RESUMEN
Dynein heavy chain (DYNC1H1) mutations can either lead to severe cerebral cortical malformations, or alternatively may be associated with the development of spinal muscular atrophy with lower extremity predominance (SMA-LED). To assess the origin of such differences, we studied a new Dync1h1 knock-in mouse carrying the cortical malformation p.Lys3334Asn mutation. Comparing with an existing neurodegenerative Dync1h1 mutant (Legs at odd angles, Loa, p.Phe580Tyr/+), we assessed Dync1h1's roles in cortical progenitor and especially radial glia functions during embryogenesis, and assessed neuronal differentiation. p.Lys3334Asn /+ mice exhibit reduced brain and body size. Embryonic brains show increased and disorganized radial glia: interkinetic nuclear migration occurs in mutants, however there are increased basally positioned cells and abventricular mitoses. The ventricular boundary is disorganized potentially contributing to progenitor mislocalization and death. Morphologies of mitochondria and Golgi apparatus are perturbed in vitro, with different effects also in Loa mice. Perturbations of neuronal migration and layering are also observed in p.Lys3334Asn /+ mutants. Overall, we identify specific developmental effects due to a severe cortical malformation mutation in Dync1h1, highlighting the differences with a mutation known instead to primarily affect motor function.
Asunto(s)
Dineínas , Atrofia Muscular Espinal , Humanos , Ratones , Animales , Dineínas/genética , Dineínas Citoplasmáticas/genética , Dineínas Citoplasmáticas/metabolismo , Atrofia Muscular Espinal/genética , Tamaño de los Órganos , Mutación/genética , Encéfalo/metabolismo , Células MadreRESUMEN
BACKGROUND: The centrosome regulates cell spatial organisation by controlling the architecture of the microtubule (MT) cytoskeleton. Conversely, the position of the centrosome within the cell depends on cytoskeletal networks it helps organizing. In mammalian cells, centrosome positioning involves a population of MT stably anchored at centrioles, the core components of the centrosome. An MT-anchoring complex containing the proteins ninein and Cep170 is enriched at subdistal appendages (SAP) that decorate the older centriole (called mother centriole) and at centriole proximal ends. Here, we studied the role played at the centrosome by hVFL3/CCDC61, the human ortholog of proteins required for anchoring distinct sets of cytoskeletal fibres to centrioles in unicellular eukaryotes. RESULTS: We show that hVFL3 co-localises at SAP and at centriole proximal ends with components of the MT-anchoring complex, and physically interacts with Cep170. Depletion of hVFL3 increased the distance between mother and daughter centrioles without affecting the assembly of a filamentous linker that tethers the centrioles and contains the proteins rootletin and C-Nap1. When the linker was disrupted by inactivating C-Nap1, hVFL3-depletion exacerbated centriole splitting, a phenotype also observed following depletion of other SAP components. This supported that hVFL3 is required for SAP function, which we further established by showing that centrosome positioning is perturbed in hVFL3-depleted interphase cells. Finally, we found that hVFL3 is an MT-binding protein. CONCLUSIONS AND SIGNIFICANCE: Together, our results support that hVFL3 is required for anchoring MT at SAP during interphase and ensuring proper centrosome cohesion and positioning. The role of the VFL3 family of proteins thus appears to have been conserved in evolution despite the great variation in the shape of centriole appendages in different eukaryotic species.
Asunto(s)
Proteínas Portadoras/metabolismo , Centriolos , Centrosoma , Tubulina (Proteína)/metabolismo , Animales , Sistemas CRISPR-Cas , Proteínas Portadoras/genética , Línea Celular , Centriolos/metabolismo , Centriolos/ultraestructura , Centrosoma/metabolismo , Centrosoma/ultraestructura , Cilios/ultraestructura , Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , Humanos , Microscopía Electrónica , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , ARN Interferente PequeñoRESUMEN
It has been well documented that neurotrophins, including brain-derived neurotrophic factor (BDNF), are severely affected in Alzheimer's disease (AD), but their administration faces a myriad of technical challenges. Here we took advantage of the early astrogliosis observed in an amyloid mouse model of AD (5xFAD) and used it as an internal sensor to administer BDNF conditionally and locally. We first demonstrate the relevance of BDNF release from astrocytes by evaluating the effects of coculturing WT neurons and BDNF-deficient astrocytes. Next, we crossed 5xFAD mice with pGFAP:BDNF mice (only males were used) to create 5xFAD mice that overexpress BDNF when and where astrogliosis is initiated (5xF:pGB mice). We evaluated the behavioral phenotype of these mice. We first found that BDNF from astrocytes is crucial for dendrite outgrowth and spine number in cultured WT neurons. Double-mutant 5xF:pGB mice displayed improvements in cognitive tasks compared with 5xFAD littermates. In these mice, there was a rescue of BDNF/TrkB downstream signaling activity associated with an improvement of dendritic spine density and morphology. Clusters of synaptic markers, PSD-95 and synaptophysin, were also recovered in 5xF:pGB compared with 5xFAD mice as well as the number of presynaptic vesicles at excitatory synapses. Additionally, experimentally evoked LTP in vivo was increased in 5xF:pGB mice. The beneficial effects of conditional BDNF production and local delivery at the location of active neuropathology highlight the potential to use endogenous biomarkers with early onset, such as astrogliosis, as regulators of neurotrophic therapy in AD.SIGNIFICANCE STATEMENT Recent evidence places astrocytes as pivotal players during synaptic plasticity and memory processes. In the present work, we first provide evidence that astrocytes are essential for neuronal morphology via BDNF release. We then crossed transgenic mice (5xFAD mice) with the transgenic pGFAP-BDNF mice, which express BDNF under the GFAP promoter. The resultant double-mutant mice 5xF:pGB mice displayed a full rescue of hippocampal BDNF loss and related signaling compared with 5xFAD mice and a significant and specific improvement in all the evaluated cognitive tasks. These improvements did not correlate with amelioration of ß amyloid load or hippocampal adult neurogenesis rate but were accompanied by a dramatic recovery of structural and functional synaptic plasticity.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Astrocitos/metabolismo , Factor Neurotrófico Derivado del Encéfalo/administración & dosificación , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Espinas Dendríticas/metabolismo , Hipocampo/metabolismo , Trastornos de la Memoria/metabolismo , Plasticidad Neuronal , Enfermedad de Alzheimer/complicaciones , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Hipocampo/efectos de los fármacos , Masculino , Trastornos de la Memoria/etiología , Trastornos de la Memoria/prevención & control , Ratones Noqueados , Plasticidad Neuronal/efectos de los fármacosRESUMEN
The nodes of Ranvier are essential regions for action potential conduction in myelinated fibers. They are enriched in multimolecular complexes composed of voltage-gated Nav and Kv7 channels associated with cell adhesion molecules. Cytoskeletal proteins ankyrin-G (AnkG) and ßIV-spectrin control the organization of these complexes and provide mechanical support to the plasma membrane. IQCJ-SCHIP1 is a cytoplasmic protein present in axon initial segments and nodes of Ranvier. It interacts with AnkG and is absent from nodes and axon initial segments of ßIV-spectrin and AnkG mutant mice. Here, we show that IQCJ-SCHIP1 also interacts with ßIV-spectrin and Kv7.2/3 channels and self-associates, suggesting a scaffolding role in organizing nodal proteins. IQCJ-SCHIP1 binding requires a ßIV-spectrin-specific domain and Kv7 channel 1-5-10 calmodulin-binding motifs. We then investigate the role of IQCJ-SCHIP1 in vivo by studying peripheral myelinated fibers in Schip1 knock-out mutant mice. The major nodal proteins are normally enriched at nodes in these mice, indicating that IQCJ-SCHIP1 is not required for their nodal accumulation. However, morphometric and ultrastructural analyses show an altered shape of nodes similar to that observed in ßIV-spectrin mutant mice, revealing that IQCJ-SCHIP1 contributes to nodal membrane-associated cytoskeleton organization, likely through its interactions with the AnkG/ßIV-spectrin network. Our work reveals that IQCJ-SCHIP1 interacts with several major nodal proteins, and we suggest that it contributes to a higher organizational level of the AnkG/ßIV-spectrin network critical for node integrity.
Asunto(s)
Ancirinas/metabolismo , Proteínas Portadoras/metabolismo , Nódulos de Ranvier/metabolismo , Animales , Biopolímeros/metabolismo , Células COS , Proteínas Portadoras/química , Chlorocebus aethiops , Ratones , Ratones Mutantes , Actividad Motora , Sistema Nervioso Periférico/fisiología , Sistema Nervioso Periférico/ultraestructuraRESUMEN
Exogenous transplanted neural precursor cells (NPCs) exhibit miscellaneous immune-modulatory effects in models of autoimmune demyelination. However, the regional interactions of NPCs with the host brain tissue in remissive inflammatory events have not been adequately studied. In this study we used the chronic MOG-induced Experimental Autoimmune Encephalomyelitis (EAE) model in C57BL/six mice. Based on previous data, we focused on neuropathology at Day 50 post-induction (D50) and studied the expression of connexin43 (Cx43) and Cx47, two of the main glial gap junction (GJ) proteins, in relation to the intraventricular transplantation of GFP(+) NPCs and their integration with the host tissue. By D50, NPCs had migrated intraparenchymally and were found in the corpus callosum at the level of the lateral ventricles and hippocampus. The majority of GFP(+) cells differentiated with simple or ramified processes expressing mainly markers of mature GLIA (GFAP and NogoA) and significantly less of precursor glial cells. GFP(+) NPCs expressed connexins and formed GJs around the hippocampus more than lateral ventricles. The presence of NPCs did not alter the increase in Cx43 GJ plaques at D50 EAE, but prevented the reduction of oligodendrocytic Cx47, increased the number of oligodendrocytes, local Cx47 levels and Cx47 GJ plaques per cell. These findings suggest that transplanted NPCs may have multiple effects in demyelinating pathology, including differentiation and direct integration into the panglial syncytium, as well as amelioration of oligodendrocyte GJ loss, increasing the supply of potent myelinating cells to the demyelinated tissue.
Asunto(s)
Encéfalo/patología , Conexina 43/metabolismo , Conexinas/metabolismo , Encefalomielitis Autoinmune Experimental/cirugía , Regulación de la Expresión Génica/fisiología , Células-Madre Neurales/trasplante , Factores de Edad , Animales , Encéfalo/citología , Diferenciación Celular , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/inducido químicamente , Encefalomielitis Autoinmune Experimental/patología , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Proteína Básica de Mielina/metabolismo , Glicoproteína Mielina-Oligodendrócito/toxicidad , Proteínas del Tejido Nervioso/metabolismo , Células-Madre Neurales/fisiología , Células-Madre Neurales/ultraestructura , Neuroglía/metabolismo , Neuroglía/patología , Neuroglía/ultraestructura , Fragmentos de Péptidos/toxicidadRESUMEN
Over the last decade, a large variety of alterations of the Contactin Associated Protein 2 (CNTNAP2) gene, encoding Caspr2, have been identified in several neuronal disorders, including neurodevelopmental disorders and peripheral neuropathies. Some of these alterations are homozygous but most are heterozygous, and one of the current challenges is to estimate to what extent they could affect the functions of Caspr2 and contribute to the development of these pathologies. Notably, it is not known whether the disruption of a single CNTNAP2 allele could be sufficient to perturb the functions of Caspr2. To get insights into this issue, we questioned whether Cntnap2 heterozygosity and Cntnap2 null homozygosity in mice could both impact, either similarly or differentially, some specific functions of Caspr2 during development and in adulthood. We focused on yet poorly explored functions of Caspr2 in axon development and myelination, and performed a morphological study from embryonic day E17.5 to adulthood of two major brain interhemispheric myelinated tracts, the anterior commissure (AC) and the corpus callosum (CC), comparing wild-type (WT), Cntnap2 -/- and Cntnap2 +/- mice. We also looked for myelinated fiber abnormalities in the sciatic nerves of mutant mice. Our work revealed that Caspr2 controls the morphology of the CC and AC throughout development, axon diameter at early developmental stages, cortical neuron intrinsic excitability at the onset of myelination, and axon diameter and myelin thickness at later developmental stages. Changes in axon diameter, myelin thickness and node of Ranvier morphology were also detected in the sciatic nerves of the mutant mice. Importantly, most of the parameters analyzed were affected in Cntnap2 +/- mice, either specifically, more severely, or oppositely as compared to Cntnap2 -/- mice. In addition, Cntnap2 +/- mice, but not Cntnap2 -/- mice, showed motor/coordination deficits in the grid-walking test. Thus, our observations show that both Cntnap2 heterozygosity and Cntnap2 null homozygosity impact axon and central and peripheral myelinated fiber development, but in a differential manner. This is a first step indicating that CNTNAP2 alterations could lead to a multiplicity of phenotypes in humans, and raising the need to evaluate the impact of Cntnap2 heterozygosity on the other neurodevelopmental functions of Caspr2.
RESUMEN
Pyk2 is a non-receptor tyrosine kinase enriched in hippocampal neurons, which can be activated by calcium-dependent mechanisms. In neurons, Pyk2 is mostly localised in the cytosol and dendritic shafts but can translocate to spines and/or to the nucleus. Here, we explore the function of a new localisation of Pyk2 in mitochondria-associated membranes (MAMs), a subdomain of ER-mitochondria surface that acts as a signalling hub in calcium regulation. To test the role of Pyk2 in MAMs' calcium transport, we used full Pyk2 knockout mice (Pyk2-/-) for in vivo and in vitro studies. Here we report that Pyk2-/- hippocampal neurons present increased ER-mitochondrial contacts along with defective calcium homeostasis. We also show how the absence of Pyk2 modulates mitochondrial dynamics and morphology. Taken all together, our results point out that Pyk2 could be highly relevant in the modulation of ER-mitochondria calcium efflux, affecting in turn mitochondrial function.
Asunto(s)
Calcio , Quinasa 2 de Adhesión Focal/metabolismo , Dinámicas Mitocondriales , Animales , Hipocampo/metabolismo , Ratones , Neuronas/metabolismoRESUMEN
Phosphorylation is involved in numerous neurodegenerative diseases. In particular, alpha-synuclein is extensively phosphorylated in aggregates in patients suffering from synucleinopathies. However, the share of this modification in the events that lead to the conversion of alpha-synuclein to aggregated toxic species needed to be clarified. The rat model that we developed through rAAV2/6-mediated expression of alpha-synuclein demonstrates a correlation between neurodegeneration and formation of small filamentous alpha-synuclein aggregates. A mutation preventing phosphorylation (S129A) significantly increases alpha-synuclein toxicity and leads to enhanced formation of beta-sheet-rich, proteinase K-resistant aggregates, increased affinity for intracellular membranes, a disarrayed network of neurofilaments and enhanced alpha-synuclein nuclear localization. The expression of a mutation mimicking phosphorylation (S129D) does not lead to dopaminergic cell loss. Nevertheless, fewer but larger aggregates are formed, and signals of apoptosis are also activated in rats expressing the phosphorylation-mimicking form of alpha-synuclein. These observations strongly suggest that phosphorylation does not play an active role in the accumulation of cytotoxic pre-inclusion aggregates. Unexpectedly, the study also demonstrates that constitutive expression of phosphorylation-mimicking forms of alpha-synuclein does not protect from neurodegeneration. The role of phosphorylation at Serine 129 in the early phase of Parkinson's disease is examined, which brings new perspective to therapeutic approaches focusing on the modulation of kinases/phosphatases activity to control alpha-synuclein toxicity.
Asunto(s)
Enfermedad de Parkinson/metabolismo , alfa-Sinucleína/metabolismo , alfa-Sinucleína/toxicidad , Animales , Núcleo Celular/química , Núcleo Celular/genética , Núcleo Celular/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Mutación Missense , Neuronas/metabolismo , Enfermedad de Parkinson/genética , Fosforilación , Transporte de Proteínas , Ratas , Ratas Wistar , alfa-Sinucleína/química , alfa-Sinucleína/genéticaRESUMEN
Oxytocin is a hypothalamic neuropeptide involved in the inhibition of nociception transmission at spinal dorsal horn (SDH) level (the first station where the incoming peripheral signals is modulated). Electrophysiological, behavioral, and pharmacological data strongly support the role of this neuropeptide and its receptor (the oxytocin receptor, OTR) as a key endogenous molecule with analgesic properties. Briefly, current data showed that oxytocin release from the hypothalamus induces OTR activation at the SDH, inducing selective inhibition of the nociceptive Aδ- and C-fibers (probably peptidergic) activity, but not the activity of proprioceptive fibers (i.e. Aß-fibers). The above inhibition could be a direct presynaptic mechanism, or a mechanism mediated by GABAergic interneurons. However, the exact anatomical localization of oxytocin and OTR remains unclear. In this context, the present study set out to analyze the role of OTRs, GABAergic cells and CGRP fibers in the SDH in rats by using electron microscopy. Ultrastructural analyses of the SDH tissue show that: (i) oxytocin and OTR are found in asymmetrical synapsis; (ii) OTR is found in GABAergic interneurons (near unmyelinated fibers), CGRPergic fibers and glial cells; (iii) whereas oxytocin is present in supraspinal descending projection fibers. These anatomical data strongly support the notion that oxytocin released at the SDH could presynaptically inhibit the nociceptive input from the peripheral primary afferent fibers. This inhibitory action could be direct or use a GABA interneuron. Furthermore, our findings that OTR is exhibited in glial tissue at the SDH requires further exploration in nociception assays.
Asunto(s)
Nocicepción , Receptores de Oxitocina , Animales , Fibras Nerviosas Amielínicas , Oxitocina , Células del Asta Posterior , Ratas , Asta Dorsal de la Médula EspinalRESUMEN
Spinal muscular atrophy (SMA) is an inborn neuromuscular disorder caused by low levels of survival motor neuron protein, and for which no efficient therapy exists. Here, we show that the slower rate of postnatal motor-unit maturation observed in type 2 SMA-like mice is correlated with the motor neuron death. Physical exercise delays motor neuron death and leads to an increase in the postnatal maturation rate of the motor-units. Furthermore, exercise is capable of specifically enhancing the expression of the gene encoding the major activating subunit of the NMDA receptor in motor neurons, namely the NR2A subunit, which is dramatically downregulated in the spinal cord of type 2 SMA-like mice. Accordingly, inhibiting NMDA-receptor activity abolishes the exercise-induced effects on muscle development, motor neuron protection and life span gain. Thus, restoring NMDA-receptor function could be a promising therapeutic approach to SMA treatment.
Asunto(s)
Neuronas Motoras/metabolismo , Condicionamiento Físico Animal/fisiología , Receptores de N-Metil-D-Aspartato/metabolismo , Atrofias Musculares Espinales de la Infancia/genética , Atrofias Musculares Espinales de la Infancia/metabolismo , Animales , Supervivencia Celular/genética , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Noqueados , Ratones Transgénicos , Neuronas Motoras/patología , Músculo Esquelético/crecimiento & desarrollo , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Receptores de N-Metil-D-Aspartato/deficiencia , Receptores de N-Metil-D-Aspartato/genética , Atrofias Musculares Espinales de la Infancia/patologíaRESUMEN
Schwannomin/merlin is the product of a tumor suppressor gene mutated in neurofibromatosis type 2 (NF2). Although the consequences of NF2 mutations on Schwann cell proliferation are well established, the physiological role of schwannomin in differentiated cells is not known. To unravel this role, we studied peripheral nerves in mice overexpressing in Schwann cells schwannomin with a deletion occurring in NF2 patients (P0-SCH-Delta39-121) or a C-terminal deletion. The myelin sheath and nodes of Ranvier were essentially preserved in both lines. In contrast, the ultrastructural and molecular organization of contacts between Schwann cells and axons in paranodal and juxtaparanodal regions were altered, with irregular juxtaposition of normal and abnormal areas of contact. Similar but more severe alterations were observed in mice with conditional deletion of the Nf2 gene in Schwann cells. The number of Schmidt-Lanterman incisures, which are cytoplasmic channels interrupting the compact myelin and characterized by distinct autotypic contacts, was increased in the three mutant lines. P0-SCH-Delta39-121 and conditionally deleted mice displayed exuberant wrapping of nonmyelinated fibers and short internodes, an abnormality possibly related to altered control of Schwann cell proliferation. In support of this hypothesis, Schwann cell number was increased along fibers before myelination in P0-SCH-Delta39-121 mice but not in those with C-terminal deletion. Schwann cell numbers were also more numerous in mice with conditional deletion. Thus, schwannomin plays an important role in the control of Schwann cell number and is necessary for the correct organization and regulation of axoglial heterotypic and glio-glial autotypic contacts.
Asunto(s)
Comunicación Celular/fisiología , Neurofibromina 2/fisiología , Células de Schwann/metabolismo , Células de Schwann/ultraestructura , Proteínas Supresoras de Tumor/fisiología , Animales , Proliferación Celular , Eliminación de Gen , Humanos , Ratones , Ratones Transgénicos , Neurofibromina 2/biosíntesis , Neurofibromina 2/deficiencia , Neurofibromina 2/genética , Nervios Periféricos/metabolismo , Nervios Periféricos/ultraestructura , Proteínas Supresoras de Tumor/genéticaRESUMEN
Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) transiently expressed in specific regions of the central and peripheral nervous systems. In this study, we focused on the rat developing dorsal root ganglion (DRG). This ganglion is composed of heterogeneous sensory neurons characterized by the expression of RTK for neurotrophic factors, such as the nerve growth factor receptor TrkA or the glial-derived neurotrophic factor family receptor Ret, which are specifically detected in nociceptive neurons. In DRG, ALK expression reached a maximum around birth. We showed that ALK is specifically present in a subtype of neurons during DRG development, and that the majority of these neurons co-expressed TrkA and Ret. Interestingly, we identified only one form (220 kDa) of ALK in DRG neurons both in vivo and in vitro. On the opposite, in transfected cells as well as in brain extracts, ALK was identified as two forms (220 and 140 kDa). The DRG is composed of neurons and glial cells, principally satellite Schwann cells. Thus, we hypothesized that the presence of satellite Schwann cells was involved in the absence of truncated ALK. Using two different cell types, HEK293 cells stably expressing ALK, and MSC80 cells, a previously described Schwann cell line, we showed that a factor secreted by the Schwann cells is likely involved in the absence of ALK cleavage. All these data hence open new perspectives concerning the role of ALK in the specification of nociceptive DRG neurons and in the neurons-Schwann cells interaction.
Asunto(s)
Ganglios Espinales/enzimología , Proteínas Tirosina Quinasas/metabolismo , Células de Schwann/enzimología , Células Receptoras Sensoriales/enzimología , Quinasa de Linfoma Anaplásico , Animales , Células COS , Comunicación Celular/genética , Células Cultivadas , Chlorocebus aethiops , Medios de Cultivo Condicionados/farmacología , Ganglios Espinales/citología , Humanos , Ratones , Nociceptores/citología , Nociceptores/metabolismo , Dolor/metabolismo , Dolor/fisiopatología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas c-ret/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas Tirosina Quinasas Receptoras , Receptor trkA/metabolismo , Células de Schwann/citología , Células Receptoras Sensoriales/citologíaRESUMEN
Deletion of murine Smn exon 7, the most frequent mutation found in spinal muscular atrophy, has been directed to either both satellite cells, the muscle progenitor cells and fused myotubes, or fused myotubes only. When satellite cells were mutated, mutant mice develop severe myopathic process, progressive motor paralysis, and early death at 1 mo of age (severe mutant). Impaired muscle regeneration of severe mutants correlated with defect of myogenic precursor cells both in vitro and in vivo. In contrast, when satellite cells remained intact, mutant mice develop similar myopathic process but exhibit mild phenotype with median survival of 8 mo and motor performance similar to that of controls (mild mutant). High proportion of regenerating myofibers expressing SMN was observed in mild mutants compensating for progressive loss of mature myofibers within the first 6 mo of age. Then, in spite of normal contractile properties of myofibers, mild mutants develop reduction of muscle force and mass. Progressive decline of muscle regeneration process was no more able to counterbalance muscle degeneration leading to dramatic loss of myofibers. These data indicate that intact satellite cells remarkably improve the survival and motor performance of mutant mice suffering from chronic myopathy, and suggest a limited potential of satellite cells to regenerate skeletal muscle.
Asunto(s)
Diferenciación Celular/genética , Músculo Esquelético/crecimiento & desarrollo , Atrofia Muscular Espinal/genética , Proteínas del Tejido Nervioso/deficiencia , Regeneración/genética , Células Satélite del Músculo Esquelético/metabolismo , Animales , Animales Recién Nacidos , Muerte Celular/genética , División Celular/genética , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones , Ratones Mutantes , Trastornos del Movimiento/genética , Trastornos del Movimiento/metabolismo , Trastornos del Movimiento/patología , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patología , Debilidad Muscular/genética , Debilidad Muscular/metabolismo , Debilidad Muscular/patología , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/terapia , Mutación/genética , Necrosis , Proteínas del Tejido Nervioso/genética , Fenotipo , Proteínas de Unión al ARN , Proteínas del Complejo SMN , Células Satélite del Músculo Esquelético/citología , Células Madre/citología , Células Madre/metabolismoRESUMEN
Apical radial glia (aRGs) are predominant progenitors during corticogenesis. Perturbing their function leads to cortical malformations, including subcortical heterotopia (SH), characterized by the presence of neurons below the cortex. EML1/Eml1 mutations lead to SH in patients, as well as to heterotopic cortex (HeCo) mutant mice. In HeCo mice, some aRGs are abnormally positioned away from the ventricular zone (VZ). Thus, unraveling EML1/Eml1 function will clarify mechanisms maintaining aRGs in the VZ. We pinpoint an unknown EML1/Eml1 function in primary cilium formation. In HeCo aRGs, cilia are shorter, less numerous, and often found aberrantly oriented within vesicles. Patient fibroblasts and human cortical progenitors show similar defects. EML1 interacts with RPGRIP1L, a ciliary protein, and RPGRIP1L mutations were revealed in a heterotopia patient. We also identify Golgi apparatus abnormalities in EML1/Eml1 mutant cells, potentially upstream of the cilia phenotype. We thus reveal primary cilia mechanisms impacting aRG dynamics in physiological and pathological conditions.
Asunto(s)
Cilios/genética , Lisencefalias Clásicas y Heterotopias Subcorticales en Banda/genética , Proteínas Asociadas a Microtúbulos/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Adolescente , Animales , Células Cultivadas , Cilios/patología , Lisencefalias Clásicas y Heterotopias Subcorticales en Banda/patología , Femenino , Aparato de Golgi/genética , Aparato de Golgi/patología , Células HEK293 , Humanos , Masculino , Ratones , Mutación , EmbarazoRESUMEN
Vacuolar H+-ATPase-dependent (V-ATPase-dependent) functions are critical for neural proteostasis and are involved in neurodegeneration and brain tumorigenesis. We identified a patient with fulminant neurodegeneration of the developing brain carrying a de novo splice site variant in ATP6AP2 encoding an accessory protein of the V-ATPase. Functional studies of induced pluripotent stem cell-derived (iPSC-derived) neurons from this patient revealed reduced spontaneous activity and severe deficiency in lysosomal acidification and protein degradation leading to neuronal cell death. These deficiencies could be rescued by expression of full-length ATP6AP2. Conditional deletion of Atp6ap2 in developing mouse brain impaired V-ATPase-dependent functions, causing impaired neural stem cell self-renewal, premature neuronal differentiation, and apoptosis resulting in degeneration of nearly the entire cortex. In vitro studies revealed that ATP6AP2 deficiency decreases V-ATPase membrane assembly and increases endosomal-lysosomal fusion. We conclude that ATP6AP2 is a key mediator of V-ATPase-dependent signaling and protein degradation in the developing human central nervous system.
Asunto(s)
Sistema Nervioso Central/fisiopatología , Enfermedades Neurodegenerativas/diagnóstico por imagen , Enfermedades Neurodegenerativas/genética , Células Madre Pluripotentes/metabolismo , Receptores de Superficie Celular/genética , ATPasas de Translocación de Protón Vacuolares/genética , Adolescente , Empalme Alternativo , Animales , Apoptosis , Encéfalo/diagnóstico por imagen , Muerte Celular , Diferenciación Celular , Supervivencia Celular , Preescolar , Eliminación de Gen , Variación Genética , Células HEK293 , Células HeLa , Humanos , Lisosomas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Células-Madre Neurales/metabolismo , Neuronas/metabolismo , ATPasas de Translocación de Protón/genética , ATPasas de Translocación de Protón/fisiología , Receptores de Superficie Celular/fisiología , ATPasas de Translocación de Protón Vacuolares/fisiologíaRESUMEN
Spinal muscular atrophy (SMA) is a common recessive autosomal disorder characterized by degeneration of motor neurons of the spinal cord. SMA is caused by mutations of the survival of motor neuron gene that encodes a multifunctional protein, and mouse models have been generated. These advances represent starting points towards an understanding of the pathophysiology of this disease and the design of therapeutic strategies in SMA.
Asunto(s)
Atrofia Muscular Espinal/genética , Proteínas del Tejido Nervioso/genética , Animales , Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Modelos Animales de Enfermedad , Conversión Génica , Eliminación de Gen , Humanos , Ratones , Atrofia Muscular Espinal/etiología , Atrofia Muscular Espinal/terapia , Proteínas del Tejido Nervioso/fisiología , Fenotipo , Proteínas de Unión al ARN , Proteínas del Complejo SMNRESUMEN
Pyk2 is a Ca2+-activated non-receptor tyrosine kinase enriched in forebrain neurons and involved in synaptic regulation. Human genetic studies associated PTK2B, the gene coding Pyk2, with risk for Alzheimer's disease (AD). We previously showed that Pyk2 is important for hippocampal function, plasticity, and spine structure. However, its potential role in AD is unknown. To address this question we used human brain samples and 5XFAD mice, an amyloid mouse model of AD expressing mutated human amyloid precursor protein and presenilin1. In the hippocampus of 5XFAD mice and in human AD patients' cortex and hippocampus, Pyk2 total levels were normal. However, Pyk2 Tyr-402 phosphorylation levels, reflecting its autophosphorylation-dependent activity, were reduced in 5XFAD mice at 8â¯months of age but not 3â¯months. We crossed these mice with Pyk2-/- mice to generate 5XFAD animals devoid of Pyk2. At 8â¯months the phenotype of 5XFAD x Pyk2-/- double mutant mice was not different from that of 5XFAD. In contrast, overexpression of Pyk2 in the hippocampus of 5XFAD mice, using adeno-associated virus, rescued autophosphorylated Pyk2 levels and improved synaptic markers and performance in several behavioral tasks. Both Pyk2-/- and 5XFAD mice showed an increase of potentially neurotoxic Src cleavage product, which was rescued by Pyk2 overexpression. Manipulating Pyk2 levels had only minor effects on Aß plaques, which were slightly decreased in hippocampus CA3 region of double mutant mice and increased following overexpression. Our results show that Pyk2 is not essential for the pathogenic effects of human amyloidogenic mutations in the 5XFAD mouse model. However, the slight decrease in plaque number observed in these mice in the absence of Pyk2 and their increase following Pyk2 overexpression suggest a contribution of this kinase in plaque formation. Importantly, a decreased function of Pyk2 was observed in 5XFAD mice, indicated by its decreased autophosphorylation and associated Src alterations. Overcoming this deficit by Pyk2 overexpression improved the behavioral and molecular phenotype of 5XFAD mice. Thus, our results in a mouse model of AD suggest that Pyk2 impairment may play a role in the symptoms of the disease.
Asunto(s)
Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/patología , Modelos Animales de Enfermedad , Quinasa 2 de Adhesión Focal/biosíntesis , Regulación Enzimológica de la Expresión Génica , Anciano de 80 o más Años , Enfermedad de Alzheimer/genética , Animales , Corteza Cerebral/enzimología , Corteza Cerebral/patología , Femenino , Quinasa 2 de Adhesión Focal/genética , Regulación Enzimológica de la Expresión Génica/fisiología , Hipocampo/enzimología , Hipocampo/patología , Humanos , Locomoción/fisiología , Masculino , Aprendizaje por Laberinto/fisiología , Ratones , Ratones Transgénicos , Placa Amiloide/enzimología , Placa Amiloide/genética , Placa Amiloide/patologíaRESUMEN
The structure and function of spines and excitatory synapses are under the dynamic control of multiple signalling networks. Although tyrosine phosphorylation is involved, its regulation and importance are not well understood. Here we study the role of Pyk2, a non-receptor calcium-dependent protein-tyrosine kinase highly expressed in the hippocampus. Hippocampal-related learning and CA1 long-term potentiation are severely impaired in Pyk2-deficient mice and are associated with alterations in NMDA receptors, PSD-95 and dendritic spines. In cultured hippocampal neurons, Pyk2 has autophosphorylation-dependent and -independent roles in determining PSD-95 enrichment and spines density. Pyk2 levels are decreased in the hippocampus of individuals with Huntington and in the R6/1 mouse model of the disease. Normalizing Pyk2 levels in the hippocampus of R6/1 mice rescues memory deficits, spines pathology and PSD-95 localization. Our results reveal a role for Pyk2 in spine structure and synaptic function, and suggest that its deficit contributes to Huntington's disease cognitive impairments.
Asunto(s)
Trastornos del Conocimiento/metabolismo , Quinasa 2 de Adhesión Focal/metabolismo , Hipocampo/metabolismo , Enfermedad de Huntington/metabolismo , Sinapsis/metabolismo , Anciano , Alelos , Animales , Conducta Animal , Encéfalo/fisiopatología , Espinas Dendríticas/metabolismo , Potenciales Postsinápticos Excitadores , Femenino , Humanos , Enfermedad de Huntington/genética , Potenciación a Largo Plazo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica , Persona de Mediana Edad , Fenotipo , Fosforilación , Receptores de N-Metil-D-Aspartato/metabolismo , Transducción de SeñalRESUMEN
Prenylated Rab acceptor family, member 2 (PRAF2) is a four transmembrane domain protein of 19 kDa that is highly expressed in particular areas of mammalian brains. PRAF2 is mostly found in the endoplasmic reticulum (ER) of neurons where it plays the role of gatekeeper for the GB1 subunit of the GABAB receptor, preventing its progression in the biosynthetic pathway in the absence of hetero-dimerization with the GB2 subunit. However, PRAF2 can interact with several receptors and immunofluorescence studies indicate that PRAF2 distribution is larger than the ER, suggesting additional biological functions. Here, we conducted an immuno-cytochemical study of PRAF2 distribution in mouse central nervous system (CNS) at anatomical, cellular and ultra-structural levels. PRAF2 appears widely expressed in various regions of mature CNS, such as the olfactory bulbs, cerebral cortex, amygdala, hippocampus, ventral tegmental area and spinal cord. Consistent with its regulatory role of GABAB receptors, PRAF2 was particularly abundant in brain regions known to express GB1 subunits. However, other brain areas where GB1 is expressed, such as basal ganglia, thalamus and hypothalamus, contain little or no PRAF2. In these areas, GB1 subunits might reach the cell surface of neurons independently of GB2 to exert biological functions distinct from those of GABAB receptors, or be regulated by other gatekeepers. Electron microscopy studies confirmed the localization of PRAF2 in the ER, but identified previously unappreciated localizations, in mitochondria, primary cilia and sub-synaptic region. These data indicate additional modes of GABAB regulation in specific brain areas and new biological functions of PRAF2.
Asunto(s)
Encéfalo/metabolismo , Médula Espinal/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animales , Encéfalo/citología , Células HEK293 , Humanos , Ratones , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Neuronas/metabolismo , Neuronas/ultraestructura , Sinapsis/metabolismo , Sinapsis/ultraestructura , Área Tegmental Ventral/metabolismo , Área Tegmental Ventral/ultraestructuraRESUMEN
Myelination allows the fast propagation of action potentials at a low energetic cost. It provides an insulating myelin sheath regularly interrupted at nodes of Ranvier where voltage-gated Na+ channels are concentrated. In the peripheral nervous system, the normal function of myelinated fibers requires the formation of highly differentiated and organized contacts between the myelinating Schwann cells, the axons and the extracellular matrix. Some of the major molecular complexes that underlie these contacts have been identified. Compact myelin which forms the bulk of the myelin sheath results from the fusion of the Schwann cell membranes through the proteins P0, PMP22 and MBP. The basal lamina of myelinating Schwann cells contains laminin-2 which associates with the glial complex dystroglycan/DPR2/L-periaxin. Non compact myelin, found in paranodal loops, periaxonal and abaxonal regions, and Schmidt-Lanterman incisures, presents reflexive adherens junctions, tight junctions and gap junctions, which contain cadherins, claudins and connexins, respectively. Axo-glial contacts determine the formation of distinct domains on the axon, the node, the paranode, and the juxtaparanode. At the paranodes, the glial membrane is tightly attached to the axolemma by septate-like junctions. Paranodal and juxtaparanodal axoglial complexes comprise an axonal transmembrane protein of the NCP family associated in cis and in trans with cell adhesion molecules of the immunoglobulin superfamily (IgSF-CAM). At nodes, axonal complexes are composed of Na+ channels and IgSF-CAMs. Schwann cell microvilli, which loosely cover the node, contain ERM proteins and the proteoglycans syndecan-3 and -4. The fundamental role of the cellular contacts in the normal function of myelinated fibers has been supported by rodent models and the detection of genetic alterations in patients with peripheral demyelinating neuropathies such as Charcot-Marie-Tooth diseases. Understanding more precisely their molecular basis now appears essential as a requisite step to further examine their involvement in the pathogenesis of peripheral neuropathies in general.