Publications by authors named "Amanda Lumsden"

20 Publications

  • Page 1 of 1

Unlocking the causal link of metabolically different adiposity subtypes with brain volumes and the risks of dementia and stroke: A Mendelian randomization study.

Neurobiol Aging 2021 Feb 21;102:161-169. Epub 2021 Feb 21.

Australian Centre for Precision Health, Unit of Clinical and Health Sciences, University of South Australia, Adelaide, Australia; South Australian Health and Medical Research Institute, Adelaide, Australia; Population, Policy and Practice, UCL Great Ormond Street Institute of Child Health, London, UK. Electronic address:

To establish causal evidence for the association of adiposity-related metabolic abnormalities with brain volumes, and the risks of dementia and stroke, we applied 1- and 2-sample Mendelian randomization (MR) analyses using up to 336,309 UK Biobank participants. We used 3 classes of genetic instruments, which all increase body mass index but are associated with different metabolic profiles (unfavorable, neutral and favorable). We validated the instruments using anthropometric and cardio-metabolic traits. Both metabolically unfavorable and metabolically neutral adiposity associated with lower gray matter volume (GMV, -9.28 cm, -12.90 to -5.66 and -12.02 cm, -20.07 to -3.97, respectively). Metabolically favorable adiposity was tentatively associated with a higher GMV (16.21 cm, -0.21 to 32.68). No causal evidence was seen for white matter and hippocampal volume, and volume of white matter hyperintensities, or with the risks of dementia and stroke (all p > 0.60). These findings suggest that obesity-related metabolic abnormalities may contribute to GMV atrophy, warranting further studies.
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http://dx.doi.org/10.1016/j.neurobiolaging.2021.02.010DOI Listing
February 2021

Relationship between Serum 25(OH)D and Depression: Causal Evidence from a Bi-Directional Mendelian Randomization Study.

Nutrients 2020 Dec 30;13(1). Epub 2020 Dec 30.

Australian Centre for Precision Health, University of South Australia Cancer Research Institute, University of South Australia, Adelaide, SA 5000, Australia.

The relationship between depression and vitamin D deficiency is complex, with evidence mostly from studies affected by confounding and reverse causality. We examined the causality and direction of the relationship between 25-hydroxyvitamin D (25(OH)D) and depression in bi-directional Mendelian randomization (MR) analyses using information from up to 307,618 white British participants from the UK Biobank and summary results from the SUNLIGHT ( = 79,366) and Psychiatric Genomics consortia (PGC 113,154 cases and 218,523 controls). In observational analysis, the odds of depression decreased with higher 25(OH)D concentrations (adjusted odds ratio (OR) per 50% increase 0.95, 95%CI 0.94-0.96). In MR inverse variance weighted (IVW) using the UK Biobank, there was no association between genetically determined serum 25(OH)D and depression (OR per 50% higher 0.97, 95%CI 0.90-1.05) with consistent null association across all MR approaches and in data from PGC consortium. In contrast, genetic liability to depression was associated with lower 25(OH)D concentrations (MR IVW -3.26%, -4.94%--1.55%), with the estimates remaining generally consistent after meta-analysing with the consortia. In conclusion, we found genetic evidence for a causal effect of depression on lower 25(OH)D concentrations, however we could not confirm a beneficial effect of nutritional vitamin D status on depression risk.
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http://dx.doi.org/10.3390/nu13010109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7823924PMC
December 2020

Experimentally Induced Bladder Permeability Evokes Bladder Afferent Hypersensitivity in the Absence of Inflammation.

Front Neurosci 2020 23;14:590871. Epub 2020 Oct 23.

Visceral Pain Research Group, Flinders Health and Medical Research Institute (FHMRI), Flinders University, Bedford Park, SA, Australia.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic urological condition characterised by urinary urgency, frequency and pelvic pain, that significantly impacts the quality of life for ∼5% of women. Bladder sensation is coordinated by primary afferent sensory neurons that innervate the bladder wall, translating bladder stretch into signals that travel to the brain via the spinal cord. Whilst the pathophysiology of IC/BPS remains unknown, an increase in the permeability of the bladder urothelium has been proposed as an initiating cause. Here we experimentally increased bladder permeability and tracked bladder afferent sensitivity for up to 28 days. We found that one day after increasing bladder epithelial permeability with bladder infusion of protamine sulfate, mechanosensitive bladder afferents exhibited significant hypersensitivity to bladder filling. This mechanical hypersensitivity was characterised by significantly increased peak afferent firing rates and a decrease in the activation threshold of individual afferents. Bladder afferent hypersensitivity occurred in the absence of inflammation and changes in bladder muscle compliance, indicating a direct sensitisation of peripheral afferent endings. Bladder afferent mechanosensitive responses to distension returned to control levels by day 7 post-protamine sulfate treatment and remained at control levels at 28-days post-treatment. Here we demonstrate, contrary to the prevailing hypothesis, that increased bladder permeability alone does not induce chronic bladder afferent sensitisation. Whilst experimentally induced changes in bladder permeability are able to induce transient bladder afferent hypersensitivity in the absence of inflammation, highly regulated homeostatic mechanisms exist to rapidly repair the urothelial barrier and normalise bladder afferent mechanosensitivity. Together, these data suggest that additional pathophysiology is required to induce chronic bladder dysfunction.
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http://dx.doi.org/10.3389/fnins.2020.590871DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7645115PMC
October 2020

Apolipoprotein E (APOE) genotype-associated disease risks: a phenome-wide, registry-based, case-control study utilising the UK Biobank.

EBioMedicine 2020 Sep 17;59:102954. Epub 2020 Aug 17.

Australian Centre for Precision Health, Cancer Research Institute, University of South Australia, Adelaide, SA 5001, Australia; South Australian Health and Medical Research Institute, Adelaide, SA 5000, Australia. Electronic address:

Background: The three main alleles of the APOE gene (ε4, ε3 and ε2) carry differential risks for conditions including Alzheimer's disease (AD) and cardiovascular disease. Due to their clinical significance, we explored disease associations of the APOE genotypes using a hypothesis-free, data-driven, phenome-wide association study (PheWAS) approach.

Methods: We used data from the UK Biobank to screen for associations between APOE genotypes and over 950 disease outcomes using genotype ε3ε3 as a reference. Data was restricted to 337,484 white British participants (aged 37-73 years).

Findings: After correction for multiple testing, PheWAS analyses identified associations with 37 outcomes, representing 18 distinct diseases. As expected, ε3ε4 and ε4ε4 genotypes associated with increased odds of AD (p ≤ 7.6 × 10), hypercholesterolaemia (p ≤ 7.1 × 10) and ischaemic heart disease (p ≤ 2.3 × 10), while ε2ε3 provided protection for the latter two conditions (p ≤ 3.7 × 10) compared to ε3ε3. In contrast, ε4-associated disease protection was seen against obesity, chronic airway obstruction, type 2 diabetes, gallbladder disease, and liver disease (all p ≤ 5.2 × 10) while ε2ε2 homozygosity increased risks of peripheral vascular disease, thromboembolism, arterial aneurysm, peptic ulcer, cervical disorders, and hallux valgus (all p ≤ 6.1 × 10). Sensitivity analyses using brain neuroimaging, blood biochemistry, anthropometric, and spirometric biomarkers supported the PheWAS findings on APOE associations with respective disease outcomes.

Interpretation: PheWAS confirms strong associations between APOE and AD, hypercholesterolaemia, and ischaemic heart disease, and suggests potential ε4-associated disease protection and harmful effects of the ε2ε2 genotype, for several conditions.

Funding: National Health and Medical Research Council of Australia.
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http://dx.doi.org/10.1016/j.ebiom.2020.102954DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7452404PMC
September 2020

Accelerated brain aging towards transcriptional inversion in a zebrafish model of the K115fs mutation of human PSEN2.

PLoS One 2020 24;15(1):e0227258. Epub 2020 Jan 24.

Alzheimer's Disease Genetics Laboratory, School of Biological Sciences, University of Adelaide, Adelaide, South Australia, Australia.

Background: The molecular changes involved in Alzheimer's disease (AD) progression remain unclear since we cannot easily access antemortem human brains. Some non-mammalian vertebrates such as the zebrafish preserve AD-relevant transcript isoforms of the PRESENILIN genes lost from mice and rats. One example is PS2V, the alternative transcript isoform of the PSEN2 gene. PS2V is induced by hypoxia/oxidative stress and shows increased expression in late onset, sporadic AD brains. A unique, early onset familial AD mutation of PSEN2, K115fs, mimics the PS2V coding sequence suggesting that forced, early expression of PS2V-like isoforms may contribute to AD pathogenesis. Here we use zebrafish to model the K115fs mutation to investigate the effects of forced PS2V-like expression on the transcriptomes of young adult and aged adult brains.

Methods: We edited the zebrafish genome to model the K115fs mutation. To explore its effects at the molecular level, we analysed the brain transcriptome and proteome of young (6-month-old) and aged (24-month-old) wild type and heterozygous mutant female sibling zebrafish. Finally, we used gene co-expression network analysis (WGCNA) to compare molecular changes in the brains of these fish to human AD.

Results: Young heterozygous mutant fish show transcriptional changes suggesting accelerated brain aging and increased glucocorticoid signalling. These early changes precede a transcriptional 'inversion' that leads to glucocorticoid resistance and other likely pathological changes in aged heterozygous mutant fish. Notably, microglia-associated immune responses regulated by the ETS transcription factor family are altered in both our zebrafish mutant model and in human AD. The molecular changes we observe in aged heterozygous mutant fish occur without obvious histopathology and possibly in the absence of Aβ.

Conclusions: Our results suggest that forced expression of a PS2V-like isoform contributes to immune and stress responses favouring AD pathogenesis. This highlights the value of our zebrafish genetic model for exploring molecular mechanisms involved in AD pathogenesis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0227258PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6980398PMC
May 2020

Activation of pruritogenic TGR5, MrgprA3, and MrgprC11 on colon-innervating afferents induces visceral hypersensitivity.

JCI Insight 2019 10 17;4(20). Epub 2019 Oct 17.

Visceral Pain Research Group, College of Medicine and Public Health, Flinders University, Bedford Park, South Australia, Australia.

Itch induces scratching that removes irritants from the skin, whereas pain initiates withdrawal or avoidance of tissue damage. While pain arises from both the skin and viscera, we investigated whether pruritogenic irritant mechanisms also function within visceral pathways. We show that subsets of colon-innervating sensory neurons in mice express, either individually or in combination, the pruritogenic receptors Tgr5 and the Mas-gene-related GPCRs Mrgpra3 and Mrgprc11. Agonists of these receptors activated subsets of colonic sensory neurons and evoked colonic afferent mechanical hypersensitivity via a TRPA1-dependent mechanism. In vivo intracolonic administration of individual TGR5, MrgprA3, or MrgprC11 agonists induced pronounced visceral hypersensitivity to colorectal distension. Coadministration of these agonists as an "itch cocktail" augmented hypersensitivity to colorectal distension and changed mouse behavior. These irritant mechanisms were maintained and enhanced in a model of chronic visceral hypersensitivity relevant to irritable bowel syndrome. Neurons from human dorsal root ganglia also expressed TGR5, as well as the human ortholog MrgprX1, and showed increased responsiveness to pruritogenic agonists in pathological states. These data support the existence of an irritant-sensing system in the colon that is a visceral representation of the itch pathways found in skin, thereby contributing to sensory disturbances accompanying common intestinal disorders.
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http://dx.doi.org/10.1172/jci.insight.131712DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6824308PMC
October 2019

Sugar Responses of Human Enterochromaffin Cells Depend on Gut Region, Sex, and Body Mass.

Nutrients 2019 Jan 22;11(2). Epub 2019 Jan 22.

College of Medicine and Public Health, Flinders University, Bedford Park SA 5042, Australia.

Gut-derived serotonin (5-HT) is released from enterochromaffin (EC) cells in response to nutrient cues, and acts to slow gastric emptying and modulate gastric motility. Rodent studies also evidence a role for gut-derived 5-HT in the control of hepatic glucose production, lipolysis and thermogenesis, and in mediating diet-induced obesity. EC cell number and 5-HT content is increased in the small intestine of obese rodents and human, however, it is unknown whether EC cells respond directly to glucose in humans, and whether their capacity to release 5-HT is perturbed in obesity. We therefore investigated 5-HT release from human duodenal and colonic EC cells in response to glucose, sucrose, fructose and α-glucoside (αMG) in relation to body mass index (BMI). EC cells released 5-HT only in response to 100 and 300 mM glucose (duodenum) and 300 mM glucose (colon), independently of osmolarity. Duodenal, but not colonic, EC cells also released 5-HT in response to sucrose and αMG, but did not respond to fructose. 5-HT content was similar in all EC cells in males, and colonic EC cells in females, but 3 to 4-fold higher in duodenal EC cells from overweight females ( < 0.05 compared to lean, obese). Glucose-evoked 5-HT release was 3-fold higher in the duodenum of overweight females ( < 0.05, compared to obese), but absent here in overweight males. Our data demonstrate that primary human EC cells respond directly to dietary glucose cues, with regional differences in selectivity for other sugars. Augmented glucose-evoked 5-HT release from duodenal EC is a feature of overweight females, and may be an early determinant of obesity.
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http://dx.doi.org/10.3390/nu11020234DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6412251PMC
January 2019

Dysregulation of Neuronal Iron Homeostasis as an Alternative Unifying Effect of Mutations Causing Familial Alzheimer's Disease.

Front Neurosci 2018 13;12:533. Epub 2018 Aug 13.

Centre for Molecular Pathology, School of Biological Sciences, University of Adelaide, Adelaide, SA, Australia.

The overwhelming majority of dominant mutations causing early onset familial Alzheimer's disease (EOfAD) occur in only three genes, , and . An effect-in-common of these mutations is alteration of production of the APP-derived peptide, amyloid β (Aβ). It is this key fact that underlies the authority of the Amyloid Hypothesis that has informed Alzheimer's disease research for over two decades. Any challenge to this authority must offer an alternative explanation for the relationship between the genes and . In this paper, we explore one possible alternative relationship - the dysregulation of cellular iron homeostasis as a common effect of EOfAD mutations in these genes. This idea is attractive since it provides clear connections between EOfAD mutations and major characteristics of Alzheimer's disease such as dysfunctional mitochondria, vascular risk factors/hypoxia, energy metabolism, and inflammation. We combine our ideas with observations by others to describe a "Stress Threshold Change of State" model of Alzheimer's disease that may begin to explain the existence of both EOfAD and late onset sporadic (LOsAD) forms of the disease. Directing research to investigate the role of dysregulation of iron homeostasis in EOfAD may be a profitable way forward in our struggle to understand this form of dementia.
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http://dx.doi.org/10.3389/fnins.2018.00533DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6099262PMC
August 2018

ICAM-1-related long non-coding RNA: promoter analysis and expression in human retinal endothelial cells.

BMC Res Notes 2018 May 9;11(1):285. Epub 2018 May 9.

Flinders University College of Medicine and Public Health, Flinders Medical Centre Room 4E-431, Flinders Drive, Bedford Park, SA, 5042, Australia.

Objective: Regulation of intercellular adhesion molecule (ICAM)-1 in retinal endothelial cells is a promising druggable target for retinal vascular diseases. The ICAM-1-related (ICR) long non-coding RNA stabilizes ICAM-1 transcript, increasing protein expression. However, studies of ICR involvement in disease have been limited as the promoter is uncharacterized. To address this issue, we undertook a comprehensive in silico analysis of the human ICR gene promoter region.

Results: We used genomic evolutionary rate profiling to identify a 115 base pair (bp) sequence within 500 bp upstream of the transcription start site of the annotated human ICR gene that was conserved across 25 eutherian genomes. A second constrained sequence upstream of the orthologous mouse gene (68 bp; conserved across 27 Eutherian genomes including human) was also discovered. Searching these elements identified 33 matrices predictive of binding sites for transcription factors known to be responsive to a broad range of pathological stimuli, including hypoxia, and metabolic and inflammatory proteins. Five phenotype-associated single nucleotide polymorphisms (SNPs) in the immediate vicinity of these elements included four SNPs (i.e. rs2569693, rs281439, rs281440 and rs11575074) predicted to impact binding motifs of transcription factors, and thus the expression of ICR and ICAM-1 genes, with potential to influence disease susceptibility. We verified that human retinal endothelial cells expressed ICR, and observed induction of expression by tumor necrosis factor-α.
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http://dx.doi.org/10.1186/s13104-018-3384-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5944171PMC
May 2018

Augmented capacity for peripheral serotonin release in human obesity.

Int J Obes (Lond) 2018 11 1;42(11):1880-1889. Epub 2018 Mar 1.

Nutrition & Metabolism, South Australian Health and Medical Research Institute (SAHMRI), Adelaide, SA, 5000, Australia.

Background/objectives: Evidence from animal studies highlights an important role for serotonin (5-HT), derived from gut enterochromaffin (EC) cells, in regulating hepatic glucose production, lipolysis and thermogenesis, and promoting obesity and dysglycemia. Evidence in humans is limited, although elevated plasma 5-HT concentrations are linked to obesity.

Subjects/methods: We assessed (i) plasma 5-HT concentrations before and during intraduodenal glucose infusion (4 kcal/min for 30 min) in non-diabetic obese (BMI 44 ± 4 kg/m, N = 14) and control (BMI 24 ± 1 kg/m, N = 10) subjects, (ii) functional activation of duodenal EC cells (immunodetection of phospho-extracellular related-kinase, pERK) in response to glucose, and in separate subjects, (iii) expression of tryptophan hydroxylase-1 (TPH1) in duodenum and colon (N = 39), and (iv) 5-HT content in primary EC cells from these regions (N = 85).

Results: Plasma 5-HT was twofold higher in obese than control responders prior to (P = 0.025), and during (iAUC, P = 0.009), intraduodenal glucose infusion, and related positively to BMI (R = 0.334, P = 0.003) and HbA1c (R = 0.508, P = 0.009). The density of EC cells in the duodenum was twofold higher at baseline in obese subjects than controls (P = 0.023), with twofold more EC cells activated by glucose infusion in the obese (EC cells co-expressing 5-HT and pERK, P = 0.001), while the 5-HT content of EC cells in duodenum and colon was similar; TPH1 expression was 1.4-fold higher in the duodenum of obese subjects (P = 0.044), and related positively to BMI (R = 0.310, P = 0.031).

Conclusions: Human obesity is characterized by an increased capacity to produce and release 5-HT from the proximal small intestine, which is strongly linked to higher body mass, and glycemic control. Gut-derived 5-HT is likely to be an important driver of pathogenesis in human obesity and dysglycemia.
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http://dx.doi.org/10.1038/s41366-018-0047-8DOI Listing
November 2018

Regional differences in nutrient-induced secretion of gut serotonin.

Physiol Rep 2017 Mar;5(6)

Department of Human Physiology and Centre for Neuroscience, Flinders University, Adelaide, Australia

Enterochromaffin (EC) cells located in the gastrointestinal (GI) tract provide the vast majority of serotonin (5-HT) in the body and constitute half of all enteroendocrine cells. EC cells respond to an array of stimuli, including various ingested nutrients. Ensuing 5-HT release from these cells plays a diverse role in regulating gut motility as well as other important responses to nutrient ingestion such as glucose absorption and fluid balance. Recent data also highlight the role of peripheral 5-HT in various pathways related to metabolic control. Details related to the manner by which EC cells respond to ingested nutrients are scarce and as that the nutrient environment changes along the length of the gut, it is unknown whether the response of EC cells to nutrients is dependent on their GI location. The aim of the present study was to identify whether regional differences in nutrient sensing capability exist in mouse EC cells. We isolated mouse EC cells from duodenum and colon to demonstrate differential responses to sugars depending on location. Measurements of intracellular calcium concentration and 5-HT secretion demonstrated that colonic EC cells are more sensitive to glucose, while duodenal EC cells are more sensitive to fructose and sucrose. Short-chain fatty acids (SCFAs), which are predominantly synthesized by intestinal bacteria, have been previously associated with an increase in circulating 5-HT; however, we find that SCFAs do not acutely stimulate EC cell 5-HT release. Thus, we highlight that EC cell physiology is dictated by regional location within the GI tract, and identify differences in the regional responsiveness of EC cells to dietary sugars.
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http://dx.doi.org/10.14814/phy2.13199DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5371566PMC
March 2017

Huntingtin-associated protein 1: Eutherian adaptation from a TRAK-like protein, conserved gene promoter elements, and localization in the human intestine.

BMC Evol Biol 2016 10 13;16(1):214. Epub 2016 Oct 13.

Centre for Neuroscience and Department of Human Physiology, Flinders University, Adelaide, South Australia, Australia.

Background: Huntingtin-associated Protein 1 (HAP1) is expressed in neurons and endocrine cells, and is critical for postnatal survival in mice. HAP1 shares a conserved "HAP1_N" domain with TRAfficking Kinesin proteins TRAK1 and TRAK2 (vertebrate), Milton (Drosophila) and T27A3.1 (C. elegans). HAP1, TRAK1 and TRAK2 have a degree of common function, particularly regarding intracellular receptor trafficking. However, TRAK1, TRAK2 and Milton (which have a "Milt/TRAK" domain that is absent in human and rodent HAP1) differ in function to HAP1 in that they are mitochondrial transport proteins, while HAP1 has emerging roles in starvation response. We have investigated HAP1 function by examining its evolution, and upstream gene promoter sequences. We performed phylogenetic analyses of the HAP1_N domain family of proteins, incorporating HAP1 orthologues (identified by genomic synteny) from 5 vertebrate classes, and also searched the Dictyostelium proteome for a common ancestor. Computational analyses of mammalian HAP1 gene promoters were performed to identify phylogenetically conserved regulatory motifs.

Results: We found that as recently as marsupials, HAP1 contained a Milt/TRAK domain and was more similar to TRAK1 and TRAK2 than to eutherian HAP1. The Milt/TRAK domain likely arose post multicellularity, as it was absent in the Dictyostelium proteome. It was lost from HAP1 in the eutherian lineage, and also from T27A3.1 in C. elegans. The HAP1 promoter from human, mouse, rat, rabbit, horse, dog, Tasmanian devil and opossum contained common sites for transcription factors involved in cell cycle, growth, differentiation, and stress response. A conserved arrangement of regulatory elements was identified, including sites for caudal-related homeobox transcription factors (CDX1 and CDX2), and myc-associated factor X (MAX) in the region of the TATA box. CDX1 and CDX2 are intestine-enriched factors, prompting investigation of HAP1 protein expression in the human duodenum. HAP1 was localized to singly dispersed mucosal cells, including a subset of serotonin-positive enterochromaffin cells.

Conclusion: We have identified eutherian HAP1 as an evolutionarily recent adaptation of a vertebrate TRAK protein-like ancestor, and found conserved CDX1/CDX2 and MAX transcription factor binding sites near the TATA box in mammalian HAP1 gene promoters. We also demonstrated that HAP1 is expressed in endocrine cells of the human gut.
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http://dx.doi.org/10.1186/s12862-016-0780-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5064798PMC
October 2016

Huntingtin-associated protein-1 is a synapsin I-binding protein regulating synaptic vesicle exocytosis and synapsin I trafficking.

J Neurochem 2016 09 18;138(5):710-21. Epub 2016 Jul 18.

Department of Human Physiology and Centre for Neuroscience, Flinders University, Adelaide, South Australia, Australia.

Huntingtin-associated protein-1 (HAP1) is involved in intracellular trafficking, vesicle transport, and membrane receptor endocytosis. However, despite such diverse functions, the role of HAP1 in the synaptic vesicle (SV) cycle in nerve terminals remains unclear. Here, we report that HAP1 functions in SV exocytosis, controls total SV turnover and the speed of vesicle fusion in nerve terminals and regulates glutamate release in cortical brain slices. We found that HAP1 interacts with synapsin I, an abundant neuronal phosphoprotein that associates with SVs during neurotransmitter release and regulates synaptic plasticity and neuronal development. The interaction between HAP1 with synapsin I was confirmed by reciprocal co-immunoprecipitation of the endogenous proteins. Furthermore, HAP1 co-localizes with synapsin I in cortical neurons as discrete puncta. Interestingly, we find that synapsin I localization is specifically altered in Hap1(-/-) cortical neurons without an effect on the localization of other SV proteins. This effect on synapsin I localization was not because of changes in the levels of synapsin I or its phosphorylation status in Hap1(-/-) brains. Furthermore, fluorescence recovery after photobleaching in transfected neurons expressing enhanced green fluorescent protein-synapsin Ia demonstrates that loss of HAP1 protein inhibits synapsin I transport. Thus, we demonstrate that HAP1 regulates SV exocytosis and may do so through binding to synapsin I. The Proposed mechanism of synapsin I transport mediated by HAP1 in neurons. HAP1 interacts with synapsin I, regulating the trafficking of synapsin I containing vesicles and/or transport packets, possibly through its engagement of microtubule motors. The absence of HAP1 reduces synapsin I transport and neuronal exocytosis. These findings provide insights into the processes of neuronal trafficking and synaptic signaling.
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http://dx.doi.org/10.1111/jnc.13703DOI Listing
September 2016

Serotonin-secreting enteroendocrine cells respond via diverse mechanisms to acute and chronic changes in glucose availability.

Nutr Metab (Lond) 2015 15;12:55. Epub 2015 Dec 15.

Department of Human Physiology and Centre for Neuroscience, Flinders University, Sturt Rd, Adelaide, SA 5042 Australia ; South Australian Health and Medical Research Institute (SAHMRI), Adelaide, 5001 Australia.

Background: Enteroendocrine cells collectively constitute our largest endocrine tissue, with serotonin (5-HT) secreting enterochromaffin (EC) cells being the largest component (~50 %). This gut-derived 5-HT has multiple paracrine and endocrine roles. EC cells are thought to act as nutrient sensors and luminal glucose is the major absorbed form of carbohydrate in the gut and activates secretion in an array of cell types. It is unknown whether EC cells release 5-HT in response to glucose in primary EC cells. Furthermore, fasting augments 5-HT synthesis and release into the circulation. However, which nutrients cause fasting-induced synthesis of EC cell 5-HT is unknown. Here we examine the effects of acute and chronic changes in glucose availability on 5-HT release from intact tissue and single EC cells.

Methods: We utilised established approaches in our laboratories measuring 5-HT release in intact mouse colon with amperometry. We then examined single EC cells function using our published protocol in guinea-pig colon. Single cell Ca(2+) imaging and amperometry were used with these cells. Real-time PCR was used along with amperometry, on primary EC cells cultured for 24 h in 5 or 25 mM glucose.

Results: We demonstrate that acute increases in glucose, at levels found in the gut lumen rather than in plasma, trigger 5-HT release from intact colon, and cause Ca(2+) entry and 5-HT release in primary EC cells. Single cell amperometry demonstrates that high glucose increases the amount of 5-HT released from individual vesicles as they undergo exocytosis. Finally, 24 h incubation of EC cells in low glucose causes an increase in the transcription of the 5-HT synthesising enzyme Tph1 as well as increasing in 5-HT secretion in EC cells.

Conclusions: We demonstrate that primary EC cells respond to acute changes in glucose availability through increases in intracellular Ca(2+) the activation of 5-HT secretion, but respond to chronic changes in glucose levels through the transcriptional regulation of Tph1 to alter 5-HT synthesis.
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http://dx.doi.org/10.1186/s12986-015-0051-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4678665PMC
December 2015

Fusion Pore Size Limits 5-HT Release From Single Enterochromaffin Cell Vesicles.

J Cell Physiol 2016 Jul 10;231(7):1593-600. Epub 2015 Dec 10.

Department of Human Physiology and Centre for Neuroscience, Flinders University, Adelaide, Australia.

Enterochromaffin cells are the major site of serotonin (5-HT) synthesis and secretion providing ∼95% of the body's total 5-HT. 5-HT can act as a neurotransmitter or hormone and has several important endocrine and paracrine roles. We have previously demonstrated that EC cells release small amounts of 5-HT per exocytosis event compared to other endocrine cells. We utilized a recently developed method to purify EC cells to demonstrate the mechanisms underlying 5-HT packaging and release. Using the fluorescent probe FFN511, we demonstrate that EC cells express VMAT and that VMAT plays a functional role in 5-HT loading into vesicles. Carbon fiber amperometry studies illustrate that the amount of 5-HT released per exocytosis event from EC cells is dependent on both VMAT and the H(+)-ATPase pump, as demonstrated with reserpine or bafilomycin, respectively. We also demonstrate that increasing the amount of 5-HT loaded into EC cell vesicles does not result in an increase in quantal release. As this indicates that fusion pore size may be a limiting factor involved, we compared pore diameter in EC and chromaffin cells by assessing the vesicle capture of different-sized fluorescent probes to measure the extent of fusion pore dilation. This identified that EC cells have a reduced fusion pore expansion that does not exceed 9 nm in diameter. These results demonstrate that the small amounts of 5-HT released per fusion event in EC cells can be explained by a smaller fusion pore that limits 5-HT release capacity from individual vesicles.
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http://dx.doi.org/10.1002/jcp.25256DOI Listing
July 2016

Gut Serotonin Is a Regulator of Obesity and Metabolism.

Gastroenterology 2015 Jul 27;149(1):253-5. Epub 2015 May 27.

South Australian Health and Medical Research Institute (SAHMRI), Adelaide, Australia; Centre for Neuroscience and Department of Human Physiology, Flinders University, Adelaide, Australia.

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http://dx.doi.org/10.1053/j.gastro.2015.05.020DOI Listing
July 2015

Deficiency of huntingtin has pleiotropic effects in the social amoeba Dictyostelium discoideum.

PLoS Genet 2011 Apr 28;7(4):e1002052. Epub 2011 Apr 28.

Molecular Neurogenetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Boston, Massachusetts, United States of America.

Huntingtin is a large HEAT repeat protein first identified in humans, where a polyglutamine tract expansion near the amino terminus causes a gain-of-function mechanism that leads to selective neuronal loss in Huntington's disease (HD). Genetic evidence in humans and knock-in mouse models suggests that this gain-of-function involves an increase or deregulation of some aspect of huntingtin's normal function(s), which remains poorly understood. As huntingtin shows evolutionary conservation, a powerful approach to discovering its normal biochemical role(s) is to study the effects caused by its deficiency in a model organism with a short life-cycle that comprises both cellular and multicellular developmental stages. To facilitate studies aimed at detailed knowledge of huntingtin's normal function(s), we generated a null mutant of hd, the HD ortholog in Dictyostelium discoideum. Dictyostelium cells lacking endogenous huntingtin were viable but during development did not exhibit the typical polarized morphology of Dictyostelium cells, streamed poorly to form aggregates by accretion rather than chemotaxis, showed disorganized F-actin staining, exhibited extreme sensitivity to hypoosmotic stress, and failed to form EDTA-resistant cell-cell contacts. Surprisingly, chemotactic streaming could be rescued in the presence of the bivalent cations Ca(2+) or Mg(2+) but not pulses of cAMP. Although hd(-) cells completed development, it was delayed and proceeded asynchronously, producing small fruiting bodies with round, defective spores that germinated spontaneously within a glassy sorus. When developed as chimeras with wild-type cells, hd(-) cells failed to populate the pre-spore region of the slug. In Dictyostelium, huntingtin deficiency is compatible with survival of the organism but renders cells sensitive to low osmolarity, which produces pleiotropic cell autonomous defects that affect cAMP signaling and as a consequence development. Thus, Dictyostelium provides a novel haploid organism model for genetic, cell biological, and biochemical studies to delineate the functions of the HD protein.
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http://dx.doi.org/10.1371/journal.pgen.1002052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3084204PMC
April 2011

Selective neuronal requirement for huntingtin in the developing zebrafish.

Hum Mol Genet 2009 Dec 29;18(24):4830-42. Epub 2009 Sep 29.

ARC Special Research Centre for the Molecular Genetics of Development and Discipline of Genetics, School of Molecular and Biomedical Sciences, The University of Adelaide, Adelaide, SA, Australia.

Huntington's disease shares a common molecular basis with eight other neurodegenerative diseases, expansion of an existing polyglutamine tract. In each case, this repeat tract occurs within otherwise unrelated proteins. These proteins show widespread and overlapping patterns of expression in the brain and yet the diseases are distinguished by neurodegeneration in a specific subset of neurons that are most sensitive to the mutation. It has therefore been proposed that expansion of the polyglutamine region in these genes may result in perturbation of the normal function of the respective proteins, and that this perturbation in some way contributes to the neuronal specificity of these diseases. The normal functions of these proteins have therefore become a focus for investigation as potential pathogenic pathways. We have used synthetic antisense morpholinos to inhibit the translation of huntingtin mRNA during early zebrafish development and have previously reported the effects of huntingtin reduction on iron transport and homeostasis. Here we report an analysis of the effects of huntingtin loss-of-function on the developing nervous system, observing distinct defects in morphology of neuromasts, olfactory placode and branchial arches. The potential common origins of these defects were explored, revealing impaired formation of the anterior-most region of the neural plate as indicated by reduced pre-placodal and telencephalic gene expression with no effect on mid- or hindbrain formation. These investigations demonstrate a specific 'rate-limiting' role for huntingtin in formation of the telencephalon and the pre-placodal region, and differing levels of requirement for huntingtin function in specific nerve cell types.
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http://dx.doi.org/10.1093/hmg/ddp455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2778375PMC
December 2009

Huntingtin-deficient zebrafish exhibit defects in iron utilization and development.

Hum Mol Genet 2007 Aug 13;16(16):1905-20. Epub 2007 Jun 13.

ARC Special Research Centre for the Molecular Genetics of Development and Discipline of Genetics, School of Molecular and Biomedical Science, The University of Adelaide, Adelaide, SA, Australia.

Huntington's disease (HD) is one of nine neurodegenerative disorders caused by expansion of CAG repeats encoding polyglutamine in their respective, otherwise apparently unrelated proteins. Despite these proteins having widespread and overlapping expression patterns in the brain, a specific and unique subset of neurons exhibits particular vulnerability in each disease. It has been hypothesized that perturbation of normal protein function contributes to the specificity of neuronal vulnerability; however, the normal biological functions of many of these proteins including the HD gene product, Huntingtin (Htt), are unclear. To explore the roles of Htt, we have used antisense morpholino oligonucleotides to observe the effects of Htt deficiency in early zebrafish development. Knockdown of Htt expression resulted in a variety of developmental defects. Most notably, Htt-deficient zebrafish had hypochromic blood due to decreased hemoglobin production, despite the presence of iron within blood cells. Furthermore, transferrin receptor 1 transcripts were increased, suggesting cellular iron starvation. Provision of iron to the cytoplasm in a bio-available form restored hemoglobin production in Htt-deficient embryos. Since erythroid cells acquire iron via receptor-mediated endocytosis of transferrin, these results suggest a role for Htt in making endocytosed iron accessible for cellular utilization. Iron is required for oxidative energy production, and defects in iron homeostasis and energy metabolism are features of HD pathogenesis that are most pronounced in the major region of neurodegeneration. It is therefore plausible that perturbation of Htt's normal role in the iron pathway (by polyglutamine tract expansion) contributes to HD pathology, and particularly to its neuronal specificity.
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http://dx.doi.org/10.1093/hmg/ddm138DOI Listing
August 2007

Molecular genetics of pseudoxanthoma elasticum: type and frequency of mutations in ABCC6.

Hum Mutat 2005 Sep;26(3):235-48

Charité, Franz Volhard Clinic, HELIOS Klinikum, Humboldt University, Berlin, Germany.

Pseudoxanthoma elasticum (PXE) is a systemic heritable disorder that affects the elastic tissue in the skin, eye, and cardiovascular system. Mutations in the ABCC6 gene cause PXE. We performed a mutation screen in ABCC6 using haplotype analysis in conjunction with direct sequencing to achieve a mutation detection rate of 97%. This screen consisted of 170 PXE chromosomes in 81 families, and detected 59 distinct mutations (32 missense, eight nonsense, and six likely splice-site point mutations; one small insertion; and seven small and five large deletions). Forty-three of these mutations are novel variants, which increases the total number of PXE mutations to 121. While most mutations are rare, three nonsense mutations, a splice donor site mutation, and the large deletion comprising exons 23-29 (c.2996_4208del) were identified as relatively frequent PXE mutations at 26%, 5%, 3.5%, 3%, and 11%, respectively. Chromosomal haplotyping with two proximal and two distal polymorphic markers flanking ABCC6 demonstrated that most chromosomes that carry these relatively frequent PXE mutations have related haplotypes specific for these mutations, which suggests that these chromosomes originate from single founder mutations. The types of mutations found support loss-of-function as the molecular mechanism for the PXE phenotype. In 76 of the 81 families, the affected individuals were either homozygous for the same mutation or compound heterozygous for two mutations. In the remaining five families with one uncovered mutation, affected showed allelic compound heterozygosity for the cosegregating PXE haplotype. This demonstrates pseudo-dominance as the relevant inheritance mechanism, since disease transmission to the next generation always requires one mutant allelic variant from each parent. In contrast to other previous clinical and molecular claims, our results show evidence only for recessive PXE. This has profound consequences for the genetic counseling of families with PXE.
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http://dx.doi.org/10.1002/humu.20206DOI Listing
September 2005