Michael D. Duffield

A Syntenic Cross Species Aneuploidy Genetic Screen Links RCAN1 Expression to β-Cell Mitochondrial Dysfunction in Type 2 Diabetes

Type 2 diabetes (T2D) is a complex metabolic disease associated with obesity, insulin resistance and hypoinsulinemia due to pancreatic β-cell dysfunction. Reduced mitochondrial function is thought to be central to β-cell dysfunction. Mitochondrial dysfunction and reduced insulin secretion are also observed in β-cells of humans with the most common human genetic disorder, Down syndrome (DS, Trisomy 21). To identify regions of chromosome 21 that may be associated with perturbed glucose homeostasis we profiled the glycaemic status of different DS mouse models. The Ts65Dn and Dp16 DS mouse lines were hyperglycemic, while Tc1 and Ts1Rhr mice were not, providing us with a region of chromosome 21 containing genes that cause hyperglycemia. We then examined whether any of these genes were upregulated in a set of ~5,000 gene expression changes we had identified in a large gene expression analysis of human T2D β-cells. This approach produced a single gene, RCAN1, as a candidate gene linking hyperglycemia and functional changes in T2D β-cells. Further investigations demonstrated that RCAN1 methylation is reduced in human T2D islets at multiple sites, correlating with increased expression. RCAN1 protein expression was also increased in db/db mouse islets and in human and mouse islets exposed to high glucose. Mice overexpressing RCAN1 had reduced in vivo glucose-stimulated insulin secretion and their β-cells displayed mitochondrial dysfunction including hyperpolarised membrane potential, reduced oxidative phosphorylation and low ATP production. This lack of β-cell ATP had functional consequences by negatively affecting both glucose-stimulated membrane depolarisation and ATP-dependent insulin granule exocytosis. Thus, from amongst the myriad of gene expression changes occurring in T2D β-cells where we had little knowledge of which changes cause β-cell dysfunction, we applied a trisomy 21 screening approach which linked RCAN1 to β-cell mitochondrial dysfunction in T2D.

Identification of unique release kinetics of serotonin from guinea-pig and human enterochromaffin cells

•  Enterochromaffin (EC) cells are enteroendocrine cells that synthesise ∼95% of the bodys total serotonin (5‐HT). •  Although 5‐HT release from EC cells plays a number of important physiological roles, primary EC cells have not been studied at the single cell level. •  This study provides the first functional characterisation of single primary guinea‐pig and human EC cells. •  EC cells release 5‐HT from large dense core vesicles in a calcium‐dependent manner with kinetics surprisingly resembling release from synaptic vesicles. •  3D modelling indicates that the quantity of 5‐HT released per vesicle fusion event is physiologically relevant to GI tract function in terms of the concentrations needed to activate local 5‐HT receptors. •  These findings represent significant advances in our understanding of EC cell function and will be of broad interest to researchers in endocrine cell biology, gastroenterology, neuroscience, exocytosis and glucose control.

Identification of Different Types of Spinal Afferent Nerve Endings That Encode Noxious and Innocuous Stimuli in the Large Intestine Using a Novel Anterograde Tracing Technique

In mammals, sensory stimuli in visceral organs, including those that underlie pain perception, are detected by spinal afferent neurons, whose cell bodies lie in dorsal root ganglia (DRG). One of the major challenges in visceral organs has been how to identify the different types of nerve endings of spinal afferents that transduce sensory stimuli into action potentials. The reason why spinal afferent nerve endings have been so challenging to identify is because no techniques have been available, until now, that can selectively label only spinal afferents, in high resolution. We have utilized an anterograde tracing technique, recently developed in our laboratory, which facilitates selective labeling of only spinal afferent axons and their nerve endings in visceral organs. Mice were anesthetized, lumbosacral DRGs surgically exposed, then injected with dextran-amine. Seven days post-surgery, the large intestine was removed. The characteristics of thirteen types of spinal afferent nerve endings were identified in detail. The greatest proportion of nerve endings was in submucosa (32%), circular muscle (25%) and myenteric ganglia (22%). Two morphologically distinct classes innervated myenteric ganglia. These were most commonly a novel class of intraganglionic varicose endings (IGVEs) and occasionally rectal intraganglionic laminar endings (rIGLEs). Three distinct classes of varicose nerve endings were found to innervate the submucosa and circular muscle, while one class innervated internodal strands, blood vessels, crypts of lieberkuhn, the mucosa and the longitudinal muscle. Distinct populations of sensory endings were CGRP-positive. We present the first complete characterization of the different types of spinal afferent nerve endings in a mammalian visceral organ. The findings reveal an unexpectedly complex array of different types of primary afferent endings that innervate specific layers of the large intestine. Some of the novel classes of nerve endings identified must underlie the transduction of noxious and/or innocuous stimuli from the large intestine.

Huntingtin-associated protein 1 regulates exocytosis, vesicle docking, readily releasable pool size and fusion pore stability in mouse chromaffin cells

Huntingtin‐associated protein 1 (HAP1) is expressed in neurons and endocrine cells, in which it is thought to regulate vesicle trafficking. HAP1 is a binding partner of the Huntingtons disease (HD)‐causing protein huntingtin, and binding is stronger in HD. Whether HAP1 regulates a significant end‐point of vesicle transport, exocytosis, and what stage of exocytosis HAP1 may regulate, is unknown. We use mouse chromaffin cells to demonstrate that HAP1 regulates exocytosis via two potentially interlinked mechanisms: control of vesicle docking and the readily releasable vesicle pool, and regulation of fusion pore stabilization. These results establish HAP1 as a significant player in exocytosis control with potential relevance for HD and for a number of neuronal and homeostatic pathways.

Huntingtin-associated protein-1 (HAP1) regulates endocytosis and interacts with multiple trafficking-related proteins

Huntingtin-associated protein 1 (HAP1) was initially identified as a binding partner of huntingtin, mutations in which underlie Huntingtons disease. Subcellular localization and protein interaction data indicate that HAP1 may be important in vesicle trafficking, cell signalling and receptor internalization. In this study, a proteomics approach was used for the identification of novel HAP1-interacting partners to attempt to shed light on the physiological function of HAP1. Using affinity chromatography with HAP1-GST protein fragments bound to Sepharose columns, this study identified a number of trafficking-related proteins that bind to HAP1. Interestingly, many of the proteins that were identified by mass spectrometry have trafficking-related functions and include the clathrin light chain B and Sec23A, an ER to Golgi trafficking vesicle coat component. Using co-immunoprecipitation and GST-binding assays the association between HAP1 and clathrin light chain B has been validated in vitro. This study also finds that HAP1 co-localizes with clathrin light chain B. In line with a physiological function of the HAP1-clathrin interaction this study detected a dramatic reduction in vesicle retrieval and endocytosis in adrenal chromaffin cells. Furthermore, through examination of transferrin endocytosis in HAP1-/- cortical neurons, this study has determined that HAP1 regulates neuronal endocytosis. In this study, the interaction between HAP1 and Sec23A was also validated through endogenous co-immunoprecipitation in rat brain homogenate. Through the identification of novel HAP1 binding partners, many of which have putative trafficking roles, this study provides us with new insights into the mechanisms underlying the important physiological function of HAP1 as an intracellular trafficking protein through its protein-protein interactions.

Homozygous mutation of STXBP5L explains an autosomal recessive infantile-onset neurodegenerative disorder

We report siblings of consanguineous parents with an infantile-onset neurodegenerative disorder manifesting a predominant sensorimotor axonal neuropathy, optic atrophy and cognitive deficit. We used homozygosity mapping to identify an ∼12-Mbp interval identical by descent (IBD) between the affected individuals on chromosome 3q13.13-21.1 with an LOD score of 2.31. We combined family-based whole-exome and whole-genome sequencing of parents and affected siblings and, after filtering of likely non-pathogenic variants, identified a unique missense variant in syntaxin-binding protein 5-like (STXBP5L c.3127G>A, p.Val1043Ile [CCDS43137.1]) in the IBD interval. Considering other modes of inheritance, we also found compound heterozygous variants in FMNL3 (c.114G>C, p.Phe38Leu and c.1372T>G, p.Ile458Leu [CCDS44874.1]) located on chromosome 12. STXBP5L (or Tomosyn-2) is expressed in the central and peripheral nervous system and is known to inhibit neurotransmitter release through inhibition of the formation of the SNARE complexes between synaptic vesicles and the plasma membrane. FMNL3 is expressed more widely and is a formin family protein that is involved in the regulation of cell morphology and cytoskeletal organization. The STXBP5L p.Val1043Ile variant enhanced inhibition of exocytosis in comparison with wild-type (WT) STXBP5L. Furthermore, WT STXBP5L, but not variant STXBP5L, promoted axonal outgrowth in manipulated mouse primary hippocampal neurons. However, the FMNL3 p.Phe38Leu and p.Ile458Leu variants showed minimal effects in these cells. Collectively, our clinical, genetic and molecular data suggest that the IBD variant in STXBP5L is the likely cause of the disorder.

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

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.

Copper modulates the large dense core vesicle secretory pathway in PC12 cells

Copper (Cu) is an essential biometal involved in a number of cell functions. Abnormal Cu homeostasis has been identified as a major factor in a number of neurodegenerative disorders. However, little is known about how cells of brain origin maintain Cu homeostasis and in particular, how they respond to an elevated Cu environment. Understanding these processes is essential to obtaining a greater insight into the pathological changes in neurodegeneration and ageing. Although previous studies have shown that Cu in neurons can be associated with synaptic function, there is little understanding of how Cu modulates the regulated secretory vesicle pathways in these cells. In this study, we examined the effect of elevated intracellular Cu on proteins associated with the regulated secretory vesicle pathway in NGF-differentiated PC12 cells that exhibit neuronal-like properties. Increasing intracellular Cu with a cell-permeable Cu-complex (Cu(II)(gtsm)) resulted in increased expression of synaptophysin and robust translocation of this and additional vesicular proteins from synaptic-like microvesicle (SLMV) fractions to chromogranin-containing putative large dense core vesicle (LDCV) fractions in density gradient preparations. The LDCV fractions also contained substantially elevated Cu levels upon treatment of cells with Cu(II)(gtsm). Expression of the H(+) pump, V-ATPase, which is essential for vesicle maturation, was increased in Cu-treated cells while inhibition of V-ATPase prevented translocation of synaptophysin to LDCV fractions. Cu treatment was found to inhibit release of LDCVs in chromaffin cells due to reduced Ca(2+)-mediated vesicle exocytosis. Our findings demonstrate that elevated Cu can modulate LDCV metabolism potentially resulting in sequestration of Cu in this vesicle pool.

Carbon-Fiber Amperometry in the Study of Exocytosis

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A new ‘dual’ in the crown for electrochemistry

nephrine play vital roles within the central nervous system. Abnormal function of these neurotransmitter systems is implicated in a range of disease states, including depression, addiction, anxiety, schizophrenia and Parkinson’s disease (see Emilien et al. 1999; Marien et al. 2004; Beaulieu and Gainetdinov 2011). Measuring the release of catecholamines in the brain requires techniques with high spatial resolution as the regions which release these substances are typically small. One technique which has proven particularly effective in furthering our understanding of the role and regulation of catecholamines is fast-scan cyclic voltammetry (FSCV). Fast-scan cyclic voltammetry utilises the innate property of catecholamines to be oxidised at low potentials to enable measurement of catecholamine concentrations with high resolution both spatially and temporally. By continuously applying high frequency voltage ramps to a carbon fibre microelectrode located in a region of interest a current ‘signature’ is obtained, allowing identification of the catecholamines being released within the vicinity of the microelectrode tip (Stamford 1990). The microelectrode is calibrated such that the output provides a measurement of catecholamine concentration at each time-point. Whilst some catecholamines have similar oxidation fingerprints, the additional use of histology and pharmacological manipulation has enabled this technique to provide significant information on the control of individual catecholamines, in particular dopamine. In the current issue, Park et al. (2011) have taken the FSCV technique into new territory by simultaneously measuring the in vivo release of both dopamine (in the anterior nucleus accumbens), and norepinephrine (in the bed nucleus of the stria terminalis) in the anaesthetised rat. Simultaneous release of catecholamines in both of these regions was accomplished through stimulation in the ventral tegmental area and substantia nigra (Park et al. 2009). FSCV microelectrode arrays have been utilised previously to perform simultaneous catecholamine measurements (e.g. Zachek et al. 2010); however, this has been limited to recording release of a single catecholamine within the same brain region. The measurement of catecholamine release in different brain regions has previously only been performed in different animals at different times, limiting result comparability. The major advantage of simultaneously measuring dopamine and norepinephrine release in the same animal (Park et al. 2011) is that it enables a direct comparison of the mechanisms controlling the release of these neurotransmitters without the potentially confounding variables associated with measurements in different animals, such as the depth of anaesthesia. Using this technique, Park et al. (2011) directly demonstrate differences in the control of norepinephrine and dopamine release in response to the same stimulation, including: (i) Slower norepinephrine release and uptake in the bed nucleus of the stria terminalis compared with that of dopamine in the anterior nucleus accumbens, as well as reduced maximal norepinephrine concentration. (ii) The presence of dopamine, but not norepinephrine, transients following pharmacological stimulation (using receptor antagonists and transport inhibitors). (iii) Elevated basal dopamine and suppressed dopamine release in the presence of amphetamine andD2 receptor antagonist (neither of which was seen with norepinephrine). (iv) Greater sensitivity of dopamine release to stimulus pulse width, as well as to inhibition of synthesis (with