Aggregation of amyloid-␤ (A␤) and Tau protein are hallmarks of Alzheimer's disease (AD), and according to the A␤-cascade hypothesis, A␤ is considered toxic for neurons and Tau a downstream target of A␤. We have investigated differentiated primary hippocampal neurons for early localized changes following exposure to A␤ 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 Ca 2ϩ , 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 GSK3␤, 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 A␤-induced effects on microtubule and mitochondria depletion, Tau missorting, and loss of spines are prevented by taxol, indicating that A␤-induced microtubule destabilization and corresponding traffic defects are key factors in incipient degeneration. By contrast, the rise in Ca 2ϩ levels, kinase activities, and Tau phosphorylation cannot be prevented by taxol. Incipient and local changes similar to those of A␤ oligomers can be evoked by cell stressors (e.g., H 2 O 2 , glutamate, serum deprivation), suggesting some common mechanism of signaling.

Mislocalization and aggregation of Ab and Tau combined with loss of synapses and microtubules (MTs) are hallmarks of Alzheimer disease. We exposed mature primary neurons to Ab oligomers and analysed changes in the Tau/ MT system. MT breakdown occurs in dendrites invaded by Tau (Tau missorting) and is mediated by spastin, an MT-severing enzyme. Spastin is recruited by MT polyglutamylation, induced by Tau missorting triggered translocalization of TTLL6 (Tubulin-Tyrosine-Ligase-Like-6) into dendrites. Consequences are spine loss and mitochondria and neurofilament mislocalization. Missorted Tau is not axonally derived, as shown by axonal retention of photoconvertible Dendra2-Tau, but newly synthesized. Recovery from Ab insult occurs after Ab oligomers lose their toxicity and requires the kinase MARK (Microtubule-AffinityRegulating-Kinase). In neurons derived from Tau-knockout mice, MTs and synapses are resistant to Ab toxicity because TTLL6 mislocalization and MT polyglutamylation are prevented; hence no spastin recruitment and no MT breakdown occur, enabling faster recovery. Reintroduction of Tau re-establishes Ab-induced toxicity in TauKO IntroductionAlzheimer disease (AD) is characterized by two types of abnormal protein aggregates in the brain, 'amyloid plaques' made up of Ab peptides, and 'neurofibrillary tangles' assembled from Tau protein Mucke and Selkoe, 2012). In familial AD, genetic evidence points to a master role of Ab aggregation, leading to downstream events including Tau hyperphosphorylation and aggregation (Haass and Selkoe, 2007;Karran et al, 2011). On the other hand, the distribution of Tau aggregates in the brain correlates better with the clinical progression of AD (Braak stages; Braak and Braak, 1991), and experiments with transgenic mice suggest a critical role for Tau in mediating Ab-induced toxicity.A key to understand the normal and toxic roles of Tau lies in the neuronal cytoskeleton, notably microtubules (MTs), the tracks of intracellular transport, and their interaction partners. Tau changes during development include (i) a shift from shorter fetal to longer adult isoforms, (ii) decreased phosphorylation (from a higher fetal to a lower adult level), and (iii) changed intracellular distribution (from ubiquitous to axonal) (Mandell and Banker, 1995;Bullmann et al, 2009). Notably, the early cytoskeletal changes in AD, such as lower MT stability, hyperphosphorylation, and missorting of Tau to the somatodendritic compartment, are reminiscent of the fetal state. In TauKO mice, the lack of Tau during development can be compensated by upregulation of other neuronal microtubule associated proteins (MAPs), for example, MAP1A (Harada et al, 1994), which may explain the mild phenotype. Depending on experimental conditions, the absence of Tau may have detrimental effects (inefficient axon outgrowth and neurodegeneration; Dawson et al, 2001;Dawson et al, 2010), or beneficial ones (reduction in Ab and excitotoxicity; King et al, 2006;Roberson et al, 2007;Ittner et al, 2010). In neuronal cell culture, ...

Missorting of Tau from axons to the somatodendritic compartment of neurons is a hallmark of Alzheimer's disease, but the mechanisms underlying normal sorting and pathological failure are poorly understood. Here, we used several Tau constructs labelled with photoconvertible Dendra2 to analyse its mobility in polarized neurons. This revealed a novel mechanism of sorting-a retrograde barrier in the axon initial segment (AIS) operating as cellular rectifier. It allows anterograde flow of axonal Tau but prevents retrograde flow back into soma and dendrites. The barrier requires binding of Tau to microtubules but does not require F-actin and thus is distinct from the sorting of membrane-associated proteins at the AIS. The barrier breaks down when Tau is phosphorylated in its repeat domain and detached from microtubules, for example, by the kinase MARK/Par1. These observations link the pathological hallmarks of Tau missorting and hyperphosphorylation in neurodegenerative diseases.

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Subcellular mislocalization of the microtubule-associated protein Tau is a hallmark of Alzheimer disease (AD) and other tauopathies. Six Tau isoforms, differentiated by the presence or absence of a second repeat or of N-terminal inserts, exist in the human CNS, but their physiological and pathological differences have long remained elusive. Here, we investigated the properties and distributions of human and rodent Tau isoforms in primary forebrain rodent neurons. We found that the Tau diffusion barrier (TDB), located within the axon initial segment (AIS), controls retrograde (axon-to-soma) and anterograde (soma-to-axon) traffic of Tau. Tau isoforms without the N-terminal inserts were sorted efficiently into the axon. However, the longest isoform (2N4R-Tau) was partially retained in cell bodies and dendrites, where it accelerated spine and dendrite growth. The TDB (located within the AIS) was impaired when AIS components (ankyrin G, EB1) were knocked down or when glycogen synthase kinase-3β (GSK3β; an AD-associated kinase tethered to the AIS) was overexpressed. Using superresolution nanoscopy and live-cell imaging, we observed that microtubules within the AIS appeared highly dynamic, a feature essential for the TDB. Pathomechanistically, amyloid-β insult caused cofilin activation and F-actin remodeling and decreased microtubule dynamics in the AIS. Concomitantly with these amyloid-β-induced disruptions, the AIS/TDB sorting function failed, causing AD-like Tau missorting. In summary, we provide evidence that the human and rodent Tau isoforms differ in axodendritic sorting and amyloid-β-induced missorting and that the axodendritic distribution of Tau depends on AIS integrity.

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In Alzheimer Disease (AD), the mechanistic connection of the two major pathological hallmarks, namely deposition of Amyloid-beta (Aβ) in the form of extracellular plaques, and the pathological changes of the intracellular protein Tau (such as phosphorylation, missorting, aggregation), is not well understood. Genetic evidence from AD and Down Syndrome (Trisomy 21), and animal models thereof, suggests that aberrant production of Aβ is upstream of Tau aggregation, but also points to Tau as a critical effector in the pathological process. Yet, the cascade of events leading from increased levels of Aβ to Tau-dependent toxicity remains a matter of debate.Using primary neurons exposed to oligomeric forms of Aβ, we have found that Tau becomes mislocalized (missorted) into the somatodendritic compartment. Missorting of Tau correlates with loss of microtubules and downstream consequences such as loss of mature spines, loss of synaptic activity, and mislocalization of mitochondria.In this cascade, missorting of Tau induces mislocalization of TTLL6 (Tubulin-Tyrosine-Ligase-Like 6) into the dendrites. TTLL6 induces polyglutamylation of microtubules, which acts as a trigger for spastin mediated severing of dendritic microtubules. Loss of microtubules makes cells unable to maintain transport of mitochondria, which in turn results in synaptic dysfunction and loss of mature spines. These pathological changes are absent in TauKO derived primary neurons. Thus, Tau mediated mislocalization of TTLL6 and spastin activation reveals a pathological gain of function for Tau and spastin in this cellular model system of AD.In contrast, in hereditary spastic paraplegia (HSP) caused by mutations of the gene encoding spastin (spg4 alias SPAST), spastin function in terms of microtubule severing is decreased at least for the gene product of the mutated allele, resulting in overstable microtubules in disease model systems. Whether total spastin severing activity or microtubule stability in human disease is also affected is not yet clear. No human disease has been associated so far with the long-chain polyglutamylation enzyme TTLL6, or the other TTLLs (1,5,11) possibly involved.Here we review the findings supporting a role for Tau, spastin and TTLL6 in AD and other tauopathies, HSP and neurodegeneration, and summarize possible therapeutic approaches for AD and HSP.

Amyloid-β peptide (Aβ) forms metastable oligomers >50 kDa, termed AβOs, that are more effective than Aβ amyloid fibrils at triggering Alzheimer’s disease-related processes such as synaptic dysfunction and Tau pathology, including Tau mislocalization. In neurons, Aβ accumulates in endo-lysosomal vesicles at low pH. Here, we show that the rate of AβO assembly is accelerated 8,000-fold upon pH reduction from extracellular to endo-lysosomal pH, at the expense of amyloid fibril formation. The pH-induced promotion of AβO formation and the high endo-lysosomal Aβ concentration together enable extensive AβO formation of Aβ42 under physiological conditions. Exploiting the enhanced AβO formation of the dimeric Aβ variant dimAβ we furthermore demonstrate targeting of AβOs to dendritic spines, potent induction of Tau missorting, a key factor in tauopathies, and impaired neuronal activity. The results suggest that the endosomal/lysosomal system is a major site for the assembly of pathomechanistically relevant AβOs.