Takashi Inui

A toxic monomeric conformer of the polyglutamine protein

Polyglutamine (polyQ) diseases are classified as conformational neurodegenerative diseases, like Alzheimer and Parkinson diseases, and they are caused by proteins with an abnormally expanded polyQ stretch. However, conformational changes of the expanded polyQ protein and the toxic conformers formed during aggregation have remained poorly understood despite their important role in pathogenesis. Here we show that a β-sheet conformational transition of the expanded polyQ protein monomer precedes its assembly into β-sheet–rich amyloid-like fibrils. Microinjection of the various polyQ protein conformers into cultured cells revealed that the soluble β-sheet monomer causes cytotoxicity. The polyQ-binding peptide QBP1 prevents the toxic β-sheet conformational transition of the expanded polyQ protein monomer. We conclude that the toxic conformational transition, and not simply the aggregation process itself, is a therapeutic target for polyQ diseases and possibly for conformational diseases in general.

Prostaglandin D2-Mediated Microglia/Astrocyte Interaction Enhances Astrogliosis and Demyelination in twitcher

Prostaglandin (PG) D2 is well known as a mediator of inflammation. Hematopoietic PGD synthase (HPGDS) is responsible for the production of PGD2 involved in inflammatory responses. Microglial activation and astrogliosis are commonly observed during neuroinflammation, including that which occurs during demyelination. Using the genetic demyelination mouse twitcher, a model of human Krabbe’s disease, we discovered that activated microglia expressed HPGDS and activated astrocytes expressed the DP1 receptor for PGD2 in the brain of these mice. Cultured microglia actively produced PGD2 by the action of HPGDS. Cultured astrocytes expressed two types of PGD2 receptor, DP1 and DP2, and showed enhanced GFAP production after stimulation of either receptor with its respective agonist. These results suggest that PGD2 plays an important role in microglia/astrocyte interaction. We demonstrated that the blockade of the HPGDS/PGD2/DP signaling pathway using HPGDS- or DP1-null twitcher mice, and twitcher mice treated with an HPGDS inhibitor, HQL-79 (4-benzhydryloxy-1-[3-(1H-tetrazol-5-yl)-propyl]piperidine), resulted in remarkable suppression of astrogliosis and demyelination, as well as a reduction in twitching and spasticity. Furthermore, we found that the degree of oligodendroglial apoptosis was also reduced in HPGDS-null and HQL-79-treated twitcher mice. These results suggest that PGD2 is the key neuroinflammatory molecule that heightens the pathological response to demyelination in twitcher mice.

Characterization of the Unfolding Process of Lipocalin-type Prostaglandin D Synthase

We found that low concentrations of guanidine hydrochloride (GdnHCl, <0.75 m) or urea (<1.5 m) enhanced the enzyme activity of lipocalin-type prostaglandin (PG) D synthase (L-PGDS) maximally 2.5- and 1.6-fold at 0.5 m GdnHCl and 1 m urea, respectively. The catalytic constants in the absence of denaturant and in the presence of 0.5 m GdnHCl or 1 m urea were 22, 57, and 30 min−1, respectively, and the K m values for the substrate, PGH2, were 2.8, 8.3, and 2.3 μm, respectively, suggesting that the increase in the catalytic constant was mainly responsible for the activation of L-PGDS. The intensity of the circular dichroism (CD) spectrum at 218 nm, reflecting the β-sheet content, was also increased by either denaturant in a concentration-dependent manner, with the maximum at 0.5 m GdnHCl or 1 m urea. By plotting the enzyme activities against the ellipticities at 218 nm of the CD spectra of L-PGDS in the presence or absence of GdnHCl or urea, we found two states in the reversible folding process of L-PGDS: one is an activity-enhanced state and the other, an inactive state. The NMR analysis of L-PGDS revealed that the hydrogen-bond network was reorganized to be increased in the activity-enhanced state formed in the presence of 0.5 m GdnHCl or 1 m urea and to be decreased but still remain in the inactive intermediate observed in the presence of 2 m GdnHCl or 4 m urea. Furthermore, binding of the nonsubstrate ligands, bilirubin or 13-cis-retinal, to L-PGDS changed from a multistate mode in the native form of L-PGDS to a simple two-state mode in the activity-enhanced form, as monitored by CD spectra of the bound ligands. Therefore, L-PGDS is a unique protein whose enzyme activity and ligand-binding property are biphasically altered during the unfolding process by denaturants.

Lipocalin-type prostaglandin D synthase (beta-trace) in cerebrospinal fluid: a useful marker for the diagnosis of normal pressure hydrocephalus.

Lipocalin-type prostaglandin D synthase (PGDS) is considered to be mainly produced in the leptomeninges and secreted into cerebrospinal fluid (CSF). We found PGDS levels in CSF of patients with normal pressure hydrocephalus (NPH) (8.99+/-2.59 microg/ml, mean+/-S.D., n=14) to be significantly lower than levels in a control (15.29+/-5.17, n=14, P<0.0001) and other dementia group (19.14+/-4.34, n=7, P=0.0003). Thus, PGDS level in CSF is a useful marker for the differential diagnosis of NPH from other diseases with dementia.

Up-Regulated Neuronal COX-2 Expression After Cortical Spreading Depression Is Involved in Non-REM Sleep Induction in Rats

Cortical spreading depression is an excitatory wave of depolarization spreading throughout cerebral cortex at a rate of 2–5 mm/min and has been implicated in various neurological disorders, such as epilepsy, migraine aura, and trauma. Although sleepiness or sleep is often induced by these neurological disorders, the cellular and molecular mechanism has remained unclear. To investigate whether and how the sleep‐wake behavior is altered by such aberrant brain activity, we induced cortical spreading depression in freely moving rats, monitoring REM and non‐REM (NREM) sleep and sleep‐associated changes in cyclooxygenase (COX)‐2 and prostaglandins (PGs). In such a model for aberrant neuronal excitation in the cerebral cortex, the amount of NREM sleep, but not of REM sleep, increased subsequently for several hours, with an up‐regulated expression of COX‐2 in cortical neurons and considerable production of PGs. A specific inhibitor of COX‐2 completely arrested the increase in NREM sleep. These results indicate that up‐regulated neuronal COX‐2 would be involved in aberrant brain excitation‐induced NREM sleep via production of PGs.

Brefeldin A inhibits osteoclastic bone resorption through induction of apoptosis.

Brefeldin A (BFA), a fungal metabolite with a macrocyclic lactone structure, has been developed for the treatment of cancer, and its major biological activity is the inhibition of intracellular protein transport from the endoplasmic reticulum to the cis-Golgi apparatus. In this study, we investigated the effect of BFA on osteoclastic pit formation in vitro. BFA reduced pit formation in a concentration-dependent manner, and the IC50 values on the pit number and the pit volume were 11.3 +/- 2.2 and 13.3 +/- 2.0 nM, respectively. In parallel with the inhibitory effect on pit formation, BFA also reduced the cell viability of osteoclasts-enriched bone cells with an IC50 value of 13.9 +/- 2.2 nM. These results suggest that the inhibition of bone resorption by BFA is caused by the induction of osteoclast cell death. BFA at a concentration of 100 nM induced DNA fragmentation in purified osteoclasts, assessed by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and DNA ladder formation, demonstrating that BFA induces cell death of osteoclasts in an apoptotic manner. In addition, the accumulation of p53 proteins to the nuclei was observed in the osteoclasts treated with 100 nM BFA. These results, taken together, suggest that BFA inhibits osteoclastic bone resorption by inducing apoptosis in osteoclasts through a p53-dependent mechanism.

Thermal unfolding mechanism of lipocalin-type prostaglandin D synthase

Lipocalin‐type prostaglandin (PG)u2003D synthase (L‐PGDS) is a dual‐functioning protein in the lipocalin family, acting as a PGD2‐synthesizing enzyme and as an extracellular transporter for small lipophilic molecules. We earlier reported that denaturant‐induced unfolding of L‐PGDS follows a four‐state pathway, including an activity‐enhanced state and an inactive intermediate state. In this study, we investigated the thermal unfolding mechanism of L‐PGDS by using differential scanning calorimetry (DSC) and CD spectroscopy. DSC measurements revealed that the thermal unfolding of L‐PGDS was a completely reversible process at pHu20034.0. The DSC curves showed no concentration dependency, demonstrating that the thermal unfolding of L‐PGDS involved neither intermolecular interaction nor aggregation. On the basis of a simple two‐state unfolding mechanism, the ratio of van’t Hoff enthalpy (ΔHvH) to calorimetric enthalpy (ΔHcal) was below 1, indicating the presence of an intermediate state (I) between the native state (N) and unfolded state (U). Then, statistical thermodynamic analyses of a three‐state unfolding process were performed. The heat capacity curves fit well with a three‐state process; and the estimated transition temperature (Tm) and enthalpy change (ΔHcal) of the N↔I and I↔U transitions were 48.2u2003°C and 190u2003kJ·mol−1, and 60.3u2003°C and 144u2003kJ·mol−1, respectively. Correspondingly, the thermal unfolding monitored by CD spectroscopy at 200, 235 and 290u2003nm revealed that L‐PGDS unfolded through the intermediate state, where its main chain retained the characteristic β‐sheet structure without side‐chain interactions.

Lipocalin-type prostaglandin D synthase in cerebrospinal fluid of patients with aneurysmal subarachnoid hemorrhage scavenges bile pigments

Abstract Lipocalin-type prostaglandin D synthase (L-PGDS) is one of the major proteins in human cerebrospinal fluid (CSF) and binds various small lipophilic substances, such as bilirubin and biliverdin. The L-PGDS concentration increased in the CSF of patients with aneurysmal subarachnoid hemorrhage (SAH) from day 1 (10.5 μg/ml) to day 5 (25.6) and returned to the basal level after 2 months (12.9). L-PGDS purified from the CSF of SAH patients showed an absorbance spectrum with a peak at 392 nm in the visible range. The absorbance at 392 nm of the purified L-PGDS increased from day 1 (0.16/mg protein) to day 5 (0.44) and almost disappeared 2 months after SAH (0.11). Conversely, the enzyme activity of the purified L-PGDS decreased from day 1 (0.71 μmol/min/mg protein) to day 5 (0.28) and recovered 2 months after SAH (0.76). These results indicate that L-PGDS binds and scavenges bile pigments that accumulate in the CSF after SAH.

Sleep in transgenic and gene-knockout mice for lipocalin-type prostaglandin D synthase

Abstract Prostaglandin (PG) D 2 is a potent sleep-inducing substance in mammals. We investigated the effects of overproduction and deficiency of endogenous PGD 2 on sleep regulation by using transgenic (TG) mice overexpressing human lipocalin-type PGD synthase (L-PGDS) gene and gene-knockout (KO) mice produced by a null mutation of the L-PGDS gene, respectively. The circadian rhythm of non-rapid eye movement (NREM) and REM sleep was almost unchanged among wild-type (WT), TG, and KO mice. However, we found that noxious stimulation by tail clipping (Tc) of TG, but not of WT, mice transiently (∼5 h) increased the amount of NREM sleep without affecting REM sleep. This increase was coupled with elevation of the PGD 2 content in the brain, suggesting that the overproduction of PGD 2 in the brain by Tc induced NREM sleep in TG mice. We also found that sleep deprivation (SD) for 6 h induced rebound of NREM sleep in WT, but not in KO, mice, and of REM sleep in both of these mice, suggesting that the L-PGDS gene is involved in the homeostasis of NREM sleep after SD. Therefore, PGD 2 endogenously produced by L-PGDS in the brain is considered to regulate NREM sleep after noxious stimulation or SD.