shank: Biological Overview | References
Gene name - shank
Synonyms - Prosap
Cytological map position - 82C1-82C5
Function - Scaffolding protein
Keywords - neuromuscular junction - regulates synaptic bouton number and maturity - regulates a noncanonical Wnt signaling pathway in the postsynaptic cell by modulating the internalization of the Wnt receptor Fz2
Symbol - shk
FlyBase ID: FBgn0040752
Genetic map position - chr2R:14,061,499-14,140,902
Classification - Src Homology 3 domain superfamily - ankyrin repeats - PDZ domain
Cellular location - cytoplasmic
Prosap/Shank scaffolding proteins regulate the formation, organization, and plasticity of excitatory synapses. Mutations in SHANK family genes are implicated in autism spectrum disorder and other neuropsychiatric conditions. However, the molecular mechanisms underlying Shank function are not fully understood, and no study to date has examined the consequences of complete loss of all Shank proteins in vivo. This study characterized the single Drosophila Prosap/Shank family homolog. Shank is enriched at the postsynaptic membrane of glutamatergic neuromuscular junctions and controls multiple parameters of synapse biology in a dose-dependent manner. Both loss and overexpression of Shank result in defects in synaptic bouton number and maturation. It was found that Shank regulates a noncanonical Wnt signaling pathway in the postsynaptic cell by modulating the internalization of the Wnt receptor Fz2. This study identifies Shank as a key component of synaptic Wnt signaling, defining a novel mechanism for how Shank contributes to synapse maturation during neuronal development (Harris, 2016).
The postsynaptic density (PSD) of excitatory synapses contains a complex and dynamic arrangement of proteins, allowing the cell to respond to neurotransmitter and participate in bidirectional signaling to regulate synaptic function. Prosap/Shank family proteins are multidomain proteins that form an organizational scaffold at the PSD. Human genetic studies have implicated SHANK family genes as causative for autism spectrum disorder (ASD) (Uchino and Waga, 2013; Guilmatre, 2014), with haploinsufficiency of SHANK3 considered one of the most prevalent causes (Betancur and Buxbaum, 2013). Investigations of Shank in animal models have identified several functions for the protein at synapses, including regulation of glutamate receptor trafficking, the actin cytoskeleton, and synapse formation, transmission, and plasticity (Grabrucker, 2011; Jiang and Ehlers, 2013). However, phenotypes associated with loss of Shank are variable, and it has been challenging to fully remove Shank protein function in vivo as a result of redundancy between three Shank family genes and the existence of multiple isoforms of each Shank. There is a single homolog of Shank in Drosophila (Liebl and Featherstone, 2008), presenting the opportunity to characterize the function of Shank at synapses in vivo in null mutant animals (Harris, 2016).
Wnt pathways play important roles in synaptic development, function, and plasticity. Like Shank and several other synaptic genes, deletions and duplications of canonical Wnt signaling components have been identified in individuals with ASD. A postsynaptic noncanonical Wnt pathway has been characterized at the Drosophila glutamatergic neuromuscular junction (NMJ), linking release of Wnt by the presynaptic neuron to plastic responses in the postsynaptic cell. In this Frizzled-2 (Fz2) nuclear import (FNI) pathway, Wnt1/Wg is secreted by the neuron and binds its receptor Fz2 in the postsynaptic membrane. Surface Fz2 is then internalized and cleaved, and a C-terminal fragment of Fz2 (Fz2-C) is imported into the nucleus in which it interacts with ribonucleoprotein particles containing synaptic transcripts. Mutations in this pathway result in defects of synaptic development at the NMJ (Harris, 2016 and references therein).
In this study, a null allele of Drosophila Shank was created, allowing investigation of the consequences of removing all Shank protein in vivo. Loss of Shank is shown to impair synaptic bouton number and maturity and results in defects in the organization of the subsynaptic reticulum (SSR), a complex system of infoldings of the postsynaptic membrane at the NMJ. It was also demonstrated that overexpression of Shank has morphological consequences similar to loss of Shank and that Shank dosage is critical to synaptic development. Finally, the results indicate that Shank regulates the internalization of Fz2 to affect the FNI signaling pathway, revealing a novel connection between the scaffolding protein Shank and synaptic Wnt signaling (Harris, 2016).
By generating Drosophila mutants completely lacking any Shank protein, this study identified a novel function of this synaptic scaffolding protein in synapse development. Aberrant expression of Shank results in defects affecting synapse number, maturity, and ultrastructure, and a subset of these defects is attributable to a downregulation of a noncanonical Wnt signaling pathway in the postsynaptic cell (Harris, 2016).
The defects observed in Shank mutants are mostly consistent with defects described from in vivo and in vitro rodent models of Shank. Synaptic phenotypes reported from Shank mutants vary, likely reflecting incomplete knockdown of Shank splice variants, and heterogeneity in the requirement for Shank between the different brain regions and developmental stages analyzed (for review, see Jiang and Ehlers, 2013). Nevertheless, taken collectively, analyses of Shank1-Shank3 mutant mice indicate that Shank genes regulate multiple parameters of the structure and function of glutamatergic synapses, including the morphology of dendritic spines and the organization of proteins in the PSD (Harris, 2016 and references therein).
By removing all Shank protein in Drosophila, this study identified essential functions for Shank at a model glutamatergic synapse. Shank mutants exhibit prominent abnormalities in synaptic structure, including a decrease in the total number of synaptic boutons, which results in an overall decrease in the number of AZs. In addition, a subset of synaptic boutons fails to assemble a postsynaptic apparatus. Finally, even in mature boutons, the SSR has fewer membranous folds and makes less frequent contact with the presynaptic membrane, indicating a defect in postsynaptic development. The SSR houses and concentrates important synaptic components near the synaptic cleft, including scaffolding proteins, adhesion molecules, and glutamate receptors. Thus, defects in SSR development can affect the assembly and regulation of synaptic signaling platforms. These findings indicate that Shank is a key regulator of synaptic growth and maturation (Harris, 2016).
The findings also indicate that gene dosage of Shank is critical for normal synapse development at Drosophila glutamatergic NMJs. The morphological phenotypes that were observed scale with the level of Shank expression, with mild phenotypes seen with both 50% loss and moderate overexpression of Shank, and severe phenotypes seen with both full loss and strong overexpression of Shank. The observation of synapse loss in heterozygotes of the Shank null allele is significant, because haploinsufficiency of SHANK3 is well established as a monogenic cause of ASD (Harris, 2016).
Consistent with the observation that excess Shank is detrimental, duplications of the SHANK3 genomic region (22q13) are known to cause a spectrum of neuropsychiatric disorders. Large duplications spanning SHANK3 and multiple neighboring genes have been reported in individuals with attention deficit-hyperactivity disorder (ADHD), schizophrenia, and ASD. Smaller duplications, spanning SHANK3 and only one or two adjacent genes, have been reported in individuals with ADHD, epilepsy, and bipolar disorder. Furthermore, duplication of the Shank3 locus in mice results in manic-like behavior, seizures, and defects in neuronal excitatory/inhibitory balance. Thus, the requirement for proper Shank dosage for normal synaptic function may be a conserved feature (Harris, 2016).
One unexpected finding from this study was the identification of a previously unappreciated aspect of Shank as a regulator of Wnt signaling. Shank regulates the internalization of the transmembrane Fz2 receptor, thus affecting transduction of Wnt signaling from the plasma membrane to the nucleus. Downregulation of this pathway is implicated in impaired postsynaptic organization, including supernumerary GBs and SSR defects. The physical proximity of Shank and Fz2 at the postsynaptic membrane suggests that Shank directly or indirectly modulates the internalization of Fz2. Shank is a scaffolding protein with many binding partners that could contribute to such an interaction. One intriguing possibility is the PDZ-containing protein Grip. Shank2 and Shank3 have been reported to bind Grip1. Furthermore, Drosophila Grip transports Fz2 to the nucleus on microtubules to facilitate the FNI pathway (Ataman, 2006). Thus, an interaction between Shank, Fz2, and Grip to regulate synaptic signaling is an attractive model (Harris, 2016).
Although loss of Shank is associated with impaired internalization of the Fz2 receptor, how excess Shank leads to FNI impairment remains an open question. One possibility is that an increase in the concentration of the Shank scaffold at the synapse physically impedes the transport of Fz2 or other components of the pathway or saturates binding partners that are essential for Fz2 trafficking. Both overexpression and loss of function of Shank ultimately lead to a failure to accumulate the cleaved Fz2 C terminus within the nucleus, in which it is required to interact with RNA binding proteins that facilitate transport of synaptic transcripts to postsynaptic compartments. Although Shank and Wnt both play important synaptic roles, this study is the first demonstration of a functional interaction between Shank and Wnt signaling at the synapse (Harris, 2016).
Intriguingly, no obvious defects were found in glutamate receptor levels or distribution in the absence of Shank. This was surprising given the role for Shank in regulating the FNI pathway, and downregulation of FNI was shown previously to lead to increased GluR field size. Several studies have reported changes in the levels of AMPA or NMDA receptor subunits in Shank mutant mice, although others have also observed no changes. Levels of metabotropic glutamate receptors are also affected in some Shank mutant models. Moreover, transfected Shank3 can recruit functional glutamate receptors in cultured cerebellar neurons. It is possible that Drosophila Shank mutants have defects in GluRs that are too subtle to detect with current methodology. Another possibility is that Shank is involved in signaling mechanisms that are secondary to FNI and that lead to compensatory changes in GluRs at individual synapses. Indeed, the results are consistent with Shank having additional functions at the synapse in addition to its role in FNI, particularly affecting synaptic bouton number. In conclusion, this study has fpind that the sole Drosophila Shank homolog functions to regulate synaptic development in a dose-dependent manner, providing a new model system to further investigate how loss of this scaffolding protein may underlie neurodevelopmental disease (Harris, 2016).
Shank (see Drosophila Shank) is a post-synaptic scaffolding protein that has many binding partners. Shank mutations and copy number variations (CNVs) are linked to several psychiatric disorders, and to synaptic and behavioral defects in mice. It is not known which Shank binding partners are responsible for these defects. This study showed that the C. elegans SHN-1/Shank binds L-type calcium channels and that increased and decreased shn-1 gene dosage alter L-channel current and activity-induced expression of a CRH-1/CREB transcriptional target (gem-4 Copine), which parallels the effects of human Shank copy number variations (CNVs) on Autism spectrum disorders and schizophrenia. These results suggest that an important function of Shank proteins is to regulate L-channel current and activity induced gene expression (Pym, 2017).
Search PubMed for articles about Drosophila Shank
Ataman, B., Budnik, V. and Thomas, U. (2006). Scaffolding proteins at the Drosophila neuromuscular junction. Int Rev Neurobiol 75: 181-216. PubMed ID: 17137929
Betancur, C. and Buxbaum, J. D. (2013). SHANK3 haploinsufficiency: a 'common' but underdiagnosed highly penetrant monogenic cause of autism spectrum disorders. Mol Autism 4: 17. PubMed ID: 23758743
Grabrucker, A. M., Schmeisser, M. J., Schoen, M. and Boeckers, T. M. (2011). Postsynaptic ProSAP/Shank scaffolds in the cross-hair of synaptopathies. Trends Cell Biol 21: 594-603. PubMed ID: 21840719
Guilmatre, A., Huguet, G., Delorme, R. and Bourgeron, T. (2014). The emerging role of SHANK genes in neuropsychiatric disorders. Dev Neurobiol 74: 113-122. PubMed ID: 24124131
Harris, K.P., Akbergenova, Y., Cho, R.W., Baas-Thomas, M.S. and Littleton, J.T. (2016). Shank modulates postsynaptic wnt signaling to regulate synaptic development. J Neurosci. 36: 5820-5832. PubMed ID: 27225771
Jiang, Y. H. and Ehlers, M. D. (2013). Modeling autism by SHANK gene mutations in mice. Neuron 78: 8-27. PubMed ID: 23583105
Liebl, F. L. and Featherstone, D. E. (2008). Identification and investigation of Drosophila postsynaptic density homologs. Bioinform Biol Insights 2: 369-381. PubMed ID: 19812789
Pym, E., Sasidharan, N., Thompson-Peer, K. L., Simon, D. J., Anselmo, A., Sadreyev, R., Hall, Q., Nurrish, S. and Kaplan, J. M. (2017). Shank is a dose-dependent regulator of Cav1 calcium current and CREB target expression. Elife 6. PubMed ID: 28477407
Uchino, S. and Waga, C. (2013). SHANK3 as an autism spectrum disorder-associated gene. Brain Dev 35: 106-110. PubMed ID: 22749736
date revised: 15 July 2017
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