Anatomy and Neuroscience - Research Publications

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    Dysferlin deficiency alters lipid metabolism and remodels the skeletal muscle lipidome in mice[S]
    Haynes, VR ; Keenan, SN ; Bayliss, J ; Lloyd, EM ; Meikle, P ; Grounds, MD ; Watt, MJ (ELSEVIER, 2019-08)
    Defects in the gene coding for dysferlin, a membrane-associated protein, affect many tissues, including skeletal muscles, with a resultant myopathy called dysferlinopathy. Dysferlinopathy manifests postgrowth with a progressive loss of skeletal muscle function, early intramyocellular lipid accumulation, and a striking later replacement of selective muscles by adipocytes. To better understand the changes underpinning this disease, we assessed whole-body energy homeostasis, skeletal muscle fatty acid metabolism, lipolysis in adipose tissue, and the skeletal muscle lipidome using young adult dysferlin-deficient male BLAJ mice and age-matched C57Bl/6J WT mice. BLAJ mice had increased lean mass and reduced fat mass associated with increased physical activity and increased adipose tissue lipolysis. Skeletal muscle fatty acid metabolism was remodeled in BLAJ mice, characterized by a partitioning of fatty acids toward storage rather than oxidation. Lipidomic analysis identified marked changes in almost all lipid classes examined in the skeletal muscle of BLAJ mice, including sphingolipids, phospholipids, cholesterol, and most glycerolipids but, surprisingly, not triacylglycerol. These observations indicate that an early manifestation of dysferlin deficiency is the reprogramming of skeletal muscle and adipose tissue lipid metabolism, which is likely to contribute to the progressive adverse histopathology in dysferlinopathies.
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    Protein kinase D and Gβγ mediate sustained nociceptive signaling by biased agonists of protease-activated receptor-2
    Zhao, P ; Pattison, LA ; Jensen, DD ; Jimenez-Vargas, NN ; Latorre, R ; Lieu, T ; Jaramillo, JO ; Lopez-Lopez, C ; Poole, DP ; Vanner, SJ ; Schmidt, BL ; Bunnett, NW (AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2019-07-05)
    Proteases sustain hyperexcitability and pain by cleaving protease-activated receptor-2 (PAR2) on nociceptors through distinct mechanisms. Whereas trypsin induces PAR2 coupling to Gαq, Gαs, and β-arrestins, cathepsin-S (CS) and neutrophil elastase (NE) cleave PAR2 at distinct sites and activate it by biased mechanisms that induce coupling to Gαs, but not to Gαq or β-arrestins. Because proteases activate PAR2 by irreversible cleavage, and activated PAR2 is degraded in lysosomes, sustained extracellular protease-mediated signaling requires mobilization of intact PAR2 from the Golgi apparatus or de novo synthesis of new receptors by incompletely understood mechanisms. We found here that trypsin, CS, and NE stimulate PAR2-dependent activation of protein kinase D (PKD) in the Golgi of HEK293 cells, in which PKD regulates protein trafficking. The proteases stimulated translocation of the PKD activator Gβγ to the Golgi, coinciding with PAR2 mobilization from the Golgi. Proteases also induced translocation of a photoconverted PAR2-Kaede fusion protein from the Golgi to the plasma membrane of KNRK cells. After incubation of HEK293 cells and dorsal root ganglia neurons with CS, NE, or trypsin, PAR2 responsiveness initially declined, consistent with PAR2 cleavage and desensitization, and then gradually recovered. Inhibitors of PKD, Gβγ, and protein translation inhibited recovery of PAR2 responsiveness. PKD and Gβγ inhibitors also attenuated protease-evoked mechanical allodynia in mice. We conclude that proteases that activate PAR2 by canonical and biased mechanisms stimulate PKD in the Golgi; PAR2 mobilization and de novo synthesis repopulate the cell surface with intact receptors and sustain nociceptive signaling by extracellular proteases.
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    The Ubiquitin Ligase Adaptor NDFIP1 Selectively Enforces a CD8+ T Cell Tolerance Checkpoint to High-Dose Antigen
    Wagle, M ; Marchingo, JM ; Howitt, J ; Tan, S-S ; Goodnow, CC ; Parish, IA (CELL PRESS, 2018-07-17)
    Escape from peripheral tolerance checkpoints that control cytotoxic CD8+ T cells is important for cancer immunotherapy and autoimmunity, but pathways enforcing these checkpoints are mostly uncharted. We reveal that the HECT-type ubiquitin ligase activator, NDFIP1, enforces a cell-intrinsic CD8+ T cell checkpoint that desensitizes TCR signaling during in vivo exposure to high antigen levels. Ndfip1-deficient OT-I CD8+ T cells responding to high exogenous tolerogenic antigen doses that normally induce anergy aberrantly expanded and differentiated into effector cells that could precipitate autoimmune diabetes in RIP-OVAhi mice. In contrast, NDFIP1 was dispensable for peripheral deletion to low-dose exogenous or pancreatic islet-derived antigen and had little impact upon effector responses to Listeria or acute LCMV infection. These data provide evidence that NDFIP1 mediates a CD8+ T cell tolerance checkpoint, with a different mechanism to CD4+ T cells, and indicates that CD8+ T cell deletion and anergy are molecularly separable checkpoints.
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    Mitochondrial dysfunction-related lipid changes occur in nonalcoholic fatty liver disease progression
    Peng, K-Y ; Watt, MJ ; Rensen, S ; Greve, JW ; Huynh, K ; Jayawardana, KS ; Meikle, PJ ; Meex, RCR (ELSEVIER, 2018-10)
    Nonalcoholic fatty liver disease (NAFLD) comprises fat-accumulating conditions within hepatocytes that can cause severe liver damage and metabolic comorbidities. Studies suggest that mitochondrial dysfunction contributes to its development and progression and that the hepatic lipidome changes extensively in obesity and in NAFLD. To gain insight into the relationship between lipid metabolism and disease progression through different stages of NAFLD, we performed lipidomic analysis of plasma and liver biopsy samples from obese patients with nonalcoholic fatty liver (NAFL) or nonalcoholic steatohepatitis (NASH) and from those without NAFLD. Congruent with earlier studies, hepatic lipid levels overall increased with NAFLD. Lipid species that differed with NAFLD severity were related to mitochondrial dysfunction; specifically, hepatic cardiolipin and ubiquinone accumulated in NAFL, and levels of acylcarnitine increased with NASH. We propose that increased levels of cardiolipin and ubiquinone may help to preserve mitochondrial function in early NAFLD, but that mitochondrial function eventually fails with progression to NASH, leading to increased acylcarnitine. We also found a negative association between hepatic odd-chain phosphatidylcholine and NAFLD, which may result from mitochondrial dysfunction-related impairment of branched-chain amino acid catabolism. Overall, these data suggest a close link between accumulation of specific hepatic lipid species, mitochondrial dysfunction, and the progression of NAFLD.
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    Kidney organoids: accurate models or fortunate accidents
    Little, MH ; Combes, AN (COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT, 2019-10-01)
    There are now many reports of human kidney organoids generated via the directed differentiation of human pluripotent stem cells (PSCs) based on an existing understanding of mammalian kidney organogenesis. Such kidney organoids potentially represent tractable tools for the study of normal human development and disease with improvements in scale, structure, and functional maturation potentially providing future options for renal regeneration. The utility of such organotypic models, however, will ultimately be determined by their developmental accuracy. While initially inferred from mouse models, recent transcriptional analyses of human fetal kidney have provided greater insight into nephrogenesis. In this review, we discuss how well human kidney organoids model the human fetal kidney and how the remaining differences challenge their utility.
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    Is more always better? How different 'doses' of exercise after incomplete spinal cord injury affects the membrane properties of deep dorsal horn interneurons
    Rank, MM ; Galea, MP ; Callister, R ; Callister, RJ (ACADEMIC PRESS INC ELSEVIER SCIENCE, 2018-02)
    Interneurons in the deep dorsal horn (DDH) of the spinal cord process somatosensory input, and form an important link between upper and lower motoneurons to subsequently shape motor output. Exercise training after SCI is known to improve functional motor recovery, but little is known about the mechanisms within spinal cord neurons that underlie these improvements. Here we investigate how the properties of DDH interneurons are affected by spinal cord injury (SCI) alone, and SCI in combination with different 'doses' of treadmill exercise training (3, 6, and 9wks). In an adult mouse hemisection model of SCI we used whole-cell patch-clamp electrophysiology to record intrinsic, AP firing and gain modulation properties from DDH interneurons in a horizontal spinal cord slice preparation. We find that neurons within two segments of the injury, both ipsi- and contralateral to the hemisection, are similarly affected by SCI and SCI plus exercise. The passive intrinsic membrane properties input resistance (Rin) and rheobase are sensitive to the effects of recovery time and exercise training after SCI thus altering DDH interneuron excitability. Conversely, select active membrane properties are largely unaffected by either SCI or exercise training. SCI itself causes a mismatch in the expression of voltage-gated subthreshold currents and AP discharge firing type. Over time after SCI, and especially with exercise training (9wks), this mismatched expression is exacerbated. Lastly, amplification properties (i.e. gain of frequency-current relationship) of DDH interneurons are altered by SCI alone and recover spontaneously with no clear effect of exercise training. These results suggest a larger 'dose' of exercise training (9wks) has a strong and selective effect on specific membrane properties, and on the output of interneurons in the vicinity of a SCI. These electrophysiological data provide new insights into the plasticity of DDH interneurons and the mechanisms by which exercise therapy after SCI can improve recovery.
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    Unidirectional monosynaptic connections from auditory areas to the primary visual cortex in the marmoset monkey
    Majka, P ; Rosa, MGP ; Bai, S ; Chan, JM ; Huo, B-X ; Jermakow, N ; Lin, MK ; Takahashi, YS ; Wolkowicz, IH ; Worthy, KH ; Rajan, R ; Reser, DH ; Wojcik, DK ; Okano, H ; Mitra, PP (SPRINGER HEIDELBERG, 2019-01)
    Until the late twentieth century, it was believed that different sensory modalities were processed by largely independent pathways in the primate cortex, with cross-modal integration only occurring in specialized polysensory areas. This model was challenged by the finding that the peripheral representation of the primary visual cortex (V1) receives monosynaptic connections from areas of the auditory cortex in the macaque. However, auditory projections to V1 have not been reported in other primates. We investigated the existence of direct interconnections between V1 and auditory areas in the marmoset, a New World monkey. Labelled neurons in auditory cortex were observed following 4 out of 10 retrograde tracer injections involving V1. These projections to V1 originated in the caudal subdivisions of auditory cortex (primary auditory cortex, caudal belt and parabelt areas), and targeted parts of V1 that represent parafoveal and peripheral vision. Injections near the representation of the vertical meridian of the visual field labelled few or no cells in auditory cortex. We also placed 8 retrograde tracer injections involving core, belt and parabelt auditory areas, none of which revealed direct projections from V1. These results confirm the existence of a direct, nonreciprocal projection from auditory areas to V1 in a different primate species, which has evolved separately from the macaque for over 30 million years. The essential similarity of these observations between marmoset and macaque indicate that early-stage audiovisual integration is a shared characteristic of primate sensory processing.
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    Transcriptomic-Proteomic Correlation in the Predation-Evoked Venom of the Cone Snail, Conus imperialis
    Jin, A-H ; Dutertre, S ; Dutt, M ; Lavergne, V ; Jones, A ; Lewis, RJ ; Alewood, PF (MDPI, 2019-03)
    Individual variation in animal venom has been linked to geographical location, feeding habit, season, size, and gender. Uniquely, cone snails possess the remarkable ability to change venom composition in response to predatory or defensive stimuli. To date, correlations between the venom gland transcriptome and proteome within and between individual cone snails have not been reported. In this study, we use 454 pyrosequencing and mass spectrometry to decipher the transcriptomes and proteomes of the venom gland and corresponding predation-evoked venom of two specimens of Conus imperialis. Transcriptomic analyses revealed 17 conotoxin gene superfamilies common to both animals, including 5 novel superfamilies and two novel cysteine frameworks. While highly expressed transcripts were common to both specimens, variation of moderately and weakly expressed precursor sequences was surprisingly diverse, with one specimen expressing two unique gene superfamilies and consistently producing more paralogs within each conotoxin gene superfamily. Using a quantitative labelling method, conotoxin variability was compared quantitatively, with highly expressed peptides showing a strong correlation between transcription and translation, whereas peptides expressed at lower levels showed a poor correlation. These results suggest that major transcripts are subject to stabilizing selection, while minor transcripts are subject to diversifying selection.
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    The α1-adrenoceptor inhibitor ρ-TIA facilitates net hunting in piscivorous Conus tulipa
    Dutt, M ; Giacomotto, J ; Ragnarsson, L ; Andersson, A ; Brust, A ; Dekan, Z ; Alewood, PF ; Lewis, RJ (NATURE PUBLISHING GROUP, 2019-11-28)
    Cone snails use separately evolved venoms for prey capture and defence. While most use a harpoon for prey capture, the Gastridium clade that includes the well-studied Conus geographus and Conus tulipa, have developed a net hunting strategy to catch fish. This unique feeding behaviour requires secretion of "nirvana cabal" peptides to dampen the escape response of targeted fish allowing for their capture directly by mouth. However, the active components of the nirvana cabal remain poorly defined. In this study, we evaluated the behavioural effects of likely nirvana cabal peptides on the teleost model, Danio rerio (zebrafish). Surprisingly, the conantokins (NMDA receptor antagonists) and/or conopressins (vasopressin receptor agonists and antagonists) found in C. geographus and C. tulipa venom failed to produce a nirvana cabal-like effect in zebrafish. In contrast, low concentrations of the non-competitive adrenoceptor antagonist ρ-TIA found in C. tulipa venom (EC50 = 190 nM) dramatically reduced the escape response of zebrafish larvae when added directly to aquarium water. ρ-TIA inhibited the zebrafish α1-adrenoceptor, confirming ρ-TIA has the potential to reverse the known stimulating effects of norepinephrine on fish behaviour. ρ-TIA may act alone and not as part of a cabal, since it did not synergise with conopressins and/or conantokins. This study highlights the importance of using ecologically relevant animal behaviour models to decipher the complex neurobiology underlying the prey capture and defensive strategies of cone snails.
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    Venomics Reveals Venom Complexity of the Piscivorous Cone Snail, Conus tulipa
    Dutt, M ; Dutertre, S ; Jin, A-H ; Lavergne, V ; Alewood, PF ; Lewis, RJ (MDPI, 2019-01)
    The piscivorous cone snail Conus tulipa has evolved a net-hunting strategy, akin to the deadly Conus geographus, and is considered the second most dangerous cone snail to humans. Here, we present the first venomics study of C. tulipa venom using integrated transcriptomic and proteomic approaches. Parallel transcriptomic analysis of two C. tulipa specimens revealed striking differences in conopeptide expression levels (2.5-fold) between individuals, identifying 522 and 328 conotoxin precursors from 18 known gene superfamilies. Despite broad overlap at the superfamily level, only 86 precursors (11%) were common to both specimens. Conantokins (NMDA antagonists) from the superfamily B1 dominated the transcriptome and proteome of C. tulipa venom, along with superfamilies B2, A, O1, O3, con-ikot-ikot and conopressins, plus novel putative conotoxins precursors T1.3, T6.2, T6.3, T6.4 and T8.1. Thus, C. tulipa venom comprised both paralytic (putative ion channel modulating α-, ω-, μ-, δ-) and non-paralytic (conantokins, con-ikot-ikots, conopressins) conotoxins. This venomic study confirms the potential for non-paralytic conotoxins to contribute to the net-hunting strategy of C. tulipa.