Mutation of FIG4 causes neurodegeneration in the pale tremor mouse and patients with CMT4J - PubMed (original) (raw)

. 2007 Jul 5;448(7149):68-72.

doi: 10.1038/nature05876. Epub 2007 Jun 17.

Yanling Zhang, James J Dowling, Natsuko Jin, Maja Adamska, Kensuke Shiga, Kinga Szigeti, Michael E Shy, Jun Li, Xuebao Zhang, James R Lupski, Lois S Weisman, Miriam H Meisler

Affiliations

Mutation of FIG4 causes neurodegeneration in the pale tremor mouse and patients with CMT4J

Clement Y Chow et al. Nature. 2007.

Abstract

Membrane-bound phosphoinositides are signalling molecules that have a key role in vesicle trafficking in eukaryotic cells. Proteins that bind specific phosphoinositides mediate interactions between membrane-bounded compartments whose identity is partially encoded by cytoplasmic phospholipid tags. Little is known about the localization and regulation of mammalian phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P2), a phospholipid present in small quantities that regulates membrane trafficking in the endosome-lysosome axis in yeast. Here we describe a multi-organ disorder with neuronal degeneration in the central nervous system, peripheral neuronopathy and diluted pigmentation in the 'pale tremor' mouse. Positional cloning identified insertion of ETn2beta (early transposon 2beta) into intron 18 of Fig4 (A530089I17Rik), the homologue of a yeast SAC (suppressor of actin) domain PtdIns(3,5)P2 5-phosphatase located in the vacuolar membrane. The abnormal concentration of PtdIns(3,5)P2 in cultured fibroblasts from pale tremor mice demonstrates the conserved biochemical function of mammalian Fig4. The cytoplasm of fibroblasts from pale tremor mice is filled with large vacuoles that are immunoreactive for LAMP-2 (lysosomal-associated membrane protein 2), consistent with dysfunction of the late endosome-lysosome axis. Neonatal neurodegeneration in sensory and autonomic ganglia is followed by loss of neurons from layers four and five of the cortex, deep cerebellar nuclei and other localized brain regions. The sciatic nerve exhibits reduced numbers of large-diameter myelinated axons, slowed nerve conduction velocity and reduced amplitude of compound muscle action potentials. We identified pathogenic mutations of human FIG4 (KIAA0274) on chromosome 6q21 in four unrelated patients with hereditary motor and sensory neuropathy. This novel form of autosomal recessive Charcot-Marie-Tooth disorder is designated CMT4J.

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Figures

Figure 1

Figure 1. Phenotypes of homozygous pale tremor mice

a, Diluted pigmentation of the pale tremor mouse (plt) compared with the wild-type (+/+) mouse. b, Abnormal limb postures. c, Juvenile lethality of F2 mice from the CAST/Ei cross (n = 50). d, RT–PCR of Fig4 transcript from brain with primers in indicated exons. e, Northern blot containing 3 mg of brain polyA+RNA, isolated at P7 before extensive neurodegeneration, using a 1 kb Fig4 cDNA probe (exons 8–15). RNA integrity and equal loading of samples is indicated by the intensity of minor bands (9–10 kb). f, Long-range PCR of genomic DNA with primers in intron 18 and exon 19. g, Three-primer genotyping assay for Fig4 produces 646 bp wild-type and 245 bp Fig4paletremor products. LTR, long terminal repeat. Molecular weight markers in e–g are given in kb. h, ETn2β retrotransposon in intron 18 of Fig4. i, Altered abundance of PtdIns(3,5)P2 in cultured fibroblasts. *Significant difference at P <0.05. Data are expressed as mean ± s.d. (n = 6).

Figure 2

Figure 2. Neuropathology in pale tremor mice

a, b, Trigeminal ganglia; c, d, dorsal root ganglia from lumbar region. Insets reveal cytoplasmic vacuoles. (Superior cervical ganglia have a similar appearance; see Supplementary Fig. 4a.) e, f, Spinal cord ventral horn. Arrow, motor neuron cell body. g, Sagittal section of the brain of a pale tremor mouse (for wild-type control lacking degeneration see Supplementary Fig. 6). V, enlarged ventricle. h, Neuronal cell bodies from regions of degeneration in P7 brain. Scale bars: a–f, 25 mm; insets in a and c, 12.5 mm; panel h, 25 μm.

Figure 3

Figure 3. Pathological abnormalities in peripheral nerves

a, b, Semi-thin sections of sciatic nerve prepared as described, demonstrating reduced density of large-diameter myelinated axons in the pale tremor mutant. Scale bars, 10 μm. c, d, Reduced sciatic nerve conduction velocity (mutant, 11.0 ± 3.4 m s−1; wild type, 21.5 ± 6.3 m s−1) and reduced amplitude of compound muscle action potentials (mutant, 2.2 ± 1.0 mA; wild type, 5.0 ± 2.1 mA) (mean ± s.d., n = 6).

Figure 4

Figure 4. Mutations of FIG4 in patients with CMT disorder

a, Sequencing chromatographs for four unrelated patients with CMT disorder. Nucleotide mutations are indicated on the _x_-axis of each chromatograph numbered from +1 for the first codon. b, Inheritance of mutant alleles in two pedigrees. Circle, female; square, male; open symbol, unaffected; filled symbol, affected. Patient BAB1079 is a compound heterozygote for protein truncation mutation F98fsX102 and missense mutation I41T. Patients BAB1372 and BAB1373 are compound heterozygotes for nonsense mutation R183X and missense mutation I41T. c, Residue isoleucine 41 is evolutionarily invariant in FIG4 from vertebrates, invertebrates and yeast. d, Location of CMT mutations in the FIG4 protein. Solid circles, protein truncation mutations. 907 amino acids are present per protein.

Figure 5

Figure 5. Yeast Fig 4Ile > Thr is defective in activation of kinase Fab1/PIKfyve

The fig 4Δ yeast strain lacking endogenous Fig 4 (refs 5, 7) was transformed with empty vector (vector), wild-type myc-Fig4 or myc-Fig4 containing I59T corresponding to human I41T (mutant). a, Yeast were labelled with FM4–64 to assess vacuole volume, an indicator of basal levels of PtdIns(3,5)P2. b, Time course of PtdIns(3,5)P2 levels after hyperosmotic shock (measured in minutes after change to hyperosmotic medium) reflects activation of Fab1 kinase. The mutant protein exhibits impaired activation at 10 min compared with the wild type (P = 0.004; mean ± s.d., n = 3). A western blot demonstrating comparable expression of wild-type and mutant constructs is presented in Supplementary Fig. 10.

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