論文

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 30 min, whereas that of wild-type PINK1 was 1 h. These results strongly suggest that Hsp90 and Cdc37 are binding partners of PINK1 which regulate its stability.

Dominant mutations in superoxide dismutase cause amyotrophic lateral sclerosis (ALS), an adult-onset neurodegenerative disease that is characterized by the loss of motor neurons. Using mice carrying a deletable mutant gene, diminished mutant expression in astrocytes did not affect onset, but delayed microglial activation and sharply slowed later disease progression. These findings demonstrate that mutant astrocytes are viable targets for therapies for slowing the progression of non-cell autonomous killing of motor neurons in ALS.

Kidney development and physiology require polarization of epithelia that line renal tubules. Genetic studies show that polarization of invertebrate epithelia requires the crumbs, partition-defective-3, and discs large complexes. These evolutionarily conserved protein complexes occur in mammalian kidney; however, their role in renal development remains poorly defined. Here, we find that mice lacking the small PDZ protein mammalian LIN-7c (MALS-3) have hypomorphic, cystic, and fibrotic kidneys. Proteomic analysis defines MALS-3 as the only known core component of both the crumbs and discs large cell polarity complexes. MALS-3 mediates stable assembly of the crumbs tight junction complex and the discs large basolateral complex, and these complexes are disrupted in renal epithelia from MALS-3 knockout mice. Interestingly, MALS-3 controls apico-basal polarity preferentially in epithelia derived from metanephric mesenchyme, and defects in kidney architecture owe solely to MALS expression in these epithelia. These studies demonstrate that defects in epithelial cell polarization can cause cystic and fibrotic renal disease.

Human and murine immune cells such as mononuclear leukocytes consisting of mainly T and B cells, bone marrow derived dendritic cells (DCs) and macrophages all express various nicotinic acetylcholine (ACh) receptor (nAChR) subunits. Activated T cells and DCs have the ability to synthesize ACh by choline acetyltransferase, suggesting the role of non-neuronal cholinergic system expressed in immune cells in the regulation of immune cell function. Stimulation of human leukemic T and B cell lines with nicotine causes a transient Ca2+-signaling that is antagonized by α-bungarotoxin, suggesting the involvement of α7 subunit. Furthermore, α7 nAChRs have been shown to negatively regulate synthesis and release of tumor necrosis factor (TNF)-α in macrophages. These findings suggest that immune cell function is regulated by its own non-neuronal cholinergic system, at least in part, via α7 nAChR-mediated pathways. In the present study, we tested the role of α7 nAChRs in the regulation of immune function by measuring total serum and antigen-specific IgG1 and IgM, and production of TNF-α, gamma interferon (IFN-γ) and interleukin (IL)-6 in activated spleen cells of nAChR α7 subunit gene knockout (α7 KO) and wild-type C57BL/6J mice immunized with ovalbumin (OVA). We found that serum levels of total and anti-OVA-specific IgG1 were significantly elevated in α7 KO mice, though there were no significant differences in serum levels of total and anti-OVA-specific IgM between the two genotypes. Production of TNF-α, IFN-γ and IL-6 in spleen cells was significantly facilitated in α7 KO mice. Expression of AChE mRNA was not different between the two genotypes. These results suggest that α7 nAChRs are involved in the regulation of cytokine production, through which modulates TNF-α, IFN-γ and IL-6 productions, leading to modification of antibody production, but are not involved in expression of cholinergic components in immune cells.

Immunological activation of T cells enhances synthesis of acetylcholine (ACh) and transcription of choline acetyltransferase (ChAT), M5 muscarinic ACh receptor (mAChR) and acetylcholinesterase (AChE). Stimulation of mAChRs on T and B cells causes oscillating Ca2+-signaling and up-regulation of c-fos expression; moreover, M1 mAChRs play a crucial role in the differentiation of CD8+ T cells into cytolytic T lymphocytes. Collectively, these findings suggest that immune cell function is regulated by its own cholinergic system. Bearing that in mind, we tested whether immune function can be regulated via mAChR-mediated pathways by immunizing combined M1 and M5 mAChR knockout (M1/M5 KO) and wild-type (WT) C57BL/6JJcl mice with ovalbumin (OVA) and measuring serum IgG1 and IgM 1 wk later. We found that serum levels of total and anti-OVA-specific IgG1 were significantly lower in M1/M5 KO than WT mice, though there was no difference in serum levels of total and anti-OVA-specific IgM between the two genotypes. Secretion of interleukin (IL)-6 from activated spleen cells was significantly reduced in M1/M5 KO mice, whereas there was no significant change in gamma interferon secretion. Expression of AChE mRNA was significantly reduced in activated spleen cells from M1/M5 KO mice. These results suggest that M1 and/or M5 mAChRs are involved in regulating cytokine (e.g., IL-6) production, leading to modulation of antibody class switching from IgM to IgG1, but are not involved in the initial generation of the antibody response. They also support the notion that a non-neuronal cholinergic system is involved in regulating immune cell function.

Using a radioimmunoassay (RIA) with high specificity and sensitivity (1 pg/tube) for acetylcholine (ACh), we have been able to measure the ACh content in samples from the bacteria, archaea and eucarya domains of the universal phylogenetic tree. We found detectable levels of ACh to be ubiquitous in bacteria (e.g., Bacillus subtilis), archaea (e.g., Thermococcus kodakaraensis KOD1), fungi (e.g., shiitake mushroom and yeast), plants (e.g., bamboo shoot and fern) and animals (e.g., bloodworm and lugworm). The levels varied considerably, however, with the highest ACh content detected in the top portion of bamboo shoot (2.9 μmol/g), which contained about 80 times that found in rat brain. In addition, using the method of Fonnum, various levels of ACh-synthesizing activity also were detected, a fraction of which was catalyzed by a choline acetyltransferase (ChAT)-like enzyme (sensitive to bromoACh, a selective ChAT inhibitor) in T. kodakaraensis KOD1 (15%), bamboo shoot (91%) and shiitake mushroom (51%), bloodworm (91%) and lugworm (81%). Taken together, these findings demonstrate the ubiquitous expression of ACh and ACh-synthesizing activity among life forms without nervous systems, and support the notion that ACh has been expressed and may be active as a local mediator and modulator of physiological functions since the early beginning of life.

A novel transduction pathway via which apoptosis of keratinocytes is regulated through nicotinic acetylcholine (ACh) receptors (nAChRs) has emerged in studies of secreted mammalian Ly6/urokinase plasminogen-type activator receptor-related protein-1 and-2 (SLURP-1 and SLURP-2, respectively). SLURP-1 reportedly binds to α7 nAChRs and enhances the amplitude of macroscopic currents induced by ACh, leading to facilitation of apoptosis, whereas SLURP-2 binds to α3 nAChRs and prevents apoptosis. These observations prompted us to test whether SLURPs are expressed in immune cells and are involved in the regulation of immune function. We initially used reverse transcription-polymerase chain reaction analysis to characterize the expression profiles of SLURP mRNAs in several murine tissues and organs. Although SLURP-1 mRNA was not expressed in the pancreas, all other tissues and organs tested, including spleen and thymus, expressed both SLURP-1 and SLURP-2 mRNAs. Expression of both mRNAs also was detected in T and B cells, bone marrow-derived dendritic cells (DCs) and macrophages. Moreover, as in keratinocytes, stimulation of MOLT-3 human leukemic T cells with recombinant human SLURP-1 evoked intracellular Ca2+ signaling. These results suggest that both SLURP-1 and SLURP-2 are expressed in various immune cells and organs, and that not only ACh but also SLURPs may be involved in regulating lymphocyte function via nAChR-mediated pathways.

It is now evident that acetylcholine (ACh) synthesized by choline acetyltransferase (ChAT) and released from T cells during antigen presentation binds to muscarinic and nicotinic ACh receptors (mAChRs and nAChRs, respectively) on T and B cells or dendritic cells, leading to modulation of their function. In the present study, we used reverse transcription-polymerase chain reaction (RT-PCR) to investigate whether mononuclear leukocytes (MNLs), bone marrow-derived dendritic cells (DCs) and macrophages from C57BL/6J mice express components of the cholinergic system. Expression of ChAT mRNA was detected in MNLs activated with ConA and DCs stimulated with LPS, but not in resting MNLs and DCs or in resting and stimulated macrophages. MNLs, DCs and macrophages all expressed mRNAs encoding the five mAChR subtypes (M1-M5) and the nAChR α2, α5, α6, α7, α10 and β2 subunits. Expression of VIP mRNA was detected in MNLs and macrophages, but not in DCs. MNLs, DCs and macrophages all expressed VIP receptor-1 (VPAC1) and -2 (VPAC2) mRNAs, as well as mRNAs encoding secreted mammalian Ly-6/urokinase-type plasminogen activator receptor-related protein (SLURP)-1 and SLURP-2, two endogenous nAChR ligands. These results suggest that the lymphocytic cholinergic system is activated by ACh via mAChR- and nAChR-mediated pathways during antigen presentation between T cells and DCs or macrophages, leading to modulation of immune cell function. Moreover, VIP released from both postganglionic cholinergic neurons and immune cells may play a role in the cholinergic anti-inflammatory reflex, acting via VPAC1 and VPAC2 on immune cells.

Mitochondrial dysfunction and oxidative damage are implicated in the pathogenesis of neurodegenerative disease. Mice deficient in the mitochondrial form of superoxide dismutase (SOD2) die during embryonic or early postnatal development, precluding analysis of a pathological role for superoxide in adult tissue. Here, we generated postnatal motor neuron-specific SOD2 knockouts by crossing mice with floxed SOD2 alleles to VAChT-Cre transgenic mice in which Cre expression is restricted to postnatal somatomotor neurons. SOD2 immunoreactivity was specifically lost in a subset of somatomotor neurons resulting in enhanced superoxide production. Yet extensive histological examination revealed no signs of oxidative damage in animals up to 1 year after birth. However, disorganization of distal nerve axons following injury was accelerated in SOD2-deficient motor neurons. These data demonstrate that postnatal motor neurons are surprisingly resistant to oxidative damage from mitochondrial-derived superoxide radicals, but that such damage may sensitize axons to disorganization following nerve injury.

Background: Dominant mutations in superoxide dismutase 1 (SOD1) cause degeneration of motor neurons in a subset of inherited amyotrophic lateral sclerosis (ALS). The pathogenetic process mediated by misfolded and/or aggregated mutant SOD1 polypeptides is hypothesized to be suppressed by protein refolding. This genetic study is aimed to test whether mutant SOD1-mediated ALS pathology recapitulated in mice could be alleviated by overexpressing a longevity-related deacetylase SIRT1 whose substrates include a transcription factor heat shock factor 1 (HSF1), the master regulator of the chaperone system. Results: We established a line of transgenic mice that chronically overexpress SIRT1 in the brain and spinal cord. While inducible HSP70 (HSP70i) was upregulated in the spinal cord of SIRT1 transgenic mice (PrP-Sirt1), no neurological and behavioral alterations were detected. To test hypothetical benefits of SIRT1 overexpression, we crossbred PrP-Sirt1 mice with two lines of ALS model mice: A high expression line that exhibits a severe phenotype (SOD1G93A-H) or a low expression line with a milder phenotype (SOD1G93A-L). The Sirt1 transgene conferred longer lifespan without altering the time of symptomatic onset in SOD1G93A-L. Biochemical analysis of the spinal cord revealed that SIRT1 induced HSP70i expression through deacetylation of HSF1 and that SOD1G93A-L/PrP-Sirt1 double transgenic mice contained less insoluble SOD1 than SOD1G93A-L mice. Parallel experiments showed that Sirt1 transgene could not rescue a more severe phenotype of SOD1G93A-H transgenic mice partly because their HSP70i level had peaked out. Conclusions: The genetic supplementation of SIRT1 can ameliorate a mutant SOD1-linked ALS mouse model partly through the activation of the HSF1/HSP70i chaperone system. Future studies shall include testing potential benefits of pharmacological enhancement of the deacetylation activity of SIRT1 after the onset of the symptom.

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.

Immune cells often express various nicotinic ACh receptor (nAChR) subtypes, including α7 nAChRs, as well as mRNA encoding secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor-related peptide (SLURP)-1, an endogenous α7 nAChR allosteric ligand. We detected SLURP-1 immunoreactivity in CD205+ dendritic cells (DCs) residing in human tonsils. Phytohemagglutinin (PHA, 10μg/ml), a T cell activator, attenuated cell proliferation and increased the ACh content of MOLT-3 human leukemic T cells compared with the vehicle control. Methyllycaconitine (MLA, 100nM), a specific α7 nAChR antagonist, abolished all effects elicited by PHA. Recombinant (r)SLURP-1 (0.5μg/ml) attenuated peripheral blood mononuclear cell proliferation and increased ChAT gene expression and the ACh content in MOLT-3 cells compared with the control, all of which were abolished by MLA. This suggests SLURP-1 activates cholinergic transmission by potentiating ACh synthesis and its action at α7 nAChRs, thereby facilitating functional development of T cells. These findings support the notion that SLURP-1 acts as a key modulator of immune responses.

In adult rats (4-9 months), chronic nicotine infusion increases the basal level of acetylcholine (ACh) release in the cerebral cortex and enhances responses of cortical ACh release and cortical vasodilation elicited by nucleus basalis of Meynert (NBM) stimulation. In the present study, we examined whether these effects of nicotine are detected in aged rats. Aged rats (27-30 months) received sustained subcutaneous nicotine (100 μg/kg/h) or saline for 14 days. Under urethane anesthesia, ACh release and regional blood flow in the parietal cortex were measured. The basal level of ACh release in the cerebral cortex was not changed by chronic nicotine. In addition, the magnitudes of ACh release and vasodilation by NBM stimulation were similar between the saline-treated and nicotine-treated groups. The lack of an effect of chronic nicotine in aged rats may be due to a decrease in nicotinic receptors in the cerebral cortex during aging (Nordberg et al., J Neurosci Res 31:103-111, 1992).

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.

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.

Although synapsin has been localized to presynaptic structures, its function remains poorly understood. In the present study, we investigated the presynaptic function of synapsin II using a synaptic vesicle recycling process using synapsin-II-overexpressing NG108-15 cells. Western blot analysis with antibodies for synaptic-vesicle-associated protein indicated that the number of synaptic vesicles was approximately doubled in synapsin II transfectants as reported previously. In differentiated synapsin-II-overexpressing and control cells, the application of high potassium induced strong intracellular calcium elevation along neurites and varicosities after differentiation and a weak calcium rise in the cell bodies. The uptake and release of the fluorescent dye FM1-43 revealed that synaptic vesicle recycling in synapsin-II-transfected cells occurred with the same kinetics in the cell body and neuritic varicosities. Furthermore, the area labeled with FM1-43 fluorescence in the synapsin-II-transfected cells was approximately twice as much as in control cells after stimulation, and ATP released after synaptic vesicle fusion with the plasma membrane in synapsin-II-expressing cells was significantly elevated relative to controls. The number of synaptic vesicles paralleled the amount of transmitter released from the cells leading to the conclusion that the number of releasable synaptic vesicles were increased by synapsin II transfection into NG108-15 cells, suggesting that synapsin II may have a role in the regulation of synaptic vesicle number in presynapse-like structures in NG108-15 cells.

Two abnormal SOD 1 mRNAs, exon 2-skipping and exon 2 and 3-skipping species, were identified from occipital brain tissue of sporadic amyotrophic lateral sclerosis (ALS) patients carrying no mutations in the SOD 1 gene. Both transcripts were ubiquitously expressed in non-neuronal as well as neuronal tissues from a subject without neurological diseases. The expression pattern did not show disease specificity or lesional selectivity associated with ALS. Transient expression studies revealed weak expression of the proteins derived from the exon 2-skipping SOD 1 cDNA in a cell-free translation system but not in cells. The putative abnormal SOD 1 protein may accumulate and exert toxic effects on motor neurons in ALS when the proteolytic system is disturbed by aging or some causal factors. (C) 2000 Lippincott Williams and Wilkins.

Synapses are highly specialized intercellular junctions organized by adhesive and scaffolding molecules that align presynaptic vesicular release with postsynaptic neurotransmitter receptors. The MALS/Veli-CASK-Mint-1 complex of PDZ proteins occurs on both sides of the synapse and has the potential to link transsynaptic adhesion molecules to the cytoskeleton. In this study, we purified the MALS protein complex from brain and found liprin-α as a major component. Liprin proteins organize the presynaptic active zone and regulate neurotransmitter release. Fittingly, mutant mice lacking all three MALS isoforms died perinatally with difficulty breathing and impaired excitatory synaptic transmission. Excitatory postsynaptic currents were dramatically reduced in autaptic cultures from MALS triple knockout mice due to a presynaptic deficit in vesicle cycling. These findings are consistent with a model whereby the MALS-CASK-liprin-α complex recruits components of the synaptic release machinery to adhesive proteins of the active zone.