GTS-21 Enhances Regulatory T Cell Development from T Cell Receptor-Activated Human CD4+ T Cells Exhibiting Varied Levels of CHRNA7 and CHRFAM7A Expression.

Immune cells such as T cells and macrophages express α7 nicotinic acetylcholine receptors (α7 nAChRs), which contribute to the regulation of immune and inflammatory responses. Earlier findings suggest α7 nAChR activation promotes the development of regulatory T cells (Tregs) in mice. Using human CD4+ T cells, we investigated the mRNA expression of the α7 subunit and the human-specific dupα7 nAChR subunit, which functions as a dominant-negative regulator of ion channel function, under resting conditions and T cell receptor (TCR)-activation. We then explored the effects of the selective α7 nAChR agonist GTS-21 on proliferation of TCR-activated T cells and Treg development. Varied levels of mRNA for both the α7 and dupα7 nAChR subunits were detected in resting human CD4+ T cells. mRNA expression of the α7 nAChR subunit was profoundly suppressed on days 4 and 7 of TCR-activation as compared to day 1, whereas mRNA expression of the dupα7 nAChR subunit remained nearly constant. GTS-21 did not alter CD4+ T cell proliferation but significantly promoted Treg development. These results suggest the potential ex vivo utility of GTS-21 for preparing Tregs for adoptive immunotherapy, even with high expression of the dupα7 subunit.

Suppression of Neuroinflammation by Coffee Component Pyrocatechol via Inhibition of NF-κB in Microglia.

According to numerous studies, it has been epidemiologically suggested that habitual coffee intake seems to prevent the onset of neurodegenerative diseases. In this study, we hypothesized that coffee consumption suppresses neuroinflammation, which is closely related to the development of neurodegenerative diseases. Using microglial BV-2 cells, we first found that the inflammatory responses induced by lipopolysaccharide (LPS) stimulation was diminished by both coffee and decaffeinated coffee through the inhibition of an inflammation-related transcription factor, nuclear factor-κB (NF-κB). Pyrocatechol, a component of roasted coffee produced by the thermal decomposition of chlorogenic acid, also exhibited anti-inflammatory activity by inhibiting the LPS-induced activation of NF-κB. Finally, in an inflammation model using mice injected with LPS into the cerebrum, we observed that intake of pyrocatechol as well as coffee decoctions drastically suppressed the accumulation of microglia and the expression of interleukin-6 (IL-6), tumor necrosis factor α (TNFα), CCL2, and CXCL1 in the inflammatory brain. These observations strongly encourage us to hypothesize that the anti-inflammatory activity of pyrocatechol as well as coffee decoction would be useful for the suppression of neurodegeneration and the prevention of the onsets of Alzheimer's (AD) and Perkinson's diseases (PD).

Regulation of Immune Functions by Non-Neuronal Acetylcholine (ACh) via Muscarinic and Nicotinic ACh Receptors.

Acetylcholine (ACh) is the classical neurotransmitter in the cholinergic nervous system. However, ACh is now known to regulate various immune cell functions. In fact, T cells, B cells, and macrophages all express components of the cholinergic system, including ACh, muscarinic, and nicotinic ACh receptors (mAChRs and nAChRs), choline acetyltransferase, acetylcholinesterase, and choline transporters. In this review, we will discuss the actions of ACh in the immune system. We will first briefly describe the mechanisms by which ACh is stored in and released from immune cells. We will then address Ca2+ signaling pathways activated via mAChRs and nAChRs on T cells and B cells, highlighting the importance of ACh for the function of T cells, B cells, and macrophages, as well as its impact on innate and acquired (cellular and humoral) immunity. Lastly, we will discuss the effects of two peptide ligands, secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor-related peptide-1 (SLURP-1) and hippocampal cholinergic neurostimulating peptide (HCNP), on cholinergic activity in T cells. Overall, we stress the fact that ACh does not function only as a neurotransmitter; it impacts immunity by exerting diverse effects on immune cells via mAChRs and nAChRs.

Physiological functions of the cholinergic system in immune cells.

T and B cells, macrophages and dendritic cells (DCs) all express most of the components necessary for a functional cholinergic system. This includes choline acetyltransferase (ChAT), muscarinic and nicotinic acetylcholine (ACh) receptors (mAChRs and nAChRs, respectively) and acetylcholinesterase (AChE). Immunological activation of T cells up-regulates cholinergic activity, including ChAT and AChE expression. Moreover, toll-like receptor agonists induce ChAT expression in DCs and macrophages, suggesting cholinergic involvement in the regulation of immune function. Immune cells express all five M1-M5 mAChR subtypes and several nAChR subtypes, including α7. Modulation of antigen-specific antibody and pro-inflammatory cytokine production in M1/M5 mAChR gene-knockout (KO) and α7 nAChR-KO mice further support the idea of a non-neuronal cholinergic system contributing to the regulation of immune function. Evidence also suggests that α7 nAChRs are involved in suppressing DC and macrophage activity, leading to suppression of T cell differentiation into effector T cells. These findings suggest the possibility that immune function could be modulated by manipulating immune cell cholinergic activity using specific agonists and antagonists. Therefore, a fuller understanding of the immune cell cholinergic system should be useful for the development of drugs and therapeutic strategies for the treatment of inflammation-related diseases and cancers.

T cells down-regulate macrophage TNF production by IRAK1-mediated IL-10 expression and control innate hyperinflammation.

Endotoxemia is caused by excessive inflammation, but the immune system has various mechanisms to avoid collateral organ damage in endotoxemia. A handful of reports have shown that innate immune responses are suppressed by the adaptive immune system. However, the molecular mechanism by which adaptive immune cells suppress innate inflammatory responses is not clear. Here, we report that T cells are shown to interact with macrophages at the early stage of enodotoxemia and to prolong survival of mice through controlling TNF and IL-10 levels by macrophage CD40 stimulation. The cross-talk between CD40 and toll-like receptor (TLR4) signaling first mediates IL-1 receptor-associated kinase 1 (IRAK1) nuclear translocation and its binding to the IL-10 gene promoter in macrophages, without interfering with the NFκB pathway. IL-10 is then detected by macrophages in an autocrine fashion to destabilize Tnfa mRNA. To induce IRAK1-mediated IL-10 expression, signals from both CD40 and TLR4 are essential. CD40 signaling induces IRAK1 sumoylation in the presence of TNF receptor-associated factor 2 (TRAF2) and intracellular isoform of osteopontin (iOPN) whereas TLR4 signaling provides IFN regulatory factor 5 (IRF5) as a chaperone for sumoylated IRAK1 nuclear translocation. Interaction of T cells with macrophages was observed in the spleen in vivo after endotoxemia induction with LPS injection. Our study demonstrates a mechanistic basis for the immunosuppressive role of macrophage CD40 in LPS endotoxemia.

Reappraisal of VAChT-Cre: Preference in slow motor neurons innervating type I or IIa muscle fibers.

VAChT-Cre.Fast and VAChT-Cre.Slow mice selectively express Cre recombinase in approximately one half of postnatal somatic motor neurons. The mouse lines have been used in various studies with selective genetic modifications in adult motor neurons. In the present study, we crossed VAChT-Cre lines with a reporter line, CAG-Syp/tdTomato, in which synaptophysin-tdTomato fusion proteins are efficiently sorted to axon terminals, making it possible to label both cell bodies and axon terminals of motor neurons. In the mice, Syp/tdTomato fluorescence preferentially co-localized with osteopontin, a recently discovered motor neuron marker for slow-twitch fatigue-resistant (S) and fast-twitch fatigue-resistant (FR) types. The fluorescence did not preferentially co-localize with matrix metalloproteinase-9, a marker for fast-twitch fatigable (FF) motor neurons. In the neuromuscular junctions, Syp/tdTomato fluorescence was detected mainly in motor nerve terminals that innervate type I or IIa muscle fibers. These results suggest that the VAChT-Cre lines are Cre-drivers that have selectivity in S and FR motor neurons. In order to avoid confusion, we have changed the mouse line names from VAChT-Cre.Fast and VAChT-Cre.Slow to VAChT-Cre.Early and VAChT-Cre.Late, respectively. The mouse lines will be useful tools to study slow-type motor neurons, in relation to physiology and pathology.

Effect of secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor-related peptide-1 (SLURP-1) on airway epithelial cells.

Acetylcholine (ACh) exerts various anti-inflammatory effects through α7 nicotinic ACh receptors (nAChRs). We have previously shown that secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor-related peptide-1 (SLURP-1), a positive allosteric modulator of α7 nAChR signaling, is down-regulated both in an animal model of asthma and in human epithelial cells treated with an inflammatory cytokine related to asthma. Our aim of this study was to explore the effect of SLURP-1, signal through α7 nAChR, in the pathophysiology of airway inflammation. Cytokine production was examined using human epithelial cells. Ciliary beat frequency of murine trachea was measured using a high speed camera. The IL-6 and TNF-α production by human epithelial cells was augmented by siRNA of SLURP-1 and α7 nicotinic ACh receptor. The cytokine production was also dose-dependently suppressed by human recombinant SLURP-1 (rSLURP-1). The ciliary beat frequency and amplitude of murine epithelial cells were augmented by PNU282987, a selective α7 nAChR agonist. Those findings suggested that SLURP-1 and stimulus through α7 nicotinic ACh receptors actively controlled asthmatic condition by stimulating ciliary beating and also by suppressing airway inflammation.

Cutting edge: critical role of intracellular osteopontin in antifungal innate immune responses.

We found that absence of osteopontin (OPN) in immunocompromised Rag2(-/-) mice, which lack T and B cells, made the mice extremely susceptible to an opportunistic fungus Pneumocystis, although immunocompetent OPN-deficient mice could clear Pneumocystis as well as wild-type mice. OPN has been studied as an extracellular protein, and the role of an intracellular isoform of OPN (iOPN) is still largely unknown. In this study, we elucidated the mechanism by which iOPN was involved in antifungal innate immunity. First, iOPN was essential for cluster formation of fungal receptors that detect Pneumocystis, including dectin-1, TLR2, and mannose receptor. Second, iOPN played a role as an adaptor molecule in TLR2 and dectin-1 signaling pathways and mediated ERK activation and cytokine production by zymosan, which simultaneously activates TLR2 and dectin-1 pathways. Third, iOPN enhanced phagocytosis and clearance of Pneumocystis. Our study suggests the critical involvement of iOPN in antifungal innate immunity.

The loss of PGAM5 suppresses the mitochondrial degeneration caused by inactivation of PINK1 in Drosophila.

PTEN-induced kinase 1 (PINK1), which is required for mitochondrial homeostasis, is a gene product responsible for early-onset Parkinson's disease (PD). Another early onset PD gene product, Parkin, has been suggested to function downstream of the PINK1 signalling pathway based on genetic studies in Drosophila. PINK1 is a serine/threonine kinase with a predicted mitochondrial target sequence and a probable transmembrane domain at the N-terminus, while Parkin is a RING-finger protein with ubiquitin-ligase (E3) activity. However, how PINK1 and Parkin regulate mitochondrial activity is largely unknown. To explore the molecular mechanism underlying the interaction between PINK1 and Parkin, we biochemically purified PINK1-binding proteins from human cultured cells and screened the genes encoding these binding proteins using Drosophila PINK1 (dPINK1) models to isolate a molecule(s) involved in the PINK1 pathology. Here we report that a PINK1-binding mitochondrial protein, PGAM5, modulates the PINK1 pathway. Loss of Drosophila PGAM5 (dPGAM5) can suppress the muscle degeneration, motor defects, and shorter lifespan that result from dPINK1 inactivation and that can be attributed to mitochondrial degeneration. However, dPGAM5 inactivation fails to modulate the phenotypes of parkin mutant flies. Conversely, ectopic expression of dPGAM5 exacerbated the dPINK1 and Drosophila parkin (dParkin) phenotypes. These results suggest that PGAM5 negatively regulates the PINK1 pathway related to maintenance of the mitochondria and, furthermore, that PGAM5 acts between PINK1 and Parkin, or functions independently of Parkin downstream of PINK1.

Osteopontin is an alpha motor neuron marker in the mouse spinal cord.

Motor neurons (MNs) are designated as alpha/gamma and fast/slow based on their target sites and the types of muscle fibers innervated; however, few molecular markers that distinguish between these subtypes are available. Here we report that osteopontin (OPN) is a selective marker of alpha MNs in the mouse spinal cord. OPN was detected in approximately 70% of postnatal choline acetyltransferase (ChAT)-positive MNs with relatively large somas, but not in those with smaller somas. OPN+/ChAT+ MNs were also positive for NeuN, an alpha MN marker, but were negative for Err3, a gamma MN marker. The size distribution of OPN+/ChAT+ cells was nearly identical to that of NeuN+/ChAT+ alpha MNs. Group Ia proprioceptive terminals immunoreactive for vesicular glutamate transporter-1 were selectively detected on the OPN+/ChAT+ cells. OPN staining was also detected at motor axon terminals at neuromuscular junctions, where the OPN+ terminals were positive or negative for SV2A, a marker distinguishing fast/slow motor endplates. Finally, retrograde labeling following intramuscular injection of fast blue indicated that OPN is expressed in both fast and slow MNs. Collectively, our findings show that OPN is an alpha MN marker present in both the soma and the endplates of alpha MNs in the postnatal mouse spinal cord.

New Pathways for the Skin's Stress Response: The Cholinergic Neuropeptide SLURP-1 Can Activate Mast Cells and Alter Cytokine Production in Mice.

Background: The alpha7 nicotinic acetylcholine receptor (Chrna7) plays an essential anti-inflammatory role in immune homeostasis and was recently found on mast cells (MC). Psychosocial stress can trigger MC hyperactivation and increases pro-inflammatory cytokines in target tissues such as the skin. If the cholinergic system (CS) and Chrna7 ligands play a role in these cascades is largely unknown. Objective: To elucidate the role of the CS in the response to psychosocial stress using a mouse-model for stress-triggered cutaneous inflammatory circuits. Methods: Key CS markers (ACh, Ch, SLURP-1, SLURP-2, Lynx1, Chrm3, Chrna7, Chrna9, ChAT, VAChT, Oct3, AChE, and BChE) in skin and its MC (sMC), MC activation, immune parameters (TNFα, IL1ß, IL10, TGFß, HIF1α, and STAT3) and oxidative stress were analyzed in skin from 24 h noise-stressed mice and in cultured MC (cMC) from C57BL/6 or Chrna7-Knockout mice. Results: First, Chrna7 and SLURP-1 mRNA were exclusively upregulated in stressed skin. Second, histomorphometry located Chrna7 and SLURP-1 in nerves and sMC and demonstrated upregulated contacts and increased Chrna7+ sMC in stressed skin, while 5 ng/mL SLURP-1 degranulated cMC. Third, IL1ß+ sMC were high in stressed skin, and while SLURP-1 alone had no significant effect on cMC cytokines, it upregulated IL1ß in cMC from Chrna7-KO and in IL1ß-treated wildtype cMC. In addition, HIF1α+ sMC were high in stressed skin and Chrna7-agonist AR-R 17779 induced ROS in cMC while SLURP-1 upregulated TNFα and IL1ß in cMC when HIF1α was blocked. Conclusions: These data infer that the CS plays a role in the regulation of stress-sensitive inflammatory responses but may have a surprising pro-inflammatory effect in healthy skin, driving IL1ß expression if SLURP-1 is involved.

Down-regulation of secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor-related peptide-1 (SLURP-1), an endogenous allosteric alpha7 nicotinic acetylcholine receptor modulator, in murine and human asthmatic conditions.

Whereas acetylcholine (ACh) acts as a bronchoconstrictor and stimulator of mucus secretion from bronchial epithelium, it acts via alpha7 nicotinic Ach receptors (nAChRs) on macrophages in the airways to exert anti-inflammatory effects by reducing synthesis of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha). Moreover, the effects of ACh are modified by secreted ly-6/urokinase-type plasminogen activator receptor-related peptide-1 (SLURP-1), a positive allosteric modulator of alpha7 nAChR signaling. Our aim was to explore the roles played by SLURP-1 in the pathophysiology of asthma by assessing SLURP-1 expression in the OVA-sensitized murine asthma model and in cultured human bronchial epithelial cells. Using real-time PCR we found that expression of SLURP-1 mRNA is down-regulated in the lungs of asthmatic model mice, as compared to healthy mice. In addition, immunohistochemical studies confirmed the diminished expression of SLURP-1 in the bronchioles of asthmatic mice, and showed it was due to extensive metaplasia of mucus-secreting cells and the concomitant loss of ciliated epithelial cells. Expression of SLURP-1 mRNA and protein was also significantly down-regulated in human epithelial cells stimulated with the pro-inflammatory cytokine interleukin-13 (IL-13), which is related to asthmatic condition. Thus SLURP-1 appears to be down-regulated in both an animal model of asthma and human epithelial cells treated with an inflammatory cytokine related to asthma. Those findings suggest that diminished expression of SLURP-1 in asthma attenuates its negative regulation of airway inflammation, and that perhaps changes in SLURP-1 expression could serve as a marker of airway damage in asthma.

Minireview: Divergent roles of α7 nicotinic acetylcholine receptors expressed on antigen-presenting cells and CD4+ T cells in the regulation of T cell differentiation.

α7 nAChRs expressed on immune cells regulate antigen-specific antibody and proinflammatory cytokine production. Using spleen cells from ovalbumin (OVA)-specific T cell receptor transgenic DO11.10 mice and the α7 nAChR agonist GTS-21, investigation of (1) antigen processing-dependent and (2) -independent, antigen presenting cell (APC)-dependent, naïve CD4+ T cell differentiation, as well as (3) non-specific APC-independent, anti-CD3/CD28 mAbs-induced CD4+ T cell differentiation, revealed the differential roles of α7 nAChRs expressed on T cells and APCs in the regulation of CD4+ T cell differentiation. GTS-21 suppressed OVA-induced antigen processing- and APC-dependent differentiation into regulatory T cells (Tregs) and effector T cells (Th1, Th2 and Th17) without affecting OVA uptake or cell viability. By contrast, GTS-21 upregulated OVA peptide-induced antigen processing-independent T cell differentiation into all lineages. During anti-CD3/CD28 mAbs-induced T cell differentiation in the presence of polarizing cytokines, GTS-21 promoted wild-type T cell differentiation into all lineages, but did not affect α7 nAChR-deficient T cell differentiation. These results demonstrate (1) that α7 nAChRs on APCs downregulate T cell differentiation by inhibiting antigen processing and thereby interfering with antigen presentation; and (2) that α7 nAChRs on T cells upregulate differentiation into Tregs and effector T cells. Thus, the divergent roles of α7 nAChRs on APCs and T cells likely regulate the intensity of immune responses. These findings suggest the possibility of using α7 nAChR agonists to harvest greater numbers of Tregs and Th1 and Th2 cells for adoptive immune therapies for treatment of autoimmune diseases and cancers.

Endogenous neurotoxin-like protein Ly6H inhibits alpha7 nicotinic acetylcholine receptor currents at the plasma membrane.

α7 nicotinic acetylcholine receptors (nAChRs) are widely expressed in the central nervous system and regarded as potential therapeutic targets for neurodegenerative conditions, such as Alzheimer's disease and schizophrenia. Yet, despite the assumed pathophysiological importance of the α7 nAChR, molecular physiological characterization remains poorly advanced because α7 nAChR cannot be properly folded and sorted to the plasma membranes in most mammalian cell lines, thus preventing the analyses in heterologous expression system. Recently, ER-resident membrane protein NACHO was discovered as a strong chaperone for the functional expression of α7 nAChR in non-permissive cells. Ly6H, a brain-enriched GPI-anchored neurotoxin-like protein, was reported as a novel modulator regulating intracellular trafficking of α7 nAChR. In this study, we established cell lines that stably and robustly express surface α7 nAChR by introducing α7 nAChR, Ric-3, and NACHO cDNA into HEK293 cells (Triple α7 nAChR/RIC-3/NACHO cells; TARO cells), and re-evaluated the function of Ly6H. We report here that Ly6H binds with α7 nAChRs on the cell membrane and modulates the channel activity without affecting intracellular trafficking of α7 nAChR.

Acetylcholine synthesis and release in NIH3T3 cells coexpressing the high-affinity choline transporter and choline acetyltransferase.

Acetylcholine (ACh) is known to be a key neurotransmitter in the central and peripheral nervous systems, but it is also produced in a variety of non-neuronal tissues and cells, including lymphocytes, placenta, amniotic membrane, vascular endothelial cells, keratinocytes, and epithelial cells in the digestive and respiratory tracts. To investigate contribution made by the high-affinity choline transporter (CHT1) to ACh synthesis in both cholinergic neurons and nonneuronal cells, we transfected rat CHT1 cDNA into NIH3T3ChAT cells, a mouse fibroblast line expressing mouse choline acetyltransferase (ChAT), to establish the NIH3T3ChAT 112-1 cell line, which stably expresses both CHT1 and ChAT. NIH3T3ChAT 112-1 cells showed increased binding of the CHT1 inhibitor [(3)H]hemicholinium-3 (HC-3) and greater [(3)H]choline uptake and ACh synthesis than NIH3T3ChAT 103-1 cells, a CHT1-negative control cell line. HC-3 significantly inhibited ACh synthesis in NIH3T3ChAT 112-1 cells but did not affect synthesis in NIH3T3ChAT 103-1 cells. ACh synthesis in NIH3T3ChAT 112-1 cells was also reduced by amiloride, an inhibitor of organic cation transporters (OCTs) involved in low-affinity choline uptake, and by procaine and lidocaine, two local anesthetics that inhibit plasma membrane phospholipid metabolism. These results suggest that CHT1 plays a key role in ACh synthesis in NIH3T3ChAT 112-1 cells and that choline taken up by OCTs or derived from the plasma membrane is also utilized for ACh synthesis in both cholinergic neurons and nonneuronal cholinergic cells, such as lymphocytes.

Expression of SLURP-1, an endogenous alpha7 nicotinic acetylcholine receptor allosteric ligand, in murine bronchial epithelial cells.

Mammalian secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor-related peptide-1 (SLURP-1) is a positive allosteric ligand for alpha7 nicotinic acetylcholine (ACh) receptors (alpha7 nAChRs) that potentiates responses to ACh and elicits proapoptotic activity in human keratinocytes. Mutations in the gene encoding SLURP-1 have been detected in patients with Mal de Meleda, a rare autosomal recessive skin disorder characterized by transgressive palmoplantar keratoderma. On the basis of these findings, SLURP-1 is postulated to be involved in regulating tumor necrosis factor-alpha (TNF-alpha) release from keratinocytes and macrophages via alpha7 nAChR-mediated pathways. In the present study, we assessed SLURP-1 expression in lung tissue from C57BL/6J mice to investigate the functions of SLURP-1 in pulmonary physiology and pathology. Immunohistochemical and in situ hybridization analyses revealed expression of SLURP-1 protein and mRNA, respectively, exclusively in ciliated bronchial epithelial cells. This was supported by Western blotting showing the presence of the 9.5-kDa SLURP-1 protein in whole-lung tissue and trachea. In addition, high-affinity choline transporter (CHT1) was detected in apical regions of bronchial epithelial cells and in neurons located in the lamina propria of the bronchus, suggesting that bronchial epithelial cells are able to synthesize both SLURP-1 and ACh. We also observed direct contact between F4/80-positive macrophages and bronchial epithelial cells and the presence of invading macrophages in close proximity to CHT1-positive nerve elements. Collectively, these results suggest that SLURP-1 contributes to the maintenance of bronchial epithelial cell homeostasis and to the regulation of TNF-alpha release from macrophages in bronchial tissue.

Distinct Roles of α7 nAChRs in Antigen-Presenting Cells and CD4+ T Cells in the Regulation of T Cell Differentiation.

It is now apparent that immune cells express a functional cholinergic system and that α7 nicotinic acetylcholine receptors (α7 nAChRs) are involved in regulating T cell differentiation and the synthesis of antigen-specific antibodies and proinflammatory cytokines. Here, we investigated the specific function α7 nAChRs on T cells and antigen presenting cells (APCs) by testing the effect of GTS-21, a selective α7 nAChR agonist, on differentiation of CD4+ T cells from ovalbumin (OVA)-specific TCR transgenic DO11.10 mice activated with OVA or OVA peptide323-339 (OVAp). GTS-21 suppressed OVA-induced antigen processing-dependent development of CD4+ regulatory T cells (Tregs) and effector T cells (Th1, Th2, and Th17). By contrast, GTS-21 up-regulated OVAp-induced antigen processing-independent development of CD4+ Tregs and effector T cells. GTS-21 also suppressed production of IL-2, IFN-γ, IL-4, IL-17, and IL-6 during OVA-induced activation but, with the exception IL-2, enhanced their production during OVAp-induced activation. In addition, during antigen-nonspecific, APC-independent anti-CD3/CD28 antibody-induced CD4+ polyclonal T cell activation in the presence of respective polarizing cytokines, GTS-21 promoted development of all lineages, which indicates that GTS-21 also acts via α7 nAChRs on T cells. These results suggest 1) that α7 nAChRs on APCs suppress CD4+ T cell activation by interfering with antigen presentation through inhibition of antigen processing; 2) that α7 nAChRs on CD4+ T cells up-regulate development of Tregs and effector T cells; and that α7 nAChR agonists and antagonists could be potentially useful agents for immune response modulation and enhancement.

Aberrant trafficking of the high-affinity choline transporter in AP-3-deficient mice.

The high-affinity choline transporter (CHT) is expressed in cholinergic neurons and efficiently transported to axon terminals where it controls the rate-limiting step in acetylcholine synthesis. Recent studies have shown that the majority of CHT is unexpectedly localized on synaptic vesicles (SV) rather than the presynaptic plasma membrane, establishing vesicular CHT trafficking as a basis for activity-dependent CHT regulation. Here, we analyse the intracellular distribution of CHT in the adaptor protein-3 (AP-3)-deficient mouse model mocha. In the mocha mouse, granular structures in cell bodies are intensely labelled with CHT antibody, indicating possible deficits in CHT trafficking from the cell body to the axon terminal. Western blot analyses reveal that CHT on SV in mocha mice is decreased by 30% compared with wild-type mice. However, no significant difference in synaptosomal choline uptake activity is detected, consistent with the existence of a large reservoir pool for CHT. To further characterize CHT trafficking, we established a PC12D-CHT cell line. In this line, CHT is found associated with a subpopulation of synaptophysin-positive synaptic-like microvesicles (SLMV). The amounts of CHT detected on SLMV are greatly reduced by treating the cell with agents that halt AP-dependent membrane trafficking. These results demonstrate that APs have important functions for CHT trafficking in neuronal cells.

L347P PINK1 mutant that fails to bind to Hsp90/Cdc37 chaperones is rapidly degraded in a proteasome-dependent manner.

Mutation of PTEN-induced kinase 1 (PINK1), which encodes a putative mitochondrial serine/threonine kinase, leads to PARK6, an autosomal recessive form of familial Parkinson's disease. Although the precise function(s) of PINK1 protein is unknown, the recessive inheritance of this form of Parkinson's disease suggests loss of PINK1 function is closely associated with its pathogenesis. Here we report that PINK1 forms a complex with the molecular chaperones Hsp90 and Cdc37/p50 within cells, which appears to enhance its stability. When cells were treated with an Hsp90 inhibitor (geldanamycin or novobiocin), levels of PINK1 were greatly diminished, reflecting its rapid degradation via ubiquitin-proteasome pathway. Similarly, the half-life of a pathogenic PINK1 mutant (L347P) that did not interact with Hsp90 or Cdc37/p50 was only 30min, whereas that of wild-type PINK1 was 1h. These results strongly suggest that Hsp90 and Cdc37 are binding partners of PINK1 which regulate its stability.

Dissociation of blood-brain barrier disruption and disease manifestation in an aquaporin-4-deficient mouse model of amyotrophic lateral sclerosis.

Aquaporin-4 (AQP4) is abundantly expressed in the central nervous system and is involved in the water balance in the cellular environment. Previous studies have reported that AQP4 expression is upregulated in rat models of amyotrophic lateral sclerosis (ALS), a fatal disease that affects motor neurons in the brain and spinal cord. In this study, we report that astrocytic AQP4 overexpression is evident during the course of disease in the spinal cord of an ALS mouse model, as well as in tissue from patients with ALS. AQP4 overexpression appears to be specifically associated with ALS because it was not induced by other experimental manipulations that produced acute or chronic gliosis. In order to examine the contribution of AQP4 to ALS disease development, we crossed AQP4 knockout mice with a mouse model of ALS. Significant improvement in blood-brain barrier (BBB) permeability was observed in the AQP4-deficient ALS mouse model. However, the time to disease onset and total lifespan were reduced in the AQP4-deficient ALS mouse model. The contradictory results suggest that changes in AQP4 may be context-dependent and further studies are required to understand the precise contribution of brain water balance in ALS.