The Folate Metabolic Enzyme ALDH1L1 Is Restricted to the Midline of the Early CNS, Suggesting a Role in Human Neural Tube Defects

THE JOURNAL OF COMPARATIVE NEUROLOGY 500:368–383 (2007) The Folate Metabolic Enzyme ALDH1L1 Is Restricted to the Midline of the Early CNS, Suggesting a Role in Human Neural Tube Defects † TODD E. ANTHONY AND NATHANIEL HEINTZ* Laboratory of Molecular Biology, Howard Hughes Medical Institute, Rockefeller University, New York, New York ABSTRACT Folate supplementation prevents up to 70% of human neural tube defects (NTDs), although the precise cellular and metabolic sites of action remain undefined. One possibility is that folate modulates the function of metabolic enzymes expressed in cellular populations involved in neural tube closure. Here we show that the folate metabolic enzyme ALDH1L1 is cell-specifically expressed in PAX3-negative radial glia at the midline of the neural tube during early murine embryogenesis. Midline restriction is not a general property of this branch of folate metabolism, as MTHFD1 displays broad and apparently ubiquitous expression throughout the neural tube. Consistent with previous work showing antiproliferative effects in vitro, ALDH1L1 upregulation during central nervous system (CNS) development correlates with reduced proliferation and most midline ALDH1L1ϩ cells are quiescent. These data provide the first evidence for localized differences in folate metabolism within the early neural tube and suggest that folate might modulate proliferation via effects on midline Aldh1l1ϩ cells. To begin addressing its role in neurulation, we analyzed a microdeletion mouse strain lacking Aldh1l1 and observed neither increased failure of neural tube closure nor detectable proliferation defects. Although these results indicate that loss-of-function Aldh1l1 mutations do not impair these processes in mice, the specific midline expression of ALDH1L1 and its ability to dominantly suppress proliferation in a folate responsive manner may suggest that mutations contributing to disease are gain-of- function, rather than loss-of-function. Moreover, a role for loss-of-function mutations in human NTDs remains possible, as Mthfr null mice do not develop NTDs even though MTHFR mutations increase human NTD risk. J. Comp. Neurol. 500:368–383, 2007. © 2006 Wiley-Liss, Inc. Indexing terms: radial glial cell; folic acid; glia; BAC transgenic; Chromosome 6; CNS injury; reactive astrocyte; NEUT2; Pax3 Neural tube defects (NTDs) are among the most com- fects of folate metabolism. This idea is supported by stud- mon human birth defects, occurring in ϳ1 in 1,000 preg- ies using the mouse mutant splotch (Sp2H), which has a nancies in the United States (Cragan et al., 1995) and an estimated 300,000 or more newborns worldwide each year (Shibuya and Murray, 1998). NTDs are polygenic, with no Grant sponsor: Howard Hughes Medical Institute (to N.H.); Grant spon- single dominant or recessive causative gene yet identified sor: National Institutes of Health Genetics Predoctoral Training Grants; (Juriloff and Harris, 2000); accordingly, the risk of recur- Grant numbers: GM0848504, GM0848505, GM0798217S1, GM0798218S1 ϳ (to T.E.A.); Grant sponsor: National Institutes of Health Molecular Biology rence in sibships is 2–5% (McBride, 1979; Campbell et Predoctoral Training Grant; Grant number: GM0798219 (to T.E.A.). al., 1986). Although folate taken during the periconcep- *Correspondence to: N. Heintz, Laboratory of Molecular Biology, tual period has been shown to prevent the recurrence of up Howard Hughes Medical Institute, Rockefeller University, 1230 York Ave., to 70% of human NTDs (Smithells et al., 1981; Wald et al., New York, NY. E-mail: [email protected] 1991; Czeizel and Dudas, 1992), the mechanisms under- †Current address: California Institute of Technology, Div. of Biology MC lying these protective effects have remained unclear. 216-76, 1200 E. California Blvd., Pasadena, CA 91125 Received 8 March 2006; Revised 5 July 2006; Accepted 25 August 2006 As folate deficiency alone is insufficient to cause NTDs DOI 10.1002/cne.21179 in mice (Heid et al., 1992), one possible mode of action is Published online in Wiley InterScience (www.interscience.wiley. that folate supplementation acts to correct inherited de- com). © 2006 WILEY-LISS, INC. The Journal of Comparative Neurology. DOI 10.1002/cne NEURAL TUBE MIDLINE RESTRICTION 369 deletion within the paired homeodomain of the transcrip- during neural tube closure has not been explored, we tion factor Pax3 (Epstein et al., 1991). Homozygous Sp2H investigated ALDH1L1 expression and function further. embryos develop exencephaly and/or spina bifida (Epstein et al., 1991; Fleming and Copp, 1998), and ϳ40% of these can be prevented when folate is delivered in utero (Flem- MATERIALS AND METHODS ing and Copp, 1998). Moreover, analysis of cultured em- Mice and BAC transgenesis bryos demonstrated that folate cycling is compromised in Sp2H/Sp2H mice but can be restored to levels indistin- Mice were maintained and utilized according to proto- guishable from wildtype mice when exogenous folic acid is cols approved by the Institutional Animal Care and Use added (Fleming and Copp, 1998). The normalization of Committee at Rockefeller University. Wildtype C57B6/J both neural tube closure and folate cycling suggests that mice were used for expression analysis. NEUT2 mice were folate might exert its protective effects in splotch mice by previously generated as described (Champion et al., 1994) supplementing impaired metabolic pathways. Although and were obtained from Charles River Laboratories (Wil- this mechanism may explain NTD prevention in some mington, MA) with the permission of Dr. Carol Giometti. cases, there is evidence that folate does more than just NEUT2 mice were bred to C57B6/J for three generations correct biochemical deficiencies. In particular, mice lack- and then the F3s intercrossed for maintenance. To restore ing the Cited2 gene develop exencephaly and die at late Aldh1l1 expression onto a NEUT2 background, the BAC gestation due to failed neural tube closure in the midbrain clone RP23-7M9 (see text and Fig, 5) was injected into Ϫ Ϫ ϫ and hindbrain (Bamforth et al., 2001; Barbera et al., oocytes generated from a NEUT2 / B6CBA F1 cross using standard transgenic techniques (Hogan et al., 1994). 2002); despite normal folate cycling in these mutants, the Positive founders were then bred to NEUT2 homozygous NTDs can be prevented by exogenous folate (Barbera et mice to generate BAC transgene positive NEUT2 homozy- al., 2002). These data suggest that, in addition to rescuing gotes; these are denoted Ϫ/Ϫ;TgN(RP23-7M9). All quan- impaired metabolism, folate supplementation could also titative analysis was done using mice from these crosses, act by modulating folate-dependent reactions in popula- and therefore have a mixed genetic background of C57B6, tions of cells that are involved in neural tube closure CBA, and BALB/C. (Barbera et al., 2002; Copp et al., 2003). Currently, it is not known upon which cellular population(s) folate acts, Immunostaining which reactions might be modulated, or how such effects can prevent NTDs. Tissues were either immersion-fixed (embryos) or per- fused (postnatal/adult) with 4% paraformaldehyde (PFA) One approach toward gaining insight into these issues and postfixed overnight at 4°C. Tissues were then washed is to study the expression and function of folate metabolic in phosphate-buffered saline (PBS, pH 7.4) and cut either enzymes present in the developing nervous system. We on a cryostat (20 ␮m) or vibratome (75 ␮m); tissues to be previously found that the expression of aldehyde dehydro- cryosectioned were first cryoprotected in 15% sucrose genase 1 family, member L1 (Aldh1l1) is developmentally overnight at 4°C. Details on source and specificity of the regulated in the cerebellum, with high mRNA levels post- antibodies used are as follows: Rabbit ␣-ALDH1L1 was natally and decreased levels in the adult (Kuhar et al., used at 1:1,000 and was a gift of Dr. Robert Cook. This 1993); this observation suggested that Aldh1l1 might ful- antiserum was generated against full-length recombinant fill a unique developmental role. ALDH1L1 (EC 1.5.1.6; rat ALDH1L1; specificity was confirmed by a complete a.k.a. 10-formyltetrahydrofolate dehydrogenase, FDH, lack of staining in NEUT2 homozygotes (Aldh1l1 null FTHFD) is a cytosolic folate binding protein that irrevers- mice, Fig. 5). Rat ␣-BrdU (1:100, Oxford Biotechnology, ibly converts 10-formyltetrahydrofolate (10-FTHF) to tet- Kidlington, UK) was generated against bromodeoxyuri- rahydrofolate (THF) and CO2 (Kutzbach and Stokstad, dine (BrdU) coupled to keyhole limpet hemocyanin (KLH), 1971; Cook, 2001a), and which may also be responsible for and yielded no staining in mice not administered BrdU. regulating cellular THF levels (Kim et al., 1996). As the Rabbit ␣-GFAP (1:500, Dako, Carpinteria, CA) was gen- reaction catalyzed by ALDH1L1 results in permanent re- erated against glial fibrillary acidic protein (GFAP) iso- moval of one-carbon units from the cellular pool, it has lated from bovine spinal cord; mouse ␣-GFAP (1:200, been suggested that ALDH1L1 may function primarily as Sternberger Monoclonals, Baltimore, MD) was generated a regulatory molecule (Krebs et al., 1976; Scrutton and against human GFAP. Both of these antibodies labeled Beis, 1979). Moreover, as 10-FTHF is required during two only cells with the morphology of fibrillary astrocytes. steps of purine synthesis, it has been proposed that Rabbit ␣-Ki67 (1:500, Novocastra, Newcastle upon Tyne, ALDH1L1 might restrict proliferation. This idea is sup- UK) was generated against a 1,086-bp Ki67

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