Mutations in Hevin/Sparcl1 and risk of auti sm spectrum disorder

Takumi Taketomi,Fuminori Tsuruta

Hevin/Sparcl1 (hereafter referred to as Hevin) is an extracellular matrix protein encoded by theSPARCL1gene.Recently,it has been revealed that Hevin has various functions,such as synapse formation,neuronal migration,inflammation,and angiogenesis (Gongidi et al.,2004;Naschberger et al.,2016;Singh et al.,2016;Liu et al.,2021).In addition,genome-wide association studies uncoveredde novoand familial mutations of theSPARCL1gene associated with a risk for autism spectrum disorder (ASD) (De Rubeis et al.,2014).However,the relationship between ASD-associated Hevin mutant and cellular phenotype has not been clarified.Recently,we have reported that ASDassociated mutation in Hevin reduces secretion efficiency and induces endoplasmic reticulum (ER)stress caused by structural instability (Taketomi et al.,2022).In this perspective,we discuss the relationship between the molecular functions of Hevin and ASD risk (Figure 1A).Also,we introduce our recent findings that link ASD-associated Hevin mutant to the cellular phenotype of ASD.

ASD is the most common neurodevelopmental disorder.ASD patients often suffer from deficits in social communication and adaptive capability.The global prevalence of ASD is estimated at approximately one in a hundred (Zeidan et al.,2022).It has been suggested that parental age is a significant risk factor for the birth of autistic children.Notably,it is known that paternal aging consistently increases the risk of ASD onset.A comprehensive study found that children of fathers over 50 years old have about 1.66 times the risk of ASD compared to those aged their 20s(Sandin et al.,2016).An interesting hypothesis to explain this phenomenon is that the probability of gene mutations is increased in spermatogonia due to increase number of replication and exposure to mutagens during aging.In fact,congenital genetic defects are the common causes of ASD risk.Currently,many genes are categorized as ASD-associated genes by several genome-wide association studies.These protein dysfunctions caused by a point mutation are highly linked to the pathogenesis of ASD.The behavior alteration in ASD may be attributed to abnormalities in neuronal migration,cerebral angiogenesis,neuroinflammation,and synaptogenesis.Particularly,abnormal synaptic formation and aberrant excitatory-inhibitory balance are considered to be the most prevalent theories.Therefore,understanding the mechanisms by which mutations in synaptic genes alter brain functions is crucial for clarification of ASD onset.A genome-wide association studies of ASD has verified that gene mutation in theSPARCL1gene is associated with ASD risk (De Rubeis et al.,2014).Hevin comprises an acidic flexible domain at the N-terminal region,a follistati n-like (FS) domain at the center region,and an extracellular calciumbinding (EC) domain,which has two EF-hands at the C-terminus.It has been reported that Hevin acts as a pre-and postsynaptic organizer via its bridging Neurexin 1α and Neuroligin 1B (NL1B)in the cerebral cortex (Singh et al.,2016).Hevin deficient mice show a reduction in the number of thalamocortical excitatory synapses (Singh et al.,2016).In addition,a recent structural analysis has clarified that the FS domain in Hevin is significant in forming a complex with Neurexins and NLs.Hevin also interacts with Collagen V,suggesting the possibility that Hevin stabilizes transsynaptic bridges by binding to collagen of the extracellular matrix (Fan et al.,2021).Interaction of Hevin with NLs and collagen V depends on Ca2+concentration,suggesting the importance of EC domain structural integrity.Importantly,Hevin also has functions in neuronal migration,inflammation,and angiogenesis.It is reported that Hevin is expressed in radial glia,which are the scaffold cells of migrating neurons in the developmental stage,and regulates termination of neuronal migration by cell detaching function(Gongidi et al.,2004).A hepatology study revealed that Hevin highly exists in the plasma and induces cytokine genes in the liver in the high fructose and high cholesterol-diet mice but not normal mice.In addition,Hevin interacts with Toll-like receptor 4,leading to activation of the nuclear factor-κB pathway in hepatocytes,supporting the potential functions that Hevin is involved in neuroinflammation (Liu et al.,2021).These observations suggest the possibility that Hevin activates inflammatory pathways in a context-dependent manner.The latest research investigated that SPARC,which is the Hevin family protein,also interacts with Toll-like receptor 4 and converts M2 macrophages to M1 (Ryu et al.,2022).SPARC has the FS-EC domain,and a small N-terminal acidic domain compared to Hevin.The FS and EC domain of SPARC has an amino acid sequence with approximately 60% identi ty to Hevin.These findings suggest that the C-terminus including FS-EC domain is an important region that underlies the interaction of Hevin with Tolllike receptor 4.A recent study revealed that Hevin is expressed in endothelial cells in the quiescent state and suppresses angiogenesis (Naschberger et al.,2016).Interestingly,we found that Hevin is expressed at the adjacent microvessels in the late embryonic brains,suggesting that Hevin regulates angiogenesis in the developing brain (unpublished data).These researches indicate that the proper Hevin functions are crucial for brain homeostasis and development (Figure 1A).Although Hevin is an ASD-related protein,the molecular mechanisms by which Hevin mutant links to ASD-associated cellular phenotype are not fully understood.

Our recent studies have reported that ASDassociated amino acid substitution of Trp 647 with Arg (W647R) in Hevin attenuates secretion efficiency and induces ER stress response(Taketomi et al.,2022).Furthermore,we conducted structural observation and found that the hydrophobic amino acids around W647 areti ghtly connected and form the hydrophobic core.The amino acids forming the hydrophobic core are D517,V520,L616,I521,F630,T628,Y588,I658,K582,and Y590.We also conducted the molecular dynamics simulation to verify the effect of ASDrelated amino acid substitution.We found that the hydrophobic core is exposed by replacing Trp with Arg.Hydrophobic amino acid is the potenti al target of interaction for BiP,which is the molecular chaperone in the ER and critical regulator of ER stress signaling.We found that Hevin W647R mutants interact with BiP compared to Hevin wildtype.Therefore,it is plausible that exposure of the hydrophobic amino acids to the surface is the key process for upregulating ER stress.Other ASDassociatedSPARCL1mutations are predominantly in the FS and EC domains (De Rubeis et al.,2014;Taketomi et al.,2022).Interestingly,the mutation in which Met 587 was replaced with Ile is next to the hydrophobic core forming Y588.Thus,it is plausible that M587I amino acid substitution in Hevin likely triggers exposure of the hydrophobic amino acids.Hence,it is likely that a single amino acid substitution in the EC domain affects the export of Hevin from the ER via structural instability,leading to the activation of ER stress response pathway.

Our study raises questions and opens the door for future studies.Our data shows that the Hevin W647R mutant presents secretion efficiency reduction,ER stress,and abnormal structure(Taketomi et al.,2022).However,how these phenomena link to ASD-associated pathogenesis is sti ll undefined.One possibility is that the W647R amino acid substitution changes Hevin molecular functions such as synaptogenesis,proper neural migration,inflammation,and angiogenesis.An interesting hypothesis is that ER stress signaling is the key mechanism that underlies ASD pathogenesis.Indeed,clinical research revealed that ER stress-related transcription factors are upregulated in the middle frontal cortex of ASD subjects (Crider et al.,2017).Moreover,several ASD-associated genes involved in synaptogenesis,such as cell adhesion molecules 1,NL3,and SLC6A1,are known to accumulate in the ER,followed by activation of the ER stress response.These genes are also known to be expressed in endothelial cells in the brain.The ER stress signaling transcription factors,such as transcription factors activating transcription factor 6 and PKRlike endoplasmic reticulum kinase in endothelial cells,are known to be the key regulator for VEGFmediated angiogenesis (Karali et al.,2014).Thus,it is possible that excessive ER stress signaling causes abnormal vessel formation in the brain.Another hypothesis is that the Hevin W647R mutant has gain-of-function by interaction with unexpected molecules.The hydrophobic region is also known to be the target of molecular interaction.Because the Hevin W647R is evidently secreted to the extracellular spaces,although the secretion efficiency is down compared to the wild type.Thus,it is possible that the secreted Hevin W647R has another partner and causes gain-of-function in the extracellular space.However,further studies are needed to clarify these possibilities in the future.

In summary,we addressed here that Hevin is one of the pivotal factors implicated in ASD risk.Our recent studies have reported that W647R mutant reduces secretory efficiency and induces ER stress by altering its structure (Figure 1B).In the future,research using genetically engineered mice will contribute to elucidating the mechanisms that lead to point mutation to the pathogenesis of ASD underlying the ASD behavioral changes.We apologize to the many authors whose papers could not be cited due to space limitations.We would like to thank the members of our laboratory members and collaborators for supporting our research and helpful discussions.

Figure 1｜The characteristics of Hevin and ASD risk.

Takumi Taketomi,Fuminori Tsuruta*

Ph.D.Program in Human Biology,School of Integrative and Global Majors,University of Tsukuba,Tsukuba,Ibaraki,Japan (Taketomi T,Tsuruta F)

Master’s and Doctoral Programs in Biology,Faculty of Life and Environmental Sciences,University of Tsukuba,Tsukuba,Ibaraki,Japan;Ph.D.Program in Humanics,School of Integrative and Global Majors,University of Tsukuba,Tsukuba,Ibaraki,Japan;Master’s and Doctoral Program in Neuroscience,Graduate School of Comprehensive Human Sciences,University of Tsukuba,Tsukuba,Ibaraki,Japan (Tsuruta F)

*Correspondence to:Fuminori Tsuruta,PhD,tsuruta.fuminori.fn@u.tsukuba.ac.jp.

https://orcid.org/0000-0002-3471-7654(Fuminori Tsuruta)

Date of submission:August 31,2022

Date of decision:October 10,2022

Date of acceptance:October 20,2022

Date of web publication:November 25,2022

https://doi.org/10.4103/1673-5374.361543

How to cite this article:Taketomi T,Tsuruta F(2023) Mutations in Hevin/Sparcl1 and risk of auti sm spectrum disorder.Neural Regen Res 18(7):1499-1500.

Open access statement:This is an open access journal,and articles are distributed under the terms of the Creative Commons AttributionNonCommercial-ShareAlike 4.0 License,which allows others to remix,tweak,and build upon the work non-commercially,as long as appropriate credit is given and the new creations are licensed under the identical terms.