Catherine Pan

DJ-1 and α-synuclein in human cerebrospinal fluid as biomarkers of Parkinson's disease

Biomarkers are urgently needed for the diagnosis and monitoring of disease progression in Parkinsons disease. Both DJ-1 and alpha-synuclein, two proteins critically involved in Parkinsons disease pathogenesis, have been tested as disease biomarkers in several recent studies with inconsistent results. These have been largely due to variation in the protein species detected by different antibodies, limited numbers of patients in some studies, or inadequate control of several important variables. In this study, the nature of DJ-1 and alpha-synuclein in human cerebrospinal fluid was studied by a combination of western blotting, gel filtration and mass spectrometry. Sensitive and quantitative Luminex assays detecting most, if not all, species of DJ-1 and alpha-synuclein in human cerebrospinal fluid were established. Cerebrospinal fluid concentrations of DJ-1 and alpha-synuclein from 117 patients with Parkinsons disease, 132 healthy individuals and 50 patients with Alzheimers disease were analysed using newly developed, highly sensitive Luminex technology while controlling for several major confounders. A total of 299 individuals and 389 samples were analysed. The results showed that cerebrospinal fluid DJ-1 and alpha-synuclein levels were dependent on age and influenced by the extent of blood contamination in cerebrospinal fluid. Both DJ-1 and alpha-synuclein levels were decreased in Parkinsons patients versus controls or Alzheimers patients when blood contamination was controlled for. In the population aged > or = 65 years, when cut-off values of 40 and 0.5 ng/ml were chosen for DJ-1 and alpha-synuclein, respectively, the sensitivity and specificity for patients with Parkinsons disease versus controls were 90 and 70% for DJ-1, and 92 and 58% for alpha-synuclein. A combination of the two markers did not enhance the test performance. There was no association between DJ-1 or alpha-synuclein and the severity of Parkinsons disease. Taken together, this represents the largest scale study for DJ-1 or alpha-synuclein in human cerebrospinal fluid so far, while using newly established sensitive Luminex assays, with controls for multiple variables. We have demonstrated that total DJ-1 and alpha-synuclein in human cerebrospinal fluid are helpful diagnostic markers for Parkinsons disease, if variables such as blood contamination and age are taken into consideration.

Detection of biomarkers with a multiplex quantitative proteomic platform in cerebrospinal fluid of patients with neurodegenerative disorders

Biomarkers are needed to assist in the diagnosis and medical management of various neurodegenerative disorders, including Alzheimers disease (AD), Parkinsons 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.

YAP/TAZ as mechanosensors and mechanotransducers in regulating organ size and tumor growth

Organ size is controlled by the concerted action of biochemical and physical processes. Although mechanical forces are known to regulate cell and tissue behavior, as well as organogenesis, the precise molecular events that integrate mechanical and biochemical signals to control these processes are not fully known. The recently delineated Hippo‐tumor suppressor network and its two nuclear effectors, YAP and TAZ, shed light on these mechanisms. YAP and TAZ are proto‐oncogene proteins that respond to complex physical milieu represented by the rigidity of the extracellular matrix, cell geometry, cell density, cell polarity and the status of the actin cytoskeleton. Here, we review the current knowledge of how YAP and TAZ function as mechanosensors and mechanotransducers. We also suggest that by deciphering the mechanical and biochemical signals controlling YAP/TAZ function, we will gain insights into new strategies for cancer treatment and organ regeneration.

Quantitative proteomics of cerebrospinal fluid from patients with Alzheimer disease

Biomarkers to assist in the diagnosis and medical management of Alzheimer disease (AD) are a pressing need. We have employed a proteomic approach, microcapillary liquid chromatography mass spectrometry of proteins labeled with isotope-coded affinity tags (ICAT), to quantify relative changes in the proteome of human cerebrospinal fluid (CSF) obtained from the lumbar cistern. Using CSF from well-characterized AD patients and age-matched controls at 2 different institutions, we quantified protein concentration ratios of 42% of the 390 CSF proteins that we have identified and found differences > or = 20% in over half of them. We confirmed our findings by western blot and validated this approach by quantifying relative levels of amyloid precursor protein and cathepsin B in 17 AD patients and 16 control individuals. Quantitative proteomics of CSF from AD patients compared to age-matched controls, as well as from other neurodegenerative diseases, will allow us to generate a roster of proteins that may serve as specific biomarker panels for AD and other geriatric dementias.

Proteomic analysis of neurofibrillary tangles in Alzheimer disease identifies GAPDH as a detergent-insoluble paired helical filament tau binding protein

We performed proteomic analysis of neurofibrillary tangles (NFTs) obtained by laser capture microdissection from pyramidal neurons in hippocampal sector CA1 in patients with Alzheimer disease (AD) using liquid chromatography (LC)‐mass spectrometry (MS)/MS. We discovered a total of 155 proteins in laser captured NFTs, 72 of which were identified by multiple unique peptides. Of these 72 proteins, 63 had previously unknown association with NFTs; one of these was glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). We validated by immunohistochemistry that GAPDH colocalized with the majority of NFTs as well as plaque‐like structures in AD brain and was co‐immunoprecipitated by antibodies to abnormal forms of tau in AD, but not tau from AD temporal cortex. Characterization of GAPDH showed that it, along with phosphorylated tau and Aβ peptides, was present in detergent‐insoluble fractions from AD temporal cortex but not from age‐matched controls. These data are the first proteomic investigation of NFTs. Moreover, our results validate this approach by demonstrating that GAPDH, a glycolytic and microtubule binding protein, not only co‐localized to NFTs and immunoprecipitated with PHF‐tau, but also is one of the few proteins known to undergo conversion to a detergent‐insoluble form in AD.

Quantitative proteomic analysis of age-related changes in human cerebrospinal fluid

Identification of cerebrospinal fluid (CSF) biomarkers of the common age-related neurodegenerative diseases would be of great value to clinicians because of the difficulties in differential diagnoses of these diseases in clinical practice. Proteins are one class of potential biomarkers currently under investigation in the hope that different ensembles of proteins will aid in the diagnosis of these diseases, as well as in the assessment of progression and response to therapy. However, before undertaking a rational approach to CSF protein biomarkers of age-related neurodegeneration, we must first systematically identify CSF proteins and determine whether their levels change with normal aging. In this study, we used a powerful shotgun proteomic method, two-dimensional microcapillary liquid chromatography electrospray ionization tandem mass spectrometry, to identify proteins in human CSF. Additionally, using pooled CSF samples, we quantitatively compared the CSF proteome of younger adults with that of older adults using isotope-coded affinity tags (ICAT). From these studies we identified more than 300 proteins in CSF and found that there were 30 proteins with >20% change in concentrations between older and younger individuals. Finally, we validated changes in concentration for two of these proteins using Western blots in CSF from a separate set of individuals. These data not only expand substantially our current knowledge regarding human CSF proteins, but also supply the necessary information to appropriately interpret protein biomarkers of age-related neurodegenerative diseases.

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.

Phosphorylated α-Synuclein in Parkinson’s Disease

An assay for detecting phosphorylated α-synuclein in CSF may help to diagnose Parkinson’s disease and determine disease severity. Tracking the Course of Neurodegeneration Parkinson’s disease (PD), a neurodegenerative disorder characterized by loss of motor function, affects millions of people worldwide. Although there are drugs that can replace dopamine and thus compensate for the loss of dopaminergic neurons of the nigrostriatal pathway, there is no treatment that can prevent neuronal degeneration. A big goal has been to discover biomarkers that could be used to distinguish PD from other parkinsonian disorders, such as multiple system atrophy (MSA) and progressive supranuclear palsy (PSP), and to follow disease progression. To date, one of the most extensively tested markers is α-synuclein, a protein that has been implicated in the pathogenesis of PD. There is a decrease in the concentration of α-synuclein in the cerebrospinal fluid (CSF) of patients with PD compared to healthy individuals. However, α-synuclein does not appear to be useful in terms of differentiating PD from other parkinsonian disorders with overlapping symptoms and does not correlate with PD severity or progression. Now, Wang and colleagues have identified an isoform of α-synuclein, phosphorylated α-synuclein (PS-129), in human CSF that may prove to be a more useful marker of PD than α-synuclein. First, the authors developed a highly sensitive and specific assay to measure PS-129 concentrations as well as total α-synuclein in CSF samples from healthy individuals and from a cohort of patients with PD, MSA, PSP, and Alzheimer’s disease. The authors discovered that the PS-129 concentration in CSF, when combined with the total α-synuclein concentration in CSF, helped to distinguish PD patients from those with MSA and PSP. Additionally, CSF PS-129 concentrations in CSF correlated with disease severity in PD patients. These early results suggest that PS-129 may be useful as a marker to assist in the differential diagnosis of PD and to monitor disease progression. This would be of value for selecting patients for clinical trials to test new PD-modifying therapies as they become available and to monitor disease in response to these treatments. However, before PS-129 can be deployed as a marker for PD, it will need to be validated in independent cohorts of PD patients, especially those with samples collected longitudinally. Phosphorylated α-synuclein (PS-129), a protein implicated in the pathogenesis of Parkinson’s disease (PD), was identified by mass spectrometry in human cerebrospinal fluid (CSF). A highly sensitive and specific assay was established and used to measure PS-129 together with total α-synuclein in the CSF of patients with PD, other parkinsonian disorders such as multiple system atrophy (MSA) and progressive supranuclear palsy (PSP), and healthy individuals (a total of ~600 samples). PS-129 CSF concentrations correlated weakly with PD severity and, when combined with total α-synuclein concentrations in CSF, contributed to distinguishing PD from MSA and PSP. Further rigorous validation in independent cohorts of patients, especially those where samples have been collected longitudinally, will determine whether the concentration of PS-129 in CSF will be useful for diagnosing PD and for monitoring PD severity and progression.

Salivary α-synuclein and DJ-1: potential biomarkers for Parkinson's disease

Sir, Parkinsons disease is a neurodegenerative disorder belonging to a group of heterogeneous diseases characterized by a progressive and relatively selective loss of anatomically or physiologically related neuronal systems (Lang and Lozano, 1998; Silvers and Som, 1998). The identification of Parkinsons disease specific biomarkers, particularly at early stages, is critical for Parkinsons disease diagnosis, monitoring disease progression and patient management as well as the development of therapeutic interventions. Thus far, the proteins α-synuclein (α-Syn) and DJ-1 have been tested rigorously in Parkinsons disease. In our recent study published in Brain (Hong et al. , 2010), where a large cohort of patients with Parkinsons disease and controls were included, we provided evidence that α-Syn, along with DJ-1, decreases in Parkinsons disease CSF, providing high sensitivity and specificity for Parkinsons disease diagnosis. However, even though CSF is close to the main site of pathology in Parkinsons disease and other neurodegenerative disorders in the CNS, it cannot be readily obtained in most clinical settings (Shi et al. , 2010). To address this issue, several groups have examined serum/plasma concentrations of α-Syn and DJ-1 as potential biomarkers of Parkinsons disease. Unfortunately, a major drawback in assessing serum/plasma α-Syn and DJ-1 levels is the fact that >95% of total blood α-Syn and DJ-1 are derived from red blood cells. After controlling for several major variables, we concluded in a recent investigation that, unlike CSF, these two markers in plasma are unable to differentiate patients with Parkinsons disease from controls (Shi et al. , 2010). Of note, blood contamination of human CSF is also a major problem when assessing levels of α-Syn and DJ-1 in CSF (Hong et al. , 2010; Shi et al. , 2010). In an effort to look for other potential sources of clinically accessible samples for Parkinsons disease diagnosis …