Llama-derived nanobodies in this study markedly reduced cellular toxicity

Flickr, Laura Ledesma

Debilitating conditions such as Alzheimer’s and Parkinson’s disease represent one of the major medical challenges of the modern world. It is estimated that more than 40 million people worldwide suffer from dementia and this figure is expected to triple by 2050. Despite the rapidly growing numbers and the increasing burden to our economy and society, there are currently no effective treatments available for these devastating conditions.

Molecular mechanisms

Alzheimer’s and Parkinson’s disease are members of a larger class of conditions characterized by the misfolding and aggregation of specific disease-related proteins. Other notable representatives include type II diabetes, Huntington’s disease and the motor neuron disease ALS.

The underlying molecular mechanism in these diseases is protein aggregation, which ends in solid macroscopically visible deposits of the aggregating protein composed of bundles of fiber-like structures, commonly known as amyloid. The amyloid deposits of Parkinson’s disease predominantly consist of alpha-synuclein protein, and their pathological deposition within living brain tissue is linked to large-scale cell death and neuro-inflammation.

The highest toxicity and the strongest ability to cause cell death is associated with the small aggregates of alpha-synuclein, termed oligomers

It is known, however, that the highest toxicity and the strongest ability to cause cell death is associated with the small aggregates of alpha-synuclein, termed oligomers, that are formed early during the aggregation process. The small oligomeric aggregates are highly mobile, very sticky, and can actively interfere with key cellular processes.

Therapeutic strategies

Substantial drug discovery efforts over the past decade have focused on the development of antibodies targeting the native and aggregated forms of disease-related proteins, and antibodies against alpha-synuclein are currently undergoing clinical trials worldwide.

One potentially promising therapeutic strategy is the search for antibodies that bind the most harmful oligomers and neutralize their toxicity. However, protein oligomers are experimentally challenging to study because these species are usually present at low concentrations, exist in a range of sizes and have different inter-converting structures and hence toxicities.

Our laboratory has previously addressed the experimental challenge of oligomer characterization by developing single-molecule fluorescence microscopy to study the transient and toxic oligomers of alpha-synuclein in great detail.

Llama derived nanobodies

In our current study published in BMC Biology, we have combined the single-molecule fluorescence technique with multiple biophysical assays to understand the effect of a novel class of therapeutic molecules, namely nanobodies, on alpha-synuclein aggregation.

Nanobodies, derived from llamas, comprise only a small part (a single domain) of conventional antibodies, yet possess full antigen-binding capacity, and have recently shown great potential both as structural tools and as therapeutic agents in a number of diseases caused by protein dysfunction.

Nanobodies, derived from llamas, comprise only a small part (a single domain) of conventional antibodies, yet possess full antigen-binding capacity

We found that the nanobodies binding to alpha-synuclein molecules are able to slow down its aggregation reaction. Moreover, our single-molecule experimental results revealed that the nanobodies cause a rapid and persistent conformational conversion from the toxic to less toxic oligomers of alpha-synuclein.

This structural re-arrangement in the oligomers, promoted by the nanobodies, results in dramatically reduced cell death, lower production of reactive oxygen species and decreased release of a pro-inflammatory cytokine in cell assays.

This novel mechanism of the nanobodies may have important consequences; namely, these molecules have the potential to halt the propagation of the disease by the toxic oligomers of alpha-synuclein, by converting the aggregates into benign and easily degradable species. This previously unknown mode of action by antibody-like molecules could be particularly significant in the context of the development of immunotherapy.

These nanobodies, however, have not yet been tested in animals, and one key hurdle that remains to be addressed, as with all other antibody-based therapeutic approaches, is the efficiency of their passage through the blood-brain barrier, although a small fraction of antibodies is known to overcome this obstacle, which may be sufficient to produce a therapeutic effect.

Other promising methods are being developed that can enhance the delivery of antibodies and other proteins to the central nervous system. Therefore, our recent insights might prompt further investigations of the use of nanobodies for the development of immunotherapies against Parkinson’s disease, as well as related protein-deposition disorders.