Lipid droplets (LDs) are the major fat storage organelles in eukaryotic cells, but how their size is regulated is unknown. Using genetic screens in C. elegans for LD morphology defects in intestinal cells, we found that mutations in atlastin, a GTPase required for homotypic fusion of endoplasmic reticulum (ER) membranes, cause not only ER morphology defects, but also a reduction in LD size. Similar results were obtained after depletion of atlastin or expression of a dominant-negative mutant, whereas overexpression of atlastin had the opposite effect. Atlastin depletion in Drosophila fat bodies also reduced LD size and decreased triglycerides in whole animals, sensitizing them to starvation. In mammalian cells, co-overexpression of atlastin-1 and REEP1, a paralog of the ER tubule-shaping protein DP1/REEP5, generates large LDs. The effect of atlastin-1 on LD size correlates with its activity to promote membrane fusion in vitro. Our results indicate that atlastin-mediated fusion of ER membranes is important for LD size regulation.

Frontotemporal dementia (FTD) is the most common cause of dementia in people under 60 yr of age and is pathologically associated with mislocalization of TAR DNA/RNA binding protein 43 (TDP-43) in approximately half of cases (FLTD-TDP). Mutations in the gene encoding progranulin (GRN), which lead to reduced progranulin levels, are a significant cause of familial FTLD-TDP. Grn-KO mice were developed as an FTLD model, but lack cortical TDP-43 mislocalization and neurodegeneration. Here, we report retinal thinning as an early disease phenotype in humans with GRN mutations that precedes dementia onset and an age-dependent retinal neurodegenerative phenotype in Grn-KO mice. Retinal neuron loss in Grn-KO mice is preceded by nuclear depletion of TDP-43 and accompanied by reduced expression of the small GTPase Ran, which is a master regulator of nuclear import required for nuclear localization of TDP-43. In addition, TDP-43 regulates Ran expression, likely via binding to its 3'-UTR. Augmented expression of Ran in progranulin-deficient neurons restores nuclear TDP-43 levels and improves their survival. Our findings establish retinal neurodegeneration as a new phenotype in progranulin-deficient FTLD, and suggest a pathological loop involving reciprocal loss of Ran and nuclear TDP-43 as an underlying mechanism.

Metabolic fingerprinting provides valuable information on the physiopathological states of cells and tissues. Traditional imaging mass spectrometry and magnetic resonance imaging are unable to probe the spatial-temporal dynamics of metabolites at the subcellular level due to either lack of spatial resolution or inability to perform live cell imaging. Here we report a complementary metabolic imaging technique that is based on hyperspectral stimulated Raman scattering (hsSRS). We demonstrated the use of hsSRS imaging in quantifying two major neutral lipids: cholesteryl ester and triacylglycerol in cells and tissues. Our imaging results revealed previously unknown changes of lipid composition associated with obesity and steatohepatitis. We further used stable-isotope labeling to trace the metabolic dynamics of fatty acids in live cells and live Caenorhabditis elegans with hsSRS imaging. We found that unsaturated fatty acid has preferential uptake into lipid storage while saturated fatty acid exhibits toxicity in hepatic cells. Simultaneous metabolic fingerprinting of deuterium-labeled saturated and unsaturated fatty acids in living C. elegans revealed that there is a lack of interaction between the two, unlike previously hypothesized. Our findings provide new approaches for metabolic tracing of neutral lipids and their precursors in living cells and organisms, and could potentially serve as a general approach for metabolic fingerprinting of other metabolites.

Variations in the gene LDAH (C2ORF43), which encodes lipid droplet-associated hydrolase (LDAH), are among few loci associated with human prostate cancer. Homologs of LDAH have been identified as proteins of lipid droplets (LDs). LDs are cellular organelles that store neutral lipids, such as triacylglycerols and sterol esters, as precursors for membrane components and as reservoirs of metabolic energy. LDAH is reported to hydrolyze cholesterol esters and to be important in macrophage cholesterol ester metabolism. Here, we confirm that LDAH is localized to LDs in several model systems. We generated a murine model in which Ldah is disrupted but found no evidence for a major function of LDAH in cholesterol ester or triacylglycerol metabolism in vivo, nor a role in energy or glucose metabolism. Our data suggest that LDAH is not a major cholesterol ester hydrolase, and an alternative metabolic function may be responsible for its possible effect on development of prostate cancer.

Exercise increases skeletal muscle glucose uptake, but the underlying mechanisms are only partially understood. The atypical protein kinase C (PKC) isoforms λ and ζ (PKC-λ/ζ) have been shown to be necessary for insulin-, AICAR-, and metformin-stimulated glucose uptake in skeletal muscle, but not for treadmill exercise-stimulated muscle glucose uptake. To investigate if PKC-λ/ζ activity is required for contraction-stimulated muscle glucose uptake, we used mice with tibialis anterior muscle-specific overexpression of an empty vector (WT), wild-type PKC-ζ (PKC-ζ(WT)), or an enzymatically inactive T410A-PKC-ζ mutant (PKC-ζ(T410A)). We also studied skeletal muscle-specific PKC-λ knockout (MλKO) mice. Basal glucose uptake was similar between WT, PKC-ζ(WT), and PKC-ζ(T410A) tibialis anterior muscles. In contrast, in situ contraction-stimulated glucose uptake was increased in PKC-ζ(T410A) tibialis anterior muscles compared to WT or PKC-ζ(WT) tibialis anterior muscles. Furthermore, in vitro contraction-stimulated glucose uptake was greater in soleus muscles of MλKO mice than WT controls. Thus, loss of PKC-λ/ζ activity increases contraction-stimulated muscle glucose uptake. These data clearly demonstrate that PKC-λζ activity is not necessary for contraction-stimulated glucose uptake.

Sphingolipids are abundant membrane components and important signaling molecules in eukaryotic cells. Their levels and localization are tightly regulated. However, the mechanisms underlying this regulation remain largely unknown. In this study, we identify the Golgi-associated retrograde protein (GARP) complex, which functions in endosome-to-Golgi retrograde vesicular transport, as a critical player in sphingolipid homeostasis. GARP deficiency leads to accumulation of sphingolipid synthesis intermediates, changes in sterol distribution, and lysosomal dysfunction. A GARP complex mutation analogous to a VPS53 allele causing progressive cerebello-cerebral atrophy type 2 (PCCA2) in humans exhibits similar, albeit weaker, phenotypes in yeast, providing mechanistic insights into disease pathogenesis. Inhibition of the first step of de novo sphingolipid synthesis is sufficient to mitigate many of the phenotypes of GARP-deficient yeast or mammalian cells. Together, these data show that GARP is essential for cellular sphingolipid homeostasis and suggest a therapeutic strategy for the treatment of PCCA2.

Calorie restriction results in leanness, which is linked to metabolic conditions that favor longevity. We show here that deficiency of the triglyceride synthesis enzyme acyl CoA:diacylglycerol acyltransferase 1 (DGAT1), which promotes leanness, also extends longevity without limiting food intake. Female DGAT1-deficient mice were protected from age-related increases in body fat, tissue triglycerides, and inflammation in white adipose tissue. This protection was accompanied by increased mean and maximal life spans of ~25% and ~10%, respectively. Middle-agedDgat1-/- mice exhibited several features associated with longevity, including decreased levels of circulating insulin growth factor 1 (IGF1) and reduced fecundity. Thus, deletion of DGAT1 in mice provides a model of leanness and extended lifespan that is independent of calorie restriction.

Acyl-CoA:diacylglycerol acyltransferase (DGAT)1 and DGAT2 catalyze triglyceride (TG) biosynthesis in humans. Biallelic loss-of-function mutations in human DGAT1 result in severe congenital diarrhea and protein-losing enteropathy. Additionally, pharmacologic inhibition of DGAT1 led to dose-related diarrhea in human clinical trials. Here we identify a previously unknown DGAT1 mutation in identical twins of South Asian descent. These male patients developed watery diarrhea shortly after birth, with protein-losing enteropathy and failure to thrive. Exome sequencing revealed a homozygous recessive mutation in DGAT1, c.314T>C, p.L105P. We show here that the p.L105P DGAT1 enzyme produced from the mutant allele is less abundant, resulting in partial loss of TG synthesis activity and decreased formation of lipid droplets in patient-derived primary dermal fibroblasts. Thus, in contrast with complete loss-of-function alleles of DGAT1, the p.L105P missense allele partially reduces TG synthesis activity and causes a less severe clinical phenotype. Our findings add to the growing recognition of DGAT1 deficiency as a cause of congenital diarrhea with protein-losing enteropathy and indicate that DGAT1 mutations result in a spectrum of diseases.

Pathways localizing proteins to their sites of action are essential for eukaryotic cell organization and function. Although mechanisms of protein targeting to many organelles have been defined, how proteins, such as metabolic enzymes, target from the endoplasmic reticulum (ER) to cellular lipid droplets (LDs) is poorly understood. Here we identify two distinct pathways for ER-to-LD protein targeting: early targeting at LD formation sites during formation, and late targeting to mature LDs after their formation. Using systematic, unbiased approaches in Drosophila cells, we identified specific membrane-fusion machinery, including regulators, a tether and SNARE proteins, that are required for the late targeting pathway. Components of this fusion machinery localize to LD-ER interfaces and organize at ER exit sites. We identified multiple cargoes for early and late ER-to-LD targeting pathways. Our findings provide a model for how proteins target to LDs from the ER either during LD formation or by protein-catalysed formation of membrane bridges.

Mammals store metabolic energy as triacylglycerols (TGs) in adipose tissue. TG synthesis is catalyzed by the evolutionarily unrelated acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes DGAT1 and DGAT2, which catalyze the same reaction and account for nearly all TG synthesis. The reasons for their convergent evolution to synthesize TGs remain unclear. Mice lacking DGAT1 are viable with reduced fat stores of TGs, whereas DGAT2 KO mice die postnatally just after birth with >90% reduction of TGs, suggesting that DGAT2 is the predominant enzyme for TG storage. To better understand the functional differences between the DGATs, we studied mice fed chow or high-fat diets lacking either enzyme in adipose tissue. Unexpectedly, mice lacking DGAT2 in adipocytes have normal TG storage and glucose metabolism on regular or high-fat diets, indicating DGAT2 is not essential for fat storage. In contrast, mice lacking DGAT1 in adipocytes have normal TG storage on a chow diet but moderately decreased body fat accompanied by glucose intolerance when challenged with a high-fat diet. The latter changes were associated with the activation of ER stress pathways. We conclude that DGAT1 and DGAT2 can largely compensate for each other for TG storage but that DGAT1 uniquely has an important role in protecting the ER from the lipotoxic effects of high-fat diets.

At the subcellular level, fat storage is confined to the evolutionarily conserved compartments termed lipid droplets (LDs), which are closely associated with the endoplasmic reticulum (ER). However, the molecular mechanisms that enable ER-LD interaction and facilitate neutral lipid loading into LDs are poorly understood. In this paper, we present evidence that FATP1/acyl-CoA synthetase and DGAT2/diacylglycerol acyltransferase are components of a triglyceride synthesis complex that facilitates LD expansion. A loss of FATP1 or DGAT2 function blocked LD expansion in Caenorhabditis elegans. FATP1 preferentially associated with DGAT2, and they acted synergistically to promote LD expansion in mammalian cells. Live imaging indicated that FATP1 and DGAT2 are ER and LD resident proteins, respectively, and electron microscopy revealed FATP1 and DGAT2 foci close to the LD surface. Furthermore, DGAT2 that was retained in the ER failed to support LD expansion. We propose that the evolutionarily conserved FATP1-DGAT2 complex acts at the ER-LD interface and couples the synthesis and deposition of triglycerides into LDs both physically and functionally.

Metabolic energy is stored in cells primarily as triacylglycerols in lipid droplets (LDs), and LD dysregulation leads to metabolic diseases. The formation of monolayer-bound LDs from the endoplasmic reticulum (ER) bilayer is poorly understood, but the ER protein seipin is essential to this process. In this study, we report a cryo-electron microscopy structure and functional characterization of Drosophila melanogaster seipin. The structure reveals a ring-shaped dodecamer with the luminal domain of each monomer resolved at ∼4.0 Å. Each luminal domain monomer exhibits two distinctive features: a hydrophobic helix (HH) positioned toward the ER bilayer and a β-sandwich domain with structural similarity to lipid-binding proteins. This structure and our functional testing in cells suggest a model in which seipin oligomers initially detect forming LDs in the ER via HHs and subsequently act as membrane anchors to enable lipid transfer and LD growth.

PKMζ is a persistently active PKC isoform proposed to maintain late-LTP and long-term memory. But late-LTP and memory are maintained without PKMζ in PKMζ-null mice. Two hypotheses can account for these findings. First, PKMζ is unimportant for LTP or memory. Second, PKMζ is essential for late-LTP and long-term memory in wild-type mice, and PKMζ-null mice recruit compensatory mechanisms. We find that whereas PKMζ persistently increases in LTP maintenance in wild-type mice, PKCι/λ, a gene-product closely related to PKMζ, persistently increases in LTP maintenance in PKMζ-null mice. Using a pharmacogenetic approach, we find PKMζ-antisense in hippocampus blocks late-LTP and spatial long-term memory in wild-type mice, but not in PKMζ-null mice without the target mRNA. Conversely, a PKCι/λ-antagonist disrupts late-LTP and spatial memory in PKMζ-null mice but not in wild-type mice. Thus, whereas PKMζ is essential for wild-type LTP and long-term memory, persistent PKCι/λ activation compensates for PKMζ loss in PKMζ-null mice.

The pathogenic mechanisms of frontotemporal dementia (FTD) remain poorly understood. Here we generated multiple induced pluripotent stem cell lines from a control subject, a patient with sporadic FTD, and an FTD patient with a novel heterozygous GRN mutation (progranulin [PGRN] S116X). In neurons and microglia differentiated from PGRN S116X induced pluripotent stem cells, the levels of intracellular and secreted PGRN were reduced, establishing patient-specific cellular models of PGRN haploinsufficiency. Through a systematic screen of inducers of cellular stress, we found that PGRN S116X neurons, but not sporadic FTD neurons, exhibited increased sensitivity to staurosporine and other kinase inhibitors. Moreover, the serine/threonine kinase S6K2, a component of the phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways, was specifically downregulated in PGRN S116X neurons. Both increased sensitivity to kinase inhibitors and reduced S6K2 were rescued by PGRN expression. Our findings identify cell-autonomous, reversible defects in patient neurons with PGRN deficiency, and provide a compelling model for studying PGRN-dependent pathogenic mechanisms and testing potential therapies.

Homologs of the Saccharomyces cerevisiae Sir2 protein, sirtuins, promote longevity in many organisms. Studies of the sirtuin SIRT3 have so far been limited to cell culture systems. Here, we investigate the localization and function of SIRT3 in vivo. We show that endogenous mouse SIRT3 is a soluble mitochondrial protein. To address the function and relevance of SIRT3 in the regulation of energy metabolism, we generated and phenotypically characterized SIRT3 knockout mice. SIRT3-deficient animals exhibit striking mitochondrial protein hyperacetylation, suggesting that SIRT3 is a major mitochondrial deacetylase. In contrast, no mitochondrial hyperacetylation was detectable in mice lacking the two other mitochondrial sirtuins, SIRT4 and SIRT5. Surprisingly, despite this biochemical phenotype, SIRT3-deficient mice are metabolically unremarkable under basal conditions and show normal adaptive thermogenesis, a process previously suggested to involve SIRT3. Overall, our results extend the recent finding of lysine acetylation of mitochondrial proteins and demonstrate that SIRT3 has evolved to control reversible lysine acetylation in this organelle.

How proteins control the biogenesis of cellular lipid droplets (LDs) is poorly understood. Using Drosophila and human cells, we show here that seipin, an ER protein implicated in LD biology, mediates a discrete step in LD formation-the conversion of small, nascent LDs to larger, mature LDs. Seipin forms discrete and dynamic foci in the ER that interact with nascent LDs to enable their growth. In the absence of seipin, numerous small, nascent LDs accumulate near the ER and most often fail to grow. Those that do grow prematurely acquire lipid synthesis enzymes and undergo expansion, eventually leading to the giant LDs characteristic of seipin deficiency. Our studies identify a discrete step of LD formation, namely the conversion of nascent LDs to mature LDs, and define a molecular role for seipin in this process, most likely by acting at ER-LD contact sites to enable lipid transfer to nascent LDs.

Lipid droplets (LDs) form in the endoplasmic reticulum by phase separation of neutral lipids. This process is facilitated by the seipin protein complex, which consists of a ring of seipin monomers, with a yet unclear function. Here, we report a structure of S. cerevisiae seipin based on cryogenic-electron microscopy and structural modeling data. Seipin forms a decameric, cage-like structure with the lumenal domains forming a stable ring at the cage floor and transmembrane segments forming the cage sides and top. The transmembrane segments interact with adjacent monomers in two distinct, alternating conformations. These conformations result from changes in switch regions, located between the lumenal domains and the transmembrane segments, that are required for seipin function. Our data indicate a model for LD formation in which a closed seipin cage enables triacylglycerol phase separation and subsequently switches to an open conformation to allow LD growth and budding.

Lipid droplets (LDs) are organelles of cellular lipid storage with fundamental roles in energy metabolism and cell membrane homeostasis. There has been an explosion of research into the biology of LDs, in part due to their relevance in diseases of lipid storage, such as atherosclerosis, obesity, type 2 diabetes, and hepatic steatosis. Consequently, there is an increasing need for a resource that combines datasets from systematic analyses of LD biology. Here, we integrate high-confidence, systematically generated human, mouse, and fly data from studies on LDs in the framework of an online platform named the "Lipid Droplet Knowledge Portal" (https://lipiddroplet.org/). This scalable and interactive portal includes comprehensive datasets, across a variety of cell types, for LD biology, including transcriptional profiles of induced lipid storage, organellar proteomics, genome-wide screen phenotypes, and ties to human genetics. This resource is a powerful platform that can be utilized to identify determinants of lipid storage.

Numerous mutations that impair retrograde membrane trafficking between endosomes and the Golgi apparatus lead to neurodegenerative diseases. For example, mutations in the endosomal retromer complex are implicated in Alzheimer's and Parkinson's diseases, and mutations of the Golgi-associated retrograde protein (GARP) complex cause progressive cerebello-cerebral atrophy type 2 (PCCA2). However, how these mutations cause neurodegeneration is unknown. GARP mutations in yeast, including one causing PCCA2, result in sphingolipid abnormalities and impaired cell growth that are corrected by treatment with myriocin, a sphingolipid synthesis inhibitor, suggesting that alterations in sphingolipid metabolism contribute to cell dysfunction and death. Here we tested this hypothesis in wobbler mice, a murine model with a homozygous partial loss-of-function mutation in Vps54 (GARP protein) that causes motor neuron disease. Cytotoxic sphingoid long-chain bases accumulated in embryonic fibroblasts and spinal cords from wobbler mice. Remarkably, chronic treatment of wobbler mice with myriocin markedly improved their wellness scores, grip strength, neuropathology, and survival. Proteomic analyses of wobbler fibroblasts revealed extensive missorting of lysosomal proteins, including sphingolipid catabolism enzymes, to the Golgi compartment, which may contribute to the sphingolipid abnormalities. Our findings establish that altered sphingolipid metabolism due to GARP mutations contributes to neurodegeneration and suggest that inhibiting sphingolipid synthesis might provide a useful strategy for treating these disorders.

Haploinsufficiency of GRN causes frontotemporal dementia (FTD). The GRN locus produces progranulin (PGRN), which is cleaved to lysosomal granulin polypeptides. The function of lysosomal granulins and why their absence causes neurodegeneration are unclear. Here we discover that PGRN-deficient human cells and murine brains, as well as human frontal lobes from GRN-mutation FTD patients have increased levels of gangliosides, glycosphingolipids that contain sialic acid. In these cells and tissues, levels of lysosomal enzymes that catabolize gangliosides were normal, but levels of bis(monoacylglycero)phosphates (BMP), lipids required for ganglioside catabolism, were reduced with PGRN deficiency. Our findings indicate that granulins are required to maintain BMP levels to support ganglioside catabolism, and that PGRN deficiency in lysosomes leads to gangliosidosis. Lysosomal ganglioside accumulation may contribute to neuroinflammation and neurodegeneration susceptibility observed in FTD due to PGRN deficiency and other neurodegenerative diseases.