Rescue of neurodegeneration in the Fig4 null mouse by a catalytically inactive FIG4 transgene

Abstract

The lipid phosphatase FIG4 is a subunit of the protein complex that regulates biosynthesis of the signaling lipid PI(3,5)P2. Mutations of FIG4 result in juvenile lethality and spongiform neurodegeneration in the mouse, and are responsible for the human disorders Charcot-Marie-Tooth disease, Yunis-Varon syndrome and polymicrogyria with seizures. We previously demonstrated that conditional expression of a wild-type FIG4 transgene in neurons is sufficient to rescue most of the abnormalities of Fig4 null mice, including juvenile lethality and extensive neurodegeneration. To evaluate the contribution of the phosphatase activity to the in vivo function of Fig4, we introduced the mutation p.Cys486Ser into the Sac phosphatase active-site motif CX5RT. Transfection of the Fig4(Cys486Ser) cDNA into cultured Fig4(-/-) fibroblasts was effective in preventing vacuolization. The neuronal expression of an NSE-Fig4(Cys486Ser) transgene in vivo prevented the neonatal neurodegeneration and juvenile lethality seen in Fig4 null mice. These observations demonstrate that the catalytically inactive FIG4 protein provides significant function, possibly by stabilization of the PI(3,5)P2 biosynthetic complex and/or localization of the complex to endolysosomal vesicles. Despite this partial rescue, later in life the NSE-Fig4(Cys486Ser) transgenic mice display significant abnormalities that include hydrocephalus, defective myelination and reduced lifespan. The late onset phenotype of the NSE-Fig4(Cys486Ser) transgenic mice demonstrates that the phosphatase activity of FIG4 has an essential role in vivo.

Figure 1.

Rescue of cell vacuolization by the Fig4 cDNA with an active-site mutation. (A) Vacuolated Fig4^−/− fibroblasts in culture. (B) Representative cell after transfection of wild-type Fig4 cDNA. (C) Representative cell after transfection of Fig4^Cys486Ser cDNA. (D) The structure of mutant Fig4 cDNA construct with chicken β-actin promoter. (E) Sanger sequence chromatogram of codon 486 from wild-type (TGT) and mutant (TCT) cDNAs. (F) Quantitation of rescue of vacuolization by wild-type Fig4 cDNA and cDNA containing the active-site mutation p.Cys486Ser. The number of vacuolated cells and total cells counted are indicated.

Figure 2.

Neuronal expression of the p.Cys486Ser Fig4 cDNA transgene. (A) The structure of the neuron-specific NSE transgene, with positions of PCR primers used for genotyping and RT-PCR. (B) Genotyping of mouse genomic DNA; control = Vac14 (6). (C) RT-PCR of brain RNA from transgenic and wild-type mice; control = Scn8a (31). (D) Western blot probed with monoclonal anti-FIG4 antibody; control = α-tubulin (32). The expression of FIG4 protein in the mutant is 45 ± 5% of endogenous wild-type expression (Image J statistics; Supplementary Material, Fig. S1). (E) Quantitation of brain RNA by qRT-PCR using a TaqMan gene expression assay.

Figure 3.

Rescue of neurodegeneration, impaired growth and juvenile lethality by the Fig4^Cys486Ser transgene. (A) Mice of the indicated genotypes were weighted daily from birth. (B) Rescue of body size at P35 by the mutant transgene. (C) Kaplan–Meier plot of survival for Fig4 null mice with and without the transgene. Open circles, currently surviving transgenic mice. (D) Rescue of neurodegeneration in CNS and PNS. Sections of brain and dorsal root ganglion were prepared at P30 from Fig4^−/−, Tg+ and Fig4^−/− littermates and stained with hematoxylin/eosin. The extensive in vivo vacuolization in the Fig4^−/− null tissues is corrected by the mutant transgene.

Figure 4.

Shortened lifespan, hydrocephalus, and pigmentation defect in Fig4^−/−,Tg^Cys486Ser transgenic mice. (A) Reduced lifespan of transgenic mice carrying the mutant transgene. (B) Reduced body weight of Fig4^−/− mice carrying the Tg^Cys486Ser transgene at 2–3 months of age. (C) Diluted pigmentation in Fig4^−/− transgenic mouse carrying Tg-C486 (red marker on tail) compared with corrected pigmentation in mouse carrying the wild-type Fig4 transgene (blue tail). (D) The saggital section of brains from Fig4^−/− transgenic mice expressing mutant or wild-type FIG4 transgenes. H&E staining. (E) The expression of the mutant transgene in brain is maintained at 8 months of age (RT-PCR).

Figure 5.

Impaired myelination and nerve conduction in Fig4^−/−,Tg^Cys486Ser transgenic mice. (A) Western blot of brain membrane proteins isolated at P21 and probed with antibodies to the myelin proteins MAG, MBP and PLP and the neuronal marker class III β-tubulin. (B–D) Quantitation of western blots normalized to βIII Tub. Results are shown as mean value ± SEM, unpaired two-tailed Student's t-test. *P < 0.03, ** < 0.01. (E–G) Representative traces of CAPs recorded at P21 reveal an increase in the population of slow conducting (non-myelinated) fibers (arrows) in optic nerve from Fig4^−/−,Tg-Cys486Ser mice. (H) The amplitude ratio for slow conducting to fast conducting peaks identified in E–G. Results are shown as mean value ± SEM, unpaired Student's t-test. ****P < 0.0001.