The study by Pigino et al. study elegantly highlights a possible mechanism by which Aβ oligomers can influence axonal transport. Though the validity of intracellular Aβ is debatable in the context of human AD pathology, Pigino et al. convincingly show that in a simple model-system of axonal transport, nanomolar levels of Aβ can influence transport; they also provide convincing evidence for the involvement of a specific signaling cascade in this process. The paper is a must-read!

View all comments by Subhojit Roy

A number of experimental observations support a role for axonal transport defects in Alzheimer¹s disease (Morfini et al., 2002). Two recent papers reporting impaired axonal transport caused by presenilin mutations and Aβ protein, respectively, lend additional support to this hypothesis. Presenilin mutations increase GSK3β activity leading to abnormal kinesin phosphorylation and impaired axonal transport (Pigino et al., 2003; see also ARF live discussion). The molecular mechanism by which Aβ inhibits fast axonal transport (FAT) in neurons is not clear. According to results by Hiruma and coworkers in this study, Aβ-mediated inhibition of FAT involves actin polymerization and aggregation; however, the study does not present evidence of the molecular mechanism(s) that might lead to changes in microfilament polymerization. One possibility is that Aβ...  Read more

A number of experimental observations support a role for axonal transport defects in Alzheimer¹s disease (Morfini et al., 2002). Two recent papers reporting impaired axonal transport caused by presenilin mutations and Aβ protein, respectively, lend additional support to this hypothesis. Presenilin mutations increase GSK3β activity leading to abnormal kinesin phosphorylation and impaired axonal transport (Pigino et al., 2003; see also ARF live discussion). The molecular mechanism by which Aβ inhibits fast axonal transport (FAT) in neurons is not clear. According to results by Hiruma and coworkers in this study, Aβ-mediated inhibition of FAT involves actin polymerization and aggregation; however, the study does not present evidence of the molecular mechanism(s) that might lead to changes in microfilament polymerization. One possibility is that Aβ abnormally activates focal adhesion signals, resulting in misregulation of actin dynamics leading to impaired axonal transport and neuritic dystrophy (Grace and Busciglio, 2003). This hypothesis is supported by the presence of activated focal adhesion proteins in dystrophic neurites surrounding Aβ plaque cores in the Alzheimer's brain. Interestingly, we found in the same study that activation of focal adhesion signaling may also lead to a direct induction of MAP kinase and GSK3β, two kinases that appear to be involved in tau hyperphosphorylation, which in turn may lead to microtubule destabilization and impaired axonal transport (see ARF comment). In addition, other studies suggest that a direct accumulation of Aβ and APP metabolic derivatives in neuronal processes may be responsible for trafficking alterations (Bayer et al., 2001; Wirths et al., 2002). Collectively, these results indicate that Aβ may alter axonal transport in neurons in a number of different ways. Future studies directed to advance our understanding of the role of axonal transport defects in AD neuropathology are warranted.

View all comments by Jorge Busciglio

This paper underscores the importance of impaired axonal transport and motor neuron deficits induced by familial mutations in PS1. We agree with the notion that the problem in AD is intraneuronal mistrafficking of different axonal proteins, and the results presented may explain some pathological features we have previously observed. We have studied two bigenic AD mouse models with abundant intraneuronal Aβ accumulation, which correlated well with the observed neuron loss, and axonal degeneration in brain and spinal cord. We agree with Lazarov et al. that these defects are likely induced by a different trafficking of APP due to expression of mutant PS1. In both models, the APP751SL/PS1M146L (Schmitz et al., 2004), and the APP/PS1KI (APP751SL and knock-in of PS1M233T and PS1L235P) (Casas et al., 2004) mouse model, we have shown that neuronal dysfunction is plaque-independent (Wirths et al., 2006a; Wirths et al., 2006b).

The APP/PS1KI mouse model is especially interesting, because 50 percent of CA1 neurons are lost at 10 months of age (Casas et al., 2004). These mice also...  Read more

This paper underscores the importance of impaired axonal transport and motor neuron deficits induced by familial mutations in PS1. We agree with the notion that the problem in AD is intraneuronal mistrafficking of different axonal proteins, and the results presented may explain some pathological features we have previously observed. We have studied two bigenic AD mouse models with abundant intraneuronal Aβ accumulation, which correlated well with the observed neuron loss, and axonal degeneration in brain and spinal cord. We agree with Lazarov et al. that these defects are likely induced by a different trafficking of APP due to expression of mutant PS1. In both models, the APP751SL/PS1M146L (Schmitz et al., 2004), and the APP/PS1KI (APP751SL and knock-in of PS1M233T and PS1L235P) (Casas et al., 2004) mouse model, we have shown that neuronal dysfunction is plaque-independent (Wirths et al., 2006a; Wirths et al., 2006b).

The APP/PS1KI mouse model is especially interesting, because 50 percent of CA1 neurons are lost at 10 months of age (Casas et al., 2004). These mice also exhibit early and robust brain and spinal cord axonal degeneration, as shown by the occurrence of axonal spheroids, together with a reduced ability to perform motor performance tasks, including balance beam, string suspension, or rotarod. Working memory deficits were also evident at that time point (6 months of age) (Wirths et al., 2007). In good agreement with Lazarov et al. we have found evidence for increased levels of phosphorylated proteins (Tau [pS199] and APP [Thr668]) in degenerating axons inducing a loss of trophic support which might explain the robust age-dependent axonal degeneration in APP/PS1KI mice.

References:
Casas C, Sergeant N, Itier JM, Blanchard V, Wirths O, van der Kolk N, Vingtdeux V, van de Steeg E, Ret G, Canton T, Drobecq H, Clark A, Bonici B, Delacourte A, Benavides J, Schmitz C, Tremp G, Bayer TA, Benoit P, Pradier L. Massive CA1/2 neuronal loss with intraneuronal and N-terminal truncated Abeta42 accumulation in a novel Alzheimer transgenic model. Am J Pathol. 2004 Oct;165(4):1289-300. Abstract

Schmitz C, Rutten BP, Pielen A, Schafer S, Wirths O, Tremp G, Czech C, Blanchard V, Multhaup G, Rezaie P, Korr H, Steinbusch HW, Pradier L, Bayer TA. Hippocampal neuron loss exceeds amyloid plaque load in a transgenic mouse model of Alzheimer's disease. Am J Pathol. 2004 Apr;164(4):1495-502. Abstract

Wirths O, Weis J, Kayed R, Saido TC, Bayer TA. Age-dependent axonal degeneration in an Alzheimer mouse model. Neurobiol Aging. 2006 Sep 8; [Epub ahead of print] Abstract

Wirths O, Weis J, Szczygielski J, Multhaup G, Bayer TA. Axonopathy in an APP/PS1 transgenic mouse model of Alzheimer's disease. Acta Neuropathol (Berl). 2006 Apr;111(4):312-9. Epub 2006 Mar 7. Abstract

Wirths O, Breyhan H, Schafer S, Roth C, Bayer TA. Deficits in working memory and motor performance in the APP/PS1ki mouse model for Alzheimer's disease. Neurobiol Aging. 2007 Jan 8; [Epub ahead of print] Abstract

View all comments by Thomas Bayer
View all comments by Oliver Wirths

This careful study rigorously tests a creative concept. We have also detected BACE and APP, though not PS-1, in the same subcellular compartment in AD neurons. That APP, BACE and PS1 are colocalized at the same subcellular site in axons is intriguing and helpful to explain some important issues, but the axonal membrane compartment may not be only major site for Aβ generation (we found one or two sites in AD neurons). While sciatic nerve is a simple, good model to test this working hypothesis, it is also important to keep in mind that sciative nerve nerve cells differ from neurons in the brain, especially cortical or hippocampal neurons. The authors use corpus callosum as an axonal model, however, hippocampal or enthorinal cortex neurons may also be worth pursuing. They all contain neuronal cell bodies and their axonal projections and are pathologically affected areas in AD. This is a very good paper.

View all comments by Yong Shen

Despite intensive study the functions of APP are unknown, however an increasing number of experiments are identifying functions of APP. This paper is interesting because it identifies a function that appears to be dependent on APP which, if true, would be a major function of APP and further the understanding of its basic biology.

View all comments by Benjamin Wolozin

The study of the biology of APP and its proteolytic products, although pioneered in the early 1990s by Eddie Koo, Joseph Buxbaum, Sam Sisodia, and others, has nevertheless remained mostly out of the limelight until the last few years. The present study from Elaine Bearer’s laboratory now illuminates part of a picture that has been taking shape in the last few years suggesting that APP is likely involved in the modulation of synaptic activity in adults (Priller et al., 2006; Yang et al., 2005; Seabrook et al., 1999), in synapse formation and function (Wang et al., 2005), and in neuronal migration and adhesion during development (Herms et al., 2004).

APP is a synaptic protein that is anterogradely transported to terminals. A few years ago Kamal et al. suggested that the C-terminus of APP could serve as a receptor for kinesin (  Read more

The study of the biology of APP and its proteolytic products, although pioneered in the early 1990s by Eddie Koo, Joseph Buxbaum, Sam Sisodia, and others, has nevertheless remained mostly out of the limelight until the last few years. The present study from Elaine Bearer’s laboratory now illuminates part of a picture that has been taking shape in the last few years suggesting that APP is likely involved in the modulation of synaptic activity in adults (Priller et al., 2006; Yang et al., 2005; Seabrook et al., 1999), in synapse formation and function (Wang et al., 2005), and in neuronal migration and adhesion during development (Herms et al., 2004).

APP is a synaptic protein that is anterogradely transported to terminals. A few years ago Kamal et al. suggested that the C-terminus of APP could serve as a receptor for kinesin (Kamal et al., 2000), but this observation was subsequently questioned by Lazarov et al. (Lazarov et al., 2005). The present study by Satpute-Krishnan et al. provides strong evidence that the C-terminus of APP may indeed contain sequences sufficient for its association with axonal transport components. The careful experiments addressed this question using a fairly well-defined system, the squid giant axon, and the investigators’ observations indicate that the C-terminal domain of APP, either through a direct interaction with kinesin or indirectly via scaffolding proteins such as JIPs, participates in fast anterograde axonal transport. Quoting their discussion, “The robust motility of C99 beads in the intact axon argues for a physiological role of APP in recruitment of anterograde transport machinery inside cells.” It certainly does, and it comes as no surprise. Although the study by Satpute-Krishnan et al. does not answer the question of whether the interaction of APP with kinesin is or is not direct, it significantly adds to the rapidly growing evidence suggesting a crucial role of the C-terminus of APP (and possibly its family members APLP1 and 2) in neuronal biology, possibly at synaptic sites.

The remarkable conservation of the C-terminal sequences of APP across phyla suggests conservation of function. Supporting this idea, the phenotypes of APP/APLP2 double and APP/APLP1/APLP2 triple knockouts and those of two prominent APP-interacting proteins (X11 and the Fe65 family) involve alterations in neuronal function, synaptic formation, function, and regulation (Wang et al., 2005; Ho et al., 2003; Yang et al., 2005; Priller et al., 2006), and in the case of the Fe65/FE65L double and APP/APLP1/APLP2 triple knockouts, result in cortical dysplasias and heterotopias (Herms et al., 2004, Guenette et al., 2006). Interestingly, it was recently shown that transgenic expression of AICD in combination with Fe65 causes alterations in signaling (Ryan and Pimplikar, 2005) and activation of proteins involved in growth cone collapse and axonal guidance.

Why is this important? Most of all, because a significant component of amyloid-β toxicity requires multimerization of APP and cleavage of its C-terminus at Asp664 (Lu et al., 2003; Lu et al., 2003; Shaked et al., 2006). This cleavage not only releases a toxic peptide, but also removes the sequences required for the formation of a multiplicity of protein complexes at APP’s cytoplasmic domain, and as Satpute-Krishnan now suggest, for fast axonal transport. Consistent with what may be an important role of the extreme C-terminal sequences of APP in transducing amyloid-β toxicity, we recently showed that stabilization of APP’s cytoplasmic tail by mutation of the Asp664 cleavage site had a dramatic effect in the development of AD-like deficits in transgenic mice (Galvan et al., 2006)—even in the presence of abundant amyloid-β. With this in mind, the question arises as to whether cleavage at Asp664 while in transit towards synaptic sites would, as expected, prevent delivery of the molecule to its destination—and if the hypothesis of Kamal et al. is correct, whether it would affect the delivery of any subset of associated axonal transport vesicles. Thus, a population of Asp664-intact (transport-competent) and Asp664-cleaved (transport-incompetent) APP molecules may exist. Satpute-Krishnan et al. estimate that 3,000 copies of APP may be associated with each motile bead in their system; although in this study they don’t address the question of what is the minimal number of APP molecules required for transport, it is conceivable that transport-incompetent (Asp664-cleaved) APP molecules may be “carried along” in vesicles containing a sufficient number of transport-competent (Asp664-intact) APP. Cleavage of APP at Asp664 would thus affect not only the transport-competence (and thus the rate of delivery) of APP to neuronal terminals, but since the motifs required for the interaction of APP with a variety of cellular functions reside downstream of Asp664, it would also affect the overall signaling ability of populations of APP molecules at their destination at synaptic sites.

View all comments by Veronica Galvan

Comment by Jorge J. Palop and Lennart Mucke
We completely agree with Dr. Ashford in that the specific connection between Aβ and tau revealed by this and our previous study (Roberson et al., 2007) deserves to be explored further. However, we believe that the potential role of Aβ-induced aberrant overexcitation in the pathogenesis of AD may have been underestimated.

As highlighted by our study, much of such activity is non-convulsive and, thus, could easily escape detection by standard clinical exams. Our study also revealed a striking compensatory remodeling and activation of inhibitory circuits, which could account for the fact that obvious convulsive seizures are not frequent in this condition.

However, convulsive seizures are probably more frequent in AD than many clinicians realize. As discussed in our paper, AD patients clearly have a higher incidence of seizures than reference populations (Amatniek et al., 2006; Hauser et al., 1986; Hesdorffer et al., 1996; Lozsadi and Larner, 2006; Mendez and Lim, 2003).

Interestingly, the risk of...  Read more

Comment by Jorge J. Palop and Lennart Mucke
We completely agree with Dr. Ashford in that the specific connection between Aβ and tau revealed by this and our previous study (Roberson et al., 2007) deserves to be explored further. However, we believe that the potential role of Aβ-induced aberrant overexcitation in the pathogenesis of AD may have been underestimated.

As highlighted by our study, much of such activity is non-convulsive and, thus, could easily escape detection by standard clinical exams. Our study also revealed a striking compensatory remodeling and activation of inhibitory circuits, which could account for the fact that obvious convulsive seizures are not frequent in this condition.

However, convulsive seizures are probably more frequent in AD than many clinicians realize. As discussed in our paper, AD patients clearly have a higher incidence of seizures than reference populations (Amatniek et al., 2006; Hauser et al., 1986; Hesdorffer et al., 1996; Lozsadi and Larner, 2006; Mendez and Lim, 2003).

Interestingly, the risk of epileptic activity is particularly high in AD patients with early-onset dementia and during the earlier stages of the disease, reaching an 87-fold increase in seizure incidence compared with an age-matched reference population (Amatniek et al., 2006; Mendez et al., 1994). Thus, aberrant neuronal overexcitation may play an important role not only in hAPP mouse models, but also in the pathogenesis of dementia in sporadic AD.

Indeed, epileptiform activity has been associated with transient episodes of amnestic wandering and disorientation in AD (Rabinowicz et al., 2000). It is interesting in this regard that the relationship between seizures and AD is even tighter in autosomal-dominant early-onset FAD. Pedigrees with epilepsy have been identified in FAD linked to mutations in presenilin-1, presenilin-2, and APP (Edwards-Lee et al., 2005; Marcon et al., 2004; Snider et al., 2005). More than 30 different mutations in presenilin-1 are associated with seizures (Larner and Doran, 2006). Our results suggest that the increased epileptic activity in sporadic and autosomal-dominant AD may be caused by Aβ-induced increases in network excitability. Future studies will need to test the hypothesis that this alteration contributes critically to the pathogenesis of AD, objectively and without preconceived notions about outcomes.

References:
Amatniek JC, Hauser WA, DelCastillo-Castaneda C, Jacobs DM, Marder K, Bell K, Albert M, Brandt J, Stern Y. Incidence and predictors of seizures in patients with Alzheimer's disease. Epilepsia. 2006 May;47(5):867-72. Abstract

Edwards-Lee T, Ringman JM, Chung J, Werner J, Morgan A, St George Hyslop P, Thompson P, Dutton R, Mlikotic A, Rogaeva E, Hardy J. An African American family with early-onset Alzheimer disease and an APP (T714I) mutation. Neurology. 2005 Jan 25;64(2):377-9. Abstract

Hauser WA, Morris ML, Heston LL, Anderson VE. Seizures and myoclonus in patients with Alzheimer's disease. Neurology. 1986 Sep;36(9):1226-30. Abstract

Hesdorffer DC, Hauser WA, Annegers JF, Kokmen E, Rocca WA. Dementia and adult-onset unprovoked seizures. Neurology. 1996 Mar;46(3):727-30. Abstract

Larner AJ, Doran M. Clinical phenotypic heterogeneity of Alzheimer's disease associated with mutations of the presenilin-1 gene. J Neurol. 2006 Feb;253(2):139-58. Epub 2005 Nov 4. Review. Abstract

Lozsadi DA, Larner AJ. Prevalence and causes of seizures at the time of diagnosis of probable Alzheimer's disease. Dement Geriatr Cogn Disord. 2006;22(2):121-4. Epub 2006 May 29. Abstract

Marcon G, Giaccone G, Cupidi C, Balestrieri M, Beltrami CA, Finato N, Bergonzi P, Sorbi S, Bugiani O, Tagliavini F. Neuropathological and clinical phenotype of an Italian Alzheimer family with M239V mutation of presenilin 2 gene. J Neuropathol Exp Neurol. 2004 Mar;63(3):199-209. Abstract

Mendez MF, Catanzaro P, Doss RC, ARguello R, Frey WH 2nd. Seizures in Alzheimer's disease: clinicopathologic study. J Geriatr Psychiatry Neurol. 1994 Oct-Dec;7(4):230-3. Abstract

Mendez M, Lim G. Seizures in elderly patients with dementia: epidemiology and management. Drugs Aging. 2003;20(11):791-803. Review. Abstract

Rabinowicz AL, Starkstein SE, Leiguarda RC, Coleman AE. Transient epileptic amnesia in dementia: a treatable unrecognized cause of episodic amnestic wandering. Alzheimer Dis Assoc Disord. 2000 Oct-Dec;14(4):231-3. Abstract

Roberson ED, Scearce-Levie K, Palop JJ, Yan F, Cheng IH, Wu T, Gerstein H, Yu GQ, Mucke L. Reducing endogenous tau ameliorates amyloid beta-induced deficits in an Alzheimer's disease mouse model. Science. 2007 May 4;316(5825):750-4. Abstract

Snyder EM, Nong Y, Almeida CG, Paul S, Moran T, Choi EY, Nairn AC, Salter MW, Lombroso PJ, Gouras GK, Greengard P. Regulation of NMDA receptor trafficking by amyloid-beta. Nat Neurosci. 2005 Aug;8(8):1051-8. Epub 2005 Jul 17. Abstract

View all comments by Lennart Mucke
View all comments by Jorge J Palop

This is a significant advance in understanding how networks are affected in AD. The recent report by Kim et al. that the α-, β-, and γ-secretases process, and regulate expression and function of, the β2 subunit of voltage-sensitive sodium channels suggests that widespread changes in neuronal excitability in AD may have a more fundamental explanation than effects on transmitter receptors.

References:
Kim DY, Carey BW, Wang H, Ingano LA, Binshtok AM, Wertz MH, Pettingell WH, He P, Lee VM, Woolf CJ, Kovacs DM; BACE1 regulates voltage-gated sodium channels and neuronal activity. Nat Cell Biol. 2007 Jul;9(7):755-64. Abstract

View all comments by Michael King

Palop et al. clearly demonstrate neural network dysfunction in hAPPFAD-mice. Our recent study also supports neural network dysfunction in AD patients, as a consequence of elevated BACE1 activity rather than a direct effect of increased Aβ levels. We found that BACE1 regulates voltage-gated sodium channel levels and surface expression through processing of its β2 subunit (Kim et al., 2007). In particular, increased BACE1 activity reduces surface Nav1.1 sodium channel expression and sodium current by 50 percent in hippocampal neurons from BACE1-transgenic mice as compared to wild-type controls. Haploinsufficiency of Nav1.1 induces epileptic seizures in mouse and human by preferentially decreasing sodium currents in GABAergic inhibitory neurons (Yu et al., 2006; for humans, see a review by Meisler and Kearney, 2005). For this reason, we predicted that elevated BACE1 activity in AD would alter sodium channel metabolism, leading to neural network dysfunctions such as seizures (Kim et al., 2007).

It will be interesting to examine the specific contribution of the two...  Read more

Palop et al. clearly demonstrate neural network dysfunction in hAPPFAD-mice. Our recent study also supports neural network dysfunction in AD patients, as a consequence of elevated BACE1 activity rather than a direct effect of increased Aβ levels. We found that BACE1 regulates voltage-gated sodium channel levels and surface expression through processing of its β2 subunit (Kim et al., 2007). In particular, increased BACE1 activity reduces surface Nav1.1 sodium channel expression and sodium current by 50 percent in hippocampal neurons from BACE1-transgenic mice as compared to wild-type controls. Haploinsufficiency of Nav1.1 induces epileptic seizures in mouse and human by preferentially decreasing sodium currents in GABAergic inhibitory neurons (Yu et al., 2006; for humans, see a review by Meisler and Kearney, 2005). For this reason, we predicted that elevated BACE1 activity in AD would alter sodium channel metabolism, leading to neural network dysfunctions such as seizures (Kim et al., 2007).

It will be interesting to examine the specific contribution of the two pathways to neural network dysfunction in AD patients: one via elevated BACE1 activity leading to voltage-gated sodium channel dysfunction, the other via elevated Aβ with unclear molecular mechanism. These two pathways may be separate, both contributing to network dysfunction in AD patients. The former may affect membrane excitability/neuronal activity in the axons, soma, and dendrites of neuronal cells while the latter may directly affect synapses. However, they can also interact with each other. Zhao et al. recently reported that amyloid plaques induce BACE1 in surrounding neurons in mice and AD brains (Zhao et al., 2007). Therefore, elevated BACE1 by Aβ plaques could also contribute to network dysfunction and non-convulsive seizure activities by altering sodium channel metabolism. Elevated BACE1 activity increases Aβ generation in AD patients as well as sodium channel dysfunction, both of which can synergistically contribute to the network dysfunction. The interaction of these two pathways will be an interesting subject to explore in relation to AD pathogenesis.

References:
Kim DY, Carey BW, Wang H, Ingano LA, Binshtok AM, Wertz MH, Pettingell WH, He P, Lee VM, Woolf CJ, Kovacs DM. BACE1 regulates voltage-gated sodium channels and neuronal activity. Nat Cell Biol. 2007 Jul 1;9(7):755-64. Abstract

Meisler MH, Kearney JA. Sodium channel mutations in epilepsy and other neurological disorders. J Clin Invest. 2005 Aug;115(8):2010-7. Review. Abstract

Yu FH, Mantegazza M, Westenbroek RE, Robbins CA, Kalume F, Burton KA, Spain WJ, McKnight GS, Scheuer T, Catterall WA. Reduced sodium current in GABAergic interneurons in a mouse model of severe myoclonic epilepsy in infancy. Nat Neurosci. 2006 Sep;9(9):1142-9. Epub 2006 Aug 20. Erratum in: Nat Neurosci. 2007 Jan;10(1):134. Abstract

Zhao J, Fu Y, Yasvoina M, Shao P, Hitt B, O'Connor T, Logan S, Maus E, Citron M, Berry R, Binder L, Vassar R. Beta-site amyloid precursor protein cleaving enzyme 1 levels become elevated in neurons around amyloid plaques: implications for Alzheimer's disease pathogenesis. J Neurosci. 2007 Apr 4;27(14):3639-49. Abstract

View all comments by Doo Yeon Kim
View all comments by Dora M. Kovacs

My colleague and I would also like to echo the importance of the connection between amyloid, tau, and neuronal dysfunction. The concept that tau levels within the neuron dictate the toxic response to Aβ clearly works in both directions. Our lab, in conjunction with the Ferreira and Binder labs, showed that primary cultures (Rapoport et al., 2002) of tau knockout neurons were resistant to Aβ-induced cell death. These same tau knockout mice were mated to APP transgenics by Mucke’s lab and they also showed that loss of tau impairs amyloid mediated damage. It stands to reason, then, that increased intraneuronal levels of hyperphosphorylated tau would promote amyloid mediated neuronal damage. Our unique bigenic mouse models (APPSw/NOS2-/- and APPSwDI/NOS2-/-) clearly demonstrate that non-mutated mouse tau becomes hyperphosphorylated at AD-like sites in the presence of amyloid deposition. Furthermore, the increased levels of amyloid and hyperphosphorylated tau are associated with profound neuronal loss in multiple brain regions (Colton et al.; Wilcock et al.). In addition to...  Read more

My colleague and I would also like to echo the importance of the connection between amyloid, tau, and neuronal dysfunction. The concept that tau levels within the neuron dictate the toxic response to Aβ clearly works in both directions. Our lab, in conjunction with the Ferreira and Binder labs, showed that primary cultures (Rapoport et al., 2002) of tau knockout neurons were resistant to Aβ-induced cell death. These same tau knockout mice were mated to APP transgenics by Mucke’s lab and they also showed that loss of tau impairs amyloid mediated damage. It stands to reason, then, that increased intraneuronal levels of hyperphosphorylated tau would promote amyloid mediated neuronal damage. Our unique bigenic mouse models (APPSw/NOS2-/- and APPSwDI/NOS2-/-) clearly demonstrate that non-mutated mouse tau becomes hyperphosphorylated at AD-like sites in the presence of amyloid deposition. Furthermore, the increased levels of amyloid and hyperphosphorylated tau are associated with profound neuronal loss in multiple brain regions (Colton et al.; Wilcock et al.). In addition to this neuronal loss, the work by Gouras and colleagues suggest that colocalization of phospho-tau and amyloid may also affect synapses in a way that could further impair neuronal function. These intimate interconnections between tau, amyloid, synapses, and neuronal loss may be a critical starting point for the downward spiral observed in AD brains.

References:
Wilcock DM, Lewis MR, Van Nostrand WE, Davis J, Previti ML, Gharkholonarehe N, Vitek MP, Colton CA Progression of amyloid pathology to Alzheimer's disease pathology in an amyloid precursor protein transgenic mouse model by removal of nitric oxide synthase 2. Neurosci. 2008 Feb 13;28(7):1537-45. Abstract

Colton CA, Vitek MP, Wink DA, Xu Q, Cantillana V, Previti ML, Van Nostrand WE, Weinberg JB, Dawson H. NO synthase 2 (NOS2) deletion promotes multiple pathologies in a mouse model of Alzheimer's disease Proc Natl Acad Sci U S A. 2006 Aug 22;103(34):12867-72. Abstract

Rapoport M, Dawson HN, Binder LI, Vitek MP, Ferreira A. Tau is essential to beta-amyloid-induced neurotoxicity. Proc Natl Acad Sci U S A. 2002 Apr 30;99(9):6364-9. Abstract

View all comments by Carol Colton
View all comments by Michael Vitek

The comment that the cleavage of neuropeptide Y to generate a biologically active fragment by neprilysin (Neutral EndoPeptidase-24.11) is the first such example for the enzyme is incorrect. At least one example has previously been reported in the metabolism of calcitonin gene-related peptide (CGRP) (Davies et al., 1992).

References:
Davies D, Medeiros MS, Keen J, Turner AJ, Haynes LW. Endopeptidase-24.11 cleaves a chemotactic factor from alpha-calcitonin gene-related peptide. Biochem Pharmacol. 1992 Apr 15;43(8):1753-6. Abstract

View all comments by Tony Turner

It has been well suggested that APP is processed during its intracellular trafficking to generate APP CTFs, Aβ, and the APP intracellular domain (AICD). However, how these APP derivatives are transported intracellularly is much less known. In this paper by Zoia Muresan and colleagues, the authors utilized various antibodies against different APP domains for immunocytochemistry and found that full-length APP and APP derivatives are sorted into distinct vesicles and transported independently, with APP CTFs preferentially entering the lamellipodium and filopodia of growth cones and becoming concentrated in regions of growth cone turning and advancement.

In some experiments, the authors used antibody 22C11 for detecting the extracellular fragment of APP and antibody 4G8 for Aβ. Since 22C11 cross-reacts with other APP family proteins (APLP1 and APLP2) while 4G8 only sees APP (and Aβ), the comparisons for the localizations of full-length APP and its derivatives (especially Aβ) may not be appropriate. Nevertheless, these results are very interesting and suggest...  Read more

It has been well suggested that APP is processed during its intracellular trafficking to generate APP CTFs, Aβ, and the APP intracellular domain (AICD). However, how these APP derivatives are transported intracellularly is much less known. In this paper by Zoia Muresan and colleagues, the authors utilized various antibodies against different APP domains for immunocytochemistry and found that full-length APP and APP derivatives are sorted into distinct vesicles and transported independently, with APP CTFs preferentially entering the lamellipodium and filopodia of growth cones and becoming concentrated in regions of growth cone turning and advancement.

In some experiments, the authors used antibody 22C11 for detecting the extracellular fragment of APP and antibody 4G8 for Aβ. Since 22C11 cross-reacts with other APP family proteins (APLP1 and APLP2) while 4G8 only sees APP (and Aβ), the comparisons for the localizations of full-length APP and its derivatives (especially Aβ) may not be appropriate. Nevertheless, these results are very interesting and suggest that a large amount of APP can be cleaved before it is sorted into axonal transport vesicles, probably at the ER and the Golgi/TGN, as we have reported before (Xu et al., 1997 and Greenfield et al., 1999). Moreover, the results indicate that full-length APP and its derivatives may be transported to different locations and exert distinct functions. Several studies from Larry Goldstein and William Mobley’s labs have already suggested that APP plays an active role in axonal transport. Our very recent study also found that both full-length APP and APP bCTF (C99) can regulate intracellular trafficking of PS1/β-secretase components for their cell surface delivery (the results, Liu et al., 2009, are also commented on in this Research News). However, whether or not the intracellular trafficking of proteins other than PS1/γ-secretase components may be regulated by APP awaits further examination. Further, whether the distinct sorting paths or localizations of APP and its derivatives may differentially direct the transport of their respective “cargos,” especially at growth cones also deserves careful scrutiny.

View all comments by Huaxi Xu
View all comments by Yunwu Zhang

We read with great interest the paper by Liu et al. (1), and would like to comment on their exciting findings. The paper proposes a novel mechanism by which the amyloid-β precursor protein (APP) could regulate the intracellular transport of a select group of proteins with emphasis on those that form the γ-secretase complex.

APP was previously proposed to regulate the intraneuronal transport by functioning as a receptor for the microtubule motor kinesin-1. However, it is still largely debated to what extent this model is relevant in vivo, and whether the interaction between APP and kinesin-1 is direct or mediated by bridging protein(s), such as JIP-1 (cJun NH2-terminal kinase-interacting protein-1). Related questions are now addressed by two papers: Liu et al. (1), and our paper, Muresan et al. (2), which are the subject of this Research News. Both articles show that the transport of APP, and the role of APP in regulating transport of other cargo proteins, are far more complex than previously anticipated.

Liu et al. (1) propose that APP could modulate the...  Read more

We read with great interest the paper by Liu et al. (1), and would like to comment on their exciting findings. The paper proposes a novel mechanism by which the amyloid-β precursor protein (APP) could regulate the intracellular transport of a select group of proteins with emphasis on those that form the γ-secretase complex.

APP was previously proposed to regulate the intraneuronal transport by functioning as a receptor for the microtubule motor kinesin-1. However, it is still largely debated to what extent this model is relevant in vivo, and whether the interaction between APP and kinesin-1 is direct or mediated by bridging protein(s), such as JIP-1 (cJun NH2-terminal kinase-interacting protein-1). Related questions are now addressed by two papers: Liu et al. (1), and our paper, Muresan et al. (2), which are the subject of this Research News. Both articles show that the transport of APP, and the role of APP in regulating transport of other cargo proteins, are far more complex than previously anticipated.

Liu et al. (1) propose that APP could modulate the delivery of the γ-secretase complex to the cell surface by regulating its exit from the trans-Golgi network (TGN). A possible mechanism is that APP interacts with the γ-secretase complex and prevents it from being recruited into cargo vesicles, at the TGN. A direct consequence of this mechanism is that APP regulates in this way the cleavage by γ-secretase of substrates localized to the cell surface, such as Notch. Implicitly, Notch signaling is regulated by APP in this way. Liu et al. (1) also show that the trafficking of γ-secretase to the cell surface is regulated not only by APP, but also by the activity of phospholipase D1 (PLD1), a lipid-modifying enzyme that converts phosphatidylcholine to phosphatidic acid. Intriguingly, PLD1 also regulates the transport of APP in a presenilin-1 (PS1, a component of the γ-secretase complex) independent manner. This is important, because several groups reported that PS1 regulates the axonal transport by mechanisms that are not fully understood. Although more work needs to be done to elucidate the complex regulation of the transport of APP and its processing machinery, Liu et al. (1) bring an exciting contribution into this picture.

As perceived from the articles of Liu et al. (1) and of Muresan et al. (2), full-length APP is largely restricted to the intracellular compartments of the early secretory pathway. Biochemical data recently obtained with mouse brain (3) certainly support this scenario. In spite of this, a fraction of full-length APP does reach the plasma membrane, and full-length APP may also have functions at the plasma membrane. However, a significant fraction of it is cleaved prior to entering the cargo vesicles that transport APP (or, rather, its fragments) to various intracellular destinations, as shown by us (2).

We would like to comment on the specificity of the antibody 22C11 (4), an issue raised by Drs. Xu and Zhang in their comment to our paper. This antibody, largely used to detect full-length APP and the soluble N-terminal fragments (sAPPs), was used in some of our immunolabeling experiments due to its high sensitivity. We are aware that this antibody may cross-react with the amyloid precursor-like proteins (APLP1 and APLP2), although with lower affinity. To circumvent this problem, along with 22C11, we employed a plethora of antibodies recognizing epitopes from various regions of APP polypeptide, including antibodies that do not cross-react with APLP1 or APLP2, such as Alz90 (Roche; recognizing residues 511-608 of APP, poorly conserved in APLPs).

With regard to antibody 4G8, which also recognizes the full-length APP, in addition to cleaved fragments, in our study we included antibodies against the cleaved C-terminal ends of Aβ40 and Aβ42, which selectively detect the Aβ fragments. Although our study was not aimed at the precise identification of the APP-derived polypeptides that are transported within neuritis, our results indicate that the transport of APP within neurites occurs to a large extent as cleaved fragments generated by proteolytic processing of APP in the cell body. Thus, the most important idea that derives from our study is that each APP fragment has a life of its own that begins early in the secretory pathway, and may thus have functions that are largely independent from that of full-length APP. Thus, we think that APP is indeed an “All Purpose Protein” (a term that we first heard from Dr. Sangram Sisodia, during a seminar talk) which functions as full-length protein and also as cleaved fragments.

References:
1. Liu, Y., et al., Intracellular trafficking of presenilin 1 is regulated by beta-amyloid precursor protein and phospholipase D1. J Biol Chem, 2009. Abstract

2. Muresan, V., et al., The cleavage products of amyloid-beta precursor protein are sorted to distinct carrier vesicles that are independently transported within neurites. J Neurosci, 2009. 29(11): p. 3565-78. Abstract

3. Bai, Y., et al., The in vivo brain interactome of the amyloid precursor protein. Mol Cell Proteomics. 2008 Jan;7(1):15-34. Abstract

4. Hilbich, C., et al., Amyloid-like properties of peptides flanking the epitope of amyloid precursor protein-specific monoclonal antibody 22C11. J Biol Chem, 1993. 268(35): p. 26571-7. Abstract

View all comments by Virgil Muresan
View all comments by Zoia Muresan

We would like to comment on the interesting results of the recent study by Morfini et al. (1). Kinesin-1, a major microtubule motor that transports cargo in the plus-end direction of microtubules, is a heterotetramer consisting of two microtubule-binding, motor polypeptides (the heavy chains; KHCs) and two cargo-binding polypeptides (the light chains; KLCs). Being a soluble, cytoplasmic protein, kinesin-1 needs to bind the cargo in order to transport it. Therefore, recruitment of kinesin-1 to the cargo vesicle, and its release from it, are important regulatory steps of axonal transport. About 10 years ago, Scott Brady’s laboratory identified the first mechanism leading to the release of kinesin-1 from vesicles. According to this model, kinesin-1 is released through the action of the chaperone HSC70, and is nucleotide-dependent and NEM-sensitive (2). One year later, work from Larry Goldstein’s laboratory suggested that the premature release of kinesin-1 from cargo vesicles in neurons could impair fast axonal transport and lead to neuronal pathology and disease (3). Although the...  Read more

We would like to comment on the interesting results of the recent study by Morfini et al. (1). Kinesin-1, a major microtubule motor that transports cargo in the plus-end direction of microtubules, is a heterotetramer consisting of two microtubule-binding, motor polypeptides (the heavy chains; KHCs) and two cargo-binding polypeptides (the light chains; KLCs). Being a soluble, cytoplasmic protein, kinesin-1 needs to bind the cargo in order to transport it. Therefore, recruitment of kinesin-1 to the cargo vesicle, and its release from it, are important regulatory steps of axonal transport. About 10 years ago, Scott Brady’s laboratory identified the first mechanism leading to the release of kinesin-1 from vesicles. According to this model, kinesin-1 is released through the action of the chaperone HSC70, and is nucleotide-dependent and NEM-sensitive (2). One year later, work from Larry Goldstein’s laboratory suggested that the premature release of kinesin-1 from cargo vesicles in neurons could impair fast axonal transport and lead to neuronal pathology and disease (3). Although the mechanisms for the release of kinesin-1 from its vesicular cargos were incompletely understood at that time, the general idea that a premature release of the motor from its cargo could be at the core of the pathology in neurodegenerative diseases turned out to be correct, and generated an increased interest for research in this direction. Thus, work from the Brady and Busciglio laboratories identified at least two pathways for release of kinesin-1 from vesicles and halt of transport, which are likely to be factors leading to the axonal pathology and synaptic failure in Alzheimer’s disease (AD) (4-6).

Both pathways lead to phosphorylation of the KLCs, followed by detachment of kinesin-1 from the cargo, and impairment of vesicle transport. They are initiated by the addition of soluble Aβ oligomers, or expression of FAD-linked presenilin 1 variants, which trigger aberrant activation of casein kinase 2 or glycogen synthase 3β, which phosphorylate the KLCs. Why the phosphorylated kinesin-1 is released from vesicles is still not fully understood.

Along with AD, kinesin-1 is a target for abnormal phosphorylation in other neurodegenerative diseases, such as spinal and bulbar muscular atrophy (SBMA) and Huntington’s disease, as revealed by the studies from the Brady laboratory, including the work featured here (1, 7). However, in this case, the phosphorylation targets the KHCs, and the activated kinase that performs the phosphorylation is the cJun-N-terminal kinease (JNK). The phosphorylation of the KHCs leads to inhibition of binding of kinesin-1 to microtubules. As a result, the kinesin-1-cargo complex is released from the microtubules, and the transport is halted. These studies showed that the abnormal activation of JNK is triggered by the pathogenic, polyglutaminated, mutant proteins characteristic for polyglutamine (polyQ) expansion diseases: polyQ-androgen receptor in SBMA) (7), and polyQ-huntingtin in Huntingon’s disease(1). As the study by Morfini et al. (1) showed, polyQ-huntingtin activates JNK3, a neuron-specific JNK, that in turn phosphorylates KHC at a serine residue critical for the microtubule-binding function of kinesin-1. While in this case JNK3 is aberrantly activated by a disease factor, it is likely that, under normal conditions, the JNK-3 pathway contributes to the regulation of axonal transport.

Interestingly, in the squid axon system used in these studies, polyQ-huntingtin inhibits, not only the anterograde (kinesin-driven), but also the retrograde (cytoplasmic dynein-driven) fast axonal transport (1). It is not clear whether this inhibition of transport in both directions is due to the fact that kinesin-1 and cytoplasmic dynein interact and coordinate each other’s function (8), or is caused by a direct effect on the dynein machinery. Other studies showed that huntingtin regulates dynein-mediated vesicle transport, and can interact with both dynein and its accessory complex, dynactin (9, 10); however, the assays used by Morfini et al. (1) did not detect an interaction of huntingtin with dynein.

Certainly, other mechanisms, besides the release of the kinesin motor from the cargo or the microtubules, could contribute to the pathogenic processes in these neurodegenerative diseases. Other potentially damaging pathways that target the intracellular transport by affecting the cytoskeleton or the supply of ATP (by disrupting mitochondrial function) have been described (reviewed in (11)). Also, the activation of the kinases is likely to lead to the abnormal phosphorylation of other protein targets as well, with detrimental consequences for the function of neurons via mechanisms that may not involve abnormal axonal transport. For now, a picture emerges where the release of kinesin-1 from either cargo or microtubules, followed by impairment of axonal transport, becomes an important component of the pathogenic process in many neurodegenerative diseases. Therefore, it is the time to think of possibilities to correct the deficiencies, or to find means to enhance the disease-inflicted axonal transport.

References:
1. Morfini GA, You YM, Pollema SL, Kaminska A, Liu K, Yoshioka K, Björkblom B, Coffey ET, Bagnato C, Han D, Huang CF, Banker G, Pigino G, Brady ST. Pathogenic huntingtin inhibits fast axonal transport by activating JNK3 and phosphorylating kinesin. Nat Neurosci. 2009 Jul;12(7):864-71. Abstract

2. Tsai MY, Morfini G, Szebenyi G, Brady ST. Release of kinesin from vesicles by hsc70 and regulation of fast axonal transport. Mol Biol Cell. 2000 Jun;11(6):2161-73. Abstract

3. Kamal A, Almenar-Queralt A, LeBlanc JF, Roberts EA, Goldstein LS. Kinesin-mediated axonal transport of a membrane compartment containing beta-secretase and presenilin-1 requires APP. Nature. 2001 Dec 6;414(6864):643-8. Abstract

4. Morfini G, Szebenyi G, Elluru R, Ratner N, Brady ST. Glycogen synthase kinase 3 phosphorylates kinesin light chains and negatively regulates kinesin-based motility. EMBO J. 2002 Feb 1;21(3):281-93. Abstract

5. Pigino G, Morfini G, Atagi Y, Deshpande A, Yu C, Jungbauer L, Ladu M, Busciglio J, Brady S. Disruption of fast axonal transport is a pathogenic mechanism for intraneuronal amyloid beta. Proc Natl Acad Sci U S A. 2009 Apr 7;106(14):5907-12. Abstract

6. Pigino G, Morfini G, Pelsman A, Mattson MP, Brady ST, Busciglio J. Alzheimer's presenilin 1 mutations impair kinesin-based axonal transport. J Neurosci. 2003 Jun 1;23(11):4499-508. Abstract

7. Morfini G, Pigino G, Szebenyi G, You Y, Pollema S, Brady ST. JNK mediates pathogenic effects of polyglutamine-expanded androgen receptor on fast axonal transport. Nat Neurosci. 2006 Jul;9(7):907-16. Abstract

8. Ligon LA, Tokito M, Finklestein JM, Grossman FE, Holzbaur EL. A direct interaction between cytoplasmic dynein and kinesin I may coordinate motor activity. J Biol Chem. 2004 Apr 30;279(18):19201-8. Abstract

9. Caviston JP, Ross JL, Antony SM, Tokito M, Holzbaur EL. Huntingtin facilitates dynein/dynactin-mediated vesicle transport. Proc Natl Acad Sci U S A. 2007 Jun 12;104(24):10045-50. Abstract

10. Zala D, Colin E, Rangone H, Liot G, Humbert S, Saudou F. Phosphorylation of mutant huntingtin at S421 restores anterograde and retrograde transport in neurons. Hum Mol Genet. 2008 Dec 15;17(24):3837-46. Abstract

11. De Vos KJ, Grierson AJ, Ackerley S, Miller CC. Role of axonal transport in neurodegenerative diseases. Annu Rev Neurosci. 2008;31:151-73. Abstract

View all comments by Virgil Muresan
View all comments by Zoia Muresan

We have recently observed that the membrane surfaces of neurons (mainly pyramidal cells) in contact with plaques lack GABAergic perisomatic synapses (Garcia-Marin et al., 2009). Indeed, a large proportion of plaques are in contact with neurons, and of the several hundred neurons that we found to come into contact with plaques, in no cases were perisomatic terminals found at the surface of the neuron that was directly touching the plaque. Since these perisomatic synapses are thought to exert a strong influence on the output of pyramidal cells, their loss may lead to the hyperactivity of the neurons in contact with plaques. These findings are consistent with the in-vivo calcium-imaging experiments of Busche et al. (2008).

References:
Busche, M.A., Eichhoff, G., Adelsberger, H., Abramowski, D., Wiederhold, K.H., Haass, C., Staufenbiel, M., Konnerth, A., and Garaschuk, O. (2008). Clusters of hyperactive neurons near amyloid plaques in a mouse model of Alzheimer's disease. Science 321, 1686-1689. Abstract

Garcia-Marin V, Blazquez-Llorca L, Rodriguez J, Boluda S, Muntane G, Ferrer I and DeFelipe J (2009) Diminished perisomatic GABAergic terminals on cortical neurons adjacent to amyloid plaques. Front. Neuroanat. 3:28. Abstract

View all comments by Javier DeFelipe

I am very enthusiastic about the paper by Ittner et al. for several reasons. First, it confirms the highly protective effects of tau reduction we observed in hAPP-J20 mice (Roberson et al., 2007 and Palop et al., 2007) in another APP transgenic line with a solid AD-like phenotype and on an independent tau knockout strain. As in our lines, tau reduction rescued memory and longevity in APP23 mice without changing Aβ levels or plaque loads. This kind of reproducibility underlines the robustness of the tau reduction effects and is reassuring to me, especially in light of a recent report suggesting that tau ablation changes Aβ levels and plaque loads in opposite directions and has adverse effects in the Tg2576 model (Dawson et al., 2010).

Second, while the biological functions of tau have so far been explored primarily in axons, Ittner et al. discovered an interesting new mechanism by which tau may modulate synaptic function and...  Read more

I am very enthusiastic about the paper by Ittner et al. for several reasons. First, it confirms the highly protective effects of tau reduction we observed in hAPP-J20 mice (Roberson et al., 2007 and Palop et al., 2007) in another APP transgenic line with a solid AD-like phenotype and on an independent tau knockout strain. As in our lines, tau reduction rescued memory and longevity in APP23 mice without changing Aβ levels or plaque loads. This kind of reproducibility underlines the robustness of the tau reduction effects and is reassuring to me, especially in light of a recent report suggesting that tau ablation changes Aβ levels and plaque loads in opposite directions and has adverse effects in the Tg2576 model (Dawson et al., 2010).

Second, while the biological functions of tau have so far been explored primarily in axons, Ittner et al. discovered an interesting new mechanism by which tau may modulate synaptic function and neuronal excitability in dendrites. Third, this mechanism involves the tyrosine kinase Fyn, which we showed sensitizes APP transgenic mice to Aβ-induced neuronal, synaptic and cognitive deficits (Chin et al., 2004 and Chin et al., 2005). Fourth, consistent with our hypothesis that tau reduction protects against Aβ by preventing neuronal overexcitation (Roberson et al., 2007), targeted perturbation of the NR/PSD-95 interaction, which prevents excitotoxicity, also prevented premature mortality and memory deficits in APP23 mice.

Taken together, these findings strongly suggest that modulating tau, its interaction with Fyn, or key proteins involved in or affected by this interaction may be of therapeutic benefit in AD. It remains possible, though, that other mechanisms also contribute to the excito-protective effects of tau reduction, including changes in presynaptic terminals or in the axonal transport of cargoes supporting synaptic functions.

View all comments by Lennart Mucke

In this manuscript, Ittner and colleagues showed that tau has a role in Aβ toxicity, which may be different from the role of tau on microtubules. Interaction of tau and Fyn is required for stabilizing the NR2/PSD95 complex. Reduction of tau, or interfering with the interaction of tau and Fyn, rescued the premature death and memory deficit in the APP Tg mouse. The results are very interesting, and suggest tau as an attractive drug target for AD therapy.

The physiological role of tau has been thought of as microtubule stabilization. However, the tau gene-deficient mouse did not show much evidence of brain dysfunction. Recently, the results of crossbreeding tau-deficient mice with the GSK3β overexpression or the APP overexpression mouse were reported. Reduction of tau level rescued both the impairment of LTP caused by GSK3β overexpression, and the memory deficits caused by APP overexpression (Gomez de Barreda et al., 2010; Roberson et al., 2007). These reports and the paper by Ittner et al. suggest that tau may have some roles in the synapse in addition to...  Read more

In this manuscript, Ittner and colleagues showed that tau has a role in Aβ toxicity, which may be different from the role of tau on microtubules. Interaction of tau and Fyn is required for stabilizing the NR2/PSD95 complex. Reduction of tau, or interfering with the interaction of tau and Fyn, rescued the premature death and memory deficit in the APP Tg mouse. The results are very interesting, and suggest tau as an attractive drug target for AD therapy.

The physiological role of tau has been thought of as microtubule stabilization. However, the tau gene-deficient mouse did not show much evidence of brain dysfunction. Recently, the results of crossbreeding tau-deficient mice with the GSK3β overexpression or the APP overexpression mouse were reported. Reduction of tau level rescued both the impairment of LTP caused by GSK3β overexpression, and the memory deficits caused by APP overexpression (Gomez de Barreda et al., 2010; Roberson et al., 2007). These reports and the paper by Ittner et al. suggest that tau may have some roles in the synapse in addition to stabilizing microtubules. The extent of memory impairment in APP Tg mice depends on hippocampal LTP level. Tau is involved in synaptic plasticity, and deficiency of tau rescues LTP in the APP Tg mouse. I am not sure whether the destabilization of NR2/PSD95 in the tau knockout mouse can explain the attenuation of LTP level in the APP Tg mouse.

There are two different tau-deficient mouse lines. One is a simple tau gene knockout, and the other involves the replacement of exon 1 of the tau gene by EGFP cDNA. The former two reports used the tau gene knockout mouse, and Ittner used the EGFP mouse as a tau-deficient mouse. We may need to give careful consideration to this difference.

References:
Gomez de Barreda, E., Perez, M., Gomez Ramos, P., de Cristobal, J., Martin-Maestro, P., Moran, A., Dawson, H.N., Vitek, M.P., Lucas, J.J., Hernandez, F. and Avila, J. (2010) Tau-knockout mice show reduced GSK3-induced hippocampal degeneration and learning deficits. Neurobiol Dis. 2010 Mar;37(3):622-9. Abstract

Roberson, E.D., Scearce-Levie, K., Palop, J.J., Yan, F., Cheng, I.H., Wu, T., Gerstein, H., Yu, G.Q. and Mucke, L. (2007) Reducing endogenous tau ameliorates amyloid beta-induced deficits in an Alzheimer's disease mouse model. Science, 316, 750-754. Abstract

View all comments by Akihiko Takashima

The important result by Ittner et al. that post-synaptic targeting of the Src kinase Fyn depends on tau should also be relevant to p75-mediated Aβ toxicity. The observed prevention of Aβ toxicity in APP23 mice with absent or truncated tau could, in part, be due to diminished p75 activity since Src kinases are required for p75 activation by Aβ aggregates (Egert et al., 2007).

References:
Egert S, Piechura H, Hambruch N, Feigel M, Blöchl A. (2007) Characterization of a peptide that specifically blocks the Ras binding domain of p75. Int J Pep Res Ther 13: 413-421. Abstract

View all comments by Rudolf Bloechl

I agree with Lennart Mucke, Akihiko Takashima, and Michel Goedert that this is a major opus by Ittner and Goetz and coworkers, and will become seminal in the long-standing question of how amyloid and tau are related to each other in the pathogenic processes in AD. The amyloid-tau relation is central by definition, as well as pathologically diagnostic for AD. Moreover, I approach the age where the matter becomes personally more and more important to be solved sooner rather than later. The issues at hand have separated "baptists" and "tauists" for too long, and for no apparent reason. I, at least, have adhered to both convictions over the last 20 years without too much negative consequences. I therefore welcome the Ittner study also in this respect.

Whether Fyn is "the" missing link in AD needs, and deserves, careful consideration, but this study will undoubtedly impact the field for some time to come. The data presented were dug out of an impressive number of cellular and mouse models by a wide range of technologies. Typical for the better studies is that they stir up more...  Read more

I agree with Lennart Mucke, Akihiko Takashima, and Michel Goedert that this is a major opus by Ittner and Goetz and coworkers, and will become seminal in the long-standing question of how amyloid and tau are related to each other in the pathogenic processes in AD. The amyloid-tau relation is central by definition, as well as pathologically diagnostic for AD. Moreover, I approach the age where the matter becomes personally more and more important to be solved sooner rather than later. The issues at hand have separated "baptists" and "tauists" for too long, and for no apparent reason. I, at least, have adhered to both convictions over the last 20 years without too much negative consequences. I therefore welcome the Ittner study also in this respect.

Whether Fyn is "the" missing link in AD needs, and deserves, careful consideration, but this study will undoubtedly impact the field for some time to come. The data presented were dug out of an impressive number of cellular and mouse models by a wide range of technologies. Typical for the better studies is that they stir up more questions and have many implications that I have yet to come to full terms with, given the mass of finer details uncovered by these findings. Here, I planned to restrict myself to aspects and questions closest to our own scientific interests, i.e., the synaptic effects of tau and its phosphorylation in vivo (by GSK3 mainly), and the relation to amyloid in various mouse models, as we recently reviewed (Jaworski et al., 2010).

First, the evidence for NR2b phosphorylation by Fyn is convincing, but I wondered about the distinction between "electric" signals at the synapse (graphically not too well depicted in Fig. 7), namely, the classic ESPCs that are normal, as opposed to the "excitotoxic" signals that are strongly affected. What are these latter signals, and what is their function in wild-type mice under normal physiological conditions? These are described for wild-type mice in Figure 7, panel A—but perhaps the caption for that panel should be "APP23 mice." The data imply at least two pools of NR2bs in the same synapse, containing sub-subtypes, truncated, or otherwise modified or adapted subunits, extra-synaptic or tethered.

Teleologically, the most active synapses that produce the most Aβ peptides (for the sake of simplicity, I used the term "Aβ" throughout to indicate all molecular forms and complexity of all the amyloid peptides) must then also be the most vulnerable to this novel mechanism. How do active synapses counteract the inherent Aβ-mediated excitotoxicity? Is this the price to pay for an "LTP-ed" synapse?

A most mind-troubling issue, even an enigma, for me, is why the protein tau remains labeled by some of the most ardent tauists as "an axonal protein," c.q. specific axonal marker? Because we are interested in the role of tau in those brain regions that are struck in AD, we deal with glutamatergic synapses that are located on dendritic spines. I permanently force my Ph.D. students and coworkers to search the literature for evidence that tau is present not only pre-synaptically in axons, but also post-synaptically in dendritic spines—so far unequivocal proof is lacking. We do see, without many problems, mouse protein tau in dendritic spines in primary neuron cultures, and definitely in transfected neurons expressing human tau. Apparently, extending this to mouse brain in vivo, i.e., demonstrating that tau in spines in wild-type mouse brain sections, is technically (too) demanding, but must be done sooner than later….

In another vein, and raising further questions, is the notion that microtubules are not permanently but dynamically based in dendritic spines (Jaworski et al., 2009). This leads us to another dissociation: If protein tau is present in dendritic spines, which we never doubted, it cannot be bound to microtubules. That leaves it free to become bound to the PSD, proposed and implicated here by Ittner et al., or become sequestered to the actin network—that other important post-synaptic scaffold in spines.

A most intriguing question, raised but discussed somewhat "en passant" by Ittner et al., is, How does protein tau get into the dendrites and post-synaptic compartments, i.e., spines, in the first place? Besides the "piggy-back transport of Fyn," the findings highlight a major “normal” physiological function of protein tau in dendrites, which could potentially become more important for neuroscience than for the AD field (this thought illustrates nicely the narrow boundary between physiology and pathology, a concept instilled in my brain in my early Ph.D. student days!). There is, nevertheless, an important pathological flipside even to this dendritic tau: the dramatic neuropil tauopathy in AD, which according to experts is quantitatively an order of magnitude more important than neurofibrillary tangles in the soma (Mitchell et al., 2000). Is that exclusively axonal and pre-synaptic—or what is the contribution of dendritic post-synaptic compartments? In this respect, the statement by Ittner et al that "...levels of tau in the dendritic compartment are much lower than in axons,..." needs to be taken cautiously and verified by proper quantitative methods—if at all possible.

We used an AAV-tau.255 vector to express the same truncated version of tau, which, in contrast to full-length tau.4R and tau.P301L, was not neurotoxic. The lack of toxicity correlated with the fact that tau.255 was largely retained to the cell soma, as opposed to full-length tau that located to dendrites (see, e.g., Fig. S5D in Jaworski et al., 2009). We proposed that the missing microtubule binding domain (MTBD) and the inherent lack of transport over microtubules (MTs) prevented tau.255 from reaching the post-synaptic compartment in the dendritic spines. Interestingly, we observed increased phosphorylation of tau residues that constitute epitope AT180, i.e., S231/S235, which align closely with the 7th PXXP motif (P233-K-S-P235) that binds Fyn (Lee et al., 1998; confirmed here by Ittner et al.). Hardly accidental is then the fact that the AT180 epitope is typically generated by GSK3, which is the subject of our current efforts to understand the contribution of these kinases to the physiology and pathology of APP and protein tau (Terwel et al., 2008).

Importantly, in our AAV-based model, even wild-type tau was neurotoxic, causing rapid and extensive degeneration of CA1/2 pyramidal neurons in wild-type mice, i.e., in absence of human amyloid peptides, and without formation of large tau aggregates in soma or neuropil. Unfortunately, Ittner et al. did not analyze or discuss the eventual neurodegeneration they might have observed in their combined mouse models. Combining the novel data with previously published data leads me to conclude that Aβ causes excitotoxicity and provokes seizures, eventually causing premature death, but does not cause neuronal cell death—an observation made in many single transgenic APP mouse models.

The novel, documented NR2b-Fyn-mediated mechanism depends on transport of Fyn by endogenous murine protein tau into the post-synaptic compartment of dendritic spines. As stated above, the transport of Fyn appears to be "piggy-backed" on protein tau into dendritic processes, which inevitably could or even must result in phosphorylation of tau at Y18 (Lee et al., 2004). Does this phosphorylation come into play in the mechanism? Moreover, Fyn is transported by full-length tau over the dendritic MT system involving the tau-MTBD domain, which implicates this is not a novel function for tau, but "a novel route." This raises again other questions as the MT system in dendrites is not polarized like the axonal MT system. How tau manages to overcome the dendritic "traffic jam" is open for experimentation, which I am sure some of us are doing already.

I agree with Akihiko Takashima that GSK3 must be accounted for in the amyloid-tau equation. I beg to disagree with him regarding the statement that "Reduction of tau rescued…the impairment of LTP caused by GSK3β overexpression…." as LTP was not measured, but the spatial cognition task in the water maze was (Gomez de Barreda et al., 2010). Moreover, the data show that tau-/- did not rescue the GSK3-imposed phenotype, as the Tet/GSK3β mice did not differ significantly from Tet/GSK3β+tau-/- mice (Fig. 2 in Gomez de Barreda et al., 2010).

With regard to GSK3, I commented on this forum on a closely related study on amyloid-induced axonal transport problems involving NMDAR implicating GSK3 in the mechanism (Decker et al., 2010). Obviously, amyloid, tau, and GSK3 play on the same team more often than not, both at the physiological or pathological side of the brain.

Technically, I am not convinced that an issue remains in terms of tau-/- mice. I agree fully with Lennart Mucke that Ittner et al. reassures us that APP-induced deficits are mitigated in a tau-/- background, even in different parental strains (Roberson et al., 2007; Ittner et al., 2010). Science is more often than not reproducible and therefore pleasing and rewarding!

Allow me to conclude by stating that all commentators agree with Ittner and colleagues that protein tau is an attractive study target—and a promising drug target for primary and secondary tauopathies.

References:
Decker H, Lo KY, Unger SM, Ferreira ST, Silverman MA. Amyloid-β peptide oligomers disrupt axonal transport through an NMDA receptor-dependent mechanism that is mediated by glycogen synthase kinase 3beta in primary cultured hippocampal neurons. J Neurosci (2010) 30:9166-71. Abstract

Gómez de Barreda E, Pérez M, Gómez Ramos P, de Cristobal J, Martín-Maestro P, Morán A, Dawson HN, Vitek MP, Lucas JJ, Hernández F, Avila J. tau-knockout mice show reduced GSK3-induced hippocampal degeneration and learning deficits. Neurobiol Dis. (2010) 37:622-9. Abstract

Jaworski T, Dewachter I, Seymour CM, Borghgraef P, Devijver H, Kügler S, Van Leuven F. Alzheimer's disease: Old problem, new views from transgenic and viral models. Biochim Biophys Acta. 2010 Mar 21. Abstract

Jaworski T, Dewachter I, Lechat B, Croes S, Termont A, Demedts D, Borghgraef P, Devijver H, Filipkowski RK, Kaczmarek L, Kügler S, Van Leuven F. AAV-tau mediates pyramidal neurodegeneration by cell-cycle re-entry without neurofibrillary tangle formation in wild-type mice. PLoS One. 2009;4(10):e7280. Abstract

Jaworski J., Kapitein LC, Gouveia SM, Dortland BR, Wulf PS, Grigoriev I, Camera P, Spangler SA, Di Stefano P, Demmers J, Krugers H, Defilippi P, Akhmanova A, Hoogenraad CC. Dynamic microtubules regulate dendritic spine morphology and synaptic plasticity. Neuron (2009) 61:85-100. Abstract

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