Parkinson's disease (PD) and other related disorders are characterized by the accumulation of fib... more Parkinson's disease (PD) and other related disorders are characterized by the accumulation of fibrillar aggregates of α-synuclein protein (α-syn) inside brain cells. It is likely that the formation of α-syn aggregates plays a seminal role in the pathogenesis of at least some of these diseases, because two different mutations in the gene encoding α-syn have been found in inherited forms of PD. α-Syn is mainly expressed by neuronal cells and is generally considered to exist as a cytoplasmic protein. Here, we report the unexpected identification of α-syn in conditioned culture media from untransfected and α-syn-transfected human neuroblastoma cells, as well as in human cerebrospinal fluid and blood plasma. The method used was immunocapture by using anti-α-syn antibodies coupled to magnetic beads, followed by detection on Western blots. In all cases, α-syn was identified as a single 15 kDa band, which co-migrated with a recombinant form of the protein and reacted with five different antibodies to α-syn. Our findings suggest that cells normally secrete α-syn into their surrounding media, both in vitro and in vivo. The detection of extracellular α-syn and/or its modified forms in body fluids, particularly in human plasma, offers new opportunities for the development of diagnostic tests for PD and related diseases.
Background: Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy... more Background: Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA) are all neurodegenerative diseases, collectively called 'synucleinopathies', which are characterised by the accumulation of fibrillar aggregates of c~ -synuclein protein (c~-syn) inside brain cells. It is likely that the formation of c~-syn aggregates plays a seminal role in the pathogenesis of at least some of these diseases, since three different mutations in the gene encoding c<-syn have been found in inherited forms of PD. It has been reported that lesions similar to those found in the PD brains can be created in transgenic animals over-express human ct-syn, and progressively develop a loss of dopaminergic cells together with motor abnormalities. Objective(s): Inhibiting and/or reversing ct-syn self-aggregation could, therefore, provide a novel approach to treating the underlying cause of these diseases. Methods: We synthesised a library of overlapping 7-met peptides spanning the entire et-syn sequence, and identified the binding region responsible for its self-association. Results:
There is substantial biochemical, pathological, and genetic evidence that α-synuclein (A-syn) is ... more There is substantial biochemical, pathological, and genetic evidence that α-synuclein (A-syn) is a principal molecule in the pathogenesis of Parkinson disease (PD). We previously reported that total A-syn levels in cerebrospinal fluid (CSF), measured with the specific enzyme-linked immunosorbent assay (ELISA) developed by ourselves, were decreased in patients with PD, and suggested the usefulness of A-syn in CSF and plasma as a biomarker for the diagnosis of PD. After our report, a considerable number of studies have investigated the levels A-syn in CSF and in blood, but have reported inconclusive results. Such discrepancies have often been attributed not only to the use of different antibodies in the ELISAs but also to interference from hemolysis. In this study we measured the levels of A-syn in CSF and plasma by using our own sandwich ELISA with or without heterophilic antibody (HA) inhibitor in 30 patients with PD and 58 age-matched controls. We thereby revealed that HA interfere...
Phosphorylation of the α-synuclein (α-syn) protein at Ser129 [P(S129)-α-] was found to be the mos... more Phosphorylation of the α-synuclein (α-syn) protein at Ser129 [P(S129)-α-] was found to be the most abundant form in intracellular inclusions in brains from Parkinson's disease (PD) patients. This finding suggests that P(S129)-α-syn plays a central role in the pathogenesis of PD. However, it is at present unclear whether P(S129)-α-syn is pathogenic driving the neurodegenerative process. Rodent studies using neither the phosphomimics of human α-syn nor co-expression of human wild-type α-syn and kinases phosphorylating α-syn at Ser129 gave consistent results. One major concern in interpreting these findings is that human α-syn was expressed above physiological levels inducing neurodegeneration in rat nigral neurons. In order to exclude this confounding factor, we took a different approach and increased the phosphorylation level of endogenous α-syn. For this purpose, we took advantage of recombinant adeno-associated viral (rAAV) vectors to deliver polo-like kinase 2 (PLK2) or PLK3 i...
The pursuit of laboratory tests that allow for the reliable and inexpensive identification of sub... more The pursuit of laboratory tests that allow for the reliable and inexpensive identification of subjects with parkinsonism represents a hot topic in translational neuroscience. This unmet need affects the counseling of presymptomatic, at-risk subjects and delays the accurate diagnosis of already symptomatic individuals. The absence of validated markers that are closely linked to the pathological disease process also compromises the objective monitoring of therapeutic interventions in clinical trials. Typical Parkinson's disease represents a heterogenous syndrome (but the majority of patients suffer from neurodegeneration) that is linked to the misprocessing of α-synuclein (α-Syn). The identification of α-Syn as a bona fide constituent of human cerebrospinal fluid and its quantification in early cross-sectional studies represent the beginning of a new chapter in Parkinson's disease research. It will determine what role, if any, cerebrospinal fluid α-Syn plays as a biomarker can...
Biochemical and biophysical research communications, Jan 13, 2006
There is ample biochemical, pathological, and genetic evidence that the metabolism of alpha-synuc... more There is ample biochemical, pathological, and genetic evidence that the metabolism of alpha-synuclein (alpha-syn) plays a crucial role in the pathogenesis of Parkinson disease (PD). To examine whether quantification of alpha-syn in cerebrospinal fluid (CSF) is potentially informative in the diagnosis of PD, we developed a specific ELISA system and measured the concentration of alpha-syn in CSF from 33 patients with PD (diagnosed according to UK PD Society Brain Bank criteria) and 38 control subjects including 9 neurologically healthy individuals. We found that PD patients had significantly lower alpha-syn levels in their CSF than the control groups (p<0.0001) even after adjusting for gender and age. Age was independently associated with lower alpha-syn levels. Logistic regression analysis showed that reduction in CSF alpha-syn served as a significant predictor of PD beyond age and gender alone (area under ROC curve, c=0.882). Furthermore, we observed a close inverse correlation b...
Parkinson disease (PD) is the second most prevalent neurodegenerative disorder after Alzheimer... more Parkinson disease (PD) is the second most prevalent neurodegenerative disorder after Alzheimer's disease (AD). The formation of the cytoplasmic inclusions named "Lewy bodies" in the brain, considered to be a marker for neuronal degeneration in PD and dementia with Lewy bodies. However, Lewy bodies (LBs) are also observed in approximately 60 percent of both sporadic and familial cases with AD. LBs consist of fibrils mainly formed by post-translational modified α-synuclein (α-syn) protein. The modifications can be truncation, phosphorylation, nitration and mono-, di-, or tri-ubiquitination. Development of disease seems to be linked to events that increase the concentration of α-syn or cause its chemical modification, either of which can accelerate α-syn aggregation. Examples of such events include increased copy number of genes, decreased rate of degradation via the proteasome or other proteases, or modified forms of α-syn. As the aggregation of α-syn in the brain has be...
15-fold increase in xCT RNA levels in the cell. Furthermore, we show that the anti-inflammatory i... more 15-fold increase in xCT RNA levels in the cell. Furthermore, we show that the anti-inflammatory interleukins (IL)-4, IL-10 and IL-13 are capable of attenuating the AIMnduced microglial glutamate secretion by more than half. Conclusions: Given these results on glutamate release and xCT gene regulation together with our earlier reports that IL-4, IL-10, and IL-13 are found in the brain and suppress cytokine and chemokine release from A~activated microglia, we propose that glutamatergic disruption in AD may in part be mediated by chronic inflammation and microglial-derived glutamate release inadequately countered by endogenous anti-inflammatory cytokines.
Elevated cerebrospinal fluid concentrations of tau discriminate Alzheimer's disease from othe... more Elevated cerebrospinal fluid concentrations of tau discriminate Alzheimer's disease from other neurodegenerative conditions. The reasons for this are unclear. While commercial assay kits are widely used to determine total-tau concentrations, little is known about their ability to detect different aggregation states of tau. We demonstrate that the leading commercial enzyme-linked immunosorbent assay reliably detects aggregated and monomeric tau and evinces good recovery of both species when added into cerebrospinal fluid. Hence, the disparity between total-tau levels encountered in Alzheimer's disease and other neurodegenerative conditions is not due to differential recognition of tau assembly forms or the extent of degeneration.
Lesions known as Lewy bodies (LBs) and Lewy neurites (LNs) characterise brains of Parkinson's... more Lesions known as Lewy bodies (LBs) and Lewy neurites (LNs) characterise brains of Parkinson's disease (PD) patients. Intracellular aggregation of alpha-synuclein (alpha-syn) appears to play a key role in the generation of LBs and LNs. Such aggregation in the presence of redox metals may initiate Fenton reaction-mediated generation of reactive oxygen species (ROS). ROS thus generated may result in cytotoxic mechanisms such as the induction of DNA single-strand breaks.
The deposition of abnormal protein fibrils is a prominent pathological feature of many different ... more The deposition of abnormal protein fibrils is a prominent pathological feature of many different 'protein conformational' diseases, including some important neurodegenerative diseases. Some of the fibril-forming proteins or peptides associated with these diseases have been shown to be toxic to cells in culture. A clear understanding of the molecular mechanisms responsible for this toxicity should shed light on the probable link between protein deposition and cell loss in these diseases. In the case of the β-amyloid (Aβ) peptide, which accumulates in the brain in Alzheimer's disease, there is good evidence that the toxic mechanism involves the production of reactive oxygen species (ROS). By means of an electron spin resonance (ESR) spin-trapping method, we have shown that solutions of Aβ liberate hydroxyl radicals when incubated in vitro, upon the addition of small amounts of Fe(II). We have also obtained similar results with α-synuclein, which accumulates in Lewy bodies in Parkinson's disease, and with the PrP (106-126) toxic fragment of the prion protein. It is becoming clear that some transition metal ions, especially Fe(III) and Cu(II), can bind to these aggregating peptides, and that some of them can reduce the oxidation state of Fe(III) and/or Cu(II). The data suggest that hydrogen peroxide accumulates during incubation of these various proteins and peptides, and is subsequently converted to hydroxyl radicals in the presence of redox-active transition metal ions. Consequently, a fundamental molecular mechanism underlying the pathogenesis of cell death in several different neurodegenerative diseases could be the direct production of ROS during formation of the abnormal protein aggregates.
Current Medicinal Chemistry - Immunology, Endocrine & Metabolic Agents, 2003
The deposition of abnormal protein fibrils is a prominent pathological feature of many different ... more The deposition of abnormal protein fibrils is a prominent pathological feature of many different 'protein conformational' diseases, including some important neurodegenerative diseases. Some of the fibril-forming proteins or peptides associated with these diseases have been shown to be toxic to cells in culture. A clear understanding of the molecular mechanisms responsible for this toxicity should shed light on the probable link between protein deposition and cell loss in these diseases. In the case of the β-amyloid (Aβ) peptide, which accumulates in the brain in Alzheimer's disease, there is good evidence that the toxic mechanism involves the production of reactive oxygen species (ROS). By means of an electron spin resonance (ESR) spin-trapping method, we have shown that solutions of Aβ liberate hydroxyl radicals when incubated in vitro, upon the addition of small amounts of Fe(II). We have also obtained similar results with α-synuclein, which accumulates in Lewy bodies in Parkinson's disease, and with the PrP (106-126) toxic fragment of the prion protein. It is becoming clear that some transition metal ions, especially Fe(III) and Cu(II), can bind to these aggregating peptides, and that some of them can reduce the oxidation state of Fe(III) and/or Cu(II). The data suggest that hydrogen peroxide accumulates during incubation of these various proteins and peptides, and is subsequently converted to hydroxyl radicals in the presence of redox-active transition metal ions. Consequently, a fundamental molecular mechanism underlying the pathogenesis of cell death in several different neurodegenerative diseases could be the direct production of ROS during formation of the abnormal protein aggregates.
A number of human neurodegenerative diseases involve aggregated amyloid proteins in the brain, e.... more A number of human neurodegenerative diseases involve aggregated amyloid proteins in the brain, e.g. Alzheimer&#39;s disease (beta-amyloid) and Parkinson&#39;s disease (alpha-synuclein). Other examples are rare familial dementias which involve the BRI gene. In a British family, mutation of the termination codon extends the reading frame of BRI to yield a furin-processed 34-residue peptide (Abri; British dementia peptide), 11 residues longer than the wild-type (WT). In a Danish family, a ten-base insertion also yields a 34-residue peptide (Adan; Danish dementia peptide). To explore the roles of Abri and Adan in neurodegeneration, we synthesized Abri and Adan in oxidized and reduced forms and generated transgenic mice colonies expressing the WT and mutated forms of BRI. We have generated transgenic mice colonies bearing the genes coding for WT-BRI, Adan and Abri under the control of the Thy1 promoter. Whereas WT-BRI transgenic mice express full-length WT-BRI protein in their brains, Adan protein is fully processed to small peptides.
Developing effective treatments for neurodegenerative diseases is one of the greatest medical cha... more Developing effective treatments for neurodegenerative diseases is one of the greatest medical challenges of the 21st century. Although many of these clinical entities have been recognized for more than a hundred years, it is only during the past twenty years that the molecular events that precipitate disease have begun to be understood. Protein aggregation is a common feature of many neurodegenerative diseases, and it is assumed that the aggregation process plays a central role in pathogenesis. In this process, one molecule (monomer) of a soluble protein interacts with other monomers of the same protein to form dimers, oligomers, and polymers. Conformation changes in three-dimensional structure of the protein, especially the formation of beta-strands, often accompany the process. Eventually, as the size of the aggregates increases, they may precipitate as insoluble amyloid fibrils, in which the structure is stabilized by the beta-strands interacting within a beta-sheet. In this revi...
Examination of the N-terminal sequence of non-Aβ component of Alzheimer's Disease amyloid (NAC) r... more Examination of the N-terminal sequence of non-Aβ component of Alzheimer's Disease amyloid (NAC) revealed a degree of similarity to regions crucial for aggregation and toxicity of three other amyloidogenic proteins, namely amyloid β peptide (Aβ), prion protein (PrP) and islet amyloid polypeptide (IAPP), leading us to believe that this might be the part of the molecule responsible for causing aggregation. Secondary structure prediction analysis of NAC indicated that the N-terminal half was likely to form a β-structure whereas the C-terminal half was likely to form an A-helix. NAC in solution altered from random coil to β-sheet structure upon ageing, a process that has previously been shown to lead to fibril formation. To delineate the region of NAC responsible for aggregation we synthesised two fragments, NAC-(1Ϫ18)-peptide and NAC-(19Ϫ35)-peptide, and examined their physicochemical properties. Upon incubation, solutions of NAC-(1Ϫ18)-peptide became congophilic and aggregated to form fibrils of diameter 5Ϫ10 nm, whereas NAC-(19Ϫ35)-peptide did not bind Congo Red and remained in solution. Circular dichroism spectroscopy was used to study the secondary structure of NAC and the two fragments. In trifluoroethanol/water mixtures, NAC and NAC-(19Ϫ35)-peptide adopted A-helical structure but NAC-(1Ϫ18)-peptide did not. NAC-(1Ϫ18)-peptide and NAC formed β-sheet in acetonitrile/water mixtures more readily than did NAC-(19Ϫ35)-peptide. CD spectra of NAC or NAC-(1Ϫ18)-peptide in aqueous solution indicate the formation of β-sheet on ageing. We propose that the N-terminal region of NAC is the principal determinant of aggregation. Our results indicate that NAC resembles Aβ, and other amyloidogenic proteins, in that aggregation is dependent upon β-sheet development. These results lend support to a role for NAC in the development of neurodegenerative disease.
A novel ELISA has been developed which detects oligomerization of beta-amyloid (A beta). Oligomer... more A novel ELISA has been developed which detects oligomerization of beta-amyloid (A beta). Oligomerization, fibrillization and neurotoxicity of native A beta associated with Alzheimer&#39;s disease (AD) type has been compared with E22Q A beta (amyloid beta-protein containing residues 1--40 with the native Glu at residue 22 changed to Gln) implicated in Dutch cerebral haemorrhage disease. Solutions of A beta rapidly yield soluble oligomers in a concentration-dependent manner, which are detected by the ELISA, and by size-exclusion gel chromatography. Conformational changes from disordered to beta-sheet occur more slowly than oligomerization, and fibrils are produced after prolonged incubation. The E22Q A beta oligomerizes, changes conformation and fibrillizes more rapidly than the native form and produces shorter stubbier fibrils. Aged fibrillar preparations of E22Q A beta are more potent than aged fibrils of native A beta in inducing apoptotic changes and toxic responses in human neuroblastoma cell lines, whereas low-molecular-mass oligomers in briefly incubated solutions are much less potent. The differences in the rates of oligomerization of the two A beta forms, their conformational behaviour over a range of pH values, and NMR data reported elsewhere, are consistent with a molecular model of oligomerization in which strands of A beta monomers initially overcome charge repulsion to form dimers in parallel beta-sheet arrangement, stabilized by intramolecular hydrophobic interactions, with amino acids of adjacent chains in register.
Amyloid deposits derived from the amylin peptide accumulate within pancreatic islet b-cells in mo... more Amyloid deposits derived from the amylin peptide accumulate within pancreatic islet b-cells in most cases of type-2 diabetes mellitus (T2Dm). Human amylin 'oligomers' are toxic to these cells. Using two different experimental techniques, we found that H 2 O 2 was generated during the aggregation of human amylin into amyloid fibrils. This process was greatly stimulated by Cu(II) ions, and human amylin was retained on a copper affinity column. In contrast, rodent amylin, which is not toxic, failed to generate any H 2 O 2 and did not interact with copper. We conclude that the formation of H 2 O 2 from amylin could contribute to the progressive degeneration of islet cells in T2Dm.
Parkinson's disease (PD) and other related disorders are characterized by the accumulation of fib... more Parkinson's disease (PD) and other related disorders are characterized by the accumulation of fibrillar aggregates of α-synuclein protein (α-syn) inside brain cells. It is likely that the formation of α-syn aggregates plays a seminal role in the pathogenesis of at least some of these diseases, because two different mutations in the gene encoding α-syn have been found in inherited forms of PD. α-Syn is mainly expressed by neuronal cells and is generally considered to exist as a cytoplasmic protein. Here, we report the unexpected identification of α-syn in conditioned culture media from untransfected and α-syn-transfected human neuroblastoma cells, as well as in human cerebrospinal fluid and blood plasma. The method used was immunocapture by using anti-α-syn antibodies coupled to magnetic beads, followed by detection on Western blots. In all cases, α-syn was identified as a single 15 kDa band, which co-migrated with a recombinant form of the protein and reacted with five different antibodies to α-syn. Our findings suggest that cells normally secrete α-syn into their surrounding media, both in vitro and in vivo. The detection of extracellular α-syn and/or its modified forms in body fluids, particularly in human plasma, offers new opportunities for the development of diagnostic tests for PD and related diseases.
Background: Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy... more Background: Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA) are all neurodegenerative diseases, collectively called 'synucleinopathies', which are characterised by the accumulation of fibrillar aggregates of c~ -synuclein protein (c~-syn) inside brain cells. It is likely that the formation of c~-syn aggregates plays a seminal role in the pathogenesis of at least some of these diseases, since three different mutations in the gene encoding c<-syn have been found in inherited forms of PD. It has been reported that lesions similar to those found in the PD brains can be created in transgenic animals over-express human ct-syn, and progressively develop a loss of dopaminergic cells together with motor abnormalities. Objective(s): Inhibiting and/or reversing ct-syn self-aggregation could, therefore, provide a novel approach to treating the underlying cause of these diseases. Methods: We synthesised a library of overlapping 7-met peptides spanning the entire et-syn sequence, and identified the binding region responsible for its self-association. Results:
There is substantial biochemical, pathological, and genetic evidence that α-synuclein (A-syn) is ... more There is substantial biochemical, pathological, and genetic evidence that α-synuclein (A-syn) is a principal molecule in the pathogenesis of Parkinson disease (PD). We previously reported that total A-syn levels in cerebrospinal fluid (CSF), measured with the specific enzyme-linked immunosorbent assay (ELISA) developed by ourselves, were decreased in patients with PD, and suggested the usefulness of A-syn in CSF and plasma as a biomarker for the diagnosis of PD. After our report, a considerable number of studies have investigated the levels A-syn in CSF and in blood, but have reported inconclusive results. Such discrepancies have often been attributed not only to the use of different antibodies in the ELISAs but also to interference from hemolysis. In this study we measured the levels of A-syn in CSF and plasma by using our own sandwich ELISA with or without heterophilic antibody (HA) inhibitor in 30 patients with PD and 58 age-matched controls. We thereby revealed that HA interfere...
Phosphorylation of the α-synuclein (α-syn) protein at Ser129 [P(S129)-α-] was found to be the mos... more Phosphorylation of the α-synuclein (α-syn) protein at Ser129 [P(S129)-α-] was found to be the most abundant form in intracellular inclusions in brains from Parkinson's disease (PD) patients. This finding suggests that P(S129)-α-syn plays a central role in the pathogenesis of PD. However, it is at present unclear whether P(S129)-α-syn is pathogenic driving the neurodegenerative process. Rodent studies using neither the phosphomimics of human α-syn nor co-expression of human wild-type α-syn and kinases phosphorylating α-syn at Ser129 gave consistent results. One major concern in interpreting these findings is that human α-syn was expressed above physiological levels inducing neurodegeneration in rat nigral neurons. In order to exclude this confounding factor, we took a different approach and increased the phosphorylation level of endogenous α-syn. For this purpose, we took advantage of recombinant adeno-associated viral (rAAV) vectors to deliver polo-like kinase 2 (PLK2) or PLK3 i...
The pursuit of laboratory tests that allow for the reliable and inexpensive identification of sub... more The pursuit of laboratory tests that allow for the reliable and inexpensive identification of subjects with parkinsonism represents a hot topic in translational neuroscience. This unmet need affects the counseling of presymptomatic, at-risk subjects and delays the accurate diagnosis of already symptomatic individuals. The absence of validated markers that are closely linked to the pathological disease process also compromises the objective monitoring of therapeutic interventions in clinical trials. Typical Parkinson's disease represents a heterogenous syndrome (but the majority of patients suffer from neurodegeneration) that is linked to the misprocessing of α-synuclein (α-Syn). The identification of α-Syn as a bona fide constituent of human cerebrospinal fluid and its quantification in early cross-sectional studies represent the beginning of a new chapter in Parkinson's disease research. It will determine what role, if any, cerebrospinal fluid α-Syn plays as a biomarker can...
Biochemical and biophysical research communications, Jan 13, 2006
There is ample biochemical, pathological, and genetic evidence that the metabolism of alpha-synuc... more There is ample biochemical, pathological, and genetic evidence that the metabolism of alpha-synuclein (alpha-syn) plays a crucial role in the pathogenesis of Parkinson disease (PD). To examine whether quantification of alpha-syn in cerebrospinal fluid (CSF) is potentially informative in the diagnosis of PD, we developed a specific ELISA system and measured the concentration of alpha-syn in CSF from 33 patients with PD (diagnosed according to UK PD Society Brain Bank criteria) and 38 control subjects including 9 neurologically healthy individuals. We found that PD patients had significantly lower alpha-syn levels in their CSF than the control groups (p<0.0001) even after adjusting for gender and age. Age was independently associated with lower alpha-syn levels. Logistic regression analysis showed that reduction in CSF alpha-syn served as a significant predictor of PD beyond age and gender alone (area under ROC curve, c=0.882). Furthermore, we observed a close inverse correlation b...
Parkinson disease (PD) is the second most prevalent neurodegenerative disorder after Alzheimer... more Parkinson disease (PD) is the second most prevalent neurodegenerative disorder after Alzheimer's disease (AD). The formation of the cytoplasmic inclusions named "Lewy bodies" in the brain, considered to be a marker for neuronal degeneration in PD and dementia with Lewy bodies. However, Lewy bodies (LBs) are also observed in approximately 60 percent of both sporadic and familial cases with AD. LBs consist of fibrils mainly formed by post-translational modified α-synuclein (α-syn) protein. The modifications can be truncation, phosphorylation, nitration and mono-, di-, or tri-ubiquitination. Development of disease seems to be linked to events that increase the concentration of α-syn or cause its chemical modification, either of which can accelerate α-syn aggregation. Examples of such events include increased copy number of genes, decreased rate of degradation via the proteasome or other proteases, or modified forms of α-syn. As the aggregation of α-syn in the brain has be...
15-fold increase in xCT RNA levels in the cell. Furthermore, we show that the anti-inflammatory i... more 15-fold increase in xCT RNA levels in the cell. Furthermore, we show that the anti-inflammatory interleukins (IL)-4, IL-10 and IL-13 are capable of attenuating the AIMnduced microglial glutamate secretion by more than half. Conclusions: Given these results on glutamate release and xCT gene regulation together with our earlier reports that IL-4, IL-10, and IL-13 are found in the brain and suppress cytokine and chemokine release from A~activated microglia, we propose that glutamatergic disruption in AD may in part be mediated by chronic inflammation and microglial-derived glutamate release inadequately countered by endogenous anti-inflammatory cytokines.
Elevated cerebrospinal fluid concentrations of tau discriminate Alzheimer's disease from othe... more Elevated cerebrospinal fluid concentrations of tau discriminate Alzheimer's disease from other neurodegenerative conditions. The reasons for this are unclear. While commercial assay kits are widely used to determine total-tau concentrations, little is known about their ability to detect different aggregation states of tau. We demonstrate that the leading commercial enzyme-linked immunosorbent assay reliably detects aggregated and monomeric tau and evinces good recovery of both species when added into cerebrospinal fluid. Hence, the disparity between total-tau levels encountered in Alzheimer's disease and other neurodegenerative conditions is not due to differential recognition of tau assembly forms or the extent of degeneration.
Lesions known as Lewy bodies (LBs) and Lewy neurites (LNs) characterise brains of Parkinson's... more Lesions known as Lewy bodies (LBs) and Lewy neurites (LNs) characterise brains of Parkinson's disease (PD) patients. Intracellular aggregation of alpha-synuclein (alpha-syn) appears to play a key role in the generation of LBs and LNs. Such aggregation in the presence of redox metals may initiate Fenton reaction-mediated generation of reactive oxygen species (ROS). ROS thus generated may result in cytotoxic mechanisms such as the induction of DNA single-strand breaks.
The deposition of abnormal protein fibrils is a prominent pathological feature of many different ... more The deposition of abnormal protein fibrils is a prominent pathological feature of many different 'protein conformational' diseases, including some important neurodegenerative diseases. Some of the fibril-forming proteins or peptides associated with these diseases have been shown to be toxic to cells in culture. A clear understanding of the molecular mechanisms responsible for this toxicity should shed light on the probable link between protein deposition and cell loss in these diseases. In the case of the β-amyloid (Aβ) peptide, which accumulates in the brain in Alzheimer's disease, there is good evidence that the toxic mechanism involves the production of reactive oxygen species (ROS). By means of an electron spin resonance (ESR) spin-trapping method, we have shown that solutions of Aβ liberate hydroxyl radicals when incubated in vitro, upon the addition of small amounts of Fe(II). We have also obtained similar results with α-synuclein, which accumulates in Lewy bodies in Parkinson's disease, and with the PrP (106-126) toxic fragment of the prion protein. It is becoming clear that some transition metal ions, especially Fe(III) and Cu(II), can bind to these aggregating peptides, and that some of them can reduce the oxidation state of Fe(III) and/or Cu(II). The data suggest that hydrogen peroxide accumulates during incubation of these various proteins and peptides, and is subsequently converted to hydroxyl radicals in the presence of redox-active transition metal ions. Consequently, a fundamental molecular mechanism underlying the pathogenesis of cell death in several different neurodegenerative diseases could be the direct production of ROS during formation of the abnormal protein aggregates.
Current Medicinal Chemistry - Immunology, Endocrine & Metabolic Agents, 2003
The deposition of abnormal protein fibrils is a prominent pathological feature of many different ... more The deposition of abnormal protein fibrils is a prominent pathological feature of many different 'protein conformational' diseases, including some important neurodegenerative diseases. Some of the fibril-forming proteins or peptides associated with these diseases have been shown to be toxic to cells in culture. A clear understanding of the molecular mechanisms responsible for this toxicity should shed light on the probable link between protein deposition and cell loss in these diseases. In the case of the β-amyloid (Aβ) peptide, which accumulates in the brain in Alzheimer's disease, there is good evidence that the toxic mechanism involves the production of reactive oxygen species (ROS). By means of an electron spin resonance (ESR) spin-trapping method, we have shown that solutions of Aβ liberate hydroxyl radicals when incubated in vitro, upon the addition of small amounts of Fe(II). We have also obtained similar results with α-synuclein, which accumulates in Lewy bodies in Parkinson's disease, and with the PrP (106-126) toxic fragment of the prion protein. It is becoming clear that some transition metal ions, especially Fe(III) and Cu(II), can bind to these aggregating peptides, and that some of them can reduce the oxidation state of Fe(III) and/or Cu(II). The data suggest that hydrogen peroxide accumulates during incubation of these various proteins and peptides, and is subsequently converted to hydroxyl radicals in the presence of redox-active transition metal ions. Consequently, a fundamental molecular mechanism underlying the pathogenesis of cell death in several different neurodegenerative diseases could be the direct production of ROS during formation of the abnormal protein aggregates.
A number of human neurodegenerative diseases involve aggregated amyloid proteins in the brain, e.... more A number of human neurodegenerative diseases involve aggregated amyloid proteins in the brain, e.g. Alzheimer&#39;s disease (beta-amyloid) and Parkinson&#39;s disease (alpha-synuclein). Other examples are rare familial dementias which involve the BRI gene. In a British family, mutation of the termination codon extends the reading frame of BRI to yield a furin-processed 34-residue peptide (Abri; British dementia peptide), 11 residues longer than the wild-type (WT). In a Danish family, a ten-base insertion also yields a 34-residue peptide (Adan; Danish dementia peptide). To explore the roles of Abri and Adan in neurodegeneration, we synthesized Abri and Adan in oxidized and reduced forms and generated transgenic mice colonies expressing the WT and mutated forms of BRI. We have generated transgenic mice colonies bearing the genes coding for WT-BRI, Adan and Abri under the control of the Thy1 promoter. Whereas WT-BRI transgenic mice express full-length WT-BRI protein in their brains, Adan protein is fully processed to small peptides.
Developing effective treatments for neurodegenerative diseases is one of the greatest medical cha... more Developing effective treatments for neurodegenerative diseases is one of the greatest medical challenges of the 21st century. Although many of these clinical entities have been recognized for more than a hundred years, it is only during the past twenty years that the molecular events that precipitate disease have begun to be understood. Protein aggregation is a common feature of many neurodegenerative diseases, and it is assumed that the aggregation process plays a central role in pathogenesis. In this process, one molecule (monomer) of a soluble protein interacts with other monomers of the same protein to form dimers, oligomers, and polymers. Conformation changes in three-dimensional structure of the protein, especially the formation of beta-strands, often accompany the process. Eventually, as the size of the aggregates increases, they may precipitate as insoluble amyloid fibrils, in which the structure is stabilized by the beta-strands interacting within a beta-sheet. In this revi...
Examination of the N-terminal sequence of non-Aβ component of Alzheimer's Disease amyloid (NAC) r... more Examination of the N-terminal sequence of non-Aβ component of Alzheimer's Disease amyloid (NAC) revealed a degree of similarity to regions crucial for aggregation and toxicity of three other amyloidogenic proteins, namely amyloid β peptide (Aβ), prion protein (PrP) and islet amyloid polypeptide (IAPP), leading us to believe that this might be the part of the molecule responsible for causing aggregation. Secondary structure prediction analysis of NAC indicated that the N-terminal half was likely to form a β-structure whereas the C-terminal half was likely to form an A-helix. NAC in solution altered from random coil to β-sheet structure upon ageing, a process that has previously been shown to lead to fibril formation. To delineate the region of NAC responsible for aggregation we synthesised two fragments, NAC-(1Ϫ18)-peptide and NAC-(19Ϫ35)-peptide, and examined their physicochemical properties. Upon incubation, solutions of NAC-(1Ϫ18)-peptide became congophilic and aggregated to form fibrils of diameter 5Ϫ10 nm, whereas NAC-(19Ϫ35)-peptide did not bind Congo Red and remained in solution. Circular dichroism spectroscopy was used to study the secondary structure of NAC and the two fragments. In trifluoroethanol/water mixtures, NAC and NAC-(19Ϫ35)-peptide adopted A-helical structure but NAC-(1Ϫ18)-peptide did not. NAC-(1Ϫ18)-peptide and NAC formed β-sheet in acetonitrile/water mixtures more readily than did NAC-(19Ϫ35)-peptide. CD spectra of NAC or NAC-(1Ϫ18)-peptide in aqueous solution indicate the formation of β-sheet on ageing. We propose that the N-terminal region of NAC is the principal determinant of aggregation. Our results indicate that NAC resembles Aβ, and other amyloidogenic proteins, in that aggregation is dependent upon β-sheet development. These results lend support to a role for NAC in the development of neurodegenerative disease.
A novel ELISA has been developed which detects oligomerization of beta-amyloid (A beta). Oligomer... more A novel ELISA has been developed which detects oligomerization of beta-amyloid (A beta). Oligomerization, fibrillization and neurotoxicity of native A beta associated with Alzheimer&#39;s disease (AD) type has been compared with E22Q A beta (amyloid beta-protein containing residues 1--40 with the native Glu at residue 22 changed to Gln) implicated in Dutch cerebral haemorrhage disease. Solutions of A beta rapidly yield soluble oligomers in a concentration-dependent manner, which are detected by the ELISA, and by size-exclusion gel chromatography. Conformational changes from disordered to beta-sheet occur more slowly than oligomerization, and fibrils are produced after prolonged incubation. The E22Q A beta oligomerizes, changes conformation and fibrillizes more rapidly than the native form and produces shorter stubbier fibrils. Aged fibrillar preparations of E22Q A beta are more potent than aged fibrils of native A beta in inducing apoptotic changes and toxic responses in human neuroblastoma cell lines, whereas low-molecular-mass oligomers in briefly incubated solutions are much less potent. The differences in the rates of oligomerization of the two A beta forms, their conformational behaviour over a range of pH values, and NMR data reported elsewhere, are consistent with a molecular model of oligomerization in which strands of A beta monomers initially overcome charge repulsion to form dimers in parallel beta-sheet arrangement, stabilized by intramolecular hydrophobic interactions, with amino acids of adjacent chains in register.
Amyloid deposits derived from the amylin peptide accumulate within pancreatic islet b-cells in mo... more Amyloid deposits derived from the amylin peptide accumulate within pancreatic islet b-cells in most cases of type-2 diabetes mellitus (T2Dm). Human amylin 'oligomers' are toxic to these cells. Using two different experimental techniques, we found that H 2 O 2 was generated during the aggregation of human amylin into amyloid fibrils. This process was greatly stimulated by Cu(II) ions, and human amylin was retained on a copper affinity column. In contrast, rodent amylin, which is not toxic, failed to generate any H 2 O 2 and did not interact with copper. We conclude that the formation of H 2 O 2 from amylin could contribute to the progressive degeneration of islet cells in T2Dm.
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Papers by Omar El-agnaf