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1.
Nat Commun ; 11(1): 6131, 2020 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-33257677

RESUMO

After a dorsal root crush injury, centrally-projecting sensory axons fail to regenerate across the dorsal root entry zone (DREZ) to extend into the spinal cord. We find that chemogenetic activation of adult dorsal root ganglion (DRG) neurons improves axon growth on an in vitro model of the inhibitory environment after injury. Moreover, repeated bouts of daily chemogenetic activation of adult DRG neurons for 12 weeks post-crush in vivo enhances axon regeneration across a chondroitinase-digested DREZ into spinal gray matter, where the regenerating axons form functional synapses and mediate behavioral recovery in a sensorimotor task. Neuronal activation-mediated axon extension is dependent upon changes in the status of tubulin post-translational modifications indicative of highly dynamic microtubules (as opposed to stable microtubules) within the distal axon, illuminating a novel mechanism underlying stimulation-mediated axon growth. We have identified an effective combinatory strategy to promote functionally-relevant axon regeneration of adult neurons into the CNS after injury.

2.
Trends Neurosci ; 43(7): 493-504, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32434664

RESUMO

Tau, a microtubule-associated protein that modifies the dynamic properties and organization of microtubules in neurons and affects axonal transport, shows remarkable heterogeneity, with multiple isoforms (45-65 kDa) generated by alternative splicing. A high-molecular-weight (HMW) isoform (110 kDa) that contains an additional large exon termed 4a was discovered more than 25 years ago. This isoform, called Big tau, is expressed mainly in the adult peripheral nervous system (PNS), but also in adult neurons of the central nervous system (CNS) that extend processes into the periphery. Surprisingly little has been learned about Big tau since its initial characterization, leaving a significant gap in knowledge about how the dramatic switch to Big tau affects the properties of neurons in the context of development, disease, or injury. Here we review what was learned about the structure and distribution of Big tau in those earlier studies, and add contemporary insights to resurrect interest in the mysteries of Big tau and thereby set a path for future studies.

3.
Cytoskeleton (Hoboken) ; 77(3-4): 39, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32259406
4.
Cytoskeleton (Hoboken) ; 76(4): 289-297, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-31108029

RESUMO

Mutations of the SPAST gene are the chief cause of hereditary spastic paraplegia. Controversy exists in the medical community as to whether the etiology of the disease is haploinsufficiency or toxic gain-of-function properties of the mutant spastin proteins. In recognition of strong reasons that support each possible mechanism, here we present a novel perspective, based in part on new studies with mouse models and in part on the largest study to date on patients with the disease. We posit that haploinsufficiency does not cause the disease but makes the corticospinal tracts vulnerable to a second hit, which is usually the mutant spastin proteins but could also be proteins generated by mutations of other genes that may or may not cause the disease on their own.


Assuntos
Paraplegia Espástica Hereditária/etiologia , Feminino , Humanos , Masculino
5.
Trends Cell Biol ; 29(6): 452-461, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30929793

RESUMO

Tau is a multifunctional microtubule-associated protein in the neuron. For decades, tau's main function in neurons has been broadly accepted as stabilizing microtubules in the axon; however, this conclusion was reached mainly on the basis of studies performed in vitro and on ectopic expression of tau in non-neuronal cells. The idea has become so prevailing that some disease researchers are even seeking to use microtubule-stabilizing drugs to treat diseases in which tau dissociates from microtubules. Recent work suggests that tau is not a stabilizer of microtubules in the axon, but rather enables axonal microtubules to have long labile domains, in part by outcompeting genuine stabilizers. This new perspective on tau challenges long-standing dogma.


Assuntos
Proteínas tau/metabolismo , Animais , Humanos , Microtúbulos/metabolismo , Neurônios/metabolismo
6.
J Neurosci ; 39(20): 3792-3811, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30804089

RESUMO

KIFC1 (also called HSET or kinesin-14a) is best known as a multifunctional motor protein essential for mitosis. The present studies are the first to explore KIFC1 in terminally postmitotic neurons. Using RNA interference to partially deplete KIFC1 from rat neurons (from animals of either gender) in culture, pharmacologic agents that inhibit KIFC1, and expression of mutant KIFC1 constructs, we demonstrate critical roles for KIFC1 in regulating axonal growth and retraction as well as growth cone morphology. Experimental manipulations of KIFC1 elicit morphological changes in the axon as well as changes in the organization, distribution, and polarity orientation of its microtubules. Together, the results indicate a mechanism by which KIFC1 binds to microtubules in the axon and slides them into alignment in an ATP-dependent fashion and then cross-links them in an ATP-independent fashion to oppose their subsequent sliding by other motors.SIGNIFICANCE STATEMENT Here, we establish that KIFC1, a molecular motor well characterized in mitosis, is robustly expressed in neurons, where it has profound influence on the organization of microtubules in a number of different functional contexts. KIFC1 may help answer long-standing questions in cellular neuroscience such as, mechanistically, how growth cones stall and how axonal microtubules resist forces that would otherwise cause the axon to retract. Knowledge about KIFC1 may help researchers to devise strategies for treating disorders of the nervous system involving axonal retraction given that KIFC1 is expressed in adult neurons as well as developing neurons.


Assuntos
Axônios/fisiologia , Microtúbulos/fisiologia , Mitose/fisiologia , beta Carioferinas/fisiologia , Animais , Células Cultivadas , Feminino , Cones de Crescimento/fisiologia , Masculino , Ratos Sprague-Dawley
7.
J Neurosci ; 39(11): 2011-2024, 2019 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-30647150

RESUMO

Fidgetin is a microtubule-severing protein that pares back the labile domains of microtubules in the axon. Experimental depletion of fidgetin results in elongation of the labile domains of microtubules and faster axonal growth. To test whether fidgetin knockdown assists axonal regeneration, we plated dissociated adult rat DRGs transduced using AAV5-shRNA-fidgetin on a laminin substrate with spots of aggrecan, a growth-inhibitory chondroitin sulfate proteoglycan. This cell culture assay mimics the glial scar formed after CNS injury. Aggrecan is more concentrated at the edge of the spot, such that axons growing from within the spot toward the edge encounter a concentration gradient that causes growth cones to become dystrophic and axons to retract or curve back on themselves. Fidgetin knockdown resulted in faster-growing axons on both laminin and aggrecan and enhanced crossing of axons from laminin onto aggrecan. Strikingly, axons from within the spot grew more avidly against the inhibitory aggrecan concentration gradient to cross onto laminin, without retracting or curving back. We also tested whether depleting fidgetin improves axonal regeneration in vivo after a dorsal root crush in adult female rats. Whereas control DRG neurons failed to extend axons across the dorsal root entry zone after injury, DRG neurons in which fidgetin was knocked down displayed enhanced regeneration of axons across the dorsal root entry zone into the spinal cord. Collectively, these results establish fidgetin as a novel therapeutic target to augment nerve regeneration and provide a workflow template by which microtubule-related targets can be compared in the future.SIGNIFICANCE STATEMENT Here we establish a workflow template from cell culture to animals in which microtubule-based treatments can be tested and compared with one another for their effectiveness in augmenting regeneration of injured axons relevant to spinal cord injury. The present work uses a viral transduction approach to knock down fidgetin from rat neurons, which coaxes nerve regeneration by elevating microtubule mass in their axons. Unlike previous strategies using microtubule-stabilizing drugs, fidgetin knockdown adds microtubule mass that is labile (rather than stable), thereby better recapitulating the growth status of a developing axon.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/fisiologia , Axônios/fisiologia , Gânglios Espinais/fisiologia , Proteínas Associadas aos Microtúbulos/fisiologia , Microtúbulos/fisiologia , Regeneração Nervosa/fisiologia , Proteínas Nucleares/fisiologia , ATPases Associadas a Diversas Atividades Celulares/genética , Agrecanas/fisiologia , Animais , Feminino , Técnicas de Silenciamento de Genes , Masculino , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/genética , Neuroglia/fisiologia , Proteínas Nucleares/genética , Ratos Sprague-Dawley
8.
Hum Mol Genet ; 28(7): 1136-1152, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30520996

RESUMO

Mutations of the SPAST gene, which encodes the microtubule-severing protein spastin, are the most common cause of hereditary spastic paraplegia (HSP). Haploinsufficiency is the prevalent opinion as to the mechanism of the disease, but gain-of-function toxicity of the mutant proteins is another possibility. Here, we report a new transgenic mouse (termed SPASTC448Y mouse) that is not haploinsufficient but expresses human spastin bearing the HSP pathogenic C448Y mutation. Expression of the mutant spastin was documented from fetus to adult, but gait defects reminiscent of HSP (not observed in spastin knockout mice) were adult onset, as is typical of human patients. Results of histological and tracer studies on the mouse are consistent with progressive dying back of corticospinal axons, which is characteristic of the disease. The C448Y-mutated spastin alters microtubule stability in a manner that is opposite to the expectations of haploinsufficiency. Neurons cultured from the mouse display deficits in organelle transport typical of axonal degenerative diseases, and these deficits were worsened by depletion of endogenous mouse spastin. These results on the SPASTC448Y mouse are consistent with a gain-of-function mechanism underlying HSP, with spastin haploinsufficiency exacerbating the toxicity of the mutant spastin proteins. These findings reveal the need for a different therapeutic approach than indicated by haploinsufficiency alone.


Assuntos
Paraplegia Espástica Hereditária/genética , Espastina/genética , Animais , Transporte Axonal/fisiologia , Axônios/metabolismo , Modelos Animais de Doenças , Mutação com Ganho de Função/genética , Haploinsuficiência , Haplótipos , Camundongos , Camundongos Transgênicos , Microtúbulos/metabolismo , Proteínas Mutantes/genética , Mutação , Neurônios/metabolismo , Paraplegia Espástica Hereditária/fisiopatologia , Espastina/fisiologia
9.
Traffic ; 20(1): 71-81, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30411440

RESUMO

KIF15, the vertebrate kinesin-12, is best known as a mitotic motor protein, but continues to be expressed in neurons. Like KIF11 (the vertebrate kinesin-5), KIF15 interacts with microtubules in the axon to limit their sliding relative to one another. Unlike KIF11, KIF15 also regulates interactions between microtubules and actin filaments at sites of axonal branch formation and in growth cones. Our original work on these motors was done on cultured rat neurons, but we are now using zebrafish to extend these studies to an in vivo model. We previously studied kif15 in zebrafish by injecting splice-blocking morpholinos injected into embryos. Consistent with the cell culture work, these studies demonstrated that axons grow faster and longer when KIF15 levels are reduced. In the present study, we applied CRISPR/Cas9-based knockout technology to create kif15 mutants and labeled neurons with Tg(mnx1:GFP) transgene or transient expression of elavl3:EGFP-alpha tubulin. We then compared by live imaging the homozygotic, heterozygotic mutants to their wildtype siblings to ascertain the effects of depletion of kif15 during Caudal primary motor neuron and Rohon-Beard (R-B) sensory neuron development. The results showed, compared to the kif15 wildtype, the number of branches was reduced while axon outgrowth was accelerated in kif15 homozygotic and heterozygotic mutants. In R-B sensory neurons, after laser irradiation, injured axons with loss of kif15 displayed significantly greater regenerative velocity. Given these results and the fact that kif15 drugs are currently under development, we posit kif15 as a novel target for therapeutically augmenting regeneration of injured axons.


Assuntos
Cinesina/genética , Mutação , Regeneração Nervosa , Crescimento Neuronal , Proteínas de Peixe-Zebra/genética , Animais , Sistemas CRISPR-Cas , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Neurônios Motores/fisiologia , Peixe-Zebra
10.
Curr Biol ; 28(13): 2181-2189.e4, 2018 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-30008334

RESUMO

It is widely believed that tau stabilizes microtubules in the axon [1-3] and, hence, that disease-induced loss of tau from axonal microtubules leads to their destabilization [3-5]. An individual microtubule in the axon has a stable domain and a labile domain [6-8]. We found that tau is more abundant on the labile domain, which is inconsistent with tau's proposed role as a microtubule stabilizer. When tau is experimentally depleted from cultured rat neurons, the labile microtubule mass of the axon drops considerably, the remaining labile microtubule mass becomes less labile, and the stable microtubule mass increases. MAP6 (also called stable tubule-only polypeptide), which is normally enriched on the stable domain [9], acquires a broader distribution across the microtubule when tau is depleted, providing a potential explanation for the increase in stable microtubule mass. When MAP6 is depleted, the labile microtubule mass becomes even more labile, indicating that, unlike tau, MAP6 is a genuine stabilizer of axonal microtubules. We conclude that tau is not a stabilizer of axonal microtubules but is enriched on the labile domain of the microtubule to promote its assembly while limiting the binding to it of genuine stabilizers, such as MAP6. This enables the labile domain to achieve great lengths without being stabilized. These conclusions are contrary to tau dogma.


Assuntos
Axônios/metabolismo , Microtúbulos/metabolismo , Proteínas tau/metabolismo , Animais , Células Cultivadas , Ratos , Ratos Sprague-Dawley
11.
Trends Neurosci ; 41(2): 77-88, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29198454

RESUMO

A longstanding question in cellular neuroscience is how microtubules in the axon become organized with their plus ends out, a pattern starkly different from the mixed orientation of microtubules in vertebrate dendrites. Recent attention has focused on a mechanism called polarity sorting, in which microtubules of opposite orientation are spatially separated by molecular motor proteins. Here we discuss this mechanism, and conclude that microtubules are polarity sorted in the axon by cytoplasmic dynein but that additional factors are also needed. In particular, computational modeling and experimental evidence suggest that static crosslinking proteins are required to appropriately restrict microtubule movements so that polarity sorting by cytoplasmic dynein can occur in a manner unimpeded by other motor proteins.


Assuntos
Axônios/metabolismo , Polaridade Celular/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Animais , Movimento Celular/fisiologia , Dendritos/metabolismo , Humanos
12.
Mol Biol Cell ; 28(23): 3271-3285, 2017 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-28978741

RESUMO

We present a computational model to test a "polarity sorting" mechanism for microtubule (MT) organization in developing axons. We simulate the motor-based axonal transport of short MTs to test the hypothesis that immobilized cytoplasmic dynein motors transport short MTs with their plus ends leading, so "mal-oriented" MTs with minus-end-out are transported toward the cell body while "correctly" oriented MTs are transported in the anterograde direction away from the soma. We find that dynein-based transport of short MTs can explain the predominately plus-end-out polarity pattern of axonal MTs but that transient attachments of plus-end-directed motor proteins and nonmotile cross-linker proteins are needed to explain the frequent pauses and occasional reversals observed in live-cell imaging of MT transport. Static cross-linkers increase the likelihood of a stalled "tug-of-war" between retrograde and anterograde forces on the MT, providing an explanation for the frequent pauses of short MTs and the immobility of longer MTs. We predict that inhibition of the proposed static cross-linker will produce disordered transport of short MTs and increased mobility of longer MTs. We also predict that acute inhibition of cytoplasmic dynein will disrupt the polarity sorting of MTs by increasing the likelihood of "incorrect" sorting of MTs by plus-end-directed motors.


Assuntos
Polaridade Celular/fisiologia , Microtúbulos/metabolismo , Transporte Axonal/fisiologia , Axônios/metabolismo , Axônios/fisiologia , Movimento Celular , Células Cultivadas , Simulação por Computador/estatística & dados numéricos , Dineínas do Citoplasma/metabolismo , Dineínas/metabolismo , Cinesina/metabolismo , Microtúbulos/fisiologia , Neurônios/metabolismo , Transporte Proteico/fisiologia
13.
Sci Rep ; 7(1): 9675, 2017 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-28852085

RESUMO

Microtubule-stabilizing drugs have gained popularity for treating injured adult axons, the rationale being that increased stabilization of microtubules will prevent the axon from retracting and fortify it to grow through inhibitory molecules associated with nerve injury. We have posited that a better approach would be not to stabilize the microtubules, but to increase labile microtubule mass to levels more conducive to axonal growth. Recent work on fetal neurons suggests this can be accomplished using RNA interference to reduce the levels of fidgetin, a microtubule-severing protein. Methods to introduce RNA interference into adult neurons, in vitro or in vivo, have been problematic and not translatable to human patients. Here we show that a novel nanoparticle approach, previously shown to deliver siRNA into tissues and organs, enables siRNA to gain entry into adult rat dorsal root ganglion neurons in culture. Knockdown of fidgetin is partial with this approach, but sufficient to increase the labile microtubule mass of the axon, thereby increasing axonal growth. The increase in axonal growth occurs on both a favorable substrate and a growth-inhibitory molecule associated with scar formation in injured spinal cord. The nanoparticles are readily translatable to in vivo studies on animals and ultimately to clinical applications.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/antagonistas & inibidores , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/metabolismo , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Microtúbulos/metabolismo , Nanopartículas/metabolismo , Neurônios/fisiologia , Fármacos Neuroprotetores/metabolismo , RNA Interferente Pequeno/metabolismo , Animais , Células Cultivadas , Regeneração Nervosa , Ratos
14.
Cell Rep ; 19(11): 2210-2219, 2017 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-28614709

RESUMO

Axonal microtubules are predominantly organized into a plus-end-out pattern. Here, we tested both experimentally and with computational modeling whether a motor-based polarity-sorting mechanism can explain this microtubule pattern. The posited mechanism centers on cytoplasmic dynein transporting plus-end-out and minus-end-out microtubules into and out of the axon, respectively. When cytoplasmic dynein was acutely inhibited, the bi-directional transport of microtubules in the axon was disrupted in both directions, after which minus-end-out microtubules accumulated in the axon over time. Computational modeling revealed that dynein-mediated transport of microtubules can establish and preserve a predominantly plus-end-out microtubule pattern as per the details of the experimental findings, but only if a kinesin motor and a static cross-linker protein are also at play. Consistent with the predictions of the model, partial depletion of TRIM46, a protein that cross-links axonal microtubules in a manner that influences their polarity orientation, leads to an increase in microtubule transport.


Assuntos
Dineínas do Citoplasma/metabolismo , Dineínas/metabolismo , Microtúbulos/metabolismo , Animais , Transporte Biológico , Movimento Celular , Ratos
15.
Neurology ; 88(20): 1968-1975, 2017 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-28507260

RESUMO

Gulf War illness (GWI), which afflicts at least 25% of veterans who served in the 1990-1991 war in the Persian Gulf, is thought to be caused by deployment exposures to various neurotoxicants, including pesticides, anti-nerve gas pills, and low-level nerve agents including sarin/cyclosarin. GWI is a multisymptom disorder characterized by fatigue, joint pain, cognitive problems, and gastrointestinal complaints. The most prominent symptoms of GWI (memory problems, poor attention/concentration, chronic headaches, mood alterations, and impaired sleep) suggest that the disease primarily affects the CNS. Development of urgently needed treatments depends on experimental models appropriate for testing mechanistic hypotheses and for screening therapeutic compounds. Rodent models have been useful thus far, but are limited by their inability to assess the contribution of genetic or epigenetic background to the disease, and because disease-vulnerable proteins and pathways may be different in humans relative to rodents. As of yet, no postmortem tissue from the veterans has become available for research. We are moving forward with a paradigm shift in the study of GWI, which utilizes contemporary stem cell technology to convert somatic cells from Gulf War veterans into pluripotent cell lines that can be differentiated into various cell types, including neurons, glia, muscle, or other relevant cell types. Such cell lines are immortal and will be a resource for GWI researchers to pursue mechanistic hypotheses and therapeutics.


Assuntos
Reprogramação Celular , Guerra do Golfo , Neurônios , Síndrome do Golfo Pérsico/patologia , Síndrome do Golfo Pérsico/fisiopatologia , Veteranos , Animais , Córtex Cerebral , Humanos , Células-Tronco Pluripotentes Induzidas , Camundongos , Neurilema , Projetos de Pesquisa
16.
Traffic ; 18(7): 433-441, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28471062

RESUMO

Many veterans of the 1990-1991 Gulf War contracted Gulf War Illness (GWI), a multisymptom disease that primarily affects the nervous system. Here, we treated cultures of human or rat neurons with diisopropyl fluorophosphate (DFP), an analog of sarin, one of the organophosphate (OP) toxicants to which the military veterans were exposed. All observed cellular defects produced by DFP were exacerbated by pretreatment with corticosterone or cortisol, which, in rat and human neurons, respectively, serves in our experiments to mimic the physical stress endured by soldiers during the war. To best mimic the disease, DFP was used below the level needed to inhibit acetylcholinesterase. We observed a diminution in the ratio of acetylated to total tubulin that was correctable by treatment with tubacin, a drug that inhibits HDAC6, the tubulin deacetylase. The reduction in microtubule acetylation was coupled with deficits in microtubule dynamics, which were correctable by HDAC6 inhibition. Deficits in mitochondrial transport and dopamine release were also improved by tubacin. Thus, various negative effects of the toxicant/stress exposures were at least partially correctable by restoring microtubule acetylation to a more normal status. Such an approach may have therapeutic benefit for individuals suffering from GWI or other neurological disorders linked to OP exposure.


Assuntos
Anilidas/farmacologia , Substâncias para a Guerra Química/toxicidade , Ácidos Hidroxâmicos/farmacologia , Isoflurofato/toxicidade , Microtúbulos/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Estresse Fisiológico , Acetilação , Animais , Transporte Biológico , Células Cultivadas , Corticosterona/farmacologia , Dopamina/metabolismo , Relação Dose-Resposta a Droga , Humanos , Hidrocortisona/farmacologia , Microtúbulos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Síndrome do Golfo Pérsico , Ratos , Tubulina (Proteína)/metabolismo
17.
Mol Biol Cell ; 28(13): 1728-1737, 2017 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-28495799

RESUMO

The SPAST gene, which produces two isoforms (M1 and M87) of the microtubule-severing protein spastin, is the chief gene mutated in hereditary spastic paraplegia. Haploinsufficiency is a popular explanation for the disease, in part because most of the >200 pathogenic mutations of the gene are truncating and expected to produce only vanishingly small amounts of shortened proteins. Here we studied two such mutations, N184X and S245X, and our results suggest another possibility. We found that the truncated M1 proteins can accumulate to notably higher levels than their truncated M87 or wild-type counterparts. Reminiscent of our earlier studies on a pathogenic mutation that generates full-length M1 and M87 proteins, truncated M1 was notably more detrimental to neurite outgrowth than truncated M87, and this was true for both N184X and S245X. The greater toxicity and tendency to accumulate suggest that, over time, truncated M1 could damage the corticospinal tracts of human patients. Curiously, the N184X mutation triggers the reinitiation of translation at a third start codon in SPAST, resulting in synthesis of a novel M187 spastin isoform that is able to sever microtubules. Thus microtubule severing may not be as reduced as previously assumed in the case of that mutation.


Assuntos
Códon sem Sentido , Paraplegia Espástica Hereditária/genética , Espastina/genética , Animais , Células Cultivadas , Haploinsuficiência , Humanos , Microtúbulos/metabolismo , Mutagênese Sítio-Dirigida , Neuritos/metabolismo , Neurônios/metabolismo , Isoformas de Proteínas , Ratos , Paraplegia Espástica Hereditária/metabolismo , Espastina/metabolismo
18.
Hum Mol Genet ; 26(12): 2321-2334, 2017 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-28398512

RESUMO

Mutations of various genes cause hereditary spastic paraplegia (HSP), a neurological disease involving dying-back degeneration of upper motor neurons. From these, mutations in the SPAST gene encoding the microtubule-severing protein spastin account for most HSP cases. Cumulative genetic and experimental evidence suggests that alterations in various intracellular trafficking events, including fast axonal transport (FAT), may contribute to HSP pathogenesis. However, the mechanisms linking SPAST mutations to such deficits remain largely unknown. Experiments presented here using isolated squid axoplasm reveal inhibition of FAT as a common toxic effect elicited by spastin proteins with different HSP mutations, independent of microtubule-binding or severing activity. Mutant spastin proteins produce this toxic effect only when presented as the tissue-specific M1 isoform, not when presented as the ubiquitously-expressed shorter M87 isoform. Biochemical and pharmacological experiments further indicate that the toxic effects of mutant M1 spastins on FAT involve casein kinase 2 (CK2) activation. In mammalian cells, expression of mutant M1 spastins, but not their mutant M87 counterparts, promotes abnormalities in the distribution of intracellular organelles that are correctable by pharmacological CK2 inhibition. Collectively, these results demonstrate isoform-specific toxic effects of mutant M1 spastin on FAT, and identify CK2 as a critical mediator of these effects.


Assuntos
Adenosina Trifosfatases/genética , Transporte Axonal/genética , Adenosina Trifosfatases/metabolismo , Animais , Transporte Axonal/fisiologia , Caseína Quinase II/metabolismo , Células Cultivadas , Decapodiformes , Modelos Animais de Doenças , Fibroblastos , Humanos , Microtúbulos/metabolismo , Neurônios Motores/metabolismo , Proteínas Mutantes/metabolismo , Mutação , Isoformas de Proteínas/genética , Transporte Proteico/fisiologia , Ratos , Paraplegia Espástica Hereditária/genética , Paraplegia Espástica Hereditária/metabolismo , Espastina
19.
Brain Res Bull ; 126(Pt 3): 217-225, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27365230

RESUMO

Microtubules are essential for the development and maintenance of axons and dendrites throughout the life of the neuron, and are vulnerable to degradation and disorganization in a variety of neurodegenerative diseases. Microtubules, polymers of tubulin heterodimers, are intrinsically polar structures with a plus end favored for assembly and disassembly and a minus end less favored for these dynamics. In the axon, microtubules are nearly uniformly oriented with plus ends out, whereas in dendrites, microtubules have mixed orientations. Microtubules in developing neurons typically have a stable domain toward the minus end and a labile domain toward the plus end. This domain structure becomes more complex during neuronal maturation when especially stable patches of polyaminated tubulin become more prominent within the microtubule. Microtubules are the substrates for molecular motor proteins that transport cargoes toward the plus or minus end of the microtubule, with motor-driven forces also responsible for organizing microtubules into their distinctive polarity patterns in axons and dendrites. A vast array of microtubule-regulatory proteins impart direct and indirect changes upon the microtubule arrays of the neuron, and these include microtubule-severing proteins as well as proteins responsible for the stability properties of the microtubules. During neurodegenerative diseases, microtubule mass is commonly diminished, and the potential exists for corruption of the microtubule polarity patterns and microtubule-mediated transport. These ill effects may be a primary causative factor in the disease or may be secondary effects, but regardless, therapeutics capable of correcting these microtubule abnormalities have great potential to improve the status of the degenerating nervous system.


Assuntos
Microtúbulos/metabolismo , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo , Animais , Humanos
20.
J Cell Biol ; 213(3): 329-41, 2016 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-27138250

RESUMO

Contemporary models for neuronal migration are grounded in the view that virtually all functionally relevant microtubules (MTs) in migrating neurons are attached to the centrosome, which occupies a position between the nucleus and a short leading process. It is assumed that MTs do not undergo independent movements but rather transduce forces that enable movements of the centrosome and nucleus. The present results demonstrate that although this is mostly true, a small fraction of the MTs are centrosome-unattached, and this permits limited sliding of MTs. When this sliding is pharmacologically inhibited, the leading process becomes shorter, migration of the neuron deviates from its normal path, and the MTs within the leading process become buckled. Partial depletion of ninein, a protein that attaches MTs to the centrosome, leads to greater numbers of centrosome-unattached MTs as well as greater sliding of MTs. Concomitantly, the soma becomes less mobile and the leading process acquires an elongated morphology akin to an axon.


Assuntos
Microtúbulos/metabolismo , Neurônios/metabolismo , Animais , Movimento Celular/fisiologia , Centrossomo/metabolismo , Centrossomo/ultraestrutura , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas do Citoesqueleto/fisiologia , Microscopia Eletrônica de Transmissão , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/fisiologia , Microtúbulos/ultraestrutura , Neurônios/fisiologia , Neurônios/ultraestrutura , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiologia , Fenótipo , Interferência de RNA , Ratos
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