Leibniz Institute for Age Research Journal of Cell Science...

Haenold et al., Adult axoneogenesis by cellular balance of RelA and p50

JOCES/2013/140731 - Revised manuscript for Journal of Cell Science 1

NF-B determines axonal re- and degeneration by cell-specific balance of RelA and p50 1

subunits in the adult CNS 2

3

Ronny Haenold1, Falk Weih1, Karl-Heinz Herrmann2, Karl-Friedrich Schmidt3,I, Katja Krempler4, 4

Christian Engelmann1, Klaus-Armin Nave5, Jürgen R. Reichenbach2, Sigrid Löwel3,I, Otto W. Witte4, and 5

Alexandra Kretz4 6

7

1 Leibniz Institute for Age Research Fritz Lipmann Institute, Beutenbergstr. 11, 07745 Jena, Germany 8

2 Friedrich Schiller University of Jena Medical School, Institute of Diagnostic and Interventional Radiology, 9

Medical Physics Group, Philosophenweg 3, 07743 Jena, Germany 10

3 Friedrich Schiller University of Jena, Institute of General Zoology and Animal Physiology, Erbertstr. 1, 07743 11

Jena, Germany 12

4 Hans Berger Department of Neurology, Jena University Hospital, Erlanger Allee 101, 07747 Jena, Germany 13

5 Max Planck Institute for Experimental Medicine, Department of Neurogenetics, Hermann-Rein-Str. 3, 37075 14

Göttingen, Germany 15

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I Present address of S. Löwel and K.-F. Schmidt: Georg August University, Bernstein Focus Neurotechnology 17

(BFNT) and Johann Friedrich Blumenbach Institute for Zoology and Anthropology, Berliner Str. 28, 37073 18

Göttingen, Germany. 19

20

Corresponding author: Ronny Haenold, Leibniz Institute for Age Research Fritz Lipmann Institute, 21

Beutenbergstr. 11, 07745 Jena, Germany, E-mail: [email protected], Phone: +49 3641 656050, Fax: +49 3641 22

656335 23

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Running title: Adult axoneogenesis by cellular balance of RelA and p50 25

Keywords: anaphase-promoting complex, axonal regeneration, Cdh1, manganese-enhanced MRI (MEMRI), NF-26

B, p50, RelA/p65, Wallerian degeneration 27

© 2014. Published by The Company of Biologists Ltd.Jo

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JCS Advance Online Article. Posted on 23 May 2014

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Abbreviations: 28

APC anaphase-promoting complex 29

CAII carbonic anhydrase II 30

Cdh1 co-activator protein for APC 31

CNR contrast-to-noise ratio 32

CNS central nervous system 33

CRALBP cellular retinaldehyde-binding protein 34

CSPG chrondroitin sulfate proteoglycans 35

CTX cholera toxin B subunit 36

EMI1 early mitotic inhibitor 1 37

β-gal β-galactosidase 38

Id2 inhibitor of DNA binding 2 39

i.vit. intravitreally 40

MBP myelin basic protein 41

MEMRI manganese-enhanced MRI 42

MG Müller glia 43

MRI magnetic resonance imaging 44

NF-B nuclear factor-B 45

NLS nuclear localization signal 46

ODC oligodendrocyte 47

ON optic nerve 48

ONI optic nerve injury 49

RGC retinal ganglion cell 50

ROI region of interest 51

SCI spinal cord injury 52

TUJ-1 β-III tubulin 53

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Summary 55

NF-B is dually involved in neurogenesis and brain pathology. Here, we addressed its role in 56

adult axoneogenesis by generating mutations of RelA and p50 heterodimers of canonical NF-B. 57

In addition to activation in astrocytes, optic nerve axonotmesis caused a hitherto unrecognized 58

RelA induction in growth inhibitory oligodendrocytes. Intraretinally, RelA was induced in 59

severed retinal ganglion cells and inferred in bystander Muller glia. Cell type-specific deletion of 60

transactivating RelA in neurons and/or macroglia considerably stimulated axonal regeneration in 61

a distinct and synergistic pattern. In contrast, deletion of the p50 suppressor subunit promoted 62

spontaneous and post-injury Wallerian degeneration. Growth effects mediated by RelA deletion 63

paralleled a downregulation of growth inhibitory Cdh1 and upregulation of the endogenous Cdh1 64

suppressor EMI1. Pro-degenerative loss of p50, however, stabilized retinal Cdh1. In vitro, RelA 65

deletion elicited opposing, pro-regenerative shifts in active nuclear and inactive cytoplasmic 66

moieties of Cdh1 and Id2. The involvement of NF-B and cell cycle regulators such as Cdh1 in 67

regenerative processes of non-replicative neurons presents novel options regarding how 68

molecular reprograming might be executed to stimulate adult axoneogenesis and treat CNS 69

axonopathies. 70

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Introduction 73

The transcription factor nuclear factor-B (NF-B) is ubiquitously expressed and is critical for 74

various neuropathologies (Kaltschmidt and Kaltschmidt, 2009). In the nervous system, it 75

establishes its role by subunit- and cell type-specific activation and post-translational 76

modifications on the RelA and p50 subunits. Moreover, tissue maturation, its activation in 77

peripheral (PNS) versus central (CNS) nervous system, and the context of injury influence 78

cellular NF-B functions. Studies on neonatal sympathetic and sensory neurons have indicated 79

that NF-B RelA and p50 can either promote or inhibit axogenesis during post-natal 80

development (Gutierrez and Davies, 2011; Gutierrez et al., 2008). Overexpression of a dominant-81

negative form of the NF-B inhibitor IBα in astrocytes indeed limited loco-regional damage 82

after spinal cord injury (SCI) and further stimulated axon sprouting and functional recovery 83

(Brambilla et al., 2005; 2009). However, the significance of individual NF-B subunits, their 84

activation in separate cell types, and their impact on axonal regeneration and Wallerian 85

degeneration currently remain undefined. Intriguingly, a putative involvement of NF-B in the 86

repulsive feature of white matter substances (Chen et al., 2000) and the anti-growth program 87

exerted by oligodendrocytes (ODC) has not yet been investigated. More importantly, stimulus-88

dependent axo-nuclear transport of NF-B as demonstrated by FRAP analysis of cultivated 89

hippocampal neurons using a RelA-GFP reporter (Meffert et al., 2003) may trigger a cell-90

intrinsic pro-/anti-regenerative program in axons themselves. 91

NF-B-induced gene expression is modulated by nuclear translocation of either complexes 92

containing transcriptionally active RelA or homodimers of the transcriptionally inactive p50 93

subunit. While interference with the upstream kinases IKKα/β/γ or overexpression of IBα 94

results in inhibition of any dimer of the classical NF-B cascade, subunit-specific knockouts 95

shift the balance between the individual moieties activated. Thus, ablation of either RelA or p50 96

can propagate dual or even opposing effects, as exemplified for post-ischemic infarct volumes in 97

murine stroke models (Inta et al., 2006; Li et al., 2008; Zhang et al., 2005). 98

In the present study, we modulated the balance between RelA and p50 subunits specifically in 99

neurons and macroglia of mutant mice using the Cre/loxP system, or by insertion of a pGK-neo 100

cassette, and investigated cell type-specific roles of individual NF-B subunits in adult 101

axoneogenesis. We show that selective suppression of RelA induction in neurons and macroglia 102

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(RelACNSKO) or in oligodendrocytes (RelAODCKO) and astrocytes (RelAASTKO) alone differentially 103

and synergistically increased axonal regeneration. In contrast, upregulation of NF-B activity by 104

ubiquitous p50 deficiency prompted Wallerian degeneration. The divergent effects of RelA and 105

p50 on axon integrity were reflected in a subunit-specific regulation of the ubiquitin E3 ligase 106

adaptor protein Cdh1, which was suppressed under axoneogenesis and upregulated under 107

Wallerian degeneration. On the subcellular level, RelA-deficient neuronal cultures exhibited an 108

inactivating shift in Cdh1 protein from the nucleus to the cytoplasm and a reciprocal nuclear 109

accumulation of the pro-regenerative Cdh1 substrate Id2 (inhibitor of DNA binding 2). In 110

summary, balanced levels of the NF-B subunits RelA and p50 and their orchestration with cell 111

cycle regulators such as Cdh1 may contribute to define the growth potential of injured CNS fiber 112

networks. 113

114

Results 115

Visual system development is not impaired in RelACNSKO and p50KO mice 116

Conditional neuro-ectodermal deletion of RelA was achieved by nestin-Cre-based recombination 117

of homozygous floxed relA alleles (RelACNSKO). In these RelACNSKO mice, robust reduction of 118

RelA protein in the retina and optic nerve (ON) was confirmed by immunoblotting (data not 119

shown). Reduced canonical NF-B activity in the CNS of RelACNSKO mice, in the absence of a 120

compensatory upregulation of alternative NF-B subunits, has recently been demonstrated by 121

reduced expression of the NF-B target gene IB (Kretz et al., 2013). Histopathological 122

analysis revealed normal layer architecture of transversal H&E-stained retina (Fig. 1A) and an 123

obviously unimpaired relation between retinal ganglion cells (RGC) and Muller glia (MG), as 124

shown in retinal whole mount preparations labeled for the RGC marker β-tubulin (TUJ-1) and 125

the MG marker cellular retinaldehyde-binding protein (CRALBP) (Fig. 1B). Myelin sheath 126

formation and structural myelin basic protein (MBP) content in naïve ON of RelACNSKO mice 127

were inconspicuous as assessed by electron microscopy (Fig. 1C) and immunoblotting (not 128

shown). RGC numbers (P=0.88) in the ganglion cell layer (GCL) and oligodendrocyte (ODC) 129

densities (P=0.43) as well as myelin sheath dimensions (g-ratio; P=0.99) in the ON were 130

indistinguishable from controls (Fig. 1D), thus excluding developmental or apoptotic cell loss by 131

RelA deletion in neurons and macroglia. Further, as shown in Fig. 1E, in vivo parameters for 132

visual acuity and contrast sensitivity were indistinguishable among RelACNSKO, RelAfl/fl;+/+ and 133

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RelA+/+;tg/+ (RelAflox allele-negative, Cre recombinase-positive) mice (P>0.05 for each compared 134

condition), thus assuring that transgene insertion alone or loss of RelA did not impair 135

functionality of the visual projection. 136

Similarly, mice with homozygous deletion of p50 (p50KO) have been described to develop 137

normally (Sha et al., 1995). However, starting at an age of six months, they become susceptible 138

to precocious neural degeneration (Lu et al., 2006) and enhanced apoptotic cell death of the 139

visual and acoustic systems (Lang et al., 2006; Takahashi et al., 2007). 140

141

Constitutive NF-B activity is increased in p50KO mice 142

To explore the consequences of transcriptionally inhibitory p50 deletion on canonical NF-B 143

activity, p50KO mice were crossed with the B-lacZ reporter line. In this line, the lacZ gene 144

encoding for β-galactosidase (β-gal) is driven by multiple B binding sites (Schmidt-Ullrich et 145

al., 1996). Compared to B-lacZ control mice with intact p50 gene expression, double transgenic 146

B-lacZ;p50KO mice revealed a four- to five-fold increase in X-Gal-positive cells in the retina 147

(P<0.02; Fig. 1F) at just three months of age, thus confirming that loss of p50 enhances 148

constitutive NF-B activity. No signal was detected in age-matched p50KO mice devoid of the 149

lacZ gene (Fig. 1F). Thus, deletion of either NF-B subunit in RelACNSKO and p50KO mice can 150

dynamically shift transcription towards suppression or activation. 151

152

Optic nerve injury induces NF-B in different cell types 153

Optic nerve injury (ONI) induced strong and topic NF-B activation in cells of superficial retinal 154

layers and in the epicenter of the squeezed ON, as detected in respective whole mount 155

preparations of B-lacZ reporter mice (X-Gal panels in Fig. 2). Retinal numbers of X-Gal-156

positive cells significantly increased by three- and 18-fold at three and 10 days after ONI, 157

respectively, as compared to naïve specimens (P<0.01; graph in Fig. 2A). As shown by 158

immunohistochemistry, NF-B-dependent induction of β-gal was located to nuclei of TUJ-1-159

positive RGC (Fig. 2B, ONI panels; inset in bottom line). Within the ON, the majority of β-gal-160

positive nuclei could be ascribed to ODC expressing the marker protein carbonic anhydrase II 161

(CAII; Fig. 2D, ONI panels; inset in bottom line). Specificity of the CAII signal was confirmed 162

by lack of immunoreactivity in the non-myelinated head of the naïve ON (Fig. 2D, dotted line in 163

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naïve panel). Thus, ONI strongly induced NF-B activity at the site of axonal damage as well as 164

in the soma of axotomized RGC. 165

Activation of the classical NF-B pathway for target gene expression involves nuclear 166

translocation of RelA, its phosphorylation on serine residues, and exposure of a nuclear 167

localization signal (NLS). Using a Ser536 phospho-specific RelA antibody, we detected 168

substantial RelA phosphorylation in extracts of the ON within 3 h after ONI (Fig. 2C, top; n=3). 169

Notably, pSer536 was not present in the naïve ON despite of its robust RelA content (Fig. 2C 170

top, loading control). The specificity of the pSer536 immunosignal was confirmed by emergence 171

of an equivalent band following TNF-treatment of hippocampal neurons (Fig. 2C, top). Since 172

post-lesional occurrence of RelA phosphorylation was almost abolished in RelACNSKO, but 173

preserved in wild type mice (Fig. 2C, bottom), its activation should derive from axons and/or 174

macroglia of the ON lesion site. 175

NLS-RelA signal in the injured ON, but not in the naïve ON, co-localized with the nuclear β-gal 176

immunoreactivity observed in CAII-positive ODC of B-lacZ reporter mice, indicating 177

perilesional cytoplasm-to-nucleus translocation of RelA in the majority of β-gal-expressing ODC 178

(Fig. 2E, left panel). Such NLS-RelA signal was absent in the nuclei of perilesional CAII-179

positive ODC of RelACNSKO mice (Fig. 2E, right panel), confirming the specificity of RelA 180

activation in ODC. Additionally, we explored nuclear translocation of RelA in the OLN-93 181

oligodendrocytic cell line by confocal laser microscopy. As detected by the activation-specific 182

NLS-RelA antibody, nuclei of non-stimulated control cells were free of RelA signal (Fig. 2F, left 183

panel). Following application of TNF, a prototypical cytokine released during tissue damage, 184

NLS-directed immunoreactivity became strikingly evident in DAPI-positive nuclei of OLN-93 185

cells (Fig. 2F, right, Z-stack). According results were achieved using an antibody directed 186

against the C-terminus of the RelA protein, thus confirming the specificity of the NLS-RelA 187

reactivity (not shown). Collectively, solid steady-state RelA expression in ODC (not shown) 188

together with induced β-gal reactivity in ODC (Fig. 2D) indicated post-lesional RelA activation 189

in ODC. 190

191

RelA and p50 subunit-dependent RGC survival after injury 192

NF-B activation studies performed on B-lacZ;p50KO reporter mice revealed a strong induction 193

of NF-B at the ON injury site, as demonstrated by intense blue color reaction in the X-Gal 194

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assay. Analysis of corresponding retina revealed an unexpected reduction in the total number of 195

X-Gal-positive cells when analyzed as soon as 9 dpi (Fig. 3A; n=3), arguing for enhanced lesion-196

induced RGC death under p50 deficiency. Since NF-B has been shown to either inhibit or 197

propagate cell death, we investigated post-lesional RGC survival in RelACNSKO and p50KO mice 198

on the base of TUJ-1 immuno-reactivity. Neuro-glial deletion of RelA modestly supported RGC 199

rescue after ONI by 7% (P<0.05), compared to controls. In contrast, loss of p50 strongly severed 200

RGC death, thus resulting in a decline by 55% of control levels (Fig. 3B; P<0.01). 201

Histologically, retinal atrophy suggested an overall increased susceptibility of CNS neurons to 202

harmful events in p50KO animals. 203

204

Axonal regeneration is stimulated in RelACNSKO mice, whereas p50 deletion triggers Wallerian 205

degeneration 206

In RelACNSKO mice, anterograde cholera toxin B subunit (CTX) tracing (suppl. Fig. 1) revealed 207

dense bundles of regenerating axons growing into the ON and towards the injury site four weeks 208

after ONI (Fig. 4A, top). Robust trans-lesional axon growth beyond the scar and into the distal 209

ON stump was achieved in the majority of ON specimens (81%; n=16). Although not implicated 210

in software-based quantifications due to their low percentage (<5%, not shown), individual axons 211

spanned a growth distance of 2–3 mm. In control specimens, a substantially lower number of 212

newly generated RGC axons reached the ON head, which almost regularly missed intra- or trans-213

lesional growth aspects (20%; n=10; Fig. 4A, middle). RelA+/+;tg/+ animals similarly showed 214

repressed growth responses (data not shown), thus ruling out a phenotype by the transgene itself. 215

Examination of corresponding retina in each group excluded impaired labeling quality to be the 216

cause of lower ingrowth results in controls; however, in RelACNSKO mice, intraretinal fascicular 217

degeneration appeared attenuated. Generally, our histological post-mortem results correlated well 218

with in vivo analysis of ON regeneration using contrast-enhanced MRI (MEMRI; suppl. results 219

and suppl. Fig. 2). The strong crush technique applied in order to avoid fiber sparing 220

corresponded to the pronounced axonal dieback as well as to the long latency and still rather 221

limited distance of regeneration behind the lamina cribrosa (suppl. Fig. 2). 222

Software-based quantitative analysis of longitudinal ON sections detected on average a five-fold 223

increase in the extent of axonal regeneration in RelACNSKO mice compared to controls (RelAflox: 224

85.91±23.95×103 µm2 versus RelACNSKO: 458.82±66.65×103 µm2; P<0.001; Fig. 4B). Considering 225

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the maximum growth responses in both groups, an even greater stimulation (10-fold) was 226

attained. Further characterization revealed trans-lesional growth areas to be 17-fold (RelAflox: 227

1.67±0.53×103 µm2 versus RelACNSKO: 29.10±5.57×103 µm2; P<0.01; Fig. 4C) and trans-lesional 228

distances to be four-fold (RelAflox: 122.55±0.53 µm versus RelACNSKO: 487.04±59.57 µm; 229

P<0.001; Fig. 4D) increased in RelACNSKO mice compared to controls. Comparable spaces of 230

approximately 240 µm between the optic disc and the lesion site in animals of both groups 231

(RelAflox: mean 223.68 µm versus RelACNSKO: mean 263.57 µm; P=0.07; Fig. 4E) excluded 232

artifacts by biased position of the injury site. Further, co-localization of GAP-43 with the CTX 233

tracer (Fig. 5A) and the emergence of GAP-43-enhanced rudimentary growth cones (Fig. 5B) 234

emphasized the axoneogenetic process in RelACNSKO mice. Intraretinally, RGC elongated MAP2-235

positive dendritic-like arbors, which grew into deeper retinal layers towards the optic disc (Fig. 236

5B, right). Such a growth pattern was exclusive to RelACNSKO mice, thus suggesting fundamental 237

growth reprograming with high axonal and dendritic plasticity at the expense of a defined 238

polarized growth shape. 239

In contrast to the growth improvement in RelACNSKO mice, injured ON of p50KO animals 240

demonstrated negligible regeneration, which was – although not significant – even lower than in 241

controls (Fig. 4A, bottom; 4B). Of note, p50KO animals are known to develop normally and 242

possess physiological RGC counts at young mature ages, however evolve age-dependent 243

alterations in axon-myelin structure (Lu et al., 2006; Takahashi et al., 2007). Now assuming 244

spontaneous destabilization of axonal integrity in p50KO mice, we applied the degeneration 245

marker Fluoro Jade and compared the ON cytoskeleton in naïve and lesioned young RelACNSKO 246

and RelAflox mice with young and aged naïve p50KO mice. RelACNSKO and control animals 247

remained devoid of any staining under naïve conditions and were indistinguishable regarding the 248

extent of fiber degeneration two weeks after axonotmesis (Fig. 5C). At a susceptibility age of 10 249

months, however, naïve p50KO mice displayed drastic fiber disintegration and neurofilament 250

breakdown, which was not discernable in age-matched naïve wild type (not shown) or naïve 251

RelACNSKO mice (Fig. 5C, furthest right). Specificity of the assay was confirmed by negative 252

signals in brain regions adjacent to the intracerebral part of the degenerating optic tract (Fig. 5C, 253

furthest right). Further, toxic axonopathy induced by intraocular TNF application (2 ng), resulted 254

in a similar staining pattern as under axonotmesis four weeks after treatment (not shown). Since 255

RGC quantifications in p50KO mice revealed a drastic decline relative to control levels (Fig. 3B), 256

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growth failure in this case is suggested to be also due to reinforced retinal atrophy including 257

RGC decimation following ONI. These observations highlight the functional polarity of RelA 258

and p50 in axonal de- and regeneration. Whilst p50KO mice display a reduced capacity for repair 259

of endogenous or injury-induced fiber damage, fiber integrity and regeneration are stabilized and 260

promoted in RelACNSKO mice. 261

262

The pro-regenerative effect of RelA deletion is cell type dependent 263

The unexpectedly high expression of RelA in naïve ODC raised the possibility of a hitherto 264

unrecognized growth-relevant RelA function executed by interaction between neurons and 265

growth inhibitory ODC. To explore whether the pro-regenerative effect in RelACNSKO mice was 266

due to deletion of RelA in RGC or rather in ODC, we deleted RelA specifically in ODC by 267

CNP1 promoter-driven Cre recombinase expression (RelAODCKO). Activity of CNP1-Cre 268

recombination in the ON was verified in Rosa26R;CNP1-Cre reporter mice using a colorimetric 269

assay (Fig. 6A). Robust deletion of RelA protein in the ON of RelAODCKO mice was further 270

confirmed by immunoblotting (data not shown). In RelAODCKO mice, outgrowth of newly 271

generated axons four weeks after ONI was significantly enhanced over controls by about nine-272

fold (RelAflox: 17.59±9.00×103 µm2 versus RelAODCKO: 155.53±46.00×103 µm2; P<0.05; Fig. 6B), 273

suggesting that RelA ablation in ODC contributes to neutralize the common white matter-derived 274

growth failure. Since the slow kinetic of Wallerian degeneration in the CNS adds to regenerative 275

failure, we investigated the progress of ONI-dependent myelin degradation by MBP protein 276

detection. In superordinate RelACNSKO constructs implicating RelA loss in ODC, structural MBP 277

decline under Wallerian degeneration was much stronger than in controls, which may explain 278

stimulated axogenesis in RelAODCKO mice (Fig. 6C; n=3). Since in RelAODCKO mice absolute 279

growth dimensions as for expanse and length remained below those in RelACNSKO mice (Fig. 4B), 280

it is suggestive that additional cell populations add to achieve maximum growth responses under 281

neuro-glial RelA deletion. 282

We further investigated axoneogenesis in mice with conditional RelA deletion selectively in 283

astrocytes (RelAASTKO). GLAST-mediated Cre targeting also to retina-specific astrocytes, i.e. 284

MG, was verified using animals with a combined tamoxifen- (TAM) inducible GLAST-285

CreERT2/loxP system (Slezak et al., 2007) and Rosa26-YFP reporter expression (YFP;GLAST-286

CreERT2; Srinivas et al., 2001). In their retinae, obviously all YFP-positive cells identified as 287

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MG by their long processes and strong CRALBP co-expression (not shown) showed a profound 288

increase in YFP reporter signal in response to TAM (Fig. 6D, arrows). Following ONI in 289

RelAASTKO mice, a 19-fold increase in elaborated axons emerged compared to controls (RelAflox: 290

17.59±9.00×103 µm2 versus RelAASTKO: 383.31±57.18×103 µm2; P<0.01; Fig. 6E), which 291

exceeded growth stimulation in RelAODCKO mice but remained below the absolute growth values 292

observed in RelACNSKO mice (Fig. 4B). Since chondroitin sulfate proteoglycans (CSPG) secreted 293

by scar-forming astrocytes are chemically repulsive and mechanically impenetrable growth 294

inhibitors, we investigated post-lesional neurocan secretion in RelACNSKO mice. Neurocan 295

production within the lesion epicenter was less dense and compacted than in controls 10 days 296

(Fig. 6F) and four weeks (not shown) after ONI. Consecutively, axons elaborated from 297

RelACNSKO mice grew straightly into and beyond the scar, whereas in controls most of them were 298

arrested when confronted with the scar region (Fig. 6G, arrows), suggesting RelA-dependent 299

expression of repulsive scar constituents. Importantly, invasion of F4/80+ CD11b+ microglia and 300

macrophages into the injury region appeared indiscernible in either line (not shown). Therefore, 301

a difference mediated by immune-privileged CNS functions seems unlikely. 302

In summary, supreme post-lesional axonal regeneration observed in RelACNSKO mice cannot be 303

explained by RelA deletion in oligodendrocytes (RelAODCKO) or astroglia (RelAASTKO) alone, but 304

argues for further cell type-specific, in particular neuron-intrinsic molecular mechanisms. 305

306

RelA-associated regeneration parallels cell type-specific Cdh1 suppression 307

Therefore, we examined whether the axonal growth regulator Cdh1, which was found expressed 308

in the naïve mature cortex and retina (Fig. 7A), where it localizes to RGC (Fig. 7B), is a target of 309

RelA-mediated growth suppression. This hypothesis was reinforced by the fact that highest 310

levels were found in myelin-enriched projecting fibers of optic and sciatic nerves (Fig. 7A). 311

Following ONI, Cdh1 levels declined by 50% in the retina of regeneration-competent RelACNSKO 312

mice but rose to 182% in non-regenerating RelAflox mice compared to uninjured controls (Fig. 313

7C). In contrast, Cdh1 was upregulated to 220% and 225%, respectively, in young adult p50KO 314

mice after ONI as well as in the spontaneously degenerating retina of aged 10-month-old p50KO 315

mice (Fig. 7C). Assuming that neuronal Cdh1 content defines the threshold for re- and 316

degeneration and that Cdh1 levels are modulated by pro- or anti-regenerative inputs from ODC 317

and astrocytes, Cdh1 levels should be equally, but gradually suppressed in RelAODCKO and 318

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RelAASTKO mice, compared to RelACNSKO mice. Accordingly, Cdh1 levels were found reduced in 319

all growth-stimulating knockout lines, with strongest suppression by 47% in the best 320

regenerating RelACNSKO and RelAASTKO mice, respectively, as compared to 20% reduction in 321

RelAODCKO mice (Fig. 7D). In addition, the physiological inhibitor of Cdh1, EMI1 (early mitotic 322

inhibitor 1), was upregulated (40%) in naïve retina of RelACNSKO mice, whilst downregulated 323

(30%) in aged and non-regenerating p50KO mice (Fig. 7E). ONI further induced a two- to three-324

fold upregulation of EMI1; however, this induction was independent of the genotype (273% in 325

RelAflox versus 221% in RelACNSKO). 326

These data show that Cdh1 levels are negatively correlated with successful regrowth in mature 327

neurons and support the notion that the APCCdh1 cascade is involved in the NF-B-dependent 328

ambivalent regulation of axonal restoration and degradation. 329

330

RelA deletion causes an inactivating shift in subcellular Cdh1 localization 331

We next examined RelA-associated post-translational modifications of Cdh1. Since 332

phosphorylation and subsequent shift of stabilized Cdh1 from the nucleus to the cytoplasm 333

indicates its inactivation (Huynh et al., 2009) we investigated its subcellular distribution in wild 334

type and RelA-deficient hippocampal neurons (HN) during axogenesis (Fig. 7F-H). According to 335

naïve RGC in vivo (Fig. 7B) hippocampal neurons of controls showed a common mixed nuclear 336

and cytoplasmic Cdh1 content (Huynh et al., 2009), however with a nuclear dominance (Fig. 7F-337

H). In the absence of RelA, a two-fold relative increase in cytoplasmic Cdh1 occurred (RelAflox: 338

19.4±1.8% versus RelACNSKO: 40.8±3.7%; P<0.001; Fig. 7F-H). Neurons with a cytoplasmic 339

Cdh1 preponderance above 70% were almost exclusively restricted to cultures of RelACNSKO mice 340

(Fig. 7H, top). Strong staining against the axo-neuronal marker TUJ-1 indicated neuronal 341

viability and axonal vitality in both conditions (Fig. 7F, top) irrespective of their RelA-dependent 342

Cdh1 content. 343

As a further target for RelA-mediated Cdh1 regulation, we investigated protein levels of HLH-344

related, pro-regenerative Id2. Since Id2 is a nuclear target, we looked for a pro-nuclear shift of 345

active Id2 in HN of RelACNSKO with reduced Cdh1 content. Immunocytochemical and subcellular 346

analysis revealed a predominant cytoplasmic localization of Id2 in wild type HN with 347

characteristic accumulation in the axon hill (Fig. 7I, arrowhead and inset) displaying a mean 348

extranuclear signal of 59.2±4.1% (Fig. 7J). In contrast, in RelACNSKO neurons, Id2 349

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immunoreactivity was concentrated to DAPI-positive nuclei (Fig. 7I), whilst the mean 350

percentage of extranuclear signal was reduced to 21.9±2.0% (Fig. 7J). Thereby, average nuclear 351

content was augmented by about 100% (RelAflox: 40.8% versus RelACNSKO: 78.1%; P<0.001), 352

suggesting that reduced Cdh1 activity stabilized nuclear Id2 steady-state levels. Since total 353

protein content may not reflect this regulation (Kim et al., 2006), immunocytochemistry was 354

preferred over immunoblot analysis of whole cell lysates. Further studies will aim to identify 355

growth-responsive target genes regulated by this RelA/p50-EMI1-APCCdh1-Id2 pathway. 356

357

Discussion 358

There is accumulating evidence that RelA is critical for axon formation during embryonic neural 359

development (Gavaldà et al., 2009). In cervical superficial ganglia, enhanced site-specific 360

Serin536 phosphorylation of RelA in the presence of p50 impairs expansion of neurite length and 361

complexity (Gutierrez et al., 2008), whereas RelA suppression by overexpression of either p50 or 362

a dominant-negative IBα super-repressor in newborn hippocampal neurons results in complete 363

growth arrest (Imielski et al., 2012). It has been suggested that modification of both IBα and 364

activated RelA determines a functional switch from growth inhibition to growth promotion 365

(Gavaldà et al., 2009; Gutierrez et al., 2008). Moreover, as recently exemplified for hippocampal 366

neurogenesis, the balance between transactivation-competent and -incompetent NF-B subunits 367

may also be crucial for axogenesis (Imielski et al., 2012). However, such previous experiments 368

were based on in vitro analysis of premature PNS and newborn hippocampal neurons. At present, 369

the relevance of NF-B in the mature post-lesional CNS for structural restoration is undefined 370

and subunit- and cell type-specific features of NF-B activation remain unclear. In this study, we 371

investigated the importance of NF-B on axonal re- and degeneration (i) in mature neurons, (ii) 372

of the CNS, (iii) in vivo, (iv) after axonal injury, (v) in a cell-autonomous manner, and (vi) under 373

consideration of the interdependence between the CNS-dominant NF-B subunits RelA and p50. 374

Apart from induction of p50 and RelA already demonstrated for severed RGC (Choi et al., 1998; 375

Takahashi et al., 2007), ONI elicited a previously unrecognized activation of RelA in macroglia. 376

To the best of our knowledge, this is the first time that ODC as prototypical CNS growth 377

inhibitors have been shown to induce RelA by injury. Thus, RelA in ODC may play a critical 378

role in the cell-autonomous regulation of axonal de- and remyelination following axonal injury. 379

This might depend on the type of injury, since in a cuprizone model, ablation of IKK2 in 380

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astrocytes – but not in ODC – was sufficient to prevent toxic demyelination (Raasch et al., 381

2011). Further, according to Brambilla et al. (2005), we could confirm that reactive astroglia of 382

the scar region are a growth-relevant source of NF-B activation. In their study, NF-B was 383

inhibited in astrocytes by GFAP-dependent overexpression of an IBα super-repressor. Whereas 384

this approach does not discriminate between subunit-specific roles, we now have specified RelA 385

as one of the subunits involved in astroglial scar formation and growth suppression. Thus, in 386

addition to the well-characterized role of NF-B in immune cells for neuron-axonal integrity 387

(Emmanouil et al., 2009), our data emphasize the significance of RelA for axonal renewal by its 388

regulation in CNS-intrinsic neuro-ectodermal neurons and macroglia. Inhibition of such RelA 389

activation either in astrocytes or ODC, or in neurons and macroglia together, elicited a graded, 390

cell type-specific stimulation of axon regrowth. The robust growth stimulation in either case of 391

RelA depletion – displaying a five- to 19-fold relative increase over controls and an absolute 392

augmentation in RelACNSKO mice that exceeded that in RelAASTKO >> RelAODCKO mice – points to 393

multimodal positive effects exerted synergistically by suppression of tonic growth inhibitors 394

from myelin, glial scar, and severed RGC and axons. The growth stimulation in RelAODCKO mice 395

coincided with pronounced degradation of MBP protein in the lesioned ON, suggesting 396

accelerated Wallerian degeneration in RelAODCKO mice and, thus, reconstitution of a more 397

permissive post-injury milieu. This is in accordance with previously described RelA-dependent 398

activation of the MBP promoter in response to TNF stimulation (Huang et al., 2002) and may 399

have implications for de- and remyelination in various myelin-related disorders. 400

The growth-promoting influence of ODC-specific RelA deletion was quantitatively exceeded by 401

its loss in astrocytes. Mechanistically, enhanced regeneration in RelAASTKO mice correlated with a 402

diminished production of CSPG at the lesion site, suggesting that loss of RelA in astrocytes 403

facilitates penetrating growth events through the glial scar and restitutes a target-directed growth 404

orientation. Brambilla and colleagues identified that expression of a dominant-negative form of 405

IBα in scar-forming astrocytes improves functional recovery following SCI (Brambilla et al., 406

2005; 2009). Here, our GLAST-Cre model suggests further benefit from RelA inhibition not 407

only in astrocytes of the ON, but also in MG of associated retina. Due to the most comprehensive 408

deletion mechanism in the nestin-Cre line, deviations from basal RGC densities calculated for 409

naïve RelACNSKO mice were not expected for RelAODCKO or RelAASTKO mice and thus are unlikely 410

to be the reason for the gradual differences in regeneration. That the most effective regeneration 411

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was observed in RelACNSKO mice implicates neuron-intrinsic RelA effects when reciprocal neuro-412

glial communication is present. Ongoing studies on RGC/neuron-specific knockout mice will 413

further elucidate temporal and spatial neuro-glial interactions. 414

The histologically evident increase in axonal outgrowth in RelACNSKO mice was confirmed by 415

preceding tract-specific, contrast-enhanced MRI techniques performed in vivo (suppl. results and 416

suppl. Fig. 2; Haenold et al., 2012; Fischer et al., 2014). Ongoing long-term studies using 417

repetitive MRI of the visual projection may delineate whether these axons become connected 418

with midbrain targets, as recently claimed by Benowitz’s group under cAMP- and PTEN-419

regulated oncomodulin effects (De Lima et al., 2012). 420

Growth modulation by NF-B was highly subunit specific since abrogation of the RelA-binding 421

partner p50 did not show any pro-regenerative effect but rather destabilized axonal integrity. 422

Moreover, naïve p50KO animals exhibited signs of precocious atrophy as early as 10 months after 423

birth, followed by severe cytoskeletal disintegration and functional visual impairments (will be 424

shown elsewhere). Such detrimental consequences are in line with the spontaneous degenerative 425

process recently described for aging p50-deficient animals (Lang et al., 2006; Takahashi et al., 426

2007). Collectively, the balance between p50 and RelA appears necessary to stabilize axons on 427

the structural and functional level. In the absence of RelA, p50 cannot compensate for the lack of 428

transcriptional regulation by RelA. Apart from such loss-of-function, growth promotion may be 429

released by novel dimerization partners of p50, such as Bcl-3 or c-Rel. We are currently 430

generating neuro-glia-specific RelA;c-Rel double-knockout mice to establish whether the pro-431

regenerative response is further enhanced or diminished. In contrast, in p50KO mice the lack of its 432

suppressor function might release pro-apoptotic/pro-degenerative gene expression profiles. 433

Whether reactive NF-B induction in p50KO mice – as shown for retina – also occurs in ODC 434

and astrocytes of the ON and thus contributes to modulated growth responses will be addressed 435

in future studies. 436

437

Transcriptional changes controlled by RelA and p50, which influence axogenesis and axonal 438

degeneration are still undefined. Interestingly, inhibition of Cdh1, a co-activator for the E3 439

ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C), in embryonic primary 440

cultures can enhance axon elaboration and override growth suppression by myelin factors 441

(Konishi et al., 2004). In cycling cells, Cdh1 promotes ubiquitylation and degradation of cell 442

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cycle regulators such as cyclins and Cdh1 itself. Transcriptional repression of Cdh1 by 443

upregulation of the Smad-interacting protein-1 (SIP1/ZEB2) influences growth arrest and 444

senescence, e.g. upon TNF-mediated NF-B activation (Chua et al., 2007; Katoh and Katoh, 445

2009). Notably, APCCdh1 expression has been shown to remain particularly high in nuclei of 446

post-mitotic neurons. Here, analysis of Cdh1 in naïve CNS tissue indicated expression in the 447

retina and cortex as well as in myelin-enriched axonal projections, thus complementing 448

previously described Cdh1 expression in astrocytes (Herrero-Mendez et al., 2009). In accordance 449

with its anti-growth function, retinal Cdh1 levels were suppressed in growth permissive 450

RelACNSKO mice following ONI. In the different cell type-specific RelA knockout lines, the best 451

pro-regenerative effect paralleled the greatest Cdh1 decline, whilst Cdh1 levels increased in 452

growth-incompetent retinae of p50KO mice. Therefore, our data suggest that Cdh1 is a CNS-453

specific downstream target of RelA and possibly of p50, which can regulate growth responses by 454

balancing intranuclear Cdh1 levels. In support of this molecular interaction, the F-box only 455

protein EMI1 that physiologically functions as a negative Cdh1 regulator was reversely governed 456

and upregulated in growth-conditioned RelACNSKO mice, but downregulated in growth-457

incompetent p50KO mice. 458

These observations make Cdh1 and its coupling to NF-B activity a strong candidate for 459

integrating extra- and intra-neuronal growth impulses in the mature CNS, followed by either a 460

positive or negative growth response. The reduced and predominantly cytoplasmic, i.e. inactive, 461

Cdh1 protein content in RelACNSKO mice supports the proposal of a RelA-dependent Cdh1 462

inactivation with consecutive growth stimulatory function. In contrast, stabilized Cdh1 levels in 463

retinae of RelAflox mice coincided with enhanced nuclear Cdh1 accumulation. As a further line of 464

evidence for this pathway, the Cdh1 substrate Id2 was stabilized in nuclei of RelACNSKO neuronal 465

cultures. Increment of steady-state levels of Id2 either by overexpression of an ubiquitylation-466

resistant D-box mutant or by Cdh1 knockdown has been reported to stimulate axonal outgrowth 467

of immature neurons and enhances regeneration of dorsal root ganglion neurons after SCI 468

(Lasorella et al., 2006; Yu et al., 2011). As for Cdh1, pro-regenerative effects may only in part 469

depend on ubiquitylation and proteasomal degradation essential for cell cycle control, e.g. by 470

cyclins, but implement CNS-specific regulatory mechanisms. Studies on fractionated cell 471

extracts will further specify the role of Id2 in growth control. 472

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In summary, our results support a novel mechanism that controls axonal self-renewal and 473

Wallerian degeneration in the adult CNS in a dual manner by highly subunit- and cell type-474

specific regulation of a RelA/p50-EMI1-APCCdh1-Id2 cascade. Whilst recent data on the 475

influence of NF-B or Cdh1 on axonal growth were acquired from cultured and immature 476

neurons, our study addresses open questions on their growth regulatory capacity in vivo and in 477

the mature CNS and extends the current knowledge on NF-B and Cdh1 to the pathophysiology 478

of neurodegenerative and traumatic CNS diseases. 479

480

Materials and Methods 481

Transgenic mice 482

For regeneration studies, 14–18-week-old mice with a homozygous Cre/loxP-based deletion of 483

relA alleles (RelAfl/fl;tg/+) and floxed littermate controls (RelAfl/fl;+/+, designated RelAflox) were 484

used. Conditional RelAflox mice were a kind gift from R. M. Schmidt (Technical University 485

Munich, Germany) (Algül et al., 2007). The nestin-Cre mouse line was obtained from The 486

Jackson Laboratory (Bar Harbor, Maine, USA). Animals were backcrossed to a C57Bl/6 (B6) 487

background for at least 10 generations. Oligodendrocyte-specific RelA deletion was achieved by 488

CNP1 promoter-driven Cre activity in RelAODCKO mice (Lappe-Siefke et al., 2003) and verified 489

in Rosa26R;CNP1-Cre double transgenic reporter animals carrying a lox-STOP-lox cassette 490

(Soriano, 1999). In these animals, the loci of Cre expression can be monitored by lacZ-sensitive 491

colorimetric assay. For astrocyte-specific RelA ablation (RelAASTKO), a GLAST-driven and 492

tamoxifen-inducible CreERT2/loxP system was employed (Slezak et al., 2007). Deletion was 493

induced as published elsewhere (Slezak et al., 2007). Constitutive knockout animals lacking the 494

p50 subunit (p50KO) by insertion of a pGK-neo cassette into exon 6 on a B6 background (Sha et 495

al., 1995), and B6 wild type controls were applied at an age of three and 10 months. Pan-NF-B 496

activation was assessed using the B-lacZ reporter line (Schmidt-Ullrich et al., 1996). 497

Animals were kept under controlled conditions in a pathogen-free environment and provided 498

with food and water ad libitum. All animal interventions were performed under deep anesthesia 499

and in accordance with the European Convention for Animal Care and Use of Laboratory 500

Animals and approved by the local ethics committee. 501

502

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Acute CNS injury and tracer applications 503

The ON was squeezed immediately behind the posterior eye pole with small, tilted forceps for 10 504

s. Eyes which developed ocular pathology/ischemia (e.g. corneal opaqueness, retinal atrophy, 505

congestive bleeding, cataract, lipofuscin deposits) were excluded. In this model of axonotmesis, 506

preserved myelin sheaths serve as guidance structures for outgrowing axons (Misantone et al., 507

1984). 508

ON fiber regeneration was evaluated by a dual anterograde tracing protocol: two to three days 509

prior to ONI, 2 μl of FITC-conjugated CTX (CTX-488; 1 µg/µl; C-22842, Molecular 510

Probes/MoBiTec, Goettingen, Germany) were intravitreally (i.vit.) injected, followed by i.vit. 511

application of complementary Cy3-labeled CTX (2 µl of CTX-594; 1 µg/µl; C-22841, Molecular 512

Probes/MoBiTec) four weeks after ONI and two to three days prior to histological dissection 513

(suppl. Fig. 1). Atraumatic i.vit. injections were performed with a 5-µl Hamilton syringe 514

connected to a 34 G needle in the infero-temporal circumference approximately 1 mm distal of 515

the corneo-scleral circumference, thereby sparing scleral vessels. To monitor needle insertion 516

and liquid inoculation and to avoid lens puncture, microinjections were accomplished under a 517

binocular microscope (Zeiss, Jena, Germany). 518

519

Immunohistochemistry and immunocytochemistry 520

NF-B RelA expression was evaluated using RelA (polyclonal C-20, 1:500, Santa Cruz, 521

Heidelberg, Germany), phospho-specific RelA (monoclonal pSer-536 clone 93H1, 1:200-1:500, 522

Cell Signaling, Frankfurt, Germany) and NLS-specific RelA (monoclonal MAB 3026 clone 523

12H11, 1:250, Millipore, Darmstadt, Germany) antibodies. Axonal de- and regeneration were 524

assessed by co-stainings against phosphorylated neurofilaments (SMI-31, monoclonal, 1:500, 525

Sternberger Monoclonals, Lutherville, ML, USA), MAP-2 (monoclonal, 1:500, Chemicon), and 526

GAP-43 (polyclonal, 1:250, Chemicon). For cellular co-localization, antibodies against RGC 527

(TUJ-1, monoclonal, 1:250, Covance, Munich, Germany), MG (CRALBP, monoclonal, 1:250, 528

Abcam, Cambridge, MA, USA; GLAST, polyclonal, 1:250, Santa Cruz), astrocytes (GFAP, 529

polyclonal, 1:500, DAKO, Hamburg, Germany), and ODC (CAII, polyclonal, 1:500, Santa Cruz) 530

were used. Reporter expression of EGFP and β-galactosidase were assessed with antibodies 531

against GFP (polyclonal, 1:100, Santa Cruz) and β-gal (polyclonal, 1:500, Chemicon). 532

Inflammatory responses (not shown) were evaluated with antibodies against CD11b 533

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(monoclonal, 1:500, Serotec, Düsseldorf, Germany), Iba1 (polyclonal, 1:250, Wako Chemicals, 534

Richmond, VA, USA) and F4/80 (polyclonal, 1:500, Dianova, Hamburg, Germany). Glial scar 535

formation was investigated applying antibodies against neurocan (monoclonal, 1:250, Abcam) 536

and GFAP (polyclonal, 1:500, DAKO). Cdh1 and Id2 in cultured hippocampal neurons were 537

detected using anti-Fizzy-related (monoclonal, 1:500, Novus Biologicals, Cambridge, UK; 538

polyclonal, 1:500, Invitrogen, Darmstadt, Germany) and anti-Id2 (polyclonal, 1:500, Santa Cruz) 539

antibodies. Suitable secondary antibodies were used to visualize primary antibody binding. 540

Photomicrographs were captured with Zeiss AxioVision 2 (Zeiss, Jena, Germany) and 541

AxioImager microscopes (software AxioVision 4.8; Zeiss). 542

543

X-Gal and Fluoro Jade B staining protocols 544

ON and retinae of B-lacZ reporter mice were fixed in 2% PFA/0.2% glutaraldehyde in PBS 545

(4°C) for 10 min, washed, and incubated in X-Gal staining solution (Roche, Mannheim, 546

Germany) for 24 h at 37°C. Samples of liver and thymus were taken as positive controls. 547

Longitudinal ON sections were immersed in 1% sodium hydroxide in 80% ethanol for 5 min, 548

followed by incubation in 70% ethanol and distilled water for 2 min, respectively. For 549

background suppression, slides were incubated in 0.06% potassium permanganate for 10 min. 550

The staining solution was prepared from a 0.01% stock of Fluoro Jade B (Chemicon) in 0.1% 551

acetic acid and freshly used at a final concentration of 0.0004%. After a 20-min incubation, 552

slides were rinsed in distilled water, dried at 50°C, cleared with xylene, and covered with 553

ImmuMountTM (Shandon, Pittsburgh, PA, USA). 554

555

Electron microscopy 556

Animals were transcardially perfused with ice-cold PBS and freshly prepared fixative containing 557

1% PFA, 3% glutaraldehyde, 0.5% acrylaldehyde, and 0.05 M CaCl2 in 0.1 M cacodylate buffer 558

(pH 7.3). ON were fixed at 4°C overnight and processed for ultrafine sectioning on an EMTP 559

tissue processor (Leica, Wetzlar, Germany). Specimens were rinsed six times (15 min/rinse) in 560

0.1 M cacodylate buffer and postfixed in 1% osmium and 1% potassium hexacyanoferrate II in 561

0.1 M cacodylate buffer at 4°C for 1 h. Following rinsing in cacodylate buffer and distilled 562

water, ON were dehydrated in acetone (30, 50, 70, 90, 95%; 30 min at each step; Merck, 563

Darmstadt, Germany), then twice in 100% acetone (45 min each time). Samples were transferred 564

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to acetone-resin (EPON) composites and embedded for polymerization. Ultrathin sections of 50-565

nm thickness were cut with Reichert Ultracut S knifes (Leica) and 35° diamond blades (Diatome, 566

Hatfield, PA, USA). Ultramicrographs were captured by a transmission electron microscope 567

(JEM 1400, Jeol, Ehing, Germany) under 80 kV using a CCD camera (Orius SC 1000, Gatan 568

Munich, Germany). 569

570

Quantification of RGC/ODC and axonal regeneration 571

Four weeks after ONI, retinae were fixed in 4% PFA/PBS (pH 7.4), flattened on glass slides, and 572

subjected to immunohistochemical procedures using the RGC marker TUJ-1. RGC densities 573

were assessed at 3–4/6 radial eccentricity under a fluorescence microscope (AxioImager, Zeiss). 574

Fluorescence microscopy was also used to quantify ODC in 10-µm longitudinal cryosections of 575

the naïve ON after labeling with CAII antibodies. 576

Serial quantification of growth responses in anterogradely traced ON was performed using the 577

AutMess imaging program (Zeiss) supplemented by the AxioVision LE module under a 40× 578

objective (AxioImager, Zeiss). Since the undiluted CTX tracer displays defined invariable signal 579

intensity, the threshold for signal detection was similar for all groups and specimens. Fields of 580

regeneration (ROI) were automatically identified and summed to define the total regenerative 581

area in each slice. The total regenerative area of each ON was determined by summing the ROIs 582

of all the slices. 583

584

Immunoblotting 585

Lysates of retinae and ON were exposed to SDS-PAGE and processed for immunoblotting 586

according to standard protocols. The following antibody concentrations were used: RelA 587

(polyclonal C-20, 1:1,000, Santa Cruz), phospho-RelA (monoclonal pSer-536 clone 93H1, 588

1:1,000, Cell Signaling), NLS-RelA (monoclonal MAB 3026 clone 12H11, 1:500, Millipore), 589

MBP (polyclonal, 1:200, Millipore), Id2 (polyclonal, 1:500, Santa Cruz), Cdh1 (Fizzy-related, 590

monoclonal, 1:500, Novus Biologicals), and EMI1 (monoclonal, 1:500, Invitrogen). β-Actin 591

(polyclonal, 1:10,000, Abcam) was applied as loading control. Results were repeated at least 592

three times for three different specimens. 593

594

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In vitro experiments 595

For immunocytochemistry on primary neurons, cultures of E16 embryonic RelACNSKO and 596

RelAflox hippocampi were stained with SMI-31, Cdh1, and Id2 antibodies and counterstained with 597

DAPI. Our protocol on embryonic, genotype-specific neuronal cultures from transgenic mice is 598

available on request. Intranuclear-to-cytoplasmic switch of the signals was semi-quantitatively 599

assessed using a Zeiss AxioImager microscope and AxioVision imaging software (Zeiss). 600

OLN-93 cells were grown as monolayers in sterile DMEM medium supplemented with 10% 601

fetal bovine serum for 5 days at 37°C, 5% CO2 and controlled humidity. To induce 602

differentiation, serum was reduced to 0.5% for 5 days. Immuno-stainings with NLS-RelA or 603

RelA (C-20) antibodies (see above) were performed 30 min after TNF (20 ng/ml; Sigma, 604

Germany) or PBS treatment. 605

606

Optometric measurement 607

Visual acuity and contrast sensitivity were investigated making use of the optokinetic reflex in a 608

virtual-reality optomotor device. Freely moving animals were subjected to moving sine wave 609

gratings of various spatial frequencies and contrasts. Gratings were varied up to the detection 610

threshold of reflexive head tilting (Prusky et al., 2004). Animals in the knockout and control 611

groups were analyzed at identical time points within the circadian rhythm. 612

613

Statistical analysis 614

Statistical analyses were performed using the Student’s t-test for single comparisons, followed 615

by post-hoc test calculation. Data are presented as mean±standard error (s.e.m.). For each 616

experiment, individual n numbers are given separately. Results reaching P≤0.05 were considered 617

statistically significant (P<0.05, *; P<0.01, **; P<0.001, ***; P> 0.05, #). 618

619

Acknowledgements 620

We appreciate the provision of the OLN-93 cell line by C. Richter-Landsberg. We thank S. 621

Tausch for technical and M. Baldauf for histological assistance. We are grateful to K. Buder for 622

the support on ELMI and S. Schmidt and I. Krumbein for MRI advice. 623

624

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Funding 625

R.H. is supported by the VELUX Foundation, Switzerland. A.K. was supported by the IZKF 626

Jena. 627

628

Author Contributions 629

R.H. and A.K. organized the study and prepared the manuscript. K.-H.H. developed the MEMRI 630

protocol. K.K. and K.-F.S. conducted the functional animal tasks. C.E. performed cell culture 631

experiments. K.-A.N. provided the Cre line for the creation of ODC specific mouse mutants. 632

F.W., O.W.W., S.L., and J.R.R. supervised and financed the study and helped with data 633

interpretation. 634

635

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Figure Legends 773

Fig. 1. Visual system characterization under modulated RelA activity. 774

(A) H&E staining of transversal retina showed unsuspicious layer morphology in RelACNSKO 775

mice. Scale bar: 100µm (B) Co-labeling of retinal whole mounts with the RGC marker TUJ-1 776

(green) and the MG marker CRALBP (red) exhibited a normal RGC and MG ratio and cell 777

contacts in superficial NFL/GCL in RelACNSKO mice as compared to controls (n=3). Scale bar: 50 778

μm. (C) Electron microscopy displayed unimpaired myelin sheaths insulating ON axons in 779

RelACNSKO mice as compared to controls (n=5/6). Scale bar in left row: 150 nm; scale bar in right 780

row: 25 nm. (D) Impaired cell differentiation and survival were ruled out by similar naïve RGC 781

densities (P=0.88), ODC numbers (P=0.43), and g-ratios (P=0.99) in the ON of RelACNSKO mice 782

and controls (n=5/6 for each group). (E) Functional parameters revealed physiological values for 783

visual acuity (P=0.21) and contrast sensitivity (P=0.61) in three-month-old RelACNSKO mice 784

(n=5). (F) Increased retinal κB-lacZ reporter activity in B-lacZ;p50KO mice compared to 785

controls indicated RelA/NF-B hyperactivity due to p50 deletion (P<0.02; n=5). Scale bar: 200 786

μm. NFL, nerve fiber layer; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner 787

nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; PR, photo receptor layer; 788

ODC, oligodendrocyte. 789

790

Fig. 2. ONI activates NF-B in a cell-autonomous manner. 791

(A) ONI in B-lacZ reporter mice increased the number of β-gal-positive cells in the retina by 792

three- (1-3 dpi) and 18-fold (4-10 dpi) thereafter. dpi, days post injury (n=3). (B) Micrographics 793

of retinal whole mount preparations depicting increased numbers of X-Gal-positive cells (blue) 794

in superficial retinal layers after ONI (X-Gal panel). Scale bar: 200 μm. Transversal retinal 795

sections indicated pan-NF-B activation in the GCL (ONI panel), and co-localization of the β-796

gal signal (red) with the TUJ-1 staining (green) evidenced NF-B activation in DAPI-positive 797

(blue) RGC nuclei (purple; inset, 40× objective) (n=4). Scale bar: 80 μm. (C) Immunoblot 798

analysis with total RelA (C-20) and phospho-specific (Ser536) RelA antibodies demonstrated 799

expression of RelA in naïve ON, and activation within 3 h after ONI in WT animals (top; n=3). 800

TNF-treated hippocampal neurons served as a positive control. Ser536 phosphorylation of RelA 801

was almost completely abolished in RelACNSKO mice (bottom; n=3). (D) Enzymatic X-Gal assay 802

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of ON in toto preparation revealed NF-B activation in the lesion epicenter (n=6). Scale bar: 100 803

μm. Co-localization of the β-gal signal (red; ONI panel) with the ODC marker CAII (green) in 804

longitudinal ON sections confirmed NF-B activation in DAPI-stained nuclei (blue) of ODC 805

(purple; inset, 40× objective) (n=3). Scale bar: 30 μm. Note the absence of CAII reactivity in the 806

non-myelinated ON head (dotted line in naïve panel). Scale bar: 100 μm. (E) Left: Co-807

localization (yellow) of NLS-RelA-positive cells (green) with β-gal signals (red) in B-lacZ 808

reporter mice showed that RelA was the dominant NF-B subunit activated by ONI in ODC 809

(n=4). Right: No NLS-RelA signal was detectable in nuclei of CAII-positive ODC of RelACNSKO 810

mice. Scale bar: 30 μm. (F) TNF-induced activation of RelA in oligodendrocytic OLN-93 cells 811

as detected by NLS-RelA antibody. Z-stack analysis confirmed nuclear localization of RelA in 812

stimulated cells. Scale bar: 20 μm. 813

814

815

Fig. 3. RelA and p50 subunits differentially control RGC survival after ONI. 816

(A) ONI performed on B-lacZ;p50KO reporter mice strongly induces B-dependent X-Gal 817

staining (blue color) at the ON injury site (right panel), which was lacking at the naïve site (left 818

panel). Asterisk, lesion site. Scale bar: 200 μm. Photomicrographs of corresponding flat-mounted 819

retinae showing substantial numbers of X-Gal-positive RGC under naïve conditions, and a 820

drastic reduction at 9 dpi. Scale bar: 200 μm. (B) RelA deletion exerts moderate, but significant 821

anti-apoptotic effects on deafferentiated RGC four weeks after ONI (n=9 for RelACNSKO; n=7 for 822

RelAflox). In contrast, p50 ablation drastically severed RGC death (n=3). 823

824

825

Fig. 4. Axonal regeneration is enhanced in RelACNSKO mice. 826

(A-D) Axonal regeneration as detected by CTX-594-positive fiber ingrowth into the ON was 827

greatly stimulated in RelACNSKO mice (n=16) as compared to controls (n=10). (A) Trans-lesional 828

fiber elongation was frequently observed in RelACNSKO mice, but rarely in controls. Asterisks, 829

lesion site. In p50KO mice (n=6), axon ingrowth into the ON head was even lower than in 830

controls. Scale bar: 100 μm. (B) Quantification of total growth area within individual ON of 831

RelACNSKO versus p50KO and control mice. Gender-related effects were not evident. (C) Selective 832

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assessment of trans-lesional growth areas. (D) Absolute trans-lesional axon lengths in RelACNSKO 833

mice versus controls. (E) Measurement of pre-lesional distances indicated comparable positions 834

of the injury site in RelACNSKO mice and controls, thus excluding artifacts in real growth 835

distances. 836

837

Fig. 5. Wallerian degeneration is severed in p50KO mice. 838

(A) CTX-594 (red) co-localized with GAP-43 (green) in elongating axons of RelACNSKO mice. 839

Note the strongest GAP-43 signal in axon tips (arrows) beyond the injury site (asterisks). Scale 840

bar: 100µm (B) Left: In RelACNSKO mice, newly elaborated axons developed rudimentary growth 841

cones at their GAP-43-positive tips (green). Scale bar: 15 μm. Right: In retinae counterstained 842

with GFAP for anatomic orientation, the appearance of aberrant MAP2-positive processes in 843

RelACNSKO mice indicated a pro-regenerative influence on RGC dendrites too (dashed box with 844

magnification). Scale bar: 30 μm. (C) ONI induced Wallerian degeneration in RelACNSKO mice 845

and RelAflox controls (n=3), as indicated by Fluoro Jade (FJ)-reactive axon bulges. However, 846

p50KO mice showed strong spontaneous fiber degeneration at a susceptible age of 10 months, as 847

demonstrated by intense FJ staining (n=3). Specificity of the signal was indicated by lack of FJ 848

reactivity in naïve ON of 10-month-old RelACNSKO mice and outside the injured optic tract (right 849

panel). Scale bar: 50 μm. 850

851

Fig. 6. RelA differentially modulates axonal regeneration in RelACNSKO, RelAODCKO, and 852

RelAASTKO mice. 853

(A) CNP1-Cre activity in the ON of RelAODCKO mice was confirmed by its intense blue staining 854

in Rosa26R;CNP1-Cre reporter mice. Scale bar: 100 μm. (B) RelA deletion selectively in ODC 855

(RelAODCKO) significantly enhanced axonal regeneration. (C) Accelerated degradation of white 856

matter MBP in RelACNSKO mice four weeks after ONI (two independent runs using pooled protein 857

of three animals). (D) Deletion of RelA in MG (arrows), i.e. specialized astrocytes of the retina, 858

was confirmed by YFP induction in YFP;GLAST-CreERT2 reporter mice after tamoxifen (TAM) 859

treatment. Scale bar: 50µm. (E) Pro-regenerative effects of RelA deletion in astrocytes 860

(RelAASTKO) exceeded the growth response in RelAODCKO mice. (F) Growth-promoting effects 861

paralleled a reduced production of repulsive neurocan at the injury site (n=3). Asterisks denote 862

scar region. Scale bar: 250 µm. (G) In RelACNSKO mice, regrowing axons penetrated the scar 863

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region more easily (asterisks), as shown by CTX labeling (n=16 for RelACNSKO; n=10 for 864

controls). Arrows delineate preferred growth direction of newly built axons. Scale bar: 150 µm. 865

866

Fig. 7. RelA controls Cdh1 levels and its nucleo-cytoplasmic shuttling. 867

(A-D) Cdh1 acts in neurons to control RelA-dependent axon growth. (A) Expression of Cdh1 868

was increased in naïve optic and sciatic nerves and, to a lesser extent, in cortex and retina (n=3; 869

pooled protein from three animals). Calculations indicate optic density relative to actin loading 870

control. (B) Within the naïve retina, Cdh1 was localized to RGC and displayed a nuclear (n; 871

arrow), cytoplasmic (c; arrowhead), or mixed (n/c; asterisk) distribution. Scale bar, 50µm. (C) 872

Cdh1 upregulation in wild type retina four weeks after ONI was suppressed in RelACNSKO mice 873

(n=4; three samples). In contrast, Cdh1 was upregulated in growth-incompetent p50KO mice four 874

weeks after ONI as well as in progeroid retina at the age of 10 months (n=3; two samples). (D) In 875

growth-conditioned retina, Cdh1 was found suppressed as a function of the cell type targeted by 876

RelA deletion. The degree of Cdh1 decline in RelACNSKO RelAASTKO > RelAODCKO mice 877

correlated with absolute growth responses (n=4; three samples). (E) The Cdh1 inhibitor EMI1 878

was antagonistically upregulation in naïve RelACNSKO mice. ONI caused no further induction 879

(n=3; three samples). Corresponding to increased Cdh1 levels, EMI1 declined with aging in 880

p50KO mice (n=3; two samples). (F-H) RelA defines nucleo-cytoplasmic shuttling of Cdh1 in 881

cultured hippocampal neurons (HN). (F) Co-staining for Cdh1 and nuclear DAPI in HN (4 div) 882

revealed predominant nuclear (active) location of Cdh1 in wild type cells (left, purple nuclei; 883

arrowhead), whereas RelA deletion caused a shift of Cdh1 to a cytoplasmic (inactive) location 884

(right, blue nuclei; arrow). Solid neuro-neuritic TUJ-1 staining indicated viability of either 885

culture (top panel; n=3). Scale bar for TUJ-1: 30 µm; scale bar for Cdh1/DAPI: 10 µm. (G) 886

Nucleo-cytoplasmic shift of Cdh1 in RelA-deficient HN was assessed by cellular 887

immunofluorescence absorption profiles. Both graphs indicate the preponderance of 888

extranuclear, inactive Cdh1 in RelACNSKO as compared to RelAflox. (H) Quantitative analysis of 889

average cytoplasmic Cdh1 signal distribution (analyzed cell number: RelAflox n=122; RelACNSKO 890

n=131). Analysis of cytoplasmic-to-nuclear signal relation revealed a shift from high cell 891

fractions (77.1%) with low cytoplasmic Cdh1 signal (0–30%) in controls to fractions (48.1%) 892

with dominant cytoplasmic Cdh1 signal (31–70%) in RelACNSKO populations. (I) Representative 893

images of cytoplasmic Id2 signal distribution in RelAflox and RelACNSKO HN (3.5 div) revealing 894

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predominant cytoplasmic accumulation of Id2 in the axon hill (left; arrowhead and inset) of wild 895

type cells, whereas RelA deletion caused its nuclear (active) concentration (right; arrow and 896

inset). Scale bar: 10 µm. (J) Quantification of subcellular signal distribution evidenced a shift 897

from dominant cytoplasmic Id2 in controls (59.2%) to a preponderance of nuclear Id2 in 898

RelACNSKO populations (78.1%) (analyzed cell number: RelAflox n=90; RelACNSKO n=130). 899

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Leibniz Institute for Age Research Journal of Cell Science...

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