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Protein aggregation is the key event in a number of human diseases such as Alzheimer's and Parkinson's disease. We present a general method to quantify and characterize protein aggregates by dual-colour scanning for intensely fluorescent targets (SIFT). In addition to high sensitivity, this approach offers a unique opportunity to study co-aggregation processes. As the ratio of two fluorescently labelled components can be analysed for each aggregate separately in a homogeneous assay, the molecular composition of aggregates can be studied even in samples containing a mixture of different types of aggregates. Using this method, we could show that wild-type alpha-synuclein forms co-aggregates with a mutant variant found in familial Parkinson's disease. Moreover, we found a striking increase in aggregate formation at non-equimolar mixing ratios, which may have important therapeutic implications, as lowering the relative amount of aberrant protein may cause an increase of protein aggregation leading to adverse effects.

The deposition of protein aggregates in neurons is a hallmark of neurodegenerative diseases caused by polyglutamine (polyQ) proteins. We analyzed the effects of the heat shock protein (Hsp) 70 chaperone system on the aggregation of fragments of huntingtin (htt) with expanded polyQ tracts. In vitro, Hsp70 and its cochaperone Hsp40 suppressed the assembly of htt into detergent-insoluble amyloid-like fibrils in an ATP-dependent manner and caused the formation of amorphous, detergent-soluble aggregates. The chaperones were most active in preventing fibrillization when added during the lag phase of the polymerization reaction. Similarly, coexpression of Hsp70 or Hsp40 with htt in yeast inhibited the formation of large, detergent-insoluble polyQ aggregates, resulting in the accumulation of detergent-soluble inclusions. Thus, the recently established potency of Hsp70 and Hsp40 to repress polyQ-induced neurodegeneration may be based on the ability of these chaperones to shield toxic forms of polyQ proteins and to direct them into nontoxic aggregates.