Galanin acts as a trophic factor to the central and peripheral nervous systems.

The neuropeptide galanin is widely, but not ubiquitously, expressed in the adult nervous system. Its expression is markedly up-regulated in many neuronal tissues after nerve injury or disease. Over the last 10 years, we have demonstrated that the peptide plays a developmental survival role to subsets of neurons in the peripheral and central nervous systems with resulting phenotypic changes in neuropathic pain and cognition. Galanin also appears to play a trophic role to adult sensory neurons following injury, via activation of GalR2, by stimulating neurite outgrowth. Furthermore, galanin also plays a neuroprotective role to the hippocampus following excitotoxic injury, again mediated by activation of GalR2. Most recently, we have shown that galanin expression is markedly up-regulated in multiple sclerosis (MS) lesions and in the experimental autoimmune encephalomyelitis (EAE) model of MS. Over-expression of galanin in transgenic mice abolishes disease in the EAE model, whilst loss-of-function mutations in galanin or GalR2 increase disease severity. In summary, these studies demonstrate that a GalR2 agonist might have clinical utility in a variety of human diseases that affect the nervous system.

Novel mRNA isoforms of the sodium channels Na(v)1.2, Na(v)1.3 and Na(v)1.7 encode predicted two-domain, truncated proteins.

The expression of voltage-gated sodium channels is regulated at multiple levels, and in this study we addressed the potential for alternative splicing of the Na(v)1.2, Na(v)1.3, Na(v)1.6 and Na(v)1.7 mRNAs. We isolated novel mRNA isoforms of Na(v)1.2 and Na(v)1.3 from adult mouse and rat dorsal root ganglia (DRG), Na(v)1.3 and Na(v)1.7 from adult mouse brain, and Na(v)1.7 from neonatal rat brain. These alternatively spliced isoforms introduce an additional exon (Na(v)1.2 exon 17A and topologically equivalent Na(v)1.7 exon 16A) or exon pair (Na(v)1.3 exons 17A and 17B) that contain an in-frame stop codon and result in predicted two-domain, truncated proteins. The mouse and rat orthologous exon sequences are highly conserved (94-100% identities), as are the paralogous Na(v)1.2 and Na(v)1.3 exons (93% identity in mouse) to which the Na(v)1.7 exon has only 60% identity. Previously, Na(v)1.3 mRNA has been shown to be upregulated in rat DRG following peripheral nerve injury, unlike the downregulation of all other sodium channel transcripts. Here we show that the expression of Na(v)1.3 mRNA containing exons 17A and 17B is unchanged in mouse following peripheral nerve injury (axotomy), whereas total Na(v)1.3 mRNA expression is upregulated by 33% (P=0.003), suggesting differential regulation of the alternatively spliced transcripts. The alternatively spliced rodent exon sequences are highly conserved in both the human and chicken genomes, with 77-89% and 72-76% identities to mouse, respectively. The widespread conservation of these sequences strongly suggests an additional level of regulation in the expression of these channels, that is also tissue-specific.

Galanin acts as a trophic factor to the central and peripheral nervous systems.

The neuropeptide galanin is widely, but not ubiquitously, expressed in the adult nervous system. Its expression is markedly upregulated in many neuronal tissues after nerve injury or disease. Over the last 10 years we have demonstrated that the peptide plays a developmental survival role to subsets of neurons in the peripheral and central nervous systems with resulting phenotypic changes in neuropathic pain and cognition. Galanin also appears to play a trophic role to adult sensory neurons following injury, via activation of GalR2, by stimulating neurite outgrowth. Furthermore, galanin also plays a neuroprotective role to the hippocampus following excitotoxic injury, again mediated by activation of GalR2. In summary, these studies demonstrate that a GalR2 agonist might have clinical utility in a variety of human diseases that affect the nervous system.

Use of genetically engineered transgenic mice to investigate the role of galanin in the peripheral nervous system after injury.

The neuropeptide galanin is present at high levels within the dorsal root ganglia (DRG) and spinal cord during development and after peripheral nerve damage in the adult. This pattern of expression suggests that it may play a role in the adaptive response of the peripheral nervous system (PNS) to injury. Several experimental paradigms have demonstrated that galanin modulates pain transmission, particularly after nerve injury. In our laboratory we have used a transgenic approach to further elucidate the functions of galanin within the somatosensory system. We have generated mice which over-express galanin (either inducibly after nerve injury, or constitutively), and knock-out (KO) mice, in which galanin is absent in all cells, throughout development and in the adult. Analysis of the nociceptive behaviour of the galanin over-expressing animals, before and after nerve injury, supports the view that galanin is an inhibitory neuromodulator of spinal cord transmission. In apparent contradiction to these findings, galanin KO animals fail to develop allodynia and hyperalgesia after nerve injury. However, further studies have shown that galanin is critical for the developmental survival of a subset of small diameter, unmyelinated sensory neurons that are likely to be nociceptors. This finding may well explain the lack of neuropathic pain-like behaviour after injury in the KO animals. Furthermore, the developmental survival role played by galanin is recapitulated in the adult where the peptide is required for optimal neuronal regeneration after injury, and in the hippocampus where it plays a neuroprotective role after excitotoxic injury.

Galanin-like peptide (GALP) is expressed in rat hypothalamus and pituitary, but not in DRG.

Galanin-like peptide (GALP) was recently purified on the basis of its preferential activation of galanin receptor subtype 2 (GALR2) compared with galanin receptor subtype 1 (GALR1). Using in situ hybridization of adult rat brain, pituitary and dorsal root ganglia (DRG) we demonstrate that GALP mRNA expression is restricted to the arcuate nucleus and median eminence of the hypothalamus, and to the posterior lobe of the pituitary. No expression was detected elsewhere in brain, or in the DRG. In adult mouse, no expression was detected in brain or in DRG either before or after axotomy, suggesting that GALP has no apparent role in the axotomy response of DRG.

Targeted disruption of the galanin gene reduces the number of sensory neurons and their regenerative capacity.

The neuropeptide galanin is expressed developmentally in the dorsal root ganglion (DRG) and is rapidly up-regulated 120-fold after peripheral nerve section in the adult. Here we report that adult mice carrying a loss-of-function mutation in the galanin gene have a 13% reduction in the number of cells in the DRG associated with a 24% decrease in the percentage of neurons that express substance P. These deficits are associated with a 2.8- and 2.6-fold increase in the number of apoptotic cells in the DRG at postnatal days 3 and 4, respectively. After crush injury to the sciatic nerve, the rate of peripheral nerve regeneration is reduced by 35% with associated long-term functional deficits. Cultured DRG neurons from adult mutant mice demonstrate similar deficits in neurite number and length. These results identify a critical role for galanin in the development and regeneration of sensory neurons.

Galanin regulates prolactin release and lactotroph proliferation.

The neuropeptide galanin is predominantly expressed by the lactotrophs (the prolactin secreting cell type) in the rodent anterior pituitary and in the median eminence and paraventricular nucleus of the hypothalamus. Prolactin and galanin colocalize in the same secretory granule, the expression of both proteins is extremely sensitive to the estrogen status of the animal. The administration of estradiol-17beta induces pituitary hyperplasia followed by adenoma formation and causes a 3,000-fold increase in the galanin mRNA content of the lactotroph. To further study the role of galanin in prolactin release and lactotroph growth we now report the generation of mice carrying a loss-of-function mutation of the endogenous galanin gene. There is no evidence of embryonic lethality and the mutant mice grow normally. The specific endocrine abnormalities identified to date, relate to the expression of prolactin. Pituitary prolactin message levels and protein content of adult female mutant mice are reduced by 30-40% compared with wild-type controls. Mutant females fail to lactate and pups die of starvation/dehydration unless fostered onto wild-type mothers. Prolactin secretion in mutant females is markedly reduced at 7 days postpartum compared with wild-type controls with an associated failure in mammary gland maturation. There is an almost complete abrogation of the proliferative response of the lactotroph to high doses of estrogen, with a failure to up-regulate prolactin release, STAT5 expression or to increase pituitary cell number. These data further support the hypothesis that galanin acts as a paracrine regulator of prolactin expression and as a growth factor to the lactotroph.

Catecholaminergic regulation of proliferation and survival in rat forebrain paraventricular germinal cells.

We have investigated the possible role of alpha1-adrenoreceptors in regulating the germination of progenitor cells cultured from embryonic rat neocortex. High binding levels of the alpha1-selective radioligand 3[H]prazosin were detected in the forebrain of the rat embryo at E13, and the greatest density of binding sites was localized to the ventricular and subventricular zones. Catecholamine-containing axon terminals were present in these zones in the same period. Germinal neuroepithelial cells retained specific 3[H]prazosin binding in culture. Approximately 25% of cells in culture displayed complex intracellular Ca2+ transients in response to phenylephrine, many of which were abolished with the alpha1B antagonist, chloroethylclonidine. Cultures exhibited concentration-dependent catecholamine stimulation of DNA synthesis mediated by alpha1 receptors in serum-limited conditions. Neuroepithelial cells were labelled via their ventricular processes by intraventricular injection of Fast blue in E13 embryos prior to transfer of the neocortex to dissociated cell culture. Many of labelled cells were present in culture in germinal foci. Some cells which migrated from these foci underwent apoptosis, as determined by TUNEL in situ hybridization. During a transitory period of up to 48 h in culture, alpha1-adrenoreceptor activation by phenylephrine or noradrenaline increased the number of surviving cells. Apoptosis was observed in vivo in both ventricular and subventricular zones of the neocortex from E13 to E15 in increasing numbers. We propose that both the supply of noradrenaline to forebrain germinal cells, and the expression of alpha1-adrenoreceptors on their surface could act to determine whether they die or continue to proliferate.

Superactivation of transglutaminase type 2 without change in enzyme level occurs during progressive neurodegeneration in the mnd mouse mutant.

We have investigated the activity of the Ca(2+)-dependent apoptosis-related transglutaminase type 2 in the mnd/mnd mouse mutant. Transglutaminase activity in mnd/mnd central nervous system (CNS) tissue homogenates was identical to that of healthy animals at 3 months of age, but at 8 months it was greater in the mnd/mnd CNS by up to four times, depending on the region. Western blot analysis showed no difference in the level of immunoreactive transglutaminase type 2 in spinal cord homogenates between mnd/mnd and healthy mice. However, a greater number of acyl donor protein substrates of transglutaminase were identified in mnd/mnd tissue. N epsilon (gamma-Glutamyl)lysine cross-linked product of transglutaminase activity was localized to the soma of degenerating motor neurons in the mnd/mnd mouse spinal cord. We conclude that neurodegeneration in the mnd/mnd mouse is accompanied by activation of transglutaminase at substrate level. Possible mechanisms of activation and its implications for cellular pathology are discussed.

Cytoplasmic chaperonin complexes enter neurites developing in vitro and differ in subunit composition within single cells.

Chaperonins containing t-complex polypeptide-1 (CCT) are cytosolic molecular chaperone particles implicated especially in the biogenesis of cytoskeletal proteins by promoting the correct folding of the major ubiquitous cytoskeletal components, tubulin and actin. We have purified cytosolic chaperonins from the ND7/23 cell line, determined their subunit composition and examined changes in the intracellular locations of their components during differentiation of ND7/23 cells to a neuronal phenotype by using immunocytochemistry and immunoblots. Chaperonins containing the CCT alpha (TCP1) subunit enter neuritic processes and are particularly noticeable at the leading edge of growth cone-like structures where they co-localise with actin. Chaperonins containing three other components (CCT beta, epsilon and gamma), however, remain predominantly restricted to perikaryal cytoplasm. These findings suggest a heterogeneous population of chaperonin particles within single differentiated ND7/23 cells and this may reflect specialisation of chaperonin function in different cytoplasmic compartments of a neurone. Further, since ribosomes do not enter neurites while CCT alpha-containing chaperonins do, the latter may play roles, subsequent to translation, which influence cytoskeletal elaboration during neuritogenesis.

A60, an axonal membrane-skeletal spectrin-binding protein.

A60 is a 60 kDa protein which is associated with the axonal cortical cytoskeleton in adult central nervous system neurons. It was originally defined by the use of a monoclonal antibody which showed that it was closely associated with the cytoplasic face of axonal plasma membranes. A60 appears to be neuron-specific and biochemical studies show that it is tightly bound to brain membranes. Affinity chromatography has revealed that A60 interacts with brain spectrin but not with erythrocyte spectrin. As erythrocyte spectrin is closely related to the isotype of spectrin that is localized in dendrites this raises the possibility that A60 is restricted to axons by interaction with the isotype of spectrin that is found in axons. During post-natal cerebellar development (days 1-13) A60 is initially located in the perikarya of precursor Purkinje cells and is then localized in the initial dendrites of these cells as well as in the white matter. In contrast, in the adult cerebellum the location of A60 is exclusively axonal. These data indicate that A60 has a spectrin-binding activity in the adult axonal membrane skeleton which is only required after initial axon growth has occurred. A60 is transiently expressed during embryonic and post-natal development of rat dorsal root ganglia (DRG). It is located in the large light DRG cells but is essentially absent from the small dark DRG cells.