RESUMEN
Local information processing in the growth cone is essential for correct wiring of the nervous system. As an axon navigates through the developing nervous system, the growth cone responds to extrinsic guidance cues by coordinating axon outgrowth with growth cone steering. It has become increasingly clear that axon extension requires proper actin polymerization dynamics, whereas growth cone steering involves local protein synthesis. However, molecular components integrating these two processes have not been identified. Here, we show that Down syndrome critical region 1 protein (DSCR1) controls axon outgrowth by modulating growth cone actin dynamics through regulation of cofilin activity (phospho/dephospho-cofilin). Additionally, DSCR1 mediates brain-derived neurotrophic factor-induced local protein synthesis and growth cone turning. Our study identifies DSCR1 as a key protein that couples axon growth and pathfinding by dually regulating actin dynamics and local protein synthesis.
Asunto(s)
Axones/metabolismo , Axones/fisiología , Conos de Crecimiento/metabolismo , Conos de Crecimiento/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Musculares/metabolismo , Actinas/metabolismo , Animales , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Proteínas de Unión al Calcio , Células Cultivadas , Hipocampo/metabolismo , Hipocampo/fisiología , Ratones , Ratones Endogámicos C57BL , Proteínas de Microfilamentos/metabolismo , Neuronas/metabolismo , Neuronas/fisiología , Biosíntesis de Proteínas/fisiologíaRESUMEN
Arsenic (As), lead (Pb) and cadmium (Cd) are the major metal contaminants of ground water in India. We have reported the toxic effect of their mixture (metal mixture, MM), at human relevant doses, on developing rat astrocytes. Astrocyte damage has been shown to be associated with myelin disintegration in CNS. We, therefore, hypothesized that the MM would perturb myelinating white matter in cerebral cortex, optic nerve (O.N.) and retina. We observed modulation in the levels of myelin and axon proteins, such as myelin basic protein (MBP), proteolipid protein, 2'-, 3'-cyclic-nucleotide-3'-phosphodiesterase, myelin-associated glycoprotein and neurofilament (NF) in the brain of developing rats. Dose and time-dependent synergistic toxic effect was noted. The MBP- and NF-immunolabeling, as well as luxol-fast blue (LFB) staining demonstrated a reduction in the area of intact myelin-fiber, and an increase in vacuolated axons, especially in the corpus-callosum. Transmission electron microscopy (TEM) of O.N. revealed a reduction in myelin thickness and axon-density. The immunolabeling with MBP, NF, and LFB staining in O.N. supported the TEM data. The hematoxylin and eosin staining of retina displayed a decrease in the thickness of nerve-fiber, plexiform-layer, and retinal ganglion cell (RGC) count. Investigating the mechanism revealed a loss in glutamine synthetase activity in the cerebral cortex and O.N., and a fall in the brain derived neurotrophic factor in retina. An enhanced apoptosis in MBP, NF and Brn3b-containing cells justified the diminution in myelinating axons in CNS. Our findings for the first time indicate white matter damage by MM, which may have significance in neurodevelopmental-pediatrics, neurotoxicology and retinal-cell biology.
Asunto(s)
Arsénico/toxicidad , Axones/efectos de los fármacos , Encéfalo/patología , Cadmio/toxicidad , Plomo/toxicidad , Vaina de Mielina/efectos de los fármacos , Nervio Óptico/efectos de los fármacos , Retina/efectos de los fármacos , Animales , Animales Recién Nacidos , Axones/patología , Encéfalo/efectos de los fármacos , Encéfalo/crecimiento & desarrollo , Femenino , Vaina de Mielina/patología , Nervio Óptico/crecimiento & desarrollo , Nervio Óptico/patología , Embarazo , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Efectos Tardíos de la Exposición Prenatal/patología , Ratas , Retina/crecimiento & desarrollo , Retina/patologíaRESUMEN
Exposure to a mixture of As, Pb and Cd induces apoptosis and morphological alterations in the cortical astrocytes of rat brain. The levels of the glial fibrillary acidic protein (GFAP) undergo a reduction. The GFAP exists in several isoforms, viz., α, ß, κ, δ and ϵ. However, contribution of the isoforms towards astrocyte damage is not understood. We investigated the effect of the metal mixture (MM) on the expression profiles of mRNAs encoding the GFAP isoforms in astrocytes. The MM was administered in drinking water to developing rats till postnatal day (PD) 60. We observed a fall (10.20 ± 1.04%, 18.91 ± 2.12% and 30.26 ± 3.21% at PD24, PD45 and PD60 respectively) in GFAPα. This may have been compensated by a rise in ß, κ, and ϵ. The GFAPδ remained unchanged. To determine the role of the GFAPα, we silenced its gene using SiRNA technology in the rat primary astrocytes. We observed a 23.73 ± 1.56% increase in the number of apoptotic cells. The cleaved PARP and Bax levels increased by 2.48 ± 0.14-fold and 3.73 ± 0.23-fold respectively, and the Bcl-2 and Bcl-xl decreased by 2.38 ± 0.08-fold and 1.76 ± 0.09-fold respectively. The change was comparable to the cells treated with MM. Moreover, silencing the GFAPα gene induced a reduction in the area (6.19 ± 0.18-folds), perimeter (12.65 ± 1.68-folds) and the number of processes (5.88 ± 1.5-folds) in the astrocytes, which closely matched the MM-treated ones. Taken together, these observations are the first to show that MM disturbs the composition of the GFAP isoforms, and a suppressed GFAPα promotes apoptosis in the matured rat astrocytes.
Asunto(s)
Arsenitos/toxicidad , Astrocitos/efectos de los fármacos , Encéfalo/efectos de los fármacos , Cloruro de Cadmio/toxicidad , Proteína Ácida Fibrilar de la Glía/metabolismo , Compuestos Organometálicos/toxicidad , Compuestos de Sodio/toxicidad , Animales , Apoptosis/efectos de los fármacos , Astrocitos/patología , Encéfalo/metabolismo , Encéfalo/patología , Regulación hacia Abajo/efectos de los fármacos , Combinación de Medicamentos , Femenino , Silenciador del Gen , Proteína Ácida Fibrilar de la Glía/genética , Masculino , Exposición Materna , Embarazo , Isoformas de Proteínas , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , Ratas , Transfección/métodosRESUMEN
Cypermethrin is reported to affect astrocytes in rat brain; however, its mechanism of action is obscure. Here, we observed an increase in apoptosis in the cortical astrocytes upon treatment of rats with cypermethrin. We then characterized the mechanism governing the apoptosis. Because the epidermal growth factor receptor (EGFR) signaling regulates the survival of astrocytes, we investigated the effect of cypermethrin on EGFR activation. The astrocytes exhibited an early and irreversible attenuation in the basal EGFR phosphorylation. Supportively, molecular docking studies revealed considerable homology in the docking mode of cypermethrin and the known EGFR inhibitors, erlotinib and AG1478, to the kinase domain of EGFR. Furthermore, treatment with cypermethrin demonstrated a downregulation in the intracellular and secreted levels of heparin-binding epidermal growth factor (HB-EGF), an EGFR ligand. AG1478 reduced the synthesis of HB-EGF, suggesting the dependence of HB-EGF on EGFR activation. In addition, a neutralizing antibody against HB-EGF diminished the basal EGFR levels, indicating ligand-dependent expression of EGFR. Likewise, cypermethrin caused irreversible suppression in the basal EGFR levels, which induced apoptosis in astrocytes. The apoptosis was prevented by exogenous HB-EGF. These data imply an autocrine/paracrine mode of action of HB-EGF-EGFR in astrocyte survival. Consequently, cypermethrin induced a mitochondria-mediated apoptosis, characterized by rise in Bax/Bcl-2 ratio and cleavage of caspase-9, -3, and -7, and the effect was prevented by HB-EGF. HB-EGF activated the extracellular signal-regulated kinases and AKT pathways that protected against apoptosis. Together, these data demonstrate that cypermethrin induces astrocyte apoptosis by disrupting the autocrine/paracrine mode of HB-EGF-EGFR signaling at two levels, irreversible loss of basal EGFR and downregulation of HB-EGF.
Asunto(s)
Apoptosis/efectos de los fármacos , Astrocitos/efectos de los fármacos , Comunicación Autocrina/efectos de los fármacos , Corteza Cerebral/efectos de los fármacos , Receptores ErbB/efectos de los fármacos , Comunicación Paracrina/efectos de los fármacos , Piretrinas/toxicidad , Transducción de Señal/efectos de los fármacos , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Astrocitos/metabolismo , Astrocitos/patología , Células Cultivadas , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Relación Dosis-Respuesta a Droga , Receptores ErbB/química , Receptores ErbB/metabolismo , Factor de Crecimiento Similar a EGF de Unión a Heparina , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ligandos , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/patología , Modelos Moleculares , Estructura Molecular , Fosforilación , Conformación Proteica , Inhibidores de Proteínas Quinasas/farmacología , Piretrinas/química , Ratas , Ratas Wistar , Relación Estructura-Actividad , Factores de TiempoRESUMEN
Neurotoxicity of individual metals is well investigated but that of metal mixture (MM), an environmental reality, in the developing brain is relatively obscure. We investigated the combinatorial effect of arsenic (As), cadmium (Cd), and lead (Pb) on rat brain development, spanning in utero to postnatal development. MM was administered by gavage to pregnant and lactating rats, and to postweaning pups till 2 months. The pups exhibited behavioral disturbances characterized by hyperlocomotion, increased grip strength, and learning-memory deficit. Disruption of the blood-brain barrier (BBB) was associated with dose-dependent increase in deposition of the metals in developing brain. Astrocytes were affected by MM treatment as evident from their reduced density, area, perimeter, compactness, and number of processes, and increased apoptosis in cerebral cortex and cerebellum. The metals induced synergistic reduction in glial fibrillary acidic protein (GFAP) expression during brain development; however, postweaning withdrawal of MM partially restored the levels of GFAP in adults. To characterize the toxic mechanism, we treated rat primary astrocytes with MM at concentrations ranging from lethal concentration (LC)(10) to LC(75) of the metals. We observed synergistic downregulation in viability and increase in apoptosis of the astrocytes, which were induced by proximal activation of extra cellular signal-regulated kinase (ERK) signaling and downstream activation of Jun N-terminal kinase (JNK) pathway. Furthermore, rise in intracellular calcium ion ([Ca(2+)](i)) and reactive oxygen species generation promoted apoptosis in the astrocytes. Taken together, these observations are the first to show that mixture of As, Cd, and Pb has the capacity to induce synergistic toxicity in astrocytes that may compromise the BBB and may cause behavioral dysfunction in developing rats.
Asunto(s)
Astrocitos/efectos de los fármacos , Contaminantes Ambientales/toxicidad , Exposición Materna/efectos adversos , Metales Pesados/toxicidad , Enfermedades del Sistema Nervioso/inducido químicamente , Neuronas/efectos de los fármacos , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Arsenitos/toxicidad , Astrocitos/patología , Conducta Animal/efectos de los fármacos , Conducta Animal/fisiología , Encéfalo/efectos de los fármacos , Encéfalo/embriología , Encéfalo/fisiología , Cloruro de Cadmio/toxicidad , Supervivencia Celular/efectos de los fármacos , Mezclas Complejas , Combinación de Medicamentos , Sinergismo Farmacológico , Femenino , Fuerza de la Mano , Lactancia/efectos de los fármacos , Locomoción/efectos de los fármacos , Aprendizaje por Laberinto/efectos de los fármacos , Enfermedades del Sistema Nervioso/patología , Neuronas/patología , Compuestos Organometálicos/toxicidad , Embarazo , Ratas , Ratas Wistar , Compuestos de Sodio/toxicidadRESUMEN
Inadequate maternal intake of omega-3-fatty acids (omega3 FAs) causes adverse neurodevelopmental outcome in the progeny; however, their molecular mechanism of action is obscure. Since omega3 FAs are known to inhibit neuronal apoptosis during neuro-degeneration, we investigated their possible contribution in regulating neuronal apoptosis during brain development. Using rat model of hypothyroidism-induced neuronal apoptosis, we provide evidence for anti-apoptotic role of omega3 FAs during cerebellar development. omega3 FAs were supplemented as a mixture of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) to pregnant and lactating rats, and primary hypothyroidism was induced by administering methimazole. The cerebella from postnatal day 16 (d16) pups were isolated, and studies on apoptosis were conducted. We observed that omega3 FA-supplementation significantly reduced DNA fragmentation and caspase-3 activation in developing cerebellum of hypothyroid pups. The protection provided by omega3 FAs was associated with their ability to prevent increases in the level of pro-apoptotic basal cell lymphoma protein-2 (Bcl-2)-associated X protein (Bax) in the cerebellum during thyroid hormone (TH) deficiency. omega3 FAs increased the levels of anti-apoptotic proteins like Bcl-2 and Bcl-extra large (Bcl-x(L)), known to be repressed in hypothyroidism. omega3 FAs also restored levels of cerebellar phospho (p)-AKT, phospho-extracellular regulated kinase (p-ERK) and phospho-c-Jun N-terminal kinase (p-JNK), which were altered by hypothyroid insults, without interfering with the expression of TH responsive gene, myelin basic protein (mbp). Taken together, these results supplement an insight into the molecular mechanism of action of omega3 FAs in developing brain that involves regulation of apoptotic signaling pathways under stress.