The NLRP3 inflammasome modulates tau pathology and neurodegeneration in a tauopathy model.

An active role of neuroinflammation and the NLRP3 inflammasome in Alzheimer's disease and related tauopathies is increasingly identified, supporting NLRP3 as an interesting therapeutic target. However, its effect on tau-associated neurodegeneration, a key-process in tauopathies, remains unknown. While tau pathology and neurodegeneration are closely correlated, different tau forms may act as culprits in both characteristics and NLRP3-dependent microglial processes may differently affect both processes, indicating the need to study the role of NLRP3 in both processes concomitantly. To study the role of NLRP3 on tau pathology, prion-like propagation and tau-associated neurodegeneration we generated crosses of NLRP3 deficient mice with tauP301S (PS19) transgenic mice. In this model we studied non-seeded tau pathology and hippocampal atrophy, reminiscent characteristics of tauopathies. Tau pathology in hippocampus and cortex was significantly decreased in tau.NLRP3-/- versus tau.NLRP3+/+ mice. Importantly, tau.NLRP3-/- mice also displayed significantly decreased hippocampal atrophy, indicating a role of NLRP3 in neurodegeneration. We furthermore assessed the effect of NLRP3 deficiency on tau propagation and associated hippocampal atrophy. NLRP3 deficiency significantly decreased prion-like seeding and propagation of tau pathology, reflected in decreased tau pathology in ipsi- and contralateral hippocampus and cortex in tau.NLRP3-/- following tau seeding. Most importantly, hippocampal atrophy was significantly less in tau-seeded tau.NLRP3-/- mice at 8 months. We here demonstrate for the first time that NLRP3 activation affects tau-associated neurodegeneration and seeded and non-seeded tau pathology, hence affecting key molecular processes in tauopathies. Our data thereby provide key-information in the validation of NLRP3 inflammasome as therapeutic target for AD and related tauopathies.

Aggregated Tau activates NLRP3-ASC inflammasome exacerbating exogenously seeded and non-exogenously seeded Tau pathology in vivo.

Brains of Alzheimer's disease patients are characterized by the presence of amyloid plaques and neurofibrillary tangles, both invariably associated with neuroinflammation. A crucial role for NLRP3-ASC inflammasome [NACHT, LRR and PYD domains-containing protein 3 (NLRP3)-Apoptosis-associated speck-like protein containing a CARD (ASC)] in amyloid-beta (Aß)-induced microgliosis and Aß pathology has been unequivocally identified. Aß aggregates activate NLRP3-ASC inflammasome (Halle et al. in Nat Immunol 9:857-865, 2008) and conversely NLRP3-ASC inflammasome activation exacerbates amyloid pathology in vivo (Heneka et al. in Nature 493:674-678, 2013), including by prion-like ASC-speck cross-seeding (Venegas et al. in Nature 552:355-361, 2017). However, the link between inflammasome activation, as crucial sensor of innate immunity, and Tau remains unexplored. Here, we analyzed whether Tau aggregates acting as prion-like Tau seeds can activate NLRP3-ASC inflammasome. We demonstrate that Tau seeds activate NLRP3-ASC-dependent inflammasome in primary microglia, following microglial uptake and lysosomal sorting of Tau seeds. Next, we analyzed the role of inflammasome activation in prion-like or templated seeding of Tau pathology and found significant inhibition of exogenously seeded Tau pathology by ASC deficiency in Tau transgenic mice. We furthermore demonstrate that chronic intracerebral administration of the NLRP3 inhibitor, MCC950, inhibits exogenously seeded Tau pathology. Finally, ASC deficiency also decreased non-exogenously seeded Tau pathology in Tau transgenic mice. Overall our findings demonstrate that Tau-seeding competent, aggregated Tau activates the ASC inflammasome through the NLRP3-ASC axis, and we demonstrate an exacerbating role of the NLRP3-ASC axis on exogenously and non-exogenously seeded Tau pathology in Tau mice in vivo. The NLRP3-ASC inflammasome, which is an important sensor of innate immunity and intensively explored for its role in health and disease, hence presents as an interesting therapeutic approach to target three crucial pathogenetic processes in AD, including prion-like seeding of Tau pathology, Aß pathology and neuroinflammation.

Tau Interacting Proteins: Gaining Insight into the Roles of Tau in Health and Disease.

Tau is most intensely studied in relation to its executive role in Tauopathies, a family of neurodegenerative disorders characterized by the accumulation of Tau aggregates [15, 21, 38, 75, 89, 111, 121, 135, 175, 176, 192]. Tau aggregation in the different Tauopathies differs in the affected cell type, the structure of aggregates and Tau isoform composition. However, in all Tauopathies, accumulation of pathological Tau in well-characterized and well-defined brain regions, correlates strongly with symptoms associated with the dysfunction of this brain region. Hence, symptoms of neurodegenerative Tauopathies can range from motoric to cognitive and behavioral symptoms, even extending to deterioration of vital functions when the disease progresses, or combinations of different symptoms governed by the affected brain regions. The most common Tauopathies are corticobasal degeneration (CBD), Pick's disease, progressive supranuclear palsy (PSP) and frontotemporal dementias with parkinsonism linked to chromosome 17 (FTDP-17). However a growing number of diseases are characterized by Tau aggregation amounting to a large family of more than 20 disorders [176]. Most Tauopathies are sporadic, and are hence linked to a combination of environmental and genetic risk factors. However, mutations in MAPT have been identified which are autosomal dominantly linked to Tauopathies, including FTDP, PSP and CBD [94, 163, 185] (Alzforum, https://www.alzforum.org/mutations/mapt ). More than 80 mutations have been identified in MAPT, both in intronic and exonic regions of the human MAPT. These mutations can be classified as missense mutations or splicing mutations. Most missense mutations cluster in or near the microtubule binding site of Tau, while most splicing mutations affect the splicing of exon 10 (encoding the R2 domain), and hence affect the 3R/4R ratio. While Alzheimer's disease (AD), is the most prevalent Tauopathy, no mutations in MAPT associated with AD have been identified. Brains of AD patients are pathologically characterized by the combined presence of amyloid plaques and neurofibrillary tangles [171]. Familial forms of AD, termed early onset familial AD (EOFAD) with clinical mutations in APP or PS1/2, have an early onset, and are invariably characterized by the combined presence of amyloid and Tau pathology [24, 80, 170]. These EOFAD cases, identify a causal link between APP/PS1 misprocessing and the development of Tau pathology and neurodegeneration [80, 170]. Furthermore, combined genetic, pathological, biomarker and in vivo modelling data, indicate that amyloid pathology precedes Tau pathology, and support a role for Aß as initiator and Tau as executor in the pathogenetic process of AD [80, 96, 97]. Hence, AD is often considered as a secondary Tauopathy (similar as for Down syndrome patients), in contrast to the primary Tauopathies described above. Tau aggregates in Tauopathies vary with respect to the ratio of different Tau isoforms (3R/4R), to the cell types displaying Tau aggregation and the structure of the aggregates. However, in all Tauopathies a strong correlation between progressive development of pathological Tau accumulation and the loss of the respective brain functions is observed.

Long-Term Treatment with Liraglutide, a Glucagon-Like Peptide-1 (GLP-1) Receptor Agonist, Has No Effect on ß-Amyloid Plaque Load in Two Transgenic APP/PS1 Mouse Models of Alzheimer's Disease.

One of the major histopathological hallmarks of Alzheimer's disease (AD) is cerebral deposits of extracellular ß-amyloid peptides. Preclinical studies have pointed to glucagon-like peptide 1 (GLP-1) receptors as a potential novel target in the treatment of AD. GLP-1 receptor agonists, including exendin-4 and liraglutide, have been shown to promote plaque-lowering and mnemonic effects of in a number of experimental models of AD. Transgenic mouse models carrying genetic mutations of amyloid protein precursor (APP) and presenilin-1 (PS1) are commonly used to assess the pharmacodynamics of potential amyloidosis-lowering and pro-cognitive compounds. In this study, effects of long-term liraglutide treatment were therefore determined in two double APP/PS1 transgenic mouse models of Alzheimer's disease carrying different clinical APP/PS1 mutations, i.e. the 'London' (hAPPLon/PS1A246E) and 'Swedish' mutation variant (hAPPSwe/PS1ΔE9) of APP, with co-expression of distinct PS1 variants. Liraglutide was administered in 5 month-old hAPPLon/PS1A246E mice for 3 months (100 or 500 ng/kg/day, s.c.), or 7 month-old hAPPSwe/PS1ΔE9 mice for 5 months (500 ng/kg/day, s.c.). In both models, regional plaque load was quantified throughout the brain using stereological methods. Vehicle-dosed hAPPSwe/PS1ΔE9 mice exhibited considerably higher cerebral plaque load than hAPPLon/PS1A246E control mice. Compared to vehicle-dosed transgenic controls, liraglutide treatment had no effect on the plaque levels in hAPPLon/PS1A246E and hAPPSwe/PS1ΔE9 mice. In conclusion, long-term liraglutide treatment exhibited no effect on cerebral plaque load in two transgenic mouse models of low- and high-grade amyloidosis, which suggests differential sensitivity to long-term liraglutide treatment in various transgenic mouse models mimicking distinct pathological hallmarks of AD.

The GLP-1 receptor agonist liraglutide reduces pathology-specific tau phosphorylation and improves motor function in a transgenic hTauP301L mouse model of tauopathy.

In addition to a prominent role in glycemic control, glucagon-like peptide 1 (GLP-1) receptor agonists exhibit neuroprotective properties. There is mounting experimental evidence that GLP-1 receptor agonists, including liraglutide, may enhance synaptic plasticity, counteract cognitive deficits and ameliorate neurodegenerative features in preclinical models of Alzheimer's disease (AD), predominantly in the context of ß-amyloid toxicity. Here we characterized the effects of liraglutide in a transgenic mutant tau (hTauP301L) mouse tauopathy model, which develops age-dependent pathology-specific neuronal tau phosphorylation and neurofibrillary tangle formation with progressively compromised motor function (limb clasping). Liraglutide (500 µg/kg/day, s.c., q.d., n=18) or vehicle (n=18) was administered to hTauP301L mice for 6 months from the age of three months. Vehicle-dosed wild-type FVB/N mice served as normal control (n=17). The onset and severity of hind limb clasping was markedly different in liraglutide and vehicle-dosed transgenic mice. Clasping behavior was observed in 61% of vehicle-dosed hTauP301L mice with a 55% survival rate in 9-month old transgenic mice. In contrast, liraglutide treatment reduced the clasping rate to 39% of hTauP301L mice, and fully prevented clasping-associated lethality resulting in a survival rate of 89%. Stereological analyses demonstrated that hTauP301L mice exhibited hindbrain-dominant neuronal accumulation of phosphorylated tau closely correlated to the severity of clasping behavior. In correspondence, liraglutide treatment significantly reduced neuronal phospho-tau load by 61.9±10.2% (p<0.001) in hTauP301L mice, as compared to vehicle-dosed controls. In conclusion, liraglutide significantly reduced tau pathology in a transgenic mouse tauopathy model.

Templated misfolding of Tau by prion-like seeding along neuronal connections impairs neuronal network function and associated behavioral outcomes in Tau transgenic mice.

Prion-like seeding and propagation of Tau-pathology have been demonstrated experimentally and may underlie the stereotyped progression of neurodegenerative Tauopathies. However, the involvement of templated misfolding of Tau in neuronal network dysfunction and behavioral outcomes remains to be explored in detail. Here we analyzed the repercussions of prion-like spreading of Tau-pathology via neuronal connections on neuronal network function in TauP301S transgenic mice. Spontaneous and GABA(A)R-antagonist-induced neuronal network activity were affected following templated Tau-misfolding using synthetic preformed Tau fibrils in cultured primary neurons. Electrophysiological analysis in organotypic hippocampal slices of Tau transgenic mice demonstrated impaired synaptic transmission and impaired long-term potentiation following Tau-seed induced Tau-aggregation. Intracerebral injection of Tau-seeds in TauP301S mice, caused prion-like spreading of Tau-pathology through functionally connected neuroanatomical pathways. Electrophysiological analysis revealed impaired synaptic plasticity in hippocampal CA1 region 6 months after Tau-seeding in entorhinal cortex (EC). Furthermore, templated Tau aggregation impaired cognitive function, measured in the object recognition test 6 months post-seeding. In contrast, Tau-seeding in basal ganglia and subsequent spreading through functionally connected neuronal networks involved in motor control, resulted in motoric deficits reflected in clasping and impaired inverted grid hanging, not significantly affected following Tau-seeding in EC. Immunostaining, biochemical and electron microscopic analysis in the different models suggested early pathological forms of Tau, including Tau-oligomers, rather than fully mature neurofibrillary tangles (NFTs) as culprits of neuronal dysfunction. We here demonstrate for the first time using in vitro, ex vivo and in vivo models, that prion-like spreading of Tau-misfolding by Tau seeds, along unique neuronal connections, causes neuronal network dysfunction and associated behavioral dysfunction. Our data highlight the potential relevance of this mechanism in the symptomatic progression in Tauopathies. We furthermore demonstrate that the initial site of Tau-seeding thereby determines the behavioral outcome, potentially underlying the observed heterogeneity in (familial) Tauopathies, including in TauP301 mutants.

CXCR3 promotes plaque formation and behavioral deficits in an Alzheimer's disease model.

Chemokines are important modulators of neuroinflammation and neurodegeneration. In the brains of Alzheimer's disease (AD) patients and in AD animal models, the chemokine CXCL10 is found in high concentrations, suggesting a pathogenic role for this chemokine and its receptor, CXCR3. Recent studies aimed at addressing the role of CXCR3 in neurological diseases indicate potent, but diverse, functions for CXCR3. Here, we examined the impact of CXCR3 in the amyloid precursor protein (APP)/presenilin 1 (PS1) transgenic mouse model of AD. We found that, compared with control APP/PSI animals, plaque burden and Aß levels were strongly reduced in CXCR3-deficient APP/PS1 mice. Analysis of microglial phagocytosis in vitro and in vivo demonstrated that CXCR3 deficiency increased the microglial uptake of Aß. Application of a CXCR3 antagonist increased microglial Aß phagocytosis, which was associated with reduced TNF-α secretion. Moreover, in CXCR3-deficient APP/PS1 mice, microglia exhibited morphological activation and reduced plaque association, and brain tissue from APP/PS1 animals lacking CXCR3 had reduced concentrations of proinflammatory cytokines compared with controls. Further, loss of CXCR3 attenuated the behavioral deficits observed in APP/PS1 mice. Together, our data indicate that CXCR3 signaling mediates development of AD-like pathology in APP/PS1 mice and suggest that CXCR3 has potential as a therapeutic target for AD.

Models of ß-amyloid induced Tau-pathology: the long and "folded" road to understand the mechanism.

The amyloid cascade hypothesis has been the prevailing hypothesis in Alzheimer's Disease research, although the final and most wanted proof i.e. fully successful anti-amyloid clinical trials in patients, is still lacking. This may require a better in depth understanding of the cascade. Particularly, the exact toxic forms of Aß and Tau, the molecular link between them and their respective contributions to the disease process need to be identified in detail. Although the lack of final proof has raised substantial criticism on the hypothesis per se, accumulating experimental evidence in in vitro models, in vivo models and from biomarkers analysis in patients supports the amyloid cascade and particularly Aß-induced Tau-pathology, which is the focus of this review. We here discuss available models that recapitulate Aß-induced Tau-pathology and review some potential underlying mechanisms. The availability and diversity of these models that mimic the amyloid cascade partially or more complete, provide tools to study remaining questions, which are crucial for development of therapeutic strategies for Alzheimer's Disease.

Combined treatment with a BACE inhibitor and anti-Aß antibody gantenerumab enhances amyloid reduction in APPLondon mice.

Therapeutic approaches for prevention or reduction of amyloidosis are currently a main objective in basic and clinical research on Alzheimer's disease. Among the agents explored in clinical trials are anti-Aß peptide antibodies and secretase inhibitors. Most anti-Aß antibodies are considered to act via inhibition of amyloidosis and enhanced clearance of existing amyloid, although secretase inhibitors reduce the de novo production of Aß. Limited information is currently available on the efficacy and potential advantages of combinatorial antiamyloid treatment. We performed a chronic study in APPLondon transgenic mice that received treatment with anti-Aß antibody gantenerumab and BACE inhibitor RO5508887, either as mono- or combination treatment. Treatment aimed to evaluate efficacy on amyloid progression, similar to preexisting amyloidosis as present in Alzheimer's disease patients. Mono-treatments with either compound caused a dose-dependent reduction of total brain Aß and amyloid burden. Combination treatment with both compounds significantly enhanced the antiamyloid effect. The observed combination effect was most pronounced for lowering of amyloid plaque load and plaque number, which suggests effective inhibition of de novo plaque formation. Moreover, significantly enhanced clearance of pre-existing amyloid plaques was observed when gantenerumab was coadministered with RO5508887. BACE inhibition led to a significant time- and dose-dependent decrease in CSF Aß, which was not observed for gantenerumab treatment. Our results demonstrate that combining these two antiamyloid agents enhances overall efficacy and suggests that combination treatments may be of clinical relevance.

Ear2 deletion causes early memory and learning deficits in APP/PS1 mice.

To assess the consequences of locus ceruleus (LC) degeneration and subsequent noradrenaline (NA) deficiency in early Alzheimer's disease (AD), mice overexpressing mutant amyloid precursor protein and presenilin-1 (APP/PS1) were crossed with Ear2(-/-) mice that have a severe loss of LC neurons projecting to the hippocampus and neocortex. Testing spatial memory and hippocampal long-term potentiation revealed an impairment in APP/PS1 Ear2(-/-) mice, whereas APP/PS1 or Ear2(-/-) mice showed only minor changes. These deficits were associated with distinct synaptic changes including reduced expression of the NMDA 2A subunit and increased levels of NMDA receptor 2B in APP/PS1 Ear2(-/-) mice. Acute pharmacological replacement of NA by L-threo-DOPS partially restored phosphorylation of ß-CaMKII and spatial memory performance in APP/PS1 Ear2(-/-) mice. These changes were not accompanied by altered APP processing or amyloid ß peptide (Aß) deposition. Thus, early LC degeneration and subsequent NA reduction may contribute to cognitive deficits via CaMKII and NMDA receptor dysfunction independent of Aß and suggests that NA supplementation could be beneficial in treating AD.

Effects of sub-chronic donepezil on brain Abeta and cognition in a mouse model of Alzheimer's disease.

RATIONALE: Acetylcholinesterase inhibitors (AChEIs) are approved to treat the symptoms of mild to moderate Alzheimer's disease by restoring acetylcholine levels at synapses where the neurotransmitter has been depleted due to neurodegeneration. This assumption is challenged by more recent clinical studies suggesting the potential for disease-modifying effects of AChEIs as well as in vitro studies showing neuroprotective effects. However, few preclinical studies have assessed whether the improvement of cognitive symptoms may be mediated by reductions in Abeta or Tau pathology. OBJECTIVES: The objective of the present study was to determine whether short-duration treatment with donepezil could improve spatial learning and memory in transgenic mice overexpressing mutant human amyloid precursor protein (hAPP) and presenilin 1 (PS1) (Dewachter et al., J Neurosci 20(17):6452-6458, 2000) after amyloid pathology has fully developed, consistent with early stages of Alzheimer'sdisease in humans. In parallel, the effect of donepezil treatment on brain amyloid, Tau, and glial endpoints was measured. RESULTS: This study showed a significant improvement in reference memory in hAPP/PS1 mice along with dose-dependent reductions in brain amyloid-ß (Aß). CONCLUSION: These results suggest that the observed cognitive improvement produced by donepezil in Alzheimer's disease may be due, at least in part, to reduction of brain Aß.

Selective loss of noradrenaline exacerbates early cognitive dysfunction and synaptic deficits in APP/PS1 mice.

BACKGROUND: Degeneration of the locus coeruleus (LC), the major noradrenergic nucleus in the brain, occurs early and is ubiquitous in Alzheimer's disease (AD). Experimental lesions to the LC exacerbate AD-like neuropathology and cognitive deficits in several transgenic mouse models of AD. Because the LC contains multiple neuromodulators known to affect amyloid ß toxicity and cognitive function, the specific role of noradrenaline (NA) in AD is not well understood. METHODS: To determine the consequences of selective NA deficiency in an AD mouse model, we crossed dopamine ß-hydroxylase (DBH) knockout mice with amyloid precursor protein (APP)/presenilin-1 (PS1) mice overexpressing mutant APP and PS1. Dopamine ß-hydroxylase (-/-) mice are unable to synthesize NA but otherwise have normal LC neurons and co-transmitters. Spatial memory, hippocampal long-term potentiation, and synaptic protein levels were assessed. RESULTS: The modest impairments in spatial memory and hippocampal long-term potentiation displayed by young APP/PS1 or DBH (-/-) single mutant mice were augmented in DBH (-/-)/APP/PS1 double mutant mice. Deficits were associated with reduced levels of total calcium/calmodulin-dependent protein kinase II and N-methyl-D-aspartate receptor 2A and increased N-methyl-D-aspartate receptor 2B levels and were independent of amyloid ß accumulation. Spatial memory performance was partly improved by treatment with the NA precursor drug L-threo-dihydroxyphenylserine. CONCLUSIONS: These results indicate that early LC degeneration and subsequent NA deficiency in AD may contribute to cognitive deficits via altered levels of calcium/calmodulin-dependent protein kinase II and N-methyl-D-aspartate receptors and suggest that NA supplementation could be beneficial in early AD.

Locus coeruleus degeneration exacerbates olfactory deficits in APP/PS1 transgenic mice.

Neuronal loss in the locus coeruleus (LC) is 1 of the early pathological events in Alzheimer's disease (AD). Projections of noradrenergic neurons of the LC innervate the olfactory bulb (OB). Because olfactory deficits have been reported in early AD, we investigated the effect of induced LC degeneration on olfactory memory and discrimination in an AD mouse model. LC degeneration was induced by treating APP/PS1 mice with N-(2-chloroethyl)-N-ethyl-bromo-benzylamine (DSP4) repeatedly between 3 and 12 months of age. Short term odor retention, ability for spontaneous habituation to an odor, and spontaneous odor discrimination were assessed by behavioral tests. DSP4 treatment in APP/PS1 mice resulted in an exacerbation of short term olfactory memory deficits and more discrete weakening of olfactory discrimination abilities, suggesting that LC degeneration contributes to olfactory deficits observed in AD. Importantly, DSP4 treatment also increased amyloid ß (Aß) deposition in the olfactory bulb of APP/PS1 mice, which correlated with olfactory memory, not with discrimination deficits.

Nitration of tyrosine 10 critically enhances amyloid ß aggregation and plaque formation.

Part of the inflammatory response in Alzheimer's disease (AD) is the upregulation of the inducible nitric oxide synthase (NOS2) resulting in increased NO production. NO contributes to cell signaling by inducing posttranslational protein modifications. Under pathological conditions there is a shift from the signal transducing actions to the formation of protein tyrosine nitration by secondary products like peroxynitrite and nitrogen dioxide. We identified amyloid ß (Aß) as an NO target, which is nitrated at tyrosine 10 (3NTyr(10)-Aß). Nitration of Aß accelerated its aggregation and was detected in the core of Aß plaques of APP/PS1 mice and AD brains. NOS2 deficiency or oral treatment with the NOS2 inhibitor L-NIL strongly decreased 3NTyr(10)-Aß, overall Aß deposition and cognitive dysfunction in APP/PS1 mice. Further, injection of 3NTyr(10)-Aß into the brain of young APP/PS1 mice induced ß-amyloidosis. This suggests a disease modifying role for NOS2 in AD and therefore represents a potential therapeutic target.

Critical role of astroglial apolipoprotein E and liver X receptor-α expression for microglial Aß phagocytosis.

Liver X receptors (LXRs) regulate immune cell function and cholesterol metabolism, both factors that are critically involved in Alzheimer's disease (AD). To investigate the therapeutic potential of long-term LXR activation in amyloid-ß (Aß) peptide deposition in an AD model, 13-month-old, amyloid plaque-bearing APP23 mice were treated with the LXR agonist TO901317. Postmortem analysis demonstrated that TO901317 efficiently crossed the blood-brain barrier. Insoluble and soluble Aß levels in the treated APP23 mice were reduced by 80% and 40%, respectively, compared with untreated animals. Amyloid precursor protein (APP) processing, however, was hardly changed by the compound, suggesting that the observed effects were instead mediated by Aß disposal. Despite the profound effect on Aß levels, spatial learning in the Morris water maze was only slightly improved by the treatment. ABCA1 (ATP-binding cassette transporter 1) and apolipoprotein E (ApoE) protein levels were increased and found to be primarily localized in astrocytes. Experiments using primary microglia demonstrated that medium derived from primary astrocytes exposed to TO901317 stimulated phagocytosis of fibrillar Aß. Conditioned medium from TO901317-treated ApoE(-/-) or LXRα(-/-) astrocytes did not increase phagocytosis of Aß. In APP23 mice, long-term treatment with TO901317 strongly increased the association of microglia and Aß plaques. Short-term treatment of APP/PS1 mice with TO901317 also increased this association, which was dependent on the presence of LXRα and was accompanied by increased ApoE lipidation. Together, these data suggest that astrocytic LXRα activation and subsequent release of ApoE by astrocytes is critical for the ability of microglia to remove fibrillar Aß in response to treatment with TO901317.

Neuroinflammatory and behavioural changes in the Atp7B mutant mouse model of Wilson's disease.

Wilson's disease (WD) is caused by mutations in the copper transporting ATPase 7B (Atp7b). Patients present with liver pathology or behavioural disturbances. Studies on rodent models for WD so far mainly focussed on liver, not brain. The effect of knockout of atp7b on sensori-motor and cognitive behaviour, as well as neuronal number, inflammatory markers, copper and synaptic proteins in brain were studied in so-called toxic milk mice. Copper accumulated in striatum and hippocampus of toxic milk mice, but not in cerebral cortex. Inflammatory markers were increased in striatum and corpus callosum, but not in cerebral cortex and hippocampus, whereas neuronal numbers were unchanged. Toxic milk mice were mildly impaired in the rotarod and cylinder test and unable to acquire spatial memory in the Morris water maze. Despite the latter observation only synaptophysin of a number of synaptic proteins, was altered in the hippocampus of toxic milk mice. In addition to disturbances in neuronal signalling by increased brain copper, inflammation and inflammatory signalling from the periphery to the brain might add to the behavioural disturbances in the toxic milk mice. These mice can be used to evaluate therapeutic strategies to alleviate behavioural disturbances and cerebral pathology observed in WD.

Extracellular phosphorylation of the amyloid ß-peptide promotes formation of toxic aggregates during the pathogenesis of Alzheimer's disease.

Alzheimer's disease (AD) is the most common form of dementia and associated with progressive deposition of amyloid ß-peptides (Aß) in the brain. Aß derives by sequential proteolytic processing of the amyloid precursor protein by ß- and γ-secretases. Rare mutations that lead to amino-acid substitutions within or close to the Aß domain promote the formation of neurotoxic Aß assemblies and can cause early-onset AD. However, mechanisms that increase the aggregation of wild-type Aß and cause the much more common sporadic forms of AD are largely unknown. Here, we show that extracellular Aß undergoes phosphorylation by protein kinases at the cell surface and in cerebrospinal fluid of the human brain. Phosphorylation of serine residue 8 promotes formation of oligomeric Aß assemblies that represent nuclei for fibrillization. Phosphorylated Aß was detected in the brains of transgenic mice and human AD brains and showed increased toxicity in Drosophila models as compared with non-phosphorylated Aß. Phosphorylation of Aß could represent an important molecular mechanism in the pathogenesis of the most common sporadic form of AD.

Restraint stress increases neuroinflammation independently of amyloid ß levels in amyloid precursor protein/PS1 transgenic mice.

Both hypercortisolemia and hippocampal damage are features found in patients diagnosed of Alzheimer's disease (AD) and epidemiological evidence supports a role for stress as a risk factor for AD. It is known that immobilization stress is followed by accumulation of oxidative/nitrosative mediators in brain after the release of proinflammatory cytokines, nuclear factor kappa B activation, nitric oxide synthase-2 and cyclooxygenase-2 expression. Long-term exposure to elevated corticosteroid levels is known to affect the hippocampus which plays a central role in the regulation of the hypothalamic-pituitary-adrenal axis. We therefore studied the effect of chronic immobilization stress on amyloid precursor protein/PS1 mice. Stress exposure increased AD-induced neuroinflammation characterized by astrogliosis, increased inflammatory gene transcription and lipid peroxidation. Importantly, immobilization stress did not increase the soluble or insoluble amyloid ß levels suggesting that increased cortisol levels lower the threshold for a neuroinflammatory response, independently from amyloid ß. Since inflammation may act as a factor that contributes disease progression, the stress-inflammation relation described here may be relevant to understand the initial mechanisms in underlying the risk enhancing action of stress on AD.

Serine-409 phosphorylation and oxidative damage define aggregation of human protein tau in yeast.

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 GSK3ß 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.

Neuroinflammatory processes in Alzheimer's disease.

Generation of neurotoxic amyloid beta peptides and their deposition along with neurofibrillary tangle formation represent key pathological hallmarks in Alzheimer's disease (AD). Recent evidence suggests that inflammation may be a third important component which, once initiated in response to neurodegeneration or dysfunction, may actively contribute to disease progression and chronicity. Various neuroinflammatory mediators including complement activators and inhibitors, chemokines, cytokines, radical oxygen species and inflammatory enzyme systems are expressed and released by microglia, astrocytes and neurons in the AD brain. Degeneration of aminergic brain stem nuclei including the locus ceruleus and the nucleus basalis of Meynert may facilitate the occurrence of inflammation in their projection areas given the antiinflammatory and neuroprotective action of their key transmitters norepinephrine and acetylcholine. While inflammation has been thought to arise secondary to degeneration, recent experiments demonstrated that inflammatory mediators may stimulate amyloid precursor protein processing by various means and therefore can establish a vicious cycle. Despite the fact that some aspects of inflammation may even be protective for bystander neurons, antiinflammatory treatment strategies should therefore be considered. Non-steroidal anti-inflammatory drugs have been shown to reduce the risk and delay the onset to develop AD. While, the precise molecular mechanism underlying this effect is still unknown, a number of possible mechanisms including cyclooxygenase 2 or gamma-secretase inhibition and activation of the peroxisome proliferator activated receptor gamma may alone or, more likely, in concert account for the epidemiologically observed protection.