JNK signaling controls branching, nucleokinesis, and positioning of centrosomes and primary cilia in migrating cortical interneurons

Aberrant migration of inhibitory interneurons can alter the formation of cortical circuitry and lead to severe neurological disorders including epilepsy, autism, and schizophrenia. However, mechanisms involved in directing the migration of these cells remain incompletely understood. In the current study, we used live-cell confocal microscopy to explore the mechanisms by which the c-Jun NH2-terminal kinase (JNK) pathway coordinates leading process branching and nucleokinesis, two cell biological processes that are essential for the guided migration of cortical interneurons. Pharmacological inhibition of JNK signaling disrupts the kinetics of leading process branching, rate and amplitude of nucleokinesis, and leads to the rearward mislocalization of the centrosome and primary cilium to the trailing process. Genetic loss of Jnk from interneurons corroborates our pharmacological observations and suggests that important mechanics of interneuron migration depend on the intrinsic activity of JNK. These findings suggest that JNK signaling regulates leading process branching, nucleokinesis, and the trafficking of centrosomes and cilia during interneuron migration, and further implicates JNK signaling as an important mediator of cortical development. Summary Statement Loss of JNK signaling reduces growth cone branching frequency, limits interstitial side branch duration, alters rate and amplitude of nucleokinesis, and mislocalizes centrosomes and primary cilia in migrating cortical interneurons.


INTRODUCTION
distance and kinetics of individual nucleokinesis events were disrupted ( Fig. 3B; Fig. 3H; Movie 198 5, Clip 2). When we measured the mean distance that cell bodies advanced over time,  inhibited interneurons translocated significantly shorter distances compared to control cells 200 translocation is preceded by swelling extension, we measured the average distance from the 211 soma to the swelling before translocation and found that SP600125-treated interneurons did not 212 extend cytoplasmic swellings as far as controls (control: 13.13±0.38µm; SP600125: 213 11.34±0.30µm; p=0.002; Fig. 3F). Since JNK-inhibited interneurons paused for longer periods of 214 time, we asked if this was strictly due to delayed nuclear propulsion towards the swelling, or if 215 the dynamics of swelling extension were also affected. Interneurons treated with SP600125 216 displayed significantly longer lasting cytoplasmic swellings (control: 11.27±0.99min; SP600125: 217 18.31±1.33min; p=0.0005; Fig. 3G), indicating that swelling duration is concomitantly increased 218 with pause duration. Finally, the frequency and amplitude of nuclear translocations that exceed 219 a minimum distance of 5 microns was notably reduced when individual control and JNK-220 inhibited cells were compared (Fig. 3H). 221 Together, these data point to a role for JNK signaling in regulating the distance and kinetics of 222 nucleokinesis in migrating MGE interneurons, which likely contributes to the decrease in 223 migratory speed and displacement that occurs during JNK inhibition. 224 225 226

Complete genetic loss of JNK impairs nucleokinesis and leading process branching of 227 migrating MGE interneurons in vitro 228
Since acute pharmacological inhibition of JNK activity altered the dynamic behavior of migratory 229 cortical interneurons, we next asked whether genetic removal of JNK function from MGE 230 interneurons also impaired their migration. In order to genetically ablate all three JNK genes 231 from interneurons, we used mice containing the Dlx5/6-CIE transgene to conditionally remove 232  Figure 4G). 243 The combination of increased speed variability and decreased migratory straightness explain 244 why cTKO interneurons exhibited shorter migratory displacements. Together, these data 245 indicate that cTKO interneurons have subtle yet statistically significant deficits in their overall 246 migratory dynamics, similar to pharmacological inhibition of JNK . 247 To determine the genetic requirement for JNK signaling in branching, we analyzed leading 248 process branching dynamics of cTKO and WT interneurons (Movies 6-7). cTKO interneurons 249 displayed a significant reduction in the frequency of growth cone splitting compared to WT 250 interneurons  Fig. 4J; Movie 6-7, Clip 2). These data corroborate the findings 255 from our pharmacological analyses and further suggest a key role for JNK signaling in 256 controlling leading process branching dynamics.
Since we found alterations to overall migratory properties and branching dynamics, we next 258 analyzed migrating cTKO interneurons for defects in nucleokinesis. Although cTKO interneurons 259 engaged in nucleokinesis, the kinetics of nucleokinesis were significantly altered compared to 260 WT interneurons (Fig. 5)

Subcellular localization and dynamic behavior of the centrosome and primary cilia in 282 migrating MGE interneurons depend on intact JNK-signaling 283
The cytoplasmic swelling emerges from the cell body during nucleokinesis and contains multiple 284  ). When we measured the average maximal distance that the centrosome was displaced 309 from the somal front, the centrosome of JNK-inhibited interneurons maintained a significantly 310 closer position to the leading pole of the soma compared to controls (control: 9.93±0.99µm; 311 SP600125: 6.73±0.88µm; p=0.03; Fig. 6F). This was not surprising since the soma-to-swelling 312 distance in JNK-inhibited interneurons was decreased (Fig. 3E). However, when we compared 313 the average maximal rearward distance between the centrosome and somal front, the 314 centrosome of JNK-inhibited interneurons was significantly further behind that of 315 controls(control: 9.40±0.77µm; SP600125: 19.75±1.94µm; p=1.48x10 -5 ; Fig. 6F). 316 Since we found defects in centrosome dynamics, we wanted to determine whether primary cilia, 317 which normally extend from the mother centriole and house receptors important for the guided 318 In the present study, we demonstrated that migrating MGE interneurons rely on the JNK 339 signaling pathway to properly undergo leading process branching and nucleokinesis. While the exact mechanisms that cortical interneurons utilize to navigate their environment 473 remain to be fully elucidated, we have found that JNK signaling exerts fine-tune control over cell 474 biological processes required for proper interneuron migration. 475

Conclusions 476
Using a combination of pharmacological and genetic approaches, we found a novel requirement 477 for JNK signaling in MGE interneuron leading process branching and nucleokinesis. Our 478 findings are also the first to implicate the JNK signaling pathway as a key intracellular regulator 479 of the dynamic positioning of multiple subcellular organelles involved in interneuron migration. 480 The exact molecular mechanisms controlling JNK signaling in interneuron migration remain to disease. 484

MATERIALS AND METHODS 485
Animals 486 Cortices were dissected from the negative brains and pooled together for dissociation (Polleux 504 and Ghosh, 2002). After dissociation, 250µL of cell suspension diluted to 1680cells/µL was 505 added to each well and allowed to settle for 2 hours. MGE explants were dissected from GFP+ 506 brains and plated on top of cortical cells. Cultures were grown for 24 hours before treatments 507 and live imaging. Two E14.5 timed-pregnant dams were used for each genetic experiment.

Live Imaging Experiments 525
Cultures were treated with pre-warmed 37 o C serum-free media containing a 1:1000 dilution of 526 DMSO for vehicle control or 20 µM SP600125 pan-JNK inhibitor (Enzo Life Sciences BML-527 EI305-0010; Farmingdale, NY, USA) and immediately transferred to a Zeiss 710 Confocal 528 Microscope with stable environmental controls maintained at 37 o C with 5% humidified CO 2 . 529 Multi-position time-lapse z-series were acquired at 10-minute intervals over a 12-hour period 530 with a 20X Plan-Apo objective (Zeiss; Oberkochen, Germany) for overall migration analysis, 531 nucleokinesis distance, and swelling distance measurements. For measurements requiring 532 higher temporal and spatial resolution, such as swelling duration, branch dynamics, and 533 visualization of subcellular structures in electroporated cells, cultures were imaged using multi-534 position time-lapse z-series at 2-2.5 minute intervals over a 4-10 hour period with a 40X C-535 apochromat 1.2W M27 objective (Zeiss; Oberkochen, Germany). 536

Analysis of Live Imaging 537
4D live imaging movies were analyzed using Imaris 9.5.1 (Bitplane; Zürich, Switzerland) 538 software. Movies collected at 20X were evaluated in the first 12 h of each recording. Individual 539 interneurons were tracked for a minimum of 4 h. Tracks were discontinued if a cell remained 540 stationary for 60 contiguous minutes, or if the tracked cell could no longer be unambiguously 541 identified. All tracks from each movie were averaged together for dynamic analyses. Cortical 542 interneurons were tracked using the Spots feature of Imaris to capture migratory speed, minimum track length of 4 h. Data sets were acquired from a minimum of four experimental 545 days with genetic experiments containing 5 conditional triple knockout (cTKO) embryos. 546 Pharmacological swelling duration data was obtained from movies collected over 4 experimental 547 days. Genetic swelling duration was obtained from 3 experimental days with 3 cTKO embryos. 548 The minimum criteria for an interstitial side branch to be included in our analysis was as follows: 549 the cell had to remain in frame for a minimum of 3 hours, an interstitial side branch had to 550 persist for a minimum of 10 minutes, and the branch could not become the new leading 551 process. Two-tailed unpaired Student's t tests were used to determine statistical differences 552 between groups. 553  ., Miyamoto, Y., Hamasaki, H., Sanbe, A., Kusakawa, S., Nakamura, A.,