RESUMO
The establishment of neuronal connectivity relies on the microtubule (MT) cytoskeleton, which provides mechanical support, roads for axonal transport and mediates signalling events. Fine-tuned spatiotemporal regulation of MT functions by tubulin post-translational modifications and MT-associated proteins is critical for the coarse wiring and subsequent refinement of neuronal connectivity. The defective regulation of these processes causes a wide range of neurodevelopmental disorders associated with connectivity defects. This review focuses on recent studies unravelling how MT composition, post-translational modifications and associated proteins influence MT functions in axon guidance and/or pruning to build functional neuronal circuits. We here summarise experimental evidence supporting the key role of this network as a driving force for growth cone steering and branch-specific axon elimination. We further provide a global overview of the MT-interactors that tune developing axon behaviours, with a special emphasis on their emerging versatility in the regulation of MT dynamics/structure. Recent studies establishing the key and highly selective role of the tubulin code in the regulation of MT functions in axon pathfinding are also reported. Finally, our review highlights the emerging molecular links between these MT regulation processes and guidance signals that wire the nervous system.
Assuntos
Orientação de Axônios , Tubulina (Proteína) , Tubulina (Proteína)/metabolismo , Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Axônios/metabolismoRESUMO
The RhoGEF TRIO is known to play a major role in neuronal development by controlling actin cytoskeleton remodeling, primarily through the activation of the RAC1 GTPase. Numerous de novo mutations in the TRIO gene have been identified in individuals with neurodevelopmental disorders (NDDs). We have previously established the first phenotype/genotype correlation in TRIO-associated diseases, with striking correlation between the clinical features of the individuals and the opposite modulation of RAC1 activity by TRIO variants targeting different domains. The mutations hyperactivating RAC1 are of particular interest, as they are recurrently found in patients and are associated with a severe form of NDD and macrocephaly, indicating their importance in the etiology of the disease. Yet, it remains unknown how these pathogenic TRIO variants disrupt TRIO activity at a molecular level and how they affect neurodevelopmental processes such as axon outgrowth or guidance. Here we report an additional cohort of individuals carrying a pathogenic TRIO variant that reinforces our initial phenotype/genotype correlation. More importantly, by performing conformation predictions coupled to biochemical validation, we propose a model whereby TRIO is inhibited by an intramolecular fold and NDD-associated variants relieve this inhibition, leading to RAC1 hyperactivation. Moreover, we show that in cultured primary neurons and in the zebrafish developmental model, these gain-of-function variants differentially affect axon outgrowth and branching in vitro and in vivo, as compared to loss-of-function TRIO variants. In summary, by combining clinical, molecular, cellular and in vivo data, we provide compelling new evidence for the pathogenicity of novel genetic variants targeting the TRIO gene in NDDs. We report a novel mechanism whereby the fine-tuned regulation of TRIO activity is critical for proper neuronal development and is disrupted by pathogenic mutations.
Assuntos
Orientação de Axônios , Transtornos do Neurodesenvolvimento , Animais , Transtornos do Neurodesenvolvimento/genética , Neurônios , Fatores de Troca de Nucleotídeo Guanina Rho , Peixe-Zebra , HumanosRESUMO
Functional analyses of genes responsible for neurodegenerative disorders have unveiled crucial links between neurodegenerative processes and key developmental signalling pathways. Mutations in SPG4-encoding spastin cause hereditary spastic paraplegia (HSP). Spastin is involved in diverse cellular processes that couple microtubule severing to membrane remodelling. Two main spastin isoforms are synthesised from alternative translational start sites (M1 and M87). However, their specific roles in neuronal development and homeostasis remain largely unknown. To selectively unravel their neuronal function, we blocked spastin synthesis from each initiation codon during zebrafish development and performed rescue analyses. The knockdown of each isoform led to different motor neuron and locomotion defects, which were not rescued by the selective expression of the other isoform. Notably, both morphant neuronal phenotypes were observed in a CRISPR/Cas9 spastin mutant. We next showed that M1 spastin, together with HSP proteins atlastin 1 and NIPA1, drives motor axon targeting by repressing BMP signalling, whereas M87 spastin acts downstream of neuropilin 1 to control motor neuron migration. Our data therefore suggest that defective BMP and neuropilin 1 signalling may contribute to the motor phenotype in a vertebrate model of spastin depletion.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Neurônios Motores/citologia , Neuropilina-1/metabolismo , Espastina/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Animais , Axônios/metabolismo , Células COS , Sistemas CRISPR-Cas/genética , Linhagem Celular , Movimento Celular/genética , Chlorocebus aethiops , Proteínas de Ligação ao GTP/metabolismo , Técnicas de Inativação de Genes , Humanos , Proteínas de Membrana/metabolismo , Isoformas de Proteínas/genética , Paraplegia Espástica Hereditária/genética , Espastina/biossíntese , Proteínas de Peixe-Zebra/biossínteseRESUMO
The axon initial segment (AIS) is required for generating action potentials and maintaining neuronal polarity. Significant progress has been made in deciphering the basic building blocks composing the AIS, but the underlying mechanisms required for AIS formation remains unclear. The scaffolding protein ankyrin-G is the master-organizer of the AIS. Microtubules and their interactors, particularly end-binding proteins (EBs), have emerged as potential key players in AIS formation. Here, we show that the longest isoform of ankyrin-G (480AnkG) selectively associates with EBs via its specific tail domain and that this interaction is crucial for AIS formation and neuronal polarity in cultured rodent hippocampal neurons. EBs are essential for 480AnkG localization and stabilization at the AIS, whereas 480AnkG is required for the specific accumulation of EBs in the proximal axon. Our findings thus provide a conceptual framework for understanding how the cooperative relationship between 480AnkG and EBs induces the assembly of microtubule-AIS structures in the proximal axon. SIGNIFICANCE STATEMENT: Neuronal polarity is crucial for the proper function of neurons. The assembly of the axon initial segment (AIS), which is the hallmark of early neuronal polarization, relies on the longest 480 kDa ankyrin-G isoform. The microtubule cytoskeleton and its interacting proteins were suggested to be early key players in the process of AIS formation. In this study, we show that the crosstalk between 480 kDa ankyrin-G and the microtubule plus-end tracking proteins, EBs, at the proximal axon is decisive for AIS assembly and neuronal polarity. Our work thus provides insight into the functional mechanisms used by 480 kDa ankyrin-G to drive the AIS formation and thereby to establish neuronal polarity.
Assuntos
Anquirinas/metabolismo , Axônios/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Animais , Axônios/ultraestrutura , Células COS , Polaridade Celular/fisiologia , Células Cultivadas , Chlorocebus aethiops , Feminino , Camundongos , Neurônios/metabolismo , Neurônios/ultraestrutura , Ligação Proteica/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
The microtubule-associated protein MAP1B plays a key role in axon regeneration. We investigated the role of GSK3-mediated MAP1B phosphorylation in local fine-tuning of neurite branching and the underlying microtubule (MT) dynamics. In wildtype adult dorsal root ganglia (DRG) neurons, MAP1B phosphorylation is locally reduced at branching points, and branching dynamics from growth cones and distal neurite shafts is increased upon GSK3 inhibition. While map1b-/- neurites, that display increased branching, are not affected by GSK3 inhibition, transfection of map1b-/- neurons with full-length map1b-cDNA restores the wildtype branching phenotype, demonstrating that MAP1B is a key effector downstream of GSK3. Experiments in mutant mice lacking tyrosinated MTs indicate a preferential association of phospho-MAP1B with tyrosinated MTs. Interestingly, inhibition of GSK3-mediated MAP1B phosphorylation in map1b-cDNA-transfected fibroblasts protects both tyrosinated and acetylated MTs from nocodazole-induced depolymerization, while detyrosinated MTs are less abundant in the presence of MAP1B. Our data thus provide new insight into the molecular link between GSK3, MAP1B, neurite branching and MT stability regulation. We suggest that, at branching points, MAP1B undergoes a fine regulation of both its phosphorylation and sub-cellular amounts, in order to modulate the local balance between acetylated, detyrosinated, and tyrosinated microtubule pools.
Assuntos
Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Neuritos/metabolismo , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Neurogênese , FosforilaçãoRESUMO
Axonal transport is crucial for neuronal homeostasis, survival, and development. Indeed, axonal transport needs to be precisely regulated for developing axons to swiftly and accurately respond to their complex and evolving environment in space and time. A growing number of studies have started to unravel the diversity of regulatory and adaptor proteins required to orchestrate the axonal transport machinery. Despite some discrepancies between in vitro and in vivo axonal transport studies, most analyses aiming at deciphering these regulatory complexes, as well as their mode of action, were carried out in vitro in primary cultures of neurons, and mainly focused on their impact on axon specification and elongation, but rarely on axon navigation per se. Given the clear influence of the in vivo environment on axonal transport, including chemical and physical interactions with neighboring cells, it is essential to develop in vivo models to identify and characterize the molecular complexes involved in this key process. Here, we describe an experimental system to monitor axonal transport in vivo in developing axons of live zebrafish embryos with high spatial and temporal resolution. Due to its optical transparency and easy genetic manipulation, the zebrafish embryo is ideally suited to study such cellular dynamics at a single axon scale. Using this approach, we were able to unravel the key role of Fidgetin-like 1 in the regulation of bidirectional axonal transport required for motor axon targeting. Moreover, this protocol can be easily adapted to characterize a wide range of axonal transport regulators and components in physiological conditions and may additionally be used to screen new therapeutic compounds based on their ability to recue axonal transport defects in pathological conditions.
Assuntos
Transporte Axonal , Peixe-Zebra , Animais , Transporte Axonal/fisiologia , Axônios/metabolismo , Neurônios/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
In most mammals, retinal ganglion cell axons from each retina project to both sides of the brain. The segregation of ipsi and contralateral projections into eye-specific territories in their main brain targets-the dorsolateral geniculate nucleus and the superior colliculus-is critical for the processing of visual information. The investigation of the developmental mechanisms contributing to the wiring of this binocular map in mammals identified competitive mechanisms between axons from each retina while interactions between axons from the same eye were challenging to explore. Studies in vertebrates lacking ipsilateral retinal projections demonstrated that competitive mechanisms also exist between axons from the same eye. The development of a genetic approach enabling the differential manipulation and labeling of neighboring retinal ganglion cells in a single mouse retina revealed that binocular map development does not only rely on axon competition but also involves a cooperative interplay between axons to stabilize their terminal branches. These recent insights into the developmental mechanisms shaping retinal axon connectivity in the brain will be discussed here.
Assuntos
Corpos Geniculados , Retina , Animais , Axônios , Camundongos , Células Ganglionares da Retina , Colículos Superiores , Vias VisuaisRESUMO
Neuronal connectivity relies on molecular motor-based axonal transport of diverse cargoes. Yet the precise players and regulatory mechanisms orchestrating such trafficking events remain largely unknown. We here report the ATPase Fignl1 as a novel regulator of bidirectional transport during axon navigation. Using a yeast two-hybrid screen and coimmunoprecipitation assays, we showed that Fignl1 binds the kinesin Kif1bß and the dynein/dynactin adaptor Bicaudal D-1 (Bicd1) in a molecular complex including the dynactin subunit dynactin 1. Fignl1 colocalized with Kif1bß and showed bidirectional mobility in zebrafish axons. Notably, Kif1bß and Fignl1 loss of function similarly altered zebrafish motor axon pathfinding and increased dynein-based transport velocity of Rab3 vesicles in these navigating axons, pinpointing Fignl1/Kif1bß as a dynein speed limiter complex. Accordingly, disrupting dynein/dynactin activity or Bicd1/Fignl1 interaction induced motor axon pathfinding defects characteristic of Fignl1 gain or loss of function, respectively. Finally, pharmacological inhibition of dynein activity partially rescued the axon pathfinding defects of Fignl1-depleted larvae. Together, our results identify Fignl1 as a key dynein regulator required for motor circuit wiring.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Axônios/metabolismo , Proteínas do Citoesqueleto/metabolismo , Dineínas/metabolismo , Cinesinas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Animais , Transporte Biológico , Células COS , Células Cultivadas , Chlorocebus aethiops , Humanos , Peixe-ZebraRESUMO
During neural circuit assembly, extrinsic signals are integrated into changes in growth cone (GC) cytoskeleton underlying axon guidance decisions. Microtubules (MTs) were shown to play an instructive role in GC steering. However, the numerous actors required for MT remodeling during axon navigation and their precise mode of action are far from being deciphered. Using loss- and gain-of-function analyses during zebrafish development, we identify in this study the meiotic clade adenosine triphosphatase Fidgetin-like 1 (Fignl1) as a key GC-enriched MT-interacting protein in motor circuit wiring and larval locomotion. We show that Fignl1 controls GC morphology and behavior at intermediate targets by regulating MT plus end dynamics and growth directionality. We further reveal that alternative translation of Fignl1 transcript is a sophisticated mechanism modulating MT dynamics: a full-length isoform regulates MT plus end-tracking protein binding at plus ends, whereas shorter isoforms promote their depolymerization beneath the cell cortex. Our study thus pinpoints Fignl1 as a multifaceted key player in MT remodeling underlying motor circuit connectivity.
Assuntos
Adenosina Trifosfatases/metabolismo , Orientação de Axônios , Axônios/metabolismo , Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Adenosina Trifosfatases/química , Animais , Citoesqueleto/metabolismo , Técnicas de Silenciamento de Genes , Cones de Crescimento/metabolismo , Humanos , Larva/metabolismo , Locomoção , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios Motores/metabolismo , Proteínas Nucleares/química , Polimerização , Isoformas de Proteínas/metabolismo , Medula Espinal/metabolismoRESUMO
Mechanisms coordinating endosomal degradation and recycling are poorly understood, as are the cellular roles of microtubule (MT) severing. We show that cells lacking the MT-severing protein spastin had increased tubulation of and defective receptor sorting through endosomal tubular recycling compartments. Spastin required the ability to sever MTs and to interact with ESCRT-III (a complex controlling cargo degradation) proteins to regulate endosomal tubulation. Cells lacking IST1 (increased sodium tolerance 1), an endosomal sorting complex required for transport (ESCRT) component to which spastin binds, also had increased endosomal tubulation. Our results suggest that inclusion of IST1 into the ESCRT complex allows recruitment of spastin to promote fission of recycling tubules from the endosome. Thus, we reveal a novel cellular role for MT severing and identify a mechanism by which endosomal recycling can be coordinated with the degradative machinery. Spastin is mutated in the axonopathy hereditary spastic paraplegia. Zebrafish spinal motor axons depleted of spastin or IST1 also had abnormal endosomal tubulation, so we propose this phenotype is important for axonal degeneration.
Assuntos
Adenosina Trifosfatases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/metabolismo , Microtúbulos/metabolismo , Proteínas Oncogênicas/metabolismo , Adenosina Trifosfatases/química , Animais , Células Cultivadas , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Células HeLa , Humanos , Proteínas Oncogênicas/química , Espastina , Peixe-ZebraRESUMO
Mutations in SPG4, encoding the microtubule-severing protein spastin, are responsible for the most frequent form of hereditary spastic paraplegia (HSP), a heterogeneous group of genetic diseases characterized by degeneration of the corticospinal tracts. We previously reported that mice harboring a deletion in Spg4, generating a premature stop codon, develop progressive axonal degeneration characterized by focal axonal swellings associated with impaired axonal transport. To further characterize the molecular and cellular mechanisms underlying this mutant phenotype, we have assessed microtubule dynamics and axonal transport in primary cultures of cortical neurons from spastin-mutant mice. We show an early and marked impairment of microtubule dynamics all along the axons of spastin-deficient cortical neurons, which is likely to be responsible for the occurrence of axonal swellings and cargo stalling. Our analysis also reveals that a modulation of microtubule dynamics by microtubule-targeting drugs rescues the mutant phenotype of cortical neurons. Together, these results contribute to a better understanding of the pathogenesis of SPG4-linked HSP and ascertain the influence of microtubule-targeted drugs on the early axonal phenotype in a mouse model of the disease.
Assuntos
Adenosina Trifosfatases/deficiência , Adenosina Trifosfatases/genética , Animais , Transporte Axonal , Axônios/efeitos dos fármacos , Axônios/patologia , Células Cultivadas , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Modelos Animais de Doenças , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Camundongos , Camundongos Knockout , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Modelos Neurológicos , Mutação , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Nocodazol/farmacologia , Paclitaxel/farmacologia , Paraplegia Espástica Hereditária/tratamento farmacológico , Paraplegia Espástica Hereditária/genética , Paraplegia Espástica Hereditária/metabolismo , Paraplegia Espástica Hereditária/patologia , Espastina , Vimblastina/farmacologiaRESUMO
To better understand hereditary spastic paraplegia (HSP), we characterized the function of atlastin, a protein that is frequently involved in juvenile forms of HSP, by analyzing loss- and gain-of-function phenotypes in the developing zebrafish. We found that knockdown of the gene for atlastin (atl1) caused a severe decrease in larval mobility that was preceded by abnormal architecture of spinal motor axons and was associated with a substantial upregulation of the bone morphogenetic protein (BMP) signaling pathway. Overexpression analyses confirmed that atlastin inhibits BMP signaling. In primary cultures of zebrafish spinal neurons, Atlastin partially colocalized with type I BMP receptors in late endosomes distributed along neurites, which suggests that atlastin may regulate BMP receptor trafficking. Finally, genetic or pharmacological inhibition of BMP signaling was sufficient to rescue the loss of mobility and spinal motor axon defects of atl1 morphants, emphasizing the importance of fine-tuning the balance of BMP signaling for vertebrate motor axon architecture and stability.
Assuntos
Axônios/fisiologia , Proteínas Morfogenéticas Ósseas/metabolismo , Movimento Celular/fisiologia , Neurônios Motores/citologia , Transdução de Sinais/fisiologia , Medula Espinal/citologia , Animais , Animais Geneticamente Modificados , Comportamento Animal , Proteínas Morfogenéticas Ósseas/genética , Células Cultivadas , Embrião não Mamífero , Endossomos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Glicoproteínas/genética , Proteínas de Fluorescência Verde/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Larva , RNA Mensageiro/fisiologia , Transdução de Sinais/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Tubulina (Proteína)/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Spinal muscular atrophy (SMA) is characterized by degeneration of lower motor neurons and caused by mutations of the SMN1 gene. SMN1 is duplicated in a homologous gene called SMN2, which remains present in patients. SMN has an essential role in RNA metabolism, but its role in SMA pathogenesis remains unknown. Previous studies suggested that in neurons the protein lacking the C terminus (SMN(Delta7)), the major product of the SMN2 gene, had a dominant-negative effect. We generated antibodies specific to SMN(FL) or SMN(Delta7). In transfected cells, the stability of the SMN(Delta7) protein was regulated in a cell-dependent manner. Importantly, whatever the human tissues examined, SMN(Delta7) protein was undetectable because of the instability of the protein, thus excluding a dominant effect of SMN(Delta7) in SMA. A similar decreased level of SMN(FL) was observed in brain and spinal cord samples from human SMA, suggesting that SMN(FL) may have specific targets in motor neurons. Moreover, these data indicate that the vulnerability of motor neurons cannot simply be ascribed to the differential expression or a more dramatic reduction of SMN(FL) in spinal cord when compared with brain tissue. Improving the stability of SMN(Delta7) protein might be envisaged as a new therapeutic strategy in SMA.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Atrofia Muscular Espinal/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Anticorpos/imunologia , Linhagem Celular , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/análise , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/genética , Neurônios/química , Proteínas de Ligação a RNA/análise , Proteínas de Ligação a RNA/genética , Proteínas do Complexo SMN , Deleção de Sequência , Proteína 1 de Sobrevivência do Neurônio Motor , Proteína 2 de Sobrevivência do Neurônio MotorRESUMO
Mutations of the spastin gene (Sp) are responsible for the most frequent autosomal dominant form of spastic paraplegia, a disease characterized by the degeneration of corticospinal tracts. We show that a deletion in the mouse Sp gene, generating a premature stop codon, is responsible for progressive axonal degeneration, restricted to the central nervous system, leading to a late and mild motor defect. The degenerative process is characterized by focal axonal swellings, associated with abnormal accumulation of organelles and cytoskeletal components. In culture, mutant cortical neurons showed normal viability and neurite density. However, they develop neurite swellings associated with focal impairment of retrograde transport. These defects occur near the growth cone, in a region characterized by the transition between stable microtubules rich in detyrosinated alpha-tubulin and dynamic microtubules composed almost exclusively of tyrosinated alpha-tubulin. Here, we show that the Sp mutation has a major impact on neurite maintenance and transport both in vivo and in vitro. These results highlight the link between spastin and microtubule dynamics in axons, but not in other neuronal compartments. In addition, it is the first description of a human neurodegenerative disease which involves this specialized region of the axon.