Post-stroke dendritic arbor regrowth – a cortical repair process requiring the actin nucleator Cobl

Ischemic stroke is a major cause of death and long-term disability. We demonstrate that middle cerebral artery occlusion in mice leads to a strong decline in dendritic arborization of penumbral neurons. These defects were subsequently repaired by an ipsilateral recovery process requiring the actin nucleator Cobl. Ischemic stroke and excitotoxicity, caused by calpain-mediated proteolysis, significantly reduced Cobl levels. In an apparently unique manner among excitotoxicity-affected proteins, this Cobl decline was rapidly restored by increased mRNA expression and Cobl then played a pivotal role in post-stroke dendritic arbor repair in peri-infarct areas. In Cobl KO mice, the dendritic repair window determined to span day 2-4 post-stroke in WT strikingly passed without any dendritic regrowth. Instead, Cobl KO penumbral neurons of the primary motor cortex continued to show the dendritic impairments caused by stroke. Our results thereby highlight a powerful post-stroke recovery process and identified causal molecular mechanisms critical during post-stroke repair.


35
Five million people remain permanently disabled after stroke each year. In the infarct area, stroke leads 36 to a loss of neurons and neuronal network connections due to lack of energy, excitotoxicity, oxidative 37 stress, inflammation and apoptosis as pathophysiological events (Hossmann, 2006). Ischemic stroke 38 caused by middle cerebral artery occlusion (MCAO) accounts for approximately 70% of all infarcts 39 (Bogousslavsky et al., 1988;Musuka et al., 2015) and can also be achieved experimentally in rodents 40 (Fluri et al., 2015). The relative lesion size of survivable human stroke is usually limited to a few percent 41 of the brain (Cramer et al., 2006). In mice, such damages are very well resembled by 30 min induced independent primer pairs ( Fig. 3E; Fig. S2E). Cobl mRNA showed clear upregulations in both 6 h and 207 12 h post-MCAO tissue samples of M1 (Fig. 3E, Fig. S2E). 208 Thus, both the Ca 2+ -, calpain-and NMDA receptor-mediated decline of Cobl as well as its fast recovery 209 to normal levels driven by a transient increase in Cobl mRNA were phenomena observable in neurons of 210 a penumbral cortex area, the M1. Besides axons with their presynapses and dendritic spines harboring postsynapses, also the dendritic 215 arbor itself is a major structural element in neuronal wiring in the brain. As the formation of new 216 synaptic contacts subsequent to stroke-dependent loss (Brown et al., 2008;Wu et al., 2014) may only be 217 one aspect in the penumbra that compensates for the functions of entire brain regions lost upon ischemic 218 stroke and recently also acute penumbral dynamics of the dendritic arbor were reported after ischemic 219 stroke (Hu et al., 2019;Mauceri et al., 2020), we analyzed the dendritic arbor of the ipsilateral M1. 220 In order to obtain reliable data addressing infarct-related dendritic changes, we established a procedure 221 that enabled us to evaluate the core infarct area caused by 30 min MCAO in the striatum of each 222 individual mouse by anti-MAP2 immunostaining and to in parallel analyze morphologies of individual 223 neurons in a defined area adjacent to the damage zone, the penumbra (represented by the primary motor 224 cortex (M1)) using neighbored coronal brain sections (Fig. S3A,B). 225 Detailed morphometric analyses of both layer II/III ( Fig. 4A-C) and layer V neurons of the ipsilesional 226 M1 (Fig. 4D-F) unveiled that MCAO leads to dramatic dendritic arborization defects when compared to 227 sham-treated animals, which also underwent anesthesia and the surgical procedures but without MCAO 228 ( Fig. 4A-N). Both the number of dendritic branching points and the number of terminal points per 229 neuron declined subsequent to MCAO at specifically the ipsilateral side when brains were analyzed after 230 24 h reperfusion (Fig. 4G,H,K,L). 231 In cortex layer II/III, neurons even showed these dramatic impairments already after 6 h (Fig. 4G,H). 232 The MCAO-mediated defects in layer V neurons developed slower and were observable at 24 h ( Fig.   233 4K,L). Similar defects and onsets were observed when the entire length of the dendritic arbor was 234 examined in layer II/III and layer V of the cortex (Fig. 4I,M). Also Sholl analyses of the dendritic 235 complexity showed corresponding reductions of the dendritic arbor (Fig. 4J,N). 236 These ischemic stroke-induced defects in dendritic organization of neurons in both analyzed areas of the 237 motor cortex were restricted to the ipsilesional side. For all four parameters determined the data obtained 238 from the contralateral side of the same MCAO animals were indistinguishable from those of sham-239 treated mice (Fig. S3C-M).

241
As early as 4 days after MCAO, the discovered dendritic arborization defects caused by MCAO 242 were mostly compensated 243 We next evaluated whether the massive MCAO-induced impairment in dendritic arborization we 244 observed in the M1 (Fig. 4) was permanent and part of the lesion-based disabilities caused by stroke or 245 whether it would at some point become repaired by some thus far unidentified form of dendritic 246 dynamics, which may be inducible by ischemic stroke in mature neurons. We therefore analyzed the 247 morphologies of pyramidal neurons in the M1 of WT mice at 4 days and 7 days after MCAO in direct 248 comparison to the defects observed at 24 h and to sham controls in a blinded manner (Fig. 5A-D). 249 Strikingly, in neurons of the ipsilateral layer II/III, all defects observed at 24 h after MCAO were fully 250 compensated for at day 4 and 7 ( Fig. 5E-J). The dendritic branching points at day 4 and 7 after MCAO 251 exactly were at the levels of sham animals (Fig. 5E). Likewise, dendritic terminal points were back at 252 control levels (Fig. 5F). Total dendritic tree length and Sholl analyses also showed a full restoration of 253 normal dendritic arborization at 4 days and 7 days after MCAO ( Fig. 5G-J). 254 Both apical and basal parts of the dendritic arbor of layer II/III neurons showed about equally strong 255 defects ( Fig. S4A-K). Dendritic branch points and total dendritic length were reduced by about 30% 256 (Fig. S4D,F,I,K). In both apical and basal dendrites, the terminal point numbers declined by 25-30% but 257 failed to reach statisitcal significance for the apical dendrites (Fig. S4E,J). Also the restoration of 258 dendritic arbor complexity was as effective in apical dendrites as it was in basal dendrites ( Fig. S4D-K).

260
Layer V neurons, which showed a slower onset of dendritic defects (Fig. 4), showed a similarly clear 261 repair of MCAO-induced impairments ( Fig. 5K-P). Only the number of dendritic branching points still 262 showed some reduction at day 4 ( Fig. 5K). At day 7 after MCAO, all dendritic morphology parameters 263 quantitatively analyzed in MCAO-treated animals were no longer significantly different from those 264 obtained from sham animals ( Fig. 5K-M,P). 265 Together, our evaluations at the different time points unveiled that the dendritic arborization defects, 266 which we detected upon MCAO in both layer II/III and layer V of the M1, were fully compensated for 267 by a dendritic regrowth process, which gave rise to apparently normally branched and sized dendritic 268 trees and which occured in both layer II/III and layer V.

269
Examinations at the contralateral side showed that the ischemic stroke-triggered repair process in M1 is 270 restricted to the defective ipsilateral side and is not a brain-wide phenomenon of dendritic growth 271 induction. At the contralateral side, all parameters remained at sham levels during all time points 272 analyzed (Fig. S5). 273 Severe ischemic stroke models, such as 60 min MCAO or even a permanent blockage of blood flow in 274 mice, being rather unrelated to survivable human strokes, would not be suitable to study repair processes 275 in the penumbra, as surving animals usually lose large parts of the entire hemisphere affected and show 276 significant structural changes at distant sites or even at the contralateral side of the brain (Cramer et al., 277 2006;Winship and Murphy, 2008) and the penumbra is small or not existing (Popp et al., 2009). We did 278 not observe any structural alterations at the contralateral side ( Fig. S5) but specifically found cellular 279 alterations in the penumbra at the ipsilateral side (Figs 4 and 5). This demonstrated that our animal 280 stroke model (30 min MCAO) reliably mirrored the range of survivable human stroke and thus was 281 suitable to unveil repair processes in peri-infarct areas.

283
The actin nucleator Cobl promotes and is critical for dendritic arborization in cortical neurons 284 The defects caused by MCAO in pyramidal neurons in cortical layers II/III and V of the murine primary 285 motor cortex seemed somewhat related to those observed for Cobl loss-of-function in immature rat 286 hippocampal neurons in culture (Ahuja et al., 2007;Izadi et al., 2021). We therefore addressed whether 287 Cobl may also play some important role in shaping cortical neurons and whether furthermore Cobl may 288 not do so not only in developing neurons in culture but also in adult neurons in the cortex of mice with a 289 demand for remapping neuronal circuits subsequent to stroke.

290
Adressing the first hypothesis, we overexpressed Cobl in developing rat cortical neurons. GFP-Cobl 291 overexpression from DIV4 to DIV6 clearly resulted in increased dendritic arborization when compared 292 to GFP control. All parameters affected upon MCAO in mice were elevated significantly ( Fig. S6A-F). 293 In line with these results, Cobl loss-of-function experiments in developing primary rat cortical neurons 294 unveiled that Cobl does not only have the ability to modulate the dendritic trees of cortical neurons but, 295 but also is critical for this process during dendritic arbor developement ( Fig. 6A-C). Dendritic branching 296 points, terminal points and the total dendritic length were significantly reduced and also Sholl analyses 297 highlighted a loss of dendritic complexity when Cobl was lacking ( Fig. 6D-G). All of these Cobl loss-of-298 function phenotypes were specific, as all of them could be rescued by reexpression of RNAi-insensitive Taken together, these two different experimental lines clearly proved the first hypothesis and unveiled 301 that the actin nucleator Cobl played an important role in the dendritic arborization of cortical neurons.

303
Cobl KO completely ablates the ischemic stroke-induced dendritic regrowth processes, which allow 304 for restoration of proper dendritic arborization after MCAO 305 The second hypothesis, that, as reflected by the identified modulations of Cobl levels acutely following 306 ischemia and during stroke recovery, the actin nucleator Cobl may be a crucial player in the repair of the branching points, terminal points and tree extension (Fig. 7H,N). In general, all individual defects in dendritic branching points (Fig. 7E,K), nor dendritic terminal points (Fig. 7F,L) nor dendritic tree length 325 (Fig. 7G,M) nor dendritic complexity, as visualized by Sholl analyses (Fig. 7I,O), were restored to 326 proper levels in Cobl KO animals. This phenotype became obvious by comparision to sham-treated 327 animals ( Fig. 7) but also by comparing the absolute numbers of all parameters with those of WT animals 328 (Fig. 7 vs. Fig. 5). 329 At day 4, this complete lack of repair of MCAO-induced defects was observed in both layer II/III 330 neurons (Fig. 7C,E-G,I) and in layer V neurons of Cobl KO mice ( Fig. 7K-M,O). The lack of repair at 331 day 4 also occurred irrespective of the dendritic orientation, as MCAO-induced defects in Cobl KO 332 neurons were observed in both the apical and the basal dendritic arbor (Fig. S8). 333 Even at day 7 after MCAO and again in strong contrast to WT animals, Cobl KO mice still showed 334 massive impairments in dendritic arborization (Fig. 7D,E-G,J,K-M). Only Sholl analyses of layer V 335 neurons failed to visualize the differences in dendritic complexity between sham-and MCAO-treated 336 Cobl KO mice in a manner supported by statistical significances (Fig. 7P). Yet, all other seven 337 quantitative examinations still clearly demonstrated the lack of any dendritic arbor restoration in Cobl 338 KO mice even a full week after MCAO ( Fig. 7E-P). The dendritic branching points, the terminal points 339 and the summarized length of the dendritic arbor remained significantly reduced when compared to 340 sham-treated animals at day 7. The defects at day 7 thereby still remained similar to the defects observed 341 at 24 h and 4 d ( Fig. 7E-P). Taken together, the levels of the actin nucleator Cobl are responsive to 342 MCAO-induced brain damage and Cobl is critically needed for an acute process of dendritic regrowth 343 and branching that is triggered by ischemic stroke conditions and is powerful enough to fully repair the 344 strong defects in dendritic arborization, which we observed as consequences of ischemic stroke in the 345 cortex.

Discussion
Regaining of neurophysiological functions in peri-infarct areas (the penumbra) and remapping processes 350 in functionally related cortical tissues are thought to represent important aspects underlying recovery 351 from stroke in human patients. However, such processes are far from being understood at the cellular and 352 molecular level. 353 We demonstrated that ischemic stroke in mice leads to a strong decline in the entire dendritic 354 arborization of neurons in the penumbra and by carefully comparing different reperfusion times with 355 each other and sets of corresponding controls demonstrate a subsequent process of dendritic arbor repair.

356
In the cortex of mice subjected to 30 min MCAO-induced ischemic stroke in the striatum, this repair 357 process showed a net growth that was able to fully compensate for the massive loss of dendritic 358 extension and complexity observed at the first day after ischemic stroke. Importantly, with the actin 359 nucleator Cobl, we identified a cytoskeletal driving force for this recovery. Dendritic arbor repair was  (Hu et al., 2020). Also three repetitive VEGFD applications over 48 h leading to a net dendritic growth of more than a third within 5 days post In this respect, it is also very interesting that post photothromic ischemia in vivo imaging showed a 374 massive remodeling of dendrites in the somatosensory cortex at 2 weeks after stroke but no net growth 375 (Brown et al. 2020). This may suggest that the massive induction of net dendritic growth and the branch 376 induction we observed when studying much earlier time points (day 2-4) actually still continue after 377 reaching net recovery at day 4 but that at later stages of cortical remapping are marked by counteraction 378 by dendritic pruning processes operating in parallel.

379
Such a biphasic repair process would also explain apparently different findings concerning the effects 380 within the dendritic arbor. During the day 2-4 time window of Cobl-dependent acute ischemic stroke-381 induced repair, all aspects of dendritic arborization were promoted with equal efficiency. The induced 382 repair also did not discriminate between apical and basal dendrites or between the proximal and distal 383 arbor but promoted neuronal morphology in all dendritic areas equally well (this study). In contrast, later 384 dendritic dynamics did not result in any net growth anymore, as dendritic pruning processes operating in 385 parallel lead to higher net growth away from the infarct area and to a net loss of dendrites oriented 386 towards the infarct area (Brown et al., 2020). Together, such a biphasic response would very effectively 387 remap neuronal circuits in the neighborhood of infarct areas.

388
The observed readdition of on average 5-6 dendritic branching points and of about 400 µm of dendritic 389 arbor per neuron in peri-infarct areas in a time window of repair, which opens at or after day 2 after 390 ischemic stroke, obviously provides a powerful mechanism for not only reconnecting cells that anyway 391 are close enough for synapse formation but also allows for making connections over larger ranges.  What is so special about the actin nucleator Cobl that it is employed in repair processes aiming at 401 regaining brain functions after ischemic stroke in the cortex? Cobl is a powerful actin nucleator that can  In line with an acute demand of Cobl for dendritic arbor repair after ischemic stroke, Cobl protein levels 438 lost upon the Ca 2+ /calpain-mediated proteolysis were restored to normal levels already 24 h after MCAO.

439
The discovery of increasing Cobl mRNA levels during the hours prior to this restoration of Cobl protein 440 levels strongly suggests that this replenishment of Cobl levels is achieved by the observed rise in mRNA 441 level. In line with a decline of Cobl protein levels in M1, also the transient elevation of Cobl mRNA 442 levels could be detected in the M1, i.e. in the prenumbra. While nothing is known yet about how the 443 expression of Cobl is controlled, it seems likely that the mRNA increase will be linked to signaling 444 cascades triggered by high glutamate, such as massive increases in cytosolic and thereby also nuclear Taken together, our study unveiled that ischemic stroke causes damages in dendritic arborization in peri-457 infarct areas, which then are repaired by processes of dendritic regrowth relying on the actin nucleator 458 Cobl, whose levels upon ischemic stroke first are negatively affected but then are rapidly restored. The 459 excitotoxicity-induced degradation of Cobl was caused by glutamate and NMDA receptor-mediated Ca 2+ 460 influx and calpain-mediated proteolysis. Subsequent to a rapid restoration of normal Cobl levels, the 461 actin nucleator then powered dendritic repair during a time window opening between day 2 to 4 after 462 ischemic stroke. We show that Cobl-dependent post-stroke dendritic arbor regrowth is a powerful cell 463 biological process that adds to the well-known structural plasticity of post-synapses/dendritic spines. Importantly, Cobl-dependent post-stroke dendritic arbor regrowth represents a much more long-range 465 mechanism of post-stroke repair inside of peri-infarct areas.

466
The high conservation of Cobl between mice and man suggests that related Cobl-dependent processes of 467 repair after ischemic stroke may also exist in human patients and would be worthwile to exploit in the 468 identified post-stroke time window.

533
During the MCA occlusion body temperature was maintained at physiological level using a heating pad.

534
After 30 min of occlusion the suture was withdrawn to restore the blood flow and allow for reperfusion.

535
Sham animals underwent anesthesia and the same surgical procedures except the occlusion of MCA. were quickly removed and separated into two parts using a Precision Brain Slicer (Braintree Scientific infarct validation by anti-MAP2 staining (see below), and the posterior part was used for Golgi-Cox 544 staining using FD Rapid GolgiStain kits (FD NeuroTechnologies, Inc), essentially following a protocol 545 described previously (Schneider et al., 2014;Koch et al., 2020). In brief, the posterior brain part was 546 immersed in solution A+B of the FD Rapid GolgiStain kit and kept in the dark at RT for 21 days. Brains 547 were then incubated in solution C of the kit for another 5 days (RT; dark). The brains were then dipped 548 very slowly into dry ice-precooled isopentane and stored at -80°C.

549
Cryosectioning was performed at -28°C using a Leica CM 3050 S (Leica Biosystems Nussloch GmbH).   The basal dendrites and the arbor originating from the apical dendrite were analyzed accordingly by 590 dissecting the Imaris tracings of evaluated layer II/III WT and Cobl KO neurons.
The analyses were conducted in a blinded manner. Quantitative parameters were analyzed for data 592 distribution and statistical significances with Prism 6 software (SCR_002798; GraphPad).  were subjected to anti-MAP2 immunostainings as described above.

622
The anti-MAP2 stained brain sections were imaged on a light table using a digital CCD camera 623 (Hamamatsu Photonics) and the sizes of the infarct area as well as of each hemisphere and of the total 624 brain section were measured by Scion Image software (Scion Corporation). The area of the infarct was 625 traced and quantified on every twelfth section. Infarct volumes were calculated as percentage of the total 626 brain. were prepared as described previously (Sieber et al., 2010). In brief, brains were cut into three coronal 632 segments using a Precision Brain Slicer (Braintree Scientific Inc.): from +2.8 to +0.8 mm to bregma 633 (rostral segment), from +0.8 to -1.2 mm to bregma (middle segment), and from -1.2 to -3.2 mm to 634 bregma (caudal segment). The ipsilateral part (ischemic hemisphere) and the contralateral part (non-635 ischemic hemisphere) of the middle brain segment were separated and quick frozen in liquid nitrogen 636 and stored at -80°C for later processing.
In further animal experiments (after 6 h, 12 h and 24 h reperfusion time), the middle brain segment was 638 dissected further and the primary motor cortex M1 was isolated and snap-frozen for subsequent analyses 639 of exclusively penumbral ipsilateral and contralateral brain areas.  Table 1. 664 Amplification was performed using a Rotor-Gene 6000 (Qiagen GmbH) (cycle conditions: 3 min 665 polymerase activation, 40 amplification cycles of 95°C for 10 s and 60°C for 15 s each).

674
Protein isolation from mouse brain tissue for quantitative immunoblotting analyses 676 The ipsilateral part and the contralateral part, respectively of the middle brain segment (+0.8 and -1.2 677 mm to bregma) as well as ipsilateral and contralateral M1 tissue samples were prepared as described 678 above, homogenized and immunoblotted.

679
In order to improve comparability, samples of the same condition were analyzed on the same blotting

716
The immunosignals were analyzed quantitatively with a LI-COR Odyssey System. This study includes no data deposited in external repositories.

1136
Accession codes and unique identifiers are given wherever useful.

1138
The following figures have associated raw data: Fig. 1-6 and Fig. S1-5 have associated numerical data;