Deep insight into the tau inoculum

The biochemical and biophysical characterization of tau inoculum is the first vital step toward comprehending one half of the pathological process involved in its propagation. Based on its origin, the tau inoculum utilized in experimental tau propagation models can be categorized as follows:

1.

Human brain-derived insoluble tau (free oligomeric or fibrillar tau)

2.

Human brain-derived insoluble tau encapsulated in exosomes

3.

Rodent brain-derived insoluble tau (free oligomeric or fibrillar tau)

4.

Rodent brain-derived insoluble tau encapsulated in exosomes

5.

Synthetic pre-formed fibrils.

Human brain-derived inocula were extracted from the isocortex, allocortex, amygdala, or subcortical nuclei of human Alzheimer’s disease [4, 7, 11, 15, 19, 29, 45, 47, 48, 58, 65, 67, 69, 71, 74, 96, 107, 115, 116, 120, 138, 140, 156, 161], Down syndrome (DS) indistinguishable from AD (DSAD) [19], argyrophilic grain disease (AGD) [29, 48], Pick’s disease (PiD) [29, 45, 71], tangle-only dementia (TD) [29], globular glial tauopathies (GGT) [45, 49], corticobasal degeneration (CBD) [19, 29, 71, 115, 116, 171], primary age related tauopathy (PART) [45], aging-related tau astrogliopathy (ARTAG) [45], frontotemporal dementia with parkinsonism linked to chromosome 17 (FTLD-17) [45, 161], and progressive supranuclear palsy (PSP) [29, 45, 71, 115, 116] brains (Fig. 2). The choice of region from which the inoculum was isolated was based on the anatomical distribution of tau inclusions, which varies greatly depending on the disease and its stage. In fact, tau from a single brain can exhibits variation in its proteopathic potency based on the region [82], or within the region whether it is derived from grey or white matter [165], moreover fractions show differences even within a single isolation [102]. Additionally, the tau inclusions have distinct morphological characteristics and cell-specificities [28, 114] and are composed of either 3R, 4R, or both isoforms with disease-specific filament folds [43, 44, 137, 147, 176].

Fig. 2

Inoculation-based tau propagation models. The graphical abstract illustrates the pivotal elements highlighted in this review. The central theme revolves around the transcellular propagation of aberrantly modified tau protein along the functional brain network. The depicted framework emphasizes the critical steps for establishing robust models, including the judicious selection and comprehensive characterization of tau inocula through functional, biochemical, and biophysical analyses (1, 2). Key considerations involve the careful choice of animal models (3), optimal inoculation sites (4), the crucial validation of fibrillary pathology using confirmatory staining techniques (5) and downstream assessment (6). The proposed framework serves as a practical guide for researchers, offering a systematic approach to establish benchmark models for preclinical testing of potential disease-modifying drugs (DMTs)

Rodent brain-derived inocula were extracted from the cortices, brainstems, or spinal cords of transgenic mice (P301ST43 [1, 30, 80], rTg4510 [146]), or rats (SHR24 [100], SHR72 [100]), taking into account the regional distribution of tau inclusions. These inclusions are driven by the transgenic expression of either human mutant tau such as P301S [5], or P301L [128, 134] or by truncated tau (aa151-391) found in sporadic AD [51, 178] and are composed of both transgenic and if not knocked out, endogenous rodent tau. The 3R/4R isoform and the filament-fold are either reported to be NFT-like or remains to be determined at large.

From both human and rodent brains, extracellular vesicles, particularly exosomes, were derived from prodromal AD, mild cognitive impairment (MCI), AD, PSP or PiD [99, 131] or from the iPSCs derived from AD patients [12, 163], as well as from brain extracts from Tg rodent models of tauopathy [13]. Given that the neuronal cells are recognized for producing exosomes that transport cargo such as proteins, RNA-binding proteins (RBPs), and RNAs to neighbouring cells, exosomes were initially considered and subsequently confirmed to be vehicles facilitating the cell-to-cell transmission of oligomeric tau [16, 125]. The 3R/4R isoform composition of tau with characteristic filament-folds within these exosomes is specific to the disease or model.

Human/rodent brain-derived insoluble tau inoculums were prepared as either enriched detergent-insoluble protein aggregates or, in some cases, as crude protein extracts (Tables 1, 2, & 3). It is imperative to underscore that within the widely utilized sarkosyl-insoluble protein aggregate fraction, tau comprises a mere 10% of the total proteins [39]. This fraction encompasses additional constituents, including β-amyloid (Aβ), snRNP70 (U1-70 K), apolipoprotein E (ApoE) and complement component 4 (C4-A), among others [39, 64]. With the aim of obtaining a homogenous sample of aggregated tau, certain studies have also incorporated, additional fractionation steps, employing techniques such as immunoprecipitation (IP) or fast protein liquid chromatography (FPLC) [9]. These fractions also consisted of a diverse pool of fibrillary, filamentous, multimeric, and oligomeric units of pathological tau.

The propagation potential of oligomers, ranging from 3 to 100 molecules (< 30 nm long fibrils) [26, 80, 110, 160] along with larger insoluble tau aggregates [80] is ongoing. Although, it remains unclear which molecular entity among these is capable of propagation, tau aggregates were broken down into smaller fragments through differential sonication protocols before inoculation. This step is aligns with the prevailing notion that a soluble, high-molecular-weight (HMW) oligomeric form of tau may exhibit comparable or even heightened bioactivity in terms of propagation across neural networks [106, 146] (Tables 1, 2 and 3).

The comprehensive characterization of brain-derived tau inocula involved a multifaceted approach employing various techniques. Initial assessments utilized Western blot (WB) [15, 29, 45,