Wei Ping Gai

Cerebral amyloid angiopathy (CAA) refers to the deposition of β-amyloid (Aβ) peptides in the wall of brain vasculature, commonly involving capillaries and arterioles. Also being considered a part of CAA is the Aβ deposition in leptomeninge. The cellular origin of angiopathic Aβ and the pathogenic course of CAA remain incompletely understood. The present study was aimed to explore the pathogenic course of CAA in the human cerebrum via examination of changes in β-secretase-1 (BACE1), the obligatory Aβ producing enzyme, relative to Aβ and other cellular markers, by neuroanatomical and biochemical characterizations with postmortem brain samples and primary cell cultures. Immunoreactivity (IR) for BACE1 was essentially not visible at vasculature in cases without cerebral amyloidosis (control group, n = 15, age = 86.1 ± 10.3 year). In cases with brain amyloid pathology (n = 15, age = 78.7 ± 12.7 year), increased BACE1 IR was identified locally at capillaries, arterioles and along the pia, localizing to endothelia, perivascular dystrophic neurites and meningeal cells, and often coexisting with vascular iron deposition. Double immunofluorescence with densitometric analysis confirmed a site-specific BACE1 elevation at cerebral arterioles in the development of vascular Aβ deposition. Levels of BACE1 protein, activity and its immediate product (C99) were elevated in leptomeningeal lysates from cases with CAA relative to controls. The expression of BACE1 and other amyloidogenic proteins in the endothelial and meningeal cells was confirmed in primary cultures prepared from human leptomeningeal and arteriolar biopsies. These results suggest that BACE1 elevation in the endothelia and perivascular neurites may be involved in angiopathic Aβ deposition, while BACE1 elevation in meningeal cells might contribute Aβ to leptomeningeal amyloidosis.

Fluorescent probe, TPE-TPP, can differentiate monomeric, oligomeric and fibrillar α-synuclein which was previously difficult to achieve.

The accumulation and aggregation of alpha-synuclein (α-syn) in several tissue including the brain is a major pathological hallmark in Parkinson’s disease (PD). In this study, we show that α-syn can be taken up by primary human cortical neurons, astrocytes and skin-derived fibroblastsin vitro. Our findings that brain and peripheral cells exposed to α-syn can lead to impaired mitochondrial function, leading to cellular degeneration and cell death, provides additional evidence for the involvement of mitochondrial dysfunction as a mechanism of toxicity of α-syn in human cells.

Background: The A53T mutation in the α-synuclein gene causes autosomal-dominant Lewy body Parkinson's disease (PD). Cultured cell models have linked this mutation to increased cell macroautophagy, although evidence of enhanced macroautophagy in patients with this mutation has not been assessed. Objective: To determine whether macroautophagy is increased by the A53T α-synuclein gene mutation in PD patients and cell models. Methods: Formalin-fixed paraffin-embedded 10 μm-thick tissue sections from the substantia nigra and anterior cingulate cortex of two PD patients with the A53T α-synuclein gene mutation were compared with four sporadic PD cases and four controls obtained from the Sydney Brain Bank. Lewy bodies were isolated from frontal cortex of a case with late stage PD (recruited from South Australian Brain Bank). Immunohistochemistry was performed for α-synuclein and the macroautophagy markers autophagy-specific gene (ATG) 5, ATG6/Beclin1 and ATG8/LC3. SH-SY5Y cells were transfected with wild type or A53T mutant α-synuclein plasmids and observable changes in macroautophagy marker protein levels assessed using Western blotting. Results: α-Synuclein immunoreactive neurites and dots were more numerous in patients with A53T mutations compared with late stage sporadic PD patients, and perinuclear cytoplasmic α-synuclein aggregates were observed in the α-synuclein A53T gene transfected SH-SY5Y cells compared to wild type transfections. All PD patients (with or without A53T mutations) had increased immunohistochemical evidence for macroautophagy compared with controls, and the levels of the ATG5 complex were equally increased in wild type and A53T α-synuclein gene transfected cells compared to controls. Conclusion: Despite increased α-synuclein accumulation with A53T mutations, macroautophagy is not increased above that observed in sporadic patients with PD or in cells transfected with wild type α-synuclein, suggesting that mutated α-synuclein protein is not removed by macroautophagy.

Recently, we demonstrated that soluble 30–50 nm-sized nnular α-synuclein oligomers are released by mild detergent treatment from glial cytoplasmic inclusions (GCIs) purified from multiple system atrophy brain tissue (Pountneyet al., J. Neurochem. 90:502, 2004). Dynamic antibody recognition imaging using a specific anti-α-synuclein antibody confirmed that the annular structures were positive for α-synuclein. This showed that pathological α-synucleinopathy aggregates can be a source of annular α-synuclein species. In contrast to pathological α-synuclein, recombinant α-synuclein yielded only spherical oligomers after detergent treatment, indicating a greater propensity of the pathological protein to form stable annular oligomers.In vitro, we found that Ca²⁺ binding to monomeric α-synuclein, specifically amongst a range of different metal ions, induced the rapid formation of annular oligomers (Loweet al., Protein Sci., 13:3245, 2004). Hence, α-synuclein speciation may also be influenced by the intracytoplasmic Ca²⁺ concentration. We also showed that annular α-synuclein oligomers can nucleate filament formation. We hypothesize that soluble α-synuclein annular oligomers may be cytotoxic species, either by interacting with cell membranes or components of the ubiquitin proteasome system. The equilibrium between α-synuclein species may be influenced by intracellular Ca²⁺ status, interaction with lipid vesicles or other factors.

Multiple system atrophy (MSA) is characterized by the formation of oligodendroglial cytoplasmic inclusions (GCIs) consisting of α-synuclein filaments. αB-crystallin, a small chaperone protein that binds to unfolded proteins and inhibits aggregation, has been documented in GCIs. We investigated the relative abundance and speciation of αB-crystallin in GCIs in MSA brains. We also examined the influence of αB-crystallin on the formation of cytoplasmic inclusions in cultured glial cells. Immunohistochemistry and confocal microscopy revealed αB-crystallin is a prominent component of GCIs, more abundant than in Lewy bodies in Lewy body dementia. One- and two-dimensional gel electrophoresis and mass spectrometric analysis of GCIs immunopurified from MSA brains indicated that αB-crystallin is a major protein component with multiple post-translationally modified species. In cultured C6 glioma cells treated with the proteasomal inhibitor, lactacystin, to induce accumulation of ubiquitinated proteins, a subset of cells showed increased cytoplasmic staining for αB-crystallin. Proteasomeinhibited cells transfected with GFP-tagged α-synuclein resulted in ubiquitin- and αB-crystallin-positive aggregates resembling GCIs in MSA brains. Our results indicate that αB-crystallin is a major chaperone in MSA, and suggest a role of the protein in the formation of inclusion bodies in glial cells.

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.