Proper synaptic function in neural circuits requires precise pairings between correct pre- and post-synaptic partners. Errors in this process may underlie development of neuropsychiatric disorders, such as autism spectrum disorder (ASD). Development of ASD can be influenced by genetic factors, including copy number variations (CNVs). In this study, we focused on a CNV occurring at the 16p11.2 locus in the human genome and investigated potential defects in synaptic connectivity caused by reduced activities of genes located in this region at
Precise coordination between correct pre- and post-synaptic partners in the nervous system is important for its normal functions in both vertebrates and invertebrates. Synaptic contacts between neurons are established during the embryonic period as differentiating neurons navigate through the extracellular environment and locate their appropriate targets (see Christensen et al., 2013 for a recent review). As a presynaptic neuron approaches its postsynaptic target, an individual presynaptic axon is defasciculated from an axon bundle consisting of multiple axons that travel together toward their specific target to form a synapse.
A number of studies have been performed in the last few decades to unravel the identities of proteins that are critical for specific coordination between pre- and post-synaptic partners (Christensen et al., 2013). Recently, it has been suggested that dysfunction in these critical components may underlie the pathogenesis of various neuropsychiatric diseases, including schizophrenia, bipolar disorder, and autism spectrum disorder (ASD), affecting a significant fraction of the world’s population. For example, schizophrenia-associated mutations in reelin (RELN) and disrupted in schizophrenia1 (DISC1) can cause defects in neuronal migration (Kamiya et al., 2005), dendritic organization, and remodeling of synapses (Arnold, 1999). In addition, protocadherin 12 (PCDH12) linked to schizophrenia (Gregorio et al., 2009) has been implicated in neuronal differentiation and synaptogenesis (John et al., 2015). In line with these findings, another report has suggested the possibility of neuropsychiatric diseases associated with genetic perturbations in the cell adhesion molecule (CAM) pathway, including NRXN1, CNTNAP, and CASK (Redies et al., 2012).
Among the major neuropsychiatric disorders, ASD is characterized by defects in social communication and language development as well as restricted and repetitive behaviors that develops by the age of three (Abrahams and Geschwind, 2008; Reiss et al., 1986; Zoghbi and Bear, 2012). The clinical aspects for diagnosis and behavioral treatments of ASD have been mostly considered in the earlier studies, but a greater attention is now being given to genetic abnormalities that could potentially contribute to the development of ASD. Due to its complex nature, as suggested by multi-layered genetic interactions (see Bourgeron, 2015 for a recent review), it is very difficult to delineate individual candidate genes and relevant molecular pathways of which defects may underlie the pathogenesis of ASD. Recent attempts to elucidate such complex genetic contributions to ASD have employed population- and genome-based analyses, including linkage studies and genomic-wide association studies. As a result, novel genetic components have been identified, ranging from individual genes to chromosomal regions encompassing multiple genes (Bourgeron, 2015). Important outcomes of these genome-based studies in patients with ASD include significant correlations with repetitive deletion or duplication of specific chromosomal regions that leads to changes in the copy number of genes enclosed in the region, called copy number variations (CNVs). Indeed, CNVs at multiple chromosomal loci, including 2p16.3, 3p26.c, 15q11-q13, 16p11.2, and 22q13.33, appear to have strong associations with ASD (Sebat et al., 2007).
Among these CNV loci, frequent deletion or duplication events in the 16p11.2 region have been linked to ASD and schizophrenia in a series of familial studies (Kumar et al., 2008; Marshall et al., 2008; Portmann et al., 2014; Weiss et al., 2008). Furthermore, these events have been estimated to be responsible for up to 1% of ASD cases according to a previous study (5 out of 512 children) (Weiss et al., 2008). The 16p11.2 locus includes 27 annotated genes within an approximately 500Kb segment (Horev et al., 2011; Pucilowska et al., 2015), many of which have been implicated in neural development (Golzio et al., 2012; Kumar et al., 2008; Miyazaki et al., 2006). A recent study using a zebrafish model has demonstrated significant structural abnormalities induced by defects in zebrafish genes homologous to human counterparts at the 16p11.2 locus, including a change in brain size and eye structure as well as formation of abnormal axonal tracts (Blaker-Lee et al., 2012). In addition, dysfunction of extracellular signal–regulated kinase 1 (ERK1) encoded by
We have recently conducted a RNAi-based genetic screen using
All crosses and stocks were raised on standard media at 24°C with 45%–60% humidity. The Canton-S and
Male third instar larvae were dissected in 1X Phosphate-buffered saline (PBS) and prepared for immunofluorescent staining, as previously described (Lee and Wu, 2010). The morphology of neuromuscular junctions (NMJs) was visualized with Alexa 594-conjugated goat anti-HRP antibody (1:250; Jackson ImmunoResearch Laboratories, USA). For direct comparison of absolute FasII density among
The Z-stack images of body-wall muscles 12 (M12) and 13 (M13) were obtained from the abdominal segments A2 - A3 using a confocal microscope (Zeiss, LSM700; Carl Zeiss, Germany) and processed with ZEN software (Carl Zeiss). The pattern of axonal targeting was monitored in M13 that is normally innervated with type Ib, Is and II axon branches, but not with type III axons. These types of axon branches innervate target muscles by forming synaptic boutons of different sizes and shapes (Atwood et al., 1993). The presence of synaptic boutons originating from type III axons was counted as an ectopic innervation of type III axons at a specific synapse. The frequency or the ratio of ectopic type III axon innervations at M13 NMJs (the number of M13 NMJs with ectopic type III synaptic boutons over total number of NMJs examined) was then calculated for each genotype for further analyses. In addition, the defasciculation status of axonal bundles was monitored at the boundary of M13 and M12. The premature defasciculation was then defined as early separation of axon fibers within a single axon bundle before it made contact with M12. The frequency or the ratio of premature defasciculation (the number of NMJs with premature defasciculation over total number of NMJs examined) was then calculated for each genotype for further analyses.
For direct comparison of FasII immunoreactivities, the same confocal scanning protocols were applied to all samples that were treated together in the same tube during an immunostaining procedure. The FasII density was then measured as pixel intensity from at least six type Ib synaptic boutons innervating muscle 12 and connecting axon braches using the ImageJ package (NIH, USA).
Total RNA was extracted from heads of WT and mutant flies using RNeasy Mini Kit (QIAGEN Korea Ltd., Korea), followed by synthesis of cDNA from 2 μg of extracted RNA using QuantiTect Reverse Transcription Kit (QIAGEN). The real-time reverse transcription qPCR reaction (RT-qPCR) was performed using the SYBR Green qPCR master mix (Enzynomics, Korea) in a 96-well plate. The reaction cycles were initiated with a denaturation step (95°C for 10 min), followed by 40 cycles consisting of denaturation (95°C for 15 s), annealing (60°C for 20 s) and extension steps (72°C for 30 s). The RT-qPCR analysis was conducted using Applied Biosystems 7500 Real
To compare the proportional differences in ectopic axon branches between two groups, the data were categorized in the 2 × 2 contingency tables (with phenotypes
CNVs at the 16p11.2 locus have been implicated in familial cases of ASD (Kumar et al., 2008; Marshall et al., 2008; Portmann et al., 2014; Weiss et al., 2008). However, the identities of the genes responsible for the pathological features of ASD linked to these CNVs are not well understood in experimental settings. To systematically investigate the roles of individual human genes in this region, we recently performed an RNAi-based genetic screen using
When innervation patterns of presynaptic motor axons were examined in muscles 13 (M13) and 12 (M12) of the abdominal body-wall musculature in WT and
In addition to such an aberrant innervation pattern in
Abnormal targeting of type III axons and premature defasciculation of axon bundles in
Then, we examined the effect of
Taken together, our results indicate significant contributions of both neuronal and muscular Rolled MAPK3 in finding a proper target for type III axons and tightly controlling axonal defasciculation. These data also suggest differential molecular mechanisms implicated in the regulation of proper synaptic connectivity and defasciculation events of axon bundles, both of which may involve MAPK3 activities.
A previous proteomic analysis has suggested strong functional links between Rolled MAPK3 and diverse classes of proteins, including cyclin-dependent kinase 2 (Cdk2), G-protein alpha-q (Gαq), and glycoprotein 93 (Gp93) (Friedman et al., 2011). It is possible that the activities of these functional networks coordinate proper targeting and defasciculation of motor axons. If that is the case, dysfunction of these network components other than Rolled MAPK3 would also lead to similar abnormalities observed in
Importantly, ectopic type III axons in M13 were frequently detected in
Our results demonstrating similar mutational phenotypes in mutants defective in Rolled MAPK3 as well as Cdk2, Gαq, and Gp93 suggest that they may interact with each other and thus participate in similar signaling pathways. We tested this idea by monitoring the presence of genetic interactions between
Our results described above demonstrate a critical role of Rolled MAPK3 and relevant protein networks in regulating the proper selection of synaptic targets. Based upon the phenotypic similarities in mutants and the presence of genetic interactions, it is plausible that reduced activity of Rolled MAPK3 may underlie expression of the consistent mutant phenotypes we observed. We verified this idea by analyzing the level of the
It has been suggested that Rolled MAPK3 regulates the expression of fasciclin II (FasII) at
Recent studies on ASD have revealed an important link to CNVs at multiple genomic loci (Sebat et al., 2007). In the present study, we investigated the roles of individual genes at the 16p11.2 locus, one of the CNV spots frequently mapped to ASD, in the regulation of synaptic architecture. We took advantage of the stereotypic and identifiable
Recent multi-directional research efforts have yielded significant advances in our understanding about the pathophysiology of neuropsychiatric disorders, including schizophrenia, major depression and ASD. As a result, greater attention has been given to the potential involvement of structural abnormalities in the nervous system (Amaral et al., 2008; Arnold, 1999). For example, macroscopic post-mortem analyses of the brain from patients with schizophrenia revealed a significant reduction in its volume, particularly in the areas of the posterior superior temporal gyrus, amygdalahippocampal complex and hippocampus (Steen et al., 2006; Yoshida et al., 2009). In addition to a gross change in brain size, microscopic changes at the level of individual neurons, such as a decrease in the number and volume of dendritic spines, have been reported in the CA3 region of the hippocampus in patients with schizophrenia (Kolomeets et al., 2005). Similar microscopic changes were also evident in patients with a mood disorder (Law et al., 2014), along with a reduced density of pyramidal neurons and glia in the CA regions of the hippocampus (Stockmeier et al., 2004).
In line with these reports, a body of experimental evidences supports the idea that structural abnormalities in the nervous system confer susceptibility to ASD (see Amaral et al., 2008 for a recent review). Indeed, anatomical differences in several neurocognitive networks have been reported in patients with ASD (Ecker et al., 2012), including greater brain size and enhanced neuronal proliferation in the prefrontal cortex as well as disrupted cellular architecture in the cerebellar cortex (Bauman and Kemper, 2005; Courchesne et al., 2011). Furthermore, brain imaging studies have revealed atypical white- and gray-matter connectivity (Ecker et al., 2012), in line with our results demonstrating abnormal synaptic targeting induced by a mutation in the gene located at an ASD-mapped CNV locus. Taken together, these results demonstrate consistent structural abnormalities in patients suffering from neuropsychiatric disorders and provide useful insights into our understanding of their pathophysiology.
While a strong association has been suggested between abnormal neuronal architecture and susceptibility to ASD, the underlying molecular networks involving protein-protein interactions remain poorly understood. In this study, we demonstrated abnormal axonal innervation caused by a mutation in
In our study, we present a novel phenotype of altered synaptic architecture, including ectopic targeting of presynaptic axons to unnatural postsynaptic partners and early defasciculation of commonly traveling axon bundles that are frequently observed following loss of MAPK3 activity in
In addition to Rolled MAPK3, our results shown in Fig. 4 suggest contributions of other molecules presumably linked to MAPK3, including Cdk2, Gαq, and Gp93 (Friedman et al., 2011), to specific targeting of presynaptic axons. Cdk2 is a serine/threonine kinase that is important for controlling the cell cycle and thus proliferation by modulating the G1/S transition phase (Tsai et al., 1993). When Cdk2 forms a complex with MAPK in the cytoplasm, activation of MAPK induces translocation of this complex into the nucleus, allowing Cdk2 to regulate transition of the cell cycle from the G1 to the S phase in mammalian cells (Blanchard et al., 2000; Wang et al., 2009). However, whether Cdk2 activity is required during neural development other than the stage of neurogenesis remains unclear. Thus, the significance of the functional interaction between MAPK3 and Cdk2 in the regulation of neuronal architecture awaits further investigation.
Gαq, a part of the α subunits of G proteins, activates phospholipase Cβ that hydrolyzes PIP2 and eventually to diacylglycerol and inositol-1,4,5-triphosphate (see Sánchez-Fernández et al., 2014 for a recent review). In addition to a potential correlation between Gαq activity and other neuro-psychiatric disorders such as schizophrenia (Levitt et al., 2006), a recent report indicated a significantly altered
Among various proteins that are potentially linked to MAPK3, our genetic analysis indicates an apparent synergistic interaction between
Despite the differential degrees of genetic interactions between
A previous report in
Studies on molecular mechanisms of ASD have revealed consistent defects in other synaptic proteins, including Neuroligin (NLG), Neurexin (NRX) and SHANK family proteins in ASD patients and animal models (see Chen et al., 2014 for a recent review). Presynaptic NRXs bind to appropriate postsynaptic NLGs to form a trans-synaptic complex and to promote formation and stabilization of the synapse (Chen et al., 2014). A recent report has further confirmed a coordination among NRX, NLG, and Wishful Thinking as important for controlling synaptic architecture and growth at NMJs (Banerjee et al., 2016). In addition to NLG and NRX, SHANK family members are postsynaptic scaffolding proteins that play a critical role in dendritic spine morphogenesis and synaptic plasticity. For example, overexpression and knockdown of SHANK3, one of the SHANK family members, induce changes in dendritic spine morphology in opposite directions (Betancur et al., 2009). Considering their association with ASD, these data together strongly support the idea that synaptic abnormalities induced by dysfunction of NLG, NRX, and SHANK can predispose affected individuals to ASD. However, it should be noted that altered synapse morphology caused by the loss of NLG, NRX, and SHANK is qualitatively different from the aberrant nerve innervation and defasciculation patterns we observed in
In summary, we report that the loss of Rolled MAPK3 located in the ASD-mapped 16p11.2 region induces an error in axon targeting and fasciculation. Similar errors can be detected in mutants defective in other proteins functionally associated with MAPK3, presumably via transcriptional regulation of MAPK3. Our findings provide a unique opportunity to study the contributions of individual genes located at ASD-linked CNV hotspots to the regulation of neuronal architecture, or more specifically, proper synaptic connectivity during development. Taking advantage of the well-defined stereotypic nature of synapses, future studies in