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Hereditary spastic paraplegia (HSP) denotes genetically heterogeneous disorders characterized by leg spasticity due to degeneration of corticospinal axons. SPG11 and SPG15 have a similar clinical course and together are the most prevalent autosomal recessive HSP entity. The respective proteins play a role for macroautophagy/autophagy and autophagic lysosome reformation (ALR). Here, we report that spg11 and zfyve26 KO mice developed motor impairments within the same course of time. This correlated with enhanced accumulation of autofluorescent material in neurons and progressive neuron loss. In agreement with defective ALR, tubulation events were diminished in starved KO mouse embryonic fibroblasts (MEFs) and lysosomes decreased in neurons of KO brain sections. Confirming that both proteins act in the same molecular pathway, the pathologies were not aggravated upon simultaneous disruption of both. We further show that PI4K2A (phosphatidylinositol 4-kinase type 2 alpha), which phosphorylates phosphatidylinositol to phosphatidylinositol-4-phosphate (PtdIns4P), accumulated in autofluorescent deposits isolated from KO but not WT brains. Elevated PI4K2A abundance was already found at autolysosomes of neurons of presymptomatic KO mice. Immunolabelings further suggested higher levels of PtdIns4P at LAMP1-positive structures in starved KO MEFs. An increased association with LAMP1-positive structures was also observed for clathrin and DNM2/dynamin 2, which are important effectors of ALR recruited by phospholipids. Because PI4K2A overexpression impaired ALR, while its knockdown increased tubulation, we conclude that PI4K2A modulates phosphoinositide levels at autolysosomes and thus the recruitment of downstream effectors of ALR. Therefore, PI4K2A may play an important role in the pathogenesis of SPG11 and SPG15. Abbreviations: ALR: autophagic lysosome reformation; AP-5: adaptor protein complex 5; BFP: blue fluorescent protein; dKO: double knockout; EBSS: Earle’s balanced salt solution; FBA: foot base angle; GFP: green fluorescent protein; HSP: hereditary spastic paraplegia; KO: knockout; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3B/LC3: microtubule-associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; SQSTM1/p62: sequestosome 1; PI4K2A: phosphatidylinositol 4-kinase type 2 alpha; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns4P: phosphatidylinositol-4-phosphate; RFP: red fluorescent protein; SPG: spastic paraplegia gene; TGN: trans-Golgi network; WT: wild type

Inherited cutis laxa, or inelastic, sagging skin is a genetic condition of premature and generalised connective tissue ageing, affecting various elastic components of the extracellular matrix. Several cutis laxa syndromes are inborn errors of metabolism and lead to severe neurological symptoms. In a patient with cutis laxa, a choreoathetoid movement disorder, dysmorphic features and intellectual disability we performed exome sequencing to elucidate the underlying genetic defect. We identified the amino acid substitution R275W in phosphatidylinositol 4‐kinase type IIα, caused by a homozygous missense mutation in the PI4K2A gene. We used lipidomics, complexome profiling and functional studies to measure phosphatidylinositol 4‐phosphate synthesis in the patient and evaluated PI4K2A deficient mice to define a novel metabolic disorder. The R275W residue, located on the surface of the protein, is involved in forming electrostatic interactions with the membrane. The catalytic activity of PI4K2A in patient fibroblasts was severely reduced and lipid mass spectrometry showed that particular acyl‐chain pools of PI4P and PI(4,5)P2 were decreased. Phosphoinositide lipids play a major role in intracellular signalling and trafficking and regulate the balance between proliferation and apoptosis. Phosphatidylinositol 4‐kinases such as PI4K2A mediate the first step in the main metabolic pathway that generates PI4P, PI(4,5)P2 and PI(3,4,5)P3. Although neurologic involvement is common, cutis laxa has not been reported previously in metabolic defects affecting signalling. Here we describe a patient with a complex neurological phenotype, premature ageing and a mutation in PI4K2A, illustrating the importance of this enzyme in the generation of inositol lipids with particular acylation characteristics.

The minor phospholipid, phosphatidylinositol 4-phosphate (PI4P), is emerging as a key regulator of lipid transfer in ER-membrane contact sites. Four different phosphatidylinositol 4-kinase (PI4K) enzymes generate PI4P in different membrane compartments supporting distinct cellular processes, many of which are crucial for the maintenance of cellular integrity but also hijacked by intracellular pathogens. While type III PI4Ks have been targeted by small molecular inhibitors, thus helping decipher their importance in cellular physiology, no inhibitors are available for the type II PI4Ks, which hinders investigations into their cellular functions. Here, we describe the identification of small molecular inhibitors of PI4K type II alpha (PI4K2A) by implementing a large scale small molecule high-throughput screening. A novel assay was developed that allows testing of selected inhibitors against PI4K2A in intact cells using a bioluminescence resonance energy transfer approach adapted to plate readers. The compounds disclosed here will pave the way to the optimization of PI4K2A inhibitors that can be used in cellular and animal studies to better understand the role of this enzyme in both normal and pathological states.

Nucleoredoxin (NXN) is a redox-regulating protein potentially targeted by reactive oxygen species (ROS). It regulates molecular pathways that participate in several key cellular processes. However, the role of NXN in the alcohol liver disease (ALD) redox regulation has not been fully understood. Here, we investigated the effects of ethanol and ethanol plus lipopolysaccharide, a two-hit liver injury model (Ethanol/LPS), on NXN/dishevelled (DVL) interaction and on DVL-dependent phosphoinositides production both in mouse liver and in a co-culture system consisting of human hepatic stellate cells (HSC) and ethanol metabolizing-VL17A human hepatocyte cells. Ethanol and two-hit model increased Nxn protein and mRNA expression, and 4-hydroxynonenal adducts. Two-hit model promoted Nxn nuclear translocation and Dvl/Phosphatidylinositol 4-kinase type-IIα (Pi4k2a) interaction ratio but surprisingly decreased Dvl protein and mRNA levels and reverted ethanol-induced Nxn/Dvl and Dvl/frizzled (Fzd) interaction ratios. Ethanol resulted in a significant increase of Dvl protein and mRNA expression, and decreased Nxn/Dvl interaction ratio but promoted the interaction of Dvl with Fzd and Pi4k2a; formation of this complex induced phosphatidylinositol 4-phosphate [PI(4)P] production. Ethanol and LPS treatments provoked similar alterations on NXN/DVL interaction and its downstream effect in HSC/VL17A co-culture system. Interestingly, ROS and glutathione levels as well as most of ethanol-induced alterations were modified by NXN overexpression in the co-culture system. In conclusion, two-hit model of ethanol exposure disrupts NXN/DVL homeostatic status to allow DVL/FZD/PI4K2A complex formation and stimulates PI(4)P production. These results provide a new mechanism showing that NXN also participates in the regulation of phosphoinositides production that is altered by ethanol during alcoholic liver disease progression.

Phosphatidylinositol 4-kinases (PI 4-kinases) catalyze the conversion of phosphatidylinositol to phosphatidylinositol 4-phosphate (PtdIns4P). The four known mammalian PI 4-kinases, PI4KA, PI4KB, PI4K2A, and PI4K2B have roles in intracellular lipid and protein trafficking. PI4KA and PI4KB also assist in the replication of several positive-sense RNA viruses. The identification of selective inhibitors of these kinases would be facilitated by assays suitable for high-throughput screening. We describe a homogeneous and nonisotopic assay for PI 4-kinase activity based on the bioluminescent detection of the ADP produced by kinase reactions. We have evaluated this assay with known nonselective inhibitors of PI 4-kinases and show that it performs similar to radiometric assay formats previously described in the literature. In addition, this assay generates Z-factor values of >0.7 for PI4KA in a 384-well format, demonstrating its suitability for high-throughput screening applications.

Specificity of membrane fusion in vesicular trafficking is dependent on proper subcellular distribution of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). Although SNARE complexes are fairly promiscuous in vitro, substantial specificity is achieved in cells owing to the spatial segregation and shielding of SNARE motifs prior to association with cognate Q-SNAREs. In this study, we identified phosphatidylinositol 4-kinase IIα (PI4K2A) as a binding partner of vesicle-associated membrane protein 3 (VAMP3), a small R-SNARE involved in recycling and retrograde transport, and found that the two proteins co-reside on tubulo-vesicular endosomes. PI4K2A knockdown inhibited VAMP3 trafficking to perinuclear membranes and impaired the rate of VAMP3-mediated recycling of the transferrin receptor. Moreover, depletion of PI4K2A significantly decreased association of VAMP3 with its cognate Q-SNARE Vti1a. Although binding of VAMP3 to PI4K2A did not require kinase activity, acute depletion of phosphatidylinositol 4-phosphate (PtdIns4P) on endosomes significantly delayed VAMP3 trafficking. Modulation of SNARE function by phospholipids had previously been proposed based on in vitro studies, and our study provides mechanistic evidence in support of these claims by identifying PI4K2A and PtdIns4P as regulators of an R-SNARE in intact cells.

Numerous genes causing autosomal recessive hereditary spastic paraplegia (AR HSP) have been described. Despite this, in many families the causative gene and mutation are unknown. In this study we sequenced the Pi4k2a gene, whose knockout has been shown to cause a typical HSP model in mice, in 24 index cases of autosomal recessive HSP not known to be linked to any other HSP locus. No pathogenic changes were identified in exons or splice sites, suggesting the Pi4k2a gene may not be a cause of AR HSP in humans.