Franz-Werner Schwaiger

Peripheral but not central axotomy induces changes in Janus kinases (JAK) and signal transducers and activators of transcription (STAT)

Nerve injury leads to the release of a number of cytokines which have been shown to play an important role in cellular activation after peripheral nerve injury. The members of the signal transducer and activator of transcription (STAT) gene family are the main mediators in the signal transduction pathway of cytokines. After phosphorylation, STAT proteins are transported into the nucleus and exhibit transcriptional activity. Following axotomy in rat regenerating facial and hypoglossal neurons, a transient increase of mRNA for JAK2, JAK3, STAT1, STAT3 and STAT5 was detected using in situ hybridization and semi‐quantitative polymerase chain reaction (PCR). Of the investigated STAT molecules, only STAT3 protein was significantly increased. In addition, activation of STAT3 by phosphorylation on position Tyr705 and enhanced nuclear translocation was found within 3 h in neurons and after 1 day in astrocytes. Unexpectedly, STAT3 tyrosine phosphorylation was obvious for more than 3 months. In contrast, none of these changes was found in response to axotomy of non‐regenerating Clarkes nucleus neurons, although all the investigated models express c‐Jun and growth‐associated protein‐43 (GAP‐43) in response to axonal injury. Increased expression of Janus kinase (JAK) and STAT molecules after peripheral nerve transection suggests changes in the responsiveness of the neurons to signalling molecules. STAT3 as a transcription factor, which is expressed early and is activated persistently until the time of reinnervation, might be involved in the switch from the physiological gene expression to an ‘alternative program’ activated only after peripheral nerve injury.

Neuronal MCP-1 Expression in Response to Remote Nerve Injury

Direct injury of the brain is followed by inflammatory responses regulated by cytokines and chemoattractants secreted from resident glia and invading cells of the peripheral immune system. In contrast, after remote lesion of the central nervous system, exemplified here by peripheral transection or crush of the facial and hypoglossal nerve, the locally observed inflammatory activation is most likely triggered by the damaged cells themselves, that is, the injured neurons. The authors investigated the expression of the chemoattractants monocyte chemoattractant protein MCP-1, regulation on activation normal T-cell expressed and secreted (RANTES), and interferon-gamma inducible protein IP10 after peripheral nerve lesion of the facial and hypoglossal nuclei. In situ hybridization and immunohistochemistry revealed an induction of neuronal MCP-1 expression within 6 hours postoperation, reaching a peak at 3 days and remaining up-regulated for up to 6 weeks. MCP-1 expression was almost exclusively confined to neurons but was also present on a few scattered glial cells. The authors found no alterations in the level of expression and cellular distribution of RANTES or IP10, which were both confined to neurons. Protein expression of the MCP-1 receptor CCR2 did not change. MCP-1, expressed by astrocytes and activated microglia, has been shown to be crucial for monocytic, or T-cell chemoattraction, or both. Accordingly, expression of MCP-1 by neurons and its corresponding receptor in microglia suggests that this chemokine is involved in neuron and microglia interaction.

Exonic polymorphism vs intronic simple repeat hypervariability in MHC-DRB genes

Gene products encoded by the major histocompatibility complex often exhibit a high degree of polymorphism. In humans the HLA-DR polymorphism is due to more than 50 alleles with varying exon 2 sequences. Each group of DRB alleles contains a certain form of the basic simple repeat motif (gt)n(ga)m in intron 2. Identical alleles can be differentiated on the basis of the hypervariable repeat. In this study focused on cattle (Bos taurus) we identified different Bota-DRB alleles in a limited survey by amplification via polymerase chain reaction and sequencing. In addition DRB exon 2 sequences were also obtained from eight additional hoofed animal species (seven horned artiodactyls and one pig) revealing artiodactyl-specific polymorphic and nonpolymorphic substitutions. In the genus Bos the intronic simple repeat variability was compared with exonic DRB polymorphism. As in humans all Bota-DRB exons were always associated with specifically organized basic simple repeat structures. Yet the extent of simple repeat variability was lower in cattle compared to humans. Selective breeding in the process of domestication might be responsible for the diminished intronic hypervariability. Nevertheless, the hypermutable simple repeat sequences have been preserved in the same position and with the same principal structure for at least 70 × 106 years of evolution. Unexpectedly, the rate of intronic simple repeat and exonic changes appear quite similar.

Identification of regeneration-associated genes after central and peripheral nerve injury in the adult rat

BackgroundIt is well known that neurons of the peripheral nervous system have the capacity to regenerate a severed axon leading to functional recovery, whereas neurons of the central nervous system do not regenerate successfully after injury. The underlying molecular programs initiated by axotomized peripheral and central nervous system neurons are not yet fully understood.ResultsTo gain insight into the molecular mechanisms underlying the process of regeneration in the nervous system, differential display polymerase chain reaction has been used to identify differentially expressed genes following axotomy of peripheral and central nerve fibers. For this purpose, axotomy induced changes of regenerating facial nucleus neurons, and non-regenerating red nucleus and Clarkes nucleus neurons have been analyzed in an intra-animal side-to-side comparison. One hundred and thirty five gene fragments have been isolated, of which 69 correspond to known genes encoding for a number of different functional classes of proteins such as transcription factors, signaling molecules, homeobox-genes, receptors and proteins involved in metabolism. Sixty gene fragments correspond to genomic mouse sequences without known function. In situ-hybridization has been used to confirm differential expression and to analyze the cellular localization of these gene fragments. Twenty one genes (~15%) have been demonstrated to be differentially expressed.ConclusionsThe detailed analysis of differentially expressed genes in different lesion paradigms provides new insights into the molecular mechanisms underlying the process of regeneration and may lead to the identification of genes which play key roles in functional repair of central nervous tissues.

Neuregulin-1 isoforms are differentially expressed in the intact and regenerating adult rat nervous system.

Our knowledge on Neuregulin-1 (Nrg-1) during development of the nervous system is increasing rapidly, but little is known about Nrg-1-ErbB signaling in the adult brain. Nrg-1 is involved in determination, proliferation, differentiation, and migration of neurons and glial cells in the developing brain. In the peripheral nervous system, Nrg-1 signaling is required for Schwann cell differentiation and myelination, and establishment of neuromuscular junctions (NMJs). Multiple alternative splicing of Nrg-1 was shown, but correlation of its structural and functional diversity was rarely addressed. Therefore, we investigated the expression of Nrg-1 isoforms in the rat brain and brain-derived cell types, and their involvement in regeneration of the adult brain, using immunohistochemistry, in situ hybridization, and semiquantitative RT-PCR. We found expression of at least 12 distinct Nrg-1 isoforms in the brain and altered expression of several isoforms in the facial motor nucleus after peripheral transection of the seventh cranial nerve. An upregulation of Nrg-1 type-I mRNA, probably type-I-α, was observed in reactive astrocytes of the facial nucleus 1 d postaxotomy. Nrg-1 type-III and the splice variants β1 and β5 are dramatically downregulated in axotomized motoneurons, which lack contact to their target tissue. Baseline expression levels were reestablished when the first axons reached the facial muscles and reformed NMJs. Nrg-1-β1 and -β5 might act in maintenance of NMJs. The splice variants β2 and β4 display an initial downregulation of mRNA levels, followed by an increase during the period of axon remyelination. Thus, Nrg-1-β2 and -β4 might be involved in myelination.

Exonic MHC-DRB Polymorphisms and Intronic Simple Repeat Sequenees: Janus’Faces of DNA Sequence Evolution

The evolution of highly polymorphic gene loci is following routes that cannot be extrapolated from the existing knowledge of single copy genes. In addition, interpreting the evolution of the most polymorphic loci in vertebrates requires a plethora of data from different taxa. We evaluate here the rules for the evolution of Major Histocompatibility Complex (MHC‐)DRB genes recently established in humans and other primates on the basis of sequences from several artiodactyl species. MHC genes encode essential molecules for self/altered‐self/non‐self discrimination in the interaction of the organism with its environment. The necessity to effectively present various different antigens to immunocompetent cells causes positive selection pressure on the variability of these genes in the population. Artiodactyls represent the third mammalian order in which this phenomenon was evidenced independently. A further incentive to investigate also the surroundings of MHC‐DRB loci was the presence of a particular repetitive sequence stretch in the vicinity of the polymorphic exon ‐ in addition to the evolutionarily old alleles, ancient polymorphisms and the mechanisms for their generation and/or maintenance. Besides their utility for indirect gene diagnosis (MHC‐DRB typing), the closely linked stretches of simple repetitive DNA in the neighborhood of the highly polymorphic MHC‐DRB genes are also interesting remains of the evolutionary history. Evolutionary developmenl is different in genetically inert intron‐ic DNA compared to the exonic counterparts, despite their close vicinity. The persistence of these simple repeats over nearly 100 million years in one location preserving the same basic motif structure is startling. Indirect evidence is weighed that biological meaning should be considered for these elements. The combined analysis of the polymorphic DRB genes and the (highly variable but persistent) simple repeat stretches deepen our understanding of the complexities within a unique genomic compartment encoding essential molecules for self/non‐self dif‐ferentiation in the interaction of the organism with its environment.

Regulation of the LIM-type homeobox gene islet-1 during neuronal regeneration.

Peripheral nerve lesion leads to prominent changes in gene expression in the injured neurons, a process co-ordinated by transcription factors. During development the transcription factor islet-1 plays an important role in differentiation and axogenesis. In axotomized adult neurons a process of axonal regrowth and re-establishment of the neuronal function has to be activated. Thus, we studied changes in the expression of islet-1 after axotomy, under the assumption that frequently developmentally regulated factors are reactivated during neuronal regeneration. We investigated the regulation of islet-1 expression with (i) semi-quantitative reverse transcription polymerase chain reaction and (ii) confocal microscopy in combination with quantitative image analysis. Islet-1 expression was suprisingly down-regulated in motoneurons and sensory neurons of adult rats after axotomy. A maximal reduction in the expression level was reached between day 3 and 7 after nerve lesion, a period of extensive axonal sprouting. Islet-1 expression attained control level at day 42 after lesion, a time-point at which target reinnervation takes place. The decreased expression of islet-1 during axonal regeneration is in contrast to the high levels of islet-1 expression during axogenesis in the developing nervous system. Thus, the proposed role of islet-1 in axonal target finding during axogenesis could not be confirmed in the adult rat. The observed down-regulation of islet-1 rather suggests that the activation of downstream genes important for the embryonic pattern of axonal path finding is suppressed. Moreover, in the adult nervous system islet-1 might be one of the transcription factors regulating the expression of proteins significant for the physiological intact neuronal phenotype.

Cellular activation in neuroregeneration.

Publisher Summary A peripheral nerve lesion leads to a characteristic retrograde reaction with substantial changes in morphology, metabolism, and gene expression of the injured neurons and the surrounding glial cells. The process causing the induction of axonal regrowth and re-establishment of the contact to the target is gradually becoming understood. Stepwise changes seem to lead to a transition from the “normal” physiological status to the induction of sets of genes necessary for successful re-innervation in the involved cell types: neurons, astrocytes and microglial cells. This molecular activation is cell-specific and selective. In addition, reaction to injury involves cellular interaction resulting in the modification of intracellular signaling cascades. During the regeneration process the neuron recapitulates parts of the embryonic program associated with axonal outgrowth. Hence, re-activation of regulatory mechanisms controlling neuronal differentiation and axonal outgrowth during development has been observed. The astrocytic metabolism is shifted to support survival and regeneration of the neuron. This includes the biosynthesis of growth factors and cytokines, as well as the remodeling of the extracellular matrix. The microglial cell, as the intrinsic immune cell of the brain, exhibits its activation program as an unspecific repertoire to defend brain injury.

Semiquantitative analysis of low levels of mRNA expression from small amounts of brain tissue by nonradioactive reverse transcriptase-polymerase chain reaction

We have developed an easy and fast method to semiquantify low levels of mRNA from small amounts of brain tissues based on nonradioactive reverse transcription-polymerase chain reaction (RT-PCR). The regulation of mRNA for the growth associated protein GAP-43/B-50 and the homeodomain protein islet-1 was examined in the facial nucleus of the rat after a unilateral transection of the nerve. In both cases a similar sensitivity for radioactive and nonradioactive RT-PCR methods was found. The expression of the housekeeping gene, cyclophilin A was used to normalize total mRNA amounts and PCR conditions. After amplification the PCR products were separated electrophoretically on polyacrylamide gels. For nonradioactive semiquantification gels were stained with ethidium bromide and recorded using a CCD camera and transillumination. The recordings were evaluated with specialized software. Using nonradioactive RT-PCR, the increase in GAP-43/B-50 mRNA in response to axotomy was easily detectable in the small volume of tissue obtained from the facial nucleus. In contrast, the low expression of islet-1 mRNA made it necessary to develop a two-step amplification procedure in order to provide a reliable semiquantitative analysis. The procedure included preamplification of the cDNA and subsequent purification of the cDNA. Using this method, the down-regulation of islet-1 could be demonstrated with a similar sensitivity to that previously shown with radioactive RT-PCR.

Regulation of stearoyl-CoA desaturase-1 after central and peripheral nerve lesions.

BackgroundInterruption of mature axons activates a cascade of events in neuronal cell bodies which leads to various outcomes from functional regeneration in the PNS to the failure of any significant regeneration in the CNS. One factor which seems to play an important role in the molecular programs after axotomy is the stearoyl Coenzyme A-desaturase-1 (SCD-1). This enzyme is needed for the conversion of stearate into oleate. Beside its role in membrane synthesis, oleate could act as a neurotrophic factor, involved in signal transduction pathways via activation of protein kinases C.ResultsIn situ hybridization and immunohistochemistry demonstrated a strong up-regulation of SCD at mRNA and protein level in regenerating neurons of the rat facial nucleus whereas non-regenerating Clarkes and Red nucleus neurons did not show an induction of this gene.ConclusionThis differential expression points to a functionally significant role for the SCD-1 in the process of regeneration.