Daniel McLean (Doctoral Candidate)
Professor Molly Shoichet, Professor Paul Fraser, Professor Peter St. George Hyslop
Introduction
Alzheimer’s disease is a progressive neurodegenerative condition that slowly robs its victims of the ability to think, remember and understand. This devastating disease is made worse by the crude tools available to doctors when diagnosing and monitoring patients. Today, doctors rely upon observations of patient behaviour in order to diagnose and monitor the progression of Alzheimer’s. The limitations of simple observation are exasperated by inconsistent patient behaviour, which can change dramatically from day to day. Thus measuring the effectiveness of new therapies to treat Alzheimer’s is difficult and ultimately limits their approval.
The goal of this project is to image the senile plaques associated with Alzheimer’s disease and to correlate these images with the progression of the disease. Current imaging agents do not selectively bind with the plaques associated with Alzheimer’s disease, thereby leading to false positives and false negatives. By selectively binding with the Alzheimer’s plaques, we will be able to both better detect Alzheimer’s disease at an earlier stage and follow effectiveness of the treatment.
Specifically, we propose to develop a derivative of a monoclonal antibody that will cross the blood-brain-barrier, thereby allowing specific binding with the senile plaques in the Alzheimer’s patient brain. By coupling a radioactive tracer to this molecule, we will use positron emission tomography (PET) imaging to determine the location of the Alzheimer’s plaques. In the same way PET images are used to monitor tumour growth in cancer patients, we propose to develop a molecule that will illuminate those regions of the brain in which Alzheimer’s is developing. The ‘senile plaques’ observed in patients suffering from Alzheimer’s disease comprise millions of tiny proteins, called amyloid-beta, that have been slightly misfolded. In this project we aim to use antibodies that can detect and attach to these misfolded proteins. The location and amount of misfolded protein will be visualized using PET imaging, thereby giving the health care professional an unbiased assessment of the patient’s condition.
Thus far, we have focused on identifying antibodies that are capable of binding to amyloid-beta deposits in living tissue. We have currently circumvented the blood-brain barrier by injecting anti-amyloid-beta antibodies directly into the cortex of transgenic and wild type mice. Our results have shown clear binding to amyloid-beta deposits and wide distribution and clearance of the antibody.
Methods
8 month old transgenic (TgCRND8) and wild type mice were injected with fluorescently labelled (Alexa488) anti-amyloid-beta antibody (6E10). 500nL of 6E10-Alexa488 was injected into the left hemisphere (Coordinates: +1mm bragma, -2mm midline, -1mm deep). Incision was closed and animal left to recover for 4 hours.
Brains were cryo-sectioned into 100 mm coronal sections and viewed under a fluorescent microscope. To confirm the composition of the fluorescent deposits, brain slices were stained with a primary rabbit polyclonal anti-amyloid-beta antibody and secondary goat anti-rabbit antibody conjugated to an Alexa-568 fluorophore. Brain slices were viewed under a confocal microscope with GFP and rhodamine lasers.
Results
Upon viewing coronal sections from the transgenic mouse, numerous fluorescent deposits were observed as shown in Figure 1. Interestingly, neither fluorescent deposits were observed in the coronal sections harvested from the wild type animal, nor was any residual fluorescence from the injection present. We hypothesized that the fluorescent deposits found in the transgenic brain are senile plaques to which the 6E10 antibody bound in vivo. We aimed to clarify the chemical composition of the fluorescent deposits by staining the coronal sections with a polyclonal anti-amyloid-beta antibody. Figure 2 demonstrates that a different anti-amyloid-beta antibody binds to the same deposits that 6E10 was found to bind. This confirms that these deposits are indeed senile plaques.
In addition to demonstrating 6E10 binding to senile plaques, it is exciting that the antibody is able to penetrate the brain tissue to large distances from the injection site in only 4 hours. Also, unbound antibody is virtually entirely removed from the parenchyma at this short time period. An important requirement for a diagnostic imaging tool is to have the contrast agent permeate the entire tissue and unbound agent to clear rapidly from the tissue. We have shown that 6E10 can provide an unbiased near-global assessment of the amyloid-beta content in the brain of our mouse model.
Next Phase
Now that we have demonstrated convincing binding of 6E10 to amyloid-beta plaques using fluorescence, we aim to translate these results to Positron Emission Tomography. This will involve 3 steps: (1) Synthesis of positron emitting 6E10 conjugates (2) Intracerebral injection of positron emitting 6E10 conjugates (3) Comparison of positron emitting 6E10 conjugates to existing PET contrast agents for imaging amyloid-beta.
We will covalently couple a metal chelating agent (DOTA) to 6E10 using well-defined NHS chemistry. The metal chelating agent will bind a positron emitting radionuclide, Copper-64, immediately prior to injection into the brains of 8 month old transgenic and wild type animals. A four hour dynamic PET scan will reveal the clearance kinetics of the antibody-radionuclide conjugate as well as provide a bulk global assessment of the amyloid-beta content in the brain. In separate animals, we will also inject a complimentary anti-amyloid-beta antibody (4G8) and a control antibody that has no target in the central nervous system.
In addition, we aim to compare our amyloid-betaimaging agents to the most widely studied PET contrast agent, Pittsburgh compound B (PIB). PIB is widely studied because it has high binding to various isoforms of amyloid-beta, but its lack of specificity and binding in some confirmed
