ABSTRACT
Introduction: Aromatic (Ar)-turmerone is a bioactive component of turmeric oil obtained from Curcuma longa. We recently identified a novel analog (A2) of ar-turmerone that protects dopaminergic neurons from toxic stimuli by activating nuclear factor erythroid 2-related factor 2 (Nrf2). D-cysteine increases Nrf2, leading to the activation of chaperone-mediated autophagy (CMA), a pathway in the autophagy-lysosome protein degradation system, in primary cultured cerebellar Purkinje cells. In this study, we attempted to identify novel analogs of ar-turmerone that activate Nrf2 more potently and investigated whether these analogs activate CMA. Methods: Four novel analogs (A4-A7) from A2 were synthesized. We investigated the effects of A2 and novel 4 analogs on Nrf2 expression via immunoblotting and CMA activity via fluorescence observation. Results: Although all analogs, including A2, increased Nrf2 expression, only A4 activated CMA in SH-SY5Y cells. Additionally, A4-mediated CMA activation was not reversed by Nrf2 inhibition, indicating that A4 activated CMA via mechanisms other than Nrf2 activation. We focused on p38, which participates in CMA activation. Inhibition of p38 significantly prevented A4-mediated activation of CMA. Although all novel analogs significantly increased the phosphorylation of p38 6 h after drug treatment, only A4 significantly increased phosphorylation 24 h after treatment. Finally, we revealed that A4 protected SH-SY5Y cells from the cytotoxicity of rotenone, and that this protection was reversed by inhibiting p38. Conclusion: These findings suggest that the novel ar-turmerone analog, A4, activates CMA and protects SH-SY5Y cells through the persistent activation of p38.
ABSTRACT
Niemann-Pick disease type C (NPC) is a lysosomal lipid storage disorder characterized by progressive neurodegeneration and hepatic dysfunction. A cyclic heptasaccharide, 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD), is currently under clinical investigation for NPC, but its adverse events remain problematic. We previously identified that a cyclic octasaccharide, 2-hydroxypropyl-γ-cyclodextrin (HP-γ-CD), also ameliorated NPC manifestations with higher biocompatibility than HP-ß-CD. However, preclinical studies describing the associations between the biodistribution and pharmacodynamics of these compounds, which are essential for clinical application, are still lacking. Here, we investigated these properties of HP-γ-CD by measuring its organ biodistribution and therapeutic effect after systemic and central administration. The effect of HP-γ-CD on disturbed cholesterol homeostasis appeared within several hours after exposure and persisted for several days in NPC model cells and mice. Tissue distribution indicated that only a small fraction of subcutaneously administered HP-γ-CD rapidly distributed to peripheral organs and contributed to disease amelioration. We found that a subcutaneous dose of HP-γ-CD negligibly ameliorated neurological characteristics because it has limited penetration of the blood-brain barrier; however, an intracerebroventricular microdose unexpectedly attenuated hepatic dysfunction without the detection of HP-γ-CD in the liver. These results demonstrate that central administration of HP-γ-CD can indirectly attenuate peripheral manifestations of NPC.
Subject(s)
Disease Models, Animal , Liver , Niemann-Pick Disease, Type C , gamma-Cyclodextrins , Animals , Niemann-Pick Disease, Type C/drug therapy , Niemann-Pick Disease, Type C/pathology , Mice , Liver/drug effects , Liver/metabolism , Liver/pathology , gamma-Cyclodextrins/pharmacology , Tissue Distribution , Cholesterol/metabolism , Male , Mice, Inbred BALB CABSTRACT
Hemorrhage-induced injury of the corticospinal tract (CST) in the internal capsule (IC) causes severe neurological dysfunction in both human patients and rodent models of intracerebral hemorrhage (ICH). A nuclear receptor Nurr1 (NR4A2) is known to exert anti-inflammatory and neuroprotective effects in several neurological disorders. Previously we showed that Nurr1 ligands prevented CST injury and alleviated neurological deficits after ICH in mice. To prove direct effect of Nurr1 on CST integrity, we examined the effect of Nurr1 overexpression in neurons of the primary motor cortex on pathological consequences of ICH in mice. ICH was induced by intrastriatal injection of collagenase type VII, where hematoma invaded into IC. Neuron-specific overexpression of Nurr1 was induced by microinjection of synapsin I promoter-driven adeno-associated virus (AAV) vector into the primary motor cortex. Nurr1 overexpression significantly alleviated motor dysfunction but showed only modest effect on sensorimotor dysfunction after ICH. Nurr1 overexpression also preserved axonal structures in IC, while having no effect on hematoma-associated inflammatory events, oxidative stress, and neuronal death in the striatum after ICH. Immunostaining revealed that Nurr1 overexpression increased the expression of Ret tyrosine kinase and phosphorylation of Akt and ERK1/2 in neurons in the motor cortex. Moreover, administration of Nurr1 ligands 1,1-bis(3'-indolyl)-1-(p-chlorophenyl)methane or amodiaquine increased phosphorylation levels of Akt and ERK1/2 as well as expression of glial cell line-derived neurotrophic factor and Ret genes in the cerebral cortex. These results suggest that the therapeutic effect of Nurr1 on striatal ICH is attributable to the preservation of CST by acting on cortical neurons.
ABSTRACT
Menaquinone-4 (MK-4) is an isoform of vitamin K2 that has been shown to exert various biological actions besides its functions in blood coagulation and bone metabolism. Here we examined the effect of MK-4 on a mouse model of intracerebral hemorrhage (ICH). Daily oral administration of 200 mg/kg MK-4 starting from 3 h after induction of ICH by intrastriatal collagenase injection significantly ameliorated neurological deficits. Unexpectedly, MK-4 produced no significant effects on various histopathological parameters, including the decrease of remaining neurons and the increase of infiltrating neutrophils within the hematoma, the increased accumulation of activated microglia/macrophages and astrocytes around the hematoma, as well as the injury volume and brain swelling by hematoma formation. In addition, ICH-induced increases in nitrosative/oxidative stress reflected by changes in the immunoreactivities against nitrotyrosine and heme oxygenase-1 as well as the contents of malondialdehyde and glutathione were not significantly affected by MK-4. In contrast, MK-4 alleviated axon tract injury in the internal capsule as revealed by neurofilament-H immunofluorescence. Enhanced preservation of the corticospinal tract by MK-4 was also confirmed by retrograde labeling of neurons in the primary motor cortex innervating the spinal cord. These results suggest that MK-4 produces therapeutic effect on ICH by protecting structural integrity of the corticospinal tract.
Subject(s)
Cerebral Hemorrhage , Pyramidal Tracts , Vitamin K 2 , Animals , Cerebral Hemorrhage/drug therapy , Cerebral Hemorrhage/metabolism , Male , Vitamin K 2/analogs & derivatives , Vitamin K 2/pharmacology , Vitamin K 2/therapeutic use , Pyramidal Tracts/drug effects , Pyramidal Tracts/metabolism , Pyramidal Tracts/pathology , Mice , Mice, Inbred C57BL , Oxidative Stress/drug effects , Neuroprotective Agents/therapeutic use , Neuroprotective Agents/pharmacology , Nervous System Diseases/etiology , Nervous System Diseases/drug therapyABSTRACT
Goreisan is a Kampo medicine used to treat headaches associated with climate change. Here, by using an implantable complementary metal-oxide-semiconductor (CMOS) device, we evaluated the effects of Goreisan and loxoprofen on cerebral blood flow (CBF) dynamics associated with barometric pressure fluctuations in freely moving mice. In the vehicle group, decreasing barometric pressure increased CBF that was prevented by Goreisan and loxoprofen. Notably, Goreisan, but not loxoprofen, reduced CBF after returning to atmospheric pressure. These results indicate that, unlike the mechanism of action of antipyretic analgesics, Goreisan normalizes CBF abnormalities associated with barometric pressure fluctuations by actively reducing CBF increase.
Subject(s)
Atmospheric Pressure , Cerebrovascular Circulation , Drugs, Chinese Herbal , Phenylpropionates , Female , Animals , Mice , Mice, Inbred C57BLABSTRACT
Natural compounds with sulfur moiety produce various biological actions that may be beneficial for the therapies of several devastative disorders of the central nervous system. Here we investigated potential therapeutic effect of allicin, an organosulfur compound derived from garlic, in a mouse model of intracerebral hemorrhage (ICH) based on intrastriatal collagenase injection. Daily intraperitoneal administration of allicin (50 mg/kg) from 3 h after induction of ICH afforded neuroprotective effects, as evidenced by the increase of surviving neurons in the hematoma, reduction of axonal transport impairment, and prevention of axon tract injury. In addition, allicin inhibited accumulation of activated microglia/macrophages around the hematoma and infiltration of neutrophils within the hematoma. Allicin also suppressed ICH-induced mRNA upregulation of pro-inflammatory factors such as interleukin 6 and C-X-C motif ligand 2 in the brain, suggesting its anti-inflammatory effect. Moreover, ICH-induced increase of malondialdehyde as well as decrease of total glutathione in the brain was attenuated by allicin. Finally, allicin-treated mice showed better recovery of sensorimotor functions after ICH than vehicle-treated mice. These results indicate that allicin produces a therapeutic effect on ICH pathology via alleviation of neuronal damage, inflammatory responses and oxidative stress in the brain.
Subject(s)
Brain , Cerebral Hemorrhage , Mice , Animals , Cerebral Hemorrhage/drug therapy , Brain/pathology , Microglia/pathology , Hematoma/pathologyABSTRACT
BACKGROUND: Niemann-Pick disease type C (NPC) is a fatal neurodegenerative disorder caused by abnormal intracellular cholesterol trafficking. Cyclodextrins (CDs), the most promising therapeutic candidates for NPC, but with concerns about ototoxicity, are cyclic oligosaccharides with dual functions of unesterified cholesterol (UC) shuttle and sink that catalytically enhance the bidirectional flux and net efflux of UC, respectively, between the cell membrane and the extracellular acceptors. However, the properties of CDs that regulate these functions and how they could be used to improve treatments for NPC are unclear. METHODS: We estimated CD-UC complexation for nine CD derivatives derived from native α-, ß-, and γ-CD with different cavity sizes, using solubility and molecular docking analyses. The stoichiometry and complexation ability of the resulting complexes were investigated in relation to the therapeutic effectiveness and toxicity of each CD derivative in NPC experimental models. FINDINGS: We found that shuttle and sink activities of CDs are dependent on cavity size-dependent stoichiometry and substituent-associated stability of CD-UC complexation. The ability of CD derivatives to form 1:1 and 2:1 complexes with UC were correlated with their ability to normalize intracellular cholesterol trafficking serving as shuttle and with their cytotoxicity associated with cellular UC efflux acting as sink, respectively, in NPC model cells. Notably, the ability of CD derivatives to form an inclusion complex with UC was responsible for not only efficacy but ototoxicity, while a representative derivative without this ability negligibly affected auditory function, underscoring its preventability. CONCLUSIONS: Our findings highlight the importance of strategies for optimizing the molecular structure of CDs to overcome this functional dilemma in the treatment of NPC.
Subject(s)
Cyclodextrins , Niemann-Pick Disease, Type C , Ototoxicity , Humans , Cyclodextrins/pharmacology , Molecular Docking Simulation , Niemann-Pick Disease, Type C/drug therapy , CholesterolABSTRACT
Peretinoin is an acyclic retinoid that stimulates retinoic acid receptors (NR1Bs) and produces therapeutic effects on hepatocellular cancer. We have previously shown that NR1B agonists such as Am80 and all trans-retinoic acid suppress pathogenic events in intracerebral hemorrhage. The present study addressed the actions of peretinoin and Am80 against cytotoxicity of a blood protease thrombin on cortico-striatal slice cultures obtained from neonatal rat brains. Application of 100 U/ml thrombin to the slice cultures for 72 h caused cell death in the cortical region and tissue shrinkage in the striatal region. Peretinoin (50 µM) and Am80 (1 µM) counteracted these cytotoxic effects of thrombin, and the effect of peretinoin and Am80 was blocked by LE540, an NR1B antagonist. A broad-spectrum kinase inhibitor K252a (3 µM) attenuated the cytoprotective effect of peretinoin in the cortical region, whereas a specific protein kinase A inhibitor KT5720 (1 µM) attenuated the protective effect of peretinoin in the cortical and the striatal regions. On the other hand, nuclear factor-κB (NF-κB) inhibitors such as pyrrolidine dithiocarbamate (50 µM) and Bay11-7082 (10 µM) prevented thrombin-induced shrinkage of the striatal region. Peretinoin and Am80 as well as Bay11-7082 blocked thrombin-induced nuclear translocation of NF-κB in striatal microglia and loss of striatal neurons. We also found that daily administration of peretinoin reduced histopathological injury and alleviated motor deficits in a mouse model of intracerebral hemorrhage. These results indicate that NR1B agonists including peretinoin may serve as a therapeutic option for hemorrhagic brain injury.
Subject(s)
Antineoplastic Agents , Brain Injuries , Rats , Mice , Animals , Thrombin/metabolism , NF-kappa B/metabolism , Brain , Tretinoin/adverse effects , Brain Injuries/pathology , Cerebral Hemorrhage/complications , Cerebral Hemorrhage/drug therapy , Cerebral Hemorrhage/chemically induced , Antineoplastic Agents/pharmacologyABSTRACT
Missense mutations of ubiquilin 2 (UBQLN2) have been identified to cause X-linked amyotrophic lateral sclerosis (ALS). Proteasome-mediated protein degradation is reported to be impaired by ALS-associated mutations of UBQLN2. However, it remains unknown how these mutations affect autophagy-lysosome protein degradation, which consists of macroautophagy (MA), microautophagy (mA), and chaperone-mediated autophagy (CMA). Using a CMA/mA fluorescence reporter we found that overexpression of wild-type UBQLN2 impairs CMA. Conversely, knockdown of endogenous UBQLN2 increases CMA activity, suggesting that normally UBQLN2 negatively regulates CMA. ALS-associated mutant forms of UBQLN2 exacerbate this impairment of CMA. Using cells stably transfected with wild-type or ALS-associated mutant UBQLN2, we further determined that wild-type UBQLN2 increased the ratio of LAMP2A (a CMA-related protein) to LAMP1 (a lysosomal protein). This could represent a compensatory reaction to the impairment of CMA by wild-type UBQLN2. However, ALS-associated mutant UBQLN2 failed to show this compensation, exacerbating the impairment of CMA by mutant UBQLN2. We further demonstrated that ALS-associated mutant forms of UBQLN2 also impair MA, but wild-type UBQLN2 does not. These results support the view that ALS-associated mutant forms of UBQLN2 impair both CMA and MA which may contribute to the neurodegeneration observed in patients with UBQLN2-mediated ALS.
Subject(s)
Amyotrophic Lateral Sclerosis , Humans , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/pathology , Adaptor Proteins, Signal Transducing/genetics , Autophagy/genetics , Mutation , Autophagy-Related Proteins/genetics , Autophagy-Related Proteins/metabolism , Transcription Factors/metabolism , Lysosomes/metabolism , Lysosomes/pathologyABSTRACT
Matcha tea powder is believed to have various physiological benefits; however, its detailed mechanism of action has been poorly understood. Here, we investigated whether the mental state of mice, due to social isolation stress, affects the antidepressant-like effect of Matcha tea powder by using the tail suspension test. Oral administration of Matcha tea powder reduced the duration of immobility in the stress-susceptible C57BL/6J strain, but not in BALB/c strain. In C57BL/6J mice, SCH23390, a dopamine D1 receptor blocker, prevented Matcha tea powder from exerting its antidepressant-like effect. Matcha tea powder also increased the number of c-Fos-positive cells in the prefrontal cortex (PFC) region and the nucleus accumbens (NAc) region in C57BL/6J mice, but not in BALB/c mice. In contrast, Matcha tea powder did not change the number of c-Fos-positive cells in the ventral tegmental area (VTA) region. Notably, C57BL/6J mice with a shorter immobility time had a higher number of c-Fos-positive cells in the PFC, NAc, and VTA regions. However, no such correlation was observed in the stress-tolerant BALB/c mice. These results suggest that Matcha tea powder exerts an antidepressant-like effect through the activation of the dopaminergic system including the PFC-NAc-VTA circuit and that mental states are important factors affecting the physiological benefits of Matcha tea powder.
Subject(s)
Antidepressive Agents , Dopamine , Animals , Mice , Powders , Mice, Inbred C57BL , Antidepressive Agents/pharmacology , Social Isolation , Antioxidants , TeaABSTRACT
Hydrogen sulfide and polysulfides are increasingly recognized as bioactive signaling molecules to produce various actions and regulate (patho)physiological processes. Here we examined the effects of sodium sulfide (Na2S) and sodium trisulfide (Na2S3) on an experimental model of intracerebral hemorrhage (ICH) in mice. Na2S or Na2S3 (25 µmol/kg, intraperitoneally (i.p.)) was administered 30 min before ICH induction by intrastriatal injection of collagenase. We found that Na2S significantly ameliorated sensorimotor functions of mice after ICH. Histopathological examinations revealed that Na2S inhibited neuron loss in the striatum, prevented axon degeneration in the internal capsule, and ameliorated axonal transport dysfunction in the striatum and the cerebral cortex where the edge of hematoma was located. Although Na2S did not suppress accumulation of activated microglia/macrophages in the peri-hematoma region, it suppressed ICH-induced upregulation of inflammatory mediators such as C-X-C motif ligand 2. On the other hand, Na2S3 did not ameliorate ICH-induced sensorimotor dysfunction. Although the effect of Na2S3 on several parameters such as axon degeneration and axonal transport dysfunction was comparable to that of Na2S, Na2S3 did not significantly inhibit neuron loss and upregulation of inflammatory mediators. These results suggest that the regulation of multiple pathological events is involved in the effect of Na2S leading to amelioration of neurological symptoms associated with ICH.
Subject(s)
Cerebral Hemorrhage , Microglia , Mice , Animals , Cerebral Hemorrhage/drug therapy , Models, Theoretical , Hematoma/complications , Inflammation Mediators/pharmacologyABSTRACT
Niemann-Pick disease type C (NPC) is a fatal disorder with abnormal intracellular cholesterol trafficking resulting in neurodegeneration and hepatosplenomegaly. A cyclic heptasaccharide with different degrees of substitution of 2-hydroxypropyl groups, 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD), acts as a strong cholesterol solubilizer and is under investigation for treating this disease in clinical trials, but its physicochemical properties and ototoxicity remain a concern. Here, we evaluated the potential of mono-6-O-α-maltosyl-γ-CD (G2-γ-CD), a single-maltose-branched cyclic octasaccharide with a larger cavity than HP-ß-CD, for treating NPC. We identified that G2-γ-CD ameliorated NPC manifestations in model mice and showed lower ototoxicity in mice than HP-ß-CD. To investigate the molecular mechanisms of action behind the differential ototoxicity of these CDs, we performed cholesterol solubility analysis, proton nuclear magnetic resonance spectroscopy, and molecular modeling, and estimated that the cholesterol inclusion mode of G2-γ-CD maintained solely the 1:1 inclusion complex, whereas that of HP-ß-CD shifted to the highly-soluble 2:1 complex at higher concentrations. We predicted the associations of these differential complexations of CDs with cholesterol with the profile of disease attenuation and of the auditory cell toxicity using specific cell models. We proposed that G2-γ-CD can serve as a fine-tuned cholesterol solubilizer for treating NPC, being highly biocompatible and physicochemically suitable for clinical application.
Subject(s)
Hearing Loss , Niemann-Pick Disease, Type C , Ototoxicity , gamma-Cyclodextrins , Mice , Animals , Niemann-Pick Disease, Type C/drug therapy , 2-Hydroxypropyl-beta-cyclodextrin/pharmacology , 2-Hydroxypropyl-beta-cyclodextrin/therapeutic use , 2-Hydroxypropyl-beta-cyclodextrin/chemistry , Maltose/therapeutic use , Protons , Cholesterol/therapeutic use , Excipients/therapeutic use , Hearing Loss/drug therapyABSTRACT
We have previously reported that amodiaquine, a compound that binds to the ligand-binding domain of a nuclear receptor Nurr1, attenuates inflammatory responses and neurological deficits after intracerebral hemorrhage (ICH) in mice. 1,1-Bis(3'-indolyl)-1-(p-chlorophenyl)methane (C-DIM12) is another Nurr1 ligand that recognizes a domain of Nurr1 different from the ligand-binding domain. In the present study, mice were treated daily with C-DIM12 (50 or 100 mg/kg, p.o.) or amodiaquine (40 mg/kg, i.p.), or twice daily with 1400 W (20 mg/kg, i.p.), an inducible nitric oxide synthase (iNOS) inhibitor, from 3 h after ICH induction by microinjection of collagenase into the striatum. C-DIM12 improved the recovery of neurological function and prevented neuron loss in the hematoma, while suppressed activation of microglia/macrophages and expression of inflammatory mediators interleukin-6 and CC chemokine ligand 2. In addition, C-DIM12 as well as amodiaquine preserved axonal structures in the internal capsule and axonal transport function. We also found that C-DIM12 and amodiaquine suppressed the increases of iNOS mRNA expression after ICH. Moreover, 1400 W improved neurological function and prevented neuron loss, activation of microglia/macrophages and axonal transport dysfunction. These results suggest that suppression of iNOS induction contributes to several features of the therapeutic effects of Nurr1 ligands.
Subject(s)
Amodiaquine , Cerebral Hemorrhage , Encephalitis , Indoles , Nuclear Receptor Subfamily 4, Group A, Member 2 , Animals , Cerebral Hemorrhage/drug therapy , Cerebral Hemorrhage/metabolism , Encephalitis/drug therapy , Encephalitis/metabolism , Indoles/pharmacology , Ligands , Mice , Nitric Oxide Synthase Type II/antagonists & inhibitors , Nitric Oxide Synthase Type II/metabolism , Nuclear Receptor Subfamily 4, Group A, Member 2/metabolismABSTRACT
Na+/K+-ATPase (NKA) plays an important role in ion homeostasis and neurotransmitter uptake. In the retina, multidirectional communications among neurons, glia, and blood vessels (that is, neuro-glio-vascular interaction) are crucial for maintaining tissue homeostasis. We investigated the role of NKA in the elements of neuro-glio-vascular unit in neonatal and adult rat retinas. Male Sprague-Dawley rats (1- and 8-week-old) were injected intravitreally with ouabain (20 nmol/eye), an inhibitor of NKA. Morphological changes in retinal neurons, glia, and blood vessels were examined. The intravitreal injection of ouabain decreased the number of cells in the ganglion cell layer, as well as the thicknesses of the inner plexiform and inner nuclear layers in neonatal and adult rats compared to age-matched controls. The ouabain-induced neuronal cell damage was partially prevented by D-(-)-2-amino-5-phosphonopentanoic acid, an antagonist of N-methyl-D-aspartic acid receptors. In the deep retinal vascular plexus of the ouabain-injected eyes, angiogenesis was delayed in neonatal rats, whereas capillary degeneration occurred in adult rats. The immunoreactivity of glutamine synthetase and vascular endothelial growth factor (VEGF) decreased in the retinas of neonatal and adult rats injected intravitreally with ouabain. The immunoreactivity of glial fibrillary acidic protein was enhanced in the retinas of ouabain-injected adult eyes. After the ouabain injection, CD45-positive leukocytes and Iba1-positive microglia increased in the inner retinal layer of neonatal rats, whereas they increased in the middle retinal layer of adult rats. These results suggest that the inhibition of NKA induces the degeneration of neuronal and vascular cells and alteration of glial cells in both neonatal and adult retinas. In addition to the direct effects of NKA inhibition, the disturbance of retinal glutamate metabolism and decreased VEGF expression may contribute to neurovascular degeneration. The activity of NKA is crucial for maintaining elements of neuro-glio-vascular unit in the retina.
Subject(s)
Ouabain , Vascular Endothelial Growth Factor A , Adenosine Triphosphatases/metabolism , Adenosine Triphosphatases/pharmacology , Animals , Male , Neuroglia/metabolism , Ouabain/metabolism , Ouabain/pharmacology , Rats , Rats, Sprague-Dawley , Retina/metabolism , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Chaperone-mediated autophagy (CMA) is a pathway in the autophagy-lysosome protein degradation system. CMA impairment has been implicated to play a role in spinocerebellar ataxia (SCA) pathogenesis. D-cysteine is metabolized by D-amino acid oxidase (DAO), leading to hydrogen sulfide generation in the cerebellum. Although D-cysteine alleviates the disease phenotypes in SCA-model mice, it remains unknown how hydrogen sulfide derived from D-cysteine exerts this effect. In the present study, we investigated the effects of D-cysteine and hydrogen sulfide on CMA activity using a CMA activity marker that we have established. D-cysteine activated CMA in Purkinje cells (PCs) of primary cerebellar cultures where DAO was expressed, while it failed to activate CMA in DAO-deficient AD293 cells. In contrast, Na2S, a hydrogen sulfide donor, activated CMA in both PCs and AD293 cells. Nuclear factor erythroid 2-related factor 2 (Nrf2) is known to be activated by hydrogen sulfide and regulate CMA activity. An Nrf2 inhibitor, ML385, prevented CMA activation triggered by D-cysteine and Na2S. Additionally, long-term treatment with D-cysteine increased the amounts of Nrf2 and LAMP2A, a CMA-related protein, in the mouse cerebellum. These findings suggest that hydrogen sulfide derived from D-cysteine enhances CMA activity via Nrf2 activation.
Subject(s)
Chaperone-Mediated Autophagy , Hydrogen Sulfide , Animals , Mice , Cysteine/metabolism , Cysteine/pharmacology , Hydrogen Sulfide/metabolism , Hydrogen Sulfide/pharmacology , NF-E2-Related Factor 2/metabolism , Purkinje CellsABSTRACT
SIGNIFICANCE: Intrinsic optical signals (IOS) generated in the cortical tissue as a result of various interacting metabolic processes are used extensively to elucidate the underlying mechanisms that govern neurovascular coupling. However, current IOS measurements still often rely on bulky, tabletop imaging systems, and there remains a dearth of studies in freely moving subjects. Lightweight, miniature head-mounted imaging devices provide unique opportunities for investigating cortical dynamics in small animals under a variety of naturalistic behavioral settings. AIM: The aim of this work was to monitor IOS in the somatosensory cortex of wild-type mice by developing a lightweight, biocompatible imaging device that readily lends itself to animal experiments in freely moving conditions. APPROACH: Herein we describe a method for realizing long-term IOS imaging in mice using a 0.54-g, compact, CMOS-based, head-mounted imager. The two-part module, consisting of a tethered sensor plate and a base plate, allows facile assembly prior to imaging sessions and disassembly when the sensor is not in use. LEDs integrated into the device were chosen to illuminate the cortical mantle at two different wavelengths in the visible regime (λcenter: 535 and 625 nm) for monitoring volume- and oxygenation state-dependent changes in the IOS, respectively. To test whether the system can detect robust cortical responses, we recorded sensory-evoked IOS from mechanical stimulation of the hindlimbs (HL) of anesthetized mice in both acute and long-term implantation conditions. RESULTS: Cortical IOS recordings in the primary somatosensory cortex hindlimb receptive field (S1HL) of anesthetized mice under green and red LED illumination revealed robust, multiphasic profiles that were time-locked to the mechanical stimulation of the contralateral plantar hindpaw. Similar intrinsic signal profiles observed in S1HL at 40 days postimplantation demonstrated the viability of the approach for long-term imaging. Immunohistochemical analysis showed that the brain tissue did not exhibit appreciable immune response due to the device implantation and operation. A proof-of-principle imaging session in a freely behaving mouse showed minimal locomotor impediment for the animal and also enabled estimation of blood flow speed. CONCLUSIONS: We demonstrate the utility of a miniature cortical imaging device for monitoring IOS and related hemodynamic processes in both anesthetized and freely moving mice, cueing potential for applications to some neuroscientific studies of sensation and naturalistic behavior.
Subject(s)
Brain , Diagnostic Imaging , Animals , Brain/physiology , Hemodynamics , Mice , Somatosensory Cortex/diagnostic imagingABSTRACT
We examined the effect of an immunomodulator hydroxychloroquine, also known as a Nurr1 ligand and an autophagy inhibitor, on a mouse model of intracerebral hemorrhage (ICH). Daily administration of hydroxychloroquine (100 mg/kg, i.p.) from 3 h after induction of ICH alleviated neurological deficits of mice, increased the number of surviving neurons in the hematoma and prevented fragmentation of axon structures in the internal capsule. Unexpectedly, hydroxychloroquine did not inhibit either upregulation of pro-inflammatory mediators or autophagic responses in the brain. Hence, hydroxychloroquine may produce therapeutic effects on ICH primarily via neuroprotection including preservation of the axon tract integrity.
Subject(s)
Brain/drug effects , Cerebral Hemorrhage/pathology , Hydroxychloroquine/pharmacology , Motor Activity/drug effects , Neuroprotective Agents/pharmacology , Animals , Autophagy/drug effects , Inflammation/pathology , Male , Mice , Mice, Inbred ICRABSTRACT
Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra. The inflammatory activation of microglia participates in dopaminergic neurodegeneration in PD. Therefore, chemicals that inhibit microglial activation are considered to have therapeutic potential for PD. Aromatic (ar)-turmerone is a main component of turmeric oil extracted from Curcuma longa and has anti-inflammatory activity in cultured microglia. The aims of the present study are (1) to investigate whether naturally occurring S-enantiomer of ar-turmerone (S-Tur) protects dopaminergic neurons in midbrain slice cultures and (2) to examine ar-turmerone analogs that have higher activities than S-Tur in inhibiting microglial activation and protecting dopaminergic neurons. R-enantiomer (R-Tur) and two analogs showed slightly higher anti-inflammatory effects in microglial BV2 cells. S- and R-Tur and these two analogs reversed dopaminergic neurodegeneration triggered by microglial activation in midbrain slice cultures. Unexpectedly, this neuroprotection was independent of the inhibition of microglial activation. Additionally, two analogs more potently inhibited dopaminergic neurodegeneration triggered by a neurotoxin, 1-methyl-4-phenylpyridinium, than S-Tur. Taken together, we identified two ar-turmerone analogs that directly and potently protected dopaminergic neurons. An investigation using dopaminergic neuronal precursor cells suggested the possible involvement of nuclear factor erythroid 2-related factor 2 in this neuroprotection.
Subject(s)
Dopaminergic Neurons/drug effects , Ketones/pharmacology , Mesencephalon/drug effects , Neuroprotective Agents/pharmacology , Sesquiterpenes/pharmacology , Animals , Cell Line , Cells, Cultured , Ketones/chemistry , Mesencephalon/cytology , Mice , Microglia/drug effects , Neuroprotective Agents/chemistry , Rats , Rats, Wistar , Sesquiterpenes/chemistryABSTRACT
Spinocerebellar ataxia (SCA) is a group of autosomal-dominantly inherited ataxia and is classified into SCA1-48 by the difference of causal genes. Several SCA-causing proteins commonly impair dendritic development in primary cultured Purkinje cells (PCs). We assume that primary cultured PCs expressing SCA-causing proteins are available as in vitro SCA models and that chemicals that improve the impaired dendritic development would be effective for various SCAs. We have recently revealed that D-cysteine enhances the dendritic growth of primary cultured PCs via hydrogen sulfide production. In the present study, we first investigated whether D-cysteine is effective for in vitro SCA models. We expressed SCA1-, SCA3-, and SCA21-causing mutant proteins to primary cultured PCs using adeno-associated viral serotype 9 (AAV9) vectors. D-Cysteine (0.2 mM) significantly ameliorated the impaired dendritic development commonly observed in primary cultured PCs expressing these three SCA-causing proteins. Next, we investigated the therapeutic effect of long-term treatment with D-cysteine on an in vivo SCA model. SCA1 model mice were established by the cerebellar injection of AAV9 vectors, which express SCA1-causing mutant ataxin-1, to ICR mice. Long-term treatment with D-cysteine (100 mg/kg/day) significantly inhibited the progression of motor dysfunction in SCA1 model mice. Immunostaining experiments revealed that D-cysteine prevented the reduction of mGluR1 and glial activation at the early stage after the onset of motor dysfunction in SCA1 model mice. These findings strongly suggest that D-cysteine has therapeutic potential against in vitro and in vivo SCA models and may be a novel therapeutic agent for various SCAs.
Subject(s)
Ataxin-1/biosynthesis , Cerebellum/drug effects , Cerebellum/metabolism , Cysteine/therapeutic use , Spinocerebellar Ataxias/drug therapy , Spinocerebellar Ataxias/metabolism , Animals , Ataxin-1/genetics , Cells, Cultured , Cysteine/pharmacology , Female , Male , Mice , Mice, Inbred ICR , Mice, Transgenic , Pregnancy , Rats , Rats, Wistar , Spinocerebellar Ataxias/geneticsABSTRACT
BACKGROUND AND PURPOSE: Niemann-Pick disease type C (NPC) is a lysosomal storage disorder with disrupted intracellular cholesterol trafficking. A cyclic heptasaccharide, 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD), is a cholesterol solubilizer that is being developed to treat NPC, but its ototoxicity and pulmonary toxicity remain important issues. We have characterized 2-hydroxypropyl-γ-cyclodextrin (HP-γ-CD), a cyclic octasaccharide with a larger cavity than HP-ß-CD, as a candidate drug to treat NPC. However, the molecular target of HP-γ-CD with respect to NPC and its potential for clinical application are still unclear. EXPERIMENTAL APPROACH: We investigated the mode of interaction between HP-γ-CD and cholesterol by phase-solubility analysis, proton NMR spectroscopy and molecular dynamics simulations. We then evaluated the therapeutic effects of HP-γ-CD compared with HP-ß-CD using cellular and murine NPC models. Mouse auditory and pulmonary function tests were also conducted. KEY RESULTS: HP-γ-CD solely formed a 1:1 inclusion complex with cholesterol with an affinity similar to that of HP-ß-CD. In vitro, HP-γ-CD and HP-ß-CD amelioration of NPC-related manifestations was almost equivalent at lower concentrations. However, at higher concentrations, the cholesterol inclusion mode of HP-ß-CD shifted to the highly soluble 2:1 complex whereas that of HP-γ-CD maintained solely the 1:1 complex. The constant lower cholesterol solubilizing ability of HP-γ-CD conferred it with significantly reduced toxicity compared with HP-ß-CD, but equal efficacy in treating a mouse model of NPC. CONCLUSIONS AND IMPLICATIONS: HP-γ-CD can serve as a fine-tuned cholesterol solubilizer for the treatment of NPC with a wider safety margin than HP-ß-CD in terms of ototoxicity and pulmonary toxicity.