These papers provide exciting new evidence that plaque formation can occur from axonally transported APP/Aβ. Both investigations unilaterally lesioned the perforant pathway of plaque-bearing APP Swedish/PS1DE9 mice and found markedly reduced amyloid plaque burden one month postlesion in the ipsilateral hippocampus, especially in the molecular layer of the dentate gyrus. The Lazarov et al. study, with the less ideal title, additionally demonstrated provocative evidence that preplaque unilaterally lesioned mice do not have differences in hippocampal plaque burden when they were sacrificed four months post-lesion. However, both studies are consistent with our recent study demonstrating that Aβ accumulation and plaque formation occurs within neuronal processes/synaptic compartments.

View all comments by Gunnar Gouras

Tangles Come before Plaques on the Perforant Pathway
The one system where transport of APP and its relationship to amyloid deposition has now been well-established is the perforant pathway. Hyman and colleagues (Hyman et al., 1986; Hyman et al., 1988) pointed out some years ago that this pathway was one of the earliest affected in Alzheimer's disease, with the evidence based largely on the presence of tau abnormalities and tangles in the entorhinal cortex neurons projecting to the hippocampus. The studies of Hyman and of Braak and colleagues (Braak et al., 1991) make clear that at least in this pathway, tau pathology in entorhinal neurons precedes amyloid deposition in the terminal fields, and it is tempting to speculate that at least in this one case, abnormalities of APP processing, and deposition of beta amyloid in the terminal fields may be a result from the...  Read more

Tangles Come before Plaques on the Perforant Pathway
The one system where transport of APP and its relationship to amyloid deposition has now been well-established is the perforant pathway. Hyman and colleagues (Hyman et al., 1986; Hyman et al., 1988) pointed out some years ago that this pathway was one of the earliest affected in Alzheimer's disease, with the evidence based largely on the presence of tau abnormalities and tangles in the entorhinal cortex neurons projecting to the hippocampus. The studies of Hyman and of Braak and colleagues (Braak et al., 1991) make clear that at least in this pathway, tau pathology in entorhinal neurons precedes amyloid deposition in the terminal fields, and it is tempting to speculate that at least in this one case, abnormalities of APP processing, and deposition of beta amyloid in the terminal fields may be a result from the formation of tangles in the cell bodies. I wonder how often this might be the case in AD.

View all comments by Peter Davies

The two studies demonstrate that if you interrupt the supply of APP to terminals in cortical brain circuits, you abort even existing amyloid deposits outside neurons. This shows that you need a constant supply of APP to maintain the plaques, or that structural changes in the brain that follow these manipulations (what we call "plasticity") disrupt, in biochemical or even physical fashion, the microenvironment of the brain neuropil enough to "break" the plaque deposits. We used a model different from Sam and his colleagues, simply because I did not like the Scouten knife when I used it in the past, but the results are very similar.

The two studies also have slightly different emphases; we focused more on the hippocampal microanatomy, whereas Sam and his colleagues focused more on a time course of events. I am also a bit more conservative in the interpretation of findings. I believe we cannot draw conclusions on why this very interesting phenomenon happens, and that the two interpretations set up above (i.e., dynamic balance between buildup and cleansing versus plasticity of...  Read more

The two studies demonstrate that if you interrupt the supply of APP to terminals in cortical brain circuits, you abort even existing amyloid deposits outside neurons. This shows that you need a constant supply of APP to maintain the plaques, or that structural changes in the brain that follow these manipulations (what we call "plasticity") disrupt, in biochemical or even physical fashion, the microenvironment of the brain neuropil enough to "break" the plaque deposits. We used a model different from Sam and his colleagues, simply because I did not like the Scouten knife when I used it in the past, but the results are very similar.

The two studies also have slightly different emphases; we focused more on the hippocampal microanatomy, whereas Sam and his colleagues focused more on a time course of events. I am also a bit more conservative in the interpretation of findings. I believe we cannot draw conclusions on why this very interesting phenomenon happens, and that the two interpretations set up above (i.e., dynamic balance between buildup and cleansing versus plasticity of the brain) are equally compelling. I am not sure about the role of glia in this. The time course does not eliminate our need to consider plasticity factors, especially considering that the longest survival times used in the study of Sam et al. represent one-fifth to one-sixth of the life span of these mice. In fact, I am especially excited by the fact that synaptic changes may be at play, and Jin Sheng and I are planning the next wave of experiments to exploit precisely these factors. This may open up novel possibilities in dealing with amyloid deposits without having to go through immunological manipulations that may carry too much of a clinical risk.

View all comments by Vassilis Koliatsos

See a BIG picture
I read with great interest the article by Lazarov et al. As the authors state, they set "to examine whether APP transported via the perforant pathway is a major contributor to accumulation of Aβ deposits in the hippocampus." They "performed unilateral lesions of the perforant pathway of transgenic mice which express both the FAD-linked human PS1-E9 variant and a chimeric mouse-human APP Swedish (APPswe) and assessed amyloid burden in the hippocampal formation after the lesion." They further concluded that the "findings are consistent with the compelling in vivo demonstrations that, in diffuse plaques of AD patients and aged nonhuman primates, Aβ is present along neuronal dendrites and around the soma of neurons included in the plaques."

It is important to notice that the article misses the Congo red (or thioflavin) staining for plaque-like amyloid, and largely relies on the 6E10 antibody that recognizes just the human sequence of Aβ protein. Two unresolved issues are: How would the rodent’s own APP and Aβ behave under the experimental...  Read more

See a BIG picture
I read with great interest the article by Lazarov et al. As the authors state, they set "to examine whether APP transported via the perforant pathway is a major contributor to accumulation of Aβ deposits in the hippocampus." They "performed unilateral lesions of the perforant pathway of transgenic mice which express both the FAD-linked human PS1-E9 variant and a chimeric mouse-human APP Swedish (APPswe) and assessed amyloid burden in the hippocampal formation after the lesion." They further concluded that the "findings are consistent with the compelling in vivo demonstrations that, in diffuse plaques of AD patients and aged nonhuman primates, Aβ is present along neuronal dendrites and around the soma of neurons included in the plaques."

It is important to notice that the article misses the Congo red (or thioflavin) staining for plaque-like amyloid, and largely relies on the 6E10 antibody that recognizes just the human sequence of Aβ protein. Two unresolved issues are: How would the rodent’s own APP and Aβ behave under the experimental condition of perforant pathway lesioning? What would happen in wild-type animals with no transgene present? The latter experimental condition would likely match the one observed by the authors in the last set of the experiments when they "performed unilateral perforant pathway lesions in transgenic mice at four months of age, a time at which amyloid deposition is still undetectable." Four months later, brain sections showed no difference in the amyloid immunohistochemistry. However, there is no data for Aβ at a time less than one month after lesioning (when one would expect the changes to occur in these animals).

The genetically naive animals (which authors did not report on or discuss) would better represent human physiology, would not express a severe amyloid burden (a characteristic of the used APP transgenics that is irrelevant to the gradual slow onset of the most common sporadic form of Alzheimer’s), but could well have a reactive increase in amyloid staining after the lesioning.

This possibility is well-supported by a related article published a year ago¹ that deals with the "alterations in ApoE and ApoJ in relation to degeneration and regeneration in a mouse model of entorhinal cortex lesion." Its results "indicate that ApoE and ApoJ are upregulated after injury and parallel clearance of cholesterol and lipid debris from the site of injury. This coordinated alteration in apolipoproteins may redistribute lipid material to sprouting fibers to promote neurite extension, and may play an important role in long-term plasticity changes following injury."¹ Like Aβ, ApoE and ApoJ colocalize to an Alzheimer's plaque.

It is important to note that soluble Aβ is itself an apolipoprotein of the lipoproteins in the circulation and in the CNS. As an apolipoprotein. Aβ affects membrane lipid dynamics (see Ref. 2 for detailed bibliography). Particularly, Aβ modulates neuronal cholesterol esterification, influx, and efflux, and thus may regulate intracellular compartmentation and extracellular trafficking of neural cholesterol. Aβ also modulates the physical property of neuronal membrane fluidity, which is important for receptor function. Additionally, Aβ increases neural membrane lipid synthesis, in contrast to the peptide inhibitory effect, observed in human hepatic HepG2 and in HEK293 cells, in fetal rat liver and in neuronal tissue under the condition of potassium-evoked depolarization and under oxidative stress.^(3,4,5) The latter data highlight the importance of developmental, tissue, and neuronal functional specificity of the relationship between Aβ and membrane lipid biochemistry, which may vary in different brain regions and be of special importance in determining Alzheimer's specific areas of neurodegeneration as well as for interpretation of the above article. The above explains why Aβ may well follow the metabolic and functional fate described for ApoE and ApoJ.

Other than lesioning (which yields the neural repair), an increased activity-dependent plasticity burden also imposes the need for membrane rearrangements and synaptogenesis^(6,7) (see AlzForum neuroplasticity live discussion). Such a normal condition may mimic the increased demand for membrane dynamics and require an increase in neurons of Aβ, ApoE, and ApoJ.

In fact, Lazarov et al. state that they "cannot rule out the possibility that Aβ production-secretion and subsequent deposition at terminal fields of entorhinal afferents might be a function of synaptic activity-dependent release of Aβ from dendritic spines of dentate granule cells."

However, they undercite the conclusion of their past contribution that "Aβ secretion may normally function as a negative feedback to control synaptic function."⁸ The latter is supported by a number of observations, particularly by their own observation of neuronal activity-dependent secretion of natural Aβ,⁸ by an increase of synaptic amyloid precursor protein with learning capacity in rats,⁹ by upregulation of a synaptic vesicle protein transcript by Aβ1-42,¹⁰ and the amyloid precursor protein increase with development.¹¹ The modulation of APP processing that yields Aβ by several neurotransmission systems (including cholinergic,^(12,13) serotoninergic,¹⁴ and glutamatergic¹⁵) leaves little doubt regarding the vital necessity of the APP/Aβ physiology and chemistry for synaptic function.¹⁶ For further details on the role for APP/Aβ in neural function and synaptic plasticity, please see our related commentary on Pfeifer et al. (15 November 2002).

In my view, the take-home message of the important study by Lazarov et al. is a call on the neuroscience community to characterize the normal function of APP and Aβ at the synapse. The conclusion in a comment that the Lazarov study et al. provides "exciting new evidence that plaque formation can occur from axonally transported APP/Aβ" was actually not in itself tested in this study.

References:
1. White F, Nicoll JA, Horsburgh K. Alterations in ApoE and ApoJ in relation to degeneration and regeneration in a mouse model of entorhinal cortex lesion. Exp Neurol 2001 Jun;169(2):307-18. Abstract
2. Koudinov AR, Berezov TT, Koudinova NV. The levels of soluble amyloid beta in different HDL subfractions distinguish Alzheimer's and normal aging CSF: implication for brain cholesterol pathology? Neurosci Lett. 314, 115-118 (2001). Abstract
3. Koudinova NV, Koudinov AR, Yavin E. Alzheimer's Abeta1-40 peptide modulates lipid synthesis in neuronal cultures and intact rat fetal brain under normoxic and oxidative stress conditions. Neurochem Res 2000 May;25(5):653-60. Abstract
4. Koudinov AR, Koudinova NV. Essential role for cholesterol in synaptic plasticity and neuronal degeneration. FASEB J. 15, 1858-1860 (2001), originally published online June 27, 2001, 10.1096/fj.00-0815fje. Abstract
5. Koudinov AR, Koudinova NV. Brain cholesterol pathology is the cause of Alzheimer's disease. Clin Med Health Res. published online November 27, 2001, clinmed/2001100005. Abstract
6. Mesulam MM. Neuroplasticity failure in Alzheimer's disease: bridging the gap between plaques and tangles. Neuron.24, 521-529 (1999). Abstract
7. A.Koudinov. Failure of neural cholesterol dynamics is a primary pathogenic event in sporadic Alzheimer's disease. Comment. AlzForum online discussion: Cholesterol and Alzheimer's-Charging Fast but Still at a Distance From Solid Answers. Posted 15 Nov. 2002. Abstract
8. Kamenetz FR, Tomita T, Borchelt DR, Sisodia SS, Iwatsubo T, Malinow R. Activity dependent secretion of b-amyloid: roles of b -amyloid in synaptic transmission. Soc Neurosci Abstr. 26, 491 (2000). Abstract
9. Huber G, et al. Synaptic beta-amyloid precursor proteins increase with learning capacity in rats. Neurosci. 80, 313-320 (1997). Abstract
10. Heese K, Nagai Y, Sawada T. Identification of a new synaptic vesicle protein 2B mRNA transcript which is up-regulated in neurons by amyloid beta peptide fragment (1-42). Biochem Biophys Res Commun. 289, 924-928 (2001). Abstract
11. Wang C, Wurtman RJ, Lee RK. Amyloid precursor protein and membrane phospholipids in primary cortical neurons increase with development, or after exposure to nerve growth factor or Abeta(1-40). Brain Res 2000 May 26;865(2):157-67. Abstract
12. Von Der Kammer H., et al. Regulation of Gene Expression by Muscarinic Acetylcholine Receptors: A Comprehensive Approach for the Identification of Regulated Genes. Ann. N.Y. Acad. Sci., December 1, 2000; 920(1): 305–308. Abstract
13. Isacson O, Lin I. Cholinergic Modulation of Amyloid Processing and Dementia in Animal Models of Alzheimer's Disease. Ann. N.Y. Acad. Sci., December 1, 2000; 920(1): 309 - 314. Abstract
14. Nitsch RM, Deng M, Growdon JH, Wurtman RJ. Serotonin 5-HT2a and 5-HT2c receptors stimulate amyloid precursor protein ectodomain secretion. J Biol Chem. 1996 Feb 23;271(8):4188-94. Abstract
15. Nitsch RM, Deng A, Wurtman RJ, Growdon JH. Metabotropic glutamate receptor subtype mGluR1alpha stimulates the secretion of the amyloid beta protein precursor ectodomain. J Neurochem. 69, 704-712 (1997). Abstract
16. De Strooper B, Annaert W. Proteolytic processing and cell biological functions of the amyloid precursor protein. J Cell Sci 2000 Jun;113 ( Pt 11):1857-70. Abstract

View all comments by Alexei R. Koudinov

Building on their earlier provocative findings linking APP function to fast axonal transport, Stokin and colleagues, in this latest report, reinforce several important themes that are emerging from recent studies. First, significant neuronal pathobiology, especially evidence of altered vesicular trafficking, can be detected very early in Alzheimer disease (AD), before classical Alzheimer neuropathology appears. Second, these early disturbances at least partly stem from a behavior of APP or one of its processed forms; however, the issue of whether Aβ generation is an effect rather than the cause of this pathophysiology needs to be considered seriously. Finally, beyond its implications for Aβ generation, the defective vesicular transport observed in this study, and early endosomal-lysosomal dysfunction seen in other studies, are in their own right very likely to impair synapse function and axon/dendrite maintenance (Nixon, 2005). The new studies by the Goldstein group will hopefully encourage further exploration of these research themes, which are relatively understudied....  Read more

Building on their earlier provocative findings linking APP function to fast axonal transport, Stokin and colleagues, in this latest report, reinforce several important themes that are emerging from recent studies. First, significant neuronal pathobiology, especially evidence of altered vesicular trafficking, can be detected very early in Alzheimer disease (AD), before classical Alzheimer neuropathology appears. Second, these early disturbances at least partly stem from a behavior of APP or one of its processed forms; however, the issue of whether Aβ generation is an effect rather than the cause of this pathophysiology needs to be considered seriously. Finally, beyond its implications for Aβ generation, the defective vesicular transport observed in this study, and early endosomal-lysosomal dysfunction seen in other studies, are in their own right very likely to impair synapse function and axon/dendrite maintenance (Nixon, 2005). The new studies by the Goldstein group will hopefully encourage further exploration of these research themes, which are relatively understudied.

The report provides evidence for an early failure of anterograde axonal transport in AD and implicates the transport motor, kinesin-1, as one route to this failure. This could nicely explain an initial report suggesting that KLC1 polymorphisms may influence risk for AD. A more generalized defect of vesicular transport in AD could also be envisioned. A dysfunctional microtubule "track," possibly involving tau, or an altered vesicular cargo, perhaps involving post-translationally modified APP, would be expected to impair not only anterograde axonal transport, but also retrograde traffic in dendrites, where dystrophy and accumulation of vesicular cargoes is more profoundly affected than in axons. The accumulating vesicles in dystrophic neurites in the Alzheimer brain include many of lysosomal origin, as initially pointed out by Robert Terry and colleagues. At the same time, many, if not most, correspond to autophagic vacuoles, which are early and late compartments of macroautophagy, a pathway for the turnover of organelles and long-lived proteins (Nixon et al. 2005). Interestingly, autophagic vacuoles are enriched in γ-secretase activity and contain Aβ in addition to the necessary components to generate Aβ (Yu et al., 2004). In the Stokin et al. study, a proportion of the vesicles accumulating in pathologic axons of the mouse model appear to have the distinctive double limiting-membrane morphology of early autophagic vacuoles, suggesting one possible source for the extra Aβ in these mice. Endosomes, another site of amyloidogenic APP processing, are known to be abundant anterograde vesicular cargoes in axons, so it will be interesting in future studies to sort out the relative contributions of these different vesicular compartments to the Aβ effect.

References:
Nixon RA. Endosome function and dysfunction in Alzheimer's disease and other neurodegenerative diseases. Neurobiol Aging. 2005 Mar;26(3):373-82. Abstract

Nixon RA; Wegiel J, Kumar A, Yu WH, Peterhoff C, Cataldo A, Cuervo AM. Extensive Involvement of Autophagy in Alzheimer Disease: An Immuno-Electron Microscopy Study. Journal of Neuropathology and Experimental Neurology: 2005 Feb; 64(2):113-122.

Yu WH, Kumar A, Peterhoff C, Shapiro Kulkane L, Uchiyama Y, Lamb BT, Cuervo AM, Nixon RA. Autophagic vacuoles are enriched in APP-secretase activities: Implications for Aβ peptide over-production and localization in Alzheimer’s disease. International Journal of Biochemistry and Cell Biology 2004; 36:2531-2540. Abstract

See also ARF related conference report.

View all comments by Ralph Nixon

The paper by Stokin et al is most remarkable and very convincing. Reducing axonal transport enhanced axonopathy, increased intracellular Aβ levels and extracellular deposition. Stimulation of APP cleavage may be the consequence of enhanced presence of APP-containing vesicles in axonal and/or somatodendritic compartments due to mistrafficking. Increased intraneuronal Aβ accumulation as a consequence has been earlier shown to trigger neuronal death in APP/PS1 mouse models. Impaired axonal transport may be the result of age-dependent processes leading to axonal deafferentiation and loss of synaptic contacts.

In my opinion, this is a milestone paper, because it shows that intraneuronal deficits, like axonopathy, are observed prior to plaque induction. It provides further evidence for a central role of intraneuronal Aβ accumulation in the pathological processes of Alzheimer disease.

View all comments by Thomas Bayer

This paper by Stokin et al. from the lab of Larry Goldstein has some interesting and important findings. I think the finding that APPsw transgenics having half the dose of kinesin-1 have increased Aβ deposition and pathology strongly argues that normal axonal transport is involved in the development of Aβ-related pathologies in AD. This is important, as it suggests that augmentation of this function or factors that prevent axonopathy may be protective against AD.

The finding that there are neuritic swellings in very young APP transgenic mice is interesting, but whether this is relevant to AD is unclear. First, these swellings are smaller and different in appearance than the neuritic dystrophy around amyloid deposits. Second, and more importantly, the APP transgenic mice being studied overexpress mutant APP many-fold. Humans with AD of any type do not overexpress mutant APP (except in Down syndrome, in which there is APP overexpression but at a much lower level than in these mice). The overexpression of human APP increases human Aβ (required for Aβ...  Read more

This paper by Stokin et al. from the lab of Larry Goldstein has some interesting and important findings. I think the finding that APPsw transgenics having half the dose of kinesin-1 have increased Aβ deposition and pathology strongly argues that normal axonal transport is involved in the development of Aβ-related pathologies in AD. This is important, as it suggests that augmentation of this function or factors that prevent axonopathy may be protective against AD.

The finding that there are neuritic swellings in very young APP transgenic mice is interesting, but whether this is relevant to AD is unclear. First, these swellings are smaller and different in appearance than the neuritic dystrophy around amyloid deposits. Second, and more importantly, the APP transgenic mice being studied overexpress mutant APP many-fold. Humans with AD of any type do not overexpress mutant APP (except in Down syndrome, in which there is APP overexpression but at a much lower level than in these mice). The overexpression of human APP increases human Aβ (required for Aβ aggregation in mice), but also may be resulting in other biological effects of mutant APP overexpression.

It is possible that the neuritic changes described in the young APPsw mice are secondary to increased soluble Aβ. It is also possible that they are due to APPsw overexpression. Appropriate controls to sort this out might be overexpression of APPsw with the Aβ region changed in sequence or determining whether pharmacological or other inhibition of Aβ blocks the early neuritic changes. While the neuritic swellings seen in young APPsw mice are interesting and may have relevance to AD, I think this remains unclear at this point.

View all comments by David Holtzman

Kinesin molecular motor protein is involved axonal transport along microtubules. Tau protein is a major constituent of mircrotubules and thus disruption of tau (hyperphophorylation as an example) or any other part of microtubules have been shown to interfere with anterograde transport and retrograde transport. In the case of AD the research seems to point more towards APP buildup as a result of neuronal structure degradation. A drastic reduction of kinesin is merely a symptom and not directly causal of APP and amyloid beta. Presenilin mutations that affect the enzyme's activity in cutting APP are shown in a wide variety of axonal dysfucntion in AD patients.

View all comments by Jacob Mack

Aluminum could be a co-factor in the findings of Stokin and collegues. Aluminum was found to inhibit neurofilament assembly, cytoskeletal incorporation, and axonal transport by Shea et al, 1997. Deloncle et al, 2001 found that aluminum L-glutamate causes massive mitochondrial swelling in the hippocampus of younger laboratory rats that mimics similar effects of the aging process in older animals. Stokin et al. found mitochondria in the axons. Aluminum is known to interfere with ATP and is linked with neurofibrillary degeneration. Bioaccumulation of aluminum in the human brain over the lifespan exposes the aging brain to potentially significant dosages.

References:
T.B. Shea, E. Wheeler and C. Jung, Aluminum inhibits neurofilament assembly, cytoskeletal incorporation and axonal transport. Dynamic nature of aluminum-induced perikaryl neuro-filament accumulations as revealed by subunit turnover, Mol Chem Neuropathol 32(1-3)1997, 17-39

R. Deloncle, F. Huguet, B. Fernandez, N. Quellard, P. Babin and O. Guillard, Ultrastructural study of rat hippocampus after chronic adminstration of aluminum L-glutamate: an acceleration of the aging process, Exp Gerontol 36(2) 2001, 231-44

View all comments by Erik Jansson

This excellent study clearly demonstrates that axonal damage occurs long before amyloid deposition in both early stage AD and an APP mouse model. Furthermore, the authors demonstrate that reduced expression of the motor protein KCL-1 increases both the production and deposition of Aβ. However, it is unclear which comes first, the generation of soluble toxic Aβ species and then disruption of axonal transport, or disruption of transport leading to increased Aβ production and subsequent generation of toxic assemblies. A clear understanding of the pathogenic sequence is essential for the rational development of therapies and thus the temporal relationship between axonopathy and soluble Aβ species demands further investigation. Specifically, in light of the finding that anti-Aβ antibodies can lead to the clearance of early hyperphosphorylated forms of tau, it would be worthwhile determining if either passive or active immunization can rescue the pre-amyloid axonopathy.

View all comments by Dominic Walsh