New fluorescent dyes to monitor aggregation of amyloidogenic proteins

CELEJ MS, JOVIN TM

Rovinj, Croacia

Otro; IX International Summer School on Biophysics; 2006

<!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0in; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman";} @page Section1 {size:8.5in 11.0in; margin:1.0in 1.25in 1.0in 1.25in; mso-header-margin:.5in; mso-footer-margin:.5in; mso-paper-source:0;} div.Section1 {page:Section1;} --> Conformational diseases arise when a specific protein or peptide fails to fold or remain correctly folded and then aggregates to give rise to “amyloid” deposits in tissues. These intracellular inclusions are pathological hallmarks of several neurodegenerative disorders such as sporadic Parkinson’s disease (PD), Alzheimer’s disease, dementia with Lewy bodies, multiple system atrophy and amyotrophic lateral disease. PD is the second most frequent progressive neurodegenerative brain disease and the most common aged-related movement disorder. PD is characterized by the degeneration of dopaminergic neurons in the substantia nigra, and it is accompanied by the presence of cytoplasmic and neuritic inclusions in the affected and surviving dopaminergic neurons. The major component of these deposits is α-synuclein (α-syn), a monomeric, soluble protein with a presynaptic function, which belongs to the family of “natively unfolded” proteins. Upon aggregation, this and other amyloid proteins adopt a b-sheet conformation. α-syn aggregation is a nucleation-dependent first-order process progressing via oligomeric species to an elongation phase culminating in the formation of well-defined amyloid fibrils. As in other protein aggregation diseases, both neurotoxic and neuroprotective roles have been attributed to the fibrillar α-syn deposits. However, recent findings suggest that the major cytotoxic species are the early oligomers. Thus, both for understanding the molecular basis of PD and designing novel treatments for the synucleinopathies, one requires the development of new approaches for detecting the early steps of protein aggregation. These are generally not revealed by indicator dyes (e.g. thioflavin T) normally used in aggregation assays.             In our current research, we are screening extrinsic fluorescent dyes, including DAPI, ANS, and TNS derivatives, as noncovalent markers of α-syn aggregation. We are characterizing the affinities of these dyes for the various aggregated forms of α-syn. Different fluorescent properties are being analyzed during the course of aggregation,             We anticipate that some of these probes may serve as new and useful tools for high throughput screening of factors affecting (inhibiting, reversing) protein aggregation so prevalent in the major neurodegenerative diseases.