NEUROINFLAMMATION GENERATION OF NEURAL SPECIFIC KNOCKOUT Generalized and neural restricted peroxisomal MFP2 deficiency causes demyelination and motoric deficits but neuroinflammatory responses are distinct Simon Verheijden, Myriam Baes Laboratory of Cell Metabolism, KU Leuven, Belgium INTRODUCTION Inactivation of multifunctional protein 2 (MFP2), and thus peroxisomal β-oxidation, in a knockout mouse model causes severe neurological abnormalities, including progressive motor deficits 1. To elucidate the mechanisms underlying this pathology, a conditional mouse strain was generated. In the Nestin-MFP2-/- mouse model MFP2 is selectively inactivated in all neural cells, but the enzyme is preserved in nonneural cells such as microglia. We now investigated the development and progression of pathologies in the CNS of Nestin-MFP2-/- mice and compared the phenotype with general MFP2-/- mice. Onset of motoric symptoms are similar in both mouse models. However general knockout mice die before the age of 6 monhts, whereas NestinMFP2 mice live beyond this age. Histological analysis revealed demyelination in the cerebellum as possible cause of coordination problems in both mouse models. However microgliosis and pro- inflammatory gene expression are significantly lower in the NestinMFP2 model. The general MFP2 mouse model has a perturbed reaction to LPS, as evaluated in vivo and ex vivo. CONCLUSION General MFP2 deficiency causes motoric abnormalities progressing into severe ataxia and death before the age of 6 months. Motoric problems are accompanied by demyelination, axonal degeneration and neuroinflammation. The latter is evident by extensive micro- and astrogliosis and the upegulation of pro-inflammatory markers. Motoric abnormalities are similar in a conditional mouse model, which lacks MFP2 in all neural precursors but not in microglia. However, the neuroinflammatory responses are distinct in both mouse models. Moreover it is shown that the inflammatory reaction in response to LPS induction is upregulated in MFP2 KO mice. This indicates that peroxisomal β- oxidation plays a role in the regulaton of inflammatory responses in the brain. This regulatory role might be mediated by microglia. REFERENCES 1 Huyghe S. et al (2006) Am J Pathol 168(4) ACKNOWLEDGMENTS This research was supported by a grant from Fonds Wetenschappelijk Onderzoek (FWO G OT 08/040); OT KULeuven 08/40 and Association Européenne contre les leukodystropies (ELA ). MOTORIC ABNORMALITIES MYELIN/AXONAL INTEGRITYMICROGLIAL REACTIVITY IS DISTINCT GENE EXPRESSION PRO-INFLAMMATORY MARKERS RESPONSE TO LPS INDUCTION EX VIVO AND IN VIVO NESTINMFP2 MBP - blue SMI31 – green KO *** * * ** * * A.In order to create a neural-specific mutant, a targeting vector was designed in which exon 8 of the MFP2 gene is floxed. This encodes the catalytic site of the N-terminal dehydrogenase domain. Elimination of this exon was expected to yield an out of frame transcript for the hydratase and SCP2 domain. Breeding MFP2-loxP homozygotes with Nestin-Cre mice yielded offspring with inactivation of MFP2 in the neural progenitor cells B.Elimination of MFP2 transcript in NestinMFP2 in whole brain was confirmed by Northern Blot C. Absence of the MFP2 protein in whole brain was confirmed by Western Blot. A. PURKINJE CELLS * ** NESTINMFP2 MFP2 WT MFP2 NestinMFP2 B. C. A. B. A.MFP2 KO and NestinMFP2 KO mice show with similar motoric abnormalities. Both mice models show limp-clasping behaviour when lifting them by their tail (middle and right panel). Both mice developed coordination problems and became severely ataxic at the age of 4 months. B.Locomotoric behaviour was tested on an accelerating rotatrod. Both mouse models show a decrease in locomotor performance beginning at the age of 8 weeks. The MFP2 mouse model dies before the age of 6 months whereas most of the NestinMFP2 mice live beyond the age of 6 months (At least until 9 months) (data not shown) NESTINMFP2 MFP2 -/- Purkinje cell abundance is normal in both models. This was evaluated by calbindin- staining in NestinMFP2 (6months, upper panel) and MFP2 (5 months, lower panel) A.NestinMFP2 Myelination and axonal integrity was evaluated at 6 months of age by staining for myelin basic protein (MBP) (red) and phosphorylated NFs(SMI31) (green). Axons seemed to be unaffected, whereas MBP staining was slightly reduced, especially in cerebellar branches. B. MFP2 Regarding myelin (red), the same observations were made in the MFP2 KO mouse model at 5 months of age. However, axons were also affected (green) +/+ -/- +/+ -/- Gliosis was evaluated in both models by staining for F4/80 (microglia,left, green) and GFAP (astrocytes, right, red) at the age of 4 months. Both models showed with moderate to severe astrogliosis in all brain areas as seen by an increase in GFAP staining (upper and lower right panels) Staining for the microglial marker F4/80 revealed a decreased microglial reactivity in NestinMFP2 KO mice compared to MFP2 KO mice. This was most pronounced in the cerebral cortex (upper an lower left panels) A.MFP2 gene expression To evaluate neuroinflammation, gene expression of pro-inflammatory markers was measured by RT-PCR in corpus callosum. Different inflammatory markers such as TNFalpha, C1q and TLR2 were significantly upregulated at the age of 9 weeks and were even more upregulated at the age of 12 weeks. B. Comparison MFP2 vs NestinMFP2 Because of the difference in microglial reactity in both models, we compared gene expression of Mpeg-1, a macrophage marker and TNFalpha, a pro- inflammatory marker at 12 weeks of age int the cerebral cortex. Gene expression of both markers was significantly lower in NestinMFP2 KO mice compared to MFP2 KO mice A.LPS induction organotypic brain slices MFP2 KO mice Brain slices of MFP2 KO mice (P14) were cultured and induced with 100 ng/ml LPS. TNFα gene expression was evaluated at 1h – 6h and 24h post- induction. TNFα expression was significantly higher at 6h after LPS induction in MFP2 KO slices compared to WT slices. B. In vivo LPS induction MFP2 KO m MFP2 KO mice of 4 weeks old were induced with 5 mg/kg LPS intraperitoneally. After 6h mice were euthanized and dissected. Gene expression analysis of TNFα was performed on cerebral cortex. TNFα upregulation was more pronounced in LPS-induced MFP2 KO mice compared to WT mice.