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Background: Glycogen synthase kinase-3 beta (GSK3 beta) is a key regulator of cellular homeostasis. In neurons, GSK3 beta contributes to the control of neuronal transmission and plasticity, but its role in epilepsy remains to be defined. Methods: Biochemical and electrophysiological methods were used to assess the role of GSK3 beta in regulating neuronal transmission and epileptogenesis. GSK3 beta activity was increased genetically in GSK3 beta[S9A] mice. Its effects on neuronal transmission and epileptogenesis induced by kainic acid were assessed by field potential recordings in mice brain slices and video electroencephalography in vivo. The ion channel expression was measured in brain samples from mice and followed by analysis in samples from patients with temporal lobe epilepsy or focal cortical dysplasia in correlation to GSK3 beta phosphorylation. Findings: Higher GSK3 beta activity decreased the progression of kainic acid induced epileptogenesis. At the biochemical level, higher GSK3 beta activity increased the expression of hyperpolarization-activated cyclic nucleotide-gated (HCN) channel 4 under basal conditions and in the epileptic mouse brain and decreased phosphorylation of the glutamate alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluA1 at Serine 831 under basal conditions. Moreover, we found a significant correlation between higher inhibitory-GSK3 beta phosphorylation at Serine 9 and higher activating GluA1 phosphorylation at Serine 845 in brain samples from epileptic patients. Interpretation: Our data imply GSK3 beta activity in the protection of neuronal networks from hyper-activation in response to epileptogenic stimuli and indicate that the anti-epileptogenic function of GSK3 beta involves modulation of HCN4 level and the synaptic AMPA receptors pool. (c) 2018 The Authors. Published by Elsevier B.V.

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.

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.

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.

AD patients suffer epileptogenc defects. Time-line analysis of bigenic biAT mice that co-express APP.V717I and Tau.P301L in neurons and develop combined Alzheimer pathology (>10-12 months). Important precocious mortality of young biAT mice (<6 months) with epileptic symptoms was totally absent in the parental monogenic mice. Neither amyloid nor tauopathy was evident in brain of young biAT mice. Amyloid accumulation and pathology developed in biAT and parental APP.V717I mice very similar with age, while tau phosphorylation and oligomerization was more early and more extensive than in age-matched Tau.P301L mice. Surprisingly, tauopathy was less pronounced in brainstem of old biAT mice that escaped early epileptic death. Survival beyond age 12 months, the limit of Tau.P301L mice, was explained by activation of GSK3 by amyloid in the biAT mice, resorting the same effect as co-expression of GSK3β with Tau.P301L in biGT mice (Terwel et al, 2008). Despite very different timing, the biochemical pathway to tauopathy was similar: progressive tau-phosphorylation, soluble oligomers, insoluble aggregates, ending in tangles and neuropil threads. Early death of young biAT mice is by epileptogenic activity based on clinical observations in the homecage and during tests: seizures, convulsions, freezing, salivation, tongue biting, posture of corpses. Moreover, spontaneous and induced epileptogenic activity was observed during electrophysiological recordings from brain sections of young biAT mice. Additionally, young biAT mice already suffer functional and structural defects in synapses and dendritic spines in cortex and hippocampus that underlie or contribute to electric hyperactivity. The reported high incidence of epilepsy in AD patients deserves further attention by retrospective and prospective studies.

Tau.P301L transgenic mice suffer precocious mortality between ages 8 and 11 months, resulting from upper airway defects caused by tauopathy in autonomic brainstem circuits that control breathing (Dutschmann et al., 2010). In individual mice, the clinical phenotype evolves progressively and rapidly (3-6weeks) from clasping, over general motor impairment to severe reduction in body-weight into the terminal phase that announces imminent death (<3days). Surprisingly, co-expression of GSK3β with Tau.P301L significantly prolonged survival of bigenic biGT mice (Terwel et al., 2008), which we here assign to delayed development of brainstem tauopathy. Eventually, brainstem tauopathy became as prominent in old biGT mice in the specified brainstem nuclei as in the parental Tau.P301L mice, resulting in similar clinical deterioration and terminal phase preceding death, although at later age. Biochemically, in both genotypes the pathway to neurofibrillary tangles and neuropil threads was similar: phosphorylation of protein Tau and formation of soluble oligomers and insoluble aggregates, ending in the typical tangles and threads of tauopathy. The extra GSK3β activity led to expected increased phosphorylation of protein Tau, particularly at residues S262 and S396, which we must conclude to delay the aggregation of protein Tau in the brainstem of aging biGT mice. The unexpected, paradoxical alleviation of the brainstem problems in biGT mice allowed them to grow older and thereby develop more severe tauopathy in forebrain than Tau.P301L mice, which succumb at younger age.

The stratum lacunosum moleculare (SLM) is the connection hub between entorhinal cortex and hippocampus, two brain regions that are most vulnerable in Alzheimer’s disease. We recently identified a specific synaptic deficit of Nectin-3 in transgenic models for tauopathy. Here we defined cognitive impairment and electrophysiological problems in the SLM of Tau.P301L mice, which corroborated the structural defects in synapses and dendritic spines. Reduced diffusion of DiI from the ERC to the hippocampus indicated defective myelinated axonal pathways. Ultrastructurally, myelinated axons in the temporoammonic pathway (TA) that connects ERC to CA1 were damaged in Tau.P301L mice at young age. Unexpectedly, the myelin defects were even more severe in bigenic biGT mice that co-express GSK3β with Tau.P301L in neurons. Combined, our data demonstrate that neuronal expression of protein Tau profoundly affected the functional and structural organization of the entorhinal-hippocampal complex, in particular synapses and myelinated axons in the SLM. White matter pathology deserves further attention in patients suffering from tauopathy and Alzheimer’s disease.

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 microtubule associated protein tau causes primary and secondary tauopathies by unknown molecular mechanisms. Post-translational O-GlcNAc-ylation of brain proteins was demonstrated here to be beneficial for Tau.P301L mice by pharmacological inhibition of O-GlcNAc-ase. Chronic treatment of ageing Tau.P301L mice mitigated their loss in body-weight and improved their motor deficits, while the survival was 3-fold higher at the pre-fixed study endpoint at age 9.5 months. Moreover, O-GlcNAc-ase inhibition significantly improved the breathing parameters of Tau.P301L mice, which underpinned pharmacologically the close correlation of mortality and upper-airway defects. O-GlcNAc-ylation of brain proteins increased rapidly and stably by systemic inhibition of O-GlcNAc-ase. Conversely, biochemical evidence for protein Tau.P301L to become O-GlcNAc-ylated was not obtained, nor was its phosphorylation consistently or markedly affected. We conclude that increasing O-GlcNAc-ylation of brain proteins improved the clinical condition and prolonged the survival of ageing Tau.P301L mice, but not by direct biochemical action on protein tau. The pharmacological effect is proposed to be located downstream in the pathological cascade initiated by protein Tau.P301L, opening novel venues for our understanding, and eventually treating the neurodegeneration mediated by protein tau.

Background GSK3β is involved in a wide range of physiological functions, and is presumed to act in the pathogenesis of neurological diseases, from bipolar disorder to Alzheimer’s disease (AD). In contrast, the GSK3α isozyme remained largely ignored with respect to both aspects. Results We generated and characterized two mouse strains with neuron-specific or with total GSK3α deficiency. Behavioral and electrophysiological analysis demonstrated the physiological importance of neuronal GSK3α, with GSK3β not compensating for impaired cognition and reduced LTP. Interestingly, the passive inhibitory avoidance task proved to modulate the phosphorylation status of both GSK3 isozymes in wild-type mice, further implying both to function in cognition. Moreover, GSK3α contributed to the neuronal architecture of the hippocampal CA1 sub-region that is most vulnerable in AD. Consequently, practically all parameters and characteristics indicated that both GSK3 isoforms were regulated independently, but that they acted on the same physiological functions in learning and memory, in mobility and in behavior. Conclusions GSK3α proved to be regulated independently from GSK3β, and to exert non-redundant physiological neurological functions in general behavior and in cognition. Moreover, GSK3α contributes to the pathological phosphorylation of protein Tau.

Progressive aggregation of protein Tau into oligomers and fibrils correlates with cognitive decline and synaptic dysfunction, leading to neurodegeneration in vulnerable brain regions in Alzheimer's disease. The unmet need of effective therapy for Alzheimer's disease, combined with problematic pharmacological approaches, led the field to explore immunotherapy, first against amyloid peptides and recently against protein Tau. Here we adapted the liposome-based amyloid vaccine that proved safe and efficacious, and incorporated a synthetic phosphorylated peptide to mimic the important phospho-epitope of protein Tau at residues pS396/pS404. We demonstrate that the liposome-based vaccine elicited, rapidly and robustly, specific antisera in wild-type mice and in Tau.P301L mice. Long-term vaccination proved to be safe, because it improved the clinical condition and reduced indices of tauopathy in the brain of the Tau.P301L mice, while no signs of neuro-inflammation or other adverse neurological effects were observed. The data corroborate the hypothesis that liposomes carrying phosphorylated peptides of protein Tau have considerable potential as safe and effective treatment against tauopathies, including Alzheimer's disease.

Conjugation of b -N-acetylglucosamine to Ser/Thr residues is a reversible post-translational modification of many proteins, controlled by two unique enzymes: O-GlcNAc transferase (OGT) and β -N-acetyl-glucosaminidase (OGA). There physiological and pathological repercussions and significance remain largely unknown in vivo, particularly in CNS. Accumulating amyloid peptides aggravate phosphorylation of Tau by activation of GSK3a/b, modeled in our bigenic mice biAT and biGT mice (Terwel et al, 2008). Tau.P301L mice suffer cognitive deficits (4–6 mo), motor dysfunction (6–8 mo), loss of bodyweight, ending with precocious death (8–12 mo). Aggregates of Tau in brainstem correlate with upper-airway dysfunction and death (Dutschmann et al., 2010; Menuet et al, 2011a, b). We studied chronic pharmacological inhibition of OGA in old TauP301L and young biAT mice, and acute OGA inhibition in young Tau.P301L mice, and measured effects on clinical parameters, brain pathology and biochemistry Chronic treatment of ageing Tau.P301L mice mitigated loss in body-weight and motor deficits. Moreover, 3-fold more treated Tau.P301L mice survived at the pre-fixed endpoint at age 9.5 months. In addition, O-GlcNAc-ase inhibition significantly improved breathing parameters of Tau.P301L mice, which pharmacologically underpins the relation of mortality to upper-airway defects. Biochemically, O-GlcNAc-ylation of brain proteins increased rapidly and stably by systemic inhibition of OGA although the phosphorylation of Tau was only marginally affected. Similarly, chronic OGA-inhibition of young biAT mice (beginning at 3 mo) for 4 months prevented their early mortality, improved their general condition and improved their typical anxiety. But also in the biAT model, phosphorylation of protein tau was not markedly affected. Extensive biochemical analysis of Tau.P301L mice using Immunoprecipitation could not lead to evidences for Tau being O-GlcNAc-ylated in vivo. Pharmacologically increasing protein O-GlcNAc-ylation markedly improved clinical condition, and prolonged survival of ageing Tau.P301L mice with tauopathy, and young adult biAT mice with beginning combined amyloid and Tau pathology. Surprisingly, in both models the clinical benefits were not reflected by biochemical parameters of protein Tau. The pharmacological target of the OGA inhibitor is proposed to be located downstream in the pathological cascade opening a novel venue to understand - and eventually treat - neurodegeneration.

The brain of patients with Alzheimer's disease (AD) is characterized by plaques and neurofibrillary tangles (NFT) composed of misfolded protein aggregates of beta-amyloid and hyper-phosphorylated tau, respectively. Immunotherapy can alleviate the pathological impact of protein-aggregates as demonstrated in several animal models. The correlation between cognitive decline and the number of NFT in the brain in AD patients suggests phosphorylated tau (pTau) as a promising target for immunotherapy. Tau transgenic mice were immunized over a 6-month period with a liposomal pTau vaccine or a classical vaccine composed of a pTau peptide adsorbed to aluminum phosphate. Cynomolgus monkeys were immunized with the liposomal pTau vaccine by various routes of administration. The IgG antibody responses were evaluated by ELISA in which the phospho-specificity of the polyclonal antibody response was assessed by comparing the binding to pTau and the corresponding non-pTau peptide or protein. Following four immunizations with the pTau liposomal vaccine, 25 out of 25 mice showed a strong anti-pTau peptide IgG response in contrast to immunization with the classical peptide vaccine (only 4 responders out of 20 mice). The antibodies induced by the pTau liposomal vaccine were phospho-specific as they distinguished both the pTau peptide from the tau peptide and the full length pTau protein from the tau protein. No phospho-specificity was observed for the antibodies induced by the classical pTau peptide vaccine. Immunization of cynomolgus monkeys with the pTau liposomal vaccine induced a robust and persistent anti-pTau-specific IgG response in 30 out of 30 monkeys. These data suggest that liposomal pTau vaccines are more immunogenic than classical pTau peptide vaccines and induce a polyclonal antibody response which is more specific for the pTau target. The strong immunogenicity was confirmed in cynomolgus monkeys. Together with previous data showing that this pTau liposomal vaccine decreased pathological tau accumulation in a tau transgenic mouse model, this vaccine approach has the potential to become a promising immunotherapy as a treatment in AD and other tau-mediated neurodegenerative diseases.

Cell adhesion molecules are important structural substrates, required for synaptic plasticity and synaptogenesis. CAMs differ widely in their expression throughout different brain regions and their specific structural and functional roles in the brain remain to be elucidated. Here, we investigated selected cell adhesion molecules for alterations in expression levels and neuronal localization in validated mouse models for Alzheimer's disease that mimic the age-related progression of amyloid accumulation and tauopathy. Among the cell adhesion molecules analyzed, Nectin-3 expression was affected most and specifically in all mouse models with tauopathy. In particular was Nectin-3 depleted from the specific region of the hippocampus, known as the stratum lacunosum and moleculare, in mice that express wild-type or mutant human protein Tau, either chronically or sub-acutely. Tauopathy progresses from the entorhinal cortex to the hippocampus by unknown mechanisms that could involve transport by the myelinated axons of the temporoammonic and perforant pathways. The decreased expression of Nectin-3 in the stratum lacunosum moleculare is an early marker of impaired transport, and eventual synaptic problems, caused by beginning tauopathy.

ABSTRACTGlycogen 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. Synapse, 2013. © 2013 Wiley Periodicals, Inc.