Mutations in PINK1 and Parkin result in autosomal recessive Parkinson's disease (PD). Cell cu... more Mutations in PINK1 and Parkin result in autosomal recessive Parkinson's disease (PD). Cell culture and <i>in vitro</i> studies have elaborated the PINK1-dependent regulation of Parkin and defined how this dyad orchestrates the elimination of damaged mitochondria via mitophagy. PINK1 phosphorylates ubiquitin at serine 65 (Ser65) and Parkin at an equivalent Ser65 residue located within its N-terminal ubiquitin-like domain, resulting in activation; however, the physiological significance of Parkin Ser65 phosphorylation <i>in vivo</i> in mammals remains unknown. To address this, we generated a <i>Parkin</i> Ser65Ala (S65A) knock-in mouse model. We observe endogenous Parkin Ser65 phosphorylation and activation in mature primary neurons following mitochondrial depolarization and reveal this is disrupted in <i>Parkin</i><sup>S65A/S65A</sup> neurons. Phenotypically, <i>Parkin</i><sup>S65A/S65A</sup> mice exhibit selective motor dysfunction in the absence of any overt neurodegeneration or alterations in nigrostriatal mitophagy. The clinical relevance of our findings is substantiated by the discovery of homozygous PARKIN (<i>PARK2</i>) p.S65N mutations in two unrelated patients with PD. Moreover, biochemical and structural analysis demonstrates that the Parkin<sup>S65N/S65N</sup> mutant is pathogenic and cannot be activated by PINK1. Our findings highlight the central role of Parkin Ser65 phosphorylation in health and disease.
Dynamic contacts between cells within the developing neuroepithelium are poorly understood but pl... more Dynamic contacts between cells within the developing neuroepithelium are poorly understood but play important roles in cell and tissue morphology and cell signalling. Here, using live-cell imaging and electron microscopy we reveal multiple protrusive structures in neuroepithelial apical endfeet of the chick embryonic spinal cord, including sub-apical protrusions that extend laterally within the tissue, and observe similar structures in human neuroepithelium. We characterise the dynamics, shape, and cytoskeleton of these lateral protrusions and distinguish these structures from cytonemes/filopodia and tunnelling nanotubes. We demonstrate that lateral protrusions form a latticework of membrane contacts between non-adjacent cells, depend on actin but not microtubule dynamics and provide a lamellipodial-like platform for further extending fine actin-dependent filipodia. We find that lateral protrusions depend on the actin-binding protein WAVE1: mutant-WAVE1 misexpression attenuated prot...
Background:Atopic eczema is an itchy inflammatory disorder characterised by skin barrier dysfunct... more Background:Atopic eczema is an itchy inflammatory disorder characterised by skin barrier dysfunction. Loss-of-function mutations in the gene encoding filaggrin (FLG) are a major risk factor, but the mechanisms by which filaggrin haploinsufficiency leads to atopic inflammation remain incompletely understood. Skin as an organ that can be modelled using primary cellsin vitroprovides the opportunity for selected genetic effects to be investigated in detail.Methods:Primary human keratinocytes and donor-matched primary fibroblasts from healthy individuals were used to create skin organoid models with and without siRNA-mediated knockdown ofFLG. Biological replicate sets of organoids were assessed using histological, functional and biochemical measurements.Results:FLGknockdown leads to subtle changes in histology and ultrastructure including a reduction in thickness of the stratum corneum and smaller, less numerous keratohyalin granules. Immature organoids showed some limited evidence of ba...
Mutations enhancing the kinase activity of LRRK2 cause Parkinson’s disease (PD) and therapies tha... more Mutations enhancing the kinase activity of LRRK2 cause Parkinson’s disease (PD) and therapies that reduce LRRK2 kinase activity are being tested in clinical trials. Numerous rare variants of unknown clinical significance have been reported, but how the vast majority impact on LRRK2 function is unknown. Here, we investigate 100 LRRK2 variants linked to PD, including previously described pathogenic mutations. We identify 23 LRRK2 variants that robustly stimulate kinase activity, including variants within the N-terminal non-catalytic regions [ARM (E334K, A419V), ANK(R767H), LRR (R1067Q, R1325Q)], as well as variants predicted to destabilise the ROC:CORB interface [ROC (A1442P, V1447M), CORA (R1628P) CORB (S1761R, L1795F)] and COR:COR dimer interface [CORB (R1728H/L)]. Most activating variants decrease LRRK2 biomarker site phosphorylation (pSer935/pSer955/pSer973), consistent with the notion that the active kinase conformation blocks their phosphorylation. We conclude that the impact of...
www.embojournal.org Structural insights into the regulation of PDK1 by phosphoinositides and inos... more www.embojournal.org Structural insights into the regulation of PDK1 by phosphoinositides and inositol phosphates
After 2 h at 37°C, migrated or input cells were recovered from the wells, quantitated by flow cyt... more After 2 h at 37°C, migrated or input cells were recovered from the wells, quantitated by flow cytometry, and expressed as ratios of migrated or input HeN/HeJ. (B) DC were pretreated with 100 ng/ml LPS for different lengths of time before addition into the Transwell insert. Note that time = 0* data were obtained from cells where LPS was added immediately before the cells were placed onto the filter. Subsequent migration was for 2 h in all cases. The line indicates the 1:1 ratio achieved where HeN and HeJ migrate with equal efficiency. (C) Data from experiments on three or four independent DC cultures are shown. Bars represent mean data. *, P < 0.05.<b>Copyright information:</b>Taken from "TLR ligand–induced podosome disassembly in dendritic cells is ADAM17 dependent"The Journal of Cell Biology 2008;182(5):993-1005.Published online 8 Sep 2008PMCID:PMC2528573.
Mutations in PINK1 and Parkin result in autosomal recessive Parkinson's disease (PD). Cell cu... more Mutations in PINK1 and Parkin result in autosomal recessive Parkinson's disease (PD). Cell culture and <i>in vitro</i> studies have elaborated the PINK1-dependent regulation of Parkin and defined how this dyad orchestrates the elimination of damaged mitochondria via mitophagy. PINK1 phosphorylates ubiquitin at serine 65 (Ser65) and Parkin at an equivalent Ser65 residue located within its N-terminal ubiquitin-like domain, resulting in activation; however, the physiological significance of Parkin Ser65 phosphorylation <i>in vivo</i> in mammals remains unknown. To address this, we generated a <i>Parkin</i> Ser65Ala (S65A) knock-in mouse model. We observe endogenous Parkin Ser65 phosphorylation and activation in mature primary neurons following mitochondrial depolarization and reveal this is disrupted in <i>Parkin</i><sup>S65A/S65A</sup> neurons. Phenotypically, <i>Parkin</i><sup>S65A/S65A</sup> mice exhibit selective motor dysfunction in the absence of any overt neurodegeneration or alterations in nigrostriatal mitophagy. The clinical relevance of our findings is substantiated by the discovery of homozygous PARKIN (<i>PARK2</i>) p.S65N mutations in two unrelated patients with PD. Moreover, biochemical and structural analysis demonstrates that the Parkin<sup>S65N/S65N</sup> mutant is pathogenic and cannot be activated by PINK1. Our findings highlight the central role of Parkin Ser65 phosphorylation in health and disease.
Dynamic contacts between cells within the developing neuroepithelium are poorly understood but pl... more Dynamic contacts between cells within the developing neuroepithelium are poorly understood but play important roles in cell and tissue morphology and cell signalling. Here, using live-cell imaging and electron microscopy we reveal multiple protrusive structures in neuroepithelial apical endfeet of the chick embryonic spinal cord, including sub-apical protrusions that extend laterally within the tissue, and observe similar structures in human neuroepithelium. We characterise the dynamics, shape, and cytoskeleton of these lateral protrusions and distinguish these structures from cytonemes/filopodia and tunnelling nanotubes. We demonstrate that lateral protrusions form a latticework of membrane contacts between non-adjacent cells, depend on actin but not microtubule dynamics and provide a lamellipodial-like platform for further extending fine actin-dependent filipodia. We find that lateral protrusions depend on the actin-binding protein WAVE1: mutant-WAVE1 misexpression attenuated prot...
Background:Atopic eczema is an itchy inflammatory disorder characterised by skin barrier dysfunct... more Background:Atopic eczema is an itchy inflammatory disorder characterised by skin barrier dysfunction. Loss-of-function mutations in the gene encoding filaggrin (FLG) are a major risk factor, but the mechanisms by which filaggrin haploinsufficiency leads to atopic inflammation remain incompletely understood. Skin as an organ that can be modelled using primary cellsin vitroprovides the opportunity for selected genetic effects to be investigated in detail.Methods:Primary human keratinocytes and donor-matched primary fibroblasts from healthy individuals were used to create skin organoid models with and without siRNA-mediated knockdown ofFLG. Biological replicate sets of organoids were assessed using histological, functional and biochemical measurements.Results:FLGknockdown leads to subtle changes in histology and ultrastructure including a reduction in thickness of the stratum corneum and smaller, less numerous keratohyalin granules. Immature organoids showed some limited evidence of ba...
Mutations enhancing the kinase activity of LRRK2 cause Parkinson’s disease (PD) and therapies tha... more Mutations enhancing the kinase activity of LRRK2 cause Parkinson’s disease (PD) and therapies that reduce LRRK2 kinase activity are being tested in clinical trials. Numerous rare variants of unknown clinical significance have been reported, but how the vast majority impact on LRRK2 function is unknown. Here, we investigate 100 LRRK2 variants linked to PD, including previously described pathogenic mutations. We identify 23 LRRK2 variants that robustly stimulate kinase activity, including variants within the N-terminal non-catalytic regions [ARM (E334K, A419V), ANK(R767H), LRR (R1067Q, R1325Q)], as well as variants predicted to destabilise the ROC:CORB interface [ROC (A1442P, V1447M), CORA (R1628P) CORB (S1761R, L1795F)] and COR:COR dimer interface [CORB (R1728H/L)]. Most activating variants decrease LRRK2 biomarker site phosphorylation (pSer935/pSer955/pSer973), consistent with the notion that the active kinase conformation blocks their phosphorylation. We conclude that the impact of...
www.embojournal.org Structural insights into the regulation of PDK1 by phosphoinositides and inos... more www.embojournal.org Structural insights into the regulation of PDK1 by phosphoinositides and inositol phosphates
After 2 h at 37°C, migrated or input cells were recovered from the wells, quantitated by flow cyt... more After 2 h at 37°C, migrated or input cells were recovered from the wells, quantitated by flow cytometry, and expressed as ratios of migrated or input HeN/HeJ. (B) DC were pretreated with 100 ng/ml LPS for different lengths of time before addition into the Transwell insert. Note that time = 0* data were obtained from cells where LPS was added immediately before the cells were placed onto the filter. Subsequent migration was for 2 h in all cases. The line indicates the 1:1 ratio achieved where HeN and HeJ migrate with equal efficiency. (C) Data from experiments on three or four independent DC cultures are shown. Bars represent mean data. *, P < 0.05.<b>Copyright information:</b>Taken from "TLR ligand–induced podosome disassembly in dendritic cells is ADAM17 dependent"The Journal of Cell Biology 2008;182(5):993-1005.Published online 8 Sep 2008PMCID:PMC2528573.
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