Microtubule-associated Tau proteins regulate polymerization and stability of microtubules through their microtubule-binding domains and their level of phosphorylation.
In Alzheimer’s disease and related disorders, these tau proteins aggregate into filaments. Tau proteins in these filaments are phosphorylated.
Most of the phosphorylation sites are also found in physiological conditions. However, the phospho-Ser422 is considered as an abnormal site of phosphorylation and is found in several diseases with neurofibrillary degeneration (Bussière et al. 1999). This phospho-Ser422 is correlated with the severity of neuronal pathology in Alzheimer's disease (Augustinack et al. 2002) and is also an epitope encountered in numerous Tau transgenic mouse models. With such characteristics, this epitope has also been targeted in many immunotherapy approaches (Troquier et al. 2012 ; Collin et al. 2014).
The monoclonal antibody Clone 2H9 (Ref. 4BDX-1501) is directed against phospho-Ser422 and does not cross-react with the unphosphorylated Ser422 peptide by ELISA. This antibody is useful by immunoblotting to detect pS422 epitope in brain homogenates from patients presenting a Tauopathy. By immunohistochemistry, this reference has been shown to label neurofibrillary tangles in Alzheimer patients and Tau transgenic brain sections. This monoclonal antibody is also suitable for immunotherapy in experimental models of Tauopathies. This specific biomarker (Ref. 4BDX-1501) of neurofibrillary degeneration is actually the only one mouse monoclonal antibody on the market.
The pathological epitope at phosphorylated serine 422 of Tau is found in Alzheimer ‘s disease, argyrophilic grain dementia (Tolnay et al., 2002) but also several other tauopathies including down syndrome, corticobasal degeneration, progressive supranuclear palsy, pick’s disease and frontotemporal dementia linked to mutation of MAPT gene (Bussière et al., 1999 ; Sergeant et al. 1999).
Phospho-serine 422 epitope is also observed in tau aggregates in inclusion body myotisis (Maurage et al., 2004). Recently it was shown that the phosphorylation of tau at serine 422 protects tau from caspase cleavage (Sandhu et al., 2017).
In Alzheimer’s disease, phosphorylated tau at serine 422 is found in pre-tangle neurons (Guillozet-Bongaarts et al., 2016), and detection of phosphorylated serine 422 tau epitope is correlated with the disease neuropathology and cognitive decline (Vana et al., 2011).
In parallel, the Anti-Tau [pS199] antibody (Ref. 4BDX-1502) detects specifically Tau hyperphosphorylation at serine 199 at early steps of neurofibrillary degeneration in human brain tissue or in animal models of tauopathies.
Clearly, Tau protein phosphorylation at serine 199 is an early marker of neurofibrillary degeneration in Alzheimer’s patients (Maurage et al. 2003). Phosphorylated serine 199 epitope is also found in other tauopathies such as progressive supranuclear palsy (Wray et al., 2008). Interestingly, the neurons of young individuals were also shown to express the serine 199 phosphorylated tau-epitope as published in an article of Maurage et al. 2001, using the 4BioDX antibody (Ref. 4BDX-1502) in the paper entitled “A paradoxical expression of serine 199 phosphorylated tau-epitope is found in the hippocampus of young individual”.
Beta-amyloid precursor protein (APP) is a well-conserved and ubiquitous 100-140 kDa transmembrane glycoprotein that exists as several isoforms and that is strongly implicated in the pathogenesis of Alzheimer's disease. APP and APP family members APLP1 and APLP2 functions still remains elusive, but they are clearly implicated in neuronal development, signaling, intracellular transport, iron homeostasis and other aspects of neuronal homeostasis (Van der Kant and Goldstein, 2015).
APP metabolism leads to the production of amyloid-beta peptides that are the main components of amyloid deposits in Alzheimer’s disease. Amyloid-beta peptides (Ab) are produced following by sequential cleavages of APP by proteases (called secretases). APP processing by secretases produce the carboxy-terminal fragments (CTFs) among which the beta-carboxy-terminal fragments are the substrates of the gamma-secretase to produce amyloid-beta peptides.
APP processing is regulated by cytoplasmic phosphorylation at several sites (Lee et al. 2003). Phosphorylation at Thr668 (a position corresponding to the APP695 isoform) is one of the major phosphorylation site within APP.
Phosphorylated APP-CTFs are substrates of the gamma-secretase and an increase in the phosphorylation at Thr668 of APP-CTFs facilitates their processing by gamma-secretase (Vingtdeux et al. 2005). Phosphorylation of APP at Thr668 by JNK is required for γ-mediated cleavage of the C-terminal fragment of APP produced by β-secretase (Mazzitelli et al. 2011) and mechanism of phosphorylation at Thr-668 of C-terminal APP fragments regulates the localization of APP, affects its processing by γ-secretases and enhance the production of Abeta peptides (Matsushima et al. 2012).
APP is phosphorylated at Thr-668 both in vitro and in vivo by a number of kinases including glycogen synthase kinase 3β (GSK3β), Jun N-terminal kinase-3 (JNK3), Jun N-terminal kinase 1 (JNK1), cell division cycle protein (Cdc2), cyclin-dependent kinase 5 (Cdk5) and DYRK1A (Dual-specificity tyrosine(Y)-phosphorylation regulated kinase 1A) (Van der Kant and Goldstein, 2015). Phosphorylation of APP at Thr-668 is also associated with neurite extension, anterograde transport of vesicular cargo into neurites, axonal transport and in signaling to the nucleus (Vingtdeux et al. 2005 ; Chang et al. 2006).
Structurally, the phosphorylation of APP at Thr-668 results in a significant conformational change that affect interactions with binding partners and then impact its subcellular localization and metabolism (Ramelot and Nickolson, 2001). The phosphorylation status of Thr-668 either creates or destroys docking sites for intracellular proteins that interact with APP.
Through analogy to the Notch protein, APP is also described as a cell-surface receptor that signals by releasing an intracellular domain (AICD) to the nucleus which together with Fe65 and Tip60, and then regulate gene expression (Cao and Sudhof 2001 ; Vingtdeux et al. 2005). Phosphorylation at Thr-668 deliver FE65 enabling AICD nuclear translocation and gene activation that induce neurodegeneration (Vingtdeux et al. 2005 ; Kimberly et al. 2005 ; Nakaya et al. 2006). The intracellular residue Thr668 of APP is required for memory and synaptic plasticity deficits (Lombino et al. 2013) and when co-expressed with Fe65, the APP-CTFs phosphorylation at Thr-668 were shown to be reduced as well as the amyloid deposits in models of transgenic mice (Santiard-Baron et al. 2005).
The rabbit polyclonal antibody pT668 (Ref. 4BDX-1503) recognizes the phosphorylated threonine 668 of Amyloid Protein Precursor (APP) and is suitable for Western Blot, for Immunohistochemistry and Immunofluorescence. This antibody is a biomarker of neuritic processes surrounding amyloid deposits.