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Exp Neurobiol 2016; 25(1): 33-39
Published online February 29, 2016
© The Korean Society for Brain and Neural Sciences
Sang Mee Park1, J. Troy Littleton3,4, Hae Ryoun Park1,2 and Ji Hye Lee1,2,3*
1Department of Oral Pathology, School of Dentistry and 2Institute of Translational Dental Sciences, Pusan National University, Yangsan 50612, Korea 3The Picower Institute for Learning and Memory and 4Department of Biology & Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
Correspondence to: *To whom correspondence should be addressed.
TEL: 82-51-510-8259, FAX: 82-51-510-8249
Copy number variations at multiple chromosomal loci, including 16p11.2, have recently been implicated in the pathogenesis of autism spectrum disorder (ASD), a neurodevelopmental disease that affects 1~3% of children worldwide. The aim of this study was to investigate the roles of human genes at the 16p11.2 loci in synaptic development using
Keywords: Autism, Copy number variations, 16p11.2, Kinesin-2, Drosophila
Autism spectrum disorders (ASDs) are the heterogeneous group of childhood diseases defined by social impairments, defects in verbal and non-verbal communication, and repetitive behaviors . ASD phenotypes can include co-morbidity for various neuropsychiatric disorders such as epilepsy . Defining molecular pathways dysfunctional in ASD is crucial for understanding its pathophysiology. A key question is whether common molecular pathways will emerge for ASD based on defined mutations and de novo genome rearrangements that account for 5~20% of ASDs. Current evidence suggests that the disease may result from disruption in synapse formation and synaptic plasticity during development .
Recent progress in the genetics of ASD has revealed that the frequency of chromosomal rearrangements is strikingly high (~7%) in ASD patients . Linkage analyses, genome-wide association studies (GWAS), and microarray assays have revealed rare, but strong associations between ASD and frequent microdeletions or microduplications of certain chromosomal loci defined as copy number variations (CNVs) . Among the CNVs recently identified, microdeletions (as well as microduplications) of 16p11.2 have been consistently observed in several familial studies and are associated with 0.5~1% of ASD cases [6,7]. The 16p11.2 microdeletion has been sub-mapped to affect 24~27 annotated genes [8,9,10], but the loss-of-function loci responsible for the increased risk of ASD are currently unknown. Importantly, several genes at the 16p11.2 loci appear to play important roles in the neurodevelopmental processes [8,11,12,13,14]. While experimental approaches using model organisms to study the roles of these genes remain limited, a recent study using zebrafish embryos demonstrated structural changes in the brain and eyes along with formation of abnormal axonal tracts caused by defects in zebrafish homologs of human genes at the 16p11.2 loci , suggesting their significant contributions to development of the nervous system.
In this study, we used
All crosses and stocks were raised on standard media at 24℃. The
Male third instar larvae were dissected in 1X Phosphate-buffered saline (PBS) and prepared for immunofluorescent staining, as previously described . Alexa 594-conjugated anti-HRP antibody (Jackson ImmunoResearch Laboratories, Inc., USA) was used at 1:200 to visualize NMJs of wandering third instar larvae. The Z-stack images of ventral longitudinal bodywall muscles 12 and 13 in the abdominal segments A2 and A3 were obtained using a confocal microscope (Zeiss, LSM700; Carl Zeiss, Germany) and merged using ZEN software (Carl Zeiss). Following image acquisition, the pattern of aberrant axonal targeting was monitored in muscle 13 that is normally innervated by axon branches forming type Ib, Is and II boutons. The presence of ectopic innervation by axons other than these three types was counted for each genotype.
The 2×2 contingency tables including categorical data were constructed to represent the proportion of normal versus abnormal nerve innervation patterns. The data were then subjected to Fisher's exact text to compare the proportional differences between two groups. p-value less than 0.05 was considered significant.
While copy number variations at the 16p11.2 loci were linked to a subset of familial ASD cases [6,7], the genes responsible for the pathophysiology of ASD within this region have not been clearly identified in experimental settings. The 16p11.2 loci spans 500~600 Kbps and encompasses approximately at least 24 coding genes  (Fig. 1A). To investigate the roles of individual human genes located within this region, we performed a genetic screen using
In WT, muscle 13 is normally innervated by three motor axon branches (Fig. 2B, arrowheads). These branches are originated from different motoneurons located in the ventral nerve cord, including ISN, SNa and SNb/SNd, and form type Ib, Is and II boutons that are different in size, ranging from 1~2 (type Is) to 3~6 µm in diameter (type Ib) [19,20]. In contrast, type III boutons from a SNb/SNd motoneuron  are observed almost exclusively in muscle 12, but rarely in muscle 13 (Fig. 2B, red arrowhead). Such distinctive innervation patterns in neighboring muscles allowed us to analyze potential errors in specific targeting of presynaptic axons. A preliminary RNAi-based genetic screen performed for the majority of candidate genes revealed NMJ morphology similar to that observed in WT. For instance, knockdown of
Upon a detailed analysis of nerve innervation patterns in the RNAi-based screen, we also found ectopic innervation of axons forming type III boutons (hereafter type III axons) in muscle 13 along with recurrent axon branches caused by downregulation of KLP68D. Such targeting error was rarely seen in WT (Fig. 2B), but more frequently observed in animals expressing a double-stranded RNA (dsRNA) construct against
KLP68D was first described as a kinesin-like motor protein with relatively high homology to kinesin heavy chain motor domain , thus presumably participating in axonal transport [21,22]. KLP68D is considered as a component of Kinesin-2 complex, along with KLP64D and DmKAP, a non-motor accessory protein , all of which are expressed in the central nervous system as well as a subset of peripheral nervous system [21,24]. Considering a high sequence homology between KLP68D and KLP64D , we investigated if disruption of KLP64D would also induce ectopic innervation patterns observed in
When the innervation patterns were analyzed in animals expressing a double-stranded RNA construct against
Recent clinical studies on ASD have revealed gross alterations in the structure of nervous system. For instance, total brain size and the rate of neuronal proliferation in the prefrontal cortex were found to be significantly increased in ASD patients . Such structural changes may reflect altered neuronal connectivity between specific brain regions [26,27,28]. Moreover, recently proposed candidate genes responsible for ASD include various synaptic proteins that play important roles in neurite outgrowth, axonal guidance, axonal targeting and synaptogenesis [28,30,31], suggesting structural abnormalities at a synaptic level responsible for expression of ASD phenotypes. Based on these findings, we hypothesized that genetic perturbation at the 16p11.2 loci would lead to aberrant synaptic connectivity, thus underlying functional disturbances that lead to ASD. Our results demonstrate significant axon targeting errors caused by defects in KLP68D, a
Hetero-trimeric Kinesin-2 complex in
It should be noted that
In summary, we have demonstrated ectopic innervations caused by dysfunction of