ORIGINAL RESEARCH published: 03 August 2018 doi: 10.3389/fncel.2018.00217 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 1, Pushpa Khanal 1, Amr Abouelezz 1,2,3, Ville O. Paavilainen 2,4 and Pirta Hotulainen 1* 1 Minerva Foundation Institute for Medical Research, Helsinki, Finland, 2 HiLIFE, University of Helsinki, Helsinki, Finland, 3 Neuroscience Center, University of Helsinki, Helsinki, Finland, 4 Institute of Biotechnology, University of Helsinki, Helsinki, Finland Edited by: Monica Mendes Sousa, Many actin cytoskeleton-regulating proteins control dendritic spine morphology and i3S, Instituto de Investigação e density, which are cellular features often altered in autism spectrum disorder (ASD). Inovação em Saúde, Portugal Recent studies using animal models show that autism-related behavior can be rescued Reviewed by: by either manipulating actin regulators or by reversing dendritic spine density or Marco Rust, Philipps University of Marburg, morphology. Based on these studies, the actin cytoskeleton is a potential target pathway Germany for developing new ASD treatments. Thus, it is important to understand how different Jaewon Ko, Daegu Gyeongbuk Institute of Science ASD-associated actin regulators contribute to the regulation of dendritic spines and and Technology (DGIST), South Korea how ASD-associated mutations modulate this regulation. For this study, we selected Maurizio Giustetto, five genes encoding different actin-regulating proteins and induced ASD-associated Università degli Studi di Torino, Italy de novo missense mutations in these proteins. We assessed the functionality of the *Correspondence: Pirta Hotulainen wild-type and mutated proteins by analyzing their subcellular localization, and by pirta.hotulainen@helsinki.fi 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 Received: 14 February 2018 Accepted: 03 July 2018 central role in ASD, we additionally evaluated the density, size and subcellular localization Published: 03 August 2018 of inhibitory synapses. Common for all the proteins studied was the enrichment in Citation: dendritic spines. ASD-associated mutations induced changes in the localization of Hlushchenko I, Khanal P, Abouelezz A, α Paavilainen VO and Hotulainen P -actinin-4, which localized less to dendritic spines, and for SWAP-70 and SrGAP3, (2018) ASD-Associated De Novo which localized more to dendritic spines. Among the wild-type proteins studied, only Mutations in Five Actin Regulators α-actinin-4 expression caused a significant change in dendritic spine morphology Show Both Shared and Distinct Defects in Dendritic Spines and by increasing the mushroom spine density and decreasing thin spine density. We Inhibitory Synapses in Cultured hypothesized that mutations associated with ASD shift dendritic spine morphology from Hippocampal Neurons. α Front. Cell. Neurosci. 12:217. mushroom to thin spines. An M554V mutation in -actinin-4 (ACTN4) resulted in the doi: 10.3389/fncel.2018.00217 expected shift in dendritic spine morphology by increasing the density of thin spines. Frontiers in Cellular Neuroscience | www.frontiersin.org 1 August 2018 | Volume 12 | Article 217 Hlushchenko et al. ASD-Associated Mutations in Synaptic Actin Regulators 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. Keywords: autism spectrum disorder, actin cytoskeleton, dendritic spines, inhibitory synapses, de novo point mutations INTRODUCTION regulators controlling dendritic spines may play direct causal roles in ASD-related behavior. The social deficits and NMDA Autism spectrum disorder (ASD) comprises a range of receptor hypofunction displayed by Shank3-deficient mice neurological conditions characterized by social deficits, were rescued by inhibiting the activity of the actin filament- repetitive behaviors, and accompanying comorbidities, including depolymerizing protein, cofilin, or by activating Rac1, the actin intellectual disability, epilepsy, hyperactivity, and anxiety. ASD cytoskeleton master-regulator (Duffney et al., 2015). Remarkably, has a strong genetic component and almost 1000 genes are Dolan and colleagues showed that a single administration of currently associated with ASD (SFARI Gene: https://gene.sfari. p21-activated kinase inhibitor, small molecule FRAX486, was org/database/human-gene/). Many ASD-associated mutations sufficient to recover the phenotype in adult Fmr1-knockout are rare protein-disrupting de novo mutations that arose in mice, demonstrating that a post-diagnostic therapy could the germline. Mutations can be copy-number variants (CNVs) be possible for Fragile-X adults (Dolan et al., 2013). p21- or single-base-pair mutations. Numerous ASD susceptibility activated kinase is a Rac1 effector, which regulates spines genes are involved in regulating the postsynaptic site of through modulation of actin cytoskeleton dynamics. Based glutamatergic synapses (Peça and Feng, 2012; Bourgeron, on these results, the actin cytoskeleton has recently emerged 2015), the development and maturation of synaptic contacts as a potential new target for new ASD treatments. Thus, (Gilman et al., 2011), or synaptic transmission (Li et al., 2014). it is important to understand how different ASD-associated Most excitatory glutamatergic synapses are located on small actin regulators contribute to the regulation of dendritic dendritic protrusions known as dendritic spines. The formation, spines and how ASD-associated mutations modulate this maturation, and elimination of dendritic spines lie at the core of regulation. synaptic transmission and memory formation (Yang et al., 2009; When we started this study, the SFARI Gene Database was Roberts et al., 2010). Studies of postmortem human ASD brains not yet available. Therefore, to find ASD-associated missense revealed an increased spine density, which is—at least in some mutations in actin regulators, we relied on the gene and cases—the result of defective dendritic spine pruning (Tang et al., mutation lists published by (Fromer et al., 2014) , which 2014). summarized de novo mutations in genes associated with different Numerous studies have demonstrated a pivotal role for the neuropsychiatric diseases. From this list, we selected ASD- actin cytoskeleton in the formation and elimination, motility and associated genes encoding the known actin-regulating proteins: stability, and size and shape of dendritic spines (Hotulainen and SRGAP3, TRIO, MYO9B, MYO7B, MYH10, MYO15A, ACTN4, Hoogenraad, 2010). Actin filaments are polar structures with one SWAP70, NEB, and TTN. Nebulin (NEB) and titin (TTN) are end growing more rapidly (the plus or “barbed” end) than the giant proteins (nebulin 600–900 kDa, titin up to 4.2 MDa), mostly other (the minus or “pointed” end). Constant removal of the known for their function in muscle sarcomeres. Although they actin subunits from the pointed ends and addition at the barbed may also play roles in neurons, they were excluded from further ends is called actin treadmilling. Synaptic stimulation rapidly studies because of their large size, which makes molecular biology changes the actin treadmilling rate (Star et al., 2002; Okamoto approaches challenging. et al., 2004; Hlushchenko et al., 2016). The actin treadmilling Next, the expression patterns of the identified genes were rate, as well as the three-dimensional organization of actin investigated using the Allen Brain Atlas (mouse brain). MYO7B filaments, are regulated by actin-binding proteins (Hotulainen was not found in the Allen Brain Atlas, and a literature and Hoogenraad, 2010). Many actin regulators are associated search indicated that it is not expressed in the brain (Chen with ASD and these proteins are often involved in the regulation et al., 2001). MYO15A seemed to show very weak expression of the structure and function of excitatory synapses (Joensuu in the brain. Thus, these two myosins were excluded from et al., 2017). However, our knowledge of whether ASD-associated further experiments. Attempts to clone TRIO constructs were mutations in actin regulators affect their functions in dendritic unsuccessful and therefore the final study was carried out with spines or synapses is limited. five genes: ACTN4, MYO9B, SWAP70, MYH10, and SRGAP3. Recent studies using different animal models have shown The mutations we investigated are all unique, one-allele de that autistic symptoms can be rescued by either manipulating novo mutations leading to mixed expression of wild-type and actin regulators or by rescuing dendritic spine density or mutated proteins. The selected genes also have other mutations; morphology (Dolan et al., 2013; Duffney et al., 2015). Although currently, the SFARI Gene database reports 3 variants for ACTN4 it is not yet clear how aberrant dendritic spines and behavioral (inheritance pattern unknown or de novo), 27 variants for consequences are connected, these results suggest that actin MYO9B (both familial and de novo) and 4 variants for SRGAP3 Frontiers in Cellular Neuroscience | www.frontiersin.org 2 August 2018 | Volume 12 | Article 217 Hlushchenko et al.