ASD-Associated De Novo Mutations in Five Actin Regulators Show Both Shared and Distinct Defects in Dendritic Spines and Inhibitory Synapses in Cultured Hippocampal Neurons.

Iryna Hlushchenko,Ville O Paavilainen,Amr Abouelezz,Pushpa Khanal,Pirta Hotulainen

doi:10.3389/fncel.2018.00217

Iryna Hlushchenko, Ville O Paavilainen + Show 3 more

Open Access

https://doi.org/10.3389/fncel.2018.00217

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Abstract

Many actin cytoskeleton-regulating proteins control dendritic spine morphology and density, which are cellular features often altered in autism spectrum disorder (ASD). Recent studies using animal models show that autism-related behavior can be rescued by either manipulating actin regulators or by reversing dendritic spine density or morphology. Based on these studies, the actin cytoskeleton is a potential target pathway for developing new ASD treatments. Thus, it is important to understand how different ASD-associated actin regulators contribute to the regulation of dendritic spines and how ASD-associated mutations modulate this regulation. For this study, we selected five genes encoding different actin-regulating proteins and induced ASD-associated de novo missense mutations in these proteins. We assessed the functionality of the wild-type and mutated proteins by analyzing their subcellular localization, and by analyzing the dendritic spine phenotypes induced by the expression of these proteins. As the imbalance between excitation and inhibition has been suggested to have a central role in ASD, we additionally evaluated the density, size and subcellular localization of inhibitory synapses. Common for all the proteins studied was the enrichment in dendritic spines. ASD-associated mutations induced changes in the localization of α-actinin-4, which localized less to dendritic spines, and for SWAP-70 and SrGAP3, which localized more to dendritic spines. Among the wild-type proteins studied, only α-actinin-4 expression caused a significant change in dendritic spine morphology by increasing the mushroom spine density and decreasing thin spine density. We hypothesized that mutations associated with ASD shift dendritic spine morphology from mushroom to thin spines. An M554V mutation in α-actinin-4 (ACTN4) resulted in the expected shift in dendritic spine morphology by increasing the density of thin spines. In addition, we observed a trend toward higher thin spine density with mutations in myosin IXb and SWAP-70. Myosin IIb and myosin IXb expression increased the proportion of inhibitory synapses in spines. The expression of mutated myosin IIb (Y265C), SrGAP3 (E469K), and SWAP-70 (L544F) induced variable changes in inhibitory synapses.

Highlights

Autism spectrum disorder (ASD) comprises a range of neurological conditions characterized by social deficits, repetitive behaviors, and accompanying comorbidities, including intellectual disability, epilepsy, hyperactivity, and anxiety
To find ASD-associated missense mutations in actin regulators, we relied on the gene and mutation lists published by (Fromer et al, 2014), which summarized de novo mutations in genes associated with different neuropsychiatric diseases
From the distributions of width-tolength ratios of spines we can see that in cells overexpressing wild-type or mutant α-actinin-4, more spines possessed mature morphology compared to controls. This suggests that changes in spine widths and lengths occur proportionally in the same spines. These results show that mutated α-actinin-4 is less concentrated in dendritic spines and fails to mimic the effect of the wild-type protein on dendritic spine morphology

Summary

INTRODUCTION

Autism spectrum disorder (ASD) comprises a range of neurological conditions characterized by social deficits, repetitive behaviors, and accompanying comorbidities, including intellectual disability, epilepsy, hyperactivity, and anxiety. To find ASD-associated missense mutations in actin regulators, we relied on the gene and mutation lists published by (Fromer et al, 2014) , which summarized de novo mutations in genes associated with different neuropsychiatric diseases From this list, we selected ASDassociated genes encoding the known actin-regulating proteins: SRGAP3, TRIO, MYO9B, MYO7B, MYH10, MYO15A, ACTN4, SWAP70, NEB, and TTN. Our main hypothesis was that genetic mutations associated with ASD shift dendritic spine morphology from mushroom spines to thin spines To test this hypothesis, we studied how ASD-associated single base-pair de novo mutations in five selected genes affect the localization and function of the encoded proteins, and whether these mutations change the proteins’ overexpression effects on dendritic spine density and morphology in primary rat hippocampal neurons. These proteins could affect inhibitory synapse dynamics and their localization, size and density through the regulation of actin dynamics (Wierenga, 2017)

MATERIALS AND METHODS

Figures and Molecular Modeling

RESULTS

Findings

DISCUSSION