Methods for the Modulation and Analysis of NF-kappaB-dependent Adult Neurogenesis

Widera D, Müller J, Imielski Y, Heimann P, Kaltschmidt C, Kaltschmidt B. Methods for the Modulation and Analysis of NF-kappaB-dependent Adult Neurogenesis. Journal of Visualized Experiments. 2014;(84):e50870

Regrowing the adult brain: NF-kappaB controls functional circuit formation and tissue homeostasis in the dentate gyrus

Imielski Y, Schwamborn JC, Lüningschrör P, et al. Regrowing the adult brain: NF-kappaB controls functional circuit formation and tissue homeostasis in the dentate gyrus. PLoS ONE. 2012;7(2): e30838.Cognitive decline during aging is correlated with a continuous loss of cells within the brain and especially within the hippocampus, which could be regenerated by adult neurogenesis. Here we show that genetic ablation of NF-kappaB resulted in severe defects in the neurogenic region (dentate gyrus) of the hippocampus. Despite increased stem cell proliferation, axogenesis, synaptogenesis and neuroprotection were hampered, leading to disruption of the mossy fiber pathway and to atrophy of the dentate gyrus during aging. Here, NF-kappaB controls the transcription of FOXO1 and PKA, regulating axogenesis. Structural defects culminated in behavioral impairments in pattern separation. Re-activation of NF-kappaB resulted in integration of newborn neurons, finally to regeneration of the dentate gyrus, accompanied by a complete recovery of structural and behavioral defects. These data identify NF-kappaB as a crucial regulator of dentate gyrus tissue homeostasis suggesting NF-kappaB to be a therapeutic target for treating cognitive and mood disorders

Schwann Cells can be reprogrammed to multipotency by culture.

Widera D, Heimann P, Zander C, et al. Schwann Cells can be reprogrammed to multipotency by culture. STEM CELLS AND Development. 2011;20(12):2053-2064.Adult neural crest related-stem cells persist in adulthood, making them an ideal and easily accessible source of multipotent cells for potential clinical use. Recently, we reported the presence of neural crest-related stem cells within adult palatal ridges, thus raising the question of their localization in their endogenous niche. Using immunocytochemistry, reverse transcription-polymerase chain reaction, and correlative fluorescence and transmission electron microscopy, we identified myelinating Schwann cells within palatal ridges as a putative neural crest stem cell source. Palatal Schwann cells expressed nestin, p75(NTR), and S100. Correlative fluorescence and transmission electron microscopy revealed the exclusive nestin expression within myelinating Schwann cells. Palatal neural crest stem cells and nestin-positive Schwann cells isolated from adult sciatic nerves were able to grow under serum-free conditions as neurospheres in presence of FGF-2 and EGF. Spheres of palatal and sciatic origin showed overlapping expression pattern of neural crest stem cell and Schwann cell markers. Expression of the pluripotency factors Sox2, Klf4, c-Myc, Oct4, the NF-κB subunits p65, p50, and the NF-κB-inhibitor IκB-β were up-regulated in conventionally cultivated sciatic nerve Schwann cells and in neurosphere cultures. Finally, neurospheres of palatal and sciatic origin were able to differentiate into ectodermal, mesodermal, and endodermal cell types emphasizing their multipotency. Taken together, we show that nestin-positive myelinating Schwann cells can be reprogrammed into multipotent adult neural crest stem cells under appropriate culture conditions

Impaired NF-κB signalling interferes with spatial pattern separation.

<p>A <i>spatial pattern separation-Barnes Maze (SPS-BM)</i> was developed to test DG dependent spatial pattern separation (<b>a</b>). During consecutive days of training the mice had to find the food house (location F), but only one of the identical seven houses on the plate is freely accessible. Dependent on distal extramaze cues the animals have to find the open food house. The presentation of several identical objects in an environment should lead to overlapping activation in place cells. The analysis of the SPS-BM shows highly significant memory deficits in NF-κB ablated mice (<i>n</i> = 9) as compared to controls (<i>n</i> = 8). Latency (<b>b</b>), distance (<b>d</b>) and error rate (<b>c</b>) were significantly increased in IκB/tTA mice. Reactivation of NF-κB in IκB/tTA animals by doxycyline treatment (<i>n</i> = 13; lower panel) improved the performance considerably. Mice showed significantly decreased latency (<b>e</b>), the error rate (<b>f</b>) and distance covered (<b>g</b>) was comparable to control mice (<i>n</i> = 6). two-way ANOVA evaluation; error bars: SEM; S = Start position; F = food house.</p

NF-κB regulates mossy fiber projections to CA3 and replenishment of the dentate gyrus.

<p>(<b>a</b>) Immunostaining for neurofilament M (NF-M) in control mice (IκB/-) reveals a fasciculated organisation of mossy fibers, connecting granule cells to their target cells in CA3. (<b>b</b>) Impairment of mossy fiber projections after neuronal NF-κB ablation (IκB/tTA). (<b>c</b>) Statistical analysis of NF-M staining shows significantly reduced mossy fiber bundles in IκB/tTA mice (<i>n</i> = 8) as compared to controls (<i>n</i> = 4) (<i>P</i> = 0.0034); (<b>d</b>) Synapses stained for synaptophysin are organised in a laminated fashion. Strongest staining was visible in the stratum lucidum (SL), which corresponds to the termination field of mossy fibers on dendrites of CA3 neurons. (<b>e</b>) In contrast, staining is strongly reduced in the SL after NF-κB ablation, suggesting an important role of NF-κB in regulating synaptic density. (<b>f</b>) NF-κB ablation results also in a significantly decreased dentate gyrus size (white bar in <b>a</b>, <b>b</b>, <i>n</i> = 9) compared to control mice (<i>n</i> = 6) (<i>P</i> = <0.0001). (<b>g</b>, <b>h</b>) Electron microscopy shows that size and frequency of mossy fiber boutons (MFB) are reduced after NF-κB inhibition (blue, mossy fiber boutons; red, dendritic excrescences). (<b>i</b>) The size of MFB is reduced in IκB/tTA mice. (<b>j</b>, <b>k</b>) Scheme of structural defects after NF-κB ablation. Scale bars (a,b) 100 µm, (d,e) 20 µm, (g,h) 2 µm); DG-dentate gyrus, SL-stratum lucidum, PY-pyramidal layer, MFB-mossy fiber boutons. Error bars, SEM. ^**<i>P</i><0.01; ^***<i>P</i><0.0001; Unpaired t-test, two tailed.</p

Reactivation of NF-κB leads to regrowing of the dentate gyrus.

<p>(<b>A</b>) (<b>a</b>) The size of the dentate gyrus (DG) in NF-κB ablated mice is strongly reduced. Reactivation of NF-κB leads to increased number of granule cells within the DG (<b>b</b>). (<b>B</b>) Statistical analysis shows a significant decrease of granule cells after NF-κB ablation (IκB/tTA mice, <i>n</i> = 9; as compared to control mice IκB/-; <i>n</i> = 3, (<i>P</i> = <0.0001)). Note, after reactivation of NF-κB by doxycycline treatment the number of granule cells is increased in IκB/tTA mice (<i>n</i> = 7) (<i>p</i> = 0.0002). No difference in the control groups IκB/- (<i>n</i> = 3) and IκB/- +DOX (<i>n</i> = 3) was detected (<i>p</i> = 0.0605). (<b>C</b>) Doublecortin (DCX) staining marking neuronal precursors. After doxycycline treatment the number of DCX positive cells is strongly decreased in IκB/tTA mice (<i>n</i> = 3) (<b>c</b>, <b>d</b>) as compared to NF-κB ablated group (<i>n</i> = 3) (<b>a</b>, <b>b</b>). (<b>D</b>) Statistical evaluation shows significantly less DCX positive cells after doxycycline treatment in IκB/tTA mice (<i>n</i> = 3) as compared to untreated IκB/tTA group (<i>n</i> = 3) (<i>p</i> = <0.0001). No difference in control groups IκB/- (<i>n</i> = 3) and IκB/- +DOX (<i>n</i> = 3) was detected (<i>p</i> = 0.1063). (<b>E</b>) Statistical analysis of neurofilament-M (NF-M) staining shows significantly reduced mossy fiber bundles in IκB/tTA (<i>n</i> = 8) mice as compared to controls (<i>n</i> = 4) (<i>p</i> = 0.0034). Reactivation of NF-κB results in mossy fiber outgrowth of newborn neurons, thus increasing the area of mossy fibers in doxycycline treated IκB/tTA mice as compared to the untreated group (<i>p</i> = 0.0024, t-test with Welch's correction). No difference in the control groups IκB/- (<i>n</i> = 4) and IκB/- +DOX (<i>n</i> = 3) was detected ( <i>p</i> = 0.1897). Error bars: SEM; <i>p</i><0.05; ^**<i>p</i><0.01; ^***<i>p</i><0.0001.</p

NF-κB signalling can play different roles in dentate gyrus tissue homeostasis.

<p>The dentate gyrus (left) is built from stem cells that develop to neuroblasts and type 2b progenitors that still can divide. These progenitors give rise to mature granule cells, connecting CA3 pyramidal neurons via mossy fibers. NF-κB signalling plays an important role at least in two of the processes that regulate the balance between degeneration and regeneration (tissue homeostasis) of the dentate gyrus. That is firstly axon formation and maturation from a immature type 2b neuronal progenitor (DCX⁺) to a mature granule cell neuron and secondly regulation of survival and energy balance of mature granule cells. yellow = cells with NF-κB on; blue = NF-κB ablated cells, NF-κB <i>off</i>. Right side, molecular mechanisms: Our results point to a cell autonomous mechanism by which NF-κB regulates axogenesis in newborn granule cells. Critically involved are the FOXO transcription factors and the bona fide NF-κB target gene PKA (Prkaca). A second role for NF-κB in mature granule cells is neuroprotection presumably transmitted by a PKA – phospho-CREB pathway. Ablation of NF-κB (NF-κB off) results in severe structural and behavioural defects described above.</p

Increased apoptosis, neurogenesis and inflammation in the dentate gyrus of NF-κB deficient mice.

<p>(<b>a</b>, <b>b</b>) Neuronal cell death visualized by Fluoro Jade-C. Degenerating neurites (arrows) and nuclei (asterisk) are significantly increased in IκB/tTA mice (<i>n</i> = 3) as compared to control mice (<i>n</i> = 3) (<b>c</b>, <b>d</b>). (<b>e</b>) Increased cleaved caspase-3 positive cells after NF-κB ablation (<i>n</i> = 3). (<b>f</b>, <b>g</b>) DCX immunoreactivity is significantly increased in the DG after NF-κB ablation. Note that DCX⁺ cells are not arranged in the subgranular zone alone but migrate deeper into the DG. (<b>h</b>, <b>j</b>) Total number of DCX⁺ and Calretinin⁺ cells is increased in IκB/tTA mice (<i>n</i> = 3) compared to controls (<i>n</i> = 3). (<b>i</b>) Increased BrdU/DCX⁺ cells in the DG seven days after the last BrdU injections (<i>n</i> = 3). (<b>k</b>, <b>l</b>) Representative images of the distribution of glial fibrillary acidic protein (GFAP) in the hippocampus. Note significantly increased numbers of GFAP positive cells in IκB/tTA mice, particularly within the DG. Scale bars (a, b) 10 µm, (f, g) 25 µm, (k,l) 50 µm; ^*<i>P</i><0.05; ^**<i>P</i><0.01; ^***<i>P</i><0.0001; Error bars: SEM; DG dentate gyrus, GFAP glial fibrillary acidic protein, ML molecular layer, H hilus, DCX doublecortin, FJ-C fluoro Jade-C, BrdU bromodeoxyuridine.</p

NF-κB dependent regulation of PKA and FOXO1.

<p><b>A</b> (<b>a</b>, <b>b</b>) Forkhead box protein O1 (FOXO1) is expressed in the DG in control mice (IkB/-), but is downregulated in NF-κB deficient mice (<b>c</b>, <b>d</b>). (<b>e</b>, <b>f</b>) Doxycycline induced reactivation of NF-κB leads to re-expression of the transcription factor Foxo1 as shown by in situ hybridisation (encoded in false colour). <b>B</b> (<b>a</b>, <b>b</b>) The catalytic subunit alpha of Protein kinase A (PKA) is expressed in the DG of control mice (IκB/-), but is strongly downregulated in NF-κB deficient mice (<b>c</b>, <b>d</b>). Reactivation of NF-κB by doxycycline treatment results in re-expression of PKA (<b>e</b>, <b>f</b>); as shown by in situ hybridisation (encoded in false colour, data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030838#pone.0030838-Kaltschmidt2" target="_blank">[19]</a>). <b>C</b> (<b>a</b>, <b>b</b>) Phosphorylated PKA substrates are reduced in granule cells of the dentate gyrus as detected by phospho-specific antibody. (<b>c</b>) Fluorescence intensity is significantly decreased in IκB/tTA mice (<i>n</i> = 4) as compared to controls (<i>n</i> = 4) (<i>P</i> = <0.0001) (<b>d</b>) Phosphorylation of LKB1, a substrate of PKA, is reduced in hippocampal brain extracts from IκB/tTA mice as detected by Western blotting. Scale bar (a, b) 20 µm; Error bars: SEM; DG dentate gyrus.</p