Importance of Shank3 Protein in Regulating Metabotropic Glutamate Receptor 5 (mGluR5) Expression and Signaling at Synapses

Abstract

Shank3/PROSAP2 gene mutations are associated with cognitive impairment ranging from mental retardation to autism. Shank3 is a large scaffold postsynaptic density protein implicated in dendritic spines and synapse formation; however, its specific functions have not been clearly demonstrated. We have used RNAi to knockdown Shank3 expression in neuronal cultures and showed that this treatment specifically reduced the synaptic expression of the metabotropic glutamate receptor 5 (mGluR5), but did not affect the expression of other major synaptic proteins. The functional consequence of Shank3 RNAi knockdown was impaired signaling via mGluR5, as shown by reduction in ERK1/2 and CREB phosphorylation induced by stimulation with (S)-3,5-dihydroxyphenylglycine (DHPG) as the agonist of mGluR5 receptors, impaired mGluR5-dependent synaptic plasticity (DHPG-induced long-term depression), and impaired mGluR5-dependent modulation of neural network activity. We also found morphological abnormalities in the structure of synapses (spine number, width, and length) and impaired glutamatergic synaptic transmission, as shown by reduction in the frequency of miniature excitatory postsynaptic currents (mEPSC). Notably, pharmacological augmentation of mGluR5 activity using 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)-benzamide as the positive allosteric modulator of these receptors restored mGluR5-dependent signaling (DHPG-induced phosphorylation of ERK1/2) and normalized the frequency of mEPSCs in Shank3-knocked down neurons. These data demonstrate that a deficit in mGluR5-mediated intracellular signaling in Shank3 knockdown neurons can be compensated by 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)-benzamide; this raises the possibility that pharmacological augmentation of mGluR5 activity represents a possible new therapeutic approach for patients with Shank3 mutations.

Prevalence of SHANK3 variants in patients with different subtypes of autism spectrum disorders

“…Recent studies on Shank3-deficient hippocampal neurons 27,28 and mouse models [29][30][31][32] showed how this gene has an important role in modeling the PSD at the striatum level, regulating basal neurotransmission at the AMPA glutamate receptors, 38 and point mutations. [15][16][17] These results indicate that SHANK3 mutations have a frequency of about 1% in ASD cases, revealing a role for this gene as a possible major contributor in autism.…”

“…However, the knock-out model for only the a isoform (targeting the ankyrin repeats) showed minimal disruption and no behavioral issues. 28 On the other hand, deletion of the Homer-binding domain in one copy of SHANK3 is sufficient to induce a 'gain-of-function' effect with severe consequences on SHANK3 levels and function at the PSD and on NMDA receptor activity. 31 The c.1304 þ 48C4T transition affects a CpG dinucleotide in the middle of one of these highly conserved regions and bisulfate studies showed that this cytosine is supposed to be methylated and that the area surrounding the dinucleotide and encompassing part of exon 10 and part of intron 10 is methylated as well.…”

“…According to recent studies, the region containing this CpG dinucleotide appears to be critical for the regulation of a shorter isoform of SHANK3. 28,46 The loss of this specific isoform seems to cause severe synaptic disorganization and ASD-like features in mice. 28 In our study we detected some variants that were shared by ASD patients as well as unaffected parents and controls.…”

“…Moreover, recent works in neuronal cell cultures 27,28 and mouse models [29][30][31][32] demonstrated a critical role for this gene in synaptic function, social interaction, and social communication, providing a functional link between SHANK3 deficiency and ASD behavioral features. Therefore, we screened this gene for mutations in two different groups of patients with ASD from the United States (US) and Italy.…”

Dendritic spine actin cytoskeleton in autism spectrum disorder

“…In general, SHANK1 promotes dendritic spine head enlargement, and SHANK2 and 3 promote dendritic spine formation, with SHANK3 also inhibiting dendritic spine arborization by binding to Densin-180 [173][174][175][176][177] . Shank3 knockout mice suffer from a loss of cortical actin filaments, in association with reduced Rac1/p21-activated kinase (PAK) activity together with increased activity of cofilin 178 .…”