Biomedical subjects

Benoit I Giasson

Publications and source records attributed to Benoit I Giasson.

At least 37 records · Page 2Linked to original sources

PubMed2003

Novel monoclonal antibodies demonstrate biochemical variation of brain parkin with age.

Autosomal recessive juvenile parkinsonism is a movement disorder associated with the degeneration of dopaminergic neurons in substantia nigra pars compacta. The loss of functional parkin caused by parkin gene mutations is the most common single cause of juvenile parkinsonism. Parkin has been shown to aid in protecting cells from endoplasmic reticulum and oxidative stressors presumably due to ubiquitin ligase activity of parkin that targets proteins for proteasomal degradation. However, studies on parkin have been impeded because of limited reagents specific for this protein. Here we report the generation and characterization of a panel of parkin-specific monoclonal antibodies. Biochemical analyses indicate that parkin is present only in the high salt-extractable fraction of mouse brain, whereas it is present in both the high salt-extractable and RIPA-resistant, SDS-extractable fraction in young human brain. Parkin is present at decreased levels in the high salt-extractable fraction and at increased levels in the SDS-extractable fraction from aged human brain. This shift in the extractability of parkin upon aging is seen in humans but not in mice, demonstrating species-specific differences in the biochemical characteristics of murine versus human parkin. Finally, by using these highly specific anti-parkin monoclonal antibodies, it was not possible to detect parkin in alpha-synuclein-containing lesions in alpha-synucleinopathies, thereby challenging prior inferences about the role of parkin in movement disorders other than autosomal recessive juvenile parkinsonism.

Aging↗

PubMed2003

Role of alpha-synuclein carboxy-terminus on fibril formation in vitro.

Alpha-synuclein (alpha-syn) is the major component of intracellular inclusions in several neurodegenerative diseases, and the conversion of soluble alpha-syn into filamentous aggregates may contribute to disease pathogenesis. Since mechanisms leading to the formation of alpha-syn inclusions are unclear, in vitro models of alpha-syn aggregation may yield insights into this process. To that end, we examined the consequences on the progressive deletion of the carboxy-terminus of alpha-syn in regulating fibril formation, and we show here that carboxy-terminal truncated alpha-syn proteins aggregate faster than the full-length molecule. Protease digestion and immunoelectron microscopy indicate that the alpha-syn amino- and carboxy-termini are more solvent exposed than the central core and that filaments formed from carboxy-terminal truncated alpha-syn are narrower in diameter than the full-length molecule. Moreover, seeding experiments under conditions where full-length alpha-syn did not readily aggregate revealed that carboxy-truncated alpha-syn extending from amino acids 1-102 and 1-110 but not 1-120 were efficient in seeding full-length alpha-syn aggregation over a range of concentrations. Using site-directed mutagenesis, the negatively charged residues 104, 105 and 114, 115 in the carboxy-terminus were implicated in this reduced aggregation and the lack of seeding of full-length alpha-syn fibrillogenesis by 1-120. Our data support the view that the middle region of alpha-syn forms the core of alpha-syn filaments and that negative charges in the carboxy-terminus counteract alpha-syn aggregation. Thus, the carboxy-terminus of alpha-syn may regulate aggregation of full-length alpha-syn and determine the diameter of alpha-syn filaments.

Actin Cytoskeleton↗

PubMed2003

Are ubiquitination pathways central to Parkinson's disease?

Parkinson's disease (PD) is the most common neurodegenerative movement disorder. The major motor disabilities of PD are associated with the extensive loss of dopaminergic neurons in the substantia nigra pars compacta. The physiological changes and biochemical pathways involved in the selective demise of these neurons are still unclear. Recent studies have demonstrated that alterations or reductions in ubiquitin-mediated proteasome function can be causal of at least some forms of parkinsonism, and multiple lines of evidence suggest that this mechanism of protein degradation may play an important role in the etiology of PD.

Animals↗

PubMed2003

Tau and 14-3-3 in glial cytoplasmic inclusions of multiple system atrophy.

Multiple system atrophy (MSA) is a neurodegenerative disease characterized by the presence of glial cytoplasmic inclusions (GCIs), which are comprised of fibrils of the protein alpha-synuclein (alpha-syn). Increasing evidence indicate that the formation of these lesions leads to cellular dysfunction and degeneration. The events that result in the formation of GCIs remain poorly understood. It is possible that changes in the cytoplasmic milieu, perhaps the aberrant expression of alpha-syn-interacting proteins, can promote the polymerization of alpha-syn. The presence of the microtubule-binding protein, tau, in GCIs has been reported in some studies, but these findings have not been consistent, and these studies were performed prior to the availability of the more sensitive methods of detecting GCIs using anti-alpha-syn antibodies. Recently, 14-3-3 proteins, putative alpha-syn-interacting partners, have been reported in Lewy bodies, which also are pathological inclusions comprised of alpha-syn. In this study the presence of tau and 14-3-3 proteins in GCIs of 21 patients with MSA was investigated. For the majority of cases, tau and 14-3-3 proteins were detected only in a subset of GCIs. In some cases none of the GCIs contained 14-3-3 or tau. When present in GCIs, tau was in a hypophosphorylated state as demonstrated with phosphorylation-specific antibodies. Alpha-syn fibrillogenesis without 14-3-3 or tau appears to be sufficient for GCI formation, although it is possible that the accumulation of multi-functional proteins, like 14-3-3, in GCIs contribute to the disruption of cellular homeostasis.

14-3-3 Proteins↗

PubMed2003

Effects of oxidative and nitrative challenges on alpha-synuclein fibrillogenesis involve distinct mechanisms of protein modifications.

Filamentous inclusions of alpha-synuclein protein are hallmarks of neurodegenerative diseases collectively known as synucleinopathies. Previous studies have shown that exposure to oxidative and nitrative species stabilizes alpha-synuclein filaments in vitro, and this stabilization may be due to dityrosine cross-linking. To test this hypothesis, we mutated tyrosine residues to phenylalanine and generated recombinant wild type and mutant alpha-synuclein proteins. alpha-Synuclein proteins lacking some or all tyrosine residues form fibrils to the same extent as the wild type protein. Tyrosine residues are not required for protein cross-linking or filament stabilization resulting from transition metal-mediated oxidation, because higher Mr SDS-resistant oligomers and filaments stable to chaotropic agents are detected using all Tyr --> Phe alpha-synuclein mutants. By contrast, cross-linking resulting from exposure to nitrating agents required the presence of one or more tyrosine residues. Furthermore, tyrosine cross-linking is involved in filament stabilization, because nitrating agent-exposed assembled wild type, but not mutant alpha-synuclein lacking all tyrosine residues, was stable to chaotropic treatment. In addition, the formation of stable alpha-synuclein inclusions in intact cells after exposure to oxidizing and nitrating species requires tyrosine residues. These findings demonstrate that nitrative and/or oxidative stress results in distinct mechanisms of alpha-synuclein protein modifications that can influence the formation of stable alpha-synuclein fibrils.

Cell Line↗

PubMed2003

Initiation and synergistic fibrillization of tau and alpha-synuclein.

Alpha-synuclein (alpha-syn) and tau polymerize into amyloid fibrils and form intraneuronal filamentous inclusions characteristic of neurodegenerative diseases. We demonstrate that alpha-syn induces fibrillization of tau and that coincubation of tau and alpha-syn synergistically promotes fibrillization of both proteins. The in vivo relevance of these findings is grounded in the co-occurrence of alpha-syn and tau filamentous amyloid inclusions in humans, in single transgenic mice that express A53T human alpha-syn in neurons, and in oligodendrocytes of bigenic mice that express wild-type human alpha-syn plus P301L mutant tau. This suggests that interactions between alpha-syn and tau can promote their fibrillization and drive the formation of pathological inclusions in human neurodegenerative diseases.

Amyloid↗

PubMed2003

Nitration of tau protein is linked to neurodegeneration in tauopathies.

Oxidative and nitrative injury is implicated in the pathogenesis of Alzheimer's disease (AD) and Down syndrome (DS), but no direct evidence links this type of injury to the formation of neurofibrillary tau lesions. To address this, we generated a monoclonal antibody (mAb), n847, which recognizes nitrated tau and alpha-synuclein. n847 detected nitrated tau in the insoluble fraction of AD, corticobasal degeneration (CBD), and Pick's disease (PiD) brains by Western blots. Immunohistochemistry (IHC) showed that n847 labeled neurons in the hippocampus and neocortex of AD and DS brains. Double-label immunofluorescence with n847 and an anti-tau antibody revealed partial co-localization of tau and n847 positive tangles, while n847 immunofluorescence and Thioflavin-S double-staining showed that a subset of n847-labeled neurons were Thioflavin-S-positive. In addition, immuno-electron microscopy revealed that tau-positive filaments in tangle-bearing neurons were also labeled by n847 and IHC of other tauopathies showed that some of glial and neuronal tau pathologies in CBD, progressive supranuclear palsy, PiD, and frontotemporal dementia with parkinsonism linked to chromosome 17 also were n847-positive. Finally, nitrated and Thioflavin-S-positive tau aggregates were generated in a oligodendrocytic cell line after treatment with peroxynitrite. Taken together, these findings imply that nitrative injury is directly linked to the formation of filamentous tau inclusions.

Aged↗

PubMed2003

Ubiquitination of alpha-synuclein is not required for formation of pathological inclusions in alpha-synucleinopathies.

alpha-Synucleinopathies, including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy, are neurodegenerative disorders in which abnormal inclusions containing alpha-synuclein accumulate in selectively vulnerable neurons and glia. In this report, immunohistochemistry demonstrates ubiquitin in subsets of alpha-synuclein inclusions in dementia with Lewy bodies and multiple system atrophy. Biochemistry demonstrates that alpha-synuclein in the sodium dodecyl sulfate-soluble fractions of diseased brains is ubiquitinated, with mono- and di-ubiquitinated species predominating over polyubiquitinated forms. Similar immunohistochemical and biochemical characteristics were observed in an A53T mutant human alpha-synuclein transgenic mouse model of neurodegenerative alpha-synucleinopathies. Furthermore, in vitro ubiquitination of alpha-synuclein fibrils recapitulated the pattern of alpha-synuclein ubiquitination observed in human disease and the A53T alpha-synuclein mouse model. These results suggest that ubiquitination of alpha-synuclein is not required for inclusion formation and follows the fibrillization of alpha-synuclein.

Adult↗

PubMed2003

Distinct cleavage patterns of normal and pathologic forms of alpha-synuclein by calpain I in vitro.

Parkinson's disease (PD) is characterized by fibrillary neuronal inclusions called Lewy bodies (LBs) consisting largely of alpha-synuclein (alpha-syn), the protein mutated in some patients with familial PD. The mechanisms of alpha-syn fibrillization and LB formation are unknown, but may involve aberrant degradation or turnover. We examined the ability of calpain I to cleave alpha-syn in vitro. Calpain I cleaved wild-type alpha-syn predominantly after amino acid 57 and within the non-amyloid component (NAC) region. In contrast, calpain I cleaved fibrillized alpha-syn primarily in the region of amino acid 120 to generate fragments like those that increase susceptibility to dopamine toxicity and oxidative stress. Further, while calpain I cleaved wild-type alpha-syn after amino acid 57, this did not occur in mutant A53T alpha-syn. This paucity of proteolysis could increase the stability of A53T alpha-syn, suggesting that calpain I might protect cells from forming LBs by specific cleavages of soluble wild-type alpha-syn. However, once alpha-syn has polymerized into fibrils, calpain I may contribute to toxicity of these forms of alpha-syn by cleaving at aberrant sites within the C-terminal region. Elucidating the role of calpain I in the proteolytic processing of alpha-syn in normal and diseased brains may clarify mechanisms of neurodegenerative alpha-synucleinopathies.

Amino Acid Substitution↗

PubMed2003

A comparison of amyloid fibrillogenesis using the novel fluorescent compound K114.

Proteinaceous inclusions with amyloidogenic properties are a common link between many neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Histological and in vitro studies of amyloid fibrils have advanced the understanding of protein aggregation, and provided important insights into pathogenic mechanisms of these neurodegenerative brain amyloidoses. The classical amyloid dyes Congo Red (CR) and thioflavin T and S, have been used extensively to detect amyloid inclusions in situ. These dyes have also been utilized to monitor the maturation of amyloid fibrils assembled from monomer subunits in vitro. Recently, the compound (trans,trans)-1-bromo-2,5-bis-(3- hydroxycarbonyl-4-hydroxy)styrylbenzene (BSB), derived from the structure of CR, was shown to bind to a wide range of amyloid inclusions in situ. More importantly it was also used to label brain amyloids in live animals. Herein, we show that an analogue of BSB, (trans,trans)-1-bromo-2,5-bis-(4-hydroxy)styrylbenzene (K114), recognizes amyloid lesions, and has distinctive properties which allowed the quantitative monitoring of the formation of amyloid fibrils assembled from the amyloid-beta peptide, alpha-synuclein, and tau.

Amyloid↗

PubMed2003

Interactions of amyloidogenic proteins.

The various protein deposits of brain amyloidosis share common ultrastructural, biophysical, and histological properties. These amyloidogenic deposits can be composed of distinct proteins, which are conceptually associated with different neurodegenerative diseases. Amyloidogenic proteins are typically soluble monomeric precursors, which undergo remarkable conformation changes associated with the polymerization into 8- to 10-nm wide fibrils, which culminate in the formation of amyloid aggregates. Some amyloidogenic inclusions are extracellular, such as senile plaques of Alzheimer's disease, which are composed of amyloid beta (Abeta) peptides. However, intracytoplasmic amyloid aggregates, such as neurofibrillary tangles in Alzheimer's disease and Lewy bodies in Parkinson's disease, are composed of the proteins tau and alpha-synuclein, respectively. The mounting awareness that the latter proteins are directly linked to the etiology of spectrum of neurodegenerative diseases has resulted in the coining of the terms "tauopathies" and "synucleinopathies." However, emerging evidence for the overlap in the pathological and clinical features of patients with brain amyloidosis suggests that they may be linked mechanistically. Recently, it was demonstrated that alpha-synuclein, which has the ability to readily form amyloid in vitro without the need of other co-factors, can initiate tau amyloid formation. Following this initiation step, alpha-synuclein and tau can synergize the polymerization of each other. Furthermore, increased levels of Abeta peptides in brain can promote the formation of intracellular tau and alpha-synuclein amyloid aggregates, although the mechanism for this process is still unclear. These results indicate that the formation of amyloid composed of different proteins can affect each other directly or indirectly, likely contributing to the overlap in clinical and pathological features.

Amyloid beta-Peptides↗

PubMed2002

The environmental toxin arsenite induces tau hyperphosphorylation.

Abnormally hyperphosphorylated tau polymers known as paired helical filaments constitute one of the major characteristic lesions that lead to the demise of neurons in Alzheimer's disease. Here, we demonstrate that the environmental toxin arsenite causes a significant increase in the phosphorylation of several amino acid residues (Thr-181, Ser-202, Thr-205, Thr-231, Ser-262, Ser-356, Ser-396, and Ser-404) in tau, which are also hyperphosphorylated under pathological conditions. Complementary phosphopeptide mapping revealed a dramatic increase in the (32)P-labeling of many peptides in tau following arsenite treatment. Although arsenite activates extracellular-signal regulated kinases-1/-2 and stress-activated protein kinases, these enzymes did not contribute to the arsenite-increased phosphorylation, nor did they appear to normally modify tau in vivo. Tau phosphorylation induced by arsenite did not involve glycogen synthase kinase-3 or protein phosphatase-1 or -2, but the activity responsible for tau hyperphosphorylation could be inhibited with the protein kinase inhibitor roscovitine. The effects of arsenite on the phosphorylation of some tau mutations (DeltaKappa280, V337M, and R406W) associated with frontal-temporal dementia with parkinsonism linked to chromosome 17 was analyzed. The unchallenged and arsenite-induced phosphorylation of some mutant proteins, especially R406W, was altered at several phosphorylation sites, indicating that these mutations can significantly affect the structure of tau in vivo. Although the major kinase(s) involved in aberrant tau phosphorylation remains elusive, these results indicate that environmental factors, such as arsenite, may be involved in the cascade leading to deregulation of tau function associated with neurodegeneration.

Animals↗

PubMed2002

Gamma-synuclein promotes cancer cell survival and inhibits stress- and chemotherapy drug-induced apoptosis by modulating MAPK pathways.

Synucleins are a family of highly conserved small proteins predominantly expressed in neurons. Recently we and others have found that gamma-synuclein is dramatically up-regulated in the vast majority of late-stage breast and ovarian cancers and that gamma-synuclein over-expression can enhance tumorigenicity. In the current study, we have found that gamma-synuclein is associated with two major mitogen-activated kinases (MAPKs), i.e. extracellular signal-regulated protein kinases (ERK1/2) and c-Jun N-terminal kinase 1 (JNK1), and have shown that over-expression of gamma-synuclein leads to constitutive activation of ERK1/2 and down-regulation of JNK1 in response to a host of environmental stress signals, including UV, arsenate, and heat shock. We also tested the effects of gamma-synuclein on apoptosis and activation of JNK and ERK in response to several chemotherapy drugs. We have found that gamma-synuclein-expressing cells are significantly more resistant to the chemotherapeutic drugs paclitaxel and vinblastine as compared with the parental cells. The resistance to paclitaxel can be partially obliterated when ERK activity is inhibited using a MEK1/2 inhibitor. Activation of JNK and its downstream caspase-3 by paclitaxel or vinblastine is significantly down-regulated in gamma-synuclein-expressing cells, indicating that the paclitaxel- or vinblastine-activated apoptosis pathway is blocked by gamma-synuclein. In contrast to paclitaxel and vinblastine, etoposide does not activate JNK, and gamma-synuclein over-expression has no apparent effect on this drug-induced apoptosis. Taken together, our data indicate that oncogenic activation of gamma-synuclein contributes to the development of breast and ovarian cancer by promoting tumor cell survival under adverse conditions and by providing resistance to certain chemotherapeutic drugs.

Antineoplastic Agents↗

PubMed2002

The relationship between oxidative/nitrative stress and pathological inclusions in Alzheimer's and Parkinson's diseases.

Alzheimer's (AD) and Parkinson's diseases (PD) are late-onset neurodegenerative diseases that have tremendous impact on the lives of affected individuals, their families, and society as a whole. Remarkable efforts are being made to elucidate the dominant factors that result in the pathogenesis of these disorders. Extensive postmortem studies suggest that oxidative/nitrative stresses are prominent features of these diseases, and several animal models support this notion. Furthermore, it is likely that protein modifications resulting from oxidative/nitrative damage contribute to the formation of intracytoplasmic inclusions characteristic of each disease. The frequent presentation of both AD and PD in individuals and the co-occurrence of inclusions characteristic of AD and PD in several other neurodegenerative diseases suggests the involvement of a common underlying aberrant process. It can be surmised that oxidative/nitrative stress, which is cooperatively influenced by environmental factors, genetic predisposition, and senescence, may be a link between these disorders.

Alzheimer Disease↗

PubMed2002

Neuronal alpha-synucleinopathy with severe movement disorder in mice expressing A53T human alpha-synuclein.

alpha-Synucleinopathies are neurodegenerative disorders that range pathologically from the demise of select groups of nuclei to pervasive degeneration throughout the neuraxis. Although mounting evidence suggests that alpha-synuclein lesions lead to neurodegeneration, this remains controversial. To explore this issue, we generated transgenic mice expressing wild-type and A53T human alpha-synuclein in CNS neurons. Mice expressing mutant, but not wild-type, alpha-synuclein developed a severe and complex motor impairment leading to paralysis and death. These animals developed age-dependent intracytoplasmic neuronal alpha-synuclein inclusions paralleling disease onset, and the alpha-synuclein inclusions recapitulated features of human counterparts. Moreover, immunoelectron microscopy revealed that the alpha-synuclein inclusions contained 10-16 nm wide fibrils similar to human pathological inclusions. These mice demonstrate that A53T alpha-synuclein leads to the formation of toxic filamentous alpha-synuclein neuronal inclusions that cause neurodegeneration.

Animals↗

PubMed2002

Concurrence of alpha-synuclein and tau brain pathology in the Contursi kindred.

Previous genetic analysis of the familial Parkinson's disease Contursi kindred led to the identification of an Ala53Thr pathogenic mutation in the alpha-synuclein gene. We have re-examined one of the original brains from this kindred using new immunohistochemical reagents, thioflavin S staining and immunoelectron microscopy. Surprisingly, we uncovered a dense burden of alpha-synuclein neuritic pathology and rare Lewy bodies. Immunoelectron microscopy demonstrated fibrillar alpha-synuclein-immunoreactive aggregates. Unexpected tau neuritic and less frequent perikaryal inclusions were also observed. Some inclusions were comprised of both proteins with almost complete spatial disparity. We suggest that it is important to recognize that the neurodegenerative process caused by the Ala53Thr mutation in alpha-synuclein is not identical to that seen in typical idiopathic Parkinson's disease brains.

Adult↗

PubMed2002

Novel antibodies to synuclein show abundant striatal pathology in Lewy body diseases.

Intracytoplasmic inclusions composed of alpha-synuclein (alpha-syn) are characteristic of neurodegenerative Lewy body disorders. Using novel monoclonal antibodies raised against altered alpha-syn, we uncovered an unprecedented and extensive burden of alpha-syn pathology in the striatum of Lewy body disorders. The highest density of striatal pathology was observed in patients with a combination of Alzheimer's disease and dementia with Lewy bodies or pure dementia with Lewy bodies, and these alpha-syn aggregates may contribute to the parkinsonism seen in these disorders.

Antibodies, Monoclonal↗

PubMed2002

AMPA receptor-mediated toxicity in oligodendrocyte progenitors involves free radical generation and activation of JNK, calpain and caspase 3.

The molecular mechanisms underlying AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate) receptor-mediated excitotoxicity were characterized in rat oligodendrocyte progenitor cultures. Activation of AMPA receptors, in the presence of cyclothiazide to selectively block desensitization, produced a massive Ca(2+) influx and cytotoxicity which were blocked by the antagonists CNQX and GYKI 52466. A role for free radical generation in oligodendrocyte progenitor cell death was deduced from three observations: (i) treatment with AMPA agonists decreased intracellular glutathione; (ii) depletion of intracellular glutathione with buthionine sulfoximine potentiated cell death; and (iii) the antioxidant N -acetylcysteine replenished intracellular glutathione and protected cultures from AMPA receptor-mediated toxicity. Cell death displayed some characteristics of apoptosis, including DNA fragmentation, chromatin condensation and activation of caspase-3 and c-Jun N-terminal kinase (JNK). A substrate of calpain and caspase-3, alpha-spectrin, was cleaved into characteristic products following treatment with AMPA agonists. In contrast, inhibition of either caspase-3 by DEVD-CHO or calpain by PD 150606 protected cells from excitotoxicity. Our results indicate that overactivation of AMPA receptors causes apoptosis in oligodendrocyte progenitors through mechanisms involving Ca(2+) influx, depletion of glutathione, and activation of JNK, calpain, and caspase-3.

Acetylcysteine↗