Poul Henning Jensen

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.

Structural and functional characterization of two alpha-synuclein strains

α-Synuclein aggregation is implicated in a variety of diseases including Parkinsons disease, dementia with Lewy bodies, pure autonomic failure and multiple system atrophy. The association of protein aggregates made of a single protein with a variety of clinical phenotypes has been explained for prion diseases by the existence of different strains that propagate through the infection pathway. Here we structurally and functionally characterize two polymorphs of α-synuclein. We present evidence that the two forms indeed fulfil the molecular criteria to be identified as two strains of α-synuclein. Specifically, we show that the two strains have different structures, levels of toxicity, and in vitro and in vivo seeding and propagation properties. Such strain differences may account for differences in disease progression in different individuals/cell types and/or types of synucleinopathies.

Proteasomal Inhibition by α-Synuclein Filaments and Oligomers

A unifying feature of many neurodegenerative disorders is the accumulation of polyubiquitinated protein inclusions in dystrophic neurons, e.g. containing α-synuclein, which is suggestive of an insufficient proteasomal activity. We demonstrate that α-synuclein and 20 S proteasome components co-localize in Lewy bodies and show that subunits from 20 S proteasome particles, in contrast to subunits of the 19 S regulatory complex, bind efficiently to aggregated filamentous but not monomeric α-synuclein. Proteasome binding to insoluble α-synuclein filaments and soluble α-synuclein oligomers results in marked inhibition of its chymotrypsin-like hydrolytic activity through a non-competitive mechanism that is mimicked by model amyloid-Aβ peptide aggregates. Endogenous ligands of aggregated α-synuclein like heat shock protein 70 and glyceraldehyde-6-phosphate dehydrogenase bind filaments and inhibit their anti-proteasomal activity. The inhibitory effect of amyloid aggregates may thus be amenable to modulation by endogenous chaperones and possibly accessible for therapeutic intervention.

In situ and in vitro study of colocalization and segregation of α-synuclein, ubiquitin, and lipids in Lewy bodies

alpha-Synuclein and ubiquitin are two Lewy body protein components that may play antagonistic roles in the pathogenesis of Lewy bodies. We examined the relationship between alpha-synuclein, ubiquitin, and lipids in Lewy bodies of fixed brain sections or isolated from cortical tissues of dementia with Lewy bodies. Lewy bodies exhibited a range of labeling patterns for alpha-synuclein and ubiquitin, from a homogeneous pattern in which alpha-synuclein and ubiquitin were evenly distributed and overlapped across the inclusion body to a concentric pattern in which alpha-synuclein and ubiquitin were partially segregated, with alpha-synuclein labeling concentrated in the peripheral domain and ubiquitin in the central domain of the Lewy body. Lipids represented a significant component in both homogeneous and concentric Lewy bodies. These results suggest that Lewy bodies are heterogeneous in their subregional composition. The segregation of alpha-synuclein to Lewy body peripheral domain is consistent with the hypothesis that alpha-synuclein is continually deposited onto Lewy bodies.

Accumulation of toxic α-synuclein oligomer within endoplasmic reticulum occurs in α-synucleinopathy in vivo.

In Parkinsons disease (PD) and other α-synucleinopathies, prefibrillar α-synuclein (αS) oligomer is implicated in the pathogenesis. However, toxic αS oligomers observed using in vitro systems are not generally seen to be associated with α-synucleinopathy in vivo. Thus, the pathologic significance of αS oligomers to αS neurotoxicity is unknown. Herein, we show that, αS that accumulate within endoplasmic reticulum (ER)/microsome forms toxic oligomers in mouse and human brain with the α-synucleinopathy. In the mouse model of α-synucleinopathy, αS oligomers initially form before the onset of disease and continue to accumulate with the disease progression. Significantly, treatment of αS transgenic mice with Salubrinal, an anti-ER stress compound that delays the onset of disease, reduces ER accumulation of αS oligomers. These results indicate that αS oligomers with toxic conformation accumulate in ER, and αS oligomer-dependent ER stress is pathologically relevant for PD.

Calcium(II) selectively induces α-synuclein annular oligomers via interaction with the C-terminal domain

α‐Synuclein filaments are the major component of intracytoplasmic inclusion bodies characteristic of Parkinsons disease and related disorders. The process of α‐synuclein filament formation proceeds via intermediate or protofibrillar species, each of which may be cytotoxic. Because high levels of calcium(II) and other metal ions may play a role in disease pathogenesis, we investigated the influence of calcium and other metals on α‐synuclein speciation. Here we report that calcium(II) and cobalt(II) selectively induce the rapid formation of discrete annular α‐synuclein oligomeric species. We used atomic force microscopy to monitor the aggregation state of α‐synuclein after 1 d at 4°C in the presence of a range of metal ions compared with the filament formation pathway in the absence of metal ions. Three classes of effect were observed with different groups of metal ions: (1) Copper(II), iron(III), and nickel(II) yielded 0.8–4 nm spherical particles, similar to α‐synuclein incubated without metal ions; (2) magnesium(II), cadmium(II), and zinc(II) gave larger, 5–8 nm spherical oligomers; and, (3) cobalt(II) and calcium(II) gave frequent annular oligomers, 70–90 nm in diameter with calcium(II) and 22–30 nm in diameter with cobalt(II). In the absence of metal ions, annular oligomers ranging 45–90 nm in diameter were observed after 10 d incubation, short branched structures appeared after a further 3 wk and extended filaments after 2–3 mo. Previous studies have shown that α‐synuclein calcium binding is mediated by the acidic C terminus. We found that truncated α‐synuclein (1–125), lacking the C‐terminal 15 amino acids, did not form annular oligomers upon calcium addition, indicating the involvement of the calcium‐binding domain.

Natively unfolded tubulin polymerization promoting protein TPPP/p25 is a common marker of alpha-synucleinopathies

The novel basic, heat-stable tubulin polymerization promoting protein TPPP/p25 is associated with microtubules in vitro and can induce the formation of aberrant microtubule assemblies. We show by 1H-NMR spectroscopy that TPPP/p25 is natively unfolded. Antisera against peptide 186GKGKAGRVDLVDESG200NH2 (186-200) are highly specific to TPPP/p25. Immunohistochemistry and confocal microscopy demonstrates that TPPP/p25 is enriched in filamentous alpha-synuclein bearing Lewy bodies of Parkinsons (PD) and diffuse Lewy body disease (DLBD), as well as glial inclusions of multiple system atrophy (MSA). There is a correlation between TPPP/p25 and alpha-synuclein immunoreactivity in Western blot. In contrast, TPPP/p25 is not associated with abnormally phosphorylated tau in various inclusions of Picks disease (PiD), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD). However, electron microscopy confirms clusters of TPPP/p25 immunoreactivity along filaments of unstructured but not compact neurofibrillary tangles in Alzheimers disease (AD). TPPP/p25 seems to be a novel marker of alpha-synucleinopathies.

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.

Peroxiredoxin 6 in human brain: molecular forms, cellular distribution and association with Alzheimer’s disease pathology

Peroxiredoxin 6 is an antioxidant enzyme and is the 1-cys member of the peroxiredoxin family. Using two-dimensional electrophoresis and Western blotting, we have shown for the first time that, in human control and brain tissue of patient’s with Alzheimer’s disease (AD), this enzyme exists as three major and five minor forms with pIs from 5.3 to 6.1. Using specific cellular markers, we have shown that peroxiredoxin 6 is present in astrocytes with very low levels in neurons, but not detectable in microglia or oligodendrocytes. In control brains, there was a very low level of peroxiredoxin 6 staining in astrocytes that was confined to a “halo” around the nucleus. In AD, there were marked increases in the number and staining intensity of peroxiredoxin 6 positive astrocytes in both gray and white matter in the midfrontal cortex, cingulate, hippocampus and amygdala. Confocal microscopy using antibodies to Aβ peptide, tau and peroxiredoxin 6 showed that peroxiredoxin 6 positive astrocytes are closely involved with diffuse plaques and to a lesser extent with neuritic plaques, suggesting that plaques are producing reactive oxygen species. There appeared to be little astrocytic response to tau containing neurons. Although peroxiredoxin 6 positive astrocytes were seen to make multiple contacts with tau positive neurons, there was no intraneuronal colocalization. In brain tissue of patients with AD, many blood vessels exhibited peroxiredoxin 6 staining that appeared to be due to the astrocytic foot processes. These results suggest that oxidative stress conditions exist in AD and that peroxiredoxin 6 is an important antioxidant enzyme in human brain defenses.

Accumulation of oligomer-prone α-synuclein exacerbates synaptic and neuronal degeneration in vivo

In Parkinsons disease and dementia with Lewy bodies, α-synuclein aggregates to form oligomers and fibrils; however, the precise nature of the toxic α-synuclein species remains unclear. A number of synthetic α-synuclein mutations were recently created (E57K and E35K) that produce species of α-synuclein that preferentially form oligomers and increase α-synuclein-mediated toxicity. We have shown that acute lentiviral expression of α-synuclein E57K leads to the degeneration of dopaminergic neurons; however, the effects of chronic expression of oligomer-prone α-synuclein in synapses throughout the brain have not been investigated. Such a study could provide insight into the possible mechanism(s) through which accumulation of α-synuclein oligomers in the synapse leads to neurodegeneration. For this purpose, we compared the patterns of neurodegeneration and synaptic damage between a newly generated mThy-1 α-synuclein E57K transgenic mouse model that is prone to forming oligomers and the mThy-1 α-synuclein wild-type mouse model (Line 61), which accumulates various forms of α-synuclein. Three lines of α-synuclein E57K (Lines 9, 16 and 54) were generated and compared with the wild-type. The α-synuclein E57K Lines 9 and 16 were higher expressings of α-synuclein, similar to α-synuclein wild-type Line 61, and Line 54 was a low expressing of α-synuclein compared to Line 61. By immunoblot analysis, the higher-expressing α-synuclein E57K transgenic mice showed abundant oligomeric, but not fibrillar, α-synuclein whereas lower-expressing mice accumulated monomeric α-synuclein. Monomers, oligomers, and fibrils were present in α-synuclein wild-type Line 61. Immunohistochemical and ultrastructural analyses demonstrated that α-synuclein accumulated in the synapses but not in the neuronal cells bodies, which was different from the α-synuclein wild-type Line 61, which accumulates α-synuclein in the soma. Compared to non-transgenic and lower-expressing mice, the higher-expressing α-synuclein E57K mice displayed synaptic and dendritic loss, reduced levels of synapsin 1 and synaptic vesicles, and behavioural deficits. Similar alterations, but to a lesser extent, were seen in the α-synuclein wild-type mice. Moreover, although the oligomer-prone α-synuclein mice displayed neurodegeneration in the frontal cortex and hippocampus, the α-synuclein wild-type only displayed neuronal loss in the hippocampus. These results support the hypothesis that accumulating oligomeric α-synuclein may mediate early synaptic pathology in Parkinsons disease and dementia with Lewy bodies by disrupting synaptic vesicles. This oligomer-prone model might be useful for evaluating therapies directed at oligomer reduction.