Airway inflammation in a novel mouse model of asthma-COPD overlap induced by co-exposure to papain and tobacco smoke.

Asthma and chronic obstructive pulmonary disease (COPD) are respiratory diseases associated with airway inflammation, which is the main pathogenesis. Although their causes and characteristics differ, in some cases, asthma and COPD may coexist in the same patient in a condition called asthma-COPD overlap (ACO). The prognosis of ACO is more unfavourable than those of asthma or COPD alone, without any treatment strategies demonstrating efficacy. Owing to its intricate spectrum of features, the detailed pathogenesis of how ACO exacerbates respiratory features remains unclear. In this study, we exposed papain-induced asthma model mice to tobacco smoke to establish an ACO mouse model, in which features of airway inflammation observed in both asthma and COPD were incorporated. This model exhibited distinctive mixed and corticosteroid-resistant airway inflammation and emphysematous changes that are characteristic of ACO. The novel mouse model established here is expected to significantly contribute to elucidating the mechanisms of the broad pathologies of ACO and identifying potential therapeutic targets.

Quantitative Microtubule Fractionation Technique to Separate Stable Microtubules, Labile Microtubules, and Free Tubulin in Mouse Tissues.

Microtubules, composed of α/ß-tubulin dimers, are a crucial component of the cytoskeleton in eukaryotic cells. These tube-like polymers exhibit dynamic instability as tubulin heterodimer subunits undergo repetitive polymerization and depolymerization. Precise control of microtubule stability and dynamics, achieved through tubulin post-translational modifications and microtubule-associated proteins, is essential for various cellular functions. Dysfunctions in microtubules are strongly implicated in pathogenesis, including neurodegenerative disorders. Ongoing research focuses on microtubule-targeting therapeutic agents that modulate stability, offering potential treatment options for these diseases and cancers. Consequently, understanding the dynamic state of microtubules is crucial for assessing disease progression and therapeutic effects. Traditionally, microtubule dynamics have been assessed in vitro or in cultured cells through rough fractionation or immunoassay, using antibodies targeting post-translational modifications of tubulin. However, accurately analyzing tubulin status in tissues using such procedures poses challenges. In this study, we developed a simple and innovative microtubule fractionation method to separate stable microtubules, labile microtubules, and free tubulin in mouse tissues. The procedure involved homogenizing dissected mouse tissues in a microtubule-stabilizing buffer at a 19:1 volume ratio. The homogenates were then fractionated through a two-step ultracentrifugation process following initial slow centrifugation (2,400 × g) to remove debris. The first ultracentrifugation step (100,000 × g) precipitated stable microtubules, while the resulting supernatant was subjected to a second ultracentrifugation step (500,000 × g) to fractionate labile microtubules and soluble tubulin dimers. This method determined the proportions of tubulin constituting stable or labile microtubules in the mouse brain. Additionally, distinct tissue variations in microtubule stability were observed that correlated with the proliferative capacity of constituent cells. These findings highlight the significant potential of this novel method for analyzing microtubule stability in physiological and pathological conditions.

Disulfide bond formation in microtubule-associated tau protein promotes tau accumulation and toxicity in vivo.

Accumulation of microtubule-associated tau protein is thought to cause neuron loss in a group of neurodegenerative diseases called tauopathies. In diseased brains, tau molecules adopt pathological structures that propagate into insoluble forms with disease-specific patterns. Several types of posttranslational modifications in tau are known to modulate its aggregation propensity in vitro, but their influence on tau accumulation and toxicity at the whole-organism level has not been fully elucidated. Herein, we utilized a series of transgenic Drosophila models to compare systematically the toxicity induced by five tau constructs with mutations or deletions associated with aggregation, including substitutions at seven disease-associated phosphorylation sites (S7A and S7E), deletions of PHF6 and PHF6* sequences (ΔPHF6 and ΔPHF6*), and substitutions of cysteine residues in the microtubule binding repeats (C291/322A). We found that substitutions and deletions resulted in different patterns of neurodegeneration and accumulation, with C291/322A having a dramatic effect on both tau accumulation and neurodegeneration. These cysteines formed disulfide bonds in mouse primary cultured neurons and in the fly retina, and stabilized tau proteins. Additionally, they contributed to tau accumulation under oxidative stress. We also found that each of these cysteine residues contributes to the microtubule polymerization rate and microtubule levels at equilibrium, but none of them affected tau binding to polymerized microtubules. Since tau proteins expressed in the Drosophila retina are mostly present in the early stages of tau filaments self-assembly, our results suggest that disulfide bond formation by these cysteine residues could be attractive therapeutic targets.

The inducible amphisome isolates viral hemagglutinin and defends against influenza A virus infection.

The emergence of drug-resistant influenza type A viruses (IAVs) necessitates the development of novel anti-IAV agents. Here, we target the IAV hemagglutinin (HA) protein using multivalent peptide library screens and identify PVF-tet, a peptide-based HA inhibitor. PVF-tet inhibits IAV cytopathicity and propagation in cells by binding to newly synthesized HA, rather than to the HA of the parental virus, thus inducing the accumulation of HA within a unique structure, the inducible amphisome, whose production from the autophagosome is accelerated by PVF-tet. The amphisome is also produced in response to IAV infection in the absence of PVF-tet by cells overexpressing ABC transporter subfamily A3, which plays an essential role in the maturation of multivesicular endosomes into the lamellar body, a lipid-sorting organelle. Our results show that the inducible amphisomes can function as a type of organelle-based anti-viral machinery by sequestering HA. PVF-tet efficiently rescues mice from the lethality of IAV infection.

Distribution of oxidized DJ-1 in Parkinson's disease-related sites in the brain and in the peripheral tissues: effects of aging and a neurotoxin.

DJ-1 plays an important role in antioxidant defenses, and a reactive cysteine at position 106 (Cys106) of DJ-1, a critical residue of its biological function, is oxidized under oxidative stress. DJ-1 oxidation has been reported in patients with Parkinson's disease (PD), but the relationship between DJ-1 oxidation and PD is still unclear. In the present study using specific antibody for Cys106-oxidized DJ-1 (oxDJ-1), we analyzed oxDJ-1 levels in the brain and peripheral tissues in young and aged mice and in a mouse model of PD induced using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). OxDJ-1 levels in the brain, heart, and skeletal muscle were high compared with other tissues. In the brain, oxDJ-1 was detected in PD-related brain sites such as the substantia nigra (SN) of the midbrain, olfactory bulb (OB), and striatum. In aged wild-type mice, oxDJ-1 levels in the OB, striatum, and heart tended to decrease, while those in the skeletal muscle increased significantly. Expression of dopamine-metabolizing enzymes significantly increased in the SN and OB of aged DJ-1-/- mice, accompanied by a complementary increase in glutathione peroxidase 1. MPTP treatment concordantly changed oxDJ-1 levels in PD-related brain sites and heart. These results indicate that the effects of physiological metabolism, aging, and neurotoxin change oxDJ-1 levels in PD-related brain sites, heart, and skeletal muscle where mitochondrial load is high, suggesting a substantial role of DJ-1 in antioxidant defenses and/or dopamine metabolism in these tissues.

Toxic tau oligomer formation blocked by capping of cysteine residues with 1,2-dihydroxybenzene groups.

Neurofibrillary tangles, composed of hyperphosphorylated tau fibrils, are a pathological hallmark of Alzheimer's disease; the neurofibrillary tangle load correlates strongly with clinical progression of the disease. A growing body of evidence indicates that tau oligomer formation precedes the appearance of neurofibrillary tangles and contributes to neuronal loss. Here we show that tau oligomer formation can be inhibited by compounds whose chemical backbone includes 1,2-dihydroxybenzene. Specifically, we demonstrate that 1,2-dihydroxybenzene-containing compounds bind to and cap cysteine residues of tau and prevent its aggregation by hindering interactions between tau molecules. Further, we show that orally administered DL-isoproterenol, an adrenergic receptor agonist whose skeleton includes 1,2-dihydroxybenzene and which penetrates the brain, reduces the levels of detergent-insoluble tau, neuronal loss and reverses neurofibrillary tangle-associated brain dysfunction. Thus, compounds that target the cysteine residues of tau may prove useful in halting the progression of Alzheimer's disease and other tauopathies.

Immunostaining of oxidized DJ-1 in human and mouse brains.

DJ-1, the product of a causative gene of a familial form of Parkinson disease, undergoes preferential oxidation of Cys106 (cysteine residue at position 106) under oxidative stress. Using specific monoclonal antibodies against Cys106 oxidized DJ-1 (oxDJ-1), we examined oxDJ-1 immunoreactivity in brain sections from DJ-1 knockout and wild-type mice and in human brain sections from cases classified into different Lewy body stages of Parkinson disease and Parkinson disease with dementia. Oxidized DJ-1 immunoreactivity was prominently observed in neuromelanin-containing neurons and neuron processes of the substantia nigra; Lewy bodies also showed oxDJ-1 immunoreactivity. Oxidized DJ-1 was also detected in astrocytes in the striatum, in neurons and glia in the red nucleus, and in the inferior olivary nucleus, all of which are related to regulation of movement. These observations suggest the relevance of DJ-1 oxidation to homeostasis in multiple brain regions, including neuromelanin-containing neurons of the substantia nigra, and raise the possibility that oxDJ-1 levels might change during the progression of Lewy body-associated neurodegenerative diseases.

Substrate ectodomain is critical for substrate preference and inhibition of γ-secretase.

Understanding the substrate recognition mechanism of γ-secretase is a key step for establishing substrate-specific inhibition of amyloid ß-protein (Aß) production. However, it is widely believed that γ-secretase is a promiscuous protease and that its substrate-specific inhibition is elusive. Here we show that γ-secretase distinguishes the ectodomain length of substrates and preferentially captures and cleaves substrates containing a short ectodomain. We also show that a subset of peptides containing the CDCYCxxxxCxCxSC motif binds to the amino terminus of C99 and inhibits Aß production in a substrate-specific manner. Interestingly, these peptides suppress ß-secretase-dependent cleavage of APP, but not that of sialyltransferase 1. Most importantly, intraperitoneal administration of peptides into mice results in a significant reduction in cerebral Aß levels. This report provides direct evidence of the substrate preference of γ-secretase and its mechanism. Our results demonstrate that the ectodomain of C99 is a potent target for substrate-specific anti-Aß therapeutics to combat Alzheimer's disease.

Elevation of oxidized DJ-1 in the brain and erythrocytes of Parkinson disease model animals.

DJ-1, the causative gene of a familial form of Parkinson's disease (PD), has been reported undergo oxidation preferentially at the 106th cysteine residue (Cys-106) under oxidative stress. Recently, it has been found that the levels of oxidized DJ-1 in erythrocytes of unmedicated PD patients are markedly higher than those in medicated PD patients and healthy subjects. In the present study, we examined the changes in oxidized DJ-1 levels in the brain and erythrocytes of PD animal models using specific antibodies against Cys-106-oxidized DJ-1. Treatment with PD model compounds such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine significantly elevated the levels of oxidized DJ-1 in erythrocytes. Immunohistochemical analysis also revealed that the number of oxidized DJ-1 antibody-positive cells in the substantia nigra of MPTP-treated mouse increased in a dose-dependent manner. These results suggest that the oxidative modification of DJ-1 in the brain and erythrocytes is involved in the pathogenesis of PD in animal models.

Synthesis and structure-activity relationships of 5,6,7,8-tetrahydropyrido[3,4-b]pyrazine-based hydroxamic acids as HB-EGF shedding inhibitors.

HB-EGF Shedding inhibitors have been expected to become effective medicines for skin diseases caused by the proliferation of keratinocytes. In order to discover novel HB-EGF shedding inhibitors and clarify their structure-activity relationships, 5,6,7,8-tetrahydronaphthylidine-based hydroxamic acid and 5,6,7,8-tetrahydropyrido[3,4-b]pyrazine-based hydroxamic acids have been synthesized. Among the synthesized compounds, the ethoxyethoxy derivative 3o and the methoxypropoxy derivative 3p exhibited much more potent HB-EGF shedding inhibitory activity than CGS 27023A. The structural modification of 5,6,7,8-tetrahydropyrido[3,4-b]pyrazine-based hydroxamic acids enabled us to establish the following structure-activity relationships; the existence of the hydroxamic acid, the sulfonamide, and the phenyl moieties are crucial for a potent HB-EGF shedding inhibitory activity, and the stereochemistry of the alpha carbon of hydroxamic acid is also important. In addition, from the comparison of their HB-EGF shedding inhibitory activities with their MMPs inhibitory activities, we found that the S1' pocket of the responsible enzyme for HB-EGF shedding is deep unlike that of MMP-1.

Aberrant tau phosphorylation by glycogen synthase kinase-3beta and JNK3 induces oligomeric tau fibrils in COS-7 cells.

Neurofibrillary tangles (NFTs) are found in a wide range of neurodegenerative disorders, including Alzheimer's disease. The major component of NFTs is aberrantly hyperphosphorylated microtubule-associated protein tau. Because appropriate in vivo models have been lacking, the role of tau phosphorylation in NFTs formation has remained elusive. Here, we describe a new model in which adenovirus-mediated gene expression of tau, DeltaMEKK, JNK3, and GSK-3beta in COS-7 cells produces most of the pathological phosphorylation epitopes of tau including AT100. Furthermore, this co-expression resulted in the formation of tau aggregates having short fibrils that were detergent-insoluble and Thioflavin-S-reactive. These results suggest that aberrant tau phosphorylation by the combination of these kinases may be involved in "pretangle," oligomeric tau fibril formation in vivo.

New type of metalloproteinase inhibitor: design and synthesis of new phosphonamide-based hydroxamic acids.

A series of phosphonamide-based hydroxamate derivatives were synthesized, and the inhibitory activities were evaluated against various metalloproteinases in order to clarify its selectivity profile. Among the four diastereomeric isomers resulting from the chirality at the C-3 and P atoms, the compound with a (R,R)-configuration both at the C-3 position and the phosphorus atom was found to be potently active, while the other diastereomeric isomers were almost inactive. A number of (R,R)-compounds synthesized here exhibited broad spectrum activities with nanomolar K(i) values against MMP-1, -3, -9, and TACE and also showed nanomolar IC(50) values against HB-EGF shedding in a cell-based inhibition assay. The modeling study using X-ray structure of MMP-3 suggested the possible binding mode of the phosphonamide-based inhibitors.