Lynne Shanley

Small applied electric fields guide migration of hippocampal neurons.

Effectively directed neuron migration is critical for development and repair in the central nervous system (CNS). Endogenous electric fields (EFs) are widespread in developing and regenerating tissues and regulate a variety of cell behaviors including directed cell migration. Electrically‐directed neuronal migration has not been tested previously and we show that an applied EF directs migration of hippocampal neurons toward the cathode at a field strength of 120 mV/mm, close to the physiological range. Reversal of the field polarity reversed the direction of neuron migration. Neuron migration from an explant also was directed by an applied EF. Mechanistically, EF‐guided migration was transduced by activation of the second messenger molecules ROCK (Rho‐associated protein kinase) and PI3 kinase (phosphoinositide‐3 kinase) since their pharmacological inhibition decreased the directedness and speed of neuron migration. This work demonstrates that rat hippocampal neurons respond to applied EFs with directional migration and raises the possibility that EFs may be used as a cue to direct neuronal migration in novel strategies to repair the CNS. J. Cell. Physiol. 216: 527–535, 2008.

The roles of calcium signaling and ERK1/2 phosphorylation in a Pax6+/- mouse model of epithelial wound-healing delay

BackgroundCongenital aniridia caused by heterozygousity at the PAX6 locus is associated with ocular surface disease including keratopathy. It is not clear whether the keratopathy is a direct result of reduced PAX6 gene dosage in the cornea itself, or due to recurrent corneal trauma secondary to defects such as dry eye caused by loss of PAX6 in other tissues. We investigated the hypothesis that reducing Pax6 gene dosage leads to corneal wound-healing defects. and assayed the immediate molecular responses to wounding in wild-type and mutant corneal epithelial cells.ResultsPax6+/- mouse corneal epithelia exhibited a 2-hour delay in their response to wounding, but subsequently the cells migrated normally to repair the wound. Both Pax6+/+ and Pax6+/- epithelia activated immediate wound-induced waves of intracellular calcium signaling. However, the intensity and speed of propagation of the calcium wave, mediated by release from intracellular stores, was reduced in Pax6+/- cells. Initiation and propagation of the calcium wave could be largely decoupled, and both phases of the calcium wave responses were required for wound healing. Wounded cells phosphorylated the extracellular signal-related kinases 1/2 (phospho-ERK1/2). ERK1/2 activation was shown to be required for rapid initiation of wound healing, but had only a minor effect on the rate of cell migration in a healing epithelial sheet. Addition of exogenous epidermal growth factor (EGF) to wounded Pax6+/- cells restored the calcium wave, increased ERK1/2 activation and restored the immediate healing response to wild-type levels.ConclusionThe study links Pax6 deficiency to a previously overlooked wound-healing delay. It demonstrates that defective calcium signaling in Pax6+/- cells underlies this delay, and shows that it can be pharmacologically corrected. ERK1/2 phosphorylation is required for the rapid initiation of wound healing. A model is presented whereby minor abrasions, which are quickly healed in normal corneas, transiently persist in aniridic patients, compromising the corneal stroma.

Differential Activity by Polymorphic Variants of a Remote Enhancer that Supports Galanin Expression in the Hypothalamus and Amygdala: Implications for Obesity, Depression and Alcoholism

The expression of the galanin gene (GAL) in the paraventricular nucleus (PVN) and in the amygdala of higher vertebrates suggests the requirement for highly conserved, but unidentified, regulatory sequences that are critical to allow the galanin gene to control alcohol and fat intake and modulate mood. We used comparative genomics to identify a highly conserved sequence that lay 42 kb 5′ of the human GAL transcriptional start site that we called GAL5.1. GAL5.1 activated promoter activity in neurones of the PVN, arcuate nucleus and amygdala that also expressed the galanin peptide. Analysis in neuroblastoma cells demonstrated that GAL5.1 acted as an enhancer of promoter activity after PKC activation. GAL5.1 contained two polymorphisms; rs2513280(C/G) and rs2513281(A/G), that occurred in two allelic combinations (GG or CA) where the dominant GG alelle occurred in 70-83 % of the human population. Intriguingly, both SNPs were found to be in LD (R2 of 0.687) with another SNP (rs2156464) previously associated with major depressive disorder (MDD). Recreation of these alleles in reporter constructs and subsequent magnetofection into primary rat hypothalamic neurones showed that the CA allele was 40 % less active than the GG allele. This is consistent with the hypothesis that the weaker allele may affect food and alcohol preference. The linkage of the SNPs analysed in this study with a SNP previously associated with MDD together with the functioning of GAL5.1 as a PVN and amygdala specific enhancer represent a significant advance in our ability to understand alcoholism, obesity and major depressive disorder.

Influx of extracellular Ca2+ is necessary for electrotaxis in Dictyostelium

Intracellular free Ca2+ ([Ca2+]i) is a pivotal signalling element in cell migration and is thought to be required for chemotaxis of Dictyostelium. Ca2+ signalling may also be important for electrotaxis. However this suggestion has been controversial. We show that electric fields direct Dictyostelium cells to migrate cathodally and increase [Ca2+]i in Dictyostelium cells, as determined by Fluo-3 AM imaging and 45Ca2+ uptake. Omission of extracellular Ca2+([Ca2+]e) and incubation with EGTA abolished the electric-field-stimulated [Ca2+]i rise and directional cell migration. This suggests a requirement for [Ca2+]e in the electrotactic response. Deletion of iplA, a gene responsible for chemoattractant-induced [Ca2+]i increase, had only a minor effect on the electric-field-induced [Ca2+]i rise. Moreover, iplA-null Dictyostelium cells showed the same electrotactic response as wild-type cells. Therefore, iplA-independent Ca2+ influx is necessary for electrotactic cell migration. These results suggest that the [Ca2+]i regulatory mechanisms induced by electric fields are different from those induced by cAMP and folic acid in Dictyostelium cells. Different roles of the iplA gene in chemoattractant-induced and electrically induced Ca2+ signalling, and different effects of [Ca2+]i elevation on chemotaxis and electrotaxis indicate that the chemoattractant and electric cues activate distinctive initial signalling elements.

Evidence for regulatory diversity and auto-regulation at the TAC1 locus in sensory neurones

The neuropeptide substance-P (SP) is expressed from the TAC1 gene in sensory neurones where it acts as a key modulator of neurogenic inflammation. The promoter of TAC1 (TAC1prom) plays a central role in the regulation of the TAC1 gene but requires the presence of a second regulatory element; ECR2, to support TAC1 expression in sensory neurones and to respond appropriately to signalling pathways such as MAPkinases and noxious induction by capsaicin. We examined whether the effect of capsaicin on ECR2-TAC1prom activity in larger diameter neurones was cell autonomous or non- cell autonomous. We demonstrate that TRPV1 is not expressed in all the same cells as SP following capsaicin induction suggesting the presence of a non-cell autonomous mechanism for TAC1 up-regulation following capsaicin induction. In addition, we demonstrate that induction of SP and ECR1-TAC1prom activity in these larger diameter neurones can be induced by potassium depolarisation suggesting that, in addition to capsaicin induction, transgene activity may be modulated by voltage gated calcium channels. Furthermore, we show that NK1 is expressed in all SP- expressing cells after capsaicin induction and that an agonist of NK1 can activate both SP and the transgene in larger diameter neurones. These observations suggest the presence of an autocrine loop that controls the expression of the TAC1 promoter in sensory neurones. In contrast, induction of the TAC1 promoter by LPS was not dependent on ECR2 and did not occur in large diameter neurones. These studies demonstrate the diversity of mechanisms modulating the activity of the TAC1 promoter and provide novel directions for the development of new anti-inflammatory therapies.

Long-Range Regulatory Synergy Is Required to Allow Control of the TAC1 Locus by MEK/ERK Signalling in Sensory Neurones

Changes in the expression of the neuropeptide substance P (SP) in different populations of sensory neurones are associated with the progression of chronic inflammatory disease. Thus, understanding the genomic and cellular mechanisms driving the expression of the TAC1 gene, which encodes SP, in sensory neurones is essential to understanding its role in inflammatory disease. We used a novel combination of computational genomics, primary-cell culture and mouse transgenics to determine the genomic and cellular mechanisms that control the expression of TAC1 in sensory neurones. Intriguingly, we demonstrated that the promoter of the TAC1 gene must act in synergy with a remote enhancer, identified using comparative genomics, to respond to MAPK signalling that modulates the expression of TAC1 in sensory neurones. We also reveal that noxious stimulation of sensory neurones triggers this synergy in larger diameter sensory neurones – an expression of SP associated with hyperalgesia. This noxious stimulation of TAC1 enhancer-promotor synergy could be strongly blocked by antagonism of the MEK pathway. This study provides a unique insight into the role of long-range enhancer-promoter synergy and selectivity in the tissue-specific response of promoters to specific signal transduction pathways and suggests a possible new avenue for the development of novel anti-inflammatory therapies.

Postnatal emergence of mature release properties in terminals of rat fast- and slow-twitch muscles

Motor nerve terminals in adult mammalian slow‐twitch muscles have lower levels of spontaneous and evoked neurotransmitter release than terminals in fast‐twitch muscles. These reflect adaptive differences, allowing terminals in slow (postural) muscles to sustain release during the prolonged firing trains experienced in vivo. Here we ask whether these differences in terminal release properties in Sprague–Dawley rat extensor digitorum longus (EDL, fast) and soleus (slow) muscles reflect their early cytodifferentiation in the embryo or whether they might be adaptations to their distinct mature activity patterns, which emerge around two weeks postnatally. We find that the mature pattern of differences in release arise through co‐ordinated increases in presynaptically dependent release properties (quantal content, spontaneous release frequency and evoked potential amplitude), beginning at three weeks, which are particularly substantial in EDL. In contrast, other synaptic properties are either consistently greater in the same muscle throughout development (evoked potential kinetics, muscle fibre diameter) or display no systematic muscle type‐dependent differences (terminal area, input resistance, spontaneous release amplitude). Thus, the emergence of adaptive differences in terminal release properties correlates with the differentiation of locomotor activity patterns in postnatal rat hindlimb muscles.

Allele-specific differences in activity of a novel cannabinoid receptor 1 (CNR1) gene intronic enhancer in hypothalamus, dorsal root ganglia, and hippocampus.

Background: Intron 2 of CNR1 gene contains multiple disease-associated SNPs. Results: Allelic variants of a novel enhancer in CNR1 intron 2 affect its MAPK response in hypothalamus and hippocampus. Conclusion: Alleles of enhancer may be functionally linked to obesity and addictive behavior. Significance: Understanding the effects of CNR1 polymorphisms on gene regulation will accelerate understanding of human disease. Polymorphisms within intron 2 of the CNR1 gene, which encodes cannabinoid receptor 1 (CB1), have been associated with addiction, obesity, and brain volume deficits. We used comparative genomics to identify a polymorphic (rs9444584-C/T) sequence (ECR1) in intron 2 of the CNR1 gene that had been conserved for 310 million years. The C-allele of ECR1 (ECR1(C)) acted as an enhancer in hypothalamic and dorsal root ganglia cells and responded to MAPK activation through the MEKK pathway but not in hippocampal cells. However, ECR1(T) was significantly more active in hypothalamic and dorsal root ganglia cells but, significantly, and in contrast to ECR1(C), was highly active in hippocampal cells where it also responded strongly to activation of MAPK. Intriguingly, rs9444584 is in strong linkage disequilibrium with two other SNPs (rs9450898 (r2 = 0.841) and rs2023239 (r2 = 0.920)) that have been associated with addiction, obesity (rs2023239), and reduced fronto-temporal white matter volumes in schizophrenia patients as a result of cannabis misuse (rs9450898). Considering their high linkage disequilibrium and the increased response of ECR1(T) to MAPK signaling when compared with ECR1(C), it is possible that the functional effects of the different alleles of rs9444584 may play a role in the conditions associated with rs9450898 and rs2023239. Further analysis of the different alleles of ECR1 may lead to a greater understanding of the role of CNR1 gene misregulation in these conditions as well as chronic inflammatory pain.

Analysis of the effects of depression associated polymorphisms on the activity of the BICC1 promoter in amygdala neurones

The Bicaudal C Homolog 1 (BICC1) gene, which encodes an RNA binding protein, has been identified by genome wide association studies (GWAS) as a candidate gene associated with major depressive disorder (MDD). We explored the hypothesis that MDD associated single-nucleotide polymorphisms (SNPs) affected the ability of cis-regulatory elements within intron 3 of the BICC1 gene to modulate the activity of the BICC1 promoter region. We initially established that the BICC1 promoter drove BICC1 mRNA expression in amygdala, hippocampus and hypothalamus. Intriguingly, we provide evidence that MDD associated polymorphisms alter the ability of the BICC1 promoter to respond to PKA signalling within amygdala neurones. Considering the known role of amygdala PKA pathways in fear learning and mood these observations suggest a possible mechanism through which allelic changes in the regulation of the BICC1 gene in amygdala neurones may contribute to mood disorders. Our findings also suggest a novel direction for the identification of novel drug targets and the design of future personalised therapeutics.The Pharmacogenomics Journal advance online publication, 6 October 2015; doi:10.1038/tpj.2015.62

Functional effects of polymorphisms on glucocorticoid receptor modulation of human anxiogenic substance-P gene promoter activity in primary amygdala neurones

Summary Expression or introduction of the neuropeptide substance-P (SP; encoded by the TAC1 gene in humans and Tac1 in rodents) in the amygdala induces anxiety related behaviour in rodents. In addition, pharmacological antagonism of the main receptor of SP in humans; NK1, is anxiolytic. In the current study, we show that the Tac1 locus is up-regulated in primary rat amygdala neurones in response to activation of the glucocorticoid receptor (GR); a classic component of the stress response. Using a combination of bioinformatics, electrophoretic mobility shift assays (EMSA) and reporter plasmid magnetofection into rat primary amygdala neurones we identified a highly conserved GR response sequence (2GR) in the human TAC1 promoter that binds GR in response to dexamethasone (Dex) or forskolin. We also identified a second GR binding site in the human promoter that was polymorphic and whose T-allele is only found in Japanese and Chinese populations. We present evidence that the T-allele of SNPGR increases the activity of the TAC1 promoter through de-sequestration or de-repression of 2GR. The identification of Dex/forskolin response elements in the TAC1 promoter in amygdala neurones suggests a possible link in the chain of molecular events connecting GR activation and anxiety. In addition, the discovery of a SNP which can alter this response may have implications for our understanding of the role of regulatory variation in susceptibility to stress in specific populations.