An IQSEC2 Mutation Associated With Intellectual Disability and Autism Results in Decreased Surface AMPA Receptors

We have recently described an A350V mutation in IQSEC2 associated with intellectual disability, autism and epilepsy. We sought to understand the molecular pathophysiology of this mutation with the goal of developing targets for drug intervention. We demonstrate here that the A350V mutation results in interference with the binding of apocalmodulin to the IQ domain of IQSEC2. We further demonstrate that this mutation results in constitutive activation of the guanine nucleotide exchange factor (GEF) activity of IQSEC2 resulting in increased production of the active form of Arf6. In a CRISPR generated mouse model of the A350V IQSEC2 mutation, we demonstrate that the surface expression of GluA2 AMPA receptors in mouse hippocampal tissue was significantly reduced in A350V IQSEC2 mutant mice compared to wild type IQSEC2 mice and that there is a significant reduction in basal synaptic transmission in the hippocampus of A350V IQSEC2 mice compared to wild type IQSEC2 mice. Finally, the A350V IQSEC2 mice demonstrated increased activity, abnormal social behavior and learning as compared to wild type IQSEC2 mice. These findings suggest a model of how the A350V mutation in IQSEC2 may mediate disease with implications for targets for drug therapy. These studies provide a paradigm for a personalized approach to precision therapy for a disease that heretofore has no therapy

Dual role of the exocyst in AMPA receptor targeting and insertion into the postsynaptic membrane

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102104/1/emboj7601065.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/102104/2/emboj7601065-sup-0001.pd

Neurogranin targets calmodulin and lowers the threshold for the induction of long-term potentiation.

Calcium entry and the subsequent activation of CaMKII trigger synaptic plasticity in many brain regions. The induction of long-term potentiation (LTP) in the CA1 region of the hippocampus requires a relatively high amount of calcium-calmodulin. This requirement is usually explained, based on in vitro and theoretical studies, by the low affinity of CaMKII for calmodulin. An untested hypothesis, however, is that calmodulin is not randomly distributed within the spine and its targeting within the spine regulates LTP. We have previously shown that overexpression of neurogranin enhances synaptic strength in a calmodulin-dependent manner. Here, using post-embedding immunogold labeling, we show that calmodulin is not randomly distributed, but spatially organized in the spine. Moreover, neurogranin regulates calmodulin distribution such that its overexpression concentrates calmodulin closer to the plasma membrane, where a high level of CaMKII immunogold labeling is also found. Interestingly, the targeting of calmodulin by neurogranin results in lowering the threshold for LTP induction. These findings highlight the significance of calmodulin targeting within the spine in synaptic plasticity

Local Control of AMPA Receptor Trafficking at the Postsynaptic Terminal by a Small GTPase of the Rab Family

The delivery of neurotransmitter receptors into the synaptic membrane is essential for synaptic function and plasticity. However, the molecular mechanisms of these specialized trafficking events and their integration with the intracellular membrane transport machinery are virtually unknown. Here, we have investigated the role of the Rab family of membrane sorting proteins in the late stages of receptor trafficking into the postsynaptic membrane. We have identified Rab8, a vesicular transport protein associated with trans-Golgi network membranes, as a critical component of the cellular machinery that delivers AMPA-type glutamatergic receptors (AMPARs) into synapses. Using electron microscopic techniques, we have found that Rab8 is localized in close proximity to the synaptic membrane, including the postsynaptic density. Electrophysiological studies indicated that Rab8 is necessary for the synaptic delivery of AMPARs during plasticity (long-term potentiation) and during constitutive receptor cycling. In addition, Rab8 is required for AMPAR delivery into the spine surface, but not for receptor transport from the dendritic shaft into the spine compartment or for delivery into the dendritic surface. Therefore, Rab8 specifically drives the local delivery of AMPARs into synapses. These results demonstrate a new role for the cellular secretory machinery in the control of synaptic function and plasticity directly at the postsynaptic membrane.This work was supported by grants from the National Institutes of Health (Grant MH070417), National Alliance for Research on Schizophrenia and Depression, and Alzheimer's Association (to J. A. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.Peer reviewe

Analysis of Rab protein function in neurotransmitter receptor trafficking at hippocampal synapses

Members of the Rab family of small GTPases are essential regulators of intracellular membrane sorting. Nevertheless, very little is known about the role of these proteins in the membrane trafficking processes that operate at synapses, and specifically, at postsynaptic terminals. These events include the activity-dependent exocytic and endocytic trafficking of AMPA-type glutamate receptors, which underlies long-lasting forms of synaptic plasticity such as long-term potentiation (LTP) and long-term depression (LTD). This chapter summarizes different experimental methods to address the role of Rab proteins in the trafficking of neurotransmitter receptors at postsynaptic terminals in the hippocampus. These techniques include immunogold electron microscopy to ultrastructurally localize endogenous Rab proteins at synapses, molecular biology methods to express recombinant Rab proteins in hippocampal slice cultures, electrophysiological techniques to evaluate the role of Rab proteins in synaptic transmission, and confocal fluorescence imaging to monitor receptor trafficking at dendrites and spines and its dependence on Rab proteins.This work was supported by the National Institute of Mental Health (Grant MH070417 to J.A.E. and Grant F31-MH070205 to T.C.B.), the National Alliance for research on Schizophrenia and Depression (J.A.E.), the Alzheimer's Association (J.A.E.), and the Fundação para a Ciência e a Tecnologia (S.S.C.).Peer reviewe

Ng lowers LTP threshold in CA1 hippocampal pyramidal neurons.

<p>(<b>A</b>) LTP was induced by pairing 3-Hz presynaptic stimulation (300 pulses) with −20 mV postsynaptic depolarization (indicated with an arrow) in uninfected neurons (open squares, <i>n</i> = 8) and neurons expressing GFP-Ng (black squares, <i>n</i> = 7). Inserts: sample traces of evoked AMPAR-mediated synaptic responses recorded at −60 mV before pairing (gray line) and 20 min after pairing (black line) from control or infected cells as indicated. Scale bars: 20 pA, 20 ms. (<b>B</b>) Normalized average steady-state AMPAR-mediated responses in paired (LTP pathway) and unpaired (control pathways) for uninfected neurons and those expressing GFP-Ng. Pairing significantly increased AMPAR-mediated responses in both groups. Neurons expressing GFP-Ng showed higher level of LTP than control neurons (<i>p</i><0.05).</p

Ng targets and relocates dendritic CaM closer to plasma membrane of the postsynaptic compartment.

<p>(<b>A</b>) Representative images of immunogold-EM for CaM in control and Ng-overexpressing neurons. Scale bar, 100 nm. (<b>B</b>) Frequency histograms of CaM (n = 214) in control neurons and Ng-inf (n = 319) neurons. Normalized radial distance was measured the same way as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041275#pone-0041275-g001" target="_blank">Figure 1</a>. Note the increased fraction of CaM at 0.1 interval in Ng-expressed condition. (<b>C</b>) Cumulative probabilities of normalized radial distance of CaM particles located within 0.2 of the normalized radial distance, where Ng was found to be concentrated in our previous study. Ng targeted CaM closer to the plasma membrane (<i>p</i> = 0.05). (<b>D</b>) Ng did not change the global distribution of CaM in the spine (PSD: postsynaptic density; E: extrasynaptic membrane; I: intraspine). The <i>p-</i>value for comparing cumulative distribution was calculated using Kolmogorov-Smirnov test. The <i>p-</i>value for comparing global CaM distribution in different sub-compartments was calculated using Chi-squared test.</p

CaM is not randomly distributed within the synapse.

<p>(<b>A</b>) Representative images of immunogold-EM for endogenous CaM in dendritic spines of CA1 hippocampal neurons. The postsynaptic compartment (<i>s</i>) is recognized by the appearance of the prominent postsynaptic density (black arrowhead) and well-defined plasma membrane. <i>t</i>, presynaptic terminal. Arrows indicate anti-CaM immunogold particles. Scale bar, 100 nm. (<b>B</b>) Cumulative probabilities of normalized radial distance. The distance of each gold particle from the plasma membrane (within the spine) was normalized (x axis) to the corresponding radius of the spine measured through that particle. The distribution of CaM within the spine is significantly different from a theoretical random distribution (n = 214, <i>p</i><0.001).</p