Autism-associated protein kinase D2 regulates embryonic cortical neuron development

https://doi.org/10.1016/j.bbrc.2019.09.048Get rights and content

Highlights

  • 1

    Protein kinase D2 regulates embryonic cortical neuron development.

  • 2

    Protein kinase D2 harbors ASD-associated de novo mutations.

  • 3

    ASD-associated mutations in protein kinase D2 impaired its kinase activity.

  • 4

    De novo PKD2 mutations can be a risk factor for autism spectrum disorders.

Abstract

Autism spectrum disorder (ASD) is a heterogeneous neurodevelopmental disorder, characterized by impaired social interaction, repetitive behavior and restricted interests. Although the molecular etiology of ASD remains largely unknown, recent studies have suggested that de novo mutations are significantly involved in the risk of ASD. We and others recently identified spontaneous de novo mutations in PKD2, a protein kinase D family member, in sporadic ASD cases. However, the biological significance of the de novo PKD2 mutations and the role of PKD2 in brain development remain unclear. Here, we performed functional analysis of PKD2 in cortical neuron development using in utero electroporation. PKD2 is highly expressed in cortical neural stem cells in the developing cortex and regulates cortical neuron development, including the neuronal differentiation of neural stem cells and migration of newborn neurons. Importantly, we determined that the ASD-associated de novo mutations impair the kinase activity of PKD2, suggesting that the de novo PKD2 mutations can be a risk factor for the disease by loss of function of PKD2. Our current findings provide novel insight into the molecular and cellular pathogenesis of ASD.

Introduction

Autism spectrum disorder (ASD) is a group of intractable neurodevelopmental disorders with impairments in social interactions and verbal communication abilities, restricted interests and stereotyped repetitive behaviors [1]; the prevalence rate of ASD is 1 in 40 children [2]. Previous studies have suggested that aberrant brain development is critically involved in ASD [[3], [4], [5], [6]]; however, the molecular etiology underlying the pathological phenotypes of ASD remains largely unclear. Currently, there are no established treatment strategies for the core symptoms of ASD. Thus, the development of new treatment approaches based on the molecular etiology and pathophysiology of ASD is urgently needed [7,8].

Molecular genetic studies have suggested that ASD is highly heterogeneous, with many types of genetic abnormalities [9]. The concordance rate for ASD in monozygotic twins was more than 90%, which is significantly higher than the rate in dizygotic twins, suggesting that ASD has a strong genetic component [10,11]. However, numerous cases of ASD are sporadic, and the genetic cause of approximately 90% of sporadic ASD cases remains unidentified [12,13]. Recently, in addition to heritable mutations, de novo mutations, new genomic mutations found in a child but found in neither of the parent, have been investigated as a risk factor for ASD [[14], [15], [16], [17]].

Protein kinase D (PKD) is a serine/threonine protein kinase with a catalytic domain and two cysteine-rich phorbol ester binding domains similar to those of protein kinase C [18,19]. The PKD family members consist of PKD1, PKD2 and PKD3, which are ubiquitously but differentially expressed depending on the cell type and external stimulation [20,21]. In peripheral proliferative tissues, PKD2 plays important roles in cell growth and differentiation [22,23]. In addition to peripheral tissues, PKD2 is highly expressed in the brain, where it regulates the establishment and maintenance of neuronal polarity [24,25]. Recently, we and others have identified de novo mutations in PKD2 from patients with sporadic ASD [26,27], suggesting that the de novo mutations in PKD2 can be associated with the risk of ASD. Considering that ASD is suggested to be caused by aberrant cortical neuron development [[3], [4], [5], [6]], PKD2 may regulate cortical neuron development.

In this study, we conducted a functional analysis of PKD2 in cortical neuron development in vivo. We found that PKD2 was highly expressed in neural stem cells (NSCs) in the embryonic cerebral cortex and that Pkd2 knockdown disrupted neuronal development in the cerebral cortex in mouse embryos. Furthermore, we showed that the ASD-associated de novo PKD2 mutations decreased the autophosphorylation levels of PKD2 and its downstream kinases, ERK1/2, suggesting that the de novo mutations impaired the kinase activity of PKD2. Taken together, our current results strongly suggest that PKD2 regulates cortical neuron development and that ASD-associated de novo PKD2 mutations can be a risk factor for the disease by loss of function of PKD2.

Section snippets

Reverse transcription and real-time PCR

Reverse transcription of total RNA isolated from cells and tissues was performed using Superscript III (Life Technologies, CA, USA). The RNA transcription levels were measured by real-time PCR with SYBR Premix Ex Taq (TaKaRa Bio Inc., Shiga, Japan) using a CFX96 real-time PCR detection system (Bio-Rad Laboratories, CA, USA) as described previously [26]. The expression levels of Pkd2 (forward primer sequence: 5′-CTGTTCTATCGTGGACCAGAAGT-3'; reverse primer sequence: 5′-GGCTGATGTTGGGTCATGTT-3′)

Temporal, regional and cell type-specific expression patterns of Pkd2 in the brain

To reveal the function of PKD2 in the brain, we first analyzed the temporal and regional expression patterns of Pkd2 mRNA in the mouse brain. Pkd2 expression levels were elevated during embryonic neurogenesis between E10.5 and E16.5 and gradually decreased after birth (Fig. 1A). At E16.5, Pkd2 was relatively highly expressed in the ventricular and subventricular zones (VZ/SVZ) of the cerebral cortex, where radial glial cells, embryonic neural stem cells (NSCs), differentiate into young neurons

Discussion

Although previous studies have shown that PKD2 positively regulates retinoic acid-induced neuronal development in mouse neuroblastoma Neuro2a cells [26] and the establishment and maintenance of neuronal polarity in neurons [24,25], the role of PKD2 in neurogenesis is largely unknown. In this study, we found that PKD2 was highly expressed in the VZ/SVZ of the cerebral cortex during embryonic neurogenesis (Fig. 1). Our current results showed for the first time that PKD2 regulated the neuronal

Conflicts of interest

None.

Acknowledgements

This work was supported in part by JSPS KAKENHI, grant numbers JP15H04645 (T.N.), JP18H02574 (T.N.), JP17K19488 (H.H.), JP17H03989 (H.H.) and JP19K07121 (A. H-T. and H.H); the JSPS Research Fellowships for Young Scientists, grant number JP17J00152 (K.M.); MEXT KAKENHI, grant numbers JP18H05416 (H.H.), JP19H05217 (A.K.), JP19H04909 (T.N.) and JP19H05218 (T.N.); AMED, grant numbers JP19dm0107122 (H.H.), JP19dm0207061 (H.H.) and JP19am0101084; Nagai Memorial Research Scholarship, Pharmaceutical

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  • Cited by (4)

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