The team at the Microsystems Laboratory 4 (LMIS4), directed by Philippe Renaud, has developed a novel experimental method to study how Alzheimer’s Disease might spread within the brain. LMIS4’s groundbreaking in vitro method opens up the possibility of observing interactions between healthy and diseased neurons.
Alzheimer’s Disease (AD) is the most common cause of dementia and affects more than 35.6 million people worldwide, according to the World Alzheimer Report 2010. The number of people with AD is expected to reach 115.4 million by 2050. In response to this situation, Robert Meissner and Anja Kunze, members of Professor Philippe Renaud’s LMIS4 team at the EPFL, have designed a novel experimental method that opens up new ways of observing and analyzing how AD develops within the brain. Their results have been published in Biotechnology and Bioengineering. (see link in right-hand column)."Most work on AD focuses on biochemical mechanisms," says LMSI4 Director Philippe Renaud. "In fact, AD starts in a specific area of the brain, the limbic system, and then spreads to other areas. This indicates that neural networks play a role in disease propagation."
The starting point: cultured cortical neurons obtained from embryonic rats
In order to investigate just what role neural networks might in fact play in AD, the team "connected" two neuronal cell populations with each other in vitro via an active neurite network. The two groups of neurons then interacted with each other "co-pathologically," i.e., as if one group was afflicted with AD and the other healthy. The "connection" between two groups of cultured neurons took place in a complex, specially designed microfluidic-based cell culture device developed by the team. The device has three compartments (see illustration). In the two lateral compartments the team cultured cortical neuron cells obtained from embryonic rats for a period of 8 days. A neurite network was then generated in the main (central) compartment, via neurite outgrowth from each of the two lateral compartments, thereby "connecting" the two original groups of neurons. The team was able to observe indicators of these connections using immunofluorescence labeling.
Tau Proteins: a key factor in AD
Next, one of the two now-interconnected compartments needed to be deseased. Professor Renaud recounts that "in order to get one of the two compartments’ cells into what we call a ‘diseased’ state, we perfused that compartment with Okadaic Acid (OA) for 75 minutes. The neurons in that compartment began to exhibit characteristics similar to those we see in the brain cells of someone who has AD." The OA was behind this change: it causes hyperphosphorylation of the Tau proteins that normally stabilize the structure of microtubules that are essential to neuronal cell structure by adding phosphorous groups to them. This in turn leads to dissociation of the microtubules and a progressive destruction of the connections. Hyperphosphorylated Tau proteins are in fact one of the hallmarks of AD. "During the experiment, neurons were subjected to high doses of Okadaic Acid, which lead them to die within 24 to 48 hours. In the future, it would also be possible to reduce the dose, and therefore observe the phenomena for a longer period", indicates Anja Kunze, co-author of the publication.
An innovation that opens the door for further research
The brains of people with AD exhibit both areas where the cells are healthy and areas where the cells are diseased. These areas are interconnected, and Professor Renaud’s team has been able to simulate this "co-pathological" situation in vitro by perfusing one compartment, but not the other, with OA. This novel approach to AD studies would not have been possible without the innovative microfluidic cell culture device. In addition making possible the two separate controlled cell cultures in the lateral compartments, the device’s microchannels guide the neurite outgrowths, thereby "connecting" the two original cultures. "This novel experimental model represents the state of the art for the study of AD propagation mechanisms within the human brain," says Professor Renaud. "The next step, with the help of specialists in neuroscience, will be to pursue further research from a more biological angle." As for Anja Kunze, she keeps developing the experiment. She is for example working on the culture of 3D neuronal network.