What are α-synuclein oligomers in Parkinson’s Disease?
The 140-residue protein α-synuclein (α-syn) accumulates as insoluble aggregates in neuronal -and glial – cells in Parkinson’s Disease (PD) and related α-synucleinopathies. Remarkably, some brain areas affected by Lewy body (LB) pathology do not show the marked neuron loss characteristic of the catecholaminergic regions in locus coeruleus and substantia nigra seen in PD. The LB detected by classic antibodies are rather sparsely represented in most brain regions suggesting a modest impact of these α-syn macro-aggregates on the tissue. The current leading hypothesis suggest that α-syn soluble small oligomers -often invisible- are crucial in the neurodegenerative event as they can affect cellular function and integrity, e.g. by rupturing the cell and organelle membranes. This will result on impaired neurotransmission, and by extension, clinical symptoms associated with neurodegeneration and not necessarily with neuronal loss.
How do we neutralize α-syn oligomers and why is it difficult?
PD treatment will likely benefit from removing or inhibiting the toxic activities of these oligomers. Antibodies binding to oligomers may help eliminate them by targeting them for degradation via the Fc receptors on the immune cells, or they block interactions with other molecules. However, there are many different α-syn oligomers which arise as a result of e.g. different chemical modifications through various metabolic compounds or via C-terminal truncations of α-syn. All this makes oligomers a dynamic and shifting target to hit.
Our approach: raising monoclonal antibodies against a range of α-syn oligomers
We raised antibodies in mice against five different kinds of α-syn oligomers: the oligomer formed spontaneously by full-length α-syn (wt αSO), a C-terminally truncated α-syn, oligomers formed by two lipidation products (HNE and ONE) and one formed in the presence of the polyphenol EGCG. In addition, we displayed multiple copies of truncated α-syn on empty virus-like particles. In all seven cases, we were able to produce monoclonal antibodies (mAbs) which bind oligomers and we analyzed 30 of these in detail using both biophysical and immunohistochemical approaches.
The mAbs show great variability in relative and absolute binding affinity for different α-syn species, and affinities vary with binding techniques used
We used a microfluidic technique called FIDA (Flow-Induced Dispersion Analysis) to measure affinities of the mAbs, which provides true equilibrium values in solution. Most of the mAbs showed strong preference for the wt αSO over monomeric α-syn, but all the mAbs also bound to some extent to insoluble α-syn fibrils, probably because the oligomers and fibrils have many structural features in common. Also, the mAbs varied greatly in their binding affinities for oligomers and fibrils. At least 13 of the 30 mAbs recognized specific parts of the α-syn sequence; most of them bind the very flexible C-terminal tail (resides 90-140) which is highly accessible for binding. However, three mAbs prefer the NAC region of α-syn (residues 40-90) which is normally involved in intermolecular contacts, suggesting conformationally specific recognition. Our FIDA values also revealed that the commercial mAb MJF14, reported to prefer aggregated α-syn, only binds 10-fold stronger to the oligomer than monomeric α-syn in solution, whereas several of our mAbs have 80-600 fold higher oligomer affinity. We note that immobilization-based techniques such as SPR and ELISA generally do not provide simple binding data and should be used with caution.
Several mAbs block membrane disrutption and protect cells against oligomer toxicity
The mAbs were not raised specifically against fibrils, so we were not surprised to see that they only showed very weak ability to prevent α-syn from forming fibrils. Encouragingly, seven mAbs were able to essentially completely block oligomers from disrupting phospholipid vesicles, and several of them also blocked oligomer-induced formation of reactive oxygen species in SHSY5Y cells, suggesting that they can protect cells against oligomer toxicity by simple binding.
mAbs show promise in recognizing α-syn pathology in both rat and human brain tissue
Using immunofluorescence staining on brain sections from the rAAV human-α-syn rat PD model, we were able to detect α-syn pathology with 16 of our mAbs, showed by positive staining in axonal fibers and round inclusions in the striatum. This pathology resembled to some degree that of MJF14 and to a lesser degree that of pSer129, shown by calculation of Pearson Coefficient and the area of overlap. We also found pathological staining that did not co-localize or overlap with those commercial antibodies, which was quantified by calculation of the coincidence number. Moreover, three of our mAbs also found pathological rat α-syn pathology when staining sections from the rat preformed fibril (PFF) PD model, which make them suitable for future studies in the model.
From the immunofluorescence experiment five mAbs (with a low coincidence number) were chosen for immunohistochemical staining of post-mortem human brain tissue. All 5 mAbs found positive staining in brains from PD, dementia with LB (DLB) and multiple system atrophy patients. Interestingly, few ring-shaped inclusions were found in DLB and severe PD cases, which could indicate recognition of possibly early α-syn pathology. Overall, we hope our mAbs can be an addition to the existing antibody battery, creating a toolbox of mAbs that enable detection of different conformers of α-syn.
Conclusion: A promising collection of new mAbs for PD diagnosis and therapy
Most of the mAbs recognize α-syn aggregates associated with pathology in both rat and human brain tissue in ways which complement existing mAbs. We hope to be able to apply them as PET tracers for early-stage pathology indicators. We still need to test their profiles in tissues such as skin or gut and detect them in biofluids and their therapeutic potential in cell lines and animal models, but we already have preliminary evidence that they help to clear extracellular α-syn aggregates.
Molecular properties and diagnostic potential of monoclonal antibodies targeting cytotoxic α-synuclein oligomers
