Ca2+ Regulates the Drosophila Stoned-A and Stoned-B Proteins Interaction with the C2B Domain of Synaptotagmin-1

The dicistronic Drosophila stoned gene is involved in exocytosis and/or endocytosis of synaptic vesicles. Mutations in either stonedA or stonedB cause a severe disruption of neurotransmission in fruit flies. Previous studies have shown that the coiled-coil domain of the Stoned-A and the µ-homology domain of the Stoned-B protein can interact with the C2B domain of Synaptotagmin-1. However, very little is known about the mechanism of interaction between the Stoned proteins and the C2B domain of Synaptotagmin-1. Here we report that these interactions are increased in the presence of Ca2+. The Ca2+-dependent interaction between the µ-homology domain of Stoned-B and C2B domain of Synaptotagmin-1 is affected by phospholipids. The C-terminal region of the C2B domain, including the tryptophan-containing motif, and the Ca2+ binding loop region that modulate the Ca2+-dependent oligomerization, regulates the binding of the Stoned-A and Stoned-B proteins to the C2B domain. Stoned-B, but not Stoned-A, interacts with the Ca2+-binding loop region of C2B domain. The results indicate that Ca2+-induced self-association of the C2B domain regulates the binding of both Stoned-A and Stoned-B proteins to Synaptotagmin-1. The Stoned proteins may regulate sustainable neurotransmission in vivo by binding to Ca2+-bound Synaptotagmin-1 associated synaptic vesicles

Post-Translational Modifications and Lipid Binding Profile of Insect Cell-Expressed Full-Length Mammalian Synaptotagmin 1

ABSTRACT: Synaptotagmin 1 (Syt1) is a Ca2+ sensor for SNARE-mediated, Ca2+-triggered synaptic vesicle fusion in neurons. It is composed of luminal, transmembrane, linker, and two Ca2+-binding (C2) domains. Here we describe expression and purification of full-length mammalian Syt1 in insect cells along with an extensive biochemical characterization of the purified protein. The expressed and purified protein is properly folded and has increased α-helical content compared to the C2AB fragment alone. Post-translational modifications of Syt1 were analyzed by mass spectrometry, revealing the same modifications of Syt1 that were previously described for Syt1 purified from brain extract or mammalian cell lines, along with a novel modification of Syt1, tyrosine nitration. A lipid binding screen with both full-length Syt1 and the C2AB fragments of Syt1 and Syt3 isoforms revealed new Syt1−lipid interactions. These results suggest a conserved lipid binding mechanism in which Ca2+-independent interactions are mediated via a lysine rich region of the C2B domain while Ca2+-dependent interactions are mediated via the Ca2+-binding loops

Motor neuroprosthesis implanted with neurointerventional surgery improves capacity for activities of daily living tasks in severe paralysis: first in-human experience

Background: Implantable brain–computer interfaces (BCIs), functioning as motor neuroprostheses, have the potential to restore voluntary motor impulses to control digital devices and improve functional independence in patients with severe paralysis due to brain, spinal cord, peripheral nerve or muscle dysfunction. However, reports to date have had limited clinical translation. Methods Two participants with amyotrophic lateral sclerosis (ALS) underwent implant in a single-arm, open-label, prospective, early feasibility study. Using a minimally invasive neurointervention procedure, a novel endovascular Stentrode BCI was implanted in the superior sagittal sinus adjacent to primary motor cortex. The participants undertook machinelearning-assisted training to use wirelessly transmitted electrocorticography signal associated with attempted movements to control multiple mouse-click actions, including zoom and left-click. Used in combination with an eye-tracker for cursor navigation, participants achieved Windows 10 operating system control to conduct instrumental activities of daily living (IADL) tasks. Results: Unsupervised home use commenced from day 86 onwards for participant 1, and day 71 for participant 2. Participant 1 achieved a typing task average click selection accuracy of 92.63% (100.00%, 87.50%– 100.00%) (trial mean (median, Q1–Q3)) at a rate of 13.81 (13.44, 10.96–16.09) correct characters per minute (CCPM) with predictive text disabled. Participant 2 achieved an average click selection accuracy of 93.18% (100.00%, 88.19%–100.00%) at 20.10 (17.73, 12.27–26.50) CCPM. Completion of IADL tasks including text messaging, online shopping and managing finances independently was demonstrated in both participants. Conclusion: We describe the first-in-human experience of a minimally invasive, fully implanted, wireless, ambulatory motor neuroprosthesis using an endovascular stent-electrode array to transmit electrocorticography signals from the motor cortex for multiple command control of digital devices in two participants with flaccid upper limb paralysis.Thomas J Oxley, Peter E Yoo, Gil S Rind, Stephen M Ronayne, C M Sarah Lee, Christin Bird, Victoria Hampshire, Rahul P Sharma, Andrew Morokoff, Daryl L Williams, Christopher MacIsaac, Mark E Howard, Lou Irving, Ivan Vrljic, Cameron Williams, Sam E John, Frank Weissenborn, Madeleine Dazenko, Anna H Balabanski, David Friedenberg, Anthony N Burkitt, Yan T Wong, Katharine J Drummond, Patricia Desmond, Douglas Weber, Timothy Denison, Leigh R Hochberg, Susan Mathers, Terence J O, Brien, Clive N May, J Mocco, David B Grayden, Bruce C V Campbell, Peter Mitchell, Nicholas L Opi

Structural and Mutational Analysis of Functional Differentiation between Synaptotagmins-1 and -7

Synaptotagmins are known to mediate diverse forms of Ca2+-triggered exocytosis through their C2 domains, but the principles underlying functional differentiation among them are unclear. Synaptotagmin-1 functions as a Ca2+ sensor in neurotransmitter release at central nervous system synapses, but synaptotagmin-7 does not, and yet both isoforms act as Ca2+ sensors in chromaffin cells. To shed light into this apparent paradox, we have performed rescue experiments in neurons from synaptotagmin-1 knockout mice using a chimera that contains the synaptotagmin-1 sequence with its C2B domain replaced by the synaptotagmin-7 C2B domain (Syt1/7). Rescue was not achieved either with the WT Syt1/7 chimera or with nine mutants where residues that are distinct in synaptotagmin-7 were restored to those present in synaptotagmin-1. To investigate whether these results arise because of unique conformational features of the synaptotagmin-7 C2B domain, we determined its crystal structure at 1.44 Å resolution. The synaptotagmin-7 C2B domain structure is very similar to that of the synaptotagmin-1 C2B domain and contains three Ca2+-binding sites. Two of the Ca2+-binding sites of the synaptotagmin-7 C2B domain are also present in the synaptotagmin-1 C2B domain and have analogous ligands to those determined for the latter by NMR spectroscopy, suggesting that a discrepancy observed in a crystal structure of the synaptotagmin-1 C2B domain arose from crystal contacts. Overall, our results suggest that functional differentiation in synaptotagmins arises in part from subtle sequence changes that yield dramatic functional differences

Restoration of IFNγR Subunit Assembly, IFNγ Signaling and Parasite Clearance in Leishmania donovani Infected Macrophages: Role of Membrane Cholesterol

Despite the presence of significant levels of systemic Interferon gamma (IFNγ), the host protective cytokine, Kala-azar patients display high parasite load with downregulated IFNγ signaling in Leishmania donovani (LD) infected macrophages (LD-MØs); the cause of such aberrant phenomenon is unknown. Here we reveal for the first time the mechanistic basis of impaired IFNγ signaling in parasitized murine macrophages. Our study clearly shows that in LD-MØs IFNγ receptor (IFNγR) expression and their ligand-affinity remained unaltered. The intracellular parasites did not pose any generalized defect in LD-MØs as IL-10 mediated signal transducer and activator of transcription 3 (STAT3) phosphorylation remained unaltered with respect to normal. Previously, we showed that LD-MØs are more fluid than normal MØs due to quenching of membrane cholesterol. The decreased rigidity in LD-MØs was not due to parasite derived lipophosphoglycan (LPG) because purified LPG failed to alter fluidity in normal MØs. IFNγR subunit 1 (IFNγR1) and subunit 2 (IFNγR2) colocalize in raft upon IFNγ stimulation of normal MØs, but this was absent in LD-MØs. Oddly enough, such association of IFNγR1 and IFNγR2 could be restored upon liposomal delivery of cholesterol as evident from the fluorescence resonance energy transfer (FRET) experiment and co-immunoprecipitation studies. Furthermore, liposomal cholesterol treatment together with IFNγ allowed reassociation of signaling assembly (phospho-JAK1, JAK2 and STAT1) in LD-MØs, appropriate signaling, and subsequent parasite killing. This effect was cholesterol specific because cholesterol analogue 4-cholestene-3-one failed to restore the response. The presence of cholesterol binding motifs [(L/V)-X1–5-Y-X1–5-(R/K)] in the transmembrane domain of IFNγR1 was also noted. The interaction of peptides representing this motif of IFNγR1 was studied with cholesterol-liposome and analogue-liposome with difference of two orders of magnitude in respective affinity (KD: 4.27×10−9 M versus 2.69×10−7 M). These observations reinforce the importance of cholesterol in the regulation of function of IFNγR1 proteins. This study clearly demonstrates that during its intracellular life-cycle LD perturbs IFNγR1 and IFNγR2 assembly and subsequent ligand driven signaling by quenching MØ membrane cholesterol

Motor neuroprosthesis implanted with neurointerventional surgery improves capacity for activities of daily living tasks in severe paralysis: first in-human experience

BACKGROUND: Implantable brain-computer interfaces (BCIs), functioning as motor neuroprostheses, have the potential to restore voluntary motor impulses to control digital devices and improve functional independence in patients with severe paralysis due to brain, spinal cord, peripheral nerve or muscle dysfunction. However, reports to date have had limited clinical translation. METHODS: Two participants with amyotrophic lateral sclerosis (ALS) underwent implant in a single-arm, open-label, prospective, early feasibility study. Using a minimally invasive neurointervention procedure, a novel endovascular Stentrode BCI was implanted in the superior sagittal sinus adjacent to primary motor cortex. The participants undertook machine-learning-assisted training to use wirelessly transmitted electrocorticography signal associated with attempted movements to control multiple mouse-click actions, including zoom and left-click. Used in combination with an eye-tracker for cursor navigation, participants achieved Windows 10 operating system control to conduct instrumental activities of daily living (IADL) tasks. RESULTS: Unsupervised home use commenced from day 86 onwards for participant 1, and day 71 for participant 2. Participant 1 achieved a typing task average click selection accuracy of 92.63% (100.00%, 87.50%-100.00%) (trial mean (median, Q1-Q3)) at a rate of 13.81 (13.44, 10.96-16.09) correct characters per minute (CCPM) with predictive text disabled. Participant 2 achieved an average click selection accuracy of 93.18% (100.00%, 88.19%-100.00%) at 20.10 (17.73, 12.27-26.50) CCPM. Completion of IADL tasks including text messaging, online shopping and managing finances independently was demonstrated in both participants. CONCLUSION: We describe the first-in-human experience of a minimally invasive, fully implanted, wireless, ambulatory motor neuroprosthesis using an endovascular stent-electrode array to transmit electrocorticography signals from the motor cortex for multiple command control of digital devices in two participants with flaccid upper limb paralysis

Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment.

Synaptic-vesicle exocytosis is mediated by the vesicular Ca(2+) sensor synaptotagmin-1. Synaptotagmin-1 interacts with the SNARE protein syntaxin-1A and acidic phospholipids such as phosphatidylinositol 4,5-bisphosphate (PIP2). However, it is unclear how these interactions contribute to triggering membrane fusion. Using PC12 cells from Rattus norvegicus and artificial supported bilayers, we show that synaptotagmin-1 interacts with the polybasic linker region of syntaxin-1A independent of Ca(2+) through PIP2. This interaction allows both Ca(2+)-binding sites of synaptotagmin-1 to bind to phosphatidylserine in the vesicle membrane upon Ca(2+) triggering. We determined the crystal structure of the C2B domain of synaptotagmin-1 bound to phosphoserine, allowing development of a high-resolution model of synaptotagmin bridging two different membranes. Our results suggest that PIP2 clusters organized by syntaxin-1 act as molecular beacons for vesicle docking, with the subsequent Ca(2+) influx bringing the vesicle membrane close enough for membrane fusion

Assessment of safety of a fully implanted endovascular brain-computer interface for severe paralysis in 4 patients : The stentrode with thought-controlled digital switch (SWITCH) study

Importance Brain-computer interface (BCI) implants have previously required craniotomy to deliver penetrating or surface electrodes to the brain. Whether a minimally invasive endovascular technique to deliver recording electrodes through the jugular vein to superior sagittal sinus is safe and feasible is unknown. Objective To assess the safety of an endovascular BCI and feasibility of using the system to control a computer by thought. Design, Setting, and Participants The Stentrode With Thought-Controlled Digital Switch (SWITCH) study, a single-center, prospective, first in-human study, evaluated 5 patients with severe bilateral upper-limb paralysis, with a follow-up of 12 months. From a referred sample, 4 patients with amyotrophic lateral sclerosis and 1 with primary lateral sclerosis met inclusion criteria and were enrolled in the study. Surgical procedures and follow-up visits were performed at the Royal Melbourne Hospital, Parkville, Australia. Training sessions were performed at patients’ homes and at a university clinic. The study start date was May 27, 2019, and final follow-up was completed January 9, 2022. Interventions Recording devices were delivered via catheter and connected to subcutaneous electronic units. Devices communicated wirelessly to an external device for personal computer control. Main Outcomes and Measures The primary safety end point was device-related serious adverse events resulting in death or permanent increased disability. Secondary end points were blood vessel occlusion and device migration. Exploratory end points were signal fidelity and stability over 12 months, number of distinct commands created by neuronal activity, and use of system for digital device control. Results Of 4 patients included in analyses, all were male, and the mean (SD) age was 61 (17) years. Patients with preserved motor cortex activity and suitable venous anatomy were implanted. Each completed 12-month follow-up with no serious adverse events and no vessel occlusion or device migration. Mean (SD) signal bandwidth was 233 (16) Hz and was stable throughout study in all 4 patients (SD range across all sessions, 7-32 Hz). At least 5 attempted movement types were decoded offline, and each patient successfully controlled a computer with the BCI. Conclusions and Relevance Endovascular access to the sensorimotor cortex is an alternative to placing BCI electrodes in or on the dura by open-brain surgery. These final safety and feasibility data from the first in-human SWITCH study indicate that it is possible to record neural signals from a blood vessel. The favorable safety profile could promote wider and more rapid translation of BCI to people with paralysis. Trial Registration ClinicalTrials.gov Identifier: NCT0383485

Molecular machines governing exocytosis of synaptic vesicles.

Calcium-dependent exocytosis of synaptic vesicles mediates the release of neurotransmitters. Important proteins in this process have been identified such as the SNAREs, synaptotagmins, complexins, Munc18 and Munc13. Structural and functional studies have yielded a wealth of information about the physiological role of these proteins. However, it has been surprisingly difficult to arrive at a unified picture of the molecular sequence of events from vesicle docking to calcium-triggered membrane fusion. Using mainly a biochemical and biophysical perspective, we briefly survey the molecular mechanisms in an attempt to functionally integrate the key proteins into the emerging picture of the neuronal fusion machine