RESUMO
Maple syrup urine disease (MSUD) is rare autosomal recessive metabolic disorder caused by the dysfunction of the mitochondrial branched-chain 2-ketoacid dehydrogenase (BCKD) enzyme complex leading to massive accumulation of branched-chain amino acids and 2-keto acids. MSUD management, based on a life-long strict protein restriction with nontoxic amino acids oral supplementation represents an unmet need as it is associated with a poor quality of life, and does not fully protect from acute life-threatening decompensations or long-term neuropsychiatric complications. Orthotopic liver transplantation is a beneficial therapeutic option, which shows that restoration of only a fraction of whole-body BCKD enzyme activity is therapeutic. MSUD is thus an ideal target for gene therapy. We and others have tested AAV gene therapy in mice for two of the three genes involved in MSUD, BCKDHA and DBT. In this study, we developed a similar approach for the third MSUD gene, BCKDHB. We performed the first characterization of a Bckdhb-/- mouse model, which recapitulates the severe human phenotype of MSUD with early-neonatal symptoms leading to death during the first week of life with massive accumulation of MSUD biomarkers. Based on our previous experience in Bckdha-/- mice, we designed a transgene carrying the human BCKDHB gene under the control of a ubiquitous EF1α promoter, encapsidated in an AAV8 capsid. Injection in neonatal Bckdhb-/- mice at 1014 vg/kg achieved long-term rescue of the severe MSUD phenotype of Bckdhb-/- mice. These data further validate the efficacy of gene therapy for MSUD opening perspectives towards clinical translation.
Assuntos
Doença da Urina de Xarope de Bordo , Animais , Humanos , Camundongos , 3-Metil-2-Oxobutanoato Desidrogenase (Lipoamida)/química , 3-Metil-2-Oxobutanoato Desidrogenase (Lipoamida)/genética , 3-Metil-2-Oxobutanoato Desidrogenase (Lipoamida)/metabolismo , Aminoácidos de Cadeia Ramificada/metabolismo , Doença da Urina de Xarope de Bordo/genética , Doença da Urina de Xarope de Bordo/terapia , Doença da Urina de Xarope de Bordo/diagnóstico , Fenótipo , Qualidade de VidaRESUMO
Excitation-contraction coupling requires a highly specialized membrane structure, the triad, composed of a plasma membrane invagination, the T-tubule, surrounded by two sarcoplasmic reticulum terminal cisternae. Although the precise mechanisms governing T-tubule biogenesis and triad formation remain largely unknown, studies have shown that caveolae participate in T-tubule formation and mutations of several of their constituents induce muscle weakness and myopathies. Here, we demonstrate that, at the plasma membrane, Bin1 and caveolae composed of caveolin-3 assemble into ring-like structures from which emerge tubes enriched in the dihydropyridine receptor. Bin1 expression lead to the formation of both rings and tubes and we show that Bin1 forms scaffolds on which caveolae accumulate to form the initial T-tubule. Cav3 deficiency caused by either gene silencing or pathogenic mutations results in defective ring formation and perturbed Bin1-mediated tubulation that may explain defective T-tubule organization in mature muscles. Our results uncover new pathophysiological mechanisms that may prove relevant to myopathies caused by Cav3 or Bin1 dysfunction.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Cavéolas , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Canais de Cálcio Tipo L/metabolismo , Cavéolas/metabolismo , Membrana Celular/metabolismo , Retículo Sarcoplasmático/metabolismo , Animais , CamundongosRESUMO
Chromosomal aberrations including structural variations (SVs) are a major cause of human genetic diseases. Their detection in clinical routine still relies on standard cytogenetics. Drawbacks of these tests are a very low resolution (karyotyping) and the inability to detect balanced SVs or indicate the genomic localization and orientation of duplicated segments or insertions (copy number variant [CNV] microarrays). Here, we investigated the ability of optical genome mapping (OGM) to detect known constitutional chromosomal aberrations. Ultra-high-molecular-weight DNA was isolated from 85 blood or cultured cells and processed via OGM. A de novo genome assembly was performed followed by structural variant and CNV calling and annotation, and results were compared to known aberrations from standard-of-care tests (karyotype, FISH, and/or CNV microarray). In total, we analyzed 99 chromosomal aberrations, including seven aneuploidies, 19 deletions, 20 duplications, 34 translocations, six inversions, two insertions, six isochromosomes, one ring chromosome, and four complex rearrangements. Several of these variants encompass complex regions of the human genome involved in repeat-mediated microdeletion/microduplication syndromes. High-resolution OGM reached 100% concordance compared to standard assays for all aberrations with non-centromeric breakpoints. This proof-of-principle study demonstrates the ability of OGM to detect nearly all types of chromosomal aberrations. We also suggest suited filtering strategies to prioritize clinically relevant aberrations and discuss future improvements. These results highlight the potential for OGM to provide a cost-effective and easy-to-use alternative that would allow comprehensive detection of chromosomal aberrations and structural variants, which could give rise to an era of "next-generation cytogenetics."
Assuntos
Aberrações Cromossômicas , Transtornos Cromossômicos/diagnóstico , Mapeamento Cromossômico/métodos , Análise Citogenética/métodos , Variações do Número de Cópias de DNA , Genoma Humano , Análise em Microsséries/métodos , Transtornos Cromossômicos/genética , Humanos , CariotipagemRESUMO
The regulated transport of AMPA-type glutamate receptors (AMPARs) to the synaptic membrane is a key mechanism to determine the strength of excitatory synaptic transmission in the brain. In this work, we uncovered a new role for the microtubule-associated protein MAP1B in modulating access of AMPARs to the postsynaptic membrane. Using mice and rats of either sex, we show that MAP1B light chain (LC) accumulates in the somatodendritic compartment of hippocampal neurons, where it forms immobile complexes on microtubules that limit vesicular transport. These complexes restrict AMPAR dendritic mobility, leading to the intracellular trapping of receptors and impairing their access to the dendritic surface and spines. Accordingly, increasing MAP1B-LC expression depresses AMPAR-mediated synaptic transmission. This effect is specific for the GluA2 subunit of the AMPAR and requires glutamate receptor interacting protein 1 (GRIP1) interaction with MAP1B-LC. Therefore, MAP1B-LC represents an alternative link between GRIP1-AMPARs and microtubules that does not result in productive transport, but rather limits AMPAR availability for synaptic insertion, with a direct impact on synaptic transmission.SIGNIFICANCE STATEMENT The ability of neurons to modify their synaptic connections, known as synaptic plasticity, is accepted as the cellular basis for learning and memory. One mechanism for synaptic plasticity is the regulated addition and removal of AMPA-type glutamate receptors (AMPARs) at excitatory synapses. In this study, we found that a microtubule-associated protein, MAP1B light chain (MAP1B-LC), participates in this process. MAP1B-LC forms immobile complexes along dendrites. These complexes limit intracellular vesicular trafficking and trap AMPARs inside the dendritic shaft. In this manner, MAP1B restricts the access of AMPARs to dendritic spines and the postsynaptic membrane, contributing to downregulating synaptic transmission.
Assuntos
Proteínas Associadas aos Microtúbulos/fisiologia , Receptores de AMPA/fisiologia , Sinapses/fisiologia , Transmissão Sináptica/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Dendritos/efeitos dos fármacos , Espinhas Dendríticas/fisiologia , Feminino , Hipocampo/citologia , Hipocampo/metabolismo , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas Associadas aos Microtúbulos/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Ratos , Ratos Wistar , Receptores de AMPA/metabolismoRESUMO
Dendritic spines are mushroom-shaped protrusions of the postsynaptic membrane. Spines receive the majority of glutamatergic synaptic inputs. Their morphology, dynamics, and density have been related to synaptic plasticity and learning. The main determinant of spine shape is filamentous actin. Using FRAP, we have reexamined the actin dynamics of individual spines from pyramidal hippocampal neurons, both in cultures and in hippocampal organotypic slices. Our results indicate that, in cultures, the actin mobile fraction is independently regulated at the individual spine level, and mobile fraction values do not correlate with either age or distance from the soma. The most significant factor regulating actin mobile fraction was the presence of astrocytes in the culture substrate. Spines from neurons growing in the virtual absence of astrocytes have a more stable actin cytoskeleton, while spines from neurons growing in close contact with astrocytes show a more dynamic cytoskeleton. According to their recovery time, spines were distributed into two populations with slower and faster recovery times, while spines from slice cultures were grouped into one population. Finally, employing fast lineal acquisition protocols, we confirmed the existence of loci with high polymerization rates within the spine.
Assuntos
Actinas/metabolismo , Espinhas Dendríticas/metabolismo , Hipocampo/metabolismo , Polimerização , Animais , Animais Recém-Nascidos , Células Cultivadas , Hipocampo/citologia , Técnicas de Cultura de Órgãos , RatosRESUMO
Phosphoinositide 3-kinase (PI3K) and PTEN have been shown to participate in synaptic plasticity during long-term potentiation (LTP) and long-term depression (LTD), respectively. Nevertheless, the dynamics of phosphatidylinositol-(3,4,5)-trisphosphate (PIP3) and the regulation of its synthesis and degradation at synaptic compartments is far from clear. Here, we have used fluorescence resonance energy transfer (FRET) imaging to monitor changes in PIP3 levels in dendritic spines from CA1 hippocampal neurons under basal conditions and upon induction of NMDA receptor (NMDAR)-dependent LTD and LTP. We found that PIP3 undergoes constant turnover in dendritic spines. Contrary to expectations, both LTD and LTP induction trigger an increase in PIP3 synthesis, which requires NMDARs and PI3K activity. Using biochemical methods, the upregulation of PIP3 levels during LTP was estimated to be twofold. However, in the case of LTD, PTEN activity counteracts the increase in PIP3 synthesis, resulting in no net change in PIP3 levels. Therefore, both LTP and LTD signaling converge towards PIP3 upregulation, but PTEN acts as an LTD-selective switch that determines the outcome of PIP3 accumulation.
Assuntos
Espinhas Dendríticas/metabolismo , Depressão Sináptica de Longo Prazo , PTEN Fosfo-Hidrolase/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Regulação para Cima , Animais , Transferência Ressonante de Energia de Fluorescência , Potenciação de Longa Duração , PTEN Fosfo-Hidrolase/antagonistas & inibidores , Fosfatidilinositol 3-Quinases/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacosRESUMO
Ghrelin is a peptide mainly produced by the stomach and released into circulation, affecting energy balance and growth hormone release. These effects are guided largely by the expression of the ghrelin receptor growth hormone secretagogue type 1a (GHS-R1a) in the hypothalamus and pituitary. However, GHS-R1a is expressed in other brain regions, including the hippocampus, where its activation enhances memory retention. Herein we explore the molecular mechanism underlying the action of ghrelin on hippocampal-dependent memory. Our data show that GHS-R1a is localized in the vicinity of hippocampal excitatory synapses, and that its activation increases delivery of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic-type receptors (AMPARs) to synapses, producing functional modifications at excitatory synapses. Moreover, GHS-R1a activation enhances two different paradigms of long-term potentiation in the hippocampus, activates the phosphatidylinositol 3-kinase, and increases GluA1 AMPAR subunit and stargazin phosphorylation. We propose that GHS-R1a activation in the hippocampus enhances excitatory synaptic transmission and synaptic plasticity by regulating AMPAR trafficking. Our study provides insights into mechanisms that may mediate the cognition-enhancing effect of ghrelin, and suggests a possible link between the regulation of energy metabolism and learning.
Assuntos
Regulação da Expressão Gênica , Grelina/fisiologia , Hipocampo/metabolismo , Receptores de AMPA/metabolismo , Sinapses/fisiologia , Animais , Eletrofisiologia , Metabolismo Energético , Grelina/metabolismo , Aprendizagem , Potenciação de Longa Duração/fisiologia , Memória , Peptídeos/química , Fosforilação , Ratos , Transdução de Sinais , Transmissão SinápticaRESUMO
The microtubule-associated protein 1B (MAP1B) plays critical roles in neurite growth and synapse maturation during brain development. This protein is well expressed in the adult brain. However, its function in mature neurons remains unknown. We have used a genetically modified mouse model and shRNA techniques to assess the role of MAP1B at established synapses, bypassing MAP1B functions during neuronal development. Under these conditions, we found that MAP1B deficiency alters synaptic plasticity by specifically impairing long-term depression (LTD) expression. Interestingly, this is due to a failure to trigger AMPA receptor endocytosis and spine shrinkage during LTD. These defects are accompanied by an impaired targeting of the Rac1 activator Tiam1 at synaptic compartments. Accordingly, LTD and AMPA receptor endocytosis are restored in MAP1B-deficient neurons by providing additional Rac1. Therefore, these results indicate that the MAP1B-Tiam1-Rac1 relay is essential for spine structural plasticity and removal of AMPA receptors from synapses during LTD. This work highlights the importance of MAPs as signalling hubs controlling the actin cytoskeleton and receptor trafficking during plasticity in mature neurons.
Assuntos
Endocitose/fisiologia , Depressão Sináptica de Longo Prazo/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Plasticidade Neuronal/fisiologia , Receptores de AMPA/metabolismo , Sinapses/fisiologia , Animais , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Hipocampo/citologia , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/deficiência , Neuropeptídeos , Técnicas de Patch-Clamp , RNA Interferente Pequeno/genética , Coluna Vertebral/citologia , Estatísticas não Paramétricas , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T , Proteínas rac1 de Ligação ao GTPRESUMO
Microtubule-associated protein 1B (MAP1B) is prominently expressed during early stages of neuronal development, and it has been implicated in axonal growth and guidance. MAP1B expression is also found in the adult brain in areas of significant synaptic plasticity. Here, we demonstrate that MAP1B is present in dendritic spines, and we describe a decrease in the density of mature dendritic spines in neurons of MAP1B-deficient mice that was accompanied by an increase in the number of immature filopodia-like protrusions. Although these neurons exhibited normal passive membrane properties and action potential firing, AMPA receptor-mediated synaptic currents were significantly diminished. Moreover, we observed a significant decrease in Rac1 activity and an increase in RhoA activity in the post-synaptic densities of adult MAP1B(+/-) mice when compared with wild type controls. MAP1B(+/-) fractions also exhibited a decrease in phosphorylated cofilin. Taken together, these results indicate a new and important role for MAP1B in the formation and maturation of dendritic spines, possibly through the regulation of the actin cytoskeleton. This activity of MAP1B could contribute to the regulation of synaptic activity and plasticity in the adult brain.
Assuntos
Espinhas Dendríticas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Sinapses/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Camundongos , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is an important regulator of phosphatidylinositol-(3,4,5,)-trisphosphate signalling, which controls cell growth and differentiation. However, PTEN is also highly expressed in the adult brain, in which it can be found in dendritic spines in hippocampus and other brain regions. Here, we have investigated specific functions of PTEN in the regulation of synaptic function in excitatory hippocampal synapses. We found that NMDA receptor activation triggers a PDZ-dependent association between PTEN and the synaptic scaffolding molecule PSD-95. This association is accompanied by PTEN localization at the postsynaptic density and anchoring within the spine. On the other hand, enhancement of PTEN lipid phosphatase activity is able to drive depression of AMPA receptor-mediated synaptic responses. This activity is specifically required for NMDA receptor-dependent long-term depression (LTD), but not for LTP or metabotropic glutamate receptor-dependent LTD. Therefore, these results reveal PTEN as a regulated signalling molecule at the synapse, which is recruited to the postsynaptic membrane upon NMDA receptor activation, and is required for the modulation of synaptic activity during plasticity.
Assuntos
Depressão Sináptica de Longo Prazo , PTEN Fosfo-Hidrolase/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/metabolismo , Animais , Proteína 4 Homóloga a Disks-Large , Hipocampo/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Domínios PDZ , PTEN Fosfo-Hidrolase/análise , Ratos , Receptores de AMPA/metabolismo , Coluna Vertebral/ultraestrutura , Transmissão SinápticaRESUMO
Members of the striatin family are scaffolding proteins involved in numerous signaling pathways principally in neurons. Zinedin is the only member of this protein family for which the brain distribution has not been determined so far. Here, we have validated a specific antibody against zinedin and used this tool to study the localization of zinedin at cellular and sub-cellular levels in the rat brain. Zinedin is primarily expressed in neurons of the hippocampus, cerebral cortex, olfactory bulb and caudate putamen nucleus. Like other members of the striatin family, zinedin displays a polarized distribution in the somato-dendritic compartment of neurons and is enriched in dendritic spines. The rostral expression of zinedin as well as its compartmented distribution in dendritic spines may have important implications not only for zinedin function but also in the physiology of dendritic spines of a particular subset of neurons.
Assuntos
Encéfalo/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Animais , Autoantígenos/metabolismo , Encéfalo/citologia , Proteínas de Ligação a Calmodulina/genética , Linhagem Celular Transformada , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/ultraestrutura , Expressão Gênica/fisiologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microscopia Imunoeletrônica/métodos , Proteínas do Tecido Nervoso/genética , Neurônios/ultraestrutura , Ratos , Transfecção/métodosRESUMO
Striatin, SG2NA and zinedin, the three mammalian members of the striatin family are multimodular WD-repeat, calmodulin and calveolin-binding proteins. These scaffolding proteins, involved in both signaling and trafficking, are highly expressed in neurons. Using ultrastructural immunolabeling, we showed that, in Purkinje cells and hippocampal neurons, SG2NA is confined to the somatodendritic compartment with the highest density in dendritic spines. In cultured hippocampal neurons, SG2NA is also highly concentrated in dendritic spines. By expressing truncated forms of HA-tagged SG2NAbeta, we demonstrated that the coiled-coil domain plays an essential role in the targeting of SG2NA within spines. Furthermore, co-immunoprecipitation experiments indicate that this coiled-coil domain is also crucial for the homo- and hetero-oligomerization of these proteins. Thus, oligomerization of the striatin family proteins is probably an obligatory step for their routing to the dendritic spines, and hetero-oligomerization explains why all these proteins are often co-expressed in the neurons of the rat brain and spinal cord.
Assuntos
Autoantígenos/biossíntese , Proteínas de Ligação a Calmodulina/biossíntese , Espinhas Dendríticas/metabolismo , Proteínas de Membrana/biossíntese , Proteínas do Tecido Nervoso/biossíntese , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Espinhas Dendríticas/química , Técnicas de Transferência de Genes , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Estrutura Terciária de Proteína/fisiologia , Ratos , Transdução de SinaisRESUMO
Proteins of the striatin family have been identified in all multicellular animals. They are multidomain molecules containing several protein-interacting motifs. In mammals, these proteins are principally expressed in neurons with a somato-dendritic localization and high concentration in dendritic spines. Recent reports suggest that the proteins of the striatin family are molecular scaffolds that act as links between signal transduction and vesicular trafficking.