Martin D. Curran

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.

Aggregates from mutant and wild‐type α‐synuclein proteins and NAC peptide induce apoptotic cell death in human neuroblastoma cells by formation of β‐sheet and amyloid‐like filaments

α‐Synuclein (α‐syn) protein and a fragment of it, called NAC, have been found in association with the pathological lesions of a number of neurodegenerative diseases. Recently, mutations in the α‐syn gene have been reported in families susceptible to an inherited form of Parkinsons disease. We have shown that human wild‐type α‐syn, mutant α‐syn(Ala30Pro) and mutant α‐syn(Ala53Thr) proteins can self‐aggregate and form amyloid‐like filaments. Here we report that aggregates of NAC and α‐syn proteins induced apoptotic cell death in human neuroblastoma SH‐SY5Y cells. These findings indicate that accumulation of α‐syn and its degradation products may play a major role in the development of the pathogenesis of these neurodegenerative diseases.

Effects of the mutations Ala30 to Pro and Ala53 to Thr on the physical and morphological properties of α-synuclein protein implicated in Parkinson's disease

α‐Synuclein (α‐syn) protein has been found in association with the pathological lesions of a number of neurodegenerative diseases. Recently, mutations in the α‐syn gene have been reported in families susceptible to an inherited form of Parkinsons disease. We report here that human wild‐type α‐syn, PD‐linked mutant α‐syn(Ala30Pro) and mutant α‐syn(Ala53Thr) proteins can self‐aggregate and form amyloid‐like filaments. The mutant α‐syn forms more β‐sheet and mature filaments than the wild‐type protein. These findings suggest that accumulation of α‐syn as insoluble deposits of amyloid may play a major role in the pathogenesis of these neurodegenerative diseases.

Association between polymorphism in regulatory region of gene encoding tumour necrosis factor α and risk of Alzheimer's disease and vascular dementia: a case-control study

Summary Background Deposition of β-amyloid in the brains of patients with Alzheimers disease is thought to precede a chain of events that leads to an inflammatory response by the brain. We postulated that genetic variation in the regulatory region of the gene for the proinflammatory cytokine tumour necrosis factor α (TNF-α) leads to increased risk of Alzheimers disease and vascular dementia. Methods A polymorphism in the regulatory region of the TNF- α gene was analysed in a case-control study. The polymorphism (C-850T) was typed in 242 patients with sporadic Alzheimers disease, 81 patients with vascular dementia, 61 stroke patients without dementia, and 235 normal controls. These groups of individuals were also genotyped for the apolipoprotein E polymorphism, and the vascular dementia and stroke groups were typed at the HLADR locus. Findings The distribution of TNF-α genotypes in the vascular dementia group differed significantly from that in the stroke and normal control groups, giving an odds ratio of 2·51 (95% CI 1·49–4·21) for the development of vascular dementia for individuals with a CT or TT genotype. Logistic regression analysis indicated that the possession of the T allele significantly increased the risk of Alzheimers disease associated with carriage of the apolipoprotein ɛ4 allele (odds ratio 2·73 [1·68–4·44] for those with apolipoprotein E ɛ4 but no TNF-α T, vs 4·62 [2·38–8·96] for those with apolipoprotein E ɛin;4 and TNF-α T; p=0·03). Interpretation Possession of the TNF-α T allele significantly increases the risk of vascular dementia, and increases the risk of Alzheimers disease associated with apolipoprotein E. Although further research is needed, these findings suggest a potential role for anti-inflammatory therapy in vascular dementia and Alzheimers disease, and perhaps especially in patients who have had a stroke.

Natural killer cell immunoglobulin-like receptor (KIR) locus profiles in African and South Asian populations

Natural killer (NK) and some T cells express killer cell immunoglobulin-like receptors (KIRs), which interact with HLA class I expressed by target cells and consequently regulate cytolytic activity. The number of KIR loci can vary and so a range of genetic profiles is observed. We have determined the KIR genetic profiles from one African (n = 62) and two South Asian (n = 108, n = 78) populations. Several of the KIRs are present at significantly different frequencies between the two major ethnic groups (eg KIR2DS4 gene frequency 0.82 African, 0.47 S Asian. Pc < 1 × 10−6) and this is due to uneven distribution of two KIR haplotype families ‘A’ and ‘B’. All three populations described here displayed a greater degree of diversity of KIR genetic profiles than other populations investigated, which indicates further complexity of underlying haplotypes; in this respect we describe two individuals who appear homozygous for a large deletion including the previously ubiquitous 2DL4. We have also reanalysed three populations that we studied previously, for the presence of a KIR which is now known to be an indicator of the ‘B’ haplotype. South Asians had the highest overall frequencies of all KIR loci characteristic of ‘B’ haplotypes (Pc < 0.0001 to < 0.004). Furthermore, gene frequency independent deviances in the linkage disequilibrium were apparent between populations.

Mitochondrial DNA polymorphism: its role in longevity of the Irish population.

The mtDNA genome has been implicated as playing a pivotal role in determining the longevity and success of the human lifespan. A PCR-RFLP methodology was used to identify polymorphic restriction enzyme sites within a 2643 bp region of the mtDNA genome and a table of genetic haplotypes for a healthy aged and a younger control cohort of patients was constructed. Forty-six different mtDNA haplotypes and 11 groups of related haplotypes were identified across the two age groups but statistical analysis failed to show any significant associations. The European J haplogroup, previously reported to be associated with longevity, was not found at an increased frequency within the Irish aged population (P=0.36). However, the haplotypes comprising the J haplogroup could be differentiated into two distinct branches by the presence or absence of the two polymorphic restriction sites, 16,389g and 16,000g. The branch of haplotypes defined by 16,389g displayed a significant increased frequency in the aged samples (8%) compared to the controls (1%), P=0.015. Inversely, the branch of haplotypes defined by 16,000g displayed a significant decreased frequency in the aged samples (4%) compared to the controls (13%), P=0.011. The polymorphism (mt5178A) associated with longevity in the Japanese was not found in the Irish population, while the polymorphism (mt9055A) associated with successful ageing in the French centenarians was found at an increased frequency in the Irish aged population (9%) compared to the younger control group (5%), but failed to reach a level of statistical significance, P=0.164.

Natural killer cells and their receptors.

Natural killer (NK) cells have been known for a long time to be a very important component of the innate immune system. However, it is only during the last 10 years that knowledge of their receptors has emerged. Described in the present review are those receptor families killer inhibitory receptor (KIR) (belonging to the immunoglobulin superfamily), and killer lectin like receptor (KLR) CD94/NKG2, that both use HLA as a ligand and have inhibiting and activating types of receptors, and natural cytotoxic receptors (NCR) which do not associate with HLA. Association of the receptor gives rise to either an inhibiting or activating signal leading to either failure or success in lysing a target cell. The KIR receptors are very polymorphic both in the number of genes expressed in an individual and the alleles present for a gene. They would appear to have had a rapid evolution compared to the CD94/NKG2 receptors. The roles that NK cells and their receptors have with various facets of transplantation, disease, pregnancy and control of virus infection in humans are described.

mt4216C variant in linkage with the mtDNA TJ cluster may confer a susceptibility to mitochondrial dysfunction resulting in an increased risk of Parkinson's disease in the Irish

Polymorphism of the mtDNA genome has been implicated as playing a role in the development and pathogenesis of Parkinsons disease (PD). A PCR-RFLP methodology was employed to generate genetic haplotypes for a cohort of 90 PD sufferers. No association was observed between the various mtDNA haplotypes observed and PD in comparison to healthy aged controls. The longevity-associated European J haplogroup and T haplogroup were identified and were both found to be in tight linkage with the mt4216C polymorphism. The mt4216C variant was observed at a significantly increased frequency in the PD cases (28%) in comparison to the healthy aged controls (15%; p=0.014). However, when the frequency of the mt4216C variant was examined in a cohort of 200 young controls (18-45 years) a similar frequency to the PD cases (25%) was observed. The frequencies obtained for the two branches of the J haplogroup (J1 and J2) and the T haplogroup in the cohort of PD subjects also reflected those observed for the young controls used in the previous longevity study. These findings lead one to postulate that the mt4216C variant, in linkage with the mtDNA TJ cluster, may influence mitochondrial dysfunction, resulting in an increased risk of PD.

Interleukin-6-gene C/G 174 polymorphism in nonagenarian and octogenarian subjects in the BELFAST study. Reciprocal effects on IL-6, soluble IL-6 receptor and for IL-10 in serum and monocyte supernatants

In this study, we have assessed any change in the frequency of the GG homozygotes of the 174 IL-6 polymorphism with increasing age, arguing that if IL-6 tracks with functional disability and age-related diseases, then there may be attrition or reduction in the frequency of homozgyous subjects, who produce higher levels of IL-6 in serum, in older survivors in a population. We have tested this hypothesis in a large group of free-living, mentally competent, nonagenarian and octogenarian subjects from the Belfast Elderly Longitudinal Ageing Study-BELFAST study and found that the frequency of GG homozygotes with IL-6-174C/G polymorphism decreases with age by about 10%, compared with young controls. In addition we find that CC homozygotes have higher serum levels of IL-6 levels compared with GG (P=0.055), with reciprocal and significant changes in the anti-inflammatory IL-10 (P=0.05). Both IL-6 and IL-10 were spontaneously produced from separated mononuclear cell monolayers in elderly subjects, with significantly higher levels of secreted IL-10 supernatant levels (P=0.05) at 20 h, for G allele subjects carrying the IL-6-174C/G polymorphism. In conclusion, in the BELFAST study, there appears to be a reduction in the frequency of GG homozygotes in the octo/nonagenarian age group and a higher serum IL-6 level associated with CC homozygotes with reciprocal changes for the anti-inflammatory cytokine IL-10.