Biomarker

Because neurophathologica changes of AD precede symptoms by years, it is imperative to dignose AD disease as soon as possible. Biomarker is a neurochemical indicator that is used to assess the risk or presence of disease. Biomarker can not only diagnose AD ina very early stage but provide objective e and reliable measure of disease progress. It is well known [Beta Amyloid is a good indicator of AD disease which has facilitated doctors to accurately pre-dignose AD disease. When AB peptide is released by proteolytic cleavage of Amyloid precursor protein, some Aβ peptides that are solubized are detected in CSF and plasma which makes AB peptides a promising candidate for biological markers. It is proven that Beta Amyloid biomarker shows 80% or above sensitivity and specificity in distinguishing AD from other dementia. It is believed that Beta-Amyloid Biomarker will provide a future for diagnosis of AD and eventually treatment of AD which is the hope of many elderly people.

What is Beta-Amyloid?

Aβ is composed of a family of peptides produced by proteolytic cleavage of the type I transmembrane spanning glycoprotein Amyloid precursor protein (APP). Senile plaque Aβ protein species ends in residue 40 or 42, but it is suspected that Aβ42 form is crucial in the pathogenesis of AD. Although Aβ42 makes up less than 10% of total AB, it aggregates at much faster rate than Aβ40. Aβ42 is the initial and major component of senile plaque deposits. While the most prevalent hypothesis for mechanisms of Aβ-mediated “neurotoxicity” is structural damage to the synapse, various mechanisms such as oxidative stress, altered calcium homeostasis, induction of apoptosis, structural damage, chronic inflammation and neuronal formation of amyloid has been proposed. Observation of AB42/AB40 ratio has been promising biomarker for AD. However, as AB42 fails to be reliable biomarker in plasma, attention was drawn for alternative biomarkers.

Candidates

BACE1

Pros to BACE1

Various enzymatic digestion including β- and γ- secretase cleaves amyloid precursoe protein (APP) into various type of amyloid β protein. Most β-secretase activity originates from an integral membrane aspartyle protease encoded by the β-site APP-cleaving enzyme 1 gene (BACE1). According to research led by Dr. Zetterberg, he used sensitive and specific BACE1 assay to assess CSF BACE1 activity in AD. As a result increased BACE1 expression and enzymatic activity has been detected in subjects with AD. It was concluded that elevated BACE 1 activity may contribute to the amyloidgenic process in Alzheimer’s Disease. CSF BACE1 activity can be a potential candiedate biomarker to monitor amyloidogenic APP metabolism in the CNS.

Cons to BACE1

Soluble Aβ precursor protein (sAPP)

Pros to sAPP

APP is an integral membrane protein whose proteolysis generates beta amyloid ranging from 39- to 42- amino acid peptide. Although biological function of APP are not known, it is predicted that APP may play role during neuroregeneration, and regulation of neural activity, connectivity, plasticity, and memory. Recent researches have shown that large soluble APP (sAPP) that are present in CSF may serve as an novel potential biomarkers of Alzheimer’s disease. In a report posted in nature, a group led by Lewczuk performed a test to observe performance of soluble form of APP α and β. Significant increase in sAPP α and sAPP β were evident in patients with Alzheimer’s disease compared to normal subjects.

Cons to sAPP

CSF level of α-sAPP and β-sAPP has contradictory result. Although many researches have revealed that CSF level of α sAPP increases in AD patient, some report that there is no significant change, and Lannfelt argue that there is a slgiht decrease. There more studies need to be perform to confirm validity of sAPP as a biological marker for AD.

Autoantibodies

Pros to Autoantibodies

Dr. Du Y in Indiana University brought an impact discoveries AD society that will be used not only in biomarker but provide a future therapy for AD. Antibodies on Beta amyloid are found in the CSF healthy individuals. Antibodies are believed to reduce existing plaques and prevent NFP in brain. Patients suffering from AD disease have CSF Beta amyloid antibodies, but the number was significantly lower when compared to healthy subjects.

Cons to Autoantibodies

Although overall it is accepted that level of antibodies decrease in AD patients, other studies have revealed that antibody may be much higher in AD. The reason for discrepancy arises from the nature of antibody. Antibodies and antigens are in a state of dynamic equilibrium between bound and unbound forms that is concentration dependent. Therefore, there will be varied ELISA result depending on the concentration of Antigen.

Novel Approach

Recent studies primary focus on use of autoantibody not only for biological marker but for future treatment. However, there are various argument whether autoantibody method provides reliable biomarker. A number of reports show that patients with AD have lower levels of serum anti-AB antibodies than healthy individuals others have argued that level of anti-AB antibody may be higher in AD. In order to avoid provide solution for discrepancy in the existing data, Dr. Gustaw came up with novel method of dissociation sample.

Theory

In biological fluids, antibodies and antigens are in a state of dynamic equilibrium between bound and unbound forms that is concentration dependent. As antigen masks antibody, it obstructs accurate measurement of antibody-antigen detection. Dr. Gustow discovered a novel way to enhance antibody-antigen detection. Using a dissociation buffer (1.5% bovine serume albumin (BSA) and 0.2M glycine HCl pH2/5], he dissociated antigen-antibody complexes. As shown in figure below, in dissociated samples, unbound antigen-antibody complexes reveal increased disease state compared to non-diseased state.

Method

Prepare dissociation buffer: 1.4% bovine serum albumin + 0.2M glycine-HCL, pH2.5
Inclubate AB42 for 20minute
Dissolve AB42 in 500uL dissociation buffer in Microcon centrifugal device
Incubate 23C for 20 min
Centrifuge for 20 min at 16000g at 23C
Invert filger and spun for 3 min at 2000g
Bring the sample back to a neutral pH with 15-2uL 2.5M Tris pH9
Add ELISA buffer (1.5% BSA and 0.05% Tween 20 in PBS
Perform ELISA analysis

Result

Citation

Henrik Zetterberg. "Elevated Cerebrospinal Fluid BACE1 activity in Incipient Alzheimer Disease". Arch.Gen.Psychiatry. 64: 718–726.
Harald Hampel (2010). "Biological markers of amyloid B-related mechanisms in Alzheimer's disease". ELSEVIER Rep. 223: 334–346.
Katarzyna (2008). "Antigen-antibody dissociation in Alzheimer disase: novel approach to diagnosis". Journal of Neurochemistry Rep. 106: 1350–1356.
Du, Y. (2001). "Reduced levels of amyloid beta-peptide antibody in Alzheimer disease". Neurology Rep. 57: 801–805.
Seubert, P. (1992). "Isolation and quantification of soluble Alzheimer's beta-peptide from biological fluid". Nature Rep. 359: 325–327.
Hansson, O (2010). "Evaluation of plasma Abeta(40) and Abeta(42) as predictors of conversion to Alzheimer's disaese in patients with mild cognitive impairment". Neurobiol. Aging. Rep. 31: 357–367.