David Allsop

Amyloid deposition as the central event in the aetiology of Alzheimer's disease.

While there may be many causes of Alzheimers disease (AD), the same pathological sequence of events, described here by John Hardy and David Allsop, is likely to occur in all cases. The recent discovery of a pathogenic mutation in the beta-amyloid precursor protein (APP) gene on chromosome 21 suggests that APP Mismetabolism and beta-amyloid deposition are the primary events in the disease process. The occurrence of AD in Down syndrome is consistent with this hypothesis. The pathological cascade for the disease process is most likely to be: beta-amyloid deposition----tau phosphorylation and tangle formation----neuronal death. The development of a biochemical understanding of this pathological cascade will facilitate rational design of drugs to intervene in this process.

Detection of oligomeric forms of α-synuclein protein in human plasma as a potential biomarker for Parkinson’s disease

To date there is no accepted clinical diagnostic test for Parkinsons disease (PD) based on biochemical analysis of blood or cerebrospinal fluid (CSF). α‐Synuclein (α‐syn) protein has been linked to the pathogenesis of PD with the discovery of mutations in the gene encoding α‐syn in familial cases with early‐onset PD. Lewy bodies and Lewy neurites, which constitute the main pathological features in the brains of patients with sporadic PD and dementia with Lewy bodies, are formed by the conversion of soluble monomers of α‐syn into insoluble aggregates. We recently reported the presence of α‐syn in normal human blood plasma and in postmortem CSF. Here, we investigated whether α‐syn can be used as a biomarker for PD. We have developed a novel ELISA method that detects only oligomeric “soluble aggregates” of α‐syn. Using this ELISA, we report the presence of significantly elevated (P=0.002) levels of oligomeric forms of α‐syn in plasma samples obtained from 34 PD patients compared with 27 controls; 52% (95% confidence intervals 0.353–0.687) of the PD patients displayed signals >0.5 OD with our ELISA assay in comparison to only 14.8% (95% confidence intervals 0.014 –0.281) for the control cases. An analysis of the tests diagnostic value revealed a specificity of 0.852 (95% confidence intervals 0.662– 0.958), sensitivity of 0.529 (95% confidence intervals 0.351–0.702) and a positive predictive value of 0.818 (95% confidence intervals 0.597–0.948). These observations offer new opportunities for developing diagnostic tests for PD and related diseases and for testing therapeutic agents aimed at preventing or reversing the aggregation of α‐syn.—El‐Agnaf, O. M. A., Salem, S. A., Paleologou, K. W., Curran, M. D., Gibson, M. J., Court, J. A., Schlossmacher, M. G., Allsop, D. Detection of oligomeric forms of α‐synuclein protein in human plasma as a potential biomarker for Parkinsons disease. FASEB J. 20, 419 –425 (2006)

α-Synuclein implicated in Parkinson’s disease is present in extracellular biological fluids, including human plasma

Parkinsons 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.

Inflammatory mechanisms in Alzheimer's disease

Alzheimers disease is aetiologically heterogeneous, but the pathogenesis is often considered to be initiated by the deposition of amyloid fibrils, followed by neuritic tau pathology and neuronal death. A variety of inflammatory proteins has been identified in the brains of patients with Alzheimers disease post mortem. In this article, Piet Eikelenboom and colleagues review evidence to suggest that the inflammatory processes are intimately involved in several crucial events in the pathological cascade. This suggests possibilities for the treatment of Alzheimers disease with anti-inflammatory drugs.

Formation of hydrogen peroxide and hydroxyl radicals from Aβ and α-synuclein as a possible mechanism of cell death in Alzheimer's disease and Parkinson's disease

The formation of extracellular or intracellular deposits of amyloid-like protein fibrils is a prominent pathological feature of many different neurodegenerative diseases, including Alzheimers disease (AD) and Parkinsons disease (PD). In AD, the beta-amyloid peptide (A(beta)) accumulates mainly extracellularly at the center of senile plaques, whereas, in PD, the alpha-synuclein protein accumulates within neurons inside the Lewy bodies and Lewy neurites. We have shown recently that solutions of A(beta) 1-40, A(beta) 1-42, A(beta) 25-35, alpha-synuclein and non-A(beta) component (NAC; residues 61-95 of alpha-synuclein) all liberate hydroxyl radicals upon incubation in vitro followed by the addition of small amounts of Fe(II). These hydroxyl radicals were readily detected by means of electron spin resonance spectroscopy, employing 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trapping agent. Hydroxyl radical formation was inhibited by the inclusion of catalase or metal-chelators during A(beta) or alpha-synuclein incubation. Our results suggest that hydrogen peroxide accumulates during the incubation of A(beta) or alpha-synuclein, by a metal-dependent mechanism, and that this is subsequently converted to hydroxyl radicals, on addition of Fe (II), by Fentons reaction. Consequently, one of the fundamental molecular mechanisms underlying the pathogenesis of cell death in AD and PD, and possibly other neurodegenerative or amyloid diseases, could be the direct production of hydrogen peroxide during formation of the abnormal protein aggregates.

High-molecular-weight β-amyloid oligomers are elevated in cerebrospinal fluid of Alzheimer patients

There is accumulating evidence that soluble amyloid‐β (Aβ) oligomers, rather than amyloid fibrils, are the principal pathogenic species in Alzheimer disease (AD). Here, we have developed a novel enzyme‐linked immunosorbent assay (ELISA) specific for high‐molecular‐weight (HMW) Aβ oligomers. Analysis of Aβ oligomers derived from synthetic Aβ 1‐42, by size‐exclusion chromatography (SEC), revealed that our ELISA specifically detected HMW Aβ oligomers of40–200 kDa. Using this ELISA, we detected significantly higher (P<0.0001) signals in cerebrospinal fluid (CSF) samples from 25 patients with AD or mild cognitive impairment (MCI), compared to 25 age‐matched controls. As a test for discriminating between the AD/MCI and control groups, the area under the curve in receiver operating characteristic analysis for the CSF HMW Aβ oligomers was greater than that for CSF Aβx‐42. Furthermore, the CSF levels of HMW Aβ oligomers showed a negative correlation with Mini‐Mental State Examination scores in the AD/MCI group. We conclude that the CSF HMW Aβ oligomers detected by our ELISAcould be useful as a diagnostic marker for AD, and also as a potential surrogate marker for disease severity. Our results support the idea that soluble HMW Aβ oligomers play a critical role in the pathogenesis and progression of AD.—Fukumoto, H., Tokuda, T., Kasai, T., Ishigami, N., Hidaka, H., Kondo, M., Allsop, D., Nakagawa, M. High‐molecular weight β‐amyloid oligomers are elevated in cerebrospinal fluid of Alzheimer patients. FASEB J. 24, 2716–2726 (2010). www.fasebj.org

α-synuclein implicated in Parkinson’s disease catalyses the formation of hydrogen peroxide in vitro

Some rare inherited forms of Parkinsons disease (PD) are due to mutations in the gene encoding a 140-amino acid presynaptic protein called alpha-synuclein. In PD, and some other related disorders such as dementia with Lewy bodies, alpha-synuclein accumulates in the brain in the form of fibrillar aggregates, which are found inside the neuronal cytoplasmic inclusions known as Lewy bodies. By means of an electron spin resonance (ESR) spin trapping method, we show here that solutions of full-length alpha-synuclein, and a synthetic peptide fragment of alpha-synuclein corresponding to residues 61-95 (the so-called non-Abeta component or NAC), both liberate hydroxyl radicals upon incubation in vitro followed by the addition of Fe(II). We did not observe this property for the related beta- and gamma-synucleins, which are not found in Lewy bodies, and are not linked genetically to any neurodegenerative disorder. There is abundant evidence for the involvement of free radicals and oxidative stress in the pathogenesis of nigral damage in PD. Our new data suggest that the fundamental molecular mechanism underlying this pathological process could be the production of hydrogen peroxide by alpha-synuclein.

A strategy for designing inhibitors of α-synuclein aggregation and toxicity as a novel treatment for Parkinson's disease and related disorders

Convergent biochemical and genetic evidence suggests that the formation of α‐synuclein (α‐syn) protein deposits is an important and, probably, seminal step in the development of Parkinsons disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). It has been reported that transgenic animals overexpressing human α‐syn develop lesions similar to those found in the brain in PD, together with a progressive loss of dopaminergic cells and associated abnormalities of motor function. Inhibiting and/or reversing α‐syn self‐aggregation could, therefore, provide a novel approach to treating the underlying cause of these diseases. We synthesized a library of overlapping 7‐mer peptides spanning the entire α‐syn sequence, and identified amino acid residues 64‒100 of α‐syn as the binding region responsible for its self‐ association. Modified short peptides containing α‐syn amino acid sequences from part of this binding region (residues 69‒72), named α‐syn inhibitors (ASI), were found to interact with full‐ length α‐syn and block its assembly into both early oligomers and mature amyloid‐like fibrils. We also developed a cell‐permeable inhibitor of α‐syn aggregation (ASID), using the polyarginine peptide delivery system. This ASID peptide was able to inhibit the DNA damage induced by Fe(II) in neuronal cells transfected with α‐syn(A53T), a familial PD‐associated mutation. ASI peptides without this delivery system did not reverse levels of Fe(II)‐induced DNA damage. Furthermore, the ASID peptide increased (P<0.0005) the number of cells stained positive for Bcl‐2, while significantly (P<0.05) decreasing the percentage of cells stained positive for BAX. These short peptides could serve as lead compounds for the design of peptidomimetic drugs to treat PD and related disorders.

Monoclonal antibodies raised against a subsequence of senile plaque core protein react with plaque cores, plaque periphery and cerebrovascular amyloid in Alzheimer's disease

Four monoclonal antibodies (1D2/1/2, 1G10/2/3, 3B6/1/1, 4D12/2/6) were raised against a synthetic peptide consisting of residues 8-17 of a protein reported to be common to senile plaque cores, cerebrovascular amyloid and neurofibrillary tangles in Alzheimers disease. In an immunoperoxidase study of Alzheimer brain tissue, these antibodies stained plaque and vascular amyloid but not tangles, suggesting that the polypeptide chain in the region of residues 8-17 is exposed in the former two but, if present, inaccessible in the latter. In addition, staining of granular material in the plaque periphery was observed. These antibodies will be useful tools for future work on the origin of this protein.

Pick's disease is associated with mutations in the tau gene

Recently, mutations within the tau gene have been associated with some familial forms of frontotemporal dementia. To investigate whether tau gene mutations are also associated with Picks disease, we analyzed the tau gene in 30 cases of pathologically confirmed Picks disease. Two coding mutations were identified in separate cases of Picks disease. A glycine‐to‐arginine mutation at codon 389 was detected in 1 case and a lysine‐to‐threonine mutation at codon 257 was identified in another. Analysis of dephosphorylated tau from the brain of the patient with the codon 389 mutation revealed a prominent band representing tau, with four microtubule‐binding domains and no amino terminal inserts. This is in contrast to Picks disease without any tau gene mutations, which consist of tau with mainly three microtubule‐binding domains and only a trace of tau, with four microtubule‐binding domains. Functional analysis of tau with these two mutations demonstrated a reduced ability of tau to promote microtubule assembly. Surprisingly, these mutations increased taus susceptibility to calpain I digestion, suggesting that this feature may be related to the formation of a Pick type of histology. Moreover, these data suggest that Picks disease is not a separate entity but part of the frontotemporal dementia disease spectrum. Ann Neurol 2000;48:859–867