Efficacy of Chronic Paroxetine Treatment in Mitigating Amyloid Pathology and Microgliosis in APPSWE/PS1ΔE9 Transgenic Mice.

BACKGROUND: Modulation of serotonergic signaling by treatment with selective serotonin reuptake inhibitors (SSRIs) has been suggested to mitigate amyloid-ß (Aß) pathology in Alzheimer's disease, in addition to exerting an anti-depressant action. OBJECTIVE: To investigate the efficacy of chronic treatment with the SSRI paroxetine, in mitigating Aß pathology and Aß plaque-induced microgliosis in the hippocampus of 18-month-old APPswe/PS1ΔE9 mice. METHODS: Plaque-bearing APPswe/PS1ΔE9 and wildtype mice were treated with paroxetine per os at a dose of 5 mg/kg/day, from 9 to 18 months of age. The per os treatment was monitored by recording of the body weights and serum paroxetine concentrations, and by assessment of the serotonin transporter occupancy by [3H]DASB-binding in wildtype mice. Additionally, 5,7-dihydroxytryptamine was administered to 9-month-old APPswe/PS1ΔE9 mice, to examine the effect of serotonin depletion on Aß pathology. Aß pathology was evaluated by Aß plaque load estimation and the Aß42/Aß40 ratio by ELISA. RESULTS: Paroxetine treatment led to > 80% serotonin transporter occupancy. The treatment increased the body weight of wildtype mice, but not of APPswe/PS1ΔE9 mice. The treatment had no effect on the Aß plaque load (p = 0.39), the number and size of plaques, or the Aß plaque-induced increases in microglial numbers in the dentate gyrus. Three months of serotonin depletion did not significantly impact the Aß plaque load or Aß42/Aß40 ratio in APPswe/PS1ΔE9 mice at 12 months. CONCLUSION: Our results show that chronic treatment with the SSRI paroxetine does not mitigate Aß pathology and Aß plaque-induced microgliosis in the hippocampus of APPswe/PS1ΔE9 mice.

Mild Microglial Responses in the Cortex and Perivascular Macrophage Infiltration in Subcortical White Matter in Dogs with Age-Related Dementia Modelling Prodromal Alzheimer's Disease.

BACKGROUND: Microglia contribute to Alzheimer's disease (AD) pathogenesis by clearing amyloid-ß (Aß) and driving neuroinflammation. Domestic dogs with age-related dementia (canine cognitive dysfunction (CCD)) develop cerebral amyloidosis like humans developing AD, and studying such dogs can provide novel information about microglial response in prodromal AD. OBJECTIVE: The aim was to investigate the microglial response in the cortical grey and the subcortical white matter in dogs with CCD versus age-matched cognitively normal dogs. METHODS: Brains from aged dogs with CCD and age-matched controls without dementia were studied. Cases were defined by dementia rating score. Brain sections were stained for Aß, thioflavin S, hyperphosphorylated tau, and the microglial-macrophage ionized calcium binding adaptor molecule 1 (Iba1). Results were correlated to dementia rating score and tissue levels of Aß. RESULTS: Microglial numbers were higher in the Aß plaque-loaded deep cortical layers in CCD versus control dogs, while the coverage by microglial processes were comparable. Aß plaques were of the diffuse type and without microglial aggregation. However, a correlation was found between the %Iba1 area and insoluble Aß 42 and N-terminal pyroglutamate modified Aß(N3pE)-42. The %Iba1 area was higher in white matter, showing phosphorylation of S396 tau, versus grey matter. Perivascular macrophage infiltrates were abundant in the white matter particularly in CDD dogs. CONCLUSION: The results from this study of the microglial-macrophage response in dogs with CCD are suggestive of relatively mild microglial responses in the Aß plaque-loaded deep cortical layers and perivascular macrophage infiltrates in the subcortical white matter, in prodromal AD.

Tumor Necrosis Factor (TNF) Is Required for Spatial Learning and Memory in Male Mice under Physiological, but Not Immune-Challenged Conditions.

Increasing evidence demonstrates that inflammatory cytokines-such as tumor necrosis factor (TNF)-are produced at low levels in the brain under physiological conditions and may be crucial for synaptic plasticity, neurogenesis, learning and memory. Here, we examined the effects of developmental TNF deletion on spatial learning and memory using 11-13-month-old TNF knockout (KO) and C57BL6/J wild-type (WT) mice. The animals were tested in the Barnes maze (BM) arena under baseline conditions and 48 h following an injection of the endotoxin lipopolysaccharide (LPS), which was administered at a dose of 0.5 mg/kg. Vehicle-treated KO mice were impaired compared to WT mice during the acquisition and memory-probing phases of the BM test. No behavioral differences were observed between WT and TNF-KO mice after LPS treatment. Moreover, there were no differences in the hippocampal content of glutamate and noradrenaline between groups. The effects of TNF deletion on spatial learning and memory were observed in male, but not female mice, which were not different compared to WT mice under baseline conditions. These results indicate that TNF is required for spatial learning and memory in male mice under physiological, non-inflammatory conditions, however not following the administration of LPS. Inflammatory signalling can thereby modulate spatial cognition in male subjects, highlighting the importance of sex- and probably age-stratified analysis when examining the role of TNF in the brain.

Ageing and amyloidosis underlie the molecular and pathological alterations of tau in a mouse model of familial Alzheimer's disease.

Despite compelling evidence that the accumulation of amyloid-beta (Aß) promotes neocortical MAPT (tau) aggregation in familial and idiopathic Alzheimer's disease (AD), murine models of cerebral amyloidosis are not considered to develop tau-associated pathology. In the present study, we show that tau can accumulate spontaneously in aged transgenic APPswe/PS1ΔE9 mice. Tau pathology is abundant around Aß deposits, and further characterized by accumulation of Gallyas and thioflavin-S-positive inclusions, which were detected in the APPswe/PS1ΔE9 brain at 18 months of age. Age-dependent increases in argyrophilia correlated positively with binding levels of the paired helical filament (PHF) tracer [18F]Flortaucipir, in all brain areas examined. Sarkosyl-insoluble PHFs were visualized by electron microscopy. Quantitative proteomics identified sequences of hyperphosphorylated and three-repeat tau in transgenic mice, along with signs of RNA missplicing, ribosomal dysregulation and disturbed energy metabolism. Tissue from the frontal gyrus of human subjects was used to validate these findings, revealing primarily quantitative differences between the tau pathology observed in AD patient vs. transgenic mouse tissue. As physiological levels of endogenous, 'wild-type' tau aggregate secondarily to Aß in APPswe/PS1ΔE9 mice, this study suggests that amyloidosis is both necessary and sufficient to drive tauopathy in experimental models of familial AD.

Protective effect of ibuprofen in a rat model of chronic oxaliplatin-induced peripheral neuropathy.

Despite extensive preclinical and clinical investigations, a clinically relevant neuroprotective agent against oxaliplatin-induced peripheral neuropathy, which affects the quality of life following chemotherapy, has not been identified. Epidemiological data suggest that ibuprofen may reduce the risk of neuropathy. Male rats were treated with oxaliplatin (n = 6), oxaliplatin and ibuprofen (n = 5) or vehicle (n = 5) every second day for 15 days. Neuropathy was evaluated using mechanical detection thresholds (MDT) at the hind paw and sensory nerve conduction velocity (SNCV) in the tail nerve at baseline, right after and 3 weeks after the end of treatment. Intraepidermal nerve fibre density (IENFD) was evaluated in the hind paw and inflammation in the dorsal root ganglia 3 weeks after treatment. Inflammation in the dorsal root ganglia was assessed using quantitative real-time RT-PCR (qPCR) of the mRNA levels for the pro-inflammatory cytokines, TNF-α and IL-1ß, and by immunohistochemical staining for Iba1+ macrophages. SNCV was reduced in rats treated with oxaliplatin and with oxaliplatin and ibuprofen compared to control rats 3 weeks after treatment. No differences were found for MDT 3 weeks after treatment. IENFD was reduced in rats treated with oxaliplatin. There was a trend towards up-regulation of TNF-α mRNA levels in rats treated with oxaliplatin and with oxaliplatin and ibuprofen. Morphological changes of Iba1+ macrophages suggested activation, but no differences were found in area fraction or size of macrophage cell bodies. The results did not support a neuroprotective effect of ibuprofen but indicated that inflammation may play a role in oxaliplatin-induced peripheral neuropathy.

Neuroinflammation and amyloid-beta 40 are associated with reduced serotonin transporter (SERT) activity in a transgenic model of familial Alzheimer's disease.

BACKGROUND: Discrepant and often contradictory results have accumulated regarding the antidepressant and pro-cognitive effects of serotonin transporter (SERT) antagonists in Alzheimer's disease. METHODS: To address the discrepancy, we measured the activity and density of SERT in the neocortex of 3-24-month-old APPswe/PS1dE9 and wild-type littermate mice, by using [3H]DASB autoradiography and the [3H]5-HT uptake assay. Levels of soluble amyloid-ß (Aß), and pro-inflammatory cytokines that can regulate SERT function, such as interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and tumor necrosis factor (TNF), were measured in parallel. Neuroinflammation in aging APPswe/PS1dE9 mice was further evaluated by [3H]PK11195 autoradiography. RESULTS: Decreased SERT density was observed in the parietal and frontal cortex of 18-24-month-old APPswe/PS1dE9 mice, compared to age-matched, wild-type animals. The maximal velocity uptake rate (Vmax) of [3H]5-HT was reduced in neocortical preparations from 20-month-old transgenic vs. wild-type mice. The reduction was observed when the proportion of soluble Aß40 in the Aß40/42 ratio increased in the aged transgenic brain. At concentrations compatible with those measured in 20-month-old APPswe/PS1dE9 mice, synthetic human Aß40, but not Aß42, reduced the baseline Vmax of [3H]5-HT by ~ 20%. Neuroinflammation in APPswe/PS1dE9 vs. wild-type mice was evidenced by elevated [3H]PK11195 binding levels and increased concentration of IL-1ß protein, which preceded the reductions in neocortical SERT density and activity. Age-induced increases in the levels of IL-1ß, IL-6, and TNF were observed in both transgenic and wild-type animals. CONCLUSIONS: The progression of cerebral amyloidosis is associated with neuroinflammation and decreased presynaptic markers of serotonergic integrity and activity. The Aß40-induced reduction in the uptake kinetics of [3H]5-HT suggests that the activity of SERT, and potentially the effects of SERT antagonism, depend on the levels of interstitial Aß40.

Established amyloid-ß pathology is unaffected by chronic treatment with the selective serotonin reuptake inhibitor paroxetine.

INTRODUCTION: Treatment with selective serotonin reuptake inhibitors has been suggested to mitigate amyloid-ß (Aß) pathology in Alzheimer's disease, in addition to an antidepressant mechanism of action. METHODS: We investigated whether chronic treatment with paroxetine, a selective serotonin reuptake inhibitor, mitigates Aß pathology in plaque-bearing double-transgenic amyloid precursor protein (APP)swe/presenilin 1 (PS1)ΔE9 mutants. In addition, we addressed whether serotonin depletion affects Aß pathology. Treatments were assessed by measurement of serotonin transporter occupancy and high-performance liquid chromatography. The effect of paroxetine on Aß pathology was evaluated by stereological plaque load estimation and Aß42/Aß40 ratio by enzyme-linked immunosorbent assay. RESULTS: Contrary to our hypothesis, paroxetine therapy did not mitigate Aß pathology, and depletion of brain serotonin did not exacerbate Aß pathology. However, chronic paroxetine therapy increased mortality in APPswe/PS1ΔE9 transgenic mice. DISCUSSION: Our results question the ability of selective serotonin reuptake inhibitor therapy to ameliorate established Aß pathology. The severe adverse effect of paroxetine may discourage its use for disease-modifying purposes in Alzheimer's disease.

Reduced Serotonin Transporter Levels and Inflammation in the Midbrain Raphe of 12 Month Old APPswe/PSEN1dE9 Mice.

BACKGROUND: Although mood and sleep disturbances are nearly universal among patients with Alzheimer's disease (AD), brain structures involved in non-cognitive processing remain under characterized in terms of AD pathology. OBJECTIVES: This study was designed to evaluate hallmarks of AD pathology in the brainstem of the APPswe/PS1dE9 mouse model of familial AD. METHODS: Fresh-frozen sections from female, 12 month old, transgenic and control B6C3 mice (n=6/genotype) were examined for amyloid burden and neurofibrillary alterations, by using 6E10 immunohistochemistry and the Gallyas silver stain, respectively. Serotonin transporter (SERT) densities in the dorsal and the median raphe were quantified by [3H]DASB autoradiography. SERT mRNA expression was measured by RT-PCR and visualized by in situ hybridization. Neuroinflammation was evaluated by immunohistochemical staining for microglia and astrocytes, and by measuring mRNA levels of the proinflammatory cytokines TNF-α, IL-1ß and IL-6. RESULTS: No amyloid- and tau-associated lesions were observed in the midbrain raphe of 12 month old APPswe/PS1dE9 mice. SERT binding levels were reduced in transgenic animals compared to age-matched controls, and SERT mRNA levels were decreased by at least 50% from control values. Intense microglial, but not astrocytic immunoreactivity was observed in APPswe/PS1dE9 vs. wild-type mice. Levels of TNF-α mRNA were two-fold higher than control and correlated positively with SERT mRNA expression levels in transgenic animals. CONCLUSIONS: There was no amyloid accumulation and tau-associated pathology in the midbrain raphe of 12 month old APPswe/PS1dE9 mice. However, there was a local neuroinflammatory response with loss of serotonergic markers, which may partially account for some of the behavioral symptoms of AD.

Serotonin augmentation therapy by escitalopram has minimal effects on amyloid-ß levels in early-stage Alzheimer's-like disease in mice.

BACKGROUND: Dysfunction of the serotonergic (5-HTergic) system has been implicated in the cognitive and behavioural symptoms of Alzheimer's disease (AD). Accumulation of toxic amyloid-ß (Aß) species is a hallmark of AD and an instigator of pathology. Serotonin (5-HT) augmentation therapy by treatment with selective serotonin reuptake inhibitors (SSRIs) in patients with AD has had mixed success in improving cognitive function, whereas SSRI administration to mice with AD-like disease has been shown to reduce Aß pathology. The objective of this study was to investigate whether an increase in extracellular levels of 5-HT induced by chronic SSRI treatment reduces Aß pathology and whether 5-HTergic deafferentation of the cerebral cortex could worsen Aß pathology in the APPswe/PS1ΔE9 (APP/PS1) mouse model of AD. METHODS: We administered a therapeutic dose of the SSRI escitalopram (5 mg/kg/day) in the drinking water of 3-month-old APP/PS1 mice to increase levels of 5-HT, and we performed intracerebroventricular injections of the neurotoxin 5,7-dihydroxytryptamine (DHT) to remove 5-HTergic afferents. We validated the effectiveness of these interventions by serotonin transporter autoradiography (neocortex 79.7 ± 7.6%) and by high-performance liquid chromatography for 5-HT (neocortex 64% reduction). After 6 months of escitalopram treatment or housing after DHT-induced lesion, we evaluated brain tissue by mesoscale multiplex analysis and sections by IHC analysis. RESULTS: Amyloid-ß-containing plaques had formed in the neocortex and hippocampus of 9-month-old APP/PS1 mice after 6 months of escitalopram treatment and 5-HTergic deafferentation. Unexpectedly, levels of insoluble Aß42 were unaffected in the neocortex and hippocampus after both types of interventions. Levels of insoluble Aß40 increased in the neocortex of SSRI-treated mice compared with those treated with vehicle control, but they were unaffected in the hippocampus. 5-HTergic deafferentation was without effect on the levels of insoluble/soluble Aß42 and Aß40 in both the neocortex and hippocampus. However, levels of soluble amyloid precursor protein α were reduced in the neocortex after 5-HTergic deafferentation. CONCLUSIONS: Because this study shows that modulation of the 5-HTergic system has either no effect or increases levels of insoluble/soluble Aß42 and Aß40 in the cerebral cortex of APP/PS1 mice, our observations do not support 5-HT augmentation therapy as a preventive strategy for reducing Aß pathology.

Bis-pyridylethenyl benzene as novel backbone for amyloid-ß binding compounds.

Detection of cerebral ß-amyloid (Aß) by targeted contrast agents is of great interest for in vivo diagnosis of Alzheimer's disease (AD). Partly because of their planar structure several bis-styrylbenzenes have been previously reported as potential Aß imaging agents. However, these compounds are relatively hydrophobic, which likely limits their in vivo potential. Based on their structures, we hypothesized that less hydrophobic bis-pyridylethenylbenzenes may also label amyloid. We synthesized several bis-pyridylethenylbenzenes and tested whether these compounds indeed display improved solubility and lower LogP values, and studied their fluorescent properties and Aß binding characteristics. Bis-pyridylethenylbenzenes showed a clear affinity for Aß plaques on both human and murine AD brain sections. Competitive binding experiments suggested a different binding site than Chrysamine G, a well-known stain for amyloid. With a LogP value between 3 and 5, most bis-pyridylethenylbenzenes were able to enter the brain and label murine amyloid in vivo with the bis(4-pyridylethenyl)benzenes showing the most favorable characteristics. In conclusion, the presented results suggest that bis-pyridylethenylbenzene may serve as a novel backbone for amyloid imaging agents.

An integrated proteomics approach shows synaptic plasticity changes in an APP/PS1 Alzheimer's mouse model.

The aim of this study was to elucidate the molecular signature of Alzheimer's disease-associated amyloid pathology.We used the double APPswe/PS1ΔE9 mouse, a widely used model of cerebral amyloidosis, to compare changes in proteome, including global phosphorylation and sialylated N-linked glycosylation patterns, pathway-focused transcriptome and neurological disease-associated miRNAome with age-matched controls in neocortex, hippocampus, olfactory bulb and brainstem. We report that signalling pathways related to synaptic functions associated with dendritic spine morphology, neurite outgrowth, long-term potentiation, CREB signalling and cytoskeletal dynamics were altered in 12 month old APPswe/PS1ΔE9 mice, particularly in the neocortex and olfactory bulb. This was associated with cerebral amyloidosis as well as formation of argyrophilic tangle-like structures and microglial clustering in all brain regions, except for brainstem. These responses may be epigenetically modulated by the interaction with a number of miRNAs regulating spine restructuring, Aß expression and neuroinflammation.We suggest that these changes could be associated with development of cognitive dysfunction in early disease states in patients with Alzheimer's disease.

Region-specific up-regulation of oxytocin receptor binding in the brain of mice following chronic nicotine administration.

Nicotine addiction is considered to be the main preventable cause of death worldwide. While growing evidence indicates that the neurohypophysial peptide oxytocin can modulate the addictive properties of several abused drugs, the regulation of the oxytocinergic system following nicotine administration has so far received little attention. Here, we examined the effects of long-term nicotine or saline administration on the central oxytocinergic system using [(125)I]OVTA autoradiographic binding in mouse brain. Male, 7-week old C57BL6J mice were treated with either nicotine (7.8 mg/kg daily; rate of 0.5 µl per hour) or saline for a period of 14-days via osmotic minipumps. Chronic nicotine administration induced a marked region-specific upregulation of the oxytocin receptor binding in the amygdala, a brain region involved in stress and emotional regulation. These results provide direct evidence for nicotine-induced neuroadaptations in the oxytocinergic system, which may be involved in the modulation of nicotine-seeking as well as emotional consequence of chronic drug use.

Genetic deletion of the adenosine A(2A) receptor prevents nicotine-induced upregulation of α7, but not α4ß2* nicotinic acetylcholine receptor binding in the brain.

Considerable evidence indicates that adenosine A(2A) receptors (A(2A)Rs) modulate cholinergic neurotransmission, nicotinic acetylcholine receptor (nAChR) function, and nicotine-induced behavioural effects. To explore the interaction between A(2A) and nAChRs, we examined if the complete genetic deletion of adenosine A(2A)Rs in mice induces compensatory alterations in the binding of different nAChR subtypes, and whether the long-term effects of nicotine on nAChR regulation are altered in the absence of the A(2A)R gene. Quantitative autoradiography was used to measure cytisine-sensitive [¹²5I]epibatidine and [¹²5I]α-bungarotoxin binding to α4ß2* and α7 nAChRs, respectively, in brain sections of drug-naïve (n = 6) or nicotine treated (n = 5-7), wild-type and adenosine A(2A)R knockout mice. Saline or nicotine (7.8 mg/kg/day; free-base weight) were administered to male CD1 mice via subcutaneous osmotic minipumps for a period of 14 days. Blood plasma levels of nicotine and cotinine were measured at the end of treatment. There were no compensatory developmental alterations in nAChR subtype distribution or density in drug-naïve A(2A)R knockout mice. In nicotine treated wild-type mice, both α4ß2* and α7 nAChR binding sites were increased compared with saline treated controls. The genetic ablation of adenosine A(2A)Rs prevented nicotine-induced upregulation of α7 nAChRs, without affecting α4ß2* receptor upregulation. This selective effect was observed at plasma levels of nicotine that were within the range reported for smokers (10-50 ng ml⁻¹). Our data highlight the involvement of adenosine A(2A)Rs in the mechanisms of nicotine-induced α7 nAChR upregulation, and identify A(2A)Rs as novel pharmacological targets for modulating the long-term effects of nicotine on α7 receptors.

Differential region-specific regulation of α4ß2* nAChRs by self-administered and non-contingent nicotine in C57BL/6J mice.

Neuronal nAChR upregulation is the hallmark of chronic nicotine exposure. Neuroplasticity to abused drugs, however, depends on whether their administration is forced by the experimenter or is under the control of the experimental animal. Neuroadaptation to chronic nicotine self-administration was examined with a yoked-control paradigm, using nose-poking as the operating procedure. Freely moving C57BL/6J mice that responded for 0.03 mg/kg/infusion of intravenous nicotine under a continuous schedule of reinforcement (FR-1), had control over the rate and amount of drug intake that a yoked littermate passively received (n = 11). The impact of response dependency on neurobiological changes in nicotinic and dopaminergic systems was subsequently assessed using quantitative autoradiography. Cytisine-sensitive [(125)I]epibatidine binding, [³H]SCH23390, [³H]raclopride and [³H]mazindol were used to label nAChRs with α4ß2* subtype properties, D1 and D2 dopaminergic receptors, and dopamine transporters, respectively. During a period of 12 days, self-administration was reliably initiated and maintained in animals receiving response-contingent nicotine. Region specific changes in the density of α4ß2* nAChRs were found to be dependent on the contingency of nicotine treatment. Higher levels of α4ß2* receptor binding were observed in the dorsal lateral geniculate nucleus and the ventral tegmental area of self-administering mice, compared to non-contingent animals. Moreover, response-independent increases in D2 binding were observed following chronic nicotine administration. No change in D1 and DAT binding was observed among groups. These findings indicate regional specific alterations in the regulation of the nicotinic cholinergic system following contingent and non-contingent nicotine exposure, and underline the importance of response dependency on the development of nicotine addiction.