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Two closely related glycogen synthase kinase-3 (GSK-3) isoforms have been identified in mammals: GSK-3α and GSK-3β. GSK-3β is the most prominent in the central nervous system and was previously shown to control neuronal excitability. We previously demonstrated that indirubin and its structural analogue and the nonselective GSK-3 inhibitor BIO-acetoxime exerted anticonvulsant effects in acute seizure models in zebrafish, mice, and rats. We here examined for the first time the anticonvulsant effect of TCS2002, a specific and potent inhibitor of GSK-3β, in two models for limbic seizures: the pilocarpine rat model for focal seizures and the acute 6 Hz corneal mouse model for refractory seizures. Next, we additionally used the 6 Hz kindling model to establish differences in seizure susceptibility and seizure progression in mice that either overexpress human GSK-3β (GSK-3β OE) or lack GSK-3β (GSK-3β-/-) in neurons. We demonstrate that TCS2002 exerts anticonvulsant actions against pilocarpine- and 6 Hz-evoked seizures. Compared to wild-type littermates, GSK-3β OE mice are less susceptible to seizures but are more rapidly kindled. Interestingly, compared to GSK-3β+/+ mice, neuronal GSK-3β-/- mice show increased susceptibility to 6 Hz-induced seizures. These contrasting observations suggest compensatory neurodevelopmental mechanisms that alter seizure susceptibility in GSK-3β OE and GSK-3β-/- mice. Although the pronounced anticonvulsant effects of selective and acute GSK-3β inhibition in the 6 Hz model identify GSK-3β as a potential drug target for pharmacoresistant seizures, our data on the sustained disruption of GSK-3β activity in the transgenic mice suggest a role for GSK-3 in kindling and warrants further research into the long-term effects of selective pharmacological GSK-3β inhibition.

Alzheimer’s disease, characterized by brain deposits of amyloid-β plaques and neurofibrillary tangles, is also linked to neurovascular dysfunction and blood–brain barrier breakdown, affecting the passage of substances into and out of the brain. We hypothesized that treatment of neurovascular alterations could be beneficial in Alzheimer’s disease. Annexin A1 (ANXA1) is a mediator of glucocorticoid anti-inflammatory action that can suppress microglial activation and reduce blood–brain barrier leakage. We have reported recently that treatment with recombinant human ANXA1 (hrANXA1) reduced amyloid-β levels by increased degradation in neuroblastoma cells and phagocytosis by microglia. Here, we show the beneficial effects of hrANXA1 in vivo by restoring efficient blood–brain barrier function and decreasing amyloid-β and tau pathology in 5xFAD mice and Tau-P301L mice. We demonstrate that young 5xFAD mice already suffer cerebrovascular damage, while acute pre-administration of hrANXA1 rescued the vascular defects. Interestingly, the ameliorated blood–brain barrier permeability in young 5xFAD mice by hrANXA1 correlated with reduced brain amyloid-β load, due to increased clearance and degradation of amyloid-β by insulin degrading enzyme (IDE). The systemic anti-inflammatory properties of hrANXA1 were also observed in 5xFAD mice, increasing IL-10 and reducing TNF-α expression. Additionally, the prolonged treatment with hrANXA1 reduced the memory deficits and increased synaptic density in young 5xFAD mice. Similarly, in Tau-P301L mice, acute hrANXA1 administration restored vascular architecture integrity, affecting the distribution of tight junctions, and reduced tau phosphorylation. The combined data support the hypothesis that blood–brain barrier breakdown early in Alzheimer’s disease can be restored by hrANXA1 as a potential therapeutic approach., Dysfunction of the blood–brain barrier is increasingly accepted as an early biomarker for Alzheimer’s disease. Ries et al. show that the acute administration of the anti-inflammatory protein annexin A1 (ANXA1) reverses vascular damage while also reducing amyloid and tau pathology in mouse models of the disorder.

This scientific commentary refers to ‘Induction of a transmissible tau pathology by traumatic brain injury’, by Zanier et al. (doi: 10.1093/brain/awy193 ).

Chronic neuroinflammation is a hallmark of the onset and progression of brain proteinopathies such as Alzheimer disease (AD) and it is suspected to participate in the neurodegenerative process. Transcription factor NRF2, a master regulator of redox homeostasis, controls acute inflammation but its relevance in low-grade chronic inflammation of AD is inconclusive due to lack of good mouse models. We have addressed this question in a transgenic mouse that combines amyloidopathy and tauopathy with either wild type (AT-NRF2-WT) or NRF2-deficiency (AT-NRF2-KO). AT-NRF2-WT mice died prematurely, at around 14 months of age, due to motor deficits and a terminal spinal deformity but AT-NRF2-KO mice died roughly 2 months earlier. NRF2-deficiency correlated with exacerbated astrogliosis and microgliosis, as determined by an increase in GFAP, IBA1 and CD11b levels. The immunomodulatory molecule dimethyl fumarate (DMF), a drug already used for the treatment of multiple sclerosis whose main target is accepted to be NRF2, was tested in this preclinical model. Daily oral gavage of DMF during six weeks reduced glial and inflammatory markers and improved cognition and motor complications in the AT-NRF2-WT mice compared with the vehicle-treated animals. This study demonstrates the relevance of the inflammatory response in experimental AD, tightly regulated by NRF2 activity, and provides a new strategy to fight AD., This work was funded by SAF2016-76520-R of the Spanish Ministry of Economy and Competitiveness, a Pathfinder grant of the Centres of Excellence in Neurodegeneration (COEN) of the EU-Joint Program for Neurodegenerative diseases, and European Regional Development Fund, Competitiveness Operational Program 2014–2020, through the grant P_37_732/2016 REDBRAIN and a collaborative CIBERNED project PI2017/04-3. M.P. is recipient of a FPU fellowship of Autonomous University of Madrid.

Glycogen synthase kinases-3β (GSK3β) is a key regulator of cell homeostasis. In neurons, GSK3β contributes to control of neuronal transmission and plasticity. Despite extensive studies in non-neuronal cells, crosstalk between GSK3β and other signaling pathways remains not well defined in neurons. In the present study, we report that GSK3β positively affected the activity of effectors of mammalian target of rapamycin complex 1 (mTORC1) and complex 2 (mTORC2), in mature neurons in vitro and in vivo. GSK3β also promoted prosurvival signaling and attenuated kainic acid-induced apoptosis. Our study identified GSK3β as a positive regulator of prosurvival signaling, including the mTOR pathway, and indicates the possible neuroprotective role of GSK3β in models of pharmacologically induced excitotoxicity. ispartof: MOLECULAR NEUROBIOLOGY vol:55 issue:7 pages:6050-6062 ispartof: location:United States status: published

PURPOSE: Vascular dysfunction is a major hallmark of Alzheimer's disease (AD). However, studies that investigated vascular dysfunction in mice modeling AD using magnetic resonance angiography (MRA) are typically limited to qualitative and/or scoring-based paradigms, which are labor-intensive and observer-dependent. PROCEDURES: We developed and validated a semi-automatic MRA processing pipeline and applied this to high-resolution in vivo MRA images acquired on a 9.4T small animal MRI scanner. We assessed vascular morphology at 3, 6, and 12 months in wild-type (WT) and bigenic (APP.V717IxTau.P301L: biAT) mice. RESULTS: Vessel radius or length can increase with age regardless of genotype depending on the respective vessel. We also observed significantly lower internal carotid artery length in biAT mice compared to WT. CONCLUSIONS: The results demonstrate that even subtle changes in vessel morphology can be noninvasively quantified. This is of great interest for AD, but also to other models of neurodegenerative diseases involving macrovascular dysfunction. ispartof: MOLECULAR IMAGING AND BIOLOGY vol:22 issue:3 pages:578-586 ispartof: location:United States status: published

The microtubule-associated protein Tau is an intrinsically unfolded, very soluble neuronal protein. Under still unknown circumstances, Tau protein forms soluble oligomers and insoluble aggregates that are closely linked to the cause and progression of various brain pathologies, including Alzheimer's disease. Previously we reported the development of liposome-based vaccines and their efficacy and safety in preclinical mouse models for tauopathy. Here we report the use of a liposomal vaccine for the generation of a monoclonal antibody with particular characteristics that makes it a valuable tool for fundamental studies as well as a candidate antibody for diagnostic and therapeutic applications. The specificity and affinity of antibody ACI-5400 were characterized by a panel of methods: (i) measuring the selectivity for a specific phospho-Tau epitope known to be associated with tauopathy, (ii) performing a combination of peptide and protein binding assays, (iii) staining of brain sections from mouse preclinical tauopathy models and from human subjects representing six different tauopathies, and (iv) evaluating the selective binding to pathological epitopes on extracts from tauopathy brains in non-denaturing sandwich assays. We conclude that the ACI-5400 antibody binds to protein Tau phosphorylated at S396 and favors a conformation that is typically present in the brain of tauopathy patients, including Alzheimer's disease.

Autophagy is a highly coordinated process that is controlled at several levels including transcriptional regulation. Here, we identify the transcription factor NFE2L2/NRF2 (nuclear factor, erythroid 2 like 2) as a regulator of autophagy gene expression and its relevance in a mouse model of Alzheimer disease (AD) that reproduces impaired APP (amyloid β precursor protein) and human (Hs)MAPT/TAU processing, clearance and aggregation. We screened the chromatin immunoprecipitation database ENCODE for 2 proteins, MAFK and BACH1, that bind the NFE2L2-regulated enhancer antioxidant response element (ARE). Using a script generated from the JASPAR's consensus ARE sequence, we identified 27 putative AREs in 16 autophagy-related genes. Twelve of these sequences were validated as NFE2L2 regulated AREs in 9 autophagy genes by additional ChIP assays and quantitative RT-PCR on human and mouse cells after NFE2L2 activation with sulforaphane. Mouse embryo fibroblasts of nfe2l2-knockout mice exhibited reduced expression of autophagy genes, which was rescued by an NFE2L2 expressing lentivirus, and impaired autophagy flux when exposed to hydrogen peroxide. NFE2L2-deficient mice co-expressing HsAPPV717I and HsMAPTP301L, exhibited more intracellular aggregates of these proteins and reduced neuronal levels of SQSTM1/p62, CALCOCO2/NDP52, ULK1, ATG5 and GABARAPL1. Also, colocalization of HsAPPV717I and HsMAPTP301L with the NFE2L2-regulated autophagy marker SQSTM1/p62 was reduced in the absence of NFE2L2. In AD patients, neurons expressing high levels of APP or MAPT also expressed SQSTM1/p62 and nuclear NFE2L2, suggesting their attempt to degrade intraneuronal aggregates through autophagy. This study shows that NFE2L2 modulates autophagy gene expression and suggests a new strategy to combat proteinopathies.

In view of the clinical need for new antiseizure drugs (ASDs) with novel modes of action, we used a zebrafish seizure model to screen the anticonvulsant activity of medicinal plants used by traditional healers in the Congo for the treatment of epilepsy, and identified a crude plant extract that inhibited pentylenetetrazol (PTZ)-induced seizures in zebrafish larvae. Zebrafish bioassay-guided fractionation of this anticonvulsant Fabaceae species, Indigofera arrecta, identified indirubin, a compound with known inhibitory activity of glycogen synthase kinase (GSK)-3, as the bioactive component. Indirubin, as well as the more potent and selective GSK-3 inhibitor 6-bromoindirubin-3'-oxime (BIO-acetoxime) were tested in zebrafish and rodent seizure assays. Both compounds revealed anticonvulsant activity in PTZ-treated zebrafish larvae, with electroencephalographic recordings revealing reduction of epileptiform discharges. Both indirubin and BIO-acetoxime also showed anticonvulsant activity in the pilocarpine rat model for limbic seizures and in the 6-Hz refractory seizure mouse model. Most interestingly, BIO-acetoxime also exhibited anticonvulsant actions in 6-Hz fully kindled mice. Our findings thus provide the first evidence for anticonvulsant activity of GSK-3 inhibition, thereby implicating GSK-3 as a potential therapeutic entry point for epilepsy. Our results also support the use of zebrafish bioassay-guided fractionation of antiepileptic medicinal plant extracts as an effective strategy for the discovery of new ASDs with novel mechanisms of action.

Abnormal accumulation of tau aggregates in the brain is one of the hallmarks of Alzheimer disease neuropathology. We visualized tau deposition in vivo with the previously developed 2-arylquinoline derivative (18)F-THK5117 using small-animal PET in conjunction with autoradiography and immunohistochemistry gold standard assessment in 2 transgenic mouse models expressing hyperphosphorylated tau. Small-animal PET recordings were obtained in groups of P301S (n = 11) and biGT mice (n = 16) of different ages, with age-matched wild-type (WT) serving as controls. After intravenous administration of 16 ± 2 MBq of (18)F-THK5117, a dynamic 90-min emission recording was initiated for P301S mice and during 20-50 min after injection for biGT mice, followed by a 15-min transmission scan. After coregistration to the MRI atlas and scaling to the cerebellum, we performed volume-of-interest-based analysis (SUV ratio [SUVR]) and statistical parametric mapping. Small-animal PET results were compared with autoradiography ex vivo and in vitro and further validated with AT8 staining for neurofibrillary tangles. SUVRs calculated from static recordings during the interval of 20-50 min after tracer injection correlated highly with estimates of binding potential based on the entire dynamic emission recordings (R = 0.85). SUVR increases were detected in the brain stem of aged P301S mice (+11%; P < 0.001) and in entorhinal/amygdaloidal areas (+15%; P < 0.001) of biGT mice when compared with WT, whereas aged WT mice did not show increased tracer uptake. Immunohistochemical tau loads correlated with small-animal PET SUVR for both P301S (R = 0.8; P < 0.001) and biGT (R = 0.7; P < 0.001) mice, and distribution patterns of AT8-positive neurons matched voxelwise statistical parametric mapping analysis. Saturable binding of the tracer was verified by autoradiographic blocking studies. In the first dedicated small-animal PET study in 2 different transgenic tauopathy mouse models using the tau tracer (18)F-THK5117, the temporal and spatial progression could be visualized in good correlation with gold standard assessments of tau accumulation. The serial small-animal PET method could afford the means for preclinical testing of novel therapeutic approaches by accommodating interanimal variability at baseline, while detection thresholds in young animals have to be considered.

Changes in the morphology of dendritic spines are prominent during learning and in different neurological and neuropsychiatric diseases, including those in which glycogen synthase kinase-3β (GSK-3β) has been implicated. Despite much evidence of the involvement of GSK-3β in functional synaptic plasticity, it is unclear how GSK-3β controls structural synaptic plasticity (i.e., the number and shape of dendritic spines). In the present study, we used two mouse models overexpressing and lacking GSK-3β in neurons to investigate how GSK-3β affects the structural plasticity of dendritic spines. Following visualization of dendritic spines with DiI dye, we found that increasing GSK-3β activity increased the number of thin spines, whereas lacking GSK-3β increased the number of stubby spines in the dentate gyrus. Under conditions of neuronal excitation, increasing GSK-3β activity caused higher activity of extracellularly acting matrix metalloproteinase-9 (MMP-9), and MMP inhibition normalized thin spines in GSK-3β overexpressing mice. Administration of the nonspecific GSK-3β inhibitor lithium in animals with active MMP-9 and animals lacking MMP-9 revealed that GSK-3β and MMP-9 act in concert to control dendritic spine morphology. Altogether, our data demonstrate that the dysregulation of GSK-3β activity has dramatic consequences on dendritic spine morphology, implicating MMP-9 as a mediator of GSK-3β-induced synaptic alterations.

In Alzheimer's disease, the hippocampus is characterized by abundant deposition of amyloid peptides (amyloid β [Aβ]) and neuroinflammation. Adult hippocampal neurogenesis (AHN) is a form of plasticity that contributes to cognition and can be influenced by either or both pathology and neuroinflammation. Their interaction has been studied before in rapidly progressing transgenic mouse models with strong overexpression of amyloid precursor protein (APP) and/or presenilin 1. So far, changes in AHN and neuroinflammation remain poorly characterized in slower progressing models at advanced age, which approach more closely sporadic Alzheimer's disease. Here, we analyzed 10- to 26-month-old APP.V717I mice for possible correlations between Aβ pathology, microglia, and AHN. The age-related increase in amyloid pathology was closely paralleled by microglial CD68 upregulation, which was largely absent in age-matched wild-type littermates. Notably, aging reduced the AHN marker doublecortin, but not calretinin, to a similar extent in wild-type and APP.V717I mice between 10 and 26 months. This demonstrates that AHN is influenced by advanced age in the APP.V717I mouse model, but not by Aβ and microglial activation.

The microtubule-associated protein Tau is responsible for a large group of neurodegenerative disorders, known as tauopathies, including Alzheimer's disease. Tauopathy result from augmented and/or aberrant phosphorylation of Tau. Besides aging and various genetic and epigenetic defects that remain largely unknown, an important non-genetic agent that contributes is hypothermia, eventually caused by anesthesia. Remarkably, tauopathy in brains of hibernating mammals is not pathogenic, and, because it is fully reversible, is even considered to be neuroprotective. Here, we assessed the terminal phase of Tau.P301L mice and bigenic crosses with mice lacking glycogen synthase kinase 3 (GSK3)α completely, or GSK3β specifically in neurons. We also analysed biGT bigenic mice that co-express Tau.P301L with GSK3β.S9A and develop severe forebrain tauopathy with age. We found that the precocious mortality of Tau.P301L mice was typified by hypothermia that aggravated Tau phosphorylation, but, surprisingly, independently of GSK3α/β. The important contribution of hypothermia at the time of death of mice with tauopathy suggests that body temperature should be included as a parameter in the analysis of pre-clinical models, and, by extension, in patients suffering from tauopathy.

The chemokine fractalkine modulates microglial responses in neurodegenerative diseases, including tauopathies, but the mechanistic processes and their relevance in human brain pathologies is not yet known. Here, we show that hippocampal HT22 cells expressing human TAU(P301L) mutant protein produce fractalkine, which in microglia activates AKT, inhibits glycogen synthase kinase-3β and upregulates the transcription factor NRF2/NFE2L2 and its target genes including heme oxygenase 1. In a mouse model of tauopathy based on stereotaxic delivery in hippocampus of an adeno-associated viral vector for expression of TAU(P301L), we confirmed that tau-injured neurons express fractalkine. NRF2- and fractalkine receptor-knockout mice did not express heme oxygenase 1 in microglia and exhibited increased microgliosis and astrogliosis in response to neuronal TAU(P301L) expression, demonstrating a crucial role of the fractalkine/NRF2/heme oxygenase 1 pathway in attenuation of the pro-inflammatory phenotype. The hippocampus of patients with Alzheimer's disease also exhibits increased expression of fractalkine in TAU-injured neurons that recruit microglia. These events correlated with increased levels of NRF2 and heme oxygenase 1 proteins, suggesting an attempt of the diseased brain to limit microgliosis. Our combined results indicate that fractalkine mobilizes NRF2 to limit over-activation of microglia and identify this new target to control unremitting neuroinflammation in tauopathies.

Previous studies have investigated the activity and protein levels of BACE1, the β-secretase, in the brain and cerebrospinal fluid (CSF) of Alzheimer's disease (AD) patients, however, results remain contradictory. We present here a highly specific and sensitive BACE1 ELISA, which allows measuring accurately BACE1 levels in human samples. We find that BACE1 levels in CSF of AD patients and other neurological disorder (OND) patients are slightly increased when compared to those of a non-neurological disorder control group (NND). BACE1 levels in CSF were well correlated with total-tau and hyperphosphorylated tau levels in the CSF, suggesting that the recorded alterations in BACE1 levels correlate with cell death and neurodegeneration.

Glycogen synthase kinase-3β (GSK3β) activity has been previously linked to Alzheimer's disease (AD) by its phosphorylation of tau and activation by amyloid. GSK3β intracellular distribution is important in regulating its activity by restricting access to compartment-specific substrates. This study investigated regional and intracellular distribution of GSK3β in a mouse model of AD, a bigenic mouse with combined amyloid and tau pathology (BiAT), and controls (FVB). At two different ages, the entire rostrocaudal extent of each brain was examined. Young (6-months-old) FVB and BiAT mice did not differ in GSK3β expression and localization. In old (13-month-old) BiAT mice, neurons showed increased GSK3β expression only in AD-relevant brain regions as compared with modest staining in region- and age-matched controls. Two regions with the most robust changes between FVB and BiAT mice, the amygdala and piriform cortex, were quantified at the light microscopic level. In both regions, the density of darkly labeled neurons was significantly greater in the old BiAT mice vs. the old FVB mice. Electron microscopy of the piriform cortex showed neuronal GSK3β labeling in the rough endoplasmic reticulum, on ribosomes, and on microtubules in dendrites in both strains of mice. In old BiAT mice, GSK3β labeling was qualitatively more robust compared to age-matched controls, and GSK3β also appeared in neurofibrillary tangles. In conclusion, GSK3β expression was increased in specific intracellular locations and was found in tangles in old BiAT mice, suggesting that GSK3β overexpression in specific brain areas may be intrinsic to AD pathology.

Background: Traumatic brain injury (TBI), even single-impact mild TBI (mTBI), is associated with increased risk of neuronal damage and neurodegeneration, leading to dementia, in particular Alzheimer’s disease (AD). The tell-tale histopathological defects of AD, deposits of amyloid and protein tau are observed following TBI. However, little is known about the mechanisms underlying the association, and its impact on synaptic plasticity. Objectives: This study aimed to analyze whether mTBI alters or accelerates relevant changes in synaptic plasticity at early stages of tau pathology in a mouse model. Methods: A total of 24 mice were analyzed in this study, comprising Tau.P301L transgenic mice and age and background matched wild-type mice as controls. Animals received a mild single-dose closed-head impact injury or a sham surgery. We measured ex vivo parameters of basal synaptic excitability, short-term synaptic plasticity and long-term potentiation. Results: While no changes in basal synaptic excitability and presynaptic short-term plasticity were observed, long-term potentiation (LTP) in the CA1 region was severely impaired. This deficit was aggravated in the Tau.P301L mice compared to wild-type control animals. Conclusions: Our data imply a high health risk of even a single mTBI episode. Single-impact mTBI combined with genetic predisposition is proposed to trigger signals implicated in neurodegeneration.

Alzheimer's disease (AD) has been associated with altered activity of glycogen synthase kinase 3 (GSK3) isozymes, which are proposed to contribute to both neurofibrillary tangles and amyloid plaque formation. However, the molecular basis by which GSK3 affects the formation of Aβ remains unknown. Our aim was to identify the underlying mechanisms of GSK3-dependent effects on the processing of amyloid precursor protein (APP). For this purpose, N2a cells stably expressing APP carrying the Swedish mutation were treated with specific GSK3 inhibitors or transfected with GSK3α/β short interfering RNA. We show that inhibition of GSK3 leads to decreased expression of APP by enhancing its degradation via an increase in the number of lysosomes. This induction of the lysosomal/autophagy pathway was associated with nuclear translocation of transcription factor EB (TFEB), a master regulator of lysosomal biogenesis. Our data indicate that GSK3 inhibition reduces Aβ through an increase of the degradation of APP and its carboxy-terminal fragment (CTF) by activation of the lysosomal/autophagy pathway. These results suggest that an increased propensity toward autophagic/lysosomal alterations in AD patients could have consequences for neuronal function.

The transcription factor NF-E2-related factor 2 (Nrf2) is a master regulator of a genetic program, termed the phase 2 response, that controls redox homeostasis and participates in multiple aspects of physiology and pathology. Nrf2 protein stability is regulated by two E3 ubiquitin ligase adaptors, Keap1 and β-TrCP, the latter of which was only recently reported. Here, two-dimensional (2D) gel electrophoresis and site-directed mutagenesis allowed us to identify two serines of Nrf2 that are phosphorylated by glycogen synthase kinase 3β (GSK-3β) in the sequence DSGISL. Nuclear magnetic resonance studies defined key residues of this phosphosequence involved in docking to the WD40 propeller of β-TrCP, through electrostatic and hydrophobic interactions. We also identified three arginine residues of β-TrCP that participate in Nrf2 docking. Intraperitoneal injection of the GSK-3 inhibitor SB216763 led to increased Nrf2 and heme oxygenase-1 levels in liver and hippocampus. Moreover, mice with hippocampal absence of GSK-3βexhibited increased levels of Nrf2 and phase 2 gene products, reduced glutathione, and decreased levels of carbonylated proteins and malondialdehyde. This study establishes the structural parameters of the interaction of Nrf2 with the GSK-3/β-TrCP axis and its functional relevance in the regulation of Nrf2 by the signaling pathways that impinge on GSK-3, This work was supported by MICINN grant SAF2010-18722 from the Spanish Ministry of Science and Innovation. Patricia Rada is contracted under the Formación de Profesorado Universitario program of the Spanish Ministry of Science and Innovation. We acknowledge funding and infrastructural support from EEC 7th Framework Program, KU Leuven Research Fund, and KU Leuven Research & Development

The microtubule-associated protein Tau (MAPT) is a major component of the pathogenesis of a wide variety of brain-damaging disorders, known as tauopathies. These include Alzheimer's disease (AD), denoted as secondary tauopathy because of the obligatory combination with amyloid pathology. In all tauopathies, protein Tau becomes aberrantly phosphorylated, adopts abnormal conformations, and aggregates into fibrils that eventually accumulate as threads in neuropil and as tangles in soma. The argyrophilic neurofibrillary threads and tangles, together denoted as NFT, provide the postmortem pathological diagnosis for all tauopathies. In AD, neurofibrillary threads and tangles (NFTs) are codiagnostic with amyloid depositions but their separated and combined contributions to clinical symptoms remain elusive. Importantly, NFTs are now considered a late event and not directly responsible for early synaptic dysfunctions. Conversely, the biochemical and pathological timeline is not exactly known in human tauopathy, but experimental models point to smaller Tau-aggregates, termed oligomers or multimers, as synaptotoxic in early stages. The challenge is to molecularly define these Tau-isoforms that cause early cognitive and synaptic impairments. Here, we discuss relevant studies and data obtained in our mono- and bigenic validated preclinical models, with the perspective of Tau as a therapeutic target.

Cognitive demise correlates with progressive brain tauopathy in dementing patients. Improved cognition of young Tau.P301L mice contrasts with dysfunction later in life and remains unexplained (Boekhoorn et al., 2006). To unravel early mechanisms, we composed a correlative time line of clinical symptoms, cognitive defects, and biochemical and pathological traits, including comprehensive analysis of dendritic spines in specified regions of the cortex and hippocampus of young and adult Tau.P301L mice. Remarkably, young Tau.P301L mice have not more, but more mature spines than wild-type mice, revealing the anatomical substrate for their improved cognition and LTP. Spine maturation remained high in the hippocampus of adult Tau.P301L mice. However, spines regressed in length paralleling impaired cognition and increased Tau phosphorylation (Terwel et al., 2005). Conversely, spine maturation was unaffected in adult Tau.4R mice, while spine density was increased and length reduced similar to Tau.P301L mice. To explain how protein Tau promoted spinogenesis, we analyzed hippocampal synaptosomes and dendritic spines for mouse and human Tau. While synaptosomes were positive for both mouse and human Tau, weak variable reaction in spines was observed only after fixation according to Bouin. Mouse Tau was absent from spines in wild-type mice, dissociating the pathological actions of Tau in transgenic mice by relocalization into dendrites and spines from the physiological actions of protein Tau in axons only. We conclude that mutant protein Tau modulates cognition and morphology of spines similarly and in both directions, with pathology later in life coinciding with increased phosphorylation and relocalization of Tau from axons to soma and processes.

Mutations in presenilins are the major cause of familial Alzheimer's disease (FAD), leading to impairments of memory and synaptic plasticity followed by age-dependent neurodegeneration. Presenilins are the catalytic subunits of γ-secretase, which itself is critically involved in the processing of amyloid precursor protein to release neurotoxic amyloid β (Aβ). Besides Aβ generation, there is growing evidence that presenilins play an essential role in the formation and maintenance of synapses. To further elucidate the effect of presenilin1 (PS1) on synapses, we performed longitudinal in vivo two-photon imaging of dendritic spines in the somatosensory cortex of transgenic mice over-expressing either human wild-type PS1 or the FAD-mutated variant A246E (FAD-PS1). Interestingly, the consequences of transgene expression were different in two subtypes of cortical dendrites. On apical layer 5 dendrites, we found an enhanced spine density in both mice over-expressing human wild-type presenilin1 and FAD-PS1, whereas on basal layer 3 dendrites only over-expression of FAD-PS1 increased the spine density. Time-lapse imaging revealed no differences in kinetically distinct classes of dendritic spines nor was the shape of spines affected. Although γ-secretase-dependent processing of synapse-relevant proteins seemed to be unaltered, higher expression levels of ryanodine receptors suggest a modified Ca(2+) homeostasis in PS1 over-expressing mice. However, the conditional depletion of PS1 in single cortical neurons had no observable impact on dendritic spines. In consequence, our results favor the view that PS1 influences dendritic spine plasticity in a gain-of-function but γ-secretase-independent manner.

BACE1 cleaves the amyloid precursor protein (APP) at the β-cleavage site (Met(671) -Asp(672) ) to initiate the generation of amyloid peptide Aβ. BACE1 is also known to cleave APP at a much less well-characterized β'-cleavage site (Tyr(681) -Glu(682) ). We describe here the identification of a novel APP mutation E682K located at this β'-site in an early onset Alzheimer's disease (AD) case. Functional analysis revealed that this E682K mutation blocked the β'-site and shifted cleavage of APP to the β-site, causing increased Aβ production. This work demonstrates the functional importance of APP processing at the β'-site and shows how disruption of the balance between β- and β'-site cleavage may enhance the amyloidogenic processing and consequentially risk for AD. Increasing exon- and exome-based sequencing efforts will identify many more putative pathogenic mutations without conclusive segregation-based evidence in a single family. Our study shows how functional analysis of such mutations allows to determine the potential pathogenic nature of these mutations. We propose to classify the E682K mutation as probable pathogenic awaiting further independent confirmation of its association with AD in other patients. →See accompanying Closeup by Rita Guerreiro and John Hardy DOI 10.1002/emmm201100139. ispartof: EMBO Molecular Medicine vol:3 issue:5 pages:291-302 ispartof: location:England status: published

Adeno-associated virus (AAV)-mediated expression of wild-type or mutant P301L protein tau produces massive degeneration of pyramidal neurons without protein tau aggregation. We probed this novel model for genetic and structural factors and early parameters of pyramidal neurodegeneration. In yellow fluorescent protein expressing transgenic mice, intracerebral injection of AAV-tauP301L revealed early damage to apical dendrites of CA1 pyramidal neurons, whereas their somata remained normal. Ultrastructurally, more and enlarged autophagic vacuoles were contained in degenerating dendrites and manifested as dark, discontinuous, vacuolated processes surrounded by activated astrocytes. Dendritic spines were lost in AAV-tauP301L-injected yellow fluorescent protein-expressing transgenic mice, and ultrastructurally, spines appeared dark and degenerating. In CX3CR1(EGFP/EGFP)-deficient mice, microglia were recruited early to neurons expressing human tau. The inflammatory response was accompanied by extravasation of plasma immunoglobulins alpha 2-Macroglobulin, but neither albumin nor transferrin, became lodged in the brain parenchyma. Large proteins, but not Evans blue, entered the brain of mice injected with AAV-tauP301L. Ultrastructurally, brain capillaries were constricted and surrounded by swollen astrocytes with extensions that contacted degenerating dendrites and axons. Together, these data corroborate the hypothesis that neuroinflammation participates essentially in tau-mediated neurodegeneration, and the model recapitulates early dendritic defects reminiscent of "dendritic amputation" in Alzheimer's disease. (Am J Pathol 2011, 179:2001-2015; DOI: 10.1016/j.ajpath.2011.06.025)

Unraveling the biochemical and genetic alterations that control the aggregation of protein tau is crucial to understand the etiology of tau-related neurodegenerative disorders. We expressed wild type and six clinical frontotemporal dementia with parkinsonism (FTDP) mutants of human protein tau in wild-type yeast cells and cells lacking Mds1 or Pho85, the respective orthologues of the tau kinases GSK3β and cdk5. We compared tau phosphorylation with the levels of sarkosyl-insoluble tau (SinT), as a measure for tau aggregation. The deficiency of Pho85 enhanced significantly the phosphorylation of serine-409 (S409) in all tau mutants, which coincided with marked increases in SinT levels. FTDP mutants tau-P301L and tau-R406W were least phosphorylated at S409 and produced the lowest levels of SinT, indicating that S409 phosphorylation is a direct determinant for tau aggregation. This finding was substantiated by the synthetic tau-S409A mutant that failed to produce significant amounts of SinT, while its pseudophosphorylated counterpart tau-S409E yielded SinT levels higher than or comparable to wild-type tau. Furthermore, S409 phosphorylation reduced the binding of protein tau to preformed microtubules. The highest SinT levels were found in yeast cells subjected to oxidative stress and with mitochondrial dysfunction. Under these conditions, the aggregation of tau was enhanced although the protein is less phosphorylated, suggesting that additional mechanisms are involved. Our results validate yeast as a prime model to identify the genetic and biochemical factors that contribute to the pathophysiology of human tau.

Alzheimer's dementia is developing ever more as a complex syndrome with various unknown genetic and epigenetic contributions. These are compounded on and exacerbating the underlying amyloid and tau pathology that remain the basis of the pathological definition of Alzheimer's disease. Here, we present a selection of aspects of recent bigenic and virus-based mouse strains, developed as pre-clinical models for Alzheimer's disease. We discuss newer features in the context of the characteristics defined in previously validated transgenic models. We focus on specific aspects of single and multiple transgenic mouse models for Alzheimer's disease and for tauopathies, rather than providing an exhaustive list of all available models. We concentrate on the content of information related to neurodegeneration and disease mechanisms. We pay attention to aspects and defects that are predicted by the models and can be tested in humans. We discuss implications that help translate the fundamental knowledge into clinical, diagnostic and therapeutic applications. We elaborate on the increasing knowledge extracted from transgenic models and from newer adeno-associated viral models. We advocate this combination as a valuable strategy to study molecular, cellular and system-related pathogenic mechanisms in AD and tauopathies. We believe that innovative animal models remain needed to critically test current views, to identify and validate therapeutic targets, to allow testing of compounds, to help understand and eventually treat tauopathies, including Alzheimer's disease.

The amyloid precursor family of proteins are of considerable interest, both because of their role in Alzheimer's disease pathogenesis and because of their normal physiological functions. In mammals, the amyloid precursor protein (APP) has two homologs, amyloid precursor-like protein (APLP) 1 and APLP2. All three proteins undergo ectodomain shedding and regulated intramembrane proteolysis, and important functions have been attributed to the full-length proteins, shed ectodomains, C-terminal fragments and intracellular domains (ICDs). One of the proteases that is known to cleave APP and that is essential for generation of the amyloid beta-protein is the beta-site APP-cleaving enzyme 1 (BACE1). Here, we investigated the effects of genetic manipulation of BACE1 on the processing of the APP family of proteins. BACE1 expression regulated the levels and species of full-length APLP1, APP and APLP2, of their shed ectodomains, and of their membrane-bound C-terminal fragments. In particular, APP processing appears to be tightly regulated, with changes in beta-cleaved APPs (APPsbeta) being compensated for by changes in alpha-cleaved APPs (APPsalpha). In contrast, the total levels of soluble cleaved APLP1 and APLP2 species were less tightly regulated, and fluctuated with BACE1 expression. Importantly, the production of ICDs for all three proteins was not decreased by loss of BACE1 activity. These results indicate that BACE1 is involved in regulating ectodomain shedding, maturation and trafficking of the APP family of proteins. Consequently, whereas inhibition of BACE1 is unlikely to adversely affect potential ICD-mediated signaling, it may alter other important facets of amyloid precursor-like protein/APP biology.

Tauopathy comprises hyperphosphorylation of the microtubule-associated protein tau, causing intracellular aggregation and accumulation as neurofibrillary tangles and neuropil treads. Some primary tauopathies are linked to mutations in theMAPTgene coding for protein tau, but most are sporadic with unknown causes. Also, in Alzheimer's disease, the most frequent secondary tauopathy, neither the cause nor the pathological mechanisms and repercussions are understood. Transgenic mice expressing mutant Tau-P301L suffer cognitive and motor defects and die prematurely from unknown causes. Here,in situelectrophysiology in symptomatic Tau-P301L mice (7–8 months of age) revealed reduced postinspiratory discharges of laryngeal motor outputs that control laryngeal constrictor muscles. Under high chemical drive (hypercapnia), postinspiratory discharge was nearly abolished, whereas laryngeal inspiratory discharge was increased disproportionally. The latter may suggest a shift of postinspiratory laryngeal constrictor activity into inspiration.In vivodouble-chamber plethysmography of Tau-P301L mice showed significantly reduced respiratory airflow but significantly increased chest movements during baseline breathing, but particularly in hypercapnia, confirming a significant increase in inspiratory resistive load. Histological analysis demonstrated hyperphosphorylated tau in brainstem nuclei, directly or indirectly involved in upper airway motor control (i.e., the Kölliker–Fuse, periaqueductal gray, and intermediate reticular nuclei). In contrast, young Tau-P301L mice did not show breathing disorders or brainstem tauopathy. Consequently, in aging Tau-P301L mice, progressive upper airway dysfunction is linked to progressive tauopathy in identified neural circuits. Because patients with tauopathy suffer from upper airway dysfunction, the Tau-P301L mice can serve as an experimental model to study disease-specific synaptic dysfunction in well defined functional neural circuits.

Patients suffering from Alzheimer's disease (AD) are typified and diagnosed postmortem by the combined accumulations of extracellular amyloid plaques and of intracellular tauopathy, consisting of neuropil treads and neurofibrillary tangles in the somata. Both hallmarks are inseparable and remain diagnostic as described by Alois Alzheimer more than a century ago. Nevertheless, these pathological features are largely abandoned as being the actual pathogenic or neurotoxic factors. The previous, almost exclusive experimental attention on amyloid has shifted over the last 10 years in two directions. Firstly, from the "concrete" deposits of amyloid plaques to less well-defined soluble or pseudosoluble oligomers of the amyloid peptides, ranging from dimers to dodecamers and even larger aggregates. A second shift in research focus is from amyloid to tauopathy, and to their mutual relation. The role of Tau in the pathogenesis and disease progression is appreciated as leading to synaptic and neuronal loss, causing cognitive deficits and dementia. Both trends are incorporated in a modified amyloid cascade hypothesis, briefly discussed in this paper that is mainly concerned with the second aspect, that is, protein Tau and its associated fundamental questions. ispartof: International journal of Alzheimer's disease vol:2010 pages:573138- ispartof: location:United States status: published

Apolipoprotein E (apoE) is a regulator of peripheral cholesterol homeostasis, and the apoE-isoform E4 is a major risk factor for the development of Alzheimer's disease (AD). Accumulating evidence suggests a key role for aberrant cholesterol metabolism in AD. We hypothesized that apoE-deficiency in mice not only affects cholesterol homeostasis in the periphery, but also in the brain, and that this can be restored by astrocyte-specific expression of human apoE3, but not apoE4. Using gas-chromatography mass-spectrometry, we found that absence of apoE in mice does not affect brain cholesterol homeostasis although serum sterol levels increase dramatically, especially when the apoE-knockout mice are fed a high fat diet. We provide evidence suggesting that apoD and the ATP-binding Cassette Transporter A1 (ABCA1) play a compensatory role in the apoE-deficient brain. Surprisingly, astrocyte-specific expression of human apoE3 or apoE4 in brains of apoE-knockout mice significantly increases brain levels of cholesterol and its precursors compared to control mice, indicative of an increased cholesterol synthesis rate in the brain. This increase is independent of the apoE-isoform, suggesting that the detrimental effect of apoE4 on the pathogenesis of AD is unlikely to be due to an apoE-isoform effect on brain cholesterol homeostasis.