The Extracellular Matrix Proteins Tenascin-C and Tenascin-R Retard Oligodendrocyte Precursor Maturation and Myelin Regeneration in a Cuprizone-Induced Long-Term Demyelination Animal Model

Oligodendrocytes are the myelinating cells of the central nervous system. The physiological importance of oligodendrocytes is highlighted by diseases such as multiple sclerosis, in which the myelin sheaths are degraded and the axonal signal transmission is compromised. In a healthy brain, spontaneous remyelination is rare, and newly formed myelin sheaths are thinner and shorter than the former ones. The myelination process requires the migration, proliferation, and differentiation of oligodendrocyte precursor cells (OPCs) and is influenced by proteins of the extracellular matrix (ECM), which consists of a network of glycoproteins and proteoglycans. In particular, the glycoprotein tenascin-C (Tnc) has an inhibitory effect on the differentiation of OPCs and the remyelination efficiency of oligodendrocytes. The structurally similar tenascin-R (Tnr) exerts an inhibitory influence on the formation of myelin membranes in vitro. When Tnc knockout oligodendrocytes were applied to an in vitro myelination assay using artificial fibers, a higher number of sheaths per single cell were obtained compared to the wild-type control. This effect was enhanced by adding brain-derived neurotrophic factor (BDNF) to the culture system. Tnr−/− oligodendrocytes behaved differently in that the number of formed sheaths per single cell was decreased, indicating that Tnr supports the differentiation of OPCs. In order to study the functions of tenascin proteins in vivo Tnc−/− and Tnr−/− mice were exposed to Cuprizone-induced demyelination for a period of 10 weeks. Both Tnc−/− and Tnr−/− mouse knockout lines displayed a significant increase in the regenerating myelin sheath thickness after Cuprizone treatment. Furthermore, in the absence of either tenascin, the number of OPCs was increased. These results suggest that the fine-tuning of myelin regeneration is regulated by the major tenascin proteins of the CNS.


Introduction
Oligodendrocytes are the myelinating cells in the central nervous system (CNS). It is known that the production of myelin is essential for the normal functioning of the vertebrate CNS [1]. The oligodendrocytes derive from oligodendrocyte precursor cells (OPCs). These have a bipolar morphology at the beginning of their development and become progressively more complex with ongoing differentiation [2,3]. Finally, one single mature oligodendrocyte forms myelin membranes which can wrap around up to 40 different axons [4]. Oligodendrocytes fulfill many tasks, including the isolation of axons for better signal transmission with a top speed of 200 m/s [5], or the energy supplement of axons and the storage of glycogen [6]. The importance of oligodendrocytes is highlighted by demyelinating diseases such as multiple sclerosis, polyneuropathy, or neuromyelitis optica, for example [7]. Multiple sclerosis (MS) is a chronic inflammatory and demyelinating disease that affects the CNS by demyelinating the axons [8,9]. MS belongs to the

Isolation and Cultivation of Primary Murine OPCs
As described in previous studies with minor modifications murine OPCs were isolated by immunopanning with a CD140a antibody [32,33]. First, the postnatal P6-P9 brains from the different genotypes Tnc −/− , Tnc +/+ , Tnr −/− and Tnr +/+ obtained by breeding of heterozygote mice were dissected. Then, the meninges were removed and enzymatically dissected in MEM containing 30  of cultivation, a sufficient number of OPCs proliferated and were ready for further experiments. During this period media were changed every 2-3 days and PDGF-AA was added daily. To detach the OPCs the dishes were washed once with PBS, then 5 mL of 0.25% Trypsin/EDTA (Thermo Fisher Scientific Inc., Cat. No.: 25300054) was added to OPCs for trypsinization. This process was stopped by the addition of an equal amount of ovomucoid (Leibovitz's L15 medium (Cat. No.: L5520, Sigma-Aldrich), 1 mg/mL soybean trypsin inhibitor (Cat. No.: T6522, Sigma-Aldrich), 50 µg/mL BSA fraction V, 40 µg/mL DNAse I (Cat. No.: LS0020007, Worthington Biochem. Corp., Lakewood, NJ, USA)). After centrifugation for 5 min at 1000 rpm, the cell pellet was resuspended in OPC SATO medium and plated at a density of 20,000 cells per 12 mm coverslip. These were coated with 10 µg/mL (w/v) PDL beforehand. In the following cells were cultivated in the presence of 10 ng/mL PDGF-AA (Peprotech GmbH, Cat. No.: 100-13A) and 5 ng/mL NT3 (Cat. No.: 450-03, Peprotech) to maintain proliferation conditions. Alternatively, 400 ng/mL T3 (Cat. No.: T6397, Sigma-Aldrich) and 0.5% (v/v) horse serum (HS, Cat. No.: S9135, Peprotech) were added to start differentiation. The cells could be used for cultivation jointly with artificial fibers for 14 div and finally characterized by immunocytochemistry.

Immunocytochemistry
Cells were stained immunocytochemically as previously described [34,35]. Medium from primary oligodendrocytes was removed and then the cells were washed twice with PBS and subsequently fixed with 4% (w/v) paraformaldehyde (PFA) (Cat. No.:4235.1, Carl Roth) in PBS for 10 min at RT. Thereafter, fixed oligodendrocytes were washed three times with PBT1 (PBS, 1% (w/v) BSA, 0.1% (v/v) Triton-X 100 (Cat. No.: A4975, AppliChem GmbH, Darmstadt, Germany)) and primary antibodies (anti-MBP, anti-GFAP) were diluted in PBT1 and incubated with the cells for 1 h at RT. Ensuing, oligodendrocytes were washed thrice with PBS/A before species-specific antibodies coupled to AF488, Cy2, or Cy3, and the nuclear marker Hoechst, which were diluted in PBS/A, were added to the oligodendrocytes for at least 1 h at RT. In the end, three washing steps in PBS were followed before the cells were mounted with Fluoromount-G (Cat. No.: 0100-01, Southern Biotech, Birmingham, AL, USA) and subjected to microscopy.

Myelination of Electrospun Fibers
For the analysis of the impact of the EMC molecules Tnc and Tnr on myelination in vitro, Tnc +/+ , Tnc −/− , Tnr +/+ , Tnr −/− and wildtype OPCs were plated onto parallelly aligned, 2 µm thick electrospun fibers. Those fibers are composed of poly-L-lactic acid (The Electrospinning Company, Didcot, UK) and presented in 12 well dishes [36]. First, the fibers were soaked in 70% (v/v) ethanol, followed by a coating with 10 µg/mL PDL for 1 h at 37 • C. After coating, the fibers were washed three times with H 2 O to remove the excess PDL. Then, OPCs were plated onto the fibers in a density of 45,000 cells per well in myelination medium (DMEM (Cat. No.: 41966029, Thermo Fisher Scientific Inc.): Neurobasal (Cat. No.: 21103-049, Sigma-Aldrich) (1:1), Pen/Strep, B27 (Cat. No.: 17504044, Thermo Fisher Scientific), ITS supplement (Cat. No.: I3146, Sigma-Aldrich), 5 µg/mL Nacetyl-cysteine (Cat. No.: A8199, Sigma-Aldrich), 10 ng/mL D-biotin (Cat. No.: B4639, Sigma-Aldrich), and modified SATO (100 µg/mL BSA fraction V, 60 ng/mL progesterone, 16 µg/mL putrescine, 400 ng/mL tri-iodothyronine [T3]; 400 ng/mL L-thyroxine [T4]) and cultivated at 37 • C and 7.5% CO 2 for 14 days. Medium was changed three times per week. For immunocytochemical analysis, fibers were washed once in PBS and then fixed in 4% (w/v) PFA (Cat. No.:4235.1, Carl Roth) in PBS for 15 min at RT, followed by three washes in PBS. Additionally, the cells were permeabilized with 0.1% (v/v) Triton-X 100 in PBS for 15 min at RT. The primary antibodies were diluted in PBS and incubated at 4 • C overnight. On the next day, the primary antibody was removed, and fibers were washed three times with PBS. Next, the secondary antibodies were also diluted in PBS and added to the cells for 1 h at RT. After incubation, three washes with PBS followed. Last, fibers were mounted between glass slides and coverslips with Fluoromount-G (Cat. No.: 0100-01, Southern Biotech). Finally, the confocal images were obtained on a Zeiss LSM 510 Meta with an X40 oil objective. As many as necessary Z-steps of 0.37 µm were taken to image complete myelin sheaths. Analysis of the sheath lengths was determined with ImageJ by measuring the length of continuous membranes wrapping in a tube-like structure around the fibers. The number of sheaths formed by a single OL was also determined.

Cuprizone Model Induced Demyelination
In contrast to other models, the cuprizone model is a suitable model to study demyelination as well as remyelination [37]. For the analysis of demyelination and remyelination studies, 8-week-old male 129/SV, Tnc −/− and Tnr −/− mice were fed with 0.2% (w/w) cuprizone (Cat. No.: 370-81-0, Sigma-Aldrich) mixed with powdered chow for 10 weeks to induce demyelination [38], followed by a diet without cuprizone [33] for 2, 4, and 6 weeks to allow for a short-and long-term remyelination. Typically, mice are treated for 10 until 12 weeks to induce demyelination in a chronic way [39][40][41]. However, it could also be shown that after a 12-week cuprizone treatment remyelination is very sparse, resulting in a model of chronic demyelination [42] and remyelination that in some cases is insufficient, or even fails [43][44][45]. To ensure successful remyelination, we therefore decided to treat the mice for 10 weeks with cuprizone to mimic a more chronic course of demyelination. All in all, 12 mice per genotype were used for each condition, so that in total 180 mice were included in this study. However, not all individual animals were analyzed. During demyelination control mice received a normal diet of powdered chow (Suppl. Figure S2), once a week cuprizone treated mice also received a normal diet to minimize the severity of intoxication. The weight of all animals was recorded three times a week. Here, one can see that the Tnc −/− mice have the least weight loss, yet also lose a lot of weight as demyelination increases. At the end of the experiment mice of each group were perfused intracardially with 20 mL PBS under deep anesthesia (800 µL 0.9% (w/v) NaCl (Cat. No.: 7647-14-5, Thermo Fisher Scientific), 50 µL Xylazin (10 mg/mL weight) (Cat. No.: 1205, CP-Pharma, Handelsgesellschaft GmbH, Burgdorf, Germany) and 150 µL Ketamin (150 mg/mL weight) (Cat. No.: 1202, CP-Pharma, Handelsgesellschaft GmbH). Brains were removed and cut sagittally in two halves, or the whole brain was used for electron microscopy analysis. For immunohistochemical stainings, one hemisphere was fixed with 4% (w/v) PFA (Cat. No.:4235.1, Carl Roth) in PBS at 4 • C for 48 h before embedding in tissue-freezing medium for cryosectioning. The second hemisphere was frozen in liquid nitrogen for RNA analysis, in particular for RT-PCR.

Histochemistry of the Brain Slices
After the two-day fixation in 4% (w/v) PFA (Cat. No.:4235.1, Carl Roth), the hemispheres were dehydrated in 30% (w/v) sucrose (Cat. No.: 4072-01, Fisher Scientific, Waltham, MA, USA) before embedding in tissue freezing medium (Cat. No.: 14020108926, Leica Biosystems, Nussloch, Germany) on dry ice. Afterwards, cryosections of 14 µm were cut from one hemisphere on a cryostat and stored at −20 • C. Here, the area on sagittal sections was focused on the corpus callosum (CC) above the hippocampus (HC) (according to Bregma: lateral 0.32-0.48 mm), where myelination is highest [46]. In order to provide proof for the efficacy of the induced demyelination with cuprizone, the Luxol-Fast-Blue-Periodic Acid Schiff (LFB-PAS) staining was used. Luxol fast blue marked myelin in blue and Periodic Acid and Schiff reagent was applied to label the axons in red color. In addition, hematoxylin was used to stain the cell nuclei dark blue. For the Schiff reagent staining with LFB-PAS cryosections were dehydrated in an increasing alcohol series starting at 30% (v/v), up to 96% (v/v) and stained in 0.1% (w/v) LFB solution (Cat. No.: 1328-51-4, Thermo Fisher Scientific), in 96% (v/v) ethanol (Cat. No.: 64-17-5, Carl Roth) for 24 h at 60 • C. Afterwards cryosections were briefly rinsed in 96% (v/v) ethanol before they were washed for 30 s in 0.05% (w/v) Lithium-Carbonate (LiCO 3 , Cat. No.: 554-13-2, Sigma-Aldrich). A staining with 1% (w/v) periodic acid for 7 min and afterwards with Schiff's reagent (Cat. No.: 1789, Carl Roth) for 20 min followed. After cryosections had been washed hematoxylin staining was performed for 2 min. Finally, dehydration was carried out in an increasing series of alcohols from 70% (v/v) up to 100% (v/v) and cryosections were immobilized using Euparal mounting medium (Cat. No.: 7356.1, Carl Roth). For each condition and animal at least 2 sections were analyzed and at least 500 cells were examined.
For immunohistochemistry cryosections were first rehydrated in PBS before they were boiled in 0.01M citrate buffer for 70 min by 70 • C. Then the incubation with blocking solution (PBS, 1% (w/v) BSA, 0.1% (v/v) Triton-X 100, 5% (v/v) goat serum (Cat. No.: 005-000-121, Dianova GmbH) for 1 h at RT in a humid chamber followed. Primary antibodies were diluted in a blocking solution with goat serum and incubated at 4 • C overnight. After three consecutive washing steps in PBS, secondary antibodies were diluted in PBS/A (PBS, 0.1% (w/v) BSA) and incubated for 2 h at RT. Finally, stained cryosections were washed three times with PBS and mounted using ImmuMount (Cat. No.: 9990402, Thermo Fisher Scientific). The fluorescence stainings were recorded using the Axio Zoom.V16 (Cat. No.: 495010-0001-000, Carl Zeiss AG, Wetzlar, Germany), with a focus on the area of the corpus callosum. Here, the caudal part as well as the rostral part of the corpus callosum was analyzed in more detail, because these are the most affected areas. The corpus callosum is known to be subject to severe demyelination [15,47]. In order to monitor myelin-specific genes in the corpus callosum corresponding tissue was isolated from a half-frozen state of brain halves of mice exposed to cuprizone. Immediately after perfusion the isolated corpus callosum was stored at −80 • C until RNA was isolated. Total RNA from corpus callosum was obtained using the GeneE-lute™ Mammalian Total RNA MiniPrep Kit (Cat. No.: RTN350, Sigma-Aldrich) according to the manufacturer's instructions. For analysis 0.5 µg RNA was transcribed into cDNA in a volume of 40 µL using the First strand cDNA synthesis Kit (Cat. No.: K1622, Thermo Fisher Scientific). For each condition (10 weeks control, 10 weeks demyelination, 2 weeks remyelination, 4 weeks remyelination, 6 weeks remyelination) cDNA samples from three different animals were prepared. We performed RT-PCR for the analysis of several genes which are relevant for oligodendrocyte development. In all conditions the housekeeping gene β-Actin was used as a control. Moreover, in each condition four animals with the following genes were investigated: platelet-derived growth factor receptor A (PDGFRα), myelin-basic protein (MBP), and ionized calcium-binding adapter molecule 1 (Iba1). Following primers were used: β-Actin forward: 5 -tatgccaacacagtgctgtctgg-3 , β-Actin reverse: 5 -agaagcacttgcggtgcacgatg-3 , PDGFRα forward: 5 -gcaccaagtcaggtcccatt-3 , PDGFRα reverse: 5 -cttcactggtggcatggtca-3 , MBP forward: 5 -tctcagccctgacttgttcc-3 , MBP reverse: 5 -atcaaccatcacctgccttc-3 , Iba1 forward: 5 -ggatttgcagggaggaaaag-3 , Iba1 reverse: 5 -tgggatcatcgaggaattg-3 . All RT-PCR results were analyzed with the rectangle tool ImageJ. In this context, the mean gray value of each sample was measured, the background was subtracted, and the resulting values were set in relation to the actin signal.

Electron Microscopy
Following the animal experimentation license the mice were anesthetized before they were first perfused intracardially with 10 mL PBS, followed by perfusion with 4% (w/v) PFA (Cat. No.: 4235.1, Carl Roth) and 2.5% (v/v) glutaraldehyde (Cat. No.: 111-30-8, Sigma-Aldrich) in 0.1 M phosphate buffer (pH = 7.4). The Brains were removed and kept in fixative for further 4 days. Subsequently, brains were cut in the coronal plane into 1 mm thick slices and the corpora callosa were dissected between Bregma −2.12 and 1.28 [33,48].
For each condition at least 8 sections were collected. Next the dissected corpora callosa were embedded in glycidether 100 (Cat. No.: 90529-77-4, Carl Roth). In the following ultrathin sections of the corpora, callosa were prepared, before they were stained with 2% (w/v) uranyl acetate (Cat. No.: 77870.02, Serva Electrophoresis GmbH, Heidelberg, Germany) for 5 min at RT and lead citrate (1.33 g Pb(NO 3 )2, 1.76 g Na3(C 6 H 5 O 7 ) × 2 H 2 O, ad. 50 mL A. bidest) [49]. Finally, the sections were acquired on an electron microscope Sigma VP500 (Carl Zeiss AG, Wetzlar, Germany). The g-ratio (axon diameter divided by the total axon diameter including the myelin sheath) was determined. At least 200 axons for each condition per individual animal were evaluated. Overall, three animals for each condition (10 weeks control, 10 weeks demyelination, 10 weeks demyelination with 2, 4, or 6 weeks of remyelination) were examined. In each condition, the g-ratios of the knockouts were compared to the wildtypes.

Statistics
In order to examine the effects of the individual tenascins on the respective conditions, we always compared the results of the knockout situations with those of the wild types. Statistical analyses were carried out using the GraphPad Prism 7 software (GraphPad Software, San Diego, CA, USA). All results are provided as Mean ± SEM if not declared otherwise. Furthermore, the type of statistical tests and the number of performed experiments are provided in the figure legends. The significances were determined using ONE-way ANOVA with subsequent Tukey's multiple comparisons test. In pairwise comparisons also the unpaired two-tailed Student's t-test was used. The tests used are indicated in the figure legends. All statistical differences were considered as significantly different when p ≤ 0.05, p-values are referred as * for p ≤ 0.05, ** for p ≤ 0.01 and *** p ≤ 0.001.

Tenascins Intervene in Myelination of Artificial Microfibers
The reformation of myelin sheaths after damage requires the recruitment of OPCs to lesions and their local differentiation [7]. The ability to remyelinate axons seems to be an intrinsic property of oligodendrocytes [36]. In order to test if the elimination of tenascin genes affects the myelination capacity, Tnc +/+ , Tnc −/− , Tnr +/+, and Tnr −/− oligodendrocytes were tested in a fiber myelination assay ( Figure 1A). To this end, oligodendrocytes were cultivated in the presence of artificial fibers, and the cultures were investigated by immunocytochemistry using antibodies against GFAP and MBP ( Figure 1B). Myelination could be revealed in each genotype and condition. Both the average number of fibers myelinated by a single cell which identifies the amount of myelin formed, as well as the numbers of sheaths extended per single oligodendrocyte were analyzed ( Figure 1C-F). By comparing the average number of fibers ensheathed by single cells from Tnr +/+ and Tnr −/− mice, it became clear that Tnr seemed to exert no influence on the extent of myelination of artificial fibers (Tnr +/+ : 3.9 ± 0.2, Tnr −/− : 4.6 ± 0.2, Tnr −/− + BDNF: 7.2 ± 0.2, p < 0.0001; Tnr −/− vs. Tnr −/− + BDNF: p < 0.0001). However, it could be shown that Tnr seemed to have a positive influence on the number of sheaths formed per single cell. In the absence of Tnr the number of sheaths per single cell was reduced in comparison to the wildtype (Tnr +/+ : 9.7 ± 0.7, Tnr −/− : 7.8 ± 1, p = 0.0049). This could indicate that Tnr promotes the outgrowth of myelin lamellae, a support that was not available when Tnr −/− were tested. The structural homologue Tnc interferes with membrane extension by oligodendrocytes in vitro [22] and was probed for influence on myelin sheath formation in the fiber assay. When OPCs prepared from Tnc −/− or wildtype mice were compared the number of myelinated fibers did not differ ( Figure 1E) while the number of sheaths per single cell was increased ( Figure 1F).
1 Figure 1. Tnc retards whereas Tnr and BDNF promote myelination by OPCs in an artificial fiber assay. (A) OPCs from P6-P9 mice from 129/SV wild-type, Tnc −/− and Tnr −/− mice were prepared via immunopanning and cultivated for 7 div. OPCs were seeded on artificial fibers and cultivated for 14 div. Exemplary photomicrographs of artificial poly-L-lactic acid electrospun microfibres with OPCs in myelination medium for 14 div are shown (B) Immunocytochemical staining was performed with antibodies against GFAP (to exclude astrocytes) and MBP (green, to determine myelin). Scale bar: 50 µm. To determine the myelination degree in the different conditions the average sheath length of fibers per single cell (C,E) and the number of sheaths per single cell (D,F) were determined. In a pilot study, BDNF was added to Tnc −/− and Tnr −/− OPCs to analyze its impact on myelination (E,F). A minimum of 35 single cells in 3 independent experiments were analyzed (n = 35, N = 3). For statistical analysis, the unpaired two-tailed student's test was used (p ≤ 0.01 **, p ≤ 0.001 ***).
Brain-derived neurotrophic factor is known to promote myelin repair in vivo [50]. Here, we wanted to test if BDNF has an influence on the knockout conditions and if knockout effects can even be boosted. Therefore, we supplemented the co-cultures with 10 ng/mL BDNF. Under these conditions, the average number of individual myelinated fibers more than doubled (Tnc +/+ : 5.4 ± 0.2, Tnc −/− : 5.5 ± 0.2, Tnc −/− + BDNF: 14.7 ± 0.7; Tnc −/− vs. Tnc −/− + BDNF: p < 0.0001) ( Figure 1D,F). When the number of sheaths per individual cell was considered, BDNF likewise caused a significant increase (Tnc +/+ : 6.4 ± 0.4, Tnc −/− : 7.9 ± 0.5, p = 0.027; Tnc −/− + BDNF: 11.7 ± 0.1, p < 0.0001; Tnc −/− vs. Tnc −/− + BDNF: 0.0086) ( Figure 1F). Summarizing these results, the average number of ensheathed artificial fibers per cell did not depend on the genotype. However, Tnr and Tnc displayed opposite effects with regard to the number of individual sheaths per oligodendrocyte formed, in that Tnr −/− displayed a reduced and Tnc −/− OPCs an increased propensity to extend myelin lamellae. This is in agreement with our earlier report that Tnc interferes with while Tnr promotes OPC differentiation in culture [22]. The myelination capacity of OPCs could be strongly boosted by the addition of BDNF.
Concerning this parameter, our results confirmed that linear regression lines differed significantly, especially with regard to the Tnr −/− tissue ( Figure 4B-F). Interestingly, after 4 weeks of remyelination the axons of the Tnc −/− and Tnr −/− mouse lines have acquired relatively larger myelin sheaths than the wildtype ( Figure 4E). According to this parameter, the myelin sheaths in relation to the axon diameter were thinner in the wildtype than in the mutants also in the samples after demyelination and two weeks of remyelination ( Figure 4C,D). The best fit lines which were obtained by using linear regression differed in each condition significantly between Tnr −/− and the 129/SV wildtype mice. However, in the untreated control condition, during demyelination, and in the early stage of remyelination the best fit lines between Tnc −/− and 129/SV wildtype mice were not significantly different ( Figure 4B-D). Only after 4 weeks and 6 weeks of remyelination were the best fit lines between Tnc −/− and 129/SV wildtype mice significantly different ( Figure 4E,F). Concerning this parameter, our results confirmed that linear regression lines differed significantly, especially with regard to the Tnr −/− tissue ( Figure 4B-F). Interestingly, after 4 weeks of remyelination the axons of the Tnc −/− and Tnr −/− mouse lines have acquired relatively larger myelin sheaths than the wildtype ( Figure 4E). According to this parameter, the myelin sheaths in relation to the axon diameter were thinner in the wildtype than in the mutants also in the samples after demyelination and two weeks of remyelination (Figure 4C,D). The best fit lines which were obtained by using linear regression differed in each condition significantly between Tnr −/− and the 129/SV wildtype mice. However, in Interestingly, during demyelination the determined axon diameters of both Tnc −/− and Tnr −/− mice were significantly lower than in wildtype mice. (B-F) The best fit lines were also obtained by linear regression and differed significantly between Tnr −/− and wildtype mice in each treatment condition. Statistical analysis was carried out by using the ANOVA and Tukey's multiple comparison test (p ≤ 0.05 *, p ≤ 0.01 **, p ≤ 0.001 ***). (N = 3, n ≤ 594).

Tnc and Tnr Modulate Recruitment of OPCs to and Their Maturation in Myelin Lesions
To determine the role of both tenascins with regard to oligodendroglia recovery and myelin regeneration the markers Olig2 for the oligodendrocyte lineage in general and CC1 for differentiated oligodendrocytes were monitored by immunocytochemistry.  Figure 5C). The myelination process involves membrane extension, initial axonal contact, and subsequent stabilization [53]. Consequent to contact the proteolipid-protein 1 (PLP) and MBP mRNA are transported to the plasma membrane where the MBP synthesis is carried out [54,55]. The analysis of mRNA levels for MBP was performed to obtain more insight into oligodendrocyte maturation in our samples ( Figure 5D). MBP represents a well-established biomarker for the maturation of myelin membranes [56,57]. There were no apparent differences between the genotypes of the  (Figure 5C,D). The analysis of the oligodendrocyte population was complemented by an investigation of oligodendrocyte precursor cells using the marker platelet-derived growth factor alpha (PDGFRα), which is an established marker for OPCs ( Figure 6). Under control conditions more PDGFRαpositive cells were visible ( Figure 6A(a-c)) in both tenascin knockout lines (C: SV/129: 11.3 ± 1.5%, Tnc −/− : 20.2 ± 1.2%, p = 0.0019; Tnr −/− : 21.3 ± 2.6%, p = 0.0115).

The Loss of Tnc Enhances Astrocyte Reactivity in Cuprizone-Induced CNS Lesions
The reactivity of astrocytes is a hallmark of tissue lesions in the CNS [58]. The distribution of astrocytes was determined by using GFAP as a marker (Figure 7). Under control conditions, less astrocytes were counted in both knockout lines (C: SV/129: 23.45 ± 2.3%, Tnc −/− : 7.8 ± 1.8%, p = 0.0016; Tnr −/− : 9.1 ± 1.4%, p = 0.0017 ) ( Figure 7B). As the myelin was degraded by the cuprizone diet, the response to lesions was of particular interest. There, we observed a smaller fraction of GFAP-positive cells ( Figure 7A(d-f) c),B). During demyelination and the early stage of recovery, after 2 weeks of withdrawal of cuprizone treatment, in Tnc −/− significantly more astrocytes were detectable in comparison to the wildtype mice and untreated control condition (A(a-i),B). Otherwise, no significant differences between the three genotypes were visible (A(j-o),B). Data are presented as mean ± SEM and statistical significance (p ≤ 0.05 *, p ≤ 0.01 **, p ≤ 0.001 ***) was assessed using the ANOVA and Tukey's multiple comparison test (control, demyelinated, remyelinated). Four independent experiments were performed (N = 4).

Tenascins Modulate Microglia and Leucocytes in Cuprizone-Induced Lesions
Increasing evidence indicates that the ECM and tenascins regulate also the immune reactions upon lesion to the CNS [59,60]. Therefore, we investigated the local immune response including microglia and macrophages using the markers Iba1 and CD68 expression in our demyelination model (Figure 8). Interestingly, in the untreated condition the number of Iba1 positive cells appeared significantly increased in both tenascin knockouts (C: SV/129: 8.9 ± 0.8%, C: Tnc −/− : 15.3 ± 1%, p = 0.001; C: Tnr −/− : 11.74 ± 0.9%, p = 0.0443)  -c),B). During demyelination and the early stage of recovery, after 2 weeks of withdrawal of cuprizone treatment, in Tnc −/− significantly more astrocytes were detectable in comparison to the wildtype mice and untreated control condition (A(a-i),B). Otherwise, no significant differences between the three genotypes were visible (A(j-o),B). Data are presented as mean ± SEM and statistical significance (p ≤ 0.05 *, p ≤ 0.01 **, p ≤ 0.001 ***) was assessed using the ANOVA and Tukey's multiple comparison test (control, demyelinated, remyelinated). Four independent experiments were performed (N = 4).

Tenascins Modulate Microglia and Leucocytes in Cuprizone-Induced Lesions
Increasing evidence indicates that the ECM and tenascins regulate also the immune reactions upon lesion to the CNS [59,60]. Therefore, we investigated the local immune response including microglia and macrophages using the markers Iba1 and CD68 expression in our demyelination model ( Figure 8). Interestingly, in the untreated condition the number of Iba1 positive cells appeared significantly increased in both tenascin knockouts (C: SV/129: 8.9 ± 0.8%, C: Tnc −/− : 15.3 ± 1%, p = 0.001; C: Tnr −/− : 11.74 ± 0.9%, p = 0.0443) ( Figure 8B). It is known that microglia are repulsed by anti-adhesive Tnr in vitro [61].  Figure 8A(j-o),B). The microglia and macrophage compartment were investigated further using the marker CD68 that was evaluated by RT-PCR analysis ( Figure 8C). CD68 is a highly glycosylated glycoprotein which is expressed by mononuclear phagocytes and used to detect the response of activated microglia as well as macrophages that accumulate in acute lesions [62]. In the untreated situation CD68 expression was higher in Tnr   -o),B). RT-PCR analysis of CD68 to monitor the expression pattern of activated microglia was carried out. In the absence of Tnr, more activated microglia were present in the control condition (A(a-c),C). In contrast, the CD68 expression during demyelination seemed reduced in Tnr −/− (A(d-f),C). In the absence of Tnr the expression of CD68 was limited and even during demyelination and the first two remyelination stages (2 and 4 weeks) the expression increased only minimally (A(g-o),C). After 6 weeks of remyelination, the highest CD68 expression was measured in Tnc −/− . Data are presented as mean ± SEM and statistical significance (p ≤ 0.05 *, p ≤ 0.01 **, p ≤ 0.001 ***) was assessed using the ANOVA and Tukey's multiple comparison test for each group (control, demyelinated, remyelinated). Four independent experiments were performed (N = 4).

Discussion
In the present study, we investigated the influence of the glycoproteins Tnc and the structurally related Tnr on the regenerative response of oligodendroglia in a myelin lesion paradigm using cuprizone. Previous studies in the laboratory had revealed that the Tnc protein of the tenascin family of ECM glycoproteins exerts inhibitory effects on the motility and differentiation of oligodendrocyte precursor cells in vitro [22,26,63]. Although both glycoproteins have a similar structure, their expression patterns differ. Tnc is expressed by neural stem and astrocyte precursor cells and down-regulated postnatally. Tnr is restricted to the CNS and expressed postnatally by maturing oligodendrocytes and a subpopulation of neurons [22,25,64]. By comparing Tnc +/+ and Tnc −/− OPC cultures in the artificial fiber assay we obtained evidence that Tnc has a negative impact on the number of myelin sheaths per oligodendrocyte and thereby the extent of myelin formation. This is consistent with previous reports that showed an interference of Tnc with oligodendrocyte membrane extension [22]. Thus, as expected, Tnc −/− OPCs generated a higher number of myelin extensions per cell than the wildtype. This beneficial effect was strongly boosted by the addition of BDNF, a neurotrophin that is known to support myelin regeneration in vivo [50,[65][66][67]. Inhibition of membrane extension may be rooted in the fact that Tnc prevents RhoA activation [68,69], because the reduction of RhoA activation by deletion of the nucleotide exchange factor Vav3 led to reduced myelination, also in vivo [70,71]. In the artificial fiber myelination assay Tnr −/− OPCs behaved differently in that the number of myelin extensions per cell was reduced. At first sight, this was unexpected because Tnr also suppresses RhoA activation and membrane formation, similar to Tnc [22].
However, both Tnc and Tnr have opposite effects on oligodendrocyte maturation in that Tnc prevents whereas Tnr promotes the maturation of OPCs towards the expression of MBP [22,30]. Thus, the tampered ability of Tnr −/− OPCs to extend membranes may result from a maturation deficit towards the myelinating stage, a process that conversely would benefit from a deficit of Tnc. Tnc is known to modulate the adhesion of various cell types to culture substrates [72], yet the Tnc −/− mouse lines are viable and fertile, in the absence of gross morphological or functional deficits [73], and in particular the myelin compartment appeared developmentally unperturbed [52]. On the other hand, several studies revealed subtle developmental modifications in the radial glia and oligodendrocyte compartments that were compensated for in the adult CNS [74,75]. Furthermore, recent studies suggested functional roles of Tnc in response to lesions, in particular in the CNS [76][77][78][79], reviewed in [80]. Therefore, we decided to investigate the roles of Tnc and Tnr in a myelin lesion model based on the application of cuprizone to the chow [81,82]. In this approach, the newly formed myelin sheaths are generally thinner and shorter than the former ones [48,83,84]. The effects on myelin formation were monitored by electron microscopy and by examining well-established markers of oligodendrocyte progenitor maturation and the major macroglial cell types. A strong demyelination could be achieved by dispensing a cuprizone-rich diet for 6 weeks [31,81]. In our study, this effect was clearly visible after 10 weeks of treatment, which we consider to mimic chronic demyelination. Thereafter, remyelination ensued as expected [85,86]. Currently, chronic demyelination is induced by a 12-week cuprizone treatment [40,41]. However, in these cases, the remyelination fails or is insufficient [43][44][45]87] due to the reduced expression of Trem2 resulting in an impaired clearance of myelin debris from microglia. Therefore, we opted for a 10-week cuprizone treatment to mimic a chronic course of demyelination. According to the g-ratio, remyelination was more efficient in the tenascin knockouts than in the wildtype, and most effective in the absence of Tnc. This does not exclude a differential sensitivity to Cuprizone treatment. Interestingly, known effects concern the roles of Tnc in regulating the proliferation and migration of OPCs [74]. Furthermore, an anti-apoptotic effect of Tnc for oligodendrocytes in vitro has been reported [63]. Less is known in this regard about Tnr. Therefore, the question remains open whether some of the differences in de-and remyelination observed are due to a differential susceptibility of the mouse lines towards Cuprizone treatment.
The phenotype in the Tnc −/− -line most probably reflects the inhibitory properties of Tnc for maturation and membrane extension observed in vitro. This effect of Tnc was accompanied by a strong upregulation of GFAP, reflecting enhanced astrocyte reactivity in this situation. Interestingly, a strong upregulation of GFAP had also been observed in a stab wound model to the cortex of the Tnc −/− mouse line [88]. Interestingly, our laboratory has shown that the proliferation of astrocytes is augmented in the embryonic Tnc −/− spinal cord, which may be responsible for the enhanced astroglial reactivity observed in lesions [89]. Reactive astrocytes can also be considered a source for the release of Tnc in lesioned tissue [90]. Of note, it has been proposed that astrocyte-derived Tnc contributes to the inhibitory environment for remyelination in Multiple Sclerosis lesions [91,92]. It has been shown that alphaV integrins are upregulated during remyelination, in conjunction with Tnc and Tnr that may represent functional ligands in that context [27]. Integrins are considered important receptors for tenascin functions [93,94]. Beyond its role in perineuronal nets where it serves as a major scaffolding protein [95], Tnr has also been studied in lesion situations. In this context, it has been proposed as an inhibitory axonal guidance molecule, notably in the optic nerve [29] and in the spinal cord [96]. As Tnr is expressed by adult oligodendrocytes it may be part of degraded myelin that abounds in demyelinating lesions. There, it also seems to interfere with remyelination, as the Tnr −/− mice showed an improved recovery in the cuprizone model, albeit not as effectively as the Tnc −/− counterpart. Both tenascins interact with various chondroitin sulfate proteoglycans (CSPGs) of the lectican family such as versican and brevican and thus partake in the inhibitory environment that restricts regeneration of myelin, in particular in multiple sclerosis lesions [97][98][99]. The copper chelator cuprizone sets a metabolic insult that preferentially leads to the elimination of mature oligodendrocytes by apoptosis [15,82]. The elimination of oligodendrocytes was particularly effective in the Tnc −/− tissue where the lowest number of Olig2-positive cells was recorded. This is in agreement with the former report that OPC proliferation is reduced in the Tnc knockout [74]. Repair of the deficit requires the recruitment of OPCs to the lesion site, where the cells have to differentiate towards the myelin-forming stage [7,8]. Tnc is known to interfere with OPC motility in vitro [26,100] and Tnc −/− knockout mice revealed an accelerated invasion of OPCs into the optic nerve [74]. Similarly, in our current study more PDGFR-positive OPCs [101,102] were detected upon demyelination and in the first two weeks of remyelination in the absence of Tnc. Tnr embodies anti-adhesive properties for different neural cell types [103]. While its impact on migration has not explicitly been studied this may explain the increased immigration of PDGFR-positive OPCs observed upon demyelination and during the first two weeks of remyelination, analogous to albeit not as extensive as in the Tnc −/− situation. Once on site, the OPCs have to engage in differentiation in order to remyelinate the axon. Myelin basic protein (MBP) is very important for the compaction of the myelin sheaths [56]. Tnc is known to delay the maturation of OPCs towards the MBP-expressing mature state [22]. This effect is mediated by the receptor contactin-1 (Cntn1), the activation of downstream signaling pathways, and the suppression of the RNA-binding protein Sam68, an oligodendrocyte maturation factor [34]. In agreement with these inhibitory properties, we observed an increase in mature CC1-positive oligodendrocytes and MBP expression within two to four weeks of remyelination in the Tnc −/− system. Tnr, different from Tnc promotes the acquisition of MBP and has been proposed as an autocrine maturation factor of oligodendrocytes in vitro [22,30]. Notwithstanding, the maturation of OPCs in the absence of Tnr was not compromised, which might indicate that the loss of Tnr was compensated by axon-derived signals in the in vivo situation.
It is interesting to compare our results with studies that focused on autoimmune processes in tenascin knockout mutants. Thus, in an experimental autoimmune encephalitis elicited by the injection of the myelin-oligodendrocyte glycoprotein (MOG), the encephalitogenic response of Th1 and Th17 immune cells was substantially reduced in the absence of Tnc [76]. Likewise, in an autoimmune glaucoma model, the ablation of Tnc resulted in amelioration of outcome [78]. These observations conform with a recent report that Tnc belongs to the damage associated molecular patterns (DAMPs) and is as efficient regarding the activation of microglia as lipopolysaccharides [104]. These findings stage Tnc as an important modulator of immune responses in the CNS [17,60]. For example, Tnc associated with exosomes has been found to suppress T-cell activation [105] and the extracellular matrix appears involved in the immune response to ischemia [59,106]. In our studies, we saw several differences between the genotypes regarding the number of Iba1-positive cells and the expression of CD68, that was particularly upregulated after six weeks of remyelination in the Tnc −/− lesions. Interestingly, a recent report emphasized a regulatory role of Tnc for microglia surveillance and leucocyte infiltration in ischemic CNS infarct territory [106].
Summarizing our findings, it seems that the elimination of Tnc and Tnr exerts a clear impact on the remyelination of cuprizone-induced myelin degradation. Both the recruitment of OPCs to the lesion territory and their local differentiation to myelinating cells are promoted in the absence of tenascins. Consequently, remyelination is supported and myelin repair is accelerated in the mutants.

Data Availability Statement:
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.