Biomarkers are needed to assist in the diagnosis and medical management of various neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson's disease (PD), and dementia with Lewy body (DLB). We have employed a multiplex quantitative proteomics method, iTRAQ (isobaric Tagging for Relative and Absolute protein Quantification), in conjunction with multidimensional chromatography, followed by tandem mass spectrometry (MS/MS), to simultaneously measure relative changes in the proteome of cerebrospinal fluid (CSF) obtained from patients with AD, PD, and DLB compared to healthy controls. The diagnosis of AD and DLB was confirmed by autopsy, whereas the diagnosis of PD was based on clinical criteria. The proteomic findings showed quantitative changes in AD, PD, and DLB as compared to controls; among more than 1,500 identified CSF proteins, 136, 72, and 101 of the proteins displayed quantitative changes unique to AD, PD, and DLB, respectively. Eight unique proteins were confirmed by Western blot analysis, and the sensitivity at 95% specificity was calculated for each marker alone and in combination. Several panels of unique makers were capable of distinguishing AD, PD and DLB patients from each other as well as from controls with high sensitivity at 95% specificity. Although these preliminary findings must be validated in a larger and different population of patients, they suggest that a roster of proteins may be generated and developed into specific biomarkers that could eventually assist in clinical diagnosis and monitoring disease progression of AD, PD and DLB.

Functional impairment of mitochondria and proteasomes and increased oxidative damage comprise the main pathological phenotypes of Parkinson disease (PD). Using an unbiased quantitative proteomic approach, we compared nigral mitochondrial proteins of PD patients with those from age-matched controls. 119 of 842 identified proteins displayed significant differences in their relative abundance (increase/decrease) between the two groups. We confirmed that one of these, mortalin (mthsp70/GRP75, a mitochondrial stress protein), is substantially decreased in PD brains as well as in a cellular model of PD. In addition, nine candidate mortalin-binding partners were identified as potential mediators of PD pathology. Parkinson disease (PD)1 is characterized by preferential dopaminergic (DAergic) neurodegeneration in the substantia nigra pars compacta (SNpc) with subsequent DA loss in the nigrostriatal pathway and the presence of Lewy bodies in the remaining nigral neurons (1). Although the mechanisms underlying PD development remain elusive in both genetic and sporadic PD, mitochondrial and proteasomal dysfunction and oxidative stress are recognized as major contributors (2, 3).The pivotal roles of these pathways are further substantiated by the fact that all chemically induced parkinsonian models, including 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (4), rotenone (5), and more recently epoxomicin (6), lead to mitochondrial and proteasomal dysfunction as well as increased oxidative stress. Nonetheless despite decades of research, identification of molecules involved in these processes in the setting of DAergic neurodegeneration has yielded limited success. Consequently current clinical treatment of PD is at a standstill with the replacement of DA or with DAergic agonistic approaches (7,8).In this study, we used an unbiased state-of-the-art proteomic technique called shotgun proteomics multidimensional protein identification technology (MudPIT) to quantitatively profile mitochondrial proteins from pathologically verified PD patients and normal age-matched controls as well as in a cellular model of PD, i.e. DAergic cells treated with parkinsonian toxicant rotenone. MudPIT uses multidimensional LC and tandem mass spectrometry to separate and fragment peptides for protein identification (9) as well as for quantification when used in combination with ICAT and stable isotope labeling by amino acids in cell culture (SILAC) techniques (10, 11). With these approaches, we identified many novel proteins with quantitative expression differences in the SNpc of PD patients as compared with controls. One of these proteins, mortalin/mthsp70/GRP75, decreased significantly in many PD brain samples and in the cellular model of PD. Several mortalin-binding proteins likely participating in rotenone-mediated toxicity were also identified. Furthermore overexpression and silencing of mortalin expression in the cellular model of PD significantly influenced PD type pathologies. Thus, we report for the first time that a mitochondrial stress protei...

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.

The molecular mechanisms leading to neurodegeneration in Parkinson disease (PD) remain elusive, although many lines of evidence have indicated that ␣-synuclein and DJ-1, two critical proteins in PD pathogenesis, interact with each other functionally. The investigation on whether ␣-synuclein directly interacts with DJ-1 has been controversial. In the current study, we analyzed proteins associated with ␣-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 ␣-synuclein-and DJ-1-associated protein complexes, respectively. Among ␣-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 ␣-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 ␣-synuclein and DJ-1. Of those, 114 proteins displayed significant changes in the relative abundance in the complexes associated with ␣-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 ␣-synuclein and DJ-1 using confocal microscopy, Western blot, and/or immunoprecipitation. Thus, we not only confirmed that there was no direct interaction between ␣-synuclein and DJ-1 but also, for the first time, report these five novel proteins to be associating with both ␣-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 ␣-synuclein, and vice versa, in the setting of PD.

Biomarkers are pressingly needed to assist with the clinical diagnosis of neurodegenerative diseases and/or the monitoring of disease progression. Glycoproteins are enriched in bodily fluids such as human cerebrospinal fluid (CSF), an ideal source for discovering biomarkers due to its proximity to the central nervous system (CNS), and consequently can serve as diagnostic and/or therapeutic markers for CNS diseases. We report here an in-depth identification of glycoproteins in human CSF using a complementary proteomic approach which integrated hydrazide chemistry and lectin affinity column for glycoprotein enrichment, followed by multidimensional chromatography separation and tandem mass spectrometric analysis. Using stringent criteria, a total of 216 glycoproteins, including many low-abundance proteins, was identified with high confidence. Approximately one-third of these proteins was already known to be relevant to the CNS structurally or functionally. This investigation, for the first time, not only categorized many glycoproteins in human CSF but also expanded the existing overall CSF protein database.