Anatomy and Neuroscience - Research Publications
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ItemDiacetylbis(N(4)-methylthiosemicarbazonato) Copper(II) (CuII(atsm)) Protects against Peroxynitrite-induced Nitrosative Damage and Prolongs Survival in Amyotrophic Lateral Sclerosis Mouse Model(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2011-12-23)Amyotrophic lateral sclerosis (ALS) is a progressive paralyzing disease characterized by tissue oxidative damage and motor neuron degeneration. This study investigated the in vivo effect of diacetylbis(N(4)-methylthiosemicarbazonato) copper(II) (CuII(atsm)), which is an orally bioavailable, blood-brain barrier-permeable complex. In vitro the compound inhibits the action of peroxynitrite on Cu,Zn-superoxide dismutase (SOD1) and subsequent nitration of cellular proteins. Oral treatment of transgenic SOD1G93A mice with CuII(atsm) at presymptomatic and symptomatic ages was performed. The mice were examined for improvement in lifespan and motor function, as well as histological and biochemical changes to key disease markers. Systemic treatment of SOD1G93A mice significantly delayed onset of paralysis and prolonged lifespan, even when administered to symptomatic animals. Consistent with the properties of this compound, treated mice had reduced protein nitration and carbonylation, as well as increased antioxidant activity in spinal cord. Treatment also significantly preserved motor neurons and attenuated astrocyte and microglial activation in mice. Furthermore, CuII(atsm) prevented the accumulation of abnormally phosphorylated and fragmented TAR DNA-binding protein-43 (TDP-43) in spinal cord, a protein pivotal to the development of ALS. CuII(atsm) therefore represents a potential new class of neuroprotective agents targeting multiple major disease pathways of motor neurons with therapeutic potential for ALS.
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ItemEarly Development of Electrical Excitability in the Mouse Enteric Nervous System(SOC NEUROSCIENCE, 2012-08-08)Neural activity is integral to the development of the enteric nervous system (ENS). A subpopulation of neural crest-derived cells expresses pan-neuronal markers at early stages of ENS development (at E10.5 in the mouse). However, the electrical activity of these cells has not been previously characterized, and it is not known whether all cells expressing neuronal markers are capable of firing action potentials (APs). In this study, we examined the activity of "neuron"-like cells (expressing pan-neuronal markers or with neuronal morphology) in the gut of E11.5 and E12.5 mice using whole-cell patch-clamp electrophysiology and compared them to the activity of neonatal and adult enteric neurons. Around 30-40% of neuron-like cells at E11.5 and E12.5 fired APs, some of which were very similar to those of adult enteric neurons. All APs were sensitive to tetrodotoxin (TTX), indicating that they were driven by voltage-gated Na+ currents. Expression of mRNA encoding several voltage-gated Na+ channels by the E11.5 gut was detected using RT-PCR. The density of voltage-gated Na+ currents increased from E11.5 to neonates. Immature active responses, mediated in part by TTX- and lidocaine-insensitive channels, were observed in most cells at E11.5 and E12.5, but not in P0/P1 or adult neurons. However, some cells expressing neuronal markers at E11.5 or E12.5 did not exhibit an active response to depolarization. Spontaneous depolarizations resembling excitatory postsynaptic potentials were observed at E12.5. The ENS is one of the earliest parts of the developing nervous system to exhibit mature forms of electrical activity.
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ItemN-Glycosylation Determines Ionic Permeability and Desensitization of the TRPV1 Capsaicin Receptor(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2012-06-22)The balance of glycosylation and deglycosylation of ion channels can markedly influence their function and regulation. However, the functional importance of glycosylation of the TRPV1 receptor, a key sensor of pain-sensing nerves, is not well understood, and whether TRPV1 is glycosylated in neurons is unclear. We report that TRPV1 is N-glycosylated and that N-glycosylation is a major determinant of capsaicin-evoked desensitization and ionic permeability. Both N-glycosylated and unglycosylated TRPV1 was detected in extracts of peripheral sensory nerves by Western blotting. TRPV1 expressed in HEK-293 cells exhibited various degrees of glycosylation. A mutant of asparagine 604 (N604T) was not glycosylated but did not alter plasma membrane expression of TRPV1. Capsaicin-evoked increases in intracellular calcium ([Ca(2+)](i)) were sustained in wild-type TRPV1 HEK-293 cells but were rapidly desensitized in N604T TRPV1 cells. There was marked cell-to-cell variability in capsaicin responses and desensitization between individual cells expressing wild-type TRPV1 but highly uniform responses in cells expressing N604T TRPV1, consistent with variable levels of glycosylation of the wild-type channel. These differences were also apparent when wild-type or N604T TRPV1-GFP fusion proteins were expressed in neurons from trpv1(-/-) mice. Capsaicin evoked a marked, concentration-dependent increase in uptake of the large cationic dye YO-PRO-1 in cells expressing wild-type TRPV1, indicative of loss of ion selectivity, that was completely absent in cells expressing N604T TRPV1. Thus, TRPV1 is variably N-glycosylated and glycosylation is a key determinant of capsaicin regulation of TRPV1 desensitization and permeability. Our findings suggest that physiological or pathological alterations in TRPV1 glycosylation would affect TRPV1 function and pain transmission.
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ItemCellular Up-regulation of Nedd4 Family Interacting Protein 1 (Ndfip1) using Low Levels of Bioactive Cobalt Complexes(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2011-03-11)The delivery of metal ions using cell membrane-permeable metal complexes represents a method for activating cellular pathways. Here, we report the synthesis and characterization of new [Co(III)(salen)(acac)] complexes capable of up-regulating the ubiquitin ligase adaptor protein Ndfip1. Ndfip1 is a neuroprotective protein that is up-regulated in the brain after injury and functions in combination with Nedd4 ligases to ubiquitinate harmful proteins for removal. We previously showed that Ndfip1 can be increased in human neurons using CoCl(2) that is toxic at high concentration. Here we demonstrate a similar effect can be achieved by low concentrations of synthetic Co(III) complexes that are non-toxic and designed to be activated following cellular entry. Activation is achieved by intracellular reduction of Co(III) to Co(II) leading to release of Co(II) ions for Ndfip1 up-regulation. The cellular benefit of Ndfip1 up-regulation by Co(III) complexes includes demonstrable protection against cell death in SH-SY5Y cells during stress. In vivo, focal delivery of Co(III) complexes into the adult mouse brain was observed to up-regulate Ndfip1 in neurons. These results demonstrate that a cellular response pathway can be advantageously manipulated by chemical modification of metal complexes, and represents a significant step of harnessing low concentration metal complexes for therapeutic benefit.
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ItemGenome-wide discovery of human splicing branchpoints(COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT, 2015-02)During the splicing reaction, the 5' intron end is joined to the branchpoint nucleotide, selecting the next exon to incorporate into the mature RNA and forming an intron lariat, which is excised. Despite a critical role in gene splicing, the locations and features of human splicing branchpoints are largely unknown. We use exoribonuclease digestion and targeted RNA-sequencing to enrich for sequences that traverse the lariat junction and, by split and inverted alignment, reveal the branchpoint. We identify 59,359 high-confidence human branchpoints in >10,000 genes, providing a first map of splicing branchpoints in the human genome. Branchpoints are predominantly adenosine, highly conserved, and closely distributed to the 3' splice site. Analysis of human branchpoints reveals numerous novel features, including distinct features of branchpoints for alternatively spliced exons and a family of conserved sequence motifs overlapping branchpoints we term B-boxes, which exhibit maximal nucleotide diversity while maintaining interactions with the keto-rich U2 snRNA. Different B-box motifs exhibit divergent usage in vertebrate lineages and associate with other splicing elements and distinct intron-exon architectures, suggesting integration within a broader regulatory splicing code. Lastly, although branchpoints are refractory to common mutational processes and genetic variation, mutations occurring at branchpoint nucleotides are enriched for disease associations.
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ItemImproved definition of the mouse transcriptome via targeted RNA sequencing(COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT, 2016-05)Targeted RNA sequencing (CaptureSeq) uses oligonucleotide probes to capture RNAs for sequencing, providing enriched read coverage, accurate measurement of gene expression, and quantitative expression data. We applied CaptureSeq to refine transcript annotations in the current murine GRCm38 assembly. More than 23,000 regions corresponding to putative or annotated long noncoding RNAs (lncRNAs) and 154,281 known splicing junction sites were selected for targeted sequencing across five mouse tissues and three brain subregions. The results illustrate that the mouse transcriptome is considerably more complex than previously thought. We assemble more complete transcript isoforms than GENCODE, expand transcript boundaries, and connect interspersed islands of mapped reads. We describe a novel filtering pipeline that identifies previously unannotated but high-quality transcript isoforms. In this set, 911 GENCODE neighboring genes are condensed into 400 expanded gene models. Additionally, 594 GENCODE lncRNAs acquire an open reading frame (ORF) when their structure is extended with CaptureSeq. Finally, we validate our observations using current FANTOM and Mouse ENCODE resources.
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ItemElectrophysiological characterization of spontaneous recovery in deep dorsal horn interneurons after incomplete spinal cord injury(ACADEMIC PRESS INC ELSEVIER SCIENCE, 2015-09)In the weeks and months following an incomplete spinal cord injury (SCI) significant spontaneous recovery of function occurs in the absence of any applied therapeutic intervention. The anatomical correlates of this spontaneous plasticity are well characterized, however, the functional changes that occur in spinal cord interneurons after injury are poorly understood. Here we use a T10 hemisection model of SCI in adult mice (9-10 wks old) combined with whole-cell patch clamp electrophysiology and a horizontal spinal cord slice preparation to examine changes in intrinsic membrane and synaptic properties of deep dorsal horn (DDH) interneurons. We made these measurements during short-term (4 wks) and long-term (10 wks) spontaneous recovery after SCI. Several important intrinsic membrane properties are altered in the short-term, but recover to values resembling those of uninjured controls in the longer term. AP discharge patterns are reorganized at both short-term and long-term recovery time points. This is matched by reorganization in the expression of voltage-activated potassium and calcium subthreshold-currents that shape AP discharge. Excitatory synaptic inputs onto DDH interneurons are significantly restructured in long-term SCI mice. Plots of sEPSC peak amplitude vs. rise times suggest considerable dendritic expansion or synaptic reorganization occurs especially during long-term recovery from SCI. Connectivity between descending dorsal column pathways and DDH interneurons is reduced in the short-term, but amplified in long-term recovery. Our results suggest considerable plasticity in both intrinsic and synaptic mechanisms occurs spontaneously in DDH interneurons following SCI and takes a minimum of 10 wks after the initial injury to stabilize.
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ItemDeveloping a spinal cord injury research strategy using a structured process of evidence review and stakeholder dialogue. Part III: outcomes(NATURE PUBLISHING GROUP, 2015-10)STUDY DESIGN: Focus Group. OBJECTIVES: To develop a unified, regional spinal cord injury (SCI) research strategy for Australia and New Zealand. SETTING: Australia. METHODS: A 1-day structured stakeholder dialogue was convened in 2013 in Melbourne, Australia, by the National Trauma Research Institute in collaboration with the SCI Network of Australia and New Zealand. Twenty-three experts participated, representing local and international research, clinical, consumer, advocacy, government policy and funding perspectives. Preparatory work synthesised evidence and articulated draft principles and options as a starting point for discussion. RESULTS: A regional SCI research strategy was proposed, whose objectives can be summarised under four themes. (1) Collaborative networks and strategic partnerships to increase efficiency, reduce duplication, build capacity and optimise research funding. (2) Research priority setting and coordination to manage competing studies. (3) Mechanisms for greater consumer engagement in research. (4) Resources and infrastructure to further develop SCI data registries, evaluate research translation and assess alignment of research strategy with stakeholder interests. These are consistent with contemporary international SCI research strategy development activities. CONCLUSION: This first step in a regional SCI research strategy has articulated objectives for further development by the wider SCI research community. The initiative has also reinforced the importance of coordinated, collective action in optimising outcomes following SCI.
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ItemGait recovery following spinal cord injury in mice: Limited effect of treadmill training(TAYLOR & FRANCIS LTD, 2016)BACKGROUND: Several studies in rodents with complete spinal cord transections have demonstrated that treadmill training improves stepping movements. However, results from studies in incomplete spinal cord injured animals have been conflicting and questions regarding the training dosage after injury remain unresolved. OBJECTIVES: To assess the effects of treadmill-training regimen (20 minutes daily, 5 days a week) for 3, 6 or 9 weeks on the recovery of locomotion in hemisected SCI mice. METHODS: A randomized and blinded controlled experimental trial used a mouse model of incomplete spinal cord injury (SCI). After a left hemisection at T10, adult male mice were randomized to trained or untrained groups. The trained group commenced treadmill training one week after surgery and continued for 3, 6 or 9 weeks. Quantitative kinematic gait analysis was used to assess the spatiotemporal characteristics of the left hindlimb prior to injury and at 1, 4, 7 and 10 weeks post-injury. RESULTS: One week after injury there was no movement of the left hindlimb and some animals dragged their foot. Treadmill training led to significant improvements in step duration, but had limited effect on the hindlimb movement pattern. Locomotor improvements in trained animals were most evident at the hip and knee joints whereas recovery of ankle movement was limited, even after 9 weeks of treadmill training. CONCLUSION: These results demonstrate that treadmill training may lead to only modest improvement in recovery of hindlimb movement after incomplete spinal cord injury in mice.
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ItemClaustrum projections to prefrontal cortex in the capuchin monkey (Cebus apella)(FRONTIERS MEDIA SA, 2014)We examined the pattern of retrograde tracer distribution in the claustrum following intracortical injections into the frontal pole (area 10), and in dorsal (area 9), and ventral lateral (area 12) regions of the rostral prefrontal cortex in the tufted capuchin monkey (Cebus apella). The resulting pattern of labeled cells was assessed in relation to the three-dimensional geometry of the claustrum, as well as recent reports of claustrum-prefrontal connections in other primates. Claustrum-prefrontal projections were extensive, and largely concentrated in the ventral half of the claustrum, especially in the rostral 2/3 of the nucleus. Our data are consistent with a topographic arrangement of claustrum-cortical connections in which prefrontal and association cortices receive connections largely from the rostral and medial claustrum. Comparative aspects of claustrum-prefrontal topography across primate species and the implications of claustrum connectivity for understanding of cortical functional networks are explored, and we hypothesize that the claustrum may play a role in controlling or switching between resting state and task-associated cortical networks.