RESUMEN
Human peptidylarginine deiminase isoform VI (PAD6), which is predominantly limited to cytoplasmic lattices in the mammalian oocytes in ovarian tissue, is essential for female fertility. It belongs to the peptidylarginine deiminase (PAD) enzyme family that catalyzes the conversion of arginine residues to citrulline in proteins. In contrast to other members of the family, recombinant PAD6 was previously found to be catalytically inactive. We sought to provide structural insight into the human homologue to shed light on this observation. We report here the first crystal structure of PAD6, determined at 1.7â Å resolution. PAD6 follows the same domain organization as other structurally known PAD isoenzymes. Further structural analysis and size-exclusion chromatography show that PAD6 behaves as a homodimer similar to PAD4. Differential scanning fluorimetry suggests that PAD6 does not coordinate Ca2+ which agrees with acidic residues found to coordinate Ca2+ in other PAD homologs not being conserved in PAD6. The crystal structure of PAD6 shows similarities with the inactive state of apo PAD2, in which the active site conformation is unsuitable for catalytic citrullination. The putative active site of PAD6 adopts a non-productive conformation that would not allow protein-substrate binding due to steric hindrance with rigid secondary structure elements. This observation is further supported by the lack of activity on the histone H3 and cytokeratin 5 substrates. These findings suggest a different mechanism for enzymatic activation compared with other PADs; alternatively, PAD6 may exert a non-enzymatic function in the cytoplasmic lattice of oocytes and early embryos.
Asunto(s)
Dominio Catalítico , Arginina Deiminasa Proteína-Tipo 6 , Humanos , Cristalografía por Rayos X , Arginina Deiminasa Proteína-Tipo 6/metabolismo , Desiminasas de la Arginina Proteica/metabolismo , Desiminasas de la Arginina Proteica/química , Desiminasas de la Arginina Proteica/genética , Conformación Proteica , Hidrolasas/química , Hidrolasas/metabolismo , Modelos Moleculares , Calcio/metabolismoRESUMEN
Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion in the huntingtin (HTT) gene that encodes the pathologic mutant HTT (mHTT) protein with an expanded polyglutamine (polyQ) tract. Whereas several therapeutic programs targeting mHTT expression have advanced to clinical evaluation, methods to visualize mHTT protein species in the living brain are lacking. Here, we demonstrate the development and characterization of a positron emission tomography (PET) imaging radioligand with high affinity and selectivity for mHTT aggregates. This small molecule radiolabeled with 11C ([11C]CHDI-180R) allowed noninvasive monitoring of mHTT pathology in the brain and could track region- and time-dependent suppression of mHTT in response to therapeutic interventions targeting mHTT expression in a rodent model. We further showed that in these animals, therapeutic agents that lowered mHTT in the striatum had a functional restorative effect that could be measured by preservation of striatal imaging markers, enabling a translational path to assess the functional effect of mHTT lowering.
Asunto(s)
Enfermedad de Huntington , Enfermedades Neurodegenerativas , Animales , Cuerpo Estriado/diagnóstico por imagen , Cuerpo Estriado/metabolismo , Modelos Animales de Enfermedad , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Enfermedad de Huntington/diagnóstico por imagen , Enfermedad de Huntington/genética , Enfermedad de Huntington/metabolismo , Ligandos , Enfermedades Neurodegenerativas/patologíaRESUMEN
Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion in the huntingtin gene (HTT), which codes for the pathologic mutant HTT (mHTT) protein. Since normal HTT is thought to be important for brain function, we engineered zinc finger protein transcription factors (ZFP-TFs) to target the pathogenic CAG repeat and selectively lower mHTT as a therapeutic strategy. Using patient-derived fibroblasts and neurons, we demonstrate that ZFP-TFs selectively repress >99% of HD-causing alleles over a wide dose range while preserving expression of >86% of normal alleles. Other CAG-containing genes are minimally affected, and virally delivered ZFP-TFs are active and well tolerated in HD neurons beyond 100 days in culture and for at least nine months in the mouse brain. Using three HD mouse models, we demonstrate improvements in a range of molecular, histopathological, electrophysiological and functional endpoints. Our findings support the continued development of an allele-selective ZFP-TF for the treatment of HD.
Asunto(s)
Alelos , Proteína Huntingtina/genética , Enfermedad de Huntington/terapia , Mutación , Transcripción Genética , Dedos de Zinc , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Humanos , Enfermedad de Huntington/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Neuroprotección , Repeticiones de TrinucleótidosRESUMEN
Several studies have indicated that inflammation during pregnancy increases the risk for the development of neuropsychiatric disorders in the offspring. Morphological brain abnormalities combined with deviations in the inflammatory status of the brain can be observed in patients of both autism and schizophrenia. It was shown that acute infection can induce changes in maternal cytokine levels which in turn are suggested to affect fetal brain development and increase the risk on the development of neuropsychiatric disorders in the offspring. Animal models of maternal immune activation reproduce the etiology of neurodevelopmental disorders such as schizophrenia and autism. In this study the poly (I:C) model was used to mimic viral immune activation in pregnant mice in order to assess the activation status of fetal microglia in these developmental disorders. Because microglia are the resident immune cells of the brain they were expected to be activated due to the inflammatory stimulus. Microglial cell density and activation level in the fetal cortex and hippocampus were determined. Despite the presence of a systemic inflammation in the pregnant mice, there was no significant difference in fetal microglial cell density or immunohistochemically determined activation level between the control and inflammation group. These data indicate that activation of the fetal microglial cells is not likely to be responsible for the inflammation induced deficits in the offspring in this model.
RESUMEN
ΒACE1 is the major drug target for Alzheimer's disease, but we know surprisingly little about its normal function in the CNS. Here, we show that this protease is critically involved in semaphorin 3A (Sema3A)-mediated axonal guidance processes in thalamic and hippocampal neurons. An active membrane-bound proteolytic CHL1 fragment is generated by BACE1 upon Sema3A binding. This fragment relays the Sema3A signal via ezrin-radixin-moesin (ERM) proteins to the neuronal cytoskeleton. APH1B-γ-secretase-mediated degradation of this fragment stops the Sema3A-induced collapse and sensitizes the growth cone for the next axonal guidance cue. Thus, we reveal a cycle of proteolytic activity underlying growth cone collapse and restoration used by axons to find their correct trajectory in the brain. Our data also suggest that BACE1 and γ-secretase inhibition have physiologically opposite effects in this process, supporting the idea that combination therapy might attenuate some of the side effects associated with these drugs.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Endopeptidasas/metabolismo , Conos de Crecimiento/metabolismo , Secretasas de la Proteína Precursora del Amiloide/genética , Animales , Ácido Aspártico Endopeptidasas/genética , Células Cultivadas , Endopeptidasas/genética , Hipocampo/citología , Hipocampo/metabolismo , Proteínas de la Membrana , Ratones , Proteolisis , Semaforina-3A/genética , Semaforina-3A/metabolismoRESUMEN
The brain's impotence to utilize long-chain fatty acids as fuel, one of the dogmas in neuroscience, is surprising, since the nervous system is the tissue most energy consuming and most vulnerable to a lack of energy. Challenging this view, we here show in vivo that loss of the Drosophila carnitine palmitoyltransferase 2 (CPT2), an enzyme required for mitochondrial ß-oxidation of long-chain fatty acids as substrates for energy production, results in the accumulation of triacylglyceride-filled lipid droplets in adult Drosophila brain but not in obesity. CPT2 rescue in glial cells alone is sufficient to restore triacylglyceride homeostasis, and we suggest that this is mediated by the release of ketone bodies from the rescued glial cells. These results demonstrate that the adult brain is able to catabolize fatty acids for cellular energy production.
Asunto(s)
Drosophila melanogaster/metabolismo , Metabolismo Energético , Neuroglía/metabolismo , Envejecimiento/metabolismo , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Encéfalo/ultraestructura , Carnitina O-Palmitoiltransferasa/química , Carnitina O-Palmitoiltransferasa/deficiencia , Carnitina O-Palmitoiltransferasa/genética , Carnitina O-Palmitoiltransferasa/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Drosophila melanogaster/genética , Homeostasis , Humanos , Larva/metabolismo , Gotas Lipídicas/metabolismo , Metabolismo de los Lípidos , Datos de Secuencia Molecular , Mutación , Obesidad/patología , Oxidación-Reducción , Fosfolípidos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de Secuencia , Análisis de Supervivencia , Triglicéridos/metabolismoRESUMEN
A compelling amount of data is accumulating about the polyphonic role of neuronal cadherins during brain development throughout all developmental stages, starting from the involvement of cadherins in the organization of neurulation up to synapse development and plasticity. Recent work has confirmed that specifically N-cadherins play an important role in asymmetrical cellular processes in developing neurons that are at the basis of polarity. In this review we will summarize recent data, which demonstrate how N-cadherin orchestrates distinct processes of polarity establishment in neurons.
Asunto(s)
Axones/fisiología , Cadherinas/fisiología , Polaridad Celular/fisiología , Animales , Adhesión Celular , Movimiento Celular , Humanos , Ratones , Neurogénesis , Neurulación , Ratas , Transducción de SeñalRESUMEN
Deficiencies in fragile X mental retardation protein (FMRP) are the most common cause of inherited intellectual disability, fragile X syndrome (FXS), with symptoms manifesting during infancy and early childhood. Using a mouse model for FXS, we found that Fmrp regulates the positioning of neurons in the cortical plate during embryonic development, affecting their multipolar-to-bipolar transition (MBT). We identified N-cadherin, which is crucial for MBT, as an Fmrp-regulated target in embryonic brain. Furthermore, spontaneous network activity and high-resolution brain imaging revealed defects in the establishment of neuronal networks at very early developmental stages, further confirmed by an unbalanced excitatory and inhibitory network. Finally, reintroduction of Fmrp or N-cadherin in the embryo normalized early postnatal neuron activity. Our findings highlight the critical role of Fmrp in the developing cerebral cortex and might explain some of the clinical features observed in patients with FXS, such as alterations in synaptic communication and neuronal network connectivity.
Asunto(s)
Movimiento Celular/fisiología , Polaridad Celular/fisiología , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/fisiología , Red Nerviosa/fisiología , Neuronas/fisiología , Corteza Somatosensorial/fisiología , Animales , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Red Nerviosa/citología , Técnicas de Cultivo de Órganos , Embarazo , Corteza Somatosensorial/citologíaRESUMEN
Final morphological polarization of neurons, with the development of a distinct axon and several dendrites, is preceded by phases where they have a non-polarized architecture. The earliest of these phases is that of the round neuron arising from the last mitosis. A second non-polarized stage corresponds to the bipolar neuron, with two morphologically identical neurites. Both phases have their distinctive relevance in the establishment of neuronal polarity. During the round cell stage, a decision is made as to where from the cell periphery a first neurite will form, thus creating the first sign of asymmetry. At the bipolar stage a decision is made as to which of the two neurites becomes the axon in neurons polarizing in vitro, and the leading edge in neurons in situ. In this study, we analysed cytoskeletal and membrane dynamics in cells at these two 'pre-polarity' stages. By means of time lapse imaging in dissociated hippocampal neurons and ex vivo cortical slices, we show that both stages are characterized by polarized intracellular arrangements. However, the stages have distinct temporal hierarchies: polarized actin dynamics marks the site of first polarization in round cells, whereas polarized membrane dynamics precedes asymmetric growth in the bipolar stage.
Asunto(s)
Actinas/metabolismo , Membrana Celular/metabolismo , Polaridad Celular , Citoesqueleto/metabolismo , Hipocampo/crecimiento & desarrollo , Neurogénesis , Neuronas/fisiología , Animales , Células Cultivadas , Femenino , Hipocampo/citología , Ratones , Técnicas de Cultivo de Órganos , Embarazo , Transporte de Proteínas , Ratas , Ratas Endogámicas , Imagen de Lapso de TiempoRESUMEN
Neuregulin 1 (NRG1) and the γ-secretase subunit APH1B have been previously implicated as genetic risk factors for schizophrenia and schizophrenia relevant deficits have been observed in rodent models with loss of function mutations in either gene. Here we show that the Aph1b-γ-secretase is selectively involved in Nrg1 intracellular signalling. We found that Aph1b-deficient mice display a decrease in excitatory synaptic markers. Electrophysiological recordings show that Aph1b is required for excitatory synaptic transmission and plasticity. Furthermore, gain and loss of function and genetic rescue experiments indicate that Nrg1 intracellular signalling promotes dendritic spine formation downstream of Aph1b-γ-secretase in vitro and in vivo. In conclusion, our study sheds light on the physiological role of Aph1b-γ-secretase in brain and provides a new mechanistic perspective on the relevance of NRG1 processing in schizophrenia.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Endopeptidasas/metabolismo , Regulación de la Expresión Génica , Hipocampo/embriología , Neurregulina-1/metabolismo , Enfermedad de Alzheimer/genética , Animales , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Electrofisiología , Eliminación de Gen , Hipocampo/metabolismo , Proteínas de la Membrana , Ratones , Ratones Transgénicos , Mutación , Neuronas/metabolismo , Técnicas de Placa-Clamp , Esquizofrenia/metabolismo , Transducción de Señal , Sinapsis/metabolismoRESUMEN
In neuronal cells, actin remodeling plays a well known role in neurite extension but is also deeply involved in the organization of intracellular structures, such as the Golgi apparatus. However, it is still not very clear which mechanisms may regulate actin dynamics at the different sites. In this report we show that high levels of the TTC3 protein, encoded by one of the genes of the Down Syndrome Critical Region (DCR), prevent neurite extension and disrupt Golgi compactness in differentiating primary neurons. These effects largely depend on the capability of TTC3 to promote actin polymerization through signaling pathways involving RhoA, ROCK, CIT-N and PIIa. However, the functional relationships between these molecules differ significantly if considering the TTC3 activity on neurite extension or on Golgi organization. Finally, our results reveal an unexpected stage-dependent requirement for F-actin in Golgi organization at different stages of neuronal differentiation.
Asunto(s)
Actinas/metabolismo , Diferenciación Celular/fisiología , Aparato de Golgi/metabolismo , Neuronas/metabolismo , Transducción de Señal/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Células Cultivadas , Hipocampo/metabolismo , Neuritas/metabolismo , Ratas , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
The main role of Telomerase Reverse Transcriptase (TERT) is to protect telomere length from shortening during cell division. However, recent works have revealed the existence of a pool of TERT associated to mitochondria, where it plays a role in survival. We here show that in fully differentiated neurons the largest pool of cytoplasmic TERT associates to TIA1 positive RNA granules, where it binds the messenger RNA of the cyclin kinase inhibitor p15INK4B. Upon stress, p15INK4B and TERT dissociate and p15INK4B undergoes efficient translation, allowing its pro-survival function. These results unveil another mechanism implicated in the survival of fully differentiated neurons.
Asunto(s)
Ciclo Celular/fisiología , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/fisiología , Neuronas/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , ARN/metabolismo , Telomerasa/metabolismo , Animales , Ratones , Ratones Endogámicos C57BL , Ratas , Ratas WistarRESUMEN
The establishment of neuronal connectivity depends on the correct initial polarization of the young neurons. In vivo, developing neurons sense a multitude of inputs and a great number of molecules are described that affect their outgrowth. In vitro, many studies have shown the possibility to influence neuronal morphology and growth by biophysical, i.e. topographic, signaling. In this work we have taken this approach one step further and investigated the impact of substrate topography in the very early differentiation stages of developing neurons, i.e. when the cell is still at the round stage and when the first neurite is forming. For this purpose we fabricated micron sized pillar structures with highly reproducible feature sizes, and analyzed neurons on the interface of flat and topographic surfaces. We found that topographic signaling was able to attract the polarization markers of mouse embryonic neurons -N-cadherin, Golgi-centrosome complex and the first bud were oriented towards topographic stimuli. Consecutively, the axon was also preferentially extending along the pillars. These events seemed to occur regardless of pillar dimensions in the range we examined. However, we found differences in neurite length that depended on pillar dimensions. This study is one of the first to describe in detail the very early response of hippocampal neurons to topographic stimuli.
Asunto(s)
Polaridad Celular , Neuronas/citología , Neuronas/fisiología , Animales , Axones/metabolismo , Cadherinas/metabolismo , Diferenciación Celular , Células Cultivadas , Centrosoma/metabolismo , Aparato de Golgi/metabolismo , Conos de Crecimiento/metabolismo , Ratones , Neuritas/metabolismo , Fosforilación , Transporte de Proteínas , Ratas , Transducción de Señal , Tirosina/metabolismoRESUMEN
Expression of amyloid precursor protein (APP) and its two paralogues, APLP1 and APLP2 during brain development coincides with key cellular events such as neuronal differentiation and migration. However, genetic knockout and shRNA studies have led to contradictory conclusions about their role during embryonic brain development. To address this issue, we analysed in depth the role of APLP2 during neurogenesis by silencing APLP2 in vivo in an APP/APLP1 double knockout mouse background. We find that under these conditions cortical progenitors remain in their undifferentiated state much longer, displaying a higher number of mitotic cells. In addition, we show that neuron-specific APLP2 downregulation does not impact the speed or position of migrating excitatory cortical neurons. In summary, our data reveal that APLP2 is specifically required for proper cell cycle exit of neuronal progenitors, and thus has a distinct role in priming cortical progenitors for neuronal differentiation.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animales , Ciclo Celular , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Movimiento Celular , Células Cultivadas , Electroporación , Femenino , Inmunohistoquímica , Ratones , Ratones Noqueados , Ratones Transgénicos , EmbarazoRESUMEN
BACKGROUND INFORMATION: PIX proteins are exchange factors for Rac and Cdc42 GTPases that are differentially expressed in the brain, where they are implicated in neuronal morphogenesis. The PIX family includes the two members αPIX and ßPIX, and the gene of αPIX is mutated in patients with intellectual disability. RESULTS: We have analysed the expression of PIX proteins in the developing brain and addressed their role during early hippocampal neuron development. Mass spectrometry identified several ßPIX isoforms and a major p75 αPIX isoform in brain and hippocampal cultures. PIX proteins expression increased with time during neuronal differentiation in vitro. The PIX partners GIT1 and GIT2 are also found in brain and their expression was increased during neuronal differentiation. We found that αPIX, but not ßPIX, was required for proper hippocampal neuron differentiation, since silencing of αPIX specifically hampered dendritogenesis and axonal branching. Interestingly, the depletion of GIT2 but not GIT1 mimicked the phenotype observed after αPIX knock-down. Over-expression of αPIX specifically enhanced dendritic branching, while both αPIX and ßPIX over-expression affected axonal morphology. Again, only over-expression of GIT2, but not GIT1, affected neuritic morphology. CONCLUSIONS: The results indicate that αPIX and GIT2 are required for neuronal differentiation, and suggest that they are part of the same pathway, while GIT1 and ßPIX are dispensable for early hippocampal neurons development.
Asunto(s)
Axones/metabolismo , Dendritas/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Hipocampo/citología , Animales , Diferenciación Celular , Células Cultivadas , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Hipocampo/metabolismo , Ratones , Neuronas/citología , Neuronas/metabolismo , Ratas , Factores de Intercambio de Guanina Nucleótido RhoRESUMEN
The precise polarization and orientation of developing neurons is essential for the correct wiring of the brain. In pyramidal excitatory neurons, polarization begins with the sprouting of opposite neurites, which later define directed migration and axo-dendritic domains. We here show that endogenous N-cadherin concentrates at one pole of the newborn neuron, from where the first neurite subsequently emerges. Ectopic N-cadherin is sufficient to favour the place of appearance of the first neurite. The Golgi and centrosome move towards this newly formed morphological pole in a second step, which is regulated by PI3K and the actin/microtubule cytoskeleton. Moreover, loss of function experiments in vivo showed that developing neurons with a non-functional N-cadherin misorient their cell axis. These results show that polarization of N-cadherin in the immediate post-mitotic stage is an early and crucial mechanism in neuronal polarity.
Asunto(s)
Cadherinas/metabolismo , División Celular , Polaridad Celular , Proteínas del Tejido Nervioso/metabolismo , Neuronas/fisiología , Animales , Centrosoma/metabolismo , Citoesqueleto/metabolismo , Aparato de Golgi/metabolismo , Neuritas/fisiología , Fosfatidilinositol 3-Quinasa/metabolismo , RatasRESUMEN
Early in vitro and recent in vivo studies demonstrated that neuronal polarization occurs by the sequential formation of two oppositely located neurites. This early bipolar phenotype is of crucial relevance in brain organization, determining neuronal migration and brain layering. It is currently considered that the place of formation of the first neurite is dictated by extrinsic cues, through the induction of localized changes in membrane and cytoskeleton dynamics leading to deformation of the cells' curvature followed by the growth of a cylindrical extension (neurite). It is unknown if the appearance of the second neurite at the opposite pole, thus the formation of a bipolar cell axis and capacity to undergo migration, is defined by the growth at the first place, therefore intrinsic, or requires external determinants. We addressed this question by using a mathematical model based on the induction of dynamic changes in one pole of a round cell. The model anticipates that a second area of growth can spontaneously form at the opposite pole. Hence, through mathematical modeling we prove that neuronal bipolar axis of growth can be due to an intrinsic mechanism.