Qual

Bradley Monk - Qualifying Paper Proposal

For my Qualifying Paper, I will provide (1) a critical review of the postsynaptic surface regulation of AMPA-type glutamate receptors (AMPARs), and (2) propose a unified quantitative model of AMPAR lateral diffusion into synapses. This topic is of predominate interest to scientists who investigate synaptic potentiation, and is fundamentally important to the scientific investigation of memory-related conditions such as dementia and addiction. A unified model of AMPAR membrane diffusion would provide significant insight into the basic physiological underpinnings of synaptic plasticity and potentiation -- the neural correlates of learning and memory. The expression of AMPARs at excitatory synapses can be thought of, theoretically, as a final product. That is, if it were possible to take a snapshot of a brain in its current state and then quantify the synaptic densities of AMPARs, it would reveal which excitatory pathways were the most potentiated. From there, one could predict how the neural network would respond to input at a given source. Small changes in the quantity of AMPARs at synapses can directly impact the firing-activity of a neuron; because of this, AMPARs are under tight regulation involving hundreds of coordinating molecular pathways. It is not in purview of my qualifying paper to review the legion of antecedent molecular cascades involved in the final product, but rather outline what happens after AMPARs are expressed at the dendritic membrane surface. Once a pool of AMPARs are embedded into the extrasynaptic membrane, they must diffuse laterally along the dendritic surface before entering synapses. This means that any given single particle is not predestined to serve one particular synapse. This idea gives rise to a myriad of questions related to how synapses capture AMPARs from the diffusing swarm, how long they are held,

There has been a major shift over the last 5-10 years in how AMPARs are expressed at synapses. The emerging consensus among leaders in the field, including Choquet, Nicol, Svoboda, Malinow, Triller, Sabatini, Svoboda, and others is that AMPARs are not trafficked from the spine cytosol directly to the postsynaptic density (PSD), but rather diffuse laterally along the dendritic membrane before they enter synapses.

a primary questions revolve around the fate of these AMPARs as they diffuse laterally throughout the dendritic surface.

A second important question is what is the quantitative landscape that surrounds the drawing from a common pool.

This project aims to identify how receptors accumulate at synapses to produce and sustain long term potentiation (LTP), a neural correlate of long term memory [1-8]. It has been established that AMPA receptor (AMPAR) expression at synapses is fundamental to this memory-related neural plasticity [9-12]. These glutamatergic cation channels are primary mediators of excitatory neurotransmission in the CNS [13-16]. As such, characterizing the regulation of these receptors was a focus of early studies examining LTP [9, 17], followed by interest in memory maintenance mechanisms [1, 18]. Research on the induction of LTP has identified numerous molecular cascades important for the initial consolidation of memory [19]. Early efforts to characterize LTP induction worked under the assumption that AMPARs are trafficked directly to the potentiated membrane shortly after learning [20, 21]. Recent evidence suggests atypical kinases, PKMζ in particular, could help maintain LTP through constitutive site-directed receptor trafficking [26-34].

Various kinases (i.e. PKA, CaMKII, PKC) have been confirmed to be functionally important for AMPAR-related LTP induction [22-25], which has been studied extensively; however there remains uncertainty about the molecular dynamics that underlie plasticity., but subsequent work indicates that PKMζ has a non-critical role in long-term memory [35, 36]. While atypical kinases may be involved in memory maintenance, it may not be through site-directed constitutive trafficking of AMPARs as proposed [28, 29]. Receptor expression changes at synapses were thought to be mainly a function of cycling between intracellular and membrane compartments, but recent evidence suggests that receptors accumulate at synapses largely via lateral diffusion and association with scaffold associated proteins (SAP) [37]. Even atypical kinases like PKMζ may be involved with trafficking of SAPs instead of AMPARs directly: in cultured hippocampal neurons PKMζ was shown to increase PSD-95 cluster size, which then in turn increased postsynaptic expression of AMPARs [38]. PSD-95 and other SAPs contribute to the aggregation receptors at the synapse, and may have a central role in LTP maintenance. The intracellular C-terminus of the various AMPAR subunits interact with a family of SAPs with PDZ domains, including PSD-95, MAGI-2, SAP97, PICK1, and GRIP1 [39-46]. This interaction with PDZ proteins can be both direct [47-50] and indirect [51-54], and when the interaction site is mutated, potentiation is blocked [25]. Indeed, the dendritic localization of many membrane-associated proteins are regulated by PDZ-containing SAPs [55-58], which help determine the size and strength of synapses by organizing glutamate receptors at the postsynaptic density [59]. Several studies imaged the synaptic capture of AMPARs from the pool of receptors diffusing rapidly in the extra-synaptic membrane [60]; while tracking the location of new AMPARs, receptors were first shown to be distributed along dendrites, followed by their lateral translocation into a postsynaptic density. Single-particle imaging reveals the synaptic capture of laterally diffusing AMPARs is dependent on interactions between PSD-95 and Stargazin, an AMPAR auxiliary subunit [51]. When this interaction was disrupted, it strongly increased AMPAR diffusion, and prevented synaptic accumulation. Together, this indicates that LTP maintenance is a likely function of SAP proteins that continually annex diffusing AMPARs into the synaptic density. Missing from a complete model of memory maintenance, however, is (1) how neural activity modulates the expression of SAP, (2) how changes in SAP expression effect AMPAR diffusion at synaptic sites, and (3) the identity of SAPs that are important for in vivo memory maintenance. In order to address these gaps, this project has the following aims:

Specific aim 1: Use single-particle tracking to examine SAP regulation responses to neural activity Hypothesis 1: Neural activity will cause rapid redistribution of SAPs through lateral translocation or cytosolic internalization. Specific aim 2. Use single-particle tracking to examine how SAP expression alters AMPAR distribution Hypothesis 2: Synaptic loss of SAP will lead to a net loss of AMPARs, while synapses that retain and/or gain SAP will recapture the highest proportion of receptors after a burst of neural activity. Specific aim 3. The MAGUK protein PSD-95 is thought to be important for synaptic plasticity. Using a PSD-95 dominant negative antimorph, we’ll test for deficiencies in fear and addiction-related memory. Hypothesis 3: The group expressing a PSD-95 dominant negative protein will have deficits in learning and memory compared to control subjects.

REFERENCES

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Basic research aimed at defining the physiological mechanisms that underlie memory could help identify new targets for dementia and addiction treatment [1-8]. This project aims to identify how receptors accumulate at synapses to produce and sustain long term potentiation (LTP), a neural correlate of long term memory [9-16]. It has been established that AMPA receptor (AMPAR) expression at synapses is fundamental to this memory-related neural plasticity [17-20]. These glutamatergic cation channels are primary mediators of excitatory neurotransmission in the CNS [21-24]. As such, characterizing the regulation of these receptors was a focus of early studies examining LTP [17, 25], followed by interest in memory maintenance mechanisms [9, 26]. Research on the induction of LTP has identified numerous molecular cascades important for the initial consolidation of memory [27]. In the traditional scheme of LTP induction, AMPARs are trafficked directly to the potentiated membrane shortly after learning [28, 29], with various kinases (i.e. PKA, CaMKII, PKC) being functionally important for AMPAR-related LTP induction [30-33]. However it’s questionable whether continuous delivery of receptors from cytosol-to-membrane is also the primary mechanism behind memory maintenance. In this model of LTP, in order for a memory-trace to persist there would need to be molecular agents that keep synapses potentiated by continuously trafficking AMPARs to the membrane. The search for such agents has so far provided a single candidate thought to be functionally capable of this role - an atypical form of protein kinase C known as PKMζ was hypothesized to maintain LTP through constitutive site-directed receptor trafficking [34-42]. However, subsequent work indicates this atypical kinase has, at best, a non-critical role in long-term memory [43, 44]. Certainly, changes in receptor numbers at synapses can be due to cycling between surface and intracellular compartments. However, recent studies using updated single-particle tracking methods show that receptors diffuse along the membrane between synaptic and extrasynaptic locations, and localization of receptors at synapses results from their interactions with submembranous scaffold-associated proteins (SAP) [68][45]. This has changed our view of the organization of the neuronal membrane to include lateral diffusion of receptors as a key parameter in synaptic plasticity. In fact, there is now evidence that atypical kinases are more involved with the regulation of SAPs than the receptors themselves, as in one study that suggests PKMζ is involved with trafficking of the SAP molecule PSD-95, increasing its cluster size, and in turn increasing postsynaptic expression of AMPARs [46]. PSD-95 and other SAPs contribute to the aggregation receptors at the synapse, and may have a central role in LTP maintenance. The intracellular C-terminus of the various AMPAR subunits interact with a family of SAPs with PDZ domains (MAGUK proteins), including PSD-95, MAGI-2, SAP97, PICK1, and GRIP1 [47-54]. Indeed, the dendritic localization of many membrane-associated proteins are regulated by PDZ-containing SAPs [63-66], which help determine the size and strength of synapses by organizing glutamate receptors at the postsynaptic density [67]. This PDZ interaction can be direct [55-58] or indirect [59-62], and when the PDZ site is mutated, potentiation is blocked [33]. Other single-particle imaging experiments reveal the synaptic capture of laterally diffusing AMPARs is dependent on interactions between PSD-95 and Stargazin, an AMPAR auxiliary subunit [59]; blocking this interaction prevented AMPAR synaptic accumulation. Together, this indicates that LTP maintenance is a likely function of SAP proteins that continually annex diffusing AMPARs into the synaptic density. Missing from a complete model of memory maintenance, however, is (1) how neural activity modulates the expression of SAP, (2) how changes in SAP expression effect AMPAR diffusion at synaptic sites, and (3) the identity of SAPs that are important for in vivo memory maintenance. In order to address these gaps, this project has the following aims:

Specific aim 1: Use single-particle tracking to examine SAP regulation responses to neural activity Hypothesis 1: Neural activity will cause rapid redistribution of SAPs through lateral translocation or cytosolic internalization. Specific aim 2. Use single-particle tracking to examine how SAP expression alters AMPAR distribution Hypothesis 2: Synaptic loss of SAP will lead to a net loss of AMPARs, while synapses that retain and/or gain SAP will recapture the highest proportion of receptors after a burst of neural activity. Specific aim 3. The MAGUK protein PSD-95 is thought to be important for synaptic plasticity. Using a PSD-95 dominant negative antimorph, we’ll test for deficiencies in fear and addiction-related memory. Hypothesis 3: The group expressing a PSD-95 dominant negative protein will have deficits in learning and memory compared to control subjects.

Specific Aims are limited to one page. State concisely the goals of the proposed research and summarize the expected outcome(s), including the impact that the results of the proposed research will exert on the research field(s) involved. List succinctly the specific objectives of the research proposed, e.g., to test a stated hypothesis, create a novel design, solve a specific problem, challenge an existing paradigm or clinical practice, address a critical barrier to progress in the field, or develop new technology.