Additional file 1 of NF-κB is a critical mediator of post-mitotic senescence in oligodendrocytes and subsequent white matter loss
Creators: name: Schlett, Judith Stefanie
name: Mettang, Melanie
name: Skaf, Aladdin
name: Schweizer, Pavel
name: Errerd, Alina
name: Mulugeta, Ephraim Alemayehu
name: Hein, Tabea Melissa
name: Tsesmelis, Konstantinos
name: Tsesmelis, Miltiadis
name: Büttner, Ulrike F. G.
name: Wendt, Heinrich
name: Abaei, Alireza
name: Rasche, Volker
name: Prex, Vivien
name: Nespoli, Ester
name: Alami, Najwa Ouali
name: Tews, Daniel
name: Walther, Paul
name: Yilmazer-Hanke, Deniz
name: Oswald, Franz
name: Dimou, Leda
name: Wirth, Thomas
name: Baumann, Bernd
Publisher: name: figshare
Year: 2024
Subjects: subject: Medicine
subject: Microbiology
subject: FOS: Biological sciences
subject: Cell Biology
subject: Genetics
subject: Molecular Biology
subject: Neuroscience
subject: Physiology
subject: Immunology
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Cancer
subject: Mental Health

description: Additional file 1: Supplementary Figure 1. Functional characterization of the IKK2-CAPLP-CreERT2 mouse model. a Scheme of the conditional transgenic IKK2-CAPLP-CreERT2 mouse model. Expression of a constitutive-active version of IKK2 (IKK2-CA) and the IRES driven eGFP reporter gene is inhibited through a stopper cassette until removed by tamoxifen-activated Cre recombinase. Cre recombinase expression under control of the PLP promotor ensures specificity to mature OLs. b Transgene expression was induced by 3 intraperitoneal injections of tamoxifen (2 mg) followed by tamoxifen-enriched food (400mg/kg) for 14 days. c and d Immunohistochemical analysis of RFPPLP-CreERT2 mice revealed no leakiness of the transgenic system. Control and uninduced RFPPLP-CreERT2 animals (no tamoxifen treatment) revealed no RFP expression, while recombination efficiency in RFPPLP-CreERT2 animals was found at 63.2%±4.8% (n = 3). e Immunoblot analysis of midbrain and cerebellar tissue samples isolated from control and IKK2-CAPLP-CreERT2 mice at 3 wpi. Transgene activation was demonstrated by prominent IKK2 and eGFP expression, abundance of p-p65 validates functional activation of IKK/NF-κB signaling (n = 3). f Elevated Ccl5 gene expression levels in cortex, CC, cerebellar and spinal cord tissue 3 wpi indicate an inflammatory milieu within the CNS (n = 3-4). g NF-κB target genes iNos, IL1b and Lcn2 are prominently upregulated in the OL-rich corpus callosum whereas expression is unchanged in spinal cord, cortex and cerebellum. h and i Immunohistochemical analysis of Ki67+/Pdgfrα+ double positive cells revealed a significantly increased number of proliferating precursor cells. (3 wpi – Control (n = 3): 8.3±4.3 cells/mm2, IKK2-CAPLP-CreERT2 (n = 3): 30.8±3.2 cells/mm2; 20 wpi – Control (n = 3): 5.0±3.6 cells/mm2, IKK2-CAPLP-CreERT2 (n = 3): 10.1±3.8 cells/mm2). j and k Expression of the IKK2-CA transgene is restricted to mature OLs. Arrows indicate no colocalization of the GFP reporter gene with NG2 staining marking progenitor cells (cortex tissue, 3wpi). (I) Sciatic nerve tissue does not show GFP reporter gene expression in control or IKK2-CAPLP-CreERT2 animals. As positive control sciatic nerve tissue from a Sox10-GFP reporter mouse was used, which shows robust expression of GFP in Schwann cells. Data are shown as mean ± SEM. Grey bars, control; red bars, IKK2-CAPLP-CreERT2. Statistical analysis: One-way or Two-way ANOVA multiple comparison test followed by Bonferroni´s post hoc test (*p &lt; 0.05; **p &lt; 0.01; ***p &lt; 0.001; ****p &lt; 0.0001 NS, non-significant (p &gt; 0.05)). Supplementary Figure 2. Consequences of IKK2-CA activation in OLs. a Weight development was observed over the time course of 40 weeks. IKK2-CAPLP-CreERT2 mice show normal weight gain until 19 wpi, thereafter natural weight gain is decreased in both male and female mice. b IKK2-CAPLP-CreERT2 mice do not lose pre-learnt motoric skills. If animals were introduced to the rotarod task immediately after transgene activation, performance of IKK2-CAPLP-CreERT2 and control animals was comparable over the entire time course of 40 weeks. c and d IKK2-CA activation in oligodendrocytes does affect cerebellar homeostasis. Measurement of cerebellar width showed no differences between IKK2-CAPLP-CreERT2 and control animals. The black line in D indicates the measured length (n = 5-7). e Motoric deficits do not issue from purkinje cell loss. Histochemical analysis using Nissl staining of the cerebellum shows intact morphology and normal purkinje cell number as indicated by black arrows. f Immunohistochemical analysis using SMI32 did not reveal axonal pathology in IKK2CAPLPCreERT2 animals. As a positive control tissue slides from TBI treated wildtype animals were used. Data are shown as mean ± SEM. Black dots/grey bars, control; red dots/bars, IKK2-CAPLP-CreERT2. Statistical analysis: Two-way ANOVA multiple comparison test followed by Bonferroni´s post hoc test, Two-tailed Mann-Whitney-U Test (*p &lt; 0.05; **p &lt; 0.01; ***p &lt; 0.001; ****p &lt; 0.0001 NS, non-significant (p &gt; 0.05)). Supplementary Figure 3. NF-ĸB activation in OLs leads to white matter loss in the corpus callosum. a and b Ex-vivo MRI analysis of the brain at 20 wpi reveals reduced signal strength (appearing brighter) in the CC as well as reduction in width of the CC of IKK2-CAPLP-CreERT2 mice. White arrows indicate the CC. c Quantification of the width of the CC (n = 3-4). d and e Distribution of the axon diameter of myelinated axons in the CC is not changed IKK2-CAPLP-CreERT2 and control animals at 3 wpi and 20 wpi. f Diameter of unmyelinated axons depicted as single values reveals a greater scattering of the axon diameter of unmyelinated axons in the CC of IKK2-CAPLP-CreERT2 mice 20 wpi. g – i Luxol Fast Blue (LFB) staining revealed a significant decrease in the width of the CC in IKK2-CAPLP-CreERT2 animals at different bregma points at 20 wpi. Bregma points evaluated were Bregma -1mm (g), Bregma (h) and Bregma+1mm (i). j Quantification of g – i (Bregma-1mm: 3wpi: Control: 348±21.7 μm, IKK2-CAPLP-CreERT2: 344±16.8 μm; 20wpi: Control: 442±17.3 μm, IKK2-CAPLP-CreERT2: 353±16.2 μm; Bregma: 3wpi: Control: 231±8.9 μm, IKK2-CAPLP-CreERT2: 225±2.3 μm; 20wpi: Control: 235±3.1 μm, IKK2CAPLP-CreERT2: 184±13.0 μm; Bregma+1mm: 3wpi: Control: 508±40.6 μm, IKK2CAPLPCreERT2: 498±32.9 μm; 20wpi: Control: 624±3.9 μm, IKK2-CAPLP-CreERT2: 503±22.5 μm n = 3-4). Data are shown as mean ± SEM. Black lines/grey bars, control; red lines/bars, IKK2-CAPLP-CreERT2. Statistical analysis: Two-way ANOVA multiple comparison test followed by Bonferroni´s post hoc test, (*p &lt; 0.05; **p &lt; 0.01; ***p &lt; 0.001; ****p &lt; 0.0001 NS, non-significant (p &gt; 0.05)). Supplementary Figure 4. Isolation of O4+ OLs from brains of IKK2-CAPLP-CreERT2 mice and subsequent transcriptome analysis. a Schematic workflow of the consecutive isolation of primary O4+ OLs and CD11b+ microglia from adult mouse brains. After tissue dissociation, the obtained cell suspension is treated with magnetic beads labeled with O4 antibodies thus allowing separation of O4+ OLs in a magnetic field. The remaining cell suspension is again treated with magnetic beads, this time labeled with Cd11b antibodies allowing isolation of Cd11b+ microglia in a second step. b Cell number of isolated O4+ OLs does not significantly differ between IKK2-CAPLP-CreERT2 and control animals at 3 and 20 wpi (3 wpi: Control: 0.90±0.05 x106, IKK2-CAPLP-CreERT2: 0.81±0.05 x106 n = 20-23, 20 wpi: Control: 1.1±0.07 x106, IKK2-CAPLP-CreERT2: 1.3±0.09 x106, n = 10-23, two brains with identical genotype were pooled and set to n = 1). c and d qRT-PCR reveals strong upregulation of the IKK2 transgene (IKK2-CA) as well as GFP reporter gene in O4+ OLs from IKK2-CAPLP-CreERT2 animals, e western blot analysis indicates robust expression of IKK2-CA protein solely in IKK2-CAPLP-CreERT2 O4+ OLs. Cortex tissue from IKK2-CAPLP-CreERT2 mice served as positive control (n = 3). f FACS analysis of cells isolated with O4+ beads shows a strong enrichment (70%) of O4high/CD11blow cells. GFP expressing IKK2-CAPLP-CreERT2 O4+ OLs are strongly enriched within the isolated O4+ OL cell population. Quantification of the GFP+ O4+ cells 3 and 20 wpi (3 wpi: 27.8±4.1 % n = 9, 20 wpi: 9.2±1.5 % n = 5). g FACS staining of O4+ OLs with GalC and MOG as markers for intermediate and mature OLs revealed strong enrichment of myelinating OL within the bead-sorted O4 fraction without differences between control and IKK2-CAPLP-CreERT2 O4+ OLs (GalC: Control: 70.74±1.64 %, IKK2-CAPLP-CreERT2: 72.73±1.80 %; MOG: Control: 56.14±3.20 %, IKK2-CAPLP-CreERT2: 58.85±4.04 %, n = 3-5). h and i Volcano plot depicting upregulated and downregulated genes in O4+ OLs from IKK2CAPLP-CreERT2 mice compared to control mice at 3 and 20 wpi, revealed by RNA sequencing analysis. j and k RNA sequencing reveals significantly enriched NF-ĸB target gene expression (i) in IKK2-CAPLP-CreERT2 O4+ OLs whereas genes connected to the GO class myelin sheath formation (GO: 0043209) (j) are only slightly deregulated as 3 and 20 wpi (as indicated in the heatmaps). Out of the genes depicted only Mobp and Plec were found significantly deregulated at 3 and 20 wpi, while at 3 wpi additionally Tppp, Plp1 and Msn reached significance, as well as Tubb4b, Calm1 and Gsn at 20 wpi. Data are shown as mean ± SEM. Grey bars, control; red bars, IKK2-CAPLP-CreERT2. Statistical analysis: Two-tailed Mann-Whitney-U Test, (*p &lt; 0.05; **p &lt; 0.01; ***p &lt; 0.001; ****p &lt; 0.0001 NS, non-significant (p &gt; 0.05)). Supplementary Figure 5. Gene enrichment analysis of O4+OLs with activated IKK/NFκB signaling. a and b GSEA shows strong enrichment in gene sets of cellular senescence (a) and p53 signaling pathway (b) in IKK2-CAPLP-CreERT2 O4+OLs compared to control O4+OLs at 3 wpi. c Heatmap depicts strong significant overall upregulation of SASP factors in IKK2CAPLPCreERT2 O4+OLs compared to control O4+OLs both at 3 and 20 wpi. d Schematic overview of translational arrest by phosphorylation of eucaryotic initiation factor 2, subunit alpha (eIF2α). Stress-activated kinases (e.g., PERK, PKR, GCN2 and HRI) are able to phosphorylate eIF2α leading to the inhibition of GDP to GTP exchange resulting in global translational arrest. e RNA sequencing analysis reveals enrichment in proteasome (MMU03050) in IKK2CAPLPCreERT2 O4+OLs 20 wpi. Supplementary Figure 6. NF-ĸB activation in OLs initiates an aging-associated microglia phenotype. a Bead-Isolated Cd11b+ microglia were subjected to FACS analysis using Cd11b-PE labeled antibody and revealed a purity of 83%. b and c Volcano plot depicting upregulated and downregulated genes in Cd11b+ microglia from IKK2-CAPLP-CreERT2 mice compared to control mice at 3 and 20 wpi (determined by RNA sequencing analysis). d Cd11b+ microglia isolated from IKK2-CAPLP-CreERT2 animals (3 wpi) show an overlap with aged white matter associated microglia (WAM) in gene sets involved in phagocytic activity and metabolism (Safaiyan 2021). IKK2-CAPLP-CreERT2 derived Cd11b+microglia exhibit a more pronounced elevation of genes related to lipid-metabolism and phagosome activity compared to WAM and activated microglia. Genes linked to ribosomal subunits and mitochondrial genes are rather downregulated in WAM IKK2-CAPLP-CreERT2 Cd11b+microglia. e GSEA uncovers gene signatures of cellular senescence in IKK2-CAPLP-CreERT2 Cd11b+ microglia compared to control microglia at 3wpi.