Switching On Depression and Potentiation in the Cerebellum

Long-term depression (LTD) and long-term potentiation (LTP) in the cerebellum are important for motor learning. However, the signaling mechanisms controlling whether LTD or LTP is induced in response to synaptic stimulation remain obscure. Using a unified model of LTD and LTP at the cerebellar parallel fiber-Purkinje cell (PF-PC) synapse, we delineate the coordinated pre- and postsynaptic signaling that determines the direction of plasticity. We show that LTP is the default response to PF stimulation above a well-defined frequency threshold. However, if the calcium signal surpasses the threshold for CaMKII activation, then an ultrasensitive “on switch” activates an extracellular signal-regulated kinase (ERK)-based positive feedback loop that triggers LTD instead. This postsynaptic feedback loop is sustained by another, trans-synaptic, feedback loop that maintains nitric oxide production throughout LTD induction. When full depression is achieved, an automatic “off switch” inactivates the feedback loops, returning the network to its basal state and demarcating the end of the early phase of LTD

Postsynaptic Stability and Variability Described by a Stochastic Model of Endosomal Trafficking

Neurons undergo dynamic processes of constitutive AMPA-type glutamate receptor (AMPAR) trafficking, such as the insertion and internalization of AMPARs by exocytosis and endocytosis, while stably maintaining synaptic efficacy. Studies using advanced imaging techniques have suggested that the frequency of these constitutive trafficking processes, as well as the number of AMPARs that are involved in a particular event highly fluctuate. In addition, mechanisms that trigger some forms of synaptic plasticity have been shown to include not only these processes but also additional fluctuating processes, such as the sorting of AMPARs to late endosomes (LEs). Thus, the regulation of postsynaptic AMPARs by the endosomal trafficking system appears to have superficially conflicting properties between the stability or organized control of plasticity and highly fluctuating or stochastic processes. However, it is not clear how the endosomal trafficking system reconciles and utilizes such conflicting properties. Although deterministic models have been effective to describe the stable maintenance of synaptic AMPAR numbers by constitutive recycling, as well as the involvement of endosomal trafficking in synaptic plasticity, they do not take stochasticity into account. In this study, we introduced the stochasticity into the model of each crucial machinery of the endosomal trafficking system. The specific questions we solved by our improved model are whether stability is accomplished even with a combination of fluctuating processes, and how overall variability occurs while controlling long-term synaptic depression (LTD). Our new stochastic model indeed demonstrated the stable regulation of postsynaptic AMPAR numbers at the basal state and during LTD maintenance, despite fast fluctuations in AMPAR numbers as well as high variability in the time course and amounts of LTD. In addition, our analysis suggested that the high variability arising from this stochasticity is beneficial for reproducing the relatively constant timing of LE sorting for LTD. We therefore propose that the coexistence of stability and stochasticity in the endosomal trafficking system is suitable for stable synaptic transmission and the reliable induction of synaptic plasticity, with variable properties that have been observed experimentally

Structure-function analysis on the level of individual synapses

Excitatory synapses in the mammalian brain are made on small protrusions of the postsynaptic cell called dendritic spines. Dendritic spines are highly variable in their morphology and in their microanatomy (e.g. presence of subsynaptic organelles). It is unclear whether and how variability in spine morphological and anatomical properties translates into differences in synaptic function. Using two photon imaging, we analyzed how spine properties can affect synaptic signals and the potential for synaptic plasticity at single identified spine synapses. We show that synaptic signals can be tightly regulated on the level of individual synapses and that differences in spine morphology and microanatomy regulate synaptic function. We also provide evidence for the existence of functionally distinct populations of synapses in regard to their potential for synaptic plasticity. The present thesis is subdivided into three main sections. The first section is dedicated to the analysis of the function of specialized subsynaptic organelles in regulating synaptic plasticity. In the second section we studied the impact of spine morphology on synaptic signals and in the third section we examined whether critical proteins can be tagged to individual synapses in response to plasticity inducing stimuli. In pyramidal cells, only a subset of dendritic spines contains endoplasmic reticulum (ER). Spine ER often forms a ‘spine apparatus’, a specialized organelle with unknown function. It is unclear whether these specialized subsynaptic structures can affect the function of the synapse on the spine head. The possible involvement of spine ER in shaping spine calcium transients, a key trigger for synaptic plasticity, raises the possibility that spine ER could modulate the potential of a given synapse to undergo activity dependent modifications. Using a genetic approach to label the ER in living neurons, we find that the ER preferentially localizes to spines containing strong synapses. We demonstrate that spine ER represents a specialized calcium signaling machinery required for the induction of metabotropic glutamate receptor dependent long term depression at individual synapses. We demonstrate that different subsets of synapses exist in regard to their potential to undergo specific forms of plasticity. Spine ER represents the anatomical correlate for a mechanism by which strong synapses can be retuned in an activity dependent manner. Dendritic spines are separated from their parent dendrite by a thin spine neck. The spine neck slows down diffusion of molecules from the spine head to the parent dendrite, allowing spine-specific action of second messengers and activated enzymes. The resistance of the spine neck is crucial in determining whether spines can also be considered electrical compartments. Only a high enough spine neck resistance leads to electrical compartmentalization and activation of voltage gated channels in the spine in response to synaptic stimulation. We show that spine neck resistance can change in an activity dependent manner. Using single spine calcium imaging as a reporter of NMDA receptor activation and spine head depolarization, we show that spines can indeed act as electrical compartments. Using pharmacological experiments and modeling, we demonstrate that different voltage dependent channels cooperatively participate in shaping spine head depolarization and spine calcium transients. We also show that in vivo the spine neck resistance is higher compared to the situation in acutely sliced brain tissue, demonstrating that in the living animal a higher fraction of spines can be considered electrical compartments compared to the in vitro situation. We provide strong evidence that the spine neck can profoundly affect synaptic calcium signals. Biochemical and electrical compartmentalization is dynamically regulated in an activity dependent way. Spine calcium signals can activate key signaling cascades responsible for the induction of synaptic plasticity. Long term potentiation (LTP) has been shown to require the activity of CaMKII, a serine/ threonine kinase. A chemical protocol leading to LTP has been shown to induce translocation of CaMKII to dendritic spines. It is however unclear whether this molecule acts at single synapses or whether it can spread and modulate neighboring synapses in response to more physiological protocols. Using a new optical approach to induce LTP at single visualized synapses, we show that LTP induction is accompanied by a long-lasting increase of CaMKII at the stimulated synapse. This increase was specific to the stimulated spine and did not spread to neighboring spines. We provide evidence that CaMKII acts locally, on the micrometer scale, to regulate plasticity. We show that the concentration of proteins involved in regulating synaptic plasticity can be tightly regulated at the level of single synapses

9th Annual Postdoctoral Science Symposium

The mission of the Annual Postdoctoral Science Symposium (APSS) is to provide a platform for talented postdoctoral fellows throughout the Texas Medical Center to present their work to a wider audience. The MD Anderson Postdoctoral Association convened its inaugural Annual Postdoctoral Science Symposium (APSS) on August 4, 2011. The APSS provides a professional venue for postdoctoral scientists to develop, clarify, and refine their research as a result of formal reviews and critiques of faculty and other postdoctoral scientists. Additionally, attendees discuss current research on a broad range of subjects while promoting academic interactions and enrichment and developing new collaborations

Investigation Of The Spatiotemporal Dynamics Of Camp And Pka Signaling And The Role Of Hcn4 Subunits In Anxiety-Related Behavior And Memory

In the hippocampus, long-term memory and synaptic plasticity occur through a series of coordinated intracellular signaling cascades that strengthen and stabilize subsets of synaptic connections while leaving thousands of others unaltered. Therefore, understanding how molecular signals are accurately transmitted is critical to understanding how hippocampal neurons store information. Molecules like cAMP and protein kinase A are critical components of memory and plasticity, but it is unclear how these diffusible signals are dynamically regulated to achieve the spatial and temporal specificity that underlies pathway-specific plasticity. Hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels are ion channels that are modulated by cAMP and are known to regulate the spatial and temporal dynamics of excitatory postsynaptic potentials. HCN1 and HCN2 subunits have been implicated in memory, plasticity and anxiety-related behaviors, but the role for HCN4 subunits remains untested. In Chapter 1, I review the role of cAMP signaling in hippocampal synaptic plasticity and memory consolidation with emphasis on the molecular mechanisms regulating cAMP, PKA and HCN channels. In Chapter 2, I combine live two-photon imaging of genetically-encoded fluorescent FRET sensors and computational modeling to investigate the molecular mechanisms regulating the spatiotemporal dynamics of cAMP and PKA activity in hippocampal neurons during stimulation of β-adrenergic receptors. Results suggest that the ratio between adenylyl cyclase and phosphodiesterase-4 scales with neuronal compartment size to maintain basal cAMP levels and produce rapid-onset, high-amplitude cAMP transients in small compartments. Conversely, imaging experiments show that PKA activity is greater in large neuronal compartments and modeling suggests that compartmental differences in PKA activity depend on the concentration of protein phosphatase and not on the concentration of PKA substrates or PKA holoenzyme. In Chapter 3, I use recombinant adeno-associated viruses and shRNA-mediated silencing of HCN4 subunits to examine their role in anxiety, memory, and contextual fear extinction. Results from a battery of behavioral assays suggest that reduction of HCN4 subunits increases anxiety-related behavior, but does not affect object-location memory or contextual fear conditioning. Together, my thesis work provides novel insight into the molecular mechanism regulating the spatiotemporal dynamics of cAMP/PKA signaling and provides suggests a role for HCN4 subunits in anxiety-related behavior

Regulation of glutamatergic AMPA receptor stability and trafficking by ubiquitination

AMPA-type glutamate receptors (AMPARs) play a critical role in mediating the majority of fast excitatory synaptic transmission in the brain, where alterations in receptor expression, distribution and trafficking have been shown to underlie synaptic plasticity and higher brain function. However, the molecular mechanisms regulating AMPAR surface expression and turnover are still not fully understood. We report that mammalian AMPARs are subject to post-translational modification by ubiquitin, and identify Nedd4 as the E3 ligase responsible for mediating this process. AMPAR ubiquitination enhanced receptor degradation and reduced AMPAR cell-surface expression; conversely, inhibition of proteasomal activity caused AMPAR accumulation. Using site-directed mutagenesis we replaced each of four lysine residues available as putative ubiquitination sites on the AMPAR subunit GluA1 C-terminal with an arginine and identified critical residues for ubiquitination and receptor degradation. Consistent with the role of protein ubiquitination, lysine mutation reduced the efficiency of AMPAR endocytosis. We further investigated the molecular mechanisms involved in the internalization of ubiquitinated AMPARs. We find that the endocytic adaptor protein Eps15 plays a critical role in this process. siRNA-mediated suppression or overexpression of Eps15 results in changes in AMPAR surface expression. Eps15 interaction with AMPARs requires Nedd4-mediated GluA1 ubiquitination along with the ubiquitin interacting motif (UIM) of Eps15. Consistent with ubiquitination-mediated receptor internalization, knockdown of Eps15 suppresses GluA1 internalization of wild-type GluA1, but not a mutant GluA1 lacking ubiquitination sites, indicating a crucial role for Eps15 in the trafficking of ubiquitinated AMPARs. These findings reveal novel regulatory mechanisms in the control of glutamate receptor amount and distribution dynamics, which are key factors implicated in higher brain functions and neurological disorders

The role of calcium-permeable AMPA receptors and arc in secreted amyloid precursor protein alpha-mediated plasticity

The orchestrated regulation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-subtype of glutamate receptors by neuronal activity and neuromodulators is critical to the expression of both long-term potentiation (LTP) and memory. In particular, GluA1-containing, Ca2+-permeable AMPAR (CP-AMPAR) comprise a unique role in these processes due to their transient, activity-regulated expression at the synapse. Importantly, many of the mechanisms which govern these processes are negatively affected in neurodegenerative disorders such as Alzheimer’s disease, suggesting that understanding the mode of action of neuromodulatory molecules may reveal much needed novel therapeutic interventions. Secreted amyloid precursor protein-alpha (sAPPα), a metabolite of the parent amyloid precursor protein (APP) has been previously shown to enhance hippocampal LTP and facilitate memory formation. Accordingly, we hypothesised that sAPPα may act via modulation of AMPAR synthesis and cell surface expression. Using primary hippocampal neurons grown in culture, we found that sAPPα (1 nM) differentially regulates the expression of cell surface GluA1-, GluA2-, and GluA3-containing AMPAR. Interestingly, using fluorescent non-canonical amino acid tagging with proximity ligation assay (FUNCAT-PLA), we found that short-term sAPPα treatments (1 nM, 30 min) rapidly enhanced the cell surface expression of newly synthesised extrasynaptic GluA1-, but not GluA2-containing AMPAR, while long-term treatments of sAPPα (1 nM, 120 min) increased levels of pre-existing GluA1/2-containing heteromers at the cell surface, indicating a dynamic regulation of distinct AMPARs following treatment. Moreover, using electrophysiology in area CA1 of acute hippocampal slices, we provide evidence that the expression of CP-AMPAR is important in the induction of sAPPα-enhanced LTP. Using immunocytochemistry and siRNA knockdown, we provide evidence that internalization of CP-AMPARs may be governed, at least in part by sAPPα-driven expression of the activity-regulated cytoskeletal-associated protein (Arc). Further, we show that Arc expression is not induced by the related APP metabolite sAPPβ, but is dependent on synergistic activation of N-Methyl-D-Aspartate and α7-nicotinic acetylcholine receptors, as well as downstream activation of CaMKII, MAPK, and PKG. Together, these findings suggest that application of sAPPα to hippocampal neurons engages a cascade of mechanisms which enhance the synthesis and expression of AMPAR and Arc protein, in the regulation of synaptic strength and the expression of hippocampal LTP. These experiments expand upon our current knowledge underlying mechanisms of synaptic plasticity in hippocampal neurons