Shank Proteins Differentially Regulate Synaptic Transmission

Abstract

Shank proteins, one of the principal scaffolds in the postsynaptic density (PSD) of the glutamatergic synapses, have been associated with autism spectrum disorders and neuropsychiatric diseases. However, it is not known whether different Shank family proteins have distinct functions in regulating synaptic transmission, and how they differ from other scaffold proteins in this aspect. Here, we investigate the role of Shanks in regulating glutamatergic synaptic transmission at rat hippocampal SC-CA1 synapses, using lentivirus-mediated knockdown and molecular replacement combined with dual whole-cell patch clamp in hippocampal slice culture. In line with previous findings regarding PSD-MAGUK scaffold manipulation, we found that loss of scaffold proteins via knockdown of Shank1 or Shank2, but not Shank3, led to a reduction of the number but not the unitary response of AMPAR-containing synapses. Only when both Shank1 and Shank2 were knocked down, were both the number and the unitary response of active synapses reduced. This reduction was accompanied by a decrease in NMDAR-mediated synaptic response, indicating more profound deficits in synaptic transmission. Molecular replacement with Shank2 and Shank3c rescued the synaptic transmission to the basal level, and the intact sterile α-motif (SAM) of Shank proteins is required for maintaining glutamatergic synaptic transmission. We also found that altered neural activity did not influence the effect of Shank1 or Shank2 knockdown on AMPAR synaptic transmission, in direct contrast to the activity dependence of the effect of PSD-95 knockdown, revealing differential interaction between activity-dependent signaling and scaffold protein families in regulating synaptic AMPAR function.

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

“…With the shShk2, we observed a dramatic loss of most major Shank2 isoforms (Figure 10I ) along with the reduction of the third longest isoform of Shank3 (c: 58.98% Figure 10J ). The effect of shShk2 on Shank3 expression was similar to previous studies suggesting that Shank2 might be necessary for recruiting synaptic Shank3 over development (Grabrucker et al, 2011 ; Shi et al, 2017 ). On the other hand, the shShk3 produced ~50%–80% loss of the three longest Shank3 isoforms (a: 70.05%, b: 80.01%, c: 53.6%; Figure 10J ), which are the major zinc-binding isoforms.…”

Distinct Phenotypes of Shank2 Mouse Models Reflect Neuropsychiatric Spectrum Disorders of Human Patients With SHANK2 Variants

“…At postnatal day P5, SHANK is accumulated in the lamellipodium, then shifts into the cytoplasm of the cell bodies and in the growing neurites at P8, and finally to the PSD at P10, which supports the idea that it may play a role in multiple cell biological frameworks of neurons (Boeckers et al, 1999a ). Interestingly, on the electrophysiological level, SHANK1 and SHANK2, but not SHANK3 virus-mediated knockdown reduced the number of spines and the AMPAR responses at CA3-to-CA1 synapses in acute hippocampal slice cultures (Shi et al, 2017 ). Due to this functional divergence of SHANK proteins, it is not surprising that in ASD patients, SHANK2 and SHANK3 mutations are over-represented (Leblond et al, 2014 ).…”

Shank2 expression identifies a subpopulation of glycinergic interneurons involved in nociception and altered in an autism mouse model

“…Since different members of the Shank family, despite their similarity, are not considered mutually redundant (Shi et al, 2017;Schmeisser et al, 2013), the function of Shank2 high cells is predicted to be heavily impacted by Shank2 loss, despite their anatomical integrity (i.e., their normal number and positioning). In agreement with observations in other neuronal subtypes (Chung et al, 2019;Shi et al, 2017;Pappas et al, 2017), we find that loss of Shank2 causes the decrease in NMDAR expression in excitatory synapses on GlyT2+ interneurons.…”

“…Since different members of the Shank family, despite their similarity, are not considered mutually redundant (Shi et al, 2017;Schmeisser et al, 2013), the function of Shank2 high cells is predicted to be heavily impacted by Shank2 loss, despite their anatomical integrity (i.e., their normal number and positioning). In agreement with observations in other neuronal subtypes (Chung et al, 2019;Shi et al, 2017;Pappas et al, 2017), we find that loss of Shank2 causes the decrease in NMDAR expression in excitatory synapses on GlyT2+ interneurons. Although baseline neurotransmission in the pain processing circuit appears to be not affected (as the acute phase of the formalin test is comparable in Shank2-KO mice and WT littermates), the NMDAR-dependent synaptic plasticity that is thought to underlie the second phase of the behavioural response to the formalin test (Coderre et al, 1990;Asante et al, 2009) may be unbalanced, with insufficient potentiation of the inhibitory circuit.…”