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Lysophospholipid transporter LplT and acyltransferase Aas consist of a lysophospholipid-remodeling system ubiquitously found in gram-negative microorganisms. LplT flips lysophospholipids across the inner membrane, which are subsequently acylated by Aas on the cytoplasmic membrane surface. Our previous study showed that the proper functioning of this system is important to protecting E. coli from phospholipase-mediated host attack by maintaining the integrity of the bacterial cell envelope. However, the working mechanism of this system is still unclear. Herein, we report that LplT and Aas form a membrane protein complex in E. coli which allows these two enzymes to cooperate efficiently to move lysophospholipids across the bacterial membrane and catalyze their acylation. The direct interaction of LplT and Aas was demonstrated both in vivo and in vitro with a binding affinity of 2.3 µM. We found that a cytoplasmic loop of LplT adjacent to the exit of the substrate translocation pathway plays an important role in maintaining its interaction with Aas. Aas contains an acyl-acyl carrier protein synthase domain and an acyl-transferase domain. Its interaction with LplT is mediated exclusively by its transferase domain. Mutations within the three loops near the putative catalytic site of the transferase domain, respectively, disrupt its interaction with LplT and lysophospholipid acylation activity. These results support a hypothesis of the functional coupling mechanism, in which LplT directly interacts with the transferase domain of Aas for specific substrate membrane migration, providing synchronization of substrate translocation and biosynthetic events.
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INTRODUCTION: The substantia nigra pars compacta (SNc) is the key pathologic locus in neurodegenerative parkinsonian disorders. Recently, in vivo susceptibility MRI metrics were associated with postmortem glial cell density and tau burden in the SNc of parkinsonism subjects. This study investigated the red nucleus (RN), another iron-rich region adjacent to the SNc and a potential site of higher functionality in parkinsonisms. METHODS: In vivo MRI and postmortem data were obtained from 34 parkinsonism subjects and 3 controls. Neuron density, glial cell density, and percentages of area occupied by α-synuclein and tau were quantified using digitized midbrain slides. R2* and quantitative susceptibility mapping (QSM) metrics in the RN and SNc were derived from multi-gradient echo images. Histopathology data were compared between the RN and SNc using paired t-tests. MRI-histology associations were analyzed using partial Pearson correlations. RESULTS: The RN had greater neuron (t23 = 3.169, P = 0.004) and glial cell densities (t23 = 2.407, P = 0.025) than the SNc, whereas the SNc had greater α-synuclein (t28 = 4.614, P < 0.0001) and tau burden (t24 = 4.513, P = 0.0001). In both the RN (R2*: r = 0.47, P = 0.043; QSM: r = 0.52, P = 0.024) and SNc (R2*: r = 0.57, P = 0.01; QSM: r = 0.58, P = 0.009), MRI values were associated with glial cell density but not neuron density or α-synuclein (Ps > 0.092). QSM associated with tau burden (r = 0.49, P = 0.038) in the SNc, but not the RN. CONCLUSIONS: The RN is resilient to parkinsonian-related pathological processes compared to the SNc, and susceptibility MRI captured glial cell density in both regions. These findings help to further our understanding of the underlying pathophysiological processes in parkinsonisms.
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Imagen por Resonancia Magnética , Trastornos Parkinsonianos , Porción Compacta de la Sustancia Negra , Núcleo Rojo , Sustancia Negra , Humanos , Núcleo Rojo/diagnóstico por imagen , Núcleo Rojo/patología , Núcleo Rojo/metabolismo , Masculino , Anciano , Femenino , Porción Compacta de la Sustancia Negra/diagnóstico por imagen , Porción Compacta de la Sustancia Negra/patología , Porción Compacta de la Sustancia Negra/metabolismo , Anciano de 80 o más Años , Persona de Mediana Edad , Trastornos Parkinsonianos/diagnóstico por imagen , Trastornos Parkinsonianos/patología , Trastornos Parkinsonianos/metabolismo , Sustancia Negra/diagnóstico por imagen , Sustancia Negra/patología , Sustancia Negra/metabolismo , Proteínas tau/metabolismo , Neuroglía/patología , Neuroglía/metabolismo , alfa-Sinucleína/metabolismo , Neuronas/patología , Neuronas/metabolismoRESUMEN
The integrity of the plasma membrane is critical to cell function and survival. Cells have developed multiple mechanisms to repair damaged plasma membranes. A key process during plasma membrane repair is to limit the size of the damage, which is facilitated by the presence of tetraspanin-enriched rings surrounding damage sites. Here, we identify phosphatidylserine-enriched rings surrounding damaged sites of the plasma membrane, resembling tetraspanin-enriched rings. Importantly, the formation of both the phosphatidylserine- and tetraspanin-enriched rings requires phosphatidylserine and its transfer proteins ORP5 and ORP9. Interestingly, ORP9, but not ORP5, is recruited to the damage sites, suggesting cells acquire phosphatidylserine from multiple sources upon plasma membrane damage. We further demonstrate that ORP9 contributes to efficient plasma membrane repair. Our results thus unveil a role for phosphatidylserine and its transfer proteins in facilitating the formation of tetraspanin-enriched macrodomains and plasma membrane repair.
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Membrana Celular , Fosfatidilserinas , Tetraspaninas , Humanos , Células HeLa , Proteínas de la Membrana/metabolismo , Receptores de Esteroides/metabolismoRESUMEN
Background: Environmental exposure to metal mixtures is common and may be associated with increased risk for neurodegenerative disorders including Alzheimer's disease. Objective: This study examined associations of mixed metal exposures with medial temporal lobe (MTL) MRI structural metrics and neuropsychological performance. Methods: Metal exposure history, whole blood metal, and neuropsychological tests were obtained from subjects with/without a history of mixed metal exposure from welding fumes (42 exposed subjects; 31 controls). MTL structures (hippocampus, entorhinal and parahippocampal cortices) were assessed by morphologic (volume, cortical thickness) and diffusion tensor imaging [mean (MD), axial (AD), radial diffusivity (RD), and fractional anisotropy (FA)] metrics. In exposed subjects, correlation, multiple linear, Bayesian kernel machine regression, and mediation analyses were employed to examine effects of single- or mixed-metal predictor(s) and their interactions on MTL structural and neuropsychological metrics; and on the path from metal exposure to neuropsychological consequences. Results: Compared to controls, exposed subjects had higher blood Cu, Fe, K, Mn, Pb, Se, and Zn levels (p's<0.026) and poorer performance in processing/psychomotor speed, executive, and visuospatial domains (p's<0.046). Exposed subjects displayed higher MD, AD, and RD in all MTL ROIs (p's<0.040) and lower FA in entorhinal and parahippocampal cortices (p's<0.033), but not morphological differences. Long-term mixed-metal exposure history indirectly predicted lower processing speed performance via lower parahippocampal FA (p=0.023). Higher whole blood Mn and Cu predicted higher entorhinal diffusivity (p's<0.043) and lower Delayed Story Recall performance (p=0.007) without overall metal mixture or interaction effects. Discussion: Mixed metal exposure predicted MTL structural and neuropsychological features that are similar to Alzheimer's disease at-risk populations. These data warrant follow-up as they may illuminate the path for environmental exposure to Alzheimer's disease-related health outcomes.
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INTRODUCTION: Chronic excessive welding exposure may be related to higher metal accumulation and structural differences in different subcortical structures. We examined how welding affected brain structures and their associations with metal exposure and neurobehavioral consequences. METHODS: Study includes 42 welders and 31 controls without a welding history. Welding-related structural differences were assessed by volume and diffusion tensor imaging (DTI) metrics in basal ganglia, red nucleus (RN), and hippocampus. Metal exposure was estimated by both exposure questionnaires and whole blood metal levels. Brain metal accumulations were estimated by R1 (for Mn) and R2* (for Fe). Neurobehavioral status was assessed by standard neuropsychological tests. RESULTS: Compared to controls, welders displayed higher hippocampal mean (MD), axial (AD), and radial diffusivity (RD) (p's < 0.036), but similar DTI or volume in other ROIs (p's > 0.117). Welders had higher blood metal levels (p's < 0.004), higher caudate and RN R2* (p's < 0.014), and lower performance on processing/psychomotor speed, executive function, and visuospatial processing tasks (p's < 0.046). Higher caudate and RN R2* were associated with higher blood Fe and Pb (p's < 0.043), respectively. RN R2* was a significant predictor of all hippocampal diffusivity metrics (p's < 0.006). Higher hippocampal MD and RD values were associated with lower Trail Making Test-A scores (p's < 0.025). A mediation analysis of both groups revealed blood Pb indirectly affected hippocampal diffusivity via RN R2* (p's < 0.041). DISCUSSION: Welding-related higher hippocampal diffusivity metrics may be associated with higher RN R2* and lower psychomotor speed performance. Future studies are warranted to test the role of Pb exposure in these findings.
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Exposición Profesional , Soldadura , Humanos , Imagen de Difusión Tensora/métodos , Desempeño Psicomotor , Obreros Metalúrgicos , Plomo/análisis , Núcleo Rojo/química , Exposición Profesional/efectos adversos , Exposición Profesional/análisis , Hipocampo/diagnóstico por imagenRESUMEN
BACKGROUND: Susceptibility magnetic resonance imaging (MRI) is sensitive to iron-related changes in the substantia nigra pars compacta (SNc), the key pathologic locus of parkinsonisms. It is unclear, however, if iron deposition in the SNc is associated with its neurodegeneration. OBJECTIVE: The objective of this study was to test whether susceptibility MRI metrics in parkinsonisms are associated with SNc neuropathologic features of dopaminergic neuron loss, gliosis, and α-synuclein and tau burden. METHODS: This retrospective study included 27 subjects with both in vivo MRI and postmortem data. Multigradient echo imaging was used to derive the apparent transverse relaxation rate (R2*) and quantitative susceptibility mapping (QSM) in the SNc. Archived midbrain slides that were stained with hematoxylin and eosin, anti-α-synuclein, and anti-tau were digitized to quantify neuromelanin-positive neuron density, glial density, and the percentages of area occupied by positive α-synuclein and tau staining. MRI-histology associations were examined using Pearson correlations and regression. RESULTS: Twenty-four subjects had postmortem parkinsonism diagnoses (Lewy body disorder, progressive supranuclear palsy, multiple system atrophy, and corticobasal degeneration), two had only Alzheimer's neuropathology, and one exhibited only mild atrophy. Among all subjects, both R2* and QSM were associated with glial density (r ≥ 0.67; P < 0.001) and log-transformed tau burden (r ≥ 0.53; P ≤ 0.007). Multiple linear regression identified glial density and log-transformed tau as determinants for both MRI metrics (R2 ≥ 0.580; P < 0.0001). Neither MRI metric was associated with neuron density or α-synuclein burden. CONCLUSIONS: R2* and QSM are associated with both glial density and tau burden, key neuropathologic features in the parkinsonism SNc. © 2023 International Parkinson and Movement Disorder Society.
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Enfermedad de Parkinson , Trastornos Parkinsonianos , Humanos , Porción Compacta de la Sustancia Negra , Enfermedad de Parkinson/diagnóstico por imagen , Enfermedad de Parkinson/patología , Sustancia Negra/diagnóstico por imagen , Sustancia Negra/patología , Estudios Retrospectivos , Trastornos Parkinsonianos/patología , Imagen por Resonancia Magnética/métodos , HierroRESUMEN
BACKGROUND AND PURPOSE: The circuitry underlying heterogenous cognitive profiles in Parkinson's disease (PD) remains unclear. The purpose of this study is to investigate whether structural changes in frontostriatal and limbic pathways contribute to different cognitive trajectories in PD. METHODS: We obtained clinical and multimodal MRI data from 120 control and 122 PD subjects without dementia or severe motor disability. T1/T2-weighted images estimated volume, and diffusion imaging evaluated fractional anisotropy (FA) of frontostriatal (striatum and frontostriatal white matter [FSWM]) and limbic (hippocampus and fornix) structures. Montreal Cognitive Assessment (MoCA) gauged total and domain-specific (attention/executive and memory) cognitive function. Linear mixed-effects models were used to compare MRI and cognitive progression over 4.5 years between controls and PD and evaluate associations between baseline MRI and cognitive changes in PD. RESULTS: At baseline, control and PD groups were comparable, except PD participants had smaller striatal volume (p < 0.001). Longitudinally, PD showed faster decline in hippocampal volume, FSWM FA, and fornix FA (ps < .016), but not striatal volume (p = .218). Total and domain-specific MoCA scores declined faster in PD (ps < .030). In PD, lower baseline hippocampal volume (p = .005) and fornix FA (p = .032), but not striatal volume (p = .662) or FSWM FA (p = .143), were associated with faster total MoCA decline. Baseline frontostriatal metrics of striatal volume and FSWM FA were associated with faster attention/executive decline (p < .038), whereas lower baseline hippocampal volume was associated with faster memory decline (p = .005). CONCLUSION: In PD, frontostriatal structural metrics are associated with attention/executive tasks, whereas limbic changes correlated with faster global cognitive decline, particularly in memory tasks.
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Disfunción Cognitiva , Personas con Discapacidad , Trastornos Motores , Enfermedad de Parkinson , Humanos , Enfermedad de Parkinson/complicaciones , Enfermedad de Parkinson/diagnóstico por imagen , Trastornos Motores/complicaciones , Cognición , Pruebas NeuropsicológicasRESUMEN
Motor synergies, i.e., neural mechanisms that organize multiple motor elements to ensure stability of actions, are affected by several neurological condition. Asymptomatic welders showed impaired synergy controlling the stability of multi-finger action compared to non-welders and this impairment was associated with microstructural damage in the globus pallidus. We further explored the effect of welding-related metal exposure on multi-finger synergy and extended our investigation to posture-stabilizing synergy during a standing task. Occupational, MRI, and performance-stabilizing synergies during multi-finger accurate force production and load releasing while standing were obtained from 29 welders and 19 age- and sex-matched controls. R2* and R1 relaxation rate values were used to estimate brain iron and manganese content, respectively, and diffusion tensor imaging was used to reflect brain microstructural integrity. Associations of brain MRI (caudate, putamen, globus pallidus, and red nucleus), and motor synergy were explored by group status. The results revealed that welders had higher R2* values in the caudate (p = 0.03), putamen (p = 0.01), and red nucleus (p = 0.08, trend) than controls. No group effect was revealed on multi-finger synergy index during steady-state phase of action (ΔVZss). Compared to controls, welders exhibited lower ΔVZss (-0.106 ± 0.084 vs. 0.160 ± 0.092, p = 0.04) and variance that did not affect the performance variable (VUCM, 0.022 ± 0.003 vs. 0.038 ± 0.007, p = 0.03) in the load releasing, postural task. The postural synergy index, ΔVZss, was associated negatively with higher R2* in the red nucleus in welders (r = -0.44, p = 0.03), but not in controls. These results suggest that the synergy index in the load releasing during a standing task may reflect welding-related neurotoxicity in workers with chronic metals exposure. This finding may have important clinical and occupational health implications.
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Contaminantes Ocupacionales del Aire , Exposición Profesional , Soldadura , Humanos , Imagen de Difusión Tensora , Esfuerzo Físico , Exposición Profesional/efectos adversos , Exposición Profesional/análisis , Manganeso/toxicidad , Metales , Contaminantes Ocupacionales del Aire/toxicidadRESUMEN
BACKGROUND: Higher nigral iron has been reported in Parkinson's disease (PD). OBJECTIVE: The aim is to understand the dynamics of nigral iron accumulation in PD and its association with drug treatment. METHODS: Susceptibility magnetic resonance imaging data were obtained from 79 controls and 18 drug-naive (PDDN ) and 87 drug-treated (PDDT ) PD patients. Regional brain iron in basal ganglia and cerebellar structures was estimated using quantitative susceptibility mapping. Nigral iron was compared between PDDN and PDDT subgroups defined by disease duration (early [PDE, <2 years], middle [PDM, 2-6 years], and later [PDL, >6 years]). Associations with both disease duration and types of antiparkinson drugs were explored using regression analysis. RESULTS: Compared to controls, PDDN had lower iron in the substantia nigra (P = 0.018), caudate nucleus (P = 0.038), and globus pallidus (P = 0.01) but not in the putamen or red nucleus. In contrast, PDDT had higher iron in the nigra (P < 0.001) but not in other regions, compared to either controls or PDDN . Iron in the nigra increased with disease duration (PDE > PDDN [P = 0.001], PDM > PDE [P = 0.045]) except for PDM versus PDL (P = 0.226). Levodopa usage was associated with higher (P = 0.013) nigral iron, whereas lower nigral iron was correlated with selegiline usage (P = 0.030). CONCLUSION: Nigral iron is lower before the start of dopaminergic medication and then increases throughout the disease until it plateaus at late stages, suggesting increased iron may not be an etiological factor. Interestingly, PD medications may have differential associations with iron accumulation that need further investigation. © 2022 International Parkinson and Movement Disorder Society.
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Enfermedad de Parkinson , Globo Pálido/patología , Humanos , Hierro , Imagen por Resonancia Magnética/métodos , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/patología , Sustancia Negra/diagnóstico por imagen , Sustancia Negra/patologíaRESUMEN
Lead is a nonessential metal and may be a coexposure in welding fumes. Preclinical data indicate lead may affect iron regulation. The current study investigated blood lead concentrations and their association with brain iron accumulation in workers with chronic welding fume exposure, with a focus on iron-rich subcortical regions of the cerebellum and basal ganglia. Occupational exposure, whole blood metal, and brain MRI data were obtained from 29 controls and 42 welders. R2* (1/T2*) and R1 (T1 relaxation rate) values were used to estimate brain iron and manganese content, respectively. Blood metals and brain R2* (in the red nucleus [RN], dentate nucleus, caudate, putamen, globus pallidus, and substantia nigra) were compared between groups. Associations between brain R2* values and exposure metrics were tested within each group, and analyses were adjusted for potential confounders. Welders had significantly higher levels of whole blood lead, manganese, iron, and copper. Welders also had higher R2* RN (p = .002), but not R1. A 2nd-order polynomial modeled the association between R2* RN and a long-term welding exposure metric. In welders, but not controls, R2* RN was associated positively with whole blood lead (r = 0.54, p = .003), and negatively with whole blood manganese (r = -0.43, p = .02). Higher blood Pb and lower blood Mn independently accounted for variance in high RN R2*. Together, these data suggest that higher RN R2* values may mark lead exposure in welders. Because lead is a known neurotoxicant, additional studies are warranted to confirm this finding, and ascertain its scientific and public/occupational health implications.
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Contaminantes Ocupacionales del Aire , Exposición Profesional , Soldadura , Humanos , Hierro , Plomo , Manganeso , Obreros Metalúrgicos , Exposición Profesional/efectos adversos , Núcleo RojoRESUMEN
BACKGROUND: Recent randomized clinical trials using hydrophobic statins reported no influence on Parkinson's disease (PD) clinical progression. Hydrophobicity is a key determinant for blood-brain barrier penetrance. OBJECTIVE: Investigate a potential effect of statins on PD progression. METHODS: Statin use was determined at baseline and subtyped according to hydrophobicity in 125 PD patients participating in the PD Biomarker Program (PDBP, 2012-2015) at our site. Clinical (Nâ=â125) and susceptibility MRI (Nâ=â86) data were obtained at baseline and 18-months. Movement Disorders Society-Unified PD Rating Scales were used to track progression of non-motor (MDS-UPDRS-I) and motor (MDS-UPDRS-II) symptoms, and rater-based scores (MDS-UPDRS-III) of patients in the "on" drug state. R2* values were used to capture pathological progression in the substantia nigra. Associations between statin use, its subtypes, and PD progression were evaluated with linear mixed effect regressions. RESULTS: Compared to statin non-users, overall statin or lipophilic statin use did not significantly influence PD clinical or imaging progression. Hydrophilic statin users, however, demonstrated faster clinical progression of non-motor symptoms [MDS-UPDRS-I (ß=â4.8, pâ=â0.010)] and nigral R2* (ß=â3.7, pâ=â0.043). A similar trend was found for MDS-UPDRS-II (ß=â3.9, pâ=â0.10), but an opposite trend was observed for rater-based MDS-UPDRS-III (ß=â-7.3, pâ=â0.10). Compared to lipophilic statin users, hydrophilic statin users also showed significantly faster clinical progression of non-motor symptoms [MDS-UPDRS-I (ß=â5.0, pâ=â0.020)], but R2* did not reach statistical significance (ß=â2.5, pâ=â0.24). CONCLUSION: This study suggests that hydrophilic, but not lipophilic, statins may be associated with faster PD progression. Future studies may have clinical and scientific implications.
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Inhibidores de Hidroximetilglutaril-CoA Reductasas , Enfermedad de Parkinson , Progresión de la Enfermedad , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Enfermedad de Parkinson/diagnóstico por imagen , Enfermedad de Parkinson/tratamiento farmacológico , Índice de Severidad de la Enfermedad , Sustancia NegraRESUMEN
MLKL (mixed lineage kinase domain like pseudokinase) is a well-known core component of necrosome that executes necroptotic cell death upon phosphorylation by RIPK3 (receptor interacting serine/threonine kinase 3). Recent studies also implicate a role of MLKL in endosomal trafficking, which is not always dependent on RIPK3. Using mouse Neuro-2a and L929 as well as human HEK293 and HT29 cells, we show here that MLKL is phosphorylated in response to serum and amino acid deprivation from the culture medium, in a manner that depends on CAMK2/CaMKII (calcium/calmodulin dependent protein kinase II) but not RIPK3. The starvation-induced increase in MLKL phosphorylation was accompanied by decreases in levels of lipidated MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta; LC3-II) and SQSTM1/p62 (sequestosome 1), markers of autophagosomes. These changes were prevented by disrupting either MLKL or CAMK2 by pharmacology and genetic manipulations. Moreover, disrupting MLKL or CAMK2 also inhibited the incorporation of LC3-II into autolysosomes, demonstrating a role of the CAMK2-MLKL pathway in facilitating autophagic flux during short-term starvation, in contrast to necroptosis which suppressed autophagic flux. Furthermore, unlike the necroptotic pathway, the starvation-evoked CAMK2-mediated MLKL phosphorylation protected cells from starvation-induced death. We propose that upon nutrient deprivation, MLKL is activated by CAMK2, which in turn facilitates membrane scission needed for autophagosome maturation, allowing the proper fusion of the autophagosome with lysosome and the subsequent substance degradation. This novel function is independent of RIPK3 and is not involved in necroptosis, implicating new roles for this pseudokinase in cell survival, signaling and metabolism.Abbreviations: CAMK2/CaMKII: calcium/calmodulin dependent protein kinase II; DIABLO/SMAC: direct inhibitor of apoptosis-binding protein with low pI/second mitochondria-derived activator of caspase; ECS: extracellular solution; ESCRT: endosomal sorting complexes required for transport; FBS: fetal bovine serum; GSK3B: glycogen synthase kinase 3 beta; HBSS: Hanks' balanced salt solution; KO: knockout; LC3-II: lipidated microtubule associated protein 1 light chain 3 beta; LDH: lactate dehydrogenase; MLKL: mixed lineage kinase domain like pseudokinase; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; N2a: Neuro-2a neuroblastoma; Nec-1: necrostatin-1; NSA: necrosulfonamide; PBS: phosphate-buffered saline; PI: propidium iodide; PK-hLC3: pHluorin-mKate2-human LC3; RIPK1: receptor interacting serine/threonine kinase 1; RIPK3: receptor interacting serine/threonine kinase 3; ROS: reactive oxygen species; RPS6KB1/S6K: ribosomal protein S6 kinase B1; shRNA: short hairpin RNA; siRNA: small interference RNA; SQSTM1/p62: sequestosome 1; TBS: Tris-buffered saline; TNF/TNF-α: tumor necrosis factor; TSZ, treatment with TNF + DIABLO mimetics + z-VAD-FMK.
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Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Calcio , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Autofagia/fisiología , Calcio/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Células HEK293 , Humanos , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Quinasas/metabolismo , ARN Interferente Pequeño/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteína Sequestosoma-1/metabolismo , Serina , Serina-Treonina Quinasas TOR/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Glutamine is one of the most abundant amino acids in the cell. In mitochondria, glutaminases 1 and 2 (GLS1/2) hydrolyze glutamine to glutamate, which serves as the precursor of multiple metabolites. Here, we show that ammonium generated during GLS1/2-mediated glutaminolysis regulates lysosomal pH and in turn lysosomal degradation. In primary human skin fibroblasts BJ cells and mouse embryonic fibroblasts, deprivation of total amino acids for 1 h increased lysosomal degradation capacity as shown by the increased turnover of lipidated microtubule-associated proteins 1A/1B light chain 3B (LC3-II), several autophagic receptors, and endocytosed DQ-BSA. Removal of glutamine but not any other amino acids from the culture medium enhanced lysosomal degradation similarly as total amino acid starvation. The presence of glutamine in regular culture media increased lysosomal pH by >0.5 pH unit and the removal of glutamine caused lysosomal acidification. GLS1/2 knockdown, GLS1 antagonist, or ammonium scavengers reduced lysosomal pH in the presence of glutamine. The addition of glutamine or NH4Cl prevented the increase in lysosomal degradation and curtailed the extension of mTORC1 function during the early time period of amino acid starvation. Our findings suggest that glutamine tunes lysosomal pH by producing ammonium, which regulates lysosomal degradation to meet the demands of cellular activities. During the early stage of amino acid starvation, the glutamine-dependent mechanism allows more efficient use of internal reserves and endocytosed proteins to extend mTORC1 activation such that the normal anabolism is not easily interrupted by a brief disruption of the amino acid supply.
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Compuestos de Amonio , Glutamina , Animales , Ratones , Humanos , Glutamina/metabolismo , Compuestos de Amonio/farmacología , Compuestos de Amonio/metabolismo , Fibroblastos/metabolismo , Aminoácidos/metabolismo , Ácido Glutámico/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Lisosomas/metabolismo , Concentración de Iones de HidrógenoRESUMEN
Phospholipase D (PLD) generates the signaling lipid phosphatidic acid (PA) and has been known to mediate proliferation signal in vascular smooth muscle cells (VSMCs). However, it remains unclear how PLD contributes to vascular diseases. VSMC proliferation directly contributes to the development and progression of cardiovascular disease, such as atherosclerosis and restenosis after angioplasty. Using the mouse carotid artery ligation model, we find that deletion of Pld1 gene inhibits neointima formation of the injuried blood vessels. PLD1 deficiency reduces the proliferation of VSMCs in both injured artery and primary cultures through the inhibition of ERK1/2 and AKT signals. Immunohistochemical staining of injured artery and flow cytometry analysis of VSMCs shows a reduction of the levels of reactive oxygen species (ROS) in Pld1-/- VSMCs. An increase of intracellular ROS by hydrogen peroxide stimulation restored the reduced activities of ERK and AKT in Pld1-/- VSMCs, whereas a reduction of ROS by N-acetyl-l-cysteine (NAC) scavenger lowered their activity in wild-type VSMCs. These results indicate that PLD1 plays a critical role in neointima, and that PLD1 mediates VSMC proliferation signal through promoting the production of ROS. Therefore, inhibition of PLD1 may be used as a therapeutic approach to suppress neointimal formation in atherosclerosis and restenosis after angioplasty.
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Aterosclerosis/genética , Traumatismos de las Arterias Carótidas/genética , Neointima/genética , Fosfolipasa D/genética , Animales , Aterosclerosis/metabolismo , Aterosclerosis/patología , Arterias Carótidas/metabolismo , Arterias Carótidas/patología , Traumatismos de las Arterias Carótidas/patología , Modelos Animales de Enfermedad , Humanos , Ratones , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Neointima/metabolismo , Neointima/patología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
A growing body of evidence suggests nigral iron accumulation plays an important role in the pathophysiology of Parkinson's disease (PD), contributing to dopaminergic neuron loss in the substantia nigra pars compacta (SNc). Converging evidence suggests this accumulation might be related to, or increased by, serotonergic dysfunction, a common, often early feature of the disease. We investigated whether lower plasma serotonin in PD is associated with higher nigral iron. We obtained plasma samples from 97 PD patients and 89 controls and MRI scans from a sub-cohort (62 PD, 70 controls). We measured serotonin concentrations using ultra-high performance liquid chromatography and regional iron content using MRI-based quantitative susceptibility mapping. PD patients had lower plasma serotonin (p < 0.0001) and higher nigral iron content (SNc: p < 0.001) overall. Exclusively in PD, lower plasma serotonin was correlated with higher nigral iron (SNc: r(58) = - 0.501, p < 0.001). This correlation was significant even in patients newly diagnosed (< 1 year) and stronger in the SNc than any other region examined. This study reveals an early, linear association between low serotonin and higher nigral iron in PD patients, which is absent in controls. This is consistent with a serotonin-iron relationship in the disease process, warranting further studies to determine its cause and directionality.
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Hierro/metabolismo , Enfermedad de Parkinson/metabolismo , Serotonina/sangre , Sustancia Negra/metabolismo , Anciano , Estudios de Cohortes , Femenino , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Enfermedad de Parkinson/diagnóstico por imagen , Enfermedad de Parkinson/tratamiento farmacológico , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Inhibidores Selectivos de la Recaptación de Serotonina/uso terapéutico , Inhibidores de Captación de Serotonina y Norepinefrina/metabolismo , Inhibidores de Captación de Serotonina y Norepinefrina/farmacología , Inhibidores de Captación de Serotonina y Norepinefrina/uso terapéutico , Índice de Severidad de la Enfermedad , Sustancia Negra/diagnóstico por imagen , TiempoRESUMEN
Ligand-independent epidermal growth factor receptor (EGFR) endocytosis is inducible by a variety of stress conditions converging upon p38 kinase. A less known pathway involves phosphatidic acid (PA) signaling toward the activation of type 4 phosphodiesterases (PDE4) that decrease cAMP levels and protein kinase A (PKA) activity. This PA/PDE4/PKA pathway is triggered with propranolol used to inhibit PA hydrolysis and induces clathrin-dependent and clathrin-independent endocytosis, followed by reversible accumulation of EGFR in recycling endosomes. Here we give further evidence of this signaling pathway using biosensors of PA, cAMP, and PKA in live cells and then show that it activates p38 and ERK1/2 downstream the PKA inhibition. Clathrin-silencing and IN/SUR experiments involved the activity of p38 in the clathrin-dependent route, while ERK1/2 mediates clathrin-independent EGFR endocytosis. The PA/PDE4/PKA pathway selectively increases the EGFR endocytic rate without affecting LDLR and TfR constitute endocytosis. This selectiveness is probably because of EGFR phosphorylation, as detected in Th1046/1047 and Ser669 residues. The EGFR accumulates at perinuclear recycling endosomes colocalizing with TfR, fluorescent transferrin, and Rab11, while a small proportion distributes to Alix-endosomes. A non-selective recycling arrest includes LDLR and TfR in a reversible manner. The PA/PDE4/PKA pathway involving both p38 and ERK1/2 expands the possibilities of EGFR transmodulation and interference in cancer.
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Sistema de Señalización de MAP Quinasas , Ácidos Fosfatidicos , Clatrina/metabolismo , Endocitosis/fisiología , Receptores ErbB/metabolismo , Ligandos , Ácidos Fosfatidicos/metabolismo , Fosforilación , Transducción de SeñalRESUMEN
Lysophosphatidate (LPA) and sphingosine 1-phosphate (S1P) promote vasculogenesis, angiogenesis, and wound healing by activating a plethora of overlapping signaling pathways that stimulate mitogenesis, cell survival, and migration. As such, maladaptive signaling by LPA and S1P have major effects in increasing tumor progression and producing poor patient outcomes after chemotherapy and radiotherapy. Many signaling actions of S1P and LPA are not redundant; each are vital in normal physiology and their metabolisms differ. In the present work, we studied how LPA signaling impacts S1P metabolism and signaling in MDA-MB-231 and MCF-7 breast cancer cells. LPA increased sphingosine kinase-1 (SphK1) synthesis and rapidly activated cytosolic SphK1 through association with membranes. Blocking phospholipase D activity attenuated the LPA-induced activation of SphK1 and the synthesis of ABCC1 and ABCG2 transporters that secrete S1P from cells. This effect was magnified in doxorubicin-resistant MCF-7 cells. LPA also facilitated S1P signaling by increasing mRNA expression for S1P1 receptors. Doxorubicin-resistant MCF-7 cells had increased S1P2 and S1P3 receptor expression and show increased LPA-induced SphK1 activation, increased expression of ABCC1, ABCG2 and greater S1P secretion. Thus, LPA itself and LPA-induced S1P signaling counteract doxorubicin-induced death of MCF-7 cells. We conclude from the present and previous studies that LPA promotes S1P metabolism and signaling to coordinately increase tumor growth and metastasis and decrease the effectiveness of chemotherapy and radiotherapy for breast cancer treatment.
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Lisofosfolípidos , Esfingosina/análogos & derivadosRESUMEN
BACKGROUND: Parkinson's disease (PD) is marked clinically by motor symptoms and pathologically by Lewy bodies and dopamine neuron loss in the substantia nigra pars compacta (SNc). Higher iron accumulation, assessed by susceptibility MRI, also is observed as PD progresses. Recently, evidence has suggested that PD affects the retina. OBJECTIVE: To better understand retinal alterations in PD and their association to clinical and SNc iron-related imaging metrics. METHODS: Ten PD and 12 control participants (2 eyes each) from an ongoing PD imaging biomarker study underwent enhanced depth imaging optical coherence tomography evaluation. Choroidal (vascular) thickness and nerve layers were measured in 4 subregions [superior, temporal, inferior, and nasal] and at 3 foveal distances (1, 1.5, and 3âmm). These metrics were compared between PD and control groups. For significantly different metrics, their associations with clinical [levodopa equivalent daily dosage (LEDD), motor and visuospatial function] and SNc susceptibility MRI metrics [R2* and quantitative susceptibility mapping (QSM)] were explored. RESULTS: Compared to control participants, PD participants had a thicker choroid (pâ=â0.005), but no changes in nerve layers. Higher mean choroidal thickness was associated with lower LEDD (pâ<â0.01) and better visuospatial function (pâ<â0.05). Subregion analyses revealed higher choroidal thickness correlated with lower LEDD and better motor and visuospatial measures. Higher mean choroidal thickness also was associated with lower nigral iron MRI (pâ<â0.05). CONCLUSION: A small cohort of PD research participants displayed higher choroidal thickness that was related to better clinical performance and less nigral pathology. These intriguing findings warrant further investigation.
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Coroides , Enfermedad de Parkinson , Benchmarking , Coroides/diagnóstico por imagen , Humanos , Hierro , Levodopa , Enfermedad de Parkinson/diagnóstico por imagen , Proyectos Piloto , Tomografía de Coherencia ÓpticaRESUMEN
BACKGROUND: Deposition and spreading of misfolded proteins (α-synuclein and tau) have been linked to Parkinson's disease cognitive dysfunction. The glymphatic system may play an important role in the clearance of these toxic proteins via cerebrospinal fluid (CSF) flow through perivascular and interstitial spaces. Recent studies discovered that sleep-dependent global brain activity is coupled to CSF flow, which may reflect glymphatic function. OBJECTIVE: The objective of this current study was to determine if the decoupling of brain activity-CSF flow is linked to Parkinson's disease cognitive dysfunction. METHODS: Functional and structural MRI data, clinical motor (Unified Parkinson's Disease Rating Scale), and cognitive (Montreal Cognitive Assessment [MoCA]) scores were collected from 60 Parkinson's disease and 58 control subjects. Parkinson's disease patients were subgrouped into those with mild cognitive impairment (MoCA < 26), n = 31, and those without mild cognitive impairment (MoCA ≥ 26), n = 29. The coupling strength between the resting-state global blood-oxygen-level-dependent signal and associated CSF flow was quantified, compared among groups, and associated with clinical and structural measurements. RESULTS: Global blood-oxygen-level-dependent signal-CSF coupling decreased significantly (P < 0.006) in Parkinson's disease patients showing mild cognitive impairment, compared with those without mild cognitive impairment and controls. Reduced global blood-oxygen-level-dependent signal-CSF coupling was associated with decreased MoCA scores present in Parkinson's disease patients (P = 0.005) but not in controls (P = 0.65). Weaker global blood-oxygen-level-dependent signal-CSF coupling in Parkinson's disease patients also was associated with a thinner right entorhinal cortex (Spearman's correlation, -0.36; P = 0.012), an early structural change often seen in Alzheimer's disease. CONCLUSIONS: The decoupling between global brain activity and associated CSF flow is related to Parkinson's disease cognitive impairment. © 2021 International Parkinson and Movement Disorder Society.