Research Article

Autism-associated SHANK3 haploinsufficiency causes Ih channelopathy in human neurons

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Science  06 May 2016:
Vol. 352, Issue 6286, aaf2669
DOI: 10.1126/science.aaf2669

Faulty channels, not faulty synapses

SHANK3 is a widely expressed scaffolding protein that is enriched in postsynaptic specializations. In mutant mice, SHANK3 mutations cause autism-like behavioral changes and exhibit alterations in synaptic transmission. Yi et al. produced human neurons lacking SHANK3 but not other genes that are also involved in the autism-like disease Phelan-McDermid syndrome. Instead of affecting synapses, SHANK3 mutations primarily caused a channelopathy, with the major phenotype consisting of a specific impairment of HCN channels. Chronic impairment of membrane currents through channelopathy could account for the phenotypes observed in Phelan-McDermid neurons, such as alterations in cognitive functions and the predisposition to epilepsy.

Science, this issue p. 10.1126/science.aaf2669

Structured Abstract


SHANK3 is a scaffolding protein that is enriched in postsynaptic densities of excitatory synapses but ubiquitously expressed in most cells. SHANK3 gene mutations are significantly associated with autism spectrum disorders (ASDs), and deletion of SHANK3 is thought to cause the major symptoms of Phelan-McDermid syndrome. Moreover, increasing evidence links SHANK3 mutations to schizophrenia. Because SHANK3 is a synaptic protein, SHANK3 mutations are thought to predispose to neuropsychiatric disorders by impairing synaptic function. How SHANK3 mutations are pathogenic, however, remains unclear.


Human neurons derived from Phelan-McDermid syndrome patients display complex abnormalities, including synaptic deficits and altered intrinsic electrical properties. Although some of these abnormalities are reversed by SHANK3 reexpression, the altered electrical properties are difficult to reconcile with a primarily synaptic impairment. Moreover, in mice, Shank3 deletions produce behavioral changes and synaptic transmission deficits, although no cellular phenotype has been identified. Here, we explored the pathogenetic mechanism of human SHANK3 mutations with a conditional genetic approach in human neurons and correlated the results with those obtained in Shank3-mutant mouse neurons. We introduced conditional SHANK3 deletions into human embryonic stem cells and examined isogenic control and heterozygous and homozygous SHANK3-mutant neurons derived from these conditionally mutant cells. In addition, we analyzed developing mouse Shank3-mutant neurons and compared their phenotype with that of human SHANK3-mutant neurons.


Heterozygous and homozygous SHANK3-mutant human neurons displayed diverse abnormalities, ranging from a massive increase in input resistance to increased excitability, modest impairments in dendritic arborization, and decreases in synaptic transmission. Because the increased input resistance suggested an altered channel conductance as a primary impairment, we tested various conductances. We found that the SHANK3 mutations caused a profound impairment in hyperpolarization-activated cation (Ih) currents, which are mediated by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. This impairment produced the increased input resistance; moreover, chronic pharmacological inhibition of Ih currents in wild-type human neurons impaired dendritic arborization and synaptic transmission similar to the SHANK3 mutations. Mechanistically, we detected a direct interaction of HCN channels with SHANK3 protein and observed a decrease in HCN-channel proteins in SHANK3-mutant neurons. Finally, we found that developing hippocampal neurons cultured from heterozygous and homozygous Shank3-mutant mice also exhibited an increased input resistance, reduced Ih currents, and an increased excitability similar to SHANK3-mutant human neurons.


Using human neurons with conditional SHANK3 mutations, we found that SHANK3 mutations impair Ih-channel function, thereby increasing neuronal input resistance and enhancing neuronal excitability. This impairment in intrinsic electrical properties accounts, at least in part, for the decreased dendritic arborization and synaptic transmission of SHANK3-mutant neurons. The reduced Ih-current phenotype manifests early in neuronal development and is similarly observed in immature Shank3-mutant mouse neurons. We propose that, in addition to having a specifically postsynaptic function, SHANK3 protein may perform a general role during neurodevelopment by scaffolding HCN channels that mediate Ih currents in neurons and nonneuronal cells consistent with the ubiquitous expression of SHANK3. Thus, we hypothesize that SHANK3 mutations induce an Ih channelopathy that contributes to ASD pathogenesis and may be amenable to pharmacological intervention.

Conditional SHANK3 deletion in human neurons impairs Ih channel.

Comparison of isogenic control and SHANK3-deficient human neurons reveals that heterozygous and homozygous SHANK3 mutations dramatically decrease Ih-channel function, resulting in multifarious secondary impairments, including a decrease in dendritic arborization and synaptic responses and an increase in input resistance and neuronal excitability.


Heterozygous SHANK3 mutations are associated with idiopathic autism and Phelan-McDermid syndrome. SHANK3 is a ubiquitously expressed scaffolding protein that is enriched in postsynaptic excitatory synapses. Here, we used engineered conditional mutations in human neurons and found that heterozygous and homozygous SHANK3 mutations severely and specifically impaired hyperpolarization-activated cation (Ih) channels. SHANK3 mutations caused alterations in neuronal morphology and synaptic connectivity; chronic pharmacological blockage of Ih channels reproduced these phenotypes, suggesting that they may be secondary to Ih-channel impairment. Moreover, mouse Shank3-deficient neurons also exhibited severe decreases in Ih currents. SHANK3 protein interacted with hyperpolarization-activated cyclic nucleotide-gated channel proteins (HCN proteins) that form Ih channels, indicating that SHANK3 functions to organize HCN channels. Our data suggest that SHANK3 mutations predispose to autism, at least partially, by inducing an Ih channelopathy that may be amenable to pharmacological intervention.

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