Plk2 promotes synaptic destabilization through disruption of N-cadherin adhesion complexes during homeostatic adaptation to hyperexcitation.

Synaptogenesis in the brain is highly organized and orchestrated by synaptic cellular adhesion molecules (CAMs) such as N-cadherin and amyloid precursor protein (APP) that contribute to the stabilization and structure of synapses. Although N-cadherin plays an integral role in synapse formation and synaptic plasticity, its function in synapse dismantling is not as well understood. Synapse weakening and loss are prominent features of neurodegenerative diseases, and can also be observed during homeostatic compensation to neuronal hyperexcitation. Previously, we have shown that during homeostatic synaptic plasticity, APP is a target for cleavage triggered by phosphorylation by Polo-like kinase 2 (Plk2). Here, we found that Plk2 directly phosphorylates N-cadherin, and during neuronal hyperexcitation Plk2 promotes N-cadherin proteolytic processing, degradation, and disruption of complexes with APP. We further examined the molecular mechanisms underlying N-cadherin degradation. Loss of N-cadherin adhesive function destabilizes excitatory synapses and promotes their structural dismantling as a prerequisite to eventual synapse elimination. This pathway, which may normally help to homeostatically restrain excitability, could also shed light on the dysregulated synapse loss that occurs in cognitive disorders.

Haploinsufficiency of Cyfip2 Causes Lithium-Responsive Prefrontal Dysfunction.

OBJECTIVE: Genetic variants of the cytoplasmic FMR1-interacting protein 2 (CYFIP2) encoding an actin-regulatory protein are associated with brain disorders, including intellectual disability and epilepsy. However, specific in vivo neuronal defects and potential treatments for CYFIP2-associated brain disorders remain largely unknown. Here, we characterized Cyfip2 heterozygous (Cyfip2+/- ) mice to understand their neurobehavioral phenotypes and the underlying pathological mechanisms. Furthermore, we examined a potential treatment for such phenotypes of the Cyfip2+/- mice and specified a neuronal function mediating its efficacy. METHODS: We performed behavioral analyses of Cyfip2+/- mice. We combined molecular, ultrastructural, and in vitro and in vivo electrophysiological analyses of Cyfip2+/- prefrontal neurons. We also selectively reduced CYFIP2 in the prefrontal cortex (PFC) of mice with virus injections. RESULTS: Adult Cyfip2+/- mice exhibited lithium-responsive abnormal behaviors. We found increased filamentous actin, enlarged dendritic spines, and enhanced excitatory synaptic transmission and excitability in the adult Cyfip2+/- PFC that was restricted to layer 5 (L5) neurons. Consistently, adult Cyfip2+/- mice showed increased seizure susceptibility and auditory steady-state responses from the cortical electroencephalographic recordings. Among the identified prefrontal defects, lithium selectively normalized the hyperexcitability of Cyfip2+/- L5 neurons. RNA sequencing revealed reduced expression of potassium channel genes in the adult Cyfip2+/- PFC. Virus-mediated reduction of CYFIP2 in the PFC was sufficient to induce L5 hyperexcitability and lithium-responsive abnormal behavior. INTERPRETATION: These results suggest that L5-specific prefrontal dysfunction, especially hyperexcitability, underlies both the pathophysiology and the lithium-mediated amelioration of neurobehavioral phenotypes in adult Cyfip2+/- mice, which can be implicated in CYFIP2-associated brain disorders. ANN NEUROL 2020;88:526-543.

Epilepsy- and intellectual disability-associated CYFIP2 interacts with both actin regulators and RNA-binding proteins in the neonatal mouse forebrain.

Variants of the cytoplasmic FMR1-interacting protein 2 (CYFIP2) gene are associated with early-onset epileptic encephalopathy, intellectual disability, and developmental delay. However, the current understanding of the molecular functions of CYFIP2 is limited to those related to actin dynamics, and thus, the detailed mechanisms of CYFIP2-associated brain disorders remain largely unknown. Here, we isolated the neonatal forebrain CYFIP2 complex using newly generated Cyfip2-3×Flag knock-in mice, and performed mass spectrometry-based analyses to identify proteins in the complex. The CYFIP2 interactome, consisting of 140 proteins, contained not only the expected actin regulators but also 25 RNA-binding proteins (RBPs) including Argonaute proteins. Functionally, overexpression of brain disorder-associated CYFIP2 R87 variants, but not wild-type, inhibited stress granule formation in HeLa cells. Mechanistically, the CYFIP2 R87 variants formed intracellular clusters with Argonaute proteins under both basal and stress conditions, and thereby possibly preventing their assembly into stress granules. Beyond identifying CYFIP2 interactors in vivo, these results may provide novel insights for better understanding the molecular mechanisms of CYFIP2-associated brain disorders.

Shank3 regulates striatal synaptic abundance of Cyld, a deubiquitinase specific for Lys63-linked polyubiquitin chains.

The SH3 and multiple ankyrin repeat domains 3 (Shank3) proteins are core organizers of the postsynaptic density in neuronal excitatory synapses, and their defects cause various neurodevelopmental and neuropsychiatric disorders. Mechanistically, Shank3 directly and indirectly interacts with hundreds of synaptic proteins with diverse functions and potentially exerts its regulatory roles in synaptic development and function via these interactors. However, Shank3-dependent regulation of synaptic abundance has been validated in vivo for only a few Shank3 interactors. Here, using a quantitative proteomic analysis, we identified 136 proteins with altered synaptic abundance in the striatum of Shank3-overexpressing transgenic (TG) mice. By comparing these proteins with those found in a previous analysis of the postsynaptic density of Shank3 knock-out (KO) striatum, we identified and confirmed that cylindromatosis-associated deubiquitinase (Cyld), a deubiquitinase specific for Lys63-linked polyubiquitin chains, was up- and down-regulated in Shank3 TG and KO striatal synapses, respectively. Consistently, we found that the synaptic levels of Lys63-linked polyubiquitin chains were down- and up-regulated in the Shank3 TG and KO striata, respectively. Furthermore, by isolating and analyzing the synaptic Cyld complex, we generated a Cyld interactome consisting of 103 proteins, which may include Cyld substrates. Bioinformatic analyses suggested associations of the Cyld interactome with a few brain disorders and synaptic functions. Taken together, these results suggest that Shank3 regulates the synaptic abundance of Cyld in the mouse striatum and, thereby, potentially modulates the Lys63-linked polyubiquitination of striatal synaptic proteins.

IQ Motif and SEC7 Domain-containing Protein 3 (IQSEC3) Interacts with Gephyrin to Promote Inhibitory Synapse Formation.

Gephyrin is a central scaffold protein that mediates development, function, and plasticity of mammalian inhibitory synapses by interacting with various inhibitory synaptic proteins. Here, we show that IQSEC3, a guanine nucleotide exchange factor for ARF6, directly interacts with gephyrin, an interaction that is critical for the inhibitory synapse localization of IQSEC3. Overexpression of IQSEC3 increases inhibitory, but not excitatory, synapse density in a guanine nucleotide exchange factor activity-dependent manner. Conversely, knockdown of IQSEC3 decreases size of gephyrin cluster without altering gephyrin puncta density. Collectively, these data reveal that IQSEC3 acts together with gephyrin to regulate inhibitory synapse development.

Characterization of the zinc-induced Shank3 interactome of mouse synaptosome.

Variants of the SHANK3 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 ZnCl2, and analyzed them with mass spectrometry. We used strict criteria to identify 71 proteins that specifically interacted with Shank3 when extra ZnCl2 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 in vitro mouse synaptosomes provided an additional link between zinc and core synaptic proteins that have been implicated in multiple brain disorders.

Increased ribosomal protein levels and protein synthesis in the striatal synaptosome of Shank3-overexpressing transgenic mice.

The SH3 and multiple ankyrin repeat domains 3 (Shank3) protein is a core organizer of the macromolecular complex in excitatory postsynapses, and its defects cause numerous synaptopathies, including autism spectrum disorders. Although the function of Shank3 as a postsynaptic scaffold is adequately established, other potential mechanisms through which Shank3 broadly modulates the postsynaptic proteome remain relatively unexplored. In our previous quantitative proteomic analysis, six up-regulated ribosomal proteins were identified in the striatal synaptosome of Shank3-overexpressing transgenic (TG) mice. In the present study, we validated the increased levels of RPLP1 and RPL36A in synaptosome, but not in whole lysate, of the TG striatum. Moreover, protein synthesis and extracellular signaling-regulated kinase (ERK) activity were enhanced in the TG striatal synaptosome. To understand the potential contribution of increased protein synthesis to the proteomic change in the TG striatal synaptosome, we performed RNA-sequencing analyses on both whole synaptosomal and synaptic polysome-enriched fractions. Comparative analyses showed a positive correlation only between the polysome-associated transcriptome and up-regulated proteome in the TG striatal synaptosome. Our findings suggest a novel mechanism through which Shank3 may remodel the postsynaptic proteome by regulating synaptic protein synthesis, whose dysfunction can be implicated in SHANK3-associated synaptopathies.

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

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].

Emerging roles of Lys63-linked polyubiquitination in neuronal excitatory postsynapses.

In the mammalian brain, neuronal excitatory synaptic development, function, and plasticity largely rely on dynamic, activity-dependent changes in the macromolecular protein complex called the postsynaptic density (PSD). Activity-dependent Lys48-linked polyubiquitination and subsequent proteasomal degradation of key proteins in the PSD have been reported. However, investigations into the functions and regulatory mechanisms of Lys63-linked polyubiquitination, the second most abundant polyubiquitin form in synapses, have recently begun. Recent studies showed that a Lys63 linkage-specific deubiquitinase (DUB), cylindromatosis-associated DUB (CYLD) localizes to the PSD where its DUB activity is regulated by different kinases. In addition, Lys63-linked polyubiquitination of postsynaptic density 95 (PSD-95), a core scaffolding protein of the PSD, was identified and its functional significance in synaptic plasticity was characterized. In this review, we summarize these recent findings on Lys63-linked polyubiquitination in excitatory postsynapses, and also propose key questions and prospects about this emerging type of posttranslational modification of the PSD proteome.

Transcriptome analyses suggest minimal effects of Shank3 dosage on directional gene expression changes in the mouse striatum.

Both deletions and duplications of the SH3 and multiple ankyrin repeat domains 3 (SHANK3) gene, encoding excitatory postsynaptic scaffolds, are causally associated with various brain disorders, suggesting that proper Shank3 dosage is critical for normal brain development and function. In addition to its well-established synaptic functions, recent studies have suggested that Shank3 can also affect gene expression in the nucleus. However, it has not been investigated whether there are a group of genes whose directional expression is regulated in a Shank3 dosage-dependent manner (i.e. showing opposite changes in expression following Shank3 reduction and overexpression). This is an important issue to be examined for better understanding why neuronal development and function are sensitive to Shank3 dosage, and how much transcriptional changes contribute to neuronal phenotypes affected by Shank3 dosage. To examine this, we performed transcriptome analyses on the striatum of Shank3 heterozygous and knock-out mice, which identified three and 17 differentially expressed genes, respectively. We then compared the results to those of our previous striatal transcriptome analysis of Shank3 overexpressing mice and identified 31 candidate genes showing directional expression changes in a Shank3 dosage-dependent manner. However, overall, their Shank3 dosage-dependent fold changes were very subtle (average of absolute log2(fold change) was 0.139). Meanwhile, the gene set enrichment analyses of the striatal transcriptome suggested that Shank3 dosage may affect anchoring junction-related functions. Taken together, these results suggest that Shank3 dosage minimally affects directional gene expression changes in the mouse striatum.

Unexpected Compensatory Increase in Shank3 Transcripts in Shank3 Knock-Out Mice Having Partial Deletions of Exons.

Genetic variants of the SH3 and multiple ankyrin repeat domains 3 (SHANK3) gene, which encodes excitatory postsynaptic core scaffolds cause numerous brain disorders. Several lines of Shank3 knock-out (KO) mice with deletions of different Shank3 exons have previously been generated and characterized. The different Shank3 KO mouse lines have both common and line-specific phenotypes. Shank3 isoform diversity is considered a mechanism underlying phenotypic heterogeneity, and compensatory changes through regulation of Shank3 expression may contribute to this heterogeneity. However, whether such compensatory changes occur in Shank3 KO mouse lines has not been investigated in detail. Using previously reported RNA-sequencing analyses, we identified an unexpected increase in Shank3 transcripts in two different Shank3 mutant mouse lines (Shank3B and Shank3ΔC) having partial deletions of Shank3 exons. We validated an increase in Shank3 transcripts in the hippocampus, cortex, and striatum, but not in the cerebellum, of Shank3B heterozygous (HET) and KO mice, using qRT-PCR analyses. In particular, expression of the N-terminal exons 1-12, but not the more C-terminal exons 19-22, was observed to increase in Shank3B mice with deletion of exons 13-16. This suggests a selective compensatory activation of upstream Shank3 promoters. Furthermore, using domain-specific Shank3 antibodies, we confirmed that the increased Shank3 transcripts in Shank3B KO mice produced a small Shank3 isoform that was not detected in wild-type mice. Taken together, our results illustrate another layer of complexity in the regulation of Shank3 expression in the brain, which may also contribute to the phenotypic heterogeneity of different Shank3 KO mouse lines.

Inhibition of Polo-like kinase 2 ameliorates pathogenesis in Alzheimer's disease model mice.

Alzheimer disease (AD) is a neurodegenerative disorder characterized by pathological hallmarks of neurofibrillary tangles and amyloid plaques. The plaques are formed by aggregation and accumulation of amyloid ß (Aß), a cleavage fragment of amyloid precursor protein (APP). Enhanced neuronal activity and seizure events are frequently observed in AD, and elevated synaptic activity promotes Aß production. However, the mechanisms that link synaptic hyperactivity to APP processing and AD pathogenesis are not well understood. We previously found that Polo-like kinase 2 (Plk2), a homeostatic repressor of neuronal overexcitation, promotes APP ß-processing in vitro. Here, we report that Plk2 stimulates Aß production in vivo, and that Plk2 levels are elevated in a spatiotemporally regulated manner in brains of AD mouse models and human AD patients. Genetic disruption of Plk2 kinase function reduces plaque deposits and activity-dependent Aß production. Furthermore, pharmacological Plk2 inhibition hinders Aß formation, synapse loss, and memory decline in an AD mouse model. Thus, Plk2 links synaptic overactivity to APP ß-processing, Aß production, and disease-relevant phenotypes in vivo, suggesting that Plk2 may be a potential target for AD therapeutics.

Excitatory and inhibitory synaptic dysfunction in mania: an emerging hypothesis from animal model studies.

Bipolar disorder (BD) is a common psychiatric disorder characterized by recurrent mood swings between depression and mania, and is associated with high treatment costs. The existence of manic episodes is the defining feature of BD, during which period, patients experience extreme elevation in activity, energy, and mood, with changes in sleep patterns that together severely impair their ability to function in daily life. Despite some limitations in recapitulating the complex features of human disease, several rodent models of mania have been generated and characterized, which have provided important insights toward understanding its underlying pathogenic mechanisms. Among the mechanisms, neuronal excitatory and inhibitory (E/I) synaptic dysfunction in some brain regions, including the frontal cortex, hippocampus, and striatum, is an emerging hypothesis explaining mania. In this review, we highlight recent studies of rodent manic models having impairments in the E/I synaptic development and function. We also summarize the molecular and functional changes of E/I synapses by some mood stabilizers that may contribute to the therapeutic efficacy of drugs. Furthermore, we discuss potential future directions in the study of this emerging hypothesis to better connect the outcomes of basic research to the treatment of patients with this devastating mental illness.

Spontaneous seizure and partial lethality of juvenile Shank3-overexpressing mice in C57BL/6 J background.

The SH3 and multiple ankyrin repeat domains 3 (SHANK3) gene encodes core scaffolds in neuronal excitatory postsynapses. SHANK3 duplications have been identified in patients with hyperkinetic disorders and early-onset generalized tonic-clonic seizures. Consistently, Shank3 transgenic (TG) mice, which mildly overexpress Shank3 proteins exhibit hyperkinetic behavior and spontaneous seizures. However, the seizure phenotype of Shank3 TG mice has only been investigated in adults of the seizure-sensitive strain FVB/N. Therefore, it remains unknown if spontaneous seizures occur in Shank3 TG mice from the early postnatal stages onward, or even in seizure-resistant strains. Clinically, generalized tonic-clonic seizures are the critical risk factor for epilepsy-associated mortality. However, the potential association between Shank3 overexpression and mortality, at least in mice, has not been investigated in detail. In the present study, we backcrossed Shank3 TG mice in seizure-resistant C57BL/6 J strain and monitored their home-cage activities at 3 weeks of age. Of the 15 Shank3 TG mice monitored, two exhibited spontaneous tonic-clonic seizures, and one died immediately after the seizure event. Based on this observation, we determined the survival rate of the Shank3 TG mice from 3 to 12 weeks of age. We found that approximately 40-45% of the Shank3 TG mice, both males and females, died before reaching 12 weeks of age. Notably, 53% and 70% of the total deaths in male and female Shank3 TG mice, respectively, occurred in the juvenile stages. These results suggest spontaneous seizure and partial lethality of juvenile Shank3 TG mice in seizure-resistant background, further supporting the validity of this model.

Transcriptome analysis of Shank3-overexpressing mice reveals unique molecular changes in the hypothalamus.

Various mutations in the SH3 and multiple ankyrin repeat domains 3 (SHANK3) gene are associated with neurodevelopmental and neuropsychiatric disorders. Thus far, synaptic abnormalities in multiple brain regions, including the hippocampus, prefrontal cortex, striatum, and ventral tegmental area, have been investigated in several lines of Shank3 mutant mice. However, although some reports have shown loss and gain of body weight in Shank3 knock-out and overexpressing transgenic (TG) mice, respectively, the potential functions of Shank3 in the hypothalamus, a brain region critically involved in energy intake and expenditure, are unknown. Hence, we first characterized endogenous Shank3 mRNA and protein expression in the hypothalamus of adult wild-type mice. Thereafter, we performed transcriptome analysis (RNA-sequencing) in the hypothalamus of adult Shank3 TG mice which mildly overexpress Shank3 proteins. By comparing the 174 differentially expressed genes in the hypothalamus with those previously reported in the striatum and medial prefrontal cortex (mPFC) of Shank3 TG mice, we found that 159 were hypothalamus-specific while only 15 were also observed in either the striatum or mPFC. Furthermore, gene set enrichment analysis of the RNA-sequencing analysis revealed that ribosome-related genes were enriched especially in the up-regulated genes of Shank3 TG hypothalamus, which is in contrast to the results of the Shank3 TG striatum and mPFC analyses, where ribosome-related genes were enriched in the down-regulated genes. Beyond revealing endogenous Shank3 mRNA and protein expression in the hypothalamus, our results suggest unique molecular changes in the hypothalamus of Shank3 TG mice compared with those in the striatum and mPFC.

Integrative Brain Transcriptome Analysis Reveals Region-Specific and Broad Molecular Changes in Shank3-Overexpressing Mice.

Variants of the SH3 and multiple ankyrin repeat domain 3 (SHANK3) gene, encoding excitatory postsynaptic core scaffolding proteins, are causally associated with numerous neurodevelopmental and neuropsychiatric disorders, including autism spectrum disorder (ASD), bipolar disorder, intellectual disability, and schizophrenia (SCZ). Although detailed synaptic changes of various Shank3 mutant mice have been well characterized, broader downstream molecular changes, including direct and indirect changes, remain largely unknown. To address this issue, we performed a transcriptome analysis of the medial prefrontal cortex (mPFC) of adult Shank3-overexpressing transgenic (TG) mice, using an RNA-sequencing approach. We also re-analyzed previously reported RNA-sequencing results of the striatum of adult Shank3 TG mice and of the prefrontal cortex of juvenile Shank3+/ΔC mice with a 50-70% reduction of Shank3 proteins. We found that several myelin-related genes were significantly downregulated specifically in the mPFC, but not in the striatum or hippocampus, of adult Shank3 TG mice by comparing the differentially expressed genes (DEGs) of the analyses side by side. Moreover, we also found nine common DEGs between the mPFC and striatum of Shank3 TG mice, among which we further characterized ASD- and SCZ-associated G protein-coupled receptor 85 (Gpr85), encoding an orphan Gpr interacting with PSD-95. Unlike the mPFC-specific decrease of myelin-related genes, we found that the mRNA levels of Gpr85 increased in multiple brain regions of adult Shank3 TG mice, whereas the mRNA levels of its family members, Gpr27 and Gpr173, decreased in the cortex and striatum. Intriguingly, in cultured neurons, the mRNA levels of Gpr27, Gpr85, and Gpr173 were modulated by the neuronal activity. Furthermore, exogenously expressed GPR85 was co-localized with PSD-95 and Shank3 in cultured neurons and negatively regulated the number of excitatory synapses, suggesting its potential role in homeostatic regulation of excitatory synapses in Shank3 TG neurons. Finally, we performed a gene set enrichment analysis of the RNA-sequencing results, which suggested that Shank3 could affect the directional expression pattern of numerous ribosome-related genes in a dosage-dependent manner. To sum up, these results reveal previously unidentified brain region-specific and broad molecular changes in Shank3-overexpressing mice, further elucidating the complexity of the molecular pathophysiology of SHANK3-associated brain disorders.

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

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 de novo hotspot variants in CYFIP2 from patients with early-onset epileptic encephalopathy and microcephaly, suggesting that CYFIP2 may have some functions in embryonic brain development. Although perinatal lethality of Cyfip2-null (Cyfip2 -/-) mice was reported, the exact developmental time point and cause of lethality, and whether Cyfip2 -/- embryonic mice have brain abnormalities remain unknown. We found that endogenous Cyfip2 is mainly expressed in the brain, spinal cord, and thymus of mice at late embryonic stages. Cyfip2 -/- 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 Cyfip2 +/- littermates. Meanwhile, at postnatal day 0, all identified Cyfip2 -/- mice were found dead, suggesting early postnatal lethality of the mice. Nevertheless, the brain size and cortical cytoarchitecture were comparable among WT, Cyfip2 +/-, and Cyfip2 -/- mice at E18.5. Using RNA-sequencing analyses, we identified 98 and 72 differentially expressed genes (DEGs) from the E18.5 cortex of Cyfip2 +/- and Cyfip2 -/- mice, respectively. Further bioinformatic analyses suggested that extracellular matrix (ECM)-related gene expression changes in Cyfip2 -/- 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.

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

Dysfunction of inhibitory GABAergic interneurons is considered a major pathophysiological feature of various neurodevelopmental and neuropsychiatric disorders. The variants of SHANK3 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 SHANK3-related disorders, but the limitation of these studies is that they used mainly Shank3 knock-out mice. Notably, Shank3-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 Shank3 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 Shank3 transgenic mice. We found no significant difference in the measurements performed on wild-type versus Shank3 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 Shank3 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 Shank3 transgenic mice.

Polo-like kinase 2 phosphorylation of amyloid precursor protein regulates activity-dependent amyloidogenic processing.

Alzheimer's disease (AD) is a neurodegenerative disorder with cognitive deficits. Amyloidogenic processing of amyloid precursor protein (APP) produces amyloid ß (Aß), the major component of hallmark AD plaques. Synaptic activity stimulates APP cleavage, whereas APP promotes excitatory synaptic transmission, suggesting APP participates in neuronal homeostasis. However, mechanisms linking synaptic activity to APP processing are unclear. Here we show that Polo-like kinase 2 (Plk2), an activity-inducible regulator of homeostatic plasticity, directly binds and phosphorylates threonine-668 and serine-675 of APP in vitro and associates with APP in vivo. Plk2 accelerates APP amyloidogenic cleavage by ß-secretase at synapses and is required for neuronal overactivity-stimulated Aß secretion. These findings implicate Plk2 as a novel mediator of activity-dependent APP amyloidogenic processing.