The effect of axotomy on the expression of the 57 kDa neuronal intermediate filament (IF) protein in adult rat dorsal root ganglion (DRG) neurons was examined. This IF protein is known to have an exclusively neuronal localization but is considerably more limited in its distribution in the nervous system than the neurofilament (NF) triplet proteins. The 57 kDa neuronal IF protein is similar (and perhaps identical) to the protein "peripherin" and is known to be the product of a Type III IF gene. Since the down-regulated expression of NF proteins (products of type IV IF genes) has been well established, it was of interest to determine whether the novel 57 kDa IF protein was regulated in a similar or different manner from that of the NFs in axotomized neurons. In vitro pulse-labeling of DRGs with 35S-methionine: cysteine followed by 2-dimensional gel electrophoresis/fluorography revealed that the synthesis of the 57 kDa neuronal IF protein was increased 2 weeks after sciatic nerve crush. Immunocytochemical studies using a polyclonal antibody to the 57 kDa neuronal IF protein showed that the immunodetectable levels of this protein increased in DRG neurons after peripheral axotomy. In the normal DRG, staining was localized almost exclusively to small-sized neurons. At 2 weeks after axotomy, however, large- and medium-sized neurons also became immunoreactive; in addition, the overall level of staining in the DRG was greater than normal. Quantitative analysis of in situ hybridizations of DRG neurons with a 35S-labeled cDNA probe specific for the 57 kDa neuronal IF protein revealed a significant increase in the level of 57 kDa IF mRNA in the large-sized (greater than 1000 microns2) neurons 2 weeks after axotomy; the level of 57 kDa IF mRNA in the small neurons was not different from normal at that time. Finally, using a newly developed paradigm for examining the composition of regenerating axons by axonal transport, we determined that significant amounts of the 57 kDa neuronal IF protein were conveyed into the regrowing axonal sprouts of DRG neurons. When DRG neurons were conditioned by a previous axotomy (a crush axotomy of the distal sciatic nerve 2 weeks earlier) and then stimulated to regenerate axons by a second crush axotomy located very close to the DRG, the regenerating sprouts incorporated and conveyed significantly more 57 kDa IF protein by slow axonal transport than did those elaborated by unprimed DRG neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Quantitative in situ hybridization and RNA blotting methods were used to define the time course and magnitude of changes in expression of mRNAs encoding peripherin and the neurofilament (NF) triplet proteins in rat dorsal root ganglion (DRG) neurons during axonal regeneration. mRNA levels in adult rat L4 and L5 DRGs were examined in autoradiograms after in situ hybridization with specific 35S-labeled cDNA probes 1-56 days following unilateral crush lesions of the sciatic nerve. The results of quantitative analyses indicated that peripherin mRNA levels were significantly increased in large-sized (greater than 1000 microns 2) DRG neurons at 7, 14, and 28 days after axotomy while the mRNA levels for each of the NF triplet proteins were significantly decreased at these same time points. The mRNA levels of the low (NF-L) and middle (NF-M) sized NF subunits were significantly decreased as early as 1 day postaxotomy but the mRNA level of the large NF subunit (NF-H) did not change until 7 days after axotomy. The maximal reduction in NF mRNA levels was observed at 14 days postaxotomy when NF-L mRNA levels were only 35% of those in large-sized, normal control neurons. Recovery toward normal levels of both NF and peripherin mRNAs was observed at 8 weeks postaxotomy. RNA blot analyses with total RNA obtained from DRGs at different postaxotomy times confirmed that NF-L mRNA levels were reduced in the DRG during the first 4 weeks after axotomy but, interestingly, failed to detect an increase in peripherin mRNA levels. This difference concerning peripherin mRNA levels in axotomized preparations obtained by RNA blotting vs. in situ hybridization was attributed to the fact that RNA blots utilized total DRG RNA which includes mRNAs from both small and large-sized DRG neurons. A recent in situ hybridization study showed that the small-sized DRG neurons which contain the majority of the peripherin mRNA in the DRG do not increase their peripherin mRNA levels 14 days after axotomy (Oblinger et al., 1989b). This may mask any change in the large neuron response when total RNA is examined. Overall, the results of this study demonstrate (1) that type III (peripherin) and type IV (NF) intermediate filament genes are regulated differently during axonal regeneration, and (2) that the three NF genes are down-regulated in a fairly coordinate manner during regeneration. These data suggest that an important component of the regeneration program is the alteration of the composition of the IF component of the cytoskeleton.(ABSTRACT TRUNCATED AT 400 WORDS)

Many of the structural and functional differences between axons are thought to reflect underlying differences in the biochemical composition and dynamic aspects of the axonal cytoskeleton and cytomatrix. In this study we investigated how the composition of the 2 slow components of axonal transport, SCa and SCb, which convey the cytoskeleton and cytomatrix, differs in axons that are structurally and functionally distinct. For this comparison we analyzed axons of retinal ganglion cells in the optic nerve (ON), axons of dorsal root ganglion (DRG) cells, and axons of ventral motor neurons (VMN) in adult rats. 35S-Methionine-labeled proteins transported with the peak of SCa and SCb were analyzed using high-resolution 2-dimensional polyacrylamide gels (2D-PAGE) and fluorography, and the amounts of major SCa and SCb proteins were quantified. The polypeptide composition of both SCa and SCb was found to be largely similar in DRG and VMN axons, but major qualitative as well as quantitative differences between these axons and ON axons were found. Notable among these were higher ratios of neurofilament protein to tubulin in SCa in DRG and VMN axons compared to ON axons, and significantly larger amounts of 2 microtubule-associated proteins relative to tubulin in SCa of ON axons than in both VMN and DRG axons. Tubulin was the major SCb protein in VMN and DRG axons, but it was not present in SCb in ON axons. Additionally, relatively larger amounts of 2 metabolic enzymes, creatine phosphokinase and nerve-specific enolase, were present in SCb in ON axons than in DRG or VMN axons. The results indicate that significant biochemical heterogeneity among different types of axons can be identified by examining the slow components of axonal transport.

In the mature rat dorsal root ganglion (DRG), only one tau isoform is expressed, and this protein (110 kDa in apparent molecular weight) is considerably larger in size than the predominant tau isoforms found in brain. The size of the mRNA encoding the "big" tau mRNA in DRG [approximately 8 kilobases (kb)] is also much larger than that of the major rat brain tau mRNA species (approximately 6 kb). In this study, we examined the pattern of normal developmental changes in expression of this high-molecular-weight (HMW) tau and its encoding mRNA and also determined how axonal injury of adult DRG neurons effected the expression of this gene. RNA blotting experiments revealed that higher levels of HMW tau mRNA were present in the DRG at early postnatal times than in the adult. Immunoblotting of total DRG protein using a monoclonal tau antibody revealed that the immature DRG (7 d postnatal) contained a 62-kDa tau isoform in addition to the HMW tau isoform that was expressed in the adult DRG. Neither of the tau isoforms expressed in the immature DRG was present to any significant extent in either immature or adult rat brain. To examine how tau expression changed in adult DRG neurons during regeneration, the sciatic nerves of rats were unilaterally crushed, and the L4 and L5 DRG were harvested 1, 7, and 14 d later.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in the synthesis and axonal transport of neurofilament (NF) proteins and tubulin were examined after various selective axotomies of adult rat DRG cells. For axonal transport studies, DRGs were labeled by microinjection of 35S-methionine 14 d after axonal injuries, and nerves were retrieved 7 or 14 d after labeling. Slowly transported proteins were examined by quantitative PAGE/fluorography. After distal peripheral nerve crush (50-55 mm from the DRG), the cytoskeleton that entered undamaged regions of peripheral branch DRG axons by slow axonal transport differed from normal, while the cytoskeleton that entered dorsal root axons did not. Specifically, smaller-than-normal ratios of labeled NF protein/tubulin were transported in peripheral DRG axons after distal peripheral nerve crush. This change was almost entirely due to a selective decrease in the output of labeled NF proteins rather than to an increase in the amount of tubulin transported with NF proteins. Since the efficiency of axonal regeneration is known to be lower after cut injury than after nerve crush, we compared the effect of cut versus crush axotomy of peripheral DRG axons on cytoskeletal protein output. A more substantial reduction in the labeled NF/tubulin transport resulted in peripheral DRG axons if the distal sciatic nerve was cut rather than crushed but, even under these axotomy conditions, the labeled NF/tubulin ratios in dorsal root axons were not reduced. Peripheral cut axotomy did result in a lag in the advance of the labeling peak of the NF/microtubule protein wave in dorsal root axons, suggesting either that these proteins were delayed in exiting the cell body or that a slowing of the rate of their transport occurred. Pulse-labeling DRGs in vitro using 35S-methionine, and analysis of labeled proteins by 2-dimensional PAGE-fluorography demonstrated that the incorporation of radioactivity into NF proteins was significantly reduced, while the labeling of tubulins was unchanged 14 d after distal peripheral axotomy. In contrast to the results of peripheral axotomy, dorsal root crushes made close to the DRG (2-3 mm) or considerably distal (at the CNS entry zone 28-30 mm from the DRG) did not produce detectable changes in the amount of labeled NF or tubulin transport in central or peripheral branch axons. These findings indicate that the down-regulation of NF production/output that is exhibited at 14 d after peripheral branch axotomy is not present after central branch injury.(ABSTRACT TRUNCATED AT 400 WORDS)

We have previously demonstrated that systemic administration of testosterone increases the rate of axonal regeneration following facial nerve crush in adult male hamsters. In this investigation, the molecular mechanisms by which androgens may enhance axonal regeneration were examined. Specifically, the following question was addressed using Northern blot and in situ hybridization with three cytoskeletal cDNA probes complementary to beta II-, beta III-, and alpha 1-tubulin mRNA: does exogenous testosterone augment axotomy-induced changes in tubulin mRNA expression in hamster facial motoneurons (FMN)? Adult male hamsters were subjected to unilateral facial nerve severance, with the opposite side serving as an internal control. One-half of the animals were subcutaneously implanted with Silastic capsules containing crystalline testosterone propionate and the other half implanted with blank capsules. Postoperative survival times were 2 and 7 d. At 2 d after axotomy alone, no changes in levels of any of the three tubulin mRNAs were observed in the injured FMN. However, by 7 d after axotomy, significant increases in all three tubulin mRNAs were observed. This time course of axotomy-induced changes in tubulin gene expression is consistent with findings in other injured neuronal populations. Administration of testosterone at the time of injury had two major effects on the cytoskeletal response pattern in axotomized FMN. First, testosterone differentially regulated the set of tubulin mRNAs examined, in that beta II-tubulin mRNA levels were selectively altered by the steroid, whereas beta III- or alpha 1-tubulin mRNAs were not.(ABSTRACT TRUNCATED AT 250 WORDS)

Estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) mRNAs are both expressed in rat dorsal root ganglion (DRG) neurons, but the distribution of these two mRNAs differs markedly. Radiolabeled probes highly specific to ERalpha or ERbeta mRNAs were used for in situ hybridization studies; two antibodies specific to ERalpha protein were used for immunocytochemistry and specific primers were used for reverse transcription polymerase chain reaction (RT-PCR) studies. These revealed that ERbeta mRNA is widely expressed in the DRG of both male and female rats, with virtually all neurons showing positive signals. In contrast, ERalpha mRNA, as well as nuclear localized ERalpha protein, is more restricted in its localization and is present in many, but not all, of the small-sized (<600 microm(2)) DRG neurons, but is only rarely present in larger neurons. The L6-S1 DRG levels, which contain sensory neurons that innervate reproductive tissues, are relatively enriched in ERalpha compared to L3-L5 DRG levels, which contain sensory neurons that innervate hind limb regions. Long-term estrogen treatment of ovariectomized rats (21-28 days) dramatically reduces immunocytochemically detectable ERalpha protein in the DRG relative to that in ovariectomized controls. RT-PCR studies also showed that long-term estrogen treatment of ovariectomized rats downregulates the levels of ERalpha mRNA, but upregulates the levels of ERbeta mRNA in the DRG. Interestingly, in intact cycling female rats, ERalpha and ERbeta mRNA levels in the DRG were both higher during proestrus compared to metestrus. These findings suggest that the changes in expression of estrogen receptors which occur dynamically during the estrus cycle differ from those induced by long-term estrogen treatment of ovariectomized animals.

Axotomy of the peripheral axon of dorsal root ganglion (DRG) cells is known to result in chromatolysis and changes in protein synthesis in DRG cells. We investigated whether a stimulus produced by peripheral branch axotomy would affect the regenerative properties of both the central and peripheral axon of the DRG cell equally. To examine this question, a conditioning crush lesion was made distally on the sciatic nerve 2 weeks prior to a testing lesion of either the dorsal root or peripheral branch axon near the DRG. Fast axonal transport of radioactive proteins was used to assess regeneration of DRG axons. In the adult rat, leading peripheral branch axons normally regenerate at a rate of 4.4 mm/day. If a conditioning lesion of the sciatic nerve is made 2 weeks before the test lesion, the rate of peripheral branch axonal regeneration increases by 25% to 5.5 mm/day. This effect is not limited to the fastest growing axons in the nerve since a population of more slowly growing axons also exhibits accelerated outgrowth in response to a prior peripheral axotomy. In contrast to this, the fastest growing central branch axons of DRG cells, which normally regenerate at a rate of 2.5 mm/day, are not significantly affected by a prior peripheral axotomy. A population of more slowly growing axons in the dorsal root also does not exhibit accelerated outgrowth in response to a peripheral conditioning lesion. The results of these experiments indicate that changes in the DRG neuron's metabolism induced by prior axotomy of its peripheral axon do not affect the regenerative properties of both axons equally. This raises the possibility that accelerated axonal outgrowth in only one axonal branch results from a differentially regulated supply of proteins to the two axons by the DRG cell body.