RESUMEN
Extensive evidence supports the claim that the serum neurofilament light chain (sNfL) can be used as a biomarker to monitor disease severity in patients with spinocerebellar ataxia type 3 (SCA3). However, little is known about the associations between sNfL levels and neurochemical alterations in SCA3 patients. In this study, we performed a cross-sectional study to analyze the association between sNfL and brain metabolic changes in SCA3 patients. The severity of ataxia was assessed by using the Scale for the Assessment and Rating of Ataxia (SARA) and the International Cooperative Ataxia Rating Scale (ICARS). The sNfL levels and brain metabolic changes, represented by N-acetyl aspartate (NAA)/creatine (Cr) and choline complex (Cho)/Cr ratios, were measured by a single-molecule array and proton magnetic resonance spectroscopy, respectively. In this cohort, we observed consistently elevated sNfL levels and reduced brain metabolites in the cerebellar hemispheres, dentate nucleus, and cerebellar vermis. However, this correlation was further validated in the cerebellar cortex after analysis using pairwise comparisons and a Bonferroni correction. Taken together, our results further confirmed that sNfL levels were increased in SCA3 patients and were negatively correlated with metabolic changes in the cerebellar cortex. Our data also support the idea that sNfL levels are a promising potential complementary biomarker for patients with SCA3.
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Ataxia Cerebelosa , Enfermedad de Machado-Joseph , Neuroquímica , Humanos , Estudios Transversales , Filamentos Intermedios/metabolismo , Filamentos Intermedios/patología , Proteínas de Neurofilamentos , Ataxia , BiomarcadoresRESUMEN
Vimentin is highly expressed in metastatic cancers, and its expression correlates with poor patient prognoses. However, no causal in vivo studies linking vimentin and non-small cell lung cancer (NSCLC) progression existed until now. We use three complementary in vivo models to show that vimentin is required for the progression of NSCLC. First, we crossed LSL-KrasG12D; Tp53fl/fl mice (KPV+/+) with vimentin knockout mice (KPV-/-) to demonstrate that KPV-/- mice have attenuated tumor growth and improved survival compared with KPV+/+ mice. Next, we therapeutically treated KPV+/+ mice with withaferin A (WFA), an agent that disrupts vimentin intermediate filaments (IFs). We show that WFA suppresses tumor growth and reduces tumor burden in the lung. Finally, luciferase-expressing KPV+/+, KPV-/-, or KPVY117L cells were implanted into the flanks of athymic mice to track cancer metastasis to the lung. In KPVY117L cells, vimentin forms oligomers called unit-length filaments but cannot assemble into mature vimentin IFs. KPV-/- and KPVY117L cells fail to metastasize, suggesting that cell-autonomous metastasis requires mature vimentin IFs. Integrative metabolomic and transcriptomic analysis reveals that KPV-/- cells upregulate genes associated with ferroptosis, an iron-dependent form of regulated cell death. KPV-/- cells have reduced glutathione peroxidase 4 (GPX4) levels, resulting in the accumulation of toxic lipid peroxides and increased ferroptosis. Together, our results demonstrate that vimentin is required for rapid tumor growth, metastasis, and protection from ferroptosis in NSCLC.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Ratones , Animales , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Filamentos Intermedios/metabolismo , Vimentina/genética , Vimentina/metabolismo , Modelos Animales de Enfermedad , Ratones NoqueadosRESUMEN
OBJECTIVE: To evaluate whether participating in physical contact sports is associated with a release of neurofilaments and whether such release is related to future clinical neurologic and/or psychiatric impairment. METHODS: We performed a systematic review of the PubMed, MEDLINE, and Cochrane Library databases using a combination of the search terms neurofilament(s)/intermediate filament and sport(s)/athletes. Original studies, written in English, reporting on neurofilaments in CSF and/or serum/plasma of contact sport athletes were included. This review was conducted following the Preferred Reporting Items for Systematic Review and Analyses guidelines. RESULTS: Eighteen studies in 8 different contact sports (i.e., boxing, American football, ice hockey, soccer, mixed martial arts, lacrosse, rugby, and wrestling) matched our criteria. Elevated light chain neurofilament (NfL) levels were described in 13/18 cohorts. Most compelling evidence was present in boxing and American football, where exposure-related increases were appreciable at the intraindividual level (up to 4.1- and 2.0-fold, respectively) in well-defined groups. Differences in exposure severity (including previous cumulative effects), sampling/measurement time points (with regard to expected peak values), and definitions of the baseline setting are considered as main contributors to the variability in findings. No studies were encountered that have investigated the relationship with the targeted clinical end points; therefore no NfL cutoffs exist that are associated with a poor outcome. CONCLUSION: NfL release can be seen, as a potential marker of neuronal brain damage, in participants of physical contact sports, particularly boxing and American football. The exact significance regarding the risk for future clinical impairment remains to be elucidated.
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Traumatismos en Atletas/diagnóstico , Biomarcadores/sangre , Biomarcadores/líquido cefalorraquídeo , Conmoción Encefálica/etiología , Filamentos Intermedios/metabolismo , Traumatismos en Atletas/sangre , Traumatismos en Atletas/líquido cefalorraquídeo , Boxeo/lesiones , Conmoción Encefálica/diagnóstico , Fútbol Americano/lesiones , Hockey/lesiones , Humanos , Artes Marciales/lesiones , Deportes de Raqueta/lesiones , Fútbol/lesiones , Lucha/lesionesRESUMEN
Plakin proteins form connections that link the cell membrane to the intermediate filament cytoskeleton. Their interactions are mediated by a highly conserved linker domain through an unresolved mechanism. Here analysis of the human periplakin linker domain structure reveals a bi-lobed module transected by an electropositive groove. Key basic residues within the periplakin groove are vital for co-localization with vimentin in human cells and compromise direct binding which also requires acidic residues D176 and E187 in vimentin. We propose a model whereby basic periplakin linker domain residues recognize acidic vimentin side chains and form a complementary binding groove. The model is shared amongst diverse linker domains and can be used to investigate the effects of pathogenic mutations in the desmoplakin linker associated with arrhythmogenic right ventricular cardiomyopathy. Linker modules either act solely or collaborate with adjacent plakin repeat domains to create strong and adaptable tethering within epithelia and cardiac muscle.
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Plaquinas/química , Plaquinas/metabolismo , Vimentina/química , Vimentina/metabolismo , Secuencia de Aminoácidos , Aminoácidos Acídicos/química , Aminoácidos Acídicos/genética , Aminoácidos Acídicos/metabolismo , Ácido Aspártico/metabolismo , Ácido Glutámico/metabolismo , Células HeLa , Humanos , Filamentos Intermedios/química , Filamentos Intermedios/metabolismo , Modelos Moleculares , Mutación Missense , Plaquinas/genética , Unión Proteica/genética , Dominios y Motivos de Interacción de Proteínas/genética , Estructura Cuaternaria de Proteína , Vimentina/genéticaRESUMEN
OBJECTIVES: The effect of vitamin D supplementation on the disease course of multiple sclerosis (MS) is not established. Neurofilament light chain (NFL) is a sensitive marker of axonal degeneration. The aim of this study was to establish whether high-dose vitamin D supplementation reduces serum levels of NFL. MATERIALS AND METHODS: We have performed a 96 weeks placebo-controlled randomized study of weekly supplementation with 20 000 IU vitamin D3 in 71 patients with relapsing remitting MS (RRMS). Serum levels of NFL were measured at baseline, week 48 and week 96 with a single molecule (Simoa) assay in 69 of these patients. RESULTS: Serum levels of 25-hydroxyvitamin D more than doubled in the vitamin D group. Compared to placebo, vitamin D supplementation had no overall effect on the change in serum levels of NFL from baseline (P = 0.93 at week 48 and P = 0.56 at week 96). In the subgroup of patients not receiving disease-modifying therapy, NFL decreased by 30.9% to week 48% and 32.6% to week 96 from baseline in the vitamin D group as compared to the placebo group (P = 0.06 for both time points). CONCLUSION: With a possible exception for patients not treated with disease-modifying drugs, weekly supplementation with 20 000 IU vitamin D3 did not affect NFL levels in these RRMS patients.
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Filamentos Intermedios/metabolismo , Esclerosis Múltiple Recurrente-Remitente/tratamiento farmacológico , Vitamina D/análogos & derivados , Vitaminas/uso terapéutico , Adolescente , Adulto , Suplementos Dietéticos , Método Doble Ciego , Femenino , Humanos , Masculino , Persona de Mediana Edad , Esclerosis Múltiple Recurrente-Remitente/metabolismo , Vitamina D/administración & dosificación , Vitamina D/uso terapéutico , Vitaminas/administración & dosificaciónRESUMEN
Inferior alveolar nerve (IAN) injuries may occur during various dental routine procedures, especially in the removal of impacted lower third molars, and nerve recovery in these cases is a great challenge in dentistry. Here, the IAN crush injury model was used to assess the efficacy of photobiomodulation (PBM) in the recovery of the IAN in rats following crushing injury (a partial lesion). Rats were divided into four experimental groups: without any procedure, IAN crush injury, and IAN crush injury with PBM and sham group with PBM. Treatment was started 2 days after surgery, above the site of injury, and was performed every other day, totaling 10 sessions. Rats were irradiated with GaAs Laser (Gallium Arsenide, Laserpulse, Ibramed Brazil) emitting a wavelength of 904 nm, an output power of 70 mWpk, beam spot size at target â¼0.1 cm2, a frequency of 9500 Hz, a pulse time 60 ns, and an energy density of 6 J/cm2. Nerve recovery was investigated by measuring the morphometric data of the IAN using TEM and by the expression of laminin, neurofilaments (NFs), and myelin protein zero (MPZ) using Western blot analysis. We found that IAN-injured rats which received PBM had a significant improvement of IAN morphometry when compared to IAN-injured rats without PBM. In parallel, all MPZ, laminin, and NFs exhibited a decrease after PBM. The results of this study indicate that the correlation between the peripheral nerve ultrastructure and the associated protein expression shows the beneficial effects of PBM.
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Terapia por Luz de Baja Intensidad , Nervio Mandibular/metabolismo , Nervio Mandibular/patología , Compresión Nerviosa , Neuropéptidos/metabolismo , Animales , Densitometría , Filamentos Intermedios/metabolismo , Laminina/metabolismo , Masculino , Nervio Mandibular/ultraestructura , Proteína P0 de la Mielina/metabolismo , Ratas WistarRESUMEN
There are substantial differences across species in the organization and function of the motor pathways. These differences extend to basic electrophysiological properties. Thus, in rat motor cortex, pyramidal cells have long duration action potentials, while in the macaque, some pyramidal neurons exhibit short duration "thin" spikes. These differences may be related to the expression of the fast potassium channel Kv3.1b, which in rat interneurons is associated with generation of thin spikes. Rat pyramidal cells typically lack these channels, while there are reports that they are present in macaque pyramids. Here we made a systematic, quantitative comparison of the Kv3.1b expression in sections from macaque and rat motor cortex, using two different antibodies (NeuroMab, Millipore). As our standard reference, we examined, in the same sections, Kv3.1b staining in parvalbumin-positive interneurons, which show strong Kv3.1b immunoreactivity. In macaque motor cortex, a large sample of pyramidal neurons were nearly all found to express Kv3.1b in their soma membranes. These labeled neurons were identified as pyramidal based either by expression of SMI32 (a pyramidal marker), or by their shape and size, and lack of expression of parvalbumin (a marker for some classes of interneuron). Large (Betz cells), medium, and small pyramidal neurons all expressed Kv3.1b. In rat motor cortex, SMI32-postive pyramidal neurons expressing Kv3.1b were very rare and weakly stained. Thus, there is a marked species difference in the immunoreactivity of Kv3.1b in pyramidal neurons, and this may be one of the factors explaining the pronounced electrophysiological differences between rat and macaque pyramidal neurons.
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Corteza Motora/citología , Células Piramidales/metabolismo , Canales de Potasio Shaw/metabolismo , Animales , Recuento de Células , Femenino , Filamentos Intermedios/metabolismo , Interneuronas/metabolismo , Macaca mulatta , Masculino , Microscopía Confocal , Parvalbúminas/metabolismo , Ratas , Ratas Wistar , Especificidad de la EspecieRESUMEN
BACKGROUND: The survival of motor neurons is dependent upon neurotrophic factors both during childhood and adolescence and during adult life. In disease conditions, such as in patients with amyotrophic lateral sclerosis (ALS), the mRNA levels of trophic factors like brain-derived neurotrophic factor (BDNF), insulin-like growth factor-1 (IGF-1), fibroblast growth factor-2 (FGF-2), and vascular endothelial growth factor are downregulated. This was replicated in our in vivo experimental system following the injection of cerebral spinal fluid (CSF) of sporadic ALS (ALS-CSF) patients. OBJECTIVE: To evaluate the protective role of BDNF in a model of sporadic ALS patients. METHODS: The expressions of endogenous BDNF, its receptor TrkB, the enzyme choline acetyl transferase (ChAT), and phosphorylated neurofilaments were studied in NSC-34 cells. The calcium-buffering and proapoptotic effects were assessed by calbindin-D28K and caspase-3 expression, respectively. RESULTS: ALS-CSF considerably depleted the endogenous BDNF protein, while its effect on IGF-1 and FGF-2 was inconsequential; this indirectly indicates a key role for BDNF in supporting motor neuronal survival. The exogenous supplementation of BDNF reversed autocrine expression; however, it may not be completely receptor mediated, as the TrkB levels were not restored. BDNF completely revived ChAT expression. It may inhibit apoptosis by restoring Ca2+ homeostasis, since caspase-3 and calbindin-D28K expression was back to normal. The organellar ultrastructural changes were only partially reversed. CONCLUSION: Our study provides evidence that BDNF supplementation ameliorates most but not all degenerative changes. The incomplete revival at the ultrastructural level signifies the requirement of factors other than BDNF for near-total protection of motor neurons, and, to an extent, it explains why only a partial success is achieved in clinical trials with BDNF in ALS patients.
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Esclerosis Amiotrófica Lateral/líquido cefalorraquídeo , Factor Neurotrófico Derivado del Encéfalo/farmacología , Neuronas Motoras/efectos de los fármacos , Degeneración Nerviosa/tratamiento farmacológico , Fármacos Neuroprotectores/farmacología , Recuperación de la Función/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Calcio/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Humanos , Filamentos Intermedios/efectos de los fármacos , Filamentos Intermedios/metabolismo , Filamentos Intermedios/patología , Ratones , Neuronas Motoras/fisiología , Neuronas Motoras/ultraestructura , Degeneración Nerviosa/patología , Degeneración Nerviosa/fisiopatología , Ratas Wistar , Receptor trkB/metabolismo , Recuperación de la Función/fisiología , Médula Espinal/efectos de los fármacos , Médula Espinal/patología , Médula Espinal/fisiopatologíaRESUMEN
Intermediate filament (IF) proteins have unique and complex cell and tissue distribution. Importantly, IF gene mutations cause or predispose to more than 80 human tissue-specific diseases (IF-pathies), with the most severe disease phenotypes being due to mutations at conserved residues that result in a disrupted IF network. A critical need for the entire IF-pathy field is the identification of drugs that can ameliorate or cure these diseases, particularly since all current therapies target the IF-pathy complication, such as diabetes or cardiovascular disease, rather than the mutant IF protein or gene. We describe a high-throughput approach to identify drugs that can normalize disrupted IF proteins. This approach utilizes transduction of lentivirus that expresses green fluorescent protein-tagged keratin 18 (K18) R90C in A549 cells. The readout is drug "hits" that convert the dot-like keratin filament distribution, due to the R90C mutation, to a wild-type-like filamentous array. A similar strategy can be used to screen thousands of compounds and can be utilized for practically any IF protein with a filament-disrupting mutation, and could therefore potentially target many IF-pathies. "Hits" of interest require validation in cell culture then using in vivo experimental models. Approaches to study the mechanism of mutant IF normalization by potential drugs of interest are also described. The ultimate goal of this drug screening approach is to identify effective and safe compounds that can potentially be tested for clinical efficacy in patients.
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Evaluación Preclínica de Medicamentos/métodos , Proteínas de Filamentos Intermediarios/metabolismo , Filamentos Intermedios/metabolismo , Animales , Humanos , Proteínas de Filamentos Intermediarios/genética , Filamentos Intermedios/genética , Lentivirus/genética , Mutación/genéticaRESUMEN
Glutamatergic principal neurons, GABAergic interneurons and thalamocortical axons (TCAs) are essential elements of the cerebrocortical network. Principal neurons originate locally from radial glia and intermediate progenitors (IPCs), whereas interneurons and TCAs are of extrinsic origin. Little is known how the assembly of these elements is coordinated. C-X-C motif chemokine 12 (CXCL12), which is known to guide axons outside the neural tube and interneurons in the cortex, is expressed in the meninges and IPCs. Using mouse genetics, we dissected the influence of IPC-derived CXCL12 on TCAs and interneurons by showing that Cxcl12 ablation in IPCs, leaving meningeal Cxcl12 intact, attenuates intracortical TCA growth and disrupts tangential interneuron migration in the subventricular zone. In accordance with strong CXCR4 expression in the forming thalamus and TCAs, we identified a CXCR4-dependent growth-promoting effect of CXCL12 on TCAs in thalamus explants. Together, our findings indicate a cell-autonomous role of CXCR4 in promoting TCA growth. We propose that CXCL12 signals from IPCs link cortical neurogenesis to the progression of TCAs and interneurons spatially and temporally. Significance statement: The cerebral cortex exerts higher brain functions including perceptual and emotional processing. Evolutionary expansion of the mammalian cortex is mediated by intermediate progenitors, transient amplifying cells generating cortical excitatory neurons. During the peak period of cortical neurogenesis, migrating precursors of inhibitory interneurons originating in subcortical areas and thalamic axons invade the cortex. Although defects in the assembly of cortical network elements cause neurological and mental disorders, little is known how neurogenesis, interneuron recruitment, and axonal ingrowth are coordinated. We demonstrate that intermediate progenitors release the chemotactic cytokine CXCL12 to promote intracortical interneuron migration and growth of thalamic axons via the cognate receptor CXCR4. This paracrine signal may ensure thalamocortical connectivity and dispersion of inhibitory neurons in the rapidly growing cortex.
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Corteza Cerebral/citología , Quimiocina CXCL12/metabolismo , Interneuronas/fisiología , Transducción de Señal/fisiología , Células Madre/fisiología , Tálamo/citología , Animales , Axones/metabolismo , Corteza Cerebral/embriología , Quimiocina CXCL12/genética , Embrión de Mamíferos , Regulación del Desarrollo de la Expresión Génica/genética , Filamentos Intermedios/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas del Tejido Nervioso , Vías Nerviosas/fisiología , Técnicas de Cultivo de Órganos , Receptores CXCR/genética , Receptores CXCR/metabolismo , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Tálamo/embriologíaRESUMEN
We recently showed that inter-keratin disulfide bonding plays an important role in the assembly, organization, and dynamics of keratin intermediate filaments in skin keratinocytes. In particular, cysteine 367 located in the central α-helical rod domain of keratin 14 is necessary for the formation of a stable perinuclear network of keratin filaments (with type II partner keratin 5) in skin keratinocytes analyzed by static and live cell imaging. Here, we show that two additional cysteine residues located in the non-helical head domain of K14, Cys-4 and Cys-40, also participate in inter-keratin disulfide bonding and tandemly play a key role complementary to that of Cys-367 in the assembly, organization, and dynamics of keratin filaments in skin keratinocytes in primary culture. Analysis of K14 variants with single or multiple substitutions of cysteine residues points to a spatial and temporal hierarchy in how Cys-4/Cys-40 and Cys-367 regulate keratin assembly in vitro and filament dynamics in live keratinocytes in culture. Our findings substantiate the importance and complexity of a novel determinant, namely inter-keratin disulfide bonding, for the regulation of several aspects of keratin filaments in surface epithelia.
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Filamentos Intermedios/metabolismo , Queratina-14/metabolismo , Queratinocitos/metabolismo , Piel/metabolismo , Sustitución de Aminoácidos , Animales , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Humanos , Filamentos Intermedios/química , Filamentos Intermedios/genética , Queratina-14/química , Queratina-14/genética , Queratinocitos/citología , Ratones , Mutación Missense , Estructura Secundaria de Proteína , Piel/citologíaRESUMEN
Morphine has a profound role in neurofilament (NF) expression. However, there are very few studies on the fate of NFs during morphine abstinence coinciding with periods of relapse. Mice were treated chronically with morphine to render them tolerant to and dependent on morphine and sacrificed thereafter while another group, treated similarly, was left for 2 months without morphine. A long-lasting alteration in the stoichiometric ratio of the three NFs was observed under both conditions in both the cortex and subcortex. Morphine abstinence caused significant alterations in the phosphorylated and nitrated forms of the three NF subunits. Nitrated neurofilament light polypeptide chain (NFL) was significantly increased during chronic morphine treatment which persisted even after 2 months of morphine withdrawal. Mass spectrometric analysis following two-dimensional gel electrophoresis (2DE)-gel electrophoresis of cytoskeleton fractions of both cortex and subcortex regions identified enzymes associated with energy metabolism, cytoskeleton-associated proteins as well as NFs which showed sustained regulation even after abstinence of morphine for 2 months. It is suggestive that alteration in the levels of some of these proteins may be instrumental in the increased nitration of NFL during morphine exposure. Such gross alteration in NF dynamics is indicative of a concerted biological process of neuroadaptation during morphine abstinence.
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Corteza Cerebral/metabolismo , Filamentos Intermedios/metabolismo , Dependencia de Morfina/metabolismo , Morfina/farmacología , Narcóticos/farmacología , Animales , Western Blotting , Electroforesis en Gel Bidimensional , Hipotálamo/metabolismo , Masculino , Espectrometría de Masas , Mesencéfalo/metabolismo , Ratones , Nitrocompuestos/metabolismo , Fosforilación/efectos de los fármacos , Proteómica , Tabique del Cerebro/metabolismo , Síndrome de Abstinencia a Sustancias/metabolismo , Tálamo/metabolismoRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Kai-Xin-San (KXS), a well-known traditional Chinese herbal decoction, has been widely used to treat mental depression and memory loss in China. It has a combination of four herbs: Ginseng Radix et Rhizoma (GR; root and rhizome of Panax ginseng C. A. Mey.), Polygalae Radix (PR; root of Polygala tenuifolia Wild.), Acori Tatarinowii Rhizoma (ATR; rhizome of Acorus tatarinowii Schott), and Poria (PO; sclerotium of Poriacocos (Schw.) Wolf), from which a pairing of two herbs was considered as paired-herb, such as the pairing of GR-PR and ATR-PO. The depression-induced neural cell loss is one of the major pathogenesis in depression. Here, an optimized KXS by changing the ratio of paired-herbs in KXS was demonstrated aiming at promoting neural cell differentiation. MATERIALS AND METHODS: Quantitative assessment of chemical markers in each herbal extract was determined by LC-MS. Promoters of neurofilaments, NF68 and NF200, linked with luciferase reporter gene (pNF68-Luc and pNF200-Luc) were applied in cultured pheochromocytoma (PC12) cells to study the transcriptional activation of each herbal extract. The effect of GR-PR and ATR-PO in improving NF promoter activity was analyzed by Compusyn software. The activation of PKA was indicated. RESULTS: In PC12 cells, an optimized KXS named KXS1:5 having 1:5 of GR-PR:ATR-PO had greater capability in promoting the expression of neurofilament. The synergistic effect of GR-PR and ATR-PO on the improved efficiency was further determined. Moreover, the treatment of H89, a PKA inhibitor, significantly inhibited the induced NF promoter activity. CONCLUSION: These results indicated an optimized KXS by optimizing the compatibility of paired-herb and this compatibility was proven to exert synergistic effect. Moreover, the underlying mechanism was mediated by a PKA signaling pathway.
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Medicamentos Herbarios Chinos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Filamentos Intermedios/metabolismo , Animales , Medicamentos Herbarios Chinos/química , Filamentos Intermedios/genética , Células PC12 , RatasRESUMEN
The exposure of pregnant females to stress during a critical period of fetal brain development is an environmental risk factor for the development of schizophrenia in adult offspring. Schizophrenia is a group of common mental disorders of unclear origin, affecting approximately 1% of the global population, showing a generally young age at onset. In the present study, a repeated variable stress paradigm was applied to pregnant rats during the final week of gestation. The effects of an extract of Panax ginseng C.A. Meyer (PG) on rats exposed to prenatal stress (PNS) were investigated in terms of behavioral activity and protein expression analyses. In the behavioral tests, grooming behavior in a social interaction test, line-crossing behavior in an open-field test and swimming activity in a forced-swim test were decreased in the rats exposed to PNS compared with the non-stressed offspring; the changes in behavioral activity were reversed upon oral treatment with PG (300 mg/kg). Subsequently, western blot analysis and immunohistochemical analyses of the prefrontal cortex and hippocampus revealed that the downregulation of several neurodevelopmental genes which occurred following exposure to PNS was reversed upon treatment with PG. The current findings demonstrate that the downregulation of several genes following exposure to PNS may affect subsequent behavioral changes, and that these phenomena are reversed following treatment with PG during pregnancy. Our results suggest that oral treatment with PG reduces the incidence of psychiatric disorders, such as schizophrenia.
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Exposición Materna , Panax/química , Extractos Vegetales/farmacología , Efectos Tardíos de la Exposición Prenatal , Estrés Fisiológico , Animales , Conducta Animal/efectos de los fármacos , Femenino , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Filamentos Intermedios/metabolismo , Modelos Animales , Proteínas del Tejido Nervioso/metabolismo , Corteza Prefrontal/metabolismo , Embarazo , RatasRESUMEN
The ubiquitously expressed multifunctional cytolinker protein plectin is essential for muscle fiber integrity and myofiber cytoarchitecture. Patients suffering from plectinopathy-associated epidermolysis bullosa simplex with muscular dystrophy (EBS-MD) and mice lacking plectin in skeletal muscle display pathological desmin-positive protein aggregation and misalignment of Z-disks, which are hallmarks of myofibrillar myopathies (MFMs). Here, we developed immortalized murine myoblast cell lines to examine the pathogenesis of plectinopathies at the molecular and single cell level. Plectin-deficient myotubes, derived from myoblasts, were fully functional and mirrored the pathological features of EBS-MD myofibers, including the presence of desmin-positive protein aggregates and a concurrent disarrangement of the myofibrillar apparatus. Using this cell model, we demonstrated that plectin deficiency leads to increased intermediate filament network and sarcomere dynamics, marked upregulation of HSPs, and reduced myotube resilience following mechanical stretch. Currently, no specific therapy or treatment is available to improve plectin-related or other forms of MFMs; therefore, we assessed the therapeutic potential of chemical chaperones to relieve plectinopathies. Treatment with 4-phenylbutyrate resulted in remarkable amelioration of the pathological phenotypes in plectin-deficient myotubes as well as in plectin-deficient mice. Together, these data demonstrate the biological relevance of the MFM cell model and suggest that this model has potential use for the development of therapeutic approaches for EBS-MD.
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Músculo Esquelético/patología , Mioblastos/fisiología , Fenilbutiratos/farmacología , Plectina/deficiencia , Animales , Diferenciación Celular , Células Cultivadas , Desmina/metabolismo , Evaluación Preclínica de Medicamentos , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Filamentos Intermedios/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/metabolismo , Fuerza Muscular/efectos de los fármacos , Músculo Esquelético/metabolismo , Miopatías Estructurales Congénitas/tratamiento farmacológico , Miopatías Estructurales Congénitas/metabolismo , Miopatías Estructurales Congénitas/patología , Fenilbutiratos/uso terapéutico , Plectina/genética , Estabilidad Proteica , Sarcómeros/metabolismo , Sarcómeros/patología , Regulación hacia ArribaRESUMEN
The detrimental role of leptin in experimental autoimmune encephalomyelitis (EAE) is opposite to its neuroprotective role in other neuropathologies. We hypothesize that a shifted cellular distribution of leptin receptors underlies the differential effects of leptin. A robust increase of ObR immunoreactivity was seen along glial fibrillary acidic protein (GFAP)(+) intermediate filaments in reactive astrocytes in the hippocampus and hypothalamus of mice with EAE. Although astrocyte-specific GFAP mRNA and protein were both increased, ObRa mRNA was elevated only after resolution of EAE symptoms, and ObRb mRNA was even decreased at the peak time of symptoms of EAE. A cell type-specific action of leptin may underlie its differential effects.
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Astrocitos/metabolismo , Encefalomielitis Autoinmune Experimental/metabolismo , Hipocampo/metabolismo , Hipotálamo/metabolismo , Proteínas del Tejido Nervioso/biosíntesis , Receptores de Leptina/biosíntesis , Animales , Convalecencia , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/inmunología , Regulación de la Expresión Génica , Gliosis/etiología , Gliosis/genética , Gliosis/metabolismo , Gliosis/patología , Hipocampo/patología , Hipotálamo/patología , Filamentos Intermedios/metabolismo , Leptina/fisiología , Ratones , Ratones Endogámicos , Proteína Proteolipídica de la Mielina/inmunología , Proteína Proteolipídica de la Mielina/toxicidad , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Especificidad de Órganos , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/toxicidad , Procesamiento Proteico-Postraduccional , ARN Mensajero/biosíntesis , Receptores de Leptina/genética , Transcripción Genética , Regulación hacia ArribaRESUMEN
The involvement of calcium-mediated signaling pathways in the mechanism of action of 1α,25-dihydroxyvitamin D(3) (1,25D) is currently demonstrated. In this study we found that 1,25D induces nongenomic effects mediated by membrane vitamin D receptor (VDRm) by modulating intermediate filament (IF) phosphorylation and calcium uptake through L-type voltage-dependent calcium channels (L-VDCC) in cerebral cortex of 10 day-old rats. Results showed that the mechanism of action of 1,25D involves intra- and extracellular calcium levels, as well as the modulation of chloride and potassium channels. The effects of L-VDCCs on membrane voltage occur over a broad potential range and could involve depolarizing or hyperpolarizing coupling modes, supporting a cross-talk among Ca(2+) uptake and potassium and chloride channels. Also, the Na(+)/K(+)-ATPase inactivation by ouabain mimicked the 1,25D action on (45)Ca(2+) uptake. The Na(+)/K(+)-ATPase inhibition observed herein might lead to intracellular Na(+) accumulation with subsequent L-VDCC opening and consequently increased (45)Ca(2+) (calcium, isotope of mass 45) uptake. Moreover, the 1,25D effect is dependent on the activation of the following protein kinases: cAMP-dependent protein kinase (PKA), Ca(2+)/calmodulin-dependent protein kinase (PKCaMII), phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase p38 (p38(MAPK)). The modulation of calcium entry into neural cells by the 1,25D we are highlighting, might take a role in the regulation of a plethora of intracellular processes. Considering that vitamin D deficiency can lead to brain illness, 1,25D may be a possible candidate to be used, at least as an adjuvant, in the pharmacological therapy of neuropathological conditions.
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Envejecimiento/metabolismo , Canales de Calcio Tipo L/metabolismo , Calcio/metabolismo , Corteza Cerebral/metabolismo , Filamentos Intermedios/metabolismo , Activación del Canal Iónico/efectos de los fármacos , Vitamina D/análogos & derivados , Envejecimiento/efectos de los fármacos , Animales , Antígenos Nucleares/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Corteza Cerebral/efectos de los fármacos , Canales de Cloruro/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de Filamentos Intermediarios/metabolismo , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Modelos Biológicos , Proteínas del Tejido Nervioso/metabolismo , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Canales de Potasio/metabolismo , Proteína Quinasa C/metabolismo , Ratas , Receptores de Calcitriol/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Vitamina D/farmacologíaRESUMEN
Viscum album agglutinin-I (VAA-I) is a plant lectin that possesses interesting potential therapeutic properties and immunomodulatory activities. We have recently found that VAA-I is a potent inducer of human neutrophil apoptosis, but the mechanism(s) involved require further elucidation. In this study, we found that VAA-I alters mitochondrial transmembrane potential and increases intracellular levels of reactive oxygen species (ROS). Despite these observations, treatment with the mitochondrial stabilizer, bongkrekic acid, or with catalase, known to degrade H(2)O(2), fails to reverse VAA-I-induced apoptosis. Moreover, VAA-I was found to induce apoptosis in PLB-985 cells deficient in gp91(phox), indicating that the lectin acts via an ROS-independent mechanism. Pretreatment of neutrophils with brefeldin A, an inhibitor of vesicular transport, was found to reverse VAA-I-induced apoptosis. Protein expression of Mcl-1 was decreased by VAA-I. The role of caspases in the degradation of cytoskeletal proteins during both spontaneous and VAA-I-induced neutrophil apoptosis was also investigated. Paxillin and vimentin were markedly degraded by VAA-I when compared with neutrophils that undergo spontaneous apoptosis, but not vinculin or alpha- and beta-tubulin. Caspases were involved in cytoskeletal protein degradation because preincubation with the pan-caspase inhibitor N-benzyloxycarbonyl-V-A-D-O-methylfluoromethyl ketone was found to reverse protein cleavage. We conclude that VAA-I needs to be internalized to mediate apoptosis and that its activity is not dependent on a cell surface receptor-mediated pathway. Also, we conclude that VAA-I induces apoptosis by ROS-independent and Mcl-1-dependent mechanisms and that caspases are involved in cytoskeletal protein degradation in both spontaneous and VAA-I-induced neutrophil apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Caspasas/fisiología , Proteínas del Citoesqueleto/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , Neutrófilos/metabolismo , Fosfoproteínas/metabolismo , Preparaciones de Plantas , Proteínas de Plantas , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas Proto-Oncogénicas c-bcl-2 , Toxinas Biológicas/farmacología , Vimentina/metabolismo , Brefeldino A/farmacología , Proteínas del Citoesqueleto/antagonistas & inhibidores , Citometría de Flujo , Humanos , Filamentos Intermedios/efectos de los fármacos , Filamentos Intermedios/metabolismo , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/fisiología , Potenciales de la Membrana/efectos de los fármacos , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Muérdago , Mitocondrias/efectos de los fármacos , Mitocondrias/fisiología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/metabolismo , Neutrófilos/citología , Neutrófilos/efectos de los fármacos , Neutrófilos/enzimología , Paxillin , Fosfoproteínas/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Proteínas Inactivadoras de Ribosomas , Proteínas Inactivadoras de Ribosomas Tipo 2 , Células Tumorales Cultivadas , Regulación hacia Arriba/efectos de los fármacos , Vimentina/antagonistas & inhibidoresRESUMEN
Purified assembly-competent vimentin, an intermediate filament protein, was obtained from bovine lens in this study. The effects of withangulatin A on vimentin assembly with or without phosphorylation were examined by negative-stain electron microscopy. Soluble tetrameric vimentin was assembled into irregular fibrils with lateral associations or short filaments after pretreatment with 50 or 100 microM withangulatin A, respectively. Incubation of assembled vimentin filaments with withangulatin A at 50 or 100 microM resulted in the formation of aggregates, and the degree of aggregation was concentration dependent. The appearance of vimentin filaments was slightly altered after treatment with cAMP-dependent protein kinase or protein kinase C; however, phosphorylation of filamentous vimentin by the protein kinases in the presence of withangulatin A resulted in higher degrees of aggregation of the filaments, compared with those treated only with the drug. Moreover, the level of phosphorylation of filamentous vimentin by the protein kinases was augmented in the presence of withangulatin A. Experimental results indicated that withangulatin A directly and specifically affects the conformation of the vimentin molecules, thereby resulting in alterations in assembly behavior and reactivity toward cAMP-dependent protein kinase and protein kinase C. The data observed further imply that withangulatin A, which directly causes aggregation of vimentin filaments, is a vimentin intermediate filament-targeting drug.
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Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Medicamentos Herbarios Chinos/farmacología , Filamentos Intermedios/efectos de los fármacos , Pregnenos/farmacología , Proteína Quinasa C/metabolismo , Vimentina/metabolismo , Animales , Bovinos , Técnicas In Vitro , Filamentos Intermedios/metabolismo , Filamentos Intermedios/ultraestructura , Fosforilación , Vimentina/químicaRESUMEN
A major structural component of the inner face of the nuclear envelope in vertebrates and invertebrates is the nuclear lamina, an array of 1-3 extrinsic membrane proteins, lamins A, B and C. These proteins are highly homologous to intermediate filaments and are classified as type V. We report the first purification, antigenic characterization and immunocytochemical localization of putative plant lamin proteins from pea nuclei. We conclude that plant cells contain this ancestral class of intermediate filaments in their nuclei and that regulation of nuclear envelope assembly/disassembly and mitosis in plants may be similar to that in animal cells.