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Autophagy is a cellular catabolic pathway generally thought to be neuroprotective. However, autophagy and in particular its upstream regulator, the ULK1 kinase, can also promote axonal degeneration. We examined the role and the mechanisms of autophagy in axonal degeneration using a mouse model of contusive spinal cord injury (SCI). Consistent with activation of autophagy during axonal degeneration following SCI, autophagosome marker LC3, ULK1 kinase, and ULK1 target, phospho-ATG13, accumulated in the axonal bulbs and injured axons. SARM1, a TIR NADase with a pivotal role in axonal degeneration, colocalized with ULK1 within 1 h after SCI, suggesting possible interaction between autophagy and SARM1-mediated axonal degeneration. In our in vitro experiments, inhibition of autophagy, including Ulk1 knockdown and ULK1 inhibitor, attenuated neurite fragmentation and reduced accumulation of SARM1 puncta in neurites of primary cortical neurons subjected to glutamate excitotoxicity. Immunoprecipitation data demonstrated that ULK1 physically interacted with SARM1 in vitro and in vivo and that SAM domains of SARM1 were necessary for ULK1-SARM1 complex formation. Consistent with a role in regulation of axonal degeneration, in primary cortical neurons ULK1-SARM1 interaction increased upon neurite damage. Supporting a role for autophagy and ULK1 in regulation of SARM1 in axonal degeneration in vivo, axonal ULK1 activation and accumulation of SARM1 were both decreased after SCI in Becn1+/- autophagy hypomorph mice compared to wild-type (WT) controls. These findings suggest a regulatory crosstalk between autophagy and axonal degeneration pathways, which is mediated through ULK1-SARM1 interaction and contributes to the ability of SARM1 to accumulate in injured axons.
Assuntos
Proteínas do Domínio Armadillo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Proteínas do Citoesqueleto , Traumatismos da Medula Espinal , Animais , Camundongos , Proteínas do Domínio Armadillo/genética , Proteínas do Domínio Armadillo/metabolismo , Autofagia , Axônios/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Camundongos Knockout , Traumatismos da Medula Espinal/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismoRESUMO
BACKGROUND: Understanding the differences in skin blood flow (SBF) on the plantar and dorsal foot in people with diabetes mellitus (DM) may help to assess the influence of diabetes and neuropathy on microvascular dysfunction and risks of diabetic foot ulcers in this population. However, there is no study comparing SBF oscillations between the plantar and dorsal foot in people with DM and peripheral neuropathy (PN). OBJECTIVE: The objective of this study was to compare SBF oscillations between the plantar and dorsal foot in people with DM and PN and investigate the underlying mechanisms responsible for the differences. METHODS: 18 people with Type 2 DM and PN and 8 healthy controls were recruited. Laser Doppler flowmetry (LDF) was used to measure SBF on the plantar and dorsal foot for 10â¯min when the subject was in the supine position. Wavelet analysis was used to quantify the relative amplitude of the characteristic frequency components of SBF oscillations. Sample entropy analysis was used to quantify the regularity degree of SBF oscillations. RESULTS: People with DM and PN had a higher SBF on the plantar foot compared to the dorsal foot. The relative wavelet amplitudes of metabolic and myogenic frequency components on the plantar foot were respectively higher and lower compared to the dorsal foot. Sample entropy analysis showed that SBF on the plantar foot had a higher degree of regularity compared to the dorsal foot. CONCLUSIONS: In people with DM and PN, higher SBF on the plantar foot is attributed to the metabolic and myogenic controls, and SBF on the plantar foot exhibits a higher degree of regularity compared to the dorsal foot. People with DM and PN also had higher plantar and dorsal SBF compared to the healthy controls. This study provides evidence to document differences in SBF of the plantar and dorsal foot in people with DM and PN.
Assuntos
Diabetes Mellitus Tipo 2/fisiopatologia , Pé Diabético/fisiopatologia , Microcirculação , Pele/irrigação sanguínea , Adulto , Biomarcadores/sangue , Velocidade do Fluxo Sanguíneo , Estudos de Casos e Controles , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/diagnóstico , Pé Diabético/sangue , Pé Diabético/diagnóstico , Feminino , Pé , Hemoglobinas Glicadas/metabolismo , Humanos , Fluxometria por Laser-Doppler , Masculino , Pessoa de Meia-Idade , Fluxo Sanguíneo Regional , Fatores de Tempo , Análise de Ondaletas , Adulto JovemRESUMO
Circadian rhythms optimize physiology and behavior to the varying demands of the 24 h day. The master circadian clock is located in the suprachiasmatic nuclei (SCN) of the hypothalamus and it regulates circadian oscillators in tissues throughout the body to prevent internal desynchrony. Here, we demonstrate for the first time that, under standard 12 h:12 h light/dark (LD) cycles, object, visuospatial, and olfactory recognition performance in C57BL/6J mice is consistently better at midday relative to midnight. However, under repeated exposure to constant light (rLL), recognition performance becomes desynchronized, with object and visuospatial performance better at subjective midday and olfactory performance better at subjective midnight. This desynchrony in behavioral performance is mirrored by changes in expression of the canonical clock genes Period1 and Period2 (Per1 and Per2), as well as the immediate-early gene Fos in the SCN, dorsal hippocampus, and olfactory bulb. Under rLL, rhythmic Per1 and Fos expression is attenuated in the SCN. In contrast, hippocampal gene expression remains rhythmic, mirroring object and visuospatial performance. Strikingly, Per1 and Fos expression in the olfactory bulb is reversed, mirroring the inverted olfactory performance. Temporal desynchrony among these regions does not result in arrhythmicity because core body temperature and exploratory activity rhythms persist under rLL. Our data provide the first demonstration that abnormal lighting conditions can give rise to temporal desynchrony between autonomous circadian oscillators in different regions, with different consequences for performance across different sensory domains. Such a dispersed network of dissociable circadian oscillators may provide greater flexibility when faced with conflicting environmental signals.SIGNIFICANCE STATEMENT A master circadian clock in the suprachiasmatic nuclei (SCN) of the hypothalamus regulates physiology and behavior across the 24 h day by synchronizing peripheral clocks throughout the brain and body. Without the SCN, these peripheral clocks rapidly become desynchronized. Here, we provide a unique demonstration that, under lighting conditions in which the central clock in the SCN is dampened, peripheral oscillators in the hippocampus and olfactory bulb become desynchronized, along with the behavioral processes mediated by these clocks. Multiple clocks that adopt different phase relationships may enable processes occurring in different brain regions to be optimized to specific phases of the 24 h day. Moreover, such a dispersed network of dissociable circadian clocks may provide greater flexibility when faced with conflicting environmental signals (e.g., seasonal changes in photoperiod).
Assuntos
Ritmo Circadiano/fisiologia , Percepção de Forma/fisiologia , Memória/fisiologia , Mascaramento Perceptivo/fisiologia , Reconhecimento Psicológico/fisiologia , Olfato/fisiologia , Navegação Espacial/fisiologia , Animais , Sincronização Cortical/fisiologia , Masculino , Rememoração Mental/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Reconhecimento Visual de Modelos/fisiologia , Estimulação Luminosa/métodos , Análise e Desempenho de TarefasRESUMO
Autophagy is a catabolic process that degrades cytoplasmic constituents and organelles in the lysosome, thus serving an important role in cellular homeostasis and protection against insults. We previously reported that defects in autophagy contribute to neuronal cell damage in traumatic spinal cord injury (SCI). Recent data from other inflammatory models implicate autophagy in regulation of immune and inflammatory responses, with low levels of autophagic flux associated with pro-inflammatory phenotypes. In the present study, we examined the effects of genetically or pharmacologically manipulating autophagy on posttraumatic neuroinflammation and motor function after SCI in mice. Methods: Young adult male C57BL/6, CX3CR1-GFP, autophagy hypomorph Becn1+/- mice, and their wildtype (WT) littermates were subjected to moderate thoracic spinal cord contusion. Neuroinflammation and autophagic flux in the injured spinal cord were assessed using flow cytometry, immunohistochemistry, and NanoString gene expression analysis. Motor function was evaluated with the Basso Mouse Scale and horizontal ladder test. Lesion volume and spared white matter were evaluated by unbiased stereology. To stimulate autophagy, disaccharide trehalose, or sucrose control, was administered in the drinking water immediately after injury and for up to 6 weeks after SCI. Results: Flow cytometry demonstrated dysregulation of autophagic function in both microglia and infiltrating myeloid cells from the injured spinal cord at 3 days post-injury. Transgenic CX3CR1-GFP mice revealed increased autophagosome formation and inhibition of autophagic flux specifically in activated microglia/macrophages. NanoString analysis using the neuroinflammation panel demonstrated increased expression of proinflammatory genes and decreased expression of genes related to neuroprotection in Becn1+/- mice as compared to WT controls at 3 days post-SCI. These findings were further validated by qPCR, wherein we observed significantly higher expression of proinflammatory cytokines. Western blot analysis confirmed higher protein expression of the microglia/macrophage marker IBA-1, inflammasome marker, NLRP3, and innate immune response markers cGAS and STING in Becn1+/- mice at 3 day after SCI. Flow cytometry demonstrated that autophagy deficit did not affect either microglial or myeloid counts at 3 days post-injury, instead resulting in increased microglial production of proinflammatory cytokines. Finally, locomotor function showed significantly worse impairments in Becn1+/- mice up to 6 weeks after SCI, which was accompanied by worsening tissue damage. Conversely, treatment with a naturally occurring autophagy inducer trehalose, reduced protein levels of p62, an adaptor protein targeting cargo to autophagosomes as well as the NLRP3, STING, and IBA-1 at 3 days post-injury. Six weeks of trehalose treatment after SCI led to improved motor function recovery as compared to control group, which was accompanied by reduced tissue damage. Conclusions: Our data indicate that inhibition of autophagy after SCI potentiates pro-inflammatory activation in microglia and is associated with worse functional outcomes. Conversely, increasing autophagy with trehalose, decreased inflammation and improved outcomes. These findings highlight the importance of autophagy in spinal cord microglia and its role in secondary injury after SCI.
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Proteína 3 que Contém Domínio de Pirina da Família NLR , Traumatismos da Medula Espinal , Animais , Autofagia , Citocinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Doenças Neuroinflamatórias , Traumatismos da Medula Espinal/complicações , Trealose/metabolismo , Trealose/farmacologiaRESUMO
Elderly patients with traumatic brain injury (TBI) have greater mortality and poorer outcomes than younger individuals. The extent to which old age alters long-term recovery and chronic microglial activation after TBI is unknown, and evidence for therapeutic efficacy in aged mice is sorely lacking. The present study sought to identify potential inflammatory mechanisms underlying age-related outcomes late after TBI. Controlled cortical impact was used to induce moderate TBI in young and old male C57BL/6 mice. At 12 weeks post-injury, aged mice exhibited higher mortality, poorer functional outcomes, larger lesion volumes, and increased microglial activation. Transcriptomic analysis identified age- and TBI-specific gene changes consistent with a disease-associated microglial signature in the chronically injured brain, including those involved with complement, phagocytosis, and autophagy pathways. Dysregulation of phagocytic and autophagic function in microglia was accompanied by increased neuroinflammation in old mice. As proof-of-principle that these pathways have functional importance, we administered an autophagic enhancer, trehalose, in drinking water continuously for 8 weeks after TBI. Old mice treated with trehalose showed enhanced functional recovery and reduced microglial activation late after TBI compared to the sucrose control group. Our data indicate that microglia undergo chronic changes in autophagic regulation with both normal aging and TBI that are associated with poorer functional outcome. Enhancing autophagy may therefore be a promising clinical therapeutic strategy for TBI, especially in older patients.
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Lesões Encefálicas Traumáticas , Microglia , Idoso , Animais , Encéfalo/patologia , Lesões Encefálicas Traumáticas/complicações , Lesões Encefálicas Traumáticas/genética , Lesões Encefálicas Traumáticas/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/patologia , Trealose/metabolismoRESUMO
Necroptosis, a regulated necrosis pathway mediated by the receptor-interacting protein kinases 1 and 3 (RIPK1 and RIPK3), is induced following spinal cord injury (SCI) and thought to contribute to neuronal and glial cell death. However, mechanisms leading to activation of necroptosis after SCI remain unclear. We have previously shown that autophagy, a catabolic pathway facilitating degradation of cytoplasmic proteins and organelles in a lysosome-dependent manner, is inhibited following SCI in rats. Our current data confirm that inhibition of autophagy also occurs after thoracic contusive SCI in the mouse model, as indicated by accumulation of both the autophagosome marker, LC3-II and autophagy cargo protein, p62/SQSTM1. This was most pronounced in the ventral horn neurons and was caused by rapid inhibition of lysosomal function after SCI. Interestingly, RIPK1, RIPK3, and the necroptosis effector protein MLKL also rapidly accumulated after SCI and localized to neurons with disrupted autophagy, suggesting that these events may be related. To determine if lysosomal dysfunction could contribute to induction of necroptosis, we treated PC12 cells and primary rat cortical neurons with lysosomal inhibitors. This led to rapid accumulation of RIPK1 and RIPK3, confirming that they are normally degraded by the lysosomal pathway. In PC12 cells lysosomal inhibition also sensitized cells to necroptosis induced by tumor necrosis factor α (TNFα) and caspase inhibitor. Imaging studies confirmed that RIPK1 partially localized to lysosomes in both untreated and lysosomal inhibitor treated cells. Similarly, we detected presence of RIPK1, RIPK3 and MLKL in both cytosol and at lysosomes after SCI in vivo. Furthermore, stimulation of autophagy and lysosomal function with rapamycin treatment led to decreased accumulation of RIPK1 and attenuated cell death after SCI. These data suggest that lysosomal dysfunction after SCI may contribute to both inhibition of autophagy and sensitize cells to necroptosis by promoting RIPK1 and RIPK3 accumulation.
Assuntos
Autofagia , Lisossomos/enzimologia , Neurônios/enzimologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Traumatismos da Medula Espinal/enzimologia , Animais , Lisossomos/genética , Lisossomos/patologia , Masculino , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/patologia , Células PC12 , Ratos , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/patologiaRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0191074.].
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The present study investigated the effects of pulsed electromagnetic field (PEMF) on the tensile biomechanical properties of diabetic wounds at different phases of healing. Two intensities of PEMF were adopted for comparison. We randomly assigned 111 10-week-old male streptozotocin-induced diabetic Sprague-Dawley rats to two PEMF groups and a sham control group. Six-millimetre biopsy punched full thickness wounds were made on the lateral side of their hindlimbs. The PEMF groups received active PEMF delivered at 25 Hz with intensity of either 2 mT or 10 mT daily, while the sham group was handled in a similar way except they were not exposed to PEMF. Wound tissues were harvested for tensile testing on post-wounding days 3, 5, 7, 10, 14 and 21. Maximum load, maximum stress, energy absorption capacity, Young's modulus and thickness of wound tissue were measured. On post-wounding day 5, the PEMF group that received 10-mT intensity had significantly increased energy absorption capacity and showed an apparent increase in the maximum load. However, the 10-mT PEMF group demonstrated a decrease in Young's modulus on day 14. The 10-mT PEMF groups showed a significant increase in the overall thickness of wound tissue whereas the 2-mT group showed a significant decrease in the overall maximum stress of the wounds tissue. The present findings demonstrated that the PEMF delivered at 10 mT can improve energy absorption capacity of diabetic wounds in the early healing phase. However, PEMF (both 2-mT and 10-mT) seemed to impair the material properties (maximum stress and Young's modulus) in the remodelling phase. PEMF may be a useful treatment for promoting the recovery of structural properties (maximum load and energy absorption capacity), but it might not be applied at the remodelling phase to avoid impairing the recovery of material properties.