Ronit Sharon

The Formation of Highly Soluble Oligomers of α-Synuclein Is Regulated by Fatty Acids and Enhanced in Parkinson's Disease

Accumulation of misfolded proteins as insoluble aggregates occurs in several neurodegenerative diseases. In Parkinsons disease (PD) and dementia with Lewy bodies (DLB), alpha-synuclein (alpha S) accumulates in insoluble inclusions. To identify soluble alpha S oligomers that precede insoluble aggregates, we probed the cytosols of mesencephalic neuronal (MES) cells, normal and alpha S-transgenic mouse brains, and normal, PD, and DLB human brains. All contained highly soluble oligomers of alpha S whose detection was enhanced by delipidation. Exposure of living MES neurons to polyunsaturated fatty acids (PUFAs) increased alpha S oligomer levels, whereas saturated FAs decreased them. PUFAs directly promoted oligomerization of recombinant alphaS. Transgenic mice accumulated soluble oligomers with age. PD and DLB brains had elevated amounts of the soluble, lipid-dependent oligomers. We conclude that alpha S interacts with PUFAs in vivo to promote the formation of highly soluble oligomers that precede the insoluble alpha S aggregates associated with neurodegeneration.

Parkin Localizes to the Lewy Bodies of Parkinson Disease and Dementia with Lewy Bodies

Mutations in alpha-synuclein (alpha S) and parkin cause heritable forms of Parkinson disease (PD). We hypothesized that neuronal parkin, a known E3 ubiquitin ligase, facilitates the formation of Lewy bodies (LBs), a pathological hallmark of PD. Here, we report that affinity-purified parkin antibodies labeled classical LBs in substantia nigra sections from four related human disorders: sporadic PD, inherited alphaS-linked PD, dementia with LBs (DLB), and LB-positive, parkin-linked PD. Anti-parkin antibodies also detected LBs in entorhinal and cingulate cortices from DLB brain and alphaS inclusions in sympathetic gangliocytes from sporadic PD. Double labeling with confocal microscopy of DLB midbrain sections revealed that approximately 90% of anti-alpha S-reactive LBs were also detected by a parkin antibody to amino acids 342 to 353. Accordingly, parkin proteins, including the 53-kd mature isoform, were present in affinity-isolated LBs from DLB cortex. Fluorescence resonance energy transfer and immunoelectron microscopy showed that alphaS and parkin co-localized within brainstem and cortical LBs. Biochemically, parkin appeared most enriched in cytosolic and postsynaptic fractions of adult rat brain, but also in purified, alpha S-rich presynaptic elements that additionally contained parkins E2-binding partner, UbcH7. We conclude that parkin and UbcH7 are present with alphaS in subcellular compartments of normal brain and that parkin frequently co-localizes with alpha S aggregates in the characteristic LB inclusions of PD and DLB. These results suggest that functional parkin proteins may be required during LB formation.

alpha-Synuclein occurs in lipid-rich high molecular weight complexes, binds fatty acids, and shows homology to the fatty acid-binding proteins.

α-Synuclein (αS) is a 140-residue neuronal protein that forms insoluble cytoplasmic aggregates in Parkinsons disease (PD) and several other neurodegenerative disorders. Two missense mutations (A53T and A30P) are linked to rare forms of familial PD. The normal function of αS is unknown, and cultured cell systems that model its modification from soluble monomers to aggregated forms have not been reported. Through a systematic centrifugal fractionation of mesencephalic neuronal cell lines and transgenic mouse brains expressing wild-type or A53T human αS, we observed unusual, previously unrecognized species of αS that migrate well above the 17-kDa monomeric form in denaturing gels. Incubation at 65°C of high-speed cytosols from cells or brains revealed a modified αS species migrating at ≈36 kDa and an extensive higher molecular mass αS-reactive smear. Extraction of the cytosols with chloroform/methanol or with a resin (Lipidex 1000) that binds fatty acids resulted in a similar pattern of higher molecular mass αS forms. On the basis of this effect of delipidation, we reexamined the primary structure of αS and detected a motif at the N and C termini that is homologous to a fatty acid-binding protein signature. In accord, we found that purified human αS binds oleic acid, with an apparent Kd of 12.5 μM. We also observed an enhanced association of A53T αS with microsomal membranes in both mesencephalic cells and transgenic mouse brains. We conclude that αS has biochemical properties and a structural motif that suggest it is a novel member of the fatty acid-binding protein family and may thus transport fatty acids between the aqueous and membrane phospholipid compartments of the neuronal cytoplasm.

Altered fatty acid composition of dopaminergic neurons expressing α-synuclein and human brains with α-synucleinopathies

α-Synuclein (αS) is an abundant neuronal protein that accumulates in insoluble inclusions in Parkinsons disease (PD) and the related disorder, dementia with Lewy bodies (DLB). A central question about the role of αS in the pathogenesis of PD and DLB concerns how this normally soluble protein assembles into insoluble aggregates associated with neuronal dysfunction. We recently detected highly soluble oligomers of αS in normal brain supernatants and observed their augmentation in PD and DLB brains. Further, we found that polyunsaturated fatty acids (PUFAs) enhanced αS oligomerization in intact mesencephalic neuronal cells. We now report the presence of elevated PUFA levels in PD and DLB brain soluble fractions. Higher PUFA levels were also detected in the supernatants and high-speed membrane fractions of neuronal cells over-expressing wild-type or PD-causing mutant αS. This increased PUFA content in the membrane fraction was accompanied by increased membrane fluidity in the αS overexpressing neurons. In accord, membrane fluidity and the levels of certain PUFAs were decreased in the brains of mice genetically deleted of αS. Together with our earlier observations, these results suggest that αS-PUFA interactions help regulate neuronal PUFA levels as well as the oligomerization state of αS, both normally and in human synucleinopathies.

α–Synuclein and PolyUnsaturated Fatty Acids Promote Clathrin Mediated Endocytosis and Synaptic Vesicle Recycling

α‐Synuclein (αS) is an abundant neuronal cytoplasmic protein implicated in Parkinson’s disease (PD), but its physiological function remains unknown. Consistent with its having structural motifs shared with class A1 apolipoproteins, αS can reversibly associate with membranes and help regulate membrane fatty acid composition. We previously observed that variations in αS expression level in dopaminergic cultured cells or brains are associated with changes in polyunsaturated fatty acid (PUFA) levels and altered membrane fluidity. We now report that αS acts with PUFAs to enhance the internalization of the membrane‐binding dye, FM 1‐43. Specifically, αS expression coupled with exposure to physiological levels of certain PUFAs enhanced clathrin‐mediated endocytosis in neuronal and non‐neuronal cultured cells. Moreover, αS expression and PUFA‐enhanced basal and ‐evoked synaptic vesicle (SV) endocytosis in primary hippocampal cultures of wild type (wt) and genetically depleted αS mouse brains. We suggest that αS and PUFAs normally function in endocytic mechanisms and are specifically involved in SV recycling upon neuronal stimulation.

The Transgenic Overexpression of α-Synuclein and Not Its Related Pathology Associates with Complex I Inhibition

α-Synuclein (αS) is a protein involved in the cytopathology and genetics of Parkinson disease and is thought to affect mitochondrial complex I activity. Previous studies have shown that mitochondrial toxins and specifically inhibitors of complex I activity enhance αS pathogenesis. Here we show that αS overexpression specifically inhibits complex I activity in dopaminergic cells and in A53T αS transgenic mouse brains. Importantly, our results indicate that the inhibitory effect on complex I activity is not associated with αS-related pathology. Specifically, complex I activity measured in purified mitochondria from A53T αS transgenic mouse brains was not affected by mouse age; Parkinson disease-like symptoms; levels of αS soluble oligomers; levels of insoluble, lipid-associated αS; or αS intraneuronal depositions in vivo. Likewise, no correlation was found between complex I activity and polyunsaturated fatty acid-induced αS depositions in Lewy body-like inclusions in cultured dopaminergic cells. We further show that the effect of αS on complex I activity is not due to altered mitochondrial protein levels or affected complex I assembly. Based on the results herein, we suggest that αS expression negatively regulates complex I activity as part of its normal, physiological role.

Increased Neuronal α-Synuclein Pathology Associates with Its Accumulation in Oligodendrocytes in Mice Modeling α-Synucleinopathies

Multiple system atrophy (MSA) is a progressive neurodegenerative disorder characterized by striatonigral degeneration and olivo-pontocerebellar atrophy. The histopathological hallmark of MSA is glial cytoplasmic inclusions (GCI) within oligodendrocytes, accompanied by neuronal degeneration. MSA is a synucleinopathy, and α-Synuclein (α-Syn) is the major protein constituent of the GCI. It is unclear how the neuronal α-Syn protein accumulates in oligodendrocytes. We tested the hypothesis that oligodendrocytes can take up neuronal-secreted α-Syn as part of the pathogenic mechanisms leading to MSA. We report that increases in the degree of α-Syn soluble oligomers or intracellular α-Syn levels, enhance its secretion from cultured MN9D dopaminergic cells, stably expressing the protein. In accord, we show that primary oligodendrocytes from rat brain and oligodendroglial cell lines take-up neuronal-secreted or exogenously added α-Syn from their conditioning medium. This uptake is concentration-, time-, and clathrin-dependent. Utilizing the demonstrated effect of polyunsaturated fatty acids (PUFA) to enhance α-Syn neuropathology, we show an in vivo effect for brain docosahexaenoic acid (DHA) levels on α-Syn localization to oligodendrocytes in brains of a mouse model for synucleinopathies, expressing human A53T α-Syn cDNA under the PrP promoter. Hence, pathogenic mechanisms leading to elevated levels of α-Syn in neurons underlie neuronal secretion and subsequent uptake of α-Syn by oligodendrocytes in MSA.

Β-Synuclein occurs in vivo in lipid-associated oligomers and forms hetero-oligomers with α-synuclein

α‐synuclein (αS) and β‐synuclein (βS) are homologous proteins implicated in Parkinson’s disease and related synucleinopathies. While αS is neurotoxic and its aggregation and deposition in Lewy bodies is related to neurodegeneration, βS is considered as a potent inhibitor of αS aggregation and toxicity. No mechanism for the neuroprotective role of βS has been described before. Here, we report that similar to αS, βS normally occurs in lipid‐associated, soluble oligomers in wild‐type (WT) mouse brains. We partially purified βS and αS proteins from whole mouse brain by size exclusion followed by ion exchange chromatography and found highly similar elution profiles. Using this technique, we were able to partially separate βS from αS and further separate βS monomer from its own oligomers. Importantly, we show that although αS and βS share high degree of similarities, βS oligomerization is not affected by increasing cellular levels of polyunsaturated fatty acids (PUFAs), while αS oligomerization is dramatically enhanced by PUFA. We show the in vivo occurrence of hetero‐oligomers of αS and βS and suggest that βS expression inhibits PUFA‐enhanced αS oligomerization by forming hetero‐oligomers up to a quatramer that do not further propagate.

α‐Synuclein Neuropathology is Controlled by Nuclear Hormone Receptors and Enhanced by Docosahexaenoic Acid in A Mouse Model for Parkinson's Disease

α‐Synuclein (α‐Syn) is a neuronal protein that accumulates progressively in Parkinsons disease (PD) and related synucleinopathies. Attempting to identify cellular factors that affect α‐Syn neuropathology, we previously reported that polyunsaturated fatty acids (PUFAs) promote α‐Syn oligomerization and aggregation in cultured cells. We now report that docosahexaenoic acid (DHA), a 22:6 PUFA, affects α‐Syn oligomerization by activating retinoic X receptor (RXR) and peroxisome proliferator‐activated receptor γ2 (PPARγ2). In addition, we show that dietary changes in brain DHA levels affect α‐Syn cytopathology in mice transgenic for the PD‐causing A53T mutation in human α‐Syn. A diet enriched in DHA, an activating ligand of RXR, increased the accumulation of soluble and insoluble neuronal α‐Syn, neuritic injury and astrocytosis. Conversely, abnormal accumulations of α‐Syn and its deleterious effects were significantly attenuated by low dietary DHA levels. Our results suggest a role for activated RXR/PPARγ 2, obtained by elevated brain PUFA levels, in α‐Syn neuropathology.

Induced Neuroprotection Independently From PrPSc Accumulation in a Mouse Model for Prion Disease Treated With Simvastatin

BACKGROUND The misfolding and aggregation of specific proteins has emerged as a key feature of several neurodegenerative diseases. In prion diseases, progressive disease and neuronal loss are associated with the accumulation of PrP(Sc), the misfolded isoform of PrP(C). Previous in vitro studies suggest that cholesterol-lowering drugs inhibit the conversion of PrP(C) to PrP(Sc) and the accumulation of the latter, possibly through the disturbance of cholesterol-rich membrane domains (lipid rafts). OBJECTIVE To examine the effect of simvastatin, a cholesterol-lowering drug, on prion disease progression and survival. DESIGN Controlled animal study. SETTING University medical center research laboratory. SUBJECTS Female mice from the FVB/N strain. INTERVENTIONS Peripheral and central nervous system inoculations with scrapie Rocky Mountain Laboratory inoculum. MAIN OUTCOME MEASURES Clinical, immunological, pathological, and molecular assays were performed. RESULTS Simvastatin delayed disease progression, leading to increased survival in peripheral as well as central nervous system inoculations. Simvastatins beneficial effect is mediated through the l-mevalonate pathway; however, it is independent of brain cholesterol levels. Interestingly, simvastatin treatment induced PrP(Sc) accumulation in parallel with an induced neuroprotective effect. In accordance, we found that simvastatin induced immunomodulatory mechanisms in the brains of infected mice, affecting expression levels of specific microglial chemokines and cytokines. CONCLUSIONS Simvastatin delays prion disease progression and increases survival in vivo, independently of the pathogenic conversion of PrP(C) to PrP(Sc). We show that simvastatins effects on neuroprotection are correlated with downregulation of Cox2 levels and induction of microglial activation in prion-infected mouse brains.