E XP ER I ME NTAL C E LL RE S E ARCH

34 35 36 37 38 39 40 41 42 43 44 45 46 47 48Q1 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93

] (]]]]) ]]]–]]]

Available online at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/yexcr

Review Article

Regulation of neuronal polarity Giovanna Lallin Wolfson Centre for Age-Related Diseases, King's College London, Guy's Campus, London SE1 1UL, UK

article information

abstract

Article Chronology:

The distinctive polarized morphology of neuronal cells is essential for the proper wiring of the

Received 30 April 2014

nervous system. The rodent hippocampal neuron culture established about three decades ago has

Received in revised form

provided an amenable in vitro system to uncover the molecular mechanisms underlying neuronal

24 July 2014

polarization, a process relying on highly regulated cytoskeletal dynamics, membrane traffic and

Accepted 26 July 2014

localized protein degradation. More recent research in vivo has highlighted the importance of the extracellular environment and cell–cell interactions in neuronal polarity. Here, I will review some

Keywords:

key signaling pathways regulating neuronal polarization and provide some insights on the

Neuronal polarity

complexity of this process gained from in vivo studies.

Axon

& 2014 Published by Elsevier Inc.

Dendrite Cytoskeleton Membrane traffic

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The cytoskeleton in neuronal polarization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Centrosome positioning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Signaling mechanisms in neuronal polarization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The PI3K-Akt signaling cascade . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The cAMP-LKB1 pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The PAR complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Membrane traffic in axon initiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Local protein degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neuronal polarization in vivo. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Distinct modes of neuronal polarization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Extracellular cues regulating neuronal polarity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

n

Fax: þ44 20 7848 6816. E-mail address: [email protected]

http://dx.doi.org/10.1016/j.yexcr.2014.07.033 0014-4827/& 2014 Published by Elsevier Inc.

Please cite this article as: G. Lalli, Regulation of neuronal polarity, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.07.033

1 2 2 3 4 4 4 4 5 6 6 6 6 8 8

94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117

2

118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177

E XP E RI ME N TAL CE L L R ES E ARC H

Introduction The neuron is a classical example of a polarized cell, usually characterized by a long, thin axon and thick, shorter dendrites. The establishment and maintenance of neuronal polarity are essential for the correct development and function of the nervous system and rely on the exquisite coordination between membrane transport and cytoskeletal dynamics. Disruption of these fundamental processes has been linked to intellectual disability and a variety of neurological diseases [1]. The last four decades have seen great progress in understanding how neuronal polarity is established and maintained, and recent technological advances have enabled scientists to study these events in vivo using a range of animal model systems. While the role of the cytoskeleton has been the focus of much effort in the initial studies on neuronal polarity, new concepts such as selective protein degradation, the function of mechanical tension and the interplay with the extracellular environment in an in vivo context have been explored in more recent years. After summarizing some key signaling mechanisms underlying neuronal polarization, this mini-review will highlight the latest advances in this fascinating field, with a particular emphasis on the events involved in regulating axon/ dendrite establishment in vivo.

The cytoskeleton in neuronal polarization The hippocampal neuron culture established by Dotti et al. in the eighties [2] have been an invaluable tool to dissect signaling pathways regulating neuronal polarization. These neurons, isolated from E18 rat embryos, go through a series of stereotypical stages (Fig. 1A): 1) initial extension/retraction of filopodia; 2) extension of short neurites; 3) extension of the presumptive axon; 4) further growth and branching of the axonal process, dendritic maturation with appearance of spines; 5) formation of functional synaptic contacts. Not surprisingly, dynamic rearrangements of the actin and microtubule cytoskeleton accompany these different stages. The growth cone of the presumptive axon is characterized by a very dynamic actin cytoskeleton. Indeed, local actin destabilization is sufficient to cause axon initiation [3] by allowing microtubule “invasion” of the central domain of the growth cone, followed by microtubule bundling and consolidation [4]. This process transforms the growth cone of a neurite into the shaft of the nascent axon and continues to be required for axon elongation. The small GTPase Rac acts as a crucial regulator of actin cytoskeletal dynamics in lamellipodia by triggering two major signaling cascades, the P21-activated kinase (PAK)-cofilin pathway and the WAVE-Arp2/3 pathway. While distinct Rac isoforms are likely to act redundantly in different types of neurons [5], the Rac effectors WAVE and PAK1 appear to participate in neuronal polarization, even though with different requirements in distinct types of neurons. For example, overexpression of an active, membrane-tethered WAVE mutant partially rescues axonogenesis in cerebellar granule neurons from Rac1 knockout mice [6] while PAK1 is necessary and sufficient for polarization of hippocampal neurons [7]. The small GTPase Cdc42, another modulator of actin dynamics, can also bind PAK1, which in turn activates LIM kinase (LIMK), leading to phosphorylation and inactivation of the actin-depolymerizing factor cofilin (Fig. 1B). Indeed, Cdc42

] (]]]]) ]]]–]]]

conditional knockout mice show substantial defects in axonogenesis in the cortex and striatum and display abnormally high levels of phosphorylated cofilin [8]. Taken together, these observations strongly support the requirement for a dynamic cycle of actin polymerization/depolymerization in the perspective axon. Microtubule stabilization is essential for axon initiation, and, surprisingly, is also sufficient to cause extension of supernumerary axons from dendrites of mature neurons [9]. Stabilization of the nascent axon occurs through several microtubule-associated proteins (MAPs), including Collapsin Response Mediator Protein 2 (CRMP2), Map1b and Tau, together with plus-end tip (þTIPS) binding proteins Adenomatous Polyposis Coli (APC), Cytoplasmic Linker Proteins (CLIP)-115 and CLIP-170 [4]. Overexpression of CRMP2 leads to the appearance of multiple axons, and is even sufficient to switch a mature dendrite into an axon [10]. This important molecule contributes to axonogenesis by performing multiple functions: carrying tubulin heterodimers to promote microtubule assembly and stabilization, promoting selective transport of the Sra-1/WAVE actin-regulating complex towards the growing axon, and binding to Numb, a protein involved in clathrin-mediated endocytosis, to regulate endocytosis of the neural cell adhesion molecule L1 in the growth cone of the nascent axon [11]. The function of þTIPs is to allow protrusion of microtubules into the leading edge of the growth cone, facilitating axon initiation and growth. Both CLIP-115 and CLIP-170 are necessary and sufficient for axon formation in vitro [12]. APC, CRMP2, Map1b and Tau are substrates of Glycogen Synthase Kinase 3 (GSK3), a key kinase in neuronal polarization [13–16], which phosphorylates them and reduces their microtubule-binding ability. Therefore, local inhibition of GSK3 leads to microtubule stabilization and axon initiation by allowing the binding of these MAPs to microtubules. Notably, MAPs contain multiple phosphorylation sites and are likely to be targets for multiple kinases, some of which are still unknown. Thus, we are still far away from completely understanding how microtubule dynamics is regulated in neuronal polarization.

Centrosome positioning While centrosome positioning plays a crucial role in many polarization events, its involvement in neuronal polarity may be different depending on the neuronal cell type and the developmental context. For example, in zebrafish retinal ganglion cells axons emerge from the basal side of the cell body, opposite to the apical location of the centrosome [17]. Other reports have instead correlated the axon initiation site to centrosome localization [18,19], suggesting an instructive role for the centrosome in determining axon initiation. However, later live imaging studies in Drosophila sensory neurons showed that centrosome positioning follows initial polarization, specified by clustering of adherens junction components [20]. In the developing cortex neurons shift from a multipolar to a bipolar morphology during their migration towards the brain surface, with the centrosome aligning towards the extending axon in multipolar-stage neurons, but opposite to the nascent axon in bipolar-stage neurons. In this case the location of the centrosome is associated with the predominant, most protrusive processes rather than with axonal identity, arguing against an instructive role for this organelle in specifying axon initiation [21].

Please cite this article as: G. Lalli, Regulation of neuronal polarity, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.07.033

178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237

E X PE R IM EN TA L C ELL R E S EA RC H

238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297

Signaling mechanisms in neuronal polarization The process of axon initiation can be triggered by extracellular cues such as growth factors and extracellular matrix components able to activate different signaling cascades leading to the asymmetric

] (]]]]) ]]]–]]]

3

distribution of polarity regulators (Fig. 1B). In this context, determinants of axon specification necessary and sufficient for neuronal polarization include activation of phosphoinositide 3-kinase (PI3K) in the growth cone of the nascent axon, causing local accumulation of phosphatidylinositol 3,4,5 triphosphate (PIP3) [16,22,23], localized cAMP/Protein Kinase A (PKA) activity leading to phosphorylation of

Fig. 1 – Key signaling pathways in neuronal polarization. (A) Hippocampal neuronal polarization in vitro. Immediately after plating (stage 1), round neurons extend dynamic filopodia and lamellipodia, which is followed by the extension of multiple immature neurites (stage 2). About 12 h after plating (stage 3), one of the neurites starts to grow to become the axon, thanks to microtubule stabilization in the shaft (blue) and highly dynamic actin/microtubule cytoskeleton in the growth cone (red). In the following 4–7 days (stage 4), the axon continues to extend while the other neurites mature into dendrites, which will subsequently develop spines (yellow) and establish functional synaptic contacts during the following 2 weeks (stage 5). (B) Schematic depiction of key signaling events regulating neuronal polarity. Following Ras activation after growth factor/adhesion signals, PI3K activation and PIP3 accumulation trigger a series of signaling events: 1) Akt activation and GSK3 inhibition resulting in microtubule stabilization; 2) activation of Rac regulating actin and microtubule dyamics; 3) Rheb-Rap1b-Cdc42 activation leading to activation of aPKC in the PAR complex, promoting actin cytoskeletal remodeling and polarized membrane delivery. Local activation of RalA and the exocyst complex also favor proper localization of PAR complex at the tip of the nascent axon. cAMP-dependent activation of PKA leads to activation of LKB1, which in turn phosphorylates SAD kinases and MARK/PAR1, regulating microtubule dynamics via modulating the affinity of MAPs for microtubules. The Wnt pathway may regulate polarity via Dvl by activating aPKC and/or inihibiting GSK3. Local degradation of some proteins (encircled in red) via proteasome, calpain, and Smurf ubiquitin ligases also contributes to neuronal polarization. See text for further details. Please cite this article as: G. Lalli, Regulation of neuronal polarity, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.07.033

298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357

4

358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417

E XP E RI ME N TAL CE L L R ES E ARC H

Liver Kinase B1 (LKB1) [24–26], and asymmetric distribution of Plasma Membrane Ganglioside Sialidase (PMGS) causing increased levels of GM1 ganglioside in one neurite and activation of the neurotrophin receptor TrkA [27]. These signaling cascades involve the participation of members of the PAR family (PAR-1, PAR-4, PAR-3 and PAR-6), originally discovered in a screen for regulators of asymmetric cell division in Caenorhabditis elegans [28] (see below).

The PI3K-Akt signaling cascade Extracellular matrix components and growth factor receptors may lead to local activation of several members of the Ras GTPase family, including H-, R-, K- and N-Ras in the perspective axon [11]. Recruitment of the Ras effector PI3K causes accumulation of PIP3 in one neurite, triggering the activation of kinases like integrinlinked kinase and Akt, both of which inactivate GSK3 [14,22,29] (Fig. 1B). GSK3 inhibition activates microtubule-binding proteins normally repressed by GSK3, ultimately leading to microtubule stabilization (see above). Interestingly, double knock-in mice lacking the Akt phosphorylation sites in both GSK3 isoforms (α and β) do not show neuronal polarization defects [30], suggesting that Akt-independent modes of GSK3 inhibition could also regulate axon specification in vivo. One of these could be via Wnt signaling, which inhibits GSK3 via the Frizzled receptor and Dishevelled (Dvl). Wnt5a could contribute to axon growth by promoting binding of Dvl to atypical protein kinase C (aPKC) [31], resulting in aPKC-dependent phosphorylation and inhibition of the microtubule-destabilizing protein MARK2, one of the mammalian orthologues of the PAR1 gene [32]. PI3K activation also leads to activation of the Ras family member Rheb and its target mammalian Target Of Rapamycin (mTOR), which increase translation of the Ras-related GTPase Rap1b in the perspective axon [33]. Interestingly, Rap1b is necessary and sufficient for axon specification in vitro by acting upstream Cdc42, ultimately influencing actin dynamics at the growth cone [34]. In addition, PI3K-dependent PIP3 accumulation in the nascent axon recruits the Rac activator DOCK7, leading to regulation of actin dynamics and inhibition of the microtubuledestabilizer stathmin [35]. Therefore, local activation of PI3K signaling plays a central role in neuronal polarization by regulating several downstream events controlling cytoskeletal dynamics.

The cAMP-LKB1 pathway Recent studies have shown that Brain Derived Neurotrophic Factor (BDNF) signaling or elevation of intracellular cAMP levels favours axon initiation via PKA-dependent phosphorylation of LKB1 (the mammalian orthologue of the C. elegans par-4 gene), a kinase essential for axon formation in vitro and in vivo [24,25]. LKB1 translocates from the nucleus and is activated by heterodimerization with one of two related pseudokinases known as Stradα and β. Overexpression of LKB1 and its coactivator Strad is sufficient to induce the formation of multiple axons [24,25]. Local phosphorylation of LKB1 on Ser431 in one neurite activates this kinase, which in turn phosphorylates SAD-A/B kinases and MARK2, a MAP modulator. Importantly, knocking out both SAD-A and SAD-B in mice abolishes axon formation [36]. The function of SAD kinases remains unclear, but may be related to 1) promoting the traffic of pre-synaptic vesicles in the nascent axon, based on experiments in C. elegans [37] and 2) to modulating the activity of

] (]]]]) ]]]–]]]

MAPs such as MAP2, MAP4 and Tau [38]. SAD- or MARK2dependent phosphorylation decreases the affinity of MAPs for microtubules, therefore destabilizing them. It remains to be clarified how SAD and MARK2 activities are coordinated with signals promoting microtubule stabilization (like GSK3 inhibition) at the tip of the future axon. Intriguingly, a recent report showed that, surprisingly, homozygous LKB1S431A/S431A knockin mice are viable, fertile, have no overt phenotype and retain normal SAD-A/B kinase activity [39]. Other compensatory kinases or phosphorylation of other sites on LKB1 may lead to activation of SAD kinases in vivo and these issues will require further examination.

The PAR complex The PAR-3, PAR-6 and aPKC complex is a crucial scaffold recruiting signaling players in many polarization events, including axon specification. Depletion of PAR-3 and PAR-6 impairs neuronal polarization, while overexpression of PAR-3 causes the appearance of multiple axons [22]. Active Cdc42, acting downstream a PI3K-Rap1b signaling cascade, binds PAR-6, activating aPKC in the PAR complex and triggering several key events promoting axon initiation (Fig. 1B). These include aPKC-mediated phosphorylation of Lethal Giant Larvae 1 (Lgl), promoting membrane insertion at the nascent axon [40] and inactivation of MARK-2, causing microtubule stabilization and growth [32]. Moreover, the active Cdc42-PAR-aPKC complex recruits the guanine nucleotide exchange factors (GEFs) Tiam1 and STEF to promote activation of Rac in the nascent axon. Overexpression of Tiam1 in cultured neurons leads to multiple axons [41]. Different Rac isoforms could regulate neuronal polarization in vivo, as shown by the lack of neuronal polarity defects in Rac1-depleted Drosophila or mammalian neurons [42]. Intriguingly, the PAR complex appears dispensable for axonogenesis in Drosophila [43], probably due to species-specific differences with mammalian systems.

Membrane traffic in axon initiation Polarized membrane traffic plays an essential role in the establishment of an axon, which, in contrast with dendrites, contains bundled microtubules uniformly oriented with their plus tips at the distal end. Selective vectorial membrane flow appears to even precede morphological differentiation of an axon, and leads to an enlarged growth cone in stage 2 hippocampal neurons [44]. Interestingly, in stage-2 hippocampal neurons the plus-directed motor kinesin-1 (KIF5) preferentially localizes in one of the neurites, where it allows the selective transport of αβ-tubulin heterodimers via CRMP2 [45–47]. In addition, in stage-2 neurons the kinesin-3 motor protein GAKIN/KIF13B transports PIP3 towards one of the neurites, thus providing spatial restriction of signaling cascades promoting axonogenesis [48]. Finally, KIF3 (kinesin-2) transports Par3 in the nascent axon, allowing formation of the PAR/aPKC scaffolding complex crucial for neuronal polarization [49]. Once the axo-dendritic polarity has been established, kinesin motors and dynein are responsible for targeting cargos to dendrites, while the unipolar microtubule organization in the axon leads to kinesin-dependent anterograde transport towards the distal side of the axon and dynein-dependent retrograde

Please cite this article as: G. Lalli, Regulation of neuronal polarity, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.07.033

418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477

E X PE R IM EN TA L C ELL R E S EA RC H

478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537

transport towards the cell body. How various cargoes are bound to specific motors for selective targeting to the dendritic or axonal compartment is still the focus of intense investigation and may vary depending on the nature of the vesicular cargo, but undoubtedly the existence of such selective membrane traffic pathways is crucial for the maintenance of neuronal polarity [50]. Interestingly, disruption of actin polymerization causes mislocalization of dendritic proteins, supporting the idea of a ‘F-actin diffusion barrier’ present in the proximal section of newly formed axons. The appearance of this area, characterized by a peculiar F-actin network [51,52], is likely to precede the formation of the axon initial segment (AIS), a specialized region essential for the maintenance of neuronal polarity. The AIS is defined proximally by clustered Ankyrin G molecules and is enriched in spectrins, voltage-gated ion channels and cell adhesion molecules. Several studies have now convincingly shown a crucial role for the AIS not only in action potential initiation and modulation, but also in maintaining protein traffic segregation between the axonal and dendritic compartments [53]. Interestingly, recent report has proposed that during axon specification, a distal “exclusion” mechanism involving the assembly of a submembranous axonal cytoskeleton (comprised of Ankyrin B, αII-spectrin and βII-spectrin) helps define a boundary restricting AnkyrinG to the AIS in the proximal axon [54]. The exact molecular mechanisms underlying this process, however, remain to be elucidated. So far the molecular details of the membrane traffic pathways contributing to axonogenesis have not been completely dissected. An interesting report has highlighted an important signaling cascade regulating polarized membrane insertion in the axon. Following Cdc42-mediated binding to the Par complex, active aPKC phosphorylates the Par3/6 binding partner Lgl1, which in turn leads to activation of Rab10, a small GTPase promoting membrane delivery essential for axon initiation in vitro and in vivo [40]. Importantly, active (GTP-loaded) Rab10 shows enhanced binding to the c-Jun N-terminal kinase-interacting protein 1 (JIP1), an adapter involved in kinesin-1-dependent axonal transport [55]. This interesting finding provides a molecular mechanism controlling the directional transport of plasmalemmal precursor vesicles downstream Cdc42 activation during neuronal polarization. The Ras-like GTPase RalA also contributes to axonogenesis via one of its effectors, the exocyst, a multisubunit protein complex essential for polarized membrane traffic in several cellular types. Depletion of either RalA or exocyst subunits leads to unpolarized neurons and to mislocalization of PAR-3 [56]. Active RalA directly interacts with the Exo84 exocyst subunit, and this is likely to promote a conformational change in Exo84 promoting direct recruitment of PAR-6 via an internal, non-canonical PDZ domain [57]. Local activation of RalA can therefore result in the assembly of a functional exocyst complex as well as in the proper localization of Par complex components. In parallel with polarized membrane delivery, local protein secretion can help create positive feedback loops favouring axon initiation. For example, autocrine neurotrophin signaling can promote insertion of neurotrophin receptor in the plasma membrane and establish a neurotrophin-induced neurotrophin release cycle leading to self-amplification of a growth signal as in the case of the BDNF receptor TrkB [58]. However, confirmation of this attractive model in vivo may be hindered by potential compensatory effects of other neurotrophin

] (]]]]) ]]]–]]]

5

factors with similar functions. The situation in vivo may be more complex, as highlighted for example in the developing cortex, where multiple neurotrophins may act via distinct signaling pathways involving different Trk receptors [59]. More recent results have pointed to an important role for p75NTR, a receptor able to bind to all members of the neurotrophin family, in axonal specification in vitro and in vivo. Localized exposure to BDNF causes polarized localization of p75NTR in an undifferentiated neurite of cultured hippocampal neurons, suggesting the existence of a positive neurotrophin-p75NTR feedback loop for axon initiation [60].

Local protein degradation Increasing evidence has highlighted additional mechanisms involving localized protein degradation in neuronal polarity. Indeed, prolonged treatment with the proteasome inhibitor lactacystin leads to the formation of multiple axons [61]. Proteasomemediated degradation can help restrict activity of key proteins to the presumptive axon. This is the case of inactive (less phosphorylated) Akt found in the somatodendritic compartment, which is degraded by the proteasome resulting in the enrichment of active Akt in the process destined to become the axon [61]. Similarly, inactive Rap1b found in the processes that will become dendrites is targeted for degradation by the Smurf2 ubiquitin ligase, leaving active Rap1b in the future axon [62]. Intriguingly, an interaction between the HECT domain of Smurf2 and the third PDZ domain of PAR-3 allows coupling to the KIF3A subunit of kinesin-2, resulting in PAR3-mediated targeting of Smurf2 in the axon. This interaction appears to regulate neuronal polarity, since its disruption leads to increased levels of active Rap1b in all neurites and axon loss [63]. Exactly how the dynamics of Smurf2 traffic is regulated in the polarizing neuron remains to be clarified. Localized protein degradation promoting neuronal polarization could also affect the Rac GEF Tiam1 [64], which is found in the nascent axon and could be targeted for calpain-mediated degradation following ERK-dependent phosphorylation [65]. Post-translational modification can modulate the sensitivity of substrates to E3 ubiquitin ligases. For example, PKA-mediated phosphorylation of LKB1 on Ser431 in the future axon reduces its ubiquitination [66], probably due to the formation of the LKB1/ STRAD stable complex [25]. On the other hand, post-translational modification can also modulate the substrate specificity of E3 ligases themselves downstream extracellular stimuli. Indeed, PKA-mediated phosphorylation of Smurf1 downstream BDNF stimulation leads to a substrate switch resulting in preferential degradation of growth-inhibiting RhoA and reduced proteolysis of axon-promoting PAR-6 [66]. In other cases, proteolysis modulates substrate activity. For example, calpain cuts the N-terminal segment of GSK3β, leading to a constitutively active kinase activity [13,67]. Calpain can also cut the C-terminal region of CRMP2, inhibiting its microtubule growth-promoting ability [68], and modulate the activity of Kidins220/ARMS, a protein kinase D (PKD) substrate able to regulate MAPs and neuronal polarity [69]. It will be important to uncover how calpain activity is regulated in the axon and verify the functional role of these calpain-mediated proteolytic events in vivo.

Please cite this article as: G. Lalli, Regulation of neuronal polarity, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.07.033

538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597

6

598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657

E XP E RI ME N TAL CE L L R ES E ARC H

Neuronal polarization in vivo Distinct modes of neuronal polarization While cultured hippocampal neurons provide an easily accessible model to elucidate the molecular mechanisms of neuronal polarization, the dynamics of axon initiation in vivo may be more complex and vary depending on the neuronal cell type [42]. Many neurons polarize already after exiting the cell cycle during migration towards their final target sites. In some cases they may inherit their polarity directly from the initial apico-basal polarity of their progenitors, as in the case of retinal ganglion cells and bipolar cells of the developing vertebrate retina (Fig. 2A and B). Upon asymmetric division, neural progenitors detach their apical process, which will later become the dendrite. In parallel, their nucleus translocates away from the apical surface, with the basal process becoming an axon while extending in the basal membrane (Fig. 2A). Instead, bipolar neuroepithelial progenitors in the mouse retina first detach their basal process, which will branch and become the axon, while the apical process branches before detaching from the surface to give rise to the dendritic compartment (Fig. 2B). In other cases, upon cell cycle exit migrating neurons progress through a series of stereotypical morphological changes before the clear emergence of the axonal and dendritic compartments. This is the case of cerebellar granule neurons and cortical pyramidal neurons, which are the best-studied models of neuronal polarization in vivo (Fig. 2C and D). Cerebellar granule neurons first adopt a bipolar morphology before migrating tangentially (i. e. parallel to the surface) with a leading and a trailing process. Subsequently, a perpendicular protrusion originates from the cell body and becomes the leading process, allowing migration of the cerebellar neuron towards the inner granule layer. After arrival to their target area, the leading process gives rise to the dendritic compartment, while the trailing process becomes a T-shaped axon (Fig. 2C). In the developing cortex, radial glial progenitors located in the subventricular zone extend a long basal process attached to the basal membrane of the pial surface and a shorter apical process linked to the ventricle (Fig. 2D). Asymmetric division of these progenitors, which also involves some key regulators of in vitro neuronal polarization [70], gives rise to postmitotic pyramidal neurons undergoing a “multipolar transition phase” characterized by the extension of multiple neurites (Fig. 2D). Extracellular signals are likely to mediate the transformation of one of these neurites into the leading process, promoting radial glia-guided migration of the neuron towards the cortical plate. While the leading process gives rise to a highly branched dendritic tree, the trailing process extending into the intermediate zone becomes the axon.

Extracellular cues regulating neuronal polarity In vivo neuronal polarization occurs in a complex environment, and likely requires the concerted actions of multiple determinants. These could trigger intracellular cascades including feedback loops promoting local signal amplification, ultimately resulting into the cytoskeletal rearrangements and membrane traffic events necessary for polarization. Extracellular matrix

] (]]]]) ]]]–]]]

components are likely to make key contributions to neuronal polarization, as shown in retinal ganglion cells, where the contact of processes with the basal protein laminin1 is necessary and sufficient for the neuron to polarize the axon towards the basal lamina [71]. In C. elegans, Wnt and Netrin are important regulators of neuronal polarity [72–74]. Notably, genetic deletion of many factors shown to regulate neuronal polarization in vitro does not lead to polarization defects in mice due to compensatory effects. However, mice lacking the type II TGF-β receptor (TβRII) display severe axon formation defects (even though axons are not completely absent), pointing to a likely important role for this morphogen secreted in a gradient fashion at the ventricular zone of the developing cortex [75]. In the developing cortex, the extracellular protein Reelin activates Rap1 to achieve the initial polarization of multipolar neurons, a process required for their net migration towards the upper cortical layers [76]. Importantly, the regulated activity of Rap1 seems necessary to maintain proper levels of surface N-cadherin and ensure the polarized migration in multipolar stage neurons. N-cadherin degradation, likely mediated by the Rab GTPase-regulated endocytic pathway [77] would then come into play in the final phase of migration in the upper cortical layer. Other small GTPases such as Ral, Rac and Cdc42 are likely to be involved downstream of Rap1 in the control of N-cadherin trafficking [76], although their exact role remains to be clarified. Interestingly, a recent study highlighted an important function for N-cadherin localization in even earlier phases of neuronal polarization, right after progenitor division in the subventricular zone. Here, N-cadherin appears to be localized in distinct patches aligned along the apical-basal axis in the newborn neurons, and could function as an early “landmark” for symmetry breaking, triggering the recruitment of intracellular organelles to promote the polarized outgrowth of the initial neurites along the migration axis in the cortex [78]. More recently, cell–cell interaction mediated by another cell adhesion molecule, transient axonal glycoprotein (TAG-1) was shown to regulate axon initiation from multipolar neurons contacting TAG-1-expressing cortical efferent axons. TAG-1-mediated interaction between pre-existing axons and multipolar neurons may help stabilize a single neurite in the latter cells, possibly acting via downstream activation of the Src family kinase Lyn and Rac [79]. However, the lack of neuronal polarization defects in TAG-1 knockout mice provides additional evidence for the presence of multiple compensatory mechanisms ensuring correct neuronal development. In the future it will be important to dissect the signaling events orchestrating the functions and possible cross-talk among cell adhesion molecules in neuronal polarization.

Concluding remarks A large number of studies based on the hippocampal neuron culture system have uncovered an intricate network of signaling pathways controlling cytoskeletal dynamics and membrane traffic to achieve neuronal polarization. The challenge ahead will be to validate these findings in vivo using innovative experimental approaches, genetic model systems and advances in imaging technology. It will be important to analyze how intracellular signaling cascades are triggered by the coordinated action of extracellular cues and clarify how the surrounding environment

Please cite this article as: G. Lalli, Regulation of neuronal polarity, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.07.033

658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717

E X PE R IM EN TA L C ELL R E S EA RC H

718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777

] (]]]]) ]]]–]]]

7

Fig. 2 – Modes of neuronal polarization in vivo. Distinct modes of neuronal polarization in vivo. In all panels the axonal and dendritic compartments are depicted in blue and red, respectively. (A) Mouse retinal ganglion cells derive from asymmetric division of neuroepithelial progenitors extending an apical and a basal attachment. Following division, the apical process detaches and will become the dendrite, while the nucleus undergoes a basal translocation with concomitant extension of the basal process that will become the axon. (B) In the mouse retina, bipolar cells derive from neuroepithelial progenitors, which first lose their basal attachment that subsequently branches in the inner plexiform layer (IPL) and becomes the axon. The apical process starts branching in the outer plexiform layer (OPL) before detaching to become the dendrite. (C) Polarization in the mammalian cerebellum. After their final division in the external granular layer (EGL), newborn granule cell neurons tangentially migrate with a leading and a trailing process, before extending a third process orthogonal to the cell body which will become a leading process for migration towards the inner granule layer (IGL). The trailing process will become a T-shaped axon, while the leading process will give rise to the dendritic compartment. (D) Schematic diagram of polarization in the mammalian developing cortex. New neurons (green) generated from radial glial cells in the ventricular zone (VZ) move to the intermediate zone (IZ) as bipolar cells and switch to a multipolar morphology. Neurons then resume bipolar migration in the upper IZ and the cortical plate (CP). The trailing process will become the axon, while the leading process will give rise to the dendritic compartment branching in the marginal zone (MZ). Please cite this article as: G. Lalli, Regulation of neuronal polarity, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.07.033

778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 833 834 835 836 837

8

838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888 889 890 891 892 893 894 895 896 897

E XP E RI ME N TAL CE L L R ES E ARC H

and cell–cell interactions contribute to neuronal polarization keeping in mind that, differently from cultured hippocampal neurons, axon formation sometimes follows dendrite specification. Given the constraints associated to the organization of neuronal tissue, investigating how extracellular signals couple with mechanical forces to promote axon initiation and growth will add a new perspective on the process of neuronal polarization. More surprising findings are still to come, and this new knowledge will undoubtedly help us understand how axon initiation and growth is regulated in vivo and how it can be promoted in injury and neurodegeneration.

Acknowledgments I apologize to those researchers whose work could not be cited due to space limitations. I thank Giovanni Lesa for valuable comments on this manuscript.

references [1] T. Yoshimura, N. Arimura, K. Kaibuchi, Molecular mechanisms of axon specification and neuronal disorders, Ann. N. Y. Acad. Sci. 1086 (2006) 116–125. [2] C.G. Dotti, C.A. Sullivan, G.A. Banker, The establishment of polarity by hippocampal neurons in culture, J. Neurosci. 8 (4) (1988) 1454–1468. [3] F. Bradke, C.G. Dotti, The role of local actin instability in axon formation, Science 283 (5409) (1999) 1931–1934. [4] D. Neukirchen, F. Bradke, Neuronal polarization and the cytoskeleton, Semin. Cell Dev. Biol. 22 (8) (2011) 825–833. [5] A. Hall, G. Lalli, Rho and Ras GTPases in axon growth, guidance, and branching, Cold Spring Harb. Perspect. Biol. 2 (2) (2010) a001818. [6] S. Tahirovic, et al., Rac1 regulates neuronal polarization through the WAVE complex, J. Neurosci. 30 (20) (2010) 6930–6943. [7] T. Jacobs, et al., Localized activation of p21-activated kinase controls neuronal polarity and morphology, J. Neurosci. 27 (32) (2007) 8604–8615. [8] B.K. Garvalov, et al., Cdc42 regulates cofilin during the establishment of neuronal polarity, J. Neurosci. 27 (48) (2007) 13117– 13129. [9] S. Gomis-Ruth, C.J. Wierenga, F. Bradke, Plasticity of polarization: changing dendrites into axons in neurons integrated in neuronal circuits, Curr. Biol. 18 (13) (2008) 992–1000. [10] N. Inagaki, et al., CRMP-2 induces axons in cultured hippocampal neurons, Nat. Neurosci. 4 (8) (2001) 781–782. [11] N. Arimura, K. Kaibuchi, Neuronal polarity: from extracellular signals to intracellular mechanisms, Nat. Rev. Neurosci. 8 (3) (2007) 194–205. [12] D. Neukirchen, F. Bradke, Cytoplasmic linker proteins regulate neuronal polarization through microtubule and growth cone dynamics, J. Neurosci. 31 (4) (2011) 1528–1538. [13] S.H. Shi, et al., APC and GSK-3beta are involved in mPar3 targeting to the nascent axon and establishment of neuronal polarity, Curr. Biol. 14 (22) (2004) 2025–2032. [14] F.Q. Zhou, et al., NGF-induced axon growth is mediated by localized inactivation of GSK-3beta and functions of the microtubule plus end binding protein APC, Neuron 42 (6) (2004) 897–912. [15] T. Yoshimura, et al., GSK-3beta regulates phosphorylation of CRMP-2 and neuronal polarity, Cell 120 (1) (2005) 137–149. [16] H. Jiang, et al., Both the establishment and the maintenance of neuronal polarity require active mechanisms: critical roles of GSK-3beta and its upstream regulators, Cell 120 (1) (2005) 123–135.

] (]]]]) ]]]–]]]

[17] F.R. Zolessi, et al., Polarization and orientation of retinal ganglion cells in vivo, Neural Dev. 1 (2006) 2. [18] J.F. Zmuda, R.J. Rivas, The Golgi apparatus and the centrosome are localized to the sites of newly emerging axons in cerebellar granule neurons in vitro, Cell Motil. Cytoskelet. 41 (1) (1998) 18–38. [19] F.C. de Anda, et al., Centrosome localization determines neuronal polarity, Nature 436 (7051) (2005) 704–708. [20] G. Pollarolo, et al., Cytokinesis remnants define first neuronal asymmetry in vivo, Nat. Neurosci. 14 (12) (2011) 1525–1533. [21] A. Sakakibara, et al., Dynamics of centrosome translocation and microtubule organization in neocortical neurons during distinct modes of polarization, Cereb. Cortex 24 (5) (2014) 1301–1310. [22] S.H. Shi, L.Y. Jan, Y.N. Jan, Hippocampal neuronal polarity specified by spatially localized mPar3/mPar6 and PI 3-kinase activity, Cell 112 (1) (2003) 63–75. [23] T. Yoshimura, et al., Ras regulates neuronal polarity via the PI3-kinase/Akt/GSK-3beta/CRMP-2 pathway, Biochem. Biophys. Res. Commun. 340 (1) (2006) 62–68. [24] A.P. Barnes, et al., LKB1 and SAD kinases define a pathway required for the polarization of cortical neurons, Cell 129 (3) (2007) 549–563. [25] M. Shelly, et al., LKB1/STRAD promotes axon initiation during neuronal polarization, Cell 129 (3) (2007) 565–577. [26] M. Shelly, et al., Local and long-range reciprocal regulation of cAMP and cGMP in axon/dendrite formation, Science 327 (5965) (2010) 547–552. [27] J.S. Da Silva, et al., Asymmetric membrane ganglioside sialidase activity specifies axonal fate, Nat. Neurosci. 8 (5) (2005) 606–615. [28] K.J. Kemphues, et al., Identification of genes required for cytoplasmic localization in early C. elegans embryos, Cell 52 (3) (1988) 311–320. [29] I. Oinuma, H. Katoh, M. Negishi, R-Ras controls axon specification upstream of glycogen synthase kinase-3beta through integrinlinked kinase, J. Biol. Chem. 282 (1) (2007) 303–318. [30] A. Gartner, X. Huang, A. Hall, Neuronal polarity is regulated by glycogen synthase kinase-3 (GSK-3beta) independently of Akt/PKB serine phosphorylation, J. Cell Sci. 119 (19) (2006) 3927–3934. [31] X. Zhang, et al., Dishevelled promotes axon differentiation by regulating atypical protein kinase C, Nat. Cell Biol. 9 (7) (2007) 743–754. [32] Y.M. Chen, et al., Microtubule affinity-regulating kinase 2 functions downstream of the PAR-3/PAR-6/atypical PKC complex in regulating hippocampal neuronal polarity, Proc. Natl. Acad. Sci. USA 103 (22) (2006) 8534–8539. [33] Y.H. Li, H. Werner, A.W. Puschel, Rheb and mTOR regulate neuronal polarity through Rap1B, J. Biol. Chem. 283 (48) (2008) 33784–33792. [34] J.C. Schwamborn, A.W. Puschel, The sequential activity of the GTPases Rap1B and Cdc42 determines neuronal polarity, Nat. Neurosci. 7 (9) (2004) 923–929. [35] M. Watabe-Uchida, et al., The Rac activator DOCK7 regulates neuronal polarity through local phosphorylation of stathmin/ Op18, Neuron 51 (6) (2006) 727–739. [36] M. Kishi, et al., Mammalian SAD kinases are required for neuronal polarization, Science 307 (5711) (2005) 929–932. [37] J.G. Crump, et al., The SAD-1 kinase regulates presynaptic vesicle clustering and axon termination, Neuron 29 (1) (2001) 115–129. [38] F. Polleux, W. Snider, Initiating and growing an axon, Cold Spring Harb. Perspect. Biol. 2 (4) (2010) a001925. [39] V.P. Houde, et al., Investigation of LKB1 Ser431 phosphorylation and Cys433 farnesylation using mouse knockin analysis reveals an unexpected role of prenylation in regulating AMPK activity, Biochem. J. 458 (1) (2014) 41–56. [40] T. Wang, et al., Lgl1 activation of rab10 promotes axonal membrane trafficking underlying neuronal polarization, Dev. Cell 21 (3) (2011) 431–444.

Please cite this article as: G. Lalli, Regulation of neuronal polarity, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.07.033

898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 921 922 923 924 925 926 927 928 929 930 931 932 933 934 935 936 937 938 939 940 941 942 943 944 945 946 947 948 949 950 951 952 953 954 955 956 957

E X PE R IM EN TA L C ELL R E S EA RC H

958 959 960 961 962 963 964 965 966 967 968 969 970 971 972 973 974 975 976 977 978 979 980 981 982 983 984 985 986 987 988 989 990 991 992 993 994 995 996 997 998 999 1000 1001 1002 1003 1004 1005 1006

[41] T. Nishimura, et al., PAR-6-PAR-3 mediates Cdc42-induced Rac activation through the Rac GEFs STEF/Tiam1, Nat. Cell Biol. 7 (3) (2005) 270–277. [42] A.P. Barnes, F. Polleux, Establishment of axon-dendrite polarity in developing neurons, Annu. Rev. Neurosci. 32 (2009) 347–381. [43] M.M. Rolls, C.Q. Doe, Baz, Par-6 and aPKC are not required for axon or dendrite specification in Drosophila, Nat. Neurosci. 7 (12) (2004) 1293–1295. [44] F. Bradke, C.G. Dotti, Neuronal polarity: vectorial cytoplasmic flow precedes axon formation, Neuron 19 (6) (1997) 1175–1186. [45] Y. Fukata, et al., CRMP-2 binds to tubulin heterodimers to promote microtubule assembly, Nat. Cell Biol. 4 (8) (2002) 583–591. [46] T. Kimura, et al., Tubulin and CRMP-2 complex is transported via Kinesin-1, J. Neurochem. 93 (6) (2005) 1371–1382. [47] C. Jacobson, B. Schnapp, G.A. Banker, A change in the selective translocation of the Kinesin-1 motor domain marks the initial specification of the axon, Neuron 49 (6) (2006) 797–804. [48] K. Horiguchi, et al., Transport of PIP3 by GAKIN, a kinesin-3 family protein, regulates neuronal cell polarity, J. Cell Biol. 174 (3) (2006) 425–436. [49] T. Nishimura, et al., Role of the PAR-3-KIF3 complex in the establishment of neuronal polarity, Nat. Cell Biol. 6 (4) (2004) 328–334. [50] L.C. Kapitein, C.C. Hoogenraad, Which way to go? Cytoskeletal organization and polarized transport in neurons, Mol. Cell. Neurosci. 46 (1) (2011) 9–20. [51] K. Watanabe, et al., Networks of polarized actin filaments in the axon initial segment provide a mechanism for sorting axonal and dendritic proteins, Cell Rep. 2 (6) (2012) 1546–1553. [52] K. Xu, G. Zhong, X. Zhuang, Actin, spectrin, and associated proteins form a periodic cytoskeletal structure in axons, Science 339 (6118) (2013) 452–456. [53] Ho, T. Szu-Yu, M.N. Rasband, Maintenance of neuronal polarity, Dev. Neurobiol. 71 (6) (2011) 474–482. [54] M.R. Galiano, et al., A distal axonal cytoskeleton forms an intraaxonal boundary that controls axon initial segment assembly, Cell 149 (5) (2012) 1125–1139. [55] C.Y. Deng, et al., JIP1 mediates anterograde transport of Rab10 cargos during neuronal polarization, J. Neurosci. 34 (5) (2014) 1710–1723. [56] G. Lalli, RalA and the exocyst complex influence neuronal polarity through PAR-3 and aPKC, J. Cell Sci. 122 (10) (2009) 1499–1506. [57] A. Das, et al., RalA promotes a direct exocyst-Par6 interaction to regulate polarity in neuronal development, J. Cell Sci. 127 (3) (2014) 686–699. [58] P.L. Cheng, et al., Self-amplifying autocrine actions of BDNF in axon development, Proc. Natl. Acad. Sci. USA 108 (45) (2011) 18430–18435. [59] S. Nakamuta, et al., Local application of neurotrophins specifies axons through inositol 1,4,5-trisphosphate, calcium, and Ca2þ/calmodulindependent protein kinases, Sci. Signal. 4 (199) (2011) ra76. [60] E. Zuccaro, et al., Polarized expression of p75(NTR) specifies axons during development and adult neurogenesis, Cell Rep. 7 (1) (2014) 138–152.

] (]]]]) ]]]–]]]

9

[61] D. Yan, L. Guo, Y. Wang, Requirement of dendritic Akt degradation by the ubiquitin-proteasome system for neuronal polarity, J. Cell Biol. 174 (3) (2006) 415–424. [62] J.C. Schwamborn, et al., Ubiquitination of the GTPase Rap1B by the ubiquitin ligase Smurf2 is required for the establishment of neuronal polarity, EMBO J. 26 (5) (2007) 1410–1422. [63] J.C. Schwamborn, M.R. Khazaei, A.W. Puschel, The interaction of mPar3 with the ubiquitin ligase Smurf2 is required for the establishment of neuronal polarity, J. Biol. Chem. 282 (48) (2007) 35259–35268. [64] P. Kunda, et al., Evidence for the involvement of Tiam1 in axon formation, J. Neurosci. 21 (7) (2001) 2361–2372. [65] S.A. Woodcock, et al., SRC-induced disassembly of adherens junctions requires localized phosphorylation and degradation of the rac activator tiam1, Mol. Cell 33 (5) (2009) 639–653. [66] P.L. Cheng, et al., Phosphorylation of E3 ligase Smurf1 switches its substrate preference in support of axon development, Neuron 69 (2) (2011) 231–243. [67] P. Goni-Oliver, et al., N-terminal cleavage of GSK-3 by calpain: a new form of GSK-3 regulation, J. Biol. Chem. 282 (31) (2007) 22406–22413. [68] W. Liu, et al., Calpain-truncated CRMP-3 and -4 contribute to potassium deprivation-induced apoptosis of cerebellar granule neurons, Proteomics 9 (14) (2009) 3712–3728. [69] A.M. Higuero, et al., Kidins220/ARMS modulates the activity of microtubule-regulating proteins and controls neuronal polarity and development, J. Biol. Chem. 285 (2) (2010) 1343–1357. [70] S.A. Fietz, W.B. Huttner, Cortical progenitor expansion, selfrenewal and neurogenesis-a polarized perspective, Curr. Opin. Neurobiol. 21 (1) (2011) 23–35. [71] O. Randlett, et al., The oriented emergence of axons from retinal ganglion cells is directed by laminin contact in vivo, Neuron 70 (2) (2011) 266–280. [72] C.E. Adler, R.D. Fetter, C.I. Bargmann, UNC-6/Netrin induces neuronal asymmetry and defines the site of axon formation, Nat. Neurosci. 9 (4) (2006) 511–518. [73] M.A. Hilliard, C.I. Bargmann, Wnt signals and frizzled activity orient anterior-posterior axon outgrowth in C. elegans, Dev. Cell 10 (3) (2006) 379–390. [74] M. Montcouquiol, E.B. Crenshaw 3rd, M.W. Kelley, Noncanonical Wnt signaling and neural polarity, Annu. Rev. Neurosci. 29 (2006) 363–386. [75] J.J. Yi, et al., TGF-beta signaling specifies axons during brain development, Cell 142 (1) (2010) 144–157. [76] Y. Jossin, J.A. Cooper, Reelin, Rap1 and N-cadherin orient the migration of multipolar neurons in the developing neocortex, Nat. Neurosci. 14 (6) (2011) 697–703. [77] T. Kawauchi, et al., Rab GTPases-dependent endocytic pathways regulate neuronal migration and maturation through N-cadherin trafficking, Neuron 67 (4) (2010) 588–602. [78] A. Gartner, et al., N-cadherin specifies first asymmetry in developing neurons, EMBO J. 31 (8) (2012) 1893–1903. [79] T. Namba, et al., Pioneering axons regulate neuronal polarization in the developing cerebral cortex, Neuron 81 (4) (2014) 814–829.

Please cite this article as: G. Lalli, Regulation of neuronal polarity, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.07.033

1007 1008 1009 1010 1011 1012 1013 1014 1015 1016 1017 1018 1019 1020 1021 1022 1023 1024 1025 1026 1027 1028 1029 1030 1031 1032 1033 1034 1035 1036 1037 1038 1039 1040 1041 1042 1043 1044 1045 1046 1047 1048 1049 1050 1051 1052 1053 1054