Robert Pope

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.

Mice deficient for galanin receptor 2 have decreased neurite outgrowth from adult sensory neurons and impaired pain-like behaviour.

Expression of the neuropeptide galanin is markedly up‐regulated within the adult dorsal root ganglia (DRG) following peripheral nerve injury. We have previously demonstrated that galanin knockout (Gal‐KO) mice have a developmental loss of a subset of DRG neurons. Galanin also plays a trophic role in the adult animal, and the rate of peripheral nerve regeneration and neurite outgrowth is reduced in adult Gal‐KO mice. Here we describe the characterization of mice with an absence of GalR2 gene transcription (GalR2‐MUT) and demonstrate that they have a 15% decrease in the number of calcitonin gene‐related peptide (CGRP) expressing neuronal profiles in the adult DRG, associated with marked deficits in neuropathic and inflammatory pain behaviours. Adult GalR2‐MUT animals also have a one third reduction in neurite outgrowth from cultured DRG neurons that cannot be rescued by either galanin or a high‐affinity GalR2/3 agonist. Galanin activates extracellular signal‐regulated kinase (ERK) and Akt in adult wild‐type (WT) mouse DRG. Intact adult DRG from GalR2‐MUT animals have lower levels of pERK and higher levels of pAkt than are found in WT controls. These data suggest that a lack of GalR2 activation in Gal‐KO and GalR2‐MUT animals is responsible for the observed developmental deficits in the DRG, and the decrease in neurite outgrowth in the adult.

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.

Endogenous galanin potentiates spinal nociceptive processing following inflammation

&NA; We have undertaken a series of experiments using galanin null mutant mice to better define the role of endogenous galanin in spinal excitability following inflammation and in response to centrally sensitizing stimuli. We have employed a behavioural paradigm, the formalin test, as a model of tonic nociception in both galanin knock‐out (gal−/−) and wild‐type (gal+/+) mice. In this model, we find that gal−/− mice are markedly hypo‐responsive, especially in the second phase response. Additionally, we have examined the thermal hyperalgesia which develops following peripheral injection of carrageenan into the plantar surface of one hindpaw. In this inflammatory paradigm, thermal hyperalgesia is markedly attenuated in gal−/− mice. These behavioural findings suggest that endogenous galanin contributes to nociceptive processing. We have tested this hypothesis further by employing an electrophysiological measure of spinal excitability, the flexor withdrawal reflex in gal−/− and gal+/+ mice. We found no differences in acute reflex responses to single stimuli at C‐fibre strength or in the time course and magnitude of wind‐up induced by a short conditioning train between non‐inflamed gal+/+ and gal−/− mice. However, the long‐lasting post‐conditioning enhancement of reflex excitability was only seen in gal+/+ mice. Moreover, following carrageenan inflammation, there was a marked increase in spinal nociceptive reflex excitability in the inflamed gal+/+ mice, but this enhanced excitability was absent in gal−/− animals. These findings illustrate that endogenous galanin is necessary for the full expression of central sensitization, and as such, plays a critical role in the development of hyperalgesia following peripheral tissue injury.

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.

ARF6-Dependent Regulation of P2Y Receptor Traffic and Function in Human Platelets

Adenosine diphosphate (ADP) is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors, the P2Y1 and P2Y12 purinoceptors. Recently, we demonstrated that P2Y1 and P2Y12 purinoceptor activities are rapidly and reversibly modulated in human platelets, revealing that the underlying mechanism requires receptor internalization and subsequent trafficking as an essential part of this process. In this study we investigated the role of the small GTP-binding protein ADP ribosylation factor 6 (ARF6) in the internalization and function of P2Y1 and P2Y12 purinoceptors in human platelets. ARF6 has been implicated in the internalization of a number of GPCRs, although its precise molecular mechanism in this process remains unclear. In this study we show that activation of either P2Y1 or P2Y12 purinoceptors can stimulate ARF6 activity. Further blockade of ARF6 function either in cell lines or human platelets blocks P2Y purinoceptor internalization. This blockade of receptor internalization attenuates receptor resensitization. Furthermore, we demonstrate that Nm23-H1, a nucleoside diphosphate (NDP) kinase regulated by ARF6 which facilitates dynamin-dependent fission of coated vesicles during endocytosis, is also required for P2Y purinoceptor internalization. These data describe a novel function of ARF6 in the internalization of P2Y purinoceptors and demonstrate the integral importance of this small GTPase upon platelet ADP receptor function.

Arrestin Scaffolds NHERF1 to the P2Y12 Receptor to Regulate Receptor Internalization

Background: The PDZ-binding motif of the P2Y12 receptor regulates correct receptor traffic in human platelets. Results: The PDZ-binding protein NHERF1 binds to the P2Y12 receptor to promote agonist-dependent internalization. Conclusion: Arrestin scaffolds NHERF1 to the P2Y12 receptor to facilitate effective NHERF1-dependent receptor internalization. Significance: A novel model of arrestin-dependent GPCR internalization. We have recently shown in a patient with mild bleeding that the PDZ-binding motif of the platelet G protein-coupled P2Y12 receptor (P2Y12R) is required for effective receptor traffic in human platelets. In this study we show for the first time that the PDZ motif-binding protein NHERF1 exerts a major role in potentiating G protein-coupled receptor (GPCR) internalization. NHERF1 interacts with the C-tail of the P2Y12R and unlike many other GPCRs, NHERF1 interaction is required for effective P2Y12R internalization. In vitro and prior to agonist stimulation P2Y12R/NHERF1 interaction requires the intact PDZ binding motif of this receptor. Interestingly on receptor stimulation NHERF1 no longer interacts directly with the receptor but instead binds to the receptor via the endocytic scaffolding protein arrestin. These findings suggest a novel model by which arrestin can serve as an adaptor to promote NHERF1 interaction with a GPCR to facilitate effective NHERF1-dependent receptor internalization.

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.