Altered phenotypes due to genetic interaction between the mouse phosphoinositide biosynthesis genes Fig4 and Pip4k2c

Abstract Loss-of-function mutations of FIG4 are responsible for neurological disorders in human and mouse that result from reduced abundance of the signaling lipid PI(3,5)P2. In contrast, loss-of-function mutations of the phosphoinositide kinase PIP4K2C result in elevated abundance of PI(3,5)P2. These opposing effects on PI(3,5)P2 suggested that we might be able to compensate for deficiency of FIG4 by reducing expression of PIP4K2C. To test this hypothesis in a whole animal model, we generated triallelic mice with genotype Fig 4−/−, Pip4k2c+/−; these mice are null for Fig 4 and haploinsufficient for Pip4k2c. The neonatal lethality of Fig 4 null mice in the C57BL/6J strain background was rescued by reduced expression of Pip4k2c. The lysosome enlargement characteristic of Fig 4 null cells was also reduced by heterozygous loss of Pip4k2c. The data demonstrate interaction between these two genes, and suggest that inhibition of the kinase PIPK4C2 could be a target for treatment of FIG4 deficiency disorders such as Charcot-Marie-Tooth Type 4J and Yunis-Varón Syndrome.


Introduction
Recessively inherited, loss-of-function variants of the phosphoinositide phosphatase FIG4 are responsible for several rare genetic disorders. Complete loss-of-function of FIG4 results in Yunis-Varón Syndrome (OMIM 216340), a lethal multisystem disorder affecting development of the skeleton and nervous system (Campeau et al. 2013). Partial loss of FIG4 function results in the peripheral neuropathy Charcot-Marie-Tooth Type 4J (CMT4J) (OMIM 611228), most often caused by compound heterozygosity for a null allele and the partial loss-of-function variant p.Ile41Thr (Chow et al. 2007;Nicholson et al. 2011). The p.Ile41Thr variant is present at an allele frequency of 0.001 in European populations, and homozygous individuals were recently described (LaFontaine et al. 2021). Other partial loss-of-function variants of FIG4 result in polymicrogyria with epilepsy (OMIM 612619) (Baulac et al. 2014) and pediatric neurodegeneration with hypomyelination (Lenk et al. 2019a), related to the requirement for PI(3,5)P 2 during oligodendrocyte maturation (Mironova et al. 2016). Deficiency of the FIG4 binding partner VAC14 results in similar neurological disorders (OMIM 617054) de Gusmao et al. 2019).
Chemical inhibition of the phosphoinositide kinase PIP4K2C is one intervention that increases intracellular levels of PI(3,5)P 2 in cultured cells (Al-Ramahi et al. 2017). To determine whether this observation could be reproduced in the whole animal and compensate for loss of Fig 4, we carried out crosses between mice with mutations in the two genes. The Pip4k2c null mouse exhibits normal growth and viability (Shim et al. 2016). The Fig 4 null mouse has a lethal phenotype that includes neurodegeneration, diluted pigmentation, and tremor (Chow et al. 2007;Ferguson et al. 2012;Bissig et al. 2019). A partial loss-of-function model of Fig 4, which is seen in most patients, is not currently available in the mouse . Therefore, to evaluate the effect of reduced Pip4k2c, we generated FIG4 and PIP4K2C are components of the phosphoinositide metabolic pathway (Fig. 1). PI(3)P is converted to PI(3,5)P 2 by a biosynthetic complex that includes the kinase PIKFYVE, the phosphatase FIG4, and the scaffold protein VAC14. The 3-phosphate is removed from PI(3,5)P 2 by the myotubularins (MTMRs) to generate PI(5)P (Hnia et al. 2012). Subsequent phosphorylation by the kinase PIP4K2C generates PI(4,5)P 2 , an abundant phosphoinositide in cell membranes and synapses (Al-Ramahi et al. 2017).
We hypothesized that the increase in PI(3,5)P 2 resulting from inactivation of Pip4k2c might compensate for the deficiency of PI(3,5)P 2 in

Pip4k2c +/− genotype also rescues enlarged lysosomes in mouse embryonic fibroblasts
To investigate the mechanism of rescued viability, we examined the appearance of lysosomes from mouse embryonic fibroblasts (MEFs) cultured at E13.5 from F2 mice. Most of the Fig 4 null cultured cells exhibit extensive enlargement of LAMP-positive, acidic vacuoles ( Fig. 3a) (Ferguson et al. 2009;Lenk et al. 2019b). In contrast, many of the

Discussion
The experiments described here demonstrate interaction between Fig 4 and Pip4k2c in regulation of lysosome function. The kinase PIP4K2C phosphorylates PI5P to generate PI(4,5)P 2 (Fig. 1). Inhibition of PIP4K2C alters the relative proportions of these phosphoinositides (Al-Ramahi et al. 2017). The resulting elevation of PI(3,5)P 2 may reflect the buildup of precursors due to the downstream block in PIP4K2C enzymatic activity. Alternatively, PIK4K2C is known to inhibit the kinase PIP5K by direct protein interaction (Wang et al. 2019). If PIP4K2C also inhibits the kinase PIKfyve, then relief of that inhibition in heterozygous null PIP4K2C cells could directly increase production of PI(3,5)P 2 . Regardless of the underlying mechanism, reduction of PIP4K2C provides an intervention for in vivo elevation of PI(3,5)P 2 . Knockout of the phosphatase MTMR2 (Fig. 1)  In Fig 4 null fibroblasts, the level of PI(3,5)P 2 is reduced to 50% of that of wildtype cells (Chow et al. 2007). The low level of PI(3,5)P 2 results in lysosomal vacuolization and neonatal lethality. We have demonstrated that vacuolization and neonatal lethality can be corrected by 50% reduction of Pip4k2c via heterozygous knockout in Fig 4 null mice. This observation introduces a new paradigm for treatment of FIG4 deficiency.
Although striking, the beneficial effects in Fig 4 −/− , Pip4k2c +/− mice are transient and maximal survival was observed at 2 weeks postnatal. This temporal limitation may be a consequence of the normal developmental increase in Pip4k2c expression that occurs between 1 and 2 weeks postnatal (Clarke et al. 2009). The developmental increase could elevate Pip4k2c level in the heterozygous null mouse beyond a critical threshold and eliminate the clearly beneficial effects seen during the first week of postnatal life. Alternatively, in vivo correction in neurons may be less efficient than observed in cultured cells.
The F2 cross that we carried out also generated double null mice with the genotype. The active site of the kinase Pip4k2c is a "druggable target" that has been investigated as a modifier of protein turnover and metastasis. The PIP4K2C inhibitor phenazopyridine is a widely used over-the-counter drug for treatment of urinary tract pain    Lenk et al. 2016. (Preynat-Seauve et al. 2021. The selective inhibitor NCT-504 alters the proportions of phosphoinositides in cultured cells and enhances autophagy; this inhibitor was identified in a screen for enhanced degradation of mutant huntingtin (Al-Ramahi et al. 2017). The covalent Pip4k2c inhibitor THZ-P1-2 causes defects in autophagy similar to those caused by inactivation of the gene (Sivakumaren et al. 2020). Our studies suggest that the new generation of pharmacological inhibitors of PIP4K2C could be applied to the PI(3,5)P 2 deficiency disorders caused by mutations of FIG4 and VAC14.
We observed positive effects of reduced Pip4k2c in mice with a severe disorder due to complete loss of