ghua Jin

Proteomic Identification of Novel Proteins in Cortical Lewy Bodies

Lewy body (LB) inclusions are one of the pathological hallmarks of Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). One way to better understand the process leading to LB formation and associated pathogenesis responsible for neurodegeneration in PD and DLB is to examine the content of LB inclusions. Here, we performed a proteomic investigation of cortical LBs, obtained by laser capture microdissection from neurons in the temporal cortex of dementia patients with cortical LB disease. Analysis of over 2500 cortical LBs discovered 296 proteins; of those, 17 had been associated previously with brainstem and/or cortical LBs. We validated several proteins with immunohistochemical staining followed by confocal microscopy. The results demonstrated that heat shock cognate 71 kDa protein (also known as HSC70, HSP73, or HSPA10) was indeed not only colocalized with the majority of LBs in the temporal cortex but also colocalized to LBs in the frontal cortex of patients with diffuse LB disease. Our investigation represents the first extensive proteomic investigation of cortical LBs, and it is expected that characterization of the proteins in the cortical LBs may reveal novel mechanisms by which LB forms and pathways leading to neurodegeneration in DLB and/or advanced PD. Further investigation of these novel candidates is also necessary to ensure that the potential proteins in cortical LBs are not identified incorrectly because of incomplete current human protein database.

Identification of Novel Proteins Associated with Both α-Synuclein and DJ-1

The molecular mechanisms leading to neurodegeneration in Parkinson disease (PD) remain elusive, although many lines of evidence have indicated that α-synuclein and DJ-1, two critical proteins in PD pathogenesis, interact with each other functionally. The investigation on whether α-synuclein directly interacts with DJ-1 has been controversial. In the current study, we analyzed proteins associated with α-synuclein and/or DJ-1 with a robust proteomics technique called stable isotope labeling by amino acids in cell culture (SILAC) in dopaminergic MES cells exposed to rotenone versus controls. We identified 324 and 306 proteins in the α-synuclein- and DJ-1-associated protein complexes, respectively. Among α-synuclein-associated proteins, 141 proteins displayed significant changes in the relative abundance (increase or decrease) after rotenone treatment; among DJ-1-associated proteins, 119 proteins displayed significant changes in the relative abundance after rotenone treatment. Although no direct interaction was observed between α-synuclein and DJ-1, whether analyzed by affinity purification followed by mass spectrometry or subsequent direct co-immunoprecipitation, 144 proteins were seen in association with both α-synuclein and DJ-1. Of those, 114 proteins displayed significant changes in the relative abundance in the complexes associated with α-synuclein, DJ-1, or both after rotenone treatment. A subset of these proteins (mortalin, nucleolin, grp94, calnexin, and clathrin) was further validated for their association with both α-synuclein and DJ-1 using confocal microscopy, Western blot, and/or immunoprecipitation. Thus, we not only confirmed that there was no direct interaction between α-synuclein and DJ-1 but also, for the first time, report these five novel proteins to be associating with both α-synuclein and DJ-1. Further characterization of these docking proteins will likely shed more light on the mechanisms by which DJ-1 modulates the function of α-synuclein, and vice versa, in the setting of PD.

Prostaglandin E2 receptor subtype 2 (EP2) regulates microglial activation and associated neurotoxicity induced by aggregated α-synuclein

BackgroundThe pathogenesis of idiopathic Parkinsons disease (PD) remains elusive, although evidence has suggested that neuroinflammation characterized by activation of resident microglia in the brain may contribute significantly to neurodegeneration in PD. It has been demonstrated that aggregated α-synuclein potently activates microglia and causes neurotoxicity. However, the mechanisms by which aggregated α-synuclein activates microglia are not understood fully.MethodsWe investigated the role of prostaglandin E2 receptor subtype 2 (EP2) in α-synuclein aggregation-induced microglial activation using ex vivo, in vivo and in vitro experimental systems.ResultsResults demonstrated that ablation of EP2(EP2-/-) significantly enhanced microglia-mediated ex vivo clearance of α-synuclein aggregates (from mesocortex of Lewy body disease patients) while significantly attenuating neurotoxicity and extent of α-synuclein aggregation in mice treated with a parkinsonian toxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Furthermore, we report that reduced neurotoxicity by EP2-/- microglia could be attributed to suppressed translocation of a critical cytoplasmic subunit (p47-phox) of NADPH oxidase (PHOX) to the membranous compartment after exposure to aggregated α-synuclein.ConclusionThus, it appears that microglial EP2 plays a critical role in α-synuclein-mediated neurotoxicity.

Microglial Activation Induced by Neurodegeneration A Proteomic Analysis

Neuroinflammation mediated by microglial activation appears to play an essential role in the pathogenesis of Parkinson disease; however, the mechanisms by which microglia are activated are not fully understood. Thus, we first evaluated the effects of two parkinsonian toxicants, manganese ethylene bisdithiocarbamate (Mn-EBDC) and 1-methyl-4-phenylpyridine (MPP+), on microglial activation as well as associated dopaminergic (DAergic) neurotoxicity in primary cell culture systems. The results demonstrated that, when rat primary mesencephalic neuron-enriched or neuron-microglia mixed cultures were treated with Mn-EBDC at 2–8 μm or MPP+ at 0.25–5 μm, respectively, for 7 days, both toxicants were capable of inducing DAergic neurodegeneration as well as activating microglia via a mechanism secondary to DAergic neurodegeneration. Furthermore activated microglia subsequently enhanced DAergic neurotoxicity induced by Mn-EBDC or MPP+. Detailed scrutiny of neuron-microglia interactions identified a fraction of the conditioned media derived from a DAergic cell line treated with Mn-EBDC or MPP+ that potently activated microglia. To further define potential mediators leading to microglial activation secondary to neurodegeneration, we utilized a quantitative proteomic technique termed SILAC (for stable isotope labeling by amino acids in cell culture) to compare the protein profiles of MPP+-treated cellular fraction that mediated microglial activation as compared with controls. The search revealed numerous novel proteins that are potentially important in neurodegeneration-mediated microglial activation, a process believed to be critical in Parkinson disease progression.

Mortalin: a protein associated with progression of Parkinson disease?

Abstract Parkinson disease (PD) is a progressive neurodegenerative disorder that is considered to affect the brainstem at its early stages and other brain regions, including the limbic system and isocortex, in advanced stages. It has been suggested that PD progression is characterized pathologically by the spreading of Lewy body deposition. To identify novel proteins involved in PD progression, we prepared subcellular fractions from the frontal cortex of pathologically verified PD patients at different stages of disease and Lewy body deposition and from age-matched controls. Protein expression profiles were compared using a robust quantitative proteomic technique called isobaric tagging for relative and absolute quantification in conjunction with mass spectrometry. Approximately 200 proteins were found to display significant differences in their relative abundance between PD patients at various stages and controls. Gene ontology analysis indicated that these altered proteins belonged to many categories (e.g. mitochondrial function and neurotransmission) that were likely critically involved in the pathogenesis of PD. Of those, mortalin, a mitochondrial protein, was decreased in the advanced PD cases and was further validated to be decreased using independent techniques. These results suggest a role for mortalin in PD progression.

Proteomics Identification of Proteins in Human Cortex Using Multidimensional Separations and MALDI Tandem Mass Spectrometer

It is essential to characterize the proteome of various regions of human brain because most, if not all, neurodegenerative diseases are region-specific. Here we report an in-depth proteomics identification of proteins extracted from the frontal cortex, a region playing a critical role in cognitive function. The integrated proteomics analytical flow consisted of biochemical fractionation, strong cation exchange chromatography, reverse phase liquid chromatography, and MALDI-TOF/TOF mass spectrometric analysis. In total, 812 proteins were confidently identified with two or more peptides. These proteins demonstrated diverse isoelectric points and molecular weights and are involved in several molecular functions, including protein binding, catalytic activity, transport, structure, and signal transduction. A number of proteins known to be associated with neurodegenerative diseases were also identified. Detailed characterization of these proteins will supply the necessary information to appropriately interpret proteins associated with aging and/or age-related neurodegenerative diseases. Finally 140 proteins found in the cortical proteome were present in the proteome of cerebrospinal fluid, providing tissue-specific candidates for biomarker discovery in body fluid.

Identification of novel proteins affected by rotenone in mitochondria of dopaminergic cells

BackgroundMany studies have shown that mitochondrial dysfunction, complex I inhibition in particular, is involved in the pathogenesis of Parkinsons disease (PD). Rotenone, a specific inhibitor of mitochondrial complex I, has been shown to produce neurodegeneration in rats as well as in many cellular models that closely resemble PD. However, the mechanisms through which complex I dysfunction might produce neurotoxicity are as yet unknown. A comprehensive analysis of the mitochondrial protein expression profile affected by rotenone can provide important insight into the role of mitochondrial dysfunction in PD.ResultsHere, we present our findings using a recently developed proteomic technology called SILAC (stable isotope labeling by amino acids in cell culture) combined with polyacrylamide gel electrophoresis and liquid chromatography-tandem mass spectrometry to compare the mitochondrial protein profiles of MES cells (a dopaminergic cell line) exposed to rotenone versus control. We identified 1722 proteins, 950 of which are already designated as mitochondrial proteins based on database search. Among these 950 mitochondrial proteins, 110 displayed significant changes in relative abundance after rotenone treatment. Five of these selected proteins were further validated for their cellular location and/or treatment effect of rotenone. Among them, two were confirmed by confocal microscopy for mitochondrial localization and three were confirmed by Western blotting (WB) for their regulation by rotenone.ConclusionOur findings represent the first report of these mitochondrial proteins affected by rotenone; further characterization of these proteins may shed more light on PD pathogenesis.

Characterization of Proteome of Human Cerebrospinal Fluid

Publisher Summary Human cerebrospinal fluid (CSF) is an ideal source for identifying biomarkers for neurodegenerative diseases such as Alzheimers disease (AD), Parkinsons disease (PD), and dementia with Lewy bodies (DLB). Proteomics has been used to analyze CSF in order to discover disease-associated proteins and elucidate the basic molecular mechanisms that either cause, or result from, central nervous system disorders. To identify as many CSF proteins in well-characterized healthy young subjects as possible, sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) was used to prefractionate the CSF proteins before further separation by multidimensional liquid chromatography and analyzed with LCQ or LTQ-FT mass spectrometry (MS). LCQ-MS/ MS identified 466 proteins and LTQ-FTMS/MS identified 608 proteins, which was 30% over those identified by LCQ-MS/MS. Issues related to sample preparation, proteomic instrumentation, and database search are discussed further in the context of characterization of human CSF proteome.