1. Pittsburgh Fly Meeting, July 29, 2011
Nanometer-Resolution Characterization of Axonal Transport Defects in a Drosophila Model of Alzheimer’s Disease
Ge Yang1,2,3
1Department of Biomedical Engineering
2Lane Center for Computational Biology
3Department of Biological Sciences
Carnegie Mellon University
Pittsburgh Fly Meeting, July 29, 2011
I would like to thank the organizers for providing this opportunity. My lab uses imaging and Drosophila genetics with computational analysis to study the molecular mechanism of axonal transport in neuronal cells. Today I would like to present some preliminary data on characterizing the phenotype of axonal transport in a Drosophila model of Alzheimer’s disease. Specifically, this line that models the microtubule associated protein tau related pathology of Alzheimer’s disease.

2. An Overview of Axonal Transport
From Brain Facts, Society for Neuroscience
Hirokawa N., JCB, 94:129, 1982
Bars: 0.1m
Axonal transport is critical to survival and function of neurons.
Transport defects have been implicated in many neurodegenerative diseases.
Axonal transport is a powerful model of intracellular transport.
Neurons are polarized cells. A hallmark of its polarized structure is a long and thin cytoplasmic process called the axon. In the human nervous system the axon can extend more than a meter.
The polarized structure is necessary for neurons to carry out its function of information processing and transmission. However, this also creates a logistic challenge because protein synthesis is undertaken almost exclusively within the neuronal cell body. Neurons rely on an active bidirection transport within the axons for their survive and function.
This transport process is challenging.
It is not completely surprising that defects of this process are strongly implicated in many aging-related human neurodegenerative diseases.
From a broader cell biology perspective, axonal transport also provides a good model to study general intracellular transport because of its simple and well defined geometry.

3. Molecular Machinery of Axonal Transport
It is now clear that individual cargoes are moved by molecular motors.
The molecular motor kinesin is responsible for the transport towards the distal end. Different kinesin families are involved. What is shown here is a conventional kinesin, which has two types of polypeptide chains, the kinesin heavy chain and the kinesin light chain.
In the other direction, the molecular motor that is responsible for the transport is cytoplasmic dynein.
Adapted from Schliwa & Woehlke, Nature, 422:759, 2003

4. Potential Mechanisms of Axonal Transport Defects
It is now clear that individual cargoes are moved by molecular motors.
The molecular motor kinesin is responsible for the transport towards the distal end. Different kinesin families are involved. What is shown here is a conventional kinesin, which has two types of polypeptide chains, the kinesin heavy chain and the kinesin light chain.
In the other direction, the molecular motor that is responsible for the transport is cytoplasmic dynein.

5. Microtubule Associated Protein Tau in Axonal Transport
It is now clear that individual cargoes are moved by molecular motors.
The molecular motor kinesin is responsible for the transport towards the distal end. Different kinesin families are involved. What is shown here is a conventional kinesin, which has two types of polypeptide chains, the kinesin heavy chain and the kinesin light chain.
In the other direction, the molecular motor that is responsible for the transport is cytoplasmic dynein.
Ballatore et al, Nat. Rev. Neurosci. 2007
Morris et al, Neuron, 2011

6. Coordinated Activation
Kinesin and Dynein Compete and Coordinate
Exclusive Presence
Tug-of-War
I would like to start with an example that we are all familiar with. What we are looking at is a cell going through division. This is one of most fundamental cellular processes. Chromosomes get aligned and then segregated so that the genetic information of the mother cell can be transmitted to the two daughter cells. Within our body at every second, there are about 25 million cells undergoing division. As you would imagine, this process must be executed with great precision and robustness.
To achieve this level of precision and robustness, hundreds and thousands of molecules must work closely together in space and time. The message here is that basic cellular processes must be understood at the molecular level in space and time.
It is for this reason that this movie, as impressive as it is, is not very informative in the sense that it gives us no molecular details. Instead, we need fluorescence imaging techniques to specifically label molecules we are interested in and follow their activities.
Coordinated Activation

7. How does tau modulate axonal transport?
It is now clear that individual cargoes are moved by molecular motors.
The molecular motor kinesin is responsible for the transport towards the distal end. Different kinesin families are involved. What is shown here is a conventional kinesin, which has two types of polypeptide chains, the kinesin heavy chain and the kinesin light chain.
In the other direction, the molecular motor that is responsible for the transport is cytoplasmic dynein.
[tau]

8. A Drosophila Model of Alzheimer’s Disease
Two pathological hallmarks of AD: Aβ plaques & tau tangles
Control: SG26.1 GAL4/+; UAS-APPYFP/+  transport is driven by kinesin SG26.1 GAL4/+; UAS-SynGFP  transport is driven by kinesin-3
Mutants:
SG26.1 GAL4/+; UAS-APPYFP/+; UAS-wt hTau/+
SG26.1 GAL4/+; UAS-APPYFP/+; UAS-R406W hTau/+
It is now clear that individual cargoes are moved by molecular motors.
The molecular motor kinesin is responsible for the transport towards the distal end. Different kinesin families are involved. What is shown here is a conventional kinesin, which has two types of polypeptide chains, the kinesin heavy chain and the kinesin light chain.
In the other direction, the molecular motor that is responsible for the transport is cytoplasmic dynein.
Wittmann et al, Science, 2001

9. Imaging Axonal Transport
It is now clear that individual cargoes are moved by molecular motors.
The molecular motor kinesin is responsible for the transport towards the distal end. Different kinesin families are involved. What is shown here is a conventional kinesin, which has two types of polypeptide chains, the kinesin heavy chain and the kinesin light chain.
In the other direction, the molecular motor that is responsible for the transport is cytoplasmic dynein.

10. Tau Overexpression Affects Axonal Transport
It is now clear that individual cargoes are moved by molecular motors.
The molecular motor kinesin is responsible for the transport towards the distal end. Different kinesin families are involved. What is shown here is a conventional kinesin, which has two types of polypeptide chains, the kinesin heavy chain and the kinesin light chain.
In the other direction, the molecular motor that is responsible for the transport is cytoplasmic dynein.

11. Nanometer Resolution Cargo Detection
(2, 2)
(2, 2)

12. Detection Resolution Validation
Q-dots
F-beads

13. Cargo Population Analysis
It is now clear that individual cargoes are moved by molecular motors.
The molecular motor kinesin is responsible for the transport towards the distal end. Different kinesin families are involved. What is shown here is a conventional kinesin, which has two types of polypeptide chains, the kinesin heavy chain and the kinesin light chain.
In the other direction, the molecular motor that is responsible for the transport is cytoplasmic dynein.

14. Cargo Velocity & Switching Frequency
It is now clear that individual cargoes are moved by molecular motors.
The molecular motor kinesin is responsible for the transport towards the distal end. Different kinesin families are involved. What is shown here is a conventional kinesin, which has two types of polypeptide chains, the kinesin heavy chain and the kinesin light chain.
In the other direction, the molecular motor that is responsible for the transport is cytoplasmic dynein.

15. Pause Frequency & Duration
It is now clear that individual cargoes are moved by molecular motors.
The molecular motor kinesin is responsible for the transport towards the distal end. Different kinesin families are involved. What is shown here is a conventional kinesin, which has two types of polypeptide chains, the kinesin heavy chain and the kinesin light chain.
In the other direction, the molecular motor that is responsible for the transport is cytoplasmic dynein.

16. Axon Swelling & Accumulation of APP Vesicles
It is now clear that individual cargoes are moved by molecular motors.
The molecular motor kinesin is responsible for the transport towards the distal end. Different kinesin families are involved. What is shown here is a conventional kinesin, which has two types of polypeptide chains, the kinesin heavy chain and the kinesin light chain.
In the other direction, the molecular motor that is responsible for the transport is cytoplasmic dynein.

17. Axon Swelling & Accumulation of SYNT Vesicles
It is now clear that individual cargoes are moved by molecular motors.
The molecular motor kinesin is responsible for the transport towards the distal end. Different kinesin families are involved. What is shown here is a conventional kinesin, which has two types of polypeptide chains, the kinesin heavy chain and the kinesin light chain.
In the other direction, the molecular motor that is responsible for the transport is cytoplasmic dynein.

18. Velocity Mode Analysis & Comparison
anterograde
retrograde
It is now clear that individual cargoes are moved by molecular motors.
The molecular motor kinesin is responsible for the transport towards the distal end. Different kinesin families are involved. What is shown here is a conventional kinesin, which has two types of polypeptide chains, the kinesin heavy chain and the kinesin light chain.
In the other direction, the molecular motor that is responsible for the transport is cytoplasmic dynein.

19. Summary
Tau modulates axonal cargo transport through affecting kinesin driven cargo movement.
Dynein mediated cargo movement is indirectly affected through the interaction between kinesin and dynein.
Tau overexpression induces axon swelling and cargo aggregation.
Regional variation of axonal transport is consistent with a spatial gradient of tau.
Axonal transport defects started to develop in the early stage of fly development.

20. Acknowledgement Minhua Qiu Khyti Dave Jacob Heng-Kai Sheu
Breanna Stillo
Yiyi Yu
Jonathan Minden (CMU)
Brooke McCartney (CMU)
Larry Goldstein (UCSD & HHMI)
Shermali Gunawardena (University of Buffalo)
Mel Feany (Harvard Medical School)
Funding
National Science Foundation Samuel & Emma Winters Foundation