Epitope: Conformational epitope including amino acids at both 5-15 and 312-322
Immunogen: Homogenates of Alzheimer’s brains
Clonality: Monoclonal
Isotype: IgM
Host: Mouse
Reactivity: Human; Mouse; Rat; Non human primates; Bovine; Sheep

Overview

This monoclonal antibody recognizes a tau conformation that precedes the appearance of neurofibrillary tangles. Similar to monoclonal antibody MC-1, Alz-50 detects a discontinuous epitope that includes amino acids in tau’s N-terminal and third repeat regions. However, Alz-50 and MC-1 are not identical, as Alz-50 recognizes the FAC1 protein (see below), but MC-1 does not. The Alz-50 and MC-1 epitopes are present in all six tau isoforms.

Alz-50 was generated against homogenates of Alzheimer’s brains and was initially described as recognizing a protein much more abundant in AD brains than normal brains (Wolozin et al., 1986). In AD brains, Alz-50 stains tangle-bearing neurons, neuritic dystrophies, and neuropil threads, as well as some normal-appearing neurons (Wolozin et al., 1986; Hyman et al., 1988). It has been suggested that these apparently normal neurons in AD brains are on their way to forming neurofibrillary tangles (Hyman et al., 1988; Goedert et al., 1991). Alz-50 immunoreactivity also has been observed in normal brains, although the degree varies among studies, and in the brains of subjects with neurodegenerative diseases other than AD, as described below.

Alz-50 recognizes tau filaments. Electron micrograph showing Alz-50-immunoreactive filaments in the hippocampus  of an aged 3xTg-AD mouse. Immunoperoxidase technique. Right panel is a magnified view of the region outlined by the box in the left panel. Scale bars: left,  0.5 μm; right, 200 nm. From Oh et al., 2010; Figure 14i,j; licensed under Creative Commons BY 3.0.

Generation and epitope mapping

The Alz-50 antibody was generated against pooled homogenates from the basal forebrains of Alzheimer’s subjects. The antibody was initially described as recognizing a 68-kDa protein on western blots of AD brains, as well as small amounts of a lower-molecular-weight protein in the brains of normal subjects (Wolozin et al., 1986).

Subsequent studies pointed toward tau as the target of Alz-50. The antibody reacted with tau biochemically isolated from AD brains (Nukina et al., 1988) as well as from normal human brains and bovine brains, (Ksiezak-Reding et al., 1988), and it immunoprecipitated anti-tau-immunoreactive proteins from AD brains (Nukina et al., 1988).

The Alz-50 epitope was defined by deletion mapping using recombinant tau protein, and it was found that two discontinuous regions are required for antibody binding—amino acids 7-9 near the N terminus and a sequence between amino acids 313 and 322 in the third repeat domain—and that the epitope is formed by intramolecular interactions (Carmel et al., 1996; Jicha et al., 1997; Jicha et al., 1999). While not essential for antibody binding, some part of the 300-amino acid intervening sequence is needed for full immunoreactivity, perhaps allowing the protein space to fold into the shape that brings the two sections of the epitope together. The Alz-50 epitope is sensitive to denaturation, supporting the conclusion that Alz-50 is a conformation-selective antibody (Carmel et al., 1996).

The interaction of Alz-50 with recombinant tau suggests that post-translational modifications are not required to generate the Alz-50 epitope (Jicha et al., 1997). However, the affinity of Alz-50 for paired helical filament (PHF) tau in solution is nearly 100 times greater than for recombinant human tau (Carmel et al., 1996), and it remains possible that post-translational modifications influence the ability of tau to adopt or maintain the conformation seen by Alz-50. It was reported that Alz-50 immunoreactivity—assessed on western blots of AD brain homogenates or by immunohistochemical staining of AD brain sections—was decreased by acid phosphatase treatment and that the inclusion of phosphatase inhibitors during sample preparation increased Alz-50 immunoreactivity on immunoblots (Uéda et al., 1990). By contrast, another study did not find any diminution of Alz-50 immunoreactivity when PHFs were treated with hydrofluoric acid (Greenberg et al., 1992). It has also been suggested that the creation or stabilization of the Alz-50 epitope is influenced by oxidative damage: Treatment of normal tau with 4-hydroxy-2-nonenal, a reactive product of lipid peroxidation, increased Alz-50 immunoreactivity on immunoblots more than 10-fold, an effect that was phosphorylation-dependent (Takeda et al., 2000; Liu et al., 2005). Consistent with the hypothesis that tau adopts the Alz-50 conformation under conditions of oxidative stress, in one study, all Alz-50-immunoreactive neurons in AD brains also stained for the antioxidant heme oxygenase-1 (Takeda et al., 2000).

Post-translational modifications of tau increase Alz-50 immunoreactivity. A) Tau biochemically isolated from normal human brain was treated with 4-hydroxy-2-nonenal (HNE) and Alz-50 was used to probe dot blots of the resulting product. HNE treatment increased Alz-50 immunoreactivity in a concentration-dependent manner. B) The effect of HNE on Alz-50 immunoreactivity is phosphorylation-dependent. Upper panel, Dephosphorylation of tau with alkaline phosphatase (AP) or hydrofluoric acid (HF) decreases the effect of HNE treatment on Alz-50 immunoreactivity; unt, sample not treated with AP or HF. Middle panel, Confirmation that treatment with AP or HF dephosphorylates tau: both treatments increase immunoreactivity to monoclonal antibody tau-1 (τ-1), which recognizes a non-phosphorylated epitope between amino acids 192 and 204 (Szendrei et al., 1993). Lower panel shows that treatment of tau with HNE, AP, or HF does not impair binding to the membrane. [From Takeda et al., 2000 (A, Fig. 2; B, Fig. 4); Journal of Neurochemistry. © 2000 International Society for Neurochemistry.]

Specificity

While Alz-50 is often used to detect a “pathological” conformation of tau that precedes the appearance of neurofibrillary tangles, the extent to which Alz-50 reacts with tau from normal brains is debatable. Alz-50 immunoreactivity in homogenates of Alzheimer's brains was more than 50-fold that of control brains, measured using a direct ELISA (Wolozin and Davies, 1987). Early immunohistochemical studies found Alz-50 staining in neurons of individuals with Alzheimer’s disease or certain other tauopathies, but immunostaining was absent or rare in controls (Wolozin and Davies, 1987; Hyman et al., 1988). Soon thereafter, considerable Alz-50 staining was reported in the brains of non-demented elderly (Price et al., 1991) and normal human brains across the lifespan (Rye et al., 1993). The antibody detected proteins in the brains of normal subjects on Western blots, although the molecular weights were lower and the amounts less than the Alz-50-immunoreactive species found in AD brains (Wolozin et al., 1986; Wolozin and Davies, 1987). One study found no difference in the amount of Alz-50-immunoreactive material in the temporal cortices of Alzheimer’s cases (Braak stages 4-6) and controls (Braak stages 0-3), assessed using dot blots (Koss et al., 2016). There was a slight, but statistically significant, correlation between Braak stage and Alz-50 reactivity in this study. It has been suggested that blotting conditions may artificially create the tau conformation detected by Alz-50—perhaps through tau interacting with charged membranes (Jicha et al., 1999).

Alz-50 immunoreactivity has also been observed in the brains of subjects with Pick’s disease (Wolozin and Davies, 1987), Guam-Parkinson dementia complex (Wolozin and Davies, 1987; Love et al., 1988), Kufs’ disease (Love et al., 1988), progressive supranuclear palsy (Love et al., 1988; Yamada et al., 1993), Down syndrome (Sparks and Hunsaker, 1992; Perez et al., 2019), Parkinson’s disease (de la Monte et al., 1992), corticobasal degeneration (Ksiezak-Reding et al., 1994), Gerstmann-Sträussler-Scheinker disease (Giaccone et al., 1990), and ischemic stroke (Uchihara et al., 1995). However, the quantity and quality (e.g., cell type, neuropil versus soma) of immunoreactivity differs among these conditions.

Alz-50 reactivity in human brains. A) Alz-50 immunoreactivity in lysates of temporal cortices from subjects with neuropathologically confirmed Alzheimer’s disease (Braak stages 4-6) and controls (Braak stages 0-3). i) Sample blot.  ii-iv) Quantification of dot blot signals. B) Western blot of lysates of temporal cortices . Lanes: A) Alzheimer’s disease, B) Control, C, D) Pick’s disease, E) Guam-Parkinson dementia complex. C) Immunohistochemistry. Alz-50 staining in brain sections from a) AD, b) normal, c) Pick’s disease, d) Guam-Parkinson dementia complex, e) Pantothenate kinase-associated neurodegeneration. [A) From Koss et al., 2016; Figure 3a; licensed under Creative Commons BY 4.0. B and C) From Wolozin and Davies, 1987, Figures 3 and 1, respectively. Annals of Neurology. © 1987 American Neurological Association.]

Species

Alz-50-immunoreactive neurons were found in the cortices and hippocampi of western lowland gorillas ranging from 22 to 55 years of age (Perez et al., 2013). As mentioned above, Alz-50 was shown to react with biochemically isolated bovine tau (Ksiezak-Reding et al., 1988; Ksiezak-Reding et al., 1990). The antibody was also reported to recognize tau from sheep brain on western blots and to stain somatostatin-positive medium aspiny neurons in the striatum; however, the reported cross-reactivity of Alz-50 with fragments of the somatostatin precursor protein (see below) confounds the interpretation of this finding (Nelson et al., 1993). Alz-50 was reported to be “weakly reactive” with samples from normal rat and mouse brains on western blots (Ksiezak-Reding et al., 1990). Alz-50-immunoreactive neurons were observed in the brains of mice expressing human APP (Higgins et al., 1994; Higgins et al., 1995) or murine Psen1 (Tanemura et al., 2006) with AD-linked mutations—animals that express endogenous mouse tau—as well as transgenic mice expressing human tau with the P301L mutation linked to frontotemporal dementia (Kopeikina et al., 2013; Oh et al., 2010).

Cross-reactivity

In the fetal brain, Alz-50 detects the zinc-finger protein FAC1 (Fetal  Alz-50-Reactive Clone 1, also known as Nucleosome-remodeling factor subunit BPTF), discovered during screening of a human fetal brain cDNA library to identify targets of Alz-50 (Bowser et al., 1995). Sequence homologies in tau and FAC1 within the Alz-50 epitopes—tau amino acids 7-9 and 326-330—likely account for this cross reactivity (Jicha et al., 1997).

Alz-50 detected denatured bovine and human serum albumins on western blots (Davis and Johnson, 1994).

Upon observing Alz-50 immunoreactivity in somatostatin-containing neurons in the hypothalamus of control brains, van de Nes et al. tested whether the antibody recognizes the somatostatin peptide itself, and found that the antibody did bind fragments of the somatostatin precursor in dot blots (van de Nes et al., 1994).

Validation

In the absence of defined reagents that specifically mimic or disrupt the Alz-50 tau conformational epitope, it has not been possible to validate this antibody. In two studies, Alz-50 immunoreactivity was eliminated by preincubation of the antibody with peptides containing amino terminal sequences of tau (Goedert et al., 1991; Nelson et al., 1993).

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References

Research Models Citations

1. 3xTg

Paper Citations

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External Citations

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