Shank and Zinc Mediate an AMPA Receptor Subunit Switch in Developing Neurons

Abstract

During development, pyramidal neurons undergo dynamic regulation of AMPA receptor (AMPAR) subunit composition and density to help drive synaptic plasticity and maturation. These normal developmental changes in AMPARs are particularly vulnerable to risk factors for Autism Spectrum Disorders (ASDs), which include loss or mutations of synaptic proteins and environmental insults, such as dietary zinc deficiency. Here, we show how Shank2 and Shank3 mediate a zinc-dependent regulation of AMPAR function and subunit switch from GluA2-lacking to GluA2-containing AMPARs. Over development, we found a concomitant increase in Shank2 and Shank3 with GluA2 at synapses, implicating these molecules as potential players in AMPAR maturation. Since Shank activation and function require zinc, we next studied whether neuronal activity regulated postsynaptic zinc at glutamatergic synapses. Zinc was found to increase transiently and reversibly with neuronal depolarization at synapses, which could affect Shank and AMPAR localization and activity. Elevated zinc induced multiple functional changes in AMPAR, indicative of a subunit switch. Specifically, zinc lengthened the decay time of AMPAR-mediated synaptic currents and reduced their inward rectification in young hippocampal neurons. Mechanistically, both Shank2 and Shank3 were necessary for the zinc-sensitive enhancement of AMPAR-mediated synaptic transmission and act in concert to promote removal of GluA1 while enhancing recruitment of GluA2 at pre-existing Shank puncta. These findings highlight a cooperative local dynamic regulation of AMPAR subunit switch controlled by zinc signaling through Shank2 and Shank3 to shape the biophysical properties of developing glutamatergic synapses. Given the zinc sensitivity of young neurons and its dependence on Shank2 and Shank3, genetic mutations and/or environmental insults during early development could impair synaptic maturation and circuit formation that underlie ASD etiology.

Transcriptional signatures of participant-derived neural progenitor cells and neurons implicate altered Wnt signaling in Phelan McDermid syndrome and autism

“…The transcriptional signatures of PMS in iNeurons point to altered postsynaptic density, glutamatergic synaptic and GABAergic genes. Our results are in line with evidence supporting a role for SHANK3 prior to synaptogenesis and neural circuit formation, specifically in early morphogenesis and excitatory/ inhibitory balance [32, 65, 71–74]. For example, a zebrafish model of PMS that utilized morpholinos to disrupt shank3a and shank3b resulted in delayed mid- and hindbrain development, disruptions in motor behaviors, and seizure-like behaviors [73].…”

Neural markers of vulnerability to anxiety outcomes following traumatic brain injury

“…Four bilateral immunofluorescent labeled sections were taken in each mouse (8 images per animal per region, per marker), sections were approximately 200 um apart. Images were pre-processed using FIJI, and then the number and intensity of puncta were calculated using the IMFLAN3D analysis package (Tai et al, 2007, Schindelin et al, 2012, Ha et al, 2018). (A ‘punctum’ was defined as a cluster of ‘connected’ pixels identified in an objective, automated manner using the bwlabeln function in MATLAB with the ‘eight-connected neighborhood’ criterion (Tai et al, 2007, Schindelin et al, 2012, Ha et al, 2018))…”

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

“…Short hairpin RNA (shRNA) oligonucleotides targeted for rat Shank3 mRNA (sh‐Shank3) were synthesized and cloned into a pSUPER and pFUGW H1 by the pZOFF vector (Leal‐Ortiz et al, ): 5′‐GATC CCC G GTT CTT CGC AAT GGC GGT TTC AAG AGA ACC GCC ATT GCG AAG AAC C TTTTT GG AAA‐3′ and 5′‐AGCT TTT CC AAAAA G GTT CTT CGC AAT GGC GGT TCT CTT GAA ACC GCC ATT GCG AAG AAC CGGG‐3′, corresponding to nucleotide region 732–752 within the Ankyrin repeat domain of rat Shank3 (Arons et al, ; Roussignol et al, ). This shRNA has previously been characterized in publications from our laboratories and others (Arons et al, ; Bidinosti et al, ; Ha et al, ; Verpelli et al, ). No off target effects were evident, as Shank3 downregulation had no effect on the abundance of other presynaptic (VGluT1, synaptophysin, synapsin, VAMP2, piccolo, Munc13) or postsynaptic (PSD95, Homer1) proteins, or the size of the total recycling pool of synaptic vesicles (Arons et al, ).…”