Central nervous system rather than immune cell-derived BDNF mediates axonal protective effects early in autoimmune demyelination
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Brain-derived neurotrophic factor (BDNF) is involved in neuronal and glial development and survival. While neurons and astrocytes are its main cellular source in the central nervous system (CNS), bioactive BDNF is also expressed in immune cells and in lesions of multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). Previous data revealed that BDNF exerts neuroprotective effects in myelin oligodendrocyte glycoprotein-induced EAE. Using a conditional knock-out model with inducible deletion of BDNF, we here show that clinical symptoms and structural damage are increased when BDNF is absent during the initiation phase of clinical EAE. In contrast, deletion of BDNF later in the disease course of EAE did not result in significant changes, either in the disease course or in axonal integrity. Bone marrow chimeras revealed that the deletion of BDNF in the CNS alone, with no deletion of BDNF in the infiltrating immune cells, was sufficient for the observed effects. Finally, the therapeutic effect of glatiramer acetate, a well-characterized disease-modifying drug with the potential to modulate BDNF expression, was partially reversed in mice in which BDNF was deleted shortly before the onset of disease. In summary, our data argue for an early window of therapeutic opportunity where modulation of BDNF may exert neuroprotective effects in experimental autoimmune demyelination.
KeywordsNeurotrophins BDNF EAE Conditional knock-out mice Neuroimmunology
Brain-derived neurotrophic factor (BDNF) is a neurotrophin involved in neuronal survival, differentiation and function as well as axonal growth, modulation of neuronal activity, and activity-dependent synaptic and dendritic plasticity in the central nervous system (CNS) (reviewed in ). In several experimental models it was shown to exert neuroprotective effects [8, 12, 26]. The importance of BDNF for brain function and maintenance is underscored by the fact that BDNF-deficient mice already die during the first weeks of life . Even heterozygous mice with 50% reduced levels of BDNF expression display some behavioral deficits such as aggressiveness, hyperactivity, hyperphagia and obesity [14, 22, 33].
BDNF also plays a role in autoimmune neuroinflammation. Recently, we showed that BDNF indeed has a neuroprotective role in the myelin oligodendrocyte glycoprotein (MOG)-induced animal model experimental autoimmune encephalomyelitis (EAE) . This was particularly highlighted by a more severe clinical disease course in mice in which BDNF was conditionally deleted either in cells of the immune system (T cells and monocytes/macrophages) or in astrocytes/neuronal subpopulations. A higher amount of axonal damage was detected in these mice without accompanying alteration of the inflammatory process . Immune cell-derived BDNF could be of relevance for the observed protective effects. Indeed, it had been observed before that this neurotrophin is expressed in T cells, B cells and activated monocytes/macrophages [15, 17]. Moreover, BDNF was found in lesions of multiple sclerosis (MS) patients and also in EAE . Conditional BDNF-deficient mice specifically lacking BDNF in T cells and monocytes/macrophages displayed a progressive motor impairment in the late phase of EAE accompanied by an increase in axonal damage . Furthermore, supplementation of BDNF by injection of BDNF over-expressing MOG-specific T cells resulted in amelioration of EAE symptoms and reduced axonal damage . These results support the concept of BDNF being a molecular correlate for a neuroprotective immune response .
The disadvantage of the aforementioned approaches is that they are all based on the permanent ablation of BDNF early during development. The permanent absence of BDNF may result in compensatory up-regulation of other neurotrophines or cytokines or disruption of fine-tuned regulatory networks within the target cells, thus making it difficult to isolate the function of BDNF. In this regard it has been recently reported that heterozygous BDNF +/− mice have a diminished peripheral Th1/Th17 antigen-specific response after immunization with the myelin basic protein–proteolipid protein fusion protein MP4 and consequently reduced EAE clinical course and histopathology . Another disadvantage is that previously a reduction of BDNF expression within the CNS could only be achieved in distinct cell types such as astrocytes and as yet poorly defined neuronal subpopulations but not over the entire CNS. Finally, these approaches did not allow one to define the relevant time period of BDNF action in the course of EAE to mediate neuroprotective effects. Thus, it is of great interest to study the role of endogenous BDNF in a setting where the factor is present during the development and normal homeostasis but can be specifically deleted at different time points after EAE induction. In this study, we employed tamoxifen-inducible Cre mice  to yield an inducible reduction of BDNF levels in cells both of the CNS and the immune system. In general, we could confirm the neuroprotective role of BDNF. An induced reduction of BDNF early (after the priming phase but before the onset of clinical symptoms) but not later in the course of EAE resulted in a more severe disease course. These results were reproduced in bone marrow chimeras with BDNF levels reduced within the CNS but spared in the hematopoetic compartment, thus arguing for the relevance of BDNF production by CNS resident cells for the observed effects.
Materials and methods
Mice with two loxP sites flanking exon 8 in the BDNF locus (BDNFfl/fl) are already described elsewhere [18, 25] and were bred in our colony in IVC cages. ESR-Cre mice were purchased from Jackson (stock number 004682) . The two lines were crossed, resulting in ESR-Cre/BDNFfl/fl mice (designated as BDNFind−/− mice). BDNFfl/fl littermates served as controls in all experiments. All mice were backcrossed on a C57BL/6 background for at least ten generations. Animal experiments were approved by the responsible authorities in Lower Saxonia.
BDNF deletion and EAE induction
Tamoxifen was purchased from Sigma, Deisenhofen, Germany and dissolved in sun flower oil. Mice received three injections of 3 mg (in a total volume of 150 μl) every other day. In EAE experiments, mice received 3 mg tamoxifen at days 7, 9 and 11 after immunization (p.i., early deletion) or at days 14, 16 and 18 p.i. (late deletion). In the very first control experiment on the effect of tamoxifen in EAE development, control mice received sun flower oil only.
EAE induction and scoring are described elsewhere [18, 19]. Briefly, mice were immunized with 50 μg MOG35–55 in an equal amount of complete Freund’s adjuvant and received 200 ng pertussis toxin (List Biochemicals, Campbell, CA, USA) i.p. on days 0 and 2 p.i. The clinical evaluation was performed on a 10-point scale ranging from 0 (healthy) to 10 (dead) . Mice were scored and weighed daily. In some experiments, mice were treated with 500 μg glatiramer acetate s.c. at the time of immunization .
Mice were killed on the indicated days and mRNA was prepared from the cerebrum, cerebellum, spinal cord, T cells and macrophages using an RNeasy Mini kit (Qiagen, Hilden, Germany). T cells were purified from a single cell suspension of splenocytes using a Pan T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and macrophages were isolated from the peritoneum by lavage with two times 2–3 ml ice-cold PBS, followed by plating for 6 h in medium at 37°C, removal of non-adherent cells and further cultivation overnight at 37°C before harvesting and RNA preparation. cDNA was prepared using superscript™ II reverse transcriptase (Invitrogen, Karlsruhe, Germany). Real-time PCR was used to amplify the coding sequence of BDNF with mBDNF S (5′-GGGCCGGATGCTTCCTT-3′), mBDNF AS (5′-GCAACCGAAGTATGAAATAACCATAG-3′), and mBDNF Son (5′-TTCCACCAGGTGAGAAGAGTGATGACCAT-3′) as primers. All reactions were performed on a 7500 Sequence Detection System (Applied Biosystems, Darmstadt, Germany) with 2 min at 50°C, 2 min at 95°C and 40–50 cycles with 15 s at 95°C and 1 min at 60°C. β-Actin served as endogenous control for relative quantification according to the ΔΔC t-method [18, 21]. Individual values for each sample are calculated setting BDNF mRNA levels from the cerebrum of control mice arbitrarily to “1”.
For the early deletion of BDNF with the tamoxifen treatment starting on day 7 p.i., mice were killed on day 15 p.i., perfused with 4% paraformaldehyde and then the lumbar, thoracal and cervical part of their spinal cord was embedded in paraffin. For the late BDNF deletion with tamoxifen treatment starting on day 14 p.i., the tissue was prepared on day 30 p.i. In experiments including glatiramer acetate treatment, histological work-up was performed on day 50 p.i. Spinal cord cross-sections were stained with Luxol fast Blue (LFB) to assess demyelination. Bielschowsky silver impregnation or staining for amyloid precursor protein (APP; 1:1,000, MAB348; Millipore, Schwabach, Germany) was employed to analyze axonal damage and loss . T cells were labeled by rat anti-CD3 (Serotec; Wiesbaden, Germany; 1:300) and macrophages/microglia by rat anti-mouse Mac-3 (BD Pharmingen; 1:200) and quantified as described earlier . Confocal laser scanning microscopy after double-label immunohistochemistry for 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase; 1:4,000, Millipore) and the neurofilament antibodies SMI31 or SMI32 (1:1,000, Invitrogen) with anti-mouse Alexa 488- and Alexa 647-conjugated secondary antibodies or double-label immunohistochemistry for SMI32 and APP were carried out according to techniques described elsewhere .
Generation of bone marrow chimeric mice
12-Week-old mice were lethally irradiated. Radiation treatment at a dose rate of 1 Gy/min was delivered by a RS 225 X-Ray Research System (Gulmay Medical Systems, Camberley, Surrey, UK) operated at 200 kV, 15 mA and with 0.5-mm Cu filtration. Animals were placed in a customized Perspex box and received a total body dose of 12.5 Gy. The following day, bone marrow cells were removed from the femurs of donor mice, resuspended in PBS and injected intravenously into the irradiated recipients at a concentration of 2 × 106 cells/mouse. The chimeric mice were treated with neomycin at a dose of 0.15 mg/ml in their drinking water for 4 weeks. Chimeric mice were generated either by reconstituting BDNFfl/fl mice positive for ESR-Cre or not with CD45.1 congenic C57BL/6 bone marrow or, alternatively, CD45.1 congenic C57BL/6 mice with bone marrow from BDNFfl/fl mice positive or negative for ESR-Cre. Reconstitution of the mice was tested 4–5 weeks after bone marrow transfer by FACS analysis of peripheral blood using antibodies against CD45.1 and CD45.2 and routinely yielded a reconstitution efficiency of more than 90%. 8 weeks after reconstitution, EAE was induced by immunization with 50 μg MOG35–55 and one injection of 200 ng pertussis toxin. For deletion of BDNF, mice were treated with 3 mg tamoxifen on days 7, 9 and 11 p.i.
Lymphocytes were isolated from the spinal cord by density centrifugation following perfusion of mice with NaCl as previously described . Subsequently, cells were stained with the following antibodies: anti-CD3e (145-2C11), anti-CD4 (RM4-5), anti-CD44 (IM7), anti-CD11a/LFA-1 (2D7), anti-CD8 (53-6.7) (all from BD Biosciences) and F4/80 (Serotec). The antibodies were either directly labeled with FITC, PE, PE-Cy7, APC or APC-Cy7 or biotinylated followed by streptavidin-PE-Cy5. All analyses were performed on a FACS Aria II Sorp device allowing for the detection of six fluorescent dyes (BD Biosciences).
Histological quantification and statistical analyses
For the analysis of EAE disease course, group and time effects as well as their interaction on EAE disease score were studied using twofold non-parametric analysis of variance for repeated measures . In this analysis, we were particularly interested in the group × time interaction. A significant interaction indicates a different time course for the two groups with **P < 0.01. The significance level for all P values was chosen to be α = 5%. All analyses were performed using the free software R (version 2.12, http://www.r-project.org).
Histological quantification was performed by a blinded observer by means of overlaying a stereological grid onto the sections and counting T cells, macrophages/microglia, APP positive profiles and axonal densities on up to 10–12 lesions per mouse from representative spinal cord cross-sections comprising cervical, thoracic and lumbar spinal cord as described previously . Axonal densities were counted using a 24-point eyepiece from Olympus (Hamburg, Germany), and the number of points crossing axons was measured as a fraction of the total number of points on the sterological grid . Demyelination was analyzed semi-automatically with the CellD software (Olympus).
Differences in the histological parameters between the groups were calculated using the non-parametric Mann–Whitney test not assuming Gauss-distributions with *P < 0.05 and **P < 0.01.
Rapid deletion of BDNF in the central nervous system after tamoxifen administration
BDNF deletion in the preclinical phase results in enhanced clinical course and more severe axonal loss
Estrogens, in general, including the synthetic derivative tamoxifen, are known to have the capacity to modulate the susceptibility and severity of mice against EAE induction [2, 31]. Therefore, we first tested if tamoxifen given alone would influence the clinical course of EAE in C57BL/6 wild type mice. To this end, EAE was induced in a cohort of female C57BL/6 mice and the animals were treated either with tamoxifen (3 mg i.p./treatment) or with sun flower oil as vehicle at days 7, 9 and 11 p.i. There was no significant difference in onset of disease and clinical score (mean clinical scores on day 24: 3.7 ± 2.5 for controls vs. 4.5 ± 3.4 for tamoxifen-treated mice, n.s., n = 5–6 per group), indicating that this tamoxifen treatment protocol did not influence the clinical outcome.
BDNF deletion after the peak of EAE influences neither the clinical course nor the extent of axonal damage
The therapeutic efficacy of glatiramer acetate is limited after inducible BDNF deletion
Enhanced motor impairment after early BDNF deletion in bone marrow chimeric mice
BDNF is a neurotrophic factor with pleiotropic properties within the nervous system and is involved in neuronal survival and differentiation as well as synaptic plasticity . In addition, BDNF is expressed in activated immune cells (T cells, B cells and monocytes)  and is histologically observed to be in close proximity to lesions in the CNS of MS patients , suggesting a possible protective role in this setting. Indeed, we and others showed that BDNF is neuroprotective in MOG35–55-induced EAE [18, 23, 24].
Our observations extend these previous studies by addressing (a) the time point at which BDNF action seems to be critical for its neuroprotective effects in EAE and (b) the relevant tissue (CNS vs. hematopoetic system) responsible for BDNF production. To this end, we employed a conditional knockout system with a significant reduction in global BDNF levels by administration of tamoxifen. Notably, the neuroprotective effect was most prominent when BDNF was deleted early, i.e. at a time point after T cell priming (to avoid possible immunological effects) and shortly before the occurrence of first clinical symptoms. In contrast, BDNF deletion after the first peak of disease resulted in only marginal effects on the clinical disease course and on histopathological parameters of tissue destruction. These data suggest that the neuroprotective effects of BDNF in the experimental model of MS are particularly relevant during the early phase of EAE. This is in line with the observations both in EAE and MS patients that key features of neurodegeneration already occur in early disease phases [10, 11, 36]. Thus, it seems that neuro-axonal injury during this early phase may also impact on the functional outcome of disease at later time points, and that an interference with neurodegeneration during this critical phase, i.e. by survival-promoting neurotrophins such as BDNF as in the current study, is a prerequisite for BDNF’s action. Importantly, early deletion of BDNF also affected the efficacy of glatiramer acetate treatment which was previously shown to exert neuroprotective effects via BDNF . This confirms that the observed effects are indeed mediated via the reduction of BDNF levels and not secondary to tamoxifen treatment.
Our experiments using bone marrow chimeras in which BDNFind−/− mice were reconstituted with wild-type bone marrow revealed that CNS-derived BDNF is even more critical than immune cell-derived BDNF for a neuroprotective effect in autoimmune demyelination. This is in line with the results observed for tissue-specific deletion of BDNF. The deletion of BDNF in astrocytes/neuronal subpopulations resulted in an enhanced motor impairment early in the course of EAE, whereas BDNF deletion in immune cells resulted in a similar phenotype only at much later time points . Together, the data argue for an intrinsic self-protective mechanism of the CNS that is already called into action at a very early time point of autoimmune inflammation.
The deletion of BDNF was highest in the CNS followed by T cells while reduction of BDNF levels was not detectable in macrophages. Of note, Cre expression as measured by real-time PCR was ten times higher in the CNS compared to the thymus and even lower in the spleen, giving an explanation for the weaker recombination of cells in these organs (unpublished results). When compared with CNS tissue and T cells, macrophages express BDNF at low levels , thus making the detection of recombination difficult.
Since MS and EAE are reported to be influenced by estrogen receptor signaling [29, 30], we wondered whether tamoxifen treatment per se impacts on the disease course of EAE. In our control experiments using female C57BL/6 mice, we could not detect significant differences between the clinical EAE course of mice treated with tamoxifen at days 7, 9 and 11 p.i. and controls only receiving sun flower oil. Yet, upon administration of tamoxifen as early as 14 days prior to immunization, we found protective effects of tamoxifen in EAE (data not shown) which is in line with previous observations by Bebo et al.  and precludes an unambiguous analysis of an even earlier tamoxifen-induced BDNF deletion during EAE. While tamoxifen may reduce T cell proliferation and MHC class II expression early during EAE, it seems conceivable that such immunomodulatory effects are only of minor importance if tamoxifen treatment is started as late as day 7 p.i. In addition, in our experimental setting all mice were treated with tamoxifen including controls. Therefore, potential minor effects of tamoxifen on the immune response would affect all animals and do not explain our observations.
Mice heterozygous for BDNF display behavioral abnormalities such as learning deficiencies , aggressiveness, hyperactivity and also hyperphagia accompanied by obesity [14, 22]. Furthermore, mice in which BDNF was deleted in parts of the CNS shortly after birth using a conditional system showed higher levels of anxiety and obesity . A recent approach resulting in a more global deletion of BDNF in the CNS starting around birth also resulted in features of hyperactivity and obesity . In the current study, we could confirm these observations. BDNFind−/− mice exhibited behavioral abnormalities starting 3–4 days after the first tamoxifen treatment. This included hyperactivity, restlessness, sudden jumps and somersaults, as already described by others [14, 33]. Most strikingly, these symptoms were even evident in mice suffering from EAE (unpublished observations). In addition, tamoxifen-treated BDNFind−/− mice gained weight even in the presence of hyperactivity. Over time, their body weight was significantly higher than that of their control littermates (unpublished observations). These observations are well in line with the concept that physiological BDNF levels are not only necessary during the development but also play a steady-state role in the regulation of motor activity and eating behavior in the adult mouse.
While BDNF is well known as a survival factor for neurons, the molecular mechanisms linking BDNF to axonal damage are not yet well defined. BDNF has been described as acting on immune cells, e.g. regulating IL-2 production of T cells  and MHC II expression of microglia , presumably via activation of the transcription factor “cAMP responsive element binding protein” (CREB) . In neurons, CREB is a major mediator in the BDNF-TrkB signaling cascade activated either via the PLCγ/Ca2+/CamK IV or, alternatively, through the Ras/ERK/Rsk pathway and regulates BDNF-induced gene expression . Whether CREB is really instrumental for the described axonal preservation in EAE and what role, if any, GA plays in this pathway deserves further investigation.
In summary, BDNF plays a protective role in the disease course of EAE. In particular, it is BDNF produced by the CNS that is the main source for BDNF’s immediate neuroprotective action while BDNF from infiltrating immune cells seems to play, if any, rather a minor role. Moreover, BDNF is critically involved already early in the disease course at a time when the first symptoms occur and the inflammatory assault to the CNS is initiated. This observation may have important implications on the timing of neuroprotective drug treatment strategies in MS patients. Our data argue for a therapeutic window of opportunity not only for immunomodulatory drugs  but also for neuroprotective treatment approaches aiming at the modulation of neurotrophic factors.
We thank M. Weig, B. Curdt and N. Meyer for excellent technical assistance, C. Ludwig for language corrections and Dr. A. Junker for help with the RT-PCR. We wish to thank Prof Michael Wegner and Dr. Claus Stolt, Dept. of Biochemistry, University of Erlangen for help with confocal laser scanning microscopy. F.L., R.L. and R.G. were supported by the Gemeinnützige Hertie-Stiftung (AZ 1.01.1/05/009), R.G. and F.L. by the Deutsche Stifterverband, Fritz und Hildegard Berg-Stiftung (AZ T 155-15.284), F.L. and A.F. were supported by the Bundesministerium für Bildung und Forschung (BMBF) (competence network multiple sclerosis, ‘Understand MS’) and the Deutsche Forschungsgemeinschaft (DFG, SFB-TR-43 TP B11).
Conflict of interest
The authors declare that they have no conflict of interest.
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