Alzheimer's disease (AD) is characterized by enhanced ␤-amyloid peptide (␤A) deposition along with glial activation in senile plaques, selective neuronal loss, and cognitive deficits. Cannabinoids are neuroprotective agents against excitotoxicity in vitro and acute brain damage in vivo. This background prompted us to study the localization, expression, and function of cannabinoid receptors in AD and the possible protective role of cannabinoids after ␤A treatment, both in vivo and in vitro. Here, we show that senile plaques in AD patients express cannabinoid receptors CB 1 and CB 2 , together with markers of microglial activation, and that CB 1 -positive neurons, present in high numbers in control cases, are greatly reduced in areas of microglial activation. In pharmacological experiments, we found that G-protein coupling and CB 1 receptor protein expression are markedly decreased in AD brains. Additionally, in AD brains, protein nitration is increased, and, more specifically, CB 1 and CB 2 proteins show enhanced nitration. Intracerebroventricular administration of the synthetic cannabinoid WIN55,212-2 to rats prevent ␤A-induced microglial activation, cognitive impairment, and loss of neuronal markers. Cannabinoids (HU-210, WIN55,212-2, and JWH-133) block ␤A-induced activation of cultured microglial cells, as judged by mitochondrial activity, cell morphology, and tumor necrosis factor-␣ release; these effects are independent of the antioxidant action of cannabinoid compounds and are also exerted by a CB 2 -selective agonist. Moreover, cannabinoids abrogate microglia-mediated neurotoxicity after ␤A addition to rat cortical cocultures. Our results indicate that cannabinoid receptors are important in the pathology of AD and that cannabinoids succeed in preventing the neurodegenerative process occurring in the disease.

Because chronic neuroinflammation is a hallmark of neurodegenerative diseases and compromises neuron viability, it is imperative to discover pharmacologic targets to modulate the activation of immune brain cells, the microglia. In this study, we identify the transcription factor Nrf2, guardian of redox homeostasis, as such target in a model of LPS-induced inflammation in mouse hippocampus. Nrf2 knockout mice were hypersensitive to the neuroinflammation induced by LPS, as determined by an increase in F4/80 mRNA and protein, indicative of an increase in microglial cells, and in the inflammation markers inducible NO synthase, IL-6, and TNF-α, compared with the hippocampi of wild-type littermates. The aliphatic isothiocyanate sulforaphane elicited an Nrf2-mediated antioxidant response in the BV2 microglial cell line, determined by flow cytometry of cells incubated with the redox sensitive probe dihydrodichlorofluorescein diacetate, and by the Nrf2-dependent induction of the phase II antioxidant enzyme heme oxygenase-1. Animals treated with sulforaphane displayed a 2–3-fold increase in heme oxygenase-1, a reduced abundance of microglial cells in the hippocampus and an attenuated production of inflammation markers (inducible NO synthase, IL-6, and TNF-α) in response to LPS. Considering that release of reactive oxygen species is a property of activated microglia, we propose a model in which late induction of Nrf2 intervenes in the down-regulation of microglia. This study opens the possibility of targeting Nrf2 in brain as a means to modulate neuroinflammation.

Autophagy is a highly coordinated process that is controlled at several levels including transcriptional regulation. Here, we identify the transcription factor NFE2L2/NRF2 (nuclear factor, erythroid 2 like 2) as a regulator of autophagy gene expression and its relevance in a mouse model of Alzheimer disease (AD) that reproduces impaired APP (amyloid β precursor protein) and human (Hs)MAPT/TAU processing, clearance and aggregation. We screened the chromatin immunoprecipitation database ENCODE for 2 proteins, MAFK and BACH1, that bind the NFE2L2-regulated enhancer antioxidant response element (ARE). Using a script generated from the JASPAR's consensus ARE sequence, we identified 27 putative AREs in 16 autophagy-related genes. Twelve of these sequences were validated as NFE2L2 regulated AREs in 9 autophagy genes by additional ChIP assays and quantitative RT-PCR on human and mouse cells after NFE2L2 activation with sulforaphane. Mouse embryo fibroblasts of nfe2l2-knockout mice exhibited reduced expression of autophagy genes, which was rescued by an NFE2L2 expressing lentivirus, and impaired autophagy flux when exposed to hydrogen peroxide. NFE2L2-deficient mice co-expressing HsAPPV717I and HsMAPTP301L, exhibited more intracellular aggregates of these proteins and reduced neuronal levels of SQSTM1/p62, CALCOCO2/NDP52, ULK1, ATG5 and GABARAPL1. Also, colocalization of HsAPPV717I and HsMAPTP301L with the NFE2L2-regulated autophagy marker SQSTM1/p62 was reduced in the absence of NFE2L2. In AD patients, neurons expressing high levels of APP or MAPT also expressed SQSTM1/p62 and nuclear NFE2L2, suggesting their attempt to degrade intraneuronal aggregates through autophagy. This study shows that NFE2L2 modulates autophagy gene expression and suggests a new strategy to combat proteinopathies.