Florey Department of Neuroscience and Mental Health - Research Publications
Now showing items 1-12 of 565
A rare functional haplotype of the P2RX4 and P2RX7 genes leads to loss of innate phagocytosis and confers increased risk of age-related macular degeneration
(FEDERATION AMER SOC EXP BIOL, 2013-04-01)
Age-related macular degeneration (AMD) is a leading cause of blindness in Western countries and is diagnosed by the clinical appearance of yellow subretinal deposits called drusen. Genetic changes in immune components are clearly implicated in the pathology of this disease. We have previously shown that the purinergic receptor P2X7 can act as a scavenger receptor, mediating phagocytosis of apoptotic cells and insoluble debris. We performed a genetic association study of functional polymorphisms in the P2RX7 and P2RX4 genes in a cohort of 744 patients with AMD and 557 age-matched Caucasian control subjects. The P2X4 Tyr315Cys variant was 2-fold more frequent in patients with AMD compared to control subjects, with the minor allele predicting susceptibility to disease. Pairwise linkage disequilibrium was observed between Tyr315Cys in the P2RX4 gene and Gly150Arg in the P2RX7 gene, and these two minor alleles formed a rare haplotype that was overrepresented in patients with AMD (n=17) compared with control subjects (n=3) (odds ratio 4.05, P=0.026). Expression of P2X7 (wild type or variant 150Arg) in HEK293 cells conferred robust phagocytosis toward latex beads, whereas coexpression of the P2X7 150Arg with P2X4 315Cys variants almost completely inhibited phagocytic capacity. Fresh human monocytes harboring this heterozygous 150Arg-315Cys haplotype showed 40% reduction in bead phagocytosis. In the primate eye, immunohistochemistry indicated that P2X7 and P2X4 receptors were coexpressed on microglia and macrophages, but neither receptor was seen on retinal pigment epithelial cells. These results demonstrate that a haplotype including two rare variants in P2RX7 and P2RX4 confers a functional interaction between these two variant receptors that impairs the normal scavenger function of macrophages and microglia. Failure of this P2X7-mediated phagocytic pathway may impair removal of subretinal deposits and predispose individuals toward AMD.-Gu, B. J., Baird, P. N., Vessey, K. A., Skarratt, K. K., Fletcher, E. L., Fuller, S. J., Richardson, A. J., Guymer, R. H., Wiley, J. S. A rare functional haplotype of the P2RX4 and P2RX7 genes leads to loss of innate phagocytosis and confers increased risk of age related macular degeneration. FASEB J. 27, 1479-1487 (2013). www.fasebj.org
Highly impulsive rats: modelling an endophenotype to determine the neurobiological, genetic and environmental mechanisms of addiction
(COMPANY OF BIOLOGISTS LTD, 2013-03-01)
Impulsivity describes the tendency of an individual to act prematurely without foresight and is associated with a number of neuropsychiatric co-morbidities, including drug addiction. As such, there is increasing interest in the neurobiological mechanisms of impulsivity, as well as the genetic and environmental influences that govern the expression of this behaviour. Tests used on rodent models of impulsivity share strong parallels with tasks used to assess this trait in humans, and studies in both suggest a crucial role of monoaminergic corticostriatal systems in the expression of this behavioural trait. Furthermore, rodent models have enabled investigation of the causal relationship between drug abuse and impulsivity. Here, we review the use of rodent models of impulsivity for investigating the mechanisms involved in this trait, and how these mechanisms could contribute to the pathogenesis of addiction.
Links between L-glutamate transporters, Na+/K+-ATPase and cytoskeleton in astrocytes: evidence following inhibition with rottlerin
Astrocytes are plastic cells that play key roles in brain physiology and pathology, including via their glutamate transporters, excitatory amino acid transporter (EAAT)1 and EAAT2, maintaining low extracellular glutamate concentrations and protecting against excitotoxic neuronal injury. Alterations in cell surface expression of EAATs and astrocytic cytoskeleton are important for regulating transporter activity. This study employed the actions of rottlerin, to interrogate the regulation of EAAT activity, expression and localization, and interfaces with Na+/K+-ATPase and astrocytic morphology. EAAT activity and expression were determined in primary cultures of mouse astrocytes in the presence of and after rottlerin removal, with or without trafficking inhibitors, using uptake ([H-3]D-aspartate, Rb-86(+)) and molecular analyses. Astrocytic morphology and EAAT localization were investigated using Western blotting and immunocytochemistry, in concert with image analysis of glial fibrillary acidic protein, F-actin and EAAT1/2. Rottlerin induced a time-dependent inhibition of glutamate transport (V-max). Rapid changes in cytoskeletal arrangement were observed and immunoblotting revealed increases in EAAT2 total and cell surface expression, despite reduced EAAT activity. Rottlerin-induced inhibition was reversible and its rate was increased by monensin co-treatment. Rottlerin inhibited, while monensin stimulated Na+/K+-ATPase. Removal of rottlerin rapidly elevated Na+/K+-ATPase activity beyond control levels, while co-treatment with monensin failed to stimulate the Na+/K+-ATPase. These data reveal inhibition of EAAT activity by rottlerin is not associated with loss of EAATs at the cell surface, but rather linked to cytoskeletal rearrangement, and inhibition of the Na+/K+-ATPase. Rapid recovery of Na+/K+-ATPase activity, and subsequent restoration of glutamate uptake indicates that astrocytic morphology and EAAT activity are co-regulated by a tightly coupled, homeostatic relationship between L-glutamate uptake, the electrochemical gradient and the activity of the Na+/K+-ATPase.
Ceruloplasmin dysfunction and therapeutic potential for Parkinson disease
Ceruloplasmin is an iron-export ferroxidase that is abundant in plasma and also expressed in glia. We found a approximate to 80% loss of ceruloplasmin ferroxidase activity in the substantia nigra of idiopathic Parkinson disease (PD) cases, which could contribute to the pro-oxidant iron accumulation that characterizes the pathology. Consistent with a role for ceruloplasmin in PD etiopathogenesis, ceruloplasmin knockout mice developed parkinsonism that was rescued by iron chelation. Additionally, peripheral infusion of ceruloplasmin attenuated neurodegeneration and nigral iron elevation in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model for PD. These findings show, in principle, that intravenous ceruloplasmin may have therapeutic potential in PD. Ann Neurol 2013;73:554-559
Sex differences in the adolescent developmental trajectory of parvalbumin interneurons in the hippocampus: A role for estradiol
Objective: Gender differences in the neurodevelopmental disorder, schizophrenia, have been described for nearly all features of the illness. Reduced hippocampal expression of the GABAergic interneuron marker, parvalbumin (PV), and GABA synthesizing enzyme, GAD67, are consistently reported in schizophrenia. However, little is known of the expression patterns of hippocampal PV and GAD67 during adolescence and their interaction with sex steroid hormones during adolescent development. This study examined the effects of altered sex steroid hormone levels during adolescence on protein levels of PV, GAD67 and estrogen receptors (ER alpha/beta) in the hippocampus of mice.Methods: Protein expression of PV and GAD67 was measured in the dorsal (DHP) and ventral (VHP) hippocampus of female and male C57Bl/6 mice by Western blot in a week by week analysis from pre-pubescence to adulthood (week 3-12). Fluorescent immunohistochemistry (INC) was used to investigate the relationship between ERs and Pr cells in the hippocampus of female mice at young adulthood (week 10-11). To further examine the role of sex steroid hormones on PV and GAD67 expression, gonadectomy and hormone replacement was done at 5 weeks of age.Results: Female mice showed a significant gradual increase in PV expression from 3 to 12 weeks of age in the DHP and VHP which correlated with serum 17 beta-estradiol levels. Fluorescent IHC showed approximately 30-50% co-localization of ER-alpha in PV+ cells in the female DHP and VHP (dentate gryus/hilus and CA1 CA3). Adolescent ovariectomy significantly reduced PV expression in the DHP but not VHP of female mice, while 17 beta-estradiol replacement prevented this deficit in DHP PV levels. ER-alpha expression, but not ER-beta, was also reduced in the DHP following ovariectomy with no significant effect of 17 beta-estradiol replacement. In contrast to female mice, male mice did not show any significant changes in hippocampal PV/GAD67 expression throughout adolescent development. Furthermore, adolescent castration and treatment with testosterone or dihydrotestosterone produced no changes in PV/GAD67 expression.Conclusions: Our data suggest a differential developmental trajectory of PV expression between the sexes and manipulating circulating levels of sex steroid hormones by ovariectomy alters this trajectory in a region-dependent manner. This may be mediated via ER-a signaling as this receptor was found to be co-localized with PV+ cells in the female mouse hippocampus. Alternative mechanisms of 17 beta-estradiol-induced regulation of PV expression are also discussed herein. Together, results from the present study may offer more insight into neurodevelopmental disorders, including schizophrenia, where sex steroid hormones and GABAergic markers are implicated in the pathophysiology of the illness.
Behavioral endophenotypes of drug addiction: Etiological insights from neuroimaging studies
This article reviews recent advances in the elucidation of neurobehavioral endophenotypes associated with drug addiction made possible by the translational neuroimaging techniques magnetic resonance imaging (MRI) and positron emission tomography (PET). Increasingly, these non-invasive imaging approaches have been the catalyst for advancing our understanding of the etiology of drug addiction as a brain disorder involving complex interactions between pre-disposing behavioral traits, environmental influences and neural perturbations arising from the chronic abuse of licit and illicit drugs. In this article we discuss the causal role of trait markers associated with impulsivity and novelty-/sensation-seeking in speeding the development of compulsive drug administration and in facilitating relapse. We also discuss the striking convergence of imaging findings from these behavioural traits and addiction in rats, monkeys and humans with a focus on biomarkers of dopamine neurotransmission, and highlight areas where further research is needed to disambiguate underlying causal mechanisms. This article is part of a Special Issue entitled 'NIDA 40th Anniversary Issue' .
Central injection of relaxin-3 receptor (RXFP3) antagonist peptides reduces motivated food seeking and consumption in C57BL/6J mice
Behavioural arousal in mammals is regulated by various interacting central monoamine- and peptide-neurotransmitter/receptor systems, which function to maintain awake, alert and active states required for performance of goal-directed activities essential for survival, including food seeking. Existing anatomical and functional evidence suggests the highly-conserved neuropeptide, relaxin-3, which signals via its cognate G(i/o)-protein coupled receptor, RXFP3, contributes to behavioural arousal and feeding behaviour in rodents. In studies to investigate this possibility further, adult male C57BL/6J mice were treated with the selective RXFP3 antagonist peptides, R3(B1-22)R/I5(A) and R3(B1-22)R, and motivated food seeking and consumption was assessed as a reflective output of behavioural arousal. Compared to vehicle treatment, intracerebroventricular (icv) injection of RXFP3 antagonists reduced: (i) food anticipatory activity before meal time during food restriction; (ii) consumption of highly palatable food; (iii) consumption of regular chow during the initial dark phase, and; (iv) consumption of regular chow after mild (similar to 4-h) food deprivation. Effects were not due to sedation and appeared to be specifically mediated via antagonism of relaxin-3/RXFP3 signalling, as RXFP3 antagonist treatment did not alter locomotor activity in wild-type mice or reduce palatable food intake in relaxin-3 deficient (knock-out) mice. Notably, in contrast to similar studies in the rat, icy injection of RXFP3 agonists and infusion into the paraventricular hypothalamic nucleus did not increase food consumption in mice, suggesting species differences in relaxin-3/RXFP3-related signalling networks. Together, our data provide evidence that endogenous relaxin-3/RXFP3 signalling promotes motivated food seeking and consumption, and in light of the established biological and translational importance of other arousal systems, relaxin-3/RXFP3 networks warrant further experimental investigation.
Central orexin (hypocretin) 2 receptor antagonism reduces ethanol self-administration, but not cue-conditioned ethanol-seeking, in ethanol-preferring rats
(OXFORD UNIVERSITY PRESS, 2013-10-27)
Orexins are hypothalamic neuropeptides which bind to two G-protein-coupled receptors, orexin-1 (OX1R) and orexin-2 (OX2R) receptor. While a role for OX1R has been established in both ethanol reinforcement and ethanol-seeking behaviour, the role of OX2R in these behaviours is relatively less-studied. The aim of this study was to determine the role of central OX2R in ethanol-taking and ethanol-seeking behaviour. Indiana ethanol-preferring rats were trained to self-administer ethanol (10% w/v) or sucrose (0.7-1% w/v) in the presence of reward-associated cues before being implanted with indwelling guide cannulae. The selective OX2R antagonist TCS-OX2-29 was administered i.c.v. to assess its effect on operant self-administration and cue-induced reinstatement following extinction. Following i.c.v. injection TCS-OX2-29 reduced self-administration of ethanol, but not sucrose. Despite reducing ethanol self-administration, TCS-OX2-29 had no impact on cue-induced reinstatement of ethanol seeking. To determine where in the brain OX2R were acting to modulate ethanol self-administration, TCS-OX2-29 was microinjected into either the shell or core of the nucleus accumbens (NAc). Intra-NAc core, but not shell, infusions of TCS-OX2-29 decreased responding for ethanol. Importantly, the doses of TCS-OX2-029 used were non-sedating. Collectively, these findings implicate OX2R in the NAc in mediating the reinforcing effects of ethanol. This effect appears to be drug-specific as antagonism of central OX2R had no impact on sucrose self-administration. Thus, OX2R in addition to OX1R may represent a potential therapeutic target for the treatment of ethanol-use disorders. However, unlike OX1R, no impact of OX2R antagonism was observed on cue-induced reinstatement, suggesting a more prominent role for OX2R in ethanol self-administration compared to cue-conditioned ethanol-seeking.
Efficient expansion and dopaminergic differentiation of human fetal ventral midbrain neural stem cells by midbrain morphogens
Human fetal midbrain tissue grafting has provided proof-of-concept for dopamine cell replacement therapy (CRT) in Parkinson's disease (PD). However, limited tissue availability has hindered the development and widespread use of this experimental therapy. Here we present a method for generating large numbers of midbrain dopaminergic (DA) neurons based on expanding and differentiating neural stem/progenitor cells present in the human ventral midbrain (hVM) tissue. Our results show that hVM neurospheres (hVMN) with low cell numbers, unlike their rodent counterparts, expand the total number of cells 3-fold, whilst retaining their capacity to differentiate into midbrain DA neurons. Moreover, Wnt5a promoted DA differentiation of expanded cells resulting in improved morphological maturation, midbrain DA marker expression, DA release and electrophysiological properties. This method results in cell preparations that, after expansion and differentiation, can contain 6-fold more midbrain DA neurons than the starting VM preparation. Thus, our results provide evidence that by improving expansion and differentiation of progenitors present in the hVM it is possible to greatly enrich cell preparations for DA neurons. This method could substantially reduce the amount of human fetal midbrain tissue necessary for CRT in patients with PD, which could have major implications for the widespread adoption of this approach.
N-linked glycosylation modulates dimerization of protein disulfide isomerase family A member 2 (PDIA2)
Protein disulfide isomerase (PDI) family members are important enzymes for the correct folding and maturation of proteins that transit or reside in the endoplasmic reticulum (ER). The human PDI family comprises at least 19 members that differ in cell type expression, substrate specificity and post-translational modifications. PDI family A member 2 (PDIA2, previously known as PDIp) has a similar domain structure to prototypical PDI (also known as PDIA1), but the function and post-translational modifications of PDIA2 remain poorly understood. Unlike most PDI family members, PDIA2 contains three predicted N-linked glycosylation sites. By site-directed mutagenesis and enzymatic deglycosylation, we show here that all three Asn residues within the potential N-linked glycosylation sites of human PDIA2 (N127, N284 and N516) are glycosylated in human cells. Furthermore, mutation of N284 to glycosylation-null Gln increases formation of a highly stable disulfide-bonded PDIA2 dimer. Nevertheless, in HeLa cells, both wild-type and N127/284/516Q mutant PDIA2 proteins localize to the ER, but not the ERGolgi intermediate compartment, suggesting that glycosylation is important for PDIA2 proteinprotein interactions but not subcellular localization. Finally, we identified human major histocompatibility complex class 1 antigens (HLA-A,B,C) as potential binding partners of PDIA2, suggesting an involvement for PDIA2 in antigen presentation in addition to its previously described roles in autoimmunity and Parkinson's disease. These results further characterize this poorly defined member of the PDI family. Structured digital abstract Calreticulin and PDIA2 colocalize by fluorescence microscopy (View interaction) PDIA2 and PDIA1 colocalize by fluorescence microscopy (View interaction)
Reflex control of inflammation by sympathetic nerves, not the vagus
Key pointsIt is believed that the CNS controls inflammation via the autonomic nervous system, but the strength of this action and the neural pathways responsible are unclear.In anaesthetized rats we measured the inflammatory response to lipopolysaccharide (LPS, 60 mu gkg(-1), i.v.) by plasma tumour necrosis factor alpha (TNF alpha) levels 90min later.Bilateral section of the splanchnic sympathetic nerves before LPS treatment resulted in a 5-fold increase in the plasma TNF alpha response, but bilateral vagotomy had no effect.LPS treatment strongly increased efferent activity in the splanchnic sympathetic nerve and its splenic branch; vagotomy did not affect this.These results show that, besides directly stimulating inflammation, LPS engages a powerful anti-inflammatory reflex that can inhibit the plasma TNF alpha response by 80%.The reflex efferent arm is in the splanchnic sympathetic nerves; the vagi play no part.We investigated a neural reflex that controls the strength of inflammatory responses to immune challenge - the inflammatory reflex. In anaesthetized rats challenged with intravenous lipopolysaccharide (LPS, 60 mu gkg(-1)), we found strong increases in plasma levels of the key inflammatory mediator tumour necrosis factor alpha (TNF alpha) 90min later. Those levels were unaffected by previous bilateral cervical vagotomy, but were enhanced approximately 5-fold if the greater splanchnic sympathetic nerves had been cut. Sham surgery had no effect, and plasma corticosterone levels were unaffected by nerve sections, so could not explain this result. Electrophysiological recordings demonstrated that efferent neural activity in the splanchnic nerve and its splenic branch was strongly increased by LPS treatment. Splenic nerve activity was dependent on inputs from the splanchnic nerves: vagotomy had no effect on the activity in either nerve. Together, these data demonstrate that immune challenge with this dose of LPS activates a neural reflex that is powerful enough to cause an 80% suppression of the acute systemic inflammatory response. The efferent arm of this reflex is in the splanchnic sympathetic nerves, not the vagi as previously proposed. As with other physiological responses to immune challenge, the afferent pathway is presumptively humoral: the present data show that vagal afferents play no measurable part. Because inflammation sits at the gateway to immune responses, this reflex could play an important role in immune function as well as inflammatory diseases.