Tau is a microtubule-associated protein found mainly in the axons of neurons in the brain. Abnormal changes in Tau (e.g., aggregation, hyperphosphorylation) are hallmarks of Alzheimer’s disease. Two processes of relocalization of Tau may be related to early states of the pathology and have received much attention: (1) the redistribution of Tau within cells (termed “somatodendritic missorting”) and (2) the release and reuptake of Tau from donor to acceptor cells (termed “spreading”). Because of the tripartite nature of neurons (cell body, dendrites, axons), these changes can be studied by microfluidic chambers (MFCs) which allow separation and observation of Tau in neuronal compartments. In this chapter, we present some methods and research results obtained by using microfluidic devices.

Significant efforts have been channeled into developing antibodies for the treatment of CNS indications. Disappointment with the first generation of clinical Tau antibodies in Alzheimer's disease has highlighted the challenges in understanding whether an antibody can reach or affect the target in the compartment where it is involved in pathological processes. Here, we highlight different aspects essential for improving translatability of Tau-based immunotherapy.

Intracellular accumulation of tau is a hallmark pathology in Alzheimer disease (AD) and the related tauopathies, thus targeting tau could be promising for drug development. Proteolysis Targeting Chimera (PROTAC) is a novel drug discovery strategy for selective protein degradation from within cells. Methods: A novel small-molecule PROTAC, named as C004019 with a molecular mass of 1,035.29 dalton, was designed to simultaneously recruite tau and E3-ligase (Vhl) and thus to selectively enhance ubiquitination and proteolysis of tau proteins. Western blotting, immunofluoresence and immunohistochemical staining were employed to verify the effects of C004019 in cell models (HEK293 and SH-SY5Y) and mouse models (hTau-transgenic and 3xTg-AD), respectively. The cognitive capacity of the mice was assessed by a suite of behavior experiments. Electrophysiology and Golgi staining were used to evaluate the synaptic plasticity. Results: C004019 induced a robust tau clearance via promoting its ubiquitination-proteasome-dependent proteolysis in HEK293 cells with stable or transient overexpression of human tau (hTau), and in SH-SY5Y that constitutively overexpress hTau. Furthermore, intracerebral ventricular infusion of C004019 induced a robust tau clearance in vivo. Most importantly, both single-dose and multiple-doses (once per 6 days for a total 5 times) subcutaneous administration of C004019 remarkably decreased tau levels in the brains of wild-type, hTau-transgenic and 3xTg-AD mice with improvement of synaptic and cognitive functions. Conclusions: The PROTAC (C004019) created in the current study can selectively and efficiently promote tau clearance both in vitro and in vivo, which provides a promising drug candidate for AD and the related tauopathies.

Tau aggregation is a hallmark of a group of neurodegenerative diseases termed Tauopathies. Reduction of aggregation-prone Tau has emerged as a promising therapeutic approach. Here, we show that an anti-aggregant Tau fragment (F3ΔKPP, residues 258–360) harboring the ΔK280 mutation and two proline substitutions (I277P & I308P) in the repeat domain can inhibit aggregation of Tau constructs in vitro, in cultured cells and in vivo in a Caenorhabditis elegans model of Tau aggregation. The Tau fragment reduced Tau-dependent cytotoxicity in a N2a cell model, suppressed the Tau-mediated neuronal dysfunction and ameliorated the defective locomotion in C. elegans. In vitro the fragment competes with full-length Tau for polyanionic aggregation inducers and thus inhibits Tau aggregation. Our combined in vitro and in vivo results suggest that the anti-aggregant Tau fragment may potentially be used to address the consequences of Tau aggregation in Tauopathies.

Missorting of MAPT/Tau represents one of the early signs of neurodegeneration in Alzheimer disease. The triggers for this are still a matter of debate. Here we investigated the sorting mechanisms of endogenous MAPT in mature primary neurons using microfluidic chambers (MFCs) where cell compartments can be observed separately. Blocking protein degradation pathways with proteasomal or autophagy inhibitors dramatically increased the missorting of MAPT in dendrites on the neuritic side, suggesting that degradation of MAPT in dendrites is a major determinant for the physiological axonal distribution of MAPT. Such missorted dendritic MAPT differed in its phosphorylation pattern from axonal MAPT. By contrast, enhancing autophagy or proteasomal pathways strongly reduced MAPT missorting, thereby confirming the role of protein degradation pathways in the polar distribution of MAPT. Dendritic missorting of MAPT by blocking protein degradation resulted in the loss of spines but not in overall cell toxicity. Inhibition of local protein synthesis in dendrites eliminated the missorting of MAPT, indicating that the accumulation of dendritic MAPT is locally generated. In support of this, a substantial fraction of Mapt/Tau mRNA was detected in dendrites. Taken together, our results indicate that the autophagy and proteasomal pathways play important roles in fine-tuning dendritic MAPT levels and thereby prevent synaptic toxicity caused by MAPT accumulation. Abbreviations Ani: anisomycin; Baf: bafilomycin A1; BSA: bovine serum albumin; cAMP: cyclic adenosine monophosphate; CHX: cycloheximide; DMSO: dimethyl sulfoxide; DIV: days in vitro; Epo: epoxomicin; E18: embryonic day 18; FISH: fluorescence in situ hybridization; IgG: immunoglobulin; kDa: kilodalton; Lac: lactacystin; LDH: lactate dehydrogenase; MFC: microfluidic chambers; MAPs: microtubule-associated proteins; MAPT/Tau: microtubule-associated protein tau; PVDF: polyvinylidene difluoride; PBS: phosphate-buffered saline; PRKA: protein kinase AMP-activated; RD150: round device 150; RT: room temperature; SDS: sodium dodecyl sulfate; SEM: standard error of the mean; Wor: wortmannin.

Tau is a microtubule-associated protein that has a role in stabilizing neuronal microtubules and thus in promoting axonal outgrowth. Structurally, tau is a natively unfolded protein, is highly soluble and shows little tendency for aggregation. However, tau aggregation is characteristic of several neurodegenerative diseases known as tauopathies. The mechanisms underlying tau pathology and tau-mediated neurodegeneration are debated, but considerable progress has been made in the field of tau research in recent years, including the identification of new physiological roles for tau in the brain. Here, we review the expression, post-translational modifications and functions of tau in physiology and in pathophysiology.

Focal epilepsies often originate in the hippocampal formation of the temporal lobe (temporal lobe epilepsy) and are generally acquired after transient brain insults. Such insults induce cellular and structural reorganization processes of the hippocampus, referred to as epileptogenesis that finally convert the brain spontaneous epileptic. Here, we developed a new molecular imaging strategy in a state-of-the-art animal model to provide insights into key epileptogenic mechanisms. Our new approach combines recombinant adeno-associated virus (rAAV) gene delivery with in vivo bioluminescence imaging. rAAV particles harboring the luciferase reporter gene under control of the minimal T type Ca(2+)-channel subunit Ca V 3.2-promoter were generated and injected stereotaxically in the hippocampal region of mice. Bioluminescent signals, corresponding to Ca V 3.2 promoter activation, were imaged in vivo in the pilocarpine model of status epilepticus (SE). We detected activation of key Ca V 3.2 promoter motifs at 3 and 10 days after SE but not after the onset of chronic seizures. These data suggest Ca V 3.2 promoter activation as novel anti-epileptogenic target. In more general terms, we have established an experimental approach that allows to follow cerebral gene promoter dynamics longitudinally and to correlate this activity to behavioral parameters in the same mice.

Tau aggregates are present in several neurodegenerative diseases and correlate with the severity of memory deficit in AD (Alzheimer's disease). However, the triggers of tau aggregation and tau-induced neurodegeneration are still elusive. The impairment of protein-degradation systems might play a role in such processes, as these pathways normally keep tau levels at a low level which may prevent aggregation. Some proteases can process tau and thus contribute to tau aggregation by generating amyloidogenic fragments, but the complete clearance of tau mainly relies on the UPS (ubiquitin-proteasome system) and the ALS (autophagy-lysosome system). In the present paper, we focus on the regulation of the degradation of tau by the UPS and ALS and its relation to tau aggregation. We anticipate that stimulation of these two protein-degradation systems might be a potential therapeutic strategy for AD and other tauopathies.

Modulating the Tau level may represent a therapeutic target for Alzheimer Disease, as accumulating evidence shows that Abeta-induced neurodegeneration is mediated by Tau. It is therefore important to understand the expression and degradation of Tau in neurons. Recently we showed that over expressed mutant Tau and Tau aggregates are degraded via the autophagic pathway in an N2a cell model, and how autophagy contributes to Tau cleavage, aggregation, and degradation (Wang et al., HMG2009). Here we investigated how different aspects of autophagy contribute to the degradation of Tau in cultured primary neurons. We further examined whether the stimulation of autophagy can reduce aggregation and alleviate cytotoxicity in a regulable N2a cell model of Tauopathy. Autophagy was induced by Trehalose in Neuroblastoma cells and rat primary neurons. The activation of autophagy was validated by Flux assays and fluorescence microscopy. Tau level was analysed by western blot with a pan-Tau antibody and a series of phosphorylation dependent antibodies: 12E8, PHF1, AT8 and AT180.Tau aggregates were separated with Sarkosyl extraction and visualized with Thioflavin S staining. Trehalose activated autophagy in rat cortical neurons, as seen by (1) elevated LC3-II levels, (2) an increased number of autophagosomes, (3) stimulated flux and (4) reduced p62 level, a known substrate of autophagy. At the same time Trehalose significantly reduced the endogenous Tau level. Phosphorylation seems to have no effect on Tau degradation by autophagy, as Tau phosphorylated at 12E8, PHF1, AT8 and AT180 sites was also decreased upon Trehalose treatment. In a N2a cell model of Tauopathy, Trehalose induced autophagy, which caused the reduction of Tau aggregation and toxicity. The activation of autophagy reduced endogenous Tau regardless of phosphorylation in primary cortical neurons and Tau aggregates in a N2a cell model of Tauopathy, indicating that stimulation of autophagy could serve as a therapeutic target.

The amyloid cascade hypothesis of AD posits that Aß triggers the neurofibrillary pathology of Tau. It was proposed that a toxic “17 kD” fragment of Tau, generated by the protease calpain, was induced by exposure of neurons to Aß. Its size (residues 45-230, MW=18.7kD) was estimated on the basis of potential cleavage sites in Tau by calpain (Park & Ferreira, J. Neurosci. 2005). In the current study, we sought to identify the exact nature of the “17 kD” fragment of Tau generated by calpain and its mode of toxicity. The limited proteolysis of Tau by calpain in vitro or in cultured neurons caused by treatment with Aß oligomers was analysed by western blotting. Calpain-induced fragments of Tau were identified by protein sequencing, mass spectrometry, and antibody labelling. Cytotoxicity of Tau fragments was monitored by LDH release assay or apoptotic assay. The presence of fragments in Alzheimer or control brains was assessed by immunoblotting. The “Mr=17 kD” fragment is actually much smaller than assumed so far, containing only residues 125-230 (MW=10.7kD). On SDS gels it runs at Mr∼17kD because of the anomalous behavior of Tau. Inducing Tau hyperphosphorylation by okadaic acid or mimicking phosphorylation by multiple Glu mutations did not prevent the generation of this fragment. The fragment was induced not only by Aß oligomers, but also by other cell stressors, e.g. thapsigargin or glutamate in cortical neurons. However, overexpression of neither Tau45-230 nor Tau125-230 fragment was toxic to neurons. Finally, the calpain-induced fragment was observed both in AD brains and in control normal human brains. The Mr=17 kD Tau fragment is a metastable cleavage product of calpain and comprises residues 125-230. The fragment is induced by cell stress, including exposure to Aß, but it is not a mediator of Aß-induced toxicity. This suggests that events upstream of calpain activation might cause both Tau fragmentation and toxicity. - Supported by: EU/Memosad, BMBF/KNDD, Breuer Fund, Metlife Fund.

Modulating the tau level may represent a therapeutic target for Alzheimer's disease (AD), as accumulating evidence shows that Abeta-induced neurodegeneration is mediated by tau. It is therefore important to understand the expression and degradation of tau in neurons. Recently we showed that overexpressed mutant tau and tau aggregates are degraded via the autophagic pathway in an N2a cell model. Here we investigated whether autophagy is involved in the degradation of endogenous tau in cultured primary neurons. We activated this pathway in primary neurons with trehalose, an enhancer of autophagy. This resulted in the reduction of endogenous tau protein. Tau phosphorylation at several sites elevated in AD pathology had little influence on its degradation by autophagy. Furthermore, by using a neuronal cell model of tauopathy, we showed that activation of autophagy suppresses tau aggregation and eliminates cytotoxicity. Notably, apart from activating autophagy, trehalose also inhibits tau aggregation directly. Thus, trehalose may be a good candidate for developing therapeutic strategies for AD and other tauopathies.

The amyloid cascade hypothesis of Alzheimer's disease (AD) posits that the generation of β-amyloid (Aβ) triggers Tau neurofibrillary pathology. Recently a "17 kD" calpain-induced Tau fragment, comprising residues 45-230 (molecular weight [MW], 18.7 kD), was proposed to mediate Aβ-induced toxicity. Here, we demonstrate that the "17 kD" fragment is actually much smaller, containing residues 125-230 (molecular weight, 10.7 kD). Inducing Tau phosphorylation by okadaic acid or mimicking phosphorylation by Glu mutations at the epitopes of Alzheimer-diagnostic antibodies AT100/AT8/PHF1 could not prevent the generation of this fragment. The fragment can be induced not only by Aβ oligomers, but also by other cell stressors, e.g., thapsigargin (a Ca(2+)-ATPase inhibitor) or glutamate (an excitatory neurotransmitter). However, overexpression of neither Tau(45-230) nor Tau(125-230) fragment is toxic to Chinese hamster ovary (CHO) cells, neuroblastoma cells (N2a) or primary hippocampal neurons. Finally, the calpain-induced fragment can be observed both in Alzheimer's disease brains and in control normal human brains. We conclude that the 17 kD Tau fragment is not a mediator of Aβ-induced toxicity, leaving open the possibility that upstream calpain activation might cause both Tau fragmentation and toxicity.

Tau aggregation characterizes a series of neurodegenerative diseases including AD and other tauopathies. The distribution of Tau deposits correlates with the loss of neurons in these neurodegenerative diseases, and Tau-induced toxicity depends on its ability to aggregate. We have used an inducible cell model to study the expression of Tau variants, the buildup of aggregates, and their removal by the autophagy-lysosomal system. Incomplete chaperone-mediated autophagy of Tau generates amyloidogenic fragments that promote aggregation. The Tau aggregates are removed from cells by macroautophagy. Thus the two autophagic pathways could become possible therapeutic targets.

Aggregation and cleavage are two hallmarks of Tau pathology in Alzheimer disease (AD), and abnormal fragmentation of Tau is thought to contribute to the nucleation of Tau paired helical filaments. Clearance of the abnormally modified protein could occur by the ubiquitin-proteasome and autophagy-lysosomal pathways, the two major routes for protein degradation in cells. There is a debate on which of these pathways contributes to clearance of Tau protein and of the abnormal Tau aggregates formed in AD. Here, we demonstrate in an inducible neuronal cell model of tauopathy that the autophagy-lysosomal system contributes to both Tau fragmentation into pro-aggregating forms and to clearance of Tau aggregates. Inhibition of macroautophagy enhances Tau aggregation and cytotoxicity. The Tau repeat domain can be cleaved near the N terminus by a cytosolic protease to generate the fragment F1. Additional cleavage near the C terminus by the lysosomal protease cathepsin L is required to generate Tau fragments F2 and F3 that are highly amyloidogenic and capable of seeding the aggregation of Tau. We identify in this work that components of a selective form of autophagy, chaperone-mediated autophagy, are involved in the delivery of cytosolic Tau to lysosomes for this limited cleavage. However, F1 does not fully enter the lysosome but remains associated with the lysosomal membrane. Inefficient translocation of the Tau fragments across the lysosomal membrane seems to promote formation of Tau oligomers at the surface of these organelles which may act as precursors of aggregation and interfere with lysosomal functioning.

Tau is a highly soluble protein, and yet it aggregates abnormally in Alzheimer's disease. This begs the question of what factor(s) cause the pathological aggregation. It is known that in vitro, tau fragments derived from the microtubule-binding repeat domain aggregate more readily than full-length tau. This suggests the possibility that fragmentation of tau by proteolysis could lead to the nucleation and aggregation of full-length tau; however, the mechanism and pathway of such a fragmentation has remained ill-defined. We have addressed this problem in neuronal cell models (N2a cells) expressing the repeat domain of tau or tau mutants in an inducible fashion. The cells show abnormal aggregation of tau similar to Alzheimer aggregates, but notably the aggregation is preceded by fragmentation. We have followed the fate of the pre-aggregation fragments and determined the cleavage sites. Fragments are generated in a step-wise fashion, starting with a cleavage near the N-terminus of the tau construct, followed by successive cleavages near the C-terminus. The N-terminal cleavage is a prerequisite for further processing, suggesting that it opens up a conformation which becomes more susceptible to further cleavage. Fragments generated by cleavage near both ends are highly competent to aggregate and can nucleate the aggregation of full-length tau, both in vitro and in the cell models. Both the C-terminal truncation and the co-aggregation of fragments with full-length tau depend on the propensity for β-structure. If beta structure is eliminated, e.g. by proline-mutations in the hexapeptide motifs that are responsible for nucleating the aggregation, no C-termina cleavage and no aggregation takes place. Thus the toxicity of tau in cells can be prevented by inhibiting either aggregation or proteolysis. Supported by MPG and DFG.

The pathological aggregation of tau into paired helical filaments is a hallmark of several neurodegenerative diseases, including Alzheimer's disease. We have generated cell models of tau aggregation in order to study mechanisms involving abnormal changes of tau. In the cell models the repeat domain of tau (tau(RD)) and some of its variants are expressed in a regulated fashion, e. g. the 4-repeat domain of tau with the wild-type sequence, the repeat domain with the Delta K280 mutation ("pro-aggregation mutant"), or the repeat domain with additional proline mutations ("anti-aggregation mutant"). The aggregation of tauRD is toxic to the cells, but aggregation and toxicity can be prevented by low molecular weight compounds identified by a screen for inhibitors. Thus the cell models are suitable for developing aggregation inhibitor drugs and testing their cellular roles.

The activity of protein phosphatase (PP)‐2A and PP‐1 decreased in the brains of Alzheimer’s disease and inhibition of the phosphatases led to spatial memory deficit in rats. However, the molecular basis underlying memory impairment of the phosphatase inhibition is elusive. In the present study, we observed a selective inhibition of PP‐2A and PP‐1 with Calyculin A (CA) not only caused hyperphosphorylation of cytoskeletal proteins, but also impaired the transport of pEGFP‐labeled neurofilament‐M subunit in the axon‐like processes of neuroblastoma N2a cells and resulted in accumulation of neurofilament in the cell bodies. To analyze the morphological alteration of the cells during inhibition of the phosphatases, we established a cell model showing steady outgrowth of axon‐like cell processes and employed a stereological system to analyze the retraction of the processes. We found CA treatment inhibited outgrowth of the cell processes and prolonged treatment with CA caused retraction of the processes and meanwhile, the early neurodegenerative varicosities were also obvious in the CA‐treated cells. We conclude suppression of PP‐2A and PP‐1 by CA not only damages intracellular transport but also leads to cell degeneration, which may serve as the functional and structural elements for the memory deficits induced by suppression of the phosphatases.

In the course of our studies to develop drugs that prevent tau protein aggregation and degeneration of neurons in tauopathies, we recently generated several cell models of tauopathy. They are based on the N2a neuroblastoma cell line and express different variants of the repeat domain of tau (tauRD) in an inducible fashion (Tet–on): the wild–type sequence, a pro–aggregation mutant (ΔK280), and an anti–aggregation mutant, containing additional proline residues. The cells expressing K18ΔK280 mutant show robust aggregation of tau. The aggregates are toxic to cells and their removal is beneficial. We also found that fragmentation of tauRD is important for initiation of aggregation and that phosphorylation in the repeat domain cannot be considered as a precursor of PHF–tauRD. To find out which protease(s) are responsible for the fragments generated from K18ΔK280 in N2a Tet–On cells, we used different protease inhibitors against caspases, proteasome, calpain and thrombin. Only the thrombin inhibitor PPACK inhibited the fragmentation of tauRD suggesting the involvement of a thrombin–like activity in tau aggregation in this cell system. We have now obtained a significantly improved N2a cell model generating tau aggregates composed of full length tau molecules. In order to evaluate the influence of the phosphorylation at SP or TP motifs (in the flanking region of the repeats, targets of proline–directed kinases) on aggregation, we generated a new cell model expressing the full–length isoform htau40ΔK280 and observed its aggregation in the N2a cell model. The aggregation was characterized by three methods: 1) on the biochemical level, the presence of aggregates was demonstrated by sarcosyl extraction of the cells and analysis of soluble and insoluble components, 2) fluorescence microscopy using ThS fluorescence which reports on the propensity of the protein to form β–structure, and finally 3) we visualized PHFs by electron microscopy after density gradient enrichment and gold labeling.

Here we report the generation of several cell models of tau pathology in order to study tau's mechanisms of neurodegeneration in Alzheimer's and other brain diseases. N2a neuroblastoma cell lines were created that inducibly express different variants of tau when exposed to doxycyclin. Three variants of tauRD were chosen, based on the repeat domain of full–length htau40: the 4R wild–type repeat domain (K18), the 4R repeat domain with the deletion mutation delK280 known from frontotemporal dementia and highly prone to spontaneous aggregation (pro–aggregation mutant), and the 4R repeat domain with delK280 and two further proline point mutations that strongly inhibit aggregation (anti–aggregation mutant). The comparison of these wild–type, pro–aggregation and anti–aggregation tau constructs shows: (1) Tau aggregation into paired helical filaments is toxic to cells, (2) the degree of aggregation and toxicity strongly depends on tau's propensity for beta–structure, (3) soluble tau mutants that cannot aggregate are also not toxic; (4) tau phosphorylation in the repeat domain (at KXGS motifs) precedes aggregation but is not correlated with the degree of aggregation; (5) tau aggregates disappear when the tau expression is silenced, showing that aggregation is reversible; (6) Tau aggregation can be prevented by drugs, and even pre–formed aggregates can be dissolved again by drugs. Thus, the cell models open up new insights into the relationship between tau structure, expression, phosphorylation, aggregation, and toxicity that can be used in the screening drugs for AD and other tauopathies.