Integrative Analysis of Brain Region-specific Shank3 Interactomes for Understanding the Heterogeneity of Neuronal Pathophysiology Related to <i>SHANK3</i> Mutations

Lee, Yeunkum,Kang, Hyojin,Lee, Bokyoung,Zhang, Yinhua,Kim, Yoonhee,Kim, Shinhyun,Kim, Won-Ki,Han, Kihoon Frontiers Media S.A. 2017 Frontiers in molecular neuroscience Vol.10 No.-

<P>Recent molecular genetic studies have identified 100s of risk genes for various neurodevelopmental and neuropsychiatric disorders. As the number of risk genes increases, it is becoming clear that different mutations of a single gene could cause different types of disorders. One of the best examples of such a gene is <I>SHANK3</I>, which encodes a core scaffold protein of the neuronal excitatory post-synapse. Deletions, duplications, and point mutations of <I>SHANK3</I> are associated with autism spectrum disorders, intellectual disability, schizophrenia, bipolar disorder, and attention deficit hyperactivity disorder. Nevertheless, how the different mutations of <I>SHANK3</I> can lead to such phenotypic diversity remains largely unknown. In this study, we investigated whether Shank3 could form protein complexes in a brain region-specific manner, which might contribute to the heterogeneity of neuronal pathophysiology caused by <I>SHANK3</I> mutations. To test this, we generated a medial prefrontal cortex (mPFC) Shank3 <I>in vivo</I> interactome consisting of 211 proteins, and compared this protein list with a Shank3 interactome previously generated from mixed hippocampal and striatal (HP+STR) tissues. Unexpectedly, we found that only 47 proteins (about 20%) were common between the two interactomes, while 164 and 208 proteins were specifically identified in the mPFC and HP+STR interactomes, respectively. Each of the mPFC- and HP+STR-specific Shank3 interactomes represents a highly interconnected network. Upon comparing the brain region-enriched proteomes, we found that the large difference between the mPFC and HP+STR Shank3 interactomes could not be explained by differential protein expression profiles among the brain regions. Importantly, bioinformatic pathway analysis revealed that the representative biological functions of the mPFC- and HP+STR-specific Shank3 interactomes were different, suggesting that these interactors could mediate the brain region-specific functions of Shank3. Meanwhile, the same analysis on the common Shank3 interactors, including Homer and GKAP/SAPAP proteins, suggested that they could mainly function as scaffolding proteins at the post-synaptic density. Lastly, we found that the mPFC- and HP+STR-specific Shank3 interactomes contained a significant number of proteins associated with neurodevelopmental and neuropsychiatric disorders. These results suggest that Shank3 can form protein complexes in a brain region-specific manner, which might contribute to the pathophysiological and phenotypic diversity of disorders related to <I>SHANK3</I> mutations.</P>

Reduced CYFIP2 Stability by Arg87 Variants Causing Human Neurological Disorders

Lee, Yeunkum,Zhang, Yinhua,Ryu, Jae Ryun,Kang, Hyae Rim,Kim, Doyoun,Jin, Chunmei,Kim, Yoonhee,Sun, Woong,Han, Kihoon John WileySons, Inc. 2019 Annals of neurology Vol.86 No.5

Age-dependent decrease of GAD65/67 mRNAs but normal densities of GABAergic interneurons in the brain regions of <i>Shank3</i>-overexpressing manic mouse model

Lee, Bokyoung,Zhang, Yinhua,Kim, Yoonhee,Kim, Shinhyun,Lee, Yeunkum,Han, Kihoon Elsevier 2017 Neuroscience Letters Vol.649 No.-

<P><B>Abstract</B></P> <P>Dysfunction of inhibitory GABAergic interneurons is considered a major pathophysiological feature of various neurodevelopmental and neuropsychiatric disorders. The variants of <I>SHANK3</I> gene, encoding a core scaffold protein of the excitatory postsynapse, have been associated with numerous brain disorders. It has been suggested that abnormalities of GABAergic interneurons could contribute to the <I>SHANK3</I>-related disorders, but the limitation of these studies is that they used mainly <I>Shank3</I> knock-out mice. Notably, <I>Shank3</I>-overexpressing transgenic mice, modeling human hyperkinetic disorders, also show reduced inhibitory synaptic transmission, abnormal electroencephalography, and spontaneous seizures. However, it has not been investigated whether these phenotypes of <I>Shank3</I> transgenic mice are associated with GABAergic interneuron dysfunction, or solely due to the cell-autonomous postsynaptic changes of principal neurons. To address this issue, we investigated the densities of parvalbumin- and somatostatin-positive interneurons, and the mRNA and protein levels of GAD65/67 GABA-synthesizing enzymes in the medial prefrontal cortex, striatum, and hippocampus of adult <I>Shank3</I> transgenic mice. We found no significant difference in the measurements performed on wild-type versus <I>Shank3</I> transgenic mice, except for the decreased GAD65 or GAD67 mRNAs in these brain regions. Interestingly, only GAD65 mRNA was decreased in the hippocampus, but not mPFC and striatum, of juvenile <I>Shank3</I> transgenic mice which, unlike the adult mice, did not show behavioral hyperactivity. Together, our results suggest age-dependent decrease of GAD65/67 mRNAs but normal densities of certain GABAergic interneurons in the <I>Shank3</I> transgenic mice.</P> <P><B>Highlights</B></P> <P> <UL> <LI> The densities of PV<SUP>+</SUP> and SOM<SUP>+</SUP> interneurons are normal in the <I>Shank3</I> transgenic mice. </LI> <LI> The protein levels of GAD65 and GAD67 are normal in the <I>Shank3</I> transgenic mice. </LI> <LI> The mRNA levels of GAD65 and GAD67 are age-dependently decreased in the <I>Shank3</I> transgenic mice. </LI> </UL> </P>

Phosphorylation of CYFIP2, a component of the WAVE-regulatory complex, regulates dendritic spine density and neurite outgrowth in cultured hippocampal neurons potentially by affecting the complex assembly

Lee, Yeunkum,Kim, Doyoun,Ryu, Jae Ryun,Zhang, Yinhua,Kim, Shinhyun,Kim, Yoonhee,Lee, Bokyoung,Sun, Woong,Han, Kihoon Lippincott Williams & Wilkins 2017 NeuroReport Vol.28 No.12

<P>Actin dynamics is a critical mechanism underlying many cellular processes in neurons. The heteropentameric WAVE-regulatory complex (WRC), consisting of WAVE, CYFIP1/2, Nap, Abi, and HSPC300, is a key regulator of actin dynamics that activates the Arp2/3 complex to initiate actin polymerization and branching. The WRC is basally inactive because of intermolecular interactions among the components, which can be modulated by bindings of phospholipids and Rac1, and phosphorylations of WAVE and Abi. However, the phosphorylation of other components of WRC and their functional significance remain largely unknown. To address this issue, we focused on CYFIP1/2, in which we found two brain-specific phosphorylation sites (S582 of CYFIP2 and T1068/T1067 of CYFIP1/2) from a publicly available phosphoproteome database. To understand their functional effects, we overexpressed wild-type, phospho-blocking, or phospho-mimetic mutants of CYFIP2 in cultured hippocampal neurons, and found that only T1067A CYFIP2 decreased the density of stubby spines. Moreover, overexpression of wild-type CYFIP2 increased neurite length, but T1067A did not exert this effect. To understand the mechanism, we modeled CYFIP2 phosphorylation in the crystal structure of WRC and found that T1067 phosphorylation could weaken the interaction between CYFIP2 and Nap1 by inducing conformational changes of CYFIP2 -helical bundles. In the co-immunoprecipitation assay, however, wild-type, T1067A, and T1067E CYFIP2 showed similar interaction levels to Nap1, suggesting that T1067 phosphorylation alone is not sufficient to disrupt the interaction. Considering that the activation of WRC requires disassembly of the complex, our results suggest that T1067 phosphorylation, together with other factors, could contribute toward the activation process.</P>

Striatal Transcriptome and Interactome Analysis of <i>Shank3</i> -overexpressing Mice Reveals the Connectivity between Shank3 and mTORC1 Signaling

Lee, Yeunkum,Kim, Sun Gyun,Lee, Bokyoung,Zhang, Yinhua,Kim, Yoonhee,Kim, Shinhyun,Kim, Eunjoon,Kang, Hyojin,Han, Kihoon Frontiers Media S.A. 2017 Frontiers in molecular neuroscience Vol.10 No.-

<P>Mania causes symptoms of hyperactivity, impulsivity, elevated mood, reduced anxiety and decreased need for sleep, which suggests that the dysfunction of the striatum, a critical component of the brain motor and reward system, can be causally associated with mania. However, detailed molecular pathophysiology underlying the striatal dysfunction in mania remains largely unknown. In this study, we aimed to identify the molecular pathways showing alterations in the striatum of SH3 and multiple ankyrin repeat domains 3 (Shank3)-overexpressing transgenic (TG) mice that display manic-like behaviors. The results of transcriptome analysis suggested that mammalian target of rapamycin complex 1 (mTORC1) signaling may be the primary molecular signature altered in the <I>Shank3</I> TG striatum. Indeed, we found that striatal mTORC1 activity, as measured by mTOR S2448 phosphorylation, was significantly decreased in the <I>Shank3</I> TG mice compared to wild-type (WT) mice. To elucidate the potential underlying mechanism, we re-analyzed previously reported protein interactomes, and detected a high connectivity between Shank3 and several upstream regulators of mTORC1, such as tuberous sclerosis 1 (TSC1), TSC2 and Ras homolog enriched in striatum (Rhes), via 94 common interactors that we denominated “Shank3-mTORC1 interactome”. We noticed that, among the 94 common interactors, 11 proteins were related to actin filaments, the level of which was increased in the dorsal striatum of <I>Shank3</I> TG mice. Furthermore, we could co-immunoprecipitate Shank3, Rhes and Wiskott-Aldrich syndrome protein family verprolin-homologous protein 1 (WAVE1) proteins from the striatal lysate of <I>Shank3</I> TG mice. By comparing with the gene sets of psychiatric disorders, we also observed that the 94 proteins of Shank3-mTORC1 interactome were significantly associated with bipolar disorder (BD). Altogether, our results suggest a protein interaction-mediated connectivity between Shank3 and certain upstream regulators of mTORC1 that might contribute to the abnormal striatal mTORC1 activity and to the manic-like behaviors of <I>Shank3</I> TG mice.</P>

Characterization of the zinc-induced Shank3 interactome of mouse synaptosome

Lee, Yeunkum,Ryu, Jae Ryun,Kang, Hyojin,Kim, Yoonhee,Kim, Shinhyun,Zhang, Yinhua,Jin, Chunmei,Cho, Hyo Min,Kim, Won-Ki,Sun, Woong,Han, Kihoon Academic Press 2017 Biochemical and biophysical research communication Vol. No.

<P><B>Abstract</B></P> <P>Variants of the <I>SHANK3</I> gene, which encodes a core scaffold protein of the postsynaptic density of excitatory synapses, have been causally associated with numerous brain disorders. Shank3 proteins directly bind zinc ions through their C-terminal sterile α motif domain, which enhances the multimerization and synaptic localization of Shank3, to regulate excitatory synaptic strength. However, no studies have explored whether zinc affects the protein interactions of Shank3, which might contribute to the synaptic changes observed after zinc application. To examine this, we first purified Shank3 protein complexes from mouse brain synaptosomal lysates that were incubated with different concentrations of ZnCl<SUB>2</SUB>, and analyzed them with mass spectrometry. We used strict criteria to identify 71 proteins that specifically interacted with Shank3 when extra ZnCl<SUB>2</SUB> was added to the lysate. To characterize the zinc-induced Shank3 interactome, we performed various bioinformatic analyses that revealed significant associations of the interactome with subcellular compartments, including mitochondria, and brain disorders, such as bipolar disorder and schizophrenia. Together, our results showing that zinc affected the Shank3 protein interactions of <I>in vitro</I> mouse synaptosomes provided an additional link between zinc and core synaptic proteins that have been implicated in multiple brain disorders.</P> <P><B>Highlights</B></P> <P> <UL> <LI> The synaptosomal Shank3 complexes incubated with different ZnCl<SUB>2</SUB> concentrations were analyzed by mass spectrometry. </LI> <LI> The 71 proteins that specifically interacted with Shank3 under extra ZnCl<SUB>2</SUB> conditions were identified. </LI> <LI> The zinc-induced Shank3 interactome is associated with mitochondria and brain disorders. </LI> </UL> </P>

기후변화 요인이 의약품 이상사례 보고에 미치는 영향에 관한 연구 : 설사병 및 호흡기계 질환을 중심으로

이성현(Sunghyun Lee),장보원(Bowon Jang),이연금(YeunKum Lee) 대한약학회 2021 약학회지 Vol.65 No.6

The relationship between diarrhea and respiratory disorders and climate change has been previously elucidated. Diarrhea and respiratory diseases have been reported to the Korea Institute of Drug Safety and Risk Management (KIDS) as adverse drug events (ADEs) and as serious adverse events (SAEs) due to the occurrences of hospitalization, death, and malformations. ADEs are reported by patients, hospitals, clinics, pharmacies, manufacturers (importers), and regional drug safety centers. However, it has not been determined that the reported adverse events of diarrhea and respiratory diseases are caused by whether climate change or drugs. Here, we investigate the number of ADEs of diarrhea and respiratory diseases that were reported to the Korea Institute of Drug Safety and Risk Management (KIDS) from September 1, 2012 to October 4, 2020, and analyze them with respect to climatic factors including average temperature, daily precipitation, relative humidity, and fine dust concentration (PM10, PM2.5). Our findings suggest that none of the climate change variables are statistically associated with the reported ADEs or SAEs, and climate change has no effect on the number of reported ADEs and SAEs.

Neuronal function and dysfunction of CYFIP2: from actin dynamics to early infantile epileptic encephalopathy

( Yinhua Zhang ),( Yeunkum Lee ),( Kihoon Han ) 생화학분자생물학회(구 한국생화학분자생물학회) 2019 BMB Reports Vol.52 No.5

The cytoplasmic FMR1-interacting protein family (CYFIP1 and CYFIP2) are evolutionarily conserved proteins originally identified as binding partners of the fragile X mental retardation protein (FMRP), a messenger RNA (mRNA)-binding protein whose loss causes the fragile X syndrome. Moreover, CYFIP is a key component of the heteropentameric WAVE regulatory complex (WRC), a critical regulator of neuronal actin dynamics. Therefore, CYFIP may play key roles in regulating both mRNA translation and actin polymerization, which are critically involved in proper neuronal development and function. Nevertheless, compared to CYFIP1, neuronal function and dysfunction of CYFIP2 remain largely unknown, possibly due to the relatively less well established association between CYFIP2 and brain disorders. Despite high amino acid sequence homology between CYFIP1 and CYFIP2, several in vitro and animal model studies have suggested that CYFIP2 has some unique neuronal functions distinct from those of CYFIP1. Furthermore, recent whole-exome sequencing studies identified de novo hot spot variants of CYFIP2 in patients with early infantile epileptic encephalopathy (EIEE), clearly implicating CYFIP2 dysfunction in neurological disorders. In this review, we highlight these recent investigations into the neuronal function and dysfunction of CYFIP2, and also discuss several key questions remaining about this intriguing neuronal protein. [BMB Reports 2019; 52(5): 304-311]

Smaller Body Size, Early Postnatal Lethality, and Cortical Extracellular Matrix-Related Gene Expression Changes of <i>Cyfip2</i> -Null Embryonic Mice

Zhang, Yinhua,Kang, Hyojin,Lee, Yeunkum,Kim, Yoonhee,Lee, Bokyoung,Kim, Jin Yong,Jin, Chunmei,Kim, Shinhyun,Kim, Hyun,Han, Kihoon Frontiers Media S.A. 2018 Frontiers in molecular neuroscience Vol.11 No.-

<P>Cytoplasmic FMR1-interacting protein 2 (CYFIP2) is a key component of the WAVE regulatory complex (WRC) which regulates actin polymerization and branching in diverse cellular compartments. Recent whole exome sequencing studies identified <I>de novo</I> hotspot variants in <I>CYFIP2</I> from patients with early-onset epileptic encephalopathy and microcephaly, suggesting that CYFIP2 may have some functions in embryonic brain development. Although perinatal lethality of <I>Cyfip2</I>-null (<I>Cyfip2</I><SUP>−/−</SUP>) mice was reported, the exact developmental time point and cause of lethality, and whether <I>Cyfip2</I><SUP>−/−</SUP> embryonic mice have brain abnormalities remain unknown. We found that endogenous <I>Cyfip2</I> is mainly expressed in the brain, spinal cord, and thymus of mice at late embryonic stages. <I>Cyfip2</I><SUP>−/−</SUP> embryos did not show lethality at embryonic day 18.5 (E18.5), but their body size was smaller than that of wild-type (WT) or <I>Cyfip2</I><SUP>+/−</SUP> littermates. Meanwhile, at postnatal day 0, all identified <I>Cyfip2</I><SUP>−/−</SUP> mice were found dead, suggesting early postnatal lethality of the mice. Nevertheless, the brain size and cortical cytoarchitecture were comparable among WT, <I>Cyfip2</I><SUP>+/−</SUP>, and <I>Cyfip2</I><SUP>−/−</SUP> mice at E18.5. Using RNA-sequencing analyses, we identified 98 and 72 differentially expressed genes (DEGs) from the E18.5 cortex of <I>Cyfip2</I><SUP>+/−</SUP> and <I>Cyfip2</I><SUP>−/−</SUP> mice, respectively. Further bioinformatic analyses suggested that extracellular matrix (ECM)-related gene expression changes in <I>Cyfip2</I><SUP>−/−</SUP> embryonic cortex. Together, our results suggest that CYFIP2 is critical for embryonic body growth and for early postnatal survival, and that loss of its expression leads to ECM-related gene expression changes in the embryonic cortex without severe gross morphological defects.</P>

Synaptic adhesion molecule IgSF11 regulates synaptic transmission and plasticity

Jang, Seil,Oh, Daeyoung,Lee, Yeunkum,Hosy, Eric,Shin, Hyewon,van Riesen, Christoph,Whitcomb, Daniel,Warburton, Julia M,Jo, Jihoon,Kim, Doyoun,Kim, Sun Gyun,Um, Seung Min,Kwon, Seok-kyu,Kim, Myoung-Hwa NATURE AMERICA 2016 NATURE NEUROSCIENCE Vol.19 No.1

<P>Synaptic adhesion molecules regulate synapse development and plasticity through mechanisms that include trans-synaptic adhesion and recruitment of diverse synaptic proteins. We found that the immunoglobulin superfamily member 11 (IgSF11), a homophilic adhesion molecule that preferentially expressed in the brain, is a dual-binding partner of the postsynaptic scaffolding protein PSD-95 and AMPA glutamate receptors (AMPARs). IgSF11 required PSD-95 binding for its excitatory synaptic localization. In addition, IgSF11 stabilized synaptic AMPARs, as determined by IgSF11 knockdown-induced suppression of AMPAR-mediated synaptic transmission and increased surface mobility of AMPARs, measured by high-throughput, single-molecule tracking. IgSF11 deletion in mice led to the suppression of AMPAR-mediated synaptic transmission in the dentate gyrus and long-term potentiation in the CA1 region of the hippocampus. IgSF11 did not regulate the functional characteristics of AMPARs, including desensitization, deactivation or recovery. These results suggest that IgSF11 regulates excitatory synaptic transmission and plasticity through its tripartite interactions with PSD-95 and AMPARs.</P>