Overview of Alzheimer’s Disease Antibody Targets

Alzheimer’s disease (AD) is a chronic, progressive neurodegenerative disease that accounts for 60-80% of dementia cases. Although it is not a part of normal aging, AD tends to affect people over 65 years of age and it is estimated that there are over 40 million people currently living with some form of the disease. The most common, early symptom of Alzheimer’s disease is memory loss characterized by forgetting recent events and conversations. With progression of the disease, affected persons will develop severe memory loss and impaired social and behavioral abilities. These conditions gradually worsen with time eventually affecting daily living, thinking, reasoning, and bodily functions, ultimately leading to death.

The exact cause of AD is poorly understood, although scientists believe it to be triggered by a combination of genetic, environmental, and lifestyle factors. Research shows that age, familial history of AD, brain injury, cardiovascular disease, and lifestyle factors such as smoking, diabetes, and obesity can increase the risk of having Alzheimer’s disease. AD is typically identified using a combination of memory and behavior evaluations, brain imaging, and laboratory tests to look for protein markers.

Using antibodies to study protein changes in AD offers the best insight into disease etiology and helps scientists predict ways to slow disease progress. Antibodies are considered workhorses of biological research and can be used in a variety of applications including western blotting, immunofluorescence, ELISA, flow cytometry, and immunoprecipitation to study protein interactions and functions, both qualitatively and quantitatively. Antibodies can also be used as markers to identify and/or isolate specific cell types and study cellular differentiation.

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The cleavage of healthy amyloid precursor protein (APP) to pathogenic beta Amyloid (Aβ) peptides is brought about by β and γ secretases. This occurs via two distinct pathways - the non-amyloidogenic pathway and the amyloidogenic pathway. The non-amyloidogenic pathway provides beneficial neurotrophic effects and the amyloidogenic pathway produces neurotoxic Aβ peptides. These peptides can misfold and aggregate into deposits that are characteristic of Alzheimer’s disease pathology. The key to slowing the progression of AD is to target the accumulation of beta-Amyloid by inhibiting its production. This can be brought about by blocking APP cleavage using β secretase (BACE) inhibitors.

A major roadblock to decoding the role of Aβ in Alzheimer’s is the lack of correlation between Aβ in the brain of Alzheimer’s patients and their cognitive ability. This is possibly due to the structural variations of Aβ deposits that show up at different times in different parts of patient brains.

Invitrogen’s antibodies against beta Amyloid protein are tested for specificity in a variety of applications, including western blot, immunohistochemistry, ELISA, and immunoprecipitation. The figure below highlights some of Invitrogen’s antibodies against beta Amyloid, which are verified for specificity using relative expression across cell lines.

Figure 1. beta Amyloid antibodies in immunofluorescence and western blot.(A) Immunofluorescence staining of Amyloid Precursor Protein (APP) using Amyloid Precursor Protein Monoclonal Antibody (mAbP2-1) (Cat. No. OMA1-03132). (B) Immunofluorescence staining of beta Amyloid protein using beta Amyloid Monoclonal Antibody (DE2B4) (Cat. No. MA1-24966). The intended proteins are clearly picked up in the PC3 cell line, which is positive for beta Amyloid (green), and the signal is absent in K562 cells. (C) Western blot analysis of beta Amyloid using a beta Amyloid Polyclonal Antibody (Cat. No. 36-6900). (D) Relative expression of beta Amyloid in PC3, K562, and HEL 92.1.7 cell lines using beta Amyloid Monoclonal Antibody (DE2B4) (Cat. No. MA1-24966). (E) Relative expression of APP in western blot using Amyloid Precursor Protein Polyclonal Antibody (Cat. No. PA1-84165). Signals in the immunofluorescence experiments were detected using Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Cat. No. A32766, 1:2000 dilution) for 45 minutes at room temperature. Nuclei were stained with ProLong Diamond Antifade Mountant with DAPI (Cat. No. P36962). F-actin was stained with Rhodamine Phalloidin (Cat. No. R415, 1:300 dilution). For the western blot analyses, either Goat anti–Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, HRP (Cat. No. G-21234) or Goat Anti–Mouse IgG (H+L) Secondary Antibody, HRP (Cat. No. 62-6520) were used and detected using Pierce ECL Western Blotting Substrate (Cat. No. 32106) and imaged using the iBright FL1500 Imaging System (Cat. No. A44115).

Tau proteins are found in neurons and one of their major functions is to modulate stability of axonal microtubules. They belong to a group of six highly-soluble proteins that are encoded for by the MAPT (Microtubule Associated Protein Tau) gene. Hyperphosphorylated insoluble aggregates of tau, referred to as neurofibrillary tangles (NFT), are found to be associated with pathologies of the nervous system, including Alzheimer’s and Parkinson’s disease.

The tau protein contains around 85 phosphorylation sites. Under normal circumstances, the phosphorylation sites function to regulate microtubule dynamics, axonal transport, and neurite outgrowth. Disruption in normal phosphorylation in tau is a hallmark of neurodegenerative diseases like Alzheimer’s disease. Abnormal tau phosphorylation not only results in a toxic loss of function (for example, decreased microtubule binding), but is also thought to result in gain of toxic function (for example, increased tau-tau interactions). Identification of the protein kinases that phosphorylate tau could potentially help find therapeutic targets for the treatment of AD.

Delving into the interactions of tau with other proteins provides insight into the Alzheimer’s brain. Antibodies specific to tau can help understand the link between tau aggregation and resulting pathologies associated with the disease. Invitrogen offers antibodies against total tau as well as several phosphorylated forms. The figure below represents data from validation experiments of tau antibodies.

Figure 2. Tau Antibodies in immunofluorescence and western blot.(A) Western blot analysis of Tau Monoclonal Antibody (TAU-5) (Cat. No. MA5-12808) across multiple cell lines. (B) Immunofluorescence analysis in human iPSC-derived forebrain organoids at day 40 using Tau Monoclonal Antibody (TAU-5) (green) (Cat. No. AHB0042) and Phospho-Tau (Ser396) Polyclonal Antibody (red) (Cat. No. 44-752G), and counterstained with DAPI (blue) (Cat. No. D1306). (C) Western blot analysis of the specificity of Phospho-Tau (Ser199, Ser202) Polyclonal Antibody (Cat. No. 44-768G) binding in the presence of the specific phosphor-peptide target (lane 2), the corresponding non-phosphorylated peptide (lane 3), and other phosphoserine-containing peptides (lanes 4-6); signal loss was only seen with the specific peptide. (D) Western blot analysis of Phospho-Tau (Ser262) Polyclonal Antibody (Cat. No. 44-750G) in mouse brain, rat brain, and mouse kidney lysates. For the western blot analyses, either Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, HRP (Cat. No. G-21234) or Goat Anti–Mouse IgG (H+L) Secondary Antibody, HRP (Cat. No. 62-6520) were used and detected using Pierce ECL Western Blotting Substrate (Cat. No. 32106) and imaged using the iBright FL1500 Imaging System (Cat. No. A44115).

In a healthy brain, a type of glial cell called microglia are responsible for the engulfment and destruction of wastes and toxins. In Alzheimer’s disease, microglia are thought to have lost this function and they fail to remove waste and protein aggregates, including the beta Amyloid plaques. This leads to a buildup of glial cells in the brain, which ultimately causes chronic inflammation. TREM2 is one of the key proteins that instruct microglia to clear plaques from the brain and is therefore a protein of value to Alzheimer’s research. TREM2 is a transmembrane receptor expressed on myeloid cells and its expression is regulated by anti- and pro-inflammatory cytokines. The figure below shows representative data from TREM2 antibodies offered by Invitrogen for Alzheimer’s disease research. These antibodies have been tested for specificity using techniques such as relative expression and cell treatment.

Figure 3. TREM2 antibodies used in immunofluorescence and western blot.(A) Western blot performed using TREM2 Polyclonal Antibody (Cat. No. PA5-46978) to detect a 30 kDa band corresponding to TREM2 in the condition media of THP1 and Raw 264.7 cells. (B) Immunofluorescence analysis with the same antibody, showing detection in only RAW 264.7 cells upon PTI (green) treatment in comparison to NIH/3T3 with PTI treatment. (C) Western blot using TREM2 Recombinant Rabbit Monoclonal Antibody (4H42L9) (Cat. No. 703422), showing increased expression of protein TREM2 (glycosylated) in THP-1 cells upon differentiation into macrophages and dendritic cells. (D) Immunofluorescence analysis of TREM2 using TREM2 Recombinant Rabbit Monoclonal Antibody (9H4L26) (Cat. No. 702886) detects TREM2 in the membrane of dendritic cells in comparison to lower expression in undifferentiated THP-1 cells.

Apart from the proteins described above, a few others have emerged as potential targets in Alzheimer’s disease research.

Presenilin 1 (PSEN1) has been identified as a major contributor for autosomal dominant AD and some of the youngest patients of familial AD (28-60 years) have shown to have a mutation in the gene. Mutations in a second related gene – Presenilin 2 (PSEN2) – contributes to AD pathogenesis with a later and more variable age of onset. Both genes encode production of γ secretase, which is responsible for the cleavage of amyloid precursor.

The ε4 allele of Apolipoprotein E (APOE) has been identified as a major genetic risk factor for Alzheimer’s disease. Although numerous attempts have been made to identify the underlying mechanism for this increased risk, its influence in the onset and progression of AD is yet to be proven. However, there is increasing evidence that the differential effects of APOE isoforms on Aβ aggregation and clearance play a major role in AD pathogenesis.

Sirtuin 6 (SIRT6) is a protein that has been implicated in telomere maintenance, DNA repair, metabolism, and inflammation. It has recently been found to be lacking in Alzheimer’s disease patients and has emerged as a potential therapeutic target. In AD, SIRT6 is thought to directly interact with and deacetylate tau, reducing the latter’s phosphorylation and has been shown to promote non-amyloidogenic α-secretase-mediated APP cleavage.

The figure below illustrates representative data using Invitrogen antibodies against these targets. These antibodies are validated for specificity using techniques such as siRNA-silencing and relative expression.

Figure 4. Emerging Alzheimer’s disease antibody targets in western blot.(A) Western blot analysis on untransfected and siRNA-transfected T-47D cells to verify antibody specificity of Presenilin 1 Monoclonal Antibody (APS 18) (Cat. No. MA1-752). (B) Relative expression of PSEN2 observed in THP-1 cells when compared to Raji and Jurkat cell lines using PSEN2 Monoclonal Antibody (APS 26) (Cat. No. MA1-754) in western blot. (C) A549 cells were transfected with SIRT6 siRNA and a reduction of signal was observed in western blot using SIRT6 Polyclonal Antibody (Cat. No. PA5-17215). For each of the western blot analyses, either Goat anti–Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, HRP (Cat. No. G-21234) or Goat Anti–Mouse IgG (H+L) Secondary Antibody, HRP (Cat. No. 62-6520) were used and detected using Pierce ECL Western Blotting Substrate (Cat. No. 32106) and imaged using the iBright FL1500 Imaging System (Cat. No. A44115).

Alzheimer’s disease is a complex neurodegenerative disorder caused by a combination of genetic, physiological, and environmental factors. While current treatment can temporarily improve memory loss, they cannot fix the underlying death of brain cells. Extensive genetic and molecular studies are required to understand disease processes and antibodies can prove useful tools in doing so. Antibodies that are specific to target proteins can help elucidate protein-protein interactions and reveal the underlying mechanisms of disease pathology. The antibodies discussed in this article are against well-documented, as well as emerging proteins, important to Alzheimer’s disease research. These antibodies have been extensively validated in biologically relevant cell/tissue models and antibody specificity has been demonstrated using siRNA knockdown or biological variations.
 

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