Andrea C. Klaver

α-Synuclein and Anti-α-Synuclein Antibodies in Parkinson’s Disease, Atypical Parkinson Syndromes, REM Sleep Behavior Disorder, and Healthy Controls

α-synuclein is thought to play a key role in Parkinson’s disease (PD) because it is the major protein in Lewy bodies, and because its gene mutations, duplication, and triplication are associated with early-onset PD. There are conflicting reports as to whether serum and plasma concentrations of α-synuclein and anti-α-synuclein antibodies differ between PD and control subjects. The objectives of this study were to compare the levels of α-synuclein and its antibodies between individuals with typical PD (nu200a=u200a14), atypical Parkinson syndromes (nu200a=u200a11), idiopathic rapid eye movement sleep behavior disorder (nu200a=u200a10), and healthy controls (nu200a=u200a9), to assess the strength of association between these serum proteins, and to determine group sizes needed for a high probability (80% power) of detecting statistical significance for 25% or 50% differences between typical PD and control subjects for these measurements. Analysis of log-transformed data found no statistically significant differences between groups for either α-synuclein or its antibodies. The concentrations of these proteins were weakly correlated (Spearman rhou200a=u200a0.16). In subjects with typical PD and atypical Parkinson syndromes, anti-α-synuclein antibody levels above 1.5 µg/ml were detected only in subjects with no more than four years of clinical disease. Power analysis indicated that 236 and 73 samples per group would be required for an 80% probability that 25% and 50% differences, respectively, in mean α-synuclein levels between typical PD and control subjects would be statistically significant; for anti-α-synuclein antibodies, 283 and 87 samples per group would be required. Our findings are consistent with those previous studies which suggested that serum concentrations of α-synuclein and its antibodies are not significantly altered in PD.

Intravenous immunoglobulin products contain specific antibodies to recombinant human tau protein.

Intravenous immunoglobulin (IVIG) products are prepared from plasma immunoglobulins from healthy donors. Pilot studies suggest that IVIG may stabilize cognitive functioning in patients with mild-to-moderate Alzheimers disease. This study measured antibodies to recombinant human tau protein in the IVIG products Gammagard (Baxter), Gamunex (Talecris), and Flebogamma (Grifols). Anti-tau antibodies were measured by ELISA, subtracting IVIGs polyvalent binding from its binding to tau-coated wells to calculate specific anti-tau antibody levels. Because polyvalent binding of IVIG products may interfere with ELISA measurement of their specific antibody levels, the percentage of binding of each IVIG product to tau-coated wells that was specific for tau was also determined. Specific anti-tau antibodies were detected in all three IVIG products, with significant differences between these products (p<0.001) even when Flebogammas anti-tau antibodies were doubled to account for its preparation as a 5% solution vs. 10% solutions for Gammagard and Gamunex (means: Gammagard, 3.1 μg/ml; Gamunex, 2.5 μg/ml; Flebogamma, 1.2 μg/ml). The percentages of each IVIG products specific binding to tau-coated wells also varied between the various products (p<0.001) and between all pairs of IVIG products (means: Gammagard, 73.1%; Flebogamma, 54.5%; Gamunex, 37.4%; p<0.01 for all pairwise comparisons). These findings indicate that IVIG products contain specific anti-tau antibodies. The concentrations of these antibodies and the percentages of specific binding of IVIG to tau-coated wells vary between IVIG products. Further studies are indicated to determine if IVIG also contains antibodies to pathologic forms of tau.

Kinetic analysis of IgG antibodies to beta-amyloid oligomers with surface plasmon resonance

Surface plasmon resonance was used to investigate the kinetics, affinity, and specificity of binding between anti-Aβ (beta-amyloid) IgG antibodies and oligomeric Aβ. Two factors were needed to accurately characterize the IgG binding kinetics. First, a bivalent model was necessary to properly fit the kinetic association and dissociation sensograms. Second, a high concentration of IgG was necessary to overcome a significant mass transport limitation that existed regardless of oligomer density on the sensor surface. Using high IgG concentrations and bivalent fits, consistent kinetic parameters were found at varying sensor surface ligand densities. A comparison of binding specificity, affinity, and kinetic flux between monoclonal and natural human anti-Aβ IgG antibodies revealed the following findings. First, monoclonal antibodies 6E10 and 4G8 single-site binding affinity is similar between Aβ oligomers and monomers. Second, natural human anti-Aβ IgG binding readily binds Aβ oligomers but does not bind monomers. Third, natural human anti-Aβ IgG binds Aβ oligomers with a higher affinity and kinetic flux than 6E10 and 4G8. Both the current analytical methodology and antibody binding profiles are important for advances in antibody drug development and kinetic biomarker applications for Alzheimers disease.

Comparison of ELISA measurements of anti-Aβ concentrations and percentages of specific binding to Aβ between unfractionated intravenous immunoglobulin products and their purified anti-Aβ antibodies

Intravenous immunoglobulin (IVIG) products are being investigated as possible therapeutic agents for mild cognitive impairment and Alzheimers disease (AD). Anti-Aβ antibodies have been measured by ELISA in unfractionated IVIG products and in affinity-purified antibodies from these products, but it is unclear if similar results are obtained with these two approaches. Measurements of anti-Aβ antibodies in unfractionated IVIG may be confounded by the presence of polyvalent antibodies which can bind to multiple antigens, including those on ELISA plates; whether this is an issue when measuring anti-Aβ antibodies in purified antibody eluates from IVIG is also unknown. The objective of this study was to clarify these issues. The concentrations of specific antibodies to Aβ1-42 monomer and the percentages of specific binding to it were compared via ELISA between three unfractionated IVIG products (Gamunex [Talecris], Gammagard [Baxter], and Flebogamma [Grifols]) and their affinity-purified anti-Aβ antibodies. The concentrations of anti-Aβ antibodies in unfractionated IVIG products were higher than in their respective purified anti-Aβ eluates, and the rank order of the IVIG products with respect to their anti-Aβ concentrations differed between the two types of samples. The percentages of specific binding to Aβ were lower for unfractionated IVIG than for purified anti-Aβ eluates. These findings indicate that ELISA measurements of specific anti-Aβ antibodies and percentages of specific binding to Aβ produce different results depending upon whether these measurements are made in unfractionated IVIG products or their purified anti-Aβ antibodies. Polyvalent binding occurs even with purified anti-Aβ antibodies eluated from IVIG products, but it is less extensive than with unfractionated IVIG.

Effects of External Beam Radiation on In Vitro Formation of Abeta1-42 Fibrils and Preformed Fibrils

Abstract Plaques containing fibrillar amyloid-beta (Abeta) are a characteristic finding in Alzheimers disease. Although plaque counts correlate poorly with the extent of cognitive deficits in this disorder, fibrillar Abeta can promote neuronal damage through a variety of mechanisms. External beam radiotherapy has been reported to be an effective treatment for tracheobronchial amyloidosis, in which amyloid is deposited as submucosal plaques and tumor-like masses in the trachea and/or bronchi. Radiotherapys effectiveness in this disorder is thought to be due to its toxicity to plasma cells, but direct effects of radiotherapy on amyloid may also be involved. On this basis, whole-brain radiotherapy has been suggested as a treatment for Alzheimers disease. The objective of this study was to determine the effects of external beam radiation on preformed Abeta1-42 fibrils and on the formation of these fibrils. Using the Thioflavin-T assay, no effects of radiation were found on either of these parameters. Our results in this in vitro study suggest that whole-brain irradiation is unlikely to directly reduce plaque counts in the Alzheimers disease brain. This treatment might still lower plaque counts indirectly, but any potential benefits would need to be weighed against its possible neurotoxic effects, which could induce further cognitive deficits.

Effects of antibodies to phosphorylated and non-phosphorylated tau on in vitro tau phosphorylation at Serine-199: Preliminary report

UNLABELLEDnPhosphorylation of multiple amino acids on tau protein (hyperphosphorylation) is required for the development of tau pathology in Alzheimers disease. Administration of anti-tau antibodies to transgenic tauopathy mice has been shown to reduce their tau pathology but the mechanisms responsible are unclear. To examine the effects of anti-tau antibodies on tau phosphorylation, we used western blots to study the effects of three antibodies to phosphorylated tau (pTau), namely anti-pTau S199, T231, and S396, and three antibodies to non-phosphorylated tau on in vitro phosphorylation of recombinant human tau-441 at S199. Inclusion of an anti-pTau T231 antibody in the phosphorylation reaction reduced the intensity of monomeric pTau S199 in western blots of denaturing gels, but the other antibodies had no apparent effects on this process. Surprisingly, including all three anti-phospho-tau antibodies in the reaction did not reduce the intensity of the monomer band, possibly due to steric hindrance between the antibodies.nnnCONCLUSIONSnThese preliminary findings suggest that anti-tau antibodies may have minimal direct effects on tau phosphorylation. Limitations of using western blots to examine the effects of anti-tau antibodies on this process were found to include between-experiment variability in pTau band densities and poor resolution of high molecular weight pTau oligomers. The presence of bands representing immunoglobulins as well as pTau may also complicate interpretation of the western blots. Further studies are indicated to examine the effects of anti-pTau antibodies on phosphorylation of other tau amino acids in addition to S199.

ELISA measurement of specific antibodies to phosphorylated tau in intravenous immunoglobulin products

The therapeutic effects of intravenous immunoglobulin (IVIG) products were recently studied in Alzheimers disease (AD) patients. Pilot studies produced encouraging results but phase II and III trials gave disappointing results; a further study is in progress. IVIG products contain antibodies to tau protein, the main component of neurofibrillary tangles (NFTs). The tau used to detect IVIGs anti-tau antibodies in previous studies was non-phosphorylated recombinant human tau-441, but NFT-associated tau is extensively phosphorylated. The objective of this study was to determine if various IVIG products contain specific antibodies to phosphorylated tau (anti-pTau antibodies). ELISAs were used to evaluate binding of six IVIG products to a 12 amino acid peptide, tau 196-207, which was phosphorylated (pTau peptide) or non-phosphorylated (non-pTau peptide) at Serine-199 and Serine-202. Both amino acid residues are phosphorylated in AD NFTs. Each IVIGs anti-pTau antibody ratio was calculated by dividing its binding to the pTau peptide by its binding to the non-pTau peptide. Seven experiments were performed and data were pooled, with each experiment contributing one data point from each IVIG product. Mean anti-pTau antibody ratios greater than 1.0, suggesting specific antibodies to phosphorylated tau, were found for three IVIG products. Because administration of antibodies to phosphorylated tau has been found to reduce tau-associated pathology in transgenic mouse models of tauopathy, increasing the levels of anti-pTau antibodies, together with other selected antibodies such as anti-Aβ, in IVIG might increase its ability to slow ADs progression.

CSF lamp2 concentrations are decreased in female Parkinson’s disease patients with LRRK2 mutations

Lysosome-associated membrane glycoprotein 2 (lamp2) plays critical roles in chaperone-mediated autophagy (CMA) and macroautophagy. Its isoform lamp2a is decreased in Parkinsons disease (PD) substantia nigra. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most known common cause of late-onset PD; although LRRK2 is thought to regulate macroautophagy, the influence of LRRK2 mutations on lamp2 concentrations in the CNS is unknown. To examine this issue we compared lamp2 levels in cerebrospinal fluid (CSF) between sporadic PD (sPD) patients (nu202f=u202f31), LRRK2 PD patients (nu202f=u202f20), and healthy control subjects with or without LRRK2 mutations (LRRK2 CTLu202f=u202f30, CTLu202f=u202f27). We also examined lamp2s correlations with age, oxidative stress, PD progression, and PD duration. Median lamp2 concentrations (pg/mL) were LRRK2 PDu202f=u202f127, sPDu202f=u202f333, CTLu202f=u202f436, and LRRK2 CTLu202f=u202f412. Log-transformed lamp2 concentrations, adjusting for gender effects (and excluding male LRRK2 PD patients because of low number), were lower in female LRRK2 PD patients than in LRRK2 CTL (pu202f=u202f0.002) and CTL (pu202f=u202f0.005) subjects (pu202f=u202f0.06 for lamp2 comparison between female LRRK2 PD patients and sPD patients). Lamp2 did not appear to be associated with age, PD progression, or PD duration; however, three of four Spearman rho values for correlations between lamp2 and oxidative stress markers in PD subjects were ≥0.30. These findings suggest that CSF lamp2 concentrations may be decreased in female LRRK2 PD patients compared to healthy individuals with or without LRRK2 mutations.

Specific serum antibody binding to phosphorylated and non-phosphorylated tau in non-cognitively impaired, mildly cognitively impaired, and Alzheimer’s disease subjects: an exploratory study

BackgroundTau vaccination and administration of anti-tau antibodies can prevent pathology and cognitive impairment in transgenic mouse models of tauopathy, suggesting that therapies which increase anti-tau antibodies might slow the development and/or progression of Alzheimer’s disease (AD). The extent to which individuals with no cognitive impairment (NCI) possess serum anti-tau antibodies, and whether their concentrations of these antibodies differ from anti-tau antibody levels in persons with mild cognitive impairment (MCI) or AD, are unclear. Previous studies measuring these antibodies did not account for antibody polyvalent binding, which can be extensive, nor that antibody binding to phosphorylated tau peptides could be due to binding to non-phosphorylated epitopes on those peptides.MethodsAn ELISA controlling for these factors was used to measure the specific binding of serum IgG and IgM to phosphorylated (“pTau;” phosphorylated at Serine-199 and Serine-202) and non-phosphorylated (“non-pTau”) tau 196-207 in subjects with NCI, MCI, or AD (nu2009=u200910/group). Between-group differences in these antibody levels were evaluated for statistical significance, and correlations were examined in pooled data from all subjects between these antibody levels and subject age, global cognitive functioning, and NFT counts.ResultsSpecific IgG binding to pTau and non-pTau was detected in all subjects except for one NCI control. Specific IgM binding was detected to pTau in all subjects except for two AD patients, and to non-pTau in all subjects. Mean pTau IgG was increased in MCI subjects by 53% and 70% vs. AD and NCI subjects respectively (both pxa0<xa00.05), while no significant differences were found between groups for non-pTau IgG (pu2009=u20090.052), pTau IgM, or non-pTau IgM. Non-pTau IgG was negatively associated with global cognition (Spearman rhou2009=u2009−0.50).ConclusionsSpecific binding of serum IgG and IgM to phosphorylated and non-phosphorylated tau may be present in older persons regardless of their cognitive status. Serum IgG to phosphorylated tau may be increased in individuals with MCI, but this unexpected finding requires confirmation. The approach used in this study to measure specific serum antibodies to phosphorylated tau should be useful for measuring antibodies to other post-translationally-modified proteins that are of relevance to neurodegenerative disorders.

Aβ anti-idiotypic antibodies are present in intravenous immunoglobulin and are produced in mice following its administration

Abstract The effects of intravenous immunoglobulin (IVIG) products were recently examined in patients with Alzheimer’s disease (AD). Although encouraging results were obtained in pilot studies, later trials produced negative results. The rationale for these studies was that IVIG contains antibodies to amyloid-beta (Aβ). However, if Aβ anti-idiotypic antibodies (antibodies which bind to anti-Aβ antibodies) are present in IVIG or induced by its administration, these antibodies could potentially reduce its neuroprotective effects in AD. The objective of this study was to determine if IVIG contained such antibodies. Enzyme-linked immunosorbent assays (ELISAs) measured specific binding of IVIG Gamunex to purified human anti-Aβ IgG. The mean concentration of its Aβ anti-idiotypic antibodies in four experiments was 1.85u2009μg/mL (18.5u2009μg/g IgG; rangeu2009=u20091.82–1.89u2009μg/mL [18.2–18.9u2009μg/g IgG]), and their mean percentage of specific binding was 72.2% (rangeu2009=u200968.3–75.3%). We then performed ELISAs to determine if antibodies to purified human anti-Aβ were produced in C57BL/6 mice injected with the IVIG product Gammagard in an earlier study. After subtracting the expected immune response to normal human immunoglobulins, the median concentrations of these antibodies were 15.6u2009ng/mL (rangeu2009=u20091.2–108.2u2009ng/mL) in pre-treatment sera and 2419.4u2009ng/mL (rangeu2009=u2009327.4–8478.4u2009ng/mL) in post-treatment sera. These results indicate that specific Aβ anti-idiotypic antibodies are detectable in IVIG and may be induced in mice by its administration. The presence of Aβ anti-idiotypic antibodies in IVIG products might decrease neuroprotective effects of their anti-Aβ antibodies in AD.