A model organism pipeline provides insight into the clinical heterogeneity of TARS1 loss-of-function variants.

Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed, essential enzymes that complete the first step of protein translation: ligation of amino acids to cognate tRNAs. Genes encoding ARSs have been implicated in myriad dominant and recessive phenotypes, the latter often affecting multiple tissues but with frequent involvement of the central and peripheral nervous systems, liver, and lungs. Threonyl-tRNA synthetase (TARS1) encodes the enzyme that ligates threonine to tRNA in the cytoplasm.

Biallelic loss-of-function variants of SLC12A9 cause lysosome dysfunction and a syndromic neurodevelopmental disorder.

Purpose: Pathogenic variants of FIG4 generate enlarged lysosomes and neurological and developmental disorders. To identify additional genes regulating lysosomal volume, we carried out a genome-wide activation screen to detect suppression of enlarged lysosomes in FIG4 cells.

Methods: The CRISPR-a gene activation screen utilized sgRNAs from the promoters of protein-coding genes.

The chloride antiporter CLCN7 is a modifier of lysosome dysfunction in FIG4 and VAC14 mutants.

The phosphatase FIG4 and the scaffold protein VAC14 function in the biosynthesis of PI(3,5)P2, a signaling lipid that inhibits the lysosomal chloride transporter ClC-7. Loss-of-function mutations of FIG4 and VAC14 reduce PI(3,5)P2 and result in lysosomal disorders characterized by accumulation of enlarged lysosomes and neurodegeneration. Similarly, a gain of function mutation of CLCN7 encoding ClC-7 also results in enlarged lysosomes.

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

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.

Chloroquine corrects enlarged lysosomes in FIG4 null cells and reduces neurodegeneration in Fig4 null mice.

Loss-of-function mutations of FIG4 impair the biosynthesis of PI(3,5)P and are responsible for rare genetic disorders including Yunis-Varón Syndrome and Charcot-Marie-Tooth Disease Type 4 J. Cultured cells deficient in FIG4 accumulate enlarged lysosomes with hyperacidic pH, due in part to impaired regulation of lysosomal ion channels and elevated intra-lysosomal osmotic pressure. We evaluated the effects of the FDA approved drug chloroquine, which is known to reduce lysosome acidity, on FIG4 deficient cell culture and on a mouse model.

AAV9-mediated FIG4 delivery prolongs life span in Charcot-Marie-Tooth disease type 4J mouse model.

Charcot-Marie-Tooth disease type 4J (CMT4J) is caused by recessive, loss-of-function mutations in FIG4, encoding a phosphoinositol(3,5)P2-phosphatase. CMT4J patients have both neuron loss and demyelination in the peripheral nervous system, with vacuolization indicative of endosome/lysosome trafficking defects. Although the disease is highly variable, the onset is often in childhood and FIG4 mutations can dramatically shorten life span.

Scn8a Antisense Oligonucleotide Is Protective in Mouse Models of SCN8A Encephalopathy and Dravet Syndrome.

Objective: SCN8A encephalopathy is a developmental and epileptic encephalopathy (DEE) caused by de novo gain-of-function mutations of sodium channel Na 1.6 that result in neuronal hyperactivity. Affected individuals exhibit early onset drug-resistant seizures, developmental delay, and cognitive impairment.

CRISPR knockout screen implicates three genes in lysosome function.

Defective biosynthesis of the phospholipid PI(3,5)P underlies neurological disorders characterized by cytoplasmic accumulation of large lysosome-derived vacuoles. To identify novel genetic causes of lysosomal vacuolization, we developed an assay for enlargement of the lysosome compartment that is amenable to cell sorting and pooled screens. We first demonstrated that the enlarged vacuoles that accumulate in fibroblasts lacking FIG4, a PI(3,5)P biosynthetic factor, have a hyperacidic pH compared to normal cells'.

Cerebral hypomyelination associated with biallelic variants of FIG4.

The lipid phosphatase gene FIG4 is responsible for Yunis-Varón syndrome and Charcot-Marie-Tooth disease Type 4J, a peripheral neuropathy. We now describe four families with FIG4 variants and prominent abnormalities of central nervous system (CNS) white matter (leukoencephalopathy), with onset in early childhood, ranging from severe hypomyelination to mild undermyelination, in addition to peripheral neuropathy. Affected individuals inherited biallelic FIG4 variants from heterozygous parents.

The PIKfyve complex regulates the early melanosome homeostasis required for physiological amyloid formation.

The metabolism of PI(3,5)P2 is regulated by the PIKfyve, VAC14 and FIG4 complex, mutations in which are associated with hypopigmentation in mice. These pigmentation defects indicate a key, but as yet unexplored, physiological relevance of this complex in the biogenesis of melanosomes. Here, we show that PIKfyve activity regulates formation of amyloid matrix composed of PMEL protein within the early endosomes in melanocytes, called stage I melanosomes.

Protective role of the lipid phosphatase Fig4 in the adult nervous system.

The signaling lipid phosphatidylinositol 3,5-bisphosphate, PI(3,5)P2, functions in vesicular trafficking through the endo-lysosomal compartment. Cellular levels of PI(3,5)P2 are regulated by an enzyme complex comprised of the kinase PIKFYVE, the phosphatase FIG4, and the scaffold protein VAC14. Mutations of human FIG4 cause inherited disorders including Charcot-Marie-Tooth disease type 4J, polymicrogyria with epilepsy, and Yunis-Varón syndrome.

Biallelic Mutations of VAC14 in Pediatric-Onset Neurological Disease.

In the PI(3,5)P2 biosynthetic complex, the lipid kinase PIKFYVE and the phosphatase FIG4 are bound to the dimeric scaffold protein VAC14, which is composed of multiple heat-repeat domains. Mutations of FIG4 result in the inherited disorders Charcot-Marie-Tooth disease type 4J, Yunis-Varón syndrome, and polymicrogyria with seizures. We here describe inherited variants of VAC14 in two unrelated children with sudden onset of a progressive neurological disorder and regression of developmental milestones.

PI(3,5)P2 biosynthesis regulates oligodendrocyte differentiation by intrinsic and extrinsic mechanisms.

Proper development of the CNS axon-glia unit requires bi-directional communication between axons and oligodendrocytes (OLs). We show that the signaling lipid phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2] is required in neurons and in OLs for normal CNS myelination. In mice, mutations of Fig4, Pikfyve or Vac14, encoding key components of the PI(3,5)P2 biosynthetic complex, each lead to impaired OL maturation, severe CNS hypomyelination and delayed propagation of compound action potentials.

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

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.

Loss of Fig4 in both Schwann cells and motor neurons contributes to CMT4J neuropathy.

Mutations of FIG4 are responsible for Yunis-Varón syndrome, familial epilepsy with polymicrogyria, and Charcot-Marie-Tooth type 4J neuropathy (CMT4J). Although loss of the FIG4 phospholipid phosphatase consistently causes decreased PtdIns(3,5)P₂ levels, cell-specific sensitivity to partial loss of FIG4 function may differentiate FIG4-associated disorders. CMT4J is an autosomal recessive neuropathy characterized by severe demyelination and axonal loss in human, with both motor and sensory involvement.

Whole exome sequencing identifies three recessive FIG4 mutations in an apparently dominant pedigree with Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth disease (CMT) is genetically heterogeneous and classification based on motor nerve conduction velocity and inheritance is used to direct genetic testing. With the less common genetic forms of CMT, identifying the causative genetic mutation by Sanger sequencing of individual genes can be time-consuming and costly. Next-generation sequencing technologies show promise for clinical testing in diseases where a similar phenotype is caused by different genes.

Role of the phosphoinositide phosphatase FIG4 gene in familial epilepsy with polymicrogyria.

Objective: The aim of this study was to identify the causal gene in a consanguineous Moroccan family with temporo-occipital polymicrogyria, psychiatric manifestations, and epilepsy, previously mapped to the 6q16-q22 region.

Methods: We used exome sequencing and analyzed candidate variants in the 6q16-q22 locus, as well as a rescue assay in Fig4-null mouse fibroblasts and immunohistochemistry of Fig4-null mouse brains.

Results: A homozygous missense mutation (p.

Mouse models of PI(3,5)P2 deficiency with impaired lysosome function.

The endolysosomal system and autophagy are essential components of macromolecular turnover in eukaryotic cells. The low-abundance signaling lipid PI(3,5)P2 is a key regulator of this pathway. Analysis of mouse models with defects in PI(3,5)P2 biosynthesis has revealed the unique dependence of the mammalian nervous system on this signaling pathway.

Altered cardiac electrophysiology and SUDEP in a model of Dravet syndrome.

Objective: Dravet syndrome is a severe form of intractable pediatric epilepsy with a high incidence of SUDEP: Sudden Unexpected Death in epilepsy. Cardiac arrhythmias are a proposed cause for some cases of SUDEP, yet the susceptibility and potential mechanism of arrhythmogenesis in Dravet syndrome remain unknown. The majority of Dravet syndrome patients have de novo mutations in SCN1A, resulting in haploinsufficiency.

Murine Fig4 is dispensable for muscle development but required for muscle function.

Background: Phosphatidylinositol phosphates (PIPs) are low-abundance phospholipids that participate in a range of cellular processes, including cell migration and membrane traffic. PIP levels and subcellular distribution are regulated by a series of lipid kinases and phosphatases. In skeletal muscle, PIPs and their enzymatic regulators serve critically important functions exemplified by mutations of the PIP phosphatase MTM1 in myotubular myopathy (MTM), a severe muscle disease characterized by impaired muscle structure and abnormal excitation-contraction coupling.

A TRP channel in the lysosome regulates large particle phagocytosis via focal exocytosis.

Phagocytosis of large extracellular particles such as apoptotic bodies requires delivery of the intracellular endosomal and lysosomal membranes to form plasmalemmal pseudopods. Here, we identified mucolipin TRP channel 1 (TRPML1) as the key lysosomal Ca2+ channel regulating focal exocytosis and phagosome biogenesis. Both particle ingestion and lysosomal exocytosis are inhibited by synthetic TRPML1 blockers and are defective in macrophages isolated from TRPML1 knockout mice.