Objective —The growth factor progranulin (PGRN) is a widely expressed protein with various biological functions, and it is known to have an anti-inflammatory effect. The aim of this study was to investigate the possible ameliorative effects of PGRN against cerebral ischemia-reperfusion (I/R) injury. Methods —In vivo ischemic stroke was induced in 4-week-old male ddY mice by 2 h of middle cerebral artery occlusion (MCAO) followed by 22 h of reperfusion. The following 4 experimental protocols were devised: (1) to determine the expression level of PGRN in the I/R brain (n = 4 per group); (2) to establish the dose-response effects of intracerebroventricularly administered recombinant PGRN (r-PGRN; 0.1 to 1.0 ng) on cerebral I/R injury (n = 6 to 8 per group); (3) to establish the therapeutic time window of r-PGRN treatment (n = 8 to 9 per group); and (4) to investigate the effects of r-PGRN treatment on the expression of inflammatory cytokines in the I/R brain (n = 5 per group). Results —We found that the expression level of PGRN was significantly reduced in the I/R brain (P < 0.01, vs. Sham; t test). The administration of 1.0 ng of r-PGRN at 2 h after MCAO resulted in a reduction in the infarct volume and brain swelling at 24 h after MCAO (P < 0.01, vs. Vehicle, in each; Dunnett test), and this led to an improvement in neurological scores (P < 0.05; Wilcoxon signed-rank test). Delayed administration of r-PGRN at 6 h after MCAO did not reduce the infarct volume, but significantly reduced brain swelling (P < 0.05; t test). We also confirmed that r-PGRN administration significantly reduced the phosphorylation of NF-κB and expression of MMP-9 in the I/R brain (P < 0.05, vs. Vehicle, in each; t test). Conclusion —r-PGRN treatment exerts ameliorative effects on experimental ischemic stroke, and these effects are due to the prevention of harmful inflammatory reactions following cerebral I/R. This study suggests the feasibility of r-PGRN administration as a novel therapeutic strategy for treatment in the acute phase of ischemic stroke.
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder with no effective treatment. Fasudil hydrochloride (fasudil), a potent rho kinase (ROCK) inhibitor, is useful for the treatment of ischaemic diseases. In previous reports, fasudil improved pathology in mouse models of Alzheimer's disease and spinal muscular atrophy, but there is no evidence in that it can affect ALS. We therefore investigated its effects on experimental models of ALS.In mice motor neuron (NSC34) cells, the neuroprotective effect of hydroxyfasudil (M3), an active metabolite of fasudil, and its mechanism were evaluated. Moreover, the effects of fasudil, 30 and 100 mg·kg(-1), administered via drinking water to mutant superoxide dismutase 1 (SOD1(G93A)) mice were tested by measuring motor performance, survival time and histological changes, and its mechanism investigated.M3 prevented motor neuron cell death induced by SOD1(G93A). Furthermore, M3 suppressed both the increase in ROCK activity and phosphorylated phosphatase and tensin homologue deleted on chromosome 10 (PTEN), and the reduction in phosphorylated Akt induced by SOD1(G93A). These effects of M3 were attenuated by treatment with a PI3K inhibitor (LY294002). Moreover, fasudil slowed disease progression, increased survival time and reduced motor neuron loss, in SOD1(G93A) mice. Fasudil also attenuated the increase in ROCK activity and PTEN, and the reduction in Akt in SOD1(G93A) mice.These findings indicate that fasudil may be effective at suppressing motor neuron degeneration and symptom progression in ALS. Hence, fasudil may have potential as a therapeutic agent for ALS treatment.
Glycoprotein nonmetastatic melanoma protein B (GPNMB) has a neuroprotective effect against neuronal cell death caused by the accumulation of abnormal mutated proteins. It is known that the accumulation of pathological proteins induces endoplasmic-reticulum (ER) stress leading to cell damage. The aim of this study was to determine the role of GPNMB in the ER stress response. GPNMB was greatly up-regulated by thapsigargin-induced ER stress. Under the ER stress conditions, GPNMB relocated to the nucleus and specifically up-regulated expression of BiP at the mRNA level by promoting the BiP pre-mRNA splicing, not through the pathways initiated by the three major transducers of the unfolded protein response: IRE1, PERK, and ATF6. Furthermore, we found that the protein level of BiP and the infarction were increased and attenuated, respectively, in Gpnmb-transgenic mice after occlusion of the middle cerebral artery, in comparison with wild-type mice. Thus, our findings indicate that GPNMB enhances the BiP expression by promoting the splicing (thereby preventing cell death caused by ER stress) and could be a therapeutic target in ER stress-related disorders.
Huntington's disease (HD) is an inherited genetic disorder, characterized by cognitive dysfunction and abnormal body movements, and at present there is no effective treatment for HD. Therapeutic options for HD are limited to symptomatic treatment approaches and there is no cure for this devastating disease. Here, we examined whether SUN N8075, (2S)-1-(4-amino-2,3,5-trimethylphenoxy)-3-{4-[4-(4-fluorobenzyl)phenyl]-1-piperazinyl}-2-propanol dimethanesulfonate, which exerts neuroprotective effects by antioxidant effects and induction of VGF nerve growth factor inducible (VGF), has beneficial effects in STHdh cells derived from striatum of knock-in HD mice and R6/2 HD mice. In an in vitro study, SUN N8075 inhibited the cell death caused by mutant huntingtin (mHtt) and upregulated the VGF mRNA level via the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). Furthermore, 30 amino acid of VGF C-terminal peptide, AQEE-30 inhibited the cell death and the aggregation of mHtt. In an in vivo study, SUN N8075 improved the survival and the clasping response in the R6/2 mice. Furthermore, SUN N8075 increased the number of surviving neurons in the striatum of the R6/2 mice. These findings suggest that SUN N8075 may be an effective candidate for HD treatments.
Hypoxic stress is a risk factor of ocular neovascularization. Hypoxia visualization may provide clues regarding the underlying cause of angiogenesis. Recently, we developed a hypoxia-specific probe, protein transduction domain-oxygen-dependent degradation domain-HaloTag-Rhodamine (POH-Rhodamine). In this study, we observed the localization of HIF-1α proteins by immunohistochemistry and the fluorescence of POH-Rhodamine on RPE-choroid flat mounts. Moreover, we compared the localization of POH-Rhodamine with pimonidazole which is a standard reagent for detecting hypoxia. Next, we investigated the effects of triamcinolone acetonide (TAAC) against visual function that was evaluated by recording electroretinogram (ERG) and choroidal neovascularization (CNV) development. Mice were given laser-induced CNV using a diode laser and treated with intravitreal injection of TAAC. Finally, we investigated POH-Rhodamine on CNV treated with TAAC. In this study, the fluorescence of POH-Rhodamine and HIF-1α were co-localized in laser-irradiated sites, and both the POH-Rhodamine and pimonidazole fluorescent areas were almost the same. Intravitreal injection of TAAC restored the reduced ERG b-wave but not the a-wave and decreased the mean CNV area. Furthermore, the area of the POH-Rhodamine-positive cells decreased. These findings indicate that POH-Rhodamine is useful for evaluating tissue hypoxia in a laser-induced CNV model, suggesting that TAAC suppressed CNV through tissue hypoxia improvement.
To elucidate the effect of physicochemical properties of nanocarrier systems on drug delivery efficiency to the retina by eyedrop administration in mice, rabbits, and monkeys.Submicron-sized liposomes (ssLips) of different particle size, cholesterol content, surface charge, and multilamellar vesicles (MLV) were prepared by the hydrationFluorescence probe (coumarin-6)-incorporated liposomes, lipid emulsions, and FITC-labeled polystyrene particles were used to investigate their intraocular behavior after eyedrop administration, using epifluorescence microscopy in mice, rabbits, and monkeys.Delivery efficiency of fluorescent probes to the mouse retina from dropped liposomes was extensively improved by reducing their particle size (<600 nm) and cholesterol content, whereas negligible improvement was observed in the case of MLV. Furthermore, FITC-labeled polystyrene particles and coumarin-6-incorporated lipid emulsions showed an insufficient effect on retinal delivery in mice even if their size was controlled at 110 nm. The highest accumulation of the fluorescent probe in the retina was observed around 30 minutes with any type of ssLip used, followed by the prompt disappearance of their fluorescence within 120 minutes in mice. Changes in the fluorescence intensity of coumarin-6 in rabbits and monkeys were observed in a manner similar to that described in mice. Retinal flat-mount images suggest that coumarin-6 incorporated in ssLip diffused from the iris and ciliary body side to the optic disc side in the retina after eyedrop administration.The delivery efficiency of coumarin-6 to the retina was altered depending on particle size, constituents, and rigidity. ssLips with appropriate features would be promising drug carriers for retinal delivery through eyedrops.
Diacylglycerol kinase (DGK) is an enzyme that converts diacylglycerol to phosphatidic acid. Several DGK isoforms have been implicated in the pathogenesis of seizure, but the role of DGKβ in seizure is unknown. In the present study, we investigated the involvement of DGKβ in seizure using DGKβ knockout (KO) mice. Seizures were more severe in DGKβ KO mice than in wild-type (WT) mice after pentylenetetrazol (PTZ) treatment and after kainic acid treatment, but there were no differences in latency to seizure. The expression levels of DGKβ in the hippocampal CA1, CA3, or DG areas did not differ between PTZ (60 mg/kg) treatment and saline treatment. There were fewer parvalbumin-positive interneurons in the hippocampal CA3 area in DGKβ KO mice than in control WT mice, which might partly account for the increased seizure susceptibility displayed by DGKβ KO mice. These results suggest that DGKβ may play a pivotal role in the development of the relevant interneurons, and that on inherent deficiency of DGKβ increases the animal's sensitivity to seizure-inducing stimuli.
