Lipase regulation of cellular fatty acid homeostasis as a Parkinson's disease therapeutic strategy.

Synucleinopathy (Parkinson's disease (PD); Lewy body dementia) disease-modifying treatments represent a huge unmet medical need. Although the PD-causing protein α-synuclein (αS) interacts with lipids and fatty acids (FA) physiologically and pathologically, targeting FA homeostasis for therapeutics is in its infancy. We identified the PD-relevant target stearoyl-coA desaturase: inhibiting monounsaturated FA synthesis reversed PD phenotypes. However, lipid degradation also generates FA pools. Here, we identify the rate-limiting lipase enzyme, LIPE, as a candidate target. Decreasing LIPE in human neural cells reduced αS inclusions. Patient αS triplication vs. corrected neurons had increased pSer129 and insoluble αS and decreased αS tetramer:monomer ratios. LIPE inhibition rescued all these and the abnormal unfolded protein response. LIPE inhibitors decreased pSer129 and restored tetramer:monomer equilibrium in αS E46K-expressing human neurons. LIPE reduction in vivo alleviated αS-induced dopaminergic neurodegeneration in Caenorhabditis elegans. Co-regulating FA synthesis and degradation proved additive in rescuing PD phenotypes, signifying co-targeting as a therapeutic strategy.

A Brain-Penetrant Stearoyl-CoA Desaturase Inhibitor Reverses α-Synuclein Toxicity.

Increasing evidence has shown that Parkinson's disease (PD) impairs midbrain dopaminergic, cortical and other neuronal subtypes in large part due to the build-up of lipid- and vesicle-rich α-synuclein (αSyn) cytotoxic inclusions. We previously identified stearoyl-CoA desaturase (SCD) as a potential therapeutic target for synucleinopathies. A brain-penetrant SCD inhibitor, YTX-7739, was developed and has entered Phase 1 clinical trials. Here, we report the efficacy of YTX-7739 in reversing pathological αSyn phenotypes in various in vitro and in vivo PD models. In cell-based assays, YTX-7739 decreased αSyn-mediated neuronal death, reversed the abnormal membrane interaction of amplified E46K ("3K") αSyn, and prevented pathological phenotypes in A53T and αSyn triplication patient-derived neurospheres, including dysregulated fatty acid profiles and pS129 αSyn accumulation. In 3K PD-like mice, YTX-7739 crossed the blood-brain barrier, decreased unsaturated fatty acids, and prevented progressive motor deficits. Both YTX-7739 treatment and decreasing SCD activity through deletion of one copy of the SCD1 gene (SKO) restored the physiological αSyn tetramer-to-monomer ratio, dopaminergic integrity, and neuronal survival in 3K αSyn mice. YTX-7739 efficiently reduced pS129 + and PK-resistant αSyn in both human wild-type αSyn and 3K mutant mice similar to the level of 3K-SKO. Together, these data provide further validation of SCD as a PD therapeutic target and YTX-7739 as a clinical candidate for treating human α-synucleinopathies.

Pathogenic Mechanisms of Cytosolic and Membrane-Enriched α-Synuclein Converge on Fatty Acid Homeostasis.

α-Synuclein (αS) plays a key role in Parkinson's disease. Although Parkinson's disease is typically "sporadic," inherited αS missense mutations provide crucial insights into molecular mechanisms. Here, we examine two clinical mutants, E46K and G51D, which are both in the conserved N-terminus that mediates transient αS-membrane interactions. However, E46K increases and G51D decreases αS-membrane interactions. Previously, we amplified E46K via the 11-residue repeat motifs, creating "3K" (E35K+E46K+E61K). Here, we engineered these motifs to amplify G51D (V40D+G51D+V66D = "3D") and systematically compared E46K/3K versus G51D/3D. We found that G51D increased cytosolic αS in neural cells and 3D aggravates this. G51D, and 3D even more, reduced αS multimer-to-monomer (αS60:αS14) ratio. Both amplified variants caused cellular stress in rat primary neurons and reduced growth in human neuroblastoma cells. Importantly, both 3K- and 3D-induced stress was ameliorated by pharmacologically inhibiting stearoyl-CoA desaturase or by conditioning the cells in palmitic (16:0) or myristic (14:0) acid. SCD inhibition lowered lipid-droplet accumulation in both 3D- and 3K-expressing cells and benefitted G51D by normalizing multimer:monomer ratio, as reported previously for E46K. Our findings suggest that, despite divergent cytosol/membrane partitioning, both G51D and E46K neurotoxicity can be prevented by decreasing fatty-acid unsaturation as a common therapeutic approach.SIGNIFICANCE STATEMENT α-Synuclein (αS) dyshomeostasis is linked to Parkinson's disease. Here we focus on two contrasting familial-Parkinson's disease αS mutants, E46K and G51D, that alter αS membrane association in opposite directions (E46K increases, G51D decreases it). Taking advantage of αS repeat structure, here we designed αS "3D," an amplified G51D variant (V40D+G51D+V66D). αS 3D further enhanced G51D's cytosolic enrichment. Systematic comparison of G51D/3D with membrane-enriched E46K/its amplified variant 3K revealed that both can elicit stress in human neural cells and primary rodent neurons. This toxicity can be ameliorated by inhibiting stearoyl-CoA desaturase or by saturated fatty acid conditioning. Thus, despite divergent membrane binding, both G51D and E46K αS dyshomeostasis are mitigated by altering fatty acid saturation as a shared target.

Excess membrane binding of monomeric alpha-, beta- and gamma-synuclein is invariably associated with inclusion formation and toxicity.

α-Synuclein (αS) has been well-documented to play a role in human synucleinopathies such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB). First, the lesions found in PD/DLB brains-Lewy bodies and Lewy neurites-are rich in aggregated αS. Second, genetic evidence links missense mutations and increased αS expression to familial forms of PD/DLB. Third, toxicity and cellular stress can be caused by αS under certain experimental conditions. In contrast, the homologs ß-synuclein (ßS) and γ-synuclein (γS) are not typically found in Lewy bodies/neurites, have not been clearly linked to brain diseases and have been largely non-toxic in experimental settings. In αS, the so-called non-amyloid-ß component of plaques (NAC) domain, constituting amino acids 61-95, has been identified to be critical for aggregation in vitro. This domain is partially absent in ßS and only incompletely conserved in γS, which could explain why both homologs do not cause disease. However, αS in vitro aggregation and cellular toxicity have not been firmly linked experimentally, and it has been proposed that excess αS membrane binding is sufficient to induce neurotoxicity. Indeed, recent characterizations of Lewy bodies have highlighted the accumulation of lipids and membranous organelles, raising the possibility that ßS and γS could also become neurotoxic if they were more prone to membrane/lipid binding. Here, we increased ßS and γS membrane affinity by strategic point mutations and demonstrate that these proteins behave like membrane-associated monomers, are cytotoxic and form round cytoplasmic inclusions that can be prevented by inhibiting stearoyl-CoA desaturase.

Lipotoxicity Downstream of α-Synuclein Imbalance: A Relevant Pathomechanism in Synucleinopathies?

Neuronal loss in Parkinson's disease and related brain diseases has been firmly linked to the abundant neuronal protein α-synuclein (αS). However, we have gained surprisingly little insight into how exactly αS exerts toxicity in these diseases. Hypotheses of proteotoxicity, disturbed vesicle trafficking, mitochondrial dysfunction and other toxicity mechanisms have been proposed, and it seems possible that a combination of different mechanisms may drive pathology. A toxicity mechanism that has caught increased attention in the recent years is αS-related lipotoxicity. Lipotoxicity typically occurs in a cell when fatty acids exceed the metabolic needs, triggering a flux into harmful pathways of non-oxidative metabolism. Genetic and experimental approaches have revealed a significant overlap between lipid storage disorders, most notably Gaucher's disease, and synucleinopathies. There is accumulating evidence for lipid aberrations causing synuclein misfolding as well as for αS excess and misfolding causing lipid aberration. Does that mean the key problem in synucleinopathies is lipotoxicity, the accumulation of harmful lipid species or alteration in lipid equilibrium? Here, we review the existing literature in an attempt to get closer to an answer.

A Stearoyl-Coenzyme A Desaturase Inhibitor Prevents Multiple Parkinson Disease Phenotypes in α-Synuclein Mice.

OBJECTIVE: Parkinson disease (PD) has useful symptomatic treatments that do not slow the neurodegenerative process, and no significant disease-modifying treatments are approved. A key therapeutic target in PD is α-synuclein (αS), which is both genetically implicated and accumulates in Lewy bodies rich in vesicles and other lipid membranes. Reestablishing αS homeostasis is a central goal in PD. Based on previous lipidomic analyses, we conducted a mouse trial of a stearoyl-coenzyme A desaturase (SCD) inhibitor ("5b") that prevented αS-positive vesicular inclusions and cytotoxicity in cultured human neurons. METHODS: Oral dosing and brain activity of 5b were established in nontransgenic mice. 5b in drinking water was given to mice expressing wild-type human αS (WT) or an amplified familial PD αS mutation (E35K + E46K + E61K ["3K"]) beginning near the onset of nigral and cortical neurodegeneration and the robust PD-like motor syndrome in 3K. Motor phenotypes, brain cytopathology, and SCD-related lipid changes were quantified in 5b- versus placebo-treated mice. Outcomes were compared to effects of crossing 3K to SCD1-/- mice. RESULTS: 5b treatment reduced αS hyperphosphorylation in E46K-expressing human neurons, in 3K neural cultures, and in both WT and 3K αS mice. 5b prevented subtle gait deficits in WT αS mice and the PD-like resting tremor and progressive motor decline of 3K αS mice. 5b also increased αS tetramers and reduced proteinase K-resistant lipid-rich aggregates. Similar benefits accrued from genetically deleting 1 SCD allele, providing target validation. INTERPRETATION: Prolonged reduction of brain SCD activity prevented PD-like neuropathology in multiple PD models. Thus, an orally available SCD inhibitor potently ameliorates PD phenotypes, positioning this approach to treat human α-synucleinopathies. ANN NEUROL 2021;89:74-90.

Vesicle trafficking and lipid metabolism in synucleinopathy.

The neuronal protein α-synuclein (αS) is central to the pathogenesis of Parkinson's disease and other progressive brain diseases such as Lewy body dementia and multiple system atrophy. These diseases, collectively referred to as 'synucleinopathies', have long been considered purely proteinopathies: diseases characterized by the misfolding of a protein into small and large aggregates mainly consisting of that protein (in this case: α-synuclein). However, recent morphological insights into Lewy bodies, the hallmark neuropathology of human synucleinopathies, suggests these lesions are also rich in vesicles and other membranous organelles. Moreover, αS physiology and pathology are both strongly associated with various aspects of intracellular vesicle trafficking and lipid biology. αS physiologically binds to synaptic and other small vesicles, and several functions of αS in regulating vesicle biology have been proposed. Familial PD-linked αS excess and missense mutations have been shown to impair vesicle trafficking and alter lipid homeostasis. On the other hand, vesicle trafficking and lipid-related genes have emerged as Parkinson's risk factors, suggesting a bidirectional relationship. The answer to the question "Does abnormal αS accumulation cause impaired vesicle trafficking and lipid dyshomeostasis or is αS aggregation the consequence of such impairments?" may be "Both". Here, we review current knowledge of the αS-lipid and αS-vesicle trafficking interplay, with a special focus on Parkinson's disease and Lewy body dementia.

Rapid Alpha-Synuclein Toxicity in a Neural Cell Model and Its Rescue by a Stearoyl-CoA Desaturase Inhibitor.

Genetic and biochemical evidence attributes neuronal loss in Parkinson's disease (PD) and related brain diseases to dyshomeostasis of the 14 kDa protein α-synuclein (αS). There is no consensus on how αS exerts toxicity. Explanations range from disturbed vesicle biology to proteotoxicity caused by fibrillar aggregates. To probe these mechanisms further, robust cellular toxicity models are needed, but their availability is limited. We previously reported that a shift from dynamic multimers to monomers is an early event in αS dyshomeostasis, as caused by familial PD (fPD)-linked mutants such as E46K. Excess monomers accumulate in round, lipid-rich inclusions. Engineered αS '3K' (E35K+E46K+E61K) amplifies E46K, causing a PD-like, L-DOPA-responsive motor phenotype in transgenic mice. Here, we present a cellular model of αS neurotoxicity after transducing human neuroblastoma cells to express yellow fluorescent protein (YFP)-tagged αS 3K in a doxycycline-dependent manner. αS-3K::YFP induction causes pronounced growth defects that accord with cell death. We tested candidate compounds for their ability to restore growth, and stearoyl-CoA desaturase (SCD) inhibitors emerged as a molecule class with growth-restoring capacity, but the therapeutic window varied among compounds. The SCD inhibitor MF-438 fully restored growth while exerting no apparent cytotoxicity. Our αS bioassay will be useful for elucidating compound mechanisms, for pharmacokinetic studies, and for compound/genetic screens.

Parkinson's disease: proteinopathy or lipidopathy?

Lipids play a more significant role in Parkinson's disease and its related brain disorders than is currently recognized, supporting a "lipid cascade". The 14 kDa protein α-synuclein (αS) is strongly associated with Parkinson's disease (PD), dementia with Lewy bodies (DLB), other synucleinopathies such as multiple system atrophy, and even certain forms of Alzheimer's disease. Rigorously deciphering the biochemistry of αS in native systems is the key to developing treatments. αS is highly expressed in the brain, the second most lipid-rich organ, and has been proposed to be a lipid-binding protein that physiologically interacts with phospholipids and fatty acids (FAs). αS-rich cytoplasmic inclusions called Lewy bodies and Lewy neurites are the hallmark lesions of synucleinopathies. Excess αS-membrane interactions may trigger proteinaceous αS aggregation by stimulating its primary nucleation. However, αS may also exert its toxicity prior to or independent of its self-aggregation, e.g., via excessive membrane interactions, which may be promoted by certain lipids and FAs. A complex αS-lipid landscape exists, which comprises both physiological and pathological states of αS. As novel insights about the composition of Lewy lesions occur, new lipid-related PD drug candidates emerge, and genome-wide association studies (GWAS) increasingly validate new hits in lipid-associated pathways, it seems timely to review our current knowledge of lipids in PD and consider the roles for these pathways in synucleinopathies.Fig. 1αS ↔ lipid interplay: aspects of cellular αS homeostasis (blue oval), aspects of lipid homeostasis (green oval), and overlapping aspects.Pathological states are labeled in red. Simplified schematic of both select αS and select lipid species. Several existing publications suggest αS effects on lipids and vice versa, as indicated by arrows. DG diglyceride, ER endoplasmic reticulum, FA fatty acid, LD, lipid droplet, TG triglyceride.

Cell models of lipid-rich α-synuclein aggregation validate known modifiers of α-synuclein biology and identify stearoyl-CoA desaturase.

Microscopy of Lewy bodies in Parkinson's disease (PD) suggests they are not solely filamentous deposits of α-synuclein (αS) but also contain vesicles and other membranous material. We previously reported the existence of native αS tetramers/multimers and described engineered mutations of the αS KTKEGV repeat motifs that abrogate the multimers. The resultant excess monomers accumulate in lipid membrane-rich inclusions associated with neurotoxicity exceeding that of natural familial PD mutants, such as E46K. Here, we use the αS "3K" (E35K+E46K+E61K) engineered mutation to probe the mechanisms of reported small-molecule modifiers of αS biochemistry and then identify compounds via a medium-throughput automated screen. αS 3K, which forms round, vesicle-rich inclusions in cultured neurons and causes a PD-like, l-DOPA-responsive motor phenotype in transgenic mice, was fused to YFP, and fluorescent inclusions were quantified. Live-cell microscopy revealed the highly dynamic nature of the αS inclusions: for example, their rapid clearance by certain known modulators of αS toxicity, including tacrolimus (FK506), isradipine, nilotinib, nortriptyline, and trifluoperazine. Our automated 3K cellular screen identified inhibitors of stearoyl-CoA desaturase (SCD) that robustly prevent the αS inclusions, reduce αS 3K neurotoxicity, and prevent abnormal phosphorylation and insolubility of αS E46K. SCD inhibition restores the E46K αS multimer:monomer ratio in human neurons, and it actually increases this ratio for overexpressed wild-type αS. In accord, conditioning 3K cells in saturated fatty acids rescued, whereas unsaturated fatty acids worsened, the αS phenotypes. Our cellular screen allows probing the mechanisms of synucleinopathy and refining drug candidates, including SCD inhibitors and other lipid modulators.

Genome Sequence for Candida albicans Clinical Oral Isolate 529L.

The diploid heterozygous yeast Candida albicans is the most common cause of fungal infection. Here, we report the genome sequence assembly of the clinical oral isolate 529L. As this isolate grows as a commensal, this genome will serve as a reference for experimental and genetic studies of mucosal colonization.

Studying α-Synuclein Conformation by Intact-Cell Cross-Linking.

ß-Sheet-rich aggregates of α-synuclein (αS) are the hallmark neuropathology of Parkinson's disease (PD) and related synucleinopathies, whereas the native conformations of αS in healthy cells are under debate. Cross-linking analyses in intact cells detect a large portion of endogenous αS in apparent multimeric states, most notably as putative tetramers (αS60) that run around 60 kDa on SDS-PAGE, but also point at the dynamic nature of cellular αS states. Standardization of αS cross-linking methods will facilitate efforts to study the effects of genetic, pharmacological, and environmental factors on αS conformation. Here, we present detailed protocols for cross-linking cellular αS multimers in cultured cells and brain tissues. These protocols will benefit future studies aimed at characterizing αS conformation in its cellular environment, both at steady state and upon perturbation, be it chronic or acute.

Lipidomic Analysis of α-Synuclein Neurotoxicity Identifies Stearoyl CoA Desaturase as a Target for Parkinson Treatment.

In Parkinson's disease (PD), α-synuclein (αS) pathologically impacts the brain, a highly lipid-rich organ. We investigated how alterations in αS or lipid/fatty acid homeostasis affect each other. Lipidomic profiling of human αS-expressing yeast revealed increases in oleic acid (OA, 18:1), diglycerides, and triglycerides. These findings were recapitulated in rodent and human neuronal models of αS dyshomeostasis (overexpression; patient-derived triplication or E46K mutation; E46K mice). Preventing lipid droplet formation or augmenting OA increased αS yeast toxicity; suppressing the OA-generating enzyme stearoyl-CoA-desaturase (SCD) was protective. Genetic or pharmacological SCD inhibition ameliorated toxicity in αS-overexpressing rat neurons. In a C. elegans model, SCD knockout prevented αS-induced dopaminergic degeneration. Conversely, we observed detrimental effects of OA on αS homeostasis: in human neural cells, excess OA caused αS inclusion formation, which was reversed by SCD inhibition. Thus, monounsaturated fatty acid metabolism is pivotal for αS-induced neurotoxicity, and inhibiting SCD represents a novel PD therapeutic approach.

Translocon Declogger Ste24 Protects against IAPP Oligomer-Induced Proteotoxicity.

Aggregates of human islet amyloid polypeptide (IAPP) in the pancreas of patients with type 2 diabetes (T2D) are thought to contribute to ß cell dysfunction and death. To understand how IAPP harms cells and how this might be overcome, we created a yeast model of IAPP toxicity. Ste24, an evolutionarily conserved protease that was recently reported to degrade peptides stuck within the translocon between the cytoplasm and the endoplasmic reticulum, was the strongest suppressor of IAPP toxicity. By testing variants of the human homolog, ZMPSTE24, with varying activity levels, the rescue of IAPP toxicity proved to be directly proportional to the declogging efficiency. Clinically relevant ZMPSTE24 variants identified in the largest database of exomes sequences derived from T2D patients were characterized using the yeast model, revealing 14 partial loss-of-function variants, which were enriched among diabetes patients over 2-fold. Thus, clogging of the translocon by IAPP oligomers may contribute to ß cell failure.

FKBP12 contributes to α-synuclein toxicity by regulating the calcineurin-dependent phosphoproteome.

Calcineurin is an essential Ca2+-dependent phosphatase. Increased calcineurin activity is associated with α-synuclein (α-syn) toxicity, a protein implicated in Parkinson's Disease (PD) and other neurodegenerative diseases. Calcineurin can be inhibited with Tacrolimus through the recruitment and inhibition of the 12-kDa cis-trans proline isomerase FK506-binding protein (FKBP12). Whether calcineurin/FKBP12 represents a native physiologically relevant assembly that occurs in the absence of pharmacological perturbation has remained elusive. We leveraged α-syn as a model to interrogate whether FKBP12 plays a role in regulating calcineurin activity in the absence of Tacrolimus. We show that FKBP12 profoundly affects the calcineurin-dependent phosphoproteome, promoting the dephosphorylation of a subset of proteins that contributes to α-syn toxicity. Using a rat model of PD, partial elimination of the functional interaction between FKBP12 and calcineurin, with low doses of the Food and Drug Administration (FDA)-approved compound Tacrolimus, blocks calcineurin's activity toward those proteins and protects against the toxic hallmarks of α-syn pathology. Thus, FKBP12 can endogenously regulate calcineurin activity with therapeutic implications for the treatment of PD.

Loss of native α-synuclein multimerization by strategically mutating its amphipathic helix causes abnormal vesicle interactions in neuronal cells.

α-Synuclein (αS) forms round cytoplasmic inclusions in Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Evidence suggests a physiological function of αS in vesicle trafficking and release. In contrast to earlier tenets, recent work indicates that αS normally exists in cells in a dynamic equilibrium between monomers and tetramers/multimers. We engineered αS mutants incapable of multimerization, leading to excess monomers at vesicle membranes. By EM, such mutants induced prominent vesicle clustering, leading to round cytoplasmic inclusions. Immunogold labeling revealed abundant αS intimately associated with vesicles of varied size. Fluorescence microscopy with marker proteins showed that the αS-associated vesicles were of diverse endocytic and secretory origin. An αS '3K' mutant (E35K + E46K + E61K) that amplifies the PD/DLB-causing E46K mutation induced αS-rich vesicle clusters resembling the vesicle-rich areas of Lewy bodies, supporting pathogenic relevance. Mechanistically, E46K can increase αS vesicle binding via membrane-induced amphipathic helix formation, and '3K' further enhances this effect. Another engineered αS variant added hydrophobicity to the hydrophobic half of αS helices, thereby stabilizing αS-membrane interactions. Importantly, substituting charged for uncharged residues within the hydrophobic half of the stabilized helix not only reversed the strong membrane interaction of the multimer-abolishing αS variant but also restored multimerization and prevented the aberrant vesicle interactions. Thus, reversible αS amphipathic helix formation and dynamic multimerization regulate a normal function of αS at vesicles, and abrogating multimers has pathogenic consequences.

Genome-Scale Networks Link Neurodegenerative Disease Genes to α-Synuclein through Specific Molecular Pathways.

Numerous genes and molecular pathways are implicated in neurodegenerative proteinopathies, but their inter-relationships are poorly understood. We systematically mapped molecular pathways underlying the toxicity of alpha-synuclein (α-syn), a protein central to Parkinson's disease. Genome-wide screens in yeast identified 332 genes that impact α-syn toxicity. To "humanize" this molecular network, we developed a computational method, TransposeNet. This integrates a Steiner prize-collecting approach with homology assignment through sequence, structure, and interaction topology. TransposeNet linked α-syn to multiple parkinsonism genes and druggable targets through perturbed protein trafficking and ER quality control as well as mRNA metabolism and translation. A calcium signaling hub linked these processes to perturbed mitochondrial quality control and function, metal ion transport, transcriptional regulation, and signal transduction. Parkinsonism gene interaction profiles spatially opposed in the network (ATP13A2/PARK9 and VPS35/PARK17) were highly distinct, and network relationships for specific genes (LRRK2/PARK8, ATXN2, and EIF4G1/PARK18) were confirmed in patient induced pluripotent stem cell (iPSC)-derived neurons. This cross-species platform connected diverse neurodegenerative genes to proteinopathy through specific mechanisms and may facilitate patient stratification for targeted therapy.

Mutational analysis of essential septins reveals a role for septin-mediated signaling in filamentation.

Septin proteins are conserved structural proteins that often demarcate regions of cell division. The essential nature of the septin ring, composed of several septin proteins, complicates investigation of the functions of the ring, although careful analysis in the model yeast Saccharomyces cerevisiae has elucidated the role that septins play in the cell cycle. Mutation analysis of nonessential septins in the pathogenic fungus Candida albicans has shown that septins also have vital roles in cell wall regulation (CWR), hyphal formation, and pathogenesis. While mutations in nonessential septins have been useful in establishing phenotypes, the septin defect is so slight that identifying causative associations between septins and downstream effectors has been difficult. In this work, we describe decreased abundance by mRNA perturbation (DAmP) alleles of essential septins, which display a septin defect more severe than the defect observed in deletions of nonessential septins. The septin DAmP alleles have allowed us to genetically separate the roles of septins in hyphal growth and CWR and to identify the cyclic AMP pathway as a pathway that likely acts in a parallel manner with septins in hyphal morphogenesis.

Candida albicans Czf1 and Efg1 coordinate the response to farnesol during quorum sensing, white-opaque thermal dimorphism, and cell death.

Quorum sensing by farnesol in Candida albicans inhibits filamentation and may be directly related to its ability to cause both mucosal and systemic diseases. The Ras1-cyclic AMP signaling pathway is a target for farnesol inhibition. However, a clear understanding of the downstream effectors of the morphological farnesol response has yet to be unraveled. To address this issue, we screened a library for mutants that fail to respond to farnesol. Six mutants were identified, and the czf1Δ/czf1Δ mutant was selected for further characterization. Czf1 is a transcription factor that regulates filamentation in embedded agar and also white-to-opaque switching. We found that Czf1 is required for filament inhibition by farnesol under at least three distinct environmental conditions: on agar surfaces, in liquid medium, and when embedded in a semisolid agar matrix. Since Efg1 is a transcription factor of the Ras1-cyclic AMP signaling pathway that interacts with and regulates Czf1, an efg1Δ/efg1Δ czf1Δ/czf1Δ mutant was tested for filament inhibition by farnesol. It exhibited an opaque-cell-like temperature-dependent morphology, and it was killed by low farnesol levels that are sublethal to wild-type cells and both efg1Δ/efg1Δ and czf1Δ/czf1Δ single mutants. These results highlight a new role for Czf1 as a downstream effector of the morphological response to farnesol, and along with Efg1, Czf1 is involved in the control of farnesol-mediated cell death in C. albicans.