Class A plexin expression in axotomized rubrospinal and facial motoneurons.

The semaphorin family of guidance molecules plays a role in many aspects of neural development, and more recently semaphorins have been implicated to contribute to the failure of injured CNS neurons to regenerate. While semaphorin expression patterns after neural injury are partially understood, little is known about the expression of their signal transducing transmembrane receptors, the plexins. Therefore, in this study, we compared the expression patterns of all class A plexins (Plxn-A1, A2, A3, A4) in mouse CNS (rubrospinal) and peripheral nervous system (PNS)-projecting (facial) motoneurons for up to two weeks following axonal injury. Using in situ hybridization, immunohistochemistry, and Western blot analysis, in rubrospinal neurons, Plxn-A1 mRNA and protein and Plxn-A4 expression did not change as a result of injury while Plxn-A2 mRNA increased and Plxn-A3 mRNA was undetectable. In facial motoneurons, Plxn-A1, -A3 and -A4 mRNA expression increased, Plxn-A2 mRNA decreased while Plxn-A1 protein expression did not change following injury. We demonstrate that with the exception of the absence of Plxn-A3 mRNA in rubrospinal neurons, both injured rubrospinal (CNS) and facial (PNS) neurons maintain expression of all plexin A family members tested. Hence, there are distinct expression patterns of the individual plexin-A family members suggesting that regenerating rubrospinal and facial motoneurons have a differential ability to transduce semaphorin signals.

Regulation of neuronal and glial galectin-1 expression by peripheral and central axotomy of rat primary afferent neurons.

Galectin-1 (Gal1) is an endogenously-expressed protein important for the embryonic development of the full complement of primary sensory neurons and their synaptic connections in the spinal cord. Gal1 also promotes axonal regeneration following peripheral nerve injury, but the regulation of Gal1 by axotomy in primary afferent neurons has not yet been examined. Here, we show by immunohistochemistry and in situ hybridization that Gal1 expression is differentially regulated by peripheral nerve injury and by dorsal rhizotomy. Following peripheral nerve injury, the proportion of Gal1-positive DRG neurons was increased. An increase in the proportion of large-diameter DRG neurons immunopositive for Gal1 was paralleled by an increase in the depth of immunoreactivity in the dorsal horn, where Gal1-positive terminals are normally restricted to laminae I and II. Dorsal rhizotomy did not affect the proportions of neurons containing Gal1 mRNA or protein, but did deplete the ipsilateral dorsal horn of Gal1 immunoreactivity, indicating that it is transported centrally by dorsal root axons. Dorsal rhizotomy also resulted in an increase in Gal1 mRNA the nerve peripheral to the PNS-CNS interface (likely within Schwann cells and/or macrophages), and to a lesser extent within deafferented spinal cord regions undergoing Wallerian degeneration. This latter increase was notable in the dorsal columns and along the prior trajectories of myelinated afferents into the deeper dorsal horn. These results show that neuronal and glial expressions of Gal1 are tightly correlated with regenerative success. Thus, the differential expression pattern of Gal1 following peripheral axotomy and dorsal rhizotomy suggests that endogenous Gal1 may be a factor important to the regenerative response of injured axons.

Altered primary afferent anatomy and reduced thermal sensitivity in mice lacking galectin-1.

The transmission of nociceptive information occurs along non-myelinated, or thinly myelinated, primary afferent axons. These axons are generally classified as peptidergic (CGRP-expressing) or non-peptidergic (IB4-binding), although there is a sub-population that is both CGRP-positive and IB4-binding. During neuronal development and following injury, trophic factors and their respective receptors regulate their survival and repair. Recent reports also show that the carbohydrate-binding protein galectin-1 (Gal1), which is expressed by nociceptive primary afferent neurons during development and into adulthood, is involved in axonal pathfinding and regeneration. Here we characterize anatomical differences in dorsal root ganglia (DRG) of Gal1 homozygous null mutant mice (Gal1(-/-)), as well as behavioural differences in tests of nociception. Gal1(-/-) mice have a significantly reduced proportion of IB4-binding DRG neurons, an increased proportion of NF200-immunoreactive DRG neurons, increased depth of central terminals of IB4-binding and CGRP-immunoreactive axons in the dorsal horn, and a reduced number of Fos-positive second order neurons following thermal (cold or hot) stimulation. While there is no difference in the total number of axons in the dorsal root of Gal1(-/-) mice, there are an increased number of myelinated axons, suggesting that in the absence of Gal1, neurons that are normally destined to become IB4-binding instead become NF200-expressing. In addition, mice lacking Gal1 have a decreased sensitivity to noxious thermal stimuli. We conclude that Gal1 is involved in nociceptive neuronal development and that the lack of this protein results in anatomical and functional deficits in adulthood.

Galectin-1 in regenerating motoneurons.

The exogenous application of recombinant galectin-1 has recently been shown to promote the rate of peripheral nerve regeneration. Endogenous neuronal galectin-1 expression has recently been demonstrated to increase after axotomy. Here we demonstrate a significant increase in the endogenous neuronal expression of galectin-1 mRNA in facial motoneurons after either a nerve resection or crush injury in mice. This increase in galectin-1 expression was due in part to the loss of target-derived factor(s) as indicated by both the return of galectin-1 expression to control levels following target re-innervation and the increase in galectin-1 expression after blockade of axonal transport by an interneuronal colchicine injection. Furthermore, interneuronal injections of glial-derived neurotrophic factor into the uninjured nerve also increased galectin-1 mRNA expression within facial motoneurons suggesting that positive signals may also be involved in the regulation of galectin-1 expression. Galectin-1 null mutant mice showed an attenuated rate of functional recovery of whisking movement after a facial nerve crush.

Galectin-1 expression correlates with the regenerative potential of rubrospinal and spinal motoneurons.

Axotomized spinal motoneurons are able to regenerate to their peripheral targets, whereas injured rubrospinal neurons that lie completely within the CNS fail to regenerate. The differing cell body reactions to axotomy of these two neuronal populations have been implicated in their disparate regenerative ability. Recently, the lectin galectin-1 has been shown to be involved in both spinal motoneurons and primary afferent regeneration. Using in situ hybridization, we compared the endogenous galectin-1 mRNA expression in spinal motoneurons and rubrospinal neurons after axotomy. We found that 7 and 14 days after axotomy, galectin-1 mRNA increased in spinal motoneurons but decreased in rubrospinal neurons. Infusion of the brain-derived neurotrophic factor into the vicinity of the injured rubrospinal nucleus, which we have previously shown to increase the regenerative capacity of rubrospinal neurons, significantly increased galectin-1 mRNA compared with uninjured control levels. Thus, the expression of galectin-1 in neurons correlates with the regenerative propensity.

Endogenous expression of inhibitor of apoptosis proteins in facial motoneurons of neonatal and adult rats following axotomy.

The inhibitor of apoptosis protein family members inhibit cell death resulting from a variety of apoptotic stimuli. However, the endogenous expression of neuronal inhibitor of apoptosis proteins following axonal injury has not been thoroughly examined. Neonatal facial motoneurons are highly susceptible to axotomy-induced apoptosis, whereas adult facial motoneurons survive axotomy. We hypothesized that the endogenous expression of inhibitor of apoptosis proteins may be involved in the differential susceptibility of adult and neonatal facial motoneurons to axonal injury. In this study, we examined the expression of two endogenous inhibitor of apoptosis proteins, neuronal apoptosis inhibitory protein and x-linked inhibitory apoptosis protein, in adult and neonatal rat facial motoneurons following axotomy. Analyses using reverse-transcription polymerase chain reaction and in situ hybridization indicated that neuronal apoptosis inhibitory protein mRNA was increased in neonatal facial nuclei 24 h post axotomy. In the adult, neuronal apoptosis inhibitory protein mRNA expression increased at 1, 3, 7 and 14 days post axotomy, while little change in the expression of X-linked inhibitory apoptosis protein mRNA was detected at any age or time point time point analyzed. Interestingly, immunohistochemistry using antibodies for neuronal apoptosis inhibitory protein and X-linked inhibitory apoptosis protein, revealed the level of these proteins was higher in the neonatal motoneurons when compared with the adult. Furthermore, immunohistochemistry and western blot for neuronal apoptosis inhibitory protein revealed, in contrast to the observed increase in neuronal apoptosis inhibitory protein mRNA, a decline in the expression of neuronal apoptosis inhibitory protein following axotomy in the adult, whereas no change in neuronal apoptosis inhibitory protein was detected in neonatal facial motoneurons. X-linked inhibitory apoptosis protein, as analyzed by immunohistochemistry and western blot, remained unchanged by axotomy in neonatal motoneurons and adult motoneurons. These results indicate differential expression and/or turnover of inhibitor of apoptosis proteins in neonatal versus adult facial motoneurons, and suggest the level of inhibitor of apoptosis protein expression alone is not an indicator of cell fate following axotomy.