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1.
J Cell Biol ; 223(6)2024 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-38568173

RESUMO

Disruption of synapse assembly and maturation leads to a broad spectrum of neurodevelopmental disorders. Presynaptic proteins are largely synthesized in the soma, where they are packaged into precursor vesicles and transported into distal axons to ensure precise assembly and maintenance of presynapses. Due to their morphological features, neurons face challenges in the delivery of presynaptic cargos to nascent boutons. Thus, targeted axonal transport is vital to build functional synapses. A growing number of mutations in genes encoding the transport machinery have been linked to neurodevelopmental disorders. Emerging lines of evidence have started to uncover presynaptic mechanisms underlying axonal transport defects, thus broadening the view of neurodevelopmental disorders beyond postsynaptic mechanisms. In this review, we discuss presynaptic perspectives of neurodevelopmental disorders by focusing on impaired axonal transport and disturbed assembly and maintenance of presynapses. We also discuss potential strategies for restoring axonal transport as an early therapeutic intervention.


Assuntos
Transporte Axonal , Transtornos do Neurodesenvolvimento , Terminações Pré-Sinápticas , Humanos , Axônios , Corpo Celular , Mutação , Transtornos do Neurodesenvolvimento/genética
2.
J Vis Exp ; (204)2024 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-38436410

RESUMO

Axonal transport is a prerequisite to deliver axonal proteins from their site of synthesis in the neuronal cell body to their destination in the axon. Consequently, loss of axonal transport impairs neuronal growth and function. Studying axonal transport therefore improves our understanding of neuronal cell biology. With recent improvements in CRISPR Cas9 genome editing, endogenous labeling of axonal cargos has become accessible, enabling to move beyond ectopic expression-based visualization of transport. However, endogenous labeling often comes at the cost of low signal intensity and necessitates optimization strategies to obtain robust data. Here, we describe a protocol to optimize the visualization of axonal transport by discussing acquisition parameters and a bleaching approach to improve the signal of endogenous labeled cargo over diffuse cytoplasmic background. We apply our protocol to optimize the visualization of synaptic vesicle precursors (SVPs) labeled by green fluorescent protein (GFP)-tagged RAB-3 to highlight how fine-tuning acquisition parameters can improve the analysis of endogenously labeled axonal cargo in Caenorhabditis elegans (C. elegans).


Assuntos
Transporte Axonal , Caenorhabditis elegans , Corantes Verde de Lissamina , Animais , Axônios , Microscopia de Fluorescência
3.
J Cell Biol ; 223(6)2024 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-38512027

RESUMO

Gain-of-function mutations in the LRRK2 gene cause Parkinson's disease (PD), characterized by debilitating motor and non-motor symptoms. Increased phosphorylation of a subset of RAB GTPases by LRRK2 is implicated in PD pathogenesis. We find that increased phosphorylation of RAB3A, a cardinal synaptic vesicle precursor (SVP) protein, disrupts anterograde axonal transport of SVPs in iPSC-derived human neurons (iNeurons) expressing hyperactive LRRK2-p.R1441H. Knockout of the opposing protein phosphatase 1H (PPM1H) in iNeurons phenocopies this effect. In these models, the compartmental distribution of synaptic proteins is altered; synaptophysin and synaptobrevin-2 become sequestered in the neuronal soma with decreased delivery to presynaptic sites along the axon. We find that RAB3A phosphorylation disrupts binding to the motor adaptor MADD, potentially preventing the formation of the RAB3A-MADD-KIF1A/1Bß complex driving anterograde SVP transport. RAB3A hyperphosphorylation also disrupts interactions with RAB3GAP and RAB-GDI1. Our results reveal a mechanism by which pathogenic hyperactive LRRK2 may contribute to the altered synaptic homeostasis associated with characteristic non-motor and cognitive manifestations of PD.


Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Doença de Parkinson , Vesículas Sinápticas , Proteína rab3A de Ligação ao GTP , Humanos , Transporte Axonal , Axônios , Homeostase , Cinesinas , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Doença de Parkinson/genética , Fosforilação , Proteína rab3A de Ligação ao GTP/genética
4.
J Cell Biol ; 223(5)2024 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-38470363

RESUMO

Mitochondria transport is crucial for axonal mitochondria distribution and is mediated by kinesin-1-based anterograde and dynein-based retrograde motor complexes. While Miro and Milton/TRAK were identified as key adaptors between mitochondria and kinesin-1, recent studies suggest the presence of additional mechanisms. In C. elegans, ric-7 is the only single gene described so far, other than kinesin-1, that is absolutely required for axonal mitochondria localization. Using CRISPR engineering in C. elegans, we find that Miro is important but is not essential for anterograde traffic, whereas it is required for retrograde traffic. Both the endogenous RIC-7 and kinesin-1 act at the leading end to transport mitochondria anterogradely. RIC-7 binding to mitochondria requires its N-terminal domain and partially relies on MIRO-1, whereas RIC-7 accumulation at the leading end depends on its disordered region, kinesin-1, and metaxin2. We conclude that transport complexes containing kinesin-1 and RIC-7 polarize at the leading edge of mitochondria and are required for anterograde axonal transport in C. elegans.


Assuntos
Transporte Axonal , Cinesinas , Animais , Axônios , Caenorhabditis elegans/citologia , Caenorhabditis elegans/metabolismo , Cinesinas/metabolismo , Mitocôndrias/metabolismo
5.
J Cell Biol ; 223(5)2024 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-38407313

RESUMO

Axonal transport is essential for neuronal survival. This is driven by microtubule motors including dynein, which transports cargo from the axon tip back to the cell body. This function requires its cofactor dynactin and regulators LIS1 and NDEL1. Due to difficulties imaging dynein at a single-molecule level, it is unclear how this motor and its regulators coordinate transport along the length of the axon. Here, we use a neuron-inducible human stem cell line (NGN2-OPTi-OX) to endogenously tag dynein components and visualize them at a near-single molecule regime. In the retrograde direction, we find that dynein and dynactin can move the entire length of the axon (>500 µm). Furthermore, LIS1 and NDEL1 also undergo long-distance movement, despite being mainly implicated with the initiation of dynein transport. Intriguingly, in the anterograde direction, dynein/LIS1 moves faster than dynactin/NDEL1, consistent with transport on different cargos. Therefore, neurons ensure efficient transport by holding dynein/dynactin on cargos over long distances but keeping them separate until required.


Assuntos
Transporte Axonal , Axônios , Complexo Dinactina , Dineínas , Neurônios , Humanos , Complexo Dinactina/genética , Dineínas/genética , Células-Tronco Neurais
6.
Mol Neurodegener ; 19(1): 13, 2024 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-38282024

RESUMO

BACKGROUND: Bioenergetic maladaptations and axonopathy are often found in the early stages of neurodegeneration. Nicotinamide adenine dinucleotide (NAD), an essential cofactor for energy metabolism, is mainly synthesized by Nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2) in CNS neurons. NMNAT2 mRNA levels are reduced in the brains of Alzheimer's, Parkinson's, and Huntington's disease. Here we addressed whether NMNAT2 is required for axonal health of cortical glutamatergic neurons, whose long-projecting axons are often vulnerable in neurodegenerative conditions. We also tested if NMNAT2 maintains axonal health by ensuring axonal ATP levels for axonal transport, critical for axonal function. METHODS: We generated mouse and cultured neuron models to determine the impact of NMNAT2 loss from cortical glutamatergic neurons on axonal transport, energetic metabolism, and morphological integrity. In addition, we determined if exogenous NAD supplementation or inhibiting a NAD hydrolase, sterile alpha and TIR motif-containing protein 1 (SARM1), prevented axonal deficits caused by NMNAT2 loss. This study used a combination of techniques, including genetics, molecular biology, immunohistochemistry, biochemistry, fluorescent time-lapse imaging, live imaging with optical sensors, and anti-sense oligos. RESULTS: We provide in vivo evidence that NMNAT2 in glutamatergic neurons is required for axonal survival. Using in vivo and in vitro studies, we demonstrate that NMNAT2 maintains the NAD-redox potential to provide "on-board" ATP via glycolysis to vesicular cargos in distal axons. Exogenous NAD+ supplementation to NMNAT2 KO neurons restores glycolysis and resumes fast axonal transport. Finally, we demonstrate both in vitro and in vivo that reducing the activity of SARM1, an NAD degradation enzyme, can reduce axonal transport deficits and suppress axon degeneration in NMNAT2 KO neurons. CONCLUSION: NMNAT2 ensures axonal health by maintaining NAD redox potential in distal axons to ensure efficient vesicular glycolysis required for fast axonal transport.


Assuntos
Transporte Axonal , NAD , Nicotinamida-Nucleotídeo Adenililtransferase , Animais , Camundongos , Trifosfato de Adenosina/metabolismo , Proteínas do Domínio Armadillo/metabolismo , Axônios/metabolismo , Proteínas do Citoesqueleto/metabolismo , Glicólise , Homeostase , NAD/metabolismo , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo
7.
Cells ; 13(1)2024 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-38201307

RESUMO

Autophagy is a major degradative pathway that plays a key role in sustaining cell homeostasis, integrity, and physiological functions. Macroautophagy, which ensures the clearance of cytoplasmic components engulfed in a double-membrane autophagosome that fuses with lysosomes, is orchestrated by a complex cascade of events. Autophagy has a particularly strong impact on the nervous system, and mutations in core components cause numerous neurological diseases. We first review the regulation of autophagy, from autophagosome biogenesis to lysosomal degradation and associated neurodevelopmental/neurodegenerative disorders. We then describe how this process is specifically regulated in the axon and in the somatodendritic compartment and how it is altered in diseases. In particular, we present the neuronal specificities of autophagy, with the spatial control of autophagosome biogenesis, the close relationship of maturation with axonal transport, and the regulation by synaptic activity. Finally, we discuss the physiological functions of autophagy in the nervous system, during development and in adulthood.


Assuntos
Autofagia , Macroautofagia , Autofagossomos , Transporte Axonal , Lisossomos
8.
Commun Biol ; 7(1): 57, 2024 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-38191649

RESUMO

The lemur family of protein kinases has gained much interest in recent years as they are involved in a variety of cellular processes including regulation of axonal transport and endosomal trafficking, modulation of synaptic functions, memory and learning, and they are centrally placed in several intracellular signalling pathways. Numerous studies have also implicated role of the lemur kinases in the development and progression of a wide range of cancers, cystic fibrosis, and neurodegenerative diseases. However, parallel discoveries and inaccurate prediction of their kinase activity have resulted in a confusing and misleading nomenclature of these proteins. Herein, a group of international scientists with expertise in lemur family of protein kinases set forth a novel nomenclature to rectify this problem and ultimately help the scientific community by providing consistent information about these molecules.


Assuntos
Fibrose Cística , Lemur , Animais , Proteínas Quinases , Fosforilação , Transporte Axonal
9.
Cytoskeleton (Hoboken) ; 81(1): 47-52, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37694806

RESUMO

Tau is a microtubule-associated protein that is enriched in the axonal process of neurons. Post-translational modifications of tau have been implicated in the development of tauopathies characterized by defects in axonal transport, neuronal atrophy, and microtubule disassembly. Although tau is almost quantitatively bound to microtubules under physiological conditions, it does not significantly affect axonal transport. Furthermore, acute or chronic tau deficiency does not result in significant destabilization of neuronal microtubules, challenging the classical view that disease-related tau modifications directly cause axonal microtubule collapse. Here, we discuss how the rapid interaction kinetics of the tau-microtubule interaction, which we previously termed the kiss-and-hop interaction, explains why tau does not affect microtubule-dependent axonal transport but still allows tau to modulate microtubule polymerization. In contrast, tau modifications that slow down the kinetics of the tau-microtubule interaction and increase the residence time of tau at a microtubule interaction site can disrupt axonal transport and cause dendritic atrophy. We discuss the consequences of such a gain-of-toxicity mechanism in terms of the development of disease-modulating drugs that target the tau protein.


Assuntos
Tauopatias , Proteínas tau , Humanos , Transporte Axonal , Microtúbulos/metabolismo , Tauopatias/metabolismo , Atrofia/metabolismo
10.
Biochem Biophys Res Commun ; 691: 149246, 2024 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-38029540

RESUMO

Huntington's disease (HD) is a progressive genetic neurodegenerative disease caused by an abnormal expansion of a cytosine-adenine-guanine trinucleotide repeat in the huntingtin gene. One pathological feature of HD is neuronal loss in the striatum. Despite many efforts, mechanisms underlying neuronal loss in HD striatum remain elusive. It was suggested that the mutant huntingtin protein interacts mitochondrial proteins and causes mitochondrial dysfunction in striatal neurons. However, whether axonal transport of mitochondria is altered in HD striatal neurons remains controversial. Here, we examined axonal transport of single mitochondria labelled with Mito-DsRed2 in cultured striatal neurons of zQ175 knock-in mice (a knock-in mouse model of HD). We observed decreased anterograde axonal transport of proximal mitochondria in HD striatal neurons compared with wild-type (WT) striatal neurons. Decreased anterograde transport in HD striatal neurons was prevented by overexpressing mitochondrial Rho GTPase 1 (Miro1). Our results offer a new insight into mechanisms underlying neuronal loss in the striatum in HD.


Assuntos
Doença de Huntington , Doenças Neurodegenerativas , Camundongos , Animais , Doença de Huntington/metabolismo , Transporte Axonal , Camundongos Transgênicos , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Mitocôndrias/metabolismo , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo
11.
Cytoskeleton (Hoboken) ; 81(1): 10-15, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37578198

RESUMO

Over the last 50 years the different isoforms of tau proteins (45-60 kDa) have been a focus of research because of their roles in modulating the dynamic properties of microtubules shaping the structure and function of neurons but also becoming a center of attention in the pathology of neurodegeneration associated with tauopathies. Much less attention has been given to Big tau, a unique isoform containing exon 4a encoding about 250 amino acids to form a much longer projection domain of a protein of 110 kDa. Big tau is expressed in peripheral neurons and selective regions of the central nervous system in a defined transition during postnatal developmental stages. Although Big tau was discovered 30 years ago, there has been a persistent gap of knowledge regarding its physiological properties and pathological implications. This Perspective summarizes the progress so far in defining the structure and expression of Big tau within and outside the nervous system, proposes a role for Big tau in improving axonal transport in projecting axons, considers its potential in averting tau aggregation in tauopathies and highlights the need for further progress.


Assuntos
Tauopatias , Proteínas tau , Humanos , Proteínas tau/genética , Proteínas tau/química , Tauopatias/genética , Tauopatias/metabolismo , Tauopatias/patologia , Axônios , Neurônios/metabolismo , Transporte Axonal/fisiologia
12.
J Cell Biol ; 222(12)2023 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-37909920

RESUMO

Neuronal autophagosomes form and engulf cargos at presynaptic sites in the axon and are then transported to the soma to recycle their cargo. Autophagic vacuoles (AVs) mature en route via fusion with lysosomes to become degradatively competent organelles; transport is driven by the microtubule motor protein cytoplasmic dynein, with motor activity regulated by a sequential series of adaptors. Using lysate-based single-molecule motility assays and live-cell imaging in primary neurons, we show that JNK-interacting proteins 3 (JIP3) and 4 (JIP4) are activating adaptors for dynein that are regulated on autophagosomes and lysosomes by the small GTPases ARF6 and RAB10. GTP-bound ARF6 promotes formation of the JIP3/4-dynein-dynactin complex. Either knockdown or overexpression of RAB10 stalls transport, suggesting that this GTPase is also required to coordinate the opposing activities of bound dynein and kinesin motors. These findings highlight the complex coordination of motor regulation during organelle transport in neurons.


Assuntos
Autofagossomos , Transporte Axonal , Dineínas , Axônios , Dineínas/genética , Cinesinas , Proteínas rab de Ligação ao GTP/genética
13.
J Math Biol ; 88(1): 1, 2023 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-38006409

RESUMO

In this study, we consider axonal transport of large cargo vesicles characterised by transient expansion of the axon shaft. Our goal is to formulate a mathematical model which captures the dynamic mechanical interaction of such cargo vesicles with the membrane associated periodic cytoskeletal structure (MPS). It consists of regularly spaced actin rings that are transversal to the longitudinal direction of the axon and involved in the radial contraction of the axon. A system of force balance equations is formulated by which we describe the transversal rings as visco-elastic Kelvin-Voigt elements. In a homogenisation limit, we reformulate the model as a free boundary problem for the interaction of the submembranous MPS with the large vesicle. We derive a non-linear force-velocity relation as a quasi-steady state solution. Computationally we analyse the vesicle size dependence of the transport speed and use an asymptotic approximation to formulate it as a power law that can be tested experimentally.


Assuntos
Transporte Axonal , Axônios , Axônios/metabolismo , Actinas/metabolismo , Modelos Biológicos
14.
Cells ; 12(19)2023 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-37830617

RESUMO

The amyloid precursor protein (APP) is a key molecular component of Alzheimer's disease (AD) pathogenesis. Proteolytic APP processing generates various cleavage products, including extracellular amyloid beta (Aß) and the cytoplasmic APP intracellular domain (AICD). Although the role of AICD in the activation of kinase signaling pathways is well established in the context of full-length APP, little is known about intracellular effects of the AICD fragment, particularly within discrete neuronal compartments. Deficits in fast axonal transport (FAT) and axonopathy documented in AD-affected neurons prompted us to evaluate potential axon-autonomous effects of the AICD fragment for the first time. Vesicle motility assays using the isolated squid axoplasm preparation revealed inhibition of FAT by AICD. Biochemical experiments linked this effect to aberrant activation of selected axonal kinases and heightened phosphorylation of the anterograde motor protein conventional kinesin, consistent with precedents showing phosphorylation-dependent regulation of motors proteins powering FAT. Pharmacological inhibitors of these kinases alleviated the AICD inhibitory effect on FAT. Deletion experiments indicated this effect requires a sequence encompassing the NPTY motif in AICD and interacting axonal proteins containing a phosphotyrosine-binding domain. Collectively, these results provide a proof of principle for axon-specific effects of AICD, further suggesting a potential mechanistic framework linking alterations in APP processing, FAT deficits, and axonal pathology in AD.


Assuntos
Doença de Alzheimer , Precursor de Proteína beta-Amiloide , Humanos , Precursor de Proteína beta-Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Transporte Axonal , Doença de Alzheimer/metabolismo , Axônios/metabolismo
15.
eNeuro ; 10(10)2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37816595

RESUMO

Lysosomes are acidic organelles that traffic throughout neurons delivering catabolic enzymes to distal regions of the cell and maintaining degradative demands. Loss of function mutations in the gene GBA encoding the lysosomal enzyme glucocerebrosidase (GCase) cause the lysosomal storage disorder Gaucher's disease (GD) and are the most common genetic risk factor for synucleinopathies like Parkinson's disease (PD) and dementia with Lewy bodies (DLB). GCase degrades the membrane lipid glucosylceramide (GlcCer) and mutations in GBA, or inhibiting its activity, results in the accumulation of GlcCer and disturbs the composition of the lysosomal membrane. The lysosomal membrane serves as the platform to which intracellular trafficking complexes are recruited and activated. Here, we investigated whether lysosomal trafficking in axons was altered by inhibition of GCase with the pharmacological agent Conduritol B Epoxide (CBE). Using live cell imaging in human male induced pluripotent human stem cell (iPSC)-derived forebrain neurons, we demonstrated that lysosomal transport was similar in both control and CBE-treated neurons. Furthermore, we tested whether lysosomal rupture, a process implicated in various neurodegenerative disorders, was affected by inhibition of GCase. Using L-leucyl-L-leucine methyl ester (LLoME) to induce lysosomal membrane damage and immunocytochemical staining for markers of lysosomal rupture, we found no difference in susceptibility to rupture between control and CBE-treated neurons. These results suggest the loss of GCase activity does not contribute to neurodegenerative disease by disrupting either lysosomal transport or rupture.


Assuntos
Células-Tronco Pluripotentes Induzidas , Doenças Neurodegenerativas , Masculino , Humanos , Glucosilceramidase/genética , Glucosilceramidase/metabolismo , Doenças Neurodegenerativas/metabolismo , Transporte Axonal , Neurônios/metabolismo , Prosencéfalo/metabolismo , Lisossomos/metabolismo , alfa-Sinucleína/metabolismo
16.
Science ; 382(6667): 223-230, 2023 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-37824668

RESUMO

Neurons relay information via specialized presynaptic compartments for neurotransmission. Unlike conventional organelles, the specialized apparatus characterizing the neuronal presynapse must form de novo. How the components for presynaptic neurotransmission are transported and assembled is poorly understood. Our results show that the rare late endosomal signaling lipid phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] directs the axonal cotransport of synaptic vesicle and active zone proteins in precursor vesicles in human neurons. Precursor vesicles are distinct from conventional secretory organelles, endosomes, and degradative lysosomes and are transported by coincident detection of PI(3,5)P2 and active ARL8 via kinesin KIF1A to the presynaptic compartment. Our findings identify a crucial mechanism that mediates the delivery of synaptic vesicle and active zone proteins to developing synapses.


Assuntos
Transporte Axonal , Neurônios , Fosfatos de Fosfatidilinositol , Vesículas Sinápticas , Humanos , Transporte Axonal/fisiologia , Cinesinas/metabolismo , Neurônios/metabolismo , Vesículas Sinápticas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo
17.
Int J Numer Method Biomed Eng ; 39(12): e3770, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37688421

RESUMO

Recent publications report that although the mitochondria population in an axon can be quickly replaced by a combination of retrograde and anterograde axonal transport (often within less than 24 hours), the axon contains much older mitochondria. This suggests that not all mitochondria that reach the soma are degraded and that some are recirculating back into the axon. To explain this, we developed a model that simulates mitochondria distribution when a portion of mitochondria that return to the soma are redirected back to the axon rather than being destroyed in somatic lysosomes. Utilizing the developed model, we studied how the percentage of returning mitochondria affects the mean age and age density distributions of mitochondria at different distances from the soma. We also investigated whether turning off the mitochondrial anchoring switch can reduce the mean age of mitochondria. For this purpose, we studied the effect of reducing the value of a parameter that characterizes the probability of mitochondria transition to the stationary (anchored) state. The reduction in mitochondria mean age observed when the anchoring probability is reduced suggests that some injured neurons may be saved if the percentage of stationary mitochondria is decreased. The replacement of possibly damaged stationary mitochondria with newly synthesized ones may restore the energy supply in an injured axon. We also performed a sensitivity study of the mean age of stationary mitochondria to the parameter that determines what portion of mitochondria re-enter the axon and the parameter that determines the probability of mitochondria transition to the stationary state. The sensitivity of the mean age of stationary mitochondria to the mitochondria stopping probability increases linearly with the number of compartments in the axon. High stopping probability in long axons can significantly increase mitochondrial age.


Assuntos
Axônios , Neurônios , Axônios/fisiologia , Neurônios/metabolismo , Mitocôndrias/metabolismo , Transporte Axonal/fisiologia
18.
Neurosci Res ; 197: 25-30, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37734449

RESUMO

Kinesin motor proteins play crucial roles in anterograde transport of cargo vesicles in neurons, moving them along axons from the cell body towards the synaptic region. Not only the transport force and velocity of single motor protein, but also the number of kinesin molecules involved in transporting a specific cargo, is pivotal for synapse formation. This collective transport by multiple kinesins ensures stable and efficient cargo transport in neurons. Abnormal increases or decreases in the number of engaged kinesin molecules per cargo could potentially act as biomarkers for neurodegenerative diseases such as Alzheimer's, Parkinson's, amyotrophic lateral sclerosis (ALS), spastic paraplegia, polydactyly syndrome, and virus transport disorders. We review here a model constructed using physical measurements to quantify the number of kinesin molecules associated with their cargo, which could shed light on the molecular mechanisms of neurodegenerative diseases related to axonal transport.


Assuntos
Esclerose Amiotrófica Lateral , Cinesinas , Humanos , Cinesinas/metabolismo , Transporte Axonal/fisiologia , Axônios/metabolismo , Dineínas/metabolismo , Esclerose Amiotrófica Lateral/metabolismo
19.
Dev Cell ; 58(19): 1847-1863.e12, 2023 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-37751746

RESUMO

An actin-spectrin lattice, the membrane periodic skeleton (MPS), protects axons from breakage. MPS integrity relies on spectrin delivery via slow axonal transport, a process that remains poorly understood. We designed a probe to visualize endogenous spectrin dynamics at single-axon resolution in vivo. Surprisingly, spectrin transport is bimodal, comprising fast runs and movements that are 100-fold slower than previously reported. Modeling and genetic analysis suggest that the two rates are independent, yet both require kinesin-1 and the coiled-coil proteins UNC-76/FEZ1 and UNC-69/SCOC, which we identify as spectrin-kinesin adaptors. Knockdown of either protein led to disrupted spectrin motility and reduced distal MPS, and UNC-76 overexpression instructed excessive transport of spectrin. Artificially linking spectrin to kinesin-1 drove robust motility but inefficient MPS assembly, whereas impairing MPS assembly led to excessive spectrin transport, suggesting a balance between transport and assembly. These results provide insight into slow axonal transport and MPS integrity.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Espectrina , Animais , Transporte Axonal , Axônios/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Cinesinas/metabolismo , Espectrina/metabolismo
20.
Mol Biol Cell ; 34(11): ar110, 2023 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-37585286

RESUMO

Alcadein α (Alcα) and amyloid-ß protein precursor (APP) are cargo receptors that associate vesicles with kinesin-1. These vesicles, which contain either Alcα or APP, transport various proteins/cargo molecules into axon nerve terminals. Here, we analyzed immune-isolated Alcα- and APP-containing vesicles of adult mouse brains with LC-MS/MS and identified proteins present in vesicles that contained either Alcα or APP. Among these proteins, Frizzled-5 (Fzd5), a Wnt receptor, was detected mainly in Alcα vesicles. Although colocalization ratios of Fzd5 with Alcα are low in the neurites of differentiating neurons by a low expression of Fzd5 in embryonic brains, the suppression of Alcα expression decreased the localization of Fzd5 in neurites of primary cultured neurons. Furthermore, Fzd5-EGFP expressed in primary cultured neurons was preferentially transported in axons with the transport velocities of Alcα vesicles. In synaptosomal fractions of adult-mice brains that express higher levels of Fzd5, the amount of Fzd5 and the phosphorylation level of calcium/calmodulin-dependent protein kinase-II were reduced in the Alcα-deficient mice. These results suggest that reduced transport of Fzd5 by Alcα-containing vesicles associated with kinesin-1 in axon terminals may impair the response to Wnt ligands in the noncanonical Ca2+-dependent signal transduction pathway at nerve terminals of mature neurons.


Assuntos
Transporte Axonal , Cinesinas , Animais , Camundongos , Precursor de Proteína beta-Amiloide/metabolismo , Transporte Axonal/fisiologia , Cromatografia Líquida , Cinesinas/metabolismo , Espectrometria de Massas em Tandem
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