Individuals with DS develop dementia and AD pathology at a variable age, but earlier than in LOAD and perhaps earlier than in EOAD. Given that as much as 90% of individuals with DS develop AD-like dementia by the fifth decade of life [126], one might hypothesize that Tau seeding and spread within the DS brain may occur decades earlier. Recent improvements in measuring P-Tau by immunoassays, seeding assays and Positron Emission tomography (PET) have enabled this hypothesis to be tested and led to several breakthroughs, while producing new questions about when Tau gains seeding “prion-like” capacities.

With the recent development of new sensitive assays such as the single molecule array (Simoa [127]), investigators are now capable of measuring even minute amounts of biomarkers in plasma in different neurological conditions. We performed a study focused on exosome biomarkers in individuals with DS, showing increased amyloid exosomal levels already in early childhood, which increased through adulthood [19]. However, we also unexpectedly found an increase in p-Tau (S396 and T181) when comparing all DS and all control participants at an early age, and noted an increase of p-Tau with age (T181) [20]. These findings suggested that misfolding and phosphorylation of Tau may be an early event in DS and could be used to predict the onset of dementia, and also improve treatment efficacy for this population. Measuring biomarkers in exosomes proved to be a sensitive and possibly less variable assay of dementia-related biomarkers than when the same biomarkers were applied in plasma, which was due to the relatively low levels of AD-related proteins observed in plasma.

In engaging with older adults with DS, Alzheimer Biomarkers Consortium—Down Syndrome (ABC–DS) demonstrated early changes in persons with DS by using different p-Tau isoforms in plasma [128]. The Petersen et al. group [128] studied more than 300 participants with DS at different ages, showing that plasma levels of total Tau and neurofilament light (NfL) were highly predictive of both AD pathology and clinical status in those with DS at different ages. In another recent biomarker study for the European DS clinical network Horizon 21, Carmona and collaborators examined neurofilament light (NfL) [129], and found that NfL plasma levels had excellent diagnostic performance and a highly similar temporal distribution of change, compared to that seen in autosomal dominant AD [129]. Phospho-Tau biomarkers in plasma have also been examined by using ultrasensitive methods [130,131]. And Plasma p-Tau (T217), glial fibrillary acidic protein (GFAP), amyloid beta peptides 42 and 40 (Aβ42/Aβ40), NfL, and total Tau (t-Tau) were assayed. The study, which included 300 participants with DS and 37 non-DS siblings, found that higher p-Tau (T217) levels and no other biomarkers were associated with worse performance in the DS mental status examination and cued recall test, suggesting that plasma p-Tau (T217) is an accurate blood-based biomarker of both Tau and Aβ pathological brain changes in DS that could be used to include individuals with DS in AD clinical trials, especially when combined with age as a covariate [130]. Since plasma levels of AD-related pathology are easier to accomplish in the clinic than in NDE assessments, this newly developed measure may lend itself better to large clinical studies than to more cumbersome exosomal biomarker studies [20,132].

Promising techniques have been developed for the quantitation of Tau seeding activity in human biofluid. Holme et al. (2014) developed a cell-based assay, where a Tau-containing biosample is incubated with a cell line overexpressing tau linked to a fluorescent protein [108]. Upon aggregation, energy transfers between fluorescent proteins (FRET) allowed for the detection of seeding capacity signals using flow cytometry. Using this FRET method, robust Tau seeding activity could be observed one month before histopathological stains showed NFTs, suggesting that tau seeding is an early signature of tauopathy. Cell-free assays have been developed by real-time quaking-induced conversion (RT-QuIC) to measure prion seeding [133] and, more recently, Tau seeding [134]. Tau seed-containing material from biofluids or brain samples is incubated with recombinant tau substrate and thioflavin T in optimized conditions. When RT-QuIC is used, seeding-competent material induces the aggregation of the substrate, which generates a fluorescent signal.

Jin et al. (2022) recently developed a Tau seeding activity assay using truncated HA-tagged Tau151-391 peptide and cellular transformation [135]. Cells transiently express the HA-tagged Tau151-391 peptide, which is readily captured and aggregated by oligomeric Tau derived from postmortem AD brain samples. The captured Tau is then quantified using traditional immunoblot methods. They employed these assays on AD and DS brain samples in two different studies [136] and discovered that only brain extracts from AD or DS brain samples contained hyperphosphorylated Tau seeded Tau aggregation in cultured cells. Interestingly, Tau extracts of DS corpus callosum showed low tau seeding activity, and no detectable tau seeding activity was observed in DS cerebellar cortex. Again, Tau seeding ability was found to be highly correlated with phosphorylation, but the group did not study the effects of other Tau PTMs in this assay.

Tau PET imaging is emerging as an important clinical tool for early diagnosis of AD and other tauopathies, as p-Tau appears to correlate more closely than amyloid with symptomatic dementia progression. In the last ten years, multiple Tau PET ligands have been developed. Tau-specific ligands for use in PET include first-generation ligands and, consequently, second-generation ligands (see Leuzy et al. [137]). Several of these Tau PET ligands have been tested in patients with DS and AD including, for example, the [18F]-AV-1451 Tau PET ligand [138]. Rafii et al. demonstrated that amyloid-negative participants who were DS imaged were all Tau-negative, and that both amyloid and Tau burden correlated with age [138]. They also found that Tau binding in the brain correlated significantly with cognitive decline, suggesting that this clinical measure can be used to predict the onset or progression of dementia in DS-AD. Recently, Dr. Bradley Christian and his research group have performed longitudinal PET analyses in a DS cohort with the aim of better defining a timeline for the progression of amyloid and Tau burden through the conventional Braak stages [139,140,141]. By using PET scans, they revealed early and rapid Tau elevation following the onset of amyloid-positive PET imaging. While Tau elevation is highly variable in AD cohorts, individuals with DS displayed uniform increases in Tau within 2.5 years of the onset of amyloid binding in the brain. Although very early in life, the spatial progression of neurofibrillary Tau tangles and paired helical filament Tau in DS closely follows the hierarchical staging pattern outlined by Braak and Braak for LOAD studies [142]. This pattern has been well described by Davidson et al., who observe that Tau pathology usually commences after 35-years-of-age, in the entorhinal cortex, hippocampal formation, and in subcortical brainstem regions such as the locus coeruleus and dorsal raphe nucleus. Later, Tau pathology spreads throughout the neocortex, sparing the occipital lobes until the mid-50 years of age [18]. Although this pattern is generally recognized in individuals with DS, there are reports showing that dysregulation of Tau phosphorylation can occur quite early in life in those with DS [143], which is something that cannot be detected by Tau PET imaging, which serves to demonstrate the need for detailed postmortem tissue studies.

To further quantify the binding of Tau PET ligands in specific brain regions, we performed an autoradiographic binding study of Tau PET ligand binding in postmortem, fixed and paraffin-embedded brain tissue sections from individuals that received a neuropathological diagnosis of either LOAD, EOAD, or DS-AD, and compared them against age-matched controls [144]. Interestingly, we found that the binding of both the first generation (THK5117) and the second generation (MK6240) Tau PET ligand exhibited a significant increase in frontal cortex (middle frontal gyrus) in DS–AD postmortem cases, compared to both EOAD and LOAD cases. In addition, autoradiographic binding of both of these Tau ligands correlated significantly with AT8 p-Tau immunostaining of adjacent sections, strongly suggesting that on-target binding was more prevalent than off-target binding, at least in these fixed and paraffin-embedded sections [144]. Other studies have suggested that in fresh frozen materials and in vivo, there is off-target binding of Tau ligands to, for example, monoamine oxidase B (MAO-B) [145], which did not appear to be the case in our study using fixed materials. Investigators have identified a binding site for all the Tau tracers on MAO-B [145], which could make clinical studies difficult to undertake unless a better understanding of off-target binding is first achieved. However, it was reported by Murugan et al [145] that a second-generation Tau PET ligand, MK6240, has a lower affinity for MAO-B than the first-generation tracers. Nevertheless, in our binding study, we found a significant correlation between p-Tau immunostaining (AT8 antibodies), with both the first and second-generation Tau ligands used in this study [144], suggesting less off-target binding in fixed tissues than what was previously reported in vivo.