RESUMO
The neuromuscular junction (NMJ) is a crucial structure that connects the cholinergic motor neurons to the muscle fibers and allows for muscle contraction and movement. Despite the interruption of the supraspinal pathways that occurs in spinal cord injury (SCI), the NMJ, innervated by motor neurons below the injury site, has been found to remain intact. This highlights the importance of studying the NMJ in rodent models of various nervous system disorders, such as amyotrophic lateral sclerosis (ALS), Charcot-Marie-Tooth disease (CMT), spinal muscular atrophy (SMA), and spinal and bulbar muscular atrophy (SBMA). The NMJ is also involved in myasthenic disorders, such as myasthenia gravis (MG), and is vulnerable to neurotoxin damage. Thus, it is important to analyze the integrity of the NMJ in rodent models during the early stages of the disease, as this may allow for a better understanding of the condition and potential treatment options. The spinal cord also plays a crucial role in the functioning of the NMJ, as the junction relays information from the spinal cord to the muscle fibers, and the integrity of the NMJ could be disrupted by SCI. Therefore, it is vital to study SCI and muscle function when studying NMJ disorders. This review discusses the formation and function of the NMJ after SCI and potential interventions that may reverse or improve NMJ dysfunction, such as exercise, nutrition, and trophic factors.
Assuntos
Esclerose Lateral Amiotrófica , Traumatismos da Medula Espinal , Humanos , Junção Neuromuscular/metabolismo , Neurônios Motores/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Medula Espinal/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Traumatismos da Medula Espinal/metabolismoRESUMO
Spinal cord injury (SCI) causes significant mortality and morbidity. Currently, no FDA-approved pharmacotherapy is available for treating SCI. Previously, low doses of estrogen (17ß-estradiol, E2) were shown to improve the post-injury outcome in a rat SCI model. However, the range of associated side effects makes advocating its therapeutic use difficult. Therefore, this study aimed at investigating the therapeutic efficacy of Premarin (PRM) in SCI. PRM is an FDA-approved E2 (10%) formulation, which is used for hormone replacement therapy with minimal risk of serious side effects. The effects of PRM on SCI were examined by magnetic resonance imaging, immunofluorescent staining, and western blot analysis in a rat model. SCI animals treated with vehicle alone, PRM, E2 receptor antagonist (ICI), or PRM + ICI were graded in a blinded way for locomotor function by using the Basso-Beattie-Bresnahan (BBB) locomotor scale. PRM treatment for 7 days decreased post-SCI lesion volume and attenuated neuronal cell death, inflammation, and axonal damage. PRM also altered the balance of pro- and anti-apoptotic proteins in favor of cell survival and improved angiogenesis and microvascular growth. Increased expression of estrogen receptors (ERs) ERα and ERß following PRM treatment and their inhibition by ER inhibitor indicated that the neuroprotection associated with PRM treatment might be E2-receptor mediated. The attenuation of glial activation with decreased inflammation and cell death, and increased angiogenesis by PRM led to improved functional outcome as determined by the BBB locomotor scale. These results suggest that PRM treatment has significant therapeutic implications for the improvement of post-SCI outcome.
Assuntos
Estrogênios Conjugados (USP)/farmacologia , Doenças Neurodegenerativas/tratamento farmacológico , Traumatismos da Medula Espinal/tratamento farmacológico , Animais , Axônios/efeitos dos fármacos , Axônios/metabolismo , Modelos Animais de Doenças , Estradiol/metabolismo , Estrogênios/metabolismo , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Locomoção/efeitos dos fármacos , Masculino , Atividade Motora/efeitos dos fármacos , Doenças Neurodegenerativas/metabolismo , Neuroproteção/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica/efeitos dos fármacos , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Traumatismos da Medula Espinal/metabolismoRESUMO
Spinal cord injury (SCI) is associated with devastating neurological deficits affecting more than 11,000 Americans each year. Although several therapeutic agents have been proposed and tested, no FDA-approved pharmacotherapy is available for SCI treatment. We have recently demonstrated that estrogen (E2) acts as an antioxidant and anti-inflammatory agent, attenuating gliosis in SCI. We have also demonstrated that nanoparticle-mediated focal delivery of E2 to the injured spinal cord decreases lesion size, reactive gliosis, and glial scar formation. The current study tested in vitro effects of E2 on reactive oxygen species (ROS) and calpain activity in microglia, astroglia, macrophages, and fibroblasts, which are believed to participate in the inflammatory events and glial scar formation after SCI. E2 treatment decreased ROS production and calpain activity in these glial cells, macrophages, and fibroblast cells in vitro. This study also tested the efficacy of fast- and slow-release nanoparticle-E2 constructs in a rat model of SCI. Focal delivery of E2 via nanoparticles increased tissue distribution of E2 over time, attenuated cell death, and improved myelin preservation in injured spinal cord. Specifically, the fast-release nanoparticle-E2 construct reduced the Bax/Bcl-2 ratio in injured spinal cord tissues, and the slow-release nanoparticle-E2 construct prevented gliosis and penumbral demyelination distal to the lesion site. These data suggest this novel E2 delivery strategy to the lesion site may decrease inflammation and improve functional outcomes following SCI.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Estrogênios/administração & dosagem , Bainha de Mielina/efeitos dos fármacos , Nanopartículas/administração & dosagem , Fármacos Neuroprotetores/administração & dosagem , Traumatismos da Medula Espinal/tratamento farmacológico , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Humanos , Masculino , Camundongos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/patologia , Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Células RAW 264.7 , Ratos , Ratos Sprague-Dawley , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia , Vértebras Torácicas/lesõesRESUMO
Spinal Cord Injury (SCI) is a debilitating condition characterized by damage to the spinal cord, resulting in loss of function, mobility, and sensation. Although increasingly prevalent in the US, no FDA-approved therapy exists due to the unfortunate complexity of the condition, and the difficulties of SCI may be furthered by the development of SCI-related complications, such as osteoporosis. SCI demonstrates two crucial stages for consideration: the primary stage and the secondary stage. While the primary stage is suggested to be immediate and irreversible, the secondary stage is proposed as a promising window of opportunity for therapeutic intervention. Enolase, a metabolic enzyme upregulated after SCI, performs non-glycolytic functions, promoting inflammatory events via extracellular degradative actions and increased production of inflammatory cytokines and chemokines. Neuron-specific enolase (NSE) serves as a biomarker of functional damage to neurons following SCI, and the inhibition of NSE has been demonstrated to reduce signs of secondary injury of SCI and to ameliorate dysfunction. This Viewpoint article involves enolase activation in the regulation of RANK-RANKL pathway and summarizes succinctly the mechanisms influencing osteoclast-mediated resorption of bone in SCI. Our laboratory proposes that inhibition of enolase activation may reduce SCI-induced inflammatory response and decrease osteoclast activity, limiting the chances of skeletal tissue loss in SCI.
RESUMO
This study examines the cytokine/chemokine profile of a 62-year-old African American male with progressive multiple sclerosis (MS). MRI images of the MS patient demonstrated generalized white matter involvement with multiple lesions in the periventricular area. A 42-plex Discovery Assay® (Eve Technologies) of the patient's plasma and peripheral blood mononuclear cells (PBMCs) supernatant or PBMC-derived T cell supernatant samples from two separate clinic visits revealed vastly differing cytokine/chemokine levels. In addition, certain cytokine/chemokine profiles had notable differences when compared to the larger patient group or patients' PBMCs treated with a calpain inhibitor in vitro. Interestingly, large numbers of cytokines/chemokines and growth factors in MS PBMCs are modulated by calpain inhibition, suggesting the clinical significance of these findings in designing better therapeutics against progressive MS.
Assuntos
Calpaína/sangue , Quimiocinas/sangue , Citocinas/sangue , Glicoproteínas/uso terapêutico , Esclerose Múltipla Crônica Progressiva/sangue , Esclerose Múltipla Crônica Progressiva/tratamento farmacológico , Biomarcadores/sangue , Calpaína/antagonistas & inibidores , Quimiocinas/antagonistas & inibidores , Citocinas/antagonistas & inibidores , Glicoproteínas/farmacologia , Humanos , Interferon beta-1a/farmacologia , Interferon beta-1a/uso terapêutico , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla Crônica Progressiva/diagnóstico por imagemRESUMO
The cytokine/chemokine expression signature of a 60-year-old African American male with relapsing-remitting multiple sclerosis (RRMS) was analyzed using patient blood samples obtained from two separate visits to the clinic. Thirty-six different cytokines, chemokines, and growth factors were detected in the plasma of the RRMS patient using a multiplexed bead-based immunoassay. Results indicated that at least ten of these factors with a concentration of > 100 pg/mL are identified as pro-inflammatory. Calpain inhibition led to an anti-inflammatory effect, as indicated by a decrease in expression of pro-inflammatory cytokines/chemokines such as GM-CSF, IFNγ, and IL-17A, and a relative increase in two of the anti-inflammatory cytokines (IL-13 and IL-4) in the peripheral blood mononuclear cells activated with anti-CD3/CD28. Overall, these results suggest that the unique cytokine/chemokine pattern observed in the plasma of the RRMS patient can be used as a prognostic marker and calpain inhibition may be used as a novel therapeutic strategy for treating excessive inflammatory response specific to RRMS patients.
Assuntos
Quimiocinas/biossíntese , Inibidores de Cisteína Proteinase/uso terapêutico , Citocinas/biossíntese , Dipeptídeos/uso terapêutico , Leucócitos Mononucleares/metabolismo , Esclerose Múltipla Recidivante-Remitente/sangue , Quimiocinas/genética , Inibidores de Cisteína Proteinase/farmacologia , Citocinas/genética , Dipeptídeos/farmacologia , Expressão Gênica , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológicoRESUMO
Spinal cord injury (SCI) is a complex debilitating condition leading to permanent life-long neurological deficits. The complexity of SCI suggests that a concerted multi-targeted therapeutic approach is warranted to optimally improve function. Damage to spinal cord is complicated by an increased detrimental response from secondary injury factors mediated by activated glial cells and infiltrating macrophages. While elevation of enolase especially neuron specific enolase (NSE) in glial and neuronal cells is believed to trigger inflammatory cascades in acute SCI, alteration of NSE and its subsequent effects in acute SCI remains unknown. This study measured NSE expression levels and key inflammatory mediators after acute SCI and investigated the role of ENOblock, a novel small molecule inhibitor of enolase, in a male Sprague-Dawley (SD) rat SCI model. Serum NSE levels as well as cytokines/chemokines and metabolic factors were evaluated in injured animals following treatment with vehicle alone or ENOblock using Discovery assay. Spinal cord samples were also analyzed for NSE and MMPs 2 and 9 as well as glial markers by Western blotting. The results indicated a significant decrease in serum inflammatory cytokines/chemokines and NSE, alterations of metabolic factors and expression of MMPs in spinal cord tissues after treatment with ENOblock (100 µg/kg, twice). These results support the hypothesis that activation of glial cells and inflammation status can be modulated by regulation of NSE expression and activity. Analysis of SCI tissue samples by immunohistochemistry confirmed that ENOblock decreased gliosis which may have occurred through reduction of elevated NSE in rats. Overall, elevation of NSE is deleterious as it promotes extracellular degradation and production of inflammatory cytokines/chemokines and metabolic factors which activates glia and damages neurons. Thus, reduction of NSE by ENOblock may have potential therapeutic implications in acute SCI.
Assuntos
Neurônios/efeitos dos fármacos , Fosfopiruvato Hidratase/farmacologia , Traumatismos da Medula Espinal/tratamento farmacológico , Medula Espinal/efeitos dos fármacos , Doença Aguda , Animais , Biomarcadores/sangue , Citocinas/metabolismo , Modelos Animais de Doenças , Gliose/tratamento farmacológico , Gliose/metabolismo , Masculino , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neurônios/metabolismo , Ratos Sprague-Dawley , Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologiaRESUMO
Multiple sclerosis (MS) is a demyelinating disorder characterized by massive neurodegeneration and profound axonal loss. Since myelin is enriched with sphingolipids and some of them display toxicity, biological function of sphingolipids in demyelination has been investigated in MS brain tissues. An elevation of sphingosine with a decrease in monoglycosylceramide and psychosine (myelin markers) was observed in MS white matter and plaque compared to normal brain tissue. This indicated that sphingosine toxicity might mediate oligodendrocyte degeneration. To explain the source of sphingosine accumulation, total sphingolipid profile was investigated in Lewis rats after inducing experimental autoimmune encephalomyelitis (EAE) and also in human oligodendrocytes in culture. An intermittent increase in ceramide followed by sphingosine accumulation in EAE spinal cord along with a stimulation of serine-palmitoyltransferase (SPT) activity was observed. Apoptosis was identified in the lumbar spinal cord, the most prominent demyelinating area, in the EAE rats. TNFα and IFNγ stimulation of oligodendrocytes in culture also led to an accumulation of ceramide with an elevation of sphingosine. Ceramide elevation was drastically blocked by myriocin, an inhibitor of SPT, and also by FTY720. Myriocin treatment also protected oligodendrocytes from cytokine mediated apoptosis or programmed cell death. Hence, we propose that sphingosine toxicity may contribute to demyelination in both EAE and MS, and the intermittent ceramide accumulation in EAE may, at least partly, be mediated via SPT activation, which is a novel observation that has not been previously reported.
Assuntos
Ceramidas/farmacologia , Encefalomielite Autoimune Experimental/tratamento farmacológico , Esclerose Múltipla/tratamento farmacológico , Serina C-Palmitoiltransferase/metabolismo , Esfingosina/toxicidade , Animais , Modelos Animais de Doenças , Cloridrato de Fingolimode/farmacologia , Humanos , Bainha de Mielina/metabolismo , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/metabolismo , Ratos , Medula Espinal/metabolismoRESUMO
Optic neuritis (ON), inflammation of the optic nerve, is strongly associated with multiple sclerosis. ON pathology is characterized by attack of autoreactive T cells against optic nerve antigens, resulting in demyelination, death of retinal ganglion cells, and cumulative visual impairment. A model of experimental autoimmune encephalomyelitis (EAE) was utilized to study the onset and progression of ON and the neuroprotective efficacy of oral treatment with the calpain inhibitor SNJ 1945. EAE was actively induced in B10.PL mice with myelin basic protein on Days 0 and 2, and mice received twice daily oral dosing of SNJ 1945 from Day 9 until sacrificing (Day 26). Visual function was determined by electroretinogram recordings and daily measurement of optokinetic responses (OKR) to a changing pattern stimulus. Optic nerve and retinal histopathology was investigated by immunohistochemical and luxol fast blue staining. EAE mice manifested losses in OKR thresholds, a measurement of visual acuity, which began early in the disease course. There was a significant bias toward unilateral OKR impairment among EAE-ON eyes. Treatment with SNJ 1945, initiated after the onset of OKR threshold decline, improved visual acuity, pattern electroretinogram amplitudes, and paralysis, with attenuation of retinal ganglion cell death. Furthermore, calpain inhibition spared oligodendrocytes, prevented degradation of axonal neurofilament protein, and attenuated reactive astrocytosis. The trend of early, unilateral visual impairment in EAE-ON parallels the clinical presentation of ON exacerbations associated with multiple sclerosis. Calpain inhibition may represent an ideal candidate therapy for the preservation of vision in clinical ON. As in multiple sclerosis (MS) patients, optic neuritis (ON) and early, primarily monocular loss in spatial acuity is observed in a rodent model (EAE, experimental autoimmune encephalomyelitis). Daily oral treatment with the calpain inhibitor SNJ 1945 preserves visual acuity and preserves retinal ganglion cells (Brn3a, brain-specific homeobox/POU domain protein 3A) and their axons (MOSP, myelin oligodendrocyte-specific protein). Calpain inhibition may represent a candidate therapy for the preservation of vision in ON.
Assuntos
Calpaína/antagonistas & inibidores , Carbamatos/farmacologia , Carbamatos/uso terapêutico , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Neurite Óptica/tratamento farmacológico , Células Ganglionares da Retina/efeitos dos fármacos , Animais , Morte Celular/efeitos dos fármacos , Eletrorretinografia/efeitos dos fármacos , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/patologia , Gliose/prevenção & controle , Masculino , Camundongos , Proteína Básica da Mielina/metabolismo , Nistagmo Optocinético/efeitos dos fármacos , Neurite Óptica/etiologia , Neurite Óptica/fisiopatologia , Estimulação Luminosa , Acuidade Visual/efeitos dos fármacosRESUMO
Spinal cord injury (SCI) causes loss of neurological function and, depending upon the severity of injury, may lead to paralysis. Currently, no FDA-approved pharmacotherapy is available for SCI. High-dose methylprednisolone is widely used, but this treatment is controversial. We have previously shown that low doses of estrogen reduces inflammation, attenuates cell death, and protects axon and myelin in SCI rats, but its effectiveness in recovery of function is not known. Therefore, the goal of this study was to investigate whether low doses of estrogen in post-SCI would reduce inflammation, protect cells and axons, and improve locomotor function during the chronic phase of injury. Injury (40 g.cm force) was induced at thoracic 10 in young adult male rats. Rats were treated with 10 or 100 µg 17ß-estradiol (estrogen) for 7 days following SCI and compared with vehicle-treated injury and laminectomy (sham) controls. Histology (H&E staining), immunohistofluorescence, Doppler laser technique, and Western blotting were used to monitor tissue integrity, gliosis, blood flow, angiogenesis, the expression of angiogenic factors, axonal degeneration, and locomotor function (Basso, Beattie, and Bresnahan rating) following injury. To assess the progression of recovery, rats were sacrificed at 7, 14, or 42 days post injury. A reduction in glial reactivity, attenuation of axonal and myelin damage, protection of cells, increased expression of angiogenic factors and microvessel growth, and improved locomotor function were found following estrogen treatment compared with vehicle-treated SCI rats. These results suggest that treatment with a very low dose of estrogen has significant therapeutic implications for the improvement of locomotor function in chronic SCI. Experimental studies with low dose estrogen therapy in chronic spinal cord injury (SCI) demonstrated the potential for multi-active beneficial outcomes that could ameliorate the degenerative pathways in chronic SCI as shown in (a). Furthermore, the alterations in local spinal blood flow could be significantly alleviated with low dose estrogen therapy. This therapy led to the preservation of the structural integrity of the spinal cord (b), which in turn led to the improved functional recovery as shown (c).
Assuntos
Indutores da Angiogênese/administração & dosagem , Estradiol/administração & dosagem , Locomoção/efeitos dos fármacos , Recuperação de Função Fisiológica/efeitos dos fármacos , Traumatismos da Medula Espinal/tratamento farmacológico , Animais , Doença Crônica , Estrogênios/administração & dosagem , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/patologia , Locomoção/fisiologia , Masculino , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica/fisiologia , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologiaRESUMO
Spinal cord injury (SCI) is a debilitating condition with neurological deficits and loss of motor function that, depending on the severity, may lead to paralysis. The only treatment currently available is methylprednisolone, which is widely used and renders limited efficacy in SCI. Therefore, other therapeutic agents must be developed. The neuroprotective efficacy of estrogen in SCI was studied with a pre-clinical and pro-translational perspective. Acute SCI was induced in rats that were treated with low doses of estrogen (1, 5, 10, or 100 µg/kg) and compared with vehicle-treated injured rats or laminectomy control (sham) rats at 48 h post-SCI. Changes in gliosis and other pro-inflammatory responses, expression and activity of proteolytic enzymes (e.g., calpain, caspase-3), apoptosis of neurons in SCI, and cell death were monitored via Western blotting and immunohistochemistry. Negligible pro-inflammatory responses or proteolytic events and very low levels of neuronal death were found in sham rats. In contrast, vehicle-treated SCI rats showed profound pro-inflammatory responses with reactive gliosis, elevated expression and activity of calpain and caspase-3, elevated Bax:Bcl-2 ratio, and high levels of neuronal death in lesion and caudal regions of the injured spinal cord. Estrogen treatment at each dose reduced pro-inflammatory and proteolytic activities and protected neurons in the caudal penumbra in acute SCI. Estrogen treatment at 10 µg was found to be as effective as 100 µg in ameliorating the above parameters in injured animals. Results from this investigation indicated that estrogen at a low dose could be a promising therapeutic agent for treating acute SCI. Experimental studies with low dose estrogen therapy in acute spinal cord injury (SCI) demonstrated the potential for multi-active beneficial outcomes. Estrogen has been found to ameliorate several degenerative pathways following SCI. Thus, such early protective effects may even lead to functional recovery in long term injury. Studies are underway in chronic SCI in a follow up manuscript.
Assuntos
Estrogênios/administração & dosagem , Estrogênios/farmacologia , Gliose/tratamento farmacológico , Neurônios/efeitos dos fármacos , Recuperação de Função Fisiológica/efeitos dos fármacos , Traumatismos da Medula Espinal/tratamento farmacológico , Animais , Morte Celular/efeitos dos fármacos , Modelos Animais de Doenças , Gliose/patologia , Masculino , Fármacos Neuroprotetores/administração & dosagem , Fármacos Neuroprotetores/farmacologia , Ratos Sprague-Dawley , Medula Espinal/fisiopatologia , Medula Espinal/cirurgia , Traumatismos da Medula Espinal/patologiaRESUMO
Activated microglia release pro-inflammatory factors and calpain into the extracellular milieu, damaging surrounding neurons. However, mechanistic links to progressive neurodegeneration in disease such as multiple sclerosis (MS) remain obscure. We hypothesize that persistent damaged/dying neurons may also release cytotoxic factors and calpain into the media, which then activate microglia again. Thus, inflammation, neuronal damage, and microglia activation, i.e., bi-directional interaction between neurons and microglia, may be involved in the progressive neurodegeneration. We tested this hypothesis using two in vitro models: (i) the effects of soluble factors from damaged primary cortical neurons upon primary rat neurons and microglia and (ii) soluble factors released from CD3/CD28 activated peripheral blood mononuclear cells of MS patients on primary human neurons and microglia. The first model indicated that neurons due to injury with pro-inflammatory agents (IFN-γ) release soluble neurotoxic factors, including COX-2, reactive oxygen species, and calpain, thus activating microglia, which in turn released neurotoxic factors as well. This repeated microglial activation leads to persistent inflammation and neurodegeneration. The released calpain from neurons and microglia was confirmed by the use of calpain inhibitor calpeptin or SNJ-1945 as well as µ- and m-calpain knock down using the small interfering RNA (siRNA) technology. Our second model using activated peripheral blood mononuclear cells, a source of pro-inflammatory Th1/Th17 cytokines and calpain released from auto-reactive T cells, corroborated similar results in human primary cell cultures and confirmed calpain to be involved in progressive MS. These insights into reciprocal paracrine regulation of cell injury and calpain activation in the progressive phase of MS, Parkinson's disease, and other neurodegenerative diseases suggest potentially beneficial preventive and therapeutic strategies, including calpain inhibition.
Assuntos
Calpaína/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Microglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Animais , Calpaína/antagonistas & inibidores , Calpaína/genética , Carbamatos/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Dipeptídeos/farmacologia , Ativação Enzimática/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Inflamação/induzido quimicamente , Inflamação/patologia , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/patologia , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Doenças Neurodegenerativas/induzido quimicamente , Doenças Neurodegenerativas/patologia , Fármacos Neuroprotetores/farmacologia , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Células Th1/metabolismo , Células Th17/metabolismoRESUMO
Radiation-induced necrosis (RN) is a relatively common side effect of radiation therapy for glioblastoma. However, the molecular mechanisms involved and the ways RN mechanisms differ from regulated cell death (apoptosis) are not well understood. Here, we compare the molecular mechanism of cell death (apoptosis or necrosis) of C6 glioma cells in both in vitro and in vivo (C6 othotopically allograft) models in response to low and high doses of X-ray radiation. Lower radiation doses were used to induce apoptosis, while high-dose levels were chosen to induce radiation necrosis. Our results demonstrate that active caspase-8 in this complex I induces apoptosis in response to low-dose radiation and inhibits necrosis by cleaving RIP1 and RI. When activation of caspase-8 was reduced at high doses of X-ray radiation, the RIP1/RIP3 necrosome complex II is formed. These complexes induce necrosis through the caspase-3-independent pathway mediated by calpain, cathepsin B/D, and apoptosis-inducing factor (AIF). AIF has a dual role in apoptosis and necrosis. At high doses, AIF promotes chromatinolysis and necrosis by interacting with histone H2AX. In addition, NF-κB, STAT-3, and HIF-1 play a crucial role in radiation-induced inflammatory responses embedded in a complex inflammatory network. Analysis of inflammatory markers in matched plasma and cerebrospinal fluid (CSF) isolated from in vivo specimens demonstrated the upregulation of chemokines and cytokines during the necrosis phase. Using RIP1/RIP3 kinase specific inhibitors (Nec-1, GSK'872), we also establish that the RIP1-RIP3 complex regulates programmed necrosis after either high-dose radiation or TNF-α-induced necrosis requires RIP1 and RIP3 kinases. Overall, our data shed new light on the relationship between RIP1/RIP3-mediated programmed necrosis and AIF-mediated caspase-independent programmed necrosis in glioblastoma.
Assuntos
Raios gama/efeitos adversos , Glioblastoma/radioterapia , Necrose/metabolismo , Necrose/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Lesões por Radiação/metabolismo , Lesões por Radiação/patologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/metabolismo , Western Blotting , Caspases , Proliferação de Células , Glioblastoma/metabolismo , Glioblastoma/patologia , Técnicas Imunoenzimáticas , Masculino , Necrose/etiologia , Lesões por Radiação/etiologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
To clarify the molecular changes of sublesional muscle in the acute phase of spinal cord injury (SCI), a moderately severe injury (40 g cm) was induced in the spinal cord (T10 vertebral level) of adult male Sprague-Dawley rats (injury) and compared with sham (laminectomy only). Rats were sacrificed at 48 h (acute) post injury, and gastrocnemius muscles were excised. Morphological examination revealed no significant changes in the muscle fiber diameter between the sham and injury rats. Western blot analyses performed on the visibly red, central portion of the gastrocnemius muscle showed significantly higher expression of muscle specific E3 ubiquitin ligases (muscle ring finger-1 and muscle atrophy f-box) and significantly lower expression of phosphorylated Akt-1/2/3 in the injury group compared to the sham group. Cyclooxygenase 2, tumor necrosis factor alpha (TNF-α), and caspase-1, also had a significantly higher expression in the injury group; although, the mRNA levels of TNF-α and IL-6 did not show any significant difference between the sham and injury groups. These results suggest activation of protein degradation, deactivation of protein synthesis, and development of inflammatory reaction occurring in the sublesional muscles in the acute phase of SCI before overt muscle atrophy is seen.
Assuntos
Perfilação da Expressão Gênica , Músculo Esquelético/metabolismo , Traumatismos da Medula Espinal/genética , Animais , Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Masculino , Músculo Esquelético/patologia , Ratos , Ratos Sprague-Dawley , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologiaRESUMO
Enolase is a multifunctional protein, which is expressed abundantly in the cytosol. Upon stimulatory signals, enolase can traffic to cell surface and contribute to different pathologies including injury, autoimmunity, infection, inflammation, and cancer. Cell-surface expression of enolase is often detected on activated macrophages, microglia/macrophages, microglia, and astrocytes, promoting extracellular matrix degradation, production of pro-inflammatory cytokines/chemokines, and invasion of inflammatory cells in the sites of injury and inflammation. Inflammatory stimulation also induces translocation of enolase from the cytosolic pool to the cell surface where it can act as a plasminogen receptor and promote extracellular matrix degradation and tissue damage. Spinal cord injury (SCI) is a devastating debilitating condition characterized by progressive pathological changes including complex and evolving molecular cascades, and insights into the role of enolase in multiple inflammatory events have not yet been fully elucidated. Neuronal damage following SCI is associated with an elevation of neuron specific enolase (NSE), which is also known to play a role in the pathogenesis of hypoxic-ischemic brain injury. Thus, NSE is now considered as a biomarker in ischemic brain damage, and it has recently been suggested to be a biomarker in traumatic brain injury (TBI), stroke and anoxic encephalopathy after cardiac arrest and acute SCI as well. This review article gives an overview of the current basic research and clinical studies on the role of multifunctional enolase in neurotrauma, with a special emphasis on NSE in acute SCI.
Assuntos
Neurônios/metabolismo , Fosfopiruvato Hidratase/metabolismo , Traumatismos da Medula Espinal/metabolismo , Medula Espinal/metabolismo , HumanosRESUMO
Recurrent meningiomas constitute an uncommon but significant problem after standard (surgery and radiation) therapy failure. Current chemotherapies (hydroxyurea, RU-486, and interferon-α) are only of marginal benefit. There is an urgent need for more effective treatments for meningioma patients who have failed surgery and radiation therapy. Limonin, Tangeritin, Zerumbone, 6-Gingerol, Ganoderic Acid A, and Ganoderic Acid DM are some of the plant derivatives that have anti-tumorgenic properties and cause cell death in meningioma cells in vitro. Due to its ease of administration, long-term tolerability, and low incidence of long-term side effects, we explored its potential as a therapeutic agent against meningiomas by examining their efficacy in vitro against meningioma cells. Treatment effects were assessed using MTT assay, Western blot analysis, caspases assay, and DNA fragmentation assay. Results indicated that treatments of IOMM-Lee and CH157MN meningioma cells with Limonin, Tangeritin, Zerumbone, 6-Gingerol, Ganoderic Acid A, and Ganoderic Acid DM induced apoptosis with enhanced phosphorylation of glycogen synthase kinase 3 ß (GSK3ß) via inhibition of the Wnt5/ß-catenin pathway. These drugs did not induce apoptosis in normal human neurons. Other events in apoptosis included downregulation of tetraspanin protein (TSPAN12), survival proteins (Bcl-XL and Mcl-1), and overexpression apoptotic factors (Bax and caspase-3). These results provide preliminary strong evidence that medicinal plants containing Limonin, Tangeritin, 6-Gingerol, Zerumbone, Ganoderic Acid A, and Ganoderic Acid DM can be applied to high-grade meningiomas as a therapeutic agent, and suggests that further in vivo studies are necessary to explore its potential as a therapeutic agent against malignant meningiomas.
Assuntos
Catecóis/administração & dosagem , Álcoois Graxos/administração & dosagem , Flavonas/administração & dosagem , Ácidos Heptanoicos/administração & dosagem , Lanosterol/análogos & derivados , Limoninas/administração & dosagem , Meningioma/tratamento farmacológico , Sesquiterpenos/administração & dosagem , Triterpenos/administração & dosagem , Apoptose/efeitos dos fármacos , Catecóis/química , Linhagem Celular Tumoral , Fragmentação do DNA/efeitos dos fármacos , Álcoois Graxos/química , Flavonas/química , Quinase 3 da Glicogênio Sintase/biossíntese , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Ácidos Heptanoicos/química , Humanos , Lanosterol/administração & dosagem , Lanosterol/química , Limoninas/química , Meningioma/genética , Meningioma/patologia , Sesquiterpenos/química , Triterpenos/química , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
A need exists for the effective treatment of individuals suffering from spinal cord injury (SCI). Recent advances in the understanding of the pathophysiological mechanisms occurring in SCI have resulted in an expansion of new therapeutic targets. This review summarizes both preclinical and clinical findings investigating the mechanisms and cognate pharmacologic therapeutics targeted to modulate hypoxia, ischemia, excitotoxicity, inflammation, apoptosis, epigenetic alterations, myelin regeneration and scar remodeling. Successful modulation of these targets has been demonstrated in both preclinical and clinical studies with agents such as Oxycyte, Minocycline, Riluzole, Premarin, Cethrin, and ATI-355. The translation of these agents into clinical studies highlights the progress the field has made in the past decade. SCI proves to be a complex condition; the numerous pathophysiological mechanisms occurring at varying time points suggests that a single agent approach to the treatment of SCI may not be optimal. As the field continues to mature, the hope is that the knowledge gained from these studies will be applied to the development of an effective multi-pronged treatment strategy for SCI.
Assuntos
Traumatismos da Medula Espinal/tratamento farmacológico , Animais , Humanos , Inflamação/patologia , Degeneração Neural , Fármacos Neuroprotetores/farmacologia , Traumatismos da Medula Espinal/patologiaRESUMO
Insulin-like growth factor-1 (IGF-1) is a neuroprotective growth factor that promotes neuronal survival by inhibition of apoptosis. To examine whether IGF-1 exerts cytoprotective effects against extracellular inflammatory stimulation, ventral spinal cord 4.1 (VSC4.1) motoneuron cells were treated with interferon-gamma (IFN-γ). Our data demonstrated apoptotic changes, increased calpain:calpastatin and Bax:Bcl-2 ratios, and expression of apoptosis-related proteases (caspase-3 and -12) in motoneurons rendered by IFN-γ in a dose-dependent manner. Post-treatment with IGF-1 attenuated these changes. In addition, IGF-1 treatment of motoneurons exposed to IFN-γ decreased expression of inflammatory markers (cyclooxygenase-2 and nuclear factor-kappa B:inhibitor of kappa B ratio). Furthermore, IGF-1 attenuated the loss of expression of IGF-1 receptors (IGF-1Rα and IGF-1Rß) and estrogen receptors (ERα and ERß) induced by IFN-γ. To determine whether the protective effects of IGF-1 are associated with ERs, ERs antagonist ICI and selective siRNA targeted against ERα and ERß were used in VSC4.1 motoneurons. Distinctive morphological changes were observed following siRNA knockdown of ERα and ERß. In particular, apoptotic cell death assessed by TUNEL assay was enhanced in both ERα and ERß-silenced VSC4.1 motoneurons following IFN-γ and IGF-1 exposure. These results suggest that IGF-1 protects motoneurons from inflammatory insult by a mechanism involving pivotal interactions with ERα and ERß.
Assuntos
Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Interferon gama/metabolismo , Neurônios Motores/metabolismo , Receptor Cross-Talk/fisiologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Calpaína/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Receptor alfa de Estrogênio/genética , Receptor beta de Estrogênio/genética , Fator de Crescimento Insulin-Like I/farmacologia , Interferon gama/farmacologia , Camundongos , Neurônios Motores/citologia , Neurônios Motores/efeitos dos fármacos , Neuroblastoma , Fármacos Neuroprotetores/metabolismo , Fármacos Neuroprotetores/farmacologia , RNA Interferente Pequeno/genética , Ratos , Receptor Cross-Talk/efeitos dos fármacos , Medula Espinal/citologiaRESUMO
Complex pathophysiology of Parkinson's disease involves multiple CNS cell types. Degeneration in spinal cord neurons alongside brain has been shown to be involved in Parkinson's disease and evidenced in experimental parkinsonism. However, the mechanisms of these degenerative pathways are not well understood. To unravel these mechanisms SH-SY5Y neuroblastoma cells were differentiated into dopaminergic and cholinergic phenotypes, respectively, and used as cell culture model following exposure to two parkinsonian neurotoxicants MPP(+) and rotenone. SNJ-1945, a cell-permeable calpain inhibitor was tested for its neuroprotective efficacy. MPP(+) and rotenone dose-dependently elevated the levels of intracellular free Ca(2+) and induced a concomitant rise in the levels of active calpain. SNJ-1945 pre-treatment significantly protected cell viability and preserved cellular morphology following MPP(+) and rotenone exposure. The neurotoxicants elevated the levels of reactive oxygen species more profoundly in SH-SY5Y cells differentiated into dopaminergic phenotype, and this effect could be attenuated with SNJ-1945 pre-treatment. In contrast, significant levels of inflammatory mediators cyclooxygenase-2 (Cox-2 and cleaved p10 fragment of caspase-1) were up-regulated in the cholinergic phenotype, which could be dose-dependently attenuated by the calpain inhibitor. Overall, SNJ-1945 was efficacious against MPP(+) or rotenone-induced reactive oxygen species generation, inflammatory mediators, and proteolysis. A post-treatment regimen of SNJ-1945 was also examined in cells and partial protection was attained with calpain inhibitor administration 1-3 h after exposure to MPP(+) or rotenone. Taken together, these results indicate that calpain inhibition is a valid target for protection against parkinsonian neurotoxicants, and SNJ-1945 is an efficacious calpain inhibitor in this context. SH-SY5Y cells, differentiated as dopaminergic (TH positive) and cholinergic (ChAT positive), were used as in vitro models for Parkinson's disease. MPP+ and rotenone induced up-regulation of calpain, expression, and activity as a common mechanism of neurodegeneration. SNJ-1945, a novel calpain inhibitor, protected both the cell phenotypes against MPP+ and rotenone.