βIII spectrin controls the planarity of Purkinje cell dendrites by modulating perpendicular axon-dendrite interaction

The mechanism underlying the geometrical patterning of axon and dendrite wiring remains elusive, despite its critical importance in the formation of functional neural circuits. Cerebellar Purkinje cell (PC) arborizes a typical planar dendrite, which forms an orthogonal network with granule cell (GC) axons. By using electrospun nanofiber substrates, we reproduce the perpendicular contacts between PC dendrites and GC axons in culture. In the model system, PC dendrites show preference to grow perpendicular to aligned GC axons, which presumably contribute to the planar dendrite arborization in vivo. We show that βIII spectrin, a causal gene for spinocerebellar ataxia type 5 (SCA5), is required for the biased growth of dendrites. βIII spectrin deficiency causes actin mislocalization and excessive microtubule invasion in dendritic protrusions, resulting in abnormally oriented branch formation. Furthermore, disease-associated mutations affect the ability of βIII spectrin to control dendrite orientation. These data indicate that βIII spectrin organizes the dendritic cytoskeleton and thereby regulates the oriented growth of dendrites with respect to the afferent axons. Summary statement βIII spectrin suppress the microtubule dynamics at the neuronal dendrite to inhibit the abnormal lateral branching, which causes misoriented branch formation.

Earlier studies have demonstrated that the planarity of PC dendrites is disturbed in loss-of-114 function mutants of βIII spectrin (Gao et al., 2011). We first confirmed the cell-autonomous role 115 of βIII spectrin in PC dendrite formation by short hairpin RNA (shRNA) knockdown in wildtype 116 background in vivo. We used a plasmid encoding an shRNA targeting βIII spectrin (βIII spectrin 117 kd) that efficiently knocks down βIII spectrin expression in PCs in a dissociation culture (Fig. S1). 118 We sparsely delivered the plasmid into PC precursors in the 4th ventricle via in utero 119 electroporation at embryonic day 11.5 (E11.5). Consistent with previous observations using βIII 120 spectrin knockout mice (Gao et al., 2011), we observed almost no differences in the total length 121 of dendrites between control (ctr) and βIII spectrin knockdown cells at postnatal day 14 (P14) 122 ( Fig. 1A-C). Compared to the normal planar dendrites in control PCs, βIII spectrin knockdown 123 14 was markedly increased (Fig. 4A, B). Similar to control PCs, most of the dendritic retractions 218 (~90%) in βIII spectrin knockdown cells occurred after the dendritic collisions. However, the 219 frequency of dendrite retraction after a collision was reduced to 39% in βIII spectrin knockdown 220 cells, suggesting that the contact-dependent dendritic retraction is dysregulated in βIII spectrin 221 deficient cells (Fig. 4D). However, the orientation of retracted dendrites was strongly biased 222 parallel to the axons, negating that the increase in misoriented dendrites in βIII spectrin 223 knockdown cells was caused by suppression of dendrite retraction of wrong arbors. Therefore, 224 we focused on how βIII Spectrin deficiency leads to the dysregulation of dendrite growth 225

orientation. 226
Our previous studies have shown that PCs form dendritic branches primarily via the 227 bifurcation of growing terminals (terminal branching), while they rarely extend collaterals from 228 the shaft (lateral branching) (Fujishima et al., 2012). Accordingly, control PC dendrites on aligned 229 fibers mainly displayed terminal branching (Fig. 4E, I). In contrast, lateral branching was 230 increased by more than 10-fold in βIII spectrin knockdown dendrites compared with control 231 dendrites (Fig. 4F, I), although the branching frequency was only slightly altered (Fig. 4J). The 232 deflection angle between the bifurcated terminal branches ranged within approximately ±30°, 233 while that of lateral branching was greater than 60° in both the control and βIII spectrin deficient 234 dendrites (Fig. 4G, H). These results suggest that βIII spectrin regulates the perpendicular growth 235 of dendrites by inhibiting lateral branching. Thus, the frequent lateral branching may contribute 236 to the increase in misoriented dendrites in the βIII spectrin deficient cells. 237 238 Lateral branch formation in βIII spectrin-knockdown dendrites. 239 We next observed how dendritic planarity was affected in βIII spectrin knockdown cells in vivo. 240 Control PC dendrites transfected with GFP aligned in a parasagittal plane in the molecular layer 241 parallel to the neighboring PC dendrites (Fig. 5A, B). These PCs rarely exhibited dendritic 242 branches growing in lateral (coronal) directions. Neighboring dendrites were separated by gaps 243 of approximately 1-3 µm, showing minimal crossing with adjacent branches. Similar to control 244 PCs, the main dendritic arbors of βIII spectrin knockdown PCs were mostly parallel to 245 neighboring PC dendrites in coronal sections (Fig. 5A, B, βIII kd), although some dendrites bent 246 or tilted into incorrect planes (white arrows in Fig. 5A). Notably, βIII spectrin knockdown 247 dendrites exhibited an increased number of laterally oriented branches growing into the territories 248 of the neighboring PC dendrites (yellow arrows in Fig. 5A, Fig. 5C). These misoriented branches 249 often turned and extended parasagittally into the gaps between PC dendrites (yellow arrowhead 250 in Fig. 5A). These misoriented lateral branches likely contribute to the disruption of planar 251 dendrite in βIII spectrin deficient PCs. 252 It has been demonstrated that the growing PC dendrites are covered with numerous 253 dendritic protrusions, including dendritic filopodia and immature spines (Kawabata Galbraith et 254 al., 2018; Shimada et al., 1998). As dendritic protrusions are known to serve as dendritic branch 255 precursors in some neurons, we next analyzed the dendritic protrusions in PCs with or without 256 βIII spectrin expression. Control dendrites presented numerous dendritic protrusions emanating 257 from the shaft with a mean length of 1.48 ± 0.04 μm (mean ± SEM, n = 231) (Fig. 5D, E). In 258 contrast, βIII spectrin knockdown dendrites exhibited significantly longer protrusions (2.21 ±0.17 259 μm, n = 102) at a lower density, in agreement with previous studies (Fig. 5D-F) (Efimova et al.,260 2017; Gao et al., 2011). Notably, some dendritic protrusions in βIII spectrin knockdown cells were 261 abnormally elongated (>5 µm) in lateral directions away from the main sagittal plane of the 262 dendritic shaft (arrows in Fig. 5D, Fig. 5G). These long lateral protrusions seemed to serve as 263 precursors of the ectopic lateral branches in βIII spectrin deficient cells. 264 265 Abnormal formation of dendritic protrusions in βIII spectrin-knockdown dendrites 266 To further analyze the implications of βIII spectrin in the formation of dendritic protrusions and 267 branches, we observed the dendritic structures in neurons grown on aligned nanofibers. Control 268 PCs bore highly dense protrusions that covered the lateral surface of the dendritic shaft, similar 269 to those observed in vivo (Fig. 6A). These protrusions expressed glutamate receptor δ2 (GluD2), 270 which functions as a synaptic glue by binding with presynaptic neurexin and cbln1 in GC axonal terminals (Matsuda et al., 2010;Uemura et al., 2010), suggesting that these protrusions are 272 dendritic spines or immature spine precursors (Fig. 6A). 273 Compared to control cells, the distal dendrites of βIII spectrin knockdown cells were 274 significantly thinner (ctr: 1.80 ± 0.15 μm, n= 19, βIII kd: 0.90± 0.06 μm, n= 22, mean ± SEM, p 275 = 7 x 10 -6 , Student's t-test) (Fig. 6B). In contrast to the dendritic protrusions in control cells, which 276 presented a relatively constant length of 1-3 µm, those in βIII spectrin knockdown cells presented 277 various lengths at a lower density, with an average length that was significantly longer than that 278 in control cells (Fig. 6C, D). We found some abnormally elongated protrusions of nearly 10 µm 279 in βIII spectrin knockdown dendrites that extended parallel to the orientation of GC axons. Other that were irregularly arranged along their lengths (Fig. 6B ROI2). The orientation of the 284 protrusions in the control and βIII spectrin knockdown dendrites was mostly parallel to GC axons 285 (Fig. 6E). These results suggested that dendritic protrusions in βIII spectrin deficient dendrites 286 abnormally extend along GC axons, which possibly induce disoriented branch formation (Fig.  287

6F). 288
We next analyzed the subcellular localization of βIII spectrin in growing dendrites in 289 PCs cultured on coverslips. In agreement with previous reports (Efimova et al., 2017;Gao et al., 290 2011), βIII spectrin was strongly localized on the surface of the dendritic shaft and the base of 291 dendritic protrusions, while it was excluded from the protrusion tips (Fig. 6G, H). Actin showed 292 an inverse gradient along the dendritic protrusions such that it was densely localized at the tip and 293 sharply declined in the base of the protrusion and the dendritic shaft (Fig. 6H, I). In contrast, in 294 βIII spectrin knockdown PCs, actin was more widely distributed along the entire length of both 295 long and thin (Type 1 in Fig. 6J) and short and stubby (Type 2 in Fig. 6J) dendritic protrusions 296 and was often dispersed in the shaft of distal dendrites (Type 1 in Fig. 6J, Fig. 6K). These results 297 imply that βIII spectrin might be involved in the formation of the structural boundary between the 298 dendritic shaft and protrusions that confines actin filaments within dendritic protrusions. blocking microtubule entry into dendritic protrusions, we monitored microtubule polymerization 322 in growing dendrites by transfecting EB3-EGFP, a plus-end marker of dynamic microtubules. We 323 analyzed the growing terminals of dendrites (within 10 μm from the terminal) and more proximal 324 dendrites (more than 10 μm away from the terminal) separately to examine the regional difference 20 in EB3 dynamics. 326 In control cells, 48 ± 5% (mean ± SEM, 226 protrusions from 18 dendrites) of dendritic 327 protrusions around the growing terminal were targeted by EB3-EGFP within 150 seconds of 328 observation (Fig. 7A, C). In contrast, a significantly lower proportion of protrusions were invaded 329 by EB3 in proximal dendrites (5 ± 2%, 149 protrusions from 11 dendrites), in line with the notion 330 that microtubule entry into filopodia triggers neurite extension at dendritic tips. 331 We found that βIII spectrin knockdown significantly increased the proportion of EB3-332 targeted protrusions in proximal regions (41 ± 6%, 91 protrusions from 10 dendrites) (Fig. 7B, 333 C), while only a slight increase was observed in the distal area (67 ± 4%, 102 protrusions from 334 13 dendrites). These data support the idea that βIII spectrin interferes with microtubule invasion 335 into dendritic protrusions in proximal dendrites. Furthermore, we often observed that EB3-336 positive puncta tipped the filopodia and promoted their aberrant extension in βIII spectrin 337 knockdown cells (Fig. 7D). The speed of microtubule polymerization was instead slightly 338 downregulated in βIII spectrin-knockdown cells, negating that excessive microtubule entry in βIII 339 spectrin-deficient cells was caused by increased microtubule polymerization activity (Fig. 7E). 340 These results suggest that βIII spectrin controls directed dendritic arborization by suppressing 341 microtubule invasion and ectopic branch formation from proximal dendritic protrusions (Fig. 7F).

Mutations causing spinocerebellar ataxia type 5 (SCA5) 344
Mutations in βIII spectrin are known to cause spinocerebellar ataxia type 5 (SCA5). Hence, we 345 wondered if the disease mutations affect biased dendrite growth perpendicular to GC axons. We 346 focused on three mutations identified in earlier studies (Fig. 8A) (Ikeda et al., 2006). The first is 347 a point mutation found in a German family that results in a leucine to proline substitution (L253P) 348 in the calponin homology domain; the second is an in-frame 39-bp deletion (E532-M544 del) in 349 the third spectrin repeat found in an American family; and the third is a 15-bp deletion in the third 350 spectrin repeat (5-amino acid deletion with the insertion of tryptophan, L629-R634 delinsW) 351 found in a French family (Fig. 8A). The amino acid sequences related to these mutations are 352 conserved among humans and mice. Thus, we generated mouse βIII spectrin mutant constructs 353 harboring the corresponding mutations. These constructs were designed to be shRNA resistant 354 and were tagged with a myc epitope at their N-termini for imaging. 355 Dissociated PCs on aligned nanofiber substrates were knocked down for endogenous 356 βIII spectrin and concomitantly transfected with wild-type βIII spectrin or disease-related mutants. 357 Wild-type βIII spectrin was distributed in the somatodendritic area up to the most distal dendritic 358 regions. In sharp contrast, the L253P mutant form was localized in intracellular vesicular 359 structures in the somatic area, while almost no signal was observed in dendrites (Fig. 8B). On the 360 other hand, mutants with deletions in the third spectrin repeat (E532-M544 del and L629-R634 delinsW) localized to the dendritic plasma membrane to a lesser extent than wild-type molecules. 362 Quantitative image analysis revealed the differential localization of mutants in PC dendrites (Fig.  363

8C). 364
PCs expressing the shRNA-resistant wild-type molecule exhibited normal perpendicular 365 dendrites. In contrast, all disease mutants were defective in the regulation of perpendicular 366 dendrite formation. L253P and E532-M544 del were completely incompetent in perpendicular 367 guidance, while the L629-R634 delinsW mutation, which showed modest mislocalization, 368 retained weak but significant guidance activity (Fig. 8D). 369 To confirm the effect of the L253P and E532-M544 del mutations in the planar dendrite 370 arborization in vivo, we delivered βIII spectrin knockdown plasmid and shRNA resistant βIII 371 spectrin wild-type, L253P or E532-M544 del mutant to immature PCs at E11.5 by in utero 372 electroporation. In agreement with in vitro observation, L253P and E532-M544 del mutants 373 exhibited abnormal localization in vesicular structures in the soma and proximal dendritic surface, 374 respectively, in contrast to the wild-type molecule spreading over the entire dendritic surface (Fig.  375 8E). PCs expressing wild-type molecule showed planar dendrites, while the cells expressing either 376 L253P or E532-M544 del mutant displayed disorganized dendrites growing away from the main 377 dendritic plane (Fig. 8E, F). These results suggest that these disease mutations of βIII spectrin 378 disrupt dendritic configuration in PCs. 379

380
In the present study, we established a simplified 2D model of axon-dendrite topology 381 using aligned nanofibers and confirmed that PC dendrites grew preferentially in the direction 382 perpendicular to the bundles of afferent parallel fiber axons. The directional arborization is likely 383 a prerequisite for the planar dendrite formation in the cerebellar tissue. Moreover, we revealed 384 that biased dendrite arborization was affected by the loss of αII/βIII spectrins. In control PCs, 385 dendritic branches were formed mainly by terminal bifurcation, with only a few collateral 386 branches emerging from proximal dendrites (Fujishima et al., 2012). In contrast, lateral branching 387 events were significantly increased in βIII spectrin knockdown cells (Fig. 4G). protein GluD2, suggesting that these lateral protrusions at the proximal dendrites are immature 392 spine precursors. Notably, βIII spectrin knockdown dendrites abnormally extended some 393 proximal protrusions to a length indistinguishable from that of dendritic branches. These 394 elongated protrusions bore multiple GluD2 puncta along their length. Thus, the loss of βIII 395 spectrin seems to alter the fate of proximal dendritic protrusions from immature spines to branch 396 precursors, which become misoriented branches extruded from the main parasagittal plane ( frequently entered these proximal protrusions and promoted their abnormal elongation in βIII 416 spectrin knockdown PCs (Fig. 7). 417

SCA5-related mutations affect dendrite growth in PCs 419
SCA5 is one of the autosomal dominant cerebellar ataxias caused by the heterozygous 420 mutation in βIII spectrin gene (Ikeda et al., 2006). Although our experiment may not completely 421 mimic the disease condition, SCA5 related mutants misexpressed in βIII spectrin knockdown cells 422 failed to substitute for wildtype βIII spectrin to control the dendrite orientation. L253P mutant 423 proteins were not delivered to dendritic membranes nor did they replace the function of wild-type 424 molecules in regulating the oriented growth of PC dendrites. This is consistent with previous 425 studies showing that the L253P mutation affects the trafficking of β-spectrin from the Golgi 426 apparatus (Clarkson et al., 2010). It is also suggested that L253P mutation may reduce the plasticity 427 of actin-spectrin network by enhancing the actin-spectrin affinity. This change might affect the 428 proper dendritic localization of βIII spectrin (Avery et al., 2017). In contrast, the L629-R634 429 delinsW mutation had only minor effects on dendritic localization and the oriented arborization 430 of dendrites. Interestingly, the E532-M544 del mutant was severely defective in controlling 431 dendrite growth orientation despite its relatively normal dendritic localization except for the 432 distalmost region. It has been proposed that the deletion of E532-M544 possibly affects the triple 433 alpha-helical structures of the spectrin repeats, which might result in the alteration of overall 434 alpha/beta structures (Ikeda et al., 2006). We assume that the E532-M544 del mutation may 435 affect the stabilization of the spectrin architecture in dendrites and interfere with dendrite growth 436 in the normal direction. 437 The SCA5 patients and βIII spectrin knockout animals exhibit progressive 438 neurodegeneration that has been attributed to the excitotoxicity due to mislocalization and the

Remaining questions in the perpendicular axon-dendrite interactions 455
We demonstrate that PC dendrites grow perpendicular to parallel fibers, which 456 seemingly contribute to the planar dendrite arborization in vivo. We further present that βIII 457 spectrin is required for the perpendicular dendrite arborization by suppressing the ectopic branch 458 formation in an incorrect direction. However, considering that main dendritic frameworks still 459 form a planar pattern in the βIII spectrin deficient PCs (Fig. 5B), βIII spectrin may function as a 460 gatekeeper to maintain the perpendicular and planar dendrites but not as the main determinant 461 regulating the directional dendrite extension. 462 The perpendicular interactions may serve as a permissive mechanism for the planar 463 arborization of PC dendrites in parasagittal planes. However, other mechanisms should also be 464 involved in the spatial organization of PC dendrites, as perpendicular contacts would not assure  The pAAV-CAG-GFP (or mCherry)-hH1 vector, including the human H1 promoter, was used to 494 express shRNA to knockdown target gene expression as previously described (Fukumitsu et al.,495 2015). The targeting sequences were designed by using the web-based software siDirect (Naito 496 et al., 2009): control shRNA (5'-GCATCTCCATTAGCGAACATT-3'), βIII-spectrin shRNA (5'-497 GTCAATGTGCACAACTTTACC-3'), αII spectrin shRNA (5'-498 GTAAAGACCTCACTAATGTCC-3'). To generate resistant mutants of αII spectrin and βIII-499 spectrin that contained three silent mutations within shRNA target sequences, the cDNA of mouse 500 αII spectrin or βIII-spectrin was cloned from a mouse brain cDNA library and mutagenized by 501 using a PCR-based method. To generate the L253P, E532-M544 del and L629-R634 502 delinsW βIII spectrin mutants, PCR-based mutagenesis was performed by using the 503 resistant mutant of βIII-spectrin as a template. αII spectrin tagged with HA at the N-504 terminus and βIII-spectrin wild-type and mutant sequences tagged with myc at the N-505 terminus were cloned into the pCAGGS vector. To generate the EB3-EGFP construct, the 506 coding sequence of EB3 was amplified from a mouse brain cDNA library and inserted 507 into the pAAV-CAG-EGFP plasmid. For the CRISPR/Cas9-based knockout of βIII-spectrin, 508 the guide RNA sequence was selected by using the web-based software CRISPRdirect (Naito et  The primary culture of cerebellar neurons was performed as previously described (Fujishima et  To separate large cell fraction (containing PCs, interneurons and glia) and small cell 548 fraction (containing GCs), dissociate cells from P0 cerebellar tissue were purified with a step 549 gradient of Percoll (Hatten, 1985). Isolated GCs from P0 mice were plated on the aligned fiber in 550 a density of 10 x 10 5 , 7 x 10 5, and 5 x 10 5 cells/cm 2 . Then, a large cell fraction including PCs was 551 plated on the culture with a density of 10 x 10 4 , 7 x 10 4, and 5 x 10 4 cells/cm 2 , respectively. 552 For the coculture of cerebellar microexplant and PCs, a microexplant culture from 553 cerebellar tissues was prepared as described previously (Nakatsuji and Nagata, 1989). Briefly, the 554 external granular layer of the cerebellar cortex from P2 mice was dissected into 300-500 μm 555 pieces and plated on the poly-d-lysin and laminin-coated coverslips. One day after plating, the 556 isolated large cell fraction including PCs from P0 mice was added to the culture.

Quantification of dendrite morphology 582
For the analysis of dendritic flatness, captured confocal z-stacked images were binarized and 583 skeletonized in ImageJ ("Skeletonize (2D/3D)" plugin). To identify the plane with the closest fit 584 to the given dendritic arbor, a principal component analysis was performed in MATLAB software 585 (Fig. S2). To analyze the distance of the dendritic branches from the fitted plane, 3000 points (for 586 Fig. 1A, B) or 100 points (for Fig. 1D, 3E, 8E) in the skeletonized dendritic images from each 587 cell were randomly selected, and the distance between each point and the fitted plane was 588

calculated. 589
For the morphometric analysis of PC dendrites on aligned fibers, Z-projected images 590 were binarized and skeletonized in the ImageJ plugin or MATLAB software. To analyze the 591 branch angle, dendritic branches were divided into 3.5 μm segments, and the angle between each 592 segment and fiber was quantified (Fig. S3). 593

STED imaging 595
We used a Leica TCS SP8 STED with an oil immersion 100x objective lens with NA 1.4 (HC-596 PL-APO 100x/1.4 OIL, Leica) to analyze the subcellular localization of βIII spectrin. The protein 597 was labeled with anti-βIII spectrin (Santa Cruz, SC-28273) and a secondary antibody conjugated 598 with Alexa555 (Thermo Fisher). The fluorophore was excited with a white laser tuned to 555 nm 599 and depleted with a 660 nm STED laser. A time gate window of 0.35-3.85 ns was used to 600 maximize the STED resolution.