Novel diffusion barrier for axonal retention of Tau in neurons and its failure in neurodegeneration
2011; Springer Nature; Volume: 30; Issue: 23 Linguagem: Inglês
10.1038/emboj.2011.376
ISSN1460-2075
AutoresXiaoyu Li, Yatender Kumar, Hans Zempel, Eva‐Maria Mandelkow, Jacek Biernat, Eckhard Mandelkow�,
Tópico(s)Nerve injury and regeneration
ResumoArticle18 October 2011free access Novel diffusion barrier for axonal retention of Tau in neurons and its failure in neurodegeneration Xiaoyu Li Xiaoyu Li Max-Planck-Unit for Structural Molecular Biology, c/o DESY, Hamburg, GermanyPresent address: Olympus Soft Imaging Solutions GmbH, Johann-Krane-Weg 39, 48149 Münster, Germany Search for more papers by this author Yatender Kumar Yatender Kumar Max-Planck-Unit for Structural Molecular Biology, c/o DESY, Hamburg, Germany German Center for Neurodegenerative Diseases (DZNE) and CAESAR, Ludwig-Erhard-Allee 2, Bonn, Germany Search for more papers by this author Hans Zempel Hans Zempel Max-Planck-Unit for Structural Molecular Biology, c/o DESY, Hamburg, Germany German Center for Neurodegenerative Diseases (DZNE) and CAESAR, Ludwig-Erhard-Allee 2, Bonn, Germany Search for more papers by this author Eva-Maria Mandelkow Eva-Maria Mandelkow Max-Planck-Unit for Structural Molecular Biology, c/o DESY, Hamburg, Germany German Center for Neurodegenerative Diseases (DZNE) and CAESAR, Ludwig-Erhard-Allee 2, Bonn, Germany Search for more papers by this author Jacek Biernat Jacek Biernat Max-Planck-Unit for Structural Molecular Biology, c/o DESY, Hamburg, Germany German Center for Neurodegenerative Diseases (DZNE) and CAESAR, Ludwig-Erhard-Allee 2, Bonn, Germany Search for more papers by this author Eckhard Mandelkow Corresponding Author Eckhard Mandelkow Max-Planck-Unit for Structural Molecular Biology, c/o DESY, Hamburg, Germany German Center for Neurodegenerative Diseases (DZNE) and CAESAR, Ludwig-Erhard-Allee 2, Bonn, Germany Search for more papers by this author Xiaoyu Li Xiaoyu Li Max-Planck-Unit for Structural Molecular Biology, c/o DESY, Hamburg, GermanyPresent address: Olympus Soft Imaging Solutions GmbH, Johann-Krane-Weg 39, 48149 Münster, Germany Search for more papers by this author Yatender Kumar Yatender Kumar Max-Planck-Unit for Structural Molecular Biology, c/o DESY, Hamburg, Germany German Center for Neurodegenerative Diseases (DZNE) and CAESAR, Ludwig-Erhard-Allee 2, Bonn, Germany Search for more papers by this author Hans Zempel Hans Zempel Max-Planck-Unit for Structural Molecular Biology, c/o DESY, Hamburg, Germany German Center for Neurodegenerative Diseases (DZNE) and CAESAR, Ludwig-Erhard-Allee 2, Bonn, Germany Search for more papers by this author Eva-Maria Mandelkow Eva-Maria Mandelkow Max-Planck-Unit for Structural Molecular Biology, c/o DESY, Hamburg, Germany German Center for Neurodegenerative Diseases (DZNE) and CAESAR, Ludwig-Erhard-Allee 2, Bonn, Germany Search for more papers by this author Jacek Biernat Jacek Biernat Max-Planck-Unit for Structural Molecular Biology, c/o DESY, Hamburg, Germany German Center for Neurodegenerative Diseases (DZNE) and CAESAR, Ludwig-Erhard-Allee 2, Bonn, Germany Search for more papers by this author Eckhard Mandelkow Corresponding Author Eckhard Mandelkow Max-Planck-Unit for Structural Molecular Biology, c/o DESY, Hamburg, Germany German Center for Neurodegenerative Diseases (DZNE) and CAESAR, Ludwig-Erhard-Allee 2, Bonn, Germany Search for more papers by this author Author Information Xiaoyu Li1, Yatender Kumar1,2,‡, Hans Zempel1,2,‡, Eva-Maria Mandelkow1,2, Jacek Biernat1,2 and Eckhard Mandelkow 1,2 1Max-Planck-Unit for Structural Molecular Biology, c/o DESY, Hamburg, Germany 2German Center for Neurodegenerative Diseases (DZNE) and CAESAR, Ludwig-Erhard-Allee 2, Bonn, Germany ‡These authors contributed equally to this work *Corresponding author. Max-Planck-Unit for Structural Molecular Biology, c/o DESY, Notkestrasse 85, Hamburg, 22607 Germany. Tel.: +49 40 8998 2810; Fax: 49 40 8971 6822; E-mail: [email protected] and [email protected] The EMBO Journal (2011)30:4825-4837https://doi.org/10.1038/emboj.2011.376 There is a Have you seen? (November 2011) associated with this Article. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Missorting of Tau from axons to the somatodendritic compartment of neurons is a hallmark of Alzheimer's disease, but the mechanisms underlying normal sorting and pathological failure are poorly understood. Here, we used several Tau constructs labelled with photoconvertible Dendra2 to analyse its mobility in polarized neurons. This revealed a novel mechanism of sorting—a retrograde barrier in the axon initial segment (AIS) operating as cellular rectifier. It allows anterograde flow of axonal Tau but prevents retrograde flow back into soma and dendrites. The barrier requires binding of Tau to microtubules but does not require F-actin and thus is distinct from the sorting of membrane-associated proteins at the AIS. The barrier breaks down when Tau is phosphorylated in its repeat domain and detached from microtubules, for example, by the kinase MARK/Par1. These observations link the pathological hallmarks of Tau missorting and hyperphosphorylation in neurodegenerative diseases. Introduction An early sign of neurodegeneration in Alzheimer's disease is the missorting of Tau protein. In normal mature neurons, it has a mostly axonal distribution (Binder et al, 1985; Kosik and Finch, 1987; Migheli et al, 1988; Mandell and Banker, 1995), but in disease it appears in the somatodendritic compartment. This occurs before Tau aggregation and correlates with the incipient loss of synapses (Coleman and Yao, 2003; Ballatore et al, 2007; Haass and Selkoe, 2007; Pimplikar et al, 2010). There has been a debate on the causes of the polarized distribution of microtubule-associated proteins (MAPs) in mature neurons, notably axonal Tau and somatodendritic MAP2. Suggested contributing factors include the preferential routing of Tau or MAP2 mRNAs towards their appropriate compartment (Garner et al, 1988; Aronov et al, 2002), selective protection of Tau or MAP2 protein against degradation, and directional cues based on the interplay of microtubules, motors, and cargo (Hirokawa et al, 1996; Nakata and Hirokawa, 2003; Konishi and Setou, 2009). The sorting of Tau can break down in abnormal situations, for example, following excitotoxicity, cell stress, or exposure to amyloid-β peptide (Mattson, 2004; Roberson et al, 2007; Zempel et al, 2010). This causes the accumulation of Tau in cell bodies and dendrites and heralds the gradual pathological aggregation. Considering Tau's role in the disease process it would be important to gain a detailed understanding of the sorting pathway: How does it function in normal neurons? What causes it to break down in disease? Where does the missorted dendritic Tau come from, what is its fate? The establishment of polarity in a neuron is essential for its function. It involves cytoskeletal fibres, notably microtubules and motor proteins of the kinesin superfamily (KIFs) for selective long-haul delivery of cargoes to somatodendritic and axonal compartments (e.g., dendritic neurotransmitter receptors or axonal synaptic vesicles; Burack et al, 2000; Lazarov et al, 2007; Hirokawa et al, 2010; Scott et al, 2011). A further element in maintaining polarity is the buildup of selective barriers (Ledesma and Dotti, 2003; Witte and Bradke, 2008). The best-known example for neurons is the axon initial segment (AIS), located just beyond the axon hillock. It has been characterized as an F-actin-based diffusion barrier for membrane-associated proteins and lipids, and for large (>70 kDa) cytosolic particles (Winckler et al, 1999; Nakada et al, 2003; Kole et al, 2008; Song et al, 2009). Because of the key role of Tau in AD, we and others studied tau transport, diffusion, and MT binding in the axon by GFP-tagged proteins and FRAP (Samsonov et al, 2004; Konzack et al, 2007; Weissmann et al, 2009). Rapid diffusion appears to be a major mechanism for axonal Tau distribution in the short-to-intermediate range, while microtubule-dependent transport of Tau dominates over long distances. Here, we describe a new approach, photoconversion of Tau tagged with Dendra2, to directly visualize and compare the movement of different Tau variants over long distances in differentiated cortical neurons. This revealed a novel barrier mechanism that allows Tau to enter the axon but prevents it from flowing back to the somatodendritic compartment. We show that this barrier is uniquely localized in the same initial segment as the actin-based barrier of membrane components, but it depends on microtubules rather than F-actin and is regulated by Tau phosphorylation. Results Tau diffuses rapidly in neurons, but is restrained by a retrograde barrier in the initial axon One of the enigmas of Tau in AD is its early redistribution from axons into the somatodendritic compartments. This begs two questions: how is Tau sorted into axons but not into dendrites, and why does it stay in axons in spite of its ability to diffuse rapidly? To study the redistribution of Tau, a common approach is to transfect neurons with GFP-tagged Tau into cells and then observe its redistribution after photobleaching (Samsonov et al, 2004; Konzack et al, 2007). In this approach, a bleached zone must be observed against a bright background, which limits the signal/noise. To overcome these limitations, we have employed new photoconvertible variants of GFP that enable one to observe the overall distribution at any time point, and then, after photoconversion in a defined region, reveal the spreading with high contrast, in spite of an apparent steady state. We chose Dendra2 which is monomeric (230 residues), highly photostable, matures rapidly at 37°C and displays bright fluorescence before and after photoconversion (Gurskaya et al, 2006; Gauthier and Brandt, 2010). An overview figure depicting the raw data and ratiometric analysis before and after photoconversion is shown in Supplementary Figure S1, illustrating a photoconversion efficiency of >60% in our experiments. When Dendra2 is transfected by lipofectamine into mature rat cortical neurons (7–11 DIV), the protein becomes visible in the cell body by its intrinsic green fluorescence (Figure 1A). In Figure 1B–D, Dendra2 is photoconverted from green to red fluorescence by irradiation at 405 nm in the cell body, and its spreading can be traced in real time (Figure 1C and D). Within seconds, Dendra2 diffuses into the proximal cell processes, and within minutes it appears along the length of dendrites and axons that can be distinguished by their length, diameter, and brightness (Figure 1D; Supplementary Movie S1). The rate of spreading is roughly consistent with the free diffusion of monomeric Dendra2 in the cytosol (D∼25 μm2/s, for details see Konzack et al, 2007). Figure 1.Photoconversion of TauDendra2 in transfected neurons. Top: Bar diagram of human tau40 with domains, phosphorylation sites, and antibody epitopes. The C-terminal half contains the repeat domain (repeats R1–R4, red, R2 alternatively spliced) plus flanking regions (green), responsible for microtubule binding. The N-terminal half (‘projection domain’) does not bind to microtubules and contains alternatively spliced inserts I1 and I2 (blue). Major target sites of kinases MARK (KXGS motifs with S262, S293, S324, and S356) and PKA (S214) that control binding of Tau to MT are labelled in red. Seventeen major target sites of proline-directed kinases (in the flanking regions of the repeats and projection domain) and some of the responsible kinases are labelled in green. Epitopes of some phosphorylation-dependent antibodies (12E8, AT8, and PHF1) are indicated. Dendra2 is linked to the N-terminus of Tau as indicated. Bottom: (A) Cortical neuron transfected with Dendra2 for 2 days, showing the green fluorescence before photoconversion. Arrow indicates the axon and arrowhead indicates the somatodendritic compartment. (B) Photoconversion of Dendra2 by UV illumination of the soma (ROI1, boxed in B–D). Images were taken in the red fluorescent channel (B–D) at the indicated time points (0–8 min). (C, D) Time-lapse images trace the rapid redistribution of photoconverted Dendra2 from the soma (ROI1) into the dendrites (arrowhead in D) and the axon (arrow in D). (E) Distribution of TauD2 in a cortical neuron transfected for 2 days before photoconversion. TauD2 appears both in the somatodendritic compartment (arrowheads, top) and in the axon (arrow). (F) Photoconversion of TauD2 by UV illumination of the soma (ROI1, boxed in F–H). (G, H) Time-lapse images in red fluorescent channel trace the redistribution of photoconverted TauD2 from the soma into the dendrites (arrowhead in H) and the axon (arrow in H). Compared with Dendra2 (B–D), the spreading of TauD2 is much slower (images at 0–95 min). The asterisk indicates the non-photoconverted neuron. (I) Intensity versus time in ROI2 in axon (∼150 μm from the soma, red circle in D or H). The signal of Dendra2 reaches a half-maximal level at ∼300 s, whereas TauD2 takes much longer (∼5300 s). Data shown here are representations of at least three independent experiments. Download figure Download PowerPoint Figure 1E–H shows the same type of experiment with full-length human Tau (441 residues, ‘2N4R’), N-terminally fused to Dendra2 (688 aa residues, including a 17 aa linker, construct termed TauD2; Figure 1, top). As expected from the higher mass, TauD2 spreads more slowly than Dendra2, but also fills dendrites and axons within ∼48 h, as seen either by the green fluorescence of Dendra2 or by immunostaining. This behaviour is unexpected since mature neurons possess a sorting machinery that routes Tau into axons but not into dendrites (Binder et al, 1985; Mandell and Banker, 1995), and indeed endogenous rat Tau is sorted axonally if expressed alone (see below). On the other hand, overexpression or microinjection of Tau is known to overwhelm the sorting machinery so that Tau becomes ubiquitous in neurons (Hirokawa et al, 1996), as is the case here (Figure 1E). When photoconverting the exogenous TauD2 in the cell body, the protein spreads gradually throughout the neuron, but again the rate is much slower than that of Dendra2 alone (∼20-fold), and there is a preferential filling of axons first (Figure 1F–H). The arrival of photoactivated TauD2 was monitored in selected regions of interest, for example, ∼150 μm from the soma (Figure 1H, ROI2). In this case, it took ∼70 min for TauD2 to redistribute from the soma to the examined area (Figure 1I; Supplementary Movie S2). Thus, this method provides a direct visualization of the dynamics of Tau in neurons. Since the above observations suggested that the sorting machinery of neurons was still functional, in spite of a background of unsorted Tau, we began a systematic investigation of the transfer of TauD2 and Dendra2 (as control) between different regions of mature neurons. Figure 2A1 illustrates part of an axon ∼60 μm from the soma of a Dendra2-transfected neuron (ROI1). Photoconverted Dendra2 redistributed rapidly into the distal axon and somatodendritic compartment (Figure 2A3). In these neurons, a homogeneous distribution of fluorescence was reached across the AIS (Figure 2A2 and A4, intensity profiles at t=1 and 5 min; Supplementary Movie S3). Figure 2.A barrier mechanism in the initial axon prevents axonal TauD2, but not Dendra2 alone, from entering the cell body. (A1–A4) Time-lapse red fluorescent images of Dendra2 alone (without Tau, A1) after photoconversion in the axon in ROI1 (inset in A1 shows the even overall distribution of Dendra2). The intensity profile (A2) corresponds to the fluorescent intensity in ROI2 in the initial axon (between 0 and 50 μm distance from the cell body) at 1 min. Only a low level of Dendra2 has diffused into ROI2, but fluorescence is already visible in the cell body (dashed circle). (A3, A4) At 5 min, the level of Dendra2 is increased by diffusion in ROI2, but even more pronounced in the cell body. Only a shallow intensity gradient is observed along ROI2, indicating that there is no barrier for diffusion for Dendra2. (B1–B4) A similar experiment was performed by photoconversion in the axon in ROI1of a neuron transfected with TauD2 (B1). The intensity profile of ROI2 at 5 min (B2) shows the pronounced gradient of TauD2 in the initial axon, with intensity decaying towards the soma. (B3, B4) The gradient is maintained throughout the experimental period (up to 30 min and more) and the intensity in the cell body (dashed circle) remains low, indicating that a barrier in the initial axons (ROI2) prevents the axonal TauD2 from missorting into the cell body. (C1–C4) Photoconversion of a dendrite (C1) in a neuron transfected with TauD2 (ROI1). The intensity profile in ROI2 at 5 min (C2) in the axon emanating from the cell body shows the smooth intensity flow from the somatodendritic compartment into the axon without indication of a barrier. Even at later time points, there is no appearance of a gradient of TauD2 (30 min, C3, C4). Data represent at least three independent experiments. Download figure Download PowerPoint When performing this experiment with TauD2, a very different picture emerged: the photoconverted TauD2 travelled without hindrance to the distal axon; but was prevented from redistributing towards the somatodendritic compartment (Figure 2B1 and B3). An intensity gradient of photoconverted TauD2 was observed in the AIS, decreasing towards the soma and maintained throughout the experimental period (Figure 2B2 and B4, intensity profiles at t=5 and 30 min; Supplementary Movie S4). This means that once TauD2 is localized in the axon, it cannot return retrogradely to the soma and dendrites. This indicates the presence of a barrier in the AIS, which functions akin to a ‘diode’ to allow directional entry of Tau from the soma to the axon but not reverse. In fact, photoconversion directly at the AIS also resulted in an anterograde flow of TauD2 towards the distal axon and not towards the soma (Supplementary Figure S2). We also tested the effect of photoconversion of TauD2 in the distal axons, and even after repeated photoconversion the barrier was maintained throughout the experimental period (Supplementary Figure S4). We asked whether a similar barrier exists between the soma and the dendrites and performed photoconversion in dendrites of a TauD2-expressing neuron (11 DIV) (Figure 2C1, ROI1). In contrast to axonal Tau, the dendritic TauD2 readily re-entered the somatodendritic compartment and the axon (Figure 2C3). The intensity profiles (Figure 2C2 and C4) indicate that there was no barrier within the somatodendritic compartment (see Supplementary Movie S5). The same effects were observed after photoconversion at the distal ends of dendrites (Supplementary Figure S3). The data demonstrate the unique axonal localization of the barrier mechanism in the AIS to trap Tau once it has entered the axon. The retrograde barrier depends on microtubules What cell components are responsible for the retrograde barrier? As Tau is a microtubule-binding protein, we suspected that intact microtubules are one of the requirements. This is indeed the case: once they are depolymerized by nocodazol, the barrier breaks down and allows retrograde axonal flow of TauD2 into the soma (Figure 3A–D; Supplementary Movie S6). This property of a microtubule-based barrier is distinct from the AIS barrier described previously for membrane-associated proteins, characterized by the accumulation of AnkyrinG, βIV-spectrin, ion channels and cell adhesion molecules and F-actin (Winckler et al, 1999; Ogawa and Rasband, 2008; Grubb and Burrone, 2010; Rasband, 2010). The AIS components are indeed stable against nocodazol, and therefore cannot account for the retrograde barrier of Tau (Figure 4). Likewise, the barrier of Tau is distinct from the porous filter described by Song et al (2009), which blocks axonal access for diffusible molecules greater than ∼70 kDa. Their filter is based on F-actin and operates in the anterograde direction, not retrogradely. Our results were confirmed by destroying the actin-based diffusion filter of Song et al (2009) in the AIS by latrunculin A (Supplementary Figure S5), yet the barrier for Tau remained intact (Figure 4G–I). The F-actin staining was significantly reduced after treatment with latrunculin A, including in the AIS (Supplementary Figure S5). Figure 3.The diffusion barrier depends on intact microtubules. (A) Green fluorescent image of an 11 DIV neuron transfected with TauD2 for 2 days before photoconversion. (B) Red fluorescent image of the same neuron immediately after illumination of its axon near the soma (ROI1) with UV light. (C) Time-lapse images up to 30 min show the anterograde diffusion of axonal Tau but retrograde blockage at the initial axon (arrowhead) so that TauD2 does not enter the cell body (dashed circle). At 30 min, cells were treated with 5 μM nocodazol to disrupt microtubules. (D) After addition of nocodazol, there is diffusion of TauD2 across the barrier in the initial axon (arrowhead) into the somatodendritic compartment (dashed circle). Data represent at least three independent experiments. Download figure Download PowerPoint Figure 4.The axonal diffusion barrier for Tau coincides with the axon initial segment (AIS) but depends on a different mechanism. (A) Cortical neuron (10 DIV) transfected with TauD2. Note the even distribution in axon (arrow), soma, and dendrites. (B) Same neuron, fixed and stained for AnkyrinG, a marker of the AIS, (C) merged images. (D) Cortical neuron (10 DIV) transfected with TauD2 and then treated with 10 μM nocodazol. This treatment destroys microtubules but not the localization of AnkyrinG in the AIS (E, F). (G) Green fluorescent image of a neuron (10 DIV) expressing TauD2 and incubated in 2.5 μM latrunculin A for 1 h to disrupt the actin cytoskeleton before photoconversion. (H) Red fluorescent image of the same neuron in the presence of latrunculin A where part of its axon was photoconverted (ROI1) (circles indicate the soma in H and I). (I) Time-lapse image after 75 min showing the blockage at the AIS (arrowhead) for TauD2 in the presence of latrunculin A, indicating that the barrier for Tau does not depend on the actin cytoskeleton. Download figure Download PowerPoint In order to test the spatial relationship of the Tau diffusion barrier with the AIS, we stained the AIS with AnkyrinG after photoconversion in the axon. The data indicate that the classical AIS, as defined by AnkyrinG, and the Tau diffusion barrier overlap significantly. The Tau diffusion barrier is localized within the broad decreasing gradient of the AnkyrinG staining beginning from the axon hillock to the more distal parts of the axon (Supplementary Figure S6). The retrograde barrier depends on Tau's microtubule affinity and state of phosphorylation Tau occurs in several isoforms and phospho-variants, and therefore we asked whether all forms of Tau are restrained by the retrograde barrier in the same way, and whether the affinity for microtubules plays a role. This was tested by varying the phosphorylation state of Tau (which in turn alters the affinity for microtubules), or by changing the balance of kinases and phosphatases. The latter is achieved by the phosphatase inhibitor okadaic acid (OA), resulting in a gradual hyperphosphorylation of Tau at multiple sites similar to AD Tau, and loss of MT binding (Lichtenberg-Kraag et al, 1992). Figure 5A–C illustrates a neuron with a stable barrier, but after exposure to 0.5 μM OA at 55 min the barrier breaks down quickly (Figure 5D). The blots (Figure 5E) illustrate that Tau becomes highly phosphorylated at the phospho-epitopes AT8, PHF1, 12E8, and others upon exposure to OA. Figure 5.Hyperphosphorylation by okadaic acid (OA) treatment allows TauD2 to pass the barrier. (A) Green fluorescent image of a 9 DIV neuron expressing TauD2 before photoconversion. (B) Red fluorescent image of the same neuron where part of its axon was photoconverted (ROI1). Time-lapse image after 20 min showing the anterograde spreading of TauD2 but blockage at the AIS near the cell body (arrowheads in B–D indicate the AIS). (C) The same protocol of photoconversion as in (B) was repeated in the same axonal region (ROI1) 55 min after the first illumination; at this time, 500 nM OA was added. The diffusion barrier at the AIS is still visible (arrowhead). (D) Redistribution of axonal Tau to the somatodendritic compartment after OA treatment. Increasing fluorescence intensity in the soma is observed shortly after addition of OA, and the gradient in distribution of axonal Tau in the AIS disappears. Dashed circles indicate the soma in (B–D). (E) Western blot of extracts of cortical neurons (11 DIV) treated with OA (500 nM, 1 h) using phosphorylation-dependent Tau antibodies AT8, PHF1, and 12E8. The signals of hyperphosphorylated Tau increase strongly after phosphatases are inhibited by OA treatment (right lanes), especially in the case of 12E8 (pS262/pS356 in the repeats, right panel); whereas in the untreated cells the signals of phosphorylated Tau at these epitopes remain low (left lanes). Note the clear upward shift of the bands of phosphorylated Tau in the OA-treated cells. A blot with anti-actin antibody was used as loading control. Download figure Download PowerPoint In the OA experiment, the phosphorylation of Tau is heterogeneous and includes sites of minor importance for MT binding (e.g., some of the SP or TP motifs). In order to demonstrate a direct and specific relationship between the barrier and the Tau-MT affinity, we transfected TauD2 modified at the four KXGS motifs in the repeat: the ‘4KXGE’ mutant mimics constitutive phosphorylation, whereas the ‘4KXGA’ is non-phosphorylatable at these motifs. The 4KXGE mutant binds to MT only weakly (Biernat and Mandelkow, 1999), and indeed there is no retrograde barrier for this Tau mutant (Figure 6A and B). By contrast, the 4KXGA mutant retains high affinity to MT, and the barrier remains intact (Figure 6C and D). This illustrates that the barrier depends not on the overall degree of phosphorylation, but on specific phosphorylation sites that detach Tau from microtubules. Figure 6.Effect of pseudo-phosphorylation of TauD2 and MARK activity on axonal Tau redistribution and diffusion barrier. (A, B) Cortical neuron transfected with pseudo-phosphorylated TauD2-4KXGE (at all four KXGS → E motifs in the repeat domain, thus weakening the binding to microtubules). After photoconversion in the axon at time t=0 (ROI1 in A) the soma (dashed circles) lights up in a time-dependent manner, indicating that loss of microtubule-binding enables TauD2-4KXGE to pass the retrograde diffusion barrier between the axon and the cell body. The inset in (A) shows the green fluorescent image of the neuron before photoconversion. (C, D) Similar experiment to (A, B) performed in neurons transfected with TauD2-4KXGA (non-phosphorylatable in the repeat domain and therefore tightly binding to microtubules). ROI1 in (C) indicates the axonal region for UV illumination. There is no retrograde flow of TauD2-4KXGA into the soma of the neuron (D, 30 min). (E) Time-lapse images taken in the red fluorescent channel at 1 h after photoconversion in the axon (ROI1) of a neuron co-transfected with CFP-MARK2 and TauD2. Note that the soma shows strong fluorescence since the phosphorylated TauD2, not bound to microtubules, can pass the barrier. The intensity profile of the AIS (ROI2 in E) (inset) shows no gradient of Tau distribution in this region, indicating that TauD2 can pass through the barrier after phosphorylation by MARK2. (F) Same experimental procedure as in (E), performed in a neuron co-transfected with the inactive mutant CFP-MARK2T208A+S212A and TauD2. ROI1 indicates the axonal region for UV illumination. The fluorescent intensity in the somatodendritic compartment remains low compared with (E), indicating that the kinase-dead mutant of MARK could not phosphorylate TauD2 at the KXGS motifs in the repeat domain, and therefore TauD2 still binds strongly to microtubules and cannot pass the barrier. The intensity profile in the AIS (ROI2 in F) (inset) shows the gradient of TauD2 typical of an intact barrier. Data represent at least three independent experiments. Download figure Download PowerPoint The KXGS motifs of Tau, notably S262, are phosphorylated early in neurodegeneration by kinases of the MARK/Par-1 family which play a role in establishing cell polarity (Matenia and Mandelkow, 2009). We, therefore, transfected TauD2 together with MARK2CFP. The kinase became distributed in the cell body, axon, and dendrites (Figure 6E, inset), Tau became strongly phosphorylated at the KXGS motifs (Thies and Mandelkow, 2007), and the barrier indeed broke down, allowing axonal TauD2 to flow into the cell body (Figure 6E). When the same experiment was done with inactive MARK2CFP (with mutations T208A+S212A; Timm et al, 2008), Tau remained in a low state of phosphorylation, and the barrier remained intact (Figure 6F). Together, the data show that the retrograde barrier restrains Tau only when its affinity to microtubules is high, but disappears when microtubules break down or when Tau becomes detached by phosphorylation. In the above experiments, we focused on factors that determine whether a retrograde barrier is present or not. However, the question arises whether there are intrinsic differences in the mobilities of axonal Tau, independent or superimposed on the properties of the barrier. For this purpose, we tested the spreading of photoactivated T
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