Wei Ping Gai

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.

Phosphorylation at S87 is enhanced in synucleinopathies, inhibits α-synuclein oligomerization and influences synuclein-membrane interactions.

Increasing evidence suggests that phosphorylation may play an important role in the oligomerization, fibrillogenesis, Lewy body (LB) formation, and neurotoxicity of α-synuclein (α-syn) in Parkinson disease. Herein we demonstrate that α-syn is phosphorylated at S87 in vivo and within LBs. The levels of S87-P are increased in brains of transgenic (TG) models of synucleinopathies and human brains from Alzheimer disease (AD), LB disease (LBD), and multiple system atrophy (MSA) patients. Using antibodies against phosphorylated α-syn (S129-P and S87-P), a significant amount of immunoreactivity was detected in the membrane in the LBD, MSA, and AD cases but not in normal controls. In brain homogenates from diseased human brains and TG animals, the majority of S87-P α-syn was detected in the membrane fractions. A battery of biophysical methods were used to dissect the effect of S87 phosphorylation on the structure, aggregation, and membrane-binding properties of monomeric α-syn. These studies demonstrated that phosphorylation at S87 expands the structure of α-syn, increases its conformational flexibility, and blocks its fibrillization in vitro. Furthermore, phosphorylation at S87, but not S129, results in significant reduction of α-syn binding to membranes. Together, our findings provide novel mechanistic insight into the role of phosphorylation at S87 and S129 in the pathogenesis of synucleinopathies and potential roles of phosphorylation in α-syn normal biology.

The solubility of α-synuclein in multiple system atrophy differs from that of dementia with Lewy bodies and Parkinson's disease

Intracellular inclusions containing α‐synuclein (αSN) are pathognomonic features of several neurodegenerative disorders. Inclusions occur in oligodendrocytes in multiple system atrophy (MSA) and in neurons in dementia with Lewy bodies (DLB) and Parkinsons disease (PD). In order to identify disease‐associated changes of αSN, this study compared the levels, solubility and molecular weight species of αSN in brain homogenates from MSA, DLB, PD and normal aged controls. In DLB and PD, substantial amounts of detergent‐soluble and detergent‐insoluble αSN were detected compared with controls in grey matter homogenate. Compared with controls, MSA cases had significantly higher levels of αSN in the detergent‐soluble fraction of brain samples from pons and white matter but detergent‐insoluble αSN was not detected. There was an inverse correlation between buffered saline‐soluble and detergent‐soluble levels of αSN in individual MSA cases suggesting a transition towards insolubility in disease. The differences in solubility of αSN between grey and white matter in disease may result from different processing of αSN in neurons compared with oligodendrocytes. Highly insoluble αSN is not involved in the pathogenesis of MSA. It is therefore possible that buffered saline‐soluble or detergent‐soluble forms of αSN are involved in the pathogenesis of other αSN‐related diseases.

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.

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.

Alpha-synuclein aggregation and Ser-129 phosphorylation-dependent cell death in oligodendroglial cells.

Multiple system atrophy is a neurodegenerative disorder characterized by accumulation of aggregated Ser-129-phosphorylated α-synuclein in oligodendrocytes. p25α is an oligodendroglial protein that potently stimulates α-synuclein aggregation in vitro. To model multiple system atrophy, we coexpressed human p25α and α-synuclein in the rat oligodendroglial cell line OLN-93 and observed a cellular response characterized by a fast retraction of microtubules from the cellular processes to the perinuclear region followed by a protracted development of apoptosis. This response was dependent on phosphorylation at Ser-129 in α-synuclein as demonstrated by site-directed mutagenesis. Treatment of the cells with the kinase inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H benzimidazole that targets kinases like casein kinase 2, and polo-like kinases abrogated the toxicity. The polo-like kinase inhibitor BI 2536 caused apoptosis in the model. Ser-129 phosphorylation was linked to the formation of phosphorylated oligomers detectable by immunoblotting, and their formation was inhibited by 2-dimethylamino-4,5,6,7-tetrabromo-1H benzimidazole. The process of microtubule retraction was also dependent on aggregation as demonstrated by the protective effect of treating the cells with the specific peptide inhibitor of α-synuclein aggregation ASI1D and the non-selective inhibitors Congo Red and baicalein. The fast microtubule retraction was followed by the development of the apoptotic markers: activated caspase-3, phosphatidylserine externalization, nuclear condensation, and fragmentation. These markers could all be blocked by the inhibitors of phosphorylation, aggregation, and caspase-3. Hence, the model predicts that both Ser-129 phosphorylation and aggregation control the toxic α-syn pathway in oligodendroglial cells and may represent therapeutic intervention points in multiple system atrophy.

Alpha-synuclein is upregulated in neurones in response to chronic oxidative stress and is associated with neuroprotection

Chronic oxidative stress has been linked to the neurodegenerative changes characteristic of Parkinsons disease, particularly alpha-synuclein accumulation and aggregation. However, it remains contentious whether these alpha-synuclein changes are cytotoxic or neuroprotective. The current study utilised long-term primary neural culture techniques with antioxidant free media to study the cellular response to chronic oxidative stress. Cells maintained in antioxidant free media were exquisitely more vulnerable to acute exposure to hydrogen peroxide, yet exposure of up to 10 days in antioxidant free media did not lead to morphological alterations in neurones or glia. However, a subpopulation of neurones demonstrated a significant increase in the level of alpha-synuclein expressed within the cell body and at synaptic sites. This subset of neurones was also more resistant to apoptotic changes following exposure to antioxidant free media relative to other neurones. These data indicate that increased alpha-synuclein content is associated with neuroprotection from relatively low levels of oxidative stress.

SUMO-1 marks the nuclear inclusions in familial neuronal intranuclear inclusion disease.

Neuronal intranuclear inclusion disease (NIID) is a rare neurodegenerative disorder characterized by progressive ataxia and neuronal nuclear inclusions (NIs), similar to the inclusions found in expanded CAG repeat diseases. NIID may be familial or sporadic. The cause of familial NIID is poorly understood, as no CAG expansion has been detected. We examined three cases, from two unrelated families, who had autosomal dominant NIID but normal CAG repeats in genes involved in polyglutamine neurodegenerative diseases. We found that NIs in all three cases were intensely immunopositive for SUMO-1, a protein which covalently conjugates to other proteins and targets them to the nuclear regions (nuclear bodies) responsible for nuclear proteasomal degradation. Electron microscopy demonstrated that SUMO-1 was located on the 10-nm fibrils of NIs. In cultured PC12 cells, we found that inhibition of proteasome function by specific inhibitors resulted in the appearance of SUMO-1-immunopositive nuclear inclusions. Our study suggests that recruitment of SUMO-1 modified proteins into insoluble nuclear inclusions and proteasomal dysfunction may be involved in the pathogenesis of NIs in familial NIID cases.

α-Synuclein fibrils constitute the central core of oligodendroglial inclusion filaments in multiple system atrophy

Abstract Multiple system atrophy (MSA) belongs to synucleinopathies and is characterized pathologically by oligodendroglial inclusions (GCIs) composed of 20- to 30-nm tubular filaments. α-Synuclein fibrils formed in vitro, however, range between 10 and 12 nm in diameter. To understand the relationship between α-synuclein and GCI filaments, we conducted structural analyses of GCIs in fixed brain sections and isolated from fresh-frozen MSA brains. In fixed brain sections, GCIs were composed of amorphous material-coated filaments up to 30 nm in size. The filaments were often organized in parallel bundles extending into oligodendroglial processes. In freshly isolated GCIs, progressive buffer washes removed amorphous material and revealed that GCI filaments consisted of 10-nm-sized central core fibrils that were strongly α-synuclein immunoreactive. Image analysis revealed that each core fibril was made of two subfibrils, and each subfibril was made of a string of 3- to 6-nm-sized particles probably α-synuclein oligomers. Immunogold labeling demonstrated that epitopes encompassing entire α-synuclein molecule were represented in the core fibrils, with the N-terminal 11–26 and C-terminal 108–131 amino acid residues most accessible to antibodies, probably exposed on the surface of the fibril. Our study indicates that GCI filaments are multilayered in structure, with α-synuclein oligomers forming the central core fibrils of the filaments.

Annular α‐synuclein species from purified multiple system atrophy inclusions

Oligodendroglial cytoplasmic inclusions composed of α‐synuclein filamentous aggregates are the pathological hallmark of multiple system atrophy (MSA). We found that cortical tissue from MSA cases contains increased detergent‐resistant high‐molecular‐weight α‐synuclein species. To analyse these species, we immunopurified α‐synuclein aggregates from pathological samples and examined their ultrastructures using scanning electron and atomic force microscopies. Purified aggregates consisted of bundles of filaments. After treatment with 1% sarcosine or 2% 3‐[(3‐cholamidopropyl) dimethyl‐ammonio]‐1‐propanesulfonate (CHAPS) detergents, we observed frequent 30–50 nm annular particles, probably released from pathological aggregates due to the dissociation of filaments by the detergents. Antibody recognition imaging using a specific anti‐α‐synuclein antibody confirmed that the annular structures were positive for α‐synuclein. In contrast to pathological α‐synuclein, detergent treatment of recombinant α‐synuclein yielded only smaller, 10–18 nm spherical particles. Our results demonstrate that detergent treatment of pathological MSA α‐synuclein aggregates, but not recombinant α‐synuclein, yields discrete α‐synuclein‐positive species with annular morphologies. The ability of the pathological α‐synuclein to form annular aggregates may be an important factor contributing to the toxicity of the protein in disease that may have implications in designing therapeutic strategies aimed at detoxifying α‐synuclein aggregates.