Axin2 as regulatory and therapeutic target in newborn brain injury and remyelination

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Axin2 as regulatory and therapeutic target in newborn brain injury and remyelination
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  Axin2 as regulatory and therapeutic target in newborn braininjury and remyelination Stephen P.J. Fancy 1 , Emily P. Harrington 1,2 , Tracy J. Yuen 1 , John C. Silbereis 1 , ChaoZhao 6 , Sergio E. Baranzini 3 , Charlotte C. Bruce 6 , Jose J. Otero 1,4 , Eric J. Huang 4 , RoelNusse 7 , Robin J.M. Franklin 6 , and David H. Rowitch 1,5 1 Departments of Pediatrics and Neurosurgery, Eli and Edythe Broad Institute for Stem CellResearch and Regeneration Medicine and Howard Hughes Medical Institute, University ofCalifornia San Francisco, 513 Parnassus Avenue, San Francisco, CA, 94143, USA 2 Medical Scientist Training Program, University of California San Francisco, 513 ParnassusAvenue, San Francisco, CA, 94143, USA 3 Department of Neurology, University of California San Francisco, 513 Parnassus Avenue, SanFrancisco, CA, 94143, USA 4 Department of Pathology, University of California San Francisco, 513 Parnassus Avenue, SanFrancisco, CA, 94143, USA 5 Division of Neonatology, University of California San Francisco, 513 Parnassus Avenue, SanFrancisco, CA, 94143, USA 6 MRC Centre for Stem Cell Biology and Regenerative Medicine and Department of VeterinaryMedicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK 7 Department of Developmental Biology and Howard Hughes Medical Institute, StanfordUniversity, Stanford, CA 94305. Abstract Permanent damage to white matter tracts, comprising axons and myelinating oligodendrocytes, isan important component of newborn brain injuries that cause cerebral palsy and cognitivedisabilities as well as multiple sclerosis (MS) in adults. However, regulatory factors relevant inhuman developmental myelin disorders and in myelin regeneration are unclear. Here, we reportexpression of  AXIN2  in immature oligodendrocyte progenitor cells (OLP) within white matterlesions of human newborns with neonatal hypoxic-ischemic and gliotic brain damage, as well asactive MS lesions in adults.  Axin2  is a target of Wnt transcriptional activation that feeds back negatively on the pathway, promoting β -catenin degradation. We show  Axin2  function is essentialfor normal kinetics of remyelination. Small molecule inhibitor XAV939, which targets enzymaticactivity of Tankyrase, acts to stabilize Axin2 levels in OLP from brain and spinal cord andaccelerates their differentiation and myelination after hypoxic and demyelinating injury. Together,these findings indicate that  Axin2  is an essential regulator of remyelination and that it might serveas a pharmacological checkpoint in this process. Correspondence: David H. Rowitch, MD, PhD, 533 Parnassus Avenue, U503, San Francisco, CA 94143 tele: (415) 476-7242; fax:(415) 476-9976 rowitchd@peds.ucsf.edu.Author ContributionsS.P.J.F. helped conceive of and performed all experiments and analysis, with the exception of the following. E.P.H performed andanalyzed all experiments related to in vitro  OLP cultures. T.J.Y performed and analyzed the ex vivo  cerebellar slice cultures. J.C.S.helped analyze Wnt pathway activation in murine hypoxic injury. C.Z. performed the electron microscopy and C.C.B. performed theG ratio analysis. S.E.B. performed bioinformatics. J.J.O. and E.J.H. procured human brain developmental tissue. R.J.M.F. and D.H.Rconceived the experiments and oversaw all aspects of the analysis. The paper was written by S.P.J.F., R.N., R.J.M.F., and D.H.R. NIH Public Access Author Manuscript  Nat Neurosci . Author manuscript; available in PMC 2012 February 1. Published in final edited form as: Nat Neurosci  . ; 14(8): 10091016. doi:10.1038/nn.2855. N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t    Introduction Oligodendrocytes are the myelinating cells of the central nervous system (CNS) that enableformation of myelin and saltatory nerve conduction. In humans, premyelinatingoligodendrocytes are most abundant at gestational age 23-32 weeks and are thought to beselectively vulnerable during injury in the neonatal brain 1 . Hypoxic ischemicencephalopathy (HIE) causes neuronal apoptosis as well as diffuse primary damage to sub-cortical white matter in the term infant brain, while periventricular leukomalacia (PVL)comprises focal injury to white matter tracts as well as diffuse gliotic lesions typically inpremature infants 2 . Such diffuse and focal injuries, collectively known as white matterinjury (WMI) in the newborn brain, can result in cerebral palsy (CP) and cognitivedisability. Indeed, WMI is the most reliable prognostic indicator of development of severeCP in premature infants 3 . In multiple sclerosis (MS), the most common cause of neurological disability in young adults, myelin sheaths are lost through the injury or death of mature oligodendrocytes as a result of autoimmune damage 4 .In these conditions, myelin sheaths can be regenerated by OLP that are recruited to lesionsand differentiate in a process called remyelination. Conversely, inhibition of remyelinationmay contribute to ongoing neurological dysfunction, axonal loss and disease progression 5,6 .Although oligodendrocytes are thought to be cellular targets of excitotoxic damage innewborn brain injuries 2,7 , several lines of evidence indicate failure of remyelination ascontributing to fixed demyelinated lesions 8,9 . Indeed, both PVL 8,9  and MS 5,6  lesions showpresence of non-myelinating OLP.The molecular mechanisms that might dysregulate myelination in neonatal brain injury areunknown. We have previously shown that active Wnt signaling can act to inhibitoligodendrocyte OLP differentiation during remyelination in the adult rodent CNS 10 , andseveral studies show that Wnt activation inhibits developmental myelination 10-12 . In theabsence of Wnt ligands, β -catenin levels are regulated through a phosphorylation/ degradation complex 13,14  containing glycogen synthase kinase 3 β  (GSK-3 β ) and thescaffolding proteins, Axis inhibition protein 1 (Axin1), adenomatous polyposis coli (APC)and disheveled (Dsh). The presence of Wnt ligand results in stabilization of β -catenin and itstranslocation to the nucleus, where it forms a nucleoprotein complex with Tcf/LEFtranscription factors to activate or repress expression of target genes 15,16 . While  Axin1  isexpressed ubiquitously,  Axin2/conductin  is a transcriptional target of active Wnt signalingthat also serves to auto-regulate and repress the pathway by promoting β -catenin degradationin many different tissues and systems 14,17-19 . Dual roles for signaling targets that also serveas feedback repressors are seen in other pathways such Sonic hedgehog (Shh), where therepressors Patched and Gli3 are activated by Shh 20 .We observed expression of  AXIN2  mRNA transcripts in situ  in OLP in WMI associated withneonatal HIE and PVL. Although  Axin2  function has been reported as dispensable duringbrain development 21 , we show that  Axin2  function is essential for normal myelination andremyelination. Moreover, Axin2 levels can be manipulated pharmacologically in OLP topromote accelerated differentiation, suggesting Axin2 serves as a therapeutic target insituations where OLP differentiation is delayed or stalled. Results AXIN2   mRNA marks OLP in human neonatal white matter injury We have previously shown Wnt pathway activation using the transgenic Bat-Gal reporter indeveloping OLP in vivo , and that forced activation of the canonical Wnt pathway during Fancy et al.Page 2  Nat Neurosci . Author manuscript; available in PMC 2012 February 1. N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t    development of transgenic Olig2-cre  X  floxed-Dominant-active  β  -catenin  mice inhibitsdifferentiation of OLPs to mature oligodendrocytes (OL) 10 . Expression profiling of P4spinal cord from these mice indicated significant downregulation of genes associated withOL maturation (Suppl. Tab. 1, Suppl. Fig. 1), including  MBP, PLP, CNPase, MOG, MAG, Mobp, FA2H, MAL  and transcription factor  MRF  22 . This analysis also identified knownWnt-activated targets, such as  Naked1, Notum  and  Axin2  as significantly upregulated. Incontrast, changes in expression of the Wnt pathway antagonist adenomatous polyposis coli(APC  , also known as CC1 )  were not significant, indicating that its expression in Olig2-cre  X  floxed-Dominant-active  β  -catenin  mice is uncoupled from OL differentiation.Using expression of  AXIN2  mRNA transcripts to mark Wnt-activated white matter cells insitu , we investigated two types of human neonatal brain injury characterized by white matterdamage and injury to oligodendrocyte lineage cells resulting in hypomyelination (Suppl.Fig.2). As shown, (Fig. 1a, b, c), we observed  AXIN2  mRNA expression solely in Olig2-positive cells within affected white matter in neonatal HIE and PVL (Note, in PVL staininglocalized adjacent to the cystic core of lesion) but not white matter in age-matched controls(Fig. 1a, c). Within the HIE cases,  AXIN2  mRNA was expressed in a subset of the Tcf4positive cells (Fig. 1d), consistent with activation of canonical Wnt signaling.  AXIN2  mRNAsegregated from the mature OL marker PLP in situ  (Fig. 1e), and segregated completelyfrom GFAP (Fig. 1f). Similarly, the independent Wnt activated target Naked1 (Nkd1) wasexpressed in PDGFR α -positive OLP, but not astroglia (Fig. 1g, h, Suppl. Fig. 3). Theexpression of these Wnt targets provides strong evidence that the Wnt pathway is activatedspecifically in immature, pre-myelinating OLPs in human neonatal white matter injury(Suppl. Fig. 4). We thus, went on to address possible functions of Axin2 in OLP in animalmodels. Axin2   function is required for OLP differentiation Whilst  Axin2  mRNA serves as a marker for pathway activation, Axin2 protein functions as anegative feedback mechanism to control activation of the Wnt pathway via β -catenindegradation 17,18 . Using wild type and  Axin2-lacZ   heterozygous mice 18  we characterized  Axin2  mRNA and reporter gene expression during developmental myelination (Fig. 2a-d).  Axin2  mRNA is expressed in immature Nkx2-2+ OLP but not CC1+ differentiated OL (Fig.2b). In contrast, reporter β -galactosidase proteins are first detectable at the later CC1+ stage(Fig. 2c, d; Suppl. Fig. 6).  Axin2-lacZ   homozygous null animals showed a significant delay in OLP differentiationduring developmental myelination (Fig. 2e, f; Suppl. Fig. 7). Moreover, as indicated by co-expression of β -galactosidase with PDGFR α  (Fig. 2g), the reduction in mature OL numberreflects delay in OLP maturation, rather than decreased OLP numbers. Indeed, there is nodifference in apoptosis or proliferation of the OLP pool (Suppl. Fig. 8). We also observed asignificant (  p  < 0.005) impairment of  Axin2 −  /  −  OL differentiation in vitro  (Fig. 2h, i), andstrong activation of the independent Wnt target  Notum  (Fig. 2j). Thus,  Axin1  is unable tocompensate for the loss of early  Axin2  functions in OL differentiation in vitro  or in vivo . Axin2   is expressed in MS and functions in myelin repair Previous studies indicate that lesions of both neonatal PVL and adult MS show OLP,evidently ‘stalled’ in their differentiation 5,6,8,9 , which has been proposed to result indefective repair and fixed demyelinated lesions 23 . Thus, we focused on regulatory functionsof  Axin2  in the context of primary damage to oligodendrocytes. We confirmed that  AXIN  2mRNA transcripts are expressed in OLP in active MS lesions (Fig. 3a, b), in which OL aretargeted for autoimmune attack.  Axin2  mRNA expressing cells were not seen in areas of normal appearing white matter (NAWM), nor in chronic silent plaques (Fig. 3a). A similar Fancy et al.Page 3  Nat Neurosci . Author manuscript; available in PMC 2012 February 1. N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t    pattern of expression was seen for the independent Wnt activated target Naked1 (Nkd1).Naked1 protein expressing cells were seen at active MS lesion edges (Fig. 3c, Suppl. Fig. 5),with the simple bipolar morphology characteristic of OLP (Fig. 3c), and at a similar cellulardensity to  Axin2  mRNA expressing cells (Fig. 3c). Remyelination can be investigated afteradult murine lysolecithin injury, which kills resident OL while leaving axons largely intact.Such lesions in spinal cord ventral or dorsal white matter have been extensivelycharacterized 24  and show OLP recruitment (5 days post lesion (dpl)), differentiation (10 dpl)and myelination (14 dpl) with stereotyped timing in young adult animals (Fig. 4a), allowingprecise assessment of remyelination kinetics.In adult animals, we observed robust expression of the β -gal reporter in  Axin2-lacZ  heterozygote mice 10 days after demyelination (Fig. 4b). Although mature OL numbersnormalize by 8 weeks of age in  Axin2 −  /  −  animals (Fig. 4f, Suppl. Fig. 9),  Axin2 −  /  −  null miceshowed significantly delayed remyelination compared to WT littermates (Fig. 4c, Suppl.Fig. 10), due to a delay in Nkx2.2+ OLP differentiation (with normal OLP recruitment) inlesions at 10 and 14 dpl (Fig. 4c-f). The inflammatory cell and astrocyte response in lesionswas not affected in  Axin2 −  /  −  mice (Suppl. Fig. 11), demonstrating that the delay inremyelination in  Axin2  null animals is attributable to a cell-autonomous requirement in OLP. Axin2 protein stabilization promotes OLP differentiation We next investigated whether enhanced Axin2 activity might promote acceleratedoligodendrocyte maturation. Axin2 and Axin1 were recently identified as substrates for thepoly-ADP-ribosylating enzymes Tankyrase 1 and 2 25 , which promote Axin degradationthrough the ubiquitin-proteosome pathway. Small molecule XAV939 inhibited tankyraseactivity in cell lines at 5 μ M concentration, resulting in stabilized Axin protein levels 25 .As shown (Fig 5a, b), Tankyrase is expressed in the rodent OL lineage during developmentcommencing at a mature stage, and continues to be expressed in oligodendrocytes in adultwhite matter (S. Fancy and D. Rowitch, unpublished observations). Tankyrase is alsoexpressed within early differentiating OLs following demyelination in the adult spinal cordwhite matter of the mouse (Fig. 5c and inset).Given these findings, we tested effects of XAV939 to stabilize Axin protein levels in OLP invitro . As shown (Fig. 5d), treatment with 0.01 or 0.1 μ M XAV939 for 24 hours producedincreases in the levels of both Axin2 and Axin1 proteins in OLP versus vehicle controls, andincreased protein levels of Tankyrase 1 and 2, presumably due to reduction in theirautoparsylation and self-induced degradation 25 . XAV939 treatment of OLP resulted inincreased levels of phospho- β -catenin and β -catenin degradation, as well as reduced levelsof  Axin2  mRNA transcripts (Fig. 5e) and activity of a transduced Topflash reporter (data notshown), indicating inhibition of the Wnt pathway. Moreover, in addition to early effects onWnt pathway  per se , XAV939 treatment promoted precocious OL differentiation (Fig. 5f-h),as evidenced by increased expression of the mature oligodendrocyte marker myelin basicprotein (MBP).We next confirmed Tankyrase protein expression within the human OL lineage duringneonatal white matter injury. As shown (Fig. 5i), Tankyrase was expressed in later stage OLcells that expressed NOGO-A+ and cytoplasmic Olig1+. Conversely, Tankyrase expressionwas not observed in inflammatory macrophages, microglia or reactive astrocytes withingliotic areas of white matter damage. These data suggest the potential for pharmacologicalmanipulation of Axin2 levels in OLPs through Tankyrase inhibition in human neonatalwhite matter injuries. Fancy et al.Page 4  Nat Neurosci . Author manuscript; available in PMC 2012 February 1. N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t    To address this question, we investigated XAV939 effects on OLP in mouse cerebellar slicecultures (Fig. 6) In this system, slices of P0-P1 neonatal cerebellum are cultured in thepresence of factors that can affect remyelination 26  of cerebellar white matter. We examinedthe ratio of NFH-positive axons that showed co-expression of myelin basic protein (MBP)and measured the density of Nodes of Ranvier, indicated by Caspr-positive paranodes, Asshown (Fig. 6a, a’), 0.01 μ M XAV939 treatment significantly increased myelinationcompared with controls. Moreover, we observed that acute hypoxic exposure (2% oxygenfor 24 hrs) reduced myelination to levels significantly below controls (Fig. 6b, a’), Wesuggest the impact of hypoxia in this system is primarily due to inhibition of OLPmaturation because Nkx2.2-Olig2 double positive OLP numbers were significantlyincreased without detectable reduction in Olig2-positive cells (Fig. 6b’). Treatment withXAV939 reversed the impact of hypoxia, and indeed, increased the extent of myelinatedaxons to well above control levels. To demonstrate that XAV939 treatment can act oncerebellar OLP to promote remyelination, we also tested its effects after addition of 0.5%lysolecithin to culture medium to induce toxic injury to oligodendrocytes (Fig. 6c). In thisparadigm, XAV939 also promoted a significant enhancement of myelin regeneration (Fig.6c’).As illustrated above (Fig. 4a), the kinetics of remyelination in vivo  in focal toxic injurymodels is tightly regulated and to our knowledge no drugs have been reported that accelerateOLP differentiation. To test whether pharmacologic stabilization of Axin might promoteOLP differentiation during remyelination in vivo , we co-injected young adult (8-10 week old) mouse spinal cord lysolecithin lesions with 0.1 μ M XAV939. As shown (Fig. 7a, c), weobserved a striking increase in PLP + differentiated OLs as early as 6 dpl in XAV939-treateddorsal and ventral lesions compared to vehicle treated controls. Moreover, such XAV939effects are Axin2-dependent, as  Axin2  null mice show significantly less OLP differentiationat 6dpl following lysolecithin treatment versus controls (Fig. 7b, c). This increase in matureOL number at 6 dpl was due to a precocious differentiation of recruited OLP rather than anincreased survival of existing OL (Fig. 7g). Whilst total Olig2+ cells were similar at 6 dpl inXAV939 treated lesions and controls, XAV939 produced a shift in the proportion of Olig2+cells that expressed the mature PLP + OL versus immature Nkx2.2+ OLP (Fig. 7d, e).XAV939 treatment did not affect the inflammatory cell or astrocyte infiltration into lesions(Fig. 7f), nor early OLP recruitment into or proliferation within the lesion (Fig. 7h, i). Suchprecocious OLP differentiation in XAV939 treated lesions was associated with significantlythicker myelin sheaths (smaller g ratios) compared to controls at 10 dpl when myelin sheathformation is on-going (Fig. 7j). Myelin thickness in XAV939-treated remyelinated lesionswas similar to controls when lesions in both groups are fully remyelinated at 28 dpl (Suppl.Fig. 12). These findings indicate that XAV939 treatment significantly accelerates theprocesses of OLP differentiation and compact myelin formation in vivo . Discussion Despite advances in neonatal intensive care in developed countries, newborn brain injuriessuch as HIE in full term infants and PVL in very premature infants remain leading causes of cerebral palsy and cognitive disability. Indeed, the incidence of these conditions is rising 27 ,owing to the increasing survival of extremely low birth weight premature infants born atgestational ages <28 weeks. While WMI and demyelination is primary in MS, it is also asignificant component of HIE and PVL. Progression in MS lesions to a stage of chronicdemyelination is thought to reflect both autoimmune-mediated damage to oligodendrocytesas well as inefficient repair. In both MS 5,6  and PVL 8,9  there is evidence for ‘stalled’ OLprecursors that fail to engage in remyelination. Fancy et al.Page 5  Nat Neurosci . Author manuscript; available in PMC 2012 February 1. N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t  
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