Presence of physiologically stimulated RET in adult rat brain: induction of RET expression during nerve regeneration.

The product of the RET proto-oncogene is a protein belonging to the receptor-like tyrosine kinase superfamily. RET is expressed in several neural crest-derived cell lineages and has been implicated in the correct development of the peripheral nervous system. To gain further insight into RET function, we investigated the presence of active RET in adult rat tissues. We show, by immunoblotting, that the products of the RET proto-oncogene (p155ret) are present in specific regions of adult rat brain, including the cerebellum, striatum, brainstem, hypothalamus, hippocampus, and olfactory bulb. Moreover, in the cerebellum, p155ret is phosphorylated in tyrosine residues, thus indicating that this brain structure contains p155ret in an activated state. Finally, the presence of RET in motoneurons prompted us to analyze the effects of hypoglossal nerve section on its expression. We observed a dramatic increase in p155ret in the motoneuron nuclei, thus suggesting that RET tyrosine kinase plays a role in the neuronal response to axotomy and/or during nerve regeneration.

Behavioural pain-related disorders and contribution of the saphenous nerve in crush and chronic constriction injury of the rat sciatic nerve.

This study evaluated the pain-related behaviours induced by 2 models of peripheral sciatic nerve injuries in the rat: transient nerve crush and chronic constriction injury (CCI). Various lesions of the saphenous nerve were performed in order to investigate the role of saphenous innervation in behavioural disorders induced by these nerve injuries. Behavioural testing included assessment of responses to phasic stimulation (mechanical and thermal) and observation of 'spontaneous' pain-related behaviour. Results confirmed that the model of CCI induces marked and prolonged phasic and spontaneous pain-related disorders (up to week 7). Rats with crush injury exhibited moderate and transient hyperalgesia and allodynia to mechanical and thermal stimulation on the lesioned side (with a maximum at day 3 and a recovery by week 1). Section plus ligation of the ipsilateral saphenous nerve on the day of surgery prevented nociceptive behaviours and induced persistent mechanical and thermal anaesthesia or hypoesthesia of the lesioned paw in both models (lasting up to 3-4 weeks). Section without ligation of the saphenous nerve induced comparable results in rats with sciatic crush, but did not significantly modify nociceptive behaviours in rats with CCI. These data emphasise the role of adjacent saphenous nerve in the mechanisms of pain-related disorders induced by these peripheral nerve lesions. On the contralateral paw, pain-related modifications were also observed in both models, suggesting that unilateral nerve lesions induce remote modifications extending beyond the site of the injured nerve.

Axonal transport of type III intermediate filament protein peripherin in intact and regenerating motor axons of the rat sciatic nerve.

Slow axonal transport of peripherin has been studied in the motor axons of both intact and regenerating rat sciatic nerves 7 days post-crush. The studies were done by two-dimensional gel electrophoresis after intraspinal injection of 35S-methionine. In the first experiment, the sciatic nerves were removed 3 weeks after the radiolabeling pulse and cut into 6 mm segments. Each nerve segment was submitted to two-dimensional gel electrophoresis and analyzed by an original procedure which allowed us to study the distribution along the nerve of the radioactivity associated with several proteins of the cytoskeleton, especially the intermediate filament proteins, peripherin, and the low molecular mass neurofilament protein, NF-L. Peripherin was transported at two main rates: 66% of the total radiolabeled peripherin moved at 1.42 mm/day and the remainder moved at 2.28 mm/day. The radioactivity associated with NF-L exhibited a similar pattern. In the second experiment, similar intraspinal injections were made 7 days after a unilateral crush of the sciatic nerve. Regenerating nerves exhibited a clear SCa wave. However, in contrast to the intact nerves, the SCb wave could not be precisely defined in the regenerating nerves. Thus, the changes in the amount of transported proteins were analyzed in the SCa wave only. Autoradiograms of 2D-PAGE revealed that in the regenerating axons, the quantity of transported peripherin in SCa was increased by 3.5-fold. In contrast, the quantity of transported NF-L was decreased by 1.6-fold. The regenerating motor axons conveyed significantly greater (approximately twofold) amounts of labeled tubulins and actin than did intact motor axons. Our results suggest that peripherin, although mainly conveyed by SCa, plays a role during the elongation process in addition to actin and tubulin.

Identification of a peripherin dimer: changes during axonal development and regeneration of the rat sciatic nerve.

Western blotting of rat dorsal root ganglion (DRG) and sciatic nerve under nonreducing conditions revealed that a peripherin-specific antibody recognized a protein species of 116/130 kDa, pI 5.6, in addition to peripherin (56 kDa, pI 5.6). We showed that this 116/130 kDa protein is a disulfide dimer of peripherin, because it gave rise to a single protein band comigrating with peripherin under reducing conditions and yielded the same proteolytic pattern as peripherin upon N-chlorosuccinimide digestion. In addition, the immunological characteristics of the resulting peptides were identical to those of peripherin. We investigated the changes in peripherin monomer and dimer protein levels during axonal development and regeneration. During postnatal development, quantitative analysis of western blots of DRG proteins showed a significant increase in peripherin monomer (+52%) and dimer (+33%) levels from the day of birth [postnatal day 0 (P0)] to P7. The monomer levels remained high until P14 and then decreased so that at P21 and later ages, the monomer levels were similar to those observed at birth. In contrast, the dimer levels decreased continuously after P7, and in the adult, its level represented only 30% of the level at birth. Changes in [35S]methionine incorporation into adult DRG proteins were studied during regeneration of axotomized sciatic axons. Quantitative analysis of proteins showed a strong increase in labeling of both peripherin monomer (+56%) and dimer (+88%) 7 days after the crush. These levels, which remained high until 28 days after the axotomy, had returned to normal 70 days post axotomy. Our results show that peripherin monomer and dimer greatly increase during DRG fiber development and regeneration, suggesting that the two forms are involved in the growth of axons.

Proteins of slow axonal transport in sciatic motoneurones of rats with streptozotocin-induced diabetes or galactosaemia.

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.

Modifications in the cytoskeleton transported by slow component A during axonal regeneration.

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.

Slow axonal transport impairment of cytoskeletal proteins in streptozocin-induced diabetic neuropathy.

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.

Differential axonal transport of isotubulins in the motor axons of the rat sciatic nerve.

The axonal transport of the diverse isotubulins in the motor axons of the rat sciatic nerve was studied by two-dimensional polyacrylamide gel electrophoresis after intraspinal injection of [35S]methionine. 3 wk after injection, the nerve segments carrying the labeled axonal proteins of the slow components a (SCa) and b (SCb) of axonal transport were homogenized in a cytoskeleton-stabilizing buffer and two distinct fractions, cytoskeletal (pellet, insoluble) and soluble (supernatant), were obtained by centrifugation. About two-thirds of the transported-labeled tubulin moved with SCa, the remainder with SCb. In both waves, tubulin was found to be associated mainly with the cytoskeletal fraction. The same isoforms of tubulin were transported with SCa and SCb; however, the level of a neuron-specific beta-tubulin subcomponent, termed beta', composed of two related isotubulins beta'1 and beta'2, was significantly greater in SCb than in SCa, relative to the other tubulin isoforms. In addition, certain specific isotubulins were unequally distributed between the cytoskeletal and the soluble fractions. In SCa as well as in SCb, alpha''-isotubulins were completely soluble in the motor axons. By contrast, alpha''' and beta'2-isotubulins, both posttranslationally modified isoforms, were always recovered in the cytoskeletal fraction and thus may represent isotubulins restricted to microtubule polymers. The different distribution of isotubulins suggests that a recruitment of tubulin isoforms, including specific posttranslational modifications of defined isoforms (such as, at least, phosphorylation of beta' and acetylation of alpha'), might be involved in the assembly of distinct subsets of axonal microtubules displaying differential properties of stability, velocity and perhaps of function.

Stable and metastable cytoskeletal polymers carried by slow axonal transport.

The proteins carried by the slow axonal transport in the rat sciatic motor axons were radiolabeled by injecting 35S-methionine into the spinal cord, and the distribution of their solubility through the 2 main components of slow transport (SCa and SCb) was considered. For this purpose, a cytoskeleton-stabilizing buffer was designed in which a pellet enriched in macromolecular and polymeric structures was separated from the solubilized proteins. The monomer/polymer ratios for tubulin were quantified in the 2 rate components. Our results indicate that 90% of the total tubulin was carried with SCa. Of this, 75% was in a polymeric state, versus only 50% of the tubulin carried with SCb. The monomeric tubulin recovered in the soluble fraction was concomitantly transported with the polymerized microtubules, suggesting that it might represent metastable regions of these microtubules. The insoluble and soluble fractions of the transported actin were measured. Actin was mostly (70%) transported with SCb. Of this, more than 80% was recovered in the soluble fraction, but we cannot say whether it was in a monomeric or polymeric state, nor if it was transported free or bound to a structure solubilized during fractionation. The other 30% of the actin, most of it transported with SCa, was recovered in the polymer-enriched fraction, probably bound to a stabilized polymer, such as the microtubules.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclic AMP metabolism in the inner and outer layers of human myometrium near term.

The metabolism of cAMP which appears to be the intracellular mediator of various relaxing agents was studied in biopsies obtained during elective caesarean section from inner and outer myometrial layers outside the placental insertion. In the inner layer, L-epinephrine, PGE1, PGE2, PGF2 alpha and PGI2 stimulated the cAMP formation process while 6-keto PGF1 alpha was ineffective. The fact that some of these prostaglandins are well-known to promote contraction, confirms that the effects of drugs on uterine motility are not necessarily related to changes in the cAMP level. On the other hand, L-epinephrine and prostaglandins did not strongly influence the cAMP formation process in the outer layer. Kinetic analysis and purification assays of phosphodiesterase (PDE) which catalyzes the degradation of cAMP revealed the presence of multiple molecular forms of the enzyme in human pregnant myometrium. Qualitative and quantitative differences between the two layers appeared in the two forms separated from the soluble fraction by DEAE-cellulose chromatography. An unequal distribution of calmodulin was also observed in the inner and outer layers. Our results support the concept of the regulatory heterogeneity of the pregnant human uterus and suggest that the myometrial inner layer plays an important role in the regulation of uterine motility at the end of pregnancy.

Altered axonal transport of cytoskeletal proteins in the mutant diabetic mouse.

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.

Quantitative analysis of axonal transport of cytoskeletal proteins in chicken oculomotor nerve.

We studied the axonal transport characteristics of major cytoskeletal proteins: tubulin, the 69,000 molecular weight protein of chicken neurofilaments, and actin. After intracerebral injection of [35S]methionine, we monitored the specific radioactivity of these proteins as they passed through a very short nerve segment of the chicken oculomotor nerve. Specific radioactivities were assessed by quantitative sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. The transport patterns obtained for tubulin and the neurofilament protein were very similar, corresponding to transport rate ranges of 1-15 and 1-10 mm/day, respectively. A narrower velocity range of 3 to 4.3 mm/day was found for actin. Tubulin and the neurofilament protein appeared to be largely dispersed during the course of their transit along the nerve. The radioactivity associated with the proteins studied persisted in the nerve segment for a long time after the bulk of the labeled molecules had swept down. Finally, none of these proteins was observed to be transported with the fast axonal transport.

Study of the 10-nm-filament fraction isolated during the standard microtubule preparation.

The cold non-depolymerizable fractions obtained during the standard procedure for the isolation of microtubules from ox brain stem-cerebral hemispheres and spinal cord have been studied. The cerebral-hemisphere preparation was composed of 10-nm filaments but also contained large amounts of membranes. The polypeptide content included tubulin, microtubule-associated proteins and minor proteins corresponding to the neurofilament triplet of proteins of mol.wt. 210 000, 160 000 and 70 000 respectively. The brain-stem preparation contained more 10-nm filaments than membranes. The polypeptide content consisted of the neurofilament triplet (35%), tubulin (30%) and minor proteins. In contrast, the spinal-cord preparation was mainly composed of 10-nm filaments, free of membranes and containing essentially the neurofilament protein triplet (64%). These filaments appeared very similar to the peripheral-nervous-system neurofilaments described by several authors. Since the best neurofilament from the central nervous system often contained less than 15% of the neurofilament protein triplet, our spinal-cord preparation is an improvement on the usual neurofilament preparation. This simple and rapid method gave large amounts of 10-nm filaments (100 mg per 100 g of spinal cord) characterized by the absence of membranous material, a low content of tubulin and the 50 000-mol.wt.-protein component, and a high content of neurofilament peptides. Thus, the presence of tubulin in 10-nm filament preparations seems to be related to the contaminant membranous material and not to be linked to the interaction in vitro of tubulin or microtubules with neurofilaments, as has been suggested previously.