Why the total phosphorylation of certain neuronal proteins decrease during development?

Why the total phosphorylation of certain neuronal proteins decrease during development?

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Looking at the effects of RIM1a which is a protein involved in neurotransmitter release. Any ideas as to why its total phosphorylation decreases as the rats develop?

Many thanks


Here are a couple Wikipedia articles that might help out:

Critical period - Critical period: the idea that certain types of plasticity in the nervous system occur only at certain "critical" times.

Long-term potentiation - LTP, talked a lot about in the paper you linked: it's a mechanism for strengthening of synaptic connections.

Synaptic pruning - # synapses/strength of synapses tend to decrease from adolescence to adulthood

The paper you linked talks a lot about the role of phosphorylated RIM1a in LTP. Without knowing more specifically about this protein, and from just a glance at the paper you linked, I'd suspect that RIM1a is phosphorylated during periods of increased LTP, particularly earlier in development, and as animals age and their brains go into the synaptic pruning phase, LTP is downregulated overall, and one of mechanisms could be the lack of phoshporylated RIM1a.

Why the total phosphorylation of certain neuronal proteins decrease during development? - Biology

Autism spectrum disorders such as Rett syndrome (RTT) have been hypothesized to arise from defects in experience-dependent synapse maturation. RTT is caused by mutations in MECP2, a nuclear protein that becomes phosphorylated at S421 in response to neuronal activation. We show here that disruption of MeCP2 S421 phosphorylation in vivo results in defects in synapse development and behavior, implicating activity-dependent regulation of MeCP2 in brain development and RTT. We investigated the mechanism by which S421 phosphorylation regulates MeCP2 function and show by chromatin immunoprecipitation-sequencing that this modification occurs on MeCP2 bound across the genome. The phosphorylation of MeCP2 S421 appears not to regulate the expression of specific genes rather, MeCP2 functions as a histone-like factor whose phosphorylation may facilitate a genome-wide response of chromatin to neuronal activity during nervous system development. We propose that RTT results in part from a loss of this experience-dependent chromatin remodeling.


► Loss of activity-dependent MeCP2 phospho-S421 in vivo alters synaptic development ► MeCP2 S421A knockin mice display abnormal behavioral responses to novelty ► MeCP2 binding across the neuronal genome is not detectably altered by stimulation ► Activity-dependent pS421 MeCP2 occurs genome-wide suggesting it has a global function


Protein palmitoylation, a classical and common lipid modification, regulates diverse aspects of neuronal protein trafficking and function. The reversible nature of palmitoylation provides a potential general mechanism for protein shuttling between intracellular compartments. The recent discovery of palmitoylating enzymes — a large DHHC (Asp-His-His-Cys) protein family — and the development of new proteomic and imaging methods have accelerated palmitoylation analysis. It is becoming clear that individual DHHC enzymes generate and maintain the specialized compartmentalization of substrates in polarized neurons. Here, we discuss the regulatory mechanisms for dynamic protein palmitoylation and the emerging roles of protein palmitoylation in various aspects of pathophysiology, including neuronal development and synaptic plasticity.


Loss of Bbs proteins affect principal neuron dendritic morphology

Given that primary cilia are required for the formation of neuronal dendrites [21], we investigated the effect of loss of ciliary Bbs proteins on the dendritic morphology of principal neurons of Bbs mouse models. We measured dendritic length, spine count, and spine density of dentate gyrus (DG), basolateral amygdala (BLA), and layer V pyramidal neurons of the frontal cortex using a Golgi-Cox impregnation method. We found that the total spine density was reduced by 55% in DG granule cells of P42 old Bbs4 −/− mice (Fig 1A and 1B and S1 Video). Total spine density on the basal and apical dendrites (further referred to as basal and apical spine density) of layer V neurons was reduced by 55% and 54% (Fig 1A and 1E), respectively, and total basal and apical spine density of BLA neurons was reduced by 23% and 22%, respectively (Fig 1A and 1J). Sholl analysis revealed a significant reduction in spine density of all branches and per 30-μm interval in DG with the exception of the most distal branch and a 300-μm circle in Bbs4 −/− mice (Fig 1C and 1D). Similar Sholl analysis results were found in apical and basal dendrites of Layer V neurons, where dendritic spine density per branch order and per 30-μm interval was affected (Fig 1F–1I). Apical and basal BLA dendrites of Bbs4 −/− mice revealed unequal patterns in spine reduction affecting only a few branches and concentric circles (Fig 1K–1N). A number of dendritic intersections in DG, Layer V, and BLA neurons were not affected when compared with control mice (S1A–S1E Fig). Total dendritic length was reduced by 48% in DG neurons and by 25% in basal dendrites of layer V cortex neurons in Bbs4 −/− mice. Change in the length of apical dendrites of layer V cortex neurons in Bbs4 −/− mice was not statistically significant. BLA apical and basal dendrites showed a statistically significant length reduction of 14% and 19%, respectively (S1F–S1J Fig). Overall, these data show significant aberrations in dendritic morphology in the Bbs mouse model.

(A) Representative images of Golgi-impregnated DG, BLA, and Layer V pyramidal neurons of P42 Bbs4 −/− and Bbs4 +/+ mice (100x scale bar, 5 μm). (B-D) Spine density of DG neurons. (B) Total spine density. (C) Spine density per branch order. (D) Spine density per 30-μm interval. (E-I) Spine density of layer V pyramidal neurons. (E) Total spine density. (F) Spine density in apical dendrites per branch order. (G) Spine density in basal dendrites per branch order. (H) Spine density in apical dendrites per 30-μm interval. (I) Spine density in basal dendrites per 30-μm interval. (J-N) Total spine density of BLA. (J) Total spine density. (K) Spine density in apical dendrites per branch order. (l) Spine density in basal dendrites per branch order. (M) Spine density in apical dendrites per 30-μm interval. (N) Spine density in basal dendrites per 30-μm interval (Nmice/WT = 5 Nmice/KO = 7, Ncells/WT = 25, Ncells /KO = 35, mean ± SD, ***P < 0.001 **P < 0.01 *P < 0.05). One-way ANOVA, Tukey post hoc test except for B, E, J, where unpaired t test was used. Underlying data are available in S1 Data. Bbs4, Bardet-Biedl syndrome 4 BLA, basolateral amygdala DG, dentate gyrus KO, knockout ns, not significant WT, wild type.

To determine when the dendritic architecture of Bbs4 −/− DG neurons begins to change, we analysed dendritic length and spine density per branch order and per 30-μm interval at E19.5 and P21. The results of P21 were similar to those obtained at P42: we observed a significant reduction in dendritic length and spine density and no significant changes in a number of dendritic intersections (S2A–S2F Fig). By contrast, at E19.5, the density of dendritic filopodia (dendritic protrusions on developing neurons) in Bbs4 −/− DG neurons were not affected. However, the dendritic length was significantly reduced at E19.5 (S2A and S2G–S2K Fig). Taken together, the Bbs4 −/− murine model shows a progressive decrease in dendritic spine density at P21 (38%) and P42 (55%), but not at late embryonic stages (S2L Fig).

To investigate whether similar dendritic abnormalities can be detected in other Bbs models, we analysed the DG dendrite morphology of P21 Bbs5 and Bbs1 M390R models. Notably, loss of the Bbs5 protein led to significant reduction in DG dendritic length (34%) and overall spine density (32%) in dentate granule cells of knockout mice (S3A–S3C Fig). Sholl analysis also revealed abnormal spine density in Bbs5 −/− mice, with a significant spine reduction from the second to fifth branch order and from the 60-μm to 150-μm interval, respectively (S3D–S3F Fig). Interestingly, Bbs1 M390R/M390R was associated with consistent but marginal abnormalities in spinogenesis of DG neurons showing only a 10% reduction in the total spine density. However, dendritic length was not affected (S3A and S3G–S3K Fig). This finding is in agreement with our clinical observations that BBS1 M390R patients have the mildest cognitive phenotype.

To determine whether specific subtypes of spines were overrepresented on DG neurons of Bbs4 −/− mice, we analysed spines based on their size and shape (S4A Fig) [22]. We observed that total spine count was reduced in all spine subtypes except ‘branched’ spines. However, when the reduction of dendritic length of Bbs4 −/− neurons was taken into account, we found that only the density of ‘thin’ spines was significantly reduced (22%) (S4B–S4E Fig).

Reduced contextual and cued fear memory but no impairment in anxiety-like behaviour in Bbs4 −/− mice

Hippocampus, amygdala, and prefrontal cortex are structures involved in learning, memory, and social interaction. To investigate whether the loss of dendritic spines of DG, BLA, and prefrontal cortex neurons correlates with behavioural changes in Bbs4 −/− mice, we performed a set of behavioural tests. Bbs mice are known to develop a number of defects, including visual impairment and obesity [23]. To minimise the effect of these confounding factors, we performed the tests in younger mice (8 weeks). According to the majority of the literature and our own assessments, retinal degenerative changes in this Bbs4 model begin to develop at 7–8 weeks, making visual impairment unlikely to account for the differences in the test. Similarly, obesity should not confound our results, as at this age there are no weight differences in Bbs4 −/− and Bbs4 +/+ mice. To assess fear memory, we performed contextual and cued fear conditioning tests. In the conditioning session at Day 1, freezing behaviour and distance travelled during the first 150 seconds without introducing a conditioned stimulus (tone) and unconditioned stimulus (footshock) were used to evaluate baseline activity in the novel environment of the contextual fear experiment. The loss of Bbs4 did not affect the percentage of freezing or distance travelled in baseline activity of Bbs4 −/− male and female mice. However, after introduction of the paired tone-foot shock stimulus at Day 1, post hoc analysis revealed a significant decrease in the percentage of freezing and an increase in the distance travelled on Day 2 of Bbs4 −/− female mice compared with female control mice. Percentage of freezing and an increase in distance travelled were not statistically significant in male mice (Fig 2A, 2B and 2D).

(A) Schematic presentation of the contextual and cued fear conditioning test. At Day 1, mice were placed in the fear conditioning chamber for 616.6 seconds. After 150 seconds, a 5-second tone is played, followed by a 0.5-second, 0.5-mA shock. The tone and shock are repeated two more times at 150-second intervals. At Day 2 mice were placed in exactly the same chamber for 300 seconds without tones or shocks. After 4 hours (Day 2), mice are placed in the altered context and left for 180 seconds. At 180 seconds, a 5-second tone is played, which is repeated twice at 60-second intervals. The first 150 seconds of the conditioning trial were used as a baseline for the context data. The first 180 seconds in the altered context were used as the baseline for the cue data. (B) Freezing (%) in the contextual memory test. (C) Freezing (%) in the cue memory test. (D) Distance travelled (cm) in the conditioning test, context test, and cued test (females: NWT = 11, NKO = 11 males: NWT = 13, NKO = 12 mean ± SD, ***P < 0.001 **P < 0.01 *P < 0.05). One-way ANOVA, Tukey post hoc test. # It was noted that there was a significant level of reduction of percent time freezing and distance travelled in Bbs4 −/− mice when unpaired, two-tailed t test (P < 0.05) was used. Underlying data are available in S3 Data. Bbs4, Bardet-Biedl syndrome 4 KO, knockout WT, wild type.

Altered context at Day 2 before the introduction of the tone was used as a baseline for the cue data. The data revealed that there was no significant change in the percentage of freezing in altered context baseline activity (Fig 2C and 2D). During the cued conditioning session with the tone (Day 2, 180–360 seconds), the percentage of freezing was significantly reduced, and distance travelled increased in Bbs4 −/− male but not in female mice (Fig 2C and 2D). This set of results indicates that loss of Bbs4 protein affects contextual and cued fear memory in a gender-task–dependent manner.

Miniature excitatory postsynaptic currents amplitude is increased in Bbs4 −/− DG neurons

The morphology of dendritic spines is highly dynamic, and their formation and maintenance depend on synaptic function and neuronal activity [24]. To assess synaptic and neuronal function in a Bbs model, we measured intrinsic and synaptic properties of hippocampal granule cells of 3–4-week-old Bbs4 −/− and Bbs4 +/+ mice in acute hippocampal slices. We found that intrinsic properties of Bbs4 −/− neurons were unaffected compared with age-matched control littermates (Fig 3A–3C). To evaluate the synaptic properties of granule cells in these two groups, we measured miniature excitatory postsynaptic currents (mEPSCs). Notably, while the frequency of mEPSCs was not different between the two groups, mEPSC amplitudes were significantly larger in Bbs4 −/− neurons (Fig 3D–3F). These data make it unlikely that decreased neuronal activity underlies diminished spine density. On the contrary, the observed increase in mEPSC amplitudes suggests an activation of compensatory mechanisms at the presynaptic and/or postsynaptic sites in response to spine loss.

(A) Comparison of firing patterns in response to current injections during current clamp recordings from hippocampal granule cells. (B) Bar graphs summarising passive membrane properties. No significant differences were found between Bbs4 −/− and Bbs4 +/+ mice in input resistance (left), membrane time constant (middle), and resting membrane potential (right). (C) Firing frequency was plotted against current injection amplitudes. No significant differences were found between Bbs4 −/− and Bbs4 +/+ mice. (D) mEPSCs were recorded from hippocampal granule cells in Bbs4 −/− and Bbs4 +/+ mice (N = 6). (E) Cumulative probability plot comparing mEPSC amplitudes between Bbs4 −/− and Bbs4 +/+ mice. mEPSC amplitudes in granule cells of Bbs4 −/− mice are significantly larger (N = 6, P < 0.05, Kolmogorov-Smirnov test). (F) Cumulative probability plot comparing inter-event intervals (IEIs) of mEPSCs between Bbs4 −/− and Bbs4 +/+ mice (N = 6 P = 0.27, Kolmogorov-Smirnov test). Underlying data are available in S3 Data. Bbs4, Bardet-Biedl syndrome 4 IEI, inter-event interval KS, Kolmogorov-Smirnov mEPSC, miniature excitatory postsynaptic current.

IGF-1R downstream signalling is dysregulated in Bbs4 −/− synaptosomes

A number of tyrosine kinase receptors (RTKs), including IGF, RET, TrkB, PDGF, and EphB are known to enhance dendritic growth and promote the formation and maintenance of dendritic spines [7,25]. To assess the signalling of RTK in the synaptosomal fractions of Bbs4 −/− and Bbs4 +/+ mice, we quantified the phosphorylation level of RTKs using a Phospho-RTK Array. Given that dendritic spine loss occurs between P1 and P21 and to capture the initial signalling changes before potential compensatory mechanisms may start taking place, we used synaptosomal fractions of P7 mice. Synaptosomal fractions from Bbs4 −/− and Bbs4 +/+ mice were incubated with the membrane containing immobilised RTK antibodies followed by detection of RTK phosphorylation by a pan anti-phospho-tyrosine antibody. Interestingly, phosphorylation levels of a number of RTKs were altered, including insulin and IGF1 receptors (Fig 4A and S5 Fig). We focused on IGF-1R/insulin receptor (IR) signalling, as it is known to have a profound effect on neuroplasticity in the CNS [7–9]. Pull-down experiments confirmed that phosphorylation of IGF-IR/InsulinR was decreased in the P7 enriched synaptosomal fraction of Bbs4 −/− mice (Fig 4B). Additionally, phosphorylation levels of Akt, a downstream target of canonical IGF signalling, were significantly reduced (Fig 4B). Next, we tested the phosphorylation level of insulin receptor substrate P53 (IRS p58), an adaptor protein that is phosphorylated by IR and IGF-1R [26]. Interestingly, IRS p58 protein has previously been shown to be highly enriched in the PSD of glutamatergic synapses, highlighting the role of this protein in neurons [27]. We found that phosphorylation of IRS p58 was significantly reduced in synaptosomal fractions of P7 Bbs4 −/− mice (Fig 4B). Furthermore, as the activities of IGF-1R and IRS p58 depend on interaction with Rho family GTPases [28, 29], we investigated the activities of Rac1 and RhoA GTPases. We observed that activity of RhoA was increased and, concurrently, Rac1 activity was decreased in the enriched synaptosomal fraction of P7 Bbs4 −/− mice (Fig 4C and 4D). We next assessed whether dysregulation of IGF-1 signalling in Bbs −/− mice affects the levels of N-methyl-D-aspartate (NMDA) and Alpha-Amino-3-Hydroxy-5-Methyl-4-Isoxazole Propionic Acid (AMPA) receptors in the total and synaptosomal fractions of Bbs4 −/− and Bbs4 +/+ mice by western blotting. We observed a significant increase in the level of NMDA and AMPA receptors in the synaptosomal fraction of P7 Bbs4 −/− mice, whereas no changes in the receptors’ levels in the total brain fraction were detected (Fig 4E). These data are consistent with our previous findings of increased mEPSC amplitudes in Bbs4 −/− neurons (Fig 3E), suggesting a compensatory mechanism in response to spine loss.

(A) Phospho-RTK array reveals significant decrease in phosphorylation of insulin and IGF1 receptors in Bbs4 −/− (N = 2, mean ± SD). (B) Pull-down analysis shows aberrant IGF-1R downstream signalling. Bbs4 −/− and Bbs4 +/+ enriched synaptosomal fractions were incubated with mouse anti-phosphotyrosine antibody overnight, followed by incubation with Dynabeads M-280 for 2 hours. Immunoblotting analysis of the proteins eluted from the beads was performed using anti-IGFR/InsR, anti-Akt, and anti-IRS p58 antibodies. Input: the total brain protein fraction before the incubation with anti-phosphotyrosine antibody, which indicates the total level of IGFR/InsR, Akt, and IRS p58 in Bbs4 −/− and Bbs4 +/+ mice. (C, D) RhoA and Rac1 G-LISA Activation Assays. Levels of activated RhoA (c) and Rac1 (d) were measured in the total brain extracts and enriched synaptosomal fraction of Bbs4 −/− and Bbs4 +/+ mice (N = 3, mean ± SD unpaired t test). (E) Representative image of western blot analysis of NMDA and AMPA receptors levels in the total brain extract and enriched synaptosomal fraction of Bbs4 −/− and Bbs4 +/+ mice. (F) Representative western blots of autophagy markers LC3-II and p62 in the synaptosomal fractions of Bbs4 −/− and Bbs4 +/+ mice at P1, P7, P14, and P21 (N = 3, mean ± SD). LC3-I is a cytosolic form of LC3. LC3-II is a LC3-phosphatidylethanolamine conjugate (LC3-II), which is recruited to autophagosomal membranes. LC3-II and p62 levels were quantified by measuring western blot band intensities using the Image J programme (N = 3, mean ± SD, unpaired t test). Housekeeping genes (actin, GAPDH, etc.) could not be used as normalisation controls due to the changes in their gene expression levels in Bbs4 −/− mice (our unpublished observations). (G) Measurement of oxidative phosphorylation (OXPHOS) complex activities in the whole brain homogenates of Bbs4 −/− and Bbs4 +/+ mice. Units: mU:U CS raw data were normalised to citrate synthase N = 4, mean ± SD ns, not significant unpaired t test. Underlying data are available in S2 Data. AMPAR, alpha-Amino-3-Hydroxy-5-Methyl-4-Isoxazole Propionic Acid receptor NMDAR, N-methyl-D-aspartate receptor Bbs4, Bardet-Biedl syndrome 4 CI, mitochondria complex I CII, mitochondria complex II CIII, mitochondria complex III CS, citrate synthase, GAPDH, Glyceraldehyde 3-phosphate dehydrogenase GluR, glutamate receptor IGF-1R, insulin-like growth factor receptor InsR, insulin receptor LC3, microtubule-associated protein 1A/1B-light chain 3 LC3-I, cytosolic form of LC3 LC3-II, LC3-phosphatidylethanolamine conjugate recruited to autophagosomal membranes OXPHOS, oxidative phosphorylation RTK, tyrosine kinase receptor SCC, succinate:cytochrome c oxidoreductase (= complex II + III combined) WB, western blot.

One of the possible mechanisms of dendritic spine pruning is macroautophagy [18], a process that is tightly regulated by IGF-1R signalling and small GTPases. To test whether autophagy is dysregulated in our Bbs model, we analysed the level of autophagy markers LC3-II and p62 in the enriched synaptosomal fractions isolated from Bbs4 −/− and Bbs4 +/+ mice brains at different postnatal stages. We observed a significant increase in LC3-II level at P1 and P7 of Bbs4 −/− mice (Fig 4F). Given the widely recognised notion that the level of p62 correlates inversely with autophagy, it was unexpected to see an increase in p62 in P1 synaptosomes in our experiment. However, it is in line with reports that p62 levels can be up-regulated during high autophagic flux due to a multifunctional role for p62 [30]. To exclude mitochondrial dysfunction and oxidative stress as triggers of autophagic induction [31], we assessed the activities of respiratory chain complexes I, II, III, IV and succinate:cytochrome c oxidoreductase (SCC complex II and III combined) in total brain homogenates of P7 Bbs4 −/− and Bbs4 +/+ mice. Oxidative phosphorylation (OXPHOS) complex activities were determined, and the results were normalised to the activity of citrate synthase (CS). We found no significant differences in the activities of OXPHOS between Bbs4 −/− and Bbs4 +/+ mice (Fig 4G), thus ruling out mitochondrial dysfunction as a cause of autophagy in BBS. Together, our findings suggest that aberrant IGF-1 signalling may lead to dysregulation of various cellular pathways that are known to control dendritic spine morphology and plasticity.

Synaptic localisation of BBS proteins

The role of Bbs proteins in the regulation of primary cilia has been recently broadened by studies showing that Bbs proteins are involved in microtubular stabilisation, actin remodelling, transcriptional regulation, and endosomal trafficking [16,17,32]. Taking into account this broad spectrum of Bbs functions as well as our current results elaborating the role of Bbs, such as reduction in dendritic spine density along with aberrant synaptic IGF receptor signalling and altered neurotransmitter receptor levels (NMDA and AMPA), we hypothesised that Bbs proteins may play a vital role in neuronal synapses. Re-evaluation of our earlier mass spectrometric analyses of synaptosomal [33,34] and crude synaptosomal fractions [35] of the rat cortex, dorsal striatum, and DG revealed the presence of Bbs1, Bbs2, Bbs4, Bbs5, Bbs7, and Bbs10 proteins (S1 Table).

To elaborate on synaptic localisation of BBS proteins biochemically, we enriched the cytosolic, detergent-soluble synaptosomal (DSS pre-synapse enriched), and PSD fractions of synaptosomal preparations from adult rat hippocampi using a previously described method (S6 Fig) [36]. Label-free MS1 intensity-based LC-MS quantitation revealed a high abundance of Bbs1, Bbs2, Bbs5, and Bbs9 proteins in the PSD fraction, whereas Bbs7 was present mostly in the cytosolic fraction (Fig 5A and 5B). A low level of Bbs4 protein was also unambiguously identified in the PSD fractions (Fig 5A and 5B). Immunofluorescence analysis of Bbs4 and Bbs5 localisation confirmed the presence of Bbs punctae throughout the entire dendritic tree of mouse dissociated hippocampal neurons (Fig 5C and S7A–S7C Fig). Collectively, these data clearly indicate the presence of Bbs proteins in neuronal processes and PSDs.

(A) Proteomic profile of the BBS proteins in biochemical fractions using nano-LC-MS/MS analysis. Protein levels of Bbs proteins and synaptic markers were estimated by label-free LC-MS analyses from following biochemical fractions of the rat hippocampi: cytosolic, detergent-soluble synaptosomal preparation (DSS, pre-synapse enriched), and postsynaptic density preparation (PSD). Protein levels of the presynaptic (VGLU1, SYP) and postsynaptic (NMDAR1, PSD95) protein markers are enriched in DSS and PSD preparations, respectively. (B) The protein abundances illustrated in the heat map are obtained from the total MS1 peptide intensities scaled to the mean of all the samples. Protein levels of the presynaptic (VGLU1, SYP) and postsynaptic (NMDAR1, PSD95) protein markers are enriched in DSS and PSD preparations, respectively. (C) Representative image of immunolabelling of Bbs proteins. Cultured mouse hippocampal neurons at low density were immunolabelled with Bbs4 and Bbs5 antibodies (red), phalloidin (green), and beta-III tubulin (green) after 6 days in vitro (DIV6). Scale bar, 20 μm (top panel) and 5 μm (bottom panels). Underlying data are available in S4 Data. BBS, Bardet-Biedl syndrome DIV6, six days in vitro hippocampal culture DSS, detergent-soluble synaptosomal LC-MS/MS, liquid chromatography- tandem mass spectrometry MS, mass spectrometry NMDAR1, N-methyl-D-aspartate receptor 1 PSD, postsynaptic density SYP, synaptophysin TUBB3, β-tubulin III encoded by TIBB3 gene VGLU1, vesicular glutamate transporter 1.


Process Formation Induced by tau Protein in Sf9 cells Is Enhanced or Suppressed by Different Phosphorylation Sites

Our aim in this study was to use the baculovirus-transfected Sf9 cell system to probe the role of tau phosphorylation in establishing cell polarity. Tau is a neuronal MAP that is involved in supporting the outgrowth of axons and in stabilizing them (Drubin and Kirschner, 1986Lee et al., 1988 Barlow et al., 1994Esmaeli-Azad et al., 1994). Cell processes are also induced when tau is transfected into nonneuronal cells, e.g., COS cells (Kanaiet al., 1989) or Sf9 insect cells (Baas et al., 1991, 1994 Knops et al., 1991). The efficiency of the baculovirus transfection system makes it an attractive model for studying cellular reactions. The use of this system for studying MAPs and their variants is well established (for review, see Kosik and McConlogue, 1994). However, to extend these studies into the area of phosphorylation, we had to ascertain first that Sf9 cells contain endogenous kinases capable of phosphorylating tau after transfection.

We chose several constructs of tau as probes that had been characterized extensively in vitro, using several criteria such as affinity for microtubules and capacity to nucleate or stabilize microtubules, to alter their dynamic instability, or to induce microtubule bundling (Butner and Kirschner, 1991 Gustke et al., 1994 Panda et al., 1995 Trinczek et al., 1995 Goode et al., 1997). Tau has many phosphorylation sites and can be phosphorylated by various kinases, but there are two classes of phosphorylation sites that are particularly interesting. One class comprises the SP and TP motifs, which are the targets of several proline-directed kinases. This type of phosphorylation is developmentally regulated i.e., it is enhanced in fetal tissue (Bramblett et al., 1993), and it is prominent in the pathological conditions of Alzheimer’s disease (for review, seeJohnson and Jenkins, 1996 Mandelkow and Mandelkow, 1998). We therefore made constructs in which some or all SP or TP sites were mutated into AP and thus were no longer phosphorylatable. The smallest (fetal) human tau isoform contains 14 such sites, all of which were turned into AP in construct tau23/AP (Figure 1A). Some of these SP and TP motifs can be monitored conveniently, because there are a number of mAbs that recognize them in a phosphorylation-dependent manner (several of these antibodies were originally generated against Alzheimer tau, e.g., AT-8 [Mercken et al., 1992] and PHF-1 [Greenberg et al., 1992 for review, see Friedhoff and Mandelkow, 1999]). Another class of phosphorylation sites comprises the KXGS motifs in the repeat domain (X = I or C). The phosphorylation of the first of these (at Ser262) has a pronounced effect on the binding of tau to microtubules (Biernat et al., 1993) and is elevated in Alzheimer tau (Morishima-Kawashimaet al., 1995 Seubert et al., 1995). The KXGS motifs are targets of the microtubule affinity–regulating kinase MARK as well as PKA (Drewes et al., 1995, 1997Zheng-Fischhöfer et al., 1998). We therefore made constructs in which the KXGS motif in repeat 1 of tau23 was turned into KXGA (construct KXGA/R1), in repeats 1 and 4 (KXGA/R1/4), or in all three repeats, R1, R3, and R4, of tau23 (KXGA/R1/3/4 Figure 1A note that the nomenclature of repeats and the sequence numbering is derived from the longest isoform, tau40).

Fig. 1. Diagrams of tau isoforms or constructs and antibody epitopes. (A) Constructs: 1) htau40, the largest isoform of tau in the human CNS, containing four 31-residue repeats in the C-terminal half (numbered boxes) and two inserts near the N-terminus (411 residues). The regions flanking the repeats are labeled P1-P2 and R′ (hatched). 2) htau23, the smallest of the six isoforms generated by alternative splicing (352 residues). It lacks the N-terminal inserts and the second repeat. 3) Construct KXGA/R1, in which Ser262 in the KXGS motif of the first repeat is replaced by Ala. 4) Construct KXGA/R1/4, in which the serines in the KXGS motifs of repeats 1 and 4 are replaced by Ala (S262A and S356A). 5) Construct KXGA/R1/3/4, in which the serines in the KXGS motifs of repeats 1, 3, and 4 are replaced by Ala (S262A, S324A, and S356A). 6) Construct tau23/AP, in which all SP and TP motifs are replaced by AP. Construct tau23/AP/R1/4 is similar, but in addition Ser262 and Ser356 are changed into Ala. 7) Construct K19 represents the repeats only. (B) Antibody epitopes. Most phosphorylation-dependent antibodies react with SP and TP motifs in the flanking domains before or after the repeats.

Cells were infected with tau-expressing baculovirus and observed in a monolayer under the microscope. The cell bodies have a round shape with a diameter of ∼16–22 μm (determined in a hemocytometer corresponding volume, ∼2–4 pl), which remains roughly constant throughout their life time. Processes begin to appear after an incubation time of >30 h. Typically there is a single process per cell, of uniform diameter (1–2 μm) and up to 100 μm long (Figure2). The frequency of processes increases linearly with time (Figure 3A), parallel to the increase in tau protein concentration. However, the efficiency of process induction varies considerably. The case of tau23 serves as a standard process formation starts at ∼34 h and increases at a pace of 1.4%/h, reaching a level of ∼60% after 75 h. With construct tau23/AP, in which the SP and TP motifs cannot be phosphorylated, the efficiency is much higher (t0 = 32 h slope, 1.9%/h final level, ∼80%). Conversely, when two or three KXGS motifs in the repeats of tau23 are nonphosphorylatable, as in KXGA/R1/4 or KXGA/R1/3/4, the efficiency drops steeply (late onset, t0 = 38 h and fivefold slower rise, 0.3%/h). These different behaviors are not related to protein expression, which reaches similar levels in all cases (∼45 μg/10 6 cells Table1). Thus the differences are due to the nature of the transfected proteins.

Fig. 2. Immunofluorescence of Sf9 cells transfected with different tau constructs. Cells were transfected with htau23 (A), KXGA/R1 (B), or KXGA/R1/3/4 (C) and immunostained 60 h after infection with antibodies DM1A (for tubulin, left) and K9JA (for tau, right). Transfection with htau23 (A) leads to many processes when Ser262 is mutated to Ala (B), one still finds processes similar to tau23, but when all three KXGS motifs are changed into KXGA (C), the formation of processes is almost completely inhibited. Note that in the case of tau staining the cell bodies appear overexposed to image the more weakly stained processes. Bar, 50 μm.

Fig. 3. Time course of process formation. (A) Quantitation from monolayer culture. Processes begin to appear at ∼30–40 h after infection their frequency rises linearly thereafter. For tau23 (filled circles), the extrapolated onset is at t0 = 34 h the increase of process-bearing cells is 1.4% cells/h. Construct tau23/AP (open squares) is more efficient processes appear earlier and increase faster (t0 = 32 h slope, 1.9%/h), showing that when the SP and TP sites remain unphosphorylated, the formation of processes is enhanced. Construct KXGA/R1/3/4 (filled triangles) is much less efficient (t0 = 38 h slope, 0.3%/h), showing that the phosphorylation of KXGS motifs enhances process formation. Construct KXGA/R1 behaves similar to tau23, whereas KXGA/R1/4 (open circles) behaves similar to KXGA/R1/3/4, indicating that two or more KXGS motifs must cooperate to change the response of the cells. Similarly, process formation is also inhibited with construct tau23/AP/R1/4 (diamonds), showing that the dominant effect of phosphorylation is in the repeats, not in the flanking regions. (B) Quantitation of process formation from suspension culture. This overemphasizes cells with stable processes, because labile ones are lost more easily by shear forces. Thus processes are less numerous, but qualitatively the trends are the same as in A i.e., construct tau23/AP is most efficient, whereas KXGA/R1/3/4 induces almost no processes. Note the fourfold difference between tau23 and tau23/AP, indicating the greater stability of the processes, because the SP and TP motifs cannot be phosphorylated. Bars, standard deviations.

Table 1. Tau protein concentrations in Sf9 cells as a function of time after infection

Note that the concentrations are comparable for all constructs so that the differences in cell extensions are accounted for by mutations rather than the concentration.

The strong suppression of processes described above was initially observed when the three KXGS motifs were mutated to KXGA simultaneously. Because a single site (Ser262 in the first repeat) had a predominant effect on tau’s affinity for microtubules (Biernat et al., 1993), we asked whether this is also the case in the assays used here. We find, however, that the single site mutation to Ala262 (construct KXGA/R1) has no effect, whereas two such mutations in repeats 1 and 4 (Ala262 and Ala356, construct KXGA/R1/4) have the same inhibitory effect as mutations in all three motifs (Ala262, Ala324, and Ala356, construct KXGA/R1/3/4 Figure 3A). We conclude that the KXGA mutations in repeats 1 and 4 are necessary and sufficient for the full inhibitory effect on process formation. These sites coincide with the strongest phosphorylation sites for the kinase MARK (Ser262 and Ser356 Drewes et al., 1995) they can be phosphorylated by PKA as well and occur in Sf9 cells (see Figure 6, A and D).

Because the mutations at SP or TP sites have the opposite effect to the KXGA mutants, we also asked which of these mutations dominate when they are mixed. In one mutant, all SP or TP sites were mutated into AP, plus the KXGA sites in repeats 1 and 4 (construct tau23/AP/R1/4). Process formation was as strongly suppressed as in construct KXGA/R1/4 (Figure3A). This shows that the KXGA mutations in the repeats dominate over the AP mutations in the flanking regions.

When the cell processes are determined from suspensions, one obtains a qualitatively similar picture, except that the differences between the tau constructs become much more pronounced (Figure 3B). For example, at 70 h after infection, ∼3% of the cells transfected with tau23 develop processes. For cells with the mutant tau23/AP the frequency is fivefold higher (∼15%), whereas cells with the mutant KXGA/R1/3/4 have almost none. Because the processes in suspension are subject to shear forces, which tend to break them, these data argue that constructs that generate processes more efficiently (in terms of early onset and rapid increase) also make them mechanically more stable.

Phosphorylation of tau in Sf9 Cells

The interpretation that process formation is influenced by tau phosphorylation depends on which sites are actually targeted by the endogenous kinases of Sf9 cells. This issue was determined by several methods: gel shift, antibody reactions, and phosphopeptide analysis (Figures 4-6). An approximate survey can be obtained from the upward shift of tau in the SDS gel, which can reach an apparent increase of ≥5 kDa, depending on the phosphorylation site (Biernat et al., 1993). This shift is also characteristic of Alzheimer tau (A68 protein, Lee et al., 1991). Figure 4 shows that tau from transfected Sf9 cells also displays a pronounced shift (distributed over several bands), indicating that tau is phosphorylated by endogenous kinases in Sf9 cells. The shift is visible with wild-type tau23, with the “repeat” mutants KXGA/R1 and KXGA/R1/3/4 but not with the “flank” mutant tau23/AP (which runs as a homogeneous band, essentially as unphosphorylated, bacterially expressed tau). This confirms our previous observations that SP and TP sites are mainly involved in the shift and shows that endogenous, proline-directed kinases are active in Sf9 cells.

Fig. 4. Phosphorylation of tau and tau constructs transfected in Sf9 cells. (A) 10% SDS-PAGE. Tau protein or mutants (tau23, KXGA/R1, KXGA/R1/3/4, and tau23/AP) were isolated from equal quantities of Sf9 cells transfected with the appropriate tau constructs and prepared in equal volumes of sample buffer. Equal volumes of protein solution representing the different harvest time points were loaded onto the SDS gel (10%). The amounts of loaded proteins correspond approximately to 0.4 μg (48 h), 2.5 μg (66 h), and 3 μg (73.5 h). As seen from the multiple and strongly shifted bands, tau23 is highly phosphorylated in a heterogeneous manner. The same applies to contructs KXGA/R1 and KXGA/R1/3/4 (which lack only one or three phosphorylation sites at KXGS motifs but retain all SP or TP sites). By contrast, construct tau23/AP shows very little phosphorylation and no shift, because it lacks most phosphorylation sites (note that the KXGS sites do not induce a shift Drewes et al., 1995). By implication, the strong shift sites S409 and S416 (targets of PKA or CaMKII) are also not phosphorylated in Sf9 cells. (B) The same samples were immunoblotted with antibody 5E2, which reacts independently of phosphorylation and reflects the total amount of tau protein. Lane M, marker proteins.

A number of phosphorylation-sensitive antibodies against tau are available, which can be used as diagnostic tools (Figure 1B). As a reference, the cell extracts of transfected Sf9 cells were immunoblotted with the phosphorylation-independent antibody 5E2 (Kosik et al., 1988), which indicates the total amount of tau. As seen in the Western blots (Figure5), Tau-1 recognizes weakly all tau23 derivatives expressed in Sf9 cells, indicating that a small fraction is not phosphorylated around residue 200. The complementary antibody AT-8 reveals a strong signal by htau23, KXGA/R1, and KXGA/R1/3/4. These repeat mutants are also clearly recognized by other antibodies sensitive to proline-directed phosphorylation (AT-270, AT-180, SMI-34, and PHF-1), indicating phosphorylation at Thr181, Thr231, Ser235, Ser396, and Ser404 (Figure 1B). A particularly interesting example is that of the antibody AT-100, which is uniquely specific for Alzheimer tau (Matsuo et al., 1994). The epitope is formed by sequential phosphorylation first of Thr212 by GSK-3 and then of Ser214 by PKA and requires a PHF-like conformation induced by polyanions (Zheng-Fischhöfer et al., 1998). This epitope is present on tau in the transfected Sf9 cells (Figure 5). Similarly, Ser262/Ser356 is clearly phosphorylated, as seen from the reaction with antibody 12E8. The phosphorylation at the epitopes of AT-100 and 12E8 is particularly sensitive to phosphatases because they disappear rapidly during the initial steps of preparation.

Fig. 5. Phosphorylation sites of baculovirus-expressed tau and tau constructs. Sf9 cell lysates (72 h after infection) were blotted and incubated with different phosphorylation-dependent and -independent mAbs. The tau constructs are, from left to right: 1) tau23/AP, 2) KXGA/R1, 3) htau23, 4) KXGA/R1/3/4, 5) untransfected Sf9 cell extract (control without tau), 6) Sf9 cells transfected with wild-type baculovirus (control without tau), 7) htau23 expressed inE. coli (unphosphorylated control), and 8) htau23 expressed in E. coli and phosphorylated with brain extract kinase activity (affecting largely the SP and TP sites and inducing a strong Mr shift see Biernatet al., 1993). Antibodies, from top to bottom: 5E2 (a pan-tau antibody) recognizes all preparations that contain tau. Tau-1 shows only a moderate reaction with tau constructs expressed in Sf9 cells, because a single P site in the vicinity of residue 200 suffices to reduce the binding, but there is a strong reaction with unphosphorylated tau expressed in E. colior tau23/AP. The antibodies AT-8 to SMI-34 are specific for different phosphorylated SP and TP motifs and therefore show no reaction with the tau23/AP mutant or with E. coli–expressed htau23. Antibody 12E8 recognizes only those constructs that contain Ser262 and/or Ser356 in the repeats. AT-100 recognizes tau phosphorylated at Thr212 and Ser214 this reaction is highly specific for Alzheimer tau but occurs in Sf9 cells as well, provided both sites are phosphorylatable (e.g., not in the tau23/AP mutant).

Metabolic Labeling of tau and Analysis of Phosphopeptides

The detection of phosphorylation sites by antibodies suffers from two drawbacks: 1) there may be sites for which there are no antibodies, and 2) the antibody staining is not a reliable indicator of the extent of the phosphorylation (because the antibody affinities are variable and often unknown). For further characterization of phosphorylation sites of htau23 and its derivatives, we performed metabolic labeling of Sf9 cells using [ 32 P]orthophosphoric acid. The labeled tau protein was isolated, digested with trypsin, and then processed for 2D peptide analysis (Boyle et al., 1991). To identify the peptides, tau expressed in Escherichia coli was phosphorylated radioactively using different kinases in vitro and digested with trypsin, and the peptides were purified by HPLC and identified by matrix-assisted laser desorption and ionization, phosphopeptide sequencing, and phosphopeptide mapping (for details, seeDrewes et al., 1995 Illenberger et al., 1998Zheng-Fischhöfer et al., 1998). Figure6A shows the phosphopeptide map found with tau23 phosphorylated in Sf9 cells, in which the main spots are identified by their phosphorylation site (for details on the identification, see Illenberger et al., 1998). The experiments with the mutants enabled us to define the phosphorylation sites by exclusion of the corresponding spots from the 2D map (Figure6, B and C). The majority of spots represent SP or TP sites, containing ∼80% of the total radioactivity. These spots disappear in the case of the tau23/AP mutant, in which only the non-SP or TP sites remain, among them S214, S262, S320, S356, and two unidentified spots (Figure 6B). It is remarkable that the distribution and intensity of phosphorylation sites of tau23 is quite similar to that of other cultured cells during interphase, including neuronal ones (Illenbergeret al., 1998). This shows that the balance of kinases and phosphatases is similar in these cells, and it provides an additional rationale for using baculovirus-transfected Sf9 cells as a model system.

Fig. 6. Two-dimensional phosphopeptide maps of tau constructs expressed in Sf9 cells and phosphorylated by endogenous kinases. (A) htau23 (higher magnification) (B) htau23/AP (C) KXGA/R1/3/4 (D) construct K19 phosphorylated with PKA (E) mixture of tau23 and K19 phosphorylated with PKA. In A the majority of spots are generated by phosphopeptides containing one or more of the 14 SP and TP motifs. The four phosphorylation sites in the repeats are highlighted and underlined (three KXGS motifs with S262, S324, and S356, plus S320). In B all SP or TP motifs are changed into AP so that the remaining major phosphorylation sites are Ser214 and those in the repeats, plus some unknown spots. In C the KXGS motifs in the repeats are changed into KXGA and are therefore absent from the map besides the phosphorylated SP and SP motifs one observes S214 and S320. In D the repeat construct K19 was phosphorylated with PKA, showing only the sites S262, S324, S356, and S320. This sample was run together with tau23 in E to identify the sites. For details on analysis of phosphopeptides, see Illenberger et al. (1998).

Figure 6C shows the phosphopeptide map for the mutant KXGA/R/1/3/4, where the three sites S262, S324, and S356 had been replaced by Ala and therefore no longer appear on the map (these spots are normally weak compared with the SP and TP sites and visible only at longer exposures cf. Figure 6A). We confirmed the phosphorylation at the sites in the repeats by mixing the phosphopeptides from metabolically labeled tau23 in Sf9 cells with phosphopeptides from construct K19 (three repeats only) phosphorylated in vitro with MARK or PKA, which resulted in the overlapping of spots representing the repeat phosphorylation sites (S262, S324, and S356 plus S320 Figure 6, D and E).

Tau Phosphorylation in Alzheimer's Disease and Other Tauopathies

The 𠇊myloid cascade hypothesis” was formulated after an amyloid precursor protein (APP) mutation was reported in a family with AD-typical histology and proposes that accumulation of an APP cleavage product, beta amyloid (Aβ), induces the biochemical, histologic, and clinical changes AD patients manifest (Hardy and Higgins, 1992). Later, Aβ oligomers were suggested to trigger neurotoxicity in AD probably via tau phosphorylation. Glycogen synthase kinase-3β (GSK-3β) activation was proposed as mediator of A𻉂 oligomer-induced effects on tau phosphorylation in P301L mice (Selenica et al., 2013).

The Role of Phosphorylation on Tau Cytotoxicity and Aggregation

Tau, in its longest isoform, contains 35 threonine, 45 serine, and 5 tyrosine residues meaning that nearly 20% of the tau protein has the potential to be phosphorylated. Early studies revealed that tau is more efficient at promoting microtubules (MT) assembly in a more unphosphorylated state (Lindwall and Cole, 1984). A few years later, tau was demonstrated to make up the paired-helical filaments (PHFs) which form the neurofibrillary tangles (NFTs) found in AD brain and to be abnormally phosphorylated in these structures (Grundke-Iqbal et al., 1986 Goedert et al., 1988 Kosik et al., 1988 Wischik et al., 1988). Further analyses revealed that PHF-tau is phosphorylated at “pathological” sites, which was assumed to contribute to pathological processes in AD. Enhanced immunoreactivity in human AD tissue was observed with the phosphorylation-dependent antibodies AT8 (epitope pS199/pS202/pT205), PHF-1 (epitope pS396/pS404), and pS262 (Gu et al., 2013a Mondragon-Rodriguez et al., 2014). Hyperphosphorylation of tau was shown to be involved in tau aggregation and cytotoxicity (Table 2) (Kosik and Shimura, 2005 Noble et al., 2013).

Table 2. Tau phosphorylation sites and effects.

Abnormal high levels of intracellular tau are frequently observed in AD patients and may be directly implicated in tau aggregation, PHF formation, and neuron loss (Gomez-Isla et al., 1997). It was speculated that the hyperphosphorylation of tau precedes NFT pathology and, more important, is a key event for the integration of tau into fibrils (Bancher et al., 1991). The staging of AD-related neurofibrillary pathology using a silver stain technique was revised using immunostaining for hyperphosphorylated tau at the AT8 epitope (Braak et al., 2006). Several studies addressed the question whether the pattern of tau hyperphosphorylation correlates with the progression of neuronal cytopathology and the formation of higher order tau species in AD. Brain tissue was classified into pre-NFTs, intra-neuronal NFTs and extra-neuronal NFTs, and was examined regarding the most prominent staining of phosphorylation-dependent tau antibodies. Epitopes that were associated with pretangle, non-fibrillar tau include pS199, pS202, pT231, pS262, pT153, and S409. Intraneuronal fibrillar structures were stained with antibodies recognizing pS46, pT175/pT181, pT231, pS262/pS356 (12E8 epitope), pS396, pS422, and pS214. Epitopes associated with extracellular filamentous tau include AT8, AT100 (pT212/pS214), and PHF-1 (Morishima-Kawashima et al., 1995b Kimura et al., 1996 Augustinack et al., 2002). Notably, with progression of the disease, tau is phosphorylated at pathological multiple-site epitopes (AT8, AT100, AT180, PHF-1, 12E8). Tau inclusions were observed in other neurodegenerative disorders such as MSA (Giasson et al., 2003b), familial and sporadic PD (Ishizawa et al., 2003 Rajput et al., 2006), and in Down syndrome (Flament et al., 1990 Mondragon-Rodriguez et al., 2014). Elevated levels of AT180 (pT231/pS235)-phosphorylated tau were detected in the cerebrospinal fluid (CSF) of patients with mild cognitive impairment who later went on to develop AD (Arai et al., 2000a).

Several animal models were generated to recapitulate hyperphosphorylation of tau and the formation of NFTs as key aspects of tauopathies (Ribeiro et al., 2013). Some studies showed that the overexpression of human mutant tau in transgenic mice led to increased phosphorylation of tau and the formation of tau inclusions, aggregates, and fibrils. Phosphorylation of tau was detected at the well-known disease-related epitopes S202, T205, S212, S216, T231, S262, S356, S422, AT100 (Kohler et al., 2013 Nilsen et al., 2013 Sahara et al., 2013). Likewise, overexpression of LRRK2 or p25/Cyclin-dependent kinase-5 (Cdk5) in mice resulted in hyperphosphorylation of tau, tau aggregation into NFT-like structures, and neuronal death (Cruz et al., 2003 Noble et al., 2003 Bailey et al., 2013). Other models took advantage of the co-expression of other disease-associated proteins such as APP and presenilin 1 (Oddo et al., 2003 Grueninger et al., 2010), or made use of the injection of Aβ fibrils (Gotz et al., 2001).

Almost all currently available animal models in AD are based on the over-expression of pathogenic mutant tau forms. Therefore, it debatable how well these models recapitulate AD cases where there are no mutations in either tau or APP. However, the first models of tauopathy, based on the overexpression of either 3-repeat or 4-repeat human WT tau, presented tau hyperphosphorylation but no NFT formation. Expression of tau-P301L, often in conjunction with other disease-associated proteins, is the most widely used and most successful approach to recapitulate key aspects of AD such as tau hyperphosphorylation, aggregation, and filament formation as well as neuron death. In these models, it is often not clear what drives tau hyperphosphorylation. In vitro studies may help to decipher the impact of specific pathogenic mutations on tau phosphorylation but existing data are not consistent. The well-known FTDP-17-associated missense tau mutations R406W, V337M, G272V, and P301L were shown to make tau a more favorable substrate for phosphorylation by rat brain kinases, in comparison to WT tau protein (Alonso Adel et al., 2004). In another study, the same mutations were shown to promote or inhibit phosphorylation at specific sites (Han et al., 2009). In vitro phosphorylation by recombinant GSK-3b exerted reduced phosphorylation of the R406W mutation, probably through long-range conformational changes. Conversely, P301L and V337M mutations had no effect (Connell et al., 2001). Similar results were obtained in cell culture (Dayanandan et al., 1999). In contrast, several other studies using cell culture models and human brain tissue indicate that the R406W mutation reduces tau phosphorylation, not only at the neighboring PHF1 epitope but at several positions (Miyasaka et al., 2001 Deture et al., 2002 Tackenberg and Brandt, 2009 Gauthier-Kemper et al., 2011). However, depending on the cellular context, R406W was also shown to increase phosphorylation, and other mutations, such as V337M, reduced phosphorylation of tau at specific sites (Deture et al., 2002 Krishnamurthy and Johnson, 2004). Alterations in the phosphorylation state can have tremendous effects on the structural properties, function, and pathology of tau as discussed below.

In vitro data imply that phosphorylation of tau at certain epitopes directly impacts on local structural properties or the global conformation of tau which in turn may affect its assembly into PHFs. Different sites were suggested to be important for the aggregation propensity and filament formation of tau including AT8, AT100, AT180, PHF-1, and S305 upstream of the PHF6-hexapeptide motif which is known to be important for tau fibrillization (Sun and Gamblin, 2009 Bibow et al., 2011 Inoue et al., 2012). Some studies suggested that the compaction of the paperclip conformation of tau becomes tighter or looser depending on phosphorylation at the AT8, PHF-1, and AT100 epitopes (Jeganathan et al., 2008 Bibow et al., 2011). Likewise, phosphorylation within the repeat region, particularly at KXGS motifs, induced specific conformational changes that altered the MT binding properties of tau (Fischer et al., 2009). In other cases, structural changes were localized in the proximity of the phosphorylation sites without affecting the global conformation (Schwalbe et al., 2013).

Despite intensive research in the field, the contribution of phosphorylation to the formation of tau aggregates is still controversial (Table 2). Recent results from in vitro experiments, showing that recombinant unphosphorylated tau induced fibril formation similar to AD-derived PHFs, questioned the necessity of tau phosphorylation for the fibrillization process (Morozova et al., 2013). Furthermore, altered spine morphology and spine loss in tissue of AD cases were attributed to the content of tangles rather than to the amount of phosphorylated tau (Merino-Serrais et al., 2013). In a study using PS19 mice (tauP301S mutation), synthetic tau fibrils induced NFT pathology in the absence of tau hyperphosphorylation (Iba et al., 2013). The introduction of “pro-” and 𠇊nti-” aggregation mutations revealed that hexapeptide motifs of tau may function as a core to form local β-sheet structure and, subsequently, to induce PHF formation (Von Bergen et al., 2000 Eckermann et al., 2007). Enhanced tau levels, via stabilization of tau mRNA, may contribute to tau pathology independent of tau phosphorylation (Qian et al., 2013).

Other PTMs of tau might interfere with its phosphorylation, thereby influencing the structure, function and regulation of the protein, but the data are not consistent. KXGS motifs were found to be hypoacetylated and hyperphosphorylated in patients with AD, consistent with in vitro data showing that the acetylation of tau prevents its phosphorylation and inhibits tau aggregation (Irwin et al., 2013 Cook et al., 2014). In contrast, acetylation of tau at K280 was associated with phosphorylation at the AT8 epitope in tau aggregates of tau transgenic mice, and detected in post-mortem tissue of cases with AD or other tauopathies (Min et al., 2010 Cohen et al., 2011 Irwin et al., 2012). Recent evidence was provided that tau itself possesses acetyltransferase activity, and is capable of catalyzing self-acetylation (Cohen et al., 2013). In vitro, O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) at S400 was inversely correlated with tau phosphorylation at S396 (Smet-Nocca et al., 2011). However, treatment of tau transgenic mice with an O-GlcNAcase inhibitor increased tau O-GlcNAcylation, hindered the formation of tau aggregates, and slowed neurodegeneration without affecting the phosphorylation of tau (Yuzwa et al., 2012 Graham et al., 2013).

Phosphorylation of tau at several residues mediates cellular toxicity (Table 2). Many data implicate that phosphorylation of tau needs to be well balanced. It was hypothesized that the detachment of tau from MTs results in impaired MT stability and excess amount of unbound hyperphosphorylated tau in the cytosol, thereby contributing to toxic insult. In vitro experiments provided evidence that phosphorylation of tau at S262, T231, and S214 is necessary for the full detachment of tau from MTs (Illenberger et al., 1998 Sengupta et al., 1998). Consistently, enhanced phosphorylation at S262 and T231 resulted in MT instability and cytotoxicity in cell and animal models (Steinhilb et al., 2007 Qureshi et al., 2013). Moreover, pathological processes were rescued by overexpression and activation of microtubule-affinity-regulating kinases (MARKs) that phosphorylate tau at KXGS motifs of the repeat domains (Mandelkow et al., 2004 Thies and Mandelkow, 2007). Aberrant phosphorylation of tau at pathological sites may result in altered tau-MT binding, thereby affecting the organization and dynamics of MT networks. This in turn may compromise axoplasmic flow and proper neuronal function, and ultimately cause cell death. The phosphorylation within KXGS motifs, especially at S262, and GSK-3β seem to take key roles among the phosphorylation sites and tau kinases, respectively.

Mitochondrial dysfunction and oxidative stress are both intimately associated with cell death in neurodegeneration. Mitochondrial oxidative stress in superoxide dismutase 2-deficient and APP expressing mice exacerbated amyloid burden and the hyperphosphorylation of tau at S396. Treatment with high doses of antioxidants prevented from tau hyperphosphorylation and neuropathology (Melov et al., 2007). Triple AD mice expressing mutant tau, APP and presenilin 1 developed tangles and Aβ plaques, and displayed deregulation of several mitochondrial proteins suggesting synergistic effects of Aβ and tau in perishing mitochondria (Rhein et al., 2009).

Undoubtedly, tau hyperphosphorylation is an important phenomenon in AD and other tauopathies and parallels the appearance of tau aggregates and NFTs, but despite great efforts, the underlying mechanisms that ultimately lead to toxicity and neurodegeneration remain elusive (Papanikolopoulou et al., 2010 Ambegaokar and Jackson, 2011). In recent years, it was hypothesized that the segregation of tau in intracellular aggregates is an escape route for the cell from excess amount of protein. Instead, tau oligomers were considered as toxic species that harm the cell and, ultimately, lead to cell death (Sahara and Avila, 2014).

Accumulation of phosphorylated (AT8, PHF-1, S422) tau oligomers was detected at human AD synapses concomitant with dysfunction of the UPS (Henkins et al., 2012 Tai et al., 2012). Use of a tau oligomer-specific antibody in human AD brain samples revealed that tau oligomers appear at early stages in AD, either before or after the manifestation of tau phosphorylation at specific epitopes (Lasagna-Reeves et al., 2012). Thus, aggregation of the hyperphosphorylated forms of tau into PHF structures could be neurotoxic by sequestering important cellular proteins, but it could also be neuroprotective by avoiding accumulation of toxic oligomeric tau.

Physiological and Pathological Implications of Tau Phosphorylation

At normal levels of phosphorylation, tau contains 2𠄳 moles phosphate/mole of protein and is a soluble cytosolic protein (Khatoon et al., 1992). From the overall 85 phosphorylatable residues, approximately 30 residues are phosphorylated in normal tau proteins (Morishima-Kawashima et al., 1995a Hanger et al., 2009a). Most of the tau phosphorylation sites are clustered in the proline-rich region, the microtubule binding repeats (MTBR) or MTBR-flanking domains.

Tau expression and phosphorylation are developmentally regulated. A single tau isoform is expressed in fetal human brain whereas six isoforms are expressed in adult human brain, with fetal tau corresponding to the shortest adult tau isoform. The degree of tau phosphorylation decreases during embryogenesis (Mawal-Dewan et al., 1994), which might be related to increasing neuronal plasticity in the early developmental process (Brion et al., 1993 Hanger et al., 2009a). PHF-tau contains 3𠄴 fold phosphates over the normal adult tau (Khatoon et al., 1992 Iqbal et al., 2013). In immature brain, as in PHFs, tau is phosphorylated at a large number of sites (Kenessey and Yen, 1993 Morishima-Kawashima et al., 1995b). However, as in adult brain, the phosphorylation in fetal tau is only partial. Phosphorylation of tau in PHFs is denominated as “hyperphosphorylation” which takes into account that other sites than the physiological ones are phosphorylated. This state is also referred to as �normal” or “pathological” phosphorylation.

MT dynamics are dependent on a balanced ratio between tau molecules and MT tracks. Either excess or poor binding of tau molecules, e.g., through dysregulation of the tau phosphorylation state, results in destabilization and breakdown of MT networks. This has a direct impact on MT function in the formation of the cytoskeletal architecture and as track for axonal and organelle transport, and is resumed in the “Tau-microtubule hypothesis” (Alonso et al., 1994).

Early studies clearly demonstrated that tau plays an important role in the establishment of neuronal polarity and axonal outgrowth (Caceres and Kosik, 1990). Neurite extension and retraction may be regulated by MARK and GSK-3β-mediated tau phosphorylation (Biernat et al., 2002 Sayas et al., 2002). It was speculated that phosphorylation of tau within the MTBR is necessary for appropriate neurite outgrowth whereas phosphorylation at SP and TP motifs within flanking domains retards neuronal differentiation (Biernat and Mandelkow, 1999). Tau, a cargo of kinesin, may displace other kinesin-based cargo indicating that the development and stabilization of axons are dependent on a balance of cytoskeletal elements (Dubey et al., 2008).

Overexpression of tau is known to compromise MT-dependent axonal transport in a phosphorylation-dependent manner (Sato-Harada et al., 1996). Co-expression of constitutively active GSK-3β exacerbated, whereas GSK-3β inhibition rescued vesicle aggregation and locomotor dysfunction in a Drosophila model (Mudher et al., 2004 Cowan et al., 2010b). Phosphorylation of tau at Y18 by the Fyn kinase was suggested to prevent the activation of the GSK-3β signaling cascade, thereby counteracting tau's inhibitory effect on anterograde fast axonal transport (Kanaan et al., 2012). These data suggest that the pathological over-activation of GSK-3β inhibits axonal transport through hyperphosphorylation of tau. In contrast, other studies showed that the inhibition of tau phosphorylation by GSK-3β inhibitors was associated with decreased mitochondrial transport and motility and increased mitochondrial clustering in cells (Tatebayashi et al., 2004 Llorens-Martin et al., 2011). Tau may control intracellular trafficking by affecting the frequencies of attachment and detachment of motors, in particular kinesin, to the MT tracks (Trinczek et al., 1999 Morfini et al., 2007). It was speculated that excess tau acts as transport block for vesicles and organelles which is reversed by removal of tau through MARK-mediated tau phosphorylation and subsequent detachment of tau from MTs (Thies and Mandelkow, 2007). However, detachment of tau from MT may also contribute to axonal transport blockage and neurodegeneration (Iijima-Ando et al., 2012). Dephosphorylation and phosphorylation cycles of tau, through the interplay of tau kinases and phosphatases, may serve as general mechanism to regulate tau's function to maintain a dynamic MT network for neurite outgrowth and axonal transport (Fuster-Matanzo et al., 2012 Mandelkow and Mandelkow, 2012). Interestingly, improper distribution of overexpressed tau in the somatodendritic compartment was shown to result in more numerous and densely packed MTs in axons and dendrites. Phosphomimic mutations of the AT8 epitope caused expansion of the space between MTs and may thereby contribute to axonal transport and mitochondrial movement defects (Thies and Mandelkow, 2007 Shahpasand et al., 2012). Furthermore, phosphorylated tau may sequester normal tau in neurites away from MTs leading to disruption of the microtubular cytoskeleton and demise of axonal transport (Niewiadomska et al., 2005 Cowan et al., 2010a Iqbal et al., 2013).

Extracellular Aβ, shown to exacerbate the hyperphosphorylation of tau and NFT formation, was also suggested to modulate N-methyl-D-aspartate receptor (NMDAR) function and to induce excitotoxicity (Lauren et al., 2009). However, among the plethora of known Aβ-interacting molecules, the specific Aβ target and the intracellular propagation of the signal remain elusive. Prion protein was proposed as binding partner of Aβ but there is still controversy about the significance of this interaction (Balducci et al., 2010 Kessels et al., 2010 Chen et al., 2013a). Aβ induces the activation of Fyn which, in turn, increases the phosphorylation of a subunit of NMDARs dependent on the status of tau phosphorylation and tau localization at the post-synapse. After an initial increase, the number of surface NMDARs declined which resulted in dendritic spine loss and excitotoxicity (Um et al., 2012). The interaction of Fyn and tau, both forming a complex together with NMDAR, seems to modulate synaptic plasticity and to sensitize synapses to glutamate excitoxicity in AD (Ittner et al., 2010).

Phosphorylation of tau was also linked to altered turnover and proteolysis. The detection of ubiquitin immunoreactivity in tau inclusions was interpreted as failure of the ubiquitin proteasome system (UPS) to proteolytically degrade excess tau (Bancher et al., 1989). Proteasomal inhibition resulted in the accumulation of particularly hyperphosphorylated tau species (Shimura et al., 2004) and disruption of neuritic transport (Agholme et al., 2013). Inhibition of autophagy in neurons resulted in 3-fold accumulation of phosphomimic tau over wild type tau indicating that both, autophagic and proteasomal pathways, are responsible for the clearance of phosphorylated tau species (Rodriguez-Martin et al., 2013). Biochemical and morphological analysis of AD cortices revealed that tau becomes hyperphosphorylated and misfolded at presynaptic and postsynaptic terminals, in association with an increase in ubiquitinated substrates and proteasome components (Tai et al., 2012).

Many other mechanisms were suggested for the implication of tau hyperphosphorylation in tauopathies. Cell death was accompanied by expression of cell-cycle regulatory proteins in aged mice expressing human tau isoforms on a knockout background (Andorfer et al., 2003). Inappropriate re-entry to the cell cycle plays a role in AD and might be linked to hyperphosphorylation of tau via activation of cell-cycle relevant kinases (Delobel et al., 2002 Absalon et al., 2013). Abnormal interaction with the mitochondrial fission protein Drp1 might be causative for mitochondrial dysfunction and neuronal damage (Manczak and Reddy, 2012). DNA damage resulted in the activation of the checkpoint kinases Chk1 and 2, subsequent tau phosphorylation at AD-related sites, and enhancement of tau-induced neurodegeneration in human tau expressing Drosophila (Iijima-Ando et al., 2010). Immunohistochemical analysis of AD brains revealed that tau is truncated at D421, and that this cleavage occurs after conformational changes detected by the Alz-50 antibody but precedes cleavage at E391 (Guillozet-Bongaarts et al., 2005). Accumulation of D421 and E391-truncated species occurs early in the disease and correlates with the progression in AD (Basurto-Islas et al., 2008). In transgenic mice, truncation of tau was shown to drive pre-tangle pathology (McMillan et al., 2011). In cells, hyperphosphorylation of tau at several residues and cleavage of tau at D421, the preferential cleavage site of caspase-3, enhanced the secretion of tau. This was suggested as potential mechanisms for the propagation of tau pathology in the brain and tau accumulation in the CSF (Plouffe et al., 2012).

Kinases Involved in Tau Phosphorylation

Similar to the pattern of tau hyperphosphorylation, the idea of a distinct signature-specific pattern of tau kinase activation emerged (Duka et al., 2013). Several attempts were done to identify the responsible kinases and the corresponding phosphorylation sites of tau (Table 2). However, most of the kinases phosphorylate several residues of tau, and most tau phosphorylation sites are targets of more than one kinase (Figure 3). In addition, the existence of priming, meaning that the phosphorylation at one site facilitates phosphorylation at another site, and feedback events to regulate the overall level of tau phosphorylation, hamper the assignment of a specific phosphorylation site to a particular (dys-)function of tau (Bertrand et al., 2010 Kiris et al., 2011).

Figure 3. Schematic representation illustrating the various residues in the longest isoform of tau that can be phosphorylated. SP/TP motifs (represented in blue), KXGS motifs (represented in yellow), and other sites (represented in gray) can be phosphorylated by proline-directed kinases (represented in blue) and non-proline directed Ser/Thr kinases (represented in green). Antibody epitopes AT8, AT100, AT180, and PHF-1 comprise dual and triple serine/threonine residues (indicated by brackets). Some mutations associated with FTDP-17 are shown in red. Alternative splicing of N1, N2, and R2 generates the six different isoforms of tau. N1, N2, N-terminal inserts 1 and 2 R1-R4,MT binding repeats 1𠄴 GSK-3β, Glycogen synthase kinase 3β Cdk5, Cyclin-dependent kinase 5 CK, casein kinase MARK, microtubule affinity-regulating kinase LRRK2, leucine-rich repeat kinase 2 DAPK, Death-associated protein kinase Dyrk1A, dual-specificity protein kinase.

Numerous kinases, including more than 20 serine/threonine kinases, were shown to phosphorylate tau in vitro but their relevance in AD is still under investigation (Hanger et al., 2009b Cavallini et al., 2013).

The proline-directed kinase GSK-3β was particularly associated with the formation of PHFs and NFTs and proposed as key mediator in the pathogenesis of AD (Hooper et al., 2008 Terwel et al., 2008 Ma, 2014 Medina and Avila, 2014). GSK-3β targets tau at SP/TP sites, including the epitopes PHF-1, AT8, AT180, AT100, S404 and S413 (Pei et al., 1999 Medina and Avila, 2014). Alterations in GSK-3β levels were associated with changes in tau phosphorylation in several cell and animal models (Hernandez et al., 2013). Stress stimuli such as mitochondrial toxins or oxidative stress to mimic conditions in neurodegenerative disorders resulted in increased GSK-3β-mediated phosphorylation of tau, reduced cell metabolic activity and MT destabilization (Hongo et al., 2012 Selvatici et al., 2013). Other studies position GSK-3β as prominent player in the pathogenesis of AD beyond its role as tau phosphorylating kinase. Tau-P301Lx GSK-3β mice developed severe forebrain tauopathy with tangles in the majority of neurons but in the absence of tau hyperphosphorylation (Muyllaert et al., 2006). In a Drosophila model, co-expression of a GSK-3β homolog and human tau led to increased toxicity more likely due to the fact that GSK-3β is a pro-apoptotic protein than due to increased tau phosphorylation (Jackson et al., 2002).

The serine/threonine kinase Cdk5 plays important roles in neuronal development and migration, neurite outgrowth, and synaptic transmission, and is implicated in the pathogenesis of AD (Cheung and Ip, 2012 Shukla et al., 2012). Immunoreactivity of Cdk5 in several brain regions in AD was associated with pre-tangle and early NFT stages, and colocalized with AT8-positive tau in a subset of neurons (Pei et al., 1998 Augustinack et al., 2002). Cdk5 activity was found to be higher in AD than control cases probably due to the conversion of the Cdk5 activator p35 into the constitutive active form p25 (Lee et al., 1999 Patrick et al., 1999 Shukla et al., 2012).

Mice overexpressing human p25/Cdk5 displayed enhanced Cdk5 activity, hyperphosphorylation of tau, and cytoskeletal disorganization (Ahlijanian et al., 2000). The activation of Cdk5 along with overexpression of mutant tau was associated with tau hyperphosphorylation and tangle formation (Noble et al., 2003). APPswe mice showed increased Cdk5 activity due to increases in p25 levels, and substantial phosphorylation of tau at AT8 and PHF-1 epitopes linking Aβ pathology to tau hyperphosphorylation via increased Cdk5 activity (Otth et al., 2002). Furthermore, Cdk5 was suggested to be linked to GSK-3β. Mice expressing human p25 showed elevated Aβ levels but decreased phospho-tau levels and reduced GSK-3β activity. Administration of Cdk5 inhibitors reduced Aβ production but did not alter the phosphorylation of tau suggesting that Cdk5 predominantly regulates APP processing, whereas GSK-3β plays a dominant role in tau phosphorylation (Wen et al., 2008 Engmann and Giese, 2009). The crosstalk between Cdk5 and mitogen-activated protein kinase (MAPK) pathways suggests a connection with neuronal apoptosis and survival signaling (Sharma et al., 2002 Zheng et al., 2007). Dysregulation of the MAPK signaling pathways, comprising the three signaling cascades extracellular signal regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK), was suggested to be implicated in AD and other neurodegenerative disorders (Kim and Choi, 2010). In the course of AD, ERK and JNK are activated throughout all stages, and p38 in mild to severe cases (Braak stages III to VI) (Pei et al., 2001 Zhu et al., 2001). p38 and JNK immunoreactivity were associated with neurons containing neuritic plaques, neuropil threads, and NFTs, structures that were also recognized by antibodies raised against phosphorylated PHF-tau (Hensley et al., 1999 Atzori et al., 2001).

The kinases MARK1-4 are non-proline directed kinases that are involved in the establishment of neuronal polarity and the regulation of neurite outgrowth (Biernat et al., 2002 Matenia and Mandelkow, 2009 Reiner and Sapir, 2014). MARKs are named after their ability to regulate the affinity of tau to MTs through phosphorylation (Drewes et al., 1997). Importantly, MARKs phosphorylate tau within the KXGS motifs, particularly at S262, which phosphorylation is detected early in the course of AD. Expression of MARK2 and MARK4, as well as the interactions of these kinases with tau, were significantly enhanced in AD brains, correlated with the Braak stages of the disease, and were associated with NFTs (Chin et al., 2000 Gu et al., 2013a).

In transgenic Drosophila, overexpression of the Drosophila homolog Par-1 was associated with increased phosphorylation and enhanced toxicity of human tau. Loss of Par-1 function and mutation of tau at the Par-1 directed phosphorylation sites (S262, S356) rescued from tau-induced toxicity. Interestingly, Par-1 phosphorylation of tau was a prerequisite for downstream phosphorylation through GSK-3β and Cdk5, and the generation of disease-associated phosphorylation epitopes (Nishimura et al., 2004). Activation of MARK2 rescued from synaptic decay caused by overexpression and improper distribution of tau in the somatodendritic compartment (Mandelkow et al., 2004 Thies and Mandelkow, 2007). However, overexpression of MARK4 resulted in tau hyperphosphorylation and loss of spines, which also manifested after Aβ treatment. Therefore, MARKs may have regulatory functions in spine morphology and synaptic transmission, but may also act as critical mediators in Aβ-induced toxicity on synapses and dendritic spines (Zempel et al., 2010 Hayashi et al., 2011 Yu et al., 2012). Furthermore, the phosphorylation of tau by MARK was suggested to inhibit tau's assembly into PHFs (Schneider et al., 1999), contradictory to the hypothesis that the pool of hyperphosphorylated, MT-unbound tau assembles into PHFs. Phosphorylation of tau at SP/TP sites has low impact on the tau-MT binding and is observed in AD, dissociating the detachment of tau from MTs from the likability to assemble into PHFs. GSK-3β was shown to phosphorylate MARK2 at two different sites, the activatory T208 and the inhibitory S212, thereby modulating the phosphorylation of tau, particularly at S262 (Kosuga et al., 2005 Timm et al., 2008). MARK1/2 activity was also regulated by the death domain of DAPK. DAPK activated MARK and promoted the phosphorylation of tau but also seems to act via MARK-independent pathways on T231, S262, and S396 of tau (Wang et al., 2010 Duan et al., 2013). Moreover, DAPK induced rough eye and loss of photoreceptor neurons in a Drosophila model, in part through the activation of the Drosophila ortholog Par-1 (Wu et al., 2011b). PKC was described as negative regulator of MARK2, playing an important role in neuronal polarity (Chen et al., 2006).

CK1 and CK2 are serine/threonine-selective protein kinases. Overall CK2 immunoreactivity is reduced in the brain of AD cases although NFTs stain very strong with anti-CK2 antibodies (Iimoto et al., 1990). CK-1δ is upregulated in AD brain, correlating with the degree of regional pathology. CK-1δ colocalizes with NFTs, neuropil threads and dystrophic neurites (Yasojima et al., 2000). In cells, CK1δ inhibition reduced the phosphorylation of tau at S396/S404 by more than 70%. Exogenous expression of CK1δ increased tau phosphorylation at S202/T205 and S396/S404 and reduced tau-MT binding (Lee and Leugers, 2012).

Several sites in PHF-tau are targeted by CK1 in concert with other kinases such as GSK-3β and protein kinase A (PKA). Moreover, three sites, S113, S238, and S433, were phosphorylated only by the action of CK1δ suggesting a relevant role of this kinase in tau pathology (Hanger et al., 2007). Synthetic Aβ was reported to stimulate the activities of CK1 and CK2 and to mediate phosphorylation of the substrate casein in vitro (Chauhan et al., 1993). Aβ production was increased in cells with exogenous expression of constitutively active CK1 and reduced by CK1 specific inhibition (Flajolet et al., 2007).

Dyrk1A is upregulated in AD, Down's syndrome, and Pick's disease. Dyrk1A immunoreactivity was observed in the cytoplasm and nucleus of scattered neurons, and detected in sarkosyl-insoluble PHF fractions. Overexpression of Dyrk1A in transgenic mice led to increased tau levels in the brain and accumulation in NFTs (Wegiel et al., 2008). Direct phosphorylation of tau at T212 by Dyrk1A still lacks evidence in vivo.

LRRK2, a putative kinase, gained interest in recent years due to its genetic association with both, inherited and sporadic PD, and a possible overlap to AD (Zhao et al., 2011 Ujiie et al., 2012). The first hints for an involvement of LRRK2 in tau pathology were given in transgenic mice expressing mutant LRRK2. These animals showed increased tau phosphorylation at T149, T153, and the AT8, CP13, 12E8 and PHF-1 epitopes, tau mislocalization in cell bodies and the neuropil, and tau aggregation (Li et al., 2009, 2010 Melrose et al., 2010 Bailey et al., 2013). Correspondingly, phosphorylation of tau at the AT8 epitope was decreased in LRRK2 knock-out mice (Gillardon, 2009). Several studies imply that LRRK2 phosphorylates and activates other kinases and signal transduction pathways, thereby contributing to enhanced tau phosphorylation, mislocalization, and dendritic degeneration (Gloeckner et al., 2009 Lin et al., 2010 Chen et al., 2012).

Several other kinases may be implicated in the pathology of AD, anticipated from their aberrant activity in human brain (Martin et al., 2013). In general, dysregulation of kinases is likely to be responsible for the hyperphosphorylation of tau, abnormal tau-MT binding, tau mislocalization, and tau assembly into PHFs. However, it is still not known, which phosphorylation sites of tau are the most critical ones, which kinases are the main players, and how these processes are mechanistically linked to toxicity in AD and other neurodegenerative disorders.


Scientists observed the thermogenic activity in brown adipose tissue, which eventually led to the discovery of UCP1, initially known as "Uncoupling Protein". [3] The brown tissue revealed elevated levels of mitochondria respiration and another respiration not coupled to ATP synthesis, which symbolized strong thermogenic activity. [3] UCP1 was the protein discovered responsible for activating a proton pathway that was not coupled to ADP phosphorylation (ordinarily done through ATP Synthase). [3]

There are five UCP homologs known in mammals. While each of these performs unique functions, certain functions are performed by several of the homologs. The homologs are as follows:

  • UCP1, also known as thermogenin or SLC25A7 , also known as SLC25A8 , also known as SLC25A9
  • UCP4, also known as SLC25A27
  • UCP5, also known as SLC25A14

Maintaining body temperature Edit

The first uncoupling protein discovered, UCP1, was discovered in the brown adipose tissues of hibernators and small rodents, which provide non-shivering heat to these animals. [3] These brown adipose tissues are essential to maintaining the body temperature of small rodents, and studies with (UCP1)-knockout mice show that these tissues do not function correctly without functioning uncoupling proteins. [3] In fact, these studies revealed that cold-acclimation is not possible for these knockout mice, indicating that UCP1 is an essential driver of heat production in these brown adipose tissues. [7] [8]

Elsewhere in the body, uncoupling protein activities are known to affect the temperature in micro-environments. [9] [10] This is believed to affect other proteins' activity in these regions, though work is still required to determine the true consequences of uncoupling-induced temperature gradients within cells. [9]

Role in ATP concentrations Edit

The effect of UCP2 and UCP3 on ATP concentrations varies depending on cell type. [9] For example, pancreatic beta cells experience a decrease in ATP concentration with increased activity of UCP2. [9] This is associated with cell degeneration, decreased insulin secretion, and type II diabetes. [9] [11] Conversely, UCP2 in hippocampus cells and UCP3 in muscle cells stimulate production of mitochondria. [9] [12] The larger number of mitochondria increases the combined concentration of ADP and ATP, actually resulting in a net increase in ATP concentration when these uncoupling proteins become coupled (i.e. the mechanism to allow proton leaking is inhibited). [9] [12]

Maintaining concentration of reactive oxygen species Edit

The entire list of functions of UCP2 and UCP3 is not known. [13] However, studies indicate that these proteins are involved in a negative-feedback loop limiting the concentration of reactive oxygen species (ROS). [14] Current scientific consensus states that UCP2 and UCP3 perform proton transportation only when activation species are present. [15] Among these activators are fatty acids, ROS, and certain ROS byproducts that are also reactive. [14] [15] Therefore, higher levels of ROS directly and indirectly cause increased activity of UCP2 and UCP3. [14] This, in turn, increases proton leak from the mitochondria, lowering the proton-motive force across mitochondrial membranes, activating the electron transport chain. [13] [14] [15] Limiting the proton motive force through this process results in a negative feedback loop that limits ROS production. [14] Especially, UCP2 decreases the transmembrane potential of mitochondria, thus decreasing the production of ROS. Thus, cancer cells may increase the production of UCP2 in mitochondria. [16] This theory is supported by independent studies which show increased ROS production in both UCP2 and UCP3 knockout mice. [15]

This process is important to human health, as high-concentrations of ROS are believed to be involved in the development of degenerative diseases. [15]

Functions in neurons Edit

By detecting the associated mRNA, UCP2, UCP4, and UCP5 were shown to reside in neurons throughout the human central nervous system. [17] These proteins play key roles in neuronal function. [9] While many study findings remain controversial, several findings are widely accepted. [9]

For example, UCPs alter the free calcium concentrations in the neuron. [9] Mitochondria are a major site of calcium storage in neurons, and the storage capacity increases with potential across mitochondrial membranes. [9] [18] Therefore, when the uncoupling proteins reduce potential across these membranes, calcium ions are released to the surrounding environment in the neuron. [9] Due to the high concentrations of mitochondria near axon terminals, this implies UCPs play a role in regulating calcium concentrations in this region. [9] Considering calcium ions play a large role in neurotransmission, scientists predict that these UCPs directly affect neurotransmission. [9]

As discussed above, neurons in the hippocampus experience increased concentrations of ATP in the presence of these uncoupling proteins. [9] [12] This leads scientists to hypothesize that UCPs improve synaptic plasticity and transmission. [9]


Neurite Outgrowth in N2a Cells Requires Phosphorylation at KXGS Motifs in the Repeat Domain of Tau

One aim of our studies was to determine the role of tau protein, its phosphorylation, and its protein kinases on the elaboration of neurites. To achieve this one needs an experimental system that allows both cell biological and biochemical observations. We focused on the mouse neuroblastoma cell line N2a because it can be readily differentiated with retinoic acid after serum deprivation to form neurite-like cell processes (Figure 2). Differentiation of neuronal cells requires endogenous tau (Drubin and Kirschner 1986 Caceres and Kosik, 1990 Esmaeli-Azad et al., 1994) in N2a cells tau is only present at low concentrations, below the detectability by immunofluorescence. We transfected human fetal tau isoform htau23 (352 residues) transiently into wild-type N2a cells that induces them to develop long neurites after a differentiation stimulus. In the example of Figure 2a, ∼60% of transfected cells have processes longer than twice the cell body diameter. As a control, among cells not expressing exogenous htau23 only 20% develop extended processes (Figure 2, a, b, and f).

Fig. 2. Neurite outgrowth by N2a cells and dependence on tau phosphorylation. N2a cells were differentiated by serum withdrawal and retinoic acid and then transiently transfected with tau or tau mutants, and scored for the development of extended neurites longer than two cell body diameters (>40 μm). (a and c) Tau staining (antibody K9JA). (b and d) Tubulin staining (DM1A). (a and b) Cells transfected transiently with htau23 (50% efficiency). Among the cells not expressing exogenous tau, only 20% develop extended neurites (control Figure 2e). In contrast, among the cells expressing exogenous htau23 the fraction with extended neurites rises to 60% (Figure 2, a and b, arrows, and f). (c and d) Cells transfected with the KXGA mutant of htau23: Among the cells expressing exogenous tau mutant (three cells highlighted by arrows in 2, c and d) only few develop neurites longer than two cell body diameters (compare other untransfected cells in d). This shows that the phosphorylation at KXGS motifs is important for neurite outgrowth. (e) Differentiated control N2a cells (no transfection with tau). (f) Histogram of neurite formation with different tau constructs: Only 20% of control cells show extended neurites, but 60% of cells transfected with htau23. Cells transfected with the KXGA mutant of htau23 are comparable with controls (20%, no phosphorylation by MARK2 possible). Cells transfected with the AP mutant are comparable with transfection by htau23 (60%, no phosphorylation by proline-directed kinases). The data show that transfection with htau23 and the AP mutant are similarly efficient in promoting neurite outgrowth, whereas transfection with the KXGA mutant has no effect. Error bars show SE.

Tau contains many Ser or Thr residues that can be phosphorylated by several kinases. To probe whether the outgrowth of neurites depends on the phosphorylation of tau we generated several tau constructs in which certain Ser or Thr residues were mutated into Ala, thus making them inaccessible to phosphorylation. Among the phosphorylation sites one can distinguish different classes: 1) The S-P or T-P motifs (14 in isoform htau23, mostly in the domains flanking the repeats Figure 1) can be phosphorylated by proline-directed kinases such as GSK-3β and cdk5. They are thought to play a role in neurodegeneration (Imahori and Uchida, 1997 Mandelkow and Mandelkow, 1998), they induce the epitopes of several antibodies characteristic of Alzheimer tau (e.g., AT-8, AT-100, AT-180, and PHF-1), but have only a modest influence on tau-microtubule interactions (Biernat et al., 1993). 2) The KXGS motifs (one per repeat, three in htau23, four in htau40 Figure 1) are targets of nonproline-directed kinases, primarily MARK (and less efficiently PKA Biernat et al., 1993 Drewes et al., 1997), which has a pronounced effect on detaching tau from microtubules (particularly Ser262 in the first repeat) and render them dynamic in vitro and in vivo (Ebneth et al., 1999). We therefore made modified constructs of htau23, one with all 14 S-P or T-P motifs mutated into A-P (AP-tau), and one with the KXGS motifs mutated into KXGA (nonphosphorylatable KXGA-tau) (Figure 1). When AP-tau was transfected into N2a cells, the effect was similar to that of wild-type tau, i.e., a strong induction of neurites, with about one-half of the transfected cells displaying extended processes (Figure2f). Because proline-directed kinases are active in the cells (see below), this result means that the phosphorylation at SP or TP motifs is of lesser importance for neurite outgrowth.

In contrast, when repeating the experiment with KXGA-tau the outgrowth of extended neurites after a differentiation stimulus was nearly abolished. As a control, the effect was limited to the cells that actually express the tau mutant (Figure 2c, cells with arrows), whereas the others obtain normal processes after differentiation (Figure 2d, cells without arrows). Thus, the KXGA mutations in the repeats essentially abrogate tau's ability to induce neurites (Figure 2f). We note that the KXGA mutant has the same ability to bind and polymerize microtubules as wild-type unphosphorylated tau, in contrast to tau phosphorylated at KXGS motifs (Biernat et al., 1993 our unpublished data). These results suggest that N2a cells contain active kinase(s) that phosphorylate the KXGS motifs and that the phosphorylation at these motifs is important for neurite outgrowth.

Neurite Outgrowth Is Promoted by Activity of MARK2

Given that the KXGS motifs of tau are important for neurite outgrowth, the next question was to identify the responsible kinase(s). We had shown previously that the kinase that phosphorylates the KXGS motifs in tau and related MAPs most efficiently is MARK (Dreweset al., 1995 Illenberger et al., 1996). We therefore approached the identification of the kinase by asking how MARK influences neurite outgrowth and the phosphorylation of tau in N2a cells. Surprisingly, transient transfection of MARK2 into N2a cells caused differentiation without further stimuli (such as serum withdrawal and retinoic acid Figure 3a, b, and e), although the transfection rate was low (1–2%). In contrast, the majority of N2a cells expressing the dominant negative mutant of MARK2 (Figure 3, c, d, and e) were not able to form extended neurites, even after a differentiation stimulus. As shown previously (Drewes et al., 1997), this mutant was rendered inactive in vitro and in cells by replacing phosphorylatable residues in the regulatory loop by alanines (T208A and S212A). Wild-type N2a cells contain endogenous tau only at the low level of 24 ng/10 7 cell (using an enzyme-linked immunosorbent assay developed by Ackmann et al., 2000). We therefore generated N2a cells stably expressing htau40, the largest tau isoform in the CNS, at a ∼40-fold higher level (∼1 μg/10 7 cells), to analyze the phosphorylation state of tau biochemically. These cells were transiently transfected with dnMARK2. Cells expressing the inactive mutant of MARK2 could not form neurites (Figure 4, a–d, arrows), whereas cells not expressing dnMARK2 were able to differentiate (Figure4, a–d, asterisks). This argues that MARK2 or a closely related isoform might be important for neurite outgrowth and for the phosphorylation of the KXGS motifs in tau. To verify that MARK2 operates via tau phosphorylation, N2a cells were cotransfected with GFP-MARK2 and KGXA-htau23 (Figure 4, e–h). In the cases where the cells express the KXGA mutant of tau, or both MARK2 and the KXGA mutant, formation of extended neurites is strongly reduced. In other words, the neurite-promoting effects of tau can be obliterated either by mutating the KXGS target motif on tau (KXGA-tau Figures 2c and 4, e–g), or by inactivating the kinase MARK2 (Figure 3, c–e).

Fig. 3. Transfection of N2a cells with MARK2 promotes neurite formation, and dominant negative MARK2 inhibits it. N2a cells were transiently transfected with MARK2 (a and b) or a dominant negative mutant of MARK2 (c and d). (a and c) Staining for MARK2 (antibody 12CA5). (b and d) Staining for tubulin (antibody DM1A). (a and b) Although cells containing only the low levels of endogenous tau show no neurites in serum before differentiation (see rounded-up cells in b, asterisks) and would require differentiation conditions to develop processes (serum withdrawal, retinoic acid, etc), transfection with MARK2 overcomes this barrier and allows 60% of MARK2 transfected cells even without serum withdrawal and retinoic acid to differentiate and extend neurites (see cell at center in a and b, arrow and see histogram e). (c and d) When N2a cells are transfected with dnMARK2, only a small fraction of the cells expressing dnMARK2 (6%) form extended neurites after differentiation (Figure 3e).

Fig. 4. Inhibition of neurite outgrowth by inactive MARK2 (dnMARK2) or nonphosphorylatable Tau (KXGA). N2a cells stably transfected with htau40 can be differentiated by serum withdrawal and retinoic acid (see b and c, asterisks). However, dnMARK2 prevents neurite formation (see a, arrows). (d) Histogram showing that dnMARK2 suppresses neurites (ca. 90% of transfected cells fail to develop neurites). (a–c) Expression of a dominant negative mutant (dnMARK2) in N2a/htau40 cells. (a) Staining for dnMARK2 (antibody 12CA5). (b) Staining for tau (K9JA). (c) Staining for tubulin (DM1A). (e–g) N2a cells cotransfected transiently with GFP-MARK2 and KXGA/htau23 and then differentiated. The doubly transfected cells (arrows) show no neurites, whereas the untransfected cell has extended neurites. (h) Histogram showing the fraction of N2a with extended neurites after transfection: control N2a cells, cells transfected with htau23, MARK2, htau23-KXGA mutant, double transfection with htau23-KXGA and MARK2. The data illustrate that htau23 and MARK2 promote neurite outgrowth, htau23-KXGA abolishes this activation, and even MARK2 is not able to rescue the inhibitory effect of the KXGA-tau mutant on neurite formation after differentiation, because the phosphorylation of tau at its KXGS motifs is blocked.

To show that MARK2 indeed phosphorylates the KXGS motifs of tau in living cells we transiently transfected GFP-MARK2 (or its dominant negative mutant) into N2a cells stably transfected with tau. Differentiated cells were analyzed by GFP fluorescence (showing MARK2 Figure 5a), immunofluorescence with the antibody p-MARK against active MARK2 (Figure 5b), and phospho-KXGS motifs in tau (antibody 12E8 Seubert et al., 1995 Figure5c). The three patterns were similar, suggesting that active MARK2 localizes in the same compartments as tau throughout the cell and would therefore be able to phosphorylate it. In contrast, transfection with dnMARK2 showed no differentiation, no staining for active MARK2, and no phospho-KXGS tau (our unpublished data). To corroborate these findings the proteins were isolated from the N2a cells, and phosphorylation sites were determined by phospho-sensitive antibodies of known specificities in Western blots (Figure 5, d and e). The pan-tau antibody K9JA detects tau regardless of phosphorylation and serves as a standard for the protein concentration. Antibody 12E8 detects the phosphorylated KXGS motifs in the tau-repeats 1 and 4 (containing S262 and S356). Its signal is enhanced when MARK2 is transfected (Figure 5d, lane 2) but weak with no transfection or after transfection with dnMARK2 (Figure 5d, lanes 1 and 3). The weak reaction in lane 1 presumably reflects the residual activity of the endogenous MARK isoforms. As a control, the expression of MARK2 (active or inactive) is revealed by the antibody against the HA-tag (Figure 5, d and e). Finally, the state of activation of MARK2 is shown by the rabbit polyclonal peptide antibody p-MARK (SA6941) raised against a peptide of the phosphorylated activating loop of MARK2 (with phosphorylated T208 and S212). This antibody was affinity purified and characterized in detail Figure 5g shows that recombinant MARK2 can be phosphorylated and activated 10-fold by a kinase activity in brain extract (Dreweset al., 1997). This phosphorylation of MARK causes a shift in the SDS gel and the reaction with p-MARK antibody (Figure 5f). The p-MARK antibody shows a pronounced signal in the blot after transfection of the cells with active MARK2, but only a weak signal without MARK2 transfection or with dnMARK2 (Figure 5d, lanes 1–3). The data argue that the elaboration of neurites is achieved by MARK2 phosphorylating the KXGS motifs on tau. The effect can be demonstrated even more clearly when the experiment is repeated, but cells are incubated with the phosphatase inhibitor okadaic acid (0.2 μM, 30 min) before harvesting. In this case the phosphorylation at the KXGS motifs seen by antibody 12E8 (against p-Tau) in blots is particularly pronounced after transfection with MARK2 (Figure 5e, lane 2), but not with dnMARK2 (Figure 5e, lane 3), indicating that other kinases do not phosphorylate the KXGS motifs in the presence of okadaic acid.

Fig. 5. Phosphorylation of tau induced by MARK2. (a–c) N2a/htau40 cells transiently transfected with GFP-MARK2, differentiated, fixed after 24 h, and analyzed by fluorescence microscopy. (a) Distribution of GFP-MARK2. (b) Immunofluorescence of p-MARK antibody against active MARK2 (phosphorylated T-loop). (c) Antibody 12E8 against phospho-KXGS motifs of tau. Note that active MARK2 and phosphorylated tau at KXGS sites colocalize (see below). (d and e) N2a/htau40 cells were transiently transfected with MARK2 or a dominant negative mutant (dnMARK2), differentiated for 6 h, and analyzed by preparing a cell lysate and probing by Western blotting with a panel of antibodies against phosphorylated MARK2 and tau. The data show that MARK2 causes the phosphorylation of the KXGS motifs of tau and dnMARK2 inhibits this. (d) Lane 1, control N2a/htau40 cell extract. Lane 2, transfection with MARK2. Lane 3, transfection with dominant negative MARK2 (T208A and S212A). Antibody K9JA (“Tau”) stains tau independently of phosphorylation. The three samples contain the same amount of tau. Antibody 12E8 against the phosphorylated KXGS motifs in tau repeats 1 and 4 (phospho-S262 and -S356, “p-Tau”) shows stronger staining in the case of MARK2 transfection (lane 2), but only weak staining without MARK2 transfection (lane 1), and no staining with dnMARK2 (lane 3). The antibody against hemagglutinin-tag (“MARK”) reveals the expression of exogenous MARK2 or dnMARK2 (lanes 2 and 3) but is absent from untransfected cells (lane 1). The antibody SA6941 against phosphorylated T-loop peptide on MARK2 (pT208, pS212, corresponding to activated MARK2) shows pronounced staining after transfection of exogenous MARK2 (lane 2) but only weak staining in controls (lane 1), corresponding to the activity of endogenous MARK-like kinases, and no staining in the presence of dnMARK. (e) N2a/htau40 cells were transiently transfected with MARK2 or a dominant negative mutant, dnMARK2, differentiated for 6 h, and before harvesting treated for 30 min with 0.2 μM okadaic acid. Note that only the sample expressing the transfected wt MARK2, but not dnMARK2, has clearly increased phosphorylation at KXGS motifs shown by reaction with antibody 12E8 (“p-Tau”, lane 2). This means that MARK2 but not other endogenous kinases such as PKA are responsible for tau phosphorylation at KXGS motifs in these conditions. (f and g) Western blot analysis demonstrating the specificity of the p-MARK antibody (polyclonal antibody SA6941) against the posphorylation sites T208 and S212 in the activating loop of kinase MARK2. Top, MARK2 protein before (lane 1) and after (lane 2) phosphorylation by brain extract kinase activity (see MATERIALS AND METHODS). Bottom, antibody p-MARK (SA6941) recognizes exclusively the phosphorylated MARK2 (lane 2) whose increased activity is demonstrated in the histogram (g). (h–j) N2a/htau40 cells transiently transfected with HA-tagged MARK2, fixed 24 h after differentiation and analyzed by confocal fluorescence microscopy. (h) Distribution of MARK2 by using fluorescent HA antibody. (i) Fluorescence of phalloidin-labeled actin. (j) Merge of h and i. Note that MARK2 and actin largely colocalize.

As seen in Figure 5, a–c, the differentiated N2a cells showed numerous filopodia and microspikes emanating from the cell body and the neurites, suggesting that active MARK2 and phospho-tau might colocalize with the actin network. We therefore transfected MARK2 transiently into N2a-htau40 cells and checked the distribution of MARK2 and actin by immunofluorescence. Figure 5, h–j, shows by confocal microscopy that the pattern of MARK2 coincides largely with that of actin. We hypothesize that tau phosphorylated at KXGS motifs detaches from microtubules and partly translocates to the actin network during neurite outgrowth, consistent with recent observations with MAP2 phosphorylated at KXGS motifs (Ozer and Halpain, 2000).

MARK Is Potently Inhibited by Hymenialdisine

Tau contains multiple phosphorylation sites that could affect its function in different ways. The majority (95%) of tau's endogenous phosphorylation in cells occurs on SP or TP motifs in the regions flanking the repeats (Illenberger et al., 1998 Biernat and Mandelkow, 1999). These sites are phosphorylated by proline-directed kinases such as cdk5 and GSK-3β, which are known to play a role in differentiating neurons. To analyze the role of these kinases in process outgrowth we used two novel potent inhibitors of both cdk5 and GSK-3β, FL and HD (Meijer et al., 2000 Leclerc et al., 2001), and applied them to N2a cells stably transfected with htau40. Figure 6a shows that control cells develop extended neurites (∼25%) in differentiation medium. When the cells were exposed to 50 μM FL, neurite outgrowth was similar or somewhat enhanced (Figure 6, b and d), consistent with our previous observation that phosphorylation of tau at SP or TP motifs was neutral or somewhat inhibitory (Biernat and Mandelkow, 1999). In contrast, when N2a/htau40 cells were treated with the kinase inhibitor HD (50 μM), neurite outgrowth was strongly inhibited (∼3% Figure6, c and d). How can this apparent discrepancy between the two kinase inhibitors be explained? To answer this question the affected phosphorylation sites on tau had to be determined in more detail. This cannot be achieved with N2a or N2a/htau40 cells because their level of tau is too low for biochemical analysis. We therefore turned to the Sf9 cell system that generates sufficient quantities of protein for the biochemical analysis of transfected tau. The justification is the observation that the interplay between tau and tau kinases during process formation is qualitatively similar between neuronal and nonneuronal cells (Biernat and Mandelkow, 1999).

Fig. 6. Effect of kinase inhibitors FL and HD on tau-induced neurite outgrowth in N2a cells stably transfected with htau40. (a) Control N2a/htau40 cells were differentiated and stained for immunofluorescence with tau antibody K9JA ∼25% of cells develop extended cell processes. (b) N2a/htau40 cells differentiated and treated with 50 μM FL, a strong inhibitor of cdk5 and GSK-3β (Leclerc et al., 2001). Neurite outgrowth is somewhat enhanced (∼35%). (c) N2a/htau40 cells differentiated and treated with 50 μM HD, also a strong inhibitor of cdk5 and GSK-3β (Meijeret al., 2000). Neurite outgrowth is strongly inhibited (∼3%). (d) Histogram illustrating the fraction of N2a/htau40 cells bearing extended neurites after differentiation and treatment with kinase inhibitors HD and FL. HD strongly reduces the extent of neurite formation, and FL causes a slight increase.

Figure 7 illustrates untreated Sf9 cells (diameter around 20 μm, no cell processes Figure 7a) and cells transfected with htau23 (Figure 7b). After 30 h of transfection, ∼25% of these cells were enlarged and developed a single cell process of uniform diameter. Next, we exposed the cells to the drug FL (50 μM). This resulted in a pronounced threefold increase in cells with processes (∼75% Figure 7, c and e), arguing that proline-directed phosphorylation of tau by cdk5 and GSK-3β is inhibitory for cell processes. However, when the cells were exposed to HD, process formation was strongly reduced (∼10% Figure 7, d and e). The results showed that both N2a cells and tau-transfected Sf9 cells had a similar response to the kinase inhibitors FL and HD.

Fig. 7. Effect of kinase inhibitors FL and HD on tau-induced process outgrowth in Sf9 cells. (a) Untreated Sf9 cells (control), showing round shapes) and no cell processes. (b) Sf9 cells transfected with htau23 by baculovirus vector ∼30% grow extended cell processes up to 100 μm. They also have larger cell bodies (2- to 3-fold). (c) Sf9 cells transfected with htau23 and treated with the kinase inhibitor FL (50 μM). The number of cells with processes increases strongly (∼3-fold). (d) Sf9 cells transfected with htau23 and treated with the kinase inhibitor HD (50 μM). The number of cells with processes drops to ∼10%. (e) Histogram displaying the efficiency of process formation by Sf9 cells in the presence of kinase inhibitors. HD inhibits them strongly, LiCl and H89 promote modestly, and FL promotes strongly.

The phosphorylation sites on tau were determined using site-specific antibodies on Western blots, and by 2D phosphopeptide mapping (Figure8). As shown previously (Illenbergeret al., 1998 Godemann et al., 1999), major targets of GSK-3β on tau are S404, followed by S396 (which together make up the epitope of PHF-1), and to a lesser extent S202 and T205 (antibody AT-8). The major targets of cdk5 are S235 followed by T231 (epitope of antibody AT-180), S202/T205 (AT-8 epitope), whereas the reaction with PHF-1 is very weak because S404 is a major site but not S396. The Sf9 cells contain very active kinases so that all phosphorylation-dependent antibody reactions are observed on the transfected tau (Biernat and Mandelkow, 1999). Figure 8a (lane 1) illustrates this for the antibodies 12E8 (pS262 and pS356), AT-100 (pT212 and pS214), AT-180 (pT231 and pS235), AT-8 (pS202 and pT205), and PHF-1 (pS396 and pS404). When the inhibitor FL is added to the cells (Figure 8a, lane 3), the reactions of tau in Western blots with antibodies AT-100, AT-180, AT-8, and PHF-1 are strongly suppressed. The same is true for the inhibitor HD (Figure 8a, lane 2). However, there was an unexpected difference with regard to antibody 12E8, diagnostic for the KXGS motifs containing phospho-S262/S356. FL allows phosphorylation at these sites, and HD inhibits it, as seen in the Western blot (Figure 8a, lanes 2 and 3). Because neither GSK-3β nor cdk5 phosphorylate S262 or S356 (Godemann et al., 1999), the result suggests that HD is also an inhibitor of MARK2, the kinase phosphorylating these sites most efficiently. This was tested directly by kinase activity assays performed with recombinant MARK2 and GSK-3β in vitro (Figure 9). In the case of GSK-3β, we found comparably strong inhibition by HD (IC50 = 0.13 μM) and FL (IC50 = 0.55 μM Figure 9). In the case of MARK2, only HD is a strong inhibitor (IC50 = 0.67 μM).

Fig. 8. Effect of kinase inhibitors on phosphorylation of htau23 in Sf9 cells. (a and b) Antibody blots. (c and f) 2D phosphopeptide maps. (a) Lane 1, no inhibitor (control). Lane 2, 50 μM HD. Lane 3, 50 μM FL. From top to bottom: Coomassie-stained gel (control) showing roughly equal amounts of tau Western blot with antibody K9JA that recognizes tau independently of phosphorylation antibody 12E8 (against phosphorylated KXGS motifs). The inhibitor HD prevents phosphorylation (lane 2) because it inhibits MARK and related kinases, and not because of its inhibition of GSK-3β (see below). LiCl and PKA inhibitors have no pronounced effect (Figure8b, lanes 1 and 2). Antibody AT-100 (against phospho-T212 and S214). This epitope requires the activity of GSK-3β (to phosphorylate T212), and PKA (to phosphorylate S214 Zheng-Fischhofer et al., 1998). Therefore, the epitope is blocked by both HD and FL (lanes 2 and 3). Antibody AT-180 (against phospho-T231 and S235). This is an epitope induced by cdk5 and GSK-3β, and thus the signal is blocked by both HD and FL (lanes 2 and 3). Antibody AT-8 (against phospho-S202 and T205). This is an epitope induced mainly by cdk5, and therefore the signal is blocked by both HD and FL (lanes 2 and 3). Antibody PHF-1 (against phospho-S396 and S404). This epitope is phophorylated mainly by GSK-3β, and therefore the signal is inhibited by HD, but less by FL. (b) Lane 1, 50 mM LiCl. Lane 2, 50 μM H89, a PKA inhibitor. (c–f) Two-dimensional phosphopeptide maps of htau23 protein expressed in Sf9 cells and phosphorylated by endogenous kinases without (c), or in the presence of kinase inhibitors (d–f). (c) Control, tau expressed in Sf9 cells. (d) Inhibition by 50 μM HD to inhibit cdk5, GSK-3β, and MARK. (e) Inhibition by 50 mM LiCl to inhibit GSK-3β. (f) Inhibition by 50 μM FL to inhibit cdk5 and GSK3β note that c, e, and f show spots for phosphorylated S262 (circle, also see insert in e at higher exposure), d does not because HD inhibits MARK activity.

Fig. 9. In vitro inhibition assay of kinases of tau by inhibitors HD and FL. (a) MARK2. (b) GSK3β. Note that GSK-3β is inhibited by HD (IC50 = 0.13 μM) and FL (IC50 = 0.55 μM), but MARK2 is inhibited mainly by HD (IC50 = 0.67 μM), whereas FL is only a weak inhibitor (IC50 = 38 μM).

Additional control experiments on Sf9 cells were done with LiCl, a specific inhibitor of GSK-3β (Stambolic et al., 1996), and H89, an inhibitor of PKA (Chijiwa et al., 1990). These experiments were prompted by our previous observations that PKA can phosphorylate the KXGS motifs of tau in vitro, albeit with low efficiency (Schneider et al., 1999), and by conflicting reports that GSK-3β could also phosphorylate KXGS motifs (Morenoet al., 1995 Godemann et al., 1999). We tested these two kinases with their inhibitors (LiCl for GSK-3β, H89 for PKA) and found that the reaction with the antibody 12E8 against pSer262 was not affected (Figure 8b, lanes 1 and 2, compare with control in Figure 8a, lane 1) in contrast, 50 μM HD suppressed the reaction with antibody 12E8 (Figure 8a, lane 2). This was confirmed by phosphopeptide mapping of tau in Sf9 cells after treatment with 50 mM LiCl to inhibit GSK-3β (see below, Figure 8e, circled spot). Furthermore, the quantification of tau-induced cell processes of Sf9 cells after treatment with inhibitors LiCl or H89 showed a slight increase, rather than the pronounced decrease observed with the MARK inhibitor HD (Figure 7e). This suggests that GSK-3β and PKA are not responsible for the phosphorylation of KXGS motifs in tau during cell process formation.

Finally, we performed metabolic labeling of Sf9 cells with 32 P and analyzed the phosphorylation state of tau by phosphopeptide mapping (Figure 8, c–f). The reference map of tryptic phosphopeptides of tau expressed in Sf9 cells is shown in Figure 8c the identification of the spots is described in detail elsewhere (Illenberger et al., 1998 Biernat and Mandelkow, 1999). The spots of pS262 and pS356 are rather weak compared with those of the SP or TP motifs. Nevertheless, the spot of pS262 is clearly visible in the maps obtained with LiCl or FL (Figure 8, e and f, circles, see inset), but not with HD (Figure 8d, circle). This confirms that HD is also an inhibitor of MARK2, and that GSK-3β or cdk5 are not responsible for the phosphorylation of tau at S262 in Sf9 cells.

The results with kinase inhibitors explain the antibody reactions (disappearance of 12E8 staining in the presence of inhibitor HD), but more importantly they explain the response of the cells to inhibitor treatment. As noted above, phosphorylation at KXGS motifs is essential for process formation. Because this is achieved by MARK2, the inhibitor HD (but not FL) suppresses the cell processes (Figure 7, d and e) because it is also an inhibitor of MARK2 and not because it is also an inhibitor of GSK-3β and cdk5.


Fertilization releases the meiotic arrest and initiates the events that prepare the egg for the ensuing developmental program. Protein degradation and phosphorylation are known to regulate protein activity during this process. However, the full extent of protein loss and phosphoregulation is still unknown. We examined absolute protein and phosphosite dynamics of the fertilization response by mass spectrometry-based proteomics in electroactivated eggs. To do this, we developed an approach for calculating the stoichiometry of phosphosites from multiplexed proteomics that is compatible with dynamic, stable, and multisite phosphorylation. Overall, the data suggest that degradation is limited to a few low-abundance proteins. However, this degradation promotes extensive dephosphorylation that occurs over a wide range of abundances during meiotic exit. We also show that eggs release a large amount of protein into the medium just after fertilization, most likely related to the blocks to polyspermy. Concomitantly, there is a substantial increase in phosphorylation likely tied to calcium-activated kinases. We identify putative degradation targets and components of the slow block to polyspermy. The analytical approaches demonstrated here are broadly applicable to studies of dynamic biological systems.

For decades, the highly synchronous events of fertilization have provided a useful system for the study of many aspects of cellular regulation, especially protein degradation and phosphorylation. The destruction of Cyclin-B and other proteins is catalyzed by the anaphase-promoting complex (APC/C), which promotes M-phase exit (1). The two activators of the APC/C are Cdc20 and Cdh1. In the egg, the cell cycle typically involves only Cdc20 (2). While the list of known Cdh1 substrates continues to grow (3), the Cdc20 target list remains small (4 ⇓ –6). It is of considerable interest to characterize the minimal set of Cdc20 substrates that powers the early cell cycle. Kinase activity is equally important to this regulation. Cyclin-B degradation promotes mitotic exit by inhibiting the activity of Cyclin-dependent kinase 1 (Cdk1). There is a bulk loss of phosphorylation following egg activation (7). The identities of the vast majority of these phosphoproteins remain undiscovered. However, there is a strong expectation that this regulation reflects the reversal of Cdk1 phosphorylation. Fertilization employs additional regulation not common to other cell cycles. There are timed waves of phosphorylation that correspond to the release of cortical granules just after fertilization as part of the slow block to polyspermy. This release is calcium sensitive and may reflect increases in the activity of protein kinase C (PKC) (8, 9) and CaMKII (10). An account of the secreted proteins, their function, and their upstream signaling components is limited. To investigate these unknown aspects of degradation, release, and modification of proteins at fertilization comprehensively, we employed mass spectrometry (MS) in Xenopus laevis eggs. Recent advances in multiplexed proteomics allow quantitative comparisons of multiple conditions using tandem mass tags (TMTs) (11 ⇓ –13). Our recent work demonstrates the power of proteomics in Xenopus for studies of early development (14 ⇓ –16). Although fertilization has been studied before with MS (17 ⇓ ⇓ –20), the studies were either qualitative or did not measure phosphorylation. With an enrichment step (21 ⇓ –23), it is feasible to measure relative changes in a large number of phosphoproteins. However, such studies are of limited utility without measuring site stoichiometry. Relying on fold change alone for phosphorylation studies will not distinguish between 10-fold relative changes from 1% occupancy to 10% versus 10 to 100%. Several approaches are available (24 ⇓ –26), but none of these is able to determine occupancies of peptides measured with multiplexed MS or that have multiple phosphorylated residues. This paper introduces biological findings about fertilization and cell cycle progression as well as methods for measuring absolute stoichiometry of phosphosites that are widely applicable to MS protein modification studies.


In this study, we asked how the function of tau, a protein known to initiate axonal outgrowth, is regulated by phosphorylation, and what kinases are responsible for it. We argue that the phosphorylation of tau at its KXGS motifs by the kinase MARK is critical in neurons and nonneuronal cell models (N2a and Sf9 cells). Because these cell types contain little or no endogenous tau, cell processes can be strongly enhanced by transfection with exogenous tau. The evidence for the importance of MARK is based on experiments where we changed either the activity of the regulator kinase MARK or the sites on its effector protein tau. The function of tau was changed by point mutations at the target sites of MARK by replacing the KXGS motifs with nonphosphorylatable KXGA motifs. KXGA-tau was not able to stimulate neurite outgrowth in N2a cells. The activity of MARK was changed in three ways: 1) transient transfection of cells with MARK2, one of the MARK isoforms, which can be activated by phosphorylation at the activating loop (Drewes et al., 1997) 2) transfection with a dominant negative MARK lacking the regulatory phosphorylation sites (T208A and S212A) and 3) inactivation of MARK by hymenialdisine, a new kinase inhibitor (Meijer et al., 2000). When MARK was inactivated, no stimulation of neurite outgrowth occurred.

Because tau contains many phosphorylation sites, an obvious issue is to distinguish their effects on the functions of tau. This is a common problem in the field because tau's phosphorylation in cells is often detected by antibodies that may vary in affinity and specificity. Many of the “Alzheimer-diagnostic” antibodies are directed against phospho-SP or -TP motifs, suggesting the activity of proline-directed kinases (e.g., MAP kinase, GSK-3β, or cdk5). In our case, we can rule out a major role of these kinases for tau-induced neurite outgrowth because the AP-tau mutant (where all SP or TP motifs were turned into AP) shows a similar or greater stimulation of neurites than normal tau, contrary to the KXGA-mutant of tau. This agrees with previous observations on Sf9 cells (Biernat and Mandelkow, 1999). Other, non-KXGS or non-SP/TP phosphorylation sites are also of minor importance because they represent a minor fraction of cellular phosphorylation sites (Watanabe et al., 1993, Illenbergeret al., 1998), and they are not phosphorylated by MARK so that they cannot explain the effects of MARK inhibition (Dreweset al., 1995). A special issue is the possible phosphorylation of KXGS motifs by two other kinases, GSK-3β or PKA. In one study GKS-3β was thought to phosphorylate tau at S262 (Morenoet al., 1995), but our subsequent analysis showed that this was due to other activities in the kinase preparation (Godemannet al., 1999). This is confirmed herein by the LiCl experiment that inhibits the GKS-3β targets on tau (SP or TP motifs Figure 8b, lane 1, PHF1 staining) without affecting the KXGS sites (Figure 8b, lane 1, staining with 12E8, and Figure 8e, spot of phospho-S262 in inset). PKA remains another possibility it indeed phosphorylates tau at several sites, including the KXGS motifs (albeit much less efficiently than MARK Drewes et al., 1995Schneider et al., 1999). However, inhibition of PKA by H89 did not inhibit process formation (Figure 7e), contrary to inhibition of MARK by HD and did not inhibit the phosphorylation of KXGS motifs (Figure 8b, lane 2, staining with 12E8). We also showed in an in vitro activity assay (Figure 9) that hymenialdisine is able to inhibit MARK, but not PKA. Furthermore, the treatment of N2a/htau40 cells with okadaic acid led to the increase in phosphorylation at KXGS motifs only after transfecting the cells with MARK2 (but not with dnMARK2), nor in cells without transfected MARK2 (Figure 5e, lanes 1–3). This suggests that no other kinase phosphorylates the KXGS-motifs of tau in differentiating N2a cells even under the conditions of okadaic acid, pointing to MARK2 as the responsible kinase. Finally, in living cells GFP-MARK2 (but not dnMARK2) colocalizes with MARK2 activity (as shown by the p-MARK antibody) and phospho-KXGS tau (Figure 5, a–c). Collectively, the biochemical and immunofluorescence evidence suggests that MARK2 or a kinase of the MARK family is the kinase phosphorylating tau at KXGS motifs in differentiating N2a cells.

Microtubule stability is considered to be necessary for neurites because they provide mechanical strength and tracks for the intracellular transport. The phosphorylation of the KXGS motifs of tau during neurite outgrowth tends to detach tau from microtubules and thus renders microtubules less stable. This is the opposite of what one would expect intuitively, and therefore the question arises why this should be important for neurite outgrowth. The answer may lie in a more subtle role of microtubules: As the growth cone advances, actin filaments prepare the ground in the form of transient lamellipodia and filopodia. This ground is probed by pioneering microtubules that make temporary excursions into the actin meshwork and retract again, until finally a decision is made to stabilize them, form bundles, and allow the growth cone to advance (reviewed in Borisy and Svitkina, 2000Bradke and Dotti, 2000 Goode et al., 2000). Thus, microtubules must be dynamically instable before neurites can extend. This explains why the suppression of dynamic instability suffices to block the growth of neurites, even when the polymer mass is not changed (Baas and Ahmad, 1993 Liao et al., 1995 Tanaka et al., 1995 Rochlin et al., 1996 Kaverina et al., 1998). The cell seems to use tau or related MAPs for this regulation (Drubin and Kirschner, 1986 Panda et al., 1999). Tau is present in the growth cone, but it is largely detached from microtubules (Black et al., 1996). Why should this pool of tau not stabilize microtubules so that they can advance out of the shaft of the neurite? We suggest that tau is locally phosphorylated at the KXGS motifs, is therefore unable to bind to microtubules (thus rendering them unstable), and relocates to the actin network (Figure5c Ozer and Halpain, 2000, for the analogous case of MAP2). Only a small fraction of microtubules are highly dynamic (Waterman-Storer and Salmon, 1997 Kabir et al., 2001) this explains why the overall extent of KXGS-phosphorylation is low in normal cells, in spite of its crucial role (Biernat and Mandelkow, 1999).

It is interesting to compare our results with two other studies on the phosphorylation of tau in neurons. Mandell and Banker (1996) argued that tau phosphorylation was lowest at the distal end of the neurite where microtubules are most dynamic. This seemed puzzling because unphosphorylated tau is often considered tantamount to stable microtubules. The finding can be explained by noting that the authors had used the antibody AT-8, which senses two phosphorylated SP and TP motifs outside the repeat domain (S202 and T205), but does not recognize the phosphorylation at KXGS motifs. We have shown elsewhere that the phosphorylation at the SP or TP motifs sites has only a modest effect on the tau-microtubule binding (Biernat et al., 1993), and indeed all proline-directed sites combined have no major influence on neurite outgrowth (Figure 2f), in contrast to the KXGS motifs. Overall, the role of proline-directed phosphorylation of tau is poorly understood and may be important in other contexts, for example, protection against degradation (Litersky and Johnson, 1995), cell division (Ookata et al., 1997 Illenberger et al., 1998), and compartmentalization in neurons (Binder et al., 1985 Hirokawa et al., 1996).

In a related study, Ozer and Halpain (2000) investigated the phosphorylation of MAP2 in HeLa cells. This MAP is similar to tau and the endogenous HeLa MAP4 by having homologous repeats, including KXGS motifs, and is sorted into the somatodendritic compartment of neurons (in contrast to axonal tau). The authors found that the KXGS motifs of MAP2 could be phosphorylated by PKA, resulting in the dissociation of MAP2 from microtubules and translocation to the actin network. This would be consistent with the fact that PKA binds to MAP2 through its regulatory RII subunit (Obar et al., 1989), which is not the case for tau. The common denominator for their results and ours would be that the cell needs to control the dynamics of microtubules by phosphorylating the locally available MAPs at their KXGS motifs. This could be tau in axons, MAP2 in dendrites, and presumably MAP4 in other cell types. The phosphorylation could be achieved by different kinases (e.g., MARK or PKA), with the same result of detaching the MAPs from the microtubules. Alternatively, it is possible that there is a more complex mechanism involving activating kinases and/or phosphatases. In our experimental system we found no evidence for a major role of PKA in initiating neurite outgrowth, consistent with a role of kinases of the MARK/PAR-1 family in establishing cell polarity (Drewes et al., 1998 Kemphues, 2000, Shulman et al., 2000Tomancak et al., 2000).

Neurons contain several MAPs with different distributions and partially overlapping functions. For example, tau and MAP1b can substitute for one another during axonal growth in different extracellular environments (DiTella et al., 1996). A transgenic mouse lacking only tau is viable, but a mouse model lacking both tau and MAP1b shows severe defects in axonogenesis (Harada et al., 1994 Takei et al., 2000). The complementarity extends even to molecular properties: Phosphorylation of tau at certain sites detaches tau from microtubules and makes them more dynamic, whereas certain phosphorylations of MAP1b promote the binding to microtubules, and in both cases there is a gradient along axons, with the lower affinity species near the growth cone (Ulloa et al., 1994). Down-regulation of GSK-3β by the WNT signaling pathway or inhibition by lithium induces the dephosphorylation of MAP1b, its detachment from microtubules, and hence axonal remodeling such as increased growth cone size and branching (Lucas et al., 1998). Because hymenialdisine is an inhibitor not only of MARK but also of GSK-3β (Meijer et al., 2000), one could argue that its effect on neurite outgrowth might be mediated by GSK-3β and possibly MAP1b. This can however be ruled out because two other inhibitors of GSK-3β, lithium and flavopiridol, do not reproduce the effects of HD. Furthermore, the effects of MARK inhibition mediated by tau (i.e., inhibition of neurite outgrowth) are opposite from the effects of GSK-3β inhibition mediated by MAP1b (growth cone remodeling). In other words, the inhibition of MARK reduces microtubule dynamics, whereas the inhibition of GSK-3β enhances dynamics.

Finally, we note that there is growing evidence for an interplay between microtubule and actin dynamics mediated by phosphorylation of accessory components during neurite outgrowth. Several kinases have been described that are docked on one or both of these fiber systems and promote their organization, e.g., c-Abl, PAK5, or protein kinase C (Kabir et al., 2001 Dan et al., 2002 Woodringet al., 2002), and MARK would be another case in point. Usually, this takes place in tight coordination with small G proteins of the rho family (Daub et al., 2001 Palazzo et al., 2001). The phosphorylation of MAPs could regulate different subaspects of microtubule behavior in the growth cone, such as microtubule bundling in the shaft, dynamic instability of the pioneer microtubules, activation of tubulin scavengers such as stathmin, and anchoring at focal contacts. Furthermore, MAPs can interact with components other than microtubules such as the actin cytoskeleton (Griffith and Pollard, 1982 DiTella et al., 1994Cunningham et al., 1997 Ozer and Halpain, 2000). This may explain why different kinases have overlapping effects on growth cone behavior, presumably in response to different external signals whose nature remains to be elucidated.

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  1. Terika

    I absolutely agree with you. There is something in this and an excellent idea, I agree with you.

  2. Jagur

    Happens... Such casual concurrence

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