Antibodies against Alpha-Synuclein Reduce Oligomerization in Living Cells

Recent research implicates soluble aggregated forms of α-synuclein as neurotoxic species with a central role in the pathogenesis of Parkinson's disease and related disorders. The pathway by which α-synuclein aggregates is believed to follow a step-wise pattern, in which dimers and smaller oligomers are initially formed. Here, we used H4 neuroglioma cells expressing α-synuclein fused to hemi:GFP constructs to study the effects of α-synuclein monoclonal antibodies on the early stages of aggregation, as quantified by Bimolecular Fluorescence Complementation assay. Widefield and confocal microscopy revealed that cells treated for 48 h with monoclonal antibodies internalized antibodies to various degrees. C-terminal and oligomer-selective α-synuclein antibodies reduced the extent of α-synuclein dimerization/oligomerization, as indicated by decreased GFP fluorescence signal. Furthermore, ELISA measurements on lysates and conditioned media from antibody treated cells displayed lower α-synuclein levels compared to untreated cells, suggesting increased protein turnover. Taken together, our results propose that extracellular administration of monoclonal antibodies can modify or inhibit early steps in the aggregation process of α-synuclein, thus providing further support for passive immunization against diseases with α-synuclein pathology

Characterization of α-synuclein oligomers : Implications for Lewy Body Disorders

Parkinson’s disease, dementia with Lewy bodies and multiple system atrophy are disorders featuring accumulation of Lewy bodies in brain. The main component of these large insoluble intracellular inclusions is the presynaptic protein alpha-synuclein (α-synuclein). It is generally believed that α-synuclein monomers adopt an abnormal conformation that favors the formation of soluble oligomers or protofibrils and, eventually, insoluble fibrils depositing as Lewy bodies. Notably, the intermediately sized oligomers/protofibrils seem to have particular neurotoxic effects. Several factors may influence the formation of α-synuclein oligomers/protofibrils, e.g. the reactive aldehydes 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE) formed during oxidative stress. The overall aims of this thesis were to investigate biophysical and biochemical properties of in vitro generated α-synuclein oligomers, characterize their functional effects on cell and animal disease models as well as to explore whether their formation could be prevented in a cell culture model for oligomerization.  Here, it was found that α-synuclein rapidly formed oligomers after incubation with both ONE and HNE. The resulting oligomers were stable and did not continue to form insoluble fibrils. By comparing HNE- and ONE induced α-synuclein oligomers biochemically they were both found to exhibit extensive β-beta sheet structure and had a molecular size of ~2000 kDa. However, they differed in morphology; the ONE induced α-synuclein oligomers described round amorphous species whereas the HNE induced α-synuclein oligomers appeared as elongated protofibril-like structures. Both these oligomers were cell internalized to varying degrees and induced toxicity in neuroblastoma cells. In addition, the ONE induced α-synuclein oligomers seemed to initiate aggregation of monomeric α-synuclein in vitro, but failed to do so in vivo. Finally, treatment of α-synuclein overexpressing cells with monoclonal antibodies specific for α-synuclein significantly reduced aggregation and lowered levels of the protein, suggesting increased turnover in these cells.  To conclude, this thesis has characterized different oligomeric α-synuclein species, which may have properties similar to soluble species central to the pathogenesis of Parkinson’s disease and other disorders with α-synuclein pathology. For therapeutic strategies it is important to selectively target such harmful protein species and avoid interaction with other forms of α-synuclein, which may have vital physiological cellular functions

Characterization of α-synuclein oligomers : Implications for Lewy Body Disorders

Parkinson’s disease, dementia with Lewy bodies and multiple system atrophy are disorders featuring accumulation of Lewy bodies in brain. The main component of these large insoluble intracellular inclusions is the presynaptic protein alpha-synuclein (α-synuclein). It is generally believed that α-synuclein monomers adopt an abnormal conformation that favors the formation of soluble oligomers or protofibrils and, eventually, insoluble fibrils depositing as Lewy bodies. Notably, the intermediately sized oligomers/protofibrils seem to have particular neurotoxic effects. Several factors may influence the formation of α-synuclein oligomers/protofibrils, e.g. the reactive aldehydes 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE) formed during oxidative stress. The overall aims of this thesis were to investigate biophysical and biochemical properties of in vitro generated α-synuclein oligomers, characterize their functional effects on cell and animal disease models as well as to explore whether their formation could be prevented in a cell culture model for oligomerization.  Here, it was found that α-synuclein rapidly formed oligomers after incubation with both ONE and HNE. The resulting oligomers were stable and did not continue to form insoluble fibrils. By comparing HNE- and ONE induced α-synuclein oligomers biochemically they were both found to exhibit extensive β-beta sheet structure and had a molecular size of ~2000 kDa. However, they differed in morphology; the ONE induced α-synuclein oligomers described round amorphous species whereas the HNE induced α-synuclein oligomers appeared as elongated protofibril-like structures. Both these oligomers were cell internalized to varying degrees and induced toxicity in neuroblastoma cells. In addition, the ONE induced α-synuclein oligomers seemed to initiate aggregation of monomeric α-synuclein in vitro, but failed to do so in vivo. Finally, treatment of α-synuclein overexpressing cells with monoclonal antibodies specific for α-synuclein significantly reduced aggregation and lowered levels of the protein, suggesting increased turnover in these cells.  To conclude, this thesis has characterized different oligomeric α-synuclein species, which may have properties similar to soluble species central to the pathogenesis of Parkinson’s disease and other disorders with α-synuclein pathology. For therapeutic strategies it is important to selectively target such harmful protein species and avoid interaction with other forms of α-synuclein, which may have vital physiological cellular functions

Characterization of α-synuclein oligomers : Implications for Lewy Body Disorders

Parkinson’s disease, dementia with Lewy bodies and multiple system atrophy are disorders featuring accumulation of Lewy bodies in brain. The main component of these large insoluble intracellular inclusions is the presynaptic protein alpha-synuclein (α-synuclein). It is generally believed that α-synuclein monomers adopt an abnormal conformation that favors the formation of soluble oligomers or protofibrils and, eventually, insoluble fibrils depositing as Lewy bodies. Notably, the intermediately sized oligomers/protofibrils seem to have particular neurotoxic effects. Several factors may influence the formation of α-synuclein oligomers/protofibrils, e.g. the reactive aldehydes 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE) formed during oxidative stress. The overall aims of this thesis were to investigate biophysical and biochemical properties of in vitro generated α-synuclein oligomers, characterize their functional effects on cell and animal disease models as well as to explore whether their formation could be prevented in a cell culture model for oligomerization.  Here, it was found that α-synuclein rapidly formed oligomers after incubation with both ONE and HNE. The resulting oligomers were stable and did not continue to form insoluble fibrils. By comparing HNE- and ONE induced α-synuclein oligomers biochemically they were both found to exhibit extensive β-beta sheet structure and had a molecular size of ~2000 kDa. However, they differed in morphology; the ONE induced α-synuclein oligomers described round amorphous species whereas the HNE induced α-synuclein oligomers appeared as elongated protofibril-like structures. Both these oligomers were cell internalized to varying degrees and induced toxicity in neuroblastoma cells. In addition, the ONE induced α-synuclein oligomers seemed to initiate aggregation of monomeric α-synuclein in vitro, but failed to do so in vivo. Finally, treatment of α-synuclein overexpressing cells with monoclonal antibodies specific for α-synuclein significantly reduced aggregation and lowered levels of the protein, suggesting increased turnover in these cells.  To conclude, this thesis has characterized different oligomeric α-synuclein species, which may have properties similar to soluble species central to the pathogenesis of Parkinson’s disease and other disorders with α-synuclein pathology. For therapeutic strategies it is important to selectively target such harmful protein species and avoid interaction with other forms of α-synuclein, which may have vital physiological cellular functions

Lipoprotein lipase in hemodialysis patients: indications that low molecular weight heparin depletes functional stores, despite low plasma levels of the enzyme

Abstract Background Lipoprotein lipase (LPL) has a central role in the catabolism of triglyceride-rich lipoproteins. The enzyme is anchored to the vascular endothelium through interaction with heparan sulphate proteoglycans and is displaced from this interaction by heparin. When heparin is infused, there is a peak of LPL activity accompanied by a reduction in triglycerides (TG) during the first hour, followed by a decrease in LPL activity to a stable plateau during the remaining session while TG increase towards and beyond baseline. This suggests that tissue stores of LPL become depleted. It has been argued that low molecular weight (LMW) heparins cause less disturbance of the LPL system than conventional heparin does. Methods We have followed LPL activity and TG during a dialysis-session with a LMW heparin (dalteparin) using the same patients and regime as in a previous study with conventional heparin, i.e. a primed infusion. Results The shape of the curve for LPL activity resembled that during the earlier dialyses with conventional heparin, but the values were lower during dialysis with dalteparin. The area under the curve for LPL activity during the peak period (0–180 minutes) was only 27% and for the plateau period (180–240 minutes) it was only 36% of that observed with conventional heparin (p Conclusion These results indicate that LMW heparins disturb the LPL system as much or more than conventional heparin does.</p

A Capped Peptide of the Aggregation Prone NAC 71-82 Amino Acid Stretch of α-Synuclein Folds into Soluble β-Sheet Oligomers at Low and Elevated Peptide Concentrations

Although Lewy bodies and Lewy neurites are hallmarks of Parkinson's disease (PD) and dementia with Lewy bodies (DLB), misfolded α-synuclein oligomers are nowadays believed to be key for the development of these diseases. Attempts to target soluble misfolded species of the full-length protein have been limited so far, probably due to the fast aggregation kinetics and burial of aggregation prone segments in final cross-β-sheet fibrils. A previous characterisation study of fibrils prepared from a capped peptide of the non-amyloid β-component (NAC) 71-82 amino acid stretch of α-synuclein demonstrated an increased aggregation propensity resulting in a cross-β-structure that is also found in prion proteins. From this, it was suggested that capped NAC 71-82 peptide oligomers would provide interesting motifs with a capacity to regulate disease development. Here, we demonstrated, from a series of circular dichroism spectroscopic measurements and molecular dynamics simulations, the molecular-environment-sensitive behaviour of the capped NAC 71-82 peptide in a solution phase and the formation of β-sheet oligomeric structures in the supernatant of a fibrillisation mixture. These results highlighted the use of the capped NAC 71-82 peptide as a motif in the preparation of oligomeric β-sheet structures that potentially could be used in therapeutic strategies in the fight against progressive neurodegenerative disorders, such as PD and DLB