Penny Vanderplank

Activation of the galanin receptor 2 (GalR2) protects the hippocampus from neuronal damage.

Expression of the neuropeptide galanin is up‐regulated in many brain regions following nerve injury and in the basal forebrain of patients with Alzheimers disease. We have previously demonstrated that galanin modulates hippocampal neuronal survival, although it was unclear which receptor subtype(s) mediates this effect. Here we report that the protective role played by galanin in hippocampal cultures is abolished in animals carrying a loss‐of‐function mutation in the second galanin receptor subtype (GalR2‐MUT). Exogenous galanin stimulates the phosphorylation of the serine/threonine kinase Akt and extracellular signal‐regulated kinase (ERK) in wild‐type (WT) cultures by 435 ± 5% and 278 ± 2%, respectively. The glutamate‐induced activation of Akt was abolished in cultures from galanin knockout animals, and was markedly attenuated in GalR2‐MUT animals, compared with WT controls. In contrast, similar levels of glutamate‐induced ERK activation were observed in both loss‐of‐function mutants, but were further increased in galanin over‐expressing animals. Using specific inhibitors of either ERK or Akt confirms that a GalR2‐dependent modulation in the activation of the Akt and ERK signalling pathways contributes to the protective effects of galanin. These findings imply that the rise in endogenous galanin observed either after brain injury or in various disease states is an adaptive response that reduces apoptosis by the activation of GalR2, and hence Akt and ERK.

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.

Transgenic overexpression of galanin in the dorsal root ganglia modulates pain-related behavior

The neuropeptide galanin is expressed in the dorsal root ganglia (DRG) and spinal cord and is thought to be involved in the modulation of pain processing. However, its mechanisms of action are complex and poorly understood, as both facilitatory and inhibitory effects have been described. To understand further the role played by galanin in nociception, we have generated two transgenic lines that overexpress galanin in specific populations of primary afferent DRG neurons in either an inducible or constitutive manner. In the first line, a previously defined enhancer region from the galanin locus was used to target galanin to the DRG (Gal-OE). Transgene expression recapitulates the spatial endogenous galanin distribution pattern in DRG neurons and markedly overexpresses the peptide in the DRG after nerve injury but not in the uninjured state. In the second line, an enhancer region of the c-Ret gene was used to constitutively and ectopically target galanin overexpression to the DRG (Ret-OE). The expression of this second transgene does not alter significantly after nerve injury. Here, we report that intact Ret-OE, but not Gal-OE, animals have significantly elevated mechanical and thermal thresholds. After nerve damage, using a spared nerve-injury model, mechanical allodynia is attenuated markedly in both the Gal-OE and Ret-OE mice compared with WT controls. These results support an inhibitory role for galanin in the modulation of nociception both in intact animals and in neuropathic pain states.

Galanin – 25 years with a multitalented neuropeptide

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.

A role for galanin in human and experimental inflammatory demyelination

The neuropeptide galanin is widely expressed by many differing subsets of neurons in the nervous system. There is a marked upregulation in the levels of the peptide in a variety of nerve injury models and in the basal forebrain of humans with Alzheimers disease. Here we demonstrate that galanin expression is specifically and markedly upregulated in microglia both in multiple sclerosis (MS) lesions and shadow plaques. Galanin expression is also upregulated in the experimental autoimmune encephalomyelitis (EAE) model of MS, although solely in oligodendrocytes. To study whether the observed increase in expression of galanin in inflammatory demyelination might modulate disease activity, we applied the EAE model to a panel of galanin transgenic lines. Over-expression of galanin in transgenic mice (Gal-OE) abolishes disease in the EAE model, whilst loss-of-function mutations in galanin or galanin receptor-2 (GalR2) increase disease severity. The pronounced effects of altered endogenous galanin or GalR2 expression on EAE disease activity may reflect a direct neuroprotective effect of the neuropeptide via activation of GalR2, similar to that previously described in a number of neuronal injury paradigms. Irrespective of the mechanism(s) by which galanin alters EAE disease activity, our findings imply that galanin/GalR2 agonists may have future therapeutic implications for MS.

Intra-neural administration of fractalkine attenuates neuropathic pain-related behaviour.

There is increasing evidence that a number of cytokines and their receptors are involved in the processes that lead to the development and maintenance of neuropathic pain states. Here we demonstrate that levels of CX3CR1 (the receptor for the chemokine fractalkine) mRNA in lumbar dorsal root ganglia (DRG) increase 5.8‐fold 7 days after sciatic nerve axotomy, and 1.7‐ and 2.9‐fold, 3 and 7 days respectively, after the spared nerve injury (SNI) model of neuropathic pain. In contrast, no significant change in the levels of fractalkine mRNA is apparent in the DRG after axotomy or SNI. The increase in CX3CR1 mRNA is paralleled by a 3.9‐ and 2.1‐fold increase in the number of CX3CR1‐positive macrophages in the DRG 7 days after axotomy and SNI, respectively. Expression of CX3CR1 in macrophages is also markedly increased in the sciatic nerve proximal to site of injury, by 25.7‐fold after axotomy and 16.2‐fold after SNI, 7 days after injury. Intra‐neural injection into the sciatic nerve of 400 ng or 100 ng of fractalkine in adult 129OlaHsd mice significantly delayed the development of allodynia for 3 days following SNI. Further, CX3CR1 knockout (KO) mice display an increase in allodynia for three weeks after SNI compared to strain‐matched Balb/c controls. Taken together, these results suggest an anti‐allodynic role for fractalkine and its receptor in the mouse.

Galanin stimulates neurite outgrowth from sensory neurons by inhibition of Cdc42 and Rho GTPases and activation of cofilin

We and others have previously shown that the neuropeptide galanin modulates neurite outgrowth from adult sensory neurons via activation of the second galanin receptor; however, the intracellular signalling pathways that mediate this neuritogenic effect have yet to be elucidated. Here, we demonstrate that galanin decreases the activation state in adult sensory neurons and PC12 cells of Rho and Cdc42 GTPases, both known regulators of filopodial and growth cone motility. Consistent with this, activated levels of Rho and Cdc42 levels are increased in the dorsal root ganglion of adult galanin knockout animals compared with wildtype controls. Furthermore, galanin markedly increases the activation state of cofilin, a downstream effector of many of the small GTPases, in the cell bodies and growth cones of sensory neurons and in PC12 cells. We also demonstrate a reduction in the activation of cofilin, and alteration in growth cone motility, in cultured galanin knockout neurons compared with wildtype controls. These data provide the first evidence that galanin regulates the Rho family of GTPases and cofilin to stimulate growth cone dynamics and neurite outgrowth in sensory neurons. These findings have important therapeutic implications for the treatment of peripheral sensory neuropathies.

The generation of knock-in mice expressing fluorescently tagged galanin receptors 1 and 2

The neuropeptide galanin has diverse roles in the central and peripheral nervous systems, by activating the G protein-coupled receptors Gal1, Gal2 and the less studied Gal3 (GalR1–3 gene products). There is a wealth of data on expression of Gal1–3 at the mRNA level, but not at the protein level due to the lack of specificity of currently available antibodies. Here we report the generation of knock-in mice expressing Gal1 or Gal2 receptor fluorescently tagged at the C-terminus with, respectively, mCherry or hrGFP (humanized Renilla green fluorescent protein). In dorsal root ganglia (DRG) neurons expressing the highest levels of Gal1-mCherry, localization to the somatic cell membrane was detected by live-cell fluorescence and immunohistochemistry, and that fluorescence decreased upon addition of galanin. In spinal cord, abundant Gal1-mCherry immunoreactive processes were detected in the superficial layers of the dorsal horn, and highly expressing intrinsic neurons of the lamina III/IV border showed both somatic cell membrane localization and outward transport of receptor from the cell body, detected as puncta within cell processes. In brain, high levels of Gal1-mCherry immunofluorescence were detected within thalamus, hypothalamus and amygdala, with a high density of nerve endings in the external zone of the median eminence, and regions with lesser immunoreactivity included the dorsal raphe nucleus. Gal2-hrGFP mRNA was detected in DRG, but live-cell fluorescence was at the limits of detection, drawing attention to both the much lower mRNA expression than to Gal1 in mice and the previously unrecognized potential for translational control by upstream open reading frames (uORFs).

Galanin-expression and galanin-dependent sensory neurons are not required for itch

BackgroundGalanin is a key modulator of nociception, and it is also required for the developmental survival of a subset of C-fibre sensory neurons which are critical to the mediation of neuropathic and inflammatory pain. However, the potential modulatory roles played by galanin, or the galanin-dependent neurons, in pruritoceptive mechanisms underlying the sensation of itch have not been investigated.FindingsHere we report that mice carrying a loss-of-function mutation in the galanin gene (Gal-KO) show no differences in spontaneous behavioural itch responses compared to wild-type (WT) controls. Similarly, the responses to a range of pruritogens are not significantly different between the two genotypes.ConclusionsThese results suggest that neither galanin expression, nor the galanin-dependent subpopulation of sensory neurons is required for itch-related behaviours.

Targeted disruption of the orphan receptor Gpr151 does not alter pain-related behaviour despite a strong induction in dorsal root ganglion expression in a model of neuropathic pain

Background: Gpr151 is an orphan GPCR whose function is unknown. The restricted pattern of neuronal expression in the habenula, dorsal horn of the spinal cord and dorsal root ganglion plus homology with the galanin family of receptors imply a role in nociception. Results: Real‐time quantitative RT‐PCR demonstrated a 49.9 ± 2.9 fold highly significant (P < 0.001) increase in Gpr151 mRNA expression in the dorsal root ganglion 7 days after the spared nerve injury model of neuropathic pain. Measures of acute, inflammatory and neuropathic pain behaviours were not significantly different using separate groups of Gpr151 loss‐of‐function mutant mice and wild‐type controls. Galanin at concentrations between 100 nM and 10 &mgr;M did not induce calcium signalling responses in ND7/23 cells transfected with Gpr151. Conclusions: Our results indicate that despite the very large upregulation in the DRG after a nerve injury model of neuropathic pain, the Gpr151 orphan receptor does not appear to be involved in the modulation of pain‐related behaviours. Further, galanin is unlikely to be an endogenous ligand for Gpr151. HighlightsGpr151 is an orphan GPCR of unknown function.RT‐PCR demonstrated a 50‐fold increase in Gpr151 mRNA in the DRG 7 days after a nerve injury model of neuropathic pain.Acute, inflammatory and neuropathic pain behaviours were unchanged between Gpr151 mutant mice and wild‐type controls.Galanin did not induce calcium signalling responses in ND7/23 cells transfected with Gpr151.