Pathogenic mechanism of the FIG4 mutation responsible for Charcot-Marie-Tooth disease CMT4J - PubMed (original) (raw)

. 2011 Jun;7(6):e1002104.

doi: 10.1371/journal.pgen.1002104. Epub 2011 Jun 2.

Cole J Ferguson, Clement Y Chow, Natsuko Jin, Julie M Jones, Adrienne E Grant, Sergey N Zolov, Jesse J Winters, Roman J Giger, James J Dowling, Lois S Weisman, Miriam H Meisler

Affiliations

Pathogenic mechanism of the FIG4 mutation responsible for Charcot-Marie-Tooth disease CMT4J

Guy M Lenk et al. PLoS Genet. 2011 Jun.

Abstract

CMT4J is a severe form of Charcot-Marie-Tooth neuropathy caused by mutation of the phosphoinositide phosphatase FIG4/SAC3. Affected individuals are compound heterozygotes carrying the missense allele FIG4-I41T in combination with a null allele. Analysis using the yeast two-hybrid system demonstrated that the I41T mutation impairs interaction of FIG4 with the scaffold protein VAC14. The critical role of this interaction was confirmed by the demonstration of loss of FIG4 protein in VAC14 null mice. We developed a mouse model of CMT4J by expressing a Fig4-I41T cDNA transgene on the Fig4 null background. Expression of the mutant transcript at a level 5 × higher than endogenous Fig4 completely rescued lethality, whereas 2 × expression gave only partial rescue, providing a model of the human disease. The level of FIG4-I41T protein in transgenic tissues is only 2% of that predicted by the transcript level, as a consequence of the protein instability caused by impaired interaction of the mutant protein with VAC14. Analysis of patient fibroblasts demonstrated a comparably low level of mutant I41T protein. The abundance of FIG4-I41T protein in cultured cells is increased by treatment with the proteasome inhibitor MG-132. The data demonstrate that FIG4-I41T is a hypomorphic allele encoding a protein that is unstable in vivo. Expression of FIG4-I41T protein at 10% of normal level is sufficient for long-term survival, suggesting that patients with CMT4J could be treated by increased production or stabilization of the mutant protein. The transgenic model will be useful for testing in vivo interventions to increase the abundance of the mutant protein.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Impaired interaction of FIG4-I41T mutant protein with VAC14 and reduced co-immunoprecipitation with FAB1.

A) Directed yeast two-hybrid interaction of wildtype and mutant yeast Fig4p with Vac14p. Selection for Leu and Trp prototrophy was carried out under less stringent (−3AT) or more stringent (+3AT) conditions. B) Co-immunoprecipitation of myc-tagged Fig4p and GFP (Venus) -tagged Vac14p from yeast lysates. C) Co-immunoprecipitation of myc-tagged Fig4p and GFP-tagged Fab1p. D) Directed yeast two-hybrid interaction of human FIG4 and human VAC14. AD, activation domain alone; BD, DNA binding domain alone. The numbers above the lanes in B and C were obtained by densitometry of non-saturated images and represent the ratio of Fig4p∶Vac14p and Fig4p∶Fab1p; the ratio for mutant Fig4p was normalized to the wildtype ratio.

Figure 2

Figure 2. Low level of wildtype FIG4 protein in Vac14 null mice.

A) Absence of VAC14 protein in Vac14 null mouse. Protein extracts from wildtype and Vac14 null P0 mice were probed with polyclonal antibody to VAC14 as described . B) Absence of FIG4 protein in Vac14 null mouse. The same extracts were probed with monoclonal antibody to FIG4. C, D) RT-PCR of RNA from Vac14 null mice with forward primer in Fig4 exon 3 and reverse primer in exon 4 detects a normal level of Fig4 transcripts. ΔCT, CT for Fig4 minus CT for Tbp.

Figure 3

Figure 3. Structure and expression of the _Fig4_-I41T transgene.

A) Structure of the _Fig4_-I41T transgene containing the mouse Fig4 cDNA downstream of the ubiquitously active chicken β-actin promoter. B) Quantitation of transgene mRNA by qRT-PCR using RNA from Fig4+/+ wildtype mice with the indicated transgene. RNA was isolated from brain at P28. The ratio of Fig4 transcript to Tbp (Tata binding protein) transcript is calculated as described in Methods. C) Western blots of brain and D) kidney extracts from Tg705 and Tg721 transgenic lines stained with the monoclonal anti-FIG4 antibody and compared with tissues from wildtype and Fig4 null mice. 100 ug of protein, 3 min film exposure. E) FIG4-I41T protein can be detected in 100 ug of brain extract from transgenic lines after film exposure for 30 min. F) FIG4-I41T in 100 ug of brain protein from Tg721 line is comparable to FIG4 in 10 ug of wildtype brain. G, H) VAC14 protein is present in brain from transgenic mice and Fig4 −/− null mice.

Figure 4

Figure 4. Rescue of juvenile lethality by expression of the Fig4-I41T transgene.

Kaplan-Meijer survival curves for Fig4 null mice carrying Tg705 (n = 17), Tg721 (n = 21) transgenes or lacking the transgene (n = 28). Transgene expression is indicated relative to wildtype expression.

Figure 5

Figure 5. Spongiform degeneration of brain and DRG is rescued by the Fig4-I41T transgene.

Top: Sagittal brain section, cortex, and DRG from Fig4 null mice demonstrating enlarged ventricle and spongiform degeneration (scale bar = 200 µm). Middle panels: Partial rescue of Fig4 null mice carrying Tg705 (2× expression) and Tg721 (5× expression). Bottom panel: Sagittal sections of brain from wildtype mice (left) and transgenic mice in the final stage of disease progression. Both transgenes provide protection from neurodegeneration, with greater protection in the higher expressing line.

Figure 6

Figure 6. Dose-dependent rescue of the autophagy defect in brain of transgenic mice.

A) Western blots of accumulated autophagy markers LAMP2 and p62 and astrocyte marker GFAP in brain homogenates from wildtype, transgenic and Fig4 null mice at P28; 30 ug protein per lane. B) Accumulation of of LAMP2 and p62 immunofluorescence in GFAP-positive astrocytes in the cortex of transgenic lines at P28. Astrocyte abundance is dramatically increased in Fig4 null mice (Scale bars = 200 µm).

Figure 7

Figure 7. Rescue of peripheral nerve myelination and nerve conduction velocity in transgenic Tg705 and Tg721 lines.

A) Transmission electron microscopy of cross-sections of sciatic nerve from Fig4 mutant and wildtype mice at P21. Arrows in the top panel indicate thinly myelinated axons in Fig4−/− mice. B) Quantitation of g-ratio in sciatic nerve; higher g-ratio indicates a thinner myelin sheath. Fig4 −/− (n = 89 axons); Fig4 −/−, Tg705 (n = 181 axons); Fig4 −/−, Tg721 (n = 152 axons); Fig4 +/+ (n = 109 axons). Scale bar: 5 µm. Error bars, SEM. P<0.05 for Fig4−/− versus WT, Fig4−/− versus Tg705, and Fig4−/− versus Tg712 (Student's t-test). C, D) Nerve conduction velocity was measured in sciatic nerve and sural nerve from 4 month old unaffected Fig4−/−,Tg705 mice and 14 month old unaffected Fig4−/−,Tg721 mice (mean +/− SEM).

Figure 8

Figure 8. FIG4 transcript and protein in CMT4J patient fibroblasts.

A) RT-PCR products containing exon 2 were amplified from total fibroblast RNA with a forward primer in exon 1 and reverse primer in exon 3. B) Sequence of the RT-PCR product demonstrating nucleotide c.122T (isoleucine) in the control RNA and nucleotide c.122C (threonine) in the patient RNA. C) Western blot of 60 ug of protein from patient and control fibroblasts. D) HEK293T cells transfected with wildtype mouse FIG4 cDNA transgene (Figure 3) or the corresponding I41T construct. Co-transfection of VAC14 stabilizes endogenous and transfected FIG4 proteins. E) The low level of FIG4 in primary fibroblasts from Tg721 transgenic mice is increased by culture for 8 hours in the presence of 10 uM concentration of the proteasome inhibitor MG-132. F) Incubation of wildtype fibroblasts with MG-132 increases the level of FIG4 protein.

Figure 9

Figure 9. Location of the _FIG4_-I41T mutation and effect on protein interaction.

A) Predicted structure for FIG4 phosphatase based on protein coordinates of yeast Sac1 phosphatase(18). The isoleucine residue mutated in CMT4J (red) is located near the surface of the N-terminal domain (blue) that is predicted to function in protein-protein interaction . The catalytic domain (yellow) contains the active site (green). Image courtesy of Yuxin Mao. B) Predicted orientation of the FIG4 protein in the PI(3,5)P2 biosynthetic complex . As shown in this paper, the I41T mutation of FIG4 (red) reduces interaction with both FAB1 and VAC14.

References

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