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Poststroke dendritic arbor regrowth requires the actin nucleator Cobl

['Yuanyuan Ji', 'Institute Of Biochemistry I', 'Jena University Hospital Friedrich Schiller University Jena', 'Jena', 'Dennis Koch', 'Jule González Delgado', 'Madlen Günther', 'Hans Berger Department Of Neurology', 'Jena University Hospital', 'Otto W. Witte']

Date: 2022-01 

Ischemic stroke is a major cause of death and long-term disability. We demonstrate that middle cerebral artery occlusion (MCAO) 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 poststroke dendritic arbor repair in peri-infarct areas. In Cobl knockout (KO) mice, the dendritic repair window determined to span day 2 to 4 poststroke in wild-type (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 poststroke recovery process and identified causal molecular mechanisms critical during poststroke repair.

Funding: This work was supported by grants from the DFG (Deutsche Forschungsgemeinschaft) to M.M.K. (KE685/3-2) and to B.Q. (RTG1715) as well as by the IZKF (Interdisziplinäres Zentrum für klinische Forschung des Universitätsklinikums Jena) to B.Q. and C.F. (RTG1715 SP18). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2021 Ji et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Here, we show that induction of ischemic stroke in mice leads to a rapid degradation and a subsequent reexpression of the actin nucleator Cobl. Cobl degradation is excitotoxicity-mediated. By analyzing Cobl knockout (KO) mice [ 37 ], we furthermore demonstrate that Cobl is crucially involved in a process of regrowth of the dendritic arbor, which we unveil to occur in the penumbra in a narrow time window from day 2 to day 4 after ischemic stroke. With Cobl-dependent poststroke dendritic arbor regrowth, our work adds a powerful cell biological process inside of peri-infarct areas that represents an acute and long-range mechanism of poststroke repair.

The morphology of the complex dendritic arbor of neuronal cells is stabilized by microtubules; yet, it is the de novo generation of actin filaments that powers the formation of initial protrusions from dendrites that establishes the dendritic arbor [ 29 ]. The Wiskott–Aldrich domain 2-based actin nucleator Cobl [ 30 , 31 ] (gi:32251014) is widely expressed in the brain [ 32 ] and was demonstrated to be critical for dendritic branching of developing hippocampal neurons [ 30 ] and of Purkinje cells [ 32 ] together with accessory machinery [ 32 – 34 ]. Interestingly, the functions of the actin nucleator Cobl hereby show regulations by arginine methylation by PRMT2 [ 35 ] and by multiple Ca 2+ /calmodulin (CaM)-mediated mechanisms directly converging onto Cobl [ 36 ].

Most dendrites and dendritic branches become stabilized at an age of about 20 days in mice [ 14 ], and much less is known about the fate of the dendritic arbor in the penumbra during the acute phase of cerebral ischemia in mice when compared to dendritic spine dynamics. The reason is that the majority of published studies focused on putative long-term effects inside and outside of ischemic areas several weeks or even several months after (often massive) brain damage caused by cerebral ischemia induced by different means in rodents [ 6 , 15 , 19 – 23 ]. Other studies tried to identify long-term effects at the contralateral side that may represent compensational processes [ 24 – 26 ] rather than focusing on the ipsilateral penumbra neighboring the infarct area. Recent reports suggested that inside of the penumbra, acute changes in the dendritic arbor occur or can be pharmacologically induced [ 27 , 28 ], respectively.

Ischemic stroke does not only lead to neuronal death and loss of connectivity inside of infarct areas; it also causes a loss of synapses (dendritic spines) adjacent to the infarct area (penumbra) [ 8 – 10 ]. The spine loss is associated with excessive Ca 2+ influx activating numerous downstream effects. Prominent among those is the Ca 2+ -activated calpain-mediated breakdown of synaptic scaffold and receptor proteins, such as spectrin/fodrin, PSD95, and NR2B [ 11 – 13 ]. Importantly, in the penumbra, these synaptic defects are transient and are subsequently repaired, as spines remain somewhat plastic even during adulthood [ 14 ]. Dynamics of dendritic spines (also referred to as dendritic remodeling, dendritic plasticity, or synaptic plasticity) is considered as important cellular mechanism for learning but also for compensatory synaptic repair subsequent to stroke [ 9 , 10 , 15 – 18 ].

Five million people remain permanently disabled after stroke each year. In the infarct area, stroke leads to a loss of neurons and neuronal network connections due to lack of energy, excitotoxicity, oxidative stress, inflammation, and apoptosis as pathophysiological events [ 1 ]. Ischemic stroke caused by middle cerebral artery occlusion (MCAO) accounts for approximately 70% of all infarcts [ 2 , 3 ] and can also be achieved experimentally in rodents [ 4 ]. The relative lesion size of survivable human stroke is usually limited to a few percent of the brain [ 5 ]. In mice, such damages are very well resembled by 30-minute induced MCAO. By contrast, prolonged paradigms do not resemble survivable human strokes, as they lead to a loss of large parts of the entire hemisphere affected and to significant structural changes at distant or even contralateral sites [ 5 – 7 ].

Results

MCAO leads to Cobl degradation followed by an efficient Cobl level recovery in the motor cortex M1 In order to directly address whether Cobl levels indeed responded to ischemic stroke conditions in wider areas of the brain, i.e., beyond the infarct area itself, and may therefore be of thus far unrecognized importance for the pathophysiology of stroke and/or subsequent repair, we conducted further MCAO experiments and isolated specifically the ipsilateral primary motor cortex (M1)—a physiologically important part of the cortex that is adjacent to but not inside the infarct area and thereby belongs to the penumbra (Fig 1Q). Quantitative immunoblotting analyses of dissected M1 tissues clearly revealed the decline of Cobl levels at the ipsilateral side to about 75% of the corresponding contralateral control values of the respective animals 6 hours after MCAO (Fig 1R and 1T). Thus, the decline of Cobl in the whole brain hemisphere (Fig 1A–1H) can also clearly be observed in the penumbral M1 (Fig 1Q–1U). Also, the recovery of Cobl expression levels at 24 hours after MCAO was observed in quantitative immunoblotting analyses of M1. Twenty-four hours after MCAO, Cobl levels were restored to about 100% of the control value (Fig 1S and 1U). Immunofluorescence analyses of brain sections also demonstrated a clear MCAO-induced ipsilateral decline of Cobl levels in the M1 6 hours post-MCAO (Fig 1V). Even without background subtractions applied, Cobl level declined in both layer II/III and in layer V in almost the same order of magnitude as determined by biochemical analyses in M1 and in the central hemisphere segment, respectively. The decline of Cobl was highly statistically significant and about equally strong in layer II/III and in layer V (Fig 1W and 1X). Thus, Cobl protein levels in the cortex show surprisingly high short-term dynamics; these dynamics are related to stroke and can explicitly also be detected in the pyramidal cell layers II/III and V of M1.

[END]

[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001399

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