TREM2 lipid sensing sustains the microglial response in an Alzheimer's disease model.

Triggering receptor expressed on myeloid cells 2 (TREM2) is a microglial surface receptor that triggers intracellular protein tyrosine phosphorylation. Recent genome-wide association studies have shown that a rare R47H mutation of TREM2 correlates with a substantial increase in the risk of developing Alzheimer's disease (AD). To address the basis for this genetic association, we studied TREM2 deficiency in the 5XFAD mouse model of AD. We found that TREM2 deficiency and haploinsufficiency augment ß-amyloid (Aß) accumulation due to a dysfunctional response of microglia, which fail to cluster around Aß plaques and become apoptotic. We further demonstrate that TREM2 senses a broad array of anionic and zwitterionic lipids known to associate with fibrillar Aß in lipid membranes and to be exposed on the surface of damaged neurons. Remarkably, the R47H mutation impairs TREM2 detection of lipid ligands. Thus, TREM2 detects damage-associated lipid patterns associated with neurodegeneration, sustaining the microglial response to Aß accumulation.

Sex-dependent effects of acute stress on amyloid-ß in male and female mice.

The risk of developing Alzheimer's disease is mediated by a combination of genetics and environmental factors, such as stress, sleep abnormalities and traumatic brain injury. Women are at a higher risk of developing Alzheimer's disease than men, even when controlling for differences in lifespan. Women are also more likely to report high levels of stress than men. Sex differences in response to stress may play a role in the increased risk of Alzheimer's disease in women. In this study, we use in vivo microdialysis to measure levels of Aß in response to acute stress in male and female mice. We show that Aß levels are altered differently between female and male mice (APP/PS1 and wild-type) in response to stress, with females showing significantly increased levels of Aß while most males do not show a significant change. This response is mediated through ß-arrestin involvement in Corticotrophin Releasing Factor receptor signalling pathway differences in male and female mice as male mice lacking ß-arrestin show increase in Aß in response to stress similar to females.

Aqp4 stop codon readthrough facilitates amyloid-ß clearance from the brain.

Alzheimer's disease is initiated by the toxic aggregation of amyloid-ß. Immunotherapeutics aimed at reducing amyloid beta are in clinical trials but with very limited success to date. Identification of orthogonal approaches for clearing amyloid beta may complement these approaches for treating Alzheimer's disease. In the brain, the astrocytic water channel Aquaporin 4 is involved in clearance of amyloid beta, and the fraction of Aquaporin 4 found perivascularly is decreased in Alzheimer's disease. Further, an unusual stop codon readthrough event generates a conserved C-terminally elongated variant of Aquaporin 4 (AQP4X), which is exclusively perivascular. However, it is unclear whether the AQP4X variant specifically mediates amyloid beta clearance. Here, using Aquaporin 4 readthrough-specific knockout mice that still express normal Aquaporin 4, we determine that this isoform indeed mediates amyloid beta clearance. Further, with high-throughput screening and counterscreening, we identify small molecule compounds that enhance readthrough of the Aquaporin 4 sequence and validate a subset on endogenous astrocyte Aquaporin 4. Finally, we demonstrate these compounds enhance brain amyloid-ß clearance in vivo, which depends on AQP4X. This suggests derivatives of these compounds may provide a viable pharmaceutical approach to enhance clearance of amyloid beta and potentially other aggregating proteins in neurodegenerative disease.

Implications of Oligomeric Amyloid-Beta (oAß42) Signaling through α7ß2-Nicotinic Acetylcholine Receptors (nAChRs) on Basal Forebrain Cholinergic Neuronal Intrinsic Excitability and Cognitive Decline.

Neuronal and network-level hyperexcitability is commonly associated with increased levels of amyloid-ß (Aß) and contribute to cognitive deficits associated with Alzheimer's disease (AD). However, the mechanistic complexity underlying the selective loss of basal forebrain cholinergic neurons (BFCNs), a well-recognized characteristic of AD, remains poorly understood. In this study, we tested the hypothesis that the oligomeric form of amyloid-ß (oAß42), interacting with α7-containing nicotinic acetylcholine receptor (nAChR) subtypes, leads to subnucleus-specific alterations in BFCN excitability and impaired cognition. We used single-channel electrophysiology to show that oAß42 activates both homomeric α7- and heteromeric α7ß2-nAChR subtypes while preferentially enhancing α7ß2-nAChR open-dwell times. Organotypic slice cultures were prepared from male and female ChAT-EGFP mice, and current-clamp recordings obtained from BFCNs chronically exposed to pathophysiologically relevant level of oAß42 showed enhanced neuronal intrinsic excitability and action potential firing rates. These resulted from a reduction in action potential afterhyperpolarization and alterations in the maximal rates of voltage change during spike depolarization and repolarization. These effects were observed in BFCNs from the medial septum diagonal band and horizontal diagonal band, but not the nucleus basalis. Last, aged male and female APP/PS1 transgenic mice, genetically null for the ß2 nAChR subunit gene, showed improved spatial reference memory compared with APP/PS1 aged-matched littermates. Combined, these data provide a molecular mechanism supporting a role for α7ß2-nAChR in mediating the effects of oAß42 on excitability of specific populations of cholinergic neurons and provide a framework for understanding the role of α7ß2-nAChR in oAß42-induced cognitive decline.

Pimavanserin, a 5HT2A receptor inverse agonist, rapidly suppresses Aß production and related pathology in a mouse model of Alzheimer's disease.

Amyloid-ß (Aß) peptide aggregation into soluble oligomers and insoluble plaques is a precipitating event in the pathogenesis of Alzheimer's disease (AD). Given that synaptic activity can regulate Aß generation, we postulated that 5HT2A -Rs may regulate Aß as well. We treated APP/PS1 transgenic mice with the selective 5HT2A inverse agonists M100907 or Pimavanserin systemically and measured brain interstitial fluid (ISF) Aß levels in real-time using in vivo microdialysis. Both compounds reduced ISF Aß levels by almost 50% within hours, but had no effect on Aß levels in 5HT2A -R knock-out mice. The Aß-lowering effects of Pimavanserin were blocked by extracellular-regulated kinase (ERK) and NMDA receptor inhibitors. Chronic administration of Pimavanserin by subcutaneous osmotic pump to aged APP/PS1 mice significantly reduced CSF Aß levels and Aß pathology and improved cognitive function in these mice. Pimavanserin is FDA-approved to treat Parkinson's disease psychosis, and also has been shown to reduce psychosis in a variety of other dementia subtypes including Alzheimer's disease. These data demonstrate that Pimavanserin may have disease-modifying benefits in addition to its efficacy against neuropsychiatric symptoms of Alzheimer's disease. Read the Editorial Highlight for this article on page 560.

Preclinical and clinical biomarker studies of CT1812: A novel approach to Alzheimer's disease modification.

INTRODUCTION: Amyloid beta (Aß) oligomers are one of the most toxic structural forms of the Aß protein and are hypothesized to cause synaptotoxicity and memory failure as they build up in Alzheimer's disease (AD) patients' brain tissue. We previously demonstrated that antagonists of the sigma-2 receptor complex effectively block Aß oligomer toxicity. CT1812 is an orally bioavailable, brain penetrant small molecule antagonist of the sigma-2 receptor complex that appears safe and well tolerated in healthy elderly volunteers. We tested CT1812's effect on Aß oligomer pathobiology in preclinical AD models and evaluated CT1812's impact on cerebrospinal fluid (CSF) protein biomarkers in mild to moderate AD patients in a clinical trial (ClinicalTrials.gov NCT02907567). METHODS: Experiments were performed to measure the impact of CT1812 versus vehicle on Aß oligomer binding to synapses in vitro, to human AD patient post mortem brain tissue ex vivo, and in living APPSwe /PS1dE9 transgenic mice in vivo. Additional experiments were performed to measure the impact of CT1812 versus vehicle on Aß oligomer-induced deficits in membrane trafficking rate, synapse number, and protein expression in mature hippocampal/cortical neurons in vitro. The impact of CT1812 on cognitive function was measured in transgenic Thy1 huAPPSwe/Lnd+ and wild-type littermates. A multicenter, double-blind, placebo-controlled parallel group trial was performed to evaluate the safety, tolerability, and impact on protein biomarker expression of CT1812 or placebo given once daily for 28 days to AD patients (Mini-Mental State Examination 18-26). CSF protein expression was measured by liquid chromatography with tandem mass spectrometry or enzyme-linked immunosorbent assay in samples drawn prior to dosing (Day 0) and at end of dosing (Day 28) and compared within each patient and between pooled treated versus placebo-treated dosing groups. RESULTS: CT1812 significantly and dose-dependently displaced Aß oligomers bound to synaptic receptors in three independent preclinical models of AD, facilitated oligomer clearance into the CSF, increased synaptic number and protein expression in neurons, and improved cognitive performance in transgenic mice. CT1812 significantly increased CSF concentrations of Aß oligomers in AD patient CSF, reduced concentrations of synaptic proteins and phosphorylated tau fragments, and reversed expression of many AD-related proteins dysregulated in CSF. DISCUSSION: These preclinical studies demonstrate the novel disease-modifying mechanism of action of CT1812 against AD and Aß oligomers. The clinical results are consistent with preclinical data and provide evidence of target engagement and impact on fundamental disease-related signaling pathways in AD patients, supporting further development of CT1812.

Astrocytic LRP1 Mediates Brain Aß Clearance and Impacts Amyloid Deposition.

Accumulation and deposition of amyloid-ß (Aß) in the brain represent an early and perhaps necessary step in the pathogenesis of Alzheimer's disease (AD). Aß accumulation leads to the formation of Aß aggregates, which may directly and indirectly lead to eventual neurodegeneration. While Aß production is accelerated in many familial forms of early-onset AD, increasing evidence indicates that impaired clearance of Aß is more evident in late-onset AD. To uncover the mechanisms underlying impaired Aß clearance in AD, we examined the role of low-density lipoprotein receptor-related protein 1 (LRP1) in astrocytes. Although LRP1 has been shown to play critical roles in brain Aß metabolism in neurons and vascular mural cells, its role in astrocytes, the most abundant cell type in the brain responsible for maintaining neuronal homeostasis, remains unclear. Here, we show that astrocytic LRP1 plays a critical role in brain Aß clearance. LRP1 knockdown in primary astrocytes resulted in decreased cellular Aß uptake and degradation. In addition, silencing of LRP1 in astrocytes led to downregulation of several major Aß-degrading enzymes, including matrix metalloproteases MMP2, MMP9, and insulin-degrading enzyme. More important, conditional knock-out of the Lrp1 gene in astrocytes in the background of APP/PS1 mice impaired brain Aß clearance, exacerbated Aß accumulation, and accelerated amyloid plaque deposition without affecting its production. Together, our results demonstrate that astrocytic LRP1 plays an important role in Aß metabolism and that restoring LRP1 expression and function in the brain could be an effective strategy to facilitate Aß clearance and counter amyloid pathology in AD.SIGNIFICANCE STATEMENT Astrocytes represent a major cell type regulating brain homeostasis; however, their roles in brain clearance of amyloid-ß (Aß) and underlying mechanism are not clear. In this study, we used both cellular models and conditional knock-out mouse models to address the role of a critical Aß receptor, the low-density lipoprotein receptor-related protein 1 (LRP1) in astrocytes. We found that LRP1 in astrocytes plays a critical role in brain Aß clearance by modulating several Aß-degrading enzymes and cellular degradation pathways. Our results establish a critical role of astrocytic LRP1 in brain Aß clearance and shed light on specific Aß clearance pathways that may help to establish new targets for AD prevention and therapy.

Contribution of reactive oxygen species to cerebral amyloid angiopathy, vasomotor dysfunction, and microhemorrhage in aged Tg2576 mice.

Cerebral amyloid angiopathy (CAA) is characterized by deposition of amyloid ß peptide (Aß) within walls of cerebral arteries and is an important cause of intracerebral hemorrhage, ischemic stroke, and cognitive dysfunction in elderly patients with and without Alzheimer's Disease (AD). NADPH oxidase-derived oxidative stress plays a key role in soluble Aß-induced vessel dysfunction, but the mechanisms by which insoluble Aß in the form of CAA causes cerebrovascular (CV) dysfunction are not clear. Here, we demonstrate evidence that reactive oxygen species (ROS) and, in particular, NADPH oxidase-derived ROS are a key mediator of CAA-induced CV deficits. First, the NADPH oxidase inhibitor, apocynin, and the nonspecific ROS scavenger, tempol, are shown to reduce oxidative stress and improve CV reactivity in aged Tg2576 mice. Second, the observed improvement in CV function is attributed both to a reduction in CAA formation and a decrease in CAA-induced vasomotor impairment. Third, anti-ROS therapy attenuates CAA-related microhemorrhage. A potential mechanism by which ROS contribute to CAA pathogenesis is also identified because apocynin substantially reduces expression levels of ApoE-a factor known to promote CAA formation. In total, these data indicate that ROS are a key contributor to CAA formation, CAA-induced vessel dysfunction, and CAA-related microhemorrhage. Thus, ROS and, in particular, NADPH oxidase-derived ROS are a promising therapeutic target for patients with CAA and AD.

Passive immunotherapy targeting amyloid-ß reduces cerebral amyloid angiopathy and improves vascular reactivity.

Prominent cerebral amyloid angiopathy is often observed in the brains of elderly individuals and is almost universally found in patients with Alzheimer's disease. Cerebral amyloid angiopathy is characterized by accumulation of the shorter amyloid-ß isoform(s) (predominantly amyloid-ß40) in the walls of leptomeningeal and cortical arterioles and is likely a contributory factor to vascular dysfunction leading to stroke and dementia in the elderly. We used transgenic mice with prominent cerebral amyloid angiopathy to investigate the ability of ponezumab, an anti-amyloid-ß40 selective antibody, to attenuate amyloid-ß accrual in cerebral vessels and to acutely restore vascular reactivity. Chronic administration of ponezumab to transgenic mice led to a significant reduction in amyloid and amyloid-ß accumulation both in leptomeningeal and brain vessels when measured by intravital multiphoton imaging and immunohistochemistry. By enriching for cerebral vascular elements, we also measured a significant reduction in the levels of soluble amyloid-ß biochemically. We hypothesized that the reduction in vascular amyloid-ß40 after ponezumab administration may reflect the ability of ponezumab to mobilize an interstitial fluid pool of amyloid-ß40 in brain. Acutely, ponezumab triggered a significant and transient increase in interstitial fluid amyloid-ß40 levels in old plaque-bearing transgenic mice but not in young animals. We also measured a beneficial effect on vascular reactivity following acute administration of ponezumab, even in vessels where there was a severe cerebral amyloid angiopathy burden. Taken together, the beneficial effects ponezumab administration has on reducing the rate of cerebral amyloid angiopathy deposition and restoring cerebral vascular health favours a mechanism that involves rapid removal and/or neutralization of amyloid-ß species that may otherwise be detrimental to normal vessel function.

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.

Neuronal-Targeted TFEB Accelerates Lysosomal Degradation of APP, Reducing Aß Generation and Amyloid Plaque Pathogenesis.

In AD, an imbalance between Aß production and removal drives elevated brain Aß levels and eventual amyloid plaque deposition. APP undergoes nonamyloidogenic processing via α-cleavage at the plasma membrane, amyloidogenic ß- and γ-cleavage within endosomes to generate Aß, or lysosomal degradation in neurons. Considering multiple reports implicating impaired lysosome function as a driver of increased amyloidogenic processing of APP, we explored the efficacy of targeting transcription factor EB (TFEB), a master regulator of lysosomal pathways, to reduce Aß levels. CMV promoter-driven TFEB, transduced via stereotactic hippocampal injections of adeno-associated virus particles in APP/PS1 mice, localized primarily to neuronal nuclei and upregulated lysosome biogenesis. This resulted in reduction of APP protein, the α and ß C-terminal APP fragments (CTFs), and in the steady-state Aß levels in the brain interstitial fluid. In aged mice, total Aß levels and amyloid plaque load were selectively reduced in the TFEB-transduced hippocampi. TFEB transfection in N2a cells stably expressing APP695, stimulated lysosome biogenesis, reduced steady-state levels of APP and α- and ß-CTFs, and attenuated Aß generation by accelerating flux through the endosome-lysosome pathway. Cycloheximide chase assays revealed a shortening of APP half-life with exogenous TFEB expression, which was prevented by concomitant inhibition of lysosomal acidification. These data indicate that TFEB enhances flux through lysosomal degradative pathways to induce APP degradation and reduce Aß generation. Activation of TFEB in neurons is an effective strategy to attenuate Aß generation and attenuate amyloid plaque deposition in AD. SIGNIFICANCE STATEMENT: A key driver for AD pathogenesis is the net balance between production and clearance of Aß, the major component of amyloid plaques. Here we demonstrate that lysosomal degradation of holo-APP influences Aß production by limiting the availability of APP for amyloidogenic processing. Using viral gene transfer of transcription factor EB (TFEB), a master regulator of lysosome biogenesis in neurons of APP/PS1 mice, steady-state levels of APP were reduced, resulting in decreased interstitial fluid Aß levels and attenuated amyloid deposits. These effects were caused by accelerated lysosomal degradation of endocytosed APP, reflected by reduced APP half-life and steady-state levels in TFEB-expressing cells, with resultant decrease in Aß production and release. Additional studies are needed to explore the therapeutic potential of this approach.

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.

Genetic modulation of soluble Aß rescues cognitive and synaptic impairment in a mouse model of Alzheimer's disease.

An unresolved debate in Alzheimer's disease (AD) is whether amyloid plaques are pathogenic, causing overt physical disruption of neural circuits, or protective, sequestering soluble forms of amyloid-ß (Aß) that initiate synaptic damage and cognitive decline. Few animal models of AD have been capable of isolating the relative contribution made by soluble and insoluble forms of Aß to the behavioral symptoms and biochemical consequences of the disease. Here we use a controllable transgenic mouse model expressing a mutant form of amyloid precursor protein (APP) to distinguish the impact of soluble Aß from that of deposited amyloid on cognitive function and synaptic structure. Rapid inhibition of transgenic APP modulated the production of Aß without affecting pre-existing amyloid deposits and restored cognitive performance to the level of healthy controls in Morris water maze, radial arm water maze, and fear conditioning. Selective reduction of Aß with a γ-secretase inhibitor provided similar improvement, suggesting that transgene suppression restored cognition, at least in part by lowering Aß. Cognitive improvement coincided with reduced levels of synaptotoxic Aß oligomers, greater synaptic density surrounding amyloid plaques, and increased expression of presynaptic and postsynaptic markers. Together these findings indicate that transient Aß species underlie much of the cognitive and synaptic deficits observed in this model and demonstrate that significant functional and structural recovery can be attained without removing deposited amyloid.

Genetic suppression of transgenic APP rescues Hypersynchronous network activity in a mouse model of Alzeimer's disease.

Alzheimer's disease (AD) is associated with an elevated risk for seizures that may be fundamentally connected to cognitive dysfunction. Supporting this link, many mouse models for AD exhibit abnormal electroencephalogram (EEG) activity in addition to the expected neuropathology and cognitive deficits. Here, we used a controllable transgenic system to investigate how network changes develop and are maintained in a model characterized by amyloid ß (Aß) overproduction and progressive amyloid pathology. EEG recordings in tet-off mice overexpressing amyloid precursor protein (APP) from birth display frequent sharp wave discharges (SWDs). Unexpectedly, we found that withholding APP overexpression until adulthood substantially delayed the appearance of epileptiform activity. Together, these findings suggest that juvenile APP overexpression altered cortical development to favor synchronized firing. Regardless of the age at which EEG abnormalities appeared, the phenotype was dependent on continued APP overexpression and abated over several weeks once transgene expression was suppressed. Abnormal EEG discharges were independent of plaque load and could be extinguished without altering deposited amyloid. Selective reduction of Aß with a γ-secretase inhibitor has no effect on the frequency of SWDs, indicating that another APP fragment or the full-length protein was likely responsible for maintaining EEG abnormalities. Moreover, transgene suppression normalized the ratio of excitatory to inhibitory innervation in the cortex, whereas secretase inhibition did not. Our results suggest that APP overexpression, and not Aß overproduction, is responsible for EEG abnormalities in our transgenic mice and can be rescued independently of pathology.

Enhancing astrocytic lysosome biogenesis facilitates Aß clearance and attenuates amyloid plaque pathogenesis.

In sporadic Alzheimer's disease (AD), impaired Aß removal contributes to elevated extracellular Aß levels that drive amyloid plaque pathogenesis. Extracellular proteolysis, export across the blood-brain barrier, and cellular uptake facilitate physiologic Aß clearance. Astrocytes can take up and degrade Aß, but it remains unclear whether this function is insufficient in AD or can be enhanced to accelerate Aß removal. Additionally, age-related dysfunction of lysosomes, the major degradative organelles wherein Aß localizes after uptake, has been implicated in amyloid plaque pathogenesis. We tested the hypothesis that enhancing lysosomal function in astrocytes with transcription factor EB (TFEB), a master regulator of lysosome biogenesis, would promote Aß uptake and catabolism and attenuate plaque pathogenesis. Exogenous TFEB localized to the nucleus with transcriptional induction of lysosomal biogenesis and function in vitro. This resulted in significantly accelerated uptake of exogenously applied Aß42, with increased localization to and degradation within lysosomes in C17.2 cells and primary astrocytes, indicating that TFEB is sufficient to coordinately enhance uptake, trafficking, and degradation of Aß. Stereotactic injection of adeno-associated viral particles carrying TFEB driven by a glial fibrillary acidic protein promoter was used to achieve astrocyte-specific expression in the hippocampus of APP/PS1 transgenic mice. Exogenous TFEB localized to astrocyte nuclei and enhanced lysosome function, resulting in reduced Aß levels and shortened half-life in the brain interstitial fluid and reduced amyloid plaque load in the hippocampus compared with control virus-injected mice. Therefore, activation of TFEB in astrocytes is an effective strategy to restore adequate Aß removal and counter amyloid plaque pathogenesis in AD.

Levetiracetam suppresses neuronal network dysfunction and reverses synaptic and cognitive deficits in an Alzheimer's disease model.

In light of the rising prevalence of Alzheimer's disease (AD), new strategies to prevent, halt, and reverse this condition are needed urgently. Perturbations of brain network activity are observed in AD patients and in conditions that increase the risk of developing AD, suggesting that aberrant network activity might contribute to AD-related cognitive decline. Human amyloid precursor protein (hAPP) transgenic mice simulate key aspects of AD, including pathologically elevated levels of amyloid-ß peptides in brain, aberrant neural network activity, remodeling of hippocampal circuits, synaptic deficits, and behavioral abnormalities. Whether these alterations are linked in a causal chain remains unknown. To explore whether hAPP/amyloid-ß-induced aberrant network activity contributes to synaptic and cognitive deficits, we treated hAPP mice with different antiepileptic drugs. Among the drugs tested, only levetiracetam (LEV) effectively reduced abnormal spike activity detected by electroencephalography. Chronic treatment with LEV also reversed hippocampal remodeling, behavioral abnormalities, synaptic dysfunction, and deficits in learning and memory in hAPP mice. Our findings support the hypothesis that aberrant network activity contributes causally to synaptic and cognitive deficits in hAPP mice. LEV might also help ameliorate related abnormalities in people who have or are at risk for AD.

Neuronal clearance of amyloid-ß by endocytic receptor LRP1.

Alzheimer's disease (AD) is the most prevalent form of dementia in the elderly population. Accumulation, aggregation, and deposition of amyloid-ß (Aß) peptides generated through proteolytic cleavage of amyloid precursor protein (APP) are likely initiating events in the pathogenesis of AD. While Aß production is accelerated in familial AD, increasing evidence indicates that impaired clearance of Aß is responsible for late-onset AD. Because Aß is mainly generated in neurons, these cells are predicted to have the highest risk of encountering Aß among all cell types in the brain. However, it is still unclear whether they are also involved in Aß clearance. Here we show that receptor-mediated endocytosis in neurons by the low-density lipoprotein receptor-related protein 1 (LRP1) plays a critical role in brain Aß clearance. LRP1 is known to be an endocytic receptor for multiple ligands including Aß. Conditional knock-out of Lrp1 in mouse forebrain neurons leads to increased brain Aß levels and exacerbated amyloid plaque deposition selectively in the cortex of amyloid model APP/PS1 mice without affecting Aß production. In vivo microdialysis studies demonstrated that Aß clearance in brain interstitial fluid is impaired in neuronal Lrp1 knock-out mice. Because the neuronal LRP1-deletion did not affect the mRNA levels of major Aß degrading enzymes, neprilysin and insulin-degrading enzyme, the disturbed Aß clearance is likely due to the suppression of LRP1-mediated neuronal Aß uptake and degradation. Together, our results demonstrate that LRP1 plays an important role in receptor-mediated clearance of Aß and indicate that neurons not only produce but also clear Aß.

Antisense reduction of tau in adult mice protects against seizures.

Tau, a microtubule-associated protein, is implicated in the pathogenesis of Alzheimer's Disease (AD) in regard to both neurofibrillary tangle formation and neuronal network hyperexcitability. The genetic ablation of tau substantially reduces hyperexcitability in AD mouse lines, induced seizure models, and genetic in vivo models of epilepsy. These data demonstrate that tau is an important regulator of network excitability. However, developmental compensation in the genetic tau knock-out line may account for the protective effect against seizures. To test the efficacy of a tau reducing therapy for disorders with a detrimental hyperexcitability profile in adult animals, we identified antisense oligonucleotides that selectively decrease endogenous tau expression throughout the entire mouse CNS--brain and spinal cord tissue, interstitial fluid, and CSF--while having no effect on baseline motor or cognitive behavior. In two chemically induced seizure models, mice with reduced tau protein had less severe seizures than control mice. Total tau protein levels and seizure severity were highly correlated, such that those mice with the most severe seizures also had the highest levels of tau. Our results demonstrate that endogenous tau is integral for regulating neuronal hyperexcitability in adult animals and suggest that an antisense oligonucleotide reduction of tau could benefit those with epilepsy and perhaps other disorders associated with tau-mediated neuronal hyperexcitability.

Amyloid-ß oligomerization in Alzheimer dementia versus high-pathology controls.

OBJECTIVE: Although amyloid-beta (Aß) peptide deposition into insoluble plaques is a pathological hallmark of Alzheimer disease; soluble oligomeric Aß has been hypothesized to more directly underlie impaired learning and memory in dementia of the Alzheimer type. However, the lack of a sensitive, specific, and quantitative assay for Aß oligomers has hampered rigorous tests of this hypothesis. METHODS: We developed a plate-based single molecule counting fluorescence immunoassay for oligomeric Aß sensitive to low pg/ml concentrations of synthetic Aß dimers using the same Aß-specific monoclonal antibody to both capture and detect Aß. The Aß oligomer assay does not recognize monomeric Aß, amyloid precursor protein, or other non-Aß peptide oligomers. RESULTS: Aß oligomers were detected in aqueous cortical lysates from patients with dementia of the Alzheimer type and nondemented patients with Aß plaque pathology. However, Aß oligomer concentrations in demented patients' lysates were tightly correlated with Aß plaque coverage (r = 0.88), but this relationship was weaker in those from nondemented patients (r = 0.30) despite equivalent Aß plaque pathology. The ratio of Aß oligomer levels to plaque density fully distinguished demented from nondemented patients, with no overlap between groups in this derived variable. Other Aß and plaque measures did not distinguish demented from nondemented patients. Aß oligomers were not detected in cerebrospinal fluid with this assay. INTERPRETATION: The results raise the intriguing hypothesis that the linkage between plaques and oligomers may be a key pathophysiological event underlying dementia of the Alzheimer type. This Aß oligomer assay may be useful for many tests of the oligomer hypothesis.

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.