Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P₂] plays a fundamental role in clathrin-mediated endocytosis. However, precisely how PI(4,5)P₂ metabolism is spatially and temporally regulated during membrane internalization and the functional consequences of endocytosis-coupled PI(4,5)P₂ dephosphorylation remain to be explored. Using cell-free assays with liposomes of varying diameters, we show that the major synaptic phosphoinositide phosphatase, synaptojanin 1 (Synj1), acts with membrane curvature generators/sensors, such as the BAR protein endophilin, to preferentially remove PI(4,5)P₂ from curved membranes as opposed to relatively flat ones. Moreover, in vivo recruitment of Synj1's inositol 5-phosphatase domain to endophilin-induced membrane tubules results in fragmentation and condensation of these structures largely in a dynamin-dependent fashion. Our study raises the possibility that geometry-based mechanisms may contribute to spatially restricting PI(4,5)P₂ elimination during membrane internalization and suggests that the PI(4,5)P₂-to-PI4P conversion achieved by Synj1 at sites of high curvature may cooperate with dynamin to achieve membrane fission.

Vibrio cholerae, responsible for acute gastroenteritis secretes a large multifunctional-autoprocessing repeat-in-toxin (MARTX) toxin linked to evasion of host immune system, facilitating colonization of small intestine. Unlike other effector domains of the multifunctional toxin that target cytoskeleton, the function of alpha-beta hydrolase (ABH) remained elusive. This study demonstrates that ABH is an esterase/lipase with catalytic Ser-His-Asp triad. ABH binds with high affinity to phosphatidylinositol-3-phosphate (PtdIns3P) and cleaves the fatty acid in PtdIns3P at the sn1 position in vitro making it the first PtdIns3P-specific phospholipase A1 (PLA1). Expression of ABH in vivo reduces intracellular PtdIns3P levels and its PtdIns3P-specific PLA1 activity blocks endosomal and autophagic pathways. In accordance with recent studies acknowledging the potential of extracellular pathogens to evade or exploit autophagy to prevent their clearance and facilitate survival, this is the first report highlighting the role of ABH in inhibiting autophagy and endosomal trafficking induced by extracellular V. cholerae.

Neuronal synapses are the fundamental units of neural signal transduction and must maintain exquisite signal fidelity while also accommodating the plasticity that underlies learning and development. To achieve these goals, the molecular composition and spatial organization of synaptic terminals must be tightly regulated; however, little is known about the regulation of lipid composition and organization in synaptic membranes. Here we quantify the comprehensive lipidome of rat synaptic membranes during postnatal development and observe dramatic developmental lipidomic remodeling during the first 60 postnatal days, including progressive accumulation of cholesterol, plasmalogens, and sphingolipids. Further analysis of membranes associated with isolated postsynaptic densities (PSDs) suggests the PSD-associated postsynaptic plasma membrane (PSD-PM) as one specific location of synaptic remodeling. We analyze the biophysical consequences of developmental remodeling in reconstituted synaptic membranes and observe remarkably stable microdomains, with the stability of domains increasing with developmental age. We rationalize the developmental accumulation of microdomain-forming lipids in synapses by proposing a mechanism by which palmitoylation of the immobilized scaffold protein PSD-95 nucleates domains at the postsynaptic plasma membrane. These results reveal developmental changes in lipid composition and palmitoylation that facilitate the formation of postsynaptic membrane microdomains, which may serve key roles in the function of the neuronal synapse.

Defects in endolysosomal and autophagic functions are increasingly viewed as key pathological features of neurodegenerative disorders. A master regulator of these functions is phosphatidylinositol-3-phosphate (PI3P), a phospholipid synthesized primarily by class III PI 3-kinase Vps34. Here we report that disruption of neuronal Vps34 function in vitro and in vivo impairs autophagy, lysosomal degradation as well as lipid metabolism, causing endolysosomal membrane damage. PI3P deficiency also promotes secretion of unique exosomes enriched for undigested lysosomal substrates, including amyloid precursor protein C-terminal fragments (APP-CTFs), specific sphingolipids, and the phospholipid bis(monoacylglycero)phosphate (BMP), which normally resides in the internal vesicles of endolysosomes. Secretion of these exosomes requires neutral sphingomyelinase 2 and sphingolipid synthesis. Our results reveal a homeostatic response counteracting lysosomal dysfunction via secretion of atypical exosomes eliminating lysosomal waste and define exosomal APP-CTFs and BMP as candidate biomarkers for endolysosomal dysfunction associated with neurodegenerative disorders.

In the amyloidogenic pathway associated with Alzheimer disease (AD), the amyloid precursor protein (APP) is cleaved by β-secretase to generate a 99-aa C-terminal fragment (C99) that is then cleaved by γ-secretase to generate the β-amyloid (Aβ) found in senile plaques. In previous reports, we and others have shown that γ-secretase activity is enriched in mitochondria-associated endoplasmic reticulum (ER) membranes (MAM) and that ER-mitochondrial connectivity and MAM function are upregulated in AD We now show that C99, in addition to its localization in endosomes, can also be found in MAM, where it is normally processed rapidly by γ-secretase. In cell models of AD, however, the concentration of unprocessed C99 increases in MAM regions, resulting in elevated sphingolipid turnover and an altered lipid composition of both MAM and mitochondrial membranes. In turn, this change in mitochondrial membrane composition interferes with the proper assembly and activity of mitochondrial respiratory supercomplexes, thereby likely contributing to the bioenergetic defects characteristic of AD.

Accumulating evidence from genetic and biochemical studies implicates dysfunction of the autophagic-lysosomal pathway as a key feature in the pathogenesis of Parkinson's disease (PD). Most studies have focused on accumulation of neurotoxic α-synuclein secondary to defects in autophagy as the cause of neurodegeneration, but abnormalities of the autophagic-lysosomal system likely mediate toxicity through multiple mechanisms. To further explore how endolysosomal dysfunction causes PD-related neurodegeneration, we generated a murine model of Kufor-Rakeb syndrome (KRS), characterized by early-onset Parkinsonism with additional neurological features. KRS is caused by recessive loss-of-function mutations in the ATP13A2 gene encoding the endolysosomal ATPase ATP13A2. We show that loss of ATP13A2 causes a specific protein trafficking defect, and that Atp13a2 null mice develop age-related motor dysfunction that is preceded by neuropathological changes, including gliosis, accumulation of ubiquitinated protein aggregates, lipofuscinosis, and endolysosomal abnormalities. Contrary to predictions from in vitro data, in vivo mouse genetic studies demonstrate that these phenotypes are α-synuclein independent. Our findings indicate that endolysosomal dysfunction and abnormalities of α-synuclein homeostasis are not synonymous, even in the context of an endolysosomal genetic defect linked to Parkinsonism, and highlight the presence of α-synuclein-independent neurotoxicity consequent to endolysosomal dysfunction.

Lipids are major constituents of the brain largely implicated in physiological and pathological processes. The hippocampus is a complex brain structure involved in learning, memory and emotional responses, and its functioning is also affected in various disorders. Despite conserved intrinsic circuitry, behavioral and anatomical studies suggest the existence of a structural and functional gradient along the hippocampal longitudinal axis. Here, we used an unbiased mass spectrometry approach to characterize the lipid composition of distinct hippocampal subregions. In addition, we evaluated the susceptibility of each area to lipid modulation by corticosterone (CORT), an important mediator of the effects of stress. We confirmed a great similarity between hippocampal subregions relatively to other brain areas. Moreover, we observed a continuous molecular gradient along the longitudinal axis of the hippocampus, with the dorsal and ventral extremities differing significantly from each other, particularly in the relative abundance of sphingolipids and phospholipids. Also, whereas chronic CORT exposure led to remodeling of triacylglycerol and phosphatidylinositol species in both hippocampal poles, our study suggests that the ventral hippocampus is more sensitive to CORT-induced changes, with regional modulation of ceramide, dihydrosphingomyelin and phosphatidic acid. Thus, our results confirm a multipartite molecular view of dorsal-ventral hippocampal axis and emphasize lipid metabolites as candidate effectors of glucocorticoid signaling, mediating regional susceptibility to neurological disorders associated with stress.

A fundamental question is how autophagosome formation is regulated. Here we show that the PX domain protein HS1BP3 is a negative regulator of autophagosome formation. HS1BP3 depletion increased the formation of LC3-positive autophagosomes and degradation of cargo both in human cell culture and in zebrafish. HS1BP3 is localized to ATG16L1- and ATG9-positive autophagosome precursors and we show that HS1BP3 binds phosphatidic acid (PA) through its PX domain. Furthermore, we find the total PA content of cells to be significantly upregulated in the absence of HS1BP3, as a result of increased activity of the PA-producing enzyme phospholipase D (PLD) and increased localization of PLD1 to ATG16L1-positive membranes. We propose that HS1BP3 regulates autophagy by modulating the PA content of the ATG16L1-positive autophagosome precursor membranes through PLD1 activity and localization. Our findings provide key insights into how autophagosome formation is regulated by a novel negative-feedback mechanism on membrane lipids.

Tether proteins attach the endoplasmic reticulum (ER) to other cellular membranes, thereby creating contact sites that are proposed to form platforms for regulating lipid homeostasis and facilitating non-vesicular lipid exchange. Sterols are synthesized in the ER and transported by non-vesicular mechanisms to the plasma membrane (PM), where they represent almost half of all PM lipids and contribute critically to the barrier function of the PM. To determine whether contact sites are important for both sterol exchange between the ER and PM and intermembrane regulation of lipid metabolism, we generated Δ-super-tether (Δ-s-tether) yeast cells that lack six previously identified tethering proteins (yeast extended synatotagmin [E-Syt], vesicle-associated membrane protein [VAMP]-associated protein [VAP], and TMEM16-anoctamin homologues) as well as the presumptive tether Ice2. Despite the lack of ER-PM contacts in these cells, ER-PM sterol exchange is robust, indicating that the sterol transport machinery is either absent from or not uniquely located at contact sites. Unexpectedly, we found that the transport of exogenously supplied sterol to the ER occurs more slowly in Δ-s-tether cells than in wild-type (WT) cells. We pinpointed this defect to changes in sterol organization and transbilayer movement within the PM bilayer caused by phospholipid dysregulation, evinced by changes in the abundance and organization of PM lipids. Indeed, deletion of either OSH4, which encodes a sterol/phosphatidylinositol-4-phosphate (PI4P) exchange protein, or SAC1, which encodes a PI4P phosphatase, caused synthetic lethality in Δ-s-tether cells due to disruptions in redundant PI4P and phospholipid regulatory pathways. The growth defect of Δ-s-tether cells was rescued with an artificial "ER-PM staple," a tether assembled from unrelated non-yeast protein domains, indicating that endogenous tether proteins have nonspecific bridging functions. Finally, we discovered that sterols play a role in regulating ER-PM contact site formation. In sterol-depleted cells, levels of the yeast E-Syt tether Tcb3 were induced and ER-PM contact increased dramatically. These results support a model in which ER-PM contact sites provide a nexus for coordinating the complex interrelationship between sterols, sphingolipids, and phospholipids that maintain PM composition and integrity.

Membrane phase behavior has been well characterized in model membranes in vitro under thermodynamic equilibrium state. However, the widely observed differences between biological membranes and their in vitro counterparts are placing more emphasis on nonequilibrium factors, including influx and efflux of lipid molecules. The endoplasmic reticulum (ER) is the largest cellular membrane system and also the most metabolically active organelle responsible for lipid synthesis. However, how the nonequilibrium metabolic activity modulates ER membrane phase has not been investigated. Here, we studied the phase behavior of functional ER in the context of lipid metabolism. Utilizing advanced vibrational imaging technique, that is, stimulated Raman scattering microscopy, we discovered that metabolism of palmitate, a prevalent saturated fatty acid (SFA), could drive solid-like domain separation from the presumably uniformly fluidic ER membrane, a previously unknown phenomenon. The potential of various fatty acids to induce solid phase can be predicted by the transition temperatures of their major metabolites. Interplay between saturated and unsaturated fatty acids is also observed. Hence, our study sheds light on cellular membrane biophysics by underscoring the nonequilibrium metabolic status of living cell.

During its life cycle, Plasmodium falciparum undergoes rapid proliferation fueled by de novo synthesis and acquisition of host cell lipids. Consistent with this essential role, Plasmodium lipid synthesis enzymes are emerging as potential drug targets. To explore their broader potential for therapeutic interventions, we assayed the global lipid landscape during P. falciparum sexual and asexual blood stage (ABS) development. Using liquid chromatography-mass spectrometry, we analyzed 304 lipids constituting 24 classes in ABS parasites, infected red blood cell (RBC)-derived microvesicles, gametocytes, and uninfected RBCs. Ten lipid classes were previously uncharacterized in P. falciparum, and 70%-75% of the lipid classes exhibited changes in abundance during ABS and gametocyte development. Utilizing compounds that target lipid metabolism, we affirmed the essentiality of major classes, including triacylglycerols. These studies highlight the interplay between host and parasite lipid metabolism and provide a comprehensive analysis of P. falciparum lipids with candidate pathways for drug discovery efforts.

The immediate responses to inhibition of phosphatidylcholine (PC) biosynthesis in yeast are altered phospholipid levels, slow growth, and defects in the morphology and localization of ER and mitochondria. With chronic lipid imbalance, yeast adapt. Lipid droplet (LD) biogenesis and conversion of phospholipids to triacylglycerol are required for restoring some phospholipids to near-wild-type levels. We confirmed that the unfolded protein response is activated by this lipid stress and find that Hsp104p is recruited to ER aggregates. We also find that LDs form at ER aggregates, contain polyubiquitinated proteins and an ER chaperone, and are degraded in the vacuole by a process resembling microautophagy. This process, microlipophagy, is required for restoration of organelle morphology and cell growth during adaptation to lipid stress. Microlipophagy does not require ATG7 but does requires ESCRT components and a newly identified class E VPS protein that localizes to ER and is upregulated by lipid imbalance.

Cellular mechanisms that mediate steatohepatitis, an increasingly prevalent condition in the Western world for which no therapies are available, are poorly understood. Despite the fact that its synthetic agonists induce fatty liver, the liver X receptor (LXR) transcription factor remains a target of interest because of its anti-atherogenic, cholesterol removal, and anti-inflammatory activities. Here we show that tetratricopeptide repeat domain protein 39B (Ttc39b, C9orf52) (T39), a high-density lipoprotein gene discovered in human genome-wide association studies, promotes the ubiquitination and degradation of LXR. Chow-fed mice lacking T39 (T39(-/-)) display increased high-density lipoprotein cholesterol levels associated with increased enterocyte ATP-binding cassette transporter A1 (Abca1) expression and increased LXR protein without change in LXR messenger RNA. When challenged with a high fat/high cholesterol/bile salt diet, T39(-/-) mice or mice with hepatocyte-specific T39 deficiency show increased hepatic LXR protein and target gene expression, and unexpectedly protection from steatohepatitis and death. Mice fed a Western-type diet and lacking low-density lipoprotein receptor (Ldlr(-/-)T39(-/-)) show decreased fatty liver, increased high-density lipoprotein, decreased low-density lipoprotein, and reduced atherosclerosis. In addition to increasing hepatic Abcg5/8 expression and limiting dietary cholesterol absorption, T39 deficiency inhibits hepatic sterol regulatory element-binding protein 1 (SREBP-1, ADD1) processing. This is explained by an increase in microsomal phospholipids containing polyunsaturated fatty acids, linked to an LXRα-dependent increase in expression of enzymes mediating phosphatidylcholine biosynthesis and incorporation of polyunsaturated fatty acids into phospholipids. The preservation of endogenous LXR protein activates a beneficial profile of gene expression that promotes cholesterol removal and inhibits lipogenesis. T39 inhibition could be an effective strategy for reducing both steatohepatitis and atherosclerosis.

Parental behavioural traits can be transmitted by non-genetic mechanisms to the offspring. Although trait transmission via sperm has been extensively researched, epidemiological studies indicate the exclusive/prominent maternal transmission of many non-genetic traits. Since maternal conditions impact the offspring during gametogenesis and through fetal/early-postnatal life, the resultant phenotype is likely the aggregate of consecutive germline and somatic effects; a concept that has not been previously studied. Here, we dissected a complex maternally transmitted phenotype, reminiscent of comorbid generalized anxiety/depression, to elementary behaviours/domains and their transmission mechanisms in mice. We show that four anxiety/stress-reactive traits are transmitted via independent iterative-somatic and gametic epigenetic mechanisms across multiple generations. Somatic/gametic transmission alters DNA methylation at enhancers within synaptic genes whose functions can be linked to the behavioural traits. Traits have generation-dependent penetrance and sex specificity resulting in pleiotropy. A transmission-pathway-based concept can refine current inheritance models of psychiatric diseases and facilitate the development of better animal models and new therapeutic approaches.

STARD4, a member of the evolutionarily conserved START gene family, is a soluble sterol transport protein implicated in cholesterol sensing and maintenance of cellular homeostasis. STARD4 is widely expressed and has been shown to transfer sterol between liposomes as well as organelles in cells. However, STARD4 knockout mice lack an obvious phenotype, so the overall role of STARD4 is unclear. To model long term depletion of STARD4 in cells, we use short hairpin RNA technology to stably decrease STARD4 expression in human U2OS osteosarcoma cells (STARD4-KD). We show that STARD4-KD cells display increased total cholesterol, slower cholesterol trafficking between the plasma membrane and the endocytic recycling compartment, and increased plasma membrane fluidity. These effects can all be rescued by transient expression of a short hairpin RNA-resistant STARD4 construct. Some of the cholesterol increase was due to excess storage in late endosomes or lysosomes. To understand the effects of reduced STARD4, we carried out transcriptional and lipidomic profiling of control and STARD4-KD cells. Reduction of STARD4 activates compensatory mechanisms that alter membrane composition and lipid homeostasis. Based on these observations, we propose that STARD4 functions as a critical sterol transport protein involved in sterol sensing and maintaining lipid homeostasis.