In a previous study, regional reductions in cerebral glucose metabolism have been demonstrated in the tauopathy mouse model rTg4510 (Endepols et al., 2022). Notably, glucose hypometabolism was present in some brain regions without co-localized synaptic degeneration measured with [18F]UCB-H. We hypothesized that in those regions hypometabolism may reflect reduced functional connectivity rather than synaptic damage. To test this hypothesis, we performed seed-based metabolic connectivity analyses using [18F]FDG-PET data in this mouse model. Eight rTg4510 mice at the age of seven months and 8 non-transgenic littermates were injected intraperitoneally with 11.1 ± 0.8 MBq [18F]FDG and spent a 35-min uptake period awake in single cages. Subsequently, they were anesthetized and measured in a small animal PET scanner for 30 min. Three seed-based connectivity analyses were performed per group. Seeds were selected for apparent mismatch between [18F]FDG and [18F]UCB-H. A seed was placed either in the medial orbitofrontal cortex, dorsal hippocampus or dorsal thalamus, and correlated with all other voxels of the brain across animals. In the control group, the emerging correlative pattern was strongly overlapping for all three seed locations, indicating a uniform fronto-thalamo-hippocampal resting state network. In contrast, rTg4510 mice showed three distinct networks with minimal overlap. Frontal and thalamic networks were greatly diminished. The hippocampus, however, formed a new network with the whole parietal cortex. We conclude that resting-state functional networks are fragmented in the brain of rTg4510 mice. Thus, hypometabolism can be explained by reduced functional connectivity of brain areas devoid of tau-related pathology, such as the thalamus.

Ziel/Aim Transgenic mouse models are frequently used to study the mechanisms of neurodegenerative diseases and possible therapeutic options. We investigated the tauopathy mouse model TauRDΔK, which expresses the repeat domain of human tau with deletion of the amino acid K280. TauRDΔK mice show a slow disease progression which makes them a suitable model for therapy studies. Unexpectedly, the PET tracer [18F]MNI-1126, which binds to the synaptic vesicle protein SV2A, showed a significantly increased uptake in the brain of 16–18 months old TauRDΔK mice in the summed image analysis (n=9; SUVRCer 0.98±0.03) compared to controls (n=7; SUVRCer: 0.94±0.02; p<0.005). This is in contrast to studies with other tauopathy mouse models, which all demonstrated reduced SV2A binding.

The stereotypical progression of Tau pathology during Alzheimer disease has been attributed to trans-neuronal spreading of misfolded Tau proteins, followed by prion-like templated aggregation of Tau. The nature of Tau and the cellular mechanisms of Tau spreading are still under debate. We hypothesized that Tau’s propensity for aggregation would correlate with its ability to spread across synapses and propagate pathology. To study the progressive propagation of Tau proteins in brain regions relevant for Alzheimer disease, we used mice expressing near-physiological levels of full-length human Tau protein carrying pro-aggregant (TauΔK280, TauΔK) or anti-aggregant (TauΔK280-PP, TauΔK−PP) mutations in the entorhinal cortex (EC). To enhance Tau expression in the EC, we performed EC injections of adeno-associated virus (AAV) particles encoding TauΔK or TauΔK−PP. The brains of injected and non-injected EC/TauΔK and EC/TauΔK−PP mice were studied by immunohistological and biochemical techniques to detect Tau propagation to dentate gyrus (DG) neurons and Tau-induced pathological changes. Pro- and anti-aggregant mice had comparable low transgene expression ( 0.2 times endogenous mouse Tau). They accumulated human Tau at similar rates and only in expressing EC neurons, including their axonal projections of the perforant path and presynaptic terminals in the molecular layer of the DG. Pro-aggregant EC/TauΔK mice showed misfolded Tau and synaptic protein alterations in EC neurons, not observed in anti-aggregant EC/TauΔK−PP mice. Additional AAV-mediated expression of TauΔK or TauΔK−PP in EC/TauΔK or EC/TauΔK−PP mice, respectively, increased the human Tau expression to 0.65 times endogenous mouse Tau, with comparable spreading of TauΔK and TauΔK−PP throughout the EC. There was a low level of transcellular propagation of Tau protein, without pathological phosphorylation or misfolding, as judged by diagnostic antibodies. Additionally, TauΔK but not TauΔK−PP expression induced hippocampal astrogliosis. Low levels of pro- or anti-aggregant full-length Tau show equivalent distributions in EC neurons, independent of their aggregation propensity. Increasing the expression via AAV induce local Tau misfolding in the EC neurons, synaptotoxicity, and astrogliosis and lead to a low level of detectable trans-neuronal spreading of Tau. This depends on its concentration in the EC, but, contrary to expectations, does not depend on Tau’s aggregation propensity/misfolding and does not lead to templated misfolding in recipient neurons.

Microtubule-associated protein tau is a naturally unfolded protein that can modulate a vast array of physiological processes through direct or indirect binding with molecular partners. Aberrant tau homeostasis has been implicated in the pathogenesis of several neurodegenerative disorders, including Alzheimer's disease. In this study, we performed an unbiased high-content protein profiling assay by incubating recombinant human tau on microarrays containing thousands of human polypeptides. Among the putative tau-binding partners, we identify SAH hydrolase-like protein 1/inositol 1,4,5-trisphosphate receptor (IP3R)-binding protein (AHCYL1/IRBIT), a member of the SAH hydrolase family and a previously described modulator of IP3R activity. Using coimmunoprecipitation assays, we show that endogenous as well as overexpressed tau can physically interact with AHCYL1/IRBIT in brain tissues and cultured cells. Proximity ligation assay experiments demonstrate that tau overexpression may modify the close localization of AHCYL1/IRBIT to IP3R at the endoplasmic reticulum. Together, our experimental evidence indicates that tau interacts with AHCYL1/IRBIT and potentially modulates AHCYL1/IRBIT function.

Cerebral glucose hypometabolism is a typical hallmark of Alzheimer’s disease (AD), usually associated with ongoing neurodegeneration and neuronal dysfunction. However, underlying pathological processes are not fully understood and reproducibility in animal models is not well established. The aim of the present study was to investigate the regional interrelation of glucose hypometabolism measured by [18F]FDG positron emission tomography (PET) with various molecular targets of AD pathophysiology using the PET tracers [18F]PI-2620 for tau deposition, [18F]DPA-714 for TSPO expression associated with neuroinflammation, and [18F]UCB-H for synaptic density in a transgenic tauopathy mouse model. Seven-month-old rTg4510 mice (n = 8) and non-transgenic littermates (n = 8) were examined in a small animal PET scanner with the tracers listed above. Hypometabolism was observed throughout the forebrain of rTg4510 mice. Tau pathology, increased TSPO expression, and synaptic loss were co-localized in the cortex and hippocampus and correlated with hypometabolism. In the thalamus, however, hypometabolism occurred in the absence of tau-related pathology. Thus, cerebral hypometabolism was associated with two regionally distinct forms of molecular pathology: (1) characteristic neuropathology of the Alzheimer-type including synaptic degeneration and neuroinflammation co-localized with tau deposition in the cerebral cortex, and (2) pathological changes in the thalamus in the absence of other markers of AD pathophysiology, possibly reflecting downstream or remote adaptive processes which may affect functional connectivity. Our study demonstrates the feasibility of a multitracer approach to explore complex interactions of distinct AD-pathomechanisms in vivo in a small animal model. The observations demonstrate that multiple, spatially heterogeneous pathomechanisms can contribute to hypometabolism observed in AD mouse models and they motivate future longitudinal studies as well as the investigation of possibly comparable pathomechanisms in human patients.

RTP801/REDD1 is a stress-regulated protein whose upregulation is necessary and sufficient to trigger neuronal death. Its downregulation in Parkinson's and Huntington's disease models ameliorates the pathological phenotypes. In the context of Alzheimer's disease (AD), the coding gene for RTP801, DDIT4, is responsive to Aβ and modulates its cytotoxicity in vitro. Also, RTP801 mRNA levels are increased in AD patients' lymphocytes. However, the involvement of RTP801 in the pathophysiology of AD has not been yet tested. Here, we demonstrate that RTP801 levels are increased in postmortem hippocampal samples from AD patients. Interestingly, RTP801 protein levels correlated with both Braak and Thal stages of the disease and with GFAP expression. RTP801 levels are also upregulated in hippocampal synaptosomal fractions obtained from murine 5xFAD and rTg4510 mice models of the disease. A local RTP801 knockdown in the 5xFAD hippocampal neurons with shRNA-containing AAV particles ameliorates cognitive deficits in 7-month-old animals. Upon RTP801 silencing in the 5xFAD mice, no major changes were detected in hippocampal synaptic markers or spine density. Importantly, we found an unanticipated recovery of several gliosis hallmarks and inflammasome key proteins upon neuronal RTP801 downregulation in the 5xFAD mice. Altogether our results suggest that RTP801 could be a potential future target for theranostic studies since it could be a biomarker of neuroinflammation and neurotoxicity severity of the disease and, at the same time, a promising therapeutic target in the treatment of AD.

BACKGROUND:One of the major hallmarks of Alzheimer's disease (AD)is the aberrant modification and aggregation of the microtubule-associated protein Tau . The extent of Tau pathology correlates with cognitive decline, strongly implicating Tau in the pathogenesis of the disease. Because the inhibition of Tau aggregation may be a promising therapeutic target, we tested the efficacy of BSc3094, an inhibitor of Tau aggregation, in reducing Tau pathology and ameliorating the disease symptoms in transgenic mice. METHODS:Mice expressing human Tau with the P301L mutation (line rTg4510) were infused with BSc3094 into the lateral ventricle using Alzet osmotic pumps connected to a cannula that was placed on the skull of the mice, thus bypassing the blood-brain barrier (BBB) . The drug treatment lasted for 2 months, and the effect of BSc3094 on cognition and on reversing hallmarks of Tau pathology was assessed. RESULTS:BSc3094 significantly reduced the levels of Tau phosphorylation and sarkosyl-insoluble Tau. In addition, the drug improved cognition in different behavioral tasks and reduced anxiety-like behavior in the transgenic mice used in the study. CONCLUSIONS:Our in vivo investigations demonstrated that BSc3094 is capable of partially reducing the pathological hallmarks typically observed in Tau transgenic mice, highlighting BSc3094 as a promising compound for a future therapeutic approach for AD.

Abnormal changes of neuronal Tau protein, such as phosphorylation and aggregation, are considered hallmarks of cognitive deficits in Alzheimer's disease. Abnormal phosphorylation is thought to precede aggregation and therefore to promote aggregation, but the nature and extent of phosphorylation remain ill-defined. Tau contains ∼85 potential phosphorylation sites, which can be phosphorylated by various kinases because the unfolded structure of Tau makes them accessible. However, methodological limitations (e.g. in MS of phosphopeptides, or antibodies against phosphoepitopes) led to conflicting results regarding the extent of Tau phosphorylation in cells. Here we present results from a new approach based on native MS of intact Tau expressed in eukaryotic cells (Sf9). The extent of phosphorylation is heterogeneous, up to ∼20 phosphates per molecule distributed over 51 sites. The medium phosphorylated fraction Pm showed overall occupancies of ∼8 Pi (± 5) with a bell-shaped distribution; the highly phosphorylated fraction Ph had 14 Pi (± 6). The distribution of sites was highly asymmetric (with 71% of all P-sites in the C-terminal half of Tau). All sites were on Ser or Thr residues, but none were on Tyr. Other known posttranslational modifications were near or below our detection limit (e.g. acetylation, ubiquitination). These findings suggest that normal cellular Tau shows a remarkably high extent of phosphorylation, whereas other modifications are nearly absent. This implies that abnormal phosphorylations at certain sites may not affect the extent of phosphorylation significantly and do not represent hyperphosphorylation. By implication, the pathological aggregation of Tau is not likely a consequence of high phosphorylation.

Tau aggregation into amyloid fibers based on the cross-beta structure is a hallmark of several Tauopathies, including Alzheimer Disease (AD). Trans-cellular propagation of Tau with pathological conformation has been suggested as a key disease mechanism. This is thought to cause the spreading of Tau pathology in AD by templated conversion of naive Tau in recipient cells into a pathological state, followed by assembly of pathological Tau fibers, similar to the mechanism of nucleated polymerization proposed for prion pathogenesis. In cell cultures, the process is often monitored by a FRET assay where the recipient cell expresses the Tau repeat domain (TauRD) with a pro-aggregant mutation, fused to GFP-based FRET pairs. Since the size of the reporter GFP (barrel of ~ 3 nm × 4 nm) is ~ 7 times larger than the β-strand distance (0.47 nm), this points to a potential steric clash. Hence, we investigated the influence of the GFP tag on TauFL or TauRD aggregation. Using biophysical methods (light scattering, atomic force microscopy (AFM), and scanning-transmission electron microscopy (STEM)), we found that the assembly of TauRD-GFP was severely inhibited and incompatible with that of Alzheimer filaments. These observations argue against the hypothesis that the propagation of Tau pathology in AD is caused by the prion-like templated aggregation of Tau protein, transmitted via cell-to-cell spreading of Tau. Thus, even though the observed local increase of FRET in recipient cells may be a valid hallmark of a pathological reaction, our data argue that it is caused by a process distinct from assembly of TauRD filaments.

Abstract Introduction Tau, a natively unfolded soluble protein, forms abnormal oligomers and insoluble filaments in several neurodegenerative diseases, including Alzheimer disease (AD). Tau‐induced toxicity is mainly due to oligomers rather than monomers or fibrils. Methods We have developed monoclonal antibodies against purified low‐n tau oligomers of the tau repeat domain as a tool to neutralize tau aggregation and toxicity. In vitro aggregation inhibition was tested by thioflavin S, dynamic light scattering (DLS), and atomic force microscopy (AFM). Using a split‐luciferase complementation assay and fluorescence‐activated cell sorting (FACS), the inhibition of aggregation was analyzed in an N2a cell model of tauopathy. Results Antibodies inhibited tau aggregation in vitro up to ~90% by blocking tau at an oligomeric state. Some antibodies were able to block tau dimerization/oligomerization in cells, as measured by a split‐luciferase complementation assay. Antibodies applied extracellularly were internalized and led to sequestration of tau into lysosomes for degradation. Discussion Novel low‐n tau oligomer specific monoclonal antibody inhibits Tau oligomerization in cells and promotes toxic tau clearance.

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.

Background Aggregation of tau proteins is a distinct hallmark of tauopathies and has been a focus of research and clinical trials for Alzheimer’s Disease. Recent reports have pointed towards a toxic effect of soluble or oligomeric tau in the spreading of tau pathology in Alzheimer’s disease. Here we investigated the effects of expressing human tau repeat domain (tauRD) with pro- or anti-aggregant mutations in regulatable transgenic mouse models of Alzheimer’s Disease on the functional neuronal networks and the structural connectivity strength. Methods Pro-aggregant and anti-aggregant mice were studied when their mutant tauRD was switched on for 12 months to reach the stage where pro-aggregant mice show cognitive impairment, whereas anti-aggregant mice remained cognitively normal. Then, mutant tauRD was switched off by doxycycline treatment for 8 weeks so that soluble transgenic tau disappeared and cognition recovered in the pro-aggregant mice, although some aggregates remained. At these two time points, at baseline after 12 months of mutant tau expression and after 8 weeks of doxycycline treatment, resting state fMRI and diffusion MRI were used to determine functional neuronal networks and fiber connectivities. Results of the transgenic mice were compared with wildtype littermates. Results Functional connectivity was strongly reduced in transgenic animals during mutant tauRD expression, in relation to WT mice. Interestingly, transgenic mice with the non-aggregant tau mutant showed identical functional deficits as the pro-aggregant mice, even though in this case there was no cognitive decline by behavioral testing. Upon 8 weeks doxycycline treatment and transgene switch-off, functional connectivity in both transgenic groups presented complete normalization of functional connectivity strength, equivalent to the situation in WT littermates. Structural connectivity was found only marginally sensitive to mutant tau expression (both pro- and anti-aggregant tauRD) and by doxycycline treatment. Conclusions Our in vivo investigations unravel for the first time a strong reduction of functional neuronal networks by the presence of increased soluble rather than fibrillary tau, independent of its intrinsic propensity of aggregation, which is reversible by switching tau off. Our functional MRI study thus is an unexpected in vivo validation of a novel property of tau, while previous results pointed to a role of aggregation propensity for a pathological state by histopathology and cognitive decline. Our results present further evidence for early tauopathy biomarkers or a potential early stage drug target by functional networks analysis.

Mutations in the microtubule-associated protein tau (MAPT) underlie multiple neurodegenerative disorders, yet the pathophysiological mechanisms are unclear. A novel variant in MAPT resulting in an alanine to threonine substitution at position 152 (A152T tau) has recently been described as a significant risk factor for both frontotemporal lobar degeneration and Alzheimer's disease. Here we use complementary computational, biochemical, molecular, genetic and imaging approaches in Caenorhabditis elegans and mouse models to interrogate the effects of the A152T variant on tau function. In silico analysis suggests that a threonine at position 152 of tau confers a new phosphorylation site. This finding is borne out by mass spectrometric survey of A152T tau phosphorylation in C. elegans and mouse. Optical pulse-chase experiments of Dendra2-tau demonstrate that A152T tau and phosphomimetic A152E tau exhibit increased diffusion kinetics and the ability to traverse across the axon initial segment more efficiently than wild-type (WT) tau. A C. elegans model of tauopathy reveals that A152T and A152E tau confer patterns of developmental toxicity distinct from WT tau, likely due to differential effects on retrograde axonal transport. These data support a role for phosphorylation of the variant threonine in A152T tau toxicity and suggest a mechanism involving impaired retrograde axonal transport contributing to human neurodegenerative disease.

Mutations in MAPT (Tau) have been implicated in several types of tauopathy, but the pathways leading to neurodegeneration have remained elusive and are heterogeneous. Here we describe the effects of two mutations, both linked to AD or FTD, that are located in different domains of Tau and show different pathways of toxicity. The deletion mutation ΔK280 lies in the repeat domain and strongly increases β-structure and hence aggregation, whereas the mutation A152T lies in the N-terminal projection domain, has little effect on aggregation but instead on signalling. Both mutations cause presynaptic dysfunction, but in opposite ways, leading to hypoexcitability/hypoactivity vs. hyperexcitability/excitotoxicity, respectively. In organotypic slices these abnormal states can be reversed by drugs, e.g. Tau aggregation inhibitors or modulators of glutamate uptake. This information could contribute to the understanding of "normal" Tau biology and possible therapeutical strategies.

Microtubule research is becoming increasingly diverse, reflecting the many isoforms and modifications of tubulin and the many proteins with which microtubules interact. Recent advances are particularly visible in four areas: microtubule motor proteins (their structures, stepping modes, and forces); microtubule nucleation (the roles of centrosomes and gamma-tubulin); tubulin folding (mediated by cytoplasmic chaperones); and the expanding list of microtubule-associated proteins, knowledge of their phosphorylation states, and information on their effects on microtubule dynamics.

Mutations in the gene encoding Tau (MAPT-microtubule-associated protein tau) cause a group of neurodegenerative diseases called tauopathies. A recently identified Tau variant, p.A152T, has been reported as a risk factor for frontotemporal dementia-related disorders and Alzheimer disease. However, the mechanism for the pathologies still remain poorly understood. Transgenic Caenorhabditis elegans expressing mutant 2N4R-TauA152T (TauAT) panneuronally show locomotor defects, neurodegeneration and accelerated aging. Here we report that, in TauAT animals, the glutamatergic nervous system is at a high risk of progressive neuronal loss. We present genetic data that this loss occurs predominantly through necrosis. The neuronal loss is caused by several determinants, such as altered adenylyl cyclase (type AC9) pathway, prevalence of excitotoxicity-like conditions, aging-related factors and finally dyshomeostasis of intracellular calcium (Ca2+). The study provides novel insights into the mechanisms involved in selective loss of glutamatergic neurons in a TauAT tauopathy model which could point to new therapeutic targets.

Misfolding and aggregation of the neuronal, microtubule-associated protein tau is involved in the pathogenesis of Alzheimer's disease and tauopathies. It has been proposed that neuronal membranes could play a role in tau release, internalization, and aggregation and that tau aggregates could exert toxicity via membrane permeabilization. Whether and how tau interacts with lipid membranes remains a matter of discussion. Here, we characterize the interaction of full-length human tau (htau40) with supported lipid membranes (SLMs) made from brain total lipid extract by time-lapse high-resolution atomic force microscopy (AFM). We observe that tau attaches to brain lipid membranes where it self-assembles in a cation-dependent manner. Sodium triggers the attachment, self-assembly, and growth, whereas potassium inhibits these processes. Moreover, tau assemblies are stable in the presence of sodium and lithium but disassemble in the presence of potassium and rubidium. Whereas the pseudorepeat domains (R1-R4) of htau40 promote the sodium-dependent attachment to the membrane and stabilize the tau assemblies, the N-terminal region promotes tau self-assembly and growth.

A 2-dimensional high-throughput screening method is presented to select peptide sequences from large peptide libraries for precision formulation additives, having a high capacity to specifically host a drug of interest and provide tailored drug release properties. The identified sequences are conjugated with poly(ethylene glycol) (PEG) to obtain peptide-PEG conjugates that proved to be valuable as solubilizers for small organic molecule drugs to overcome limitations of poor water-solubility and low bio-availability. The 2D-screening method selects peptide sequences on both (i) high loading capacities and (ii) preferred drug-release capabilities as demonstrated on an experimental Tau-protein aggregation inhibitor/Tau- deaggregator with potentials for an anti-Alzheimer disease drug (BB17). To enable 2D-screening, a one-bead one-compound (OBOC) peptide library was immobilized on a glass slide, allocating individual beads to permanent positions. While the first screening step involved incubation of the supported OBOC library with BB17 to identify beads with high drug binding capacities by fluorescence scanner readouts, the second step reveals release properties of the high capacity binders by incubation with blood plasma protein model solutions. Efficiently peptides with high BB17 capacities and either keeper or medium or fast releaser properties can be identified by direct sequence readouts from the glass slide supported resin beads via matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. Four peptides are synthesized as peptide-PEG solubilizers representing strong, medium, weak releasers and non-binders. Loading capacities reached up to 1:3.4 (mol drug per mol carrier) and release kinetics (fast/medium/slow) are in agreement with the selection process as investigated by fluorescence anisotropy and fluorescence correlation spectroscopy. The ability of BB17/conjugate complexes to inhibit the aggregation of Tau4RDΔK (four repeat Tau ((M)Q244-E372 with deletion of K280), 129 residues) in N2a cells is studied by a Tau-pelleting assay showing the modulation of cellular Tau aggregation. Promising effects such as the reduction of 55% of total Tau load are observed for the strong releaser additive. Studies of in vitro Thioflavin S Tau-aggregation assays show half-maximal inhibitory activities (IC50 values) of BB17/conjugates in the low micro-molar range.

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.