Published papers

Differential vulnerability among motor neuron (MN) subtypes is a fundamental feature of amyotrophic lateral sclerosis (ALS): fast-fatigable (FF) MNs are more vulnerable than fast fatigue-resistant (FR) or slow (S) MNs. The reason for this selective vulnerability remains enigmatic. We report here that the extracellular matrix (ECM) protein osteopontin (OPN) is selectively expressed by FR and S MNs and ALS-resistant motor pools, whereas matrix metalloproteinase-9 (MMP-9) is selectively expressed by FF MNs. OPN is secreted and accumulated as extracellular granules in ECM in three ALS mouse models and a human ALS patient. In SOD1 G93A mice, OPN/MMP-9 double positivity marks remodeled FR and S MNs destined to compensate for lost FF MNs before ultimately dying. Genetic ablation of OPN in SOD1 G93A mice delayed disease onset but then accelerated disease progression. OPN induced MMP-9 up-regulation via αvβ3 integrin in ChAT-expressing Neuro2a cells, and also induced CD44-mediated astrocyte migration and microglial phagocytosis in a non-cell-autonomous manner. Our results demonstrate that OPN expressed by FR/S MNs is involved in the second-wave neurodegeneration by up-regulating MMP-9 through αvβ3 integrin in the mouse model of ALS. The differences in OPN/MMP-9 expression profiles in MN subsets partially explain the selective MN vulnerability in ALS.

Background SLURP1 is the causal gene for Mal de Meleda (MDM), an autosomal recessive skin disorder characterized by diffuse palmoplantar keratoderma and transgressive keratosis. Moreover, although SLURP1 likely serves as an important proliferation/differentiation factor in keratinocytes, the possible relation between SLURP1 and other skin diseases, such as psoriasis and atopic dermatitis, has not been studied, and the pathophysiological control of SLURP1 expression in keratinocytes is largely unknown. Objectives Our aim was to examine the involvement of SLURP1 in the pathophysiology of psoriasis using an imiquimod (IMQ)-induced psoriasis model mice and normal human epidermal keratinocytes (NHEKs). Results SLURP1 expression was up-regulated in the skin of IMQ-induced psoriasis model mice. In NHEKs stimulated with the inflammatory cytokines IL-17, IL-22 and TNF-α, which are reportedly expressed in psoriatic lesions, SLURP1 mRNA expression was significantly upregulated by IL-22 but not the other two cytokines. The stimulatory effect of IL-22 was completely suppressed in NHEKs treated with a STAT3 inhibitor or transfected with siRNA targeting STAT3. Because IL-22 induces production of antimicrobial proteins in epithelial cells, the antibacterial activity of SLURP1 was assessed against Staphylococcus aureus (S. aureus), which is known to be associated with disease severity in psoriasis. SLURP1 significantly suppressed the growth of S. aureus. Conclusions These results indicate SLURP1 participates in pathophysiology of psoriasis by regulating keratinocyte proliferation and differentiation, and by suppressing the growth of S. aureus.

A novel nicotinic acetylcholine (ACh) receptor (nAChR)-mediated transduction pathway, regulating keratinocyte function, has been elucidated in studies of secreted mammalian Ly6/urokinase plasminogen activator receptor-related protein (SLURP)-1 and -2. SLURPs are members of Ly6/neurotoxin superfamily (Ly6SF) of proteins containing the unique three-finger domain in their three-dimensional structure. Some endogenously expressed Ly6SF proteins (such as LYNX1, SLURP-1, and SLURP-2) modulate the function of nAChR, either as allosteric and/or orthosteric modulators, or as antagonists. Although the expression and functions of SLURP-1 and SLURP-2 in keratinocytes are well documented, the expression and the modes of action of LYNX1 in keratinocytes are unknown. Additionally, a particular hybrid transcript, LYNX1-SLURP2, which contains both LYNX1 and SLURP-2 sequences, with unknown function, has been reported. Furthermore, although SLURP2 is a gene strongly induced in psoriatic skin lesions, the mechanisms controlling SLURP2 expression are largely unknown. To better understand the function of nAChRs in keratinocytes, we investigated the expression profiles of LYNX1, LYNX1-SLURP-2, and SLURP-2 in keratinocytes under various inflammatory conditions. We found that keratinocytes express LYNX1 and SLURP2, but not LYNX1-SLURP2, at mRNA and protein levels. IL-22 treatment increased SLURP2 expression in keratinocytes, but this effect was completely abolished by IFN-γ. Furthermore, the IL-22-induced up-regulation of SLURP2 was completely suppressed by the inhibitor or siRNA for STAT3, a major transcriptional factor downstream of IL-22. These findings provide new insights into the nAChR-mediated regulatory mechanism of SLURP-2 expression in keratinocytes.

In 1929, Dale and Dudley described the first reported natural occurrence of acetylcholine (ACh) in an animal's body. They identified this ACh in the spleens of horses and oxen, which we now know suggests possible involvement of ACh in the regulation of lymphocyte activity and immune function. However, the source and function of splenic ACh were left unexplored for several decades. Recent studies on the source of ACh in the blood revealed ACh synthesis catalyzed by choline acetyltransferase (ChAT) in CD4+ T cells. T and B cells, macrophages and dendritic cells (DCs) all express all five muscarinic ACh receptor subtypes (mAChRs) and several subtypes of nicotinic AChRs (nAChRs), including α7 nAChRs. Stimulation of these mAChRs and nAChRs by their respective agonists causes functional and biochemical changes in the cells. Using AChR knockout mice, we found that M1/M5 mAChR signaling up-regulates IgG1 and pro-inflammatory cytokine production, while α7 nAChR signaling has the opposite effect. These findings suggest that ACh synthesized by T cells acts in an autocrine/paracrine fashion at AChRs on various immune cells to modulate immune function. In addition, an endogenous allosteric and/or orthosteric α7 nAChR ligand, SLURP-1, facilitates functional development of T cells and increases ACh synthesis via up-regulation of ChAT mRNA expression. SLURP-1 is expressed in CD205+ DCs residing in the tonsil in close proximity to T cells, macrophages and B cells. Collectively, these findings suggest that ACh released from T cells along with SLURP-1 regulates cytokine production by activating α7 nAChRs on various immune cells, thereby facilitating T cell development and/or differentiation, leading to immune modulation.

Purpose. The secreted Ly6/uPAR-related protein-1 (Slurp1), associated with the hyperkeratotic disorder mal de Meleda, is abundantly expressed in corneas. Here, we examine its corneal expression and functions. Methods. Gene expression was quantified by quantitative PCR (qPCR), immunoblots, and immunofluorescent staining. Effect of Kruppel-like factor 4 (Klf4) on Slurp1 promoter was evaluated by chromatin immunoprecipitation (ChIP) and transient transfections. Adenoviral vectors were used to express Slurp1 in corneas. Leukocytic infiltration in bacterial lipopolysaccharide (LPS)-, herpes simplex virus type 1 (HSV-1)-, or adenovirus (serotype 5)-treated mouse corneas was characterized by flow cytometry. Results. Corneal expression of Slurp1 increased sharply upon mouse eyelid opening, concurrent with the elevated expression of Klf4. Slurp1 was significantly decreased in Klf4 conditional null (Klf4CN) corneas that displayed elevated expression of cytokines and cytokine receptors, as well as neutrophil influx consistent with a proinflammatory environment. In additional models of corneal inflammation, Slurp1 expression was abrogated within 24 hours of LPS injection or HSV-1 or adenoviral infection, accompanied by a predominantly neutrophilic infiltrate. Neutrophilic infiltration was enhanced in HSV-1-infected Klf4CN corneas lacking Slurp1. SLURP1 promoter activity was stimulated by KLF4, suppressed by IL-4, IL-13, and TNFα, and unperturbed by IFN-γ. Slurp1 downregulation and neutrophil influx were comparable in HSV-1-infected wild-type (WT) and Ifng-/- mouse corneas. Mouse corneas infected with Slurp1-expressing adenoviral vectors displayed reduced signs of inflammation and restricted neutrophilic infiltration compared with those infected with control vectors. Conclusions. Klf4 regulates the expression of Slurp1, a key immunomodulatory peptide that is abundantly expressed in healthy corneas and is downregulated in proinflammatory conditions.

Evidence suggests that protein misfolding is crucially involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). However, controversy still exists regarding the involvement of proteasomes or autophagy in ALS due to previous conflicting results. Here, we show that impairment of the ubiquitin-proteasome system, but not the autophagy-lysosome system in motor neurons replicates ALS in mice. Conditional knock-out mice of the proteasome subunit Rpt3 in a motor neuron-specific manner (Rpt3-CKO) showed locomotor dysfunction accompanied by progressive motor neuron loss and gliosis. Moreover, diverse ALS-linked proteins, including TAR DNA-binding protein 43 kDa (TDP-43), fused in sarcoma (FUS), ubiquilin 2, and optineurin were mislocalized or accumulated in motor neurons, together with other typical ALS hallmarks such as basophilic inclusion bodies. On the other hand, motor neuron-specific knock-out of Atg7, a crucial component for the induction of autophagy (Atg7-CKO), only resulted in cytosolic accumulation of ubiquitin and p62, and no TDP-43 or FUS pathologies or motor dysfunction was observed. These results strongly suggest that proteasomes, but not autophagy, fundamentally govern the development of ALS in which TDP-43 and FUS proteinopathy may play a crucial role. Enhancement of proteasome activity may be a promising strategy for the treatment of ALS.

Lymphocytes express both muscarinic and nicotinic acetylcholine (ACh) receptors (mAChRs and nAChRs, respectively), and stimulation of mAChRs and nAChRs produces various biochemical and functional changes. Although it has been postulated that parasympathetic cholinergic nerves directly innervate immune cells, no evidence has supported this hypothesis. We measured ACh in the blood of various animal species and determined its localization in T cells using a sensitive and specific radioimmunoassay. Furthermore, we showed that T cells express choline acetyltransferase (ChAT), an ACh synthesizing enzyme. Immunological T cell activation enhances ACh synthesis through the up-regulation of ChAT expression, suggesting lymphocytic cholinergic activity is related to immunological activity. Most immune cells such as T cells, B cells, and monocytes express all five subtypes of mAChRs (M1-M5), and various subunits of the nAChR, such as α3, α5, α7, α9, and α10. Studies on serum antibody production in M1 and M 5 combined mAChR gene knockout (KO) mice immunized with ovalbumin (OVA) revealed that M1/M5 mAChRs up-regulate TNF-α, IFN-γ and IL-6 production in spleen cells, leading to an elevation of serum anti-OVA specific IgG1. In contrast, studies of nAChR α7 subunit gene KO mice immunized with OVA show that α7 nAChRs down-regulate these proinflammatory cytokines, thereby leading to a reduction of anti-OVA specific IgG1. Taken together, these findings demonstrate that both mAChRs and nAChRs modulate production of cytokines, such as TNF-α, resulting in a modification of antibody production. These findings support the notion that a non-neuronal cholinergic system is involved in the regulation of immune cell function.

It has been long speculated that specific signals are transmitted from photoreceptors to the retinal pigment epithelium (RPE). However, such signals have not been identified. In this study, we examined the retinal expression and localization of acetylcholine-related molecules as putative candidates for these signals. Previous reports revealed that α7 nicotinic acetylcholine receptors (nAChRs) are present in the microvilli of RPE cells that envelope the tips of photoreceptor outer segments (OS). Secreted mammalian leukocyte antigen 6/urokinase-type plasminogen activator receptor-related protein-1 (SLURP-1) is a positive allosteric modulator of the α7 nAChR. Therefore, we first focused on the expression of SLURP-1. SLURP-1 mRNA was expressed in the outer nuclear layer, which is comprised of photoreceptor cell bodies. SLURP-1 immunoreactivity co-localized with rhodopsin and S-opsin in photoreceptor OS, while choline acetyltransferase (ChAT) and high affinity choline transporter (CHT-1) were also expressed in photoreceptor OS. Immunoelectron microscopy identified that the majority of SLURP-1 was localized to the plasma membranes of photoreceptor OS. These results provide evidence that SLURP-1 is synthesized in photoreceptor cell bodies and transported to photoreceptor OS, where SLURP-1 may also be secreted. Our findings suggest that photoreceptor OS communicate via neurotransmitters such as ACh and SLURP-1, while RPE cells might receive these signals through α7 nAChRs in their microvilli.

Immune cells, including lymphocytes, express muscarinic and nicotinic acetylcholine (ACh) receptors (mAChRs and nAChRs, respectively), and agonist stimulation of these AChRs causes functional and biochemical changes in the cells. The origin of the ACh that acts on immune cell AChRs has remained unclear until recently, however. In 1995, we identified choline acetyltransferase mRNA and protein in human T cells, and found that immunological T cell activation potentiated lymphocytic cholinergic transmission by increasing ACh synthesis and AChR expression. We also found that M1/M5 mAChR signaling upregulates IgG1 and proinflammatory cytokine production, whereas α7 nAChR signaling has the opposite effect. These findings suggest that ACh synthesized by T cells acts as an autocrine and/or paracrine factor via AChRs on immune cells to modulate immune function. In addition, a recently discovered endogenous allosteric α7 nAChR ligand, SLURP-1, also appears to be involved in modulating normal T cell function.

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.

Brain-derived neurotrophic factor (BDNF) and its receptor tropomyosin-related kinase B (TrkB) are widely expressed in the vertebrate nervous system and play a central role in mature neuronal function. In vitro BDNF/TrkB signaling promotes neuronal survival and can help neurons resist toxic insults. Paradoxically, BDNF/TrkB signaling has also been shown, under certain in vitro circumstances, to render neurons vulnerable to insults. We show here that in vivo conditional deletion of TrkB from mature motor neurons attenuates mutant superoxide dismutase 1 (SOD1) toxicity. Mutant SOD1 mice lacking motor neuron TrkB live a month longer than controls and retain motor function for a longer period, particularly in the early phase of the disease. These effects are subserved by slowed motor neuron loss, persistence of neuromuscular junction integrity and reduced astrocytic and microglial reactivity within the spinal cord. These results suggest that manipulation of BDNF/TrkB signaling might have therapeutic efficacy in motor neuron diseases.

Cholinergic neurons are endowed with a high-affinity choline uptake system for efficient synthesis of acetylcholine at the presynaptic terminals. The high-affinity choline transporter CHT1 is responsible for choline uptake, the rate-limiting step in acetylcholine synthesis. However, endogenous physiological factors that affect CHT1 expression or function and consequently regulate the acetylcholine synthesis rate are essentially unknown. Here we demonstrate that extracellular substrate decreases the cell-surface expression of CHT1 in rat brain synaptosomes, primary cultures from the basal forebrain, and mammalian cell lines transfected with CHT1. Extracellular choline rapidly decreases cell-surface CHT1 expression by accelerating its internalization, a process that is mediated by a dynamin-dependent endocytosis pathway in HEK293 cells. Specific inhibitor hemicholinium-3 decreases the constitutive internalization rate and thereby increases cell-surface CHT1 expression. We also demonstrate that the constitutive internalization of CHT1 depends on extracellular pH in cultured cells. Our results collectively suggest that the internalization of CHT1 is induced by extracellular substrate, providing a novel feedback mechanism for the regulation of acetylcholine synthesis at the cholinergic presynaptic terminals.

The present study examined the effects of sustained nicotine exposure on the cholinergic vasodilative system originating in the nucleus basalis of Meynert (NBM) and projecting to the cerebral cortex in rats. Rats received sustained subcutaneous nicotine (100 μg/kg/h) for 14 days. Under urethane anesthesia, the vasodilation response and acetylcholine release in the parietal cortex induced by electrical stimulation of the NBM (10-200 μA) were measured. The basal level of acetylcholine release was significantly higher in nicotine-treated rats than in saline-treated control rats. In the control rats, both the acetylcholine release and blood flow were increased by NBM stimulation in a stimulus intensity-dependent manner, and a threshold of 50 μA. In nicotine-treated rats, the threshold intensity of NBM stimulation producing increases in acetylcholine release and blood flow was reduced to 20 μA. The stimulus intensity-dependent acetylcholine release and vasodilation by NBM stimulation were significantly larger in nicotine-treated rats than in control rats. We conclude that sustained subcutaneous infusion of nicotine enhances cholinergic vasodilative system in the cerebral cortex originating in the NBM.

GluR2 is a subunit of the AMPA receptor, and the adenosine for the Q/R site of its pre-mRNA is converted to inosine (A-to-I conversion) by the enzyme called adenosine deaminase acting on RNA 2 (ADAR2). Failure of A-to-I conversion at this site affects multiple AMPA receptor properties, including the Ca 2+ permeability of the receptor-coupled ion channel, thereby inducing fatal epilepsy in mice (Brusa et al., 1995; Feldmeyer et al., 1999). In addition, inefficient GluR2 Q/R site editing is a disease-specific molecular dysfunction found in the motor neurons of sporadic amyotrophic lateral sclerosis (ALS) patients (Kawahara et al., 2004). Here, we generated genetically modified mice (designated as AR2) in which the ADAR2 gene was conditionally targeted in motor neurons using the Cre/loxP system. These AR2 mice showed a decline in motor function commensurate with the slow death of ADAR2-deficient motor neurons in the spinal cord and cranial motor nerve nuclei. Notably, neurons in nuclei of oculomotor nerves, which often escape degeneration in ALS, were not decreased in number despite a significant decrease in GluR2 Q/R site editing. All cellular and phenotypic changes in AR2 mice were prevented when the mice carried endogenous GluR2 alleles engineered to express edited GluR2 without ADAR2 activity (Higuchi et al., 2000). Thus, loss of ADAR2 activity causes AMPA receptor-mediated death of motor neurons.

Whereas acetylcholine (ACh) acts as a bronchoconstrictor and stimulator of mucus secretion from bronchial epithelium, it acts via α7 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-α (TNF-α). 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 α7 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.

Defective RNA metabolism is an emerging mechanism involved in ALS pathogenesis and possibly in other neurodegenerative disorders. Here, we show that microRNA (miRNA) activity is essential for long-term survival of postmitotic spinal motor neurons (SMNs) in vivo. Thus, mice that do not process miRNA in SMNs exhibit hallmarks of spinal muscular atrophy (SMA), including sclerosis of the spinal cord ventral horns, aberrant end plate architecture, and myofiber atrophy with signs of denervation. Furthermore, a neurofilament heavy subunit previously implicated in motor neuron degeneration is specifically up-regulated in miRNA-deficient SMNs. We demonstrate that the heavy neurofilament subunit is a target of miR-9, a miRNA that is specifically down-regulated in a genetic model of SMA. These data provide evidence for miRNA function in SMN diseases and emphasize the potential role of miR-9-based regulatory mechanisms in adult neurons and neurodegenerative states.

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 [3H]hemicholinium-3 (HC-3) and greater [ 3H]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.

Mammalian secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor-related peptide-1 (SLURP-1) is a positive allosteric ligand for α7 nicotinic acetylcholine (ACh) receptors (α7 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-α (TNFα) release from keratinocytes and macrophages via α7 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-α release from macrophages in bronchial tissue.