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.

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.

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.

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.