Alzheimer's disease is a common affliction, usually experienced later in life. It leads to dementia, is incurable, and is fatal.
Protein aggregates are a common indicator of the disease. Aggregates include those of amyloid beta protein (senile plaques) and tau protein (neurofibrillary tangles).
The biochemical basis underlying how both of these two types of protein aggregates influence the progession of Alzheimer's disease, possibly giving clues to how to treat the condition, is controversial. This is partly due to an inability to selectively image one over the other through the use of cheap, fluorescent synthetic molecules.
Itaru Hamachi (Kyoto University, Japan) and coworkers have addressed this limitation. They have synthesized a fluorescent molecule that selectively forms chemical bonds with neurofibrillary tangles.
The fluorescent imaging agent.
The scientists' fluorescent molecule is based on two components. One component targets the molecule to neurofibrillary tangles, and the other component enables fluorescence imaging.
The first component of this molecule is based on a zinc-containing molecule that forms selective chemical bonds to tau proteins that are extensively appended with phosphate units, i.e., neurofibrillary tangles. Unfortunately, this molecule is only weakly fluorescent, and thus chemical binding to neurofibrillary tangles is difficult to image with fluorescence microscopy.
This limitation is addressed by the other component of the molecule. This component is based on a molecule that goes by the acronym BODIPY (formal name 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene).
The BOIPY dye is a well-known fluorescent molecule. It is bright and easily synthesized.
Ease of chemical synthesis facilitates chemical binding of the dye to other molecules. Thus, one can chemically link the dye unit (imaging component) to the protein binding unit (neurofibrillary tangle binding component).
One can stain a sample with this molecule, look at it with fluorescence microscopy, and the target of interest will be fluorescent. This is how selective imaging of the target of interest is enabled.
Biochemical targeting to neurofibrillary tangles.
The scientists needed to demonstrate that their molecule chemically binds to neurofibrillary tangles. They first used genetically engineered cells to synthesize the tau protein.
They then appended phosphate groups to the protein using a phosphorylating enzyme. They determined that, on average, there were 6.1 phosphate units appended per protein.
They then aggregated the protein into fibrils. The scientists' imaging agent molecule chemically binds to these fibrils much more strongly than nonphosphorylated aggregates.
Specifically, the scientists measured the half maximal effective concentration of biochemical targeting. This is the concentration at which chemical binding is halfway between baseline and maximal levels (a common measure of chemical binding in pharmaceutical applications).
The scientists measured this concentration to be 9 nanomolar for the phosphorylated aggregate. This is approximately 9 times lower (more efficient) than to the nonphosphorylated aggregate, and approximately 70 times lower (more efficient) than to amyloid beta fibrils, demonstrating selective biochemical targeting to neurofibrillary tangles.
Imaging neurofibrillary tangles in brain tissue.
Ultimately, the scienists needed to use their molecule to selectively image neurofibrillary tangles in brain tissue. Results were successful here as well.
Fluorescence from their molecule in a diseased brain tissue sample overlapped with that from expensive fluorescent protein imaging agents. There was little fluorescence from their molecule in a nondiseased brain tissue sample.
Equally as important, clear differences in fluorescence patterns were observed in diseased brain tissue samples possessing both senile plaques and neurofibrillary tangles. This is strongly suggestive of selective chemical targeting to neurofibrillary tangles.
Outlook.
There now exists a cheap imaging agent for neurofibrillary tangles, that is selective over other protein aggregates. These particular experiments were performed in brain tissue samples, i.e., dead patients.
However, the scientists are now progressing towards imaging neurofibrillary tangles in living organisms. Such directions may eventually help shed light on the biochemical progression of Alzheimer's disease, and other diseases characterized by neurofibrillary tangles.
for more information:
Ojida, A., Sakamoto, T., Inoue, M.-a., Fujishima, S.-h., Lippens, G., & Hamachi, I. (2009). Fluorescent BODIPY-Based Zn(II) Complex as a Molecular Probe for Selective Detection of Neurofibrillary Tangles in the Brains of Alzheimer's Disease Patients Journal of the American Chemical Society, 131 (18), 6543-6548 DOI: 10.1021/ja9008369