Abnormal accumulation of lipid droplets in neurons induces the conversion of alpha-Synuclein to proteolytic resistant forms in a Drosophila model of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by alpha-synuclein (αSyn) aggregation and associated with abnormalities in lipid metabolism. The accumulation of lipids in cytoplasmic organelles called lipid droplets (LDs) was observed in cellular models of PD. To investigate the pathophysiological consequences of interactions between αSyn and proteins that regulate the homeostasis of LDs, we used a transgenic Drosophila model of PD, in which human αSyn is specifically expressed in photoreceptor neurons. We first found that overexpression of the LD-coating proteins Perilipin 1 or 2 (dPlin1/2), which limit the access of lipases to LDs, markedly increased triacylglyclerol (TG) loaded LDs in neurons. However, dPlin-induced-LDs in neurons are independent of lipid anabolic (diacylglycerol acyltransferase 1/midway, fatty acid transport protein/dFatp) and catabolic (brummer TG lipase) enzymes, indicating that alternative mechanisms regulate neuronal LD homeostasis. Interestingly, the accumulation of LDs induced by various LD proteins (dPlin1, dPlin2, CG7900 or KlarsichtLD-BD) was synergistically amplified by the co-expression of αSyn, which localized to LDs in both Drosophila photoreceptor neurons and in human neuroblastoma cells. Finally, the accumulation of LDs increased the resistance of αSyn to proteolytic digestion, a characteristic of αSyn aggregation in human neurons. We propose that αSyn cooperates with LD proteins to inhibit lipolysis and that binding of αSyn to LDs contributes to the pathogenic misfolding and aggregation of αSyn in neurons.

Physiological and pathological roles of FATP-mediated lipid droplets in Drosophila and mice retina.

Increasing evidence suggests that dysregulation of lipid metabolism is associated with neurodegeneration in retinal diseases such as age-related macular degeneration and in brain disorders such as Alzheimer's and Parkinson's diseases. Lipid storage organelles (lipid droplets, LDs), accumulate in many cell types in response to stress, and it is now clear that LDs function not only as lipid stores but also as dynamic regulators of the stress response. However, whether these LDs are always protective or can also be deleterious to the cell is unknown. Here, we investigated the consequences of LD accumulation on retinal cell homeostasis under physiological and stress conditions in Drosophila and in mice. In wild-type Drosophila, we show that dFatp is required and sufficient for expansion of LD size in retinal pigment cells (RPCs) and that LDs in RPCs are required for photoreceptor survival during aging. Similarly, in mice, LD accumulation induced by RPC-specific expression of human FATP1 was non-toxic and promoted mitochondrial energy metabolism in RPCs and non-autonomously in photoreceptor cells. In contrast, the inhibition of LD accumulation by dFatp knockdown suppressed neurodegeneration in Aats-metFB Drosophila mutants, which carry elevated levels of reactive oxygen species (ROS). This suggests that abnormal turnover of LD may be toxic for photoreceptors cells of the retina under oxidative stress. Collectively, these findings indicate that FATP-mediated LD formation in RPCs promotes RPC and neuronal homeostasis under physiological conditions but could be deleterious for the photoreceptors under pathological conditions.

Commissureless regulation of axon outgrowth across the midline is independent of Rab function.

Nervous system function requires that neurons within neural circuits are connected together precisely. These connections form during the process of axon guidance whereby each neuron extends an axon that migrates, often large distances, through a complex environment to reach its synaptic target. This task can be simplified by utilising intermediate targets to divide the route into smaller sections. This requires that axons adapt their behaviour as they migrate towards and away from intermediate targets. In the central nervous system the midline acts as an intermediate target for commissural axons. In Drosophila commissural axons switch from attraction towards to extension away from the midline by regulating the levels of the Roundabout receptor on their cell surface. This is achieved by Commissureless which directs Roundabout to an intracellular compartment in the soma prior to reaching the midline. Once across the midline Roundabout is allowed to reach the surface and acts as a receptor for the repellent ligand Slit that is secreted by cells at the midline. Here we investigated candidate intracellular mechanisms that may facilitate the intracellular targeting of Commissureless and Roundabout within the soma of commissural neurons. Using modified forms of Commissureless or Rabs we show that neither ubiquitination nor Rab activity are necessary for the intracellular targeting of Commissureless. In addition we reveal that axon outgrowth of many populations of neurons within the Drosophila central nervous system is also independent of Rab activity.

Characterisation of recombinant human fatty aldehyde dehydrogenase: implications for Sjögren-Larsson syndrome.

Fatty aldehyde dehydrogenase (FALDH) is an NAD+-dependent oxidoreductase involved in the metabolism of fatty alcohols. Enzyme activity has been implicated in the pathology of diabetes and cancer. Mutations in the human gene inactivate the enzyme and cause accumulation of fatty alcohols in Sjögren-Larsson syndrome, a neurological disorder resulting in physical and mental handicaps. Microsomal FALDH was expressed in E. coli and purified. Using an in vitro activity assay an optimum pH of approximately 9.5 and temperature of approximately 35 degrees C were determined. Medium- and long-chain fatty aldehydes were converted to the corresponding acids and kinetic parameters determined. The enzyme showed high activity with heptanal, tetradecanal, hexadecanal and octadecanal with lower activities for the other tested substrates. The enzyme was also able to convert some fatty alcohol substrates to their corresponding aldehydes and acids, at 25-30% the rate of aldehyde oxidation. A structural model of FALDH has been constructed, and catalytically important residues have been proposed to be involved in alcohol and aldehyde oxidation: Gln-120, Glu-207, Cys-241, Phe-333, Tyr-410 and His-411. These results place FALDH in a central role in the fatty alcohol/acid interconversion cycle, and provide a direct link between enzyme inactivation and disease pathology caused by accumulation of alcohols.

Characterization of the final step in the conversion of phytol into phytanic acid.

Phytol is a branched-chain fatty alcohol that is a naturally occurring precursor of phytanic acid, a fatty acid involved in the pathogenesis of Refsum disease. The conversion of phytol into phytanic acid is generally believed to take place via three enzymatic steps that involve 1) oxidation to its aldehyde, 2) further oxidation to phytenic acid, and 3) reduction of the double bond at the 2,3 position, yielding phytanic acid. Our recent investigations of this mechanism have elucidated the enzymatic steps leading to phytenic acid production, but the final step of the pathway has not been investigated so far. In this study, we describe the characterization of phytenic acid reduction in rat liver. NADPH-dependent conversion of phytenic acid into phytanic acid was detected, although at a slow rate. However, it was shown that phytenic acid can be activated to its CoA ester and that reduction of phytenoyl-CoA is much more efficient than that of phytenic acid. Furthermore, in rat hepatocytes cultured in the presence of phytol, phytenoyl-CoA could be detected, showing that it is a bona fide intermediate of phytol degradation. Subcellular fractionation experiments revealed that phytenoyl-CoA reductase activity is present in peroxisomes and mitochondria. With these findings, we have accomplished the full elucidation of the mechanism by which phytol is converted into phytanic acid.

Identification of fatty aldehyde dehydrogenase in the breakdown of phytol to phytanic acid.

Phytol is a branched chain fatty alcohol, which is abundantly present in nature as part of the chlorophyll molecule. In its free form, phytol is metabolized to phytanic acid, which accumulates in patients suffering from a variety of peroxisomal disorders, including Refsum disease. The breakdown of phytol to phytanic acid takes place in three steps, in which first, the alcohol is converted to the aldehyde, second the aldehyde is converted to phytenic acid, and finally the double bond is reduced to yield phytanic acid. By culturing fibroblasts in the presence of phytol, increases in the levels of phytenic and phytanic acid were detected. Interestingly, fibroblasts derived from patients affected by Sjögren Larsson syndrome (SLS), known to be deficient in microsomal fatty aldehyde dehydrogenase (FALDH) were found to be deficient in this. In addition, fibroblast homogenates of these patients, incubated with phytol in the presence of NAD+ did not produce any phytenic acid. This indicates that FALDH is involved in the breakdown of phytol.

Identification of PEX7 as the second gene involved in Refsum disease.

Patients affected with Refsum disease (RD) have elevated levels of phytanic acid due to a deficiency of the peroxisomal enzyme phytanoyl-CoA hydroxylase (PhyH). In most patients with RD, disease-causing mutations in the PHYH gene have been identified, but, in a subset, no mutations could be found, indicating that the condition is genetically heterogeneous. Linkage analysis of a few patients diagnosed with RD, but without mutations in PHYH, suggested a second locus on chromosome 6q22-24. This region includes the PEX7 gene, which codes for the peroxin 7 receptor protein required for peroxisomal import of proteins containing a peroxisomal targeting signal type 2. Mutations in PEX7 normally cause rhizomelic chondrodysplasia punctata type 1, a severe peroxisomal disorder. Biochemical analyses of the patients with RD revealed defects not only in phytanic acid alpha-oxidation but also in plasmalogen synthesis and peroxisomal thiolase. Furthermore, we identified mutations in the PEX7 gene. Our data show that mutations in the PEX7 gene may result in a broad clinical spectrum ranging from severe rhizomelic chondrodysplasia punctata to relatively mild RD and that clinical diagnosis of conditions involving retinitis pigmentosa, ataxia, and polyneuropathy may require a full screen of peroxisomal functions.