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Mella C, Figueroa CD, Otth C, Ehrenfeld P. Involvement of Kallikrein-Related Peptidases in Nervous System Disorders. Front Cell Neurosci 2020; 14:166. [PMID: 32655372 PMCID: PMC7324807 DOI: 10.3389/fncel.2020.00166] [Citation(s) in RCA: 29] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2019] [Accepted: 05/18/2020] [Indexed: 12/16/2022] Open
Abstract
Kallikrein-related peptidases (KLKs) are a family of serine proteases that when dysregulated may contribute to neuroinflammation and neurodegeneration. In the present review article, we describe what is known about their physiological and pathological roles with an emphasis on KLK6 and KLK8, two KLKs that are highly expressed in the adult central nervous system (CNS). Altered expression and activity of KLK6 have been linked to brain physiology and the development of multiple sclerosis. On the other hand, altered levels of KLK6 in the brain and serum of people affected by Alzheimer's disease and Parkinson's disease have been documented, pointing out to its function in amyloid metabolism and development of synucleinopathies. People who have structural genetic variants of KLK8 can suffer mental illnesses such as intellectual and learning disabilities, seizures, and autism. Increased expression of KLK8 has also been implicated in schizophrenia, bipolar disorder, and depression. Also, we discuss the possible link that exists between KLKs activity and certain viral infections that can affect the nervous system. Although little is known about the exact mechanisms that mediate KLKs function and their participation in neuroinflammatory and neurodegenerative disorders will open a new field to develop novel therapies to modulate their levels and/or activity and their harmful effects on the CNS.
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Affiliation(s)
- Cinthia Mella
- Faculty of Medicine, Institute of Clinical Microbiology, Universidad Austral de Chile, Valdivia, Chile
- Laboratory of Cellular Pathology, Institute of Anatomy, Histology, and Pathology, Universidad Austral de Chile, Valdivia, Chile
- Center for Interdisciplinary Studies on the Nervous System (CISNe), Universidad Austral de Chile, Valdivia, Chile
| | - Carlos D. Figueroa
- Laboratory of Cellular Pathology, Institute of Anatomy, Histology, and Pathology, Universidad Austral de Chile, Valdivia, Chile
- Center for Interdisciplinary Studies on the Nervous System (CISNe), Universidad Austral de Chile, Valdivia, Chile
| | - Carola Otth
- Faculty of Medicine, Institute of Clinical Microbiology, Universidad Austral de Chile, Valdivia, Chile
- Center for Interdisciplinary Studies on the Nervous System (CISNe), Universidad Austral de Chile, Valdivia, Chile
| | - Pamela Ehrenfeld
- Laboratory of Cellular Pathology, Institute of Anatomy, Histology, and Pathology, Universidad Austral de Chile, Valdivia, Chile
- Center for Interdisciplinary Studies on the Nervous System (CISNe), Universidad Austral de Chile, Valdivia, Chile
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Thrombin contributes to the injury development and neurological deficit after acute subdural hemorrhage in rats only in collaboration with additional blood-derived factors. BMC Neurosci 2018; 19:81. [PMID: 30591020 PMCID: PMC6307215 DOI: 10.1186/s12868-018-0481-5] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2018] [Accepted: 12/15/2018] [Indexed: 12/13/2022] Open
Abstract
Background Acute subdural hemorrhage (ASDH) is a severe consequence of traumatic brain injury. The occurrence of subdural blood increases the lethality of these patients independent of the amount of blood or elevated intracranial pressure. Thrombin is one of the potential harmful blood components. Possible harmful effects of thrombin are mediated via the Protease-activated-receptor-1 (PAR1) and thus, translating the acute Thrombin release after ASDH into cell loss. The objectives of the present study were twofold, namely to examine (1) the impact of direct thrombin inhibition in the acute phase after hemorrhage on the long-term histological and functional deficits and (2) the early inhibition of PAR1 activation by thrombin with the selective antagonist SCH79797 on lesion volume at 14 days after ASDH. The effects of thrombin on the lesion size were investigated in two separate experiments via (1) direct thrombin inhibition in the subdural infused blood (Argatroban 600 µg) as well as by (2) intraventricular injection of the PAR-1 antagonist SCH79797 (1 µg or 5 µg). Lesion volume and behavior deficits using a neurological deficit score and a motor function test (beam balance test) were analyzed as outcome parameters at 14 days after injury. Results 59 Male Sprague–Dawley rats received a subdural infusion of 300 µl autologous blood or sham operation. Lesion volume at 14 days after ASDH tended to be smaller in the Argatroban-treated group when compared to the vehicle group (8.1 ± 1.1 vs. 10.1 ± 2.3 mm2, n.s.). Motor deficits in the beam balance test were not significantly less severe in the Argatroban-treated group. Animals treated with SCH79797 also showed a trend towards dose-dependent decreased lesion volume in comparison to the vehicle-treated group (1 μg: 4.3 ± 0.7 mm3; 5 μg: 3.8 ± 1.1 mm3; vehicle: 6.5 ± 2.0 mm3, n.s). Conclusions Thrombin inhibition in the subdural blood and local cerebral blockade of PAR-1 cause a tendency towards reduced lesion volume or functional recovery. All results show a trend in favor of the acute treatment on the outcome parameters. Our results suggests that thrombin could be an important blood-derived factor during acute subdural hemorrhage that translates its deleterious effects in concert with other blood-induced factors.
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Abstract
BACKGROUND Vorapaxar, a novel antiplatelet thrombin PAR-1 inhibitor, is currently approved for post myocardial infarction and peripheral artery disease indications with concomitant use of clopidogrel and/or aspirin. The vorapaxar safety profile was acceptable. However, aside from heightened bleeding risks, excesses of solid cancers and diplopia, there were more amyotrophic lateral sclerosis (ALS) diagnoses after vorapaxar. STUDY QUESTION To assess the Food and Drug Administration (FDA) reviews on the potential association of vorapaxar with ALS. STUDY DESIGN The review the public FDA records on reported adverse events after vorapaxar. MEASURES AND OUTCOMES Incidence of ALS after vorapaxar and placebo. RESULTS The ALS risk appears very small, about 1 case per 10,000 treated subjects, but quite probable. Indeed, there were overall 2 placebo and 4 vorapaxar ALS incidences in the Phase III clinical trials. CONCLUSIONS Potential adverse association of vorapaxar with ALS risks may be related to off-target neuronal PAR receptor(s) blockade beyond platelet inhibition.
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Radulovic M, Yoon H, Wu J, Mustafa K, Scarisbrick IA. Targeting the thrombin receptor modulates inflammation and astrogliosis to improve recovery after spinal cord injury. Neurobiol Dis 2016; 93:226-42. [PMID: 27145117 PMCID: PMC4930708 DOI: 10.1016/j.nbd.2016.04.010] [Citation(s) in RCA: 46] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2016] [Revised: 04/08/2016] [Accepted: 04/29/2016] [Indexed: 02/07/2023] Open
Abstract
The deregulation of serine protease activity is a common feature of neurological injury, but little is known regarding their mechanisms of action or whether they can be targeted to facilitate repair. In this study we demonstrate that the thrombin receptor (Protease Activated Receptor 1, (PAR1)) serves as a critical translator of the spinal cord injury (SCI) proteolytic microenvironment into a cascade of pro-inflammatory events that contribute to astrogliosis and functional decline. PAR1 knockout mice displayed improved locomotor recovery after SCI and reduced signatures of inflammation and astrogliosis, including expression of glial fibrillary acidic protein (GFAP), vimentin, and STAT3 signaling. SCI-associated elevations in pro-inflammatory cytokines such as IL-1β and IL-6 were also reduced in PAR1-/- mice and co-ordinate improvements in tissue sparing and preservation of NeuN-positive ventral horn neurons, and PKCγ corticospinal axons, were observed. PAR1 and its agonist's thrombin and neurosin were expressed by perilesional astrocytes and each agonist increased the production of IL-6 and STAT3 signaling in primary astrocyte cultures in a PAR1-dependent manner. In turn, IL-6-stimulated astrocytes increased expression of PAR1, thrombin, and neurosin, pointing to a model in which PAR1 activation contributes to increased astrogliosis by feedforward- and feedback-signaling dynamics. Collectively, these findings identify the thrombin receptor as a key mediator of inflammation and astrogliosis in the aftermath of SCI that can be targeted to reduce neurodegeneration and improve neurobehavioral recovery.
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Affiliation(s)
- Maja Radulovic
- Neurobiology of Disease Program, Mayo Medical and Graduate School, Rehabilitation Medicine Research Center, Rochester 55905, MN, United States
| | - Hyesook Yoon
- Department of Physical Medicine and Rehabilitation, Mayo Medical and Graduate School, Rehabilitation Medicine Research Center, Rochester, MN 55905, United States; Department of Physiology and Biomedical Engineering, Mayo Medical and Graduate School, Rehabilitation Medicine Research Center, Rochester, MN 55905, United States
| | - Jianmin Wu
- Department of Physical Medicine and Rehabilitation, Mayo Medical and Graduate School, Rehabilitation Medicine Research Center, Rochester, MN 55905, United States
| | - Karim Mustafa
- Neurobiology of Disease Program, Mayo Medical and Graduate School, Rehabilitation Medicine Research Center, Rochester 55905, MN, United States
| | - Isobel A Scarisbrick
- Neurobiology of Disease Program, Mayo Medical and Graduate School, Rehabilitation Medicine Research Center, Rochester 55905, MN, United States; Department of Physical Medicine and Rehabilitation, Mayo Medical and Graduate School, Rehabilitation Medicine Research Center, Rochester, MN 55905, United States; Department of Physiology and Biomedical Engineering, Mayo Medical and Graduate School, Rehabilitation Medicine Research Center, Rochester, MN 55905, United States.
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Citron BA, Ameenuddin S, Uchida K, Suo WZ, SantaCruz K, Festoff BW. Membrane lipid peroxidation in neurodegeneration: Role of thrombin and proteinase-activated receptor-1. Brain Res 2016; 1643:10-7. [DOI: 10.1016/j.brainres.2016.04.071] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2015] [Revised: 04/10/2016] [Accepted: 04/28/2016] [Indexed: 02/08/2023]
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Ahmad M, Zakaria A, Almutairi KM. Effectiveness of minocycline and FK506 alone and in combination on enhanced behavioral and biochemical recovery from spinal cord injury in rats. Pharmacol Biochem Behav 2016; 145:45-54. [PMID: 27106204 DOI: 10.1016/j.pbb.2016.04.003] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/06/2016] [Revised: 04/15/2016] [Accepted: 04/18/2016] [Indexed: 11/15/2022]
Abstract
Injury to the spinal cord results in immediate physical damage (primary injury) followed by a prolonged posttraumatic inflammatory disorder (secondary injury). The present study aimed to investigate the neuroprotective effects of minocycline and FK506 (Tacrolimus) individually and in combination on recovery from experimental spinal cord injury (SCI). Young adult male rats were subjected to experimental SCI by weight compression method. Minocycline (50mg/kg) and FK506 (1mg/kg) were administered orally in combination and individually to the SCI group daily for three weeks. During these three weeks, the recovery was measured using behavioral motor parameters (including BBB, Tarlov and other scorings) every other day for 29days after SCI. Thereafter, the animals were sacrificed and the segment of the spinal cord centered at the injury site was removed for the histopathological studies as well as for biochemical analysis of monoamines such as 5-hydroxytryptamine (5-HT) and 5-hydroxy-indolacetic acid (5-HIAA) and some oxidative stress indices, such as thiobarbituric acid-reactive substances (TBARS), total glutathione (GSH) and myeloperoxidase (MPO). All behavioral results indicated that both drugs induced significant recovery from SCI with respect to time. The biochemical and histopathological results supported the behavioral findings, revealing significant recovery in the regeneration of the injured spinal tissues, the monoamine levels, and the oxidative stress indices. Overall, the effects of the tested drugs for SCI recovery were as follows: FK506+minocycline>minocycline>FK506 in all studied parameters. Thus, minocycline and FK506 may prove to be a potential therapy cocktail to treat acute SCI. However, further studies are warranted.
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Affiliation(s)
- Mohammad Ahmad
- Department of Medical Surgical Nursing, College of Nursing, King Saud University, Riyadh, Saudi Arabia.
| | - Abdulrahim Zakaria
- Department of Rehabilitation Sciences, College of Applied Medical Sciences, King Saud University, Riyadh, Saudi Arabia
| | - Khalid M Almutairi
- Department of Community Health Sciences, College of Applied Medical Sciences, King Saud University, Riyadh, Saudi Arabia
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Tripathy D, Sanchez A, Yin X, Martinez J, Grammas P. Age-related decrease in cerebrovascular-derived neuroprotective proteins: effect of acetaminophen. Microvasc Res 2012; 84:278-85. [PMID: 22944728 PMCID: PMC3483357 DOI: 10.1016/j.mvr.2012.08.004] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2012] [Revised: 07/17/2012] [Accepted: 08/17/2012] [Indexed: 12/19/2022]
Abstract
As the population ages, the need for effective methods to maintain brain function in older adults is increasingly pressing. Vascular disease and neurodegenerative disorders commonly co-occur in older persons. Cerebrovascular products contribute to the neuronal milieu and have important consequences for neuronal viability. In this regard vascular derived neuroprotective proteins, Such as vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), and pituitary adenylate cyclase activating peptide (PACAP) are important for maintaining neuronal viability, especially in the face of injury and disease. The objective of this study is to measure and compare levels of VEGF, PEDF and PACAP released from isolated brain microvessels of Fischer 344 rats at 6, 12, 18, and 24 months of age. Addition of acetaminophen to isolated brain microvessels is employed to determine whether this drug affects vascular expression of these neuroprotective proteins. Experiments on cultured brain endothelial cells are performed to explore the mechanisms/mediators that regulate the effect of acetaminophen on endothelial cells. The data indicate cerebrovascular expression of VEGF, PEDF and PACAP significantly decreases with age. The age-associated decrease in VEGF and PEDF is ameliorated by addition of acetaminophen to isolated brain microvessels. Also, release of VEGF, PEDF, and PACAP from cultured brain endothelial cells decreases with exposure to the oxidant stressor menadione. Acetaminophen treatment upregulates VEGF, PEDF and PACAP in brain endothelial cells exposed to oxidative stress. The effect of acetaminophen on cultured endothelial cells is in part inhibited by the selective thrombin inhibitor hirudin. The results of this study suggest that acetaminophen may be a useful agent for preserving cerebrovascular function. If a low dose of acetaminophen can counteract the decrease in vascular-derived neurotrophic factors evoked by age and oxidative stress, this drug might be useful for improving brain function in the elderly.
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Affiliation(s)
- Debjani Tripathy
- Garrison Institute on Aging, Texas Tech University Health Sciences Center, Lubbock, Texas
| | - Alma Sanchez
- Garrison Institute on Aging, Texas Tech University Health Sciences Center, Lubbock, Texas
| | - Xiangling Yin
- Garrison Institute on Aging, Texas Tech University Health Sciences Center, Lubbock, Texas
| | - Joseph Martinez
- Garrison Institute on Aging, Texas Tech University Health Sciences Center, Lubbock, Texas
| | - Paula Grammas
- Garrison Institute on Aging, Texas Tech University Health Sciences Center, Lubbock, Texas
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Liu DZ, Ander BP. Cell cycle inhibition without disruption of neurogenesis is a strategy for treatment of aberrant cell cycle diseases: an update. ScientificWorldJournal 2012; 2012:491737. [PMID: 22547985 PMCID: PMC3323905 DOI: 10.1100/2012/491737] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2011] [Accepted: 11/17/2011] [Indexed: 12/12/2022] Open
Abstract
Since publishing our earlier report describing a strategy for the treatment of central nervous system (CNS) diseases by inhibiting the cell cycle and without disrupting neurogenesis (Liu et al. 2010), we now update and extend this strategy to applications in the treatment of cancers as well. Here, we put forth the concept of "aberrant cell cycle diseases" to include both cancer and CNS diseases, the two unrelated disease types on the surface, by focusing on a common mechanism in each aberrant cell cycle reentry. In this paper, we also summarize the pharmacological approaches that interfere with classical cell cycle molecules and mitogenic pathways to block the cell cycle of tumor cells (in treatment of cancer) as well as to block the cell cycle of neurons (in treatment of CNS diseases). Since cell cycle inhibition can also block proliferation of neural progenitor cells (NPCs) and thus impair brain neurogenesis leading to cognitive deficits, we propose that future strategies aimed at cell cycle inhibition in treatment of aberrant cell cycle diseases (i.e., cancers or CNS diseases) should be designed with consideration of the important side effects on normal neurogenesis and cognition.
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Affiliation(s)
- Da-Zhi Liu
- Department of Neurology and the MIND Institute, University of California at Davis, Sacramento, CA 95817, USA.
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Cho DC, Cheong JH, Yang MS, Hwang SJ, Kim JM, Kim CH. The effect of minocycline on motor neuron recovery and neuropathic pain in a rat model of spinal cord injury. J Korean Neurosurg Soc 2011; 49:83-91. [PMID: 21519495 DOI: 10.3340/jkns.2011.49.2.83] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2010] [Revised: 10/27/2010] [Accepted: 02/27/2011] [Indexed: 11/27/2022] Open
Abstract
OBJECTIVE Minocycline, a second-generation tetracycline-class antibiotic, has been well established to exert a neuroprotective effect in animal models and neurodegenerative disease through the inhibition of microglia. Here, we investigated the effects of minocycline on motor recovery and neuropathic pain in a rat model of spinal cord injury. METHODS To simulate spinal cord injury, the rats' spinal cords were hemisected at the 10th thoracic level (T10). Minocycline was injected intraperitoneally, and was administered 30 minutes prior surgery and every second postoperative day until sacrifice 28 days after surgery. Motor recovery was assessed via the Basso-Beattie-Bresnahan test. Mechanical hyperalgesia was measured throughout the 28-day post-operative course via the von Frey test. Microglial and astrocyte activation was assessed by immunohistochemical staining for ionized calcium binding adaptor molecule 1 (Iba1) and glial fibrillary acidic protein (GFAP) at two sites: at the level of hemisection and at the 5th lumbar level (L5). RESULTS In rats, spinal cord hemisection reduced locomotor function and induced a mechanical hyperalgesia of the ipsilateral hind limb. The expression of Iba1 and GFAP was also increased in the dorsal and ventral horns of the spinal cord at the site of hemisection and at the L5 level. Intraperitoneal injection of minocycline facilitated overall motor recovery and attenuated mechanical hyperalgesia. The expression of Iba1 and GFAP in the spinal cord was also reduced in rats treated with minocycline. CONCLUSION By inhibiting microglia and astrocyte activation, minocycline may facilitate motor recovery and attenuate mechanical hyperalgesia in individuals with spinal cord injuries.
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Affiliation(s)
- Dong Charn Cho
- Department of Neurosurgery, Hanyang University College of Medicine, Seoul, Korea
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Liu DZ, Ander BP, Sharp FR. Cell cycle inhibition without disruption of neurogenesis is a strategy for treatment of central nervous system diseases. Neurobiol Dis 2009; 37:549-57. [PMID: 19944161 DOI: 10.1016/j.nbd.2009.11.013] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2009] [Revised: 11/10/2009] [Accepted: 11/18/2009] [Indexed: 12/12/2022] Open
Abstract
Classically, the cell cycle is regarded as the process leading to cellular proliferation. However, increasing evidence over the last decade supports the notion that neuronal cell cycle re-entry results in post-mitotic death. A mature neuron that re-enters the cell cycle can neither advance to a new G0 quiescent state nor revert to its earlier G0 state. This presents a critical dilemma to the neuron from which death may be an unavoidable but necessary outcome for adult neurons attempting to complete the cell cycle. In contrast, tumor cells that undergo aberrant cell cycle re-entry divide and can survive. Thus, cell cycle inhibition strategies are of interest in cancer treatment but may also represent an important means of protecting neurons. In this review, we put forth the concept of the "expanded cell cycle" and summarize the cell cycle proteins, signal transduction events and mitogenic molecules that can drive a neuron into the cell cycle in various CNS diseases. We also discuss the pharmacological approaches that interfere with the mitogenic pathways and prevent mature neurons from attempting cell cycle re-entry, protecting them from cell death. Lastly, future attempts at blocking the cell cycle to rescue mature neurons from injury should be designed so as to not block normal neurogenesis.
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Affiliation(s)
- Da-Zhi Liu
- Department of Neurology and the M.I.N.D. Institute, University of California at Davis, Sacramento, CA 95817, USA.
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Hamill CE, Mannaioni G, Lyuboslavsky P, Sastre AA, Traynelis SF. Protease-activated receptor 1-dependent neuronal damage involves NMDA receptor function. Exp Neurol 2009; 217:136-46. [PMID: 19416668 DOI: 10.1016/j.expneurol.2009.01.023] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2008] [Revised: 01/27/2009] [Accepted: 01/27/2009] [Indexed: 01/22/2023]
Abstract
Protease-activated receptor 1 (PAR1) is a G-protein coupled receptor that is expressed throughout the central nervous system. PAR1 activation by brain-derived as well as blood-derived proteases has been shown to have variable and complex effects in a variety of animal models of neuronal injury and inflammation. In this study, we have evaluated the effects of PAR1 on lesion volume in wild-type or PAR1-/- C57Bl/6 mice subjected to transient occlusion of the middle cerebral artery or injected with NMDA in the striatum. We found that removal of PAR1 reduced infarct volume following transient focal ischemia to 57% of control. Removal of PAR1 or application of a PAR1 antagonist also reduced the neuronal injury associated with intrastriatal injection of NMDA to 60% of control. To explore whether NMDA receptor potentiation by PAR1 activation contributes to the harmful effects of PAR1, we investigated the effect of NMDA receptor antagonists on the neuroprotective phenotype of PAR1-/- mice. We found that MK801 reduced penumbral but not core neuronal injury in mice subjected to transient middle cerebral artery occlusion or intrastriatal NMDA injection. Lesion volumes in both models were not significantly different between PAR1-/- mice treated with and without MK801. Use of the NMDA receptor antagonist and dissociative anesthetic ketamine also renders NMDA-induced lesion volumes identical in PAR1-/- mice and wild-type mice. These data suggest that the ability of PAR1 activation to potentiate NMDA receptor function may underlie its harmful actions during injury.
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Affiliation(s)
- Cecily E Hamill
- Department of Pharmacology, Emory University School of Medicine, Rollins Research Center, Atlanta, GA 30322-3090, USA
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Vandell AG, Larson N, Laxmikanthan G, Panos M, Blaber SI, Blaber M, Scarisbrick IA. Protease-activated receptor dependent and independent signaling by kallikreins 1 and 6 in CNS neuron and astroglial cell lines. J Neurochem 2008; 107:855-70. [PMID: 18778305 DOI: 10.1111/j.1471-4159.2008.05658.x] [Citation(s) in RCA: 53] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/21/2022]
Abstract
While protease-activated receptors (PARs) are known to mediate signaling events in CNS, contributing both to normal function and pathogenesis, the endogenous activators of CNS PARs are poorly characterized. In this study, we test the hypothesis that kallikreins (KLKs) represent an important pool of endogenous activators of CNS PARs. Specifically, KLK1 and KLK6 were examined for their ability to evoke intracellular Ca(2+) flux in a PAR-dependent fashion in NSC34 neurons and Neu7 astrocytes. Both KLKs were also examined for their ability to activate mitogen-activated protein kinases (extracellular signal-regulated kinases, C-Jun N-terminal kinases, and p38) and protein kinase B (AKT) intracellular signaling cascades. Cumulatively, these studies show that KLK6, but not KLK1, signals through PARs. KLK6 evoked intracellular Ca(2+) flux was mediated by PAR1 in neurons and both PAR1 and PAR2 in astrocytes. Importantly, both KLK1 and KLK6 altered the activation state of mitogen-activated protein kinases and AKT, suggestive of important roles for each in CNS neuron and glial differentiation, and survival. The cellular specificity of CNS-KLK activity was underscored by observations that both proteases promoted AKT activation in astrocytes, but inhibited such signaling in neurons. PAR1 and bradykinin receptor inhibitors were used to demonstrate that KLK1-mediated activation of extracellular signal-regulated kinases in neurons occurred in a non-PAR, bradykinin 2 (B2) receptor-dependent fashion, while similar signaling by KLK6 was mediated by the combined activation of PAR1 and B2. Cumulatively results indicate KLK6, but not KLK1 is an activator of CNS PARs, and that both KLKs are poised to signal in a B2 receptor-dependent fashion to regulate multiple signal transduction pathways relevant to CNS physiologic function and dysfunction.
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Affiliation(s)
- Alexander G Vandell
- Molecular Neuroscience Program, Mayo Medical and Graduate School, Rochester, Minnesota 55905, USA
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Effect of PAR1 agonist on acetylcholine secretion in a newly formed neuromuscular synapse in mice. Bull Exp Biol Med 2008; 144:653-6. [PMID: 18683487 DOI: 10.1007/s10517-007-0396-0] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/22/2022]
Abstract
Peptide agonist of PARI in a concentration of 10 microM significantly facilitated neuromuscular transmission in newly formed synapses in mice. The absence of changes in the amplitude of miniature end-plate potentials attests to presynaptic mechanism of the effect of PAR1 agonist. The effect of the peptide was blocked by protein kinase A inhibitor H89 (1 microM). Blockade of inositol-1,4,5-trisphosphate receptors with 2-amino-ethoxydiphenylborate (30 microM) did not prevent the effects of PARI agonist. Inhibition of protein kinase C with bisindolylmaleimide (1 microM) facilitated neuromuscular transmission in newly formed synapses. Protein kinase C inhibition was associated with reversal of the object of PARI agonist: transmission inhibition instead of facilitation.
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Herrera AJ, de Pablos RM, Carreño-Müller E, Villarán RF, Venero JL, Tomás-Camardiel M, Cano J, Machado A. The intrastriatal injection of thrombin in rat induced a retrograde apoptotic degeneration of nigral dopaminergic neurons through synaptic elimination. J Neurochem 2008; 105:750-62. [DOI: 10.1111/j.1471-4159.2007.05170.x] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
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Pool M, Thiemann J, Bar-Or A, Fournier AE. NeuriteTracer: a novel ImageJ plugin for automated quantification of neurite outgrowth. J Neurosci Methods 2007; 168:134-9. [PMID: 17936365 DOI: 10.1016/j.jneumeth.2007.08.029] [Citation(s) in RCA: 198] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2007] [Revised: 08/10/2007] [Accepted: 08/30/2007] [Indexed: 01/06/2023]
Abstract
In vitro assays to measure neuronal growth are a fundamental tool used by many neurobiologists studying neuronal development and regeneration. The quantification of these assays requires accurate measurements of neurite length and neuronal cell numbers in neuronal cultures. Generally, these measurements are obtained through labor-intensive manual or semi-manual tracing of images. To automate these measurements, we have written NeuriteTracer, a neurite tracing plugin for the freely available image-processing program ImageJ. The plugin analyzes fluorescence microscopy images of neurites and nuclei of dissociated cultured neurons. Given user-defined thresholds, the plugin counts neuronal nuclei, and traces and measures neurite length. We find that NeuriteTracer accurately measures neurite outgrowth from cerebellar, DRG and hippocampal neurons. Values obtained by NeuriteTracer correlate strongly with those obtained by semi-manual tracing with NeuronJ and by using a sophisticated analysis package, MetaXpress. We reveal the utility of NeuriteTracer by demonstrating its ability to detect the neurite outgrowth promoting capacity of the rho kinase inhibitor Y-27632. Our plugin is an attractive alternative to existing tracing tools because it is fully automated and ready for use within a freely accessible imaging program.
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Affiliation(s)
- Madeline Pool
- Department of Neurology and Neurosurgery, Montreal Neurological Institute, BT-109, 3801 Rue University, Montreal, Quebec, Canada H3A 2B4
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Rao HV, Thirumangalakudi L, Desmond P, Grammas P. Cyclin D1, cdk4, and Bim are involved in thrombin-induced apoptosis in cultured cortical neurons. J Neurochem 2007; 101:498-505. [PMID: 17254021 DOI: 10.1111/j.1471-4159.2006.04389.x] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
Thrombin, a multifunctional serine protease, is neurotoxic in vitro and in vivo. Thrombin has been shown to be increased in Alzheimer's disease (AD) and other neuropathological conditions and could be a mediator of pathological neuronal cell death in the brain. The mechanisms of thrombin-induced neuronal cell death are incompletely understood. The objective of this study is to explore mechanisms that contribute to thrombin-induced neuronal apoptosis focusing on the role of cell cycle regulators and the pro-apoptotic protein Bim (Bcl-2-interacting mediator of cell death) in this process. Our data show that thrombin treatment of primary cerebral cortical cultures results in dose-dependent apoptotic cell death. Exposure of neuronal cultures to thrombin leads to induction of cell cycle proteins cyclin D1 and cyclin E, at both mRNA and protein levels. In addition, thrombin treatment causes the appearance of cyclin-dependent kinase 4 (cdk4) and expression of the pro-apoptotic protein Bim. Inhibition of cdk4 prevents both induction of Bim expression and thrombin-induced neuronal apoptosis. These data demonstrate that thrombin-induced apoptosis proceeds via cell cycle activation involving cdk4 resulting in induction of Bim. Thus, cell cycle proteins could be therapeutic targets in diseases such as AD where thrombin has been implicated.
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Affiliation(s)
- Haripriya Vittal Rao
- Garrison Institute on Aging and Department of Neuropsychiatry and Behavioral Sciences, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, USA
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Scarisbrick IA, Sabharwal P, Cruz H, Larsen N, Vandell AG, Blaber SI, Ameenuddin S, Papke LM, Fehlings MG, Reeves RK, Blaber M, Windebank AJ, Rodriguez M. Dynamic role of kallikrein 6 in traumatic spinal cord injury. Eur J Neurosci 2006; 24:1457-69. [PMID: 16987227 DOI: 10.1111/j.1460-9568.2006.05021.x] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/01/2022]
Abstract
Kallikrein 6 (K6) is a member of the kallikrein gene family that comprises 15 structurally and functionally related serine proteases. In prior studies we showed that, while this trypsin-like enzyme is preferentially expressed in neurons and oligodendroglia of the adult central nervous system (CNS), it is up-regulated at sites of injury due to expression by infiltrating immune and resident CNS cells. Given this background we hypothesized that K6 is a key contributor to the pathophysiology of traumatic spinal cord injury (SCI), influencing neural repair and regeneration. Examination of K6 expression following contusion injury to the adult rat cord, and in cases of human traumatic SCI, indicated significant elevations at acute and chronic time points, not only at the injury site but also in cord segments above and below. Elevations in K6 were particularly prominent in macrophages, microglia and reactive astrocytes. To determine potential effects of elevated K6 on the regeneration environment, the ability of neurons to adhere to and extend processes on substrata which had been exposed to recombinant K6 was examined. Limited (1 h) or excess (24 h) K6-mediated proteolytic digestion of a growth-facilitatory substrate, laminin, significantly decreased neurite outgrowth. By contrast, similar hydrolysis of a growth-inhibitory substrate, aggrecan, significantly increased neurite extension and cell adherence. These data support the hypothesis that K6 enzymatic cascades mediate events secondary to spinal cord trauma, including dynamic modification of the capacity for axon outgrowth.
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Affiliation(s)
- I A Scarisbrick
- Program for Molecular Neuroscience, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.
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Lee DY, Park KW, Jin BK. Thrombin induces neurodegeneration and microglial activation in the cortex in vivo and in vitro: proteolytic and non-proteolytic actions. Biochem Biophys Res Commun 2006; 346:727-38. [PMID: 16777064 DOI: 10.1016/j.bbrc.2006.05.174] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2006] [Accepted: 05/24/2006] [Indexed: 12/27/2022]
Abstract
The present study evaluated the role of thrombin and its receptors in neurodegeneration and microglial activation. Immunocytochemical evidence indicated that intracortical injection of thrombin resulted in a significant loss of neurons and the activation of microglia in the rat cortex in vivo. Reverse transcription PCR and double-label immunocytochemistry further demonstrated the early and transient expression of pro-inflammatory cytokines and neurotoxic factors as well as their colocalization within activated microglia. The thrombin-induced loss of cortical neurons was partially blocked by N(G)-nitro-L-arginine methyl ester hydrochloride, a nitric oxide synthase inhibitor, and by NS-398, a cyclooxygenase-2 inhibitor, indicating that the activation of microglia is involved in the neurotoxicity of thrombin in the cortex in vivo. In addition, thrombin activated cortical microglia in culture, as indicated by the expression of several pro-inflammatory cytokines and produced cell death in microglia-free, neuron-enriched cortical cultures. However, agonist peptides for thrombin receptors, including protease-activated receptor-1 (SFLLRN), -3 (TFRGAP), and -4 (GYPGKF), failed to activate microglia and were not neurotoxic in culture. Intriguingly, morphological and biochemical evidence indicated that thrombin-induced neurotoxicity but not microglial activation was prevented by hirudin, a specific inhibitor of thrombin. Collectively, the present data suggest that a non-proteolytic activity of thrombin activates microglia and that the proteolytic activity mediates its neurotoxicity.
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Affiliation(s)
- Da Yong Lee
- Neuroscience Graduate Program and Brain Disease Research Center, Ajou University School of Medicine, Suwon 443-721, Republic of Korea
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Festoff BW, Ameenuddin S, Arnold PM, Wong A, Santacruz KS, Citron BA. Minocycline neuroprotects, reduces microgliosis, and inhibits caspase protease expression early after spinal cord injury. J Neurochem 2006; 97:1314-26. [PMID: 16638021 DOI: 10.1111/j.1471-4159.2006.03799.x] [Citation(s) in RCA: 171] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Minocycline, a clinically used tetracycline for over 40 years, crosses the blood-brain barrier and prevents caspase up-regulation. It reduces apoptosis in mouse models of Huntington's disease and familial amyotrophic lateral sclerosis (ALS) and is in clinical trial for sporadic ALS. Because apoptosis also occurs after brain and spinal cord (SCI) injury, its prevention may be useful in improving recovery. We analyzed minocycline's neuroprotective effects over 28 days following contusion SCI and found significant functional recovery compared to tetracycline. Histology, immunocytochemistry, and image analysis indicated statistically significant tissue sparing, reduced apoptosis and microgliosis, and less activated caspase-3 and substrate cleavage. Since our original report in abstract form, others have published both positive and negative effects of minocycline in various rodent models of SCI and with various routes of administration. We have since found decreased tumor necrosis factor-alpha, as well as caspase-3 mRNA expression, as possible mechanisms of action for minocycline's ameliorative action. These results support reports that modulating apoptosis, caspases, and microglia provide promising therapeutic targets for prevention and/or limiting the degree of functional loss after CNS trauma. Minocycline, and more potent chemically synthesized tetracyclines, may find a place in the therapeutic arsenal to promote recovery early after SCI in humans.
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Affiliation(s)
- Barry W Festoff
- Neurobiology Research Laboratory, Heartland Veterans Health Network, Department of Veterans Affairs Medical Center, Kansas City, Missouri 64128, USA.
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Lee DY, Oh YJ, Jin BK. Thrombin-activated microglia contribute to death of dopaminergic neurons in rat mesencephalic cultures: dual roles of mitogen-activated protein kinase signaling pathways. Glia 2005; 51:98-110. [PMID: 15789435 DOI: 10.1002/glia.20190] [Citation(s) in RCA: 69] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
This study evaluated the role of thrombin-activated microglia in the neurodegeneration of mesencephalic cultures. Immunocytochemical and biochemical evidence indicated that in co-cultures consisting of rat cortical microglia and mesencephalic neurons, thrombin led to nonselective loss of mesencephalic neurons. Accompanying neurodegeneration, microglial activation was obvious, evidenced by expression of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-1beta, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) and by increasing production of TNF-alpha and nitric oxide (NO). In mesencephalic neurons treated with conditioned media (CM) taken from thrombin-activated microglia, the number of dopaminergic neurons was significantly attenuated. The neurotoxicity of the CM was diminished when it was derived from microglia co-treated with thrombin and either an extracellular signal-regulated kinase 1/2 (ERK1/2) pathway inhibitor (PD98059) or a p38-mitogen-activated protein kinase (p38-MAPK) inhibitor (SB203580). Moreover, jun N-terminal kinase (JNK) and p38-MAPK were activated in mesencephalic neurons treated with CM of thrombin-activated microglia. Inhibition of JNK and p38-MAPK rescued the dopaminergic neurons. Collectively, these results indicate that thrombin-activated microglia induce neurodegeneration in cultured mesencephalic neurons and that the MAPKs actively participate in both microglial activation and neurodegeneration. The present data carefully suggest that microglial activation triggered by thrombin may be involved in the neuropathological processes of dopaminergic neuronal cell death that occur in Parkinson's disease.
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Affiliation(s)
- Da Yong Lee
- Brain Disease Research Center, Ajou University School of Medicine, Suwon, Korea
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Citron BA, Zoloty JE, Suo Z, Festoff BW. Tissue transglutaminase during mouse central nervous system development: lack of alternative RNA processing and implications for its role(s) in murine models of neurotrauma and neurodegeneration. ACTA ACUST UNITED AC 2005; 135:122-33. [PMID: 15857675 DOI: 10.1016/j.molbrainres.2004.12.009] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2004] [Revised: 11/17/2004] [Accepted: 12/05/2004] [Indexed: 11/28/2022]
Abstract
Tissue transglutaminase (tTG) is a member of a multigene family principally involved in catalyzing the formation of protein cross-links. Unlike other members of the transglutaminase family, tTG is multifunctional since it also serves as a guanosine triphosphate (GTP) binding protein (Galpha(h)) and participates in cell adhesion. Different isoforms of tTG can be produced by proteolysis or alternative splicing. We find that tTG mRNA is expressed at low levels in the mouse CNS relative to other tissues, and at lower levels in the CNS of mouse in comparison to that of human or rat. tTG mRNA levels are higher in the heart compared to the CNS, for example, and much higher in the liver. Within the CNS, tTG message is lowest in the adult cerebellum and thalamus and highest in the frontal cortex and striatum. In the hippocampus, tTG expression is highest during embryonic development and falls off dramatically after 1 week of life. We did not find alternative splicing of the mouse tTG. At the protein level, the predominant isoform is approximately 62 kDa. In summary, tTG, an important factor in neuronal survival, is expressed at low levels in the mouse CNS and, unlike rat and human tTG, does not appear to be regulated by alternative splicing. These findings have implications for analyses of rodent tTG expression in human neurodegenerative and neurotrauma models where alternative processing may be an attractive pathogenetic mechanism. They further impact on drug discovery paradigms, where modulation of activity may have therapeutic value.
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Affiliation(s)
- Bruce A Citron
- Molecular Biology, Veterans Affairs Medical Center, 4801 Linwood Boulevard, Kansas City, MO 64128, USA
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Luo W, Wang Y, Reiser G. Two types of protease-activated receptors (PAR-1 and PAR-2) mediate calcium signaling in rat retinal ganglion cells RGC-5. Brain Res 2005; 1047:159-67. [PMID: 15907810 DOI: 10.1016/j.brainres.2005.04.040] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2005] [Revised: 04/07/2005] [Accepted: 04/15/2005] [Indexed: 10/25/2022]
Abstract
Protease-activated receptors (PARs), G-protein-coupled receptors, are widely expressed in various tissues, where they participate in physiological and pathological processes, such as hemostasis, proliferation, tissue repair, and inflammation. Recently, we found that PARs were upregulated in the rat retina following optic nerve crush injury. However, the role of PAR in retinal ganglion cells following optic nerve crush still remains unknown. Here, we studied PAR-mediated calcium signaling in retinal ganglion cells, RGC-5. Using reverse transcription-polymerase chain reaction, we demonstrate that RGC-5 cells mainly express PAR-1 and to a lower extent PAR-2, which was further confirmed by indirect immunofluorescence. Short-term stimulation of RGC-5 cells with thrombin (0.001-1 U/ml) and trypsin (1-100 nM) concentration-dependently induced a transient increase in intracellular calcium concentration ([Ca(2+)](i)). An increase in [Ca(2+)](i) was also induced by both TRag (PAR-1 activating peptide) and PAR-2 activating peptide (PAR-2 AP). The EC(50) values were 0.3 nM for thrombin, 12.0 nM for trypsin, 1.3 microM for TRag, and 1.6 microM for PAR-2 AP, respectively. Desensitization was studied using two successive pulses of agonists. The thrombin-induced calcium response was significantly reduced by PAR-1 desensitization caused by pre-challenging RGC-5 cells with thrombin or TRag, but not by PAR-2 desensitization. On the other hand, pretreatment with trypsin, TRag or PAR-2 AP desensitized the cells since the calcium response to a second exposure to trypsin was significantly reduced. Calcium source studies revealed that PAR-induced [Ca(2+)](i) rise mainly comes from intracellular stores in RGC-5 cells. Thus, we demonstrate that PAR-1 and PAR-2 are functionally expressed in retinal ganglion cells, mediating calcium mobilization mainly from intracellular stores.
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Affiliation(s)
- Weibo Luo
- Institut für Neurobiochemie, Medizinische Fakultät, Otto-von-Guericke-Universität, Magdeburg, Leipziger Strasse 44, 39120 Magdeburg, Germany
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Wang Y, Richter-Landsberg C, Reiser G. Expression of protease-activated receptors (PARs) in OLN-93 oligodendroglial cells and mechanism of PAR-1-induced calcium signaling. Neuroscience 2004; 126:69-82. [PMID: 15145074 DOI: 10.1016/j.neuroscience.2004.03.024] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 03/15/2004] [Indexed: 11/30/2022]
Abstract
Protease-activated receptors (PARs) are a group of four members of the superfamily of G protein-coupled receptors that transduce cell signaling by proteolytic activity of extracellular serine proteases, such as thrombin. Possible expression and functions of PARs in oligodendrocytes, the myelin forming cells of the CNS, are still unclear. Here, the oligodendrocyte cell line OLN-93 was used to investigate the signaling of PARs. By reverse transcription-polymerase chain reaction (RT-PCR), immunostaining and Ca(2+) imaging studies, we demonstrate that OLN-93 cells functionally express PAR-1. PAR-3 seems to be expressed without apparent activity, and PAR-2 and PAR-4 cannot be detected. Short-term stimulation of the OLN-93 cells with PAR-1 agonists, such as thrombin, trypsin and PAR-1 activating peptide, dose-dependently induced a transient rise of [Ca(2+)](i). Concentration-effect curves display a sigmoidal concentration dependence. Elevation of [Ca(2+)](i) induced by PAR-1 mainly resulted from Ca(2+) release from intracellular stores. Studies on the effects of pertussis toxin (PTX), phospholipase C antagonist and 2-APB, showed that in OLN-93 cells (i). the calcium signaling cascade from PAR-1 was mediated through PTX-insensitive G proteins, (ii). activation of phospholipase C and liberation of InsP(3) were events upstream of the Ca(2+) release from the stores. In addition, the present study analyzed PAR-1 desensitization caused by exposure to thrombin, trypsin, and PAR-1 activating peptide, elucidated the influence of the protease cathepsin G on PAR-1 activation, and also characterized PAR-1 desensitization. This is the first study, which shows that OLN-93 oligodendrocytes functionally express PAR-1, and identifies the receptor coupling to mobilization of intracellular calcium. Moreover, the expression of PAR-1 was demonstrated by RT-PCR in primary oligodendrocytes from rat brain.
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Affiliation(s)
- Y Wang
- Otto-von-Guericke-Universität Magdeburg, Medizinische Fakultät, Institut für Neurobiochemie, Leipziger Strasse 44, 39120 Magdeburg, Germany
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Choi SH, Lee DY, Ryu JK, Kim J, Joe EH, Jin BK. Thrombin induces nigral dopaminergic neurodegeneration in vivo by altering expression of death-related proteins. Neurobiol Dis 2004; 14:181-93. [PMID: 14572441 DOI: 10.1016/s0969-9961(03)00085-8] [Citation(s) in RCA: 53] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023] Open
Abstract
One week after intranigral injection of thrombin resulted in a dose-dependent loss of dopaminergic neurons (20-78%) in the rat substantia nigra (SN), as evidenced by tyrosine hydroxylase (TH) immunohistochemistry. This cell death was accompanied by localization of terminal deoxynucleotidyl transferase-mediated fluorecein UTP nick end labeling (TUNEL) staining within dopaminergic neurons, activation of caspase-3 and attenuation of dopaminergic neuronal cell death in the SN by the caspase inhibitor (zVAD-fmk), indicative of apoptosis. Furthermore, Western blot analyses and double-immunofluorescent staining showed activation of c-Jun N-terminal kinase (JNK) and p53, and a localization of p53 in the dopaminergic neurons in the SN after thrombin, respectively. Intriguingly, Western blot analyses demonstrated significant down-regulation of Bcl-2 protein, but no alteration in Bax protein expression in the SN after thrombin. Consistent with in vivo data, degeneration of dopaminergic neurons and colocalization of TUNEL and TH were observed in mesencephalic cultures, following treatment with thrombin. Cell death was almost completely abolished by the thrombin-specific inhibitor, hirudin. Thrombin receptor-activating peptides (TRAP-6 and-14) did not mimic the effects of thrombin, even at much higher (1,000 to 2,000-fold) concentrations, although expression of protease-activated receptor-1 (PAR-1) mRNA was detected using RT-PCR. Morphological evidence and molecular events in vivo and in vitro collectively suggest that thrombin induces apoptosis in dopaminergic neurons via non-PAR-1 receptors.
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Affiliation(s)
- Sang-H Choi
- Brain Disease Research Center, Ajou University School of Medicine, Suwon 442-721, Korea
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Festoff BW, Suo Z, Citron BA. Prospects for the pharmacotherapy of amyotrophic lateral sclerosis : old strategies and new paradigms for the third millennium. CNS Drugs 2003; 17:699-717. [PMID: 12873154 DOI: 10.2165/00023210-200317100-00002] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/02/2022]
Abstract
Biomedical researchers interested in amyotrophic lateral sclerosis (ALS) must invoke newly developing technologies if we are to discover pharmaceutical treatments that will help a significant population of patients with the disease. The focus of ALS research over the last 10 years has been on reactive oxygen species (ROS) and glutamate excitotoxicity, resulting in several clinical trials and the launch of the only drug currently available for the treatment of ALS, riluzole. Unfortunately, the therapeutic benefits have been minimal, at best, and the prognosis for patients with ALS has not improved beyond very modest retardation of the disease course. By emphasising ROS and glutamate excitotoxicity, current ALS research has only partially been able to attenuate the rate of motor decline and neuronal loss associated with this illness. Clues to additional therapeutic potentialities will come from an increased understanding of the mode of cell death (apoptotic or other) and the pathways leading to neuronal demise. If death is apoptotic, inhibiting caspases may be useful. The regulatory modifications for cell death at the molecular level remain to be determined and exploited to prevent neuronal loss, although novel pathways have been recently elucidated that impact on protein aggregation and processing. Oxidative stress, seen in both familial and sporadic forms of ALS, may be only one post-translational mechanism likely to affect specific proteins essential for the health and stability of motor neurons. Protein cross-linking by transglutaminase paralleling that may lead to defects in proteasome function may also be a significant mechanism. The latest capabilities to screen protein changes in specific cells represent the kinds of advances needed to combat ALS in the third millennium.
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Affiliation(s)
- Barry W Festoff
- Department of Veterans Affairs Medical Center, Heartland Veterans Integrated Service Network, Kansas City, Missouri 64128, USA.
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Festoff BW. Proteinase-activated receptors (PARs) in the nervous system: Roles in neuroplasticity and neurotrauma. Drug Dev Res 2003. [DOI: 10.1002/ddr.10321] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
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Abstract
Signaling by the protease thrombin has started to be appreciated in cell biology, especially since the gene for protease-activated receptor-1 (PAR-1) has been cloned. Apart from the central role of thrombin in blood coagulation and wound healing, thrombin also regulates cellular functions in a large variety of cells through PAR-1, PAR-3 and PAR-4. Receptors are activated by a proteolytic cleavage mechanism via G protein-coupled signaling pathways. Accumulating evidence shows that thrombin changes the morphology of neurons and astrocytes, induces glial cell proliferation, and even exerts, depending on the concentration applied, either cytoprotective or cytotoxic effects on neural cells. These effects may be mediated, through either distinct or overlapping signal transduction cascades, by activation of PARs. This review focuses on the underlying signaling events initiated by thrombin in neuronal and glial cells, to summarize our understanding of the intracellular signaling machinery linking thrombin receptors to their potential physiological and pathological functions in the CNS.
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Affiliation(s)
- Hong Wang
- Institut für Neurobiochemie, Medizinische Fakultät der Otto-von-Guericke-Universität Magdeburg, Leipziger Strasse 44, D-39120 Magdeburg, Germany
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Li Y, Lu YY. Applying a highly specific and reproducible cDNA RDA method to clone garlic up-regulated genes in human gastric cancer cells. World J Gastroenterol 2002; 8:213-6. [PMID: 11925594 PMCID: PMC4658353 DOI: 10.3748/wjg.v8.i2.213] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To develop and optimize cDNA representational difference analysis (cDNA RDA) method and to identify and clone garlic up-regulated genes in human gastric cancer (HGC) cells.
METHODS: We performed cDNA RDA method by using abundant double-stranded cDNA messages provided by two self-constructed cDNA libraries (Allitridi-treated and paternal HGC cell line BGC823 cells cDNA libraries respectively). BamH I and Xho I restriction sites harbored in the library vector were used to select representations. Northern and Slot blots analyses were employed to identify the obtained difference products.
RESULTS: Fragments released from the cDNA library vector after restriction endonuclease digestion acted as good marker indicating the appropriate digestion degree for library DNA. Two novel expressed sequence tags (ESTs) and a recombinant gene were obtained. Slot blots result showed a 8-fold increase of glia-derived nexin/protease nexin 1 (GDN/PN1) gene expression level and 4-fold increase of hepatitis B virus x-interacting protein (XIP) mRNA level in BGC823 cells after Allitridi treatment for 72 h.
CONCLUSION: Elevated levels of GDN/PN1 and XIP mRNAs induced by Allitridi provide valuable molecular evidence for elucidating the garlic's efficacies against neurodegenerative and inflammatory diseases. Isolation of a recombinant gene and two novel ESTs further show cDNA RDA based on cDNA libraries to be a powerful method with high specificity and reproducibility in cloning differentially expressed genes.
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Affiliation(s)
- Yong Li
- Beijing Institute for Cancer Research, Beijing Laboratory of Molecular Oncology, School of Oncology, Peking University, 1 Da-Hong-Luo-Chang Street, Western District, Beijing 100034, China
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Kowluru A, Morgan NG. GTP-binding proteins in cell survival and demise: the emerging picture in the pancreatic beta-cell. Biochem Pharmacol 2002; 63:1027-35. [PMID: 11931834 DOI: 10.1016/s0006-2952(02)00849-3] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
It is widely believed that guanine nucleotide-binding regulatory proteins (G-proteins) play central roles as "molecular switches" in a variety of cellular processes ranging from signal transduction to protein and vesicle trafficking. To achieve these regulatory functions, G-proteins form complexes with a wide range of effector molecules whose activities are altered upon interaction with the G-protein. These effector molecules can be either soluble or membrane bound, and it is likely that some are localized to secretory granules where they direct the movement, docking, and fusion of granules during exocytosis. The effector molecules regulated by G-proteins are diverse and include phospholipases, protein kinases, protein phosphatases, ion channels, adenylate cyclases, cytoskeletal elements, as well as secretory vesicle and plasma membrane-associated fusion-proteins. The majority of studies performed in the pancreatic beta-cell have focused on the role of G-proteins in the regulation of insulin secretion, whereas very little attention has been focused on their potential involvement in other cellular processes. Such studies have identified and implicated both heterotrimeric (comprising alpha, beta, and gamma subunits) and monomeric (low molecular mass) G-proteins in the regulation of insulin secretion, but intriguing recent evidence has also begun to emerge which favors the view that they may be involved in the maintenance of beta-cell viability. In the present commentary, we will review this evidence and discuss the current understanding of the role of G-proteins in the life and death of the beta-cell.
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Affiliation(s)
- Anjaneyulu Kowluru
- Department of Pharmaceutical Sciences, College of Pharmacy and Allied Health Professions, Wayne State University, 619 Shapero Hall, Detroit, MI 48202, USA.
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