Elevated microRNA-181c and microRNA-30d levels in the enlarged amygdala of the valproic acid rat model of autism

Highlights

• VPA rat model of autism exhibits an enlarged amygdala

• Enlarged amygdala of the VPA rats have elevated levels of miR-181c and miR-30d

• Suppression of miR-181c reduces neurite numbers and branching and results in decreased synaptic density

Abstract

Autism spectrum disorders are severe neurodevelopmental disorders, marked by impairments in reciprocal social interaction, delays in early language and communication, and the presence of restrictive, repetitive and stereotyped behaviors. Accumulating evidence suggests that dysfunction of the amygdala may be partially responsible for the impairment of social behavior that is a hallmark feature of ASD. Our studies suggest that a valproic acid (VPA) rat model of ASD exhibits an enlargement of the amygdala as compared to controls rats, similar to that observed in adolescent ASD individuals. Since recent research suggests that altered neuronal development and morphology, as seen in ASD, may result from a common post-transcriptional process that is under tight regulation by microRNAs (miRs), we examined genome-wide transcriptomics expression in the amygdala of rats prenatally exposed to VPA, and detected elevated miR-181c and miR-30d expression levels as well as dysregulated expression of their cognate mRNA targets encoding proteins involved in neuronal system development. Furthermore, selective suppression of miR-181c function attenuates neurite outgrowth and branching, and results in reduced synaptic density in primary amygdalar neurons in vitro. Collectively, these results implicate the small non-coding miR-181c in neuronal morphology, and provide a framework of understanding how dysregulation of a neurodevelopmentally relevant miR in the amygdala may contribute to the pathophysiology of ASD.

Introduction

Autism spectrum disorders (ASD) constitute a heterogeneous group of severe neurodevelopmental disorders with a genetic predisposition and involvement of multiple environmental factors during embryonic and/or early postnatal life (Muller, 2007). ASD are characterized by impaired social interaction and communication, and by restricted and repetitive behavior, features that impair normal functioning. Brain imaging and genetic studies indicate that ASD are associated with impaired connectivity at the molecular, synaptic and neuronal systems levels (Parikshak et al., 2013, van Bokhoven, 2011). Besides genetic predisposition, ASD can also be triggered during pregnancy through the maternal use of medication. The anticonvulsant and mood-stabilizing drug valproic acid (VPA) is prescribed primarily for the treatment of severe forms of epilepsy and as a mood stabilizer in affective disorders. VPA increases the risk in the development of ASD in the unborn child when used in the first trimester of the pregnancy (Markram et al., 2007, Rasalam et al., 2005). In rodents, a single intraperitoneal injection of VPA to the pregnant dam at the time of embryonic neural tube closure increases the risk for ASD-like features in the offspring. Similar to human ASD patients, affected offspring exhibit brain stem injuries, diminished cerebellar Purkinje cell number (Ingram et al., 2000), social interaction deficits, enhanced anxiety, developmental delays, lowered pain sensitivity, impaired information processing and attention, and hyperactivity with lowered exploratory path-finding (Markram et al., 2008, Schneider and Przewlocki, 2005). The VPA animal model is considered one of the best-validated models for ASD and is frequently used to study the alterations in brain development at the level of morphology, gene expression, and neuronal functioning (Silva et al., 2009, Snow et al., 2008).

The mechanism by which VPA induces ASD is not entirely understood. One of the proposed mechanisms is that VPA acts as a histone deacetylase inhibitor thereby inducing epigenetic alterations in the developing brain (Yildirim et al., 2003). In fact, studies have shown that histone acetylation modulates the transcription not only of mRNAs but also several microRNAs (miRs) (Lee et al., 2011). Given that a specific miR can affect the expression of several mRNAs while protein synthesis from a single mRNA can be repressed by several different miRs, a vast combinatorial complexity exists that may partly account for the genetic complexity associated with ASD. Indeed, such a large family of small non-coding genes (> 2800) could explain some of the missing heritability of ASD (Gogos et al., 2010). Accordingly, multiple lines of evidence suggest that altered neuronal development and aberrant synaptic plasticity and morphology, as seen in neurodevelopmental disorders such as in ASD, may result from a common post-transcriptional process that is under tight regulation by miRs (Schouten et al., 2013). However, little is known about the pattern of expression and the functions of these small RNA molecules in different brain regions of normally developing individuals or patients that suffer from ASD (Kaplan et al., 2013).

Post-mortem neuropathology studies on ASD brains have revealed anatomical abnormalities on multiple levels in various brain regions, including the amygdala (reviewed in (Markram et al., 2007)). Behaviorally, the difficulty in relating to others, anxiety and the incapability to form appropriate social interactions has been proposed to be a consequence of amygdala dysfunction in ASD (Rodriguez Manzanares et al., 2005). Amygdala volume and growth have been reported to be increased in early life in ASD (Bellani et al., 2013, Kim et al., 2010, Nordahl et al., 2012, Schumann et al., 2004). Moreover, the marked increase in amygdala size that normally occurs between the ages of 8 to 18 years is absent in ASD individuals (Schumann et al., 2004). In adolescence and adulthood, amygdala volume is reduced in autism patients compared to controls, although contrasting evidence demonstrating enlarged amygdala volumes at this stage in life has also been reported (Aylward et al., 1999, Bellani et al., 2013). At the level of neuronal functioning, the amygdala appears extremely hyperactive and hyperplastic while inhibition by interneurons is impaired in the VPA model for ASD (Markram et al., 2008).

In the present study, we examined the consequences of prenatal VPA exposure on amygdala size and transcriptome expression. The outcome of this study suggests an increase in basolateral nucleus of the amygdala (BLA) volume in animals with an ASD-like phenotype resulting from prenatal VPA exposure. Transcriptomic analysis identified a significant upregulation of miR-181c and miR-30d in the amygdala of VPA-exposed rats. Further in vitro and in silico analyses of miR-181c suggested a role for this small regulatory RNA, in modulating neuronal outgrowth and dendritic complexity as well as spine density in amygdalar neurons, by modulating an intricate gene network associated with neuronal differentiation and synaptogenesis.