Rapid in vivo measurement of ß-amyloid reveals biphasic clearance kinetics in an Alzheimer's mouse model.

Findings from genetic, animal model, and human studies support the observation that accumulation of the ß-amyloid (Aß) peptide in the brain plays a central role in the pathogenic cascade of Alzheimer's disease (AD). Human studies suggest that one key factor leading to accumulation is a defect in brain Aß clearance. We have developed a novel microimmunoelectrode (MIE) to study the kinetics of Aß clearance using an electrochemical approach. This is the first study using MIEs in vivo to measure rapid changes in Aß levels in the brains of living mice. Extracellular, interstitial fluid (ISF) Aß levels were measured in the hippocampus of APP/PS1 mice. Baseline levels of Aß40 in the ISF are relatively stable and begin to decline within minutes of blocking Aß production with a γ-secretase inhibitor. Pretreatment with a P-glycoprotein inhibitor, which blocks blood-brain barrier transport of Aß, resulted in significant prolongation of Aß40 half-life, but only in the latter phase of Aß clearance from the ISF.

A spectrum of exercise training reduces soluble Aß in a dose-dependent manner in a mouse model of Alzheimer's disease.

Physical activity has long been hypothesized to influence the risk and pathology of Alzheimer's disease. However, the amount of physical activity necessary for these benefits is unclear. We examined the effects of three months of low and high intensity exercise training on soluble Aß40 and Aß42 levels in extracellular enriched fractions from the cortex and hippocampus of young Tg2576 mice. Low (LOW) and high (HI) intensity exercise training animals ran at speeds of 15m/min on a level treadmill and 32 m/min at a 10% grade, respectively for 60 min per day, five days per week, from three to six months of age. Sedentary mice (SED) were placed on a level, non-moving, treadmill for the same duration. Soleus muscle citrate synthase activity increased by 39% in the LOW group relative to SED, and by 71% in the HI group relative to LOW, indicating an exercise training effect in these mice. Soluble Aß40 concentrations decreased significantly in an exercise training dose-dependent manner in the cortex. In the hippocampus, concentrations were decreased significantly in the HI group relative to LOW and SED. Soluble Aß42 levels also decreased significantly in an exercise training dose-dependent manner in both the cortex and hippocampus. Five proteins involved in Aß clearance (neprilysin, IDE, MMP9, LRP1 and HSP70) were elevated by exercise training with its intensity playing a role in each case. Our data demonstrate that exercise training reduces extracellular soluble Aß in the brains of Tg2576 mice in a dose-dependent manner through an up-regulation of Aß clearance.

microRNA-33 Regulates ApoE Lipidation and Amyloid-ß Metabolism in the Brain.

Dysregulation of amyloid-ß (Aß) metabolism is critical for Alzheimer's disease (AD) pathogenesis. Mounting evidence suggests that apolipoprotein E (ApoE) is involved in Aß metabolism. ATP-binding cassette transporter A1 (ABCA1) is a key regulator of ApoE lipidation, which affects Aß levels. Therefore, identifying regulatory mechanisms of ABCA1 expression in the brain may provide new therapeutic targets for AD. Here, we demonstrate that microRNA-33 (miR-33) regulates ABCA1 and Aß levels in the brain. Overexpression of miR-33 impaired cellular cholesterol efflux and dramatically increased extracellular Aß levels by promoting Aß secretion and impairing Aß clearance in neural cells. In contrast, genetic deletion of mir-33 in mice dramatically increased ABCA1 levels and ApoE lipidation, but it decreased endogenous Aß levels in cortex. Most importantly, pharmacological inhibition of miR-33 via antisense oligonucleotide specifically in the brain markedly decreased Aß levels in cortex of APP/PS1 mice, representing a potential therapeutic strategy for AD. SIGNIFICANCE STATEMENT: Brain lipid metabolism, in particular Apolipoprotein E (ApoE) lipidation, is critical to Aß metabolism and Alzheimer's disease (AD). Brain lipid metabolism is largely separated from the periphery due to blood-brain barrier and different repertoire of lipoproteins. Therefore, identifying the novel regulatory mechanism of brain lipid metabolism may provide a new therapeutic strategy for AD. Although there have been studies on brain lipid metabolism, its regulation, in particular by microRNAs, is relatively unknown. Here, we demonstrate that inhibition of microRNA-33 increases lipidation of brain ApoE and reduces Aß levels by inducing ABCA1. We provide a unique approach for AD therapeutics to increase ApoE lipidation and reduce Aß levels via pharmacological inhibition of microRNA in vivo.

In vivo measurement of apolipoprotein E from the brain interstitial fluid using microdialysis.

BACKGROUND: The APOE4 allele variant is the strongest known genetic risk factor for developing late-onset Alzheimer's disease. The link between apolipoprotein E (apoE) and Alzheimer's disease is likely due in large part to the impact of apoE on the metabolism of amyloid ß (Aß) within the brain. Manipulation of apoE levels and lipidation within the brain has been proposed as a therapeutic target for the treatment of Alzheimer's disease. However, we know little about the dynamic regulation of apoE levels and lipidation within the central nervous system. We have developed an assay to measure apoE levels in the brain interstitial fluid of awake and freely moving mice using large molecular weight cut-off microdialysis probes. RESULTS: We were able to recover apoE using microdialysis from human cerebrospinal fluid (CSF) in vitro and mouse brain parenchyma in vivo. Microdialysis probes were inserted into the hippocampus of wild-type mice and interstitial fluid was collected for 36 hours. Levels of apoE within the microdialysis samples were determined by ELISA. The levels of apoE were found to be relatively stable over 36 hours. No apoE was detected in microdialysis samples from apoE KO mice. Administration of the RXR agonist bexarotene increased ISF apoE levels while ISF Aß levels were decreased. Extrapolation to zero-flow analysis allowed us to determine the absolute recoverable concentration of apoE3 in the brain ISF of apoE3 KI mice. Furthermore, analysis of microdialysis samples by non-denaturing gel electrophoresis determined lipidated apoE particles in microdialysis samples were consistent in size with apoE particles from CSF. Finally, we found that the concentration of apoE in the brain ISF was dependent upon apoE isoform in human apoE KI mice, following the pattern apoE2>apoE3>apoE4. CONCLUSIONS: We are able to collect lipidated apoE from the brain of awake and freely moving mice and monitor apoE levels over the course of several hours from a single mouse. Our technique enables assessment of brain apoE dynamics under physiological and pathophysiological conditions and in response to therapeutic interventions designed to affect apoE levels and lipidation within the brain.

Attenuating astrocyte activation accelerates plaque pathogenesis in APP/PS1 mice.

The accumulation of aggregated amyloid-ß (Aß) in amyloid plaques is a neuropathological hallmark of Alzheimer's disease (AD). Reactive astrocytes are intimately associated with amyloid plaques; however, their role in AD pathogenesis is unclear. We deleted the genes encoding two intermediate filament proteins required for astrocyte activation-glial fibrillary acid protein (Gfap) and vimentin (Vim)-in transgenic mice expressing mutant human amyloid precursor protein and presenilin-1 (APP/PS1). The gene deletions increased amyloid plaque load: APP/PS1 Gfap(-/-)Vim(-/-) mice had twice the plaque load of APP/PS1 Gfap(+/+)Vim(+/+) mice at 8 and 12 mo of age. APP expression and soluble and interstitial fluid Aß levels were unchanged, suggesting that the deletions had no effect on APP processing or Aß generation. Astrocyte morphology was markedly altered by the deletions: wild-type astrocytes had hypertrophied processes that surrounded and infiltrated plaques, whereas Gfap(-/-)Vim(-/-) astrocytes had little process hypertrophy and lacked contact with adjacent plaques. Moreover, Gfap and Vim gene deletion resulted in a marked increase in dystrophic neurites (2- to 3-fold higher than APP/PS1 Gfap(+/+)Vim(+/+) mice), even after normalization for amyloid load. These results suggest that astrocyte activation limits plaque growth and attenuates plaque-related dystrophic neurites. These activities may require intimate contact between astrocyte and plaque.

Emergence of a seizure phenotype in aged apolipoprotein epsilon 4 targeted replacement mice.

The apolipoprotein ε4 allele is the strongest genetic risk factor for late-onset Alzheimer's disease (AD) and is associated with earlier age of onset. The incidence of spontaneous seizures has been reported to be increased in sporadic AD as well as in the early onset autosomal dominant forms of AD. We now report the emergence of a seizure phenotype in aged apolipoprotein E4 (apoE4) targeted replacement (TR) mice but not in age-matched apoE2 TR or apoE3 TR mice. Tonic-clonic seizures developed spontaneously after 5 months of age in apoE4 TR mice and are triggered by mild stress. Female mice had increased seizure penetrance compared to male mice, but had slightly reduced overall seizure severity. The majority of seizures were characterized by head and neck jerks, but 25% of aged apoE4 TR mice had more severe tonic-clonic seizures which occasionally progressed to tonic extension and death. Aged apoE4 TR mice progressed through pentylenetetrazol-induced seizure stages more rapidly than did apoE3 TR and apoE2 TR mice. Electroencephalographic (EEG) recordings revealed more frequent bursts of synchronous theta activity in the hippocampus of apoE4 TR mice than in apoE2 TR or apoE3 TR mice. Cortical EEG recordings also revealed sharp spikes and other abnormalities in apoE4 TR mice. Taken together, these findings demonstrate the emergence of an age-dependent seizure phenotype in old apoE4 TR mice in the absence of human amyloid-ß peptide (Aß) overexpression, suggesting increased central nervous system neural network excitability.

ApoE-directed therapeutics rapidly clear ß-amyloid and reverse deficits in AD mouse models.

Alzheimer's disease (AD) is associated with impaired clearance of ß-amyloid (Aß) from the brain, a process normally facilitated by apolipoprotein E (apoE). ApoE expression is transcriptionally induced through the action of the nuclear receptors peroxisome proliferator-activated receptor gamma and liver X receptors in coordination with retinoid X receptors (RXRs). Oral administration of the RXR agonist bexarotene to a mouse model of AD resulted in enhanced clearance of soluble Aß within hours in an apoE-dependent manner. Aß plaque area was reduced more than 50% within just 72 hours. Furthermore, bexarotene stimulated the rapid reversal of cognitive, social, and olfactory deficits and improved neural circuit function. Thus, RXR activation stimulates physiological Aß clearance mechanisms, resulting in the rapid reversal of a broad range of Aß-induced deficits.

Opposing synaptic regulation of amyloid-ß metabolism by NMDA receptors in vivo.

The concentration of amyloid-ß (Aß) within the brain extracellular space is one determinant of whether the peptide will aggregate into toxic species that are important in Alzheimer's disease (AD) pathogenesis. Some types of synaptic activity can regulate Aß levels. Here we demonstrate two distinct mechanisms that are simultaneously activated by NMDA receptors and regulate brain interstitial fluid (ISF) Aß levels in opposite directions in the living mouse. Depending on the dose of NMDA administered locally to the brain, ISF Aß levels either increase or decrease. Low doses of NMDA increase action potentials and synaptic transmission which leads to an elevation in synaptic Aß generation. In contrast, high doses of NMDA activate signaling pathways that lead to ERK (extracellular-regulated kinase) activation, which reduces processing of APP into Aß. This depression in Aß via APP processing occurs despite dramatically elevated synaptic activity. Both of these synaptic mechanisms are simultaneously active, with the balance between them determining whether ISF Aß levels will increase or decrease. NMDA receptor antagonists increase ISF Aß levels, suggesting that basal activity at these receptors normally suppresses Aß levels in vivo. This has implications for understanding normal Aß metabolism as well as AD pathogenesis.

Serotonin signaling is associated with lower amyloid-ß levels and plaques in transgenic mice and humans.

Aggregation of amyloid-ß (Aß) as toxic oligomers and amyloid plaques within the brain appears to be the pathogenic event that initiates Alzheimer's disease (AD) lesions. One therapeutic strategy has been to reduce Aß levels to limit its accumulation. Activation of certain neurotransmitter receptors can regulate Aß metabolism. We assessed the ability of serotonin signaling to alter brain Aß levels and plaques in a mouse model of AD and in humans. In mice, brain interstitial fluid (ISF) Aß levels were decreased by 25% following administration of several selective serotonin reuptake inhibitor (SSRI) antidepressant drugs. Similarly, direct infusion of serotonin into the hippocampus reduced ISF Aß levels. Serotonin-dependent reductions in Aß were reversed if mice were pretreated with inhibitors of the extracellular regulated kinase (ERK) signaling cascade. Chronic treatment with an SSRI, citalopram, caused a 50% reduction in brain plaque load in mice. To test whether serotonin signaling could impact Aß plaques in humans, we retrospectively compared brain amyloid load in cognitively normal elderly participants who were exposed to antidepressant drugs within the past 5 y to participants who were not. Antidepressant-treated participants had significantly less amyloid load as quantified by positron emission tomography (PET) imaging with Pittsburgh Compound B (PIB). Cumulative time of antidepressant use within the 5-y period preceding the scan correlated with less plaque load. These data suggest that serotonin signaling was associated with less Aß accumulation in cognitively normal individuals.

Acute dosing of latrepirdine (Dimebon), a possible Alzheimer therapeutic, elevates extracellular amyloid-beta levels in vitro and in vivo.

BACKGROUND: Recent reports suggest that latrepirdine (Dimebon, dimebolin), a retired Russian antihistamine, improves cognitive function in aged rodents and in patients with mild to moderate Alzheimer's disease (AD). However, the mechanism(s) underlying this benefit remain elusive. AD is characterized by extracellular accumulation of the amyloid-beta (Abeta) peptide in the brain, and Abeta-lowering drugs are currently among the most popular anti-amyloid agents under development for the treatment of AD. In the current study, we assessed the effect of acute dosing of latrepirdine on levels of extracellular Abeta using in vitro and in vivo experimental systems. RESULTS: We evaluated extracellular levels of Abeta in three experimental systems, under basal conditions and after treatment with latrepirdine. Mouse N2a neuroblastoma cells overexpressing Swedish APP were incubated for 6 hr in the presence of either vehicle or vehicle + latrepirdine (500pM-5 muM). Synaptoneurosomes were isolated from TgCRND8 mutant APP-overexpressing transgenic mice and incubated for 0 to 10 min in the absence or presence of latrepirdine (1 muM or 10 muM). Drug-naïve Tg2576 Swedish mutant APP overexpressing transgenic mice received a single intraperitoneal injection of either vehicle or vehicle + latrepirdine (3.5 mg/kg). Picomolar to nanomolar concentrations of acutely administered latrepirdine increased the extracellular concentration of Abeta in the conditioned media from Swedish mutant APP-overexpressing N2a cells by up to 64% (p = 0.01), while a clinically relevant acute dose of latrepirdine administered i.p. led to an increase in the interstitial fluid of freely moving APP transgenic mice by up to 40% (p = 0.01). Reconstitution of membrane protein trafficking and processing is frequently inefficient, and, consistent with this interpretation, latrepirdine treatment of isolated TgCRND8 synaptoneurosomes involved higher concentrations of drug (1-10 muM) and led to more modest increases in extracellular Abeta(x-42 )levels (+10%; p = 0.001); of note, however, was the observation that extracellular Abeta(x-40 )levels did not change. CONCLUSIONS: Here, we report the surprising association of acute latrepirdine dosing with elevated levels of extracellular Abeta as measured in three independent neuron-related or neuron-derived systems, including the hippocampus of freely moving Tg2576 mice. Given the reported association of chronic latrepirdine treatment with improvement in cognitive function, the effects of chronic latrepirdine treatment on extracellular Abeta levels must now be determined.