**3.6. Tau-seeding-based assays**

in differential diagnosis of four neurodegenerative dementias (AD, FTLD, DLB, CJD) [107]. While the authors concluded that non-p-tau quantification had no added diagnostic value as a CSF biomarker for the differential diagnosis of neurodegenerative dementia, it improved the discrimination of sCJD cases. Unfortunately, as t-tau levels were above the quantification limit in 17 out of 19 CJD cases analyzed in this study, no significant conclu-

In summary, preliminary observations indicate that the non-p-tau assay may be an interesting additional tool for the study of the dissociation between neuronal damage and tau pathology

A growing body of literature is pointing to tau fragments, produced by cleavage events, as major players in the onset and the progression of the pathology [126–129]. In AD brains, after an initial misfolding at early stages of the disease which involves the physical contact of the N-terminal region with the microtubule binding repeats, tau is cleaved first at residue D421 followed by cleavage at residue E391, while N-terminal cleavage appears in later stages of the disease [126, 130]. The differential enzymatic cleavages during the pathological process is mostly dependent on Caspases activation, which lead to the generation of several tau fragments, each displaying its own profile of neurotoxicity [131]. Additionally, Calpain proteases have been shown to produce a triplet of tau fragments spanning from 35 kDa to 15 kDa. Similarly to full length tau [132, 133], several reports have shown that tau fragments can be

These and other findings led researchers to investigate the presence of tau fragments in CSF and other biological fluids as potential biomarkers for the differential diagnosis of tauopathies and associated diseases. In CSF, at least 10 fragments were characterized in AD by mass spectrometry [137]. The presence of the 20 kDa caspase-6 cleavage product of tau in the CSF of AD has been reported to be associated with brain pathology and was found to be increased with the severity of the disease and the overall measure of global cognition [138]. Other reports found a 26–28 kDa fragment in both AD and strokes patients [139]. Tau bands ranging from 20 to 40 kDa were found in AD and control CSF [140, 141], which corresponded to N-terminal and mid-domain fragments, while no C-terminal fragments were found. Therefore, biomarker-based diagnosis would strongly depend on the subset of tau species analyzed.

Tau fragments have been proposed as biomarkers not only for the discrimination of AD from controls, but also for the differential diagnosis within related neurodegenerative diseases. For instance, a divergent pattern of expression of different N-terminal tau fragments was found between AD and PSP, even though such kind of studies are limited by the frequently overlapping clinical diagnosis [142]. Nonetheless, the ratio between a 33 kDa and a 55 kDa fragment in CSF was proposed as a more specific and reliable biomarker for the diagnosis of PSP [63]. Moreover, in CSF derived from TBI patients, a 30–50 kDa tau fragment was found to correlate with the extent of axonal damage [143]. Lastly, in CSF derived from either lumbar or cervical puncture from amyotrophic lateral sclerosis (ALS) patients the neurotoxic 17 kDa fragment

produced by calpain cleavage was found to be elevated compared to controls [144].

sions could be drawn on the differential diagnostic accuracy of both tests.

secreted and uptaken from cells and brain slices and mediate toxicity [134–136].

in the brain and biological fluids of neurodegenerative disorders.

**3.5. Tau truncated forms**

74 Cognitive Disorders

The development and implementation of seeding-based methodologies for disease diagnostic purposes is an emerging topic with demonstrated clinical applicability in the field of prion diseases due to the real-time quaking-induced conversion assay (RT-QuIC). This assay exploits the self-propagating replication capacity of the abnormally folded and pathogenic PrP (seed), which induce the misfolding of naive PrP molecules (template) into a similar pathogenic structure. This reaction can be amplified to detectable levels and quantified in real-time. Importantly, the use of CSF from prion disease cases as a seeding material in the RT-QuIC assay allows the discrimination of CJD from non-CJD cases with high diagnostic accuracy and almost full specificity [146, 147].

Although the precise mechanism of neurofibrillary tangle formation in the brain tissue is not fully understood, the observation of tau spreading implicates the presence of a prionlike pathogenesis, where abnormal tau forms may induce the misfolding of non-pathological forms in a regional-dependent manner. Therefore, the principles of the RT-QuIC assay could be applied to the amplification of tau pathological forms in biological tissues. Although, successful cell- and tissue-based tau seeding assays have been recently developed the presence of tau seeding activity in the CSF of a tau-related pathology has been only reported once. Saijo et al. developed a tau RT-QuIC based on the use of a 3-repeat tau fragment as a substrate, a tau isoform that preferentially accumulates in Pick bodies. The authors detected positive tau RT-QUIC signal in the CSF from Pick disease (PiD) cases, suggesting that this assay may be helpful in discriminating PiD and non-PiD cases [148].
