Anatomy and Neuroscience - Research Publications
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ItemDysferlin deficiency alters lipid metabolism and remodels the skeletal muscle lipidome in mice[S](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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ItemProtein kinase D and Gβγ mediate sustained nociceptive signaling by biased agonists of protease-activated receptor-2(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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ItemAblation of C3 modulates macrophage reactivity in the outer retina during photo-oxidative damage(MOLECULAR VISION, 2020-10-10)PURPOSE: Dysregulation of the complement cascade contributes to a variety of retinal dystrophies, including age-related macular degeneration (AMD). The central component of complement, C3, is expressed in abundance by macrophages in the outer retina, and its ablation suppresses photoreceptor death in experimental photo-oxidative damage. Whether this also influences macrophage reactivity in this model system, however, is unknown. We investigate the effect of C3 ablation on macrophage activity and phagocytosis by outer retinal macrophages during photo-oxidative damage. METHODS: Age-matched C3 knockout (KO) mice and wild-type (WT) C57/Bl6 mice were subjected to photo-oxidative damage. Measurements of the outer nuclear layer (ONL) thickness and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were used to assess pathology and photoreceptor apoptosis, respectively. Macrophage abundance and phagocytosis were assessed with immunolabeling for pan-macrophage and phagocytic markers, in conjunction with TUNEL staining in cohorts of C3 KO and WT mice. RESULTS: The C3 KO mice exhibited protection against photoreceptor cell death following photo-oxidative damage, which was associated with a reduction in immunoreactivity for the stress-related factor GFAP. In conjunction, there was a reduction in IBA1-positive macrophages in the outer retina compared to the WT mice and a decrease in the number of CD68-positive cells in the outer nuclear layer and the subretinal space. In addition, the engulfment of TUNEL-positive and -negative photoreceptors by macrophages was significantly lower in the C3 KO mice cohort following photo-oxidative damage compared to the WT cohort. CONCLUSIONS: The results show that the absence of C3 mitigates the phagocytosis of photoreceptors by macrophages in the outer retina, and the net impact of C3 depletion is neuroprotective in the context of photo-oxidative damage. These data improve our understanding of the impact of C3 inhibition in subretinal inflammation and inform the development of treatments for targeting complement activation in diseases such as AMD.
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ItemKidney organoids: accurate models or fortunate accidents(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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ItemSqualamine Restores the Function of the Enteric Nervous System in Mouse Models of Parkinson's Disease(IOS Press, 2020-10-27)Background: Parkinson’s disease (PD) is a progressive neurodegenerative disorder thought to be caused by accumulation of α-synuclein (α-syn) within the brain, autonomic nerves, and the enteric nervous system (ENS). Involvement of the ENS in PD often precedes the onset of the classic motor signs of PD by many years at a time when severe constipation represents a major morbidity. Studies conducted in vitro and in vivo, have shown that squalamine, a zwitterionic amphipathic aminosterol, originally isolated from the liver of the dogfish shark, effectively displaces membrane-bound α-syn. Objective: Here we explore the electrophysiological effect of squalamine on the gastrointestinal (GI) tract of mouse models of PD engineered to express the highly aggregating A53T human α-syn mutant. Methods: GI motility and in vivo response to oral squalamine in PD model mice and controls were assessed using an in vitro tissue motility protocol and via fecal pellet output. Vagal afferent response to squalamine was measured using extracellular mesenteric nerve recordings from the jejunum. Whole cell patch clamp was performed to measure response to squalamine in the myenteric plexus. Results: Squalamine effectively restores disordered colonic motility in vivo and within minutes of local application to the bowel. We show that topical squalamine exposure to intrinsic primary afferent neurons (IPANs) of the ENS rapidly restores excitability. Conclusion: These observations may help to explain how squalamine may promote gut propulsive activity through local effects on IPANs in the ENS, and further support its possible utility in the treatment of constipation in patients with PD.
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ItemOpen access resource for cellular-resolution analyses of corticocortical connectivity in the marmoset monkey(NATURE PUBLISHING GROUP, 2020-02-28)Understanding the principles of neuronal connectivity requires tools for efficient quantification and visualization of large datasets. The primate cortex is particularly challenging due to its complex mosaic of areas, which in many cases lack clear boundaries. Here, we introduce a resource that allows exploration of results of 143 retrograde tracer injections in the marmoset neocortex. Data obtained in different animals are registered to a common stereotaxic space using an algorithm guided by expert delineation of histological borders, allowing accurate assignment of connections to areas despite interindividual variability. The resource incorporates tools for analyses relative to cytoarchitectural areas, including statistical properties such as the fraction of labeled neurons and the percentage of supragranular neurons. It also provides purely spatial (parcellation-free) data, based on the stereotaxic coordinates of 2 million labeled neurons. This resource helps bridge the gap between high-density cellular connectivity studies in rodents and imaging-based analyses of human brains.
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ItemUnidirectional monosynaptic connections from auditory areas to the primary visual cortex in the marmoset monkey(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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ItemTranscriptomic-Proteomic Correlation in the Predation-Evoked Venom of the Cone Snail, Conus imperialis(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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ItemVenomics Reveals Venom Complexity of the Piscivorous Cone Snail, Conus tulipa(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.
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ItemThe α1-adrenoceptor inhibitor ρ-TIA facilitates net hunting in piscivorous Conus tulipa(NATURE PORTFOLIO, 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.