Background
Neuroinflammation is a common hallmark of several CNS disorders, which is characterized by the upregulation of proinflammatory cytokines and chemokines such as
Tnf, Ccl2 and
Cxcl10 as well as the infiltration of activated immune cells [
1].
The activation of the NF-κB family of transcription factors is a key step in the regulation of inflammatory and immune responses. However, these proteins also regulate gene expression in a variety of other physiological processes like cell proliferation, differentiation and survival, as well as specific CNS functions including learning and memory [
2]. In resting cells, NF-κB dimers are sequestered in the cytosol by inhibitory proteins of the IκB family. The crucial step in NF-κB activation is the phosphorylation of IκB proteins by the activating IκB kinase complex. IKK2 is the critical kinase subunit inducing the canonical signalling pathway, which is essentially involved in the regulation of inflammation. Phosphorylation of inhibitory IκB proteins initiates their ubiquitination and subsequent proteosomal degradation, followed by the release and nuclear translocation of active NF-κB dimers, which then induce the expression of NF-κB target genes [
3‐
5].
Members of the IKK/NF-κB system are widely expressed in the nervous system and different factors stimulate NF-κB activation in the CNS, including damage-associated molecular patterns, pathogen-associated molecular patterns, cytokines, chemokines, neurotransmitters, neurotrophic factors and neurotoxins. NF-κB is activated both under physiological conditions, e.g. by synaptic activity, as well as in pathological conditions [
6‐
8]. Previous studies reported that the IKK/NF-κB signalling system is deregulated in various neuroinflammatory conditions as Alzheimer’s disease (AD), Huntington’s disease (HD), stroke, hydrocephalus and schizophrenia [
9‐
15]. Depending on the cell type and pathophysiological context, both protective and deleterious roles of NF-κB signalling were found in CNS diseases, e.g. in ischemic injury [
16,
17].
We have previously shown that suppression of IKK/NF-κB signalling in neurons reduces infarct formation in an animal model of stroke. Vice versa ectopic activation of IKK2 in a similar context increases the infarct size after cerebral ischemia [
9]. These findings indicate a central role of neuronal NF-κB in the regulation of cell survival in acute stroke pathogenesis.
In order to characterize the detailed role of neuronal NF-κB in the pathogenesis of chronic neurodegenerative disorders, we analysed the consequences of persistent NF-κB activation in neurons using the IKK2
nCA model. As NF-κB activation is sufficient to induce strong inflammatory processes in various cell types and tissues [
11,
18‐
20], we particularly asked whether chronic NF-κB activation in neurons is sufficient to drive a neuroinflammatory response on its own and if so, what are the pathological consequences.
Unexpectedly, IKK2nCA animals did not show massive signs of neuroinflammation such as prominent proinflammatory cytokine expression and infiltration of immune cells. Interestingly, they exhibited downregulation of the neurotrophic factor Bdnf, which correlates with an impairment of cognitive functions and degeneration of the dentate gyrus.
Discussion
The IKK/NF-κB signalling system is proposed to be critically involved in the pathogenesis of various neurological diseases [
8]. On the one hand, it is well characterized as a central regulator of inflammatory responses by controlling the expression of multiple proinflammatory acting genes [
3,
22]. On the other hand, IKK/NF-κB signalling is crucially involved in neuronal differentiation and various CNS functions [
6‐
8]. However, due to its complex regulation in different cell types and diverse responses to different physiological and pathological conditions, the precise function of the IKK/NF-κB system in CNS physiology and pathology is only partially understood.
Former studies suggested an ambivalent role of the IKK/NF-κB system in the pathogenesis of neurological disorders [
8,
43]. Due to its proinflammatory function, NF-κB activation is able to trigger neuronal dysfunction, aging and cell death, thereby increasing severity of CNS diseases [
8,
11,
44,
45]. In contrast, NF-κB activation can also mediate neuroprotection [
6,
8,
46]. Previously, we found that IKK2/NF-κB activation in neurons increases tissue damage in a mouse model of stroke, probably by enhancing the overall neuroinflammatory process elicited by this acute insult [
9]. Therefore, we wanted to further investigate the role of IKK2-mediated neuron-specific NF-κB activation in the induction of neuroinflammatory responses using the IKK2
nCA model. We hypothesized that constitutive IKK2 activation in neurons is sufficient to induce inflammation, as it was demonstrated in several non-neural cell types as well as in astrocytes [
11,
18‐
20,
23]. However, with the exception of microgliosis and astrogliosis observed in the DG, neuron-specific IKK2 activation did not result in a prominent inflammatory phenotype including infiltration of immune cells. Consistently, typical proinflammatory NF-κB target genes like
Ccl2,
Tnf,
Ptgs2, Lcn2 and
Cxcl10 that are highly expressed in other inflammatory conditions are either moderately or not induced in the IKK2
nCA model. This argues for a specific function of IKK2/NF-κB signalling in neurons.
What could be the reason for this unexpected response? As NF-κB is activated by synaptic signalling, such kind of NF-κB activation in neurons would already create a proinflammatory environment under the physiological conditions of neurotransmission. Vice versa, inflammation-mediated NF-κB activation in neurons would lead to functional conflicts like deregulation of NF-κB-mediated neurite outgrowth and synaptic plasticity. Therefore, a functional separation of neuronal IKK/NF-κB signalling versus inflammatory IKK/NF-κB signalling in other cells could be of physiological advantage. Several studies showed important functions of NF-κB in neuronal differentiation, including neurite outgrowth, formation and remodelling of synaptic connections, axogenesis and neuronal function, e.g. hippocampal learning and memory formation [
6,
26,
28,
47‐
50]. These studies are mainly based on experimental approaches inhibiting the IKK/NF-κB signalling system in neurons, therefore it could be anticipated that neuronal IKK/NF-κB activation might result in a phenotype that improves neuronal survival and cognitive capabilities. However, this idea appears to be in stark contrast to our findings. One plausible explanation for this discrepancy could be the duration of IKK/NF-κB signalling. In our model we induce permanent IKK/NF-κB activation over weeks, whereas in the physiological context of learning and memory rather a transient or repetitive activation is known to occur which is e.g., elicited by the neurotransmitter glutamate, known to induce NF-κB in synaptic signalling [
51,
52]. As excessive glutamate signalling results in excitotoxic cell death [
53], we can speculate that the constitutive NF-κB activation in our model is probably detrimental to the DG neurons. Interestingly, pharmacological inhibition of IKK2 was able to block NMDA-induced excitotoxic cell death in hippocampal neurons and oligodendrocytes [
54]. In line with the view that especially a transient NF-κB activation kinetic improves neuronal differentiation, Russo et al. shows that stereotactic application of a virus expressing IKK2-CA to the nucleus accumbens leads to spine formation within a short time of 3 days [
50].
The adequate function of the adult dentate gyrus depends on both healthy mature granule cells as well as ongoing neurogenesis [
55] and NF-κB was shown to be critically involved in different aspects of adult neurogenesis using loss-of-function approaches [
28,
56]. Since we only see neurodegeneration in the DG, a hypothesis might be that constitutive IKK2 activity also interferes with neurogenesis, which then results in a depletion of neurons in the GCL. However, a roughly 50% reduction in the cell count at 9M is difficult to explain solely by blockade of ongoing adult neurogenesis in the IKK2
nCA model but rather suggests active neurodegeneration. Furthermore, we could observe increased evels of Ki67-positive cells upon transgene inactivation arguing for elevated neurogenesis that may account for an active regeneration process of the DG rather than simple prevention of further neurodegeneration.
Imielski et al. [
28] showed that the structural degeneration of the DG depends on apoptotic cell death, which was not detected in our model as measured by cleaved caspase-3 and TUNEL assay (Additional file
5). Instead, we could identify degenerating neurons in the DG but not in other brain regions by Fluoro-jade staining suggesting that IKK2-CA induces cell death but this cell death is independent of apoptosis or is due to a very slow rate of apoptosis that may escape detection. So far it remains largely open why this degeneration process is specific to the DG. However, our findings implicate that a combination of Bdnf decrease and Tnf increase (and possibly changes in other so far unkown factors) may account for the selective neurodegeneration of the dentate gyrus in the IKK2
nCA model. The structural restoration of the dentate gyrus after transgene inactivation in both models implies that fine balanced levels of NF-κB are required for appropriate neuronal survival and homeostasis in this brain region. Therefore, reactivation of the IKK/NF-κB system for therapeutic measures of neuro-regeneration in the context of dementia-associated diseases as suggested by Imielski et al. [
28] is apparently critical and surely dose-dependent.
The neurodegenerative effect of constitutive IKK2 signalling could be due to the composition of the activated NF-κB dimers. In IKK2
nCA mice the canonical NF-κB pathway is active, most likely leading to the nuclear translocation of p65 containing dimers that are found to regulate apoptosis associated genes [
57]. Also, there might be an under-representation of c-Rel containing dimers, which are known to promote neuronal survival by enhancing
Bcl2l1 transcription [
57]. Corresponding to this, a downregulation of pro-survival genes like
Bcl2 and
Bcl2l1 was detected at older age in IKK2
nCA mice, although both of these are regulated by NF-κB [
58,
59]. Moreover, the decreased Bdnf expression in IKK2
nCA mice can be proposed as a potential mechanism that interferes with neuronal survival [
60] because it also correlates with a decline in
Bcl2 and
Bcl2l1 levels [
42]. Bdnf is well known to regulate cognitive tasks, synaptic plasticity and neuronal survival by activating its receptor TrkB [
39,
41,
60,
61] and its expression is compromised in brain disorders as AD, HD, Rett syndrome and schizophrenia [
62,
63]. Thus, the reduced levels of Bdnf and Bdnf-regulated AMPA receptors might attribute to the impaired hippocampal learning and the atrophy of the DG observed in our model.
Nevertheless, other factors may also contribute to the impaired learning and atrophy of the dentate gyrus. There is the possibility that the microgliosis and astrogliosis observed in the DG are sufficient to cause the neurodegeneration in IKK2
nCA mice [
64,
65]. Together with the elevated Tnf levels, such kind of inflammatory processes may influence learning and memory as well as neuronal survival. This might contribute to the observed phenotype, although the importance of low-grade neuroinflammation for learning and memory and neurodegeneration is still controversially discussed [
66].
What is the underlying molecular mechanism resulting in reduced Bdnf,
Bcl2 and
Bcl2l1 expression in IKK2
nCA mice? The observed downregulation for Bdnf is rather surprising, as Bdnf is an NF-κB target gene in astrocytes [
67] and would therefore expected to be rather upregulated in neurons, too. Although we did not investigate the mechanism behind this repression of Bdnf in IKK2
nCA mice, previous studies identified a similar kind of downregulation of target genes by NF-κB, e.g. in the case of hypoxia,
Tnf-dependent EAAT2 expression, or in the regulation of anti-apoptotic genes after treatment of cells with DNA-damaging agents [
68‐
70]. Campbell et al. showed that the cytotoxic stimuli like ultraviolet light (UV-C), and daunorubicin, downregulated the expression of anti-apoptotic NF-κB target genes like
Bcl2, Bcl2l1, Xiap and
A20, thus providing the possibility that canonical NF-κB activation may account for induction and repression of target genes depending on the presence of coactivators, given cell type and induction mechanism [
69]. There is also the possibility that NF-κB mediated changes in epigenetic gene regulation may affect Bdnf expression [
71‐
73]. Moreover, IKK2 has been previously described to phosphorylate Bcl-xL, a mechanism associated with reduced expression of this gene in stressed, post-mitotic neurons [
10]. A recent publication by Zhang et al. [
45] addressed the role of IKK2/NF-κB signalling in the hypothalamus, which increases with aging and mediates suppression of hypothalamic-gonadotropin-releasing hormone (GnRH1) expression finally promoting systemic aging. They found that elevated IKK2 and NF-κB activity induces cJun/cfos and PKC levels, which are able to inhibit
Gnrh1 promoter activity. This mode of NF-κB-mediated inhibition of gene expression might also account for NF-κB-mediated Bdnf repression since Bdnf expression is known to be regulated by multiple promoters.
More importantly, the IKK2-CA transgene and Bdnf expression pattern do not coincide very well in the DG of IKK2
nCA mice (Figure
6D). Bdnf expression is located to the hilus region whereas IKK2-CA protein is detected in GCL neurons. In support of this, GABAergic interneurons present in the hilus are devoid of
Camk2a expression thereby excluding Camk2a-driven transgene expression in these neurons [
74]. This strongly argues for a scenario that IKK2-CA mediated NF-κB activation does not directly influence Bdnf expression. Rather, a so far unknown factor/s released by IKK2-CA positive neurons suppresses Bdnf production in a paracrine manner in the vicinity of the hilus, a process that necessarily does not depend on NF-κB-mediated gene regulation.
Notably, recent work by Han et al. reported that the
Camk2a-tTA transgene, also used in the present study to drive IKK2-CA expression, itself exhibits a degenerating effect on the neurons which was not recognized by the scientific community for many years. Moreover, they observed that this degeneration was permanently rescued by administration of DOX during the first 6 weeks of life [
75]. As the IKK2
nCA animals were treated with DOX up to the age of 4 weeks, most likely, we also avoid the tTA-induced degeneration. Consistent with that, the
Camk2a-tTA-induced neurodegeneration gets obvious already at the age of 2 months, whereas a IKK2
nCA animals do not show atrophy up to the age of 3 months rather develop degeneration between 3 and 6 month age periods. Furthermore, IKK2
nCA animals were bred in pure NMRI background, an outbred model, which is different from the analysed hybrid strains sensitive for tTA-induced degeneration.
Materials and methods
Transgenic mice
Mice were kept in a specific pathogen-free (SPF) animal facility at University of Ulm. Double transgenic mice (CaMK2a-tTA x luciferase-(tetO)
7-CA-IKK2) were generated by directly crossing CaMK2a-tTA mice with single transgenic mice carrying a luciferase-(tetO)
7-IKK2-CA transgene. The latter mice have a bidirectional promoter (tetO)
7 which regulates the expression of luciferase reporter gene as well as IKK2-CA [
9]. Both single transgenic mouse lines were bred on the NMRI background. In order to avoid any interference with brain development, inactivation of transgene expression was carried out by administration of DOX (0.1 g/l, MP Biomedicals) in 1% sucrose, in the drinking water to the dams during pregnancy, and to pups until 4 weeks of age. As control animals usually (tetO)7-IKK2-CA single transgenic littermates were used. Genotyping was made by PCR.
All animal experiments were performed in compliance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health and the German Animal Protection Act and was approved by the Regierungspräsidium Tübingen, Germany that is the responsible government agency for animal rights.
In vivo bioluminescence assay
Images for brain localized transgene expression were obtained with IVIS 200 System (Caliper) by performing IVIS as described by [
76] and [
77].
Luciferase assay
For the detection of transgene, the tissue samples were snap frozen in liquid nitrogen, and the extracts were prepared by homogenizing the pulverized tissue in TNT extraction buffer. Luciferase reporter assay was performed as previously described [
76].
Immunoblotting
Native tissue protein extracts were prepared as described previously [
18]. Equal amounts (50 μg) of total proteins were resolved on SDS-PAGE gels and transferred to nitrocellulose membranes by a standard western blot protocol. Membranes were then blocked with 5% non-fat dry milk in TBS buffer for 1 h at room temperature. Incubation with primary antibody (see below) was performed in blocking solution overnight at 4°C or for 2 h at room temperature. After washing with TBS, incubation with the HRP coupled secondary antibody was performed for 1 h at room temperature.
Membranes were exposed to ECL detection reagent from Invitrogen for the detection of signals. The “Intelligent Dark Box” (Fuji) was used to reveal the luminescence signals.
Histology and immunostaining
For the histopathological analysis, animals were perfused with PBS and 4% PFA and decapitated. Brains were then fixed by immersion in 4% PFA (3-4 h at room temperature), dehydrated, embedded in paraffin, and cut to 7 μm thick coronal sections using the microtome Microm HM355S (Thermo Scientific, Waldorf, Germany). For making cryosections, the brains were frozen as described by [
76] and the frozen brains were sectioned to 8 μm thick slices using cryotome Leica CM1900 (Leica Microsystems, Wetzlar, Germany). To identify the morphology of brain, nissl staining was performed with the paraffin sections. For immunofluorescent staining, after rehydration, heat mediated antigen retrieval was performed with sodium citrate (10 mM, pH 6, 0.05% Tween 20) or Tris-EDTA (10 mM Tris, 1 mM EDTA, pH 9, 0.05% Tween 20) and for full permeabilization sections were incubated with 0.5% Triton X-100 for 30 min. Sections were washed with PBS and blocked with 5% BSA with Fc Block antibody (BD Pharmingen, dilution 1:100) for 1h. Incubation with the primary antibodies (in 5% BSA) was performed overnight at 4°C, secondary antibodies were applied for 1h at room temperature with DAPI for nuclear counterstaining. For GFAP, cleaved caspase-3 and CD45 staining, the cryosections from natively frozen brains were fixed with cold methanol (-20°C). Blocking and staining was performed as described above. Fluorescence images were acquired with the Zeiss Axiovert 200 M microscope with filters for DAPI, FITC/Alexa Fluor 488, and TexasRed/Alexa Fluor 568/594 and the Zeiss Axiovision software. For every channel exposure times were adjusted separately and kept same for the complete session. Adjustment of contrast and brightness was performed distinctly for each channel, but equally in all compared pictures.
For immunohistochemistry, paraffin sections were treated with 3% hydrogen per oxide, heat mediated antigen retrieval was performed with citric acid buffer (pH 6.0), washed with TBS and blocked with 5% BSA for 1h at room temperature. Afterwards, slides were incubated with the primary antibodies against Iba1, RelA or Bdnf over night at room temperature. Biotinylated rabbit secondary antibody was applied for 30 min at room temperature, subsequently slides were treated with streptavidin HRP and the signals for Iba-1 were obtained using DAB, whereas by AEC reagent for RelA and Bdnf.
Fluoro-jade B staining was carried out with the cryosection of animals perfused with 4%PFA as described by [
78].
Images were obtained by Leica CTR5500 microscope.
Antibodies for immunostaining and immunoblotting
Goat anti-human IKK2 (sc-7329), rabbit anti-RelA (sc-372), rabbit anti-Bdnf (sc-546), rabbit anti-Erk2 (sc-154) and HRP-conjugated goat anti-rabbit or donkey anti-goat were obtained from Santa Cruz Biotechnology. Mouse anti-NeuN from Millipore (MAB 377), rabbit anti-GFAP from Abcam (Ab56777), rabbit anti-Cleaved-caspase3 from cellsignalling (9661), rat anti-CD45 from BD Pharmingen (BD550539) and rabbit anti-Iba1 was obtained from WAKO (019-19741).
Alexa Fluor labelled secondary antibodies were obtained from Invitrogen, DAPI was purchased from MERCK, and biotinylated anti-rabbit from VECTOR Laboratories U.S.A.
TUNEL assay
TUNEL Assay was performed with paraffin sections using the Calbiochem TUNEL Assay kit, according to the manufacturer’s instructions.
Kinase assay
Kinase assay was performed to measure basal IKK activity. The IKK complex was immunoprecipitated from striatal lysates with an antibody recognizing NEMO using protein A beads. The
in-vitro-kinase assay was done as described in [
9], taking recombinant GST-IκBα as substrate. Radiolabelled ATP was used, whose γ-Phosphate is transferred to the substrate GST-IκBα in the presence of the IKK complex proportional to its activity. Kinase activity was determined by detection of radiolabelled GST-IκBα after SDS-PAGE and western blot. For loading control, NEMO levels were detected in the precipitates by immunoblot.
RNA from hippocampus and cortex was isolated with the PeqGOLD Trifast (peQlab) kit from the frozen tissue pulverized with a morter and pestle under liquid nitrogen. cDNA was synthesized using Roche Transcriptor High fidelity cDNA synthesis kit with 0.8 μg of total RNA and oligo-dT-primers according to the manufacturer’s instructions. Quantitative Realtime-PCR assays were performed with the Lightcycler 480 Instrument (Roche Applied Science) with primers and hydrolysis probes designed by the Roche Universal Probe Library system. Hypoxanthine-guanine phosphoribosyltransferase gene (Hprt) was used as housekeeping gene.
Primer sequences and UPLs used for the quantitative real time PCR are as follows: Hprt (5′-GGA GCG GTA GCA CCT CCT-3′, 5′- CCT GGT TCA TCA TCG CTA ATC-3′, UPL no. 69), Tnf (5′- TGC CTA TGT CTC AGC CTC TTC-3′, 5′- GAG GCC ATT TGG GAA CTT CT-3′, UPL no. 49), Ccl2 (MCP1) (5′- CAT CCA CGT GTT GGC TCA-3′, 5′- GAT CAT CTT GCT GGT GAA TGA GT-3′), Cxcl10 (IP10) (5′- GCT GCC GTC ATT TTC TGC-3′, 5′ TCT CAC TGG CCC GTC ATC-3′ UPL no. 3), Ptgs2 (cycloxygenase 2) (5′- GAT GCT CTT CCG AGC TGT G-3′, 5′- GGA TTG GAA CAG CAA GGA TTT - 3′, UPL no. 45), Bdnf (5′-AGT CTC CAG GAC AGC AAA GC-3′, 5′-TGC AAC CGA AGT ATG AAA TAA CC-3′, UPL no. 31), Ngf-β (5′-AAT TAG GCT CCC TGG AGG TG-3′, 5′-TGG ACT GCA CGA CCA CAG-3′, UPL no. 22), Ntf3 (5′-CGA CGT CCC TGG AAA TAG TC-3′, 5′-TGG ACA TCA CCT TGT TCA CC-3′, UPL no. 29), Igf2 (5′-CGC TTC AGT TTG TCT GTT CG-3′, 5′-GCA GCA CTC TTC CAC GAT G-3′, UPL no. 40), Prkaca (PKA catalytic α) (5′-GGC TCT CGG AGT CCT CAT C-3′, 5′-CAG AGC TGA AGT GGG ATG G-3′, UPL no. 46) Gria1 (GluR1) (5′- AGG GAT CGA CAT CCA GAG AG-3′, 5′- TGC ACA TTT CCT GTC AAA CC-3′, UPL no. 62), Gria2 (GluR2) (5′- CCA ATG GGA TAA GTT CGC ATA-3′, 5′- GCA CAG CTT GCA GTG TTG A-3′, UPL no. 110), Gria3 (GluR3) (5′- AAG CCG TGT GAT ACG ATG AA-3′, 5′- TGC CAG GTT AAC AGC ATT TCT-3′, UPL no. 31), Gria4 (GluR4) (5′- CTG CCA ACA GTT TTG CTG TG-3′, 5′- AAA TGG CAA ACA CCC CTC TA -3′, UPL no. 48), Bcl2 (5′- GTA CCT GAA CCG GCA TCT G -3′, 5′-GGG GCC ATA TAG TTC CAC AA-3′, UPL no. 75), Bcl2l1 (Bcl-xL) (5′- TGA CCA CCT AGA GCC TTG GA - 3′, 5′- TGT TCC CGT AGA GAT CCA CAA - 3′, UPL no. 2), Il6 (5′- CT ACC AAA CTG GAT ATA ATC AGG A - 3′, 5′- CCA GGT AGC TAT GGT ACT CCA GAA-3′, UPL no. 6),), Ccl5 (RANTES) (5′- TGC AGA GGA CTC TGA GAC AGC - 3′, 5′- GAG TGG TGT CCG AGC CATA - 3′, UPL no. 110), Tdo2 (5′- AAT CAG AGC AGG AGC AGA CG - 3′, 5′- TTG GCT CTA AAC CAG GTG TTC -3′, UPL no. 22), Mrg1b (5′- AGA CAA GGA CGC AAT CTA TGG - 3′, 5′- GCT CGC ACT TCT CAA AAA CC -3′, UPL no. 6) and Lcn2 (5′- CCA TCT ATG AGC TAC AAG AGA ACA AT - 3′, 5′- TCT GAT CCA GTA GCG ACA GC -3′, UPL no. 58).
BDNF quantification
BDNF positive area was measured using ImageJ64 software. BDNF negative area was cut from the photomicrographs, subsequently, quantification was made at a particular threshold level by measuring BDNF positive area in the specific (red) channel.
Quantification of astrogliosis
GFAP positive area was measured using ImageJ64 software. The background was subtracted after importing the images in ImageJ64. Similar threshold level was set for every image, on the dark background (in the particular channel; texas red) and the positive signals were quantified.
Quantification of microglia and Ki67 positive cells
Iba1-positive microglia were counted in two fields of cortex, CA1 region and dentate gyrus per mouse. Ki67-positive cells were counted in two fields of dentate gyrus for every animal in each age group. Images were obtained using Leica CTR5500 microscope. Cell count was performed manually using ImageJ64 software.
Quantification of nuclear cells in the dentate gyrus and CA1 region
Cresyl violet stained images were obtained from control and transgenic mouse brain sections. Particular areas were defined in the CA1 region and in the upper and lower blades of the dentate gyrus for quantification of cell number. Nine coronal planes were selected from rostral to caudal part of brain (bregma: ~ 1.46, 1.70, 1.82, 2.06, 2.18, 2.30, 2.54, 2.80, and 3.08 mm according to ref. [
79] to ensure similar topography and avoid errors due to the differences in orientation of planes. Cells were counted in the specified areas of matched planes using ImageJ64 software. Percentage cell loss was determined with respect to the cell number in the control animals. Primary cortex was measured (bregma: ~ 4.98 mm) with ImageJ64. Images for olfactory bulbi were taken at bregma: ~ 4.28 mm.
Bdnf ELISA
ELISA for Bdnf was performed with Promega (Madison, WI, USA) ELISA kit. Protein extracts from cortex and hippocampus were made in the lysis buffer as described by Promega (1% Nonidet P-40, 20 mM Tris, pH 8.0, 137 mM NaCl, 10% glycerol, 1 mM phenyl-methylsulfonyl fluoride, protease inhibitor Complete mini (Roche), 0.5 mM sodium vanadate). The procedure was made according to the manufacturer’s instructions.
Morris water maze task
The experimental subjects were 6 and 9 month old male mice (Co group, n = 13 IKK2
nCA, (n = 13), which were housed individually in a 12 h light/dark schedule for one week before the Morris water maze (MWM) task was performed to get familiar with the place. Mice were habituated to handling by the experimenter at least for three days before the experiment. The experiment was performed as described by Vorhees and Williams [
34] with some modifications; the animals were exposed to this spatial learning paradigm for eight consecutive days with four trials per mouse each day with an inter trial interval of 15 min. Visual cues were provided outside the pool on the walls of the room to help in navigation. Position of platform was kept same throughout the training session, however, each day the start locations for the trials were random. For all days, the experiment was performed at the same time of the day with the same environmental conditions. The room was sound proof and the experimenter was blind about the genotypes of mice. Before the MWM task, a visible platform test was performed to exclude motor and visual acuity impairment. The platform was marked with a flag, and tracking length and latency to reach the platform were recorded.
All data including track length, position of the animals and latency to find the hidden platform were recorded using an automated video tracking and analysis system, Viewer II software, (Biobserve, Bonn, Germany).
Statisticical analysis
Statistical analysis was performed with the Prism-software (Graphpad). All data are shown as mean ± SEM. Statistical significances were determined by using unpaired Student’s t test or as stated in the figure legends. (* p < 0.05; ** p < 0.01; *** p < 0.001).
Competing interests
All authors of the manuscript declare that none of the authors have any financial interest related to this work.
Authors’ contribution
AM carried out in vivo imaging, luciferase assays, western blot analysis, immunofluorescent staining, immunohistochemistry, Nissl staining, performed cell counting, qRT-PCR, ELISA and MWM, and drafted the manuscript. ML conducted experiments on microgliosis, helped to conceive the study and drafted the manuscript. TW contributed to the design and conception of experiments, and drafted the manuscript. BB carried out breeding of animals, conceived the study, participated in the design and coordination, and drafted the manuscript. All authors read and approved the final manuscript.