Multiple System Atrophy (MSA) and Parkinson's Disease (PD) are neurodegenerative diseases characterized by abundant α-synuclein (αSyn) aggregation in the brain. Compared to PD patients, MSA patients have more widespread neurodegeneration and a more aggressive disease course. PD-related αSyn pathology is primarily neuronal, whereas MSA brains characteristically display oligodendroglial inclusions. The strain hypothesis poses that polymorphisms of the αSyn aggregates, so-called strains, may explain disease heterogeneity. The present study investigates the differential properties of αSyn fibrils derived from MSA and PD patients' brains using the protein misfolding cyclic amplification (PMCA) method in cultured neurons and in vivo. MSA- and PD-derived αSyn species were administered to primary murine neuronal cell cultures or injected intrastriatally into wildtype mice, along with de novo generated αSyn fibrils of the ribbon and fibril types. The potency to induce phosphorylated αSyn (pSyn) pathology, microglial reactivity, and the extent of oligodendroglial pSyn pathology were compared among the different seeding materials using immunohistochemical and immunofluorescent approaches. In summary, the various seeding materials induced pSyn pathology of distinguishable potency and morphology. PMCA-derived material from MSA brains and the fibril polymorph induced more pSyn pathology in both neuronal cultures and in mice compared to PMCA-derived material from PD brains and the ribbon polymorph. Interestingly, amplified material from MSA brains induced significantly more oligodendroglial pSyn aggregates than amplified material from PD brains. Additionally, mice injected with the fibril polymorph, showed mild changes in microglial reactivity. Our findings suggest specific properties of the MSA- and PD-derived fibrils, and overall support the strain hypothesis.

Accumulation of the microtubule-associated protein TAU into inclusions is a hallmark of tauopathies including Alzheimer's disease (AD), potentially driven by impaired protein degradation and dysregulated ubiquitination. To explore the role of deubiquitinating enzymes (DUBs), we performed siRNA knockdown screens targeting 93 murine DUBs in rTg4510 cortical cultures. Knockdown and pharmacological inhibition of the ubiquitin-specific proteases 7 (Usp7) and 10 (Usp10) significantly reduced seeded TAU aggregation without affecting soluble TAU levels. These effects were observed in both cortical and organotypic hippocampal slice cultures from rTg4510 mice, as well as in wildtype neurons seeded with AD-derived pathological TAU. Inhibition of Usp7 and Usp10 was associated with increased polyubiquitination of residual TAU inclusions in rTg4510 cortical cultures. These findings suggest that Usp7 and Usp10 contribute to pathological TAU accumulation by modulating ubiquitin-dependent degradation pathways. Targeting USP7 and USP10 may offer a novel therapeutic strategy for AD and related tauopathies.

Background: Tauopathies such as Alzheimer’s disease (AD) are characterized by abnormal hyperphosphorylation of the microtubule-associated protein tau (MAPT) aggregating into neurofibrillary tangles (NFTs). O-linked β-N-acetylglucosamine (O-GlcNAc) modifications have been suggested to regulate tau phosphorylation and aggregation and N-acetylglucosaminidase (OGA) removes GlcNAc moieties from proteins. Methods: We investigated effects of the OGA inhibitor Thiamet G in rTg4510 primary neuronal cultures and in rTg4510 mice. The rTg4510 mice overexpress human tau harboring the P301L mutation and display an age-dependent progression of tau pathology including hyperphosphorylated tau species and NFTs. Aged rTg4510 mice exhibit a non-mnemonic behavioral defect involving a hyperactive phenotype that is associated with the progression of tau pathology. Results: Thiamet G increased overall O-GlcNAc levels and crossed the blood brain barrier in rTg4510 mice. The free fraction of Thiamet G in the brain was 22-fold above the half maximal effective concentration (EC50) measured in rTg4510 primary neurons. Chronic Thiamet G treatment (18 weeks) initiated in young 6 week old rTg4510 mice increased brain O-GlcNAc levels and this corresponded with a significant reduction in soluble and insoluble hyperphosphorylated tau in aged 24 week old rTg4510 mice. Levels of normally phosphorylated P301L tau were not altered under these conditions. Reduction of hyperphosphorylated tau species by increased O-GlcNAcylation was associated with significant attenuation of hyperactivity in 24 week old rTg4510 mice. Conclusions: Our findings support the pharmacological inhibition of OGA as a potential therapeutic approach for the treatment of AD and other tauopathies.

Background: Tauopathies such as Alzheimer's disease (AD) and frontotemporal dementia (FTD) are characterized by formation of neurofibrillary tangles consisting of hyperphosphorylated tau protein. Early pathophysiological and functional changes related to neurofibrillary tangles formation are considered to occur prior to extensive neurodegeneration. Hyperphosphorylated tau has been detected in postmortem retinas of AD and FTD patients, and the visual pathway is an easily accessible system in a clinical setting. Hence, assessment of the visual function may offer the potential to detect consequences of early tau pathology in patients. Objective: The aim of this study was to evaluate visual function in a tauopathy mouse model in relation to tau hyperphosphorylation and neurodegeneration. Methods: In this study we explored the association between the visual system and functional consequences of tau pathology progression using a tauopathy rTg4510 mouse model. To this end, we recorded full-field electroretinography and visual evoked potentials in anesthetized and awake states at different ages. Results: While retinal function remained mostly intact within all the age groups investigated, we detected significant changes in amplitudes of visual evoked potential responses in young rTg4510 mice exhibiting early tau pathology prior to neurodegeneration. These functional alterations in the visual cortex were positively correlated with pathological tau levels. Conclusion: Our findings suggest that visual processing could be useful as a novel electrophysiological biomarker for early stages of tauopathy.

Background: Deposits of hyperphosphorylated tau fibrils are hallmarks of a broad spectrum of tauopathies, including Alzheimer’s disease (AD). Objective: To investigate heterogeneity of tau pathology across brain extracts from a broad selection of different tauopathies and examine the binding properties of the humanized pS396-tau antibody hC10.2 and six other anti-tau antibodies. Methods: 76 individual tauopathy tissue samples were analyzed in a battery of assays: immunohistochemistry, ELISA, tau aggregation assay, western blot, [3H]PI-2620 and [3H]MK-6240 tau tracer binding, and aggregated seeding activity in RD_P301S HEK293T Biosensor cells. The efficiency of seven anti-tau antibodies to engage with pathological tau species was directly compared. Results: Our data indicate that a strong correlation existed between the tau tracer binding, amount of tau aggregates, pS396-tau phosphorylation, and seeding activity. The hC10.2 antibody, which has entered clinical development, effectively engaged with its epitope across all individual cases of mid-stage and late AD, and primary tauopathies. hC10.2 was superior compared to other phospho- and total tau antibodies to prevent seeded tau aggregation in the biosensor cells. hC10.2 effectively depleted hyperphosphorylated and aggregated tau species across all tauopathy samples proportionally to the amount of tau aggregates. In AD samples, hC10.2 bound to ghost tangles which represent extracellular pathological tau species. Conclusion: S396 hyperphosphorylation is a feature of the formation of seeding-competent tau across different tauopathies and it is present both in intra- and extracellular pathological tau. hC10.2 represents an excellent candidate for a hyperphosphorylation-selective therapeutic tau antibody for the treatment of AD and primary tauopathies.

Deposition of extensively hyperphosphorylated tau in specific brain cells is a clear pathological hallmark in Alzheimer's disease and a number of other neurodegenerative disorders, collectively termed the tauopathies. Furthermore, hyperphosphorylation of tau prevents it from fulfilling its physiological role as a microtubule-stabilizing protein and leaves it increasingly vulnerable to self-assembly, suggestive of a central underlying role of hyperphosphorylation as a contributing factor in the etiology of these diseases. Via in vitro phosphorylation and regulation of kinase activity within cells and acute brain tissue, we reveal that the inflammation associated kinase, protein kinase R (PKR), directly phosphorylates numerous abnormal and disease-modifying residues within tau including Thr181, Ser199/202, Thr231, Ser262, Ser396, Ser404 and Ser409. Similar to disease processes, these PKR-mediated phosphorylations actively displace tau from microtubules in cells. In addition, PKR overexpression and knockdown, respectively, increase and decrease tau protein and mRNA levels in cells. This regulation occurs independent of noncoding transcriptional elements, suggesting an underlying mechanism involving intra-exonic regulation of the tau-encoding microtubule-associated protein tau (MAPT) gene. Finally, acute encephalopathy in wild type mice, induced by intracranial Langat virus infection, results in robust inflammation and PKR upregulation accompanied by abnormally phosphorylated full-length- and truncated tau. These findings indicate that PKR, independent of other kinases and upon acute brain inflammation, is capable of triggering pathological modulation of tau, which, in turn, might form the initial pathologic seed in several tauopathies such as Alzheimer's disease and Chronic traumatic encephalopathy where inflammation is severe.

BACKGROUND:Alzheimer's disease (AD) is characterized by accumulation of amyloid-β (Aβ) species and deposition of senile plaques (SPs). Clinical trials with the anti-Aβ antibody aducanumab have been completed recently.OBJECTIVE:To characterize the proteomic profile of SPs and surrounding tissue in a mouse model of AD in 10-month-old tgAPPPS1-21 mice after chronic treatment with aducanumab for four months with weekly dosing (10 mg/kg).METHODS:After observing significant reduction of SP numbers in hippocampi of aducanumab-treated mice, we applied a localized proteomic analysis by combining laser microdissection and liquid chromatography-tandem mass spectrometry (LC-MS/MS) of the remaining SPs in hippocampi. We microdissected three subregions, containing SPs, SP penumbra level 1, and an additional penumbra level 2 to follow the proteomic profile as gradient.RESULTS:In the aducanumab-treated mice, we identified 17 significantly regulated proteins that were associated with 1) mitochondria and metabolism (ACAT2, ATP5J, ETFA, EXOG, HK1, NDUFA4, NDUFS7, PLCB1, PPP2R4), 2) cytoskeleton and axons (ADD1, CAPZB, DPYSL3, MAG), 3) stress response (HIST1H1C/HIST1H1D, HSPA12A), and 4) AβPP trafficking/processing (CD81, GDI2). These pathways and some of the identified proteins are implicated in AD pathogenesis. Proteins associated with mitochondria and metabolism were mainly upregulated while proteins associated with AβPP trafficking/processing and stress response pathways were mainly downregulated, suggesting that aducanumab could lead to a beneficial proteomic profile around SPs in tgAPPPS1-21 mice.CONCLUSION:We identified novel proteomic patterns of SPs and surrounding tissue indicating that chronic treatment with aducanumab could inhibit Aβ toxicity and increase phagocytosis and cell viability.

Deposition of extensively hyperphosphorylated tau in specific brain cells is a clear pathological hallmark in Alzheimer's disease and a number of other neurodegenerative disorders, collectively termed the tauopathies. Furthermore, hyperphosphorylation of tau prevents it from fulfilling its physiological role as a microtubule‐stabilizing protein and leaves it increasingly vulnerable to self‐assembly, suggestive of a central underlying role of hyperphosphorylation as a contributing factor in the etiology of these diseases. Via in vitro phosphorylation and regulation of kinase activity within cells and acute brain tissue, we reveal that the inflammation associated kinase, protein kinase R (PKR), directly phosphorylates numerous abnormal and disease‐modifying residues within tau including Thr181, Ser199/202, Thr231, Ser262, Ser396, Ser404 and Ser409. Similar to disease processes, these PKR‐mediated phosphorylations actively displace tau from microtubules in cells. In addition, PKR overexpression and knockdown, respectively, increase and decrease tau protein and mRNA levels in cells. This regulation occurs independent of noncoding transcriptional elements, suggesting an underlying mechanism involving intra‐exonic regulation of the tau‐encoding microtubule‐associated protein tau (MAPT) gene. Finally, acute encephalopathy in wild type mice, induced by intracranial Langat virus infection, results in robust inflammation and PKR upregulation accompanied by abnormally phosphorylated full‐length‐ and truncated tau. These findings indicate that PKR, independent of other kinases and upon acute brain inflammation, is capable of triggering pathological modulation of tau, which, in turn, might form the initial pathologic seed in several tauopathies such as Alzheimer's disease and Chronic traumatic encephalopathy where inflammation is severe.

Amyloid plaques are one of the hallmarks of Alzheimer's disease (AD). The main constituent of amyloid plaques is amyloid-β peptides, but a complex interplay of other infiltrating proteins also co-localizes. We hypothesized that proteomic analysis could reveal differences between amyloid plaques and adjacent control tissue in the transgenic mouse model of AD (APPPS1-21) and in similar regions from non-transgenic littermates. Our microproteomic strategy included isolation of regions of interest by laser capture microdissection and analysis by liquid chromatography mass spectrometry-based label-free relative quantification. We consistently identified 183, 224, and 307 proteins from amyloid plaques, adjacent control and non-tg samples, respectively. Pathway analysis revealed 27 proteins that were significantly regulated when comparing amyloid plaques and corresponding adjacent control regions. We further elucidated that co-localized proteins were subjected to post-translational modifications and are the first to report 193 and 117 unique modifications associated to amyloid plaques and adjacent control extracts, respectively. The three most common modifications detected in proteins from the amyloid plaques were oxidation, deamidation, and pyroglutamylation. Together, our data provide novel information about the biological processes occurring within and around amyloid plaques in the APPPS1-21 mouse model of AD.

Rationale The molecular complexity of tissue features several signal-suppression effects which reduce the ionization of analytes significantly and thereby weakens the quality of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) imaging (MALDI imaging). We report a novel approach in MALDI imaging by reducing signal-suppression effects for the analysis of beta-amyloid (A beta) plaques, one pathological hallmark of Alzheimer's disease (AD). Methods We analyzed A beta proteoforms from postmortem AD brains and brains from transgenic mice (APPPS1-21) overexpressing familial AD mutations by combining two techniques: (1) laser capture microdissection (LCM) to accumulate A beta plaques and (2) phosphoric acid (PA) as additive to the super-2,5-dihydroxybenzoic acid matrix. Results LCM and MALDI-MS enabled tandem mass spectrometric fragmentation of stained A beta plaques. PA improved the signal-to-noise (S/N) ratio, especially of the A beta 1-42 peptide, by three-fold compared with the standard matrix additive trifluoroacetic acid. The beneficial effect of the PA matrix additive in MALDI imaging was particularly important for AD brain tissue. We identified several significant differences in A beta plaque composition from AD compared with APPPS1-21, underlining the value of reducing signal-suppressing effects in MALDI imaging. Conclusions We present a novel strategy for overcoming signal-suppression effects in MALDI imaging of A beta proteoforms.

INTRODUCTION:The abnormal hyperphosphorylation of the microtubule-associated protein tau plays a crucial role in neurodegeneration in Alzheimer's disease (AD) and other tauopathies.METHODS:Highly specific and selective anti-pS396-tau antibodies have been generated using peptide immunization with screening against pathologic hyperphosphorylated tau from rTg4510 mouse and AD brains and selection in in vitro and in vivo tau seeding assays.RESULTS:The antibody C10.2 bound specifically to pS396-tau with an IC50 of 104 pM and detected preferentially hyperphosphorylated tau aggregates from AD brain with an IC50 of 1.2 nM. C10.2 significantly reduced tau seeding of P301L human tau in HEK293 cells, murine cortical neurons, and mice. AD brain extracts depleted with C10.2 were not able to seed tau in vitro and in vivo, demonstrating that C10.2 specifically recognized pathologic seeding-competent tau.DISCUSSION:Targeting pS396-tau with an antibody like C10.2 may provide therapeutic benefit in AD and other tauopathies.

Tau antibodies have shown therapeutic potential for Alzheimer's disease and several are in clinical trials. As a microtubule-associated protein, tau relies on dynamic phosphorylation for its normal functions. In tauopathies, it becomes hyperphosphorylated and aggregates into toxic assemblies, which collectively lead to neurodegeneration. Of the phospho-epitopes, the region around Ser396 has received particular attention because of its prominence and stability in tauopathies. Here we report the first structure of a monoclonal tau antibody in complex with the pathologically important phospho-Ser396 residue. Its binding region reveals tau residues Tyr394 to phospho-Ser396 stabilized in a β-strand conformation that is coordinated by a phospho-specific antigen binding site. These details highlight a molecular switch that defines this prominent conformation of tau and ways to target it. Overall, the structure of the antibody-antigen complex clarifies why certain phosphorylation sites in tau are more closely linked to neurodegeneration than others.

In order to develop improved treatments for Alzheimer's disease (AD), it is of great importance to gain deeper insight and understanding of the biological systems that are being targeted and the mechanisms by which they contribute to AD. We present a combination of MALDI mass spectrometry imaging (MALDI-MSI) and laser capture microdissection (LCM) that together provide a useful tool for investigating the peptide composition of amyloid plaques in AD. Three 9-month old female transgenic mice (Tg APPPS1-21) were sectioned and introduced to in-house protocols for MALDI-MSI or LCM in combination with MALDI. A number of different MALDI matrices and matrix additives were utilized to study the peptidomic profile of the amyloid plaques. Microdissection was acquired with the Arcturus Veritas LCM system and data acquisitions were performed using the Ultraflextreme MALDI TOF/TOF instrument followed by data analysis in SCiLS Lab and Flex Analysis software. Interpretation of the MALDI-MSI data revealed several peaks which corresponded to the theoretical mass of different Aβ species. Furthermore, a notable difference in the degree of ionization was observed when utilizing different MALDI matrices and additives. An additional layer of shared and exclusive peaks were identified when including data obtained from the combination of LCM and MALDI. This combination also enabled the acquisition of a sufficient sequence coverage that, when compared to an in-silico fragmentation spectra, validated the presence of Aβ1-42. Selecting a proper matrix and matrix additive has a high impact on the peptidomic profile obtained with MALDI-MSI. The addition of LCM and MALDI enabled an in-depth characterization of amyloid plaques that was not achieved with MALDI-MSI alone. Thus, we recommend to 1. include several matrix combinations when studying amyloid plaques and 2. utilize a combination of LCM and MALDI to increase the peptidomic coverage obtainable.

Alzheimer's disease (AD) and tauopathies, such as frontotemporal dementia (FTD), are characterized by formation of neurofibrillary tangles consisting of hyperphosphorylated tau. Further neuropathological characteristics include synaptic loss, neurodegeneration and brain atrophy. Here, we explored the association between hyperphosphorylated tau species, brain atrophy, synaptic and neuronal loss in a mouse model (rTg4510) carrying the human tau (hTau) P301L mutation found in a familiar form of FTD. We established that hTau expression during the first 6 postnatal weeks was important for the progression of tauopathy in rTg4510 mice. Short term suppression of postnatal hTau expression delayed the onset of tau pathology by approximately 6months in this model. Early postnatal hTau expression was detrimental to CA1 neurons of the hippocampus and reduced neuronal numbers in 6-10weeks young rTg4510 mice prior to the appearance of hyperphosphorylated hTau species in the hippocampus. Hyperphosphorylated hTau species emerged from 10 to 24weeks of age and were associated with increased ubiquitin levels, gliosis, and brain atrophy and preceded the synaptic loss and CA1 neurodegeneration that occurred at 48weeks of age. We present two consequences of hTau expression in CA1 in rTg4510 mice: an early decrease in neuron number already established prior to the presence of hyperphosphorylated tau species and a later neurodegeneration dependent on hyperphosphorylated tau. Neurodegeneration and synaptic protein loss were completely prevented when hTau expression was suppressed prior to the appearance of hyperphosphorylated tau species. Suppression of hTau expression after the onset of tau hyperphosphorylation and tangle pathology initiated at 16weeks partially rescued neuronal loss at 48weeks of age, while a reduction of neurodegeneration was no longer possible when hTau suppression was introduced as late as at 24weeks of age. Our results in rTg4510 mice argue that it is promising to lower hyperphosphorylated tau species at early stages of tau pathology to protect from neurodegeneration.

Tauopathies, such as Alzheimer's disease (AD) and frontotemporal dementia (FTD), are characterized by formation of neurofibrillary tangles consisting of hyperphosphorylated tau. In addition to memory loss, patients experience behavioral symptoms such as agitation, aggression, depression, and insomnia. We explored the behavioral phenotype of a mouse model (rTg4510) carrying the human tau P301L mutation found in a familial form of FTD. We tested these mice in locomotor activity assays as well as in the Morris water maze to access spatial memory. In addition to cognitive impairments, rTg4510 mice exhibited a hyperactivity phenotype which correlated with progression of tau pathology and was dependent on P301L tau transgene expression. The hyperactive phenotype was characterized by significantly increased locomotor activity in a novel and in a simulated home cage environment together with a disturbed day/night cycle. The P301L-tau-dependent hyperactivity and agitative-like phenotype suggests that these mice may form a correlate to some of the behavioral disturbances observed in advanced AD and FTD.

The identification of the novel and selective GPR3 inverse agonist AF64394, the first small molecule inhibitor of GPR3 receptor function, is described. Structure activity relationships and syntheses based around AF64394 are reported.

BACKGROUND: BACE1 (beta site amyloid precursor protein cleaving enzyme 1) is the rate limiting protease in amyloid beta production, hence a promising drug target for the treatment of Alzheimer's disease. Inhibition of BACE1, as the major beta-secretase in vivo with multiple substrates, however is likely to have mechanism-based adverse effects. We explored the impact of long-term pharmacological inhibition of BACE1 on dendritic spine dynamics, synaptic functions, and cognitive performance of adult mice.METHODS: Sandwich enzyme-linked immunosorbent assay was used to assess A beta 40 levels in brain and plasma after oral administration of BACE1 inhibitors SCH1682496 or LY2811376. In vivo two-photon microscopy of the somatosensory cortex was performed to monitor structural dynamics of dendritic spines while synaptic functions and plasticity were measured via electrophysiological recordings of excitatory postsynaptic currents and hippocampal long-term potentiation in brain slices. Finally, behavioral tests were performed to analyze the impact of pharmacological inhibition of BACE1 on cognitive performance.RESULTS: Dose-dependent decrease of A beta 40 levels in vivo confirmed suppression of BACE1 activity by both inhibitors. Prolonged treatment caused a reduction in spine formation of layer V pyramidal neurons, which recovered after withdrawal of inhibitors. Congruently, the rate of spontaneous and miniature excitatory postsynaptic currents in pyramidal neurons and hippocampal long-term potentiation were reduced in animals treated with BACE1 inhibitors. These effects were not detected in Bace1(-/-) mice treated with SCH1682496, confirming BACE1 as the pharmacological target. Described structural and functional changes were associated with cognitive deficits as revealed in behavioral tests.CONCLUSIONS: Our findings indicate important functions to BACE1 in structural and functional synaptic plasticity in the mature brain, with implications for cognition.

The formation of neurofibrillary tangles from the assembly of hyperphosphorylated tau leads to dendritic and axonal instability, synaptic degeneration, and neuronal loss. To understand the early physiological consequences of aberrant tau expression, we characterized the physiology of CA1 pyramidal neurons in rTg4510 female mice and non-transgenic (wt) littermate controls. We studied mice at the age of 10-12 weeks where only minimal hyperphosphorylated pretangle tau was present, and 22-24 weeks old mice with significant neurofibrillary tangle pathology. Our electrophysiological analysis included input-output relation, paired-pulse facilitation, and whole cell patch-clamp recordings of neurons to measure action potential threshold and action potential properties, chord-conductance, and characterization of AMPA receptor mediated synaptic transmission. We found that the input-output relation in field (excitatory postsynaptic potentials, EPSP) and whole cell recordings (excitatory postsynaptic currents, EPSC) were impaired in rTg4510 mice compared to wt controls at both ages. We measured a diminished tail current charge after depolarizing voltage input in rTg4510 mice compared to wt in both young and aged mice. Additionally, mini-EPSC properties (peak and decay time) were essentially similar between genotypes and age groups investigated. Surprisingly, in the 22-24 week old group, the mini-EPSC frequency was significantly increased (interevent interval 0.8 ± 0.1 in wt compared to 0.3 ± 0.1 in rTg4510 mice). These data indicate that the developmentally regulated expression of human P301L tau in CA1 pyramidal neurons coincide with changes in neuronal excitability but also that significant presynaptic changes occur late during the progression of tau pathology in this mouse model.

The abnormal hyperphosphorylation of the microtubule-associated protein tau plays a crucial role in neurodegeneration in Alzheimer's disease (AD) and aggregation of hyperphosphorylated tau into neurofibrillary tangles is a hallmark brain lesion in AD. Besides kinases and phosphatases tau phosphorylation is regulated by O-GlcNAcylation, a posttranslational modification of proteins on the serine or threonine residues with β-N-acetylglucosamine (GlcNAc). O-GlcNAcylation is dynamically regulated by O-GlcNAc transferase, the enzyme catalyzing the transfer of GlcNAc to proteins, and N-acetylglucosaminidase (OGA), the enzyme catalyzing the removal of GlcNAc from proteins. Thiamet-G is a specific and brain permeable OGA inhibitor. Analysis of tau pathology and behaviour in rTg4510 mice which are overexpressing an inducible human mutant tau P301L selectively in the forebrain after acute and chronic Thiamet-G administration by the drinking water. rTg4510 displayed age-dependent progression of tau pathology with tau hyperphosphorylation at AD-relevant epitopes, accumulation of the 64 kDa tau species, and neurofibrillary tangles in cortex and hippocampus. A subset of rTg4510 mice displayed a hyperactive phenotype both in a novel environment and during the diurnal cycle in the home cage, reminiscent of agitated behaviour. The percentage of hyperactive rTg4510 mice increased with age and was prevented by suppression of tau transgene indicating that this phenotype correlates with the progressive tau pathology. Treatment of rTg4510 mice with Thiamet-G increased overall O-GlcNAcylation levels in the brain indicative for sufficient target engagement. Further, Thiamet-G increased O-GlcNAcylation levels on mutant tau isolated from rTg4510 brains. Chronic Thiamet-G treatment significantly reduced soluble and insoluble 64 kDa hyperphosphorylated tau levels and reduced the number of hyperactive rTg4510 animals. These results support OGA inhibition by Thiamet-G as possible treatment concept to reduce tau pathology and dysfunction.