Mutation of Vps54 causesmotor neuron disease anddefective spermiogenesisin the wobbler mouseThomas Schmitt-John1,5, Carsten Drepper1, Anke Mu�mann1,Phillip Hahn1,2, Melanie Kuhlmann1, Cora Thiel1,Martin Hafner3, Andreas Lengeling2, Peter Heimann1,Julie M Jones4, Miriam H Meisler4 & Harald Jockusch1

Vacuolar-vesicular protein sorting (Vps) factors are involvedin vesicular trafficking in eukaryotic cells. We identified themissense mutation L967Q in Vps54 in the wobbler mouse,an animal model of amyotrophic lateral sclerosis, and alsocharacterized a lethal allele, Vps54b-geo. Motoneuron survivaland spermiogenesis are severely compromised in the wobblermouse, indicating that Vps54 has an essential role inthese processes.

The spontaneous, autosomal recessive wobbler (wr) mutation of themouse was discovered almost 50 years ago1. The mutation is pleio-tropic, causing spinal muscular atrophy and defective spermiogen-esis2,3. The wr mutation was mapped to proximal mouse chromosome11 (ref. 4) in a region homologous to human chromosome 2p13–14(ref. 5). To identify the molecular basis of the wobbler phenotype, werefined the genetic localization of the wr locus in a large cross betweenstrains C57BL/6J-wr and CAST/Ei. Evaluation of 46,000 informativemeioses narrowed the candidate interval for wr to 0.9 Mb betweenD11Hjk30 and D11Hjk29 (Fig. 1a). We evaluated the six genes in thenonrecombinant interval as candidates for wr: Peli1, Vps54, Ugp2,NM_172792, Mdh1 and NM_145425 (Fig. 1a). We identified the exonsand splice variants of each candidate gene by RT-PCR analysis of cDNAfrom multiple tissues and then screened each exon for mutations bysequencing genomic DNA. In exon 23 of Vps54, wr/wr genomic DNAcontains an A-T transversion in the second position of codon 967that results in the amino acid substitution L967Q (Fig. 1b,c). Themutation does not seem to affect splicing of the Vps54 transcript, as allthree normal transcripts (Fig. 1b) were detected in wobbler tissues.Leu967 was present in DNA sequences from eight strains of mice (Musmusculus musculus strains 129, C57BL/6J, C57BL/10, SWR and SJL;M. m. castaneus; M. m. molossinus; and Mus spretus) and, moreover,is evolutionarily conserved among vertebrates (Fig. 1c).

To confirm that the Vps54 mutation was responsible for the wobblerphenotype, we carried out transgene-mediated rescue using BAC clone

RPCI 24-115F6 from strain C57BL/6J. This BAC contains the completegenomic sequence of Vps54 plus 63 kb upstream and 18 kb down-stream (Fig. 1a). We generated BAC transgenic mice by microinjectionof fertilized eggs (Supplementary Methods online). Four independenttransgenic lines were successfully established by crossing male founderswith C57BL/6J-wr females, and we obtained informative mice from allfour lines. All (16 of 16) F2 transgenic wobbler mice (wr/wr Vps54-tg)were phenotypically rescued: they had normal mobility on a wire gridand maintained normal body weight and grip strength during the firsttwo months after birth (Fig. 2 and Supplementary Fig. 1 online).Rescue of the neurological phenotype was also indicated by normalhistological appearance of motoneurons and astroglia (SupplementaryFig. 1). The fertility of wr/wr Vps54-tg males was demonstrated bygeneration of viable progeny in multiple matings. Spermatozoa fromwr/wr Vps54-tg mice were normal in motility and appearance underphase contrast microscopy (data not shown). Electron microscopy oftestes showed a normal number of flat (wild-type) sperm heads insections of seminiferous tubules from the transgenic mice (Supple-mentary Fig. 1). Thus, both neurological and spermatogenic defects ofwobbler homozygotes were corrected by the Vps54 transgene.

To obtain a second mutated allele of Vps54, we searched theBayGenomics Clone library and identified a b-geo gene trap insertionin intron 4 of Vps54 in embryonic stem cell line RRI497(Vps54gt(pGT10)2841Ucd or Vps54b-geo; Supplementary Fig. 2 online).The position of the insertion site predicts synthesis of a hybrid proteincontaining the first 152 amino acids of Vps54 fused to the full-lengthb-geo protein. We recovered Vps54b-geo/+ heterozygous mice, whichhad a normal phenotype (Fig. 2). X-gal staining of sections fromVps54b-geo/+ adults showed widespread low-level expression of theVps54–b-geo fusion protein (data not shown). Among more than 80offspring from mated heterozygotes, we recovered no Vps54b-geo/b-geo

pups (Fig. 2). Between embryonic day (E) 8.5 and E10.5,Vps54b-geo/b-geo embryos were indistinguishable from wild–typeembryos. At E11.5, Vps54b-geo/b-geo embryos were developmentallyretarded. From E12.5 onward, we obtained no homozygous embryosbut observed resorption sites with homozygous genotype (Supple-mentary Fig. 3 online), implying that Vps54b-geo/b-geo homozygotesdid not survive beyond this embryonic stage. To investigate the causeof lethality, we serially sectioned homozygous E11.5 embryos. Thespinal cord was underdeveloped, and dorsal root ganglia were nearlycompletely absent (Supplementary Fig. 3). In addition, the atrial andventricular myocardium had severe hypoplasia (SupplementaryFig. 3), suggesting that homozygous embryos died because of cardi-ovascular malfunction. The prenatal lethality and developmentalabnormalities indicate that Vps54 is essential during development.To prove allelism between wr and Vps54, we crossed Vps54b-geo/+ mice

Received 3 June; accepted 30 August; published online 23 October 2005; doi:10.1038/ng1661

1Developmental Biology and Molecular Pathology, Bielefeld University, Germany. 2Research Group Infection Genetics and 3Department of Experimental Immunology,German Research Center for Biotechnology, Braunschweig, Germany. 4Department of Human Genetics, University of Michigan, Ann Arbor, Michigan, USA.5Present address: Department of Molecular Biology, University of Aarhus, Denmark. Correspondence should be addressed to T.S.-J. (tsj@mb.au.dk).

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with wr/+ mice. The compound heterozy-gotes had the complete wobbler phenotype,including reduced body weight and reducedgrip strength (Fig. 2). The severity of thedefects was comparable to that of wr/wrmice. Noncomplementation of the wr andVps54b-geo mutations indicated allelism, con-firming the identification of the gene on thebasis of transgenic rescue.

The identification of Vps54 as the gene underlying wr is consistentwith the previous finding that the wr mutation is cell-autonomous6.Although Vps54 is widely expressed, the wr mutation selectively affectsmotoneuron survival and spermiogenesis, indicating that an intactexon 23 is essential for these processes. In Caenorhabditis elegans, thenull mutant of the Vps54 homolog is either infertile or nonviable(allele tm585; Wormbase), and in Drosophila melanogaster, nullmutants of the Vps54 homolog scattered (scat) have defective sperma-

togenesis7. Saccharomyces cerevisiae Vps54 forms heterotetramericcomplexes with Vps51, Vps52 and Vps53 (ref. 8) to form the Golgi-associated retrograde protein (GARP) complex, which is involved invesicular trafficking. Mammalian orthologs of yeast Vps52, Vps53 andVps54 have been identified9,10. The interaction of human Vps52 withRab6 and the SNARE syntaxin10 (ref. 10) suggests that the mamma-lian GARP complex has a cellular function similar to that of theS. cerevisiae GARP complex.

Mutation in the human gene VPS33B is responsible for the kidneysyndrome arthrogryposis, renal dysfunction and cholestasis11 (ARC;OMIM 208085). Among several mouse mutations affecting vesicletransport to lysosome-related compartments, only the mocha mutanthas neurological defects12, but it does not affect motoneurons. Thefirst links between vesicular transport and motoneuron degenerationhave been reported for alsin13 (ALS2; OMIM 205100) and VABP14

(ALS8; OMIM 608627). Our investigation of mutant Vps54 is the firstreport to our knowledge of a Vps factor causing severe impairment ofmotoneurons. The precise mechanism by which the missense muta-tion in Vps54 causes motoneuron disease is not known. We suggestthat Vps54 might be critical for retrograde vesicular transport and inparticular for axonal transport in motoneurons, which is impaired inwobbler mice15. The phenotype of the wobbler mutant indicates thatsome of the unmapped hereditary syndromes with neurodegenerationor male infertility might result from mutations affecting Vps54 orother components of the vesicular transport machinery.

Note: Supplementary information is available on the Nature Genetics website.

ACKNOWLEDGMENTSWe thank G. Gavrilina, M. Ebel, W. Muller and M. Augustin for assistance andTransgenic Animal Model Core of the University of Michigan’s BiomedicalResearch Core Facilities, Center for Organogenesis, US National Institutes ofHealth, BayGenomics, Ingenium Pharmaceuticals AG, Martinsried, DeutscheForschungsgemeinschaft, Volkswagenstiftung, Fonds der Chemischen Industrie

2322212019a

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Figure 2 Transgenic rescue of the wobbler phenotype and

noncomplementation of a gene-trap allele of Vps54. Loss of body weight

and grip strength in wr/wr mice is rescued by the BAC 115F6 transgene

containing a wild-type copy of Vps54. Mice of genotypes +/+ Vps54-tg and

Vps54b-geo/+ have normal body weight and grip strength 30 d after birth.

Vps54b-geo/b-geo mice do not survive beyond E12.5. Compound heterozygotes

(Vps54b-geo/wr) have reduced body weight and grip strength like wr/wr mice.

Data shown are mean ± standard deviation (n ¼ 10 in each group).

Figure 1 Positional cloning of the gene

underlying the wr mutation. (a) Genetic andphysical map of the wr region on proximal mouse

chromosome 11. The previously reported

nonrecombinant interval5 is marked by

arrowheads. The refined nonrecombinant region

of 894 kb (dotted lines) contains six genes,

represented by bars pointing towards their 3¢ends. Mb positions are from Ensembl (mouse

genome assembly 32). The position of BAC

115F6, used for transgenic rescue, is indicated.

(b) Exon-intron structure of the Vps54 gene and

position of the wr missense mutation in exon 23

(star). Exons on the horizontal line are included

in the major splice isoform, encoding a protein of

977 amino acids. The two alternatively spliced

exons are shown above. An arrowhead marks the

b-geo insertion site in the gene-trap allele. (c) An

A-T transversion at nucleotide 72 of exon 23 in

the wobbler mouse changes evolutionarilyconserved Leu967 to glutamine.

1214 VOLUME 37 [ NUMBER 11 [ NOVEMBER 2005 NATURE GENETICS

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and NRW International Graduate School in Bioinformatics and Genome Researchfor financial support.

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Published online at http://www.nature.com/naturegenetics/

Reprints and permissions information is available online at http://npg.nature.com/

reprintsandpermissions/

1. Falconer, D.S. Mouse News Letters 15, 22 (1956).2. Duchen, L.W. & Strich, S.J. J. Neurol. Neurosurg. Psychiatr. 31, 535–542 (1968).

3. Heimann, P. et al. Differentiation 47, 77–83 (1991).4. Kaupmann, K. et al. Genomics 13, 39–43 (1992).5. Fuchs, S. et al. BMC Genet. 3, 14 (2002).6. Augustin, M. et al. Dev. Dyn. 209, 286–295 (1997).7. Fabrizio, J.J. et al. Development 125, 1833–1843 (1998).8. Conibear, E. et al. Mol. Biol. Cell 14, 1610–1623 (2003).9. Walter, L. et al. Gene 285, 213–220 (2002).10. Liewen, H. et al. Exp. Cell Res. 306, 24–34 (2005).11. Gissen, P. et al. Nat. Genet. 36, 400–404 (2004).12. Li, W. et al. Bioessays 26, 616–628 (2004).13. Yang, Y. et al. Nat. Genet. 29, 160–165 (2001).14. Nishimura, A.L. et al. Am. J. Hum. Genet. 75, 822–831 (2004).15. Mitsumoto, H. & Bradley, W.G. Brain 105, 811–834 (1982).

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a

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Supplementary Figure 2 Characterization of the Vps54 β-geo gene trap allele.(a) Exons 2 to 6 of wild type and Vps54 β-geo gene trap allele are shown (pGT0lxf-Trap β-geo cassette 8650 bp; En2 = Engrailed-2 intron, not drawn to scale). In the upper scheme positions of primer_a and primer_b are indicated. In the lower scheme, positions of hybridization probes S1, S2, primer_a and primer_c are shown. (b) Southern blot with 10 µg of genomic DNA from +/+ and Vps54 β-geo/+mice hybridized with the 32P labelled probe S1. Fragment sizes and enzymes for restriction digests are indicated in the table below. (c) Southern blot as in (b) but hybridized with probe S2. Hybridization of probe S2 to unique restriction fragments indicates a single insertion site in the gene trap ES-cell line. (d) PCR genotyping of E9.5 embryos using primers as indicated in (a) generates a 413 bp product from the wild type Vps54 allele (primers a and b) and a 324 bp product from the Vps54 β-geo allele (primers a and c).

1

Mutation of Vps54 causes motoneuron disease and defective

spermiogenesis in the wobbler mouse

Schmitt-John et al.

Supplementary Methods

Mouse strains

The mutant stock carrying the wobbler (wr) allele was obtained in 1979 from Dr. Richard

Sidman (The Children’s Hospital, Harvard University, Boston, MA, USA) and maintained

by crossing to strain C57/BL/6J. M. m. castaneus, strain CAST/Ei was purchased in 1996

from The Jackson Laboratory (Bar Harbor, ME, USA).

Genetic manipulations

DNA from BAC RPCI24-115F6 (BAC/PAC resources, Oakland Children's Hospital, CA,

USA) was column purified using NucleoBond BAC Maxi Kit (Clontech, Palo Alto, CA).

The BAC was linearized with PvuI and concentrated over a Microcon YM-100 filter

(Millipore Corp., Bedford, MA) and used for pronucleus injection1 into fertilized

(C57BL/6J x SJL/J) F2 eggs in the Transgenic Animal Model Core of the University of

Michigan (www.med.umich.edu/tamc/). Twelve BAC transgenic founders were identified

by PCR amplification of tail DNA using BAC-specific primers flanking each vector insert

junction. Four independent male founders were used to generate wr/wr, Vps54-tg mice, all

of which were phenotypically rescued.

The ES-cell line RRI497 carrying a β-geo gene trap vector in the Vps54 locus (Vps54

gt(pGT10)2841Ucd) was identified in the BayGenomics database2 (BayGenomics, San Francisco,

CA, USA) using the full-length Vps54 cDNA sequence. A single ES-cell line was found

carrying a gene trap in intron 4 of the Vps54 gene and chimeric mice were generated by

microinjection into BALB/c blastocysts and transferring these to pseudopregnant foster

mothers. Chimeric males were mated with C57BL/6J or wr/+ females. Germ-line

transmission of the mutant gene trap allele was verified by PCR and Southern blotting.

2

Mouse genotyping

New genetic markers were established with different alleles in CAST/Ei and C57BL/6J for

recombination screening (D11Hjk26 to D11Hjk30: accession numbers listed below). The

wobbler Vps54 mutant allele is detected by amplification and digestion of a 114-base pair

(bp) fragment containing parts of exon 23 from genomic DNA using forward primer

Vps54-Ex23a and reverse primer Vps54-Ex23b. After digestion with PsuI, two wild type

fragments of 40 bp and 74 bp and one wobbler fragment of 114 bp can be detected. The

presence of BAC115F6 ends were diagnosed using primer pairs T7 and BAC115_T7, and

SP6 and BAC115_SP6. Mice harbouring the Vps54 β-geo allele were genotyped by using a

three-primer PCR (primer_a, primer_b, and primer_c), amplifying 413 bp wild type and

324 bp gene trap fragments.

Phenotype analysis

Grip strength: Mice were suspended and allowed to hold on, three times in rapid

succession with their forepaws, a thin metal bar connected to a force meter until the grip

was detached; the highest force was taken as the “grip strength”, measured in centi Newton

(cN).

Histology

Cryosectioning, Nissl staining, immunohistochemistry and electron microscopy were

performed as previously described for CNS3,4 and testis5.

3

Markers and PCR primers

The following markers were used for segregation analysis of the C57/BL/6J x CAST/Ei

crossings:

D11Hjk26, MGI:3583738

D11Hjk27, MGI:3583739

D11Hjk28, MGI:3583740

D11Hjk29, MGI:3583741

D11Hjk30, MGI:3583742

Additional primers sequences are given in Supplementary Table 1.

PCR primer Sequence from 5´to 3´

Vps54-Ex23a TTT TTA CAC TGG AAA TCT TCA AGC CTT AAA AGG CCT TAA AAA TCT

GGA TC

Vps54-Ex23b GAT GAA CGA CCT GGG TCT CCA GTC TGT CAT CAC CTC TTC TGT TCC

CAG ATT TCG GCC ATA

T7 TAA TAC GAC TCA CTA TAG GG

BAC115_T7 AGA AAA TGA AGC AAA GTG TAT GTG

SP6 CCG TCG ACA TTT AGG TGA CA

BAC115_SP6 TCA TTT TCT CCC AGG CAA AC

primer_a TCC TTA GAG AGT GAT CGT GAG AA

primer_b CTC CCA GTA GAG CCA ACA AG

primer_c GCC TGC TCA AAC CTG AAC C

Supplementary Table 1 Primer sequences

References

1. Hogan, B. et al. Cold Spring Harbor Press. New York. (1994).

2. Stryke, D. et al. Nucleic Acids Res 31, 278-281 (2003).

3. Schlomann, U. et al. J Biol Chem 277, 48210-48219 (2002).

4. Rathke-Hartlieb, S. et al. Neuroreport 10, 3411-3416 (1999).

5. Augustin, M. et al. Dev Dyn 209, 286-295 (1997).