Dopamine is a potentially toxic neurotransmitter that has long been speculated to contribute to the pathogenesis of Parkinson's disease (PD). Recent work has demonstrated the importance of proper storage of dopamine in vesicles to maintain dopamine homeostasis, thus protecting neurons from the detrimental effects of dopamine accumulation and breakdown in the cytosol. These studies suggest that factors which affect dopamine storage might increase the susceptibility of dopamine neurons to further environmental or genetic insults, exacerbating the neuronal degeneration that characterizes PD. This review seeks to revisit the pathogenicity of cytosolic dopamine and further address the critical role of neurotransmitter storage in dopamine-mediated neurotoxicity.

Wallerian degeneration slow (Wld(S)) mice express a chimeric protein that delays axonal degeneration. The N-terminal domain (N70), which is essential for axonal protection in vivo, binds valosin-containing protein (VCP) and targets both Wld(S) and VCP to discrete nuclear foci. We characterized the formation, composition and localization of these potentially important foci. Missense mutations show that the N-terminal sixteen residues (N16) of Wld(S) are essential for both VCP binding and targeting Wld(S) to nuclear foci. Removing N16 abolishes foci, and VCP binding sequences from ataxin-3 or HrdI restore them. In vitro, these puncta co-localize with proteasome subunits. In vivo, Wld(S) assumes a range of nuclear distribution patterns, including puncta, and its neuronal expression and intranuclear distribution is region-specific and varies between spontaneous and transgenic Wld(S) models. We conclude that VCP influences Wld(S) intracellular distribution, and thus potentially its function, by binding within the N70 domain required for axon protection.

Monoallelic gene expression exposes an organism to the risks associated with the unmasking of recessive mutations. A recent study by Gimelbrant and colleagues, supported by results from two methodologically different studies, demonstrated that random monoallelic expression is surprisingly widespread among autosomal genes. This raises important questions about why, when and how cells choose and tolerate monoallelism and whether functional hemizygosity might provide an unappreciated advantage.

The equatorial subsegment (EqSS) was originally identified by atomic force microscopy as a discrete region within the equatorial segment of Artiodactyl spermatozoa. In this investigation, we show that the EqSS is enriched in tyrosine phosphorylated proteins and present preliminary evidence for its presence in mouse and rat spermatozoa. The anti-phosphotyrosine monoclonal antibody (McAb) 4G10 bound strongly and discretely to the EqSS of permeabilized boar, ram, and bull spermatozoa. It also bound to a small patch on the posterior acrosomal region of permeabilized mouse and rat spermatozoa, suggesting that the EqSS is not restricted to the order Artiodactyla. An anti-HSPA1A (formerly Hsp70) antibody recognized the EqSS in boar spermatozoa. Immunogold labeling with McAb 4G10 localized the tyrosine phosphorylated proteins to the outer acrosomal membrane. This was verified by freeze-fracture electron microscopy, which identified the EqSS in three overlying membranes, the plasma membrane, outer acrosomal membrane, and inner acrosomal membrane. In all five species, tyrosine phosphorylated proteins became restricted to the EqSS during sperm maturation in the epididymis. The major tyrosine phosphorylated proteins in the EqSS of boar and ram spermatozoa were identified by mass spectrometry as orthologs of human SPACA1 (formerly SAMP32). Immunofluorescence with a specific polyclonal antibody localized SPACA1 to the equatorial segment in boar spermatozoa. We speculate that the EqSS is an organizing center for assembly of multimolecular complexes that initiate fusion competence in this area of the plasma membrane following the acrosome reaction.

The CD45 tyrosine phosphatase is required for T cell development and function by virtue of its role as a positive regulator of src family kinase activity. In addition, recent data have highlighted that CD45 also acts as a negative regulator of Lck function by dephosphorylation of critical tyrosine residues. Lck functionality and TCR responsiveness are elevated in transgenic mice expressing the CD45RO isoform at 'intermediate' (10-40% of wild type) levels, indicating that the expression level of CD45 is critical in determining the sensitivity of T cells to TCR stimulation. However, it is unclear whether such a phenotype is specific for the CD45RO isoform, typically expressed by activated T cells. In the present work, the roles of three isoforms of CD45, RO, RB and RABC, in thymocyte development, T cell responses and TCR signalling pathways were directly compared. The data demonstrate that expression of CD45RB or CD45RABC at intermediate levels also results in CD4(+) T cell hyper-reactivity, as previously published for CD45RO. These data emphasize the dual functions of CD45 as both a positive and a negative regulators of TCR signalling irrespective of specific isoform expression.

Increases in cytosolic free Ca2+ ([Ca2+]i) represent a ubiquitous signalling mechanism that controls a variety of cellular processes, including proliferation, metabolism and gene transcription, yet under certain conditions increases in intracellular Ca2+ are cytotoxic. Thus, in using Ca2+ as a messenger, cells walk a tightrope in which [Ca2+]i is strictly maintained within defined boundaries. To adhere to these boundaries and to sustain their modified phenotype, many cancer cells remodel the expression or activity of their Ca2+ signalling apparatus. Here, we review the role of Ca2+ in promoting cell proliferation and cell death, how these processes are remodelled in cancer and the opportunities this might provide for therapeutic intervention.

The LIM homeobox containing genes of the LIM-3 group, Lhx3 and Lhx4, are critical for normal development. Both genes are involved in the formation of the pituitary and the motoneuron system and loss of either gene causes perinatal lethality. Previous studies had shown that Lhx3 is overexpressed in hyperplastic placentas of mouse interspecies hybrids. To determine the role of LHX3 in the mouse placenta, we performed expression and function analyses. Our results show that Lhx3 exhibits specific spatial and temporal expression in the mouse placenta. However, deletion of Lhx3 does not produce a placental phenotype. To test whether this is due to functional substitution by Lhx4, we performed a phenotype analysis of Lhx3-/-; Lhx4-/- double-mutant placentas. A subset of Lhx3-/-; Lhx4-/- placentas exhibited abnormal structure of the labyrinth. However, absence of both LIM-3 genes did not interfere with placental transport nor consistently with expression of target genes such as Gnrhr. Thus, LHX3 and LHX4 appear to be dispensable for placental development and function.

Rapamycin is an inhibitor of mTOR, a key component of the mTORC1 complex that controls the growth and survival of cells in response to growth factors, nutrients, energy balance and stresses. The downstream targets of mTORC1 include ribosome biogenesis, transcription, translation and macroautophagy. Recently it was proposed that rapamycin and its derivatives enhance the clearance (and/or reduce the accumulation) of mutant intracellular proteins causing proteinopathies such as tau, alpha-synuclein, ataxin-3, and full-length or fragments of huntingtin containing a polyglutamine (polyQ) expansion, by upregulating macroautophagy. We tested this proposal directly using macroautophagy-deficient fibroblasts. We found that rapamycin inhibits the aggregation of a fragment of huntingtin (exon 1) containing 97 polyQs similarly in macroautophagy-proficient (Atg5(+/+)) and macroautophagy-deficient (Atg5(-/-)) cells. These data demonstrate that autophagy is not the only mechanism by which rapamycin can alleviate the accumulation of misfolded proteins. Our data suggest that rapamycin inhibits mutant huntingtin fragment accumulation due to inhibition of protein synthesis. A model illustrates how a modest reduction in polyQ synthesis can lead to a long-lasting reduction in polyQ aggregation. We propose that several mechanisms exist by which rapamycin reduces the accumulation and potential toxicity of misfolded proteins in diseases caused by protein misfolding and aggregation.

Epigenetic modifications influence gene expression pattern and provide a unique signature of a cell differentiation status. Without external stimuli or signalling events, this cell identity remains stable and unlikely to change over many cell divisions. The epigenetic signature of a particular cell fate therefore needs to be replicated faithfully in daughter cells; otherwise a cell lineage cannot be maintained. However, the mechanism of transmission of cellular memory from mother to daughter cells remains unclear. It has been suggested that the inheritance of an active or silent gene state involves different kinds of epigenetic mechanisms, e.g. DNA methylation, histone modifications, replacement of histone variants, Polycomb group (PcG) and Trithorax group (TrxG) proteins. Emerging evidence supports the role of histone variant H3.3 in maintaining an active gene status and in remodelling nucleosomal composition. Here we discuss some recent findings on the propagation of epigenetic memory and propose a model for the inheritance of an active gene state through the interaction of H3.3 with other epigenetic components.

Mammalian genomes are highly organized in the 3D space of cell nuclei, but whether this affects gene function is unclear. Three papers now show that spatial relocation of a gene directly affects expression, and surprisingly, that of its neighbors.

Cell survival is promoted by the oncoprotein Bcl-2. Previous studies have established that one of the pro-survival actions of Bcl-2 is to reduce cellular fluxes of Ca2+ within cells. In particular, Bcl-2 has been demonstrated to inhibit the release of Ca2+ from the endoplasmic reticulum. However, the mechanism by which Bcl-2 causes reduced Ca2+ release is unclear. In the accompanying paper [C.J. Hanson, M.D. Bootman, C.W. Distelhorst, T. Maraldi, H.L. Roderick, The cellular concentration of Bcl-2 determines its pro- or anti-apoptotic effect, Cell Calcium (2008)], we described that only stable expression of Bcl-2 allowed it to work in a pro-survival manner whereas transient expression did not. In this study, we have employed HEK-293 cells that stably express Bcl-2, and which are, therefore, protected from pro-apoptotic stimuli, to examine the effect of Bcl-2 on Ca2+ homeostasis and signalling. We observed that Bcl-2 expression decreased the Ca2+ responses of cells induced by application of submaximal agonist concentrations. Whereas, decreasing endogenous Bcl-2 concentration using siRNA potentiated Ca2+ responses. Furthermore, we found that Bcl-2 expression reduced mitochondrial Ca2+ uptake by raising the threshold cytosolic Ca2+ concentration required to activate sequestration. Using a number of different assays, we did not find any evidence for reduction of endoplasmic reticulum luminal Ca2+ in our Bcl-2-expressing cells. Indeed, we observed that Bcl-2 served to preserve the content of the agonist-sensitive Ca2+ pool. Endogenous Bcl-2 was found to interact with inositol 1,4,5-trisphosphate receptors (InsP3Rs) in our cells, and to modify the profile of InsP3R expression. Our data suggest that the presence of Bcl-2 in the proteome of cells has multiple effects on agonist-mediated Ca2+ signals, and can abrogate responses to submaximal levels of stimulation through direct control of InsP3Rs.

Phosphatidylinositol-3-OH kinase (PI(3)K) and the nutrient sensor mTOR are evolutionarily conserved regulators of cell metabolism. Here we show that PI(3)K and mTOR determined the repertoire of adhesion and chemokine receptors expressed by T lymphocytes. The key lymph node-homing receptors CD62L (L-selectin) and CCR7 were highly expressed on naive T lymphocytes but were downregulated after immune activation. CD62L downregulation occurred through ectodomain proteolysis and suppression of gene transcription. The p110delta subunit of PI(3)K controlled CD62L proteolysis through mitogen-activated protein kinases, whereas control of CD62L transcription by p110delta was mediated by mTOR through regulation of the transcription factor KLF2. PI(3)K-mTOR nutrient-sensing pathways also determined expression of the chemokine receptor CCR7 and regulated lymphocyte trafficking in vivo. Hence, lymphocytes use PI(3)K and mTOR to match metabolism and trafficking.

A recent spate of examples of specific interactions between loci on separate chromosomes in mammalian nuclei has illuminated another layer of complexity in gene regulation. As the specifics of the cross-talk between interacting loci are worked out, it is also important to consider exactly how, when and where loci can ever reliably find each other within such an intricate environment. Answers may lie in how the genome is organised in relation to itself and to specialised nuclear sub-compartments. Here, we discuss how such specialised nuclear bodies may have the potential to specifically sequester loci and provide a context where interchromosomal communications can occur.

The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor best known for mediating the toxicity of dioxin. Environmental factors are believed to contribute to the increased prevalence of autoimmune diseases, many of which are due to the activity of T(H)17 T cells, a new helper T-cell subset characterized by the production of the cytokine IL-17. Here we show that in the CD4+ T-cell lineage of mice AHR expression is restricted to the T(H)17 cell subset and its ligation results in the production of the T(H)17 cytokine interleukin (IL)-22. AHR is also expressed in human T(H)17 cells. Activation of AHR by a high-affinity ligand during T(H)17 cell development markedly increases the proportion of T(H)17 T cells and their production of cytokines. CD4+ T cells from AHR-deficient mice can develop T(H)17 cell responses, but when confronted with AHR ligand fail to produce IL-22 and do not show enhanced T(H)17 cell development. AHR activation during induction of experimental autoimmune encephalomyelitis causes accelerated onset and increased pathology in wild-type mice, but not AHR-deficient mice. AHR ligands may therefore represent co-factors in the development of autoimmune diseases.

The etiology of mental disorders remains largely unclear. Complex interactions between genetic and environmental factors are key to the development of such disorders. Puerperal psychosis is the most extreme form of postnatal mood disorder in women. Similarly, parturition in the pig can trigger extreme behavioral disturbances, including maternal infanticide. In this study, we have used a targeted cDNA microarray approach using the pig as a model to understand the genes and genetic pathways that are involved in these processes. Two subtracted cDNA libraries from porcine hypothalamus were constructed, which were enriched for genes that were over-expressed and under-expressed in the aberrant behavioral phenotype, compared to the matched control. In addition to this, a normalized library was constructed from hypothalamus and pituitary samples taken from pigs in a variety of reproductive states. The libraries were partially sequenced and combined represented approximately 5,159 different genes. Microarray analysis determined differences in gene expression between hypothalamus samples from nine matched pairs of infanticidal versus control animals, using a common reference design. Microarray analysis of variance (MAANOVA) identified 52 clones as being differentially expressed (P <or= 0.002) in the infanticide phenotype, a second analysis with friendly statistics package for microarray analysis (FSPMA) identified 9 genes in common to MAANOVA, and a further 16 genes. A rapid cross-species screen onto a human oligonucleotide array confirmed 3 genes and highlighted 61 more potential candidates. Some of these genes and the pathways in which they are involved were also implicated in a parallel QTL study on maternal infanticide.

There is evidence that sleep is important for memory consolidation, but the underlying neuronal changes are not well understood. We studied the effect of sleep modulation on memory and on neuronal activity in a memory system of the domestic chick brain after the learning process of imprinting. Neurons in this system become, through imprinting, selectively responsive to a training (imprinting) stimulus and so possess the properties of a memory trace.

The small GTPase Rac controls cell morphology, gene expression, and reactive oxygen species formation. Manipulations of Rac activity levels in the cerebellum result in motor coordination defects, but activators of Rac in the cerebellum are unknown. P-Rex family guanine-nucleotide exchange factors activate Rac. We show here that, whereas P-Rex1 expression within the brain is widespread, P-Rex2 is specifically expressed in the Purkinje neurons of the cerebellum. We have generated P-Rex2(-/-) and P-Rex1(-/-)/P-Rex2(-/-) mice, analyzed their Purkinje cell morphology, and assessed their motor functions in behavior tests. The main dendrite is thinned in Purkinje cells of P-Rex2(-/-) pups and dendrite structure appears disordered in Purkinje cells of adult P-Rex2(-/-) and P-Rex1(-/-)/P-Rex2(-/-) mice. P-Rex2(-/-) mice show a mild motor coordination defect that progressively worsens with age and is more pronounced in females than in males. P-Rex1(-/-)/P-Rex2(-/-) mice are ataxic, with reduced basic motor activity and abnormal posture and gait, as well as impaired motor coordination even at a young age. We conclude that P-Rex1 and P-Rex2 are important regulators of Purkinje cell morphology and cerebellar function.

The ability of vaginocervical stimulation (VCS) to promote olfactory social recognition memory at different stages of the ovarian cycle was investigated in female rats. A juvenile social recognition paradigm was used and memory retention tested at 30 and 300 min after an adult was exposed to a juvenile during three 4-min trials. Results showed that an intact social recognition memory was present at 30 min in animals with or without VCS and at all stages of the estrus cycle. However, whereas no animals in any stage of the estrus cycle showed retention of the specific recognition memory at 300 min, those in the proestrus/estrus phase that received VCS 10 min before the trial started did. In vivo microdialysis studies showed that there was a significant release of oxytocin after VCS in the olfactory bulb during proestrus. There was also increased oxytocin immunoreactivity within the olfactory bulb after VCS in proestrus animals compared with diestrus ones. Furthermore, when animals received an infusion of an oxytocin antagonist directly into the olfactory bulb, or a systemic administration of alpha or beta noradrenaline-antagonists, they failed to show evidence for maintenance of a selective olfactory recognition memory at 300 min. Animals with vagus or pelvic nerve section also showed no memory retention when tested after 300 min. These results suggest that VCS releases oxytocin in the olfactory bulb to enhance the social recognition memory and that this may be due to modulatory actions on noradrenaline release. The vagus and pelvic nerves are responsible for carrying the information from the pelvic area to the CNS.

The Kcnq1ot1 antisense noncoding RNA has been implicated in long-range bidirectional silencing, but the underlying mechanisms remain enigmatic. Here we characterize a domain at the 5' end of the Kcnq1ot1 RNA that carries out transcriptional silencing of linked genes using an episomal vector system. The bidirectional silencing property of Kcnq1ot1 maps to a highly conserved repeat motif within the silencing domain, which directs transcriptional silencing by interaction with chromatin, resulting in histone H3 lysine 9 trimethylation. Intriguingly, the silencing domain is also required to target the episomal vector to the perinucleolar compartment during mid-S phase. Collectively, our data unfold a novel mechanism by which an antisense RNA mediates transcriptional gene silencing of chromosomal domains by targeting them to distinct nuclear compartments known to be rich in heterochromatic machinery.

Extracellular signal-regulated kinase-1 and -2 (ERK1/2) are activated by dual threonine and tyrosine phosphorylation of a TEY motif. The highly related kinase ERK5 is also activated by phosphorylation at a TEY motif. Inactivation of ERK1/2 is achieved by distinct members of the dual-specificity protein phosphatase (DUSP) family, which are responsible for the specific, regulated de-phosphorylation of the TEY motif. These include both nuclear (DUSP5) and cytoplasmic (DUSP6) enzymes. DUSP6, a candidate tumour suppressor gene, is thought to be highly specific for inactivation of ERK1/2 but several reports have suggested that it may also inactivate ERK5. Here we have compared the ability of DUSP6 to regulate the ERK1/2 and ERK5 protein kinases. We find that DUSP6 binds to ERK1/2 in both yeast and human cells but fails to bind to ERK5. Recombinant ERK2 can induce catalytic activation of DUSP6 whereas ERK5 cannot. Ectopic expression of DUSP6 can de-phosphorylate a co-expressed ERK2 construct but does not de-phosphorylate ERK5. Finally, expression of DUSP6 blocks the MEK1-driven activation of GAL4-ELK1, an ERK1/2-regulated transcription factor, but fails to block the MEK5-driven activation of GAL4-MEF2D, an ERK5-regulated transcription factor. These results demonstrate that even upon over-expression DUSP6 fails to inactivate ERK5, confirming that it is indeed an ERK1/2-specific DUSP.

The establishment of T cell-mediated inflammation requires the migration of primed T lymphocytes from the blood stream and their retention in antigenic sites. While naive T lymphocyte recirculation in the lymph and blood is constitutively regulated and occurs in the absence of inflammation, the recruitment of primed T cells to nonlymphoid tissue and their retention at the site are enhanced by various inflammatory signals, including TCR engagement by antigen-displaying endothelium and resident antigen-presenting cells. In this study, we investigated whether signals downstream of TCR ligation mediated by the phosphoinositide-3-kinase (PI3K) subunit p110delta contributed to the regulation of these events. T lymphocytes from mice expressing catalytically inactive p110delta displayed normal constitutive trafficking and migratory responses to nonspecific stimuli. However, these cells lost susceptibility to TCR-induced migration and failed to localize efficiently to antigenic tissue. Importantly, we showed that antigen-induced T cell trafficking and subsequent inflammation was abrogated by selective pharmacological inhibition of PI3K p110delta activity. These observations suggest that pharmacological targeting of p110delta activity is a viable strategy for the therapy of T cell-mediated pathology.