We have demonstrated that the FIG4-I41T protein is unstable in vivo in cultured cells and in transgenic mice, and that the amount of protein in cells from CMT4J patients is extremely low. FIG4 protein expression equivalent to 10% of wildtype levels is sufficient to prevent neurodegeneration and completely rescue lethality in transgenic line Tg721. In contrast, transgenic line Tg705, with lower expression of FIG4-I41T, provides an animal model of the human disorder with neurodegeneration. These key observations suggest that increasing the expression of the FIG4-I41T allele in CMT4J patients, or stabilizing the protein, would be therapeutic.
The low level of FIG4 protein in rescued mice and in patient fibroblasts appears to be a consequence of the direct effect of the I41T mutation on interaction with the scaffold protein VAC14. In yeast the co-localization of Fig4p, Fab1p and Vac14p on the vacuolar membrane requires the presence of all three proteins, and loss of one protein prevents localization of the other two 
. Similarly, in mammalian cells expressing an shRNA to downregulate Vac14
expression, a small reduction in endogenous FIG4 protein was reported 
We confirmed the importance of the FIG4-VAC14 interaction for stability of wildtype FIG4 with the demonstration that FIG4 protein is drastically reduced in mice homozygous for a null allele of VAC14. This experiment clearly demonstrates the dependence of wildtype FIG4 protein on VAC14 for in vivo
stability. Because of its reduced affinity for VAC14, FIG4-I41T is a hypomorphic allele encoding an unstable protein, resulting in a very low steady-state level of protein in vivo
. The VAC14 protein is composed of heat-repeat domains 
and is thought to function as a scaffold for the PI(3,5)P2
biosynthetic complex. In another example of the importance of interactions between proteins in this complex, the missense mutation of Vac14
in the ingls
mouse which reduces the affinity of VAC14 for FAB1 and results in a neurodegenerative disease that closely resembles the Fig4
null mice 
. In the reciprocal experiment, VAC14 protein was not
reduced in Fig4
null mice, demonstrating the greater intrinsic stability of the mammalian scaffold protein, and/or its stabilization by interaction with other components of the complex.
This model of pathogenesis is consistent with the structure of the FIG4 protein, which was predicted by superimposition with the crystal structure of Sac1p, a closely related lipid phosphatase 
. The I41T mutation is located near the surface of the non-catalytic domain, in a hydrophobic pocket between two β-sheets (). The mutation was predicted to affect protein-protein interaction by destabilizing the SacN domain 
, consistent with our observations. The I41T mutation is located at a distance from the catalytic domain, and appears not to affect the enzymatic activity 
Location of the FIG4-I41T mutation and effect on protein interaction.
Based on our observations, increased expression or stabilization of the FIG4-I41T protein in patients with CMT4J should be therapeutic and could achieve complete rescue of this progressive neurodegenerative disorder. Inhibition of proteasome degradation with MG132 resulted in increased levels of FIG4-I41T protein in cultured fibroblasts. This non-specific agent increased the abundance of approximately 200 proteins in cultured fibroblasts 
. The proteasome inhibitor Velcade (bortezemib) has been approved for treatment of multiple myeloma, and the widely used drug disulfiram (Antabuse) was recently shown to have activity as a proteasome inhibitor 
. Pharmacological interventions like these might increase FIG4 concentration to a level sufficient to protect against loss of motor function in CMT4J patients with genotype FIG4I41T/−
. Histone deacetylase inhibitors such as sodium butyrate and tricostatin A that increase the expression of other neurological disease genes such as SMN
should also be evaluated in CMT4J fibroblasts.
The correlated stepwise rescue of autophagy, gliosis, neurodegeneration and lethality in the two transgenic lines supports our proposed model of pathogenesis in which accumulation of autophagy intermediates leads to neuronal damage and then to gliosis, neural cell death and lethality 
appears to be required at a step subsequent to the formation of the autolysosome 
, and may be involved in the regeneration of lysosomes from autolysosomes 
. A newly recognized function of PI(3,5)P2
is activation of the lysosomal calcium channel TRPML1, which is mutated in the neurodegenerative disorder mucolipidosis type IV 
. Transfection of TRPML1 into Vac14
null fibroblasts rescued the vacuolization caused by deficiency of PI(3,5)P2 
. Increased lysosomal ion concentrations resulting from PI(3,5)P2
deficiency in FIG4
mutant cells could contribute to vacuolization via osmotic retention of water. Low molecular weight activators of TRPML1 are under development and might provide another route to treatment of PI(3,5)P2
The response of various tissues to rescue of FIG4 deficiency in transgenic mice is summarized in . The dramatically improved survival of cortical and dorsal root neurons, and the complete rescue of lethality, indicate that a small amount of I41T protein is sufficient for normal function in most affected cells.
Rescue of various phenotypes of the Fig4 null mice by the Tg705 and Tg721 I41T transgenes.
Fifteen CMT4J pedigrees segregating the I41T allele have been identified to date (
Nicholson et al, unpublished data). In all of the unrelated families the I41T allele is inherited on the same chromosome haplotype, indicating inheritance of a shared founder mutation. In spite of their shared FIG4I41T/−
genotype, the clinical course in CMT4J patients is highly variable. Age of onset ranges from an early childhood form with developmental delays that resembles Dejerine-Sotas syndrome to an adult onset form with rapid progression that may be triggered by trauma 
. Background genetic variation affecting the level of expression of VAC14 or other proteins in the PI(3,5)P2
biosynthetic complex could contribute to the clinical differences in patients with identical FIG4
genotype. The Tg705
model of CMT4J, which survives for 3 to 6 months and then develops severe disease, will be useful for testing therapies designed to increase the in vivo
level of FIG4-I41T protein.