16p11.2 is required for neuronal polarity

Zhi Li, Xi He, Jiexiong Feng

Department of Pediatric Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
The F.M. Kirby Neurobiology Center, Department of Neurology, Boston Children’s Hospital, Harvard Medical School, Harvard University, Boston, USA.

DOI: 10.4236/wjns.2013.34029                

Abstract

Since Autism Spectrum Disorder (ASD) is strongly associated with chromosomal abnormalities of 16p11.2, and Autism has been linked to neuronal polarity defect, our study aimed to explore the role of 16p11.2 genes in regulating neuronal polarity. We performed a neuronal polarity assay in a high throughput manner for candidate genes at 16p11.2. Our most interesting finding was that three 16p11.2 candidate genes, DOC2a, Tbx-6 and KIF 22, affected neuronal polarity. Our research, for the first time, indicates a novel association between 16p11.2 and neuronal polarity. Our results support the hypothesis that 16p11.2 is required for neuronal polarity. Our research provides new important insights into molecular mechanisms underlying the tight association between 16p11.2 and several neural developmental disorders, including autism, epilepsy, mental retardation and schizophrenia.

Keywords

Neuronal Polarity; 16p11.2; Autism; Epilepsy; Neuronal Development

1. INTRODUCTION

Neuronal polarity, defined as neurons which are highly polarized cells, presenting two molecularly and functionally different compartment, single axon and multiple dendrites, is fundamental for neural development and function [1,2]. The formation of axon-dendrite polarity is crucial for neurons to make the proper information flow within the neural system [1]. Neuronal polarity has been studied using dissociated mice embryonic hippocampal neurons, which undergo characteristic growth stages in culture [1,3]. These neurons initially form multiple lamellipodia after plating and generating minor neurites with similar lengths at stage 1 - 2. Then, at stage 3, one of these initially equivalent neurites grows rapidly and becomes the axon, whereas the other neurite subsequently develop into dendrite at stage 4 - 5 [4].

In the past decade, numerous proteins controlling the establishment of neuronal polarity were identified, including microtubule-associated protein [5], kinesin motor proteins [6], N-cadherin [7], glycogen synthase kinase-3 beta (GSK-3β) [8], SAD kinases [9,10], scaffolding proteins [11] and actin cytoskeleton [12].

Autism is a complex childhood neurodevelopmental disorder, which is characterized by impaired social communication and restricted repetitive behavior [13]. Neuronal polarity facilitates the directional flow of information, which is fundamental not only for communication between neurons but also between neurons and effector cells. In light of this point, neuronal polarity defect leads to a group of neuropsychiatric disorders, including autism [14,15], epilepsy [16], mental retardation [17], and schizophrenia [18].

Intriguingly, autism is tightly associated with chromosomal abnormalities of 16p11.2 [14,19,20]. It is confirmed that a region of chromosome 16p11.2 influences susceptibility to autism when either deleted or duplicated [14]. Furthermore, a 600 kb deletion at 16p11.2 leads to a group of neuropsychiatric disorders [21]. 16p11.2 encompasses twenty-five genes, which spans 500 kb and is flanked by 147 kb low copy repeats that are 99% identical [20]. To sum up, 16p11.2 is a novel gene domain which is strongly associated with autism.

Since autism is tightly associated with chromosomal abnormalities of 16p11.2, and autism has been linked to neuronal polarity defect, we hypothesized that 16p11.2 might be associated with neuronal polarity. In light of these, our study aimed to explore the role of 16p11.2 genes in regulating neuronal polarity.

We performed a neuronal polarity assay in a high throughput manner for candidate genes at 16p11.2 which contains 25 candidate genes as followings: MAPK3/ DOC 2A/MAZ/QPRT/SEZ6L2/HIPIP3/SPN/ALDOA/ TaoK2/Tbx-6/ASPHD1/KCTD13/LOC124446/GDPD3/ YPEL3/KIF22/CCDC95/FLJ25404/FAM57B/C16orf53/ PPP4C/C16orf54/PRRT 2/MVP/CDIPT.

Our most interesting finding is that three 16p11.2 candidate genes, DOC2a, Tbx-6 and KIF 22, affect neuronal polarity. Our research, for the first time, indicates a novel association between 16p11.2 and neuronal polarity. Our results support the hypothesis that 16p11.2 genes involve the neuronal polarity regulation. Our finding that 16p11.2 is required for neuronal polarity lays the foundation for the mechanism underlying the new viewpoint that 16p11.2 is tightly associated with several neural developmental disorders, including autism [14,15], epilepsy [16], mental retardation [17], and schizophrenia [18].

2. METHODS

2.1. Hippocampal Neurons Culture

We constructed a stable hippocampal neurons culture system. Culture of hippocampal neurons prepared from E18 mice embryos was performed as described by Banker G. [22]. Hippocampi were dissected from embryonic 18 (E18) mice, digested with a mixture of proteases at 37˚C for 15 min and dissociated with a fire-polished Pasteur Pipette in plating medium MEM (minimal essential medium), containing 10% fetal bovine serum, 0.5% glucose, 1 mM sodium pyruvate, 25 µM glutamine, and penicillin/streptomycin.

Neurons were then plated onto glass coverslips with poly-D-lysine (Sigma) at a density around 100 - 200 neurons/mm². Neuronal culture was incubated at 37˚C with 5% CO₂. After neurons attached to the substrate (normally around 3 - 4 hr after plating), the medium was exchanged to neuronal culture medium in neurobasal medium (Invitrogen) supplemented with B-27 supplement (Invitrogen) and 1 mM glutamine. After plating, hippocampal neurons were electroporated with shRNA by Amaxa^® Mouse Neuron Nucleofector^® Kit [23].

All procedures were carried out in accordance with the Guide for the Humane Use and Care of Laboratory Animals, and the study was approved by the Animal Care and Use Committee of Boston Children’s Hospital, Harvard Medical School.

2.2. sRNAs Electroporation

We knocked down candidate genes via shRNAs (short hairpin RNAs). The shRNAs which knocked down reciprocal candidate genes were designed by Invitrogen Company. We electroporated neurons with short hairpin RNAs-encoding plasmids by Amaxa^® Mouse Neuron Nucleofector^® Kit [24]. One Nucleofection^® Sample contains 2 × 10⁶ hippocampal neurons, 3 μg short hairpin RNAs-encoding plasmids together with 2 μg pmaxGFP^® Vector and 100 μg Nucleofector^® Solution.

We prepared coated coverslips in 12-well plates by filling appropriate number of wells with 300 μl culture medium and equilibrated plates in a humidified 37˚C/ 5% CO₂ incubator, equilibrated additional volume of 500 μl per Nucleofection^®, centrifuged 2 × 10⁶ hippocampal neurons at 80 ×g for 5 minutes at room temperature and suspended the cell pellet carefully in 100 μl Nucleofector^® Solution per sample. We combined 100 μl of cell suspension with 3 μg short hairpin RNAs-encoding plasmids, 2 μg pmax GFP^® Vector, transferred cell/DNA suspension into certified cuvette, selected appropriate Nucleofect^® Program, and then inserted the cuvette with suspension into the Nucleofector^® Cuvette Holder [25]. After electroporation, we added 500 μl of the preequilibrate culture medium to the cuvette and gently transferred hippocampal neurons into the prepared culture dish with the coated coverslip. We incubated hippocampal neurons and replaced fresh culture medium until analysis [26]. 

2.3. Immunocytochemistry

Neurons were fixed with 4% paraformaldehyde for 10 min and washed with PBS. Fixed neurons were permeablized and blocked with a solution containing 3% goat serum, 3% BSA, and 0.1% Triton X-100 in PBS. Neurons were incubated with anti-Tau-1(5 μg/ml) diluted in PBS overnight at 4˚C. After washing with PBS, cells were stained with secondary antibodies at room temperature for 2 hours.

2.4. Image Acquisition and Quantification

Neuron morphology was analyzed with a fluorescence microscope equipped with Hamamatsu ORCA-ER camera. Neuronal polarity phenotypes were categorized into three groups-no-axon neuron (Figure 1), single axon neuron (Figure 2) and multiple-axon neuron (Figure 3). Neuronal polarity was assessed by determining the percentage of neurons with multiple axons. 

2.5. Statistical Analyses

Statistical analysis of neuronal polarity defects in neurons was carried out by analysis of variance (ANOVA), which was performed with GraphPad Prism 5 software (La Jolla, CA). 

3. RESULTS

Our most interesting finding is that three 16p11.2 candidate genes, DOC2a, Tbx-6 and KIF 22, affect neuronal polarity.

Figure 1. No-axon neuron.

Figure 2. single-axon neuron.

Figure 3. Multiple axon.

3.1. DOC2a Knockdown Affects Neuronal Polarity

Neuronal polarity quantification analysis showed that DOC2a knock-down via shRNA significantly increased the number of multiple-axon neurons from 9.1% to 21.6% and decreased the number of single-axon neurons from 83.1% to 70.3% (Figure 4).There was significant difference between control group and DOC2a knockdown group (p < 0.001, ANOVA).

3.2. Tbx-6 Knockdown Affects Neuronal Polarity

Similarly, Tbx-6 knockdown via shRNA significantly increased the number of multiple-axon neurons from 9.1% to 21.9% and decreased the number of single-axon neurons from 83.1% to 71.5% (Figure 5). There was significant difference between control group and Tbx-6 knock-down group (p < 0.001, ANOVA).

3.3. KIF 22 Knockdown Affects Neuronal Polarity

Furthermore, KIF 22 knockdown via shRNA signifycantly increased the number of multiple-axon neurons from 9.1% to 22.1% and decreased the number of single-axon neurons from 83.1% to 69.6% (Figure 6).There was significant difference between control group and KIF 22 knock-down group (p < 0.001, ANOVA).

4. DISCUSSION

In this study, we provides evidence that 16p11.2 is required for neuronal polarity. We perform a neuronal po-

Figure 4. Results for neuronal polarity assay 16p11.2 genes: DOC2a.

Figure 5. Results for neuronal polarity assay 16p11.2 genes: Tbx-6.

Figure 6. Results for neuronal polarity assay 16p11.2 genes: KIF 22.

larity assay in a high throughput manner for 16p11.2 candidate genes. We find that three candidate genes, DOC2a, Tbx-6 and KIF 22, significantly affect neuronal polarity. Our research, for the first time, indicates a novel association between 16p11.2 and neuronal polarity. Our results support the hypothesis that 16p11.2 is required for neuronal polarity. Our research provides new insights into molecular mechanisms underlying the tight association between 16p11.2 and several neural developmental disorders, including autism [14,15], epilepsy [16], mental retardation [17], and schizophrenia [18].

The axons and dendrites of neuronal cells differ from each other in the composition of their proteins and organelles [27]. Axons are typically long and thin, with a uniform width, and they branch at right angles from the cell body. Dendrites are relatively short; as they emerge from the cell body they appear thick, but become thinner with increased distance from the cell body and then undergo Y-shaped branching [28]. Axons contain synaptic vesicles from which they release neurotransmitters at axon terminals in response to electrical signals from the cell body. Dendrites, especially dendritic spines, contain receptors for these neurotransmitters, as well as organelles and signalling systems. These two distinct cellular structures are fundamental for neuronal function and brain development, as they enable neurons to receive and transmit electrical signals. However, the molecular mechanisms that underlie this neuronal polarization were unclear until a decade ago [2].

The polarization of axon and dendrites underlies the ability of neurons to integrate and transmit information in the brain. Many experiments using cultured embryonic hippocampal neurons have revealed that, as they develop, neurons initially generate several equivalent neurites, but then begin to polarize so that one neurite becomes an axon while the remaining neuritis become dendrites. This early asymmetric neurite outgrowth is regulated by various molecules that have established roles in cytoskeletal rearrangements and protein trafficking. A balance of positive and negative signaling regulates these cellular functions, which result in the formation of a single axon [2]. In the past decade, numerous proteins controlling the establishment of neuronal polarity were identified, including microtubule-associated protein [5], kinesin motor proteins [6], N-cadherin [7], glycogen synthase kinase-3 beta (GSK-3β) [8], SAD kinases [9,10], scaffolding proteins [11] and actin cytoskeleton [12].

Nevertheless, to date, the specific gene domain which controls neuronal polarity has never been elucidated.

Intriguingly, it has been demonstrated that 16p11.2 is strongly associated with a group of neural developmental disorders, including autism spectrum disorder 9 [14,15], schizophrenia [18], attention-deficit hyperactivity disorder [29] and abnormal head size [30]. Deletion or duplication of one copy of the 16p11.2 interval is tightly associated with impaired brain function, indicating the importance of 16p11.2 gene domain [31,32].

16p11.2 gene region includes 25 known genes, of which 22 are expressed in the developing human fetal nervous system [19,33]. As yet, the mechanisms leading to neurodevelopmental abnormalities and the broader phenotypes associated with deletion or duplication of 16p11.2 have never been clarified. 

In our experiments, we detect three 16p11.2 genes affecting neuronal polarity: DOC2a, Tbx-6 and KIF22. Doc2a is specifically expressed in neuronal cells and localized on synaptic vesicles [33,34]. In vitro experiments indicate that Doc2a is kinetically tuned to function as a Ca²⁺ sensor for asynchronous neurotransmitter release [35]. Mice deleted for Doc2a show alterations in synaptic transmission and long-term potentiation and exhibit learning and behavioral deficits that include an abnormal passive avoidance task [36].We conclude that DOC2a controls neuronal polarity (structure) and synaptic transmission (function) to facilitate information flow. 

The Tbx genes, which belong to a family of T-box transcriptional factors, expressed in the paraxial mesoderm, play essential roles during the development of posterior somites [37,38]. It has been confirmed that Tbx is required for proper neural crest migration and to stabilize spatial patterns during middle and inner ear development [39]. Furthermore, Tbx gene programs a variety of hindbrain motor neurons for migration, independent of directionality, and in facial neurons is a positive regulator of the non-canonical Wnt signaling pathway [40]. We conclude that Tbx controls neuronal polarity to regulate neural crest migration. 

KIF 22, one of kinesin superfamily, a microtubule associated motor proteins, has been confirmed to transport vesicles involved in synapses [41]. Different motor proteins of the kinesin superfamily (KIFs) are responsible for selective transport of synaptic vesicle components to the axon and of transmitter receptors to the dendrite. For example, KIF5 is preferentially transported to the axon and accumulated at the axonal tip [42], and thus used for transporting axon-targeting proteins, such as VAMP2, GAP43, apolipoprotein E receptor 2 and amyloid precursor protein [41,42]. Moreover, KIF17 is responsible for transporting dendrite-targeting NR2B [43,45]. We infer that KIF22 is a novel kinesin motor protein which is required for neuronal polarity establishment.

In summary, our analyses, for the first time, indicate a novel association between 16p11.2 and neuronal polarity. Our finding supports the hypothesis that 16p11.2 is required for neuronal polarity. Our research provides new important insights into molecular mechanisms underlying the tight association between 16p11.2 and several neural developmental disorders, including autism, epilepsy, mental retardation, and schizophrenia. Future studies will focus on these three 16p11.2 candidate genes affecting neuronal polarity development in animal model (in vivo) to highlight the crucial role of 16p11.2 in neural development and a group of neurodevelopmental diseases.

5. CONCLUSION

Our research, for the first time, indicates a novel association between 16p11.2 and neuronal polarity. Our results support the hypothesis that 16p11.2 is required for neuronal polarity. Our research provides new important insights into molecular mechanisms underlying the tight association between 16p11.2 and several neural developmental disorders, including autism, epilepsy, mental retardation and schizophrenia.

6. ACKNOWLEDGMENTS

We thank The F.M. Kirby Neurobiology Center Department of Neurology Boston Children’s Hospital Harvard Medical School for imaging facility. This work was sponsored by China Scholarship Council. 

7. AUTHOR CONTRIBUTIONS

X. H. and J. F. designed and managed the experiments. Z. L. performed the experiments. Z. L. wrote the paper. J. F. revised the manuscript.

NOTES

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Craig, A. and Banker, G. (1994) Neuronal polarity. Annual Review of Neuroscience, 17, 267-310. http://dx.doi.org/10.1146/annurev.ne.17.030194.001411
[2]	Arimura, N. and Kaibuchi, K. (2007) Neuronal polarity: From extracellular signals to intracellular mechanisms. Nature Review of Neuroscience, 8, 194-205. http://dx.doi.org/10.1038/nrn2056
[3]	Dotti, C.G., Sullivan, C.A. and Banker, G.A. (1988) The establishment of polarity by hippocampal neurons in culture. Journal of Neuroscience, 8, 1454-1468.
[4]	Goslin, K. and Banker, G. (1989) Experimental observation on the development of polarity by hippocampal neurons in culture. Journal of Cell Biology, 108, 1506-1516. http://dx.doi.org/10.1083/jcb.108.4.1507
[5]	Beffert, U., Dillon, G.M., Sullivan, J.M., Stuart, C.E., Gilbert, J.P., Kambouris, J.A. and Ho, A. (2012) Microtubule plus-end tracking protein CLASP2 regulates neuronal polarity and synaptic function. Journal of Neuroscience, 32, 13906-13916. http://dx.doi.org/10.1523/JNEUROSCI.2108-12.2012
[6]	Amato, S., Liu, X., Zheng, B., Cantley, L., Rakic, P. and Man, H.Y. (2011) AMP-activated protein kinase regulates neuronal polarization by interfering with PI 3-kinase localization. Science, 332, 247-251. http://dx.doi.org/10.1126/science.1201678
[7]	Gartner, A., Fornasiero, E.F., Munck, S., Vennekens, K., Seuntjens, E., Huttner, W.B., Valtorta, F. and Dotti, C.G. (2012) N-cadherin specifies first asymmetry in developing neurons. The EMBO Journal, 31, 1893-1903. http://dx.doi.org/10.1038/emboj.2012.41
[8]	Lee, C.Y., Jaw, T., Tseng, H.C., Chen, I.C. and Liou, H.H. (2012) Lovastatin modulates glycogen synthase kinase-3β pathway and inhibits mossy fiber sprouting after pilocarpine-induced status epilepticus. PLoS One, 7, Article ID: e38789. http://dx.doi.org/10.1371/journal.pone.0038789
[9]	Shelly, M. and Poo, M.M. (2011) Role of LKB1-SAD/ MARK pathway in neuronal polarization. Developmental Neurobiology, 71, 508-527. http://dx.doi.org/10.1002/dneu.20884
[10]	Baas, S., Sharrow, M., Kotu, V., Middleton, M., Nguyen, K., Flanagan-Steet, H., Aoki, K. and Tiemeyer, M. (2011) Sugar-free frosting, a homolog of SAD kinase, drives neural-specific glycan expression in the Drosophila embryo. Development, 138, 553-563. http://dx.doi.org/10.1242/dev.055376
[11]	Durand, C.M., Perroy, J., Loll, F., Perrais, D., Fagni, L., Bourgeron, T., Montcouquiol, M. and Sans, N. (2012) SHANK3 mutations identified in autism lead to modification of dendritic spine morphology via an actin-dependent mechanism. Molecular Psychiatry, 17, 71-84. http://dx.doi.org/10.1038/mp.2011.57
[12]	Morita, T., Mayanagi, T. and Sobue, K. (2012) Caldesmon regulates axon extension through interaction with myosin II. Journal of Biological Chemistry, 287, 3349-3356. http://dx.doi.org/10.1074/jbc.M111.295618
[13]	Baudouin, S.J., Gaudias, J., Gerharz, S., Hatstatt, L., Zhou, K., Punnakkal, P., Tanaka, K.F., Spooren, W., Hen, R.D., Zeeuw, C.I., Vogt, K. and Scheiffele, P. (2012) Shared synaptic pathophysiology in syndromic and nonsyndromic rodent models of autism. Science, 338, 128-132. http://dx.doi.org/10.1126/science.1224159
[14]	Weiss, L.A., Shen. Y., Korn. J.M., Arking, D.E., Miller, D.T., Fossdal, R., Saemundsen, E., Stefansson, H., Ferreira, M.A., Green, T., Platt, O.S., Ruderfer, D.M., Walsh, C.A., Altshuler, D., Chakravarti, A., Tanzi, R.E., Stefansson, K., Santangelo, S.L., Gusella, J.F., Sklar, P., Wu, B.L., Daly, M.J. and Autism Consortium (2008) Association between microdeletion and microduplication at 16p11.2 and autism. The New England Journal of Medicine, 358, 667-675. http://dx.doi.org/10.1056/NEJMoa075974
[15]	Marshall, C.R., Noor, A., Vincent, J.B., Lionel, A.C., Feuk, L., Skaug, J., Shago, M., Moessner, R., Pinto, D., Ren, Y., Thiruvahindrapduram, B., Fiebig, A., Schreiber, S., Friedman, J., Ketelaars, C.E., Vos, Y.J., Ficicioglu, C., Kirkpatrick, S., Nicolson, R., Sloman, L., Summers, A., Gibbons, C.A., Teebi, A., Chitayat, D., Weksberg, R., Thompson, A., Vardy, C., Crosbie, V., Luscombe, S., Baatjes, R., Zwaigenbaum, L., Roberts, W., Fernandez, B., Szatmari, P. and Scherer, S.W. (2008)Structural variation of chromosomes in autism spectrum disorder. The American Journal of Human Genetics, 82, 477-488. http://dx.doi.org/10.1016/j.ajhg.2007.12.009
[16]	Bedoyan, J.K., Kumar, R.A., Sudi, J., Silverstein, F., Ackley, T., Iyer, R.K., Christian, S.L. and Martin, D.M. (2010) Duplication 16p11.2 in a child with infantile seizure disorder. American Journal of Medical Genetics Part A, 152A, 1567-1574.
[17]	Shimojima, K., Inoue, T., Fujii, Y., Ohno, K. and Yamamoto, T. (2009) A familial 593-kb microdeletion of 16p11.2 associated with mental retardation and hemivertebrae. The European Journal of Medical Genetics, 52, 433-435. http://dx.doi.org/10.1016/j.ejmg.2009.09.007
[18]	McCarthy, S.E., Makarov, V., Kirov, G., Addington, A.M., McClellan, J., Yoon, S., Perkins, D.O., Dickel, D.E., Kusenda, M., Krastoshevsky, O., Krause, V., Kumar, R.A., Grozeva, D., Malhotra, D., Walsh, T., Zackai, E.H., Kaplan, P., Ganesh, J., Krantz, I.D., Spinner, N.B., Roccanova, P., Bhandari, A., Pavon, K., Lakshmi, B., Leotta, A., Kendall, J., Lee, Y.H., Vacic, V., Gary, S., Iakoucheva, L.M., Crow, T.J., Christian, S.L., Lieberman, J.A., Stroup, T.S., Lehtimaki, T., Puura, K., Haldeman-Englert, C., Pearl, J., Goodell, M., Willour, V.L., Derosse, P., Steele, J., Kassem, L., Wolff, J., Chitkara, N., McMahon, F.J., Malhotra, A.K., Potash, J.B., Schulze, T.G., Nothen, M.M., Cichon, S., Rietschel, M., Leibenluft, E., Kustanovich, V., Lajonchere, C.M., Sutcliffe, J.S., Skuse, D., Gill, M., Gallagher, L., Mendell, N.R., Craddock, N., Owen, M.J., O’Donovan, M.C., Shaikh, T.H., Susser, E., Delisi, L.E., Sullivan, P.F., Deutsch, C.K., Rapoport, J., Levy, D.L., King, M.C. and Sebat, J. (2009) Microduplications of 16p11.2 are associated with schizophrenia. Nature Genetics, 41, 1223-1227. http://dx.doi.org/10.1038/ng.474
[19]	Kumar, R.A., KaraMohamed, S., Sudi, J., Conrad, D.F., Brune, C., Badner, J.A., Gilliam, T.C., Nowak, N.J., Cook Jr., E.H., Dobyns, W.B. and Christian, S.L. (2008) Recurrent 16p11.2 microdeletions in autism. Human Molecular Genetics, 17, 628-638. http://dx.doi.org/10.1093/hmg/ddm376
[20]	Kumar, R.A., Marshall, C.R., Badner, J.A., Babatz, T.D., Mukamel, Z., Aldinger, K.A., Sudi, J., Brune, C.W., Goh, G., Karamohamed, S., Sutcliffe, J.S., Cook, E.H., Geschwind, D.H., Dobyns, W.B., Scherer, S.W. and Christian, S.L. (2009) Association and mutation analyses of 16p11.2 autism candidate genes. PLoS One, 4, Article ID: e4582. http://dx.doi.org/10.1371/journal.pone.0004582
[21]	Zufferey, F., Sherr, E.H., Beckmann, N.D., Hanson, E., Maillard, A.M., Hippolyte, L., Macé, A., Ferrari, C., Kutalik, Z., Andrieux, J., Aylward, E., Barker, M., Bernier, R., Bouquillon, S., Conus, P., Delobel, B., Faucett, W.A., Goin-Kochel, R.P., Grant, E., Harewood, L., Hunter, J.V., Lebon, S., Ledbetter, D.H., Martin, C.L., Mannik, K., Martinet, D., Mukherjee, P., Ramocki, M.B., Spence, S.J., Steinman, K.J., Tjernagel, J., Spiro, J.E., Reymond, A., Beckmann, J.S., Chung, W.K., Jacquemont, S. and Simons VIP Consortium, 16p11.2 European Consortium (2012) A 600 kb deletion syndrome at 16p11.2 leads to energy imbalance and neuropsychiatric disorders. Journal of Medical Genetics, 49, 660-668. http://dx.doi.org/10.1136/jmedgenet-2012-101203
[22]	Kaech, S. and Banker, G. (2006)Culturing hippocampal neurons. Nature Protocol, 1, 2406-2415. http://dx.doi.org/10.1038/nprot.2006.356
[23]	Kempermann, G. (2012) Neuroscience. Youth culture in the adult brain. Science, 335, 1175-11176. http://dx.doi.org/10.1126/science.1219304
[24]	Zeitelhofer, M., Karra, D., Vessey, J.P., Jaskic, E., Macchi, P., Thomas, S., Riefler, J., Kiebler, M. and Dahm, R. (2009) High-efficiency transfection of short hairpin RNAs-encoding plasmids into primary hippocampal neurons. Journal of Neuroscience Research, 87, 289-300. http://dx.doi.org/10.1002/jnr.21840
[25]	Golzio, M., Escoffre, J.M. and Teissié, J. (2012) shRNAmediated gene knockdown in skeletal muscle. Methods in Molecular Biology, 798, 491-501. http://dx.doi.org/10.1007/978-1-61779-343-1_29
[26]	Zeitelhofer, M., Vessey, J.P., Xie, Y., Tübing, F., Thomas, S., Kiebler, M. and Dahm, R. (2007) High-efficiency transfection of mammalian neurons via nucleofection. Nature Protocol, 2, 1692-1704. http://dx.doi.org/10.1038/nprot.2007.226
[27]	Fukata, Y., Kimura, T. and Kaibuchi, K. (2002) Axon specification in hippocampal neurons. Neuroscience Research, 43, 305-315. http://dx.doi.org/10.1016/S0168-0102(02)00062-7
[28]	Nimchinsky, E.A., Sabatini, B.L. and Svoboda, K. (2002) Structure and function of dendritic spines. Annual Review of Physiology, 64, 313-353. http://dx.doi.org/10.1146/annurev.physiol.64.081501.160008
[29]	Shiow, L.R., Paris, K., Akana, M.C., Cyster, J.G., Sorensen, R.U. and Puck, J.M. (2009) Severe combined immunodeficiency (SCID) and attention deficit hyperactivity disorder (ADHD) associated with a Coronin-1A mutation and a chromosome 16p11.2 deletion. Clinical Immunology, 131, 24-30. http://dx.doi.org/10.1016/j.clim.2008.11.002
[30]	Shinawi, M., Liu, P., Kang, S.H., Shen, J., Belmont, J.W., Scott, D.A., Probst, F.J., Craigen, W.J., Graham, B.H., Pursley, A., Clark, G., Lee, J., Proud, M., Stocco, A., Rodriguez, D.L., Kozel, B.A., Sparagana, S., Roeder, E.R., McGrew, S.G., Kurczynski, T.W., Allison, L.J., Amato, S., Savage, S., Patel, A., Stankiewicz, P., Beaudet, A.L., Cheung, S.W. and Lupski, J.R. (2010) Recurrent reciprocal 16p11.2 rearrangements associated with global developmental delay, behavioural problems, dysmorphism, epilepsy, and abnormal head size. Journal of Medical Genetics, 47, 332-341. http://dx.doi.org/10.1136/jmg.2009.073015
[31]	Bachmann-Gagescu, R., Mefford, H.C., Cowan, C., Glew, G.M., Hing, A.V., Wallace, S., Bader, P.I., Hamati, A., Reitnauer, P.J., Smith, R., Stockton, D.W., Muhle, H., Helbig, I., Eichler, E.E., Ballif, B.C., Rosenfeld, J. and Tsuchiya, K.D. (2010) Recurrent 200-kb deletions of 16p11.2 that include the SH2B1 gene are associated with developmental delay and obesity. Genetic Medicine, 12, 641-647. http://dx.doi.org/10.1097/GIM.0b013e3181ef4286
[32]	Blaker-Lee, A., Gupta, S., McCammon, J.M., De Rienzo, G. and Sive, H. (2012) Zebrafish homologs of genes within 16p11.2, a genomic region associated with brain disorders, are active during brain development, and include two deletion dosage sensor genes. Disease Models. & Mechanisms, 5, 834-851. http://dx.doi.org/10.1242/dmm.009944
[33]	Orita, S., Sasaki, T., Naito, A., Komuro, R. and Ohtsuka, T. (1995) Doc2: A novel brain protein having two repeated C2-like domains. Biochemical and Biophysical Research Communication, 206, 439-448. http://dx.doi.org/10.1006/bbrc.1995.1062
[34]	Mochida, S., Orita, S., Sakaguchi, G., Sasaki, T. and Takai, Y. (1998) Role of the Doc2 alpha-Munc13-1 interaction in the neurotransmitter release process. Proceedings of the National Academy of Sciences of the United States of America, 95, 11418-11422. http://dx.doi.org/10.1073/pnas.95.19.11418
[35]	Yao, J., Gaffaney, J.D., Kwon, S.E. and Chapman, E.R. (2011) Doc2 is a Ca2+ sensor required for asynchronous neurotransmitter release. Cell, 147, 666-677. http://dx.doi.org/10.1016/j.cell.2011.09.046
[36]	Sakaguchi, G., Manabe, T., Kobayashi, K., Orita, S. and Sasaki, T. (1999) Doc2alpha is an activity-dependent modulator of excitatory synaptic transmission. European Journal of Neuroscience, 11, 4262-4268. http://dx.doi.org/10.1046/j.1460-9568.1999.00855.x
[37]	Suzuki, T., Takeuchi, J., Koshiba-Takeuchi, K. and Ogura, T. (2004) Tbx Genes specify posterior digit identity through Shh and BMP signaling. Developmental Cell, 6, 43-53. http://dx.doi.org/10.1016/S1534-5807(03)00401-5
[38]	Pereira, L.A., Wong, M.S., Lim, S.M., Sides, A., Stanley, E.G. and Elefanty, A.G. (2011) Brachyury and related Tbx proteins interact with the Mixl1 homeodomain protein and negatively regulate Mixl1 transcriptional activity. PLoS One, 6, Artcile ID: e28394. http://dx.doi.org/10.1371/journal.pone.0028394
[39]	Moraes, F., Novoa, A., Jerome-Majewska, L.A., Papaioannou, V.E. and Mallo, M. (2005) Tbx1 is required for proper neural crest migration and to stabilize spatial patterns during middle and inner ear development. Mechanisms of Development, 122, 199-212. http://dx.doi.org/10.1016/j.mod.2004.10.004
[40]	Song, M.R., Shirasaki, R., Cai, C.L., Ruiz, E.C., Evans, S.M., Lee, S.K. and Pfaff, S.L. (2006) T-Box transcription factor Tbx20 regulates a genetic program for cranial motor neuron cell body migration. Development, 133, 4945-4955. http://dx.doi.org/10.1242/dev.02694
[41]	Song, A.H., Wang, D., Chen, G., Li, Y., Luo, J., Duan, S. and Poo, M.M. (2009) A selective filter for cytoplasmic transport at the axon initial segment. Cell, 136, 1148-1160. http://dx.doi.org/10.1016/j.cell.2009.01.016
[42]	Nakata, T. and Hirokawa, N. (2003) Microtubules provide directional cues for polarized axonal transport through interaction with kinesin motor head. Journal of Cell Biology, 162, 1045-1055. http://dx.doi.org/10.1083/jcb.200302175
[43]	Kamal, A., Stokin, G.B., Yang, Z., Xia, C.H. and Goldstein, L.S. (2000).Axonal transport of amyloid precursor protein is mediated by direct binding to the kinesin light chain subunit of kinesin-I. Neuron, 28, 449-459. http://dx.doi.org/10.1016/S0896-6273(00)00124-0
[44]	Setou, M., Nakagawa, T., Seog, D.H. and Hirokawa, N. (2000) Kinesin superfamily motor protein KIF17 and mLin-10 in NMDA receptor-containing vesicle transport. Science, 288, 1796-1802. http://dx.doi.org/10.1126/science.288.5472.1796
[45]	Guillaud, L., Setou, M. and Hirokawa, N. (2003) KIF17 dynamics and regulation of NR2B trafficking in hippocampal neurons. Journal of Neuroscience, 23, 131-140.