Matthew J. Kennedy - US grants

DESCRIPTION (provided by applicant): Mechanisms for regulating the efficacy of synaptic coupling, or synaptic strength, between neurons are required for critical brain functions such as learning and memory. The process of altering synaptic strength between neurons, broadly termed synaptic plasticity, is impaired or absent in numerous neuropsychiatric disorders and diseases. A key mechanism for one of the most robust forms of synaptic plasticity, long-term potentiation, is the addition of AMPA-type neurotransmitter receptors from internal vesicular stores known as recycling endosomes (REs), to the postsynaptic membrane. Recent work has demonstrated that a large fraction of dendritic spines, the major sites of excitatory synaptic contact, contain REs and that these REs undergo fusion with the spine plasma membrane to deposit a stable pool of AMPA receptors at or near the synapse in response to plasticity-inducing activity. These data indicate that spine REs are poised for local delivery of plasticity factors to activated synapses, but many fundamental questions remain. For example, why these organelles are found in some spines but not others, how they are mobilized to fuse with the plasma membrane by plasticity-inducing stimuli, and how fusion contributes to synaptic function and plasticity are important issues we propose to address in this project. In Aim1 we will address whether the history of synaptic activity influences the distribution REs at individual synapses and/or their AMPA receptor content. In Aim2 we will determine whether plasticity at individual synapses directly scales with spine RE content. In Aim3 we will dissect the second messengers and signaling molecules that couple synaptic activity to spine RE fusion. Combined, these independent but complementary aims will greatly advance our understanding of fundamental forms of neuronal plasticity and inform future efforts in determining how and why plasticity is disrupted in numerous neuropsychiatric disorders and diseases including Alzheimer's, autism, schizophrenia and addiction.

? DESCRIPTION: Conditionally silencing the activity of specific neural ensembles is a powerful approach for mapping the circuits responsible for specific behaviors. While microbial opsin tools currently exist to silence neural activity with light, these tools have significant limitations tha make them unsuitable for applications that require persistent silencing over the course of minutes or hours. Longer term neuronal silencing has been classically achieved by expressing the catalytic light chain of Clostridium neurotoxins, which disrupt neurotransmitter release, however this approach suffers from poor temporal and spatial control. Recent chemogenetic approaches, including designer receptors exclusively activated by designer drugs (DREADDs) have been developed for persistent silencing, but lack the spatiotemporal benefits of optogenetic approaches. Thus, there remains an unmet need for silencing tools that combine the robust and persistent silencing qualities of Clostridium neurotoxin and chemogenetic approaches, with the spatiotemporal control of optogenetics. Here we will develop a completely new toolkit for rapid, extended neural silencing with a single, brief light pulse that can be readiy activated by either single or multiphoton excitation for in vivo systems. Our tools will not only b applicable to neurotransmitter systems, but also to neuromodulatory systems and glial transmission. These powerful tools will complement and extend the existing opto- and chemogenetic silencing toolset by allowing rapid, robust and persistent silencing with a single light pulse. Because our strategy operates on a completely different principle than current tools, it could be multiplexed with existing approaches for complex remote control of circuit activity during behavior.

Regulated trafficking of newly synthesized postsynaptic neurotransmitter receptors, ion channels and cell adhesion molecules to their appropriate subcellular domain is critical for establishing, maintaining and altering synaptic strength and connectivity. For example, diverse forms of synaptic plasticity at excitatory synapses are thought to require local synthesis and trafficking of neurotransmitter receptors, which must be processed through multiple organelle networks, classically defined by the endoplasmic reticulum (ER) and Golgi apparatus (GA), to reach their functional destination at synapses. However, little is known about the trafficking route for synaptic proteins leaving the ER at remote sites in dendrites. This issue is especially puzzling considering that the GA, thought to be required for trafficking secreted and integral membrane proteins, is missing from most dendritic branches in mammalian central neurons. Thus, whether secretory molecules are locally trafficked in dendrites, and if so, the identity and spatial distribution of the organelles that mediate local trafficking remain fundamental issues. We will use a combination of novel ER sequestration/release technology coupled with live cell imaging, super resolution microscopy and electrophysiology to visually and functionally track cargo molecules as they travel from the ER to synaptic sites. The proposed experiments will fill a major gap in our understanding of how protein processing, distribution and abundance are controlled within the dendritic secretory network and provide quantitative parameters that can be used to develop, refine, and perhaps challenge current cellular models of synaptic function and plasticity.

Widely recognized as one of the primary pathological agents responsible for Alzheimer's disease (AD), ?-amyloid peptide triggers a broad host of cellular pathologies including synapse loss, tau phosphorylation, and ultimately cell death. One of the earliest manifestations of AD in the central nervous system is the loss of excitatory synapses and impaired function at remaining synapses, which precedes widespread neuronal cell death. Accumulating evidence suggests that synapse elimination and dysfunction is triggered by soluble oligomeric assemblies of the beta-amyloid peptide (A?o). Intriguingly, A?o-mediated synapse loss requires activation of NMDA-type glutamate receptors (NMDARs), which conduct Ca2+, a critical second messenger for diverse forms of synaptic plasticity required for normal cognitive function. How or whether A?o directly influences synaptic NMDAR function is unclear. We recently found that Ca2+ entry through synaptic NMDARs was potently impaired following exposure to concentrations of A?o similar to those found in the cerebrospinal fluid of healthy individuals. Here we propose to dissect the mechanisms and signaling pathways responsible for A?o-induced NMDA receptor impairment and explore the possibility that A?o could play a normal role in NMDA receptor regulation in the healthy brain. If true, this would be a completely new and important function for amyloid peptides and would yield new insights into how these proteins contribute to synaptic dysfunction when misregulated.

Project Summary Abstract Postsynaptic kinase/phosphatase networks in amyloid ?-induced synaptic dysfunction Alzheimer's disease (AD) is characterized by impaired synaptic function and synapse loss in key forebrain areas required for learning and memory, including the hippocampus. While the pathologic agent that causes AD remains contentious (amyloid-beta; A? vs. tau) there is strong genetic, biochemical, anatomical and electrophysiological evidence supporting that A? is sufficient to initiate cellular processes leading to severe synaptic pathology. For example sub-micromolar doses of A? acutely (within minutes) inhibit long-term potentiation (LTP), a form of synaptic plasticity critical for learning and memory. In addition, longer A? exposure (days to weeks) leads to depression and elimination of excitatory synapses through a process that requires NMDA receptor signaling. However, the downstream signaling networks that drive acute and chronic A?-mediated synaptic pathologies are only beginning to emerge and need to be further investigated. Strong preliminary data from our labs implicate several postsynaptic ser/thr kinases (CaMKII, DAPK1, PKA) and a phosphatase (calcineurin (CaN)) as key molecular players responsible for acute A?- induced LTP disruption, possibly through impaired NMDA receptor Ca2+ entry. It remains unclear whether these same signaling mechanisms mediate chronic A?-induced synaptic depression and elimination, but published and preliminary data presented here indicate that CaN activity is required. Importantly, all of these kinases and phosphatases interact with one another in a postsynaptic signaling network that integrates NMDAR activity to promote either LTP or LTD. Indeed, synaptic anchoring of PKA and CaN by the scaffold protein AKAP79/150 appears to be critical for promoting signaling crosstalk between PKA, CaN, DAPK1 and CaMKII at synaptic sites to establish normal LTP/LTD balance. In this multi-PI project we will test the hypothesis that A? causes acute (Aims 1 & 2) and chronic (Aims 3 & 4) synaptic dysfunction by perturbing the balance of this signaling network and its downstream effectors to favor LTD, leading to impaired LTP and synapse elimination.  

Synapses throughout the central nervous system are sculpted by neural activity through changes in their size, shape and molecular composition, which either strengthen or weaken communication between neurons. This ?plasticity? in synapse function is widely viewed as the central mechanism for information storage in the brain. While many forms of synaptic plasticity have been discovered and their molecular mechanisms intensely investigated, in many cases there is surprisingly little direct evidence linking them to the cognitive functions they are proposed to control. This has remained a challenge due to a lack of tools for rapidly and locally switching on or off the requisite biochemistry and cell biology underlying different plasticity mechanisms in real time, in vivo. We are developing new tools that fill this void with the long- term goal of addressing fundamental gaps in our knowledge concerning how synapses are modified at the molecular level through development and plasticity, how these modifications influence synapse/circuit function and ultimately the relevance of these mechanisms for important cognitive functions like learning and memory.