Shank Modulates Postsynaptic Wnt Signaling to Regulate Synaptic Development

Abstract

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 Here we characterize 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. We find 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.

Significance statement: Haploinsufficiency for SHANK3 is one of the most prevalent monogenic causes of autism spectrum disorder, making it imperative to understand how the Shank family regulates neurodevelopment and synapse function. We created the first animal model lacking all Shank proteins and used the Drosophila neuromuscular junction, a model glutamatergic synapse, to characterize the role of Shank at synapses. We identified a novel function of Shank in synapse maturation via regulation of Wnt signaling in the postsynaptic cell.

Keywords: Shank; Wnt signaling; postsynaptic scaffold; synaptic development.

Figure 1.

Shank localizes to the PSD at Drosophila NMJs. A, Genomic locus of Shank (CG30483). The region deleted in Shank^D101 is indicated in red. Coding exons are green, with noncoding exons in blue. B, The Shank locus encodes a 1871 aa protein predicted to contain Ankyrin repeats (Ank), Src homolgy 3 (SH3), PDZ domains, and a C-terminal coiled-coil motif. The region used to generate anti-Shank antisera is indicated in red. Shank protein structure is highly conserved compared with human SHANK3. SHANK3 has a proline-rich region and C-terminal SAM domain that are not conserved in Drosophila Shank. A percentage identity matrix calculated using Clustal Omega is presented comparing Drosophila Shank and human SHANK1, SHANK2, and SHANK3. C–E, Representative NMJs, stained with antibodies to Shank (green). HRP staining (magenta) marks the neuronal membrane. Arrowheads mark nonspecific staining of the muscle nuclei, which is unchanged in all genotypes. F, Representative NMJs, stained with antibodies to Shank (green) and Dlg (magenta). G, Representative NMJs of animals expressing UAS–Shank–GFP with the mef2–GAL4 driver and stained for GFP (green) and Dlg (magenta). Scale bars: C–F, G, 5 μm; F′, 2 μm.

Figure 2.

Shank regulates synaptic morphology and maturity in a dose-dependent manner. A–H, Representative NMJs stained with antibodies to Dlg (magenta) and HRP (green). Bouton number is decreased in homozygous Shank null mutants (B), transheterozygotes of the Shank null allele and a chromosomal deficiency (C), Shank heterozygotes (D), and with postsynaptic overexpression of Shank with mef2–GAL4 (E) or 24B–GAL4 (F). GBs were identified as round varicosities of HRP staining lacking Dlg staining. GB number is increased in homozygous Shank null mutants (B′), Shank/Df transheterozygotes (C′), and during strong postsynaptic overexpression of Shank with mef2–GAL4 (E′). Restoration of Shank expression in muscle with a moderate (H, H′) but not a strong (G, G′) driver rescued the Shank^D101 phenotypes. Arrowheads indicate GBs. I, J, Quantification of total bouton number, normalized to the control average (H), and total GB number, normalized to the control average (I). Gray line indicates control mean. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001. n.s., No statistically significant difference. Statistical comparisons are with control unless noted. K, Relative expression measured by qPCR. Individual data points represent biological replicates. Shank expression was normalized to the internal reference gene e1F–1A and calibrated to control sample. Data are presented as mean 2^−ΔΔCτ ± SEM (see Materials and Methods). Gray line indicates the calibrated control value (= 1). Scale bars: A–H, 20 μm; A′–H′, 10 μm.

Figure 3.

Shank regulates SSR ultrastructure but not AZ organization. A, B, Representative NMJs stained with antibodies to GluRIII (green) and Brp (magenta). C, D, Representative NMJs stained with antibodies to GluRIII (green) and GluRIIA (magenta). E, F, Representative NMJs stained with antibodies to GluRIIB (green) and GluRIII (magenta). Glutamate receptor clusters and AZs appear normal in Shank null mutants. G–I, Quantification of GluR size (G), GluR fluorescence (H), and AZ density (I). J, K, Transmission electron microscopy of a bouton and surrounding SSR in control (J) and Shank^D101 (K). Homozygous Shank mutants have reduced SSR density and gaps between the neuronal membrane and SSR (black arrowheads). T-bar structure appears normal (red arrowhead). L–N, Quantification of SSR density, calculated as the area of SSR infoldings normalized to SSR cross-sectional area (L), SSR-deficient neuronal membrane, calculated as the length of neuronal membrane without adjacent SSR membrane normalized to the total bouton perimeter (M), and cross-sectional SSR area, normalized to bouton area (N). Data are presented as mean ± SEM; **p < 0.01, ***p < 0.001. n.s., Not significant. Scale bars: A–F, 5 μm; J, K, 200 nm.

Figure 4.

Shank regulates the postsynaptic FNI pathway. A–D, Representative muscle nuclei stained with antibodies to the C terminus of Fz2. The number of Fz2-C puncta (red arrowheads) is reduced with homozygous loss of Shank (B) or postsynaptic overexpression of Shank (C). The Shank^D101 mutant defect is rescued by postsynaptic overexpression of Shank with 24B–GAL4 (D). E, Quantification of Fz2-C puncta per nucleus. F–I, Representative NMJs stained with antibodies to Dlg (magenta) and HRP (green). Expression of Fz2-C.nls gives a strong rescue of the Shank GB phenotype (H, H′). Expression of GFP.nls has no rescue effect (I, I′). Arrowheads indicate GBs. J, K, Quantification of GB number, normalized to the control average (J), and total bouton number, normalized to the control average (K). Gray line indicates control mean. L, Quantification of bouton size. M, Quantification of Futsch loops. N–O, Representative NMJs stained with Futsch to visualize microtubules. Arrowheads indicate Futsch loops. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001. n.s., Not significant. Statistical comparisons are with control unless noted. Scale bars: A–D, 5 μm; F–I, N, O, 20 μm; F′–I′, N′, O′, 10 μm.

Figure 5.

Shank regulates internalization of Fz2. A, Representative NMJ from an animal expressing Fz2–GFP (green) stained with antibodies to Shank (magenta). B, Representative NMJ from an animal expressing Shank–GFP (magenta) stained with antibodies to Fz2-C (green). C–E, Representative NMJs stained to label the internalized pool (green) and surface pool (magenta) of Fz2, with an antibody against an extracellular epitope in the N terminus. Shank^D101 animals exhibited a reduction in the internalized pool of Fz2 (D). F, G, Quantification of internalized Fz2, normalized to HRP signal (F), and surface Fz2, normalized to HRP signal (G). H, Western blot and quantification of body wall muscle extracts from control animals (the UAS–Fz2–GFP line with no driver), animals expressing Fz2–GFP (mef2>Fz2–GFP), and Shank^D101 animals expressing Fz2–GFP (Shank^D101 mef2>Fz2–GFP), probed for GFP and tubulin (Tub; loading control). The level of expression of Fz2–GFP is not affected in the Shank^D101 mutant background. Data are presented as mean ± SEM; *p < 0.05. n.s., Not significant. Statistical comparisons are with control unless noted. Scale bars: A, B, 5 μm; C–E, 10 μm.

Figure 6.

Model of Shank function. A, Shank functions in a dose-dependent manner to regulate multiple parameters of synapse biology. Both partial loss and partial overexpression of Shank (blue) result in a reduction in the number of synapses at the NMJ. Very low and very high levels of Shank (purple) produce both synapse number defects and synapse maturation defects. The synapse maturation defects are associated with downregulation of FNI signaling. In Shank mutants, the mechanism of FNI downregulation is an impairment of Fz2 internalization from the membrane, whereas for high Shank overexpression, FNI impairment occurs by a different mechanism. B, Shank localizes to the postsynaptic membrane in which it regulates internalization of the Wnt receptor Fz2 to regulate synapse maturation. In the FNI signaling pathway, Fz2 is subsequently transported on microtubules and cleaved, and Fz2-C is imported into the nucleus to regulate synaptic transcription.