, therefore, impairs post-transcriptional processing of DM allele transcripts. This phenomenon has profound effects on overall DMPK locus steady-state transcript levels in cells missing a wild type allele and does not appear to be mediated by imprinting, decreased mRNA stability, generation of aberrant splice forms, or absence of polyadenylation of the mutant allele.^ In Caucasian DM subjects, the unstable repeat is in complete linkage disequlibrium with a single haplotype composed of nine alleles within and flanking DMPK over a physical distance of 30 kb. A detailed haplotype analysis of the DM region was conducted on a Nigerian (Yoruba) DM family, the only indigenous sub-Saharan DM case reported to date. Each affected member of this family had an expanded (CTG)$\sb{n}$ repeat in one of their DMPK alleles. However, unlike all other DM populations studied thus far, disassociation of the (CTG)$\sb{n}$ repeat expansion from other alleles of the putative predisposing haplotype was found. Thus, the expanded (CTG)$\sb{n}$ repeat in this family was the result of an independent mutational event. Consequently, the origin of DM is unlikely the result of a single mutational event, and the hypothesis that a single ancestral haplotype predisposes to repeat expansion is not compelling. (Abstract shortened by UMI.) ^

Glutamate and nitric oxide as regulatory transmitters in developing rabbit retina

Glutamate is the major excitatory neurotransmitter in the retina and serves as the synaptic messenger for the three classes of neurons which constitute the vertical pathway--the photoreceptors, bipolar cells and ganglion cells. In addition, the glutamate system has been localized morphologically, pharmacologically as well as molecularly during the first postnatal week of development before synaptogenesis occurs. The role which glutamate plays in the maturing visual system is complex but ranges from mediating developmental neurotoxicity to inducing neurite outgrowth.^ Nitric oxide/cGMP is a novel intercellular messenger which is thought to act in concert with the glutamate system in regulating a variety of cellular processes in the brain as well as retina, most notably neurotoxicity. Several developmental activities including programmed cell death, synapse elimination and synaptic reorganization are possible functions of cellular regulation modulated by nitric oxide as well as glutamate.^ The purpose of this thesis is to (1) biochemically characterize the endogenous pools of glutamate and determine what fraction exists extracellularly; (2) examine the morphological expression of NO producing cells in developing retina; (3) test the functional coupling of the NMDA subtype of glutamate receptor to the NO system by examining neurotoxicity which has roles in both the maturing and adult retina.^ Biochemical sampling of perfusates collected from the photoreceptor surface of ex vivo retina demonstrated that although the total pool of glutamate present at birth is relatively modest, a high percentage resides in extracellular pools. As a result, immature neurons without significant synaptic connections survive and develop in a highly glutamatergic environment which has been shown to be toxic in the adult retina.^ The interaction of the glutamate system with the NO system has been postulated to regulate neuronal survival. We therefore examined the developmental expression of the enzyme responsible for producing NO, nitric oxide synthase (NOS), using an antibody to the constitutive form of NOS found in the brain. The neurons thought to produce the majority of NO in the adult retina, a subpopulation of widefield amacrine cells, were not immunoreactive until the end of the second postnatal week. However, a unique developmental expression was observed in the ganglion cell layer and developing outer nuclear layer of the retina during the first postnatal week. We postulate NO producing neurons may not be present in a mature configuration therefore permitting neuronal survival in a highly glutamatergic microenvironment and allowing NO to play a development-specific role at this time.^ The next set of experiments constituted a functional test of the hypothesis that the absence of the prototypic NO producing cells in developing retina protects immature neurons against glutamate toxicity. An explant culture system developed in order to examine cellular responses of immature and adult neurons to glutamate toxicity showed that immature neurons were affected by NMDA but were less responsive to NMDA and NO mediated toxicity. In contrast, adult explants exhibited significant NMDA toxicity which was attenuated by NMDA antagonists, 2-amino-5-phosphonovaleric acid (APV), dextromethorphan (Dex) and N$\rm\sp{G}$-D-methyl arginine (metARG). These results indicated that pan-retinal neurotoxicity via the NMDA receptor and/or NO activation occurred in the adult retina but was not significant in the neonate. (Abstract shortened by UMI.) ^

Confocal analysis of synaptic connectivity of the rod pathway in the rabbit retina

The retina is a specialized neuronal structure that transforms the optical image into electrical signals which are transmitted to the brain via the optic nerve. As part of the strategy to cover a stimulus range as broad as 10 log units, from dim starlight to bright sunlight, retinal circuits are broadly divided into rod and cone pathways, responsible for dark and light-adapted vision, respectively. ^ In this dissertation, confocal microscopy and immunocytochemical methods were combined to study the synaptic connectivity of the rod pathway from the level of individual synapses to whole populations of neurons. The study was focused on synaptic interactions at the rod bipolar terminal. The purpose is to understand the synaptic structure of the dyad synapse made by rod bipolar terminals, including the synaptic components and connections, and their physiological functions in the rod pathway. In addition, some additional components and connections of the rod pathway were also studied in these experiments. The major results can be summarized as following: At the dyad synapse of rod bipolar terminals, three postsynaptic components—processes of All amacrine cells and the varicosities of S1 or S2 amacrine cells express different glutamate receptor subunits, which may underlie the functional diversity of these postsynaptic neurons. A reciprocal feedback system is formed by rod bipolar terminals and S1/S2 amacrine cells. Analysis showed these two wide-field GABA amacrine cells have stereotyped synaptic connections with the appropriate morphology and distribution to perform specific functions. In addition, S1 and S2 cells have different coupling patterns and, in general, there is no coupling between the two types. Besides the classic rod pathway though rod bipolar cells and All amacrine cells, the finding of direct connections between certain types of OFF cone bipolar cells and rods indicates the presence of an alternative rod pathway in the rabbit retina. ^ In summary, this dissertation presents a detailed view of the connection and receptors at rod bipolar terminals. Based on the morphology, distribution and coupling, different functional roles were identified for S1 and S2 amacrine cells. Finally, an alternative to the classic rod pathway was found in the rabbit retina. ^

The role of sensory neuron outgrowth in long-term sensitization of the tail-elicited tail-siphon withdrawal reflex of Aplysia

Neuronal outgrowth has been proposed in many systems as a mechanism underlying memory storage. For example, sensory neuron outgrowth is widely accepted as an underlying mechanism of long-term sensitization of defensive withdrawal reflexes in Aplysia. The hypothesis is that learning leads to outgrowth and consequently to the formation of new synapses, which in turn strengthen the neural circuit underlying the behavior. However, key experiments to test this hypothesis have never been performed. ^ Four days of sensitization training leads to outgrowth of siphon sensory neurons mediating the siphon-gill withdrawal response in Aplysia . We found that a similar training protocol produced robust outgrowth in tail sensory neurons mediating the tail siphon withdrawal reflex. In contrast, 1 day of training, which effectively induces long-term behavioral sensitization and synaptic facilitation, was not associated with neuronal outgrowth. Further examination of the effect of behavioral training protocols on sensory neuron outgrowth indicated that this structural modification is associated only with the most persistent forms of sensitization, and that the induction of these changes is dependent on the spacing of the training trials over multiple days. Therefore, we suggest that neuronal outgrowth is not a universal mechanism underlying long-term sensitization, but is involved only in the most persistent forms of the memory. ^ Sensory neuron outgrowth presumably contributes to long-term sensitization through formation of new synapses with follower motor neurons, but this hypothesis has never been directly tested. The contribution of outgrowth to long-term sensitization was assessed using confocal microscopy to examine sites of contact between physiologically connected pairs of sensory and motor neurons. Following 4 days of training, the strength of both the behavior and sensorimotor synapse and the number of appositions with follower neurons was enhanced only on the trained side of the animal. In contrast, outgrowth was induced on both sides of the animal, indicating that although sensory neuron outgrowth does appear to contribute to sensitization through the formation of new synapses, outgrowth alone is not sufficient to account for the effects of sensitization. This indicates that key regulatory steps are downstream from outgrowth, possibly in the targeting of new processes and activation of new synapses. ^

Mechanisms of heterosynaptic modulation of transmission in Aplysia

Heterosynaptic plasticity has received considerable attention as a means to induce and maintain cell-wide, as opposed to synapse-specific, learning-related modifications. Modulatory neurotransmitters are thought to provide the attentional and motivational state for memory formation. However, the cellular and molecular mechanisms mediating the effects of most of these modulators on synaptic plasticity and learning remain unclear. A well established system for the study of heterosynaptic plasticity is the Aplysia sensorimotor synapse, which is subject regulation by at least two neuromodulators, serotonin (5-HT) and FMRFa. ^ 5-HT engages multiple second messenger cascades to induce short- and long-term facilitation (STF and LTF, respectively) of synaptic transmission. One mechanism proposed to be involved in STF is mobilization of synaptic vesicles from a storage pool to a releasable pool. To investigate this hypothesis, we examined the involvement of the protein synapsin, a central element in the regulation of the storage pool of vesicles in nerve terminals, in STF. 5-HT induced phosphorylation of synapsin and modified its subcellular distribution via PKA and p42/44 MAPK. Electrophysiological experiments and computer simulations suggested that synapsin can support heterosynaptic plasticity by regulating vesicle mobilization. ^ FMRFa induce short- and long-term synaptic depression in Aplysia . Long-term depression (LTD) correlates with morphological changes, the mechanisms of which remain elusive. LTD is also transcription- and translation-dependent, but little is known about the genes expressed and their regulation. We investigated the role of protein degradation via the ubiquitin-proteasome system and the regulation of one of its components, ubiquitin C-terminal hydrolase (ap-uch), in LTD. LTD was sensitive to inhibition of the proteasome and was associated with upregulation of ap-uch mRNA and protein. This upregulation appeared to be mediated by the transcription factor CREB2, which is generally regarded as a transcription repressor. These results suggest that proteasome-mediated protein degradation is engaged in LTD and that CREB2 may act as a transcription activator under certain conditions. ^ These and additional studies on the interaction of the 5-HT and FMRFa-activated pathways suggest that different neuromodulators, by activating several and sometimes overlapping signaling cascades, can exercise bidirectional control on synaptic gain and information processing.^

Statins and myasthenia gravis

Introduction. 3-hydroxy-3-methylglutaryl CoA reductase inhibitor ("statin") have been widely used for hypercholesteroremia and Statin induced myopathy is well known. Whether Statins contribute to exacerbation of Myasthenia Gravis (MG) requiring hospitalization is not well known. ^ Objectives. To determine the frequency of statin use in patients with MG seen at the neuromuscular division at University of Alabama in Birmingham (UAB) and to evaluate any association between use of statins and MG exacerbations requiring hospitalization in patients with an established diagnosis of Myasthenia Gravis. ^ Methods. We reviewed records of all current MG patients at the UAB neuromuscular department to obtain details on use of statins and any hospitalizations due to exacerbation of MG over the period from January 1, 2003 to December 31, 2006. ^ Results. Of the 113 MG patients on whom information was available for this period, 40 were on statins during at least one clinic visit. Statin users were more likely to be older (mean age 60.2 vs 53.8, p = 0.029), male (70.0% vs 43.8%, p = 0.008), and had a later onset of myasthenia gravis (mean age in years at onset 49.8 versus 42.9, p = 0.051). The total number of hospitalizations or the proportion of subjects who had at least one hospitalization during the study period did not differ in the statin versus no-statin group. However, when hospitalizations which occurred from a suspected precipitant were excluded ("event"), the proportion of subjects who had at least one such event during the study period was higher in the group using statins. In the final Cox proportional hazard model for cumulative time to event, statin use (OR = 6.44, p <0.01) and baseline immunosuppression (OR = 3.03, p = 0.07) were found to increase the odds of event. ^ Conclusions. Statin use may increase the rate of hospitalizations due to MG exacerbation, when excluding exacerbations precipitated by other suspected factors.^

Spatial and temporal aspects of synaptic plasticity

The notion that changes in synaptic efficacy underlie learning and memory processes is now widely accepted even if definitive proof of the synaptic plasticity and memory hypothesis is still lacking. When learning occurs, patterns of neural activity representing the occurrence of events cause changes in the strength of synaptic connections within the brain. Reactivation of these altered connections constitutes the experience of memory for these events and for other events with which they may be associated. These statements summarize a long-standing theory of memory formation that we refer to as the synaptic plasticity and memory hypothesis. Since activity-dependent synaptic plasticity is induced at appropriate synapses during memory formation, and is both necessary and sufficient for the information storage, we can speculate that a methodological study of the synapse will help us understand the mechanism of learning. Random events underlie a wide range of biological processes as diverse as genetic drift and molecular diffusion, regulation of gene expression and neural network function. Additionally spatial variability may be important especially in systems with nonlinear behavior. Since synapse is a complex biological system we expect that stochasticity as well as spatial gradients of different enzymes may be significant for induction of plasticity. ^ In that study we address the question "how important spatial and temporal aspects of synaptic plasticity may be". We developed methods to justify our basic assumptions and examined the main sources of variability of calcium dynamics. Among them, a physiological method to estimate the number of postsynaptic receptors as well as a hybrid algorithm for simulating postsynaptic calcium dynamics. Additionally we studied how synaptic geometry may enhance any possible spatial gradient of calcium dynamics and how that spatial variability affect plasticity curves. Finally, we explored the potential of structural synaptic plasticity to provide a metaplasticity mechanism specific for the synapse. ^

THE FUNCTIONAL ORGANIZATION OF SYNAPTIC VESICLE POOLS IN A RETINAL BIPOLAR NEURON

Ribbon synapses are found in sensory systems and are characterized by ‘ribbon-like’ organelles that tether synaptic vesicles. The synaptic ribbons co-localize with sites of calcium entry and vesicle fusion, forming ribbon-style active zones. The ability of ribbon synapses to maintain rapid and sustained neurotransmission is critical for vision, hearing and balance. At retinal ribbon synapses, three vesicle pools have been proposed. A rapid pool of vesicles that are docked at the plasma membrane, and whose fusion is limited only by calcium entry, a releasable pool of ATP-primed vesicles whose size also correlates with the number of ribbon-tethered vesicles, and a reserve pool of non-ribbon-tethered cytoplasmic vesicles. However evidence of vesicle fusion at sites away from ribbon-style active zones questions this organization. Another fundamental question underlying the mechanism of vesicle fusion at these synapses is the role of SNARE (Soluble N-ethylmaleimide sensitive factor Attachment Protein Receptor) proteins. Vesicles at conventional neurons undergo SNARE complex-mediated fusion. However a recent study has suggested that ribbon synapses involved in hearing can operate independently of neuronal SNAREs. We used the well-characterized goldfish bipolar neuron to investigate the organization of vesicle pools and the role of SNARE proteins at a retinal ribbon synapse. We blocked functional refilling of the releasable pool and then stimulated bipolar terminals with brief depolarizations that triggered the fusion of the rapid pool of vesicles. We found that the rapid pool draws vesicles from the releasable pool and that both pools undergo release at ribbon-style active zones. To assess the functional role of SNARE proteins at retinal ribbon synapses, we used peptides derived from SNARE proteins that compete with endogenous proteins for SNARE complex formation. The SNARE peptides blocked fusion of reserve vesicles but not vesicles in the rapid and releasable pools, possibly because both rapid and releasable vesicles were associated with preformed SNARE complexes. However, an activity-dependent block in refilling of the releasable pool was seen, suggesting that new SNARE complexes must be formed before vesicles can join a fusion-competent pool. Taken together, our results suggest that SNARE complex-mediated exocytosis of serially-organized vesicle pools at ribbon-style active zones is important in the neurotransmission of vision.

Novel Imaging-Based Techniques Reveal a Role for PD-1/PD-L1 in Tumor Immune Surveillance in the Lung

The binding of immune inhibitory receptor Programmed Death 1 (PD-1) on T cells to its ligand PD-L1 has been implicated as a major contributor to tumor induced immune suppression. Clinical trials of PD-L1 blockade have proven effective in unleashing therapeutic anti-tumor immune responses in a subset of patients with advanced melanoma, yet current response rates are low for reasons that remain unclear. Hypothesizing that the PD-1/PD-L1 pathway regulates T cell surveillance within the tumor microenvironment, we employed intravital microscopy to investigate the in vivo impact of PD-L1 blocking antibody upon tumor-associated immune cell migration. However, current analytical methods of intravital dynamic microscopy data lack the ability to identify cellular targets of T cell interactions in vivo, a crucial means for discovering which interactions are modulated by therapeutic intervention. By developing novel imaging techniques that allowed us to better analyze tumor progression and T cell dynamics in the microenvironment; we were able to explore the impact of PD-L1 blockade upon the migratory properties of tumor-associated immune cells, including T cells and antigen presenting cells, in lung tumor progression. Our results demonstrate that early changes in tumor morphology may be indicative of responsiveness to anti-PD-L1 therapy. We show that immune cells in the tumor microenvironment as well as tumors themselves express PD-L1, but immune phenotype alone is not a predictive marker of effective anti-tumor responses. Through a novel method in which we quantify T cell interactions, we show that T cells are largely engaged in interactions with dendritic cells in the tumor microenvironment. Additionally, we show that during PD-L1 blockade, non-activated T cells are recruited in greater numbers into the tumor microenvironment and engage more preferentially with dendritic cells. We further show that during PD-L1 blockade, activated T cells engage in more confined, immune synapse-like interactions with dendritic cells, as opposed to more dynamic, kinapse-like interactions with dendritic cells when PD-L1 is free to bind its receptor. By advancing the contextual analysis of anti-tumor immune surveillance in vivo, this study implicates the interaction between T cells and tumor-associated dendritic cells as a possible modulator in targeting PD-L1 for anti-tumor immunotherapy.

Modulation of high voltage -activated calcium channels by phosphorylation

High voltage-activated (HVA) calcium channels from rat brain and rabbit heart are expressed in Xenopus laevis oocytes and their modulation by protein kinases studied. A subtype of the HVA calcium current expressed by rat brain RNA is potentiated by the phospholipid- and calcium-dependent protein kinase (PKC). The calcium channel clone $\alpha\sb{\rm1C}$ from rabbit heart is modulated by the cAMP-dependent protein kinase (PKA), and another factor present in the cytoplasm.^ The HVA calcium channels from rat brain do not belong to the L-type subclass since they are insensensitive to dihydropyridine (DHP) agonists and antagonists. The expressed currents do contain a N-type fraction which is identified by inactivation at depolarized potentials, and a P-type fraction as defined by blockade by the venom of the funnel web spider Agelenopsis Aperta. A non N-type fraction of this current is potentiated, by using phorbol esters to activate PKC. This residual fraction of current resembles the newly described Q-type channel from cerebellar granule cells in its biophysical properties, and potentiation by activation of PKC.^ The $\alpha\sb{\rm1C}$ clone from rabbit heart is expressed in oocytes and single-channel currents are measured using the cell-attached and cell-excised patch clamp technique. The single-channel current runs down within two minutes after patch excision into normal saline bath solution. The catalytic subunit of PKA + MgATP is capable of reversing this rundown for over 15 minutes. There also appears to be an additional factor present in the cytoplasm necessary for channel activity as revealed in experiments where PKA failed to prevent rundown.^ These data are important in that these types of channels are involved in synaptic transmission at many different types of synapses. The mammalian synapse is not accessible for these types of studies, however, the oocyte expression system allows access to HVA calcium channels for the study of their modulation by phosphorylation. ^