RESUMO
Our understanding of bird song, a model system for animal communication and the neurobiology of learning, depends critically on making reliable, validated comparisons between the complex multidimensional syllables that are used in songs. However, most assessments of song similarity are based on human inspection of spectrograms, or computational methods developed from human intuitions. Using a novel automated operant conditioning system, we collected a large corpus of zebra finches' (Taeniopygia guttata) decisions about song syllable similarity. We use this dataset to compare and externally validate similarity algorithms in widely-used publicly available software (Raven, Sound Analysis Pro, Luscinia). Although these methods all perform better than chance, they do not closely emulate the avian assessments. We then introduce a novel deep learning method that can produce perceptual similarity judgements trained on such avian decisions. We find that this new method outperforms the established methods in accuracy and more closely approaches the avian assessments. Inconsistent (hence ambiguous) decisions are a common occurrence in animal behavioural data; we show that a modification of the deep learning training that accommodates these leads to the strongest performance. We argue this approach is the best way to validate methods to compare song similarity, that our dataset can be used to validate novel methods, and that the general approach can easily be extended to other species.
Assuntos
Aprendizado Profundo , Tentilhões , Vocalização Animal , Animais , Vocalização Animal/fisiologia , Tentilhões/fisiologia , Algoritmos , Biologia Computacional/métodos , Julgamento/fisiologia , Masculino , Espectrografia do Som/métodos , Condicionamento Operante/fisiologia , HumanosRESUMO
Cultural and genetic inheritance combine to enable rapid changes in trait expression, but their relative importance in determining trait expression across generations is not clear. Birdsong is a socially learned cognitive trait that is subject to both cultural and genetic inheritance, as well as being affected by early developmental conditions. We sought to test whether early-life conditions in one generation can affect song acquisition in the next generation. We exposed one generation (F1) of nestlings to elevated corticosterone (CORT) levels, allowed them to breed freely as adults, and quantified their son's (F2) ability to copy the song of their social father. We also quantified the neurogenetic response to song playback through immediate early gene (IEG) expression in the auditory forebrain. F2 males with only one corticosterone-treated parent copied their social father's song less accurately than males with two control parents. Expression of ARC in caudomedial nidopallium (NCM) correlated with father-son song similarity, and patterns of expression levels of several IEGs in caudomedial mesopallium (CMM) in response to father song playback differed between control F2 sons and those with a CORT-treated father only. This is the first study to demonstrate that developmental conditions can affect social learning and neurogenetic responses in a subsequent generation.
Assuntos
Corticosterona , Aprendizagem , Vocalização Animal , Animais , Vocalização Animal/fisiologia , Masculino , Aprendizagem/fisiologia , Corticosterona/metabolismo , Feminino , Tentilhões/fisiologia , Prosencéfalo/metabolismo , Prosencéfalo/fisiologia , Genes PrecocesRESUMO
Sound is an essential source of information in many taxa and can notably be used by embryos to programme their phenotypes for postnatal environments. While underlying mechanisms are mostly unknown, there is growing evidence for the involvement of mitochondria-main source of cellular energy (i.e. ATP)-in developmental programming processes. Here, we tested whether prenatal sound programmes mitochondrial metabolism. In the arid-adapted zebra finch, prenatal exposure to 'heat-calls'-produced by parents incubating at high temperatures-adaptively alters nestling growth in the heat. We measured red blood cell mitochondrial function, in nestlings exposed prenatally to heat- or control-calls, and reared in contrasting thermal environments. Exposure to high temperatures always reduced mitochondrial ATP production efficiency. However, as expected to reduce heat production, prenatal exposure to heat-calls improved mitochondrial efficiency under mild heat conditions. In addition, when exposed to an acute heat-challenge, LEAK respiration was higher in heat-call nestlings, and mitochondrial efficiency low across temperatures. Consistent with its role in reducing oxidative damage, LEAK under extreme heat was also higher in fast growing nestlings. Our study therefore provides the first demonstration of mitochondrial acoustic sensitivity, and brings us closer to understanding the underpinning of acoustic developmental programming and avian strategies for heat adaptation.
Assuntos
Tentilhões , Efeitos Tardios da Exposição Pré-Natal , Aclimatação , Acústica , Trifosfato de Adenosina/metabolismo , Animais , Tentilhões/fisiologia , Temperatura Alta , Mitocôndrias/metabolismo , Efeitos Tardios da Exposição Pré-Natal/metabolismo , TemperaturaRESUMO
High-quality and complete reference genome assemblies are fundamental for the application of genomics to biology, disease, and biodiversity conservation. However, such assemblies are available for only a few non-microbial species1-4. To address this issue, the international Genome 10K (G10K) consortium5,6 has worked over a five-year period to evaluate and develop cost-effective methods for assembling highly accurate and nearly complete reference genomes. Here we present lessons learned from generating assemblies for 16 species that represent six major vertebrate lineages. We confirm that long-read sequencing technologies are essential for maximizing genome quality, and that unresolved complex repeats and haplotype heterozygosity are major sources of assembly error when not handled correctly. Our assemblies correct substantial errors, add missing sequence in some of the best historical reference genomes, and reveal biological discoveries. These include the identification of many false gene duplications, increases in gene sizes, chromosome rearrangements that are specific to lineages, a repeated independent chromosome breakpoint in bat genomes, and a canonical GC-rich pattern in protein-coding genes and their regulatory regions. Adopting these lessons, we have embarked on the Vertebrate Genomes Project (VGP), an international effort to generate high-quality, complete reference genomes for all of the roughly 70,000 extant vertebrate species and to help to enable a new era of discovery across the life sciences.
Assuntos
Genoma , Genômica/métodos , Vertebrados/genética , Animais , Aves , Biblioteca Gênica , Tamanho do Genoma , Genoma Mitocondrial , Haplótipos , Sequenciamento de Nucleotídeos em Larga Escala , Anotação de Sequência Molecular , Alinhamento de Sequência , Análise de Sequência de DNA , Cromossomos Sexuais/genéticaRESUMO
Prolonged social isolation has negative effects on brain and behavior in humans and other social organisms, but neural mechanisms leading to these effects are not understood. Here we tested the hypothesis that even brief periods of social isolation can alter gene expression and DNA methylation in higher cognitive centers of the brain, focusing on the auditory/associative forebrain of the highly social zebra finch. Using RNA sequencing, we first identified genes that individually increase or decrease expression after isolation and observed general repression of gene sets annotated for neurotrophin pathways and axonal guidance functions. We then pursued 4 genes of large effect size: EGR1 and BDNF (decreased by isolation) and FKBP5 and UTS2B (increased). By in situ hybridization, each gene responded in different cell subsets, arguing against a single cellular mechanism. To test whether effects were specific to the social component of the isolation experience, we compared gene expression in birds isolated either alone or with a single familiar partner. Partner inclusion ameliorated the effect of solo isolation on EGR1 and BDNF, but not on FKBP5 and UTS2B nor on circulating corticosterone. By bisulfite sequencing analysis of auditory forebrain DNA, isolation caused changes in methylation of a subset of differentially expressed genes, including BDNF. Thus, social isolation has rapid consequences on gene activity in a higher integrative center of the brain, triggering epigenetic mechanisms that may influence processing of ongoing experience.
Assuntos
Tentilhões/genética , Prosencéfalo/metabolismo , Isolamento Social , Animais , Comportamento Animal , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Corticosterona/sangue , Metilação de DNA , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Feminino , Tentilhões/sangue , Tentilhões/fisiologia , Masculino , Proteínas de Ligação a Tacrolimo/genética , Proteínas de Ligação a Tacrolimo/metabolismoRESUMO
Songbirds communicate through learned vocalizations, using a forebrain circuit with convergent similarity to vocal-control circuitry in humans. This circuit is incomplete in female zebra finches, hence only males sing. We show that the UTS2B gene, encoding Urotensin-Related Peptide (URP), is uniquely expressed in a key pre-motor vocal nucleus (HVC), and specifically marks the neurons that form a male-specific projection that encodes timing features of learned song. UTS2B-expressing cells appear early in males, prior to projection formation, but are not observed in the female nucleus. We find no expression evidence for canonical receptors within the vocal circuit, suggesting either signalling to other brain regions via diffusion or transduction through other receptor systems. Urotensins have not previously been implicated in vocal control, but we find an annotation in Allen Human Brain Atlas of increased UTS2B expression within portions of human inferior frontal cortex implicated in human speech and singing. Thus UTS2B (URP) is a novel neural marker that may have conserved functions for vocal communication.
Assuntos
Prosencéfalo/metabolismo , Aves Canoras/fisiologia , Urotensinas/genética , Animais , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Evolução Molecular , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Caracteres Sexuais , Aves Canoras/genética , Urotensinas/metabolismo , Vocalização AnimalRESUMO
Neuropathological and genetic findings suggest that the presynaptic protein α-synuclein (aSyn) is involved in the pathogenesis of synucleinopathy disorders, including Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy. Evidence suggests that the self-assembly of aSyn conformers bound to phospholipid membranes in an aggregation-prone state plays a key role in aSyn neurotoxicity. Accordingly, we hypothesized that protein binding partners of lipid-associated aSyn could inhibit the formation of toxic aSyn oligomers at membrane surfaces. To address this hypothesis, we characterized the protein endosulfine-alpha (ENSA), previously shown to interact selectively with membrane-bound aSyn, in terms of its effects on the membrane-induced aggregation and neurotoxicity of two familial aSyn mutants, A30P and G51D. We found that wild-type ENSA, but not the non-aSyn-binding S109E variant, interfered with membrane-induced aSyn self-assembly, aSyn-mediated vesicle disruption and aSyn neurotoxicity. Immunoblotting analyses revealed that ENSA was down-regulated in the brains of synucleinopathy patients versus non-diseased individuals. Collectively, these results suggest that ENSA can alleviate neurotoxic effects of membrane-bound aSyn via an apparent chaperone-like activity at the membrane surface, and a decrease in ENSA expression may contribute to aSyn neuropathology in synucleinopathy disorders. More generally, our findings suggest that promoting interactions between lipid-bound, amyloidogenic proteins and their binding partners is a viable strategy to alleviate cytotoxicity in a range of protein misfolding disorders.
Assuntos
Membrana Celular/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Peptídeos/farmacologia , Agregação Patológica de Proteínas/tratamento farmacológico , alfa-Sinucleína/efeitos dos fármacos , Adenoviridae , Idoso , Idoso de 80 Anos ou mais , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Membrana Celular/metabolismo , Células Cultivadas , Estudos de Coortes , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Escherichia coli , Feminino , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Doença por Corpos de Lewy/metabolismo , Doença por Corpos de Lewy/patologia , Masculino , Pessoa de Meia-Idade , Fármacos Neuroprotetores/metabolismo , Peptídeos/metabolismo , Agregação Patológica de Proteínas/metabolismo , Ratos Sprague-Dawley , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Lipossomas Unilamelares/química , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMO
Misfolded α-synuclein amyloid fibrils are the principal components of Lewy bodies and neurites, hallmarks of Parkinson's disease (PD). We present a high-resolution structure of an α-synuclein fibril, in a form that induces robust pathology in primary neuronal culture, determined by solid-state NMR spectroscopy and validated by EM and X-ray fiber diffraction. Over 200 unique long-range distance restraints define a consensus structure with common amyloid features including parallel, in-register ß-sheets and hydrophobic-core residues, and with substantial complexity arising from diverse structural features including an intermolecular salt bridge, a glutamine ladder, close backbone interactions involving small residues, and several steric zippers stabilizing a new orthogonal Greek-key topology. These characteristics contribute to the robust propagation of this fibril form, as supported by the structural similarity of early-onset-PD mutants. The structure provides a framework for understanding the interactions of α-synuclein with other proteins and small molecules, to aid in PD diagnosis and treatment.
Assuntos
Amiloide/química , alfa-Sinucleína/química , Sequência de Aminoácidos , Amiloide/fisiologia , Animais , Células Cultivadas , Humanos , Ligação de Hidrogênio , Corpos de Lewy/química , Camundongos , Neurônios/fisiologia , Ressonância Magnética Nuclear Biomolecular , Doença de Parkinson/patologia , Domínios Proteicos , Dobramento de Proteína , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , alfa-Sinucleína/fisiologiaRESUMO
Parkinson's disease is associated with multiplication of the α-synuclein gene and abnormal accumulation of the protein. In animal models, α-synuclein overexpression broadly impairs synaptic vesicle trafficking. However, the exact steps of the vesicle trafficking pathway affected by excess α-synuclein and the underlying molecular mechanisms remain unknown. Therefore we acutely increased synuclein levels at a vertebrate synapse and performed a detailed ultrastructural analysis of the effects on presynaptic membranes. At stimulated synapses (20 Hz), excess synuclein caused a loss of synaptic vesicles and an expansion of the plasma membrane, indicating an impairment of vesicle recycling. The N-terminal domain (NTD) of synuclein, which folds into an α-helix, was sufficient to reproduce these effects. In contrast, α-synuclein mutants with a disrupted N-terminal α-helix (T6K and A30P) had little effect under identical conditions. Further supporting this model, another α-synuclein mutant (A53T) with a properly folded NTD phenocopied the synaptic vesicle recycling defects observed with wild type. Interestingly, the vesicle recycling defects were not observed when the stimulation frequency was reduced (5 Hz). Thus excess α-synuclein impairs synaptic vesicle recycling evoked during intense stimulation via a mechanism that requires a properly folded N-terminal α-helix.
Assuntos
Proteínas de Peixes/metabolismo , Sinapses/fisiologia , Vesículas Sinápticas/metabolismo , alfa-Sinucleína/metabolismo , Potenciais de Ação/genética , Potenciais de Ação/fisiologia , Sequência de Aminoácidos , Animais , Axônios/metabolismo , Axônios/fisiologia , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Estimulação Elétrica , Endocitose/genética , Endocitose/fisiologia , Proteínas de Peixes/química , Proteínas de Peixes/genética , Immunoblotting , Lampreias/genética , Lampreias/metabolismo , Lampreias/fisiologia , Microscopia Confocal , Microscopia Eletrônica , Dados de Sequência Molecular , Mutação , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , Sinapses/metabolismo , Vesículas Sinápticas/ultraestrutura , alfa-Sinucleína/química , alfa-Sinucleína/genéticaRESUMO
α-Synuclein (α-syn), an aggregation-prone amyloid protein, has been suggested as a potential cause of Parkinson's disease. When misfolded, α-syn aggregates as Lewy bodies in the brain, the loss of which can disrupt protein homeostasis. To investigate the potential of nanoparticle-mediated therapy for amyloid diseases, α-syn adsorption onto positively charged poly(allylamine hydrochloride) coated gold nanoparticles (PAH Au NPs) was studied. α-Syn adsorbs in multilayers onto PAH Au NPs, which with increasing α-syn/PAH Au NP ratios (>2000 α-syn/PAH Au NP) results in the flocculation and sedimentation of α-syn coated PAH Au NPs. The orientation and conformation of α-syn on PAH Au NPs were studied using trypsin digestion and circular dichroism, which showed that α-syn adopts a random orientation on PAH Au NPs, with an increase in ß-sheet and a decrease in α-helix structures. A consistent global change in α-syn's conformation was also observed regardless of PAH Au NP concentration, suggesting bound α-syn initiates conformational changes to free α-syn.
Assuntos
Ouro/química , Nanopartículas Metálicas/química , alfa-Sinucleína/química , Adsorção , Sequência de Aminoácidos , Dicroísmo Circular , Humanos , Espectrometria de Massas , Dados de Sequência Molecular , Poliaminas/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Propriedades de Superfície , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMO
The disruption of α-synuclein (α-syn) homeostasis in neurons is a potential cause of Parkinson's disease, which is manifested pathologically by the appearance of α-syn aggregates, or Lewy bodies. Treatments for neurological diseases are extremely limited. To study the potential use of gold nanoparticles (Au NPs) to limit α-syn misfolding, the binding and orientation of α-syn on Au NPs were investigated. α-Syn was determined to interact with 20 and 90 nm Au NPs via multilayered adsorption: a strong electrostatic interaction between α-syn and Au NPs in the hard corona and a weaker noncovalent protein-protein interaction in the soft corona. Spectroscopic and light-scattering titrations led to the determinations of binding constants for the Au NP α-syn coronas: for the hard corona on 20 nm Au NPs, the equilibrium association constant was 2.9 ± 1.1 × 10(9) M(-1) (for 360 ± 70 α-syn/NP), and on 90 nm Au NPs, the hard corona association constant was 9.5 ± 0.8 × 10(10) M(-1) (for 5300 ± 700 α-syn/NP). The binding of the soft corona was thermodynamically unfavorable and kinetically driven and was in constant exchange with "free" α-syn in solution. A protease digestion method was used to deduce the α-syn orientation and structure on Au NPs, revealing that α-syn absorbs onto negatively charged Au NPs via its N-terminus while apparently retaining its natively unstructured conformation. These results suggest that Au NPs could be used to sequester and regulate α-syn homeostasis.
Assuntos
Ouro/química , Nanopartículas Metálicas/química , alfa-Sinucleína/química , Adsorção , Sequência de Aminoácidos , Humanos , Dados de Sequência Molecular , Tamanho da Partícula , Ligação Proteica , Proteólise , Espectrometria de Fluorescência , Tripsina/metabolismo , alfa-Sinucleína/metabolismoRESUMO
Parkinson's disease (PD) is pathologically characterized by the presence of Lewy bodies (LBs) in dopaminergic neurons of the substantia nigra. These intracellular inclusions are largely composed of misfolded α-synuclein (AS), a neuronal protein that is abundant in the vertebrate brain. Point mutations in AS are associated with rare, early-onset forms of PD, although aggregation of the wild-type (WT) protein is observed in the more common sporadic forms of the disease. Here, we employed multidimensional solid-state NMR experiments to assess A53T and E46K mutant fibrils, in comparison to our recent description of WT AS fibrils. We made de novo chemical shift assignments for the mutants, and used these chemical shifts to empirically determine secondary structures. We observe significant perturbations in secondary structure throughout the fibril core for the E46K fibril, while the A53T fibril exhibits more localized perturbations near the mutation site. Overall, these results demonstrate that the secondary structure of A53T has some small differences from the WT and the secondary structure of E46K has significant differences, which may alter the overall structural arrangement of the fibrils.
Assuntos
Mutação , Doença de Parkinson/genética , alfa-Sinucleína/química , alfa-Sinucleína/genética , Sequência de Aminoácidos , Humanos , Corpos de Lewy/química , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/ultraestrutura , Ressonância Magnética Nuclear Biomolecular , Doença de Parkinson/metabolismo , Estrutura Secundária de Proteína , alfa-Sinucleína/ultraestruturaRESUMO
Modifications to the gene encoding human α-synuclein have been linked to the development of Parkinson's disease. The highly conserved structure of α-synuclein suggests a functional interaction with membranes, and several lines of evidence point to a role in vesicle-related processes within nerve terminals. Using recombinant fusions of human α-synuclein, including new genetic tags developed for correlated light microscopy and electron microscopy (the tetracysteine-biarsenical labeling system or the new fluorescent protein for electron microscopy, MiniSOG), we determined the distribution of α-synuclein when overexpressed in primary neurons at supramolecular and cellular scales in three dimensions (3D). We observed specific association of α-synuclein with a large and otherwise poorly characterized membranous organelle system of the presynaptic terminal, as well as with smaller vesicular structures within these boutons. Furthermore, α-synuclein was localized to multiple elements of the protein degradation pathway, including multivesicular bodies in the axons and lysosomes within neuronal cell bodies. Examination of synapses in brains of transgenic mice overexpressing human α-synuclein revealed alterations of the presynaptic endomembrane systems similar to our findings in cell culture. Three-dimensional electron tomographic analysis of enlarged presynaptic terminals in several brain areas revealed that these terminals were filled with membrane-bounded organelles, including tubulovesicular structures similar to what we observed in vitro. We propose that α-synuclein overexpression is associated with hypertrophy of membrane systems of the presynaptic terminal previously shown to have a role in vesicle recycling. Our data support the conclusion that α-synuclein is involved in processes associated with the sorting, channeling, packaging, and transport of synaptic material destined for degradation.
Assuntos
Neurônios/química , Neurônios/metabolismo , Doença de Parkinson/metabolismo , alfa-Sinucleína/análise , alfa-Sinucleína/biossíntese , Animais , Células Cultivadas , Células HEK293 , Humanos , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica/métodos , Microscopia de Polarização/métodos , Neurônios/ultraestrutura , Doença de Parkinson/patologia , Ratos , Ratos Sprague-Dawley , Frações Subcelulares/metabolismo , Frações Subcelulares/ultraestrutura , alfa-Sinucleína/genéticaRESUMO
α-Synuclein (AS) is associated with both sporadic and familial forms of Parkinson disease (PD). In sporadic disease, wild-type AS fibrillates and accumulates as Lewy bodies within dopaminergic neurons of the substantia nigra. The accumulation of misfolded AS is associated with the death of these neurons, which underlies many of the clinical features of PD. In addition, a rare missense mutation in AS, A30P, is associated with highly penetrant, autosomal dominant PD, although the pathogenic mechanism is unclear. A30P AS fibrillates more slowly than the wild-type (WT) protein in vitro and has been reported to preferentially adopt a soluble, protofibrillar conformation. This has led to speculation that A30P forms aggregates that are distinct in structure compared with wild-type AS. Here, we perform a detailed comparison of the chemical shifts and secondary structures of these fibrillar species, based upon our recent characterization of full-length WT fibrils. We have assigned A30P AS fibril chemical shifts de novo and used them to determine its secondary structure empirically. Our results illustrate that although A30P forms fibrils more slowly than WT in vitro, the chemical shifts and secondary structure of the resultant fibrils are in high agreement, demonstrating a conserved ß-sheet core.
Assuntos
Proteínas Mutantes/química , Mutação , Ressonância Magnética Nuclear Biomolecular , Multimerização Proteica , alfa-Sinucleína/química , Humanos , Cinética , Proteínas Mutantes/genética , Estrutura Secundária de Proteína , alfa-Sinucleína/genéticaRESUMO
α-Synuclein (AS) fibrils are the main protein component of Lewy bodies, the pathological hallmark of Parkinson's disease and other related disorders. AS forms helices that bind phospholipid membranes with high affinity, but no atomic level data for AS aggregation in the presence of lipids is yet available. Here, we present direct evidence of a conversion from α-helical conformation to ß-sheet fibrils in the presence of anionic phospholipid vesicles and direct conversion to ß-sheet fibrils in their absence. We have trapped intermediate states throughout the fibril formation pathways to examine the structural changes using solid-state NMR spectroscopy and electron microscopy. The comparison between mature AS fibrils formed in aqueous buffer and those derived in the presence of anionic phospholipids demonstrates no major changes in the overall fibril fold. However, a site-specific comparison of these fibrillar states demonstrates major perturbations in the N-terminal domain with a partial disruption of the long ß-strand located in the 40s and small perturbations in residues located in the "non-ß amyloid component" (NAC) domain. Combining all these results, we propose a model for AS fibrillogenesis in the presence of phospholipid vesicles.
Assuntos
Fosfolipídeos/química , alfa-Sinucleína/química , Microscopia Eletrônica , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Estrutura Secundária de ProteínaRESUMO
α-Synuclein (AS) fibrils are the major component of Lewy bodies, the pathological hallmark of Parkinson's disease (PD). Here, we use results from an extensive investigation employing solid-state NMR to present a detailed structural characterization and conformational dynamics quantification of full-length AS fibrils. Our results show that the core extends with a repeated structural motif. This result disagrees with the previously proposed fold of AS fibrils obtained with limited solid-state NMR data. Additionally, our results demonstrate that the three single point mutations associated with early-onset PD-A30P, E46K and A53T-are located in structured regions. We find that E46K and A53T mutations, located in rigid ß-strands of the wild-type fibrils, are associated with major and minor structural perturbations, respectively.
Assuntos
Corpos de Lewy/patologia , Doença de Parkinson/genética , Mutação Puntual/genética , alfa-Sinucleína/química , alfa-Sinucleína/genética , Sequência de Aminoácidos , Humanos , Corpos de Lewy/química , Corpos de Lewy/genética , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Estrutura Terciária de ProteínaRESUMO
BACKGROUND: Alpha-synuclein is a presynaptic protein with a proposed role in neurotransmission and dopamine homeostasis. Abnormal accumulation of alpha-synuclein aggregates in dopaminergic neurons of the substantia nigra is diagnostic of sporadic Parkinson's disease, and mutations in the protein are linked to early onset forms of the disease. The folded conformation of the protein varies depending upon its environment and other factors that are poorly understood. When bound to phospholipid membranes, alpha-synuclein adopts a helical conformation that mediates specific interactions with other proteins. RESULTS: To investigate the role of the helical domain in transport and localization of alpha-synuclein, eGFP-tagged constructs were transfected into rat primary hippocampal neurons at 7 DIV. A series of constructs were analyzed in which each individual exon was deleted, for comparison to previous studies of lipid affinity and alpha-helix content. A53T and A30P substitutions, representing Parkinson's disease-associated variants, were analyzed as well. Single exon deletions within the lipid-binding N-terminal domain of alpha-synuclein (exons 2, 3, and 4) partially disrupted its presynaptic localization at 17-21 DIV, resulting in increased diffuse labeling of axons. Similar results were obtained for A30P, which exhibits decreased lipid binding, but not A53T. To examine whether differences in presynaptic enrichment were related to deficiencies in transport velocity, transport was visualized via live cell microscopy. Tagged alpha-synuclein migrated at a rate of 1.85 +/- 0.09 mum/s, consistent with previous reports, and single exon deletion mutants migrated at similar rates, as did A30P. Deletion of the entire N-terminal lipid-binding domain (Delta234GFP) did not significantly alter rates of particle movement, but decreased the number of moving particles. Only the A53TGFP mutant exhibited a significant decrease in transport velocity as compared to ASGFP. CONCLUSIONS: These results support the hypothesis that presynaptic localization involves a mechanism that requires helical conformation and lipid binding. Conversely, the rate of axonal transport is not determined by lipid affinity and is not sufficient to account for differences in presynaptic localization of alpha-synuclein-eGFP variants.
RESUMO
An efficient route for delivering specific proteins and peptides into neurons could greatly accelerate the development of therapies for various diseases, especially those involving intracellular defects such as Parkinson disease. Here we report the novel use of polybutylcyanoacrylate nanoparticles for delivery of intact, functional proteins into neurons and neuronal cell lines. Uptake of these particles is primarily dependent on endocytosis via the low density lipoprotein receptor. The nanoparticles are rapidly turned over and display minimal toxicity to cultured neurons. Delivery of three different functional cargo proteins is demonstrated. When primary neuronal cultures are treated with recombinant Escherichia coli beta-galactosidase as nanoparticle cargo, persistent enzyme activity is measured beyond the period of nanoparticle degradation. Delivery of the small GTPase rhoG induces neurite outgrowth and differentiation in PC12 cells. Finally, a monoclonal antibody directed against synuclein is capable of interacting with endogenous alpha-synuclein in cultured neurons following delivery via nanoparticles. Polybutylcyanoacrylate nanoparticles are thus useful for intracellular protein delivery in vitro and have potential as carriers of therapeutic proteins for treatment of neuronal disorders in vivo.
Assuntos
Resinas Acrílicas/farmacologia , Sistemas de Liberação de Medicamentos , Nanopartículas , Neuritos/metabolismo , Doença de Parkinson/tratamento farmacológico , Receptores de LDL/agonistas , Proteínas Recombinantes/farmacocinética , Animais , Diferenciação Celular/efeitos dos fármacos , Células PC12 , Doença de Parkinson/metabolismo , Ratos , Receptores de LDL/metabolismoRESUMO
Songbirds are appreciated for the insights they provide into regulated neural plasticity. Here, we describe the comparative analysis and brain expression of two gene sequences encoding probable regulators of synaptic plasticity in songbirds: neuromodulin (GAP-43) and neurogranin (RC3). Both are members of the calpacitin family and share a distinctive conserved core domain that mediates interactions between calcium, calmodulin, and protein kinase C signaling pathways. Comparative sequence analysis is consistent with known phylogenetic relationships, with songbirds most closely related to chicken and progressively more distant from mammals and fish. The C-terminus of neurogranin is different in birds and mammals, and antibodies to the protein reveal high expression in adult zebra finches in cerebellar Purkinje cells, which has not been observed in other species. RNAs for both proteins are generally abundant in the telencephalon yet markedly reduced in certain nuclei of the song control system in adult canaries and zebra finches: neuromodulin RNA is very low in RA and HVC (relative to the surrounding pallial areas), whereas neurogranin RNA is conspicuously low in Area X (relative to surrounding striatum). In both cases, this selective downregulation develops in the zebra finch during the juvenile song learning period, 25-45 days after hatching. These results suggest molecular parallels to the robust stability of the adult avian song control circuit.
Assuntos
Proteína GAP-43/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Centro Vocal Superior/crescimento & desenvolvimento , Centro Vocal Superior/metabolismo , Neurogranina/metabolismo , Vocalização Animal/fisiologia , Fatores Etários , Animais , Animais Recém-Nascidos , Clonagem Molecular , Tentilhões , Proteína GAP-43/química , Proteína GAP-43/genética , Espaço Intracelular/metabolismo , Neurogranina/química , Neurogranina/genética , RNA Mensageiro/metabolismo , Análise de SequênciaRESUMO
Endosulfine-alpha (ENSA) is a 121-residue cAMP-regulated phosphoprotein, originally identified as an endogenous regulator of ATP-sensitive potassium channels. ENSA has been implicated in the regulation of insulin secretion, and expression of ENSA is decreased in brains of both Alzheimer's disease (AD) and Down's syndrome patients. We recently described membrane-dependent interactions between ENSA and the Parkinson's disease associated protein alpha-synuclein. Here we characterize the conformational change in ENSA that occurs upon binding to membranes. Secondary chemical shift analysis demonstrates formation of four helices in the lipid-bound state that are not present in the absence of lipid. The helical structure is maintained in several different lipid mimetics (sodium dodecyl sulfate, dodecyl phosphocholine, lyso 1-palmitoyl phosphatidylglycerol, and phospholipid vesicles). Introduction of a mutation (S109E) to mimic PKA phosphorylation of ENSA leads to a perturbation of the fourth helix and disrupts the interaction with alpha-synuclein. These data establish ENSA as an intrinsically unstructured protein that adopts a stable structure upon membrane binding, properties it shares with its binding partner alpha-synuclein.