Edda Thies

Tau blocks traffic of organelles, neurofilaments, and APP vesicles in neurons and enhances oxidative stress

We studied the effect of microtubule-associated tau protein on trafficking of vesicles and organelles in primary cortical neurons, retinal ganglion cells, and neuroblastoma cells. Tau inhibits kinesin-dependent transport of peroxisomes, neurofilaments, and Golgi-derived vesicles into neurites. Loss of peroxisomes makes cells vulnerable to oxidative stress and leads to degeneration. In particular, tau inhibits transport of amyloid precursor protein (APP) into axons and dendrites, causing its accumulation in the cell body. APP tagged with yellow fluorescent protein and transfected by adenovirus associates with vesicles moving rapidly forward in the axon (∼80%) and slowly back (∼20%). Both movements are strongly inhibited by cotransfection with fluorescently tagged tau (cyan fluorescent protein–tau) as seen by two-color confocal microscopy. The data suggests a linkage between tau and APP trafficking, which may be significant in Alzheimers disease.

Aβ Oligomers Cause Localized Ca2+ Elevation, Missorting of Endogenous Tau into Dendrites, Tau Phosphorylation, and Destruction of Microtubules and Spines

Aggregation of amyloid-β (Aβ) and Tau protein are hallmarks of Alzheimers disease (AD), and according to the Aβ-cascade hypothesis, Aβ is considered toxic for neurons and Tau a downstream target of Aβ. We have investigated differentiated primary hippocampal neurons for early localized changes following exposure to Aβ oligomers. Initial events become evident by missorting of endogenous Tau into the somatodendritic compartment, in contrast to axonal sorting in normal neurons. In missorted dendritic regions there is a depletion of spines and local increase in Ca2+, and breakdown of microtubules. Tau in these regions shows elevated phosphorylation at certain sites diagnostic of AD-Tau (e.g., epitope of antibody 12E8, whose phosphorylation causes detachment of Tau from microtubules, and AT8 epitope), and local elevation of certain kinase activities (e.g., MARK/par-1, BRSK/SADK, p70S6K, cdk5, but not GSK3β, JNK, MAPK). These local effects occur without global changes in Tau, tubulin, or kinase levels. Somatodendritic missorting occurs not only with Tau, but also with other axonal proteins such as neurofilaments, and correlates with pronounced depletion of microtubules and mitochondria. The Aβ-induced effects on microtubule and mitochondria depletion, Tau missorting, and loss of spines are prevented by taxol, indicating that Aβ-induced microtubule destabilization and corresponding traffic defects are key factors in incipient degeneration. By contrast, the rise in Ca2+ levels, kinase activities, and Tau phosphorylation cannot be prevented by taxol. Incipient and local changes similar to those of Aβ oligomers can be evoked by cell stressors (e.g., H2O2, glutamate, serum deprivation), suggesting some common mechanism of signaling.

Clogging of axons by tau, inhibition of axonal traffic and starvation of synapses

Loss of synapses and dying back of axons are considered early events in brain degeneration during Alzheimers disease. This is accompanied by an aberrant behavior of the microtubule-associated protein tau (hyperphosphorylation, aggregation). Since microtubules are the tracks for axonal transport, we are testing the hypothesis that tau plays a role in the malfunctioning of transport. Experiments with various neuronal and non-neuronal cells show that tau is capable of reducing net anterograde transport of vesicles and cell organelles by blocking the microtubule tracks. Thus, a misregulation of tau could cause the starvation of synapses and enhanced oxidative stress, long before tau detaches from microtubules and aggregates into Alzheimer neurofibrillary tangles. In particular, the transport of amyloid precursor protein is retarded when tau is elevated, suggesting a possible link between the two key proteins that show abnormal behavior in Alzheimers disease.

Missorting of Tau in Neurons Causes Degeneration of Synapses That Can Be Rescued by the Kinase MARK2/Par-1

Early hallmarks of Alzheimers disease include the loss of synapses, which precedes the loss of neurons and the pathological phosphorylation and aggregation of tau protein. Mitochondrial dysfunction has been suggested as a reason, but evidence on the role of tau was lacking. Here, we show that transfection of tau in mature hippocampal neurons leads to an improper distribution of tau into the somatodendritic compartment with concomitant degeneration of synapses, as seen by the disappearance of spines and of presynaptic and postsynaptic markers. This is accompanied by transport inhibition of vesicles and organelles, concomitant with an increase and bundling of microtubules. Mitochondria degenerate, thus causing ATP levels to decrease. The tau-induced synaptic decay can be relieved by the activation of the kinase MARK2 (microtubule-associated protein/microtubule affinity regulating kinase 2)/Par-1 (protease-activated receptor 1), which can remove tau from the microtubule tracks and reverses the transport block. This leads to the rescue of dendritic spines, synapses, mitochondrial transport and ATP levels.

MARK/PAR1 kinase is a regulator of microtubule-dependent transport in axons

Microtubule-dependent transport of vesicles and organelles appears saltatory because particles switch between periods of rest, random Brownian motion, and active transport. The transport can be regulated through motor proteins, cargo adaptors, or microtubule tracks. We report here a mechanism whereby microtubule associated proteins (MAPs) represent obstacles to motors which can be regulated by microtubule affinity regulating kinase (MARK)/Par-1, a family of kinases that is known for its involvement in establishing cell polarity and in phosphorylating tau protein during Alzheimer neurodegeneration. Expression of MARK causes the phosphorylation of MAPs at their KXGS motifs, thereby detaching MAPs from the microtubules and thus facilitating the transport of particles. This occurs without impairing the intrinsic activity of motors because the velocity during active movement remains unchanged. In primary retinal ganglion cells, transfection with tau leads to the inhibition of axonal transport of mitochondria, APP vesicles, and other cell components which leads to starvation of axons and vulnerability against stress. This transport inhibition can be rescued by phosphorylating tau with MARK.

Appearance of target-specific guidance information for regenerating axons after CNS lesions

During development of the vertebrate visual system, an orderly projection of ganglion cells from the retina onto the superior colliculus (SC) is established. Mechanisms that might govern this process include the coordinated action of guidance and corresponding receptor molecules that are specifically distributed on the axons and their targets. In birds and mammals, information for axonal guidance and targeting appears to be confined to the time when the retinocollicular projection is being formed. Here we show that putative guidance activities for temporal and nasal retinal axons, which are not detectable in the normal adult SC, appear after optic nerve transection in adult rats. Both embryonic and adult retinal axons are able to respond to these guiding cues, although the guidance activities detectable in the deafferented adult rat SC might be different from those found during development. These findings imply that it might be possible to reestablish an ordered projection after lesions in the adult mammalian visual system.

Swimming against the Tide: Mobility of the Microtubule-Associated Protein Tau in Neurons

Long-haul transport along microtubules is crucial for neuronal polarity, and transport defects cause neurodegeneration. Tau protein stabilizes microtubule tracks, but in Alzheimers disease it aggregates and becomes missorted into the somatodendritic compartment. Tau can inhibit axonal transport by obstructing motors on microtubules, yet tau itself can still move into axons. We therefore investigated tau movement by live-cell fluorescence microscopy, FRAP (fluorescence recovery after photobleaching), and FSM (fluorescence speckle microscopy). Tau is highly dynamic, with diffusion coefficients of ∼3 μm2/s and microtubule dwell times of ∼4 s. This facilitates the entry of tau into axons over distances of millimeters and periods of days. For longer distances and times, two mechanisms of tau transport are observed. At low near-physiological levels, tau is cotransported with microtubule fragments from cell bodies into axons, moving at instantaneous velocities ∼1 μm/s. At high concentrations, tau forms local accumulations moving bidirectionally at ∼0.3 μm/s. These clusters first appear at distal endings of axons and may indicate an early stage of neurite degeneration.

Inhibition of APP Trafficking by Tau Protein Does Not Increase the Generation of Amyloid‐β Peptides

Amyloid‐β, a peptide derived from the precursor protein APP, accumulates in the brain and contributes to the neuropathology of Alzheimers disease. Increased generation of amyloid‐β might be caused by axonal transport inhibition, via increased dwell time of APP vesicles and thereby higher probability of APP cleavage by secretase enzymes residing on the same vesicles. We tested this hypothesis using a neuronal cell culture model of inhibited axonal transport and by imaging vesicular transport of fluorescently tagged APP and β‐secretase (BACE1). Microtubule‐associated tau protein blocks vesicle traffic by inhibiting the access of motor proteins to the microtubule tracks. In neurons co‐transfected with CFP‐tau, APP‐YFP traffic into distal neurites was strongly reduced. However, this did not increase amyloid‐β levels. In singly transfected axons, APP‐YFP was transported in large tubules and vesicles moving very fast (on average 3 µm/s) and with high fluxes in the anterograde direction (on average 8.4 vesicles/min). By contrast, BACE1‐CFP movement was in smaller tubules and vesicles that were almost 2× slower (on average 1.6 µm/s) with ~18× lower fluxes (on average 0.5 vesicles/min). Two‐colour microscopy of co‐transfected axons confirmed that the two proteins were sorted into distinct carriers. The results do not support the above hypothesis. Instead, they indicate that APP is transported on vesicles distinct from the secretase components and that amyloid‐β is not generated in transit when transport is blocked by tau.

Quantification of Amyloid Precursor Protein and Tau for the Study of Axonal Traffic Pathways

We describe a procedure for quantifying the numbers of expressed fluorescent fusion proteins on vesicles transported in axons. The method can be used to estimate numbers of vesicle-anchored molecules moving in both anterograde and retrograde directions and is also applicable to cytosolic proteins.

Tau and Intracellular Transport in Neurons

Among the early changes in the brains of Alzheimers disease patients is the loss of synapses, which is accompanied by the abnormal phosphorylation of tau protein, its missorting into the somatodendritic compartment of neurons, and its incipient aggregation. The physiological function of tau is to stabilize axonal microtubules, which enables them to carry out their role as tracks for the transport of vesicles and organelles. By implication, perturbations in the functions of tau could be related to the loss of synapses and neuronal degeneration. Cell and trans-genic animal models of tauopathy reveal that tau can indeed cause an impairment of transport in neurons. As a result, cell processes of neurons become starved, leading first to the decay of synapses and then to the loss of axons and dendrites.