The pleiotropic roles of autophagy in Alzheimer's disease: From pathophysiology to therapy.

Autophagy is a lysosomal degradation pathway and the main clearance route of many toxic protein aggregates. The molecular pathology of Alzheimer's disease (AD) manifests in the form of protein aggregates-extracellular amyloid-ß depositions and intracellular tau neurofibrillary tangles. Perturbations at different steps of the autophagy pathway observed in cellular and animal models of AD might contribute to amyloid-ß and tau accumulation. Increased levels of autophagosomes detected in patients' brains suggest an alteration of autophagy in human disease. Autophagy is also involved in the fine-tuning of inflammation, which increases in the early stages of AD and possibly drives its pathogenesis. Mounting evidence of a causal link between impaired autophagy and AD pathology uncovers an exciting opportunity for the development of autophagy-based therapeutics.

An Erg11 lanosterol 14-α-demethylase-Arv1 complex is required for Candida albicans virulence.

Azole resistant fungal infections remain a health problem for the immune compromised. Current therapies are limited due to rises in new resistance mechanisms. Therefore, it is important to identify new drug targets for drug discovery and novel therapeutics. Arv1 (are1 are2 required for viability 1) function is highly conserved between multiple pathogenic fungal species. Candida albicans (C. albicans) cells lacking CaArv1 are azole hypersusceptible and lack virulence. Saccharomyces cerevisiae (S. cerevisiae) Scarv1 cells are also azole hypersusceptible, a phenotype reversed by expression of CaArv1, indicating conservation in the molecular mechanism for azole susceptibility. To define the relationship between Arv1 function and azole susceptibility, we undertook a structure/function analysis of ScArv1. We identified several conserved amino acids within the ScArv1 homology domain (ScAhd) required for maintaining normal azole susceptibility. Erg11 lanosterol 14-α-demethylase is the rate-limiting enzyme in sterol biosynthesis and is the direct target of azole antifungals, so we used our ScArv1 mutants in order to explore the relationship between ScArv1 and ScErg11. Specific ScArv1 mutants ectopically expressed from a low copy plasmid were unable to restore normal azole susceptibility to Scarv1 cells and had reduced Erg11 protein levels. Erg11 protein stability depended on its ability to form a heterodimeric complex with Arv1. Complex formation was required for maintaining normal azole susceptibility. Scarv1 cells expressing orthologous CaArv1 mutants also had reduced CaErg11 levels, were unable to form a CaArv1-CaErg11 complex, and were azole hypersusceptible. Scarv1 cells expressing CaArv1 mutants unable to interact with CaErg11 could not sustain proper levels of the azole resistant CaErg11Y132F F145L protein. Caarv1/Caarv1 cells expressing CaArv1 mutants unable to interact with CaErg11 were found to lack virulence using a disseminated candidiasis mouse model. Expressing CaErg11Y132F F145L did not reverse the lack of virulence. We hypothesize that the role of Arv1 in Erg11-dependent azole resistance is to stabilize Erg11 protein level. Arv1 inhibition may represent an avenue for treating azole resistance.

Function of lipid droplet-organelle interactions in lipid homeostasis.

Storage of non-polar lipids in ubiquitous eukaryotic organelles, lipid droplets (LDs), prevents the toxic consequences of unesterified fatty acids and provides a lipid reservoir that can be promptly used to satisfy cellular needs under multiple metabolic and physiological conditions. Tight temporal and spatial control of LD biogenesis and mobilization of neutral lipids is essential for the correct channelling of lipid intermediates to their various cellular destinations and the maintenance of cellular homeostasis. These functions are mediated by multiple interactions between LDs and other intracellular organelles that are required for the delivery of stored lipids. Here we review recent advances in the interactions of LDs with the endoplasmic reticulum (ER), mitochondria and vacuole/lysosome. This article is part of a Special Issue entitled: Membrane Contact Sites edited by Christian Ungermann and Benoit Kornmann.

Spatial distribution of lipid droplets during starvation: Implications for lipophagy.

Survival during starvation depends largely on metabolic energy, which is stored in the form of neutral lipids in specialized organelles known as lipid droplets. The precursors for the synthesis of neutral lipids are also used for membrane biogenesis, which is required for cell growth and proliferation. Therefore cells must possess mechanisms to preferentially channel lipid precursors toward either membrane synthesis or lipid droplet storage, in response to nutrient status. How this partitioning is spatially regulated within the endoplasmic reticulum (ER) where lipid droplets co-localize, remains poorly understood. We have recently shown that at the onset of starvation lipid droplets concentrate at a perinuclear ER subdomain flanking the nucleus-vacuole junction (NVJ) and that this is crucial for maintaining proper nuclear shape and ER membrane organization. Here we show that disruption of the NVJ does not block the translocation and internalization of lipid droplets into the vacuole for their degradation, which takes place at later stages of starvation. We propose that alternative pathways of lipid droplet translocation from the ER to the vacuole may exist to enable stationary phase-induced lipophagy.

The ceramide-activated protein phosphatase Sit4p controls lifespan, mitochondrial function and cell cycle progression by regulating hexokinase 2 phosphorylation.

Sit4p is the catalytic subunit of a ceramide-activated PP2A-like phosphatase that regulates cell cycle, mitochondrial function, oxidative stress resistance and chronological lifespan in yeast. In this study, we show that hexokinase 2 (Hxk2p) is hyperphosphorylated in sit4Δ mutants grown in glucose medium by a Snf1p-independent mechanism and Hxk2p-S15A mutation suppresses phenotypes associated with SIT4 deletion, namely growth arrest at G1 phase, derepression of mitochondrial respiration, H2O2 resistance and lifespan extension. Consistently, the activation of Sit4p in isc1Δ mutants, which has been associated with premature aging, leads to Hxk2p hypophosphorylation, and the expression of Hxk2p-S15E increases the lifespan of isc1Δ cells. The overall results suggest that Hxk2p functions downstream of Sit4p in the control of cell cycle, mitochondrial function, oxidative stress resistance and chronological lifespan.

Lipid partitioning at the nuclear envelope controls membrane biogenesis.

Partitioning of lipid precursors between membranes and storage is crucial for cell growth, and its disruption underlies pathologies such as cancer, obesity, and type 2 diabetes. However, the mechanisms and signals that regulate this process are largely unknown. In yeast, lipid precursors are mainly used for phospholipid synthesis in nutrient-rich conditions in order to sustain rapid proliferation but are redirected to triacylglycerol (TAG) stored in lipid droplets during starvation. Here we investigate how cells reprogram lipid metabolism in the endoplasmic reticulum. We show that the conserved phosphatidate (PA) phosphatase Pah1, which generates diacylglycerol from PA, targets a nuclear membrane subdomain that is in contact with growing lipid droplets and mediates TAG synthesis. We find that cytosol acidification activates the master regulator of Pah1, the Nem1-Spo7 complex, thus linking Pah1 activity to cellular metabolic status. In the absence of TAG storage capacity, Pah1 still binds the nuclear membrane, but lipid precursors are redirected toward phospholipids, resulting in nuclear deformation and a proliferation of endoplasmic reticulum membrane. We propose that, in response to growth signals, activation of Pah1 at the nuclear envelope acts as a switch to control the balance between membrane biogenesis and lipid storage.

Lipid droplet-organelle interactions: emerging roles in lipid metabolism.

Cellular homeostasis depends on the precisely coordinated use of lipids as fuels for energy production, building blocks for membrane biogenesis or chemical signals for intra-cellular and inter-cellular communication. Lipid droplets (LDs) are universally conserved dynamic organelles that can store and mobilize fatty acids and other lipid species for their multiple cellular roles. Increasing evidence suggests that contact zones between LDs and other organelles play important roles in the trafficking of lipids and in the regulation of lipid metabolism. Here we review recent advances regarding the nature and functional relevance of interactions between LDs and other organelles-particularly the endoplasmic reticulum (ER), LDs, mitochondria and vacuoles-that highlight their importance for lipid metabolism.

Transcription factor Reb1p regulates DGK1-encoded diacylglycerol kinase and lipid metabolism in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the DGK1-encoded diacylglycerol kinase catalyzes the CTP-dependent phosphorylation of diacylglycerol to form phosphatidate. This enzyme, in conjunction with PAH1-encoded phosphatidate phosphatase, controls the levels of phosphatidate and diacylglycerol for phospholipid synthesis, membrane growth, and lipid droplet formation. In this work, we showed that a functional level of diacylglycerol kinase is regulated by the Reb1p transcription factor. In the electrophoretic mobility shift assay, purified recombinant Reb1p was shown to specifically bind its consensus recognition sequence (CGGGTAA, -166 to -160) in the DGK1 promoter. Analysis of cells expressing the PDGK1-lacZ reporter gene showed that mutations (GT→TG) in the Reb1p-binding sequence caused an 8.6-fold reduction in ß-galactosidase activity. The expression of DGK1(reb1), a DGK1 allele containing the Reb1p-binding site mutation, was greatly lower than that of the wild type allele, as indicated by analyses of DGK1 mRNA, Dgk1p, and diacylglycerol kinase activity. In the presence of cerulenin, an inhibitor of de novo fatty acid synthesis, the dgk1Δ mutant expressing DGK1(reb1) exhibited a significant defect in growth as well as in the synthesis of phospholipids from triacylglycerol mobilization. Unlike DGK1, the DGK1(reb1) expressed in the dgk1Δ pah1Δ mutant did not result in the nuclear/endoplasmic reticulum membrane expansion, which occurs in cells lacking phosphatidate phosphatase activity. Taken together, these results indicate that the Reb1p-mediated regulation of diacylglycerol kinase plays a major role in its in vivo functions in lipid metabolism.

Regulation of lipid droplet and membrane biogenesis by the acidic tail of the phosphatidate phosphatase Pah1p.

Lipins are evolutionarily conserved phosphatidate phosphatases that perform key functions in phospholipid, triglyceride, and membrane biogenesis. Translocation of lipins on membranes requires their dephosphorylation by the Nem1p-Spo7p transmembrane phosphatase complex through a poorly understood mechanism. Here we identify the carboxy-terminal acidic tail of the yeast lipin Pah1p as an important regulator of this step. Deletion or mutations of the tail disrupt binding of Pah1p to the Nem1p-Spo7p complex and Pah1p membrane translocation. Overexpression of Nem1p-Spo7p drives the recruitment of Pah1p in the vicinity of lipid droplets in an acidic tail-dependent manner and induces lipid droplet biogenesis. Genetic analysis shows that the acidic tail is essential for the Nem1p-Spo7p-dependent activation of Pah1p but not for the function of Pah1p itself once it is dephosphorylated. Loss of the tail disrupts nuclear structure, INO1 gene expression, and triglyceride synthesis. Similar acidic sequences are present in the carboxy-terminal ends of all yeast lipin orthologues. We propose that acidic tail-dependent binding and dephosphorylation of Pah1p by the Nem1p-Spo7p complex is an important determinant of its function in lipid and membrane biogenesis.

Activation of the Hog1p kinase in Isc1p-deficient yeast cells is associated with mitochondrial dysfunction, oxidative stress sensitivity and premature aging.

The Saccharomyces cerevisiae Isc1p, an orthologue of mammalian neutral sphingomyelinase 2, plays a key role in mitochondrial function, oxidative stress resistance and chronological lifespan. Isc1p functions upstream of the ceramide-activated protein phosphatase Sit4p through the modulation of ceramide levels. Here, we show that both ceramide and loss of Isc1p lead to the activation of Hog1p, the MAPK of the high osmolarity glycerol (HOG) pathway that is functionally related to mammalian p38 and JNK. The hydrogen peroxide sensitivity and premature aging of isc1Δ cells was partially suppressed by HOG1 deletion. Notably, Hog1p activation mediated the mitochondrial dysfunction and catalase A deficiency associated with oxidative stress sensitivity and premature aging of isc1Δ cells. Downstream of Hog1p, Isc1p deficiency activated the cell wall integrity (CWI) pathway. Deletion of the SLT2 gene, which encodes for the MAPK of the CWI pathway, was lethal in isc1Δ cells and this mutant strain was hypersensitive to cell wall stress. However, the phenotypes of isc1Δ cells were not associated with cell wall defects. Our findings support a role for Hog1p in the regulation of mitochondrial function and suggest that constitutive activation of Hog1p is deleterious for isc1Δ cells under oxidative stress conditions and during chronological aging.

Role for Sit4p-dependent mitochondrial dysfunction in mediating the shortened chronological lifespan and oxidative stress sensitivity of Isc1p-deficient cells.

Saccharomyces cerevisiae cells lacking Isc1p, an orthologue of mammalian neutral sphingomyelinase 2, display a shortened lifespan and an increased sensitivity to oxidative stress. A lipidomic analysis revealed specific changes in sphingolipids that accompanied the premature ageing of Isc1p-deficient cells under severe calorie restriction conditions, including a decrease of dihydrosphingosine levels and an increase of dihydro-C(26) -ceramide and phyto-C(26) -ceramide levels, the latter raising the possibility of activation of ceramide-dependent protein phosphatases. Consequently, deletion of the SIT4 gene, which encodes for the catalytic subunit of type 2A ceramide-activated protein phosphatase in yeast, abolished the premature ageing and hydrogen peroxide sensitivity of isc1Δ cells. SIT4 deletion also abolished the respiratory defects and catalase A deficiency exhibited by isc1Δ mutants. These results are consistent with catabolic derepression associated with the loss of Sit4p. The overall results show that Isc1p is an upstream regulator of Sit4p and implicate Sit4p activation in mitochondrial dysfunction leading to the shortened chronological lifespan and oxidative stress sensitivity of isc1Δ mutants.