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).

Lysophosphatidic acid stimulates pericyte migration via LPA receptor 1.

Lysophosphatidic acid (LPA) is a bioactive compound known to regulate various vascular functions. However, despite the fact that many vascular functions are regulated by peri-vascular cells such as pericytes, the effect of LPA on brain pericytes has not been fully evaluated. Thus, we designed this study to evaluate the effects of LPA on brain pericytes. These experiments revealed that while LPA receptors (LPARs) are expressed in cultured pericytes from mouse brains, LPA treatment does not influence the proliferation of these cells but does have a profound impact on their migration, which is regulated via the expression of LPAR1. LPAR1 expression was also detected in human pericyte culture and LPA treatment of these cells also induced migration. Taken together these findings imply that LPA-LPAR1 signaling is one of the key mechanisms modulating pericyte migration, which may help to control vascular function during development and repair processes.

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.

A copper-deficient form of mutant Cu/Zn-superoxide dismutase as an early pathological species in amyotrophic lateral sclerosis.

Dominant mutations in the gene encoding copper and zinc-binding superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS). Abnormal accumulation of misfolded SOD1 proteins in spinal motoneurons is a major pathological hallmark in SOD1-related ALS. Dissociation of copper and/or zinc ions from SOD1 has been shown to trigger the protein aggregation/oligomerization in vitro, but the pathological contribution of such metal dissociation to the SOD1 misfolding still remains obscure. Here, we tested the relevance of the metal-deficient SOD1 in the misfolding in vivo by developing a novel antibody (anti-apoSOD), which exclusively recognized mutant SOD1 deficient in metal ions at its copper-binding site. Notably, anti-apoSOD-reactive species were detected specifically in the spinal cords of the ALS model mice only at their early pre-symptomatic stages but not at the end stage of the disease. The cerebrospinal fluid as well as the spinal cord homogenate of one SOD1-ALS patient also contained the anti-apoSOD-reactive species. Our results thus suggest that metal-deficiency in mutant SOD1 at its copper-binding site is one of the earliest pathological features in SOD1-ALS.

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.

Myelinating cocultures of rodent stem cell line-derived neurons and immortalized Schwann cells.

Myelination is one of the most remarkable biological events in the neuron-glia interactions for the development of the mammalian nervous system. To elucidate molecular mechanisms of cell-to-cell interactions in myelin synthesis in vitro, establishment of the myelinating system in cocultures of continuous neuronal and glial cell lines are desirable. In the present study, we performed co-culture experiments using rat neural stem cell-derived neurons or mouse embryonic stem (ES) cell-derived motoneurons with immortalized rat IFRS1 Schwann cells to establish myelinating cultures between these cell lines. Differentiated neurons derived from an adult rat neural stem cell line 1464R or motoneurons derived from a mouse ES cell line NCH4.3, were mixed with IFRS1 Schwann cells, plated, and maintained in serum-free F12 medium with B27 supplement, ascorbic acid, and glial cell line-derived neurotrophic factor. Myelin formation was demonstrated by electron microscopy at 4 weeks in cocultures of 1464R-derived neurons or NCH4.3-derived motoneurons with IFRS1 Schwann cells. These in vitro coculture systems utilizing the rodent stable stem and Schwann cell lines can be useful in studies of peripheral nerve development and regeneration.

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.

Selective disruption of acetylcholine synthesis in subsets of motor neurons: a new model of late-onset motor neuron disease.

Motor neuron diseases are characterized by the selective chronic dysfunction of a subset of motor neurons and the subsequent impairment of neuromuscular function. To reproduce in the mouse these hallmarks of diseases affecting motor neurons, we generated a mouse line in which ~40% of motor neurons in the spinal cord and the brainstem become unable to sustain neuromuscular transmission. These mice were obtained by conditional knockout of the gene encoding choline acetyltransferase (ChAT), the biosynthetic enzyme for acetylcholine. The mutant mice are viable and spontaneously display abnormal phenotypes that worsen with age including hunched back, reduced lifespan, weight loss, as well as striking deficits in muscle strength and motor function. This slowly progressive neuromuscular dysfunction is accompanied by muscle fiber histopathological features characteristic of neurogenic diseases. Unexpectedly, most changes appeared with a 6-month delay relative to the onset of reduction in ChAT levels, suggesting that compensatory mechanisms preserve muscular function for several months and then are overwhelmed. Deterioration of mouse phenotype after ChAT gene disruption is a specific aging process reminiscent of human pathological situations, particularly among survivors of paralytic poliomyelitis. These mutant mice may represent an invaluable tool to determine the sequence of events that follow the loss of function of a motor neuron subset as the disease progresses, and to evaluate therapeutic strategies. They also offer the opportunity to explore fundamental issues of motor neuron biology.

Loss of TDP-43 causes age-dependent progressive motor neuron degeneration.

Amyotrophic lateral sclerosis is a devastating, progressive neurodegenerative disease that affects upper and lower motor neurons. Although several genes are identified as the cause of familial cases, the pathogeneses of sporadic forms, which account for 90% of amyotrophic lateral sclerosis, have not been elucidated. Transactive response DNA-binding protein 43 a nuclear protein regulating RNA processing, redistributes to the cytoplasm and forms aggregates, which are the histopathological hallmark of sporadic amyotrophic lateral sclerosis, in affected motor neurons, suggesting that loss-of-function of transactive response DNA-binding protein 43 is one of the causes of the neurodegeneration. To test this hypothesis, we assessed the effects of knockout of transactive response DNA-binding protein 43 in mouse postnatal motor neurons using Cre/loxp system. These mice developed progressive weight loss and motor impairment around the age of 60 weeks, and exhibited degeneration of large motor axon, grouped atrophy of the skeletal muscle, and denervation in the neuromuscular junction. The spinal motor neurons lacking transactive response DNA-binding protein 43 were not affected for 1 year, but exhibited atrophy at the age of 100 weeks; whereas, extraocular motor neurons, that are essentially resistant in amyotrophic lateral sclerosis, remained preserved even at the age of 100 weeks. Additionally, ultra structural analysis revealed autolysosomes and autophagosomes in the cell bodies and axons of motor neurons of the 100-week-old knockout mice. In summary, the mice in which transactive response DNA-binding protein 43 was knocked-out specifically in postnatal motor neurons exhibited an age-dependent progressive motor dysfunction accompanied by neuropathological alterations, which are common to sporadic amyotrophic lateral sclerosis. These findings suggest that transactive response DNA-binding protein 43 plays an essential role in the long term maintenance of motor neurons and that loss-of-function of this protein seems to contribute to the pathogenesis of amyotrophic lateral sclerosis.