Several neurodegenerative diseases comprise neuronal or glial deposits consisting mainly of protein tau, such as Alzheimer’s neurofibrillary tangles (NFTs) or Pick bodies, and are therefore termed “tauopathies“. In Alzheimer’s Disease (AD), tau exhibits pathological hyperphosphorylation [1, 2], allowing both histological diagnosis by use of tau antibodies against disease specific phosphorylation sites [3, 4] and, to a certain extent, even in vivo diagnosis by determination of the protein’s phosphorylation status in cerebrospinal fluid [5, 6].
One of the most important tau kinases is Glycogen Synthase Kinase 3β (GSK-3β), which has been shown to create AD specific phospho sites on tau in vitro , in cell culture [8, 9] and in vivo [10, 11]. Some [12, 13] but not all  authors reported increased GSK levels in AD brains. GSK-3β is colocalized with NFTs , and the distribution of its active form in AD brains coincides with the appearance of tau pathology .
Tau phosphorylation by GSK-3β promotes the formation of paired helical filaments (PHF) in vitro [17–19], though data concerning the relevance of this effect vary . An enhancing impact of GSK-3β on tau aggregation was also demonstrated in cell culture and in vivo [21–23], supporting a possible role of this kinase in AD pathogenesis. Furthermore, phosphorylation influences metal ion induced tau aggregation. Several studies demonstrated that tau phosphorylation enhances Al3+ induced aggregation [24, 25] or even is a prerequisite for such aggregation [26, 27].
The influence of aluminium on tau aggregation has been extensively studied, since the metal ion was shown to induce NFT-like deposits in mammalian brain after intracerebral injection . Though aluminium levels were found to be raised in AD hippocampus  and the metal ion was colocalized with NFTs and early tau deposits in brain sections [30, 31], its relevance to AD pathogenesis is still unclear, especially due to the inconsistent outcome of epidemiological studies .
In vitro studies examining effects of ferric iron (Fe3+) yielded results resembling those obtained for aluminium. Fe3+ also induces the aggregation of phosphorylated protein tau , is colocalized with NFTs [30, 33, 34] and elevated in AD hippocampus and amygdala . Furthermore, Fe3+ induces α-synuclein (α-syn) aggregation [36–38].
Co-deposits of tau and α-syn have been found in several neurodegenerative diseases, and interactions between these two proteins recently gained increasing interest. α-Syn has been detected in NFTs of AD, progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) , whereas tau was located in Lewy bodies of patients with Dementia with Lewy bodies (DLB) . In vitro, tau in solution requires inducers like heparin for filament formation, whereas the protein readily polymerizes in presence of α-syn without inducers .
Furthermore, the minimal α-syn concentration necessary for fibril formation is reduced in presence of tau, and some of the fibrils formed in presence of both proteins comprise tau and α-syn segments . Considering that both proteins are located in the cytoplasmic compartment of neurons, and that minimal concentrations of α-syn oligomers can cross-seed tau aggregation , interactions of tau and α-syn may be relevant for pathological protein aggregation in neurodegenerative diseases.
While established methods of monitoring tau and α-syn aggregation like Thioflavin T assay or atomic force microscopy yield important insights in fibril formation and the formation of large oligomers, they are not suitable to directly monitor single protein interactions or interactions of different proteins. It was demonstrated that small oligomer species are on-pathway to tau filament formation . Furthermore, it is increasingly recognised that prefibrillar small oligomers rather than the large NFTs might be responsible for neuronal and synaptic loss [44–46]. To investigate the influence of phosphorylation on tau oliomer formation and interactions between tau and α-syn, we employed fluorescence correlation spectroscopy (FCS) and scanning for intensely fluorescent targets (SIFT) to investigate the influence of phosphorylation and trivalent metal ions on tau aggregation and its coaggregation with α-syn. These methods allow monitoring of oligomerization processes at the single molecule level even at nanomolar protein concentrations [47, 48]. Moreover, the possibility to label proteins with different dyes allows the investigation of tau and α-syn interactions at the level of individual oligomers.