FIELD OF THE INVENTION

The present invention relates to novel methods for the in vitro formation and detection of paired helical filaments (PHF) of Tau. These methods can be used for the selection of compounds capable of modulating, e.g. inhibiting pathological Tau-Tau protein association and pathological neurofilament aggregation. The methods of the present invention are particularly useful in screening compounds for the prophylaxis and treatment of Alzheimer's disease (AD).

BACKGROUND OF THE INVENTION

Dementia is a syndrome that can be caused by a number of progressive disorders that affect memory, thinking, behavior and the ability to perform everyday activities. About 36 million people worldwide are suffering from dementia today. The number of people with dementia is projected to double by 2030, and more than triple to 115.4 million people by 2050.

Alzheimer's disease (AD) is the most common type of dementia. Currently, one in nine people of age 65 and older (11 percent) and nearly half of those over age 85 have Alzheimer's disease. According to Alzheimer's Disease International, the current global costs of caring for these patients exceeds $600 billion annually. These costs are likely to rise even faster than the prevalence of disease, especially in the developing world, as more formal social care systems emerge, and rising incomes lead to higher opportunity costs.

AD is characterized by two pathological protein deposits in the brains of affected individuals, i.e. the amyloid plaques, consisting largely of amyloid fibers assembled from the beta amyloid (Aβ) peptide (a derivative of the amyloid precursor protein, APP) and the neurofibrillary tangles (NFT). NFT are formed by hyperphosphorylation of a microtubule-associated protein known as Tau, causing it to aggregate, or group, in an insoluble form. These aggregations of hyperphosphorylated Tau protein are also referred to as PHFs, or “paired helical filaments”. These aggregations are a histopathological feature not only of AD, but of many neurodegenerative diseases, which are collectively known as Tauopathies. Tauopathies include, e.g., Alzheimer's disease (AD), Pick's disease (PiD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and frontotemporal lobar degeneration (FTLD).

In addition to serving as markers for differential diagnosis and staging of the disease, Tau aggregates can also foster disease propagation. Therefore, diverse strategies for inhibiting Tau aggregation are being investigated as potential therapies against neurofibrillary lesion formation and disease progression. Several assays for in vitro formation of PHF have been described in the prior art (Friedhoff et al., Biochemistry 1998, 37: 10223-10230; Barghorn and Mandelkow, Biochemistry 2002, 41: 14885-14896; Morozova et al., Biochemistry 2013, 52:6960-6967). Thus, the assembly of Tau into filaments could be reproduced in vitro by adding compounds like polyanions, and fatty acids (and derivates). These methods however been shown to lack reproducibility and so far no reliable methods to prepare a homogenous population of Tau oligomers are available, hindering the ability to evaluate Tau oligomer toxicity and its possible role in the disease.

Therefore, there is still a need for novel and reliable methods which can be used, inter alia, for the testing of potential drugs which may interfere with the above described pathological Tau assembly, which may be useful for selecting new and effective drugs useful in the diagnosis and/or treatment of Tauopathies, such as AD.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a method for the in vitro formation of paired helical filaments (PHF) of Tau protein, comprising incubating a mixture comprising a Tau protein preparation and a polyanionic co-factor for a pre-determined period of time under conditions that promote the formation of PHFs.

In a further aspect, the invention provides an in vitro method for detecting the formation of paired helical filaments (PHFs) of Tau protein, comprising the steps of:

(a) incubating a mixture comprising a Tau protein preparation and a polyanionic co-factor for a pre-determined period of time under conditions that promote the formation of PHFs; and

(b) detecting the formed PHF by microscopial or spectroscopial means.

The invention further provides an in vitro method for the identification of a compound that is capable of inhibiting or promoting the formation of paired helical filaments of tauTau protein, comprising the steps of:

(a) performing a method as describe above in the absence and in the presence of a test compound; and

(b) comparing the kinetics of the formation of PHFs in the absence of said test compound with the kinetics of the formation of PHFs in the presence of said test compound.

The present invention provides novel and reliable methods for the formation of and detection PHF which can be useful for the identification and selection of compounds that are capable of modulating, e.g. promoting or inhibiting pathological Tau-Tau protein association and pathological neurofilament aggregation. The methods of the present invention are particularly useful in screening compounds for the prophylaxis and treatment of Alzheimer's disease (AD).

