Microtubule Organization in the Axon: TRIM46 Determines the Orientation

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Previews possibility that additional mechanisms could underlie the observed alterations in adult behavior. Indeed, Berg et al. (2015) report a microcephaly phenotype in JAKMIP1 KO mice, while a previous study observed altered neocortical neuronal migration following knockdown of JAKMIP1 in utero (Vidal et al., 2012), suggesting roles for JAKMIP1 in neurodevelopmental processes. Earlier studies also reported that JAKMIP1 could bind to GABA receptor mRNA and protein, indicating that GABA receptor signaling could be altered in these mice (Couve et al., 2004; Vidal et al., 2009). As such, future efforts will be required to fully understand the basis of the behavioral defects arising from loss of JAKMIP1. Similarly, the exact role(s) of JAKMIP1 in regulating neuronal translation remain unclear. The association of JAKMIP1 with mRNP particles and mRNAs, together with earlier reports indicating that JAKMIP1 associates with the microtubule motor protein kinesin-1 and is mobile in dendrites (Vidal et al., 2007; Vidal et al., 2012), suggest that JAKMIP1 is involved in mRNA transport to synaptic sites; however, this possibility has not yet been formally tested. Likewise, the ribosomal association of JAKMIP1, reduced protein synthesis observed in JAKMIP1 KO neurons, and reduced poly-

some association of PABPC1 and DDX5 suggest possible defects in ribosome function. Thus, whether a general defect in cellular protein synthesis or a more specific effect on local synaptic translation is responsible for the cellular and behavioral phenotypes of JAKMIP1 KO mice remains an open question. While further efforts will be necessary to fully understand the details of JAKMIP1 function, Berg et al. (2015) have made a major contribution to our understanding of JAKMIP1 biology, establishing this protein as a critical regulator of mRNA translation in the mammalian brain (Figure 1). Furthermore, the authors have uncovered numerous molecular, cellular, and electrophysiological defects in JAKMIP1 KO mice leading to ASD-related phenotypes. This novel ASD model reinforces the role of neuronal translation in ASD etiology and provides a platform for further study of these processes. JAKMIP1 likely also serves as a convergence point for a subset of molecules or pathways relevant to ASD, as well as itself regulating multiple processes important for neuronal function. REFERENCES Berg, J.M., Lee, C., Chen, L., Galvan, L., Cepeda, C., Chen, J.Y., Pen˜agarikano, O., Stein, J.L., Li,

A., Oguro-Ando, A., et al. (2015). Neuron 88, this issue, 1173–1191. Chang, J., Gilman, S.R., Chiang, A.H., Sanders, S.J., and Vitkup, D. (2015). Nat. Neurosci. 18, 191–198. Couve, A., Restituito, S., Brandon, J.M., Charles, K.J., Bawagan, H., Freeman, K.B., Pangalos, M.N., Calver, A.R., and Moss, S.J. (2004). J. Biol. Chem. 279, 13934–13943. Ebert, D.H., and Greenberg, M.E. (2013). Nature 493, 327–337. Kelleher, R.J., 3rd, and Bear, M.F. (2008). Cell 135, 401–406. Krumm, N., O’Roak, B.J., Shendure, J., and Eichler, E.E. (2014). Trends Neurosci. 37, 95–105. Malenka, R.C., and Bear, M.F. (2004). Neuron 44, 5–21. Nishimura, Y., Martin, C.L., Vazquez-Lopez, A., Spence, S.J., Alvarez-Retuerto, A.I., Sigman, M., Steindler, C., Pellegrini, S., Schanen, N.C., Warren, S.T., and Geschwind, D.H. (2007). Hum. Mol. Genet. 16, 1682–1698. Pasciuto, E., Borrie, S.C., Kanellopoulos, A.K., Santos, A.R., Cappuyns, E., D’Andrea, L., Pacini, L., and Bagni, C. (2015). Neurobiol. Learn. Mem. 124, 71–87. Vidal, R.L., Fuentes, P., Valenzuela, J.I., AlvaradoDiaz, C.P., Ramirez, O.A., Kukuljan, M., and Couve, A. (2012). Mol Cell Neurosci. 51, 1–11. Vidal, R.L., Ramirez, O.A., Sandoval, L., KoenigRobert, R., Hartel, S., and Couve, A. (2007). Mol Cell Neurosci. 35, 501–512. Vidal, R.L., Valenzuela, J.I., Luja´n, R., and Couve, A. (2009). BMC Neurosci. 10, 37.

Microtubule Organization in the Axon: TRIM46 Determines the Orientation Michele Curcio1,* and Frank Bradke1,* 1Laboratory for Axon Growth and Regeneration, German Center for Neurodegenerative Diseases (DZNE), Ludwig-Erhard-Allee 2, 53175 Bonn, Germany *Correspondence: [email protected] (M.C.), [email protected] (F.B.) http://dx.doi.org/10.1016/j.neuron.2015.12.006

The microtubule cytoskeleton is a major determinant in neuronal polarity. In this issue of Neuron, van Beuningen et al. (2015) now report that TRIM46 forms parallel microtubule bundles in the proximal axon and reveal that it is crucial for the establishment and maintenance of neuronal polarity. Differentiated neurons are highly polarized cells, compartmentalized into two distinct domains: the somatodendritic

compartment, which receives and processes incoming information, and the axon, which transmits the information to

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target cells. The first step leading to the architectural polarization observed in mature neurons is the sprouting of several

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Figure 1. A Model for Microtubule Organization at the Proximal Axon (A) Schematic of a developing neuron and composition of the proximal domain of its future axon. (B) Schematic of a polarized neuron and composition of its AIS. van Beuningen et al. (2015) found that the microtubule-associated protein TRIM46 localizes to the proximal domain of the axon, where it forms parallel microtubule arrays required for the initial axon specification and for the maintenance of neuronal polarity. Indeed, depletion of TRIM46 alters the orientation of microtubules, the distribution of Ankyrin G in developing neurons and the distribution of Dephospho-Tau (antibody clone PC1C6) in polarized neurons.

short neurites from a spherical neuron. Of these neurites, one will grow longer than the others and become the axon, while the other neurites will become the dendrites (Ca´ceres et al., 2012; Takano et al., 2015). Previous studies have shown that the neuronal cytoskeleton plays a major role in the establishment and maintenance of neuronal polarity (Hoogenraad and Bradke, 2009). In particular, the stabilization, bundling and plus-end out orientation of microtubules in the axon are key events in neuronal polarization (Baas and Lin, 2011; Witte and Bradke, 2008). Therefore, the identification of factors regulating axon specification and neuronal polarity by organizing the axonal microtubules is under intense investigation. In this issue of Neuron, van Beuningen et al. (2015) show that the tripartite motif-containing (TRIM) protein TRIM46

specifically localizes to the proximal axon, where it forms microtubule bundles oriented with their plus-end pointing outward, thus contributing to the establishment and maintenance of neuronal polarity (Figure 1). In an effort to find new factors that may contribute to neuronal polarization, van Beuningen et al. (2015) found inspiration from previous studies describing the immunoreactivity of autoimmune antibodies with the axon initial segment (AIS) in cerebellar sections (Sabater et al., 2013; Shams’ili et al., 2009) and combined biochemical and mass spectrometry approaches to identify TRIM46 as the axon-specific antigen. Detailed analysis with direct stochastic optical reconstruction microscopy (dSTORM) imaging elegantly showed that TRIM46 colocalizes in the proximal axon with

microtubule bundles, but not AIS proteins. Consistent with the dSTORM images, TRIM46 does not physically interact with proteins of the AIS in pulldown experiments, suggesting a unique position and potential function for TRIM46 in the AIS and neuronal polarization. TRIM46 belongs to the C-I TRIM subfamily of the N-terminal RING finger/ B-box/coiled-coil (RBCC)/TRIM superfamily (Short and Cox, 2006). Proteins of this subfamily have an N-terminal RBCC domain, a unique C-terminal subgroup one signature (COS) box and a C-terminal FN3 and B30.2-like domain. To determine how TRIM46 localizes to the proximal axon domain, van Beuningen et al. (2015) used deletion constructs and found that the RING finger and the COS box are required for the axon-specific localization of TRIM46, but the potential E3 ubiquitin ligase activity and the folding status of the RING finger do not affect the proper localization of TRIM46. Depolymerization of microtubules with nocodazole reduced the axonal localization of TRIM46, suggesting that microtubule association facilitates axon localization. Yet, the TRIM46 mutant lacking the RING finger is still able to associate with microtubules, likely via the COS box as it mediates microtubule association in non-neuronal cell lines but does not localize to the axon. Hence the link between microtubule binding ability and axonal localization awaits further study. The specific localization of TRIM46 in the proximal axon might reflect its contribution in the establishment and maintenance of neuronal polarity. In unpolarized neurons in vitro, TRIM46 is absent in all the minor neurites but is observed in a single neurite before the future axon grows out. In many neurons TRIM46 localizes to the neurite even before axon specification and AIS assembly, which makes TRIM46 a potential marker for neuronal polarization. Disruption of TRIM46 expression by shRNA in developing neurons markedly reduced the polarized distribution of Ankyrin G (AnkG) (Figure 1A). Pharmacological microtubule stabilization with low doses of taxol is sufficient to induce supernumerary axons in dissociated unpolarized hippocampal neurons (Witte and Bradke, 2008). Therefore, microtubule stabilization increases the number of neurites per

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Previews cell positive for TRIM46 and AnkG and time course analysis confirmed that TRIM46 localizes to the proximal segment of these neurites before AIS formation. In addition to the in vitro studies, van Beuningen et al. (2015) analyzed the effects of TRIM46 loss on axon formation and neuronal polarity in vivo, by reducing TRIM46 expression in utero. A detailed analysis of neuronal morphology showed that TRIM46, in particular its RING finger domain, is essential for the generation of a typical axon process. In mature neurons, the AIS is the place where the nerve impulse is generated, and it acts as a barrier that differentiates the somatodendritic and axonal compartments, contributing to the maintenance of neuronal polarity (Leterrier and Dargent, 2014; Rasband, 2010). Indeed, mature axons change their identity into dendrites when the AIS is disassembled by knockdown of its major component, such as the scaffolding protein AnkG (Rasband, 2010). To determine whether TRIM46 controls neuronal polarity in differentiated neurons, van Beuningen et al. (2015) knocked down TRIM46 in already polarized neurons. They found that the loss of TRIM46 only partly affects the integrity of the AIS but significantly alters the polarized distribution of axonal and dendritic markers as well as the axonal morphology (Figure 1B). Thereby, TRIM46 is an essential factor for AIS assembly but not for its maintenance. To better understand how TRIM46 controls the maintenance of neuronal polarity, van Beuningen et al. (2015) combined studies performed in non-neuronal cell lines and neurons, using live-cell imaging, laser-based microsurgery, electron microscopy, dSTORM, and fluorescence recovery after photobleaching (FRAP) studies. Expression of TRIM46 in cell lines induced bundling of microtubules oriented with their plus-end outward with microtubule plus-end dynamics taking place at the tip of TRIM46-formed bundles. By analyzing the growth of plusend tracking protein (+TIP) comets formed following laser severing of TRIM46 microtubule bundles, van Beuningen et al. (2015) found that +TIP comets moved in one direction, suggesting that TRIM46 builds parallel arrays of microtubules. As these findings were obtained in non-neuronal cell lines ex-

pressing an exogenous construct, it will be interesting to test whether purified TRIM46 is sufficient to reconstitute similar microtubule arrays in vitro. When examined in cultured neurons, FRAP approaches showed that TRIM46 forms a stable compartment in the proximal axon but is not required for microtubule stability. Indeed, depletion of TRIM46 in polarized neurons alters the orientation of +TIP comets, suggesting that TRIM46 maintains polarity in mature neurons by controlling the orientation of microtubule bundles. To analyze whether the parallel microtubule organization provided by TRIM46 plays a role in axonal trafficking, van Beuningen et al. (2015) imaged neurons depleted of TRIM46 with two different approaches: live-cell recordings of an inducible cargo trafficking assay (Kapitein et al., 2010) and high-speed live-cell microscopy of axonal vesicles. As TRIM46 knockdown only partly affects the AIS composition in mature neurons, the results obtained with these approaches mainly reflect the role of TRIM46 in the orientation of microtubules. Although depletion of TRIM46 did not influence the polarized trafficking of kinesininduced peroxisomes, the mixed orientation of microtubules altered the cargo behavior, which displayed a reduced axonal targeting and an increased number of stalled vesicles and bidirectional movements. Given the striking changes seen with loss of TRIM46 as well as its localization, van Beuningen et al. (2015) examined its potential relationship with the generation of structural hallmark of the AIS, the presence of closely spaced microtubule fascicles seen in cross section via EM as rings connected by filaments or ‘‘crossbridges’’ (Leterrier and Dargent, 2014). The authors found that expressing TRIM46 in HeLA cells led to the presence of multiple clusters of perpendicular microtubule bundles interconnected by thin cross-bridges. Hence, it suggests that TRIM46 helps promoting this unique AIS-like microtubule organization. Electron microscopy studies in cultured neurons obtained from TRIM46 transgenic mice or knockout mice in vivo could further clarify the role of TRIM46 in the establishment of this structural hallmark of the AIS.

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In addition to the microtubule cytoskeleton, actin may also play important roles at the AIS (Kevenaar and Hoogenraad, 2015; Rasband, 2010) and actin-microtubule crosstalk in neurons has been suggested to be required for cytoskeletal dynamics (Coles and Bradke, 2015). Therefore, it would be very interesting to study whether TRIM46 activity also affects actin organization and whether modulation of the actin cytoskeleton affects the localization of TRIM46. The present work also leaves open questions as to the role of TRIM46 in axon regeneration. As microtubule stabilization after injury promotes axon regeneration and functional recovery after CNS injury (Ruschel et al., 2015), it would also be interesting to test whether TRIM46 could have further beneficial effects on regeneration, by modulating the microtubule cytoskeleton at the proximal axon. In summary, the work of van Beuningen et al. (2015) demonstrates that TRIM46 forms closely spaced parallel microtubule bundles in the proximal axon and plays a major role in the axon specification and in the control of neuronal polarity. The generation of transgenic animals will contribute to further investigate the role of TRIM46 and future studies will be required to understand whether other members of the RBCC/TRIM family have a role in the organization of the neuronal cytoskeleton or in neuronal polarity as well. Previous work has shown that in unpolarized neurons, microtubules are uniformly oriented with their plus-end out in all the newly formed neurites (Baas and Lin, 2011). Might other TRIM proteins have a role in the organization of parallel microtubule arrays in these immature processes? Could a single TRIM protein be expressed in a spatiotemporal fashion to regulate the initial microtubule parallel organization? The identification of TRIM46 as a regulator of neuronal polarity opens an exciting new pathway for exploring how microtubule organization orchestrates neuronal polarization.

ACKNOWLEDGMENTS We thank Charlotte Coles and Claudia Laskowski for critically reading. We apologize to authors whose relevant work we could not cite due to space limitations.

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Previews REFERENCES Baas, P.W., and Lin, S. (2011). Dev. Neurobiol. 71, 403–418. Ca´ceres, A., Ye, B., and Dotti, C.G. (2012). Curr. Opin. Cell Biol. 24, 547–553. Coles, C.H., and Bradke, F. (2015). Curr. Biol. 25, R677–R691.

Kevenaar, J.T., and Hoogenraad, C.C. (2015). Front. Mol. Neurosci. 8, 44. Leterrier, C., and Dargent, B. (2014). Semin. Cell Dev. Biol. 27, 44–51. Rasband, M.N. (2010). Nat. Rev. Neurosci. 11, 552–562.

Hoogenraad, C.C., and Bradke, F. (2009). Trends Cell Biol. 19, 669–676.

Ruschel, J., Hellal, F., Flynn, K.C., Dupraz, S., Elliott, D.A., Tedeschi, A., Bates, M., Sliwinski, C., Brook, G., Dobrindt, K., et al. (2015). Science 348, 347–352.

Kapitein, L.C., Schlager, M.A., Kuijpers, M., Wulf, P.S., van Spronsen, M., MacKintosh, F.C., and Hoogenraad, C.C. (2010). Curr. Biol. 20, 290–299.

Sabater, L., Ho¨ftberger, R., Boronat, A., Saiz, A., Dalmau, J., and Graus, F. (2013). PLoS ONE 8, e60438.

Shams’ili, S., de Leeuw, B., Hulsenboom, E., Jaarsma, D., and Smitt, P.S. (2009). Neurosci. Lett. 467, 169–172. Short, K.M., and Cox, T.C. (2006). J. Biol. Chem. 281, 8970–8980. Takano, T., Xu, C., Funahashi, Y., Namba, T., and Kaibuchi, K. (2015). Development 142, 2088–2093. van Beuningen, S.F.B., Will, L., Harterink, M., Chazeau, A., van Battum, E.Y., Frias, C.P., Franker, M.A.M., Katrukha, E.A., Stucchi, R., Vocking, K., et al. (2015). Neuron 88, this issue, 1208–1226. Witte, H., and Bradke, F. (2008). Curr. Opin. Neurobiol. 18, 479–487.

The State of the Orbitofrontal Cortex Melissa J. Sharpe,1,2,* Andrew M. Wikenheiser,1 Yael Niv,2,3 and Geoffrey Schoenbaum1,4,5,* 1National

Institute on Drug Abuse, Baltimore, MD 21224, USA Neuroscience Institute, Princeton University, Princeton, NJ 08544, USA 3Department of Psychology, Princeton University, Princeton, NJ 08544, USA 4Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, MD 21201, USA 5Solomon H. Snyder Department of Neuroscience, The John Hopkins University, Baltimore, MD 21218, USA *Correspondence: [email protected] (M.J.S.), [email protected] (G.S.) http://dx.doi.org/10.1016/j.neuron.2015.12.004 2Princeton

State representation is fundamental to behavior. However, identifying the true state of the world is challenging when explicit cues are ambiguous. Here, Bradfield and colleagues show that the medial OFC is critical for using associative information to discriminate ambiguous states.

Some decisions are easy: you go at a green light, you stop at red. Those two states of the world are clearly different, signaling different appropriate behaviors. However, sometimes you stop even at a green light—for instance, you are going left and first need to give right-of-way to oncoming traffic. Here, the state of ‘‘green light and I intend to go left’’ is different from ‘‘green light and I intend to go straight,’’ despite the two states being perceptually identical. Appropriate state representation is fundamental to behavioral flexibility—by abstracting away superfluous information (whether the oncoming car is black or gray, are pedestrians crossing the street on your right) and adding in important unobservable information (your intention, your past actions, your knowledge of traffic rules), the brain can craft a ‘‘task state’’ that is ideal for rapid, correct, and generalizable decision making.

However, identifying the ‘‘true’’ state can be particularly challenging when explicit cues are ambiguous and the candidate states imply contradictory rules. For example, a soldier returning from deployment must be able to categorize the wartime setting differently from similar civilian contexts to avoid inappropriate behavioral responses. While there are often explicit or observable cues that distinguish these states, this is not always the case; given enough abstraction, the distinction between Bagdad and Baltimore may become difficult, and largely a matter of an internal belief. It is critical that the neural representation of this belief be able to bridge the gaps between observable distinguishing events, as dysfunction in this process, even if very brief, could contribute to phenomena such as post-traumatic stress disorder (PTSD).

Given the importance of task state information for decision making and learning, and for disturbances thereof, it is of interest to identify the neural substrates mediating the ability to recognize, maintain, and deploy state representations. We have recently proposed that the orbitofrontal cortex (OFC) might be one key area involved in this process (Takahashi et al., 2011; Wilson et al., 2014). In particular, we suggested that the OFC is critical for representing and using states that include components that are not externally observable. In the current issue of Neuron, Bradfield et al. (2015) elegantly explore this possibility, focusing specifically on the role of the medial OFC in supporting goal-directed behavior that depends on a ‘‘forward search’’ over potential upcoming (and currently unobservable) task states. In a series of experiments, they test whether the medial OFC

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