A generic method for design of oligomer-specific antibodies.

Antibodies that preferentially and specifically target pathological oligomeric protein and peptide assemblies, as opposed to their monomeric and amyloid counterparts, provide therapeutic and diagnostic opportunities for protein misfolding diseases. Unfortunately, the molecular properties associated with oligomer-specific antibodies are not well understood, and this limits targeted design and development. We present here a generic method that enables the design and optimisation of oligomer-specific antibodies. The method takes a two-step approach where discrimination between oligomers and fibrils is first accomplished through identification of cryptic epitopes exclusively buried within the structure of the fibrillar form. The second step discriminates between monomers and oligomers based on differences in avidity. We show here that a simple divalent mode of interaction, as within e.g. the IgG isotype, can increase the binding strength of the antibody up to 1500 times compared to its monovalent counterpart. We expose how the ability to bind oligomers is affected by the monovalent affinity and the turnover rate of the binding and, importantly, also how oligomer specificity is only valid within a specific concentration range. We provide an example of the method by creating and characterising a spectrum of different monoclonal antibodies against both the Aß peptide and α-synuclein that are associated with Alzheimer's and Parkinson's diseases, respectively. The approach is however generic, does not require identification of oligomer-specific architectures, and is, in essence, applicable to all polypeptides that form oligomeric and fibrillar assemblies.

Thoughts and experiences from returning to work after stroke.

OBJECTIVE: The aim of this study was to increase the understanding of stroke survivors' thoughts and experiences from returning to work after stroke. METHODS: Semi-structured interviews were carried out with twelve persons, based on a thematic interview guide. A qualitative content analysis was performed. RESULTS: The main theme; Striving for optimal function at work creates mixed feelings of appreciation and frustration, contained three categories: 1) Multiple arrangements and strategies are necessary for returning to work, 2) Work as an activity holds multiple subjective meanings that are important for the motivation to return to work, and 3) The return-to-work process generates many and mixed feelings. CONCLUSIONS: The complexity of the process involving many different actors constitutes great challenges for the affected person in addition to general and medical problems. The stroke survivor should be encouraged to be an active participant during the return-to-work process. Our findings can be used for the development of a programme, including a personal mentor, to support the person striving for returning to work. For detailed planning of such a programme further research is needed.

Cytotoxicity of superoxide dismutase 1 in cultured cells is linked to Zn2+ chelation.

Neurodegeneration in protein-misfolding disease is generally assigned to toxic function of small, soluble protein aggregates. Largely, these assignments are based on observations of cultured neural cells where the suspect protein material is titrated directly into the growth medium. In the present study, we use this approach to shed light on the cytotoxic action of the metalloenzyme Cu/Zn superoxide dismutase 1 (SOD1), associated with misfolding and aggregation in amyotrophic lateral sclerosis (ALS). The results show, somewhat unexpectedly, that the toxic species of SOD1 in this type of experimental setting is not an aggregate, as typically observed for proteins implicated in other neuro-degenerative diseases, but the folded and fully soluble apo protein. Moreover, we demonstrate that the toxic action of apoSOD1 relies on the protein's ability to chelate Zn(2+) ions from the growth medium. The decreased cell viability that accompanies this extraction is presumably based on disturbed Zn(2+) homeostasis. Consistently, mutations that cause global unfolding of the apoSOD1 molecule or otherwise reduce its Zn(2+) affinity abolish completely the cytotoxic response. So does the addition of surplus Zn(2+). Taken together, these observations point at a case where the toxic response of cultured cells might not be related to human pathology but stems from the intrinsic limitations of a simplified cell model. There are several ways proteins can kill cultured neural cells but all of these need not to be relevant for neurodegenerative disease.

Clicked tacrine conjugates as acetylcholinesterase and ß-amyloid directed compounds.

The multifaceted nature of Alzheimer's disease (AD) has led to the development of multi-targeted compounds based on the classical AD drug, tacrine, first known to inhibit the acetylcholine-degrading enzyme acetylcholinesterase (AChE). In the present work, we explore the potentiality of multimers of tacrine in this field. The synthesis using the so-called "click chemistry" and the in vitro study of the conjugates are described. Two or four copies of the tacrine molecule are "clicked" on a constrained cyclopeptide template proven to be a convenient tool for multimeric presentation. The multimers significantly inhibit self-induced amyloid fibril formation from Aß(40) at low inhibitor to Aß molar ratios at which the tacrine monomer is fully inactive (Thioflavin T assays and AFM observation). Moreover, they have the capacity to bind to Aß(40) fibrils (SPR assays) while retaining the AChE inhibitory activity of the parent tacrine.

Uptake of aggregating transthyretin by fat body in a Drosophila model for TTR-associated amyloidosis.

BACKGROUND: A functional link has been established between the severe neurodegenerative disorder Familial amyloidotic polyneuropathy and the enhanced propensity of the plasma protein transthyretin (TTR) to form aggregates in patients with single point mutations in the TTR gene. Previous work has led to the establishment of an experimental model based on transgenic expression of normal or mutant forms of human TTR in Drosophila flies. Remarkably, the severity of the phenotype was greater in flies that expressed a single copy than with two copies of the mutated gene. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we analyze the distribution of normal and mutant TTR in transgenic flies, and the ultrastructure of TTR-positive tissues to clarify if aggregates and/or amyloid filaments are formed. We report the formation of intracellular aggregates of 20 nm spherules and amyloid filaments in thoracic adipose tissue and in brain glia, two tissues that do not express the transgene. The formation of aggregates of nanospherules increased with age and was more considerable in flies with two copies of mutated TTR. Treatment of human neuronal cells with protein extracts prepared from TTR flies of different age showed that the extracts from older flies were less toxic than those from younger flies. CONCLUSIONS/SIGNIFICANCE: These findings suggest that the uptake of TTR from the circulation and its subsequent segregation into cytoplasmic quasi-crystalline arrays of nanospherules is part of a mechanism that neutralizes the toxic effect of TTR.

Amyloid-ß oligomer specificity mediated by the IgM isotype--implications for a specific protective mechanism exerted by endogenous auto-antibodies.

BACKGROUND: Alzheimers disease (AD) has been strongly linked to an anomalous self-assembly of the amyloid-ß peptide (Aß). The correlation between clinical symptoms of AD and Aß depositions is, however, weak. Instead small and soluble Aß oligomers are suggested to exert the major pathological effects. In strong support of this notion, immunological targeting of Aß oligomers in AD mice-models shows that memory impairments can be restored without affecting the total burden of Aß deposits. Consequently a specific immunological targeting of Aß oligomers is of high therapeutic interest. METHODOLOGY/PRINCIPAL FINDINGS: Previously the generation of conformational-dependent oligomer specific anti-Aß antibodies has been described. However, to avoid the difficult task of identifying a molecular architecture only present on oligomers, we have focused on a more general approach based on the hypothesis that all oligomers expose multiple identical epitopes and therefore would have an increased binding to a multivalent receptor. Using the polyvalent IgM immunoglobulin we have developed a monoclonal anti-Aß antibody (OMAB). OMAB only demonstrates a weak interaction with Aß monomers and dimers having fast on and off-rate kinetics. However, as an effect of avidity, its interaction with Aß-oligomers results in a strong complex with an exceptionally slow off-rate. Through this mechanism a selectivity towards Aß oligomers is acquired and OMAB fully inhibits the cytotoxic effect exerted by Aß(1-42) at highly substoichiometric ratios. Anti-Aß auto-antibodies of IgM isotype are frequently present in the sera of humans. Through a screen of endogenous anti-Aß IgM auto-antibodies from a group of healthy individuals we show that all displays a preference for oligomeric Aß. CONCLUSIONS/SIGNIFICANCE: Taken together we provide a simple and general mechanism for targeting of oligomers without the requirement of conformational-dependent epitopes. In addition, our results suggest that IgM anti-Aß auto-antibodies may exert a more specific protective mechanism in vivo than previously anticipated.

Quenched hydrogen/deuterium exchange NMR characterization of amyloid-beta peptide aggregates formed in the presence of Cu2+ or Zn2+.

Alzheimer's disease, a neurodegenerative disorder causing synaptic impairment and neuronal cell death, is strongly correlated with aggregation of the amyloid-beta peptide (Abeta). Divalent metal ions such as Cu(2+) and Zn(2+) are known to significantly affect the rate of aggregation and morphology of Abeta assemblies in vitro and are also found at elevated levels within cerebral plaques in vivo. The present investigation characterized the architecture of the aggregated forms of Abeta(1-40) and Abeta(1-42) in the presence or absence of either Cu(2+) or Zn(2+) using quenched hydrogen/deuterium exchange combined with solution NMR spectroscopy. The NMR analyses provide a quantitative and residue-specific structural characterization of metal-induced Abeta aggregates, showing that both the peptide sequence and the type of metal ion exert an impact on the final architecture. Common features among the metal-complexed peptide aggregates are two solvent-protected regions with an intervening minimum centered at Asn27, and a solvent-accessible N-terminal region, Asp1-Lys16. Our results suggest that Abeta in complex with either Cu(2+) or Zn(2+) can attain an aggregation-prone beta-strand-turn-beta-strand motif, similar to the motif found in fibrils, but where the metal binding to the N-terminal region guides the peptide into an assembly distinctly different from the fibril form.

Shortened constraint-induced movement therapy in subacute stroke - no effect of using a restraint: a randomized controlled study with independent observers.

OBJECTIVE: To examine the effect of using a mitt during shortened constraint-induced movement therapy for patients in the subacute phase after stroke. SUBJECTS: Twenty-four patients with stroke (mean age 57.6 (standard deviation (SD) 8.5) years; average 7 weeks post-stroke) with mild to moderate impaired hand function. METHODS: The patients were randomized to mitt use or no mitt use on the less affected hand for 90% of waking hours for 12 days. All patients received 3 h of arm and hand training per day for 2 weeks. Assessments were made by blinded observers using the modified Motor Assessment Scale, the Sollerman hand function test, the 2-Point Discrimination test and Motor Activity Log test. RESULTS: Patients in both groups showed significant improvements in arm and hand motor performance and on self-reported motor ability after 2 weeks of therapy and at 3 months follow-up. However, no statistically significant differences between the groups were found in any measures at any point in time. CONCLUSION: In this study, no effect of using a restraint in patients with subacute stroke was found. Thus, this component in the constraint-induced therapy concept seems to be of minor importance for the outcome.

Quality of life after stroke: well-being, life satisfaction, and subjective aspects of work.

Stroke incidence in those of working age has been reported to be increasing significantly, implying strong incentives for research concerning working ability after stroke. This study focused on differences in subjective aspects of work and dimensions of quality of life after having experienced stroke. Sixty-five persons answered a postal questionnaire. The median age at the time of the stroke was 54 years, (Q3 -Q1 = 8 years). "Financial aspects" and "intrinsic aspects" of work were rated as the most important by 37% and 36% of the respondents, respectively. Fewer respondents ranked "social aspects" as most important. In total, 23 persons had returned to work, but no difference in the ratings of the most important aspects of work between those who had returned to work and those who had not was found. Persons who rated "intrinsic aspects" of work as the most important were more satisfied with the subjective dimensions of quality of life "vocational situation" (p = 0.020) and "work" (p = 0.015) than the others. In conclusion, subjective aspects of work need to be explored and discussed thoroughly in rehabilitation of younger stroke patients.

Indicators for return to work after stroke and the importance of work for subjective well-being and life satisfaction.

OBJECTIVES: This study focuses on the continuation of gainful employment after experiencing stroke, addressing factors indicative of readiness for return to work, subjective well-being and life satisfaction. METHODS: The target group comprised 120 patients, studied by means of medical records and postal questionnaires. RESULTS: A total of 41% had returned to work, although there were changes concerning employers, assignments and working hours. Individuals who had returned to work reported a significantly higher level in subjective well-being and life satisfaction. Being able to walk meant the greatest chance of returning to work (odds ratio = 3.98) followed by white-collar worker (odds ratio = 2.99) and having preserved cognitive capacity (odds ratio = 2.64). CONCLUSION: Returning to work after stroke is a major factor for high subjective well-being and life satisfaction. Three factors indicative of readiness for return to work were identified, providing implications for more efficient vocational rehabilitation programmes.