XLMR protein related to neurite extension (Xpn/KIAA2022) regulates cell–cell and cell–matrix adhesion and migration

doi:10.1016/j.neuint.2013.09.011

Neurochemistry International

Volume 63, Issue 6, November 2013, Pages 561-569

https://doi.org/10.1016/j.neuint.2013.09.011 Get rights and content

Highlights

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Xpn regulates cell–cell and cell–matrix adhesion.
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Xpn regulates N-cadherin and β1-integrin expression.
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Xpn is strongly expressed in nucleus.
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Xpn is involved in cellular migration.

Abstract

X-linked mental retardation (XLMR) is a common cause of moderate to severe intellectual disability in males. XLMR protein related to neurite extension (Xpn, also known as KIAA2022) has been implicated as a gene responsible for XLMR in humans. Although Xpn is highly expressed in the developing brain and is involved in neurite outgrowth in PC12 cells and neurons, little is known about the functional role of Xpn. Here, we show that Xpn regulates cell–cell and cell–matrix adhesion and migration in PC12 cells. Xpn knockdown enhanced cell–cell and cell–matrix adhesion mediated by N-cadherin and β1-integrin, respectively. N-Cadherin and β1-integrin expression at the mRNA and protein levels was significantly increased in Xpn knockdown PC12 cells. Furthermore, overexpressed Xpn protein was strongly expressed in the nuclei of PC12 and 293T cells. Finally, depletion of Xpn perturbed cellular migration by enhancing N-cadherin and β1-integrin expression in a PC12 cell wound healing assay. We conclude that Xpn regulates cell–cell and cell–matrix adhesion and cellular migration by regulating the expression of adhesion molecules.

Introduction

Several genes have been identified as members of the X-linked mental retardation (XLMR) family (Ropers, 2006). A previous study reported an inactivating disruption of the X-linked KIAA2022 gene by a chromosomal rearrangement in two male patients with severe mental retardation (Cantagrel et al., 2004). Furthermore, a recent study reported that patients with KIAA2022 mutations had intellectual disability with autistic features and strabismus (Van Maldergem et al., 2013). Thus, it was suggested that the expression level of KIAA2022 was related to the pathogenesis of XLMR. KIAA2022 encodes a large protein of 1516 amino acids. Previous reports have shown that KIAA2022 has no functional motifs or significant homology with other known proteins (Cantagrel et al., 2004, Cantagrel et al., 2009). KIAA2022 mRNA is highly expressed in the brain, particularly during the late embryonic and perinatal stages of development. Recent studies show that KIAA2022 participates in neurite outgrowth in PC12 cells and neurons, suggesting a role for Xpn in brain development. Therefore, we termed KIAA2022 as Xpn (XLMR protein related to neurite extension) (Ishikawa et al., 2012, Van Maldergem et al., 2013). However, few reports are available on the functional role of Xpn in neuronal development.

The cadherins are a superfamily of proteins that regulate many cellular functions, including cell adhesion and motility (Takeichi, 1991, Tepass et al., 2000). Cadherins are important for neuronal development, with many members of this family being expressed in the brain (Tepass et al., 2000). N-cadherin is involved in the formation of both adherens and synaptic junctions in the nervous system. Moreover, recent reports have shown that members of the cadherin family are associated with neuropsychiatric disorders, including mental retardation (Pedrosa et al., 2008, Redies et al., 2012, Wang et al., 2009). Integrins are transmembrane receptors that function in the recognition of extracellular matrix and cell-surface proteins (Hynes, 2002). Integrins have been shown to be important in cell migration, axonal guidance and proper synapse formation during the development of the central and peripheral nervous systems (Reichardt and Tomaselli, 1991, Schmid and Anton, 2003).

In the present study, to investigate the functional role of Xpn, we studied the effect of Xpn knockdown on cell–cell and cell–matrix adhesion and migration. We report that Xpn regulates cellular adhesion and migration of PC12 cells by modulating N-cadherin and β1-integrin expression.

Section snippets

Cell culture

PC12 and 293T cells were maintained in tissue culture dishes (Thermo Fisher Scientific, Rochester, NY, USA). The PC12 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies Inc., Grand Island, NY, USA) containing 5% heat inactivated fetal bovine serum and 10% horse serum. The 293T cells were cultured in DMEM containing 10% heat inactivated fetal bovine serum. The cultures were maintained at 37 °C in an atmosphere of 95% air and 5% CO₂. The cells were observed under a

Xpn knockdown promotes cellular adhesion

To investigate the physiological functions of Xpn, RNA interference analysis was used to knock down endogenous Xpn expression in PC12 cells. The siRNA sequences targeting the rat Xpn gene have already been shown to effectively suppress rat Xpn mRNA expression in PC12 cells (Ishikawa et al., 2012). Using qRT-PCR analysis, we confirmed that the siRNA targeting Xpn suppressed the expression of Xpn mRNA. The expression level of Xpn mRNA in the siRNA transfected cells was reduced to 50% compared

Discussion

In this study, we demonstrated that knockdown of Xpn expression enhanced cell–cell and cell–matrix adhesion, which are mediated by N-cadherin and β1-integrin, respectively, in PC12 cells (Fig. 1). Knockdown of Xpn increased N-cadherin and β1-integrin expression at both the protein and mRNA levels (Fig. 2). Overexpressed Xpn protein was strongly expressed in the nuclei of PC12 and 293T cells (Fig. 3). Knockdown of Xpn impaired the migration of PC12 cells in a wound healing assay (Fig. 4).

Acknowledgements

The authors declare no conflict of interest. This work was supported in part by the MEXT Strategic Research Program for Brain Sciences, a Grant-in-Aid for Scientific Research on Innovative Areas “Neural Diversity and Neocortical Organization” from MEXT and by a grant from Dainippon Sumitomo Pharma Co., Ltd. We thank Mrs. Moriya and Mrs. Ohashi for preparing this manuscript.

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The hypothesis of cellular interference has been invoked in PCHD19 X-linked clustering epilepsy.4 NEXMIF, like PCDH19, is involved in cell–cell adhesion and neuronal migration.8-10,17 Thus cellular mosaicism, occurring in females through XCI, could disrupt normal intercellular communication and neuronal networks, potentially predisposing females to epilepsy.
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Sixty-three females and 24 males (46 new patients) with NEXMIF encephalopathy were studied, with 30 novel variants. Phenotypic features included developmental delay/ID in 86/87 (99%), seizures in 71/86 (83%) and multiple comorbidities. Generalized seizures predominated including myoclonic seizures and absence seizures (both 46/70, 66%), absence with eyelid myoclonia (17/70, 24%), and atonic seizures (30/70, 43%). Males had more severe developmental impairment; females had epilepsy more frequently, and varied from unaffected to severely affected. All NEXMIF pathogenic variants led to a premature stop codon or were deleterious structural variants. Most arose de novo, although X-linked segregation occurred for both sexes. Somatic mosaicism occurred in two males and a family with suspected parental mosaicism.
NEXMIF encephalopathy is an X-linked, generalized developmental and epileptic encephalopathy characterized by myoclonic–atonic epilepsy overlapping with eyelid myoclonia with absence. Some patients have developmental encephalopathy without epilepsy. Males have more severe developmental impairment. NEXMIF encephalopathy arises due to loss-of-function variants.
Clinical spectrum of KIAA2022/NEXMIF pathogenic variants in males and females: Report of three patients from Indian kindred with a review of published patients
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The first exon itself spans 180 kb. The third exon is also exceptionally large as compared to other exons, spanning more than 4 kb[22]. The majority of the coding regions are located in this exon.
In the last two decades, with the advent of whole-exome and whole-genome sequencing, supplemented with linkage analysis, more than 150 genes responsible for X-linked intellectual disability have been identified. Some genes like NEXMIF remain an enigmatic entity, as often the carrier females show wide phenotypic diversity ranging from completely asymptomatic to severe intellectual disability and drug-resistant epilepsy.
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Together with the presented cases, 45 cases (24 symptomatic females) were identified from 15 relevant research items for analysis. Males have demonstrated a more severe intellectual disability and increasingly delayed walking age, autistic features, central hypotonia, and gastroesophageal reflux. In contrast, females have shown a predominant presentation with drug-resistant epilepsy and mild to moderate intellectual impairment. Notably, the affected females demonstrate a higher incidence of myoclonic, absence, and atonic seizures. The majority of the variants reported are nonsense or frameshift mutations, causing loss of function of the NEXMIF gene, while a considerable proportion possesses chromosomal translocations, microdeletions, and duplications.
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All patients presented with breakpoints in the Xq13q21 region. In two patients, the X-chromosome breakpoint disrupted coding sequences (KIAA2022 and ZDHHC15 genes). Although both gene disruptions caused absence of transcription in peripheral blood, there is no evidence that supports the involvement of these genes with ovarian function. The ZDHHC15 gene belongs to a conserved syntenic region that encompasses the FGF16 gene, which plays a role in female germ line development. The break in the FGF16 syntenic block may have disrupted the interaction between the FGF16 promoter and its cis-regulatory element. In the third patient, although both breakpoints are intergenic, a gene that plays a role in the DAX1 pathway (FHL2 gene) flanks distally the autosome breakpoint. The FHL2 gene may be subject to position effect due to the attachment of an autosome segment in Xq21 region.
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¹: These authors contributed equally to this work.

View full text

XLMR protein related to neurite extension (Xpn/KIAA2022) regulates cell–cell and cell–matrix adhesion and migration

Highlights

Abstract

Introduction

Section snippets

Cell culture

Xpn knockdown promotes cellular adhesion

Discussion

Acknowledgements

Am. J. Hum. Genet.

Gene Expr. Patterns

J. Mol. Biol.

Neurosci. Lett.

Cell

Neuron

Cell

Neuroscience

Neuron

J. Biol. Chem.

Curr. Neurol. Neurosci. Rep.

Schizophr. Res.

Brain Res.

Dev. Biol.

Curr. Opin. Genet. Dev.

Refinement of cortical dysgeneses spectrum associated with TUBA1A mutations

J. Med. Genet.

The chromatin-remodeling protein ATRX is critical for neuronal survival during corticogenesis

J. Clin. Invest.

Mutations in VLDLR as a cause for autosomal recessive cerebellar ataxia with mental retardation (dysequilibrium syndrome)

J. Child Neurol.

Disruption of a new X linked gene highly expressed in brain in a family with two mentally retarded males

J. Med. Genet.

Recurrence risk of epilepsy and mental retardation in females due to parental mosaicism of PCDH19 mutations

Neurology

Neuronal migration disorders

Ment. Retard. Dev. Disabil. Res. Rev.

Characterization of mutations in the gene doublecortin in patients with double cortex syndrome

Ann. Neurol.