Amorfrutin B Compromises Hypoxia/Ischemia-induced Activation of Human Microglia in a PPARγ-dependent Manner: Effects on Inflammation, Proliferation Potential, and Mitochondrial Status.

Amorfrutin B is a selective PPARγ modulator that we demonstrated to be a promising neuroprotective compound in cellular models of stroke and perinatal asphyxia. Although neuronal mechanisms of amorfrutin B-evoked neuroprotection have been identified, none of them reflects the actions of the compound on microglia, which play a pivotal role in brain response to hypoxia/ischemia. Here, we provide evidence for amorfrutin B-induced effects on human microglia subjected to hypoxia/ischemia; the compound counteracts inflammation, and influences mitochondrial status and proliferation potential in a PPARγ-dependent manner. Post-treatment with amorfrutin B decreased the IBA1 fluorescence intensity, reduced caspase-1 activity, and downregulated IL1B/IL-1ß and TNFA but not IL10/IL-10 expression, which was upregulated. Amorfrutin B also stimulated PPARγ signaling, as evidenced by increased mRNA and/or protein levels of PPARγ and PGC1α. In addition, amorfrutin B reversed the hypoxia/ischemia-evoked effects on mitochondria-related parameters, such as mitochondrial membrane potential, BCL2/BCL2 expression and metabolic activity, which were correlated with diminished proliferation potential of microglia. Interestingly, the inhibitory effect of amorfrutin B on the proliferation potential and mitochondrial function of microglia is opposite to the stimulatory effect of amorfrutin B on mouse neuronal survival, as evidenced by increased neuronal viability and reduced neurodegeneration. In summary, this study showed for the first time that amorfrutin B compromises hypoxia/ischemia-induced activation of human microglia in a PPARγ-dependent manner, which involves inhibiting inflammation, normalizing mitochondrial status, and controlling proliferation potential. These data extend the protective potential of amorfrutin B in the pharmacotherapy of hypoxic/ischemic brain injury, targeting not only neurons but also activated microglia.

Posttreatment with PaPE-1 Protects from Aß-Induced Neurodegeneration Through Inhibiting the Expression of Alzheimer's Disease-Related Genes and Apoptosis Process That Involves Enhanced DNA Methylation of Specific Genes.

Targeting the non-nuclear estrogen receptor (ER) signaling has been postulated as novel therapeutic strategy for central nervous system pathologies. Recently, we showed that newly designed PaPE-1 (Pathway Preferential Estrogen-1), which selectively activates ER non-nuclear signaling pathways, elicited neuroprotection in a cellular model of Alzheimer's disease (AD) when it was applied at the same time as amyloid-ß (Aß). Since delayed treatment reflects clinical settings better than cotreatment does, current basic study proposes a novel therapeutic approach for AD that relies on a posttreatment with PaPE-1. In this study, mouse neuronal cell cultures treated with preaggregated Aß1-42 (10 µM) showed the presence of extracellular Aß1-42, confirming the adequacy of the AD model used. We are the first to demonstrate that a 24-h delayed posttreatment with PaPE-1 decreased the degree of Aß-induced neurodegeneration, restored neurite outgrowth, and inhibited the expression of AD-related genes, i.e., Rbfox, Apoe, Bace2, App, and Ngrn, except for Chat, which was stimulated. In addition, PaPE-1 elicited anti-apoptotic effects by inhibiting Aß-induced caspase activities as well as attenuating apoptotic chromatin condensation, and in these ways, PaPE-1 prevented neuronal cell death. Posttreatment with PaPE-1 also downregulated the Aß-affected mRNA expression of apoptosis-specific factors, such as Bax, Gsk3b, Fas, and Fasl, except for Bcl2, which was upregulated by PaPE-1. In parallel, PaPE-1 decreased the protein levels of BAX, FAS, and FASL, which were elevated in response to Aß. PaPE-1 elicited a decrease in the BAX/BCL2 ratio that corresponds to increased methylation of the Bax gene. However, the PaPE-1-evoked Bcl2 gene hypermethylation suggests other PaPE-1-dependent mechanisms to control Aß-induced apoptosis.

Posttreatment with Ospemifene Attenuates Hypoxia- and Ischemia-Induced Apoptosis in Primary Neuronal Cells via Selective Modulation of Estrogen Receptors.

Stroke and perinatal asphyxia have detrimental effects on neuronal cells, causing millions of deaths worldwide each year. Since currently available therapies are insufficient, there is an urgent need for novel neuroprotective strategies to address the effects of cerebrovascular accidents. One such recent approach is based on the neuroprotective properties of estrogen receptors (ERs). However, activation of ERs by estrogens may contribute to the development of endometriosis or hormone-dependent cancers. Therefore, in this study, we utilized ospemifene, a novel selective estrogen receptor modulator (SERM) already used in dyspareunia treatment. Here, we demonstrated that posttreatment with ospemifene in primary neocortical cell cultures subjected to 18 h of hypoxia and/or ischemia followed by 6 h of reoxygenation has robust neuroprotective potential. Ospemifene partially reverses hypoxia- and ischemia-induced changes in LDH release, the degree of neurodegeneration, and metabolic activity. The mechanism of the neuroprotective actions of ospemifene involves the inhibition of apoptosis since the compound decreases caspase-3 overactivity during hypoxia and enhances mitochondrial membrane potential during ischemia. Moreover, in both models, ospemifene decreased the levels of the proapoptotic proteins BAX, FAS, FASL, and GSK3ß while increasing the level of the antiapoptotic protein BCL2. Silencing of specific ERs showed that the neuroprotective actions of ospemifene are mediated mainly via ESR1 (during hypoxia and ischemia) and GPER1 (during hypoxia), which is supported by ospemifene-evoked increases in ESR1 protein levels in hypoxic and ischemic neurons. The results identify ospemifene as a promising neuroprotectant, which in the future may be used to treat injuries due to brain hypoxia/ischemia.

Experimental Models to Study the Functions of the Blood-Brain Barrier.

The purpose of this paper was to discuss the achievements of in vitro modeling in terms of the blood-brain barrier [BBB] and to create a clear overview of this research area, which is useful in research planning. The text was divided into three main parts. The first part describes the BBB as a functional structure, its constitution, cellular and noncellular components, mechanisms of functioning and importance for the central nervous system, in terms of both protection and nourishment. The second part is an overview of parameters important in terms of establishing and maintaining a barrier phenotype that allows for formulating criteria of evaluation of the BBB in vitro models. The third and last part discusses certain techniques for developing the BBB in vitro models. It describes subsequent research approaches and models, as they underwent change alongside technological advancement. On the one hand, we discuss possibilities and limitations of different research approaches: primary cultures vs. cell lines and monocultures vs. multicultures. On the other hand, we review advantages and disadvantages of specific models, such as models-on-a-chip, 3D models or microfluidic models. We not only attempt to state the usefulness of specific models in different kinds of research on the BBB but also emphasize the significance of this area of research for advancement of neuroscience and the pharmaceutical industry.