Increasing O-GlcNAcylation Attenuates tau Hyperphosphorylation and Behavioral Impairment in rTg4510 Tauopathy Mice.

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 ß-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.

Dual strategy for reduced signal-suppression effects in matrix-assisted laser desorption/ionization mass spectrometry imaging.

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 (Aß) plaques, one pathological hallmark of Alzheimer's disease (AD). METHODS: We analyzed Aß 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 Aß 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 Aß plaques. PA improved the signal-to-noise (S/N) ratio, especially of the Aß1-42 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 Aß 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 Aß proteoforms.

Secretome protein enrichment identifies physiological BACE1 protease substrates in neurons.

Cell surface proteolysis is essential for communication between cells and results in the shedding of membrane-protein ectodomains. However, physiological substrates of the contributing proteases are largely unknown. We developed the secretome protein enrichment with click sugars (SPECS) method, which allows proteome-wide identification of shedding substrates and secreted proteins from primary cells, even in the presence of serum proteins. SPECS combines metabolic glycan labelling and click chemistry-mediated biotinylation and distinguishes between cellular and serum proteins. SPECS identified 34, mostly novel substrates of the Alzheimer protease BACE1 in primary neurons, making BACE1 a major sheddase in the nervous system. Selected BACE1 substrates-seizure-protein 6, L1, CHL1 and contactin-2-were validated in brains of BACE1 inhibitor-treated and BACE1 knock-out mice. For some substrates, BACE1 was the major sheddase, whereas for other substrates additional proteases contributed to total substrate shedding. The new substrates point to a central function of BACE1 in neurite outgrowth and synapse formation. SPECS is also suitable for quantitative secretome analyses of primary cells and may be used for the discovery of biomarkers secreted from tumour or stem cells.

The identification of GPR3 inverse agonist AF64394; the first small molecule inhibitor of GPR3 receptor function.

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.

Visual Evoked Potentials as an Early-Stage Biomarker in the rTg4510 Tauopathy Mouse Model.

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.

Highly Specific and Sensitive Target Binding by the Humanized pS396-Tau Antibody hC10.2 Across a Wide Spectrum of Alzheimer's Disease and Primary Tauopathy Postmortem Brains.

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.

Mitochondrial translocation of cofilin is an early step in apoptosis induction.

Increasing evidence suggests that movement of key proteins in or out of mitochondria during apoptosis is essential for the regulation of apoptosis. Here, we report identification of the actin-binding protein cofilin by a proteomic approach, as such a factor translocated from cytosol into mitochondria after induction of apoptosis. We found that after induction of apoptosis, cofilin was translocated to mitochondria before release of cytochrome c. Reduction of cofilin protein levels with small-interfering RNA (siRNA) resulted in inhibition of both cytochrome c release and apoptosis. Only dephosphorylated cofilin was translocated to mitochondria, and the cofilin S3D mutant, which mimicks the phosphorylated form, suppressed mitochondrial translocation and apoptosis. Translocation was achieved through exposure of an amino-terminal mitochondrial targeting signal in combination with carboxy-terminal sequences. When correctly targeted to mitochondria, cofilin induced massive apoptosis. The apoptosis-inducing ability of cofilin, but not its mitochondrial localization, was dependent on the functional actin-binding domain. Thus, domains involved in mitochondrial targeting and actin binding are indispensable for its pro-apoptotic function. Our data suggest that cofilin has an important function during the initiation phase of apoptosis.

PKR kinase directly regulates tau expression and Alzheimer's disease-related tau phosphorylation.

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.

Anti-Aß Antibody Aducanumab Regulates the Proteome of Senile Plaques and Closely Surrounding Tissue in a Transgenic Mouse Model of Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) is characterized by accumulation of amyloid-ß (Aß) species and deposition of senile plaques (SPs). Clinical trials with the anti-Aß 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) AßPP 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 AßPP 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 Aß toxicity and increase phagocytosis and cell viability.

Gamma-secretase inhibition reduces spine density in vivo via an amyloid precursor protein-dependent pathway.

Alzheimer's disease (AD) represents the most common age-related neurodegenerative disorder. It is characterized by the invariant accumulation of the beta-amyloid peptide (Abeta), which mediates synapse loss and cognitive impairment in AD. Current therapeutic approaches concentrate on reducing Abeta levels and amyloid plaque load via modifying or inhibiting the generation of Abeta. Based on in vivo two-photon imaging, we present evidence that side effects on the level of dendritic spines may counteract the beneficial potential of these approaches. Two potent gamma-secretase inhibitors (GSIs), DAPT (N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester) and LY450139 (hydroxylvaleryl monobenzocaprolactam), were found to reduce the density of dendritic spines in wild-type mice. In mice deficient for the amyloid precursor protein (APP), both GSIs had no effect on dendritic spine density, demonstrating that gamma-secretase inhibition decreases dendritic spine density via APP. Independent of the effects of gamma-secretase inhibition, we observed a twofold higher density of dendritic spines in the cerebral cortex of adult APP-deficient mice. This observation further supports the notion that APP is involved in the modulation of dendritic spine density--shown here for the first time in vivo.

Proteomic and Unbiased Post-Translational Modification Profiling of Amyloid Plaques and Surrounding Tissue in a Transgenic Mouse Model of Alzheimer's Disease.

Amyloid plaques are one of the hallmarks of Alzheimer's disease (AD). The main constituent of amyloid plaques is amyloid-ß 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.

Differential effects of gamma-secretase and BACE1 inhibition on brain Abeta levels in vitro and in vivo.

Alzheimer's disease (AD) is hypothesized to result from elevated brain levels of beta-amyloid peptide (Abeta) which is the main component of plaques found in AD brains and which cause memory impairment in mice. Therefore, there has been a major focus on the development of inhibitors of the Abeta producing enzymes gamma-secretase and beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1). In this study, we investigated the Abeta-lowering effects of the BACE1 inhibitor LY2434074 in vitro and in vivo, comparing it to the well characterized gamma-secretase inhibitor LY450139. We sampled interstitial fluid Abeta from awake APPswe/PS1dE9 AD mice by in vivo Abeta microdialysis. In addition, we measured levels of endogenous brain Abeta extracted from wildtype C57BL/6 mice. In our in vitro assays both compounds showed similar Abeta-lowering effects. However, while systemic administration of LY450139 resulted in transient reduction of Abeta in both in vivo models, we were unable to show any Abeta-lowering effect by systemic administration of the BACE1 inhibitor LY2434074 despite brain exposure exceeding the in vitro IC(50) value several fold. In contrast, significant reduction of 40-50% of interstitial fluid Abeta and wildtype cortical Abeta was observed when infusing LY2434074 directly into the brain by means of reverse microdialysis or by dosing the BACE1 inhibitor to p-glycoprotein (p-gp) mutant mice. The effects seen in p-gp mutant mice and subsequent data from our cell-based p-gp transport assay suggested that LY2434074 is a p-gp substrate. This may partly explain why BACE1 inhibition by LY2434074 has lower in vivo efficacy, with respect to decreased Abeta40 levels, compared with gamma-secretase inhibition by LY450139.

Measurement of cellular beta-site of APP cleaving enzyme 1 activity and its modulation in neuronal assay systems.

Amyloid-beta peptide (Abeta), a putatively causative agent of Alzheimer's disease (AD), is proteolytically derived from beta-amyloid precursor protein (APP). Here we describe cellular assays to detect the activity of the key protease beta-site of APP cleaving enzyme 1 (BACE1) based on an artificial reporter construct containing the BACE1 cleavage site of APP. These methods allow identification of inhibitors and indirect modulators of BACE1. In primary neuronal cultures transfected with human APP constructs (huAPP), Abeta production was modified by BACE1 inhibitors similarly to the production of endogenous murine Abeta in wild-type cells and to that of different transgenic neurons. To further improve the assay, we substituted the extracellular domain of APP by secreted alkaline phosphatase (SEAP). SEAP was easily quantified in the cell culture supernatants after cleavage of SEAP-APP by BACE1 or alpha-secretases. To render the assay specific for BACE1, the alpha-secretase cleavage site of SEAP-APP was eliminated either by site-directed mutagenesis or by substituting the transmembrane part of APP by the membrane domain of the erythropoietin receptor (EpoR). The pharmacology of these constructs was characterized in detail in HEK293 cells (human embryonic kidney cell line), and the SEAP-APP-EpoR construct was also introduced into primary murine neurons and there allowed specific measurement of BACE1 activity.

Tau Antibody Structure Reveals a Molecular Switch Defining a Pathological Conformation of the Tau Protein.

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 ß-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.

Highly specific and selective anti-pS396-tau antibody C10.2 targets seeding-competent tau.

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.

Wide spectrum modulation by KP-544 in models relevant for neuronal survival.

Reduced neurotrophic signalling has been proposed as a part of the pathophysiology behind neuronal death and dysfunction. The small molecule KP-544 was developed with the intention to enhance nerve growth factor signalling. To characterize the actions of KP-544 pharmacologically, we used four diverse models with relevance for neuronal function and survival. We found that 300-1000 nM KP-544 enhanced the neurite outgrowth in PC12 cells in response to a suboptimal concentration of nerve growth factor. KP-544 also protected the cerebellar granule cells from excitotoxicity apoptosis induced by the mitochondrial toxin methyl-phenyl-pyridinium, and modulated inflammation by inhibiting interleukin-6 production in primary astrocytes. Chronic treatment of rats with KP-544 prevented the hyper-responsiveness to amphetamine of animals treated with methylazoxymethanol acetate, a recently described neurodevelopmental model of schizophrenia.

Attenuated amyloid-beta aggregation and neurotoxicity owing to methionine oxidation.

Aggregation of the amyloid-beta (Abeta) peptide into amyloid plaques is a characteristic feature of Alzheimer's disease neuropathogenesis. We and others have previously demonstrated delayed Abeta aggregation as a consequence of oxidizing a single methionine residue at position 35 (Met-35). Here, we examined the consequences of Met-35 oxidation on the extremely aggregation-prone peptides Abeta1-42 and Abeta1-40Arctic with respect to protofibril and oligomer formation as well as neurotoxicity. Size exclusion chromatography and mass spectrometry demonstrated that monomer/dimers prevailed over larger oligomers after oxidizing Met-35, and consequently protofibril formation and aggregation of both Abeta1-42 and Abeta1-40Arctic were delayed. The oxidized peptides completely lacked neurotoxic effects in cortical neuronal cultures under these conditions, in contrast to the neurotoxic properties of the unoxidized peptides. We conclude that oxidation of Met-35 significantly attenuates aggregation of Abeta1-42 and Abeta1-40Arctic, and thereby reduces neurotoxicity.

Early depletion of CA1 neurons and late neurodegeneration in a mouse tauopathy model.

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.

Hyperactivity with Agitative-Like Behavior in a Mouse Tauopathy Model.

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.

Pharmacological inhibition of BACE1 impairs synaptic plasticity and cognitive functions.

BACKGROUND: BACE1 (beta site amyloid precursor protein cleaving enzyme 1) is the rate limiting protease in amyloid ß production, hence a promising drug target for the treatment of Alzheimer's disease. Inhibition of BACE1, as the major ß-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 Aß40 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 Aß40 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.