Fariba Chegini

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.

αB-Crystallin is a major component of glial cytoplasmic inclusions in multiple system atrophy

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.

SUMO-1 marks subdomains within glial cytoplasmic inclusions of multiple system atrophy

Conjugation of the small ubiquitin-like modifier, SUMO-1, to target proteins is linked to the regulation of multiple cellular pathways, including nucleocytoplasmic trafficking, cell cycle progression, the ubiquitin-proteasome system and apoptosis. Recently, the accumulation of SUMOylated proteins in pathological neuronal intranuclear aggregates has been found in several neurodegenerative diseases. The aim of our study was to examine SUMO-1 in the alpha-synucleinopathy diseases, Multiple System Atrophy (MSA) and Dementia with Lewy Bodies (DLB). We conducted anti-SUMO-1 immunostaining of fixed brain tissue sections and smears of unfixed brain tissue homogenates of DLB and MSA cases. We found that oligodendroglial cytoplasmic inclusions, the alpha-synuclein-positive cytoplasmic aggregates that characterize MSA, exhibit robust punctate SUMO-1 immunostaining, marking discrete submicron-sized subdomains within the inclusion bodies. Lewy bodies in smears of DLB tissue homogenates showed similar SUMO-1-positive structures, although these were not detected in fixed tissue. In cell culture experiments, we found that the nuclear and perinuclear accumulation of SUMO-1 aggregates could be induced in glioma cells by chemical inhibition of proteasomal protein degradation.

NSF, Unc-18-1, dynamin-1 and HSP90 are inclusion body components in neuronal intranuclear inclusion disease identified by anti-SUMO-1-immunocapture

Neuronal intranuclear inclusion disease, a progressive ataxia that may be familial or sporadic, is characterized by numerous neuronal intranuclear inclusion bodies similar to those found in polyglutamine repeat diseases. Previously, we found that the intranuclear inclusion bodies are intensely immunopositive for SUMO-1, a protein which covalently conjugates to other proteins in a similar way to ubiquitin. To identify the SUMO-1-associated proteins in the inclusion bodies, we isolated intranuclear inclusion bodies from fresh, frozen brain tissue of a case with familial neuronal intranuclear inclusion disease and solubilized the proteins. SUMO-1-associated inclusion body proteins were then immunocaptured using an anti-SUMO-1 antibody. The proteins, NSF, dynamin-1 and Unc-18-1 (rbSEC1), involved in membrane trafficking of proteins, and the chaperone HSP90, were identified following anti-SUMO-1-immunocapture by using tandem mass spectrometry and database searching. Immunohistochemistry of brain sections and crude brain homogenates of three cases of familial neuronal intranuclear inclusion disease confirmed the presence of these proteins in intranuclear inclusions.

Caspase-1 causes truncation and aggregation of the Parkinson’s disease-associated protein α-synuclein

Significance The aggregation of α-synuclein (aSyn) is a pathological hallmark of Parkinson’s disease. Here we show that the enzymatic component of the innate inflammation system, known as caspase-1, hydrolyzes aSyn, rendering it aggregation-prone. The aggregation of α-synuclein (aSyn) leading to the formation of Lewy bodies is the defining pathological hallmark of Parkinson’s disease (PD). Rare familial PD-associated mutations in aSyn render it aggregation-prone; however, PD patients carrying wild type (WT) aSyn also have aggregated aSyn in Lewy bodies. The mechanisms by which WT aSyn aggregates are unclear. Here, we report that inflammation can play a role in causing the aggregation of WT aSyn. We show that activation of the inflammasome with known stimuli results in the aggregation of aSyn in a neuronal cell model of PD. The insoluble aggregates are enriched with truncated aSyn as found in Lewy bodies of the PD brain. Inhibition of the inflammasome enzyme caspase-1 by chemical inhibition or genetic knockdown with shRNA abated aSyn truncation. In vitro characterization confirmed that caspase-1 directly cleaves aSyn, generating a highly aggregation-prone species. The truncation-induced aggregation of aSyn is toxic to neuronal culture, and inhibition of caspase-1 by shRNA or a specific chemical inhibitor improved the survival of a neuronal PD cell model. This study provides a molecular link for the role of inflammation in aSyn aggregation, and perhaps in the pathogenesis of sporadic PD as well.

Macroautophagy in sporadic and the genetic form of Parkinson's disease with the A53T α-synuclein mutation

BackgroundThe A53T mutation in the α-synuclein gene causes autosomal-dominant Lewy body Parkinsons 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.ObjectiveTo determine whether macroautophagy is increased by the A53T α-synuclein gene mutation in PD patients and cell models.MethodsFormalin-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.Resultsα-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.ConclusionDespite 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.

Hybrid tetanus toxin C fragment-diphtheria toxin translocation domain allows specific gene transfer into PC12 cells

To study the mechanism by which genes can efficiently be transferred into specific cell types, we have constructed several novel, single-chain multicomponent proteins by recombining the nontoxic C fragment of tetanus toxin and the translocation domain of diphtheria toxin together with the DNA-binding fragment of GAL4 transcription factor, for transportation of plasmid DNA into neuronal cells. The C fragment of tetanus toxin provided neuronal selectivity, the translocation domain of diphtheria toxin permitted endosomal escape, and the GAL4 domain provided binding to DNA. To assess the cellular tasks of each component in gene transfer, different combinations of these fragments were produced by polymerase chain reaction, expressed in Escherichia coli, and purified under native conditions from the soluble proteins. We show that only fusion proteins bearing the C fragment of tetanus toxin bind to gangliosides and, followed by their specific binding to differentiated PC12 cells, are internalized within 10 min. These proteins delivered the green fluorescence protein gene to PC12 cells, with the highest transfection efficiency achieved with proteins containing both the C fragment and the translocation domain. Addition of chloroquine elevated the transfection efficiency, which was further increased by incorporation of a nuclear localization signal in the delivery system. In addition, the effect of different DNA-condensing materials (poly-L-lysine, protamine, lysine(n=8)-trytophan(n=2)-lysine(n=8)) on gene transfer was investigated.

Lewy Bodies and the Mechanisms of Neuronal Cell Death in Parkinson's Disease and Dementia with Lewy Bodies

Neuronal loss in specific brain regions and neurons with intracellular inclusions termed Lewy bodies are the pathologic hallmark in both Parkinsons disease (PD) and dementia with Lewy bodies (DLB). Lewy bodies comprise of aggregated intracellular vesicles and proteins and α‐synuclein is reported to be a major protein component. Using human brain tissue from control, PD and DLB and light and confocal immunohistochemistry with antibodies to superoxide dismutase 2 as a marker for mitochondria, α‐synuclein for Lewy bodies and βIII Tubulin for microtubules we have examined the relationship between Lewy bodies and mitochondrial loss. We have shown microtubule regression and mitochondrial and nuclear degradation in neurons with developing Lewy bodies. In PD, multiple Lewy bodies were often observed with α‐synuclein interacting with DNA to cause marked nuclear degradation. In DLB, the mitochondria are drawn into the Lewy body and the mitochondrial integrity is lost. This work suggests that Lewy bodies are cytotoxic. In DLB, we suggest that microtubule regression and mitochondrial loss results in decreased cellular energy and axonal transport that leads to cell death. In PD, α‐synuclein aggregations are associated with intact mitochondria but interacts with and causes nuclear degradation which may be the major cause of cell death.

Reciprocal Induction Between α-Synuclein and β-Amyloid in Adult Rat Neurons

In spite of definite roles for β-amyloid (Aβ) in familial Alzheimer’s disease (AD), the cause of sporadic AD remains unknown. Amyloid senile plaques and Lewy body pathology frequently coexist in neocortical and hippocampal regions of AD and Parkinson’s diseases. However, the relationship between Aβ and α-synuclein (α-Syn), the principle components in the pathological structures, in neuronal toxicity and the mechanisms of their interaction are not well studied. As Aβ and α-Syn accumulate in aging patients, the biological functions and toxicity of these polypeptides in the aging brain may be different from those in young brain. We examined the neurotoxicity influences of Aβ1-42 or α-Syn on mature neurons and the effects of Aβ1-42 or α-Syn on the production of endogenous α-Syn or Aβ1-40 reciprocally using a model of culture enriched with primary neurons from the hippocampus of adult rats. Treatment of neurons with high concentrations of Aβ1-42 or α-Syn caused significant apoptosis of neurons. Following Aβ1-42 treatment at sub apoptotic concentrations, both intra- and extra-cellular α-Syn levels were significantly increased. Reciprocally, the non-toxic levels of α-Syn treatment also increased intra- and extra-cellular Aβ1-40 levels. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, suppressed α-Syn-induced Aβ1-40 elevation, as well as Aβ1-42-induced α-Syn elevation. Thus, high concentrations of Aβ1-42 and α-Syn exert toxic effects on mature neurons; however, non-toxic concentration treatment of these polypeptides induced the production of each other reciprocally with possible involvement of PI3K pathway.

Purification of α-synuclein containing inclusions from human post mortem brain tissue.

UNLABELLED Comparison with existing methods. BACKGROUND Neurodegenerative disorders affect a large proportion of the elderly population. A group of disorders, known as the α-synucleinopathies, are characterised by the presence of α-synuclein-containing protein inclusions, such as Lewy Bodies (LBs) found in neurons from Parkinsons Disease (PD) and Dementia with Lewy Bodies (DLB), and Glial Cytoplasmic Inclusions (GCIs) found in oligodendrocytes from Multiple System Atrophy (MSA). The analysis of the protein composition of inclusions has been hindered by limitations of methods for isolating the inclusions from the surrounding tissue. METHOD Four modifications were made to the published method for GCI purification by Gai et al. (1999) which were: collecting the entire inclusion-containing part of the Percoll gradient; lysis of nuclei prior to DNAse digestion; limited tryptic digestion to release inclusions from the cytoskeletal meshwork; and increased antibody and magnetic bead concentrations/volumes to capture the larger amounts of inclusions. RESULTS The optimised method gave a 28-fold increase in yield compared to the published method of Gai et al. (1999). A 2D-DIGE comparison revealed a 3.8-fold increase in α-synuclein enrichment and a corresponding 5.2-fold reduction in tubulin contamination. This method was also successfully adapted to the purification of LBs from DLB tissue. A 2D-DIGE comparison of purified GCIs (n=2) revealed that GCIs consist of 11.7% α-synuclein, 1.9% α-β-crystallin and 2.3% 14-3-3 proteins compared to 8.5%, 2.0% and 1.5% in LBs, respectively. CONCLUSIONS This study has generated an improved method for the purification of α-synuclein-containing inclusions with a yield sufficient for multiple forms of analysis.