Live Discussion: From Here to There: AβPP as an Axonal Transport Receptor—How Could This Explain Neurodegeneration in AD?
Larry Goldstein led this live discussion on 15 July 2002. Readers are invited to submit additional comments by using our Comments form at the bottom of the page.
See a follow-up live discussion background text: Axonal Transport Hypothesis Moves On to Implicate Presenilin prepared by Jorge Busciglio and Scott Brady.
View Transcript of Live Discussion — Posted 28 August 2006
By Larry Goldstein
In neurons, most synthesis and modification of proteins, synthesis of membranes, and biogenesis of organelles occurs in the cell body. Thus, large quantities of material must be moved by the axonal transport machinery to supply the axon and the axonal terminus. Dendrites face analogous logistical problems. Retrograde neurotrophic and damage signals must also be transported over long distances to keep the cell body informed about distant events. In the case of mammalian CNS neurons, it is likely that as an important consequence of their extensive connectivity and complexity their axons, and perhaps dendrites, are very long, branched, and of diminished caliber in their distal regions. These features may predispose axonal and dendritic processes to blockage of transport when vesicles aggregate or become damaged. This would be analogous to what we and others have observed in molecular motor and other mutations that disrupt axonal transport in Drosophila.
These observations prompted us to explore potential connections between molecular motor proteins such as kinesins, which generate the forces needed for movement in axons and dendrites, and proteins and vesicles implicated in neurodegeneration, such as AβPP, which plays a key role in the development of Alzheimer's disease.
Recent work in my laboratory has led us propose the following ideas:
1) Anterograde axonal transport of a vesicle population containing BACE, presenilin, the trk receptor, and GAP-43 may require a direct interaction of AβPP and the kinesin light chain subunit of the kinesin-I molecular motor protein.
This proposal is based on several recent observations:
a) Kinesin light chain and AβPP exhibit a high-affinity biochemical interaction;
b) Kinesin light chain is required for normal transport of AβPP in mouse sciatic nerve axons;
c) Deletion of the AβPP homologue, APPL, in Drosophila causes axonal transport defects characterized by vesicle accumulations (clogs) similar to those found in kinesin-I and known axonal transport mutants;
d) Axonal content and transport of kinesin-I, BACE, presenilin, GAP-43, and the trk receptor are diminished in AbPP deletion mutants in the mouse PNS;
e) A vesicle population lacking ER markers but containing kinesin-I, BACE, presenilin, GAP-43, and the trk receptor can be inferred from immuno-isolation experiments using material from sciatic nerve and CNS axons. (Kamal et al., 2000; Gunawardena and Goldstein, 2001; Kamal et al., 2001)
2) Blockage of transport and/or axonal damage leads to increased AβPP proteolysis and Aβ production in an axonal vesicle compartment.
We observed that low levels of Aβ are normally present in axonal vesicles containing AβPP, presenilin, and BACE. We also found that AβPP proteolysis can be induced in the axon by nerve ligation in vivo or in axonal vesicles in vitro. This AβPP proteolysis gives rise to Aβ (confirmed by SELDI mass-spectrometry), a protein fragment with properties characteristic of a free cytoplasmic C-terminus of AβPP, and it causes kinesin-I to let go of the vesicles (Kamal et al., 2001).
3) Disruption of axonal transport in the presence of the Aβ region of AbβPP can cause neuronal apoptosis.
This conclusion is based on recent observations in Drosophila (Gunawardena and Goldstein, 2001):
a) Overexpression of APPL and AβPP in Drosophila neurons causes axonal vesicle accumulations;
b) Formation of axonal clogs and axonal transport of AβPP and APPL require the cytoplasmic C-terminus that contains the proposed kinesin-I binding domain;
c) Overexpression of AβPP, but not APPL, induces neuronal apoptosis. Induction of apoptosis appears to require the presence of the Aβ domain and the formation of axonal clogs mediated by the cytoplasmic C-terminus of AβPP.
d) AβPP-induced axonal blockages and neuronal apoptosis are coordinately enhanced by a 50 percent reduction in kinesin-I dosage but suppressed by a 50 percent reduction in cytoplasmic dynein dosage. We interpretate this to mean that overexpression of a motor receptor protein, such as AβPP, titrates kinesin-I motor function away from other critical cargoes in narrow caliber axons. This wold lead to transport dysfunction, clogging, and apoptosis if an Aβ region is present.
See related ARF news stories
Dynamitin in Motor Neurons: Dynamite for ALS Research?
Tau Accused of Blocking Transport, Causing APP to Linger and Nerve Processes to Wither
Suspects for Aβ Generation Spotted Together, En Route to Nerve Terminal
Axonal Transport Suggested as Function for APP
Live discussion held 15 July 2002 with Larry Goldstein.
Participants: Larry Goldstein Goldstein, Eckhard Mandelkow, June Kinoshita, Marius Sudol, J.Wesson Ashford, Keith Crutcher, Jorge Buscigli, Gunnar Gouras
Note: The transcript has been edited for clarity and accuracy.
Marius Sudol: Larry Goldstein, interesting work! One of my questions is whether you are aware about a knockout of APP in C. elegans worms?
Larry Goldstein: Hello, June. Marius Sudol asked about worm mutants. My answer is I have only heard rumors, but have seen no data-is it lethal?
June Kinoshita: What sort of worm mutants?
Marius Sudol: The phenotype of APP deletion in C. elegans is a pharyngeal pumping defect. Could you look at vesicle transport in the neurons of the worm mutant?
Larry Goldstein: For worms-one could in principle. Scholey, Bargmann and others have done that sort of thing
Eckhard Mandelkow: What is the percentage distribution, in a differentiated neuron, of APP in any given compartment? What is your best guess?
Larry Goldstein: We made one quick estimate based on quantitative Westerns and came up with somewhere in the one percent range in the sciatic nerve If the estimate is correct this is a lot. On the other hand, axons lack nuclei, Golgi, etc., so maybe it could be that abundant.
Larry Goldstein: There was also an older estimate from Sisodia's group in either cultured neurons or brain, I can't remember which. They came up with 0.03 percent total in that population, I think.
Eckhard Mandelkow: Redefining that question, how much of that is in ER, Golgi, axon, at the synapse?
Larry Goldstein: No clue-our estimate was on sciatic nerve only, which should lack most typical ER, lack Golgi, lack synapses, etc. There will be some contribution from Schwanns and other supporting cells, but these are pretty minor components of mass based on estimates from looking at cross-sections in the microscope.
Eckhard Mandelkow: Because, my feeling is that there is a lot of discussion on APP cleavage in ER, Golgi, etc., but your work really emphasizes a later stage-during transport, that is.
Larry Goldstein: I agree that most cleavage may occur in later stages. The evidence at this point from other groups is most convincing for lack of cleavage in ER. This also places focus on later stage compartments. Koo has argued for endosomes, and there could be some there as well. We would argue for post-Golgi and probably synapse. Very little work has been done on neurons in vivo; most is cell cultures of various sorts.
Marius Sudol: Larry, how do you connect your cytoplasmic events with nuclear events? (See related paper.) To be more specific: I have in mind the Tip60-mediated apoptosis work of Hyman and Sudhoff.
Larry Goldstein: Marius, this is interesting indeed. We are intrigued by the idea that induction of cleavage in axoplasm could lead to retrograde signals to cell body or to nucleus. The readout, i.e., death or transcriptional changes could be different depending on neuronal type. We need more data on this point.
Eckhard Mandelkow: If we are talking about most cleavage during post-Golgi and transport, then the question is... Where does that Ab go-how would it get out of the cell in order to contribute to plaques
Jorge Busciglio: Hi, everybody. Larry Goldstein, this is Jorge Busciglio. Do APP KO mice show some phenotype that you can relate as a kinesin transport defect??
Larry Goldstein: Jorge, we have published data suggesting that APP KO in mouse has transport defects in sciatic nerve and corpus callosum neurons. We have published data in flies suggesting that transport defects are present in segmental nerves.
Larry Goldstein: Eckhard, I would argue, but do not know, that secretion or lysis would lead to contribution to plaques. I have a question for you: What fraction of Ab that is produced ends up in plaques? Is this the only fate possible?
Eckhard Mandelkow: I thought Brad Hyman's work says that most of what gets out of the cell gets washed away, and only a minority sticks in the plaques.
Jorge Busciglio: In people without Alzheimer's, most Ab is degraded and does not accumulate.
Larry Goldstein: Interesting. I'll have to follow these up. Perhaps the plaque is not the most important toxic element?
Eckhard Mandelkow: Jorge, isn't this also true for people with Alzheimer's, i.e., most is degraded?
Jorge Busciglio: I'm not sure this has been proven, but I'd estimate that even in AD, most is cleared out.
Jorge Busciglio: Protofibrils and fibrils may be more toxic than condensed Ab in plaques.
Eckhard Mandelkow: So the minority fraction (whatever it is) contributes to aggregation outside the cell. I know that that's the dogma. I still debate with myself whether any Ab is toxic, but I don't dare say that in public
Jorge Busciglio: Or inside the cell, as Larry Goldstein's data suggest.
Larry Goldstein: Is there any consensus on whether the plaques form at synaptic endings or around cell bodies?
Jorge Busciglio: Within plaques you see a lot of terminals...
Larry Goldstein: So, it could be presynaptic or postsynaptic termini secreting?
Jorge Busciglio: I think so.
Gunnar Gouras: I can say that I think that Ab can be toxic within neurons and processes/synapses. To Larry Goldstein, what is the function of these post-Golgi vesicles in axons?
Larry Goldstein: Gunnar, I presume that all post-Golgi anterograde vesicles are transport vesicles of one sort or another, ferrying materials to presynaptic termini or to intermediate destinations.
Jorge Busciglio: Larry, how does the ApoE4 polymorphism fit in your hypothesis?
Larry Goldstein: Jorge, unclear, other than that ApoE may be taken up by ApoE receptors at termini and perhaps transported back. Deletion of some of these receptors leads to odd tau phosphorylation, which Eckhard and Ewa would argue to cause kinesin-based transport defects-right, Eckhard?
Eckhard Mandelkow: And the question following from that is-how much APP does a synapse need at equilibrium? I think this could be measured by looking at the flow of APP-loaded vesicles.
Larry Goldstein: Eckhard, I have no idea how much APP is needed at synapse. I have even less of an idea what APP is doing at the synapse, but there is reasonable data for an effect on synaptic morphogenesis. I agree that some better quantification and precision on these issues is in order.
Eckhard Mandelkow: When we look at APP vesicles in axons, most of them move anterograde, then seem to sit around at the terminal for some time, and then disappear. So I am wondering where the minority of retrograde-moving APP vesicles comes from...
Larry Goldstein: endocytic recycling or changes in direction?
Eckhard Mandelkow: can't tell for sure, because APP vesicles are fast and dim, so any reversals would be hard to detect.
Marius Sudol: Eckhard, Jorge, and Larry, I am lobbying for the C. elegans model; I feel that C. elegans with an APP deletion may have a problem swallowing because of transport defects in selected neurons. It should be easy to check experimentally.
Eckhard Mandelkow: I agree-what would Jon Scholey say? He looked at overall transport in the worm.
Larry Goldstein: Marius, I agree that this would be a good system to investigate. Just not one I am experimentally skilled with...
Larry Goldstein: Eckhard, could you load with an endocytic tracer and ask if the retrograde vesicles have taken up material at the terminus?
Eckhard Mandelkow: To answer these questions, we need to crank up our experimental gear-better detectors, faster detectors-to follow an individual vesicle reliably, just like we can do it with mitochondria, which are bright.
J. Wesson Ashford: There is also a consideration that ApoE modulates the size of the lipid rafts to which the APP is connected, and the size of the raft influences whether there would be a greater predisposition to a or b secretase processing. What is the relationship between the vesicles and the lipid rafts?
Larry Goldstein: Wes, I don't know the relationship between vesicles and rafts. Is there evidence for rafts in axons?
J. Wesson Ashford: Larry, the raft story just seems to be developing. There was an interesting discussion of the issue by K Jacobson in the 6/7/2002 issue of Science, but I don't know that there is any knowledge of whether the rafts or caveolae would not be present in axons.
June Kinoshita: What is causing the AbPP vesicles to disappear at the terminal? b and g cleavage?
Larry Goldstein: June, presumably that is so. This would fit with our proposal that b- and g-secretase are in the vesicular packet delivered to the synapse along with APP.
Eckhard Mandelkow: June, I think it could be degradation. Since the AbPP is green at its C-terminus, these could be stubs which then move back retrogradely.
Larry Goldstein: Eckhard, but then why would the fluorescence decrease if they are just stubs?
Eckhard Mandelkow: Not sure which fluorescence decrease you mean-what we observed after inhibiting transport with tau is just the general decrease of vesicles, and also the reversal of overall directionality.
Larry Goldstein: Eckhard, you indicated that the APP vesicles go to the synapse then sit around and disappear. Do you mean disappearance of fluorescence?
Eckhard Mandelkow: Yes.
Eckhard Mandelkow: And I am not sure whether the retrograde vesicles we see have ever been near the synapse-they could also turn around midway.
Gunnar Gouras: What about APP in dendrites-less has been described about this.
Eckhard Mandelkow: Gunnar, right. It would be interesting to see an individual vesicle transcytosing, thereby proving the concept.
Larry Goldstein: Gunnar, interesting point. We haven't done much on dendrites for experimental reasons. It is relatively easy to find bundles of axons to probe biochemically, but I know of no good source of enriched dendrites. Eckhard, do you see APP vesicles in dendrites?
Eckhard Mandelkow: Yes, APP vesicles are in dendrites as well, maybe not as dense.
Larry Goldstein: Eckhard, but it sounds as though there are many fewer retrograde than anterograde? Is their brightness distribution the same?
Eckhard Mandelkow: Yes, the brightness distribution seems to be the same in both directions, arguing that the number of fluorescent particles is similar. But the distinction is hard to quantify.
Larry Goldstein: Do dendritic vesicles have the same movement characteristics as axonal?
Eckhard Mandelkow: Superficially it seems similar, but we have not analyzed it in detail.
Larry Goldstein: I have heard somebody claim that there is no caveolin in neurons; am I remembering this incorrectly?
Jorge Busciglio: I think that flotillin is more abundant than caveolin in neurons.
Eckhard Mandelkow: General question: If, as Larry suggests, Ab is continuously being generated along the path of secretion, where, then, does that Ab become toxic, assuming it is toxic?
Larry Goldstein: I guess the question is that, for proteins that are moving from cell body to synapse and that are thought to be in rafts, do they move in rafts, or move in vesicles prior to assembly into rafts at plasma membrane? In vivo studies of APP movement sure look vesicular in axons.
Eckhard Mandelkow: Larry, I agree, practically all the fluorescence we see is inside axons, not on the plasma membrane.
Larry Goldstein: Eckhard, if it is toxic, perhaps it becomes so when intracellular or intra-axonal or intradendritic aggregates become large enough to block transport.
Jorge Busciglio: There is no evidence yet of intracellular Ab toxicity.
Eckhard Mandelkow: Quite so. I guess you are referring to traffic jams, which have bright fluorescence.
June Kinoshita: Gunnar, is there any evidence of such large intracellular aggregates? Intra-axonal, more specifically.
Gunnar Gouras: There is certainly increased suggestion of intracellular toxicity, including Jorge's recent Neuron paper (See related ARF news story). Yes, several groups have now reported that Ab can accumulate within neurons.
Larry Goldstein: What is the evidence for extracellular toxicity? By the way, aggregates don't need to be that big to potentially interfere with traffic in narrow axons.
Eckhard Mandelkow: The subcellular aggregates, in my view, are aggregates of organelles, vesicles, etc., etc., and they may be toxic because they jam the axon, but not because there is Ab around.
Jorge Busciglio: Eckhard, I agree.
Eckhard Mandelkow: Jorge, thanks for telling me that Ab is not toxic.
Jorge Busciglio: Eckhard, I didn't say that!
Larry Goldstein: Eckhard Mandelkow, but might a small aggregate, say 0.1-0.5 microns in diameter, nucleate a transport blockage in a 0.2-micron-diameter axon?
Larry Goldstein: Could the intracellular Ab be produced internally as well as taken up by endocytosis?
Eckhard Mandelkow: That's a tough question-but my guess would be yes, considering that even a little tau can block traffic if it sits right on the track.
Larry Goldstein: Eckhard, my point exactly.
Marius Sudol: Going back to the question of caveolins as not being expressed in neurons... There are solid reports on caveolins being expressed in neurons and astrocytes.
Jorge Busciglio: My recollection is that flotillin, a caveolin homologue, is much more abundant than [caveolin] in neurons.
Larry Goldstein: I am not an expert on caveolins and so have to take others' words on this.
Marius Sudol: Jorge, you are right about dominance of flotillin.
J. Wesson Ashford: Would the vesicles be the transport agent that carries the APP to the synapse? Then, does it join with the lipid raft there to meet the secretases? Certain neuronal stimuli, including acetylcholine stimulation may thus be able to modulate whether processing will be a or b.
Larry Goldstein: How strong is the evidence that a cleavage occurs at presynaptic sites?
Jorge Busciglio: Larry, I sure think that intracellular Ab is produced internally, and also aggregated intracellularly; that's what we have shown happens in Down's astrocytes.
Eckhard Mandelkow: I'd like to repeat, for my own benefit, that perhaps the main consequence of Larry Goldstein's work is that we need to think about the APP-motor connection and traffic, rather than about cleavage as such.
Larry Goldstein: Obviously I agree. I think that ApoE points in this direction, as well, given the tau phosphorylation defects in some of the receptor mutants. By the way, Herz has published jip1/2 interactions with ApoE receptors. You may recall that jip1/2 also seems to have kinesin-binding activity required for proper transport.
Eckhard Mandelkow: Jorge, is that intracellular aggregate toxic because it blocks traffic, or because it perturbs some biochemical pathway?
Jorge Busciglio: Larry, we don't know yet. It is associated with energy deficits, but we are not sure what comes first.
June Kinoshita: Eckhard, I thought Larry was also proposing that cleavage has a function in the context of axonal transport by causing kinesin to release the vesicle...
Eckhard Mandelkow: So the question about cleavage is: Does it exert its effect because it generates Ab, or does it work by decreasing APP and thereby loose some linkage to motors?
Larry Goldstein: June, we did make that suggestion. But it is speculative at present. The idea is that cleavage could play a role in vesicle release at terminus, and might also lead to release following damage.
Eckhard Mandelkow: Talking about discharging kinesin, I think a better candidate would be the kinase connection; see Morfini-Brady papers.
Larry Goldstein: Eckhard Mandelkow, perhaps it is both. An interesting idea is an autocatalytic spiral. Intra-axonal cleavage leads to vesicular Ab and stalled vesicles because of kinesin release. Both might lead to more stalling, and so on.
Jorge Busciglio: Eckhard Mandelkow, We have evidence that PS1 mutations reduce kinesin binding to vesicles via increased GSK3 activity.
Larry Goldstein: Eckhard Mandelkow, the kinases are interesting and must be important, too. Not clear to me yet just how. It is worth emphasizing that our proposal is simpler than reality is likely to turn out to be because of Ockham' razor. These proteins are almost surely in much bigger complexes with presenilin and kinases.
Eckhard Mandelkow: I still have the impression that this intracellular Ab is a minor component during traffic, and accumulation becomes noticeable only at the synapse.
Eckhard Mandelkow: In this context, kinases control the ins and outs of melanosomes in melanophores.
Larry Goldstein: Eckhard Mandelkow, I don't know of a good measurement of this. If it is generated, at least in part, during transport and also by cleavage at synapse, then major accumulation should be at synapses.
Eckhard Mandelkow: Some recent paper by... it may have been Gelfand's lab???
Larry Goldstein: You mean Ockham?
Eckhard Mandelkow: Larry, yes, and that's where it is observed.
Marius Sudol: Funny.
Larry Goldstein: Eckhard, our experience in the fly is that synaptic proteins accumulate at synapses and have low steady state levels in axons, since they are in transit. If you interfere with transport, then you see accumulations of synaptic proteins in axons.
June Kinoshita: Can you send us the citation when you track it down, Eckhard?
Eckhard Mandelkow: Yes, I will. The point is that there are different mechanisms for regulating the forward and reverse transport of vesicles. Go into Pubmed and look for "bidirectional." It can be by having different motors controlled by phosphorylation, or detachment, again possibly controlled by kinases, or perhaps even APP.
Larry Goldstein: My read of the literature is that many vesicles alternate between forward and reverse. Cleavage would not be a good way to regulate this. But [maybe] for a vesicle that is highly processive in the forward direction. Eckhard, aren't your and Dotti's APP vesicles like this? Cleavage might be a good way to regulate [these] things.
Eckhard Mandelkow: Larry, yes and no. The problem is that each vesicle probably carries a number of motors, so you would have to do a lot of cleavage. But who knows?
Eckhard Mandelkow: Has anyone looked at an axon in a Campenot chamber and tried to look at local effects of Ab? I am thinking of work by the Cotman group, or Nicotera's, who looked at local signs of apoptosis along an axon.
Larry Goldstein: Eckhard, I don't know, interesting idea.
June Kinoshita: Larry, have you looked at how FAD mutations affect the function of APP in axonal transport?
Larry Goldstein: June, we have only looked in the fly where the mutations appear to be similar to wildtype in overexpression experiments. They may differ in apoptosis induction. We need more experiments on this issue.
J. Wesson Ashford: I am thinking that there may be important differences between dendritic and axonal transport. WE Ashford, Soultanian, et al., 1999. We did not examine axons. However, this discussion seems to be focusing on APP transport, probably involving axons, but I have not seen the direct evidence that the transport of the APP is stopped mechanically.
Eckhard Mandelkow: Wes, how do you think the neuropil threads get started?
Larry Goldstein: Wes, there is no such evidence as yet. But I would argue that it is worth testing. I agree that dendrites may be as important, or more important, than the axon for transport blockage by these proteins. We just don't know as much.
Larry Goldstein: What are neuropil threads?
Eckhard Mandelkow: These are early aggregates of tau, but in the dendritic compartment, which goes against the dogma of axonal tau.
Eckhard Mandelkow: Skip Binder would love me for that.
Larry Goldstein: You are pouring gas on a fire.
Eckhard Mandelkow: Not really, because the distribution of tau is not as predetermined as it might seem. The problem is that a lot of it is difficult to detect, depending on which antibodies one uses. We are waiting for Ginsberg's or Eberwine's single-cell determinations!
J. Wesson Ashford: Interesting questions just developing. There does seem to be some tau in the dendrites, probably getting abnormally phosphorylated.
Eckhard Mandelkow: Larry, thanks for the discussion; let's continue in Hamburg. We should have another moderated live chat! I just wanted to throw in a pitch for our meeting, 8-11 September. Details on website.
Larry Goldstein: Indeed this has been interesting! The question, of course, is how to test any of this in a human with disease to see if there is a reasonable connection to AD.
June Kinoshita: Eckhard, send details to me and I'll post on the ARF. Thank you, everyone!
Reference: Ashford JW, Soultanian NS, Zhang SX, Geddes JW. Related Articles,
Neuropil threads are collinear with MAP2 immunostaining in neuronal
dendrites of Alzheimer brain. J Neuropathol Exp Neurol. 1998