Activation of Wnt signaling promotes hippocampal neurogenesis in experimental autoimmune encephalomyelitis
- Reiner Schneider1,
- Barbara Koop1,
- Friederike Schröter1, 2,
- Jason Cline1,
- Jens Ingwersen1,
- Carsten Berndt1,
- Hans-Peter Hartung1,
- Orhan Aktas†1Email author and
- Tim Prozorovski†1Email author
© The Author(s). 2016
Received: 14 November 2015
Accepted: 2 July 2016
Published: 14 July 2016
Disease progression in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), as one of its animal models, is characterized by demyelination and neuronal damage in white and gray matter structures, including the hippocampus. It is thought that dysfunction of the hippocampus, a primary locus of learning and memory consolidation, may contribute to cognitive impairment in MS patients. Previously, we reported an increased generation of hippocampal neuronal progenitors in the acute stage of EAE, whereas the microenvironmental signals triggering this process remained uninvestigated.
In the present study, we used the Wnt signaling reporter mouse Axin2LacZ, to elucidate the molecular mechanisms underlying the activation of the hippocampal neurogenic niche upon autoimmune neuroinflammation. Histological and enzymatic examinations of β-gal during the disease course of EAE, allowed us to survey hippocampal Wnt/β-catenin activity, one of the key signaling pathways of adult neurogenesis. We found that Wnt signaling is transiently upregulated in the acute stage of disease, consistent with a timely induction of canonical Wnt ligands. The enhancement of signaling coincided with hippocampal neuronal damage and local expression of immune cytokines such as TNFα and IFNγ, implicating the role of the inflammatory milieu in activation of the Wnt/β-catenin pathway. Supporting this finding, we show that transient exposure to pro-inflammatory cytokine TNFα triggers Wnt signaling in hippocampal organotypic slice cultures. Importantly, inflammation-mediated activation of the Wnt/β-catenin pathway was associated with enhanced neurogenesis in vitro and in vivo, indicating its potential role in hippocampal tissue regeneration and repair.
This study raises the possibility that enhancement of Wnt signaling may support neurogenic processes to cope with neuronal deficits upon immune-mediated neuroinflammation.
A large body of research highlights the involvement of hippocampal pathology in cognitive disability that is commonly observed in patients with multiple sclerosis (MS), an inflammatory demyelinating and neurodegenerative disease of the central nervous system (CNS) [1, 2]. Structural changes in the hippocampus were shown to be associated with MS-related memory impairment  and may occur prior to cognitive dysfunction [4, 5], suggesting a therapeutic opportunity for reversing memory decline in MS patients (reviewed in [5–7]). Examinations of neurobiological alterations in post-mortem brain tissue indicate frequent and extensive hippocampal demyelination  resulting in alterations of synaptic function, axonal transport  and neuronal loss in the Ammon’s horn 1–3 (CA1-3) regions [9, 10]. Furthermore, axonal demyelination was shown to be accompanied by drastic changes in the expression of specific neuronal genes  and neuronal miRNA  suggesting disturbance of molecular pathways involved in physiological homeostasis of hippocampal tissue. Experimental autoimmune encephalomyelitis (EAE), an animal model of MS, mimics many aspects of hippocampal pathology and its clinical expression in the human disease such as demyelination, alteration in synaptic plasticity and transmission [12, 13], neuronal loss , cognitive deficits  and thus, becomes an important model for the characterization of mechanisms involved in hippocampal neuropathology and MS-related memory deficits. Our previous data identified adult hippocampal neurogenesis, an important player in cognition , as a primary target of autoimmune-mediated neuroinflammation . The analysis of hippocampal gene expression in acute and chronic stage of EAE revealed alterations in the transcriptional profile of genes relevant for key neurogenic pathways in the adult brain, including those of Wnt signaling . Considering the potential role of the Wnt pathway in tissue renewal and regeneration [17, 18], we here examined the impact of autoimmune neuroinflammation on hippocampal Wnt/β-catenin activity by using Axin2lacZ/+ reporter mice. We found that T cell-mediated autoimmune neuroinflammation leads to hippocampal neuronal damage occurring early in disease progression and may potentially trigger the activation of Wnt signaling via upregulation of a specific set of Wnt ligands. Transient increase in the Wnt activity in the acute phase was associated with increased proliferation and generation of doublecortin-positive (Dcx+) neuronal precursor cells. Further, we identified the inflammatory mediator tumor necrosis factor alpha (TNFα) as a potent enhancer of Wnt signaling activity and subsequently, the proliferation of progenitor cells in the hippocampal neurogenic niche.
Increased Wnt activity in the acute phase of EAE
Next, we examined the activation state of Wnt signaling mediators in naive and EAE mice. The ligation of Wnts inducing the canonical pathway leads to stabilization (de-phosphorylation) of β-catenin via inactivation (phosphorylation at Ser9) of GSK-3β, a member of the β-catenin destructive complex. Supporting the results of the enzymatic assay, we found a significant increase in active β-catenin (ABC)/total β-catenin ratio at day 30 of EAE mice in comparison to their respective controls (Fig. 1e). Taken together, our results indicate a transient upregulation of hippocampal Wnt signaling in acute stages of EAE (20–30 d.p.i.). In contrast to this, sustained increase in Wnt activity was observed in the spinal cord through all EAE stages examined (Fig. 1c), which was correlated with a large number of inflammatory foci (Additional file 1: Figure S1a-b).
Induction of Wnt ligands in passive EAE
Upregulation of Wnt activity correlated with neuronal injury and inflammatory processes
Wnt signaling promotes neurogenic processes in the DG
Effects of TNFα on the Wnt signaling and hippocampal neurogenesis
Though the induction of Wnt signaling was previously reported in the context of various tissue damage paradigms [17, 18], the mechanisms triggering the Wnt pathway remain unknown. Therefore, we used the Axin2lacZ/+ reporter mice to characterize the possible contribution of particular cytokines, known to be secreted by activated microglia, to promote Wnt activity in neural tissue.
We next asked whether TNFα affects hippocampal neurogenesis and whether Wnt ligands contribute to this effect. To assess the proliferation rate of hippocampal progenitors in NestineGFP hippocampal slice cultures, we labeled mitotically active cells with BrdU prior to 6 h treatment with the individual cytokines. After a 24-h chase in cytokine-free media the quantification of BrdU-retaining cells was examined. Treatment with TNFα (Fig. 5c-d) or Wnt3a, but not IFNγ (Additional file 3: Figure S3d-e), led to a significant increase in the number of BrdU+/eGFP+ neural progenitor cells. Inhibition of Wnt ligand secretion by IWP-2 suppressed the proliferative effect of TNFα (Fig. 5c-d). We further confirmed these results using co-culture experiments. For this approach we used hippocampal microglia because of its prominent activation pattern during the EAE progression (Fig. 3b-c). CD11b-sorted hippocampal microglia cultured on transwell inserts were activated with TNFα for 6 h. After several washing steps, transwells were set onto hippocampal progenitor cultures in proliferating conditions in the presence or absence of soluble Frizzled-8-Fc protein (sFz8). This recombinant protein contains a conservative cysteine-rich region (CRD) and was efficiently used to block Wnt ligand binding in hippocampal progenitor cultures . After 24 h, mitotic cells were labeled with BrdU (2 h treatment). TNFα -primed microglia significantly enhanced mitotic activity of SOX2+ progenitor cells (Fig. 5e-f). Blockade of Wnt signaling with sFz8 decreased the proliferating rate to the level observed in progenitors co-cultured with non-activated microglia in the presence of sFz8. Taken together, these results are consistent with the observation that TNFα promotes Wnt signaling to enhance the initial steps of the hippocampal neurogenic program. In part, this effect is mediated through microglia-dependent Wnt ligand production, supporting the pro-neurogenic role of activated microglia in the hippocampus [32–34].
The discovery of Wnt pathway activity in the adult CNS in physiological and pathological conditions has raised new questions about the mechanism of its activation. Moreover, it is of therapeutic interest whether the unique involvement of the Wnt pathway in developmental processes (such as neurogenesis, oligodendrogenesis, synaptogenesis and axonal guidance) may be useful for facilitation of tissue renewal and repair.
In the present study we made use of Axin2lacZ/+ reporter mice to monitor the dynamic changes in hippocampal Wnt activity upon acute and chronic stages of EAE, an animal model of MS. Our major finding is that CNS pathology, relevant for acute tissue injury and inflammation, is associated with a transient increase of hippocampal Wnt signaling. Enhanced Wnt/β-catenin activity was correlated with transient upregulation of canonical Wnt ligand (Wnt2, Wnt3, Wnt3a, Wnt8b and Wnt9a) mRNA levels. We also show that TNFα, an inflammatory cytokine upregulated in the hippocampus of EAE mice (Fig. 3d), can be considered as a potent factor contributing to Wnt signaling activation. Furthermore, the induction of Wnt activity led to increased proliferation of neuronal progenitors and generation of Dcx+ neuronal precursor cells (also known as Type2b). Taken together, our study revealed a link between neuroinflammation and Wnt-dependent activation of hippocampal neurogenesis regarding the tissue repair program.
Our finding that activation of Wnt signaling is mediated by active inflammatory processes is in line with previous reports on MS pathology. Using different biochemical techniques, the upregulation of Wnt ligands (Wnt2, Wnt7a: microarray data ); Wnt3a: proteomic analysis ) and Wnt pathway members such as Axin2 (histological examination ), GSK-3β, β-catenin and Tcf4 (proteomic analysis ) was demonstrated in active plaques in the white matter and was absent in chronic silent plaques or normal-appearing white matter. In the EAE model, transient induction of Wnt1 was demonstrated in the subventricular zone (SVZ) of the lateral ventricle .
We found that the upregulation of Wnt/β-catenin activity in the hippocampus is associated with an induction of several canonical Wnts involved in self-renewal and cell fate determination of stem/progenitor cells, synaptic plasticity, axon guidance and tissue homeostasis (reviewed in [38, 39]). Thus, Wnt3 and Wnt3a are potent activators of self-renewal and neuronal fate in the adult hippocampus [19, 29] and cultured neural progenitor cells . Wnt2 enhances neurogenesis in the developing brain  and its induction in the dentate gyrus after electroconvulsive seizures (ECS)  was reported to be associated with increased hippocampal neurogenesis . Wnt8b regulates dorsal thalamic neurogenesis during zebrafish development  and was also shown to enhance the proliferation of cultured rat adult hippocampal progenitors . Expression of Wnt16a (human analogue of mouse Wnt16) was found in the brain . However, its role in the CNS remains to be examined. Considering the transient induction of Wnt16 and activation of Wnt/β-catenin pathway at the early stage of osteoarthritis , it is plausible to assume that Wnt16 mediates the response to neural tissue injury via promotion of β-catenin signaling. Remarkably, in addition to their role in neural plasticity, Wnts likely participate in neuroimmune interactions (reviewed in ).
Consistent with previous reports demonstrating the upregulation of different components of canonical Wnt pathway in various models of acute spinal cord pathology (such as EAE , spinal cord injury  and lysolecithin-induced demyelination ), we found the enhancement of Wnt/β-catenin signaling in the spinal cord of Axin2lacZ/+ EAE mice. Notably, in this part of the CNS Wnt activity was increased throughout the course of disease and correlated with a large number of inflammatory foci (Additional file 1: Figure S1b), indicating ongoing active inflammatory processes. Thus, it is conceivable that the new waves of inflammation-mediated damage trigger the persistent activation of Wnt signaling in the spinal cord.
In line with the idea that acute inflammation enhances Wnt signaling, we showed that TNFα is a potent inducer of Wnt activity in hippocampal slice cultures. This finding is consistent with previous reports demonstrating the interplay between those two signaling pathways in other cellular contexts [48–51]. Notably, increased proliferation and induction of Wnt genes (such as of Wnt3, Wnt5a, β-catenin) was observed upon TNFα-induced trans-differentiation of human mesenchymal stem cells towards the neural lineage . Taking advantage of hippocampal slice cultures as a useful model to study neurogenic processes ex vivo , we also demonstrated that TNFα-dependent activation of the Wnt pathway induces the proliferation of Nestin+ progenitor cells in the DG,and this effect is mediated via production of Wnt ligands. We further consider hippocampal microglia as the responder cells to TNFα-dependent enhancement of Wnt signaling, although other targets of TNFα may also mediate its effect on adult neurogenesis. While the mechanism remains to be elucidated, our data is in line with the role of TNFα-TNFR2 signaling in hippocampal neurogenesis in healthy mice  and upon various CNS pathologies. Thus, in the model of status epilepticus, deficiency for TNFR2 drastically abrogates the number of newly generated neurons in the hippocampus , though not in the SVZ . Complementary to these findings, infusion of neutralizing antibodies against TNFα had an anti-proliferative effect on hippocampal progenitors in a rat model of stroke . In cultured progenitor cells, TNFα potentially triggers the proliferation of neural stem/progenitor cells [57–59].
Understanding the role of TNFα in MS is complex and has been re-considered in the last years [60, 61]. While anti-TNFα therapy has been used successfully to treat various autoimmune pathologies, it was proven to be deleterious in MS patients . The aggravation of EAE in TNFR2-deficient mice, particularly in the chronic phase , may indicate a principal involvement of the TNFα-TNFR2 axis in processes of regeneration and tissue repair. Supporting this idea, recent findings demonstrated the beneficial effect of TNFα/TNFR2 signaling on expansion of the endogenous pool of oligodendrocyte progenitors and subsequent remyelination in a toxin-induced demyelinating model .
Activation of neurogenesis was reported in various models of CNS injuries and diseases [64, 65]. In MS brains, immature neurons were found in a subpopulation of subcortical white matter lesions, which may indicate an activation of neurogenic processes serving to compensate neuronal deficits . However, the origin of these cells is unknown. In contrast to this, specification of stem/progenitor cells seems to be shifted towards the glial lineage in the SVZ of MS patients compared to the healthy brain, leading to a diminished generation of Dcx+ neuroblasts . Increased proliferation and enhanced generation of immature neurons in the acute stage of EAE was previously reported by our  and other groups [68, 69]. Enhancement of neurogenesis in EAE is transient [16, 68] and seems to be specific to the hippocampal neurogenic niche, whereas in the SVZ, the generation of neuroblasts is reduced .
Our data raised the possibility that enhancement of Wnt activity may trigger hippocampal neurogenesis in order to replenish damaged neurons. Regarding this, it was shown that lentiviral-based overexpression of Wnt3a in the striatum enhances neurogenesis and neuronal differentiation, leading to an improved neuronal function in the model of focal ischemic injury . In addition to its neurogenic role, Wnts may promote axonal regeneration , synaptic transmission  and neuronal differentiation  in the injured CNS. Prolonged activation of Wnt signaling by Wnt-activating small molecule potentiator-1 (WASP-1) infusion into the hippocampus improves cognitive function in healthy adult mice and rescues memory loss in a mouse model of Alzheimer’s disease . Our data support these findings, which already raise a considerable interest in Wnts regarding the development of novel therapeutic approaches to treat neuronal injury.
Taken together, our data argue for a role of inflammation in transient activation of hippocampal Wnt signaling upon autoimmune mediated injury. Induction of Wnts can be accounted as a key factor triggering endogenous repair by engaging the activation of neuronal progenitors in the hippocampal neurogenic niche. Further experiments are needed to test whether therapeutic boosting of Wnt activity leads to tissue regeneration and thus provides a strategy to improve hippocampal function.
The following mice were used for experiments: 6 weeks old female SJL/J mice were purchased from Janvier (Le Genest-St-Isle, France). NestineGFP+ mice  were provided by the animal facility of the University of Düsseldorf. In these transgenic mice the second intron enhancer of the rat nestin gene was placed upstream of the minimum promoter of heat shock protein 68 (HSP68) fused to eGFP cDNA and a polyadenylation signal. Axin2lacZ/+  were received from the European Mouse Mutant Archive (EMMA). In Axin2lacZ/+ mice the lacZ gene, containing a nuclear localization signal, was introduced in frame to the endogenous Axin2 (also known as conductin or Axil) by replacing exon 2 . Axin2 is a direct target of TCF/LEF1-mediated transcriptional activation that encodes an inhibitor of the Wnt signaling pathway. All mice were housed in the animal research facility of the University of Düsseldorf under specific pathogen free conditions, a dark/light cycle of 12 h, a stable temperature of 22–24 °C and unlimited access to food and water.
All experimental procedures were conducted following the guidelines and protocols approved by the local animal welfare committee (Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen (LANUV); under protocol numbers G388-11 (active EAE in Axin2lacZ/+mice), G197-09 (BrdU injections in EAE mice), G363-09 (passive EAE in SJL/J)) and follow the animal research: reporting of in vivo experiments (ARRIVE) criteria and EAE induction guideline . For passive EAE, female SJL/J mice were subcutaneously immunized with 200 μg of recombinant proteolipid protein peptide (PLP139–151) (Pepceuticals) supplemented with 800 μg heat-inactivated Mycobacterium tuberculosis (strain H37RA, Difco) emulsified in 200 μl Complete Freund’s Adjuvant (CFA, Sigma-Aldrich) per mouse. On day 10 post immunization (p.i.), spleen and lymph node cells were isolated, and the resulting single-cell suspension was re-stimulated with PLP139–151 (10 μg/mL) in RPMI 1640 medium supplemented with 10 % fetal calf serum (FCS), penicillin/streptomycin, glutamate, and 2-mercaptoethanol (all from Invitrogen Life Technologies). After four days in culture, cells were harvested and injected intraperitoneal (i.p.) into naive female SJL/J mice (3 × 107 cells per mouse). Control mice received a single i.p. injection of PBS. Animals were weighed and scored daily, according to following clinical scale: 0 (no clinical signs), 1 (tail plegia), 2 (abnormal gait), 3 (hind limb paralysis), 4 (complete paralysis) to 5 (death or euthanasia) with intervals of 0.5 . Mice were euthanized either on day 6, 20 or 50 post-transfer. Hippocampi were dissected and stored at −80 °C for total RNA and protein isolation.
To induce active EAE in the Axin2lacZ/+ mouse strain, animals were immunized by subcutaneous injection with 200 μg of recombinant myelin oligodendrocyte glycoprotein (MOG35–55) peptide (Pepceuticals) in CFA (Sigma-Aldrich), supplemented with 800 μg heat-inactivated Mycobacterium tuberculosis (strain H37RA, Difco). Intraperitoneal injections of 200 ng pertussis toxin (PTX, Sigma-Aldrich) were performed on the day of immunization and on day 2 . The control group was treated with CFA only and with 200 ng pertussis toxin on day 0 and day 2. Mice were euthanized either on day 20, 30 or 50 post immunization.
Tissue or organotypic slices isolated from Axin2lacZ/+ mice were lysed on ice for 15 min in triton-containing lysis buffer (Applied Biosystems) and cleared by centrifugation at 10,000 g for 10 min at 4 °C. Then protein concentration was determined by BCA protein assay kit (Interchim). 50 μg of protein were applied for measuring β-galactosidase (β-gal) activity in triplicates using the “Galactostar Chemiluminescence Kit” (Applied Biosystems) in a white 96-well NUNC plate (Thermo Scientific) at room temperature (RT) for 2 h using a microplate reader (TECAN). The maximum of luminescence intensity for each individual sample was used to calculate mean of triplicate measurements. Lysates of corresponding β-gal-negative brain tissues were used to calculate background intensity.
Hippocampal tissue isolated from EAE mice was lysed on ice for 15 min in RIPA buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1 % (v/v) NP40, 0.5 % (v/v) sodium deoxycholate, 0.1 % (v/v) SDS) with protease and phosphatase inhibitors (Roche). Lysates were cleared by centrifugation at 14,000 g for 20 min at 4 °C and protein concentration was determined using a BCA protein assay Kit (Interchim). Protein samples were loaded on 8–16 % gradient “Mini-Protean TGX” gel (Biorad). Blotting onto a nitrocellulose membrane was performed using the “Trans-Blot Turbo System” (Biorad) for 7 min at 25 Volts. Afterwards, membranes were incubated with blocking buffer (5 % (v/v) skimmed milk in 0.05 % (v/v) PBS/Tween) for 1 h at RT and incubated overnight at 4 °C with following antibodies: mouse anti-neurofilament 160/200 (Sigma-Aldrich), mouse anti-PSD-95 (Abcam), rabbit anti-β-catenin (Cell Signalling), mouse anti-active-β-catenin (ABC, Cell Signalling), mouse anti-β-actin (Sigma-Aldrich). Primary antibodies were detected by incubation with IR-Dye secondary antibodies (Li-COR) for 1 h at RT and subsequently quantified using the Odyssey infrared imaging system (LI-COR). Optical density analysis was performed with ImageJ software (NIH).
RNA isolation, cDNA synthesis and qPCR
RNA was isolated with a RNA isolation Kit (Macherey-Nagel) or Trizol (Invitrogen Life Technologies). 1 μg of purified RNA was used for first-strand cDNA synthesis using “Superscript III Reverse Transcriptase” and oligo(dT) in a final volume of 20 μl according to manufacturer’s instruction (Invitrogen Life Technologies). Real-time quantification of gene expression was performed using a SYBR Green qPCR assay (Applied Biosystems) or TaqMan 5′FAM- and 3′TAMRA-labeled fluorescent probes (Eurofins MWG). PCR was performed using an “ABI 7500” real-time PCR system (Applied Biosystems) with standard cycling conditions: 40 cycles of 10 s at 95 °C, 60 s at 60 °C. Specificity of the PCR product was confirmed by examination of the dissociation reaction plot and PCR product detection on 2 % agarose gel. The samples were run in duplicates and the level of expression of each gene was normalized to the expression of the housekeeping gene glycerinaldehyd-3-phosphat-dehydrogenase (GAPDH). The sequences of all PCR primers used are available on request.
Organotypic slice cultures
Organotypic slice cultures (OSCs) were generated from 10 days old Axin2lacZ/+/nestineGFP+ pups as previously described . Briefly, the hippocampus was cut into 350 μm thick slices using a McIllwain tissue chopper (GaLa Instrumente). OSCs were dissociated in ice-cold dissecting medium (Hank’s Balanced Salt Solution (HBSS), Invitrogen Life Technologies) complemented with penicillin/streptomycin (100 U/ml, Invitrogen Life Technologies), 2.5 mg/ml glucose (Sigma Aldrich) and 10 mM kynurenic acid (Sigma Aldrich). OSCs were cultured on Millicell-CM culture plate inserts (Millipore) in culture medium (50 % (v/v) MEM, 25 % (v/v) HBSS, 25 % (v/v) heat-inactivated horse serum, 2 mM glutamine, penicillin/streptomycin (100 U/ml) (all from Invitrogen Life Technologies) and 2.5 mg/ml glucose (Sigma Aldrich) for 1 week at 37 °C in a humidified atmosphere with 5 % CO2. At this point OSCs were used for further experiments. For analysis of β-gal activity and Axin2 gene expression hippocampal Axin2lacZ/+ OSCs were cultured for seven days followed by treatment with Wnt3a (20 ng/ml; R&D Systems), interferon gamma (IFNγ, 100 U/ml; Immunotools), TNFα (1 ng/ml; Immunotools) or TNFα (1 ng/ml) with the inhibitor of Porcn function, IWP-2 (4 μM; R&D Systems). After 6 h of treatment, the medium was replaced with fresh cytokine-free medium and OSCs were cultured for additional 24 h. Afterwards, tissue was either fixed (β-gal assay; discussed below) or collected for RNA isolation. For the double treatment with IWP-2, the inhibitor was all the time present in the media.
For analysis of neurogenesis in hippocampal OSCs, cultures were first pretreated for 24 h with BrdU (0.5 μM, Sigma) to allow detection of proliferating cells . Next, recombinant cytokines: TNFα (1 ng/ml), interferon gamma (IFNγ, 100 U/ml) (both from ImmunoTools) or Wnt3a (20 ng/ml, R&D Systems) were added to the culture medium for 6 h, followed by washing and replacement with cytokine-free media for additional 24 h. IWP-2 (4 μM, R&D Systems) was added at the same time as the cytokines and was present until the end of the experiment. After this period, slice cultures were directly used to analyse β-gal activity (see above), RNA isolation or fixed with aldehydes for histological examination.
Mice were anaesthetized using isoflurane (Piramal Healthcare) and perfused with PBS. Brain and spinal cord were dissected and either directly frozen for X-gal staining or postfixed with 4 % (v/v) paraformaldehyde (PFA, Roth) for 16 h, followed by dehydration in a 25–30 % (v/v) sucrose solution. Tissue samples were cryopreserved in “TissueTek” (Sakura Fintek Europe) at −80 °C. For histological examination brains and spinal cords were cut on a cryostat (Leica). 20 μm slices were permeabilized with 0.5 % (v/v) Triton X-100 and blocked in 5 % (v/v) horse serum (Invitrogen Life Technologies) and 1 % (v/v) bovine serum albumin in PBS for 2 h. OSCs were washed two times in warm PBS, fixed for 1 h in 4 % PFA and permeabilized for 1 h with 1 % (v/v) Triton X-100 (Sigma Aldrich) in PBS. Primary antibodies were diluted in PBS containing 2.5 % (v/v) horse serum with 0.25 % (v/v) Triton X-100 and incubated with tissue samples overnight (or 48 h for OSCs) at 4 °C. Following primary antibodies were used: guinea pig anti-GFAP (1:1000; Synaptic Systems), rat anti-CD68 (1:350; Biolegend), goat anti-SOX2 (1:250; Santa Cruz); mouse anti-iNOS (1:100; BD Transduction Laboratories), rabbit anti-Iba1 (1:500, Wako Pure Chemical Industries), rabbit anti-GFP (1:1000, Abcam) goat anti-Doublecortin (Dcx, 1:250, Santa Cruz Biotech) and Hoechst 33342 dye (Molecular Probes). Visualization was performed by incubation with fluorescent Cy2-, Cy3- and Cy5-conjugated secondary antibodies (Millipore) for 1–2 h at RT. Hoechst dye 33258 (Molecular Probes) was used to counterstain nuclei followed by mounting on glass slides with “Immuno Mount” (Thermo Scientific). Immunostainings were analyzed on an Olympus BX51 microscope and overlaid using Photoshop 13.0 software (Adobe).
Quantification of Iba1+ and GFAP+ cells in the dentate gyrus has been performed in four animals per group. The number of positive cells per animal was calculated as a mean of labeled cells in mm2 of the DG area in 3–4 sections (200 μm distance between sections). The ratio of micron per pixel (1 pixel = 0.16125 μm at magnification x 40) was used to calculate the area analyzed.
For X-gal staining, 20 μm thick cryoslices or OSCs, prepared from Axin2lacZ/+ mice, were fixed with PBS containing 2 % (v/v) formaldehyde, 0.2 % (v/v) glutaraldehyde and 0.02 % (v/v) NP40 (all from Sigma Aldrich) for 5 min. After washing, slices were incubated for 2 h at 37 °C in X-gal staining solution, containing 10 mM K3Fe(CN)6, 10 mM K4Fe(CN)6, 0.02 % (v/v) NP40, 2 mM MgCl2 and X-gal (0.5 mg/ml) (all from Sigma Aldrich) dissolved in PBS. Samples were fixed with 4 % (v/v) PFA for 15 min and mounted on glass slides with “Immuno Mount” (Thermo Scientific).
BrdU treatment and immunostaining
For labeling of proliferating cells, 50 mg/kg body weight of 5-bromo-20-desoxyuridine (BrdU; Sigma Aldrich) was injected i.p. daily for five following days starting at the peak of disease (14 d.p.i.). EAE animals and healthy controls were euthanized and perfused 2 weeks after the first BrdU-injection. Mouse hippocampi were collected and cryopreserved (see histology part) and afterwards processed as previously described . Briefly, sections were incubated in 2 N HCl for 30 min at 37 °C and rinsed with 100 mM tetraborate buffer. Sections were incubated with primary rat anti-BrdU antibody (1:400; Upstate) and goat anti-Dcx antibody (1:500; Santa Cruz Biotechnology) overnight at 4 °C, followed by incubation with appropriate Cy2/Cy3-conjugated secondary antibodies (Millipore) for 1 h at RT.
Primary hippocampal cell cultures
Tissue processing and preparation of mixed glial cell population
Whole hippocampus from 2 to 5-day-old C57BL/6 mice were dissected and washed with ice cold HBSS supplemented with 0.2 % glucose. Tissue was finely minced with a razor blade and digested by StemPro Accutase (Gibco) for 20 min at 37 °C followed by 10 min digestion with 0.05 % Trypsin-EDTA (Gibco), DNase (250 U/ml, Worthington). Tissue was rinsed with DMEM supplemented with 10 % heat-inactivated fetal calve serum (FCS; Gibco), gently triturated by passing through a 1-ml serological pipette until a homogenous suspension appeared and filtered through a 40 μm cell strainer (BD Biosciences). Cells were suspended in Neurobasal medium supplemented with B27, glutamax, 1 % penicillin/streptomycin (all from Gibco) (hereinafter referred to as NB media).
Microglia and astrocyte cell cultures
Isolated hippocampal cells were plated on poly-ornithine-coated petri dishes and cultured in 10 ml of NB media (see above) supplemented with 10 % (v/v) FCS and 20 ng/ml of macrophage colony stimulating factor (M-CSF; Immunotools). 50 % of culture media was replaced every 4 days with fresh media. After 2–3 weeks incubation in a 5 % CO2 incubator at 37 °C, cells were collected by dissociation with StemPro Accutase (Gibco) for positive selection of CD11b+ cells or GLAST+ cells using magnetic microbeads (Miltenyi Biotech), according to the manufacturer’s instructions. This approach results in about 90 % purity of primary microglia cells and 95 % purity of primary astrocytes determined by immunostaining for Iba-1 and GFAP, respectively. Microglia and astrocytes were harvested, washed and plated on poly-L-ornitine coated plates or coverslips at densities appropriate for each assay.
Hippocampal progenitor cultures
Isolated hippocampal cells were cultured in serum-free proliferating conditions on uncoated Petri plates in NB supplemented with 20 ng/ml bFGF (Immunotools). After 3–4 days divided cells formed free-floating neurospheres. bFGF was added every second day. 30 % of culture media was replaced every 4 days with fresh media. After 1–2 weeks in culture neurospheres were collected and cells were dissociated by trypsin-EDTA solution. For further analysis, cells were cultured as adherent cells in proliferating medium on poly-ornithine-coated petri dishes or glasses. For gene expression analysis cells were treated for 6 h with mouse recombinant TNFα (0.2 ng/ml or 1 ng/ml; R&D) or Wnt3a (20 ng/ml; R&D).
Coculture of hippocampal progenitors with microglia
2 × 105 CD11b+ cells in 200 μl NB media were added to the upper well of each Transwelltm inserts (0.2 μm diameter holes, Ibidi) for 24 h. In parallel, 4 × 104 hippocampal progenitor cells dissociated from neurospheres were cultured separately on poly-L-ornithine coated coverslips in proliferating NB media containing 20 ng/ml bFGF. 24 h after culturing of microglia on transwells, cells were activated with TNFα (1 ng/ml) for 4 h and washed carefully with NB media. After this, the transwells containing microglia were set on hippocampal progenitor cultures and incubated for 24 h in proliferating NB media (containing 5 ng/ml bFGF) in the presence or absence of soluble Frizzled-8 (sFz8; 1 μg/ml; R&D). 10 μg/ml of BrdU was added for last 2 h of experiment to allow detection of proliferating cells. Goat anti-SOX2 (1:250; Santa Cruz) and rat anti-BrdU (1:500; Upstate) primary antibody were used to detect mitotically active cells in hippocampal progenitor cultures.
All values in the figures are shown as mean + SEM. Statistical analysis was performed with GraphPad Prism 5 (GraphPad Software). The p values of *p < 0.05; **p < 0.01; ***p < 0.001 were determined to be statistically significant by using Student’s t-test.
ABC, active-β-catenin; ARRIVE, Animal Research: Reporting of In Vivo Experiments; BrdU, 5-Bromo-20-desoxyuridine; CFA, Complete Freund’s Adjuvant; CNS, central nervous system; d.p.i., days post immunization; Dcx, doublecortin; DG, dentate gyrus; EAE, experimental autoimmune encephalomyelitis; ECS, electroconvulsive seizures; FCS, fetal calf serum; GAPDH, glycerinaldehyd-3-phosphat dehydrogenase; GSK-3β, glycogensynthase-kinase-3 beta; HBSS, hank’s balanced salt solution; HSP68, heat shock protein 68; i.p., intraperitoneal; IFNγ, interferon gamma; IL-1β, interleukin-1 beta; LANUV, Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen; Lef1, lymphoid enhancer-binding factor 1; LPC, lysolecithin; MOG, myelin oligodendrocytes glycoprotein; MS, multiple sclerosis; NeuroD1, neurogenic differentiation 1; NF-H, neurofilament-H; OSC, organotypic slice culture; PFA, paraformaldehyde; PLP, proteolipid protein peptide; Prox1, prospero homeobox 1; PSD-95, postsynaptic protein 95; PTX, pertussis toxin; RT, room temperature; SEM, standard error of mean; SGZ, subgranular zone; SVZ, subventricular zone; TCF, transcription factor; TGFβ1, transforming growth factor beta 1; TNFα, Tumor necrosis factor alpha; WASP-1, Wnt-activating small molecule potentiator-1.
We would like to thank EMMA (European Mouse Mutant Archive) for providing the Axin2lacZ/+ mice.
Our research is supported by grants from the “Forschungskommission” of the Medical Faculty (Heinrich-Heine-University Düsseldorf) to T.P. and O.A. (06/2012), the Deutsche Forschungsgemeinschaft (SPP1710, BE3529/5-1 to CB) and the Walter and Ilse Rose Foundation (to O.A.).
Availability of data and materials
All requested data are available via T.P.
RS and TP designed the experiments and performed the biochemical analysis of tissue. TP, RS, CB, JC and OA wrote the manuscript. J.I. and R.S. have performed the EAE experiments, including immunization, treatment, scoring, isolation and processing of animal tissue. F.S. and B.K. contributed to treatment of animals and tissue collection. HPH and OA provided critical input. All authors read, revised and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
All experimental procedures were conducted following the guidelines and protocols approved by the local animal welfare committee (Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen (LANUV); under protocol numbers G388-11 (active EAE in Axin2lacZ/+mice), G197-09 (BrdU injections in EAE mice), G363-09 (passive EAE in SJL/J)).
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