Early and Longitudinal Microglial Activation but Not Amyloid Accumulation Predicts Cognitive Outcome in PS2APP Mice.

Neuroinflammation may have beneficial or detrimental net effects on the cognitive outcome of Alzheimer disease (AD) patients. PET imaging with 18-kDa translocator protein (TSPO) enables longitudinal monitoring of microglial activation in vivo. Methods: We compiled serial PET measures of TSPO and amyloid with terminal cognitive assessment (water maze) in an AD transgenic mouse model (PS2APP) from 8 to 13 mo of age, followed by immunohistochemical analyses of microglia, amyloid, and synaptic density. Results: Better cognitive outcome and higher synaptic density in PS2APP mice was predicted by higher TSPO expression at 8 mo. The progression of TSPO activation to 13 mo also showed a moderate association with spared cognition, but amyloidosis did not correlate with the cognitive outcome, regardless of the time point. Conclusion: This first PET investigation with longitudinal TSPO and amyloid PET together with terminal cognitive testing in an AD mouse model indicates that continuing microglial response seems to impart preserved cognitive performance.

Microglial response to increasing amyloid load saturates with aging: a longitudinal dual tracer in vivo µPET-study.

BACKGROUND: Causal associations between microglia activation and ß-amyloid (Aß) accumulation during the progression of Alzheimer's disease (AD) remain a matter of controversy. Therefore, we used longitudinal dual tracer in vivo small animal positron emission tomography (µPET) imaging to resolve the progression of the association between Aß deposition and microglial responses during aging of an Aß mouse model. METHODS: APP-SL70 mice (N = 17; baseline age 3.2-8.5 months) and age-matched C57Bl/6 controls (wildtype (wt)) were investigated longitudinally for 6 months using Aß (18F-florbetaben) and 18 kDa translocator protein (TSPO) µPET (18F-GE180). Changes in cortical binding were transformed to Z-scores relative to wt mice, and microglial activation relative to amyloidosis was defined as the Z-score difference (TSPO-Aß). Using 3D immunohistochemistry for activated microglia (Iba-1) and histology for fibrillary Aß (methoxy-X04), we measure microglial brain fraction relative to plaque size and the distance from plaque margins. RESULTS: Aß-PET binding increased exponentially as a function of age in APP-SL70 mice, whereas TSPO binding had an inverse U-shape growth function. Longitudinal Z-score differences declined with aging, suggesting that microglial response declined relative to increasing amyloidosis in aging APP-SL70 mice. Microglial brain volume fraction was inversely related to adjacent plaque size, while the proximity to Aß plaques increased with age. CONCLUSIONS: Microglial activity decreases relative to ongoing amyloidosis with aging in APP-SL70 mice. The plaque-associated microglial brain fraction saturated and correlated negatively with increasing plaque size with aging.

Decreasing the Expression of GABAA α5 Subunit-Containing Receptors Partially Improves Cognitive, Electrophysiological, and Morphological Hippocampal Defects in the Ts65Dn Model of Down Syndrome.

Trisomy 21 or Down syndrome (DS) is the most common cause of intellectual disability of a genetic origin. The Ts65Dn (TS) mouse, which is the most commonly used and best-characterized mouse model of DS, displays many of the cognitive, neuromorphological, and biochemical anomalies that are found in the human condition. One of the mechanisms that have been proposed to be responsible for the cognitive deficits in this mouse model is impaired GABA-mediated inhibition. Because of the well-known modulatory role of GABAA α5 subunit-containing receptors in cognitive processes, these receptors are considered to be potential targets for improving the intellectual disability in DS. The chronic administration of GABAA α5-negative allosteric modulators has been shown to be procognitive without anxiogenic or proconvulsant side effects. In the present study, we use a genetic approach to evaluate the contribution of GABAA α5 subunit-containing receptors to the cognitive, electrophysiological, and neuromorphological deficits in TS mice. We show that reducing the expression of GABAA α5 receptors by deleting one or two copies of the Gabra5 gene in TS mice partially ameliorated the cognitive impairments, improved long-term potentiation, enhanced neural differentiation and maturation, and normalized the density of the GABAergic synapse markers. Reducing the gene dosage of Gabra5 in TS mice did not induce motor alterations and anxiety or affect the viability of the mice. Our results provide further evidence of the role of GABAA α5 receptor-mediated inhibition in cognitive impairment in the TS mouse model of DS.

Region-specific permeability of the blood-brain barrier upon pericyte loss.

The blood-brain barrier (BBB) regulates differing needs of the various brain regions by controlling transport of blood-borne components from the neurovascular circulation into the brain parenchyma. The mechanisms underlying region-specific transport across the BBB are not completely understood. Previous work showed that pericytes are key regulators of BBB function. Here we investigated whether pericytes influence BBB permeability in a region-specific manner by analysing the regional permeability of the BBB in the pdgf-b ret/ret mouse model of pericyte depletion. We show that BBB permeability is heterogeneous in pdgf-b ret/ret mice, being significantly higher in the cortex, striatum and hippocampus compared to the interbrain and midbrain. However, we show that this regional heterogeneity in BBB permeability is not explained by local differences in pericyte coverage. Region-specific differences in permeability were not associated with disruption of tight junctions but may result from changes in transcytosis across brain endothelial cells. Our data show that certain brain regions are able to maintain low BBB permeability despite substantial pericyte loss and suggest that additional, locally-acting mechanisms may contribute to control of transport.

Increase of TREM2 during Aging of an Alzheimer's Disease Mouse Model Is Paralleled by Microglial Activation and Amyloidosis.

Heterozygous missense mutations in the triggering receptor expressed on myeloid cells 2 (TREM2) have been reported to significantly increase the risk of developing Alzheimer's disease (AD). Since TREM2 is specifically expressed by microglia in the brain, we hypothesized that soluble TREM2 (sTREM2) levels may increase together with in vivo biomarkers of microglial activity and amyloidosis in an AD mouse model as assessed by small animal positron-emission-tomography (µPET). In this cross-sectional study, we examined a strong amyloid mouse model (PS2APP) of four age groups by µPET with [18F]-GE180 (glial activation) and [18F]-florbetaben (amyloidosis), followed by measurement of sTREM2 levels and amyloid levels in the brain. Pathology affected brain regions were compared between tracers (dice similarity coefficients) and pseudo-longitudinally. µPET results of both tracers were correlated with terminal TREM2 levels. The brain sTREM2 levels strongly increased with age of PS2APP mice (5 vs. 16 months: +211%, p < 0.001), and correlated highly with µPET signals of microglial activity (R = 0.89, p < 0.001) and amyloidosis (R = 0.92, p < 0.001). Dual µPET enabled regional mapping of glial activation and amyloidosis in the mouse brain, which progressed concertedly leading to a high overlap in aged PS2APP mice (dice similarity 67%). Together, these results substantiate the use of in vivo µPET measurements in conjunction with post mortem sTREM2 in future anti-inflammatory treatment trials. Taking human data into account sTREM2 may increase during active amyloid deposition.

Trafficking of Endogenous Immunoglobulins by Endothelial Cells at the Blood-Brain Barrier.

The Blood-Brain Barrier (BBB) restricts access of large molecules to the brain. The low endocytic activity of brain endothelial cells (BECs) is believed to limit delivery of immunoglobulins (IgG) to the brain parenchyma. Here, we report that endogenous mouse IgG are localized within intracellular vesicles at steady state in BECs in vivo. Using high-resolution quantitative microscopy, we found a fraction of endocytosed IgG in lysosomes. We observed that loss of pericytes (key components of the BBB) in pdgf-b(ret/ret) mice affects the intracellular distribution of endogenous mouse IgG in BECs. In these mice, endogenous IgG was not detected within lysosomes but instead accumulate at the basement membrane and brain parenchyma. Such IgG accumulation could be due to reduced lysosomal clearance and increased sorting to the abluminal membrane of BECs. Our results suggest that, in addition to low uptake from circulation, IgG lysosomal degradation may be a downstream mechanism by which BECs further restrict IgG access to the brain.

Striatum and entorhinal cortex atrophy in AD mouse models: MRI comprehensive analysis.

Alzheimer's disease is experimentally modeled in transgenic (Tg) mice overexpressing mutated forms of the human amyloid precursor protein either alone or combined with mutated presenilins and tau. In the present study, we developed a systematic approach to compare double (TASTPM) and triple (APP/PS2/Tau) Tg mice by serial magnetic resonance imaging and spectroscopy analysis from 4 to 26 months of age to define homologous biomarkers between mice and humans. Hippocampal atrophy was found in Tg mice compared with WT. In APP/PS2/Tau the effect was age-dependent, whereas in TASTPM it was detectable from the first investigated time point. Importantly, both mice displayed an age-related entorhinal cortex thinning and robust striatal atrophy, the latter associated with a significant loss of synaptophysin. Hippocampal magnetic resonance spectroscopy revealed lower glutamate levels in both Tg mice and a selective myo-inositol increase in TASTPM. This noninvasive magnetic resonance imaging analysis, revealed common biomarkers between humans and mice, and could, thus, be promoted as a fully translational tool to be adopted in the preclinical investigation of therapeutic approaches.

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.

Neuronal uptake of tau/pS422 antibody and reduced progression of tau pathology in a mouse model of Alzheimer's disease.

The severity of tau pathology in Alzheimer's disease brain correlates closely with disease progression. Tau immunotherapy has therefore been proposed as a new therapeutic approach to Alzheimer's disease and encouraging results have been obtained by active or passive immunization of tau transgenic mice. This work investigates the mechanism by which immunotherapy can impact tau pathology. We demonstrate the development of Alzheimer's disease-like tau pathology in a triple transgenic mouse model of Alzheimer's disease and show that tau/pS422 is present in membrane microdomains on the neuronal cell surface. Chronic, peripheral administration of anti-tau/pS422 antibody reduces the accumulation of tau pathology. The unequivocal presence of anti-tau/pS422 antibody inside neurons and in lysosomes is demonstrated. We propose that anti-tau/pS422 antibody binds to membrane-associated tau/pS422 and that the antigen-antibody complexes are cleared intracellularly, thereby offering one explanation for how tau immunotherapy can ameliorate neuronal tau pathology.

Chronic metabotropic glutamate receptor 5 inhibition corrects local alterations of brain activity and improves cognitive performance in fragile X mice.

BACKGROUND: Fragile X syndrome (FXS) is the most common genetic cause for intellectual disability. Fmr1 knockout (KO) mice are an established model of FXS. Chronic pharmacological inhibition of metabotropic glutamate receptor 5 (mGlu5) in these mice corrects multiple molecular, physiological, and behavioral phenotypes related to patients' symptoms. To better understand the pathophysiology of FXS and the effect of treatment, brain activity was analyzed using functional magnetic resonance imaging in relation to learning and memory performance. METHODS: Wild-type (WT) and Fmr1 KO animals receiving chronic treatment with the mGlu5 inhibitor CTEP or vehicle were evaluated consecutively for 1) learning and memory performance in the inhibitory avoidance and extinction test, and 2) for the levels of brain activity using continuous arterial spin labeling based functional magnetic resonance imaging. Neural activity patterns were correlated with cognitive performance using a multivariate regression analysis. Furthermore, mGlu5 receptor expression in brains of untreated mice was analyzed by autoradiography and saturation analysis using [(3)H]-ABP688. RESULTS: Chronic CTEP treatment corrected the learning deficit observed in Fmr1 KO mice in the inhibitory avoidance and extinction test and prevented memory extinction in WT and Fmr1 KO animals. Chronic CTEP treatment normalized perfusion in the amygdala and the lateral hypothalamus in Fmr1 KO mice and furthermore decreased perfusion in the hippocampus and increased perfusion in primary sensorimotor cortical areas. No significant differences in mGlu5 receptor expression levels between Fmr1 WT and KO mice were detected. CONCLUSIONS: Chronic mGlu5 inhibition corrected the learning deficits and partially normalized the altered brain activity pattern in Fmr1 KO mice.

ß-Secretase (BACE1) inhibitors with high in vivo efficacy suitable for clinical evaluation in Alzheimer's disease.

An extensive fluorine scan of 1,3-oxazines revealed the power of fluorine(s) to lower the pKa and thereby dramatically change the pharmacological profile of this class of BACE1 inhibitors. The CF3 substituted oxazine 89, a potent and highly brain penetrant BACE1 inhibitor, was able to reduce significantly CSF Aß40 and 42 in rats at oral doses as low as 1 mg/kg. The effect was long lasting, showing a significant reduction of Aß40 and 42 even after 24 h. In contrast to 89, compound 1b lacking the CF3 group was virtually inactive in vivo.

Chronic pharmacological mGlu5 inhibition corrects fragile X in adult mice.

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability. Previous studies have implicated mGlu5 in the pathogenesis of the disease, but a crucial unanswered question is whether pharmacological mGlu5 inhibition is able to reverse an already established FXS phenotype in mammals. Here we have used the novel, potent, and selective mGlu5 inhibitor CTEP to address this issue in the Fmr1 knockout mouse. Acute CTEP treatment corrects elevated hippocampal long-term depression, protein synthesis, and audiogenic seizures. Chronic treatment that inhibits mGlu5 within a receptor occupancy range of 81% ± 4% rescues cognitive deficits, auditory hypersensitivity, aberrant dendritic spine density, overactive ERK and mTOR signaling, and partially corrects macroorchidism. This study shows that a comprehensive phenotype correction in FXS is possible with pharmacological intervention starting in young adulthood, after development of the phenotype. It is of great interest how these findings may translate into ongoing clinical research testing mGlu5 inhibitors in FXS patients.

The dynamics of Aß distribution after γ-secretase inhibitor treatment, as determined by experimental and modelling approaches in a wild type rat.

Inhibition of the enzyme(s) that produce the Amyloid beta (Aß) peptide, namely BACE and γ-secretase, is considered an attractive target for Alzheimer's disease therapy. However, the optimal pharmacokinetic-pharmacodynamic modelling method to describe the changes in Aß levels after drug treatment is unclear. In this study, turnover models were employed to describe Aß levels following treatment with the γ-secretase inhibitor RO5036450, in the wild type rat. Initially, Aß level changes in the brain, cerebral spinal fluid (CSF) and plasma were modeled as separate biological compartments, which allowed the estimation of a compound IC50 and Aß turnover. While the data were well described, the model did not take into consideration that the CSF pool of Aß most likely originates from the brain via the CSF drainage pathway. Therefore, a separate model was carried out, with the assumption that CSF Aß levels originated from the brain. The optimal model that described the data involved two brain Aß 40 sub-compartments, one with a rapid turnover, from which CSF Aß 40 is derived, and a second quasi-static pool of ~20%. Importantly, the estimated in vivo brain IC50 was in a good range of the in vitro IC50 (ratio, 1.4). In conclusion, the PK/PD models presented here are well suited for describing the temporal changes in Aß levels that occur after treatment with an Aß lowering drug, and identifying physiological parameters.

Selective degeneration of septal and hippocampal GABAergic neurons in a mouse model of amyloidosis and tauopathy.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by brain accumulation of amyloid-ß peptide and neurofibrillary tangles, which are believed to initiate a pathological cascade that results in progressive impairment of cognitive functions and eventual neuronal death. To obtain a mouse model displaying the typical AD histopathology of amyloidosis and tauopathy, we generated a triple-transgenic mouse line (TauPS2APP) by overexpressing human mutations of the amyloid precursor protein, presenilin2 and tau genes. Stereological analysis of TauPS2APP mice revealed significant neurodegeneration of GABAergic septo-hippocampal projection neurons as well as their target cells, the GABAergic hippocampal interneurons. In contrast, the cholinergic medial septum neurons remained unaffected. Moreover, the degeneration of hippocampal GABAergic interneurons was dependent on the hippocampal subfield and interneuronal subtype investigated, whereby the dentate gyrus and the NPY-positive interneurons, respectively, were most strongly affected. Neurodegeneration was also accompanied by a change in the mRNA expression of markers for inhibitory interneurons. In line with the loss of inhibitory neurons, we observed functional changes in TauPS2APP mice relative to WT mice, with strongly enhanced long-term potentiation in the medial-perforant pathway input to the dentate gyrus, and stereotypic hyperactivity. Our data indicate that inhibitory neurons are the targets of neurodegeneration in a mouse model of amyloidosis and tauopathy, thus pointing to a possible role of the inhibitory network in the pathophysiological and functional cascade of Alzheimer's disease.

Morphologic and functional effects of gamma secretase inhibition on splenic marginal zone B cells.

The γ-secretase complex is a promising target in Alzheimer's disease because of its role in the amyloidogenic processing of ß-amyloid precursor protein. This enzyme also catalyzes the cleavage of Notch receptor, resulting in the nuclear translocation of intracellular Notch where it modulates gene transcription. Notch signaling is essential in cell fate decisions during embryogenesis, neuronal differentiation, hematopoiesis, and development of T and B cells, including splenic marginal zone (MZ) B cells. This B cell compartment participates in the early phases of the immune response to blood-borne bacteria and viruses. Chronic treatment with the oral γ-secretase inhibitor RO4929097 resulted in dose-dependent decreased cellularity (atrophy) of the MZ of rats and mice. Significant decreases in relative MZ B-cell numbers of RO4929097-treated animals were confirmed by flow cytometry. Numbers of MZ B cells reverted to normal after a sufficient RO4929097-free recovery period. Functional characterization of the immune response in relation to RO4929097-related MZ B cell decrease was assessed in mice vaccinated with inactivated vesicular stomatitis virus (VSV). Compared with the immunosuppressant cyclosporin A, RO4929097 caused only mild and reversible delayed early neutralizing IgM and IgG responses to VSV. Thus, the functional consequence of MZ B cell decrease on host defense is comparatively mild.

Aminothiazoles as γ-secretase modulators.

We herein report the discovery of a new γ-secretase modulator class with an aminothiazole core starting from a HTS hit (3). Synthesis and SAR of this series are discussed. These novel compounds demonstrate moderate to good in vitro potency in inhibiting amyloid beta (Aß) peptide production. Overall γ-secretase is not inhibited but the formation of the aggregating, toxic Aß42 peptide is shifted to smaller non-aggregating Aß peptides. Compound 15 reduced brain Aß42 in vivo in APPSwe transgenic mice at 30mg/kg p.o.

TAAR1 activation modulates monoaminergic neurotransmission, preventing hyperdopaminergic and hypoglutamatergic activity.

The trace amine-associated receptor 1 (TAAR1), activated by endogenous metabolites of amino acids like the trace amines p-tyramine and ß-phenylethylamine, has proven to be an important modulator of the dopaminergic system and is considered a promising target for the treatment of neuropsychiatric disorders. To decipher the brain functions of TAAR1, a selective TAAR1 agonist, RO5166017, was engineered. RO5166017 showed high affinity and potent functional activity at mouse, rat, cynomolgus monkey, and human TAAR1 stably expressed in HEK293 cells as well as high selectivity vs. other targets. In mouse brain slices, RO5166017 inhibited the firing frequency of dopaminergic and serotonergic neurons in regions where Taar1 is expressed (i.e., the ventral tegmental area and dorsal raphe nucleus, respectively). In contrast, RO5166017 did not change the firing frequency of noradrenergic neurons in the locus coeruleus, an area devoid of Taar1 expression. Furthermore, modulation of TAAR1 activity altered the desensitization rate and agonist potency at 5-HT(1A) receptors in the dorsal raphe, suggesting that TAAR1 modulates not only dopaminergic but also serotonergic neurotransmission. In WT but not Taar1(-/-) mice, RO5166017 prevented stress-induced hyperthermia and blocked dopamine-dependent hyperlocomotion in cocaine-treated and dopamine transporter knockout mice as well as hyperactivity induced by an NMDA antagonist. These results tie TAAR1 to the control of monoamine-driven behaviors and suggest anxiolytic- and antipsychotic-like properties for agonists such as RO5166017, opening treatment opportunities for psychiatric disorders.

Phosphorylation of Tau at S422 is enhanced by Abeta in TauPS2APP triple transgenic mice.

Amyloid beta peptides and microtubule-associated protein Tau are misfolded and form aggregates in brains of Alzheimer's disease patients. To examine their specific roles in the pathogenesis of Alzheimer's disease and their relevance in neurodegenerative processes, we have created TauPS2APP triple transgenic mice that express human mutated Amyloid Precursor Protein, presenilin 2 and Tau. We present a cross-sectional analysis of these mice at 4, 8, 12 and 16 months of age. By comparing with single transgenic Tau mice, we demonstrate that accumulation of Abeta in TauPS2APP triple transgenic mice impacts on Tau pathology by increasing the phosphorylation of Tau at serine 422, as determined by a novel immunodetection method that is able to reliably measure phospho-Tau species in transgenic mouse brains. The TauPS2APP triple transgenic mouse model will be very useful for studying the effect of new therapeutic paradigms on amyloid deposition and downstream neurofibrillary tangle development.

Pharmacological characterization of senktide-induced tail whips.

The tachykinin NK(3) receptor shows promise as a novel target for antipsychotics, but knowledge of downstream activity following tachykinin NK(3) receptor activation is lacking. To determine the practical utility of senktide-induced tail whips in mice as a tool for determining and characterizing downstream activity following tachykinin NK(3) receptor activation, mice were injected with 0.05 nmol of senktide i.c.v. and the number of tail whip bouts was counted for 20 min. Strain differences were observed, with NMRI mice showing a stronger tail whip response than C57Bl/6J mice. Tachykinin NK(3) receptor specificity was confirmed by the absence of the senktide-induced tail whip response in tachykinin NK(3) receptor knockout mice. Effects of tachykinin receptor pharmacological agents were tested by pretreatment with tachykinin NK(3) receptor antagonists (SB222200, talnetant and osanetant), which attenuated senktide-induced tail whips, and the tachykinin NK(1) receptor antagonist MK869, which had no effect on senktide-induced tail whips. Pharmacological interactions with other neurotransmitter systems were determined by pretreatment with dopamine D(1), D(2), and D(3) receptor antagonists, atypical antipsychotics, serotonin 5HT(1a) receptor antagonists, serotonin 5HT(2a/c) receptor antagonists, benzodiazepine and putative anxiolytics, antidepressants, and an anticholinergic. Senktide-induced tail whips were attenuated by dopamine D(2) receptor antagonists, atypical antipsychotics, serotonin 5HT(2a/c) antagonists, and benzodiazepine anxiolytics, but unaffected by drugs from other classes. Thus, the senktide-induced tail whip response is easily quantifiable, specific to the tachykinin NK(3) receptor, and provides valuable information on the downstream pharmacology of tachykinin NK(3) receptor activation.