Intravenously Injected Mesenchymal Stem Cells Penetrate the Brain and Treat Inflammation-Induced Brain Damage and Memory Impairment in Mice

Neuroinflammation is regarded as one of the pathogenic factors of Alzheimer disease (AD). Previously, we showed that mice regularly injected with bacterial lipopolysaccharide (LPS) possessed the AD-like symptoms like episodic memory decline, elevated amounts of amyloid beta (Aβ) peptide (1–42), and decreased levels of nicotinic acetylcholine receptors (nAChRs) in the brain. The use of mesenchymal stem cells (MSCs), which can differentiate into multiple cell types, including neurons, is an attractive idea of regenerative medicine, in particular, for neurodegenerative disorders like AD. In the present study, we aimed to investigate whether pathogenic effect of LPS on the brain and behavior of mice can be prevented or treated by injection of MSCs or MSC-produced soluble factors. Fluorescently-labeled MSCs, injected intravenously, were found in the brain blood vessels of LPS-treated mice. Mice co-injected with LPS and MSCs did not demonstrate episodic memory impairment, Aβ (1–42) accumulation, and nAChR decrease in the brain and brain mitochondria. Their mitochondria released less cytochrome c under the effect of Ca2+ compared to mitochondria of LPS-only-treated mice. Moreover, MSCs could reverse the pathogenic symptoms developed 3 weeks after LPS injection. Cultured MSCs produced IL-6 in response to LPS and MSCs effect in vivo was accompanied by additional stimulation of both micro- and macroglia. Xenogeneic (human) MSCs were almost as efficient as allogeneic (mouse) ones and regular injections of human MSC-conditioned medium also produced positive effect. These data allow suggesting MSCs as a potential therapeutic tool to cure neuroinflammation-related cognitive pathology

Mitochondria Express α7 Nicotinic Acetylcholine Receptors to Regulate Ca2+ Accumulation and Cytochrome c Release: Study on Isolated Mitochondria

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that mediate synaptic transmission in the muscle and autonomic ganglia and regulate transmitter release in the brain. The nAChRs composed of α7 subunits are also expressed in non-excitable cells to regulate cell survival and proliferation. Up to now, functional α7 nAChRs were found exclusively on the cell plasma membrane. Here we show that they are expressed in mitochondria and regulate early pro-apoptotic events like cytochrome c release. The binding of α7-specific antibody with mouse liver mitochondria was revealed by electron microscopy. Outer membranes of mitochondria from the wild-type and β2−/− but not α7−/− mice bound α7 nAChR-specific antibody and toxins: FITC-labeled α-cobratoxin or Alexa 555-labeled α-bungarotoxin. α7 nAChR agonists (1 µM acetylcholine, 10 µM choline or 30 nM PNU-282987) impaired intramitochondrial Ca2+ accumulation and significantly decreased cytochrome c release stimulated with either 90 µM CaCl2 or 0.5 mM H2O2. α7-specific antagonist methyllicaconitine (50 nM) did not affect Ca2+ accumulation in mitochondria but attenuated the effects of agonists on cytochrome c release. Inhibitor of voltage-dependent anion channel (VDAC) 4,4′-diisothio-cyano-2,2′-stilbene disulfonic acid (0.5 µM) decreased cytochrome c release stimulated with apoptogens similarly to α7 nAChR agonists, and VDAC was co-captured with the α7 nAChR from mitochondria outer membrane preparation in both direct and reverse sandwich ELISA. It is concluded that α7 nAChRs are expressed in mitochondria outer membrane to regulate the VDAC-mediated Ca2+ transport and mitochondrial permeability transition

Functional role of the nicotinic arm of the acetylcholine regulatory axis in human B-cell lines.

We studied the involvement of nicotinic acetylcholine receptors (nAChRs) in the inflammation-related activity of human B-cell lines. Activation of nAChRs in Daudi cells with epibatidine abolished the pansorbin-dependent upregulation of the pro-inflammatory marker Cox-2 both at the mRNA and protein levels, indicating that the nicotinergic signaling suppresses B-cell activation. While the anti-inflammatory action on B-cells was mediated predominantly through α7 nAChR, as could be judged from abolishing epibatidine effects with methyllycaconitine, both α7 and non-α7 nAChRs, such as α2-containing receptors, were involved in regulation of B-cell apoptosis. The net effect was antiapoptotic. To determine the role of nAChRs in regulating B-cell activation/plasmacytic differentiation, we measured changes in the CD38, CD138 and Bcl-6 gene expression. Epibatidine significantly (P < 0.05) upregulated CD38 at the transcriptional level and CD138 and Bcl-6 - at the translational levels. AR-R17779 significantly (P < 0.05) increased the protein levels of CD38 and CD138. In both cases, the effect of epibatidine was abolished with Mec, and that of AR-R17779 - by MLA, demonstrating a functional role of nAChRs in regulating Daudi cell differentiation. The obtained results revealed distinct contributions of α7 and non-α7 nAChRs to regulation of B-cell activation/differentiation, and suggested that signaling through the nicotinic arm of acetylcholine regulatory axis is important for B-cell involvement in inflammation

Positive Allosteric Modulators of α7* or β2* Nicotinic Acetylcholine Receptors Trigger Different Kinase Pathways in Mitochondria.

International audienceMitochondrial nicotinic acetylcholine receptors (nAChRs) regulate the early stage of mitochondria-driven apoptosis, including cytochrome c release. Mitochondrial nAChR signaling is mainly mediated by intra-mitochondrial kinases, in an ion-independent manner. To determine the relationship between specific nAChR subtypes and mitochondrial kinases, the effects of a set of nAChR subtype-selective positive allosteric modulators (PAMs) on cytochrome c release from mouse liver mitochondria stimulated by 0.9 μM Ca2+, 0.5 mM H2O2 or 1.0 μM wortmanin is studied. The results indicate that Ca2+-stimulated cytochrome c release from wild-type, but not α7-/-, mice mitochondria is attenuated by the potent agonist PNU-282987 or type II PAMs (PNU-120596, 4BP-TQS, and PAM-2-4), but not by NS-1738, a type I PAM. In contrast, wortmannin-stimulated cytochrome c release from wild-type and, to a lesser extent, α7-/- mice mitochondria is efficiently attenuated by the β2-selective PAM desformylfrustrabromine. In conclusion, the ligand-evoked α7* nAChR conformational changes required to induce intra-mitochondrial signaling can be triggered through orthosteric (agonists) and transmembrane (type II PAMs) sites, but not by the interaction with type I PAMs. The α7 and β2 nAChR subunits are responsible for the engagement of distinct kinase pathways, supporting the concept that multiple heteromeric nAChR subtypes ensure mitochondria resistance to various exogenous and endogenous apoptogenic agents

Different Effects of Nicotine and N-Stearoyl-ethanolamine on Episodic Memory and Brain Mitochondria of α7 Nicotinic Acetylcholine Receptor Knockout Mice

Nicotinic acetylcholine receptors of α7 subtype (α7 nAChRs) are involved in regulating neuroinflammation and cognitive functions. Correspondingly, α7-/- mice demonstrate pro-inflammatory phenotype and impaired episodic memory. In addition, nAChRs expressed in mitochondria regulate the release of pro-apoptotic factors like cytochrome c. Here we studied whether the cognitive deficiency of α7-/- mice can be cured by oral consumption of either nicotine or N-stearoylethanolamine (NSE), a lipid possessing anti-inflammatory, cannabimimetic and membrane-stabilizing activity. Mice were examined in Novel Object Recognition behavioral test, their blood, brains and brain mitochondria were tested for the levels of interleukin-6, various nAChR subtypes and cytochrome c released by ELISA. The data presented demonstrate that both substances stimulated the raise of interleukin-6 in the blood and improved episodic memory of α7-/- mice. However, NSE improved, while nicotine worsened the brain mitochondria sustainability to apoptogenic stimuli, as shown by either decreased or increased amounts of cytochrome c released. Both nicotine and NSE up-regulated α4β2 nAChRs in the brain; NSE up-regulated, while nicotine down-regulated α9-containing nAChRs in the brain mitochondria. It is concluded that the level of alternative nAChR subtypes in the brain is critically important for memory and mitochondria sustainability in the absence of α7 nAChRs

Mitochondrial Nicotinic Acetylcholine Receptors Support Liver Cells Viability After Partial Hepatectomy

International audienceNicotinic acetylcholine receptors (nAChRs) expressed on the cell plasma membrane are ligand-gated ion channels mediating fast synaptic transmission, regulating neurotransmitter and cytokine release and supporting the viability of many cell types. The nAChRs expressed in mitochondria regulate the release of pro-apoptotic factors, like cytochrome c, in ion channel-independent manner. Here we show that α3β2, α7β2, and α9α10 nAChR subtypes are up-regulated in rat liver mitochondria 3-6 h after partial hepatectomy resulting in increased sustainability of mitochondria to apoptogenic effects of Ca2+ and H2O2. In contrast, laparotomy resulted in down-regulation of all nAChR subunits, except α9, and decreased mitochondria sustainability to apoptogenic effects of Ca2+ and H2O2. Experiments performed in liver mitochondria from α3+/-, α7-/-, β4-/-, α7β2-/-, or wild-type C57Bl/6J mice demonstrated that the decrease of α3 or absence of α7 or α7/β2 subunits in mitochondria is compensated with β4 and α9 subunits, which could be found in α3β4, α4β4, α9β4, and α9α10 combinations. Mitochondria from knockout mice maintained their sustainability to Ca2+ but were differently regulated by nAChR subtype-specific ligands: PNU-282987, methyllycaconitine, dihydro-β-erythroidine, α-conotoxin MII, and α-conotoxin PeIA. It is concluded that mitochondrial nAChRs play an important role in supporting the viability of hepatic cells and, therefore, may be a pharmacological target for pro-survival therapy. The concerted action of multiple nAChR subtypes controlling either CaKMII- or Src-dependent signaling pathways in mitochondria ensures a reliable protection against apoptogenic factors of different nature

α7 Nicotinic acetylcholine receptor-specific antibody induces inflammation and amyloid β42 accumulation in the mouse brain to impair memory.

Nicotinic acetylcholine receptors (nAChRs) expressed in the brain are involved in regulating cognitive functions, as well as inflammatory reactions. Their density is decreased upon Alzheimer disease accompanied by accumulation of β-amyloid (Aβ42), memory deficit and neuroinflammation. Previously we found that α7 nAChR-specific antibody induced pro-inflammatory interleukin-6 production in U373 glioblastoma cells and that such antibodies were present in the blood of humans. We raised a hypothesis that α7 nAChR-specific antibody can cause neuroinflammation when penetrating the brain. To test this, C57Bl/6 mice were either immunized with extracellular domain of α7 nAChR subunit α7(1-208) or injected with bacterial lipopolysaccharide (LPS) for 5 months. We studied their behavior and the presence of α3, α4, α7, β2 and β4 nAChR subunits, Aβ40 and Aβ42 and activated astrocytes in the brain by sandwich ELISA and confocal microscopy. It was found that either LPS injections or immunizations with α7(1-208) resulted in region-specific decrease of α7 and α4β2 and increase of α3β4 nAChRs, accumulation of Aβ42 and activated astrocytes in the brain of mice and worsening of their episodic memory. Intravenously transferred α7 nAChR-specific-antibodies penetrated the brain parenchyma of mice pre-injected with LPS. Our data demonstrate that (1) neuroinflammation is sufficient to provoke the decrease of α7 and α4β2 nAChRs, Aβ42 accumulation and memory impairment in mice and (2) α7(1-208) nAChR-specific antibodies can cause inflammation within the brain resulting in the symptoms typical for Alzheimer disease

Nicotinic Acetylcholine Receptor α9 and α10 Subunits Are Expressed in the Brain of Mice

The α9 and α10 nicotinic acetylcholine receptor (nAChR) subunits are likely to be the evolutionary precursors to the entire cys-loop superfamily of ligand-gated ion channels, which includes acetylcholine, GABA, glycine and serotonin ionotropic receptors. nAChRs containing α9 and α10 subunits are found in the inner ear, dorsal root ganglia and many non-excitable tissues, but their expression in the central nervous system has not been definitely demonstrated. Here we show the presence of both α9 and α10 nAChR subunits in the mouse brain by RT-PCR and immunochemical approaches with a range of nAChR subunit-selective antibodies, which selectivity was demonstrated in the brain preparations of α7−/−, α9−/− and α10−/− mice. The α9 and α10 RNA transcripts were found in medulla oblongata (MO), cerebellum, midbrain (MB), thalamus and putamen (TP), somatosensory cortex (SC), frontal cortex (FC) and hippocampus. High α9-selective signal in ELISA was observed in the FC, SC, MO, TP and hippocampus and α10-selective signal was the highest in MO and FC. The α9 and α10 proteins were found in the brain mitochondria, while their presence on the plasma membrane has not been definitely confirmed The α7-, α9- and α10-selective antibodies stained mainly neurons and hypertrophied astrocytes, but not microglia. The α9- and α10-positive cells formed ordered structures or zones in cerebellum and superior olive (SO) and were randomly distributed among α7-positive cells in the FC; they were found in CA1, CA3 and CA4, but not in CA2 region of the hippocampus. The α9 and α10 subunits were up-regulated in α7−/− mice and both α7 and α9 subunits were down-regulated in α10−/− mice. We conclude that α9 and α10 nAChR subunits are expressed in distinct neurons of the mouse brain and in the brain mitochondria and are compensatory up-regulated in the absence of α7 subunits