Thinakaran's lab reports findings on the mechanism by which neurons distribute the enzyme which plays a central role in Alzheimer's disease pathogenesis
A new study from Gopal Thinakaran's lab reports intriguing findings on the mechanism by which neurons distribute the enzyme BACE1 (beta-site amyloid precursor protein cleaving enzyme), which plays a central role in Alzheimer's disease pathogenesis.
The study, published in the December 26 issue of Cell Reports, details how the BACE1 enzyme is conveyed through neuronal processes using unidirectional transport, a process that has not been previously seen for any protein. The study also illustrates that this transport contributes to the development of Alzheimer's disease. The findings are highly significant because BACE1 is a prime therapeutic target.
The Thinakaran Lab has been investigating the cell biology of BACE1 and a second enzyme, termed gamma-secretase; these enzymes sequentially cut a large protein, named amyloid precursor protein. The resulting beta-amyloid fragment is released from the neuron, and its accumulation in brain senile plaques is one of the two pathological hallmarks of Alzheimer’s disease. Research over the years has shown that beta-amyloid is toxic to neurons and that high-levels of beta-amyloid damages the brain a decade or more before memory and cognitive problems become evident in patients afflicted with Alzheimer's disease. Understanding the details regarding the cellular and molecular mechanisms involved in beta-amyloid production is a topic of central importance in Alzheimer's disease research.
Neurons employ complex and highly regulated mechanisms to sort and deliver proteins to the appropriate location within the neuronal cell body and processes. There is general agreement in the Alzheimer's disease field that amyloid precursor protein is transported along nerve fibers (called axons) and is proteolytically converted into beta-amyloid near axon terminals. In neurons, BACE1 localizes to dendrites and axons (the two types of neuronal processes). The mechanism through which BACE1, the enzyme that initiates beta-amyloid production, reaches the axons is not clearly understood, yet this process is expected to control a major proportion of beta-amyloid production in the brain.
Virginie Buggia-Prévot, PhD, a post-doctoral fellow and Celia Fernandez, a Neuroscience PhD student in the Thinakaran Lab began to investigate BACE1 movement through neurons in real-time. They appended the jellyfish-derived yellow fluorescent protein to BACE1 and began to visualize it under a microscope and record in real-time the dynamic movement of BACE1 through neuronal processes. They also developed a technique that allowed them to follow the path of BACE1 from the cell surface using a red or far-red fluorescence label. Their effort uncovered something never before observed - whereas BACE1 movement was bidirectional when visualized by yellow fluorescence, in the same dendrites the subset of BACE1 that was labeled at the cell surface using red or far-red fluorescence and allowed to internalize underwent one-way transport towards the cell body. Remarkably, this unidirectional mode of transport in dendrites has not been observed for any protein.
Dr. Thinakaran noted: “The discovery of this one-way traffic is exciting. The vesicles containing BACE1 are capable of probing the local microtubule environment to get on the tracks that will move them along in the same direction. Not only that, they also have a general sense of where to go - in other words they know where the cell body is located - what cues they use for this navigation is very puzzling.”
Transport of membrane proteins occurs in small vesicles or transport carriers that move along the cytoskeleton. "Motor" proteins are the driving force for vesicle movement in inside cells. Proteins such as BACE1 contain "motifs", similar to zip codes, which instruct the cellular machinery and motor proteins to take them to their ultimate destination - in the case of BACE1, to the axon.
Achieving axonal localization of a given protein can involve: 1) Selective packaging of the proteins into vesicles that directly move into the axons (direct sorting); 2) Non-selective transport of proteins to dendrites and axons, followed by selective retention of the protein at the axons while the protein in the dendrite is destroyed; or 3) Moving the proteins to the cell surface of dendrites, and then loading them into endosomes such that they can be delivered to the axons (a process called transcytosis). In the second and third scenario, protein movement in vesicles is thought to involve a "push/pull" mechanism since vesicles simultaneously can engage more than one type of motor, and the cytoskeletal tracks are not always lined up in one orientation for the motors to direct the traffic in one direction. Because of the mixed orientation of the cytoskeletal tracks, the vesicles glide along the cytoskeleton back and forth many times as they progressively make their way towards the axon.
“However, what we observe for BACE1 is quite remarkable,” commented Dr. Thinakaran. “The BACE1 molecules that reach the surface of dendrites appear to acquire some unknown property (like getting a special pass for one-way traffic) that instructs any vesicle carrying them to glide along the cytoskeleton in one direction as they processively move towards the cell body.”
The above finding was also exciting in relation to Alzheimer's disease because a large fraction of beta-amyloid in the brain is generated in neuronal vesicles, called endosomes.
Dr. Buggia-Prévot's characterization revealed that BACE1 resides and travels in membrane vesicles, called recycling endosomes, from the cell surface of dendrites towards the neuronal cell body for further sorting before delivery to the ultimate destination. In studies that followed, the group discovered that EHD proteins, which are known to facilitate endosomal vesicular transport, regulate BACE1 dynamic transport after internalization from the cell surface. Compromising the activity of EHD proteins in neurons led not only to an impairment of dynamic movement of BACE1 in dendrites, but also it reduced the delivery of BACE1 to the axon. Finally, loss of EHD protein function significantly reduced the levels of secreted beta-amyloid in neurons. Thus, the unusual mode of BACE1 transport in endosomes, termed transcytosis, was responsible for delivering BACE1 to axons and has relevance to beta-amyloid production.
Two related papers published by the Thinakaran lab (one in the same issue of Cell Reports, and another in Molecular Neurodegeneration) provide additional confirmation of these findings. The Thinakaran lab is in the process of testing whether BACE1 transcytosis contributes to the previously reported abnormal accumulation of BACE1 in nerve terminals in diseased brain, which is a major contributing factor for increased amyloid burden. The goal of the group's future research is to develop a comprehensive understanding of BACE1 transcytosis and its significance to beta-amyloid production and Alzheimer's disease pathogenesis using mouse models. Their lab is also testing the hypothesis that a dysfunction in the unusual mode of transport system used by BACE1 might be at risk in the brains of aged individuals and those suffering from Alzheimer's disease.
Buggia-Prévot V, Fernandez CG, Udayar V, Vetrivel KS, Elie A, Roseman J, Sasse VA, Lefkow M, Meckler X, Bhattacharyya S, George M, Kar S, Bindokas VP, Parent AT, Rajendran L, Band H, Vassar R and Thinakaran G: A function for EHD family proteins in unidirectional retrograde dendritic transport of BACE1 and Alzheimer's disease Aβ production. Cell Reports. 2013 Dec 26;5(6):1552-63. doi: 10.1016/j.celrep.2013.12.006.
Udayar V, Buggia-Prévot V, Guerreiro RL, Siegel G, Rambabu N, Soohoo AL, Ponnuswamy M, Siegenthaler B, Bali J, AESG, Simons M, Puthenveedu MA, Hardy J, Thinakaran G and Rajendran L: A paired RNAi and RabGAP overexpression screen identifies Rab11 as a regulator of β-amyloid production. Cell Reports. 2013 Dec 26;5(6):1536-51. doi: 10.1016/j.celrep.2013.12.005.
Buggia-Prévot V, Fernandez CG, Riordan S, Vetrivel KS, Roseman J, Waters J, Bindokas VP, Vassar R, and Thinakaran G: Axonal BACE1 dynamics and targeting in hippocampal neurons: a role for Rab11 GTPase. Molecular Neurodegeneration Mol Neurodegener. 2014 Jan 4;9(1):1. http://www.molecularneurodegeneration.com/content/9/1/1/abstract