Shank3 Is Part of a Zinc-Sensitive Signaling System That Regulates Excitatory Synaptic Strength

Abstract

Shank3 is a multidomain scaffold protein localized to the postsynaptic density of excitatory synapses. Functional studies in vivo and in vitro support the concept that Shank3 is critical for synaptic plasticity and the trans-synaptic coupling between the reliability of presynaptic neurotransmitter release and postsynaptic responsiveness. However, how Shank3 regulates synaptic strength remains unclear. The C terminus of Shank3 contains a sterile alpha motif (SAM) domain that is essential for its postsynaptic localization and also binds zinc, thus raising the possibility that changing zinc levels modulate Shank3 function in dendritic spines. In support of this hypothesis, we find that zinc is a potent regulator of Shank3 activation and dynamics in rat hippocampal neurons. Moreover, we show that zinc modulation of synaptic transmission is Shank3 dependent. Interestingly, an autism spectrum disorder (ASD)-associated variant of Shank3 (Shank3 R87C) retains its zinc sensitivity and supports zinc-dependent activation of AMPAR-mediated synaptic transmission. However, elevated zinc was unable to rescue defects in trans-synaptic signaling caused by the R87C mutation, implying that trans-synaptic increases in neurotransmitter release are not necessary for the postsynaptic effects of zinc. Together, these data suggest that Shank3 is a key component of a zinc-sensitive signaling system, regulating synaptic strength that may be impaired in ASD.

Autism‐associated Shank3 mutations alter mGluR expression and mGluR‐dependent but not NMDA receptor‐dependent long‐term depression

“…Many ASD‐associated mutations in Shank3 have been shown to significantly impact glutamatergic synaptic function, especially excitatory synaptic transmission mediated by ionotropic AMPARs and NMDARs (Arons et al, , ; Lee et al, ; Peça et al, ; Zhou et al, ). Metabotropic‐type glutamate receptors (mGluRs) play a major role in modulating the function of these ionotropic receptors and synaptic plasticity, but less is known about how different ASD‐associated Shank3 mutations can influence mGluRs.…”

“…Primary dissociated hippocampal cultures were prepared from postnatal day 0 (P0) Wistar rats of either sex using a modified Banker protocol (Kaech & Banker, ), as described in our previous studies (Arons et al, , ; Goodman et al, ). The handling of all animals was performed following strict regulations approved by the University of Auckland Animal Ethics Committee.…”

“…Paired whole‐cell patch clamp recordings were performed at DIV 12–16 on primary dissociated hippocampal neurons as described previously (Arons et al, ). Neurons plated on 13‐mm round glass coverslips were transferred to a recording chamber mounted on an Olympus BX‐51 microscope and perfused at RT with artificial cerebrospinal fluid (aCSF) containing (in mM): 119 NaCl, 2.5 KCl, 1.3 MgSO 4 , 2.5 CaCl 2 , 1 Na 2 HPO 4 , 26.2 NaHCO 3 , and 11 glucose.…”

“…To measure AMPAR‐mediated excitatory postsynaptic currents (EPSCs), postsynaptic neurons were voltage clamped at −65 mV, while presynaptic neurons were held in current clamp configuration and stimulated at 0.1 Hz with a 200–500‐pA current step for 20 ms to induce an action potential. AMPAR‐mediated EPSCs were defined as monosynaptic only when the postsynaptic current occurred within 5 ms of the peak of the presynaptic action potential (Arons et al, , ). NMDAR‐dependent LTD was induced by low‐frequency presynaptic action potentials at 1 Hz paired with postsynaptic cell depolarization at −55 mV (Montgomery, Pavlidis, & Madison, ; Zheng, Jeyifous, Munro, Montgomery, & Green, ).…”

SAP97‐mediated rescue of NMDA receptor surface distribution in a neuronal model of Huntington's disease

“…Paired and single whole‐cell patch‐clamp recordings were performed to measure evoked AMPAR‐mediated and NMDAR‐mediated excitatory postsynaptic currents (EPSCs), and total surface NMDAR‐mediated currents as described in our previous studies (Waites et al, ; Li et al, ; Arons et al, ; Arons et al, ). Hippocampal neuronal cultures plated on 12 mm round glass coverslips were transferred to a recording chamber mounted on an Olympus BX‐51 microscope, and perfused at room temperature with artificial cerebrospinal fluid [aCSF; 119 mM NaCl (Sigma, St. Louis, MO, #S7653), 2.5 mM KCl (Sigma #60128), 1 mM NaH 2 PO 4 (Fluka Chemika, St. Louis, MO #71504), 1.3 mM MgSO 4 (Fisher Scientific, Pittsburgh, PA, #M1050/53), 2.5 mM CaCl 2 (Sigma #C4901), 26.2 mM NaHCO 3 (Sigma #S6297), 11 mM d ‐(+)‐glucose (Sigma #49139), ~290 mOsM].…”