Alpha-synuclein levels in blood plasma decline with healthy aging.

There is unequivocal evidence that alpha-synuclein plays a pivotal pathophysiological role in neurodegenerative diseases, and in particular in synucleinopathies. These disorders present with a variable extent of cognitive impairment and alpha-synuclein is being explored as a biomarker in CSF, blood serum and plasma. Considering key events of aging that include proteostasis, alpha-synuclein may not only be useful as a marker for differential diagnosis but also for aging per se. To explore this hypothesis, we developed a highly specific ELISA to measure alpha-synuclein. In healthy males plasma alpha-synuclein levels correlated strongly with age, revealing much lower concentrations in older (avg. 58.1 years) compared to younger (avg. 27.6 years) individuals. This difference between the age groups was enhanced after acidification of the plasmas (p<0.0001), possibly reflecting a decrease of alpha-synuclein-antibody complexes or chaperone activity in older individuals. Our results support the concept that alpha-synuclein homeostasis may be impaired early on, possibly due to disturbance of the proteostasis network, a key component of healthy aging. Thus, alpha-synuclein may be a novel biomarker of aging, a factor that should be considered when analyzing its presence in biological specimens.

Altered serum IgG levels to α-synuclein in dementia with Lewy bodies and Alzheimer's disease.

Natural self-reactive antibodies in the peripheral blood may play a considerable role in the control of potentially toxic proteins that may otherwise accumulate in the aging brain. The significance of serum antibodies reactive against α-synuclein is not well known. We explored serum IgG levels to monomeric α-synuclein in dementia with Lewy bodies (DLB) and Alzheimer's disease (AD) with a novel and validated highly sensitive ELISA assay. Antibody levels revealed stark differences in patients compared to healthy subjects and were dependent on diagnosis, disease duration and age. Anti-α-synuclein IgG levels were increased in both patient groups, but in early DLB to a much greater extent than in AD. Increased antibody levels were most evident in younger patients, while with advanced age relatively low levels were observed, similar to healthy individuals, exhibiting stable antibody levels independent of age. Our data show the presence of differentially altered IgG levels against α-synuclein in DLB and AD, which may relate to a disturbed α-synuclein homeostasis triggered by the disease process. These observations may foster the development of novel, possibly preclinical biomarkers and immunotherapeutic strategies that target α-synuclein in neurodegenerative disease.

Up-regulation of platelet-derived growth factor by peripheral-blood leukocytes during experimental allergic encephalomyelitis.

In multiple sclerosis (MS) and its animal model, experimental allergic encephalomyelitis (EAE), clinical disease is associated with infiltration of the central nervous system (CNS) by immune cells. Subsequent remission with remyelination has been linked to an increased occurrence of oligodendrocyte progenitor (O2A) cells. Platelet-derived growth factor (PDGF) and fibroblast growth factor-2 (FGF-2) are key growth factors for O2A cells, yet little is known about their relevance in EAE and MS. We analyzed the expression of PDGF, FGF-2, and their receptors by peripheral-blood leukocytes (PBLs) and lymphocyte subsets during MBP-induced EAE. Strong up-regulation of PDGF, but not FGF-2, was observed in PBLs, with the highest expression after the disease maximum. T, NK, and NKT cells expressed PDGF, which is a novel observation because thus far only monocytes/macrophages have been reported to express PDGF. These results extend the idea that growth factors may contribute to improved CNS tissue repair, including PDGF, which is secreted by lesion-homing immune cells. The production of PDGF by lymphocytes may have potential therapeutic value when activating or modulating T-cell responses in demyelinating diseases.

Structure-based discovery of nonpeptidic small organic compounds to block the T cell response to myelin basic protein.

We have utilized a computational structure-based approach to identify nonpeptidic small organic compounds that bind to a human leukocyte antigen (HLA) DR1301 molecule (HLA-DR1301 or DR1301) and block the presentation of myelin basic protein peptide 152-165 (MBP 152-165) to T cells. A three-dimensional (3D) structure of DR1301 was derived by homology modeling followed by extensive molecular dynamics simulation for structural refinement. Computational structure-based database searching was performed to identify nonpeptidic small-molecule candidates from the National Cancer Institute (NCI) database containing over 150 000 compounds that can effectively interact with the peptide-binding groove of the HLA molecule. By in vitro testing of 106 candidate small molecules, two lead compounds were confirmed to specifically block IL-2 secretion by DR1301-restricted T cells in a dose-dependent and reversible manner. The specificity of blocking DR1301-restricted MBP presentation was further validated in a binding assay using an analogue of the most potent lead compound. Computational docking was performed to predict the three-dimensional binding model of these confirmed small molecule blockers to the DR1301 molecule and to gain structural insight into their interactions. Our results suggest that computational structure-based searching is an effective approach to discover nonpeptidic small organic compounds to block the interaction between DR1301 and T cells. The nonpeptidic small organic compounds identified in this study are useful pharmacological tools to study the interactions between HLA molecules and T cells and a starting point for the development of a novel therapeutic strategy for the treatment of multiple sclerosis (MS) or other immune-related disorders.

The human T cell response to myelin oligodendrocyte glycoprotein: a multiple sclerosis family-based study.

Myelin oligodendrocyte glycoprotein (MOG) is an encephalitogenic myelin protein and a likely autoantigen in human multiple sclerosis (MS). In this work, we describe the fine specificity and cytokine profile of T cell clones (TCC) directed against MOG in three nuclear families, comprised of four individuals affected with MS and their HLA-identical siblings. TCC were generated from PBMC by limiting dilution against a mixture of eleven 20-mer overlapping peptides corresponding to the encephalitogenic extracellular domain of human MOG (aa 1-120). The frequency of MOG peptide-reactive T cells was surprisingly high (range, 1:400 to 1:3,000) and, unexpectedly, cloning efficiencies were highest at low seeding densities of 10(2) or 10(3) PBMC per well. A total of 235 MOG peptide-reactive TCC were produced, all of which were CD4(+)CD8(-)TCRalphabeta(+)TCRgammadelta(-). All 11 MOG peptides were recognized by the TCC, and different epitopes of MOG appeared to be immunodominant in the HLA-identical siblings. The patterns of cytokine secretion by TCC from single individuals were generally similar. The healthy individuals exhibited Th2-, Th0-, and T regulatory cell 1-like cytokine profiles, whereas TCC from one sibling with MS had a striking Th1-like phenotype, producing high levels of IFN-gamma and TNF-alpha, and low IL-4 levels. Thus, MOG-reactive T cells appear to constitute an important part of the natural T cell repertoire, a finding that could contribute to the development of autoimmunity to this protein.