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.