CCL2 Accelerates Microglia-Mediated Aβ Oligomer Formation and Progression of Neurocognitive Dysfunction

Background

The linkages between neuroinflammation and Alzheimer''s disease (AD) pathogenesis are well established. What is not, however, is how specific immune pathways and proteins affect the disease. To this end, we previously demonstrated that transgenic over-expression of CCL2 enhanced microgliosis and induced diffuse amyloid plaque deposition in Tg2576 mice. This rodent model of AD expresses a Swedish β-amyloid (Aβ) precursor protein mutant.

Methodology/Principal Findings

We now report that CCL2 transgene expression accelerates deficits in spatial and working memory and hippocampal synaptic transmission in β-amyloid precursor protein (APP) mice as early as 2–3 months of age. This is followed by increased numbers of microglia that are seen surrounding Aβ oligomers. CCL2 does not suppress Aβ degradation. Rather, CCL2 and tumor necrosis factor-α directly facilitated Aβ uptake, intracellular Aβ oligomerization, and protein secretion.

Conclusions/Significance

We posit that CCL2 facilitates Aβ oligomer formation in microglia and propose that such events accelerate memory dysfunction by affecting Aβ seeding in the brain.     

Farnesyltransferase Haplodeficiency Reduces Neuropathology and Rescues Cognitive Function in a Mouse Model of Alzheimer Disease

Isoprenoids and prenylated proteins have been implicated in the pathophysiology of Alzheimer disease (AD), including amyloid-β precursor protein metabolism, Tau phosphorylation, synaptic plasticity, and neuroinflammation. However, little is known about the relative importance of the two protein prenyltransferases, farnesyltransferase (FT) and geranylgeranyltransferase-1 (GGT), in the pathogenesis of AD. In this study, we defined the impact of deleting one copy of FT or GGT on the development of amyloid-β (Aβ)-associated neuropathology and learning/memory impairments in APPPS1 double transgenic mice, a well established model of AD. Heterozygous deletion of FT reduced Aβ deposition and neuroinflammation and rescued spatial learning and memory function in APPPS1 mice. Heterozygous deletion of GGT reduced the levels of Aβ and neuroinflammation but had no impact on learning and memory. These results document that farnesylation and geranylgeranylation play differential roles in AD pathogenesis and suggest that specific inhibition of protein farnesylation could be a potential strategy for effectively treating AD.     

Apolipoprotein E-Mimetics Inhibit Neurodegeneration and Restore Cognitive Functions in a Transgenic Drosophila Model of Alzheimer's Disease

Background

Mutations of the amyloid precursor protein gene (APP) are found in familial forms of Alzheimer''s disease (AD) and some lead to the elevated production of amyloid-β-protein (Aβ). While Aβ has been implicated in the causation of AD, the exact role played by Aβ and its APP precursor are still unclear.

Principal Findings

In our study, Drosophila melanogaster transgenics were established as a model to analyze AD-like pathology caused by APP overexpression. We demonstrated that age related changes in the levels and pattern of synaptic proteins accompanied progressive neurodegeneration and impairment of cognitive functions in APP transgenic flies, but that these changes may be independent from the generation of Aβ. Using novel peptide mimetics of Apolipoprotein-E, COG112 or COG133 proved to be neuroprotective and significantly improved the learning and memory of APP transgenic flies.

Conclusions

The development of neurodegeneration and cognitive deficits was corrected by injections of COG112 or COG133, novel mimetics of apolipoprotein-E (apoE) with neuroprotective activities.     

Lithium improves hippocampal neurogenesis, neuropathology and cognitive functions in APP mutant mice

Background

Alzheimer''s disease (AD) is a neurodegenerative disorder characterized by progressive deterioration of cognitive functions, extracellular β-amyloid (Aβ) plaques and intracellular neurofibrillary tangles within neocortex and hippocampus. Adult hippocampal neurogenesis plays an important role in learning and memory processes and its abnormal regulation might account for cognitive impairments associated with AD.

Methodology/Principal Findings

The double transgenic (Tg) CRND8 mice (overexpressing the Swedish and Indiana mutations in the human amyloid precursor protein), aged 2 and 6 months, were used to examine in vivo the effects of 5 weeks lithium treatment. BrdU labelling showed a decreased neurogenesis in the subgranular zone of Tg mice compared to non-Tg mice. The decrease of hippocampal neurogenesis was accompanied by behavioural deficits and worsened with age and pathology severity. The differentiation into neurons and maturation of the proliferating cells were also markedly impaired in the Tg mice. Lithium treatment to 2-month-old Tg mice significantly stimulated the proliferation and neuron fate specification of newborn cells and fully counteracted the transgene-induced impairments of cognitive functions. The drug, by the inhibition of GSK-3β and subsequent activation of Wnt/ß-catenin signalling promoted hippocampal neurogenesis. Finally, the data show that the lithium''s ability to stimulate neurogenesis and cognitive functions was lost in the aged Tg mice, thus indicating that the lithium-induced facilitation of neurogenesis and cognitive functions declines as brain Aβ deposition and pathology increases.

Conclusions

Lithium, when given on time, stimulates neurogenesis and counteracts AD-like pathology.     

Dysregulation of the mTOR Pathway Mediates Impairment of Synaptic Plasticity in a Mouse Model of Alzheimer's Disease

Background

The mammalian target of rapamycin (mTOR) is an evolutionarily conserved Ser/Thr protein kinase that plays a pivotal role in multiple fundamental biological processes, including synaptic plasticity. We explored the relationship between the mTOR pathway and β-amyloid (Aβ)-induced synaptic dysfunction, which is considered to be critical in the pathogenesis of Alzheimer''s disease (AD).

Methodology/Principal Findings

We provide evidence that inhibition of mTOR signaling correlates with impairment in synaptic plasticity in hippocampal slices from an AD mouse model and in wild-type slices exposed to exogenous Aβ1-42. Importantly, by up-regulating mTOR signaling, glycogen synthase kinase 3 (GSK3) inhibitors rescued LTP in the AD mouse model, and genetic deletion of FK506-binding protein 12 (FKBP12) prevented Aβ-induced impairment in long-term potentiation (LTP). In addition, confocal microscopy demonstrated co-localization of intraneuronal Aβ42 with mTOR.

Conclusions/Significance

These data support the notion that the mTOR pathway modulates Aβ-related synaptic dysfunction in AD.     

Ginsenoside Rg1 Decreases Aβ1–42 Level by Upregulating PPARγ and IDE Expression in the Hippocampus of a Rat Model of Alzheimer's Disease

Background and Purpose

The present study was designed to examine the effects of ginsenoside Rg1 on expression of peroxisome proliferator-activated receptor γ (PPARγ) and insulin-degrading enzyme (IDE) in the hippocampus of rat model of Alzheimer''s disease (AD) to determine how ginsenoside Rg1 (Rg1) decreases Aβ levels in AD.

Experimental Approach

Experimental AD was induced in rats by a bilateral injection of 10 µg soluble beta-amyloid peptide 1–42 (Aβ_1–42) into the CA1 region of the hippocampus, and the rats were treated with Rg1 (10 mg·kg⁻¹, intraperitoneally) for 28 days. The Morris water maze was used to test spatial learning and memory performance. Hematoxylin-eosin staining was performed to analyze the hippocampal histopathological damage. Immunohistochemistry, western blotting, and real-time PCR were used to detect Aβ_1–42, PPARγ, and insulin-degrading enzyme (IDE) expression in the hippocampus.

Key Results

Injection of soluble Aβ_1–42 into the hippocampus led to significant dysfunction of learning and memory, hippocampal histopathological abnormalities and increased Aβ_1–42 levels in the hippocampus. Rg1 treatment significantly improved learning and memory function, attenuated hippocampal histopathological abnormalities, reduced Aβ_1–42 levels and increased PPARγ and IDE expression in the hippocampus; these effects of Rg1 could be effectively inhibited by GW9662, a PPARγ antagonist.

Conclusions and Implications

Given that PPARγ can upregulate IDE expression and IDE can degrade Aβ_1–42, these results indicate that Rg1 can increase IDE expression in the hippocampus by upregulating PPARγ, leading to decreased Aβ levels, attenuated hippocampal histopathological abnormalities and improved learning and memory in a rat model of AD.     

Oridonin Attenuates Aβ1–42-Induced Neuroinflammation and Inhibits NF-κB Pathway

Neuroinflammation induced by beta-amyloid (Aβ) plays a critical role in the pathogenesis of Alzheimer’s disease (AD), and inhibiting Aβ-induced neuroinflammation serves as a potential strategy for the treatment of AD. Oridonin (Ori), a compound of Rabdosia rubescens, has been shown to exert anti-inflammatory effects. In this study, we demonstrated that Ori inhibited glial activation and decreased the release of inflammatory cytokines in the hippocampus of Aβ_1–42-induced AD mice. In addition, Ori inhibited the NF-κB pathway and Aβ_1–42-induced apoptosis. Furthermore, Ori could attenuate memory deficits in Aβ_1–42-induced AD mice. In conclusion, our study demonstrated that Ori inhibited the neuroinflammation and attenuated memory deficits induced by Aβ_1–42, suggesting that Ori might be a promising candidate for AD treatment.     

Improved Learning and Memory in Aged Mice Deficient in Amyloid β-Degrading Neutral Endopeptidase

Background

Neutral endopeptidase, also known as neprilysin and abbreviated NEP, is considered to be one of the key enzymes in initial human amyloid-β (Aβ) degradation. The aim of our study was to explore the impact of NEP deficiency on the initial development of dementia-like symptoms in mice.

Methodology/Principal Findings

We found that while endogenous Aβ concentrations were elevated in the brains of NEP-knockout mice at all investigated age groups, immunohistochemical analysis using monoclonal antibodies did not detect any Aβ deposits even in old NEP knockout mice. Surprisingly, tests of learning and memory revealed that the ability to learn was not reduced in old NEP-deficient mice but instead had significantly improved, and sustained learning and memory in the aged mice was congruent with improved long-term potentiation (LTP) in brain slices of the hippocampus and lateral amygdala. Our data suggests a beneficial effect of pharmacological inhibition of cerebral NEP on learning and memory in mice due to the accumulation of peptides other than Aβ degradable by NEP. By conducting degradation studies and peptide measurements in the brain of both genotypes, we identified two neuropeptide candidates, glucagon-like peptide 1 and galanin, as first potential candidates to be involved in the improved learning in aged NEP-deficient mice.

Conclusions/Significance

Thus, the existence of peptides targeted by NEP that improve learning and memory in older individuals may represent a promising avenue for the treatment of neurodegenerative diseases.     

LDLR Expression and Localization Are Altered in Mouse and Human Cell Culture Models of Alzheimer's Disease

Background

Alzheimer''s disease (AD) is a chronic neurodegenerative disorder and the most common form of dementia. The major molecular risk factor for late-onset AD is expression of the ε-4 allele of apolipoprotein E (apoE), the major cholesterol transporter in the brain. The low-density lipoprotein receptor (LDLR) has the highest affinity for apoE and plays an important role in brain cholesterol metabolism.

Methodology/Principal Findings

Using RT-PCR and western blotting techniques we found that over-expression of APP caused increases in both LDLR mRNA and protein levels in APP transfected H4 neuroglioma cells compared to H4 controls. Furthermore, immunohistochemical experiments showed aberrant localization of LDLR in H4-APP neuroglioma cells, Aβ-treated primary neurons, and in the PSAPP transgenic mouse model of AD. Finally, immunofluorescent staining of LDLR and of γ- and α-tubulin showed a change in LDLR localization preferentially away from the plasma membrane that was paralleled by and likely the result of a disruption of the microtubule-organizing center and associated microtubule network.

Conclusions/Significance

These data suggest that increased APP expression and Aβ exposure alters microtubule function, leading to reduced transport of LDLR to the plasma membrane. Consequent deleterious effects on apoE uptake and function will have implications for AD pathogenesis and/or progression.     

Dynamin 1 Regulates Amyloid Generation through Modulation of BACE-1

Background

Several lines of investigation support the notion that endocytosis is crucial for Alzheimer’s disease (AD) pathogenesis. Substantial evidence have already been reported regarding the mechanisms underlying amyloid precursor protein (APP) traffic, but the regulation of beta-site APP-Cleaving Enzyme 1 (BACE-1) distribution among endosomes, TGN and plasma membrane remains unclear. Dynamin, an important adaptor protein that controls sorting of many molecules, has recently been associated with AD but its functions remain controversial. Here we studied possible roles for dynamin 1 (dyn1) in Aβ biogenesis.

Principal Findings

We found that genetic perturbation of dyn1 reduces both secreted and intracellular Aβ levels in cell culture. There is a dramatic reduction in BACE-1 cleavage products of APP (sAPPβ and βCTF). Moreover, dyn1 knockdown (KD) leads to BACE-1 redistribution from the Golgi-TGN/endosome to the cell surface. There is an increase in the amount of surface holoAPP upon dyn1 KD, with resultant elevation of α–secretase cleavage products sAPPα and αCTF. But no changes are seen in the amount of nicastrin (NCT) or PS1 N-terminal fragment (NTF) at cell surface with dyn1 KD. Furthermore, treatment with a selective dynamin inhibitor Dynasore leads to similar reduction in βCTF and Aβ levels, comparable to changes with BACE inhibitor treatment. But combined inhibition of BACE-1 and dyn1 does not lead to further reduction in Aβ, suggesting that the Aβ-lowering effects of dynamin inhibition are mainly mediated through regulation of BACE-1 internalization. Aβ levels in dyn1^−/− primary neurons, as well as in 3-month old dyn1 haploinsufficient animals with AD transgenic background are consistently reduced when compared to their wildtype counterparts.

Conclusions

In summary, these data suggest a previously unknown mechanism by which dyn1 affects amyloid generation through regulation of BACE-1 subcellular localization and therefore its enzymatic activities.     

Immunogenic properties of amyloid beta oligomers

Background

The central molecule in the pathogenesis of Alzheimer’s disease (AD) is believed to be a small-sized polypeptide – beta amyloid (Aβ) which has an ability to assemble spontaneously into oligomers. Various studies concerning therapeutic and prophylactic approaches for AD are based on the immunotherapy using antibodies against Aβ. It has been suggested that either active immunization with Aβ or passive immunization with anti-Aβ antibodies might help to prevent or reduce the symptoms of the disease. However, knowledge on the mechanisms of Aβ-induced immune response is rather limited. Previous research on Aβ1-42 oligomers in rat brain cultures showed that the neurotoxicity of these oligomers considerably depends on their size. In the current study, we evaluated the dependence of immunogenicity of Aβ1-42 oligomers on the size of oligomeric particles and identified the immunodominant epitopes of the oligomers.

Results

Mice were immunized with various Aβ1-42 oligomers. The analysis of serum antibodies revealed that small Aβ1-42 oligomers (1–2 nm in size) are highly immunogenic. They induced predominantly IgG2b and IgG2a responses. In contrast, larger Aβ1-42 oligomers and monomers induced weaker IgG response in immunized mice. The monoclonal antibody against 1–2 nm Aβ1-42 oligomers was generated and used for antigenic characterization of Aβ1-42 oligomers. Epitope mapping of both monoclonal and polyclonal antibodies demonstrated that the main immunodominant region of the 1–2 nm Aβ1-42 oligomers is located at the amino-terminus (N-terminus) of the peptide, between amino acids 1 and 19.

Conclusions

Small Aβ1-42 oligomers of size 1–2 nm induce the strongest immune response in mice. The N-terminus of Aβ1-42 oligomers represents an immunodominant region which indicates its surface localization and accessibility to the B cells. The results of the current study may be important for further development of Aβ-based vaccination and immunotherapy strategies.     

The Alzheimer's Disease-Associated Amyloid β-Protein Is an Antimicrobial Peptide

Background

The amyloid β-protein (Aβ) is believed to be the key mediator of Alzheimer''s disease (AD) pathology. Aβ is most often characterized as an incidental catabolic byproduct that lacks a normal physiological role. However, Aβ has been shown to be a specific ligand for a number of different receptors and other molecules, transported by complex trafficking pathways, modulated in response to a variety of environmental stressors, and able to induce pro-inflammatory activities.

Methodology/Principal Findings

Here, we provide data supporting an in vivo function for Aβ as an antimicrobial peptide (AMP). Experiments used established in vitro assays to compare antimicrobial activities of Aβ and LL-37, an archetypical human AMP. Findings reveal that Aβ exerts antimicrobial activity against eight common and clinically relevant microorganisms with a potency equivalent to, and in some cases greater than, LL-37. Furthermore, we show that AD whole brain homogenates have significantly higher antimicrobial activity than aged matched non-AD samples and that AMP action correlates with tissue Aβ levels. Consistent with Aβ-mediated activity, the increased antimicrobial action was ablated by immunodepletion of AD brain homogenates with anti-Aβ antibodies.

Conclusions/Significance

Our findings suggest Aβ is a hitherto unrecognized AMP that may normally function in the innate immune system. This finding stands in stark contrast to current models of Aβ-mediated pathology and has important implications for ongoing and future AD treatment strategies.     

Zinc overload enhances APP cleavage and Aβ deposition in the Alzheimer mouse brain

Background

Abnormal zinc homeostasis is involved in β-amyloid (Aβ) plaque formation and, therefore, the zinc load is a contributing factor in Alzheimer''s disease (AD). However, the involvement of zinc in amyloid precursor protein (APP) processing and Aβ deposition has not been well established in AD animal models in vivo.

Methodology/Principal Findings

In the present study, APP and presenilin 1 (PS1) double transgenic mice were treated with a high dose of zinc (20 mg/ml ZnSO4 in drinking water). This zinc treatment increased APP expression, enhanced amyloidogenic APP cleavage and Aβ deposition, and impaired spatial learning and memory in the transgenic mice. We further examined the effects of zinc overload on APP processing in SHSY-5Y cells overexpressing human APPsw. The zinc enhancement of APP expression and cleavage was further confirmed in vitro.

Conclusions/Significance

The present data indicate that excess zinc exposure could be a risk factor for AD pathological processes, and alteration of zinc homeostasis is a potential strategy for the prevention and treatment of AD.     

Extremely Low-Frequency Magnetic Exposure Appears to Have No Effect on Pathogenesis of Alzheimer’s Disease in Aluminum-Overloaded Rat

Objective

Extremely low-frequency magnetic field (ELF-MF) has been reported to be of potential pathogenetic relevance to Alzheimer''s disease (AD) for years. However, evidence confirming this function remains inconclusive. Chronic Al treatment has been identified as a contributing factor to cognitive function impairment in AD. This study aims to examine whether or not ELF-MF and Al have synergistic effects toward AD pathogenesis by investigating the effects of ELF-MF with or without chronic Al treatment on SD rats.

Methods

Sprague-Dawley (SD) rats were subjected one of the following treatments: sham (control group), oral Al (Al group), ELF-MF (100 µT at 50 Hz) with oral Al (MF+Al group), or ELF-MF (100 µT at 50 Hz) without oral Al (MF group).

Results

After 12 wk of treatment, oral Al treatment groups (Al and MF+Al groups) showed learning and memory impairment as well as morphological hallmarks, including neuronal cell loss and high density of amyloid-β (Aβ) in the hippocampus and cerebral cortex. ELF-MF without Al treatment showed no significant effect on AD pathogenesis. ELF-MF+Al treatment induced no more damage than Al treatment did.

Conclusions

Our results showed no evidence of any association between ELF-MF exposure (100 µT at 50 Hz) and AD, and ELF-MF exposure does not influence the pathogenesis of AD induced by Al overload.     

Effect of BDNF Val66Met on Memory Decline and Hippocampal Atrophy in Prodromal Alzheimer’s Disease: A Preliminary Study

Objective

Cross-sectional genetic association studies have reported equivocal results on the relationship between the brain-derived neurotrophic factor (BDNF) Val66Met and risk of Alzheimer’s disease (AD). As AD is a neurodegenerative disease, genetic influences may become clearer from prospective study. We aimed to determine whether BDNF Val66Met polymorphism influences changes in memory performance, hippocampal volume, and Aβ accumulation in adults with amnestic mild cognitive impairment (aMCI) and high Aβ.

Methods

Thirty-four adults with aMCI were recruited from the Australian, Imaging, Biomarkers and Lifestyle (AIBL) Study. Participants underwent PiB-PET and structural MRI neuroimaging, neuropsychological assessments and BDNF genotyping at baseline, 18 month, and 36 month assessments.

Results

In individuals with aMCI and high Aβ, Met carriers showed significant and large decline in episodic memory (d = 0.90, p = .020) and hippocampal volume (d = 0.98, p = .035). BDNF Val66Met was unrelated to the rate of Aβ accumulation (d = −0.35, p = .401).

Conclusions

Although preliminary due to the small sample size, results of this study suggest that high Aβ levels and Met carriage may be useful prognostic markers of accelerated decline in episodic memory, and reductions in hippocampal volume in individuals in the prodromal or MCI stage of AD.     

The Temporal Expression Pattern of Alpha-Synuclein Modulates Olfactory Neurogenesis in Transgenic Mice

Background

Adult neurogenesis mirrors the brain´s endogenous capacity to generate new neurons throughout life. In the subventricular zone/ olfactory bulb system adult neurogenesis is linked to physiological olfactory function and has been shown to be impaired in murine models of neuronal alpha-Synuclein overexpression. We analyzed the degree and temporo-spatial dynamics of adult olfactory bulb neurogenesis in transgenic mice expressing human wild-type alpha-Synuclein (WTS) under the murine Thy1 (mThy1) promoter, a model known to have a particularly high tg expression associated with impaired olfaction.

Results

Survival of newly generated neurons (NeuN-positive) in the olfactory bulb was unchanged in mThy1 transgenic animals. Due to decreased dopaminergic differentiation a reduction in new dopaminergic neurons within the olfactory bulb glomerular layer was present. This is in contrast to our previously published data on transgenic animals that express WTS under the control of the human platelet-derived growth factor β (PDGF) promoter, that display a widespread decrease in survival of newly generated neurons in regions of adult neurogenesis, resulting in a much more pronounced neurogenesis deficit. Temporal and quantitative expression analysis using immunofluorescence co-localization analysis and Western blots revealed that in comparison to PDGF transgenic animals, in mThy1 transgenic animals WTS is expressed from later stages of neuronal maturation only but at significantly higher levels both in the olfactory bulb and cortex.

Conclusions

The dissociation between higher absolute expression levels of alpha-Synuclein but less severe impact on adult olfactory neurogenesis in mThy1 transgenic mice highlights the importance of temporal expression characteristics of alpha-Synuclein on the maturation of newborn neurons.     

Longitudinal Study of CSF Biomarkers in Patients with Alzheimer's Disease

Background

The CSF biomarkers tau and Aβ42 can identify patients with AD, even during the preclinical stages. However, previous studies on longitudinal changes of tau and Aβ42 in individual patients with AD and elderly controls report somewhat inconsistent results.

Methodology/Principal Findings

We investigated the levels of tau and Aβ42 at baseline and after 1 year in 100 patients with AD. In a second cohort of 45 AD patients we measured the CSF biomarkers at baseline and after 2 years. Moreover, in 34 healthy elderly controls the CSF biomarkers were followed for 4 years. The baseline levels of tau were increased with >60% in AD patients compared to controls (p<0.001), while baseline Aβ42 levels were decreased with >50% (p<0.001). In the AD group followed for 2 years, tau increased with 16% compared to the baseline levels (p<0.05). However, the levels of tau were stable over 4 years in the controls. The levels of Aβ42 did not change significantly over time in any of the groups. In the patients with AD, tau was moderately associated with worse cognitive performance already at baseline (p<0.05).

Conclusions/Significance

Tau and Aβ42 in CSF seem to reflect the underlying disease state in both early and late stages of AD. The slight increase in tau over time observed in the patients with AD is modest when compared to the relatively large difference in absolute tau levels between AD patients and controls. Therefore, these markers maintain their usefulness as state markers over time and might serve as surrogate markers for treatment efficacy in clinical trials.     

γ-Secretase Dependent Production of Intracellular Domains Is Reduced in Adult Compared to Embryonic Rat Brain Membranes

Background

γ-Secretase is an intramembrane aspartyl protease whose cleavage of the amyloid precursor protein (APP) generates the amyloid β-peptide (Aβ) and the APP intracellular domain. Aβ is widely believed to have a causative role in Alzheimer''s disease pathogenesis, and therefore modulation of γ-secretase activity has become a therapeutic goal. Besides APP, more than 50 substrates of γ-secretase with different cellular functions during embryogenesis as well as adulthood have been revealed. Prior to γ-secretase cleavage, substrates are ectodomain shedded, producing membrane bound C-terminal fragments (CTFs).

Principal Findings

Here, we investigated γ-secretase cleavage of five substrates; APP, Notch1, N-cadherin, ephrinB and p75 neurotrophin receptor (p75-NTR) in membranes isolated from embryonic, young or old adult rat brain by analyzing the release of the corresponding intracellular domains (ICDs) or Aβ40 by western blot analysis and ELISA respectively. The highest levels of all ICDs and Aβ were produced by embryonic membranes. In adult rat brain only cleavage of APP and Notch1 could be detected and the Aβ40 and ICD production from these substrates was similar in young and old adult rat brain. The CTF levels of Notch1, N-cadherin, ephrinB and p75-NTR were also clearly decreased in the adult brain compared to embryonic brain, whereas the APP CTF levels were only slightly decreased.

Conclusions

In summary our data suggests that γ-secretase dependent ICD production is down-regulated in the adult brain compared to embryonic brain. In addition, the present approach may be useful for evaluating the specificity of γ-secretase inhibitors.     

Chronic Alcohol-Induced microRNA-155 Contributes to Neuroinflammation in a TLR4-Dependent Manner in Mice

Introduction

Alcohol-induced neuroinflammation is mediated by pro-inflammatory cytokines and chemokines including tumor necrosis factor-α (TNFα), monocyte chemotactic protein-1 (MCP1) and interleukin-1-beta (IL-1β). Toll-like receptor-4 (TLR4) pathway induced nuclear factor-κB (NF-κB) activation is involved in the pathogenesis of alcohol-induced neuroinflammation. Inflammation is a highly regulated process. Recent studies suggest that microRNAs (miRNAs) play crucial role in fine tuning gene expression and miR-155 is a major regulator of inflammation in immune cells after TLR stimulation.

Aim

To evaluate the role of miR-155 in the pathogenesis of alcohol-induced neuroinflammation.

Methods

Wild type (WT), miR-155- and TLR4-knockout (KO) mice received 5% ethanol-containing or isocaloric control diet for 5 weeks. Microglia markers were measured by q-RTPCR; inflammasome activation was measured by enzyme activity; TNFα, MCP1, IL-1β mRNA and protein were measured by q-RTPCR and ELISA; phospho-p65 protein and NF-κB were measured by Western-blotting and EMSA; miRNAs were measured by q-PCR in the cerebellum. MiR-155 was measured in immortalized and primary mouse microglia after lipopolysaccharide and ethanol stimulation.

Results

Chronic ethanol feeding up-regulated miR-155 and miR-132 expression in mouse cerebellum. Deficiency in miR-155 protected mice from alcohol-induced increase in inflammatory cytokines; TNFα, MCP1 protein and TNFα, MCP1, pro-IL-1β and pro-caspase-1 mRNA levels were reduced in miR-155 KO alcohol-fed mice. NF-κB was activated in WT but not in miR-155 KO alcohol-fed mice. However increases in cerebellar caspase-1 activity and IL-1β levels were similar in alcohol-fed miR-155-KO and WT mice. Alcohol-fed TLR4-KO mice were protected from the induction of miR-155. NF-κB activation measured by phosphorylation of p65 and neuroinflammation were reduced in alcohol-fed TLR4-KO compared to control mice. TLR4 stimulation with lipopolysaccharide in primary or immortalized mouse microglia resulted in increased miR-155.

Conclusion

Chronic alcohol induces miR-155 in the cerebellum in a TLR4-dependent manner. Alcohol-induced miR-155 regulates TNFα and MCP1 expression but not caspase-dependent IL-1β increase in neuroinflammation.