Identifying and localizing toxic oligomeric a-syn with morphology specific nanobodies

Project: Research project

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Identifying and localizing toxic oligomeric a-syn with morphology specific nanobodies Identifying and localizing toxic oligomeric a-syn with morphology specific nanobodies Increasing evidence indicates that small soluble protein aggregate forms of alphasynuclein (a-syn) are the relevant toxic species in PD similar to the toxic roles oligomeric forms of beta-amyloid (A) play in Alzheimers disease (AD). While aggregation of a-syn and A have been strongly linked to numerous neurodegenerative diseases including PD, AD, DLB and other synucleinopathies and amyloidoses, there are distinct differences between these diseases, including which areas of the brain are affected (reviewed in [1, 2]), and it would be of great value to know which aggregate forms of which proteins are important for the onset and progression of these different diseases. Numerous studies including several from our lab have shown that a variety of different intermediate oligomeric forms of both a-syn and A can exist in vitro or in vivo, and different aggregate forms may have different toxic effects on cells, and may preferentially target different types of cells. Catecholamines can alter aggregation of these proteins, and dopamine, especially oxidized products of dopamine, interact with a-syn promoting formation of toxic oligomeric structures both extracellularly (reviewed in [3, 4]) and intracellularly [5-7], potentially accounting for the increased loss of dopaminergic neurons in PD [1]. In addition, dopamine can also break down nontoxic fibrillar a-syn aggregates into toxic oligomeric species [8], again increasing susceptibility of dopaminergic neurons to toxic a-syn aggregation. Therefore different aggregate species of a-syn may be a critically important factor in the development of PD and may have value as therapeutic and diagnostic targets. We have developed novel technology in our lab that enables us to isolate antibody based ligands that can recognize specific protein aggregate morphologies [9]. We have isolated single chain antibody fragments (nanobodies) against a variety of different forms of a-syn and A, and have published studies describing nanobodies that recognize different forms of a-syn including the monomeric, fibrillar, early stage oligomeric, and later stage oligomeric forms [9-13]. In addition, we have also generated nanobodies that specifically recognize a-syn/dopamine adduct forms including monomeric and oligomeric adduct forms (Table 1). We have tested several of our nanobodies with tissue taken from healthy and diseased human brains that were provided by Banner/SHRI brain bank (BBDB). We showed that the nanobodies that specifically target early and later stage oligomeric a-syn species recognize these a-syn species in human PD brain tissue, but not in AD or healthy brain tissue [13]. We have also obtained parallel results with a nanobody against a late stage oligomeric form of A, where the nanobody specifically recognizes aggregated A species in some human AD brain tissue, but not in PD or healthy tissue [14]. We have obtained similar results with two other nanobodies that recognize other oligomeric A species. In all these studies the brain tissue was taken from the cingulate gyrus and generously provided through the BBDB. These preliminary studies clearly indicate that distinct prefibrillar protein misfolding and aggregation events occur in diseased human brain tissue. Since cellular stress induced by misfolding and aggregation of one protein such as a-syn may well lead to misfolding and aggregation of other proteins such as A and tau, the presence of multiple misfolded proteins in different regions of the brain should be expected. Therefore reagents that can target specific morphologies of specific proteins are powerful tools to characterize human tissue from PD patients. We have already generated a pool of well characterized reagents that specifically recognize different aggregated species of proteins involved in a variety of related neurodegenerative diseases, and have demonstrated that the nanobodies recognize aggregated species occurring in tissue from
StatusFinished
Effective start/end date11/1/102/28/12

Funding

  • Michael J. Fox Foundation: $156,251.00

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