RESUMEN
Exposure to neurotoxin aluminum neurotoxicity is accompanied by the perikaryal accumulation of tangles of phosphorylated neurofilaments (NFs). We examined their formation and reversibility under cell-free conditions. AlCl3 induced dose-dependent formation of NF aggregates, ultimately incorporating 100% of detectable NFs. The same concentration of CaCl2 induced approximately 25% of NFs to form longitudinal dimers and did not induce aggregation. AlCl3 induced similar percentages of aggregates in the presence or absence of CaCl2, and CaCl2 could not reduce pre-formed aggregates. CaCl(2)-induced dimers and AlCl(3)-induced aggregates were prevented by prior NF dephosphorylation. While CaCl(2)-induced dimers were dissociated by phosphatase treatment, AlCl(3)-induced aggregates were only reduced by approximately 50%, suggesting that aggregates may sequester phosphorylation sites. Since phosphatases regulate NF phosphorylation within perikarya, inhibition of NF dephosphorylation by aluminum would promote perikaryal NF phosphorylation and foster precocious phospho-dependent NF-NF associations. These findings are consistent with the notion that prolonged interactions induced among phospho-NFs by the trivalent aluminum impairs axonal transport and promotes perikaryal aggregation.
Asunto(s)
Aluminio/toxicidad , Ovillos Neurofibrilares/efectos de los fármacos , Proteínas de Neurofilamentos/efectos de los fármacos , Neuronas/efectos de los fármacos , Neurotoxinas/toxicidad , Cloruro de Aluminio , Compuestos de Aluminio/toxicidad , Animales , Transporte Axonal/efectos de los fármacos , Transporte Axonal/fisiología , Cloruro de Calcio/toxicidad , Cloruros/toxicidad , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Citoesqueleto/patología , Relación Dosis-Respuesta a Droga , Femenino , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ovillos Neurofibrilares/metabolismo , Ovillos Neurofibrilares/patología , Proteínas de Neurofilamentos/química , Proteínas de Neurofilamentos/metabolismo , Neuronas/metabolismo , Neuronas/patología , Fosforilación/efectos de los fármacos , Estructura Terciaria de Proteína/efectos de los fármacos , Estructura Terciaria de Proteína/fisiologíaRESUMEN
The phosphorylation of neurofilaments (NFs) has long been considered to regulate their axonal transport rate and in doing so to provide stability to mature axons. Axons contain a centrally situated ;bundle' of closely opposed phospho-NFs that display a high degree of NF-NF associations and phospho-epitopes, surrounded by less phosphorylated ;individual' NFs that are often associated with kinesin and microtubules (MTs). Bundled NFs transport substantially slower than the surrounding individual NFs and might represent a resident population that stabilizes axons and undergoes replacement by individual NFs. To examine this possibility, fractions enriched in bundled NFs and individual NFs were generated from mice and NB2a/d1 cells by sedimentation of cytoskeletons over a sucrose cushion. More kinesin was recovered within individual versus bundled NF fractions. Individual but not bundled NFs aligned with purified MTs under cell-free conditions. The percentage of NFs that aligned with MTs was increased by the addition of kinesin, and inhibited by anti-kinesin antibodies. Bundles dissociated following incubation with EGTA or alkaline phosphatase, generating individual NFs that retained or were depleted of phospho-epitopes, respectively. These dissociated NFs aligned with MTs at a level identical to those originally isolated as individual NFs regardless of phosphorylation state. EGTA-mediated dissociation of bundles was prevented and reversed by excess Ca(2+), whereas individual NFs did not associate in the presence of excess Ca(2+). These findings confirm that bundling competes with NF-MT association, and provide a mechanism by which C-terminal NF phosphorylation might indirectly contribute to the observed slowing in axonal transport of phospho-NFs.
Asunto(s)
Cinesinas/metabolismo , Microtúbulos/metabolismo , Proteínas de Neurofilamentos/metabolismo , Animales , Línea Celular , Citoesqueleto/metabolismo , Femenino , Filamentos Intermedios/metabolismo , Masculino , Ratones , Fosforilación , Unión ProteicaRESUMEN
Neurofilament phosphorylation has long been considered to regulate their axonal transport rate, and in doing so it provides stability to mature axons. We evaluate the collective evidence to date regarding how neurofilament C-terminal phosphorylation may regulate axonal transport. We present a few suggestions for further experimentation in this area, and expand upon previous models for axonal NF dynamics. We present evidence that the NFs that display extended residence along axons are critically dependent upon the surrounding microtubules, and that simultaneous interaction with multiple microtubule motors provides the architectural force that regulates their distribution. Finally, we address how C-terminal phosphorylation is regionally and temporally regulated by a balance of kinase and phosphatase activities, and how misregulation of this balance might contribute to motor neuron disease.
Asunto(s)
Axones/fisiología , Microtúbulos/fisiología , Proteínas de Neurofilamentos , Enfermedades del Sistema Nervioso Periférico , Animales , Transporte Biológico , Humanos , Proteínas de Neurofilamentos/fisiología , FosforilaciónRESUMEN
Neurofilament (NF) phosphorylation has long been considered to regulate axonal transport rate and in doing so to provide stability to mature axons. Studies utilizing mice in which the C-terminal region of NF subunits (which contains the vast majority of phosphorylation sites) has been deleted has prompted an ongoing challenge to this hypothesis. We evaluate the collective evidence to date for and against a role for NF C-terminal phosphorylation in regulation of axonal transport and in providing structural support for axons, including some novel studies from our laboratory. We present a few suggestions for further experimentation in this area, and expand upon previous models for axonal NF dynamics. Finally, we address how C-terminal phosphorylation is regionally and temporally regulated by a balance of kinase and phosphatase activities, and how misregulation of this balance can contribute to motor neuron disease.
Asunto(s)
Transporte Axonal/fisiología , Proteínas de Neurofilamentos/metabolismo , Fosforilación , Animales , HumanosRESUMEN
Studies from several laboratories indicate that the microtubule motors kinesin and dynein respectively participate in anterograde and retrograde axonal transport of neurofilaments. Inhibition of dynein function by transfection with a construct expressing dynamitin or intracellular delivery of anti-dynein antibodies accelerates anterograde transport, which has been interpreted to indicate that the opposing action of both motors mediates the normal distribution of neurofilaments along axons. Herein, we demonstrate that, while expression of relatively low levels of exogenous dynamitin indeed accelerated anterograde neurofilament transport along axonal neurites in culture, expression of progressively increasing levels of dynamitin induced focal accumulation of neurofilaments within axonal neurites and eventually caused neurite retraction. Inhibition of kinesin inhibited anterograde transport, but did not induce similar focal accumulations. These findings are consistent with studies indicating that perturbations in dynein activity can contribute to the aberrant accumulations of neurofilaments that accompany ALS/motor neuron disease.
Asunto(s)
Transporte Axonal/fisiología , Axones/metabolismo , Sistema Nervioso Central/metabolismo , Dineínas/metabolismo , Proteínas de Neurofilamentos/metabolismo , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Animales , Transporte Axonal/efectos de los fármacos , Axones/efectos de los fármacos , Línea Celular Tumoral , Sistema Nervioso Central/fisiopatología , Complejo Dinactina , Dineínas/antagonistas & inhibidores , Dineínas/genética , Cinesinas/antagonistas & inhibidores , Cinesinas/metabolismo , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Neurofilamentos/efectos de los fármacosRESUMEN
We examined the respective roles of dynein and kinesin in axonal transport of neurofilaments (NFs). Differentiated NB2a/d1 cells were transfected with green fluorescent protein-NF-M (GFP-M) and dynein function was inhibited by co-transfection with a construct expressing myc-tagged dynamitin, or by intracellular delivery of purified dynamitin and two antibodies against dynein's cargo domain. Monitoring of the bulk distribution of GFP signal within axonal neurites, recovery of GFP signal within photobleached regions, and real-time monitoring of individual NFs/punctate structures each revealed that pertubation of dynein function inhibited retrograde transport and accelerated anterograde, confirming that dynein mediated retrograde axonal transport, while intracellular delivery of two anti-kinesin antibodies selectively inhibited NF anterograde transport. In addition, dynamitin overexpression inhibited the initial translocation of newly-expressed NFs out of perikarya and into neurites, indicating that dynein participated in the initial anterograde delivery of NFs into neurites. Delivery of NFs to the axon hillock inner plasma membrane surface, and their subsequent translocation into neurites, was also prevented by vinblastine-mediated inhibition of microtubule assembly. These data collectively suggest that some NFs enter axons as cargo of microtubues that are themselves undergoing transport into axons via dynein-mediated interactions with the actin cortex and/or larger microtubules. C-terminal NF phosphorylation regulates motor association, since anti-dynein selectively coprecipitated extensively phosphorylated NFs, while anti-kinesin selectively coprecipitated less phosphorylated NFs. In addition, however, the MAP kinase inhibitor PD98059 also inhibited transport of a constitutively-phosphorylated NF construct, indicating that one or more additional, non-NF phosphorylation events also regulated NF association with dynein or kinesin.
Asunto(s)
Transporte Axonal , Dineínas/fisiología , Filamentos Intermedios/metabolismo , Animales , Transporte Axonal/efectos de los fármacos , Axones/química , Axones/metabolismo , Transporte Biológico/efectos de los fármacos , Dineínas/antagonistas & inhibidores , Dineínas/genética , Flavonoides/farmacología , Proteínas Fluorescentes Verdes/análisis , Cinesinas/metabolismo , Cinesinas/fisiología , Ratones , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Neuritas/química , Neuritas/metabolismo , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Células Tumorales Cultivadas , Vincristina/farmacologíaRESUMEN
Neurofilaments (NFs) are thought to provide structural support for axons. Some NFs exhibit an extended residence time along axons, the nature of which remains unclear. In prior studies in NB2a/d1 cells, hypophosphorylated NFs were demonstrated to be dispersed throughout the axon and to undergo relatively rapid axonal transport, while extensively phosphorylated NFs organized into a "bundle" localized along the center of the axon. It was not conclusively determined whether bundled NFs underwent transport or instead underwent turnover via exchange with transporting individual NFs. Herein, using transfection with multiple constructs and regional photobleaching, we demonstrate that bundled NFs undergo relatively slow transport as well as exchange with surrounding individual NFs. We also demonstrate that newly synthesized NFs disperse nonhomogenously throughout axonal neurites and perikarya. These findings provide a mechanism by which some NFs exhibit extended residence time within axons, which lessens the metabolic burden of cytoskeletal turnover.
Asunto(s)
Transporte Axonal/fisiología , Citoesqueleto/metabolismo , Proteínas de Neurofilamentos/metabolismo , Animales , Línea Celular Tumoral , Células Cultivadas , Citoesqueleto/ultraestructura , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Immunoblotting , Ratones , Microscopía Confocal , Fotoblanqueo , TransfecciónRESUMEN
Vimentin (Vm) is initially expressed by early neuronal precursors in situ and in culture. Vm is essential for neuritogenesis at least in culture and is gradually replaced by neurofilaments (NFs) because of down-regulation of Vm expression. This period is accompanied by a slowing of axonal elongation. We examined whether continued expression of Vm could foster continued axonal elongation. NB2a/d1 cells differentiated with dibutyryl cAMP were transfected with constructs expressing Vm or the middle-molecular-weight NF subunit (NF-M) each conjugated to green fluorescent protein (GFP). Axonal neurites of cells expressing GFP-Vm were 30% longer than those of nonexpressing cells, or cells expressing GFP-M, and exhibited a decrease in neurite caliber. Expression of GFP-M did not enhance axonal neurite length but significantly increased caliber. These findings provide further evidence of a role for Vm in axonal outgrowth. Culturing of nontransfected cells on laminin increased neurite length, but cells expressing GFP-Vm demonstrated an equivalent increase whether cultured on laminin or culture plastic. Axonal neurites of cells expressing GFP-Vm turned to avoid a nonfavorable substrate (nitrocellulose), but culturing of these cells on nitrocellulose did not impair axonal outgrowth. These latter findings indicate that the more robust outgrowth following reexpression of Vm is independent of a favorable or nonfavorable substrate but that axonal neurites of these cells still interact with the substrate to the extent that the substrate can influence directionality.
Asunto(s)
Neuritas/fisiología , Vimentina/biosíntesis , Vimentina/fisiología , Animales , Bucladesina/farmacología , Línea Celular Tumoral , Proteínas Fluorescentes Verdes , Laminina/fisiología , Proteínas Luminiscentes/biosíntesis , Neuritas/efectos de los fármacos , Neuritas/ultraestructura , Proteínas de Neurofilamentos/biosíntesis , TransfecciónRESUMEN
Vimentin (Vm) is initially expressed by nearly all neuronal precursors in vivo, and is replaced by neurofilaments (NFs) shortly after the immature neurons become post-mitotic. Both Vm and NFs can be transiently detected within the same neurite, and Vm is essential for neuritogenesis at least in culture. How neurons effect the orderly transition from expression of Vm as their predominant intermediate filament to NFs remains unclear. We examined this phenomenon within growing axonal neurites of NB2a/d1 cells. Transfection of cells with a construct expressing Vm conjugated to green fluorescent protein confirmed that axonal transport machinery for Vm persisted following the developmental decrease in Vm, but that the amount undergoing transport decreased in parallel to the observed developmental increase in NF transport. Immunoprecipitation from pulse-chase radiolabeled cells demonstrated transient co-precipitation of newly synthesized NF-H with Vm, followed by increasing co-precipitation with NF-L. Immunofluorescent and immuno-electron microscopic analyses demonstrated that some NF and Vm subunits were incorporated into the same filamentous profiles, but that Vm was excluded from the longitudinally-oriented "bundle" of closely-apposed NFs that accumulates within developing axons and is known to undergo slower turnover than individual NFs. These data collectively suggest that developing neurons are able to replace their Vm-rich cytoskeleton with one rich in NFs simply by down-regulation of Vm expression and upregulation of NFs, coupled with turnover of existing Vm filaments and Vm-NF heteropolymers.
Asunto(s)
Diferenciación Celular/fisiología , Proteínas de Neurofilamentos/biosíntesis , Neuronas/citología , Neuronas/ultraestructura , Vimentina/biosíntesis , Citoesqueleto/fisiología , Citoesqueleto/ultraestructura , Humanos , Microscopía Inmunoelectrónica , Neuroblastoma/ultraestructura , Proteínas de Neurofilamentos/ultraestructura , Pruebas de Precipitina , Transfección , Células Tumorales Cultivadas , Vimentina/ultraestructuraRESUMEN
Neurofilaments (NFs) are classically considered to transport in a primarily anterograde direction along axons, and to undergo bulk degradation within the synapse or growth cone (GC). We compared overall NF protein distribution with that of newly expressed NF subunits within NB2a/d1 cells by transfection with a construct encoding green fluorescent protein (GFP) conjugated NF-M subunits. GCs lacked phosphorylated NF epitopes, and steady-state levels of non-phosphosphorylated NF subunits within GC were markedly reduced compared to those of neurite shaft as indicated by conventional immunofluorescence. However, GCs contained significant levels of GFP-tagged subunits in the form of punctate or short filamentous structures that in some cases exceeded that visualized along the shaft itself, suggesting that GCs contained a relatively higher concentration of newly synthesized subunits. GFP-tagged NF subunits within GCs co-localized with non-phosphorylated NF immunoreactivity. GFP-tagged subunits were observed within GC filopodia in which steady-state levels of NF subunits were too low to be detected by conventional immunofluorescence. Selective localization of fluorescein versus rhodamine fluorescene was observed within GCs following expression of NF-M conjugated to DsRed1-E5, which shifts from fluorescein to rhodamine fluorescence within hours after expression; axonal shafts contained a more even distribution of fluorescein and rhodamine fluorescence, further indicating that GCs contained relatively higher levels of the most-recently expressed subunits. GFP-tagged structures were rapidly extracted from GCs under conditions that preserved axonal structures. These short filamentous and punctate structures underwent rapid bi-directional movement within GCs. Movement of GFP-tagged structures within GCs ceased following application of nocodazole, cytochalasin B, and the kinase inhibitor olomoucine, indicating that their motility was dependent upon microtubules and actin and, moreover, was due to active transport rather than simple diffusion. Treatment with the protease inhibitor calpeptin increased overall NF subunits, but increased those within the GC to a greater extent than those along the shaft, indicating that subunits in the GC undergo more rapid turnover than do those within the shaft. Some GCs contained coiled aggregates of GFP-tagged NFs that appeared to be contiguous with axonal NFs. NFs extended from these aggregates into the advancing GC as axonal neurites elongated. These data are consistent with the presence of a population of dynamic NF subunits within GCs that is apparently capable of participating in regional filament formation during axonal elongation, and support the notion that NF polymerization and transport need not necessarily occur in a uniform proximal-distal manner.