RESUMEN
Huntington's disease (HD) is a neurodegenerative disorder resulting from the expansion of a glutamine repeat (polyQ) in the N-terminus of the huntingtin (htt) protein. Expression of polyQ-containing proteins has been previously shown to induce various cellular stress responses. Among these, activation of the c-Jun N-terminal kinase (JNK) cascade has been observed in cellular models of HD. However, the implication of the JNK pathway has not previously been evaluated in the striatum of HD animal models. Here we report that the JNK pathway participates in HD pathology in a rat model of the disease. Increased phosphorylation of the JNK target c-Jun was observed as early as 4 weeks and persisted for 13 weeks after lentiviral-mediated expression of htt171-82Q. In order to assess the importance of this pathway in HD pathology, JNK inhibitors including dominant-negative mutants of upstream kinases (ASK1(K709R), MEKK1(D1369A)), a c-Jun mutant (Delta169c-Jun) and the active domain of the scaffold protein JIP-1/IBI (IBI-JBD) were tested for their ability to mitigate the effect of htt171-82Q. The overexpression of MEKK1(D1369A) and JIP-1/IBI reduced the polyQ-related loss of DARPP-32 expression, while the other inhibitors had no effect. In all cases, the formation of EM48-positive htt inclusions and P-c-Jun immunoreactivity were unaltered. These results suggest that JNK activation is involved in HD and that blockade of this pathway may be of benefit in counteracting HD-related neurotoxicity.
Asunto(s)
Enfermedad de Huntington/enzimología , Enfermedad de Huntington/fisiopatología , MAP Quinasa Quinasa 4/metabolismo , Transducción de Señal/fisiología , Análisis de Varianza , Animales , Proteína de Unión a CREB/metabolismo , Línea Celular Transformada , Modelos Animales de Enfermedad , Dopamina/metabolismo , Fosfoproteína 32 Regulada por Dopamina y AMPc/metabolismo , Femenino , Regulación de la Expresión Génica/fisiología , Humanos , Proteína Huntingtina , Enfermedad de Huntington/genética , Lentivirus/fisiología , Mutación/fisiología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Ratas , Ratas Wistar , Serina/metabolismo , Factores de Tiempo , Transfección/métodosRESUMEN
Sonic hedgehog (SHH) is considered to play an important role in tissue induction and patterning during development, particularly in determining neuronal cell fate in the ventral neural tube and in the embryonic forebrain. SHH precursor is autoproteolytically cleaved to an aminoterminal fragment (SHHN) which retains all known SHH biological activities. Here, we demonstrate the expression of a 22-kDa SHHN immunoreactive peptide in developing and adult hamster brain regions using a rabbit antiserum directed against a mouse SHHN fragment. Interestingly, SHHN was developmentally regulated with the highest expression observed in the adult brain, was resistant to Triton X-100 solubilization at 4 degrees C and partitioned with the raft component ganglioside GM1 during density gradient centrifugation. In rat brain, Shh transcripts were identified by double in situ hybridization in GABAergic neurons located in various basal forebrain nuclei including globus pallidus, ventral pallidum, medial septum-diagonal band complex, magnocellular preoptic nucleus and in cerebellar Purkinje cells as well as in motoneurons of several cranial nerve nuclei and of the spinal cord. We show that radiolabelled SHHN peptides are synthesized in the adult hamster retina and are transported axonally along the optic nerve to the superior colliculus in vivo. Our data indicate that SHHN is associated with cholesterol rich raft-like microdomains and anterogradely transported in the adult brain, and suggest that the roles of this extracellular protein are more diverse than originally thought.
Asunto(s)
Transporte Axonal/fisiología , Encéfalo/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Factores de Edad , Animales , Anticuerpos , Encéfalo/citología , Encéfalo/crecimiento & desarrollo , Membrana Celular/química , Colesterol/metabolismo , Cricetinae , Detergentes , Gangliósido G(M1)/análisis , Expresión Génica/fisiología , Proteínas Hedgehog , Mesocricetus , Neuronas/química , Neuronas/metabolismo , ARN Mensajero/análisis , Retina/citología , Médula Espinal/citología , Médula Espinal/crecimiento & desarrollo , Médula Espinal/metabolismo , Transactivadores/inmunologíaRESUMEN
The Connecticut State Medical Society (CSMS) reviews and accredits the continuing medical education (CME) programs offered by Connecticut's hospitals. As part of the survey process, the CSMS assesses the quality of the hospitals' libraries. In 1987, the CSMS adopted the Medical Library Association's (MLA's) "Minimum Standards for Health Sciences Libraries in Hospitals." In 1990, professional librarians were added to the survey team and, later, to the CSMS CME Committee. Librarians participating in this effort are recruited from the membership of the Connecticut Association of Health Sciences Librarians (CAHSL). The positive results of having a qualified librarian on the survey team and the invaluable impact of adherence to the MLA standards are outlined. As a direct result of this process, hospitals throughout the state have added staffing, increased space, and added funding for resources during an era of cutbacks. Some hospital libraries have been able to maintain a healthy status quo, while others have had proposed cuts reconsidered by administrators for fear of losing valuable CME accreditation status. Creating a relationship with an accrediting agency is one method by which hospital librarians elsewhere may strengthen their efforts to ensure adequate library resources in an era of downsizing. In addition, this collaboration has provided a new and important role for librarians to play on an accreditation team.
Asunto(s)
Acreditación/organización & administración , Educación Médica Continua/organización & administración , Bibliotecas de Hospitales/normas , Bibliotecas Médicas/normas , Acreditación/estadística & datos numéricos , Connecticut , Humanos , Relaciones Interprofesionales , Sociedades MédicasRESUMEN
We examined the localization of PrP(c) in normal brain using free-floating section immunohistochemistry and monclonal antibody 3F4. In the mature hamster and baboon brain, PrP(c) is localized to the neuropil with a synaptic distribution and the PrP(c) immunoreactivity is denser in regions known for ongoing plasticity. Cell bodies and major fiber tracts have little or no PrP(c) immunoreactivity. At the electron microscopic level, PrP(c) immunoreactivity decorates synaptic profiles, both pre- and postsynaptically. Results obtained with two additional antibodies, 3B5 and Pri-304, showed similar patterns of PrP(c) bands on Western blots, although Pri-304 was less sensitive. On sections through the adult hamster hippocampus, 3B5 and Pri-304 both stained the synaptic neuropil while cell bodies in the pyramidal and dentate granule cell layers were not immunoreactive. Pri-304 differentiated between synaptic layers in the hippocampus and closely resembled the pattern of staining obtained with 3F4. Preliminary results of developing brain showed that PrP(c) is initially localized along fiber tracts in the neonate brain. These results show that PrP(c) has a synaptic distribution in the adult brain and suggest that there are important changes in its distribution during brain development. These results also characterize two additional reagents for studies of PrP(c) localization.
Asunto(s)
Encéfalo/metabolismo , Proteínas PrPC/análisis , Adulto , Animales , Anticuerpos Monoclonales , Western Blotting , Encéfalo/ultraestructura , Cricetinae , Técnicas de Cultivo , Hipocampo/metabolismo , Hipocampo/ultraestructura , Humanos , Inmunohistoquímica , Microscopía Electrónica , Neurópilo/metabolismo , Especificidad de Órganos , Papio , Terminales Presinápticos/ultraestructura , Retina/embriología , Retina/metabolismo , Vesículas Sinápticas/ultraestructuraRESUMEN
We studied the axonal transport of PrP(C) in hamster retinal and sciatic nerve axons. Our results show that a novel 38kDa form is the predominant form in rapid anterograde axonal transport while the 36kDa and 33kDa PrP(C) forms, abundant in nerve and brain, appear to be either stationary or slowly transported. We did not detect any significant retrograde transport of PrP(C). These results show that 38kDa PrP(C) is the form exported from the cell body to the axonal compartment where it may represent the precursor to the more abundant PrP(C) forms after its modification in nerve fibres or terminals.
Asunto(s)
Transporte Axonal , Axones/metabolismo , Proteínas PrPC/metabolismo , Animales , Axotomía , Western Blotting , Cricetinae , Cinética , Mesocricetus , Peso Molecular , Vías Nerviosas/citología , Vías Nerviosas/metabolismo , Nervio Óptico/citología , Nervio Óptico/metabolismo , Proteínas PrPC/química , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/metabolismo , Nervio Ciático/citología , Nervio Ciático/metabolismo , Nervio Ciático/cirugía , Colículos Superiores/citología , Colículos Superiores/metabolismoRESUMEN
The presence of an abnormal, protease-resistant form of the prion protein (PrP) is the hallmark of various forms of transmissible spongiform encephalopathies (TSE) which can affect a number of mammalian species, including humans. The normal, cellular form of this protein, PrPc, while abundant in brain is also present in many tissues and a number of species. In order to address the unresolved question of the precise localization of normal cerebral PrPc, we used a free-floating immunohistochemistry procedure to localize the protein at both the light and the electron microscopic levels in the brain of three TSE-sensitive species: hamster, macaque and humans. This method shows that PrPc is abundant in synaptic terminal fields in olfactory bulb, limbic-associated structures and in the striato-nigral complex, whereas many other regions of the hamster brain are essentially devoid of immunoreactivity. With the striking exception of the olfactory nerve, in which axons are continually growing throughout life, PrPc is not abundant in fibre pathways. PrPc distribution in the primate hippocampus and cortex is very similar to the distribution observed in hamster. PrPc was present at synaptic profiles as shown by immunoelectron microscopy, but was not detectable in neuronal perikaryon either by light or electron microscopy. Our results show that PrPc is abundant in a number of brain structures known for ongoing plasticity, and are consistent with the hypothesis that the protein also plays a role in synaptic function.
Asunto(s)
Encéfalo/metabolismo , Neuronas/metabolismo , Primates/metabolismo , Priones/metabolismo , Adulto , Anciano , Animales , Encéfalo/citología , Cricetinae , Humanos , Macaca fascicularis , Mesocricetus , Microscopía Electrónica , Microscopía Inmunoelectrónica , Persona de Mediana Edad , Papio , Distribución TisularRESUMEN
This study of a mononeuropathy of 1-15 weeks (W) duration was induced in rats by setting 4 loose ligatures around the common sciatic nerve. This chronic lesion, in which the continuity of the nerve was maintained, has been introduced as a model for experimental pain. Quantitative analyses of teased nerve fibres and a morphometric analysis of semi-thin transverse sections, were performed and completed by electron microscopic examination. Morphological changes were observed mainly distal, but also proximal, to the ligatures, indicating significant axonopathy with simultaneous degeneration and regeneration. The lesions were analysed in parallel with the time course of the pain-related behaviours. Both were at their maximum 2 weeks after ligature with progressive recovery beginning between W3 and W4. However, the largest fibres had not totally recovered by W15, contrasting with the disappearance of abnormal nociceptive reactions between W8 and W10. Although the damage to unmyelinated fibres is of importance, the abnormal pain-related behaviours seen in these rats appeared to be closely linked to the presence of both degenerative and regenerative changes in the A delta-range fibres, which did not necessarily correspond to initial A delta fibres.
Asunto(s)
Conducta Animal , Degeneración Nerviosa , Fibras Nerviosas Mielínicas/patología , Regeneración Nerviosa , Dolor/fisiopatología , Enfermedades del Sistema Nervioso Periférico/fisiopatología , Nervio Ciático/fisiopatología , Animales , Ligadura , Masculino , Microscopía Electrónica , Microtomía , Fibras Nerviosas Mielínicas/ultraestructura , Dolor/patología , Dolor/psicología , Enfermedades del Sistema Nervioso Periférico/etiología , Enfermedades del Sistema Nervioso Periférico/patología , Ratas , Ratas Sprague-Dawley , Tiempo de Reacción , Nervio Ciático/patología , Nervio Ciático/ultraestructuraRESUMEN
Reversal of anterograde rapid axonal transport of four molecular forms of acetylcholinesterase (AChE) was studied in chick sciatic nerve during the 24-h period following a nerve transection. Reversal of AChE activity started approximately 1 h after nerve transection, and all the forms of the enzyme, except the monomeric ones, showed reversal of transport. The quantity of enzyme activity reversed 24 h after transection was twofold greater than that normally conveyed by retrograde transport. We observed no leakage of the enzyme at the site of the nerve transection and no reversal of AChE activity transport in the distal segment of the severed nerve, a result indicating that the material carried by retrograde axonal transport cannot be reversed by axotomy. Thus, a nerve transection induces both quantitative and qualitative changes in the retrograde axonal transport, which could serve as a signal of distal injury to the cell body. The velocity of reverse transport, measured within 6 h after transection, was found to be 213 mm/day, a value close to that of retrograde transport (200 mm/day). This suggests that the reversal taking place in severed sciatic nerve is similar to the anterograde-to-retrograde conversion process normally occurring at the nerve endings.
Asunto(s)
Acetilcolinesterasa/química , Axones/enzimología , Nervio Ciático/enzimología , Acetilcolinesterasa/metabolismo , Animales , Transporte Biológico/fisiología , Pollos , Desnervación , Cinética , Conformación Molecular , PermeabilidadRESUMEN
This study examined the distribution of axonally transported tubulin and a 68 kDa polypeptide in the sciatic nerve 34 days after injection of labelled methionine into the ventral horn of the spinal cord of control rats, rats with streptozotocin-induced diabetes mellitus and rats fed a diet containing 40% galactose. The proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of pellets produced by treatment of nerve extracts with Triton X-100 followed by differential ultra-centrifugation. The most marked effect of both diabetes and galactosaemia was to reduce the amount of activity present in tubulin transported at a rate of 1.4 to 2.1 mm/day. The distribution of activity in the 68 kDa polypeptide band was not markedly affected by either of the experimental conditions. These findings, taken together with those of other studies, indicate that the polyol pathway may contribute to the development of some defects of nerve function in diabetic rats, but is uninvolved in others.
Asunto(s)
Transporte Axonal , Diabetes Mellitus Experimental/fisiopatología , Galactosemias/fisiopatología , Neuronas Motoras/análisis , Proteínas del Tejido Nervioso/análisis , Nervio Ciático/análisis , Tubulina (Proteína)/análisis , Animales , Autorradiografía , Carbohidratos/análisis , Electroforesis en Gel de Poliacrilamida , Inositol/análisis , Masculino , Metionina/metabolismo , Peso Molecular , Ratas , Ratas Endogámicas , Valores de Referencia , Radioisótopos de AzufreRESUMEN
We studied the modifications occurring in the parent cytoskeleton carried by SCa (the slower of the two slow axonal transport subcomponents) after peripheral nerve crush. The proteins transported in rat sciatic motor axons were radiolabelled by injecting [35S]methionine into the ventral horn of the spinal cord, and the nerve was crushed so as to entrap only the proteins transported by SCa along the parent axon. Two weeks after the crush, the regenerating nerve was removed and the distributions of the polymerized and unpolymerized radiolabelled cytoskeletal proteins were compared with those in normal, non-regenerating nerves. We found that in the parent axons, most of the radioactive neurofilaments were arrested by the crush, but microtubules, soluble tubulin, insoluble and soluble actin were normally transported. Thus, when the resulting cytoskeleton transported by SCa entered the daughter axon, it was enriched in microtubules and actin, and partially depleted of neurofilaments. This cytoskeleton contained larger proportions of soluble tubulin and insoluble actin than the parent cytoskeleton, but retained its coordinated progression and transport velocity, suggesting that after axotomy, the main destiny of the parent cytoskeleton carried by SCa is to become the equivalent cytoskeleton in the daughter axons.
RESUMEN
The impairment of slow axonal transport of cytoskeletal proteins was studied in the sciatic nerves of streptozocin-diabetic rats. [35S]Methionine was unilaterally injected into the fourth lumbar ganglion and spinal cord, to label the sensory and motor axons, respectively, and then the polymerized elements of the cytoskeleton and the corresponding soluble proteins were analyzed separately. In addition, the pellet/supernatant ratio for tubulin and actin was also assessed. Our results indicate that the velocity of slow component a (SCa) of axonal transport, particularly that of neurofilaments, was strongly reduced (by 60%) in sensory axons. At the same time, a decreased pellet/supernatant ratio of tubulin, possibly owing to a depolymerization of stable microtubules, was also observed. The transport of slow component b (SCb) of axonal transport was also impaired, but the extent of this impairment could not be precisely evaluated. In contrast, motor axons showed little or no impairment of both SCa and SCb at the time studied, a result suggesting a delayed development of the neuropathy in motor axons.
Asunto(s)
Transporte Axonal , Proteínas del Citoesqueleto/fisiología , Diabetes Mellitus Experimental/fisiopatología , Neuropatías Diabéticas/fisiopatología , Nervio Ciático/fisiopatología , Animales , Femenino , Ganglios Espinales/fisiopatología , Neuronas Motoras/fisiología , Neuronas Aferentes/fisiología , Ratas , Ratas Endogámicas , Valores de ReferenciaRESUMEN
Aging in the sciatic nerve of the rat is characterized by various alterations, mainly cytoskeletal impairment, the presence of residual bodies and glycogen deposits, and axonal dystrophies. These alterations could form a mechanical blockade in the axoplasm and disturb the axoplasmic transports. However, morphometric studies on the fiber distribution indicate that the increase of the axoplasmic compartment during aging could obviate this mechanical blockade. Analysis of the axoplasmic transport, using acetylcholinesterase (AChE) molecular forms as markers, demonstrates a reduction in the total AChE flow rate, which is entirely accounted for by a significant bidirectional 40-60% decrease in the rapid axonal transport of the G4 molecular form. However, the slow axoplasmic flow of G1 + G2 forms, as well as the rapid transport of the A12 form of AChE, remain unchanged. Our results support the hypothesis that the alterations observed in aged nerves might be related either to the impairment in the rapid transport of specific factor(s) or to modified exchanges between rapidly transported and stationary material along the nerves, rather than to a general defect in the axonal transport mechanisms themselves.
Asunto(s)
Acetilcolinesterasa/metabolismo , Transporte Axonal , Nervio Ciático/crecimiento & desarrollo , Envejecimiento/metabolismo , Animales , Axones/ultraestructura , Transporte Biológico , Gránulos Citoplasmáticos/ultraestructura , Citoesqueleto/ultraestructura , Masculino , Microscopía Electrónica , Ratas , Ratas Endogámicas , Nervio Ciático/metabolismo , Nervio Ciático/ultraestructuraRESUMEN
During the development of streptozotocin-induced diabetic neuropathy in the rat, the axonal transport of 4 acetylcholinesterase molecular forms was studied by measuring their accumulation on both sides of transected sciatic nerves. Our results indicate that both the anterograde and retrograde axonal transport of all these forms remain normal between 2 and 5 weeks after the induction of diabetes by streptozotocin injection.
Asunto(s)
Acetilcolinesterasa/metabolismo , Transporte Axonal , Diabetes Mellitus Experimental/metabolismo , Nervio Ciático/metabolismo , Animales , Diabetes Mellitus Experimental/enzimología , Femenino , Conformación Molecular , Ratas , Ratas Endogámicas , Nervio Ciático/enzimología , Nervio Ciático/fisiopatología , EstreptozocinaRESUMEN
Acetylcholinesterase (AChE) activity and its distribution among different molecular forms were studied in the sciatic nerve of normal and polyarthritic rats. Axonal transport of each form was investigated on the basis of its accumulation on both sides of a transection. Although an increase in total AChE activity could be detected in the sciatic nerves of polyarthritic animals, both anterograde and retrograde axonal transport of all the molecular forms investigated were similar in normal and polyarthritic rats. This suggests that neither slow nor fast axonal transport is impaired in polyarthritic rats. Hence, the neurophysiological modifications observed at the spinal, thalamic and cortical levels of the CNS are presumably not a consequence of peripheral axonal disability.
Asunto(s)
Acetilcolinesterasa/metabolismo , Artritis Experimental/enzimología , Artritis/enzimología , Transporte Axonal , Isoenzimas/metabolismo , Nervios Periféricos/enzimología , Animales , Masculino , Ratas , Ratas Endogámicas , Factores de TiempoRESUMEN
Polypeptides in the motor axons of the sciatic nerve in 120-day-old normal and diabetic mice C57BL/Ks (db/db) were labeled by injection of [35S]methionine into the ventral horn of the spinal cord. At 8, 15, and 25 days after the injection, the distribution of radiolabeled polypeptides along the sciatic nerve was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Four major radiolabeled polypeptides, tentatively identified as actin, tubulin, and the two lightest subunits of the neurofilament triplet, were studied in both diabetic and control mice. In the diabetic animals, the two polypeptides identified as actin and tubulin showed a reduction of average velocity of migration along the sciatic nerve, resulting in a higher fraction of radioactivity in the proximal part of the sciatic nerve, whereas the front of radioactivity (advancing at maximal velocity) moved at a normal rate. In contrast, both the average and maximal velocities of the two neurofilament subunits were slower in the diabetic mice than in the control mice. These results indicate that the axonal transport of the cytoskeletal proteins is differentially affected in the course of diabetic neuropathy, and may suggest that the impairment concerns mainly the proteins carried by the slowest component of axonal transport.
Asunto(s)
Transporte Axonal , Proteínas del Citoesqueleto/metabolismo , Diabetes Mellitus Experimental/metabolismo , Ratones Mutantes/metabolismo , Actinas/metabolismo , Animales , Autorradiografía , Proteínas del Citoesqueleto/aislamiento & purificación , Citoesqueleto/metabolismo , Neuropatías Diabéticas/metabolismo , Electroforesis en Gel de Poliacrilamida , Femenino , Ratones , Ratones Endogámicos C57BL , Nervio Ciático/metabolismo , Tubulina (Proteína)/metabolismoRESUMEN
Acetylcholinesterase occurs in the frog sciatic nerve under five stable molecular forms with distinct sedimentation coefficients in sucrose gradients: 3 globular forms (3.6S, 6S and 10.5S) and two asymmetric ones (14S and 18S). Whereas in birds and mammals, the asymmetric tailed forms of acetylcholinesterase are present in trace amounts in peripheral nerves and account for only a small part of the enzyme activity submitted to a rapid axonal transport, the two asymmetric 14S and 18S forms represent nearly 50% of total activity in the frog sciatic nerve and account for 60-70% of the acetylcholinesterase activity accumulated at both sides of a nerve transection, the rest being due to an accumulation of globular molecules. We showed that the three forms, 10.5S, 14S and 18S, are all carried with the fast phase of axonal transport at a velocity of 100-120 mm/day in the anterograde direction and 20-30 mm/day in the retrograde direction. The velocity of transport for the light molecular forms 3.6S and 6S could not be calculated. In addition, we observed that large amounts not only of the 10.5S but also of the asymmetric 14S and 18S forms appear to be stationary along the frog sciatic nerve, contrary to the situation described for peripheral nerves in birds or mammals. Our results thus reveal that some axonal transport parameters for the asymmetric forms of acetylcholinesterase greatly differ in the peripheral nerves of amphibians on the one hand and of birds and mammals on the other, suggesting that these heavy molecular forms might have distinct functions in the nerves of lower and higher vertebrates.
Asunto(s)
Acetilcolinesterasa/metabolismo , Isoenzimas/metabolismo , Nervios Periféricos/enzimología , Animales , Transporte Axonal , Pollos , Rana esculenta , Ratas , Especificidad de la Especie , Factores de TiempoRESUMEN
During the development of diabetic neuropathy in the mouse C57BL/Ks (db/db), the axonal transport of AChE molecular forms was tested in the sciatic nerve, by measuring the accumulation of enzyme activity in front of a nerve transection. No alteration of the fast flow rate of G4 and A12 molecular forms was found until 220 days of age. On the other hand, a reduced flow rate of G1 and G2 molecular forms, probably conveyed by slow axoplasmic flow, was noticed in the late phase of diabetic neuropathy. This result is consistent with the view that axonal dwindling could be related to disturbances of slow axonal transport and that the reduction in conduction velocity, observed at an earlier stage, may be due to other causes.
Asunto(s)
Acetilcolinesterasa/metabolismo , Transporte Axonal , Diabetes Mellitus/metabolismo , Ratones/metabolismo , Animales , Diabetes Mellitus/genética , Femenino , Ratones Endogámicos C57BL/genéticaRESUMEN
In chick sciatic nerve, acetylcholinesterase (AChE) occurs in four main molecular forms characterized by their sedimentation coefficients in sucrose gradients, referred to as G1 (5S), G2 (7.5S), G4 (11S), and A12 (20S). Under normal conditions, we previously showed by accumulation technique that the G4 and A12 forms are rapidly transported along the axons, whereas G1 and G2 are carried much more slowly. Here, we used to the same technique to study the anterograde axonal transport of these different AChE forms during normal axonal growth and experimental regeneration. During the first 2 months after hatching, G4 and A12 transport virtually doubled, whereas G1 + G2 transport increased only slightly. After nerve cutting, crushing, or freezing, the flow rates of G1 + G2 and G4 in the regenerating proximal stump decreased by 75% at 4 to 7 days compared with control values and that of A12, by 90 to 95%. In crushed and frozen nerves the transport of all four AChE forms slowly recovered thereafter, but failed to attain control values even after 7 weeks. In cut nerves, on the contrary, no significant recovery of G1 + G2, or G4 transport occurred, but A12 transport began to recover by day 7. Taken together, our results show that axonal transport of G1 + G2, G4, and A12 is selectively regulated in chick sciatic nerve, and suggest that the A12 form of AChE might have a special role and/or destination in regenerating axons.
Asunto(s)
Acetilcolinesterasa/metabolismo , Transporte Axonal , Regeneración Nerviosa , Nervio Ciático/metabolismo , Acetilcolinesterasa/análisis , Animales , Cromatografía , Peso Molecular , Compresión Nerviosa , Nervio Ciático/fisiologíaRESUMEN
Butyrylcholinesterase was found in chick sciatic nerve in four main molecular forms--G1, G2, G4 and A12--distinguishable by thier sedimentation coefficients in sucrose gradients (4.2S, 6.4S, 11.3S and 19S, respectively). Axonal transport of butyrylcholinesterase was studied by measuring the accumulation of its molecular forms on each side of a transected sciatic nerve. Twenty-four hours after transection, butyrylcholinesterase activity had risen by about 32% at the extremity of the proximal stump, and by 20% at the extremity of the distal stump. Proximal accumulation was due to a two-fold rise in G4 activity and to a six-fold rise in A12 activity, whereas distal accumulation was exclusively due to a 50% increase in G4 activity, accompanied by the complete loss of A12. The activities of G1 and G2 remained stable in both directions. Under our experimental conditions, the accumulation of butyrylcholinesterase activity cannot be attributable to local protein synthesis, cross-contamination with accumulated acetylcholinesterase or the presence of plasma butyrylcholinesterase. Hence we conclude that all A12 butyrylcholinesterase molecules were carried in the anterograde direction, moving at 11.6 +/- 4.2 mm/day, and that probably some of the G4 molecules were slowly transported in both directions. These findings suggest that some of the butyrylcholinesterase is located in the axonal mitochondria and/or axolemma.