Effects of UCH-L1 on α-synuclein over-expression mouse model of Parkinson's disease

The rare inherited form of Parkinson's disease (PD), PARK5 , is caused by a missense mutation in ubiquitin carboxy-terminal hydrolase-L1 ( UCH-L1 ) gene, resulting in Ile93Met substitution in its gene product (UCH-L1^Ile93Met). PARK5 is inherited in an autosomal-dominant mode, but whether the Ile93Met mutation gives rise to a gain-of-toxic-function or loss-of-function of UCH-L1 protein remains controversial. Here, we investigated the selective vulnerabilities of dopaminergic (DA) neurons in UCH-L1-transgenic (Tg) and spontaneous UCH-L1-null gracile axonal dystrophy mice to an important PD-causing insult, abnormal accumulation of α-synuclein (αSyn). Immunohistochemistry of midbrain sections of a patient with sporadic PD showed αSyn- and UCH-L1-double-positive Lewy bodies in nigral DA neurons, suggesting physical and/or functional interaction between the two proteins in human PD brain. Recombinant adeno-associated viral vector-mediated over-expression of αSyn for 4 weeks significantly enhanced the loss of nigral DA cell bodies in UCH-L1^Ile93Met-Tg mice, but had weak effects in age-matched UCH-L1^wild-type-Tg mice and non-Tg littermates. In contrast, the extent of αSyn-induced DA cell loss in gracile axonal dystrophy mice was not significantly different from wild-type littermates at 13-weeks post-injection. Our results support the hypothesis that PARK5 is caused by a gain-of-toxic-function of UCH-L1^Ile93Met mutant, and suggest that regulation of UCH-L1 in nigral DA cells could be a future target for treatment of PD.     

Dietary DHA supplementation causes selective changes in phospholipids from different brain regions in both wild type mice and the Tg2576 mouse model of Alzheimer's disease

Alzheimer's disease (AD) is of major concern in ageing populations and we have used the Tg2576 mouse model to understand connections between brain lipids and amyloid pathology. Because dietary docosahexaenoic acid (DHA) has been identified as beneficial, we compared mice fed with a DHA-supplemented diet to those on a nutritionally-sufficient diet.Major phospholipids from cortex, hippocampus and cerebellum were separated and analysed. Each phosphoglyceride had a characteristic fatty acid composition which was similar in cortex and hippocampus but different in the cerebellum. The biggest changes on DHA-supplementation were within ethanolamine phospholipids which, together with phosphatidylserine, had the highest proportions of DHA. Reciprocal alterations in DHA and arachidonate were found. The main diet-induced alterations were found in ethanolamine phospholipids, (and included their ether derivatives), as were the changes observed due to genotype. Tg mice appeared more sensitive to diet with generally lower DHA percentages when on the standard diet and higher relative proportions of DHA when the diet was supplemented. All four major phosphoglycerides analysed showed age-dependent decreases in polyunsaturated fatty acid contents.These data provide, for the first time, a detailed evaluation of phospholipids in different brain areas previously shown to be relevant to behaviour in the Tg2576 mouse model for AD. The lipid changes observed with genotype are consistent with the subtle alterations found in AD patients, especially for the ethanolamine phospholipid molecular species. They also emphasise the contrasting changes in fatty acid content induced by DHA supplementation within individual phospholipid classes.     

Lipid Composition in Scrapie-Infected Mouse Brain: Prion Infection Increases the Levels of Dolichyl Phosphate and Ubiquinone

Abstract: The neutral and phospholipid composition of mouse brain infected with scrapie prions was investigated. During the later stages of this disease, the level of dolichol decreased by 30% whereas the level of dolichyl phosphate increased by 30%. In terminally ill mice, there was also a 2.5-fold increase in both total ubiquinone and its reduced form. Furthermore, α-tocopherol was elevated at this stage by 50%. In contrast, no changes were observed in phospholipid amount, in phospholipid composition, and in phosphatidylethanolamine plasmalogen content during the entire disease process. The fatty acid and aldehyde composition of individual phospholipids remained unaltered as well. No modifications could be detected in cholesterol content. Thus, the majority of membrane lipids in scrapie-infected mouse brain are modified in neither quantity nor structure, but specific changes occur to a few polyisoprenoid lipids. This specificity indicates that, although prions accumulate in lysosomes, the infection process is not associated with a general membrane destruction caused by lysosomal enzyme leakage.     

Domain <Emphasis Type="Italic">a</Emphasis>’ of protein disulfide isomerase plays key role in inhibiting α-synuclein fibril formation

α-Synuclein (αSyn) is the main component of Lewy bodies formed in midbrain dopaminergic neurons which is a pathological characteristic of Parkinson's disease. It has been recently showed to induce endoplasmic reticulum (ER) stress and impair ER functions. However, the mechanism of how ER responds to αSyn toxicity is poorly understood. In the present study, we found that protein disulfide isomerase (PDI), a stress protein abundant in ER, effectively inhibits αSyn fibril formation in vitro. In PDI molecule with a structure of abb’xa’c, domain a’ was found to be essential and sufficient for PDI to inhibit αSyn fibril formation. PDI was further found to be more avid for binding with intermediate species formed during αSyn fibril formation, and the binding was more intensive in the later lag phase. Our results provide new insight into the role of PDI in protecting ER from the deleterious effects of misfolded protein accumulation in many neurodegenerative diseases.     

Lipid droplet–mediated ER homeostasis regulates autophagy and cell survival during starvation

Lipid droplets (LDs) are conserved organelles for intracellular neutral lipid storage. Recent studies suggest that LDs function as direct lipid sources for autophagy, a central catabolic process in homeostasis and stress response. Here, we demonstrate that LDs are dispensable as a membrane source for autophagy, but fulfill critical functions for endoplasmic reticulum (ER) homeostasis linked to autophagy regulation. In the absence of LDs, yeast cells display alterations in their phospholipid composition and fail to buffer de novo fatty acid (FA) synthesis causing chronic stress and morphologic changes in the ER. These defects compromise regulation of autophagy, including formation of multiple aberrant Atg8 puncta and drastically impaired autophagosome biogenesis, leading to severe defects in nutrient stress survival. Importantly, metabolically corrected phospholipid composition and improved FA resistance of LD-deficient cells cure autophagy and cell survival. Together, our findings provide novel insight into the complex interrelation between LD-mediated lipid homeostasis and the regulation of autophagy potentially relevant for neurodegenerative and metabolic diseases.     

Statins reduce neuronal alpha-synuclein aggregation in in vitro models of Parkinson's disease

Aggregation of α-synuclein (α-syn) is believed to play a critical role in the pathogenesis of disorders such as dementia with Lewy bodies and Parkinson's disease. The function of α-syn remains unclear, although several lines of evidence suggest that α-syn is involved in synaptic vesicle trafficking probably via lipid binding. Moreover, interactions with cholesterol and lipids have been shown to be involved in α-syn aggregation. In this context, the main objective of this study was to determine if statins – cholesterol synthesis inhibitors – might interfere with α-syn accumulation in cellular models. For this purpose, we studied the effects of lovastatin, simvastatin, and pravastatin on the accumulation of α-syn in a stably transfected neuronal cell line and in primary human neurons. Statins reduced the levels of α-syn accumulation in the detergent insoluble fraction of the transfected cells. This was accompanied by a redistribution of α-syn in caveolar fractions, a reduction in oxidized α-syn, and enhanced neurite outgrowth. In contrast, supplementation of the media with cholesterol increased α-syn aggregation in detergent insoluble fractions of transfected cells and was accompanied by reduced neurite outgrowth. Taken together, these results suggest that regulation of cholesterol levels with cholesterol inhibitors might be a novel approach for the treatment of Parkinson's disease.     

Correlation of phospholipid composition and physicochemical characteristics of lipids in tissues of the root vole <Emphasis Type="Italic">Microtus oeconomus</Emphasis> of different ages

There was performed a comparative analysis of phospholipids and of lipid physicochemical characteristics in spleen, blood erythrocytes, and liver of the root voles caught in the natural environmental habitation at different phases of the population cycle, as well of the animals born and raised throughout the entire live in vivarium, depending on the animals’ sex and age. The age-related changes in the phospholipid composition have been established to correlate with lipid physicochemical characteristics, while the scale and direction of the changes depend on the tissue functional role, the initial level of the antioxidative activity (AOA) of its lipids, and sex of the animals. In the process of the organism aging, parameters of the system of regulation of lipid peroxidation change not unidirectionally; in group of animals of the same age, individuals with different biochemical characteristics can be present. Under natural conditions of habitation the degree of expression of age-related changes is modified by populational factors.     

Exploring lipid-dependent conformations of membrane-bound α-synuclein with the VDAC nanopore

Regulation of VDAC by α-synuclein (αSyn) is a rich and instructive example of protein-protein interactions catalyzed by a lipid membrane surface. αSyn, a peripheral membrane protein involved in Parkinson's disease pathology, is known to bind to membranes in a transient manner. αSyn's negatively charged C-terminal domain is then available to be electromechanically trapped by the VDAC β-barrel, a process that is observed in vitro as the reversible reduction of ion flow through a single voltage-biased VDAC nanopore. Binding of αSyn to the lipid bilayer is a prerequisite of the channel-protein interaction; surprisingly, however, we find that the strength of αSyn binding to the membrane does not correlate in any simple way with its efficiency of blocking VDAC, suggesting that the lipid-dependent conformations of the membrane-bound αSyn control the interaction. Quantitative models of the free energy landscape governing the capture and release processes allow us to discriminate between several αSyn (sub-) conformations on the membrane surface. These results, combined with known structural features of αSyn on anionic lipid membranes, point to a model in which the lipid composition determines the fraction of αSyn molecules for which the charged C terminal domain is constrained to be close, but not tightly bound, to the membrane surface and thus readily captured by the VDAC nanopore. We speculate that changes in the mitochondrial membrane lipid composition may be key regulators of the αSyn-VDAC interaction and consequently of VDAC-facilitated transport of ions and metabolites in and out of mitochondria and, i.e. mitochondrial metabolism.     

Interactions of Synphilin-1 with phospholipids and lipid membranes

Synphilin-1 is an alpha-synuclein binding protein that is involved in the pathogenesis of Parkinson's disease. The present study investigated the phospholipid-binding capacity of Synphilin-1. The C-terminus of Synphilin-1 was found to selectively bind to acidic phospholipids, including phosphatidic acid, phosphatidylserine, and phosphatidylglycerol, but not to naturally charged phospholipids. Synphilin-1 was targeted to cytoplasmic lipid droplets in mammalian cells. The amino acid sequence 610-640 was found to represent the primary determinant site for phospholipid binding. Moreover, the R621C mutation identified in Parkinson's disease abolished Synphilin-1 association with lipid droplets. The lipophilicity of Synphilin-1 might prove relevant to its physiologic function.     

Alpha-synuclein gene deletion decreases brain palmitate uptake and alters the palmitate metabolism in the absence of alpha-synuclein palmitate binding

Alpha-synuclein is an abundant protein in the central nervous system that is associated with a number of neurodegenerative disorders, including Parkinson's disease. Its physiological function is poorly understood, although recently it was proposed to function as a fatty acid binding protein. To better define a role for alpha-synuclein in brain fatty acid uptake and metabolism, we infused awake, wild-type, or alpha-synuclein gene-ablated mice with [1-(14)C]palmitic acid (16:0) and assessed fatty acid uptake and turnover kinetics in brain phospholipids. Alpha-synuclein deficiency decreased brain 16:0 uptake 35% and reduced its targeting to the organic fraction. The incorporation coefficient for 16:0 entering the brain acyl-CoA pool was significantly decreased 36% in alpha-synuclein gene-ablated mice. Because incorporation coefficients alone are not predictive of fatty acid turnover in individual phospholipid classes, we calculated kinetic values for 16:0 entering brain phospholipid pools. Alpha-synuclein deficiency decreased the incorporation rate and fractional turnover of 16:0 in a number of phospholipid classes, but also increased the incorporation rate and fractional turnover of 16:0 in the choline glycerophospholipids. No differences in incorporation rate or turnover were observed in liver phospholipids, confirming that these changes in lipid metabolism were brain specific. Using titration microcalorimetry, we observed no binding of 16:0 or oleic acid to alpha-synuclein in vitro. Thus, alpha-synuclein has effects on 16:0 uptake and metabolism similar to those of an FABP, but unlike FABP, it does not directly bind 16:0; hence, the mechanism underlying these effects is different from that of a classical FABP.     

The role of ethanolamine phosphate phospholyase in regulation of astrocyte lipid homeostasis

Dietary lipid composition has been shown to impact brain morphology, brain development, and neurologic function. However, how diet uniquely regulates brain lipid homeostasis compared with lipid homeostasis in peripheral tissues remains largely uncharacterized. To evaluate the lipid response to dietary changes in the brain, we assessed actively translating mRNAs in astrocytes and neurons across multiple diets. From this data, ethanolamine phosphate phospholyase (Etnppl) was identified as an astrocyte-specific fasting-induced gene. Etnppl catabolizes phosphoethanolamine (PEtN), a prominent headgroup precursor in phosphatidylethanolamine (PE) also found in other classes of neurologically relevant lipid species. Altered Etnppl expression has also previously been associated with humans with mood disorders. We evaluated the relevance of Etnppl in maintaining brain lipid homeostasis by characterizing Etnppl across development and in coregulation with PEtN-relevant genes, as well as determining the impact to the brain lipidome after Etnppl loss. We found that Etnppl expression dramatically increased during a critical window of early brain development in mice and was also induced by glucocorticoids. Using a constitutive knockout of Etnppl (Etnppl^KO), we did not observe robust changes in expression of PEtN-related genes. However, loss of Etnppl altered the phospholipid profile in the brain, resulting in increased total abundance of PE and in polyunsaturated fatty acids within PE and phosphatidylcholine species in the brain. Together, these data suggest that brain phospholipids are regulated by the phospholyase action of the enzyme Etnppl, which is induced by dietary fasting in astrocytes.     

alpha-Synuclein membrane interactions and lipid specificity

With the discovery of missense mutations (A53T and A30P) in alpha-synuclein (alpha-Syn) in several families with early onset familial Parkinson's disease, alpha-Syn aggregation and fibril formation have been thought to play a role in the pathogenesis of alpha-synucleinopathies, such as Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. As previous reports have suggested that alpha-Syn plays a role in lipid transport and synaptic membrane biogenesis, we investigated whether alpha-Syn binds to a specific lipid ligand using thin layer chromatography overlay and examined the changes in its secondary structure using circular dichroism spectroscopy. alpha-Syn was found to bind to acidic phospholipid vesicles and this binding was significantly augmented by the presence of phosphatidylethanolamine, a neutral phospholipid. We further examined the interaction of alpha-Syn with lipids by in situ atomic force microscopy. The association of soluble wild-type alpha-Syn with planar lipid bilayers resulted in extensive bilayer disruption and the formation of amorphous aggregates and small fibrils. The A53T mutant alpha-Syn disrupted the lipid bilayers in a similar fashion but at a slower rate. These results suggest that alpha-Syn membrane interactions are physiologically important and the lipid composition of the cellular membranes may affect these interactions in vivo.     

Expression profiling and comparative sequence derived insights into lipid metabolism

Expression profiling and genomic DNA sequence comparisons are increasingly being applied to the identification and analysis of the genes that are involved in lipid metabolism. Not only has genome-wide expression profiling aided in the identification of novel genes that are involved in important processes in lipid metabolism such as sterol efflux, but also the utilization of information from these studies has added to our understanding of the regulation of pathways that participate in the process. Coupled with these gene expression studies, cross-species comparison (a technique used to search for sequences that are conserved through evolution) has proven to be a powerful tool to identify important noncoding regulatory sequences and novel genes that are relevant to lipid biology. An example of the value of this approach was the recent chance discovery of a new apolipoprotein gene (that which encodes apolipoprotein AV) that has dramatic effects on triglyceride metabolism in mice and humans.     

Agmatine transport into spinal nerve terminals is modulated by polyamine analogs

Because alpha-synuclein (Snca) has a role in brain lipid metabolism, we determined the impact that Snca deletion had on whole brain lipid composition. We analysed masses of individual phospholipid (PL) classes and neutral lipid mass as well as PL acyl chain composition in brains from wild-type and Snca-/- mice. Although total brain PL mass was not altered, cardiolipin and phosphatidylglycerol mass decreased 16% and 27%, respectively, in Snca-/- mice. In addition, no changes were observed in plasmalogen or polyphosphoinositide mass. In ethanolamine glycerophospholipids and phosphatidylserine, docosahexaenoic acid (22 : 6n-3) was decreased 7%, while 16 : 0 was increased 1.1-fold and 1.4-fold, respectively. Surprisingly, brain cholesterol, cholesteryl ester, and triacylglycerol mass were increased 1.1-fold, 1.6-fold, and 1.4-fold, respectively in Snca-/- mice. In isolated myelin, cholesterol mass was also increased 1.3-fold, but because there was also a net increase in myelin PL mass, the cholesterol to PL ratio was unaltered. No changes in the expression of cholesterogenic enzymes were observed, suggesting these did not account for the observed changes in cholesterol. These data extend our previous results in astrocytes and kinetic studies in vivo demonstrating a role for Snca in brain lipid metabolism and demonstrate a clear impact on brain neutral lipid metabolism.     

Central mechanisms of the development of motor disorders during human aging

This paper deals with the functional state of the brain and its descending regulatory influences on the brain stem-spinal formations in the elderly. The role of changes revealed in the formation of the clinical syndrome of age-related extrapyramidal insufficiency (EPI) as a risk factor of Parkinson's disease, has been shown. 274 apparently healthy subjects aged from 20 to 102 and 136 patients with early stages of Parkinson's disease were examined. The program of the neurophysiological investigation included: frequency-integrative analysis of EEG, visual and somatosensory potentials, simple motor reaction time and stimulating electroneuromyography (H reflex). It has been found that in aging and in Parkinson's disease one-directional changes in the CNS function that form age-related and pathologic EPI occur. The complex of neurophysiological indices is a reflection of the CNS influence on the underlying spinal formations, and it can be used for early diagnostics of the motor disorders in aging.     

Membrane adaptation in phospholipids and cholesterol in the widely distributed,freeze-tolerant wood frog,Rana sylvatica

Maintaining proper membrane phase and fluidity is important for preserving membrane structure and function, and by altering membrane lipid composition many organisms can adapt to changing environmental conditions. We compared the phospholipid and cholesterol composition of liver and brain plasma membranes in the freeze-tolerant wood frog, Rana sylvatica, from southern Ohio and Interior Alaska during summer, fall, and winter. We also compared membranes from winter-acclimatized frogs from Ohio that were either acclimated to 0, 4, or 10 °C, or frozen to ?2.5 °C and sampled before or after thawing. Lipids were extracted from isolated membranes, separated by one-dimensional thin-layer chromatography, and analyzed via densitometry. Liver membranes underwent seasonal changes in phospholipid composition and lipid ratios, including a winter increase in phosphatidylethanolamine, which serves to increase fluidity. However, whereas Ohioan frogs decreased phosphatidylcholine and increased sphingomyelin, Alaskan frogs only decreased phosphatidylserine, indicating that these phenotypes use different adaptive strategies to meet the functional needs of their membranes. Liver membranes showed no seasonal variation in cholesterol abundance, though membranes from Alaskan frogs contained relatively less cholesterol, consistent with the need for greater fluidity in a colder environment. No lipid changed seasonally in brain membranes in either population. In the thermal acclimation experiment, cold exposure induced an increase in phosphatidylethanolamine in liver membranes and a decrease in cholesterol in brain membranes. No changes occurred during freezing and thawing in membranes from either organ. Wood frogs use tissue-specific membrane adaptation of phospholipids and cholesterol to respond to changing environmental factors, particularly temperature, though not with freezing.     

Skeletal-muscle sarcolemma from normal and dystrophic mice. Isolation, characterization and lipid composition.

1. Mouse skeletal-muscle sarcolemma was isolated, and the preparations obtained from normal mouse muscle and from muscle of mice with hereditary muscular dystrophy were characterized with respect to appearance under the optical and electron microscopes, distribution of marker enzymes, histochemical properties and biochemical composition. 2. The sarcolemmal membranes from normal and dystrophic muscle were subjected to detailed lipied analysis. Total lipid content was shown to increase in sarcolemma from dystrophic mice as a result of a large increase in neutral lipid and a smaller increase in total phospholipids. Further analysis of the neutral-lipid fraction showed that total acylglycerols increased 6-fold, non-esterified fatty acid 4-fold and cholesterol esters 2-fold, whereas the amount of free cholesterol remained unchanged in sarcolemma from dystrophic muscle. Significant increases were found in lysophosphatidylcholine, phosphatidylcholine and phosphatidylethanolamine in dystrophic-muscle sarcolemma; however, the relative composition of the phospholipid fraction remained essentially the same as in the normal case. 3. The overall result of alterations in lipid composition of the sarcolemma in mouse muscular dystrophy was an increase in neutral lipid compared with total phospholipid, and a 4-fold decrease in the relative amount of free cholesterol in the membrane. The possible impact of these changes on membrane function is discussed.