Cannabinoid receptor 1 deficiency in a mouse model of Alzheimer's disease leads to enhanced cognitive impairment despite of a reduction in amyloid deposition.

Alzheimer's disease (AD) is characterized by amyloid-ß deposition in amyloid plaques, neurofibrillary tangles, inflammation, neuronal loss, and cognitive deficits. Cannabinoids display neuromodulatory and neuroprotective effects and affect memory acquisition. Here, we studied the impact of cannabinoid receptor type 1 (CB1) deficiency on the development of AD pathology by breeding amyloid precursor protein (APP) Swedish mutant mice (APP23), an AD animal model, with CB1-deficient mice. In addition to the lower body weight of APP23/CB1(-/-) mice, most of these mice died at an age before typical AD-associated changes become apparent. The surviving mice showed a reduced amount of APP and its fragments suggesting a regulatory influence of CB1 on APP processing, which was confirmed by modulating CB1 expression in vitro. Reduced APP levels were accompanied by a reduced plaque load and less inflammation in APP23/CB1(-/-) mice. Nevertheless, compared to APP23 mice with an intact CB1, APP23/CB1(-/-) mice showed impaired learning and memory deficits. These data argue against a direct correlation of amyloid plaque load with cognitive abilities in this AD mouse model lacking CB1. Furthermore, the findings indicate that CB1 deficiency can worsen AD-related cognitive deficits and support a potential role of CB1 as a pharmacologic target.

Oxidative stress resistance in hippocampal cells is associated with altered membrane fluidity and enhanced nonamyloidogenic cleavage of endogenous amyloid precursor protein.

Reactive oxygen species (ROS) have important roles as signaling molecules in the regulation of a variety of biological processes. On the other hand, chronic oxidative stress exerted by ROS is widely considered a causative factor in aging. Therefore, cells need to be able to adapt to a chronic oxidative challenge and do so to a certain cell-type-specific extent. Recently, we have shown in oxidative-stress-resistant cell lines, HT22(H2O2) and HT22(Glu), derived from the neuronal cell line HT22 by chronic exposure to sublethal concentrations of H(2)O(2) and glutamate, that, in addition to the known antioxidant defense mechanisms, e.g., activation of antioxidant enzymes or up-regulation of heat-shock proteins, oxidative stress resistance depends on the composition of cellular membranes. Here, we extend our previous investigations and report increased membrane fluidity in HT22(H2O2) and HT22(Glu) cells compared to the parental HT22(WT) cells. The increased membrane fluidity correlates with a redistribution of cholesterol, sphingomyelin, and membrane-associated proteins involved in APP processing between detergent-resistant and detergent-soluble membrane subdomains. The altered membrane properties were associated with drastic changes in the metabolism of the Alzheimer disease-associated amyloid precursor protein (APP), particularly toward enhanced production of soluble APP alpha, which is a known neuroprotective factor. Thus our -data provide a link between chronic oxidative stress, alterations in membrane fluidity and composition of membrane subdomains, stress adaptation, and APP processing.

Adaptation of neuronal cells to chronic oxidative stress is associated with altered cholesterol and sphingolipid homeostasis and lysosomal function.

Chronic oxidative stress has been causally linked to several neurodegenerative disorders. As sensitivity for oxidative stress greatly differs between brain regions and neuronal cell types, specific cellular mechanisms of adaptation to chronic oxidative stress should exist. Our objective was to identify molecular mechanisms of adaptation of neuronal cells after applying chronic sublethal oxidative stress. We demonstrate that cells resistant to oxidative stress exhibit altered cholesterol and sphingomyelin metabolisms. Stress-resistant cells showed reduced levels of molecules involved in cholesterol trafficking and intracellular accumulation of cholesterol, cholesterol precursors, and metabolites. Moreover, stress-resistant cells exhibited reduced SMase activity. The altered lipid metabolism was associated with enhanced autophagy. Treatment of stress-resistant cells with neutral SMase reversed the stress-resistant phenotype, whereas it could be mimicked by treatment of neuronal cells with a specific inhibitor of neutral SMase. Analysis of hippocampal and cerebellar tissue of mouse brains revealed that the obtained cell culture data reflect the in vivo situation. Stress-resistant cells in vitro showed similar features as the less vulnerable cerebellum in mice, whereas stress-sensitive cells resembled the highly sensitive hippocampal area. These findings suggest an important role of the cell type-specific lipid profile for differential vulnerabilities of different brain areas toward chronic oxidative stress.

Increased connexin 43 expression as a potential mediator of the neuroprotective activity of the corticotropin-releasing hormone.

CRH is a major central stress mediator, but also a potent neuroprotective effector. The mechanisms by which CRH mediates its neuroprotective actions are largely unknown. Here, we describe that the gap junction molecule connexin43 (Cx43) mediates neuroprotective effects of CRH toward experimentally induced oxidative stress. An enhanced gap junction communication has been reported to contribute to neuroprotection after neurotoxic insults. We show that CRH treatment up-regulates Cx43 expression and gap junctional communication in a CRH receptor-dependent manner in IMR32 neuroblastoma cells, primary astrocytes, and organotypic hippocampal slice cultures. MAPKs and protein kinase A-cAMP response element binding protein -coupled pathways are involved in the signaling cascade from CRH to enhanced Cx43 function. Inhibition of CRH-promoted gap junction communication by the gap junction inhibitor carbenoxolone could prevent neuroprotective actions of CRH in cell and tissue culture models suggesting that gap junction molecules are involved in the neuroprotective effects of CRH. The extent of oxidative stress-induced protein carbonylation and cell death inversely correlated with Cx43 protein levels as shown by Cx43 small interfering RNA knockdown experiments. Coculture studies of primary neurons and astrocytes revealed that astrocytic Cx43 likely contributes to the neuroprotective effects of CRH. To our knowledge this is the first description of Cx43 as a potential mediator of the neuroprotective actions of CRH.

Effects of sulindac sulfide on the membrane architecture and the activity of gamma-secretase.

gamma-Secretase is a membrane-embedded multi-protein complex that catalyzes the final cut of the Alzheimer's disease-related amyloid precursor protein (APP) to amyloid-beta peptides of variable length (37-43 amino acids) via an unusual intramembrane cleavage. Recent findings propose that some commonly used non-steroidal anti-inflammatory drugs (NSAIDs) have the ability to modulate specifically gamma-secretase activity without inhibiting the enzyme as a whole. These drugs may shift the processing of APP from the longer amyloid-beta 42 peptide towards shorter, less fibrillogenic and less toxic amyloid-beta species. We hypothesize that gamma-secretase activity, as an enzyme that is strictly associated with cellular membranes, is sensitive to alterations of the hydrophobic membrane environment. Here, we show that the gamma-secretase modulator and amyloid-beta 42-lowering drug sulindac sulfide alters the physical state of the membrane and strongly decreases fluidity of cellular membranes. Furthermore, sulindac sulfide changed the protein composition of membrane microdomains, the so-called lipid rafts. Most significantly, APP C-terminal fragments (CTFs) were redistributed from rafts towards non-raft membrane domains. This could be demonstrated also in cell-free assays, where in addition presenilin-1, the catalytic subunit of the gamma-secretase complex, was shifted out of lipid rafts. Together, these findings suggest that sulindac sulfide directly alters the membrane architecture and shifts the gamma-secretase-mediated cleavage of APP towards a hydrophobic environment where the enzyme-substrate complex is in a conformation for processing preferentially shorter amyloid-beta peptides.

Cholesterol-like effects of selective cyclooxygenase inhibitors and fibrates on cellular membranes and amyloid-beta production.

Strong evidence suggests a mechanistic link between cholesterol metabolism and the formation of amyloid-beta peptides, the principal constituents of senile plaques found in the brains of patients with Alzheimer's disease. Here, we show that several fibrates and diaryl heterocycle cyclooxygenase inhibitors, among them the commonly used drugs fenofibrate and celecoxib, exhibit effects similar to those of cholesterol on cellular membranes and amyloid precursor protein (APP) processing. These drugs have the same effects on membrane rigidity as cholesterol, monitored here by an increase in fluorescence anisotropy. The effect of the drugs on cellular membranes was also reflected in the inhibitory action on the sarco(endo)plasmic reticulum Ca(2+)-ATPase, which is known to be inhibited by excess ordering of membrane lipids. The drug-induced decrease of membrane fluidity correlated with an increased association of APP and its beta-site cleaving enzyme BACE1 with detergent-resistant membranes (DRMs), which represent membrane clusters of substantial rigidity. DRMs are hypothesized to serve as platforms for the amyloidogenic processing of APP. According to this hypothesis, both cholesterol and the examined compounds stimulated the beta-secretase cleavage of APP, resulting in a massive increase of secreted amyloid-beta peptides. The membrane-ordering potential of the drugs was observed in a cell-free assay, suggesting that the amyloid-beta promoting effect was analog to cholesterol due to primary effect on membrane rigidity. Because fenofibrate and celecoxib are widely used in humans as hypolipidemic drugs for prevention of atherosclerosis and as anti-inflammatory drugs against arthritis, possible side effects should be considered upon long-term clinical application.

Functional disassociation of the central and peripheral fatty acid amide signaling systems.

Fatty acid amides (FAAs) constitute a large class of endogenous signaling lipids that modulate several physiological processes, including pain, feeding, blood pressure, sleep, and inflammation. Although FAAs have been proposed to evoke their behavioral effects through both central and peripheral mechanisms, these distinct signaling pathways have remained experimentally challenging to separate. Here, we report a transgenic mouse model in which the central and peripheral FAA systems have been functionally uncoupled. Mice were generated that express the principle FAA-degrading enzyme FAA hydrolase (FAAH) specifically in the nervous system (FAAH-NS mice) by crossing FAAH(-/-) mice with transgenic mice that express FAAH under the neural specific enolase promoter. FAAH-NS mice were found to possess wild-type levels of FAAs in the brain and spinal cord, but significantly elevated concentrations of these lipid transmitters in peripheral tissues. This anatomically restricted biochemical phenotype correlated with a reversion of the reduced pain sensitivity of FAAH(-/-) mice, consistent with the FAA anandamide producing this effect by acting on cannabinoid receptors in the nervous system. Interestingly, however, FAAH-NS mice still exhibited an antiinflammatory phenotype similar in magnitude to FAAH(-/-) mice, indicating that this activity, which was not blocked by cannabinoid receptor antagonists, was mediated by peripherally elevated FAAs. These data suggest that the central and peripheral FAA signaling systems regulate discrete behavioral processes and may be targeted for distinct therapeutic gain.

Increased seizure susceptibility and proconvulsant activity of anandamide in mice lacking fatty acid amide hydrolase.

A number of recent in vitro studies have described a role for endogenous cannabinoids ("endocannabinoids") as transsynaptic modulators of neuronal activity in the hippocampus and other brain regions. However, the impact that endocannabinoid signals may have on activity-dependent neural events in vivo remains mostly unknown and technically challenging to address because of the short half-life of these chemical messengers in the brain. Mice lacking the enzyme fatty acid amide hydrolase [FAAH (-/-) mice] are severely impaired in their ability to degrade the endocannabinoid anandamide and therefore represent a unique animal model in which to examine the function of this signaling lipid in vivo. Here, we show that the administration of anandamide dramatically augments the severity of chemically induced seizures in FAAH (-/-) mice but not in wild-type mice. Anandamide-enhanced seizures in FAAH (-/-) mice resulted in significant neuronal damage in the CA1 and CA3 regions of the hippocampus for the bicuculline and kainate models, respectively. Notably, in the absence of anandamide treatment, FAAH (-/-) mice exhibited enhanced seizure responses to high doses of kainate that correlated with greatly elevated endogenous levels of anandamide in the hippocampus of these animals. Collectively, these studies suggest that both exogenously administered and endogenously produced anandamide display FAAH-regulated proconvulsant activity and do not support a general neuroprotective role for this endocannabinoid in response to excitotoxic stimuli in vivo. More generally, these findings demonstrate that the disinhibitory actions of endocannabinoids observed in hippocampal slices in vitro may also occur in vivo.