Non-alcoholic fatty liver disease induces signs of Alzheimer's disease (AD) in wild-type mice and accelerates pathological signs of AD in an AD model.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease afflicting about one third of the world's population and 30 % of the US population. It is induced by consumption of high-lipid diets and is characterized by liver inflammation and subsequent liver pathology. Obesity and consumption of a high-fat diet are known to increase the risk of Alzheimer's disease (AD). Here, we investigated NAFLD-induced liver inflammation in the pathogenesis of AD. METHODS: WT and APP-Tg mice were fed with a standard diet (SD) or a high-fat diet (HFD) for 2, 5 months, or 1 year to induce NAFLD. Another set of APP-Tg mice were removed from HFD after 2 months and put back on SD for 3 months. RESULTS: During acute phase NAFLD, WT and APP-Tg mice developed significant liver inflammation and pathology that coincided with increased numbers of activated microglial cells in the brain, increased inflammatory cytokine profile, and increased expression of toll-like receptors. Chronic NAFLD induced advanced pathological signs of AD in both WT and APP-Tg mice, and also induced neuronal apoptosis. We observed decreased brain expression of low-density lipoprotein receptor-related protein-1 (LRP-1) which is involved in ß-amyloid clearance, in both WT and APP-Tg mice after ongoing administration of the HFD. LRP-1 expression correlated with advanced signs of AD over the course of chronic NAFLD. Removal of mice from HFD during acute NAFLD reversed liver pathology, decreased signs of activated microglial cells and neuro-inflammation, and decreased ß-amyloid plaque load. CONCLUSIONS: Our findings indicate that chronic inflammation induced outside the brain is sufficient to induce neurodegeneration in the absence of genetic predisposition.

Hydroxycarboxylic acid receptor 2 mediates dimethyl fumarate's protective effect in EAE.

Taken orally, the drug dimethyl fumarate (DMF) has been shown to improve functional outcomes for patients with MS; however, it is unclear how DMF mediates a protective effect. DMF and, more so, its active metabolite, monomethyl fumarate, are known agonists of the hydroxycarboxylic acid receptor 2 (HCA2), a G protein-coupled membrane receptor. Here, we evaluated the contribution of HCA2 in mediating the protective effect afforded by DMF in EAE, a mouse model of MS. DMF treatment reduced neurological deficit, immune cell infiltration, and demyelination of the spinal cords in wild-type mice, but not in Hca2⁻/⁻ mice, indicating that HCA2 is required for the therapeutic effect of DMF. In particular, DMF decreased the number of infiltrating neutrophils in a HCA2-dependent manner, likely by interfering with neutrophil adhesion to endothelial cells and chemotaxis. Together, our data indicate that HCA2 mediates the therapeutic effects of DMF in EAE. Furthermore, identification of HCA2 as a molecular target may help to optimize MS therapy.

Functional outcome of pannexin-deficient mice after cerebral ischemia.

Pannexin (Px, Panx) channels have been implicated in several physiological and pathological processes. We recently studied the potential contribution of pannexins in ischemic brain damage using Px1(-/-) Px2(-/-) mice and provided evidence that (1) the release of IL-1ß and hemichannel function in astrocytes are, in contrast to published data, not affected by the absence of Px1 and Px2, (2) channel function in neurons lacking Px1 and Px2 is impaired and (3) Px1(-/-) Px2(-/-) mice had a better functional outcome and smaller infarcts than wild-type mice when subjected to ischemic stroke. Here, we further investigate the neurological outcome of wild-type and pannexin double-knockout mice 48 h after permanent occlusion of the distal middle cerebral artery (MCAO). Pannexin double-knockout mice (Px1(-/-) Px2(-/-)) were less impaired in parameters such as exploration, anxiety, sensorimotor function and behavioral symmetry.

A2A adenosine receptor signaling in lymphocytes and the central nervous system regulates inflammation during experimental autoimmune encephalomyelitis.

Extracellular adenosine has an important role in regulating the severity of inflammation during an immune response. Although there are four adenosine receptor (AR) subtypes, the A2AAR is both highly expressed on lymphocytes and known as a prime mediator of adenosine's anti-inflammatory effects. To define the importance of A2AAR signaling during neuroinflammatory disease progression, we used the experimental autoimmune encephalomyelitis (EAE) animal model for multiple sclerosis. In EAE induction experiments, A2AAR antagonist treatment protected mice from disease development and its associated CNS lymphocyte infiltration. However, A2AAR(-/-) mice developed a more severe acute EAE phenotype characterized by more proinflammatory lymphocytes and activated microglia/macrophages. Interestingly, very high levels of A2AAR were expressed on the choroid plexus, a well-established CNS lymphocyte entry point. To determine the contribution of A2AAR signaling in lymphocytes and the CNS during EAE, we used bone marrow chimeric mice. Remarkably, A2AAR(-/-) donor hematopoietic cells potentiated severe EAE, whereas lack of A2AAR expression on nonhematopoietic cells protected against disease development. Although no defect in the suppressive ability of A2AAR(-/-) regulatory T cells was observed, A2AAR(-/-) lymphocytes were shown to proliferate more and produced more IFN-γ following stimulation. Despite this more proinflammatory phenotype, A2AAR antagonist treatment still protected against EAE when A2AAR(-/-) lymphocytes were adoptively transferred to T cell-deficient A2AAR(+/+) mice. These results indicate that A2AAR expression on nonimmune cells (likely in the CNS) is required for efficient EAE development, while A2AAR lymphocyte expression is essential for limiting the severity of the inflammatory response.

Pannexins in ischemia-induced neurodegeneration.

Pannexin 1 (Px1, Panx1) and pannexin 2 (Px2, Panx2) form large-pore nonselective channels in the plasma membrane of cells and were suggested to play a role in the pathophysiology of cerebral ischemia. To directly test a potential contribution of pannexins in ischemia-related mechanisms, we performed experiments in Px1(-/-), Px2(-/-), and Px1(-/-)Px2(-/-) knockout mice. IL-1ß release, channel function in astrocytes, and cortical spreading depolarization were not altered in Px1(-/-)Px2(-/-) mice, indicating that, in contrast to previous concepts, these processes occur normally in the absence of pannexin channels. However, ischemia-induced dye release from cortical neurons was lower, indicating that channel function in Px1(-/-)Px2(-/-) neurons was impaired. Furthermore, Px1(-/-)Px2(-/-) mice had a better functional outcome and smaller infarcts than wild-type mice when subjected to ischemic stroke. In conclusion, our data demonstrate that Px1 and Px2 underlie channel function in neurons and contribute to ischemic brain damage.

Adenosine receptor signaling modulates permeability of the blood-brain barrier.

The blood-brain barrier (BBB) is comprised of specialized endothelial cells that form the capillary microvasculature of the CNS and is essential for brain function. It also poses the greatest impediment in the treatment of many CNS diseases because it commonly blocks entry of therapeutic compounds. Here we report that adenosine receptor (AR) signaling modulates BBB permeability in vivo. A(1) and A(2A) AR activation facilitated the entry of intravenously administered macromolecules, including large dextrans and antibodies to ß-amyloid, into murine brains. Additionally, treatment with an FDA-approved selective A(2A) agonist, Lexiscan, also increased BBB permeability in murine models. These changes in BBB permeability are dose-dependent and temporally discrete. Transgenic mice lacking A(1) or A(2A) ARs showed diminished dextran entry into the brain after AR agonism. Following treatment with a broad-spectrum AR agonist, intravenously administered anti-ß-amyloid antibody was observed to enter the CNS and bind ß-amyloid plaques in a transgenic mouse model of Alzheimer's disease (AD). Selective AR activation resulted in cellular changes in vitro including decreased transendothelial electrical resistance, increased actinomyosin stress fiber formation, and alterations in tight junction molecules. These results suggest that AR signaling can be used to modulate BBB permeability in vivo to facilitate the entry of potentially therapeutic compounds into the CNS. AR signaling at brain endothelial cells represents a novel endogenous mechanism of modulating BBB permeability. We anticipate these results will aid in drug design, drug delivery and treatment options for neurological diseases such as AD, Parkinson's disease, multiple sclerosis and cancers of the CNS.

Influenza virus infection aggravates stroke outcome.

BACKGROUND AND PURPOSE: Stroke is triggered by several risk factors, including influenza and other respiratory tract infections. However, it is unknown how and in which way influenza infection affects stroke outcome. METHODS: We infected mice intranasally with human influenza A (H1N1) virus and occluded the middle cerebral artery to induce ischemic strokes. Infarct volume and intracerebral hemorrhage were determined by histology. To evaluate the integrity of the blood-brain barrier and inflammation, we measured various cytokines in vivo and in vitro and performed immunohistochemistry of leukocyte markers, collagen IV, immunoglobulins, and matrix metalloproteinase-9. RESULTS: Influenza virus infection increased infarct size. Whereas changes in cardiovascular parameters did not explain this effect, we found evidence for an inflammatory mechanism. In influenza virus infection, the respiratory tract released cytokines into the blood, such as RANTES that induced macrophage inflammatory protein-2 and other inflammatory mediators in the ischemic brain. In infected mice, there was an increased number of neutrophils expressing the matrix metalloproteinase-9 in the ischemic brain. This was accompanied by severe disruption of the blood-brain barrier and an increased rate of intracerebral hemorrhages after tissue plasminogen activator treatment. To investigate the role of cytokines, we blocked cytokine release by using GTS-21, a selective agonist of the α7 nicotinic acetylcholine receptor. GTS-21 ameliorated ischemic brain damage and improved survival. CONCLUSIONS: Influenza virus infection triggers a cytokine cascade that aggravates ischemic brain damage and increases the risk of intracerebral hemorrhage after tissue plasminogen activator treatment. Blockade of cytokine production by α7 nicotinic acetylcholine receptor agonists is a novel therapeutic option to treat stroke in a proinflammatory context.

Aggregate formation and toxicity by wild-type and R621C synphilin-1 in the nigrostriatal system of mice using adenoviral vectors.

Synphilin-1 was described as a protein interacting with alpha-synuclein and is commonly found in Lewy bodies, the pathological hallmark of Parkinson's disease (PD). Our group has previously described and characterized in vitro a mutation in the synphilin-1 gene (R621C) in PD patients. Providing the first characterization of synphilin-1 expression in an animal model, we here used adenoviral gene transfer to study the effects of wild-type (WT) and R621C synphilin-1 in dopaminergic neurons in mouse brain. As synphilin-1 is commonly used to trigger aggregation of alpha-synuclein in cell culture, we investigated not only non-transgenic C57Bl/6 mice but also A30P-alpha-synuclein transgenic animals. Both WT synphilin-1 and R621C synphilin-1 led to the formation of Thioflavine-S positive inclusions in C57Bl/6 mice and degeneration of dopaminergic neurons in the substantia nigra. R621C synphilin-1 induced more aggregate formation than WT synphilin-1 in A30P-alpha-synuclein transgenic mice, consistent with the role of the R621C mutation as a susceptibility factor for PD. Synphilin-1 expression may be used to improve current mouse models of PD, as it induced both the formation of aggregates and degeneration of dopaminergic neurons, two core characteristics of PD that have not been well reproduced with expression of alpha-synuclein.

Accumulation and clearance of alpha-synuclein aggregates demonstrated by time-lapse imaging.

Aggregates of alpha-synuclein are the pathological hallmark of sporadic Parkinson's disease (PD), and mutations in the alpha-synuclein gene underlie familial forms of the disease. To characterize the formation of alpha-synuclein aggregates in living cells, we developed a new strategy to visualize alpha-synuclein by fluorescence microscopy: alpha-synuclein was tagged with a six amino acid PDZ binding motif and co-expressed with the corresponding PDZ domain fused to enhanced green fluorescent protein (EGFP). In contrast to the traditional approach of alpha-synuclein-EGFP fusion proteins, this technique provided several-fold higher sensitivity; this allowed us to compare alpha-synuclein variants and perform time-lapse imaging. A C-terminally truncated alpha-synuclein variant showed the highest prevalence of aggregates and toxicity, consistent with stabilization of the alpha-synuclein monomer by its C-terminus. Time-lapse imaging illustrated how cells form and accumulate aggregates of alpha-synuclein. A substantial number of cells also reduced their aggregate load, primarily through formation of an aggresome, which could itself be cleared from the cell. The molecular chaperone Hsp70 not only prevented the formation of aggregates, but also increased their reduction and clearance, underlining the therapeutic potential of similar strategies. In contrast to earlier assumptions build-up, reduction and clearance of alpha-synuclein aggregation thus appear a highly dynamic process.