ORCID Profile
0000-0001-6400-1107
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Research Topics
In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Top 5 Research Topics
ANZSRC Field of Research (FoR)
Biochemistry and Cell Biology | Protein Trafficking | Membrane Biology | Cellular Nervous System | Cell Neurochemistry | Receptors and Membrane Biology | Signal Transduction | Protein Targeting And Signal Transduction | Biochemistry and cell biology | Optical Physics | Zoology | Structural Biology (incl. Macromolecular Modelling) | Biological Physics | Cellular Nervous System | Proteomics and Intermolecular Interactions (excl. Medical Proteomics) | Central Nervous System | Peripheral Nervous System | Animal Neurobiology | Cell Physiology | Cell Neurochemistry | Neurosciences | Enzymes | Cell neurochemistry | Lasers and Quantum Electronics
ANZSRC Socio-Economic Objective (SEO)
Expanding Knowledge in the Biological Sciences | Nervous system and disorders | Endocrine organs and diseases (incl. diabetes) | Biological sciences | Expanding Knowledge in the Physical Sciences | Nervous System and Disorders | Neurodegenerative Disorders Related to Ageing | Expanding Knowledge in the Medical and Health Sciences | Diabetes | Health and support services not elsewhere classified | Mental health | Skeletal system and disorders (incl. arthritis) |
Related Links
Publications
Hericerin derivatives activates a pan‐neurotrophic pathway in central hippocampal neurons converging to ERK1 /2 signaling enhancing spatial memory
Publisher: Wiley
Date: 31-01-2023
DOI: 10.1111/JNC.15767
Abstract: The traditional medicinal mushroom Hericium erinaceus is known for enhancing peripheral nerve regeneration through targeting nerve growth factor (NGF) neurotrophic activity. Here, we purified and identified biologically new active compounds from H. erinaceus , based on their ability to promote neurite outgrowth in hippoc al neurons. N ‐de phenylethyl isohericerin (NDPIH), an isoindoline compound from this mushroom, together with its hydrophobic derivative hericene A, were highly potent in promoting extensive axon outgrowth and neurite branching in cultured hippoc al neurons even in the absence of serum, demonstrating potent neurotrophic activity. Pharmacological inhibition of tropomyosin receptor kinase B (TrkB) by ANA‐12 only partly prevented the NDPIH‐induced neurotrophic activity, suggesting a potential link with BDNF signaling. However, we found that NDPIH activated ERK1/2 signaling in the absence of TrkB in HEK‐293T cells, an effect that was not sensitive to ANA‐12 in the presence of TrkB. Our results demonstrate that NDPIH acts via a complementary neurotrophic pathway independent of TrkB with converging downstream ERK1/2 activation. Mice fed with H. erinaceus crude extract and hericene A also exhibited increased neurotrophin expression and downstream signaling, resulting in significantly enhanced hippoc al memory. Hericene A therefore acts through a novel pan‐neurotrophic signaling pathway , leading to improved cognitive performance. image
A role for SNARE s in neuronal survival?
Publisher: Wiley
Date: 02-04-2014
DOI: 10.1111/JNC.12699
Neuropilin-1 facilitates SARS-CoV-2 cell entry and infectivity
Publisher: American Association for the Advancement of Science (AAAS)
Date: 13-11-2020
Abstract: Virus-host interactions determine cellular entry and spreading in tissues. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the earlier SARS-CoV use angiotensin-converting enzyme 2 (ACE2) as a receptor however, their tissue tropism differs, raising the possibility that additional host factors are involved. The spike protein of SARS-CoV-2 contains a cleavage site for the protease furin that is absent from SARS-CoV (see the Perspective by Kielian). Cantuti-Castelvetri et al. now show that neuropilin-1 (NRP1), which is known to bind furin-cleaved substrates, potentiates SARS-CoV-2 infectivity. NRP1 is abundantly expressed in the respiratory and olfactory epithelium, with highest expression in endothelial and epithelial cells. Daly et al. found that the furin-cleaved S1 fragment of the spike protein binds directly to cell surface NRP1 and blocking this interaction with a small-molecule inhibitor or monoclonal antibodies reduced viral infection in cell culture. Understanding the role of NRP1 in SARS-CoV-2 infection may suggest potential targets for future antiviral therapeutics. Science , this issue p. 856 , p. 861 see also p. 765
Interrogation and validation of the interactome of neuronal Munc18-interacting Mint proteins with AlphaFold2
Publisher: Cold Spring Harbor Laboratory
Date: 21-02-2023
DOI: 10.1101/2023.02.20.529329
Abstract: Munc18-interacting proteins (Mints) are multi-domain adaptors that regulate neuronal membrane trafficking, signalling and neurotransmission. Mint1 and Mint2 are highly expressed in the brain and play overlapping roles in the regulation of synaptic vesicle fusion required for neurotransmitter release by interacting with the essential synaptic protein Munc18-1. The mechanism by which Mint1 mediates the interaction of Munc18-1 with Syntaxin1a (Sx1a) to control formation of the fusogenic SNARE complex and facilitate neurotransmission remains unclear. Here, we have studied both the specific interactions of the neuronal Mint proteins with Munc18-1 as well as their wider interactome. Using biochemical and biophysical binding approaches we identify a short acidic α -helical motif (AHM) within Mint1 and Mint2 that is necessary and sufficient for specific binding to Munc18-1. In silico modelling of this interaction with AlphaFold2 and extensive validation by structure-based mutagenesis showed that the AHM in Mint forms an α -helical structure that binds a conserved surface on Munc18-1 domain3b. In Munc18-1/2 double knockout cells we show that mutating the Mint-binding site significantly reduces the ability of Munc18-1 re-expression to rescue exocytosis. Although Munc18-1 can interact with Mint and Sx1a proteins simultaneously we find they have mutually reduced affinities, suggesting an allosteric coupling between the two proteins. Using AlphaFold2 we then examined the entire cellular network of putative interactions governed by the Mint scaffolds. Although this suggests many proposed binders do not associate directly with Mints, our analyses provide a structural model for their assembly with a variety of known and novel regulatory and cargo proteins including ARF3/ARF4 small GTPases, and the AP3 clathrin adaptor complex. Validation of Mint1 interaction with a new predicted binder TJAP1 provides experimental support that AlphaFold2 can correctly predict interactions across large-scale datasets. Overall, our data provides insights into the ersity of interactions mediated by the Mint family and shows that Mints may help facilitate a key trigger point in SNARE complex assembly and vesicle fusion.
Frontotemporal dementia mutant Tau promotes aberrant Fyn nanoclustering in hippocampal dendritic spines
Publisher: eLife Sciences Publications, Ltd
Date: 25-06-2019
DOI: 10.7554/ELIFE.45040
Abstract: The Src kinase Fyn plays critical roles in memory formation and Alzheimer’s disease. Its targeting to neuronal dendrites is regulated by Tau via an unknown mechanism. As nanoclustering is essential for efficient signaling, we used single-molecule tracking to characterize the nanoscale distribution of Fyn in mouse hippoc al neurons, and manipulated the expression of Tau to test whether it controls Fyn nanoscale organization. We found that dendritic Fyn exhibits at least three distinct motion states, two of them associated with nanodomains. Fyn mobility decreases in dendrites during neuronal maturation, suggesting a dynamic synaptic reorganization. Removing Tau increases Fyn mobility in dendritic shafts, an effect that is rescued by re-expressing wildtype Tau. By contrast, expression of frontotemporal dementia P301L mutant Tau immobilizes Fyn in dendritic spines, affecting its motion state distribution and nanoclustering. Tau therefore controls the nanoscale organization of Fyn in dendrites, with the pathological Tau P301L mutation potentially contributing to synaptic dysfunction by promoting aberrant Fyn nanoclustering in spines.
<em>In Vivo</em> Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal
Publisher: MyJove Corporation
Date: 14-01-2018
DOI: 10.3791/56952
STXBP1 encephalopathy
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 20-06-2019
DOI: 10.1212/WNL.0000000000007786
Abstract: De novo pathogenic variants in STXBP1 encoding syntaxin1-binding protein (STXBP1, also known as Munc18-1) lead to a range of early-onset neurocognitive conditions, most commonly early infantile epileptic encephalopathy type 4 (EIEE4, also called STXBP1 encephalopathy), a severe form of epilepsy associated with developmental delay/intellectual disability. Other neurologic features include autism spectrum disorder and movement disorders. The progression of neurologic symptoms has been reported in a few older affected in iduals, with the appearance of extrapyramidal features, reminiscent of early onset parkinsonism. Understanding the pathologic process is critical to improving therapies, as currently available antiepileptic drugs have shown limited success in controlling seizures in EIEE4 and there is no precision medication approach for the other neurologic features of the disorder. Basic research shows that genetic knockout of STXBP1 or other presynaptic proteins of the exocytic machinery leads to widespread perinatal neurodegeneration. The mechanism that regulates this effect is under scrutiny but shares intriguing hallmarks with classical neurodegenerative diseases, albeit appearing early during brain development. Most critically, recent evidence has revealed that STXBP1 controls the self-replicating aggregation of α-synuclein, a presynaptic protein involved in various neurodegenerative diseases that are collectively known as synucleinopathies, including Parkinson disease. In this review, we examine the tantalizing link among STXBP1 function, EIEE, and the neurodegenerative synucleinopathies, and suggest that neural development in EIEE could be further affected by concurrent synucleinopathic mechanisms.
Profiling of Free Fatty Acids Using Stable Isotope Tagging Uncovers a Role for Saturated Fatty Acids in Neuroexocytosis
Publisher: Elsevier BV
Date: 11-2015
DOI: 10.1016/J.CHEMBIOL.2015.09.010
Abstract: The phospholipase-catalyzed release of free fatty acids (FFAs) from phospholipids is implicated in many critical biological processes such as neurotransmission, inflammation, and cancer. However, determining the in idual change in FFAs generated during these processes has remained challenging due to the limitations of current methods, and has h ered our understanding of these key mediators. Here, we developed an "iTRAQ"-like method for profiling FFAs by stable isotope tagging (FFAST), based on the differential labeling of the carboxyl group and designed to resolve analytical variance, through a multiplexed assay in cells and subcellular fractions. With nanomolar sensitivity, this method revealed a spectrum of saturated FFAs elicited during stimulation of exocytosis that was identical in neurons and neurosecretory cells. Purified secretory vesicles also generated these FFAs when challenged with cytosol. Our multiplex method will be invaluable to assess the range of FFAs generated in other physiological and pathological settings.
Phospholipases in neuronal function: A role in learning and memory?
Publisher: Wiley
Date: 07-02-2020
DOI: 10.1111/JNC.14918
Abstract: Despite the human brain being made of nearly 60% fat, the vast majority of studies on the mechanisms of neuronal communication which underpin cognition, memory and learning, primarily focus on proteins and/or (epi)genetic mechanisms. Phospholipids are the main component of all cellular membranes and function as substrates for numerous phospholipid-modifying enzymes, including phospholipases, which release free fatty acids (FFAs) and other lipid metabolites that can alter the intrinsic properties of the membranes, recruit and activate critical proteins, and act as lipid signalling molecules. Here, we will review brain specific phospholipases, their roles in membrane remodelling, neuronal function, learning and memory, as well as their disease implications. In particular, we will highlight key roles of unsaturated FFAs, particularly arachidonic acid, in neurotransmitter release, neuroinflammation and memory. In light of recent findings, we will also discuss the emerging role of phospholipase A
The Munc18-1 domain 3a loop is essential for neuroexocytosis but not for syntaxin-1A transport to the plasma membrane
Publisher: The Company of Biologists
Date: 06-2013
DOI: 10.1242/JCS.126813
Abstract: Munc18-1 plays a dual role in transporting syntaxin-1A (Sx1a) to the plasma membrane and regulating SNARE-mediated membrane fusion. As impairment of either function leads to a common exocytic defect, assigning specific roles for various Munc18-1 domains has proved difficult. Structural analyses predict that a loop region in Munc18-1 domain 3a could catalyse the conversion of Sx1a from a ‘closed’, fusion-incompetent to an ‘open’, fusion-competent conformation. As this conversion occurs at the plasma membrane, mutations in this loop could potentially separate the chaperone and exocytic functions of Munc18-1. Expression of a Munc18-1 deletion mutant lacking 17 residues of the domain 3a loop (Munc18-1Δ317–333) in PC12 cells deficient in endogenous Munc18 (DKD-PC12 cells) fully rescued transport of Sx1a to the plasma membrane, but not exocytic secretory granule fusion. In vitro binding of Munc18-1Δ317–333 to Sx1a was indistinguishable from that of full-length Munc18-1, consistent with the critical role of the closed conformation in Sx1a transport. However, in DKD-PC12 cells, Munc18-1Δ317–333 binding to Sx1a was greatly reduced compared to that of full-length Munc18-1, suggesting that closed conformation binding contributes little to the overall interaction at the cell surface. Furthermore, we found that Munc18-1Δ317–333 could bind SNARE complexes in vitro, suggesting that additional regulatory factors underpin the exocytic function of Munc18-1 in vivo. Together, these results point to a defined role for Munc18-1 in facilitating exocytosis linked to the loop region of domain 3a that is clearly distinct from its function in Sx1a transport.
ENA/VASP proteins regulate exocytosis by mediating myosin VI-dependent recruitment of secretory granules to the cortical actin network
Publisher: Elsevier BV
Date: 10-2017
DOI: 10.1016/J.MCN.2017.07.005
Abstract: In neurosecretory cells, myosin VI associated with secretory granules (SGs) mediates their activity-dependent recruitment to the cortical actin network and is necessary to sustain exocytosis. The mechanism by which myosin VI interacts with SGs is unknown. Using a myosin VI pull-down assay and mass spectrometry we identified Mena, a member of the ENA/VASP family, as a myosin VI binding partner in PC12 cells, and confirmed that Mena colocalized with myosin VI on SGs. Using a knock-sideways approach to inactivate the ENA/VASP family members by mitochondrial relocation, we revealed a concomitant redistribution of myosin VI. This was ensued by a reduction in the association of myosin VI with SGs, a decreased SG mobility and density in proximity to the plasma membrane as well as decreased evoked exocytosis. These data demonstrate that ENA/VASP proteins regulate SG exocytosis through modulating the activity of myosin VI.
Ciguatoxin-induced catecholamine secretion in bovine chromaffin cells: Mechanism of action and reversible inhibition by brevenal
Publisher: Elsevier BV
Date: 10-2010
DOI: 10.1016/J.TOXICON.2009.08.002
Abstract: Ciguatoxin (P-CTX-1B) from the dinoflagellate Gambierdiscus toxicus, belongs to the family of polyether neurotoxins responsible for the neurological poisoning disorder ciguatera. Although it is the most widespread marine-borne disease affecting humans, there is no current FDA-approved treatment available except for symptomatic therapies. In this paper, we report that P-CTX-1B promotes catecholamine secretion from bovine chromaffin cells, an effect that is insensitive to concomitant activation of capacitative Ca(2+) entry. Moreover, we confirm that brevenal, a polyether from the dinoflagellate Karenia brevis, blocks P-CTX-1B-induced catecholamine secretion. This effect is partially reversible. Our results therefore raise the prospect of finding functional antagonists for P-CTX-1B that could be useful for the treatment of ciguatera.
The Munc18-1 domain 3a hinge-loop controls syntaxin-1A nanodomain assembly and engagement with the SNARE complex during secretory vesicle priming
Publisher: Rockefeller University Press
Date: 19-09-2016
Abstract: Munc18-1 and syntaxin-1A control SNARE-dependent neuroexocytosis and are organized in nanodomains on the plasma membrane of neurons and neurosecretory cells. Deciphering the intra- and intermolecular steps via which they prepare secretory vesicles (SVs) for fusion is key to understanding neuronal and hormonal communication. Here, we demonstrate that expression of a priming-deficient mutant lacking 17 residues of the domain 3a hinge-loop (Munc18-1Δ317-333) in PC12 cells engineered to knockdown Munc18-1/2 markedly prolonged SV docking. Single-molecule analysis revealed nonhomogeneous diffusion of Munc18-1 and syntaxin-1A in and out of partially overlapping nanodomains. Whereas Munc18-1WT mobility increased in response to stimulation, syntaxin-1A became less mobile. These Munc18-1 and syntaxin-1A diffusional switches were blocked by the expression of Munc18-1Δ317-333, suggesting that a conformational change in the Munc18-1 hinge-loop controls syntaxin-1A and subsequent SNARE complex assembly. Accordingly, syntaxin-1A confinement was prevented by expression of botulinum neurotoxin type E. The Munc18-1 domain 3a hinge-loop therefore controls syntaxin-1A engagement into SNARE complex formation during priming.
Author response: Frontotemporal dementia mutant Tau promotes aberrant Fyn nanoclustering in hippocampal dendritic spines
Publisher: eLife Sciences Publications, Ltd
Date: 21-05-2019
Synapsin2a tetramerization selectively controls the presynaptic nanoscale organisation of reserve synaptic vesicles.
Publisher: Research Square Platform LLC
Date: 31-03-2023
DOI: 10.21203/RS.3.RS-2722570/V1
Abstract: Neurotransmitter release relies on the regulated fusion of synaptic vesicles (SVs) that are tightly packed within the presynapse of neurons. The mechanism by which SVs are anchored at the presynapse while preserving their ability to dynamically recycle thereby supporting neuronal communication remains unknown. Synapsin2a tetramerization was recently suggested to cluster SV in presynapses. Here, we used Dual-pulse sub-diffractional Tracking of Internalised Molecules (DsdTIM) to simultaneously track SVs from the recycling and reserve pools, in live hippoc al neurons. The reserve pool displays a lower presynaptic mobility compared to the recycling pool and exhibits a more mobile axonal pool. Synapsin1-3 triple knockout (SynTKO) selectively increased the reserve pool mobility. Re-expression of wild-type Synapsin2a, but not the tetramerization-deficient mutant K337Q, fully rescued these effects. Tracking Synapsin2a K337Q -mEos3.2 revealed altered synapsin activity-dependent presynaptic translocation and nanoclustering. Synapsin2a tetramerization therefore controls its own presynaptic nanoclustering allowing dynamic immobilisation of the reserve pool at the presynapse.
Increased neurovirulence of omicron BA.5 and XBB variants over BA.1 in K18-hACE2 mice and human brain organoids
Publisher: Cold Spring Harbor Laboratory
Date: 25-12-2022
DOI: 10.1101/2022.12.22.521696
Abstract: A frequently repeated premise is that viruses evolve to become less pathogenic. This appears also to be true for SARS-CoV-2, although the increased level of immunity in human populations makes it difficult to distinguish between reduced intrinsic pathogenicity and increasing protective immunity. The reduced pathogenicity of the omicron BA.1 sub-lineage compared to earlier variants is well described and appears to be due to reduced utilization of TMPRRS2. That this reduced pathogenicity remains true for omicron BA.5 was recently reported. In sharp contrast, we show that a BA.5 isolate was significantly more pathogenic in K18-hACE2 mice than a BA.1 isolate, with BA.5 infection showing increased neurovirulence, encephalitis and mortality, similar to that seen for an original ancestral isolate. BA.5 also infected human cortical brain organoids to a greater extent than a BA.1 and original ancestral isolate. Neurons were the target of infection, with increasing evidence of neuron infection in COVID-19 patients. These results argue that while omicron virus may be associated with reduced respiratory symptoms, BA.5 shows increased neurovirulence compared to earlier omicron sub-variants.
Combining Single Molecule Super-Resolution Imaging Techniques to Unravel the Nanoscale Organization of the Presynapse
Publisher: Springer US
Date: 22-11-2020
A role for tropomyosins in activity-dependent bulk endocytosis?
Publisher: Elsevier BV
Date: 10-2017
DOI: 10.1016/J.MCN.2017.04.003
Abstract: Bulk endocytosis allows stimulated neurons to take up a large portion of the presynaptic plasma membrane in order to regenerate synaptic vesicle pools. Actin, one of the most abundant proteins in eukaryotic cells, plays an important role in this process, but a detailed mechanistic understanding of the involvement of the cortical actin network is still lacking, in part due to the relatively small size of nerve terminals and the limitation of optical microscopy. We recently discovered that neurosecretory cells display a similar, albeit much larger, form of bulk endocytosis in response to secretagogue stimulation. This allowed us to identify a novel highly dynamic role for the acto-myosin II cortex in generating constricting rings that precede the fission of nascent bulk endosomes. In this review we focus on the mechanism underpinning this dramatic switch in the organization and function of the cortical actin network. We provide additional experimental data that suggest a role of tropomyosin Tpm3.1 and Tpm4.2 in this process, together with an emerging model of how actin controls bulk endocytosis.
Visualizing endocytic recycling and trafficking in live neurons by subdiffractional tracking of internalized molecules
Publisher: Springer Science and Business Media LLC
Date: 30-11-2017
Abstract: Our understanding of endocytic pathway dynamics is restricted by the diffraction limit of light microscopy. Although super-resolution techniques can overcome this issue, highly crowded cellular environments, such as nerve terminals, can also dramatically limit the tracking of multiple endocytic vesicles such as synaptic vesicles (SVs), which in turn restricts the analytical dissection of their discrete diffusional and transport states. We recently introduced a pulse-chase technique for subdiffractional tracking of internalized molecules (sdTIM) that allows the visualization of fluorescently tagged molecules trapped in in idual signaling endosomes and SVs in presynapses or axons with 30- to 50-nm localization precision. We originally developed this approach for tracking single molecules of botulinum neurotoxin type A, which undergoes activity-dependent internalization and retrograde transport in autophagosomes. This method was then adapted to localize the signaling endosomes containing cholera toxin subunit-B that undergo retrograde transport in axons and to track SVs in the crowded environment of hippoc al presynapses. We describe (i) the construction of a custom-made microfluidic device that enables control over neuronal orientation (ii) the 3D printing of a perfusion system for sdTIM experiments performed on glass-bottom dishes (iii) the dissection, culturing and transfection of hippoc al neurons in microfluidic devices and (iv) guidance on how to perform the pulse-chase experiments and data analysis. In addition, we describe the use of single-molecule-tracking analytical tools to reveal the average and the heterogeneous single-molecule mobility behaviors. We also discuss alternative reagents and equipment that can, in principle, be used for sdTIM experiments and describe how to adapt sdTIM to image nanocluster formation and/or tubulation in early endosomes during sorting events. The procedures described in this protocol take ∼1 week.
An inverted CAV1 (caveolin 1) topology defines novel autophagy-dependent exosome secretion from prostate cancer cells
Publisher: Informa UK Limited
Date: 20-09-2020
The 18th International Symposia on Chromaffin Cell Biology
Publisher: Wiley
Date: 06-2016
DOI: 10.1111/JNC.13631
An Epilepsy-Associated SV2A Mutation Disrupts Synaptotagmin-1 Expression and Activity-Dependent Trafficking
Publisher: Society for Neuroscience
Date: 27-04-2020
DOI: 10.1523/JNEUROSCI.0210-20.2020
Abstract: The epilepsy-linked gene SV2A , has a number of potential roles in the synaptic vesicle (SV) life cycle. However, how loss of SV2A function translates into presynaptic dysfunction and ultimately seizure activity is still undetermined. In this study, we examined whether the first SV2A mutation identified in human disease (R383Q) could provide information regarding which SV2A-dependent events are critical in the translation to epilepsy. We utilized a molecular replacement strategy in which exogenous SV2A was expressed in mouse neuronal cultures of either sex, which had been depleted of endogenous SV2A to mimic the homozygous human condition. We found that the R383Q mutation resulted in a mislocalization of SV2A from SVs to the plasma membrane, but had no effect on its activity-dependent trafficking. This SV2A mutant displayed reduced mobility when stranded on the plasma membrane and reduced binding to its interaction partner synaptotagmin-1 (Syt1). Furthermore, the R383Q mutant failed to rescue reduced expression and dysfunctional activity-dependent trafficking of Syt1 in the absence of endogenous SV2A. This suggests that the inability to control Syt1 expression and trafficking at the presynapse may be key in the transition from loss of SV2A function to seizure activity. SIGNIFICANCE STATEMENT SV2A is a synaptic vesicle (SV) protein, the absence or dysfunction of which is linked to epilepsy. However, the series of molecular events that result in this neurological disorder is still undetermined. We demonstrate here that the first human mutation in SV2A identified in an in idual with epilepsy displays reduced binding to synaptotagmin-1 (Syt1), an SV protein essential for synchronous neurotransmitter release. Furthermore, this mutant cannot correct alterations in both Syt1 expression and trafficking when expressed in the absence of endogenous SV2A (to mimic the homozygous human condition). This suggests that the inability to control Syt1 expression and trafficking may be key in the transition from loss of SV2A function to seizure activity.
Phosphatidylinositol(4,5)bisphosphate coordinates actin-mediated mobilization and translocation of secretory vesicles to the plasma membrane of chromaffin cells
Publisher: Springer Science and Business Media LLC
Date: 04-10-2011
DOI: 10.1038/NCOMMS1500
Abstract: Neurosecretory vesicles undergo docking and priming before Ca(2+)-dependent fusion with the plasma membrane. Although de novo synthesis of phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P(2)) is required for exocytosis, its precise contribution is still unclear. Here we show that inhibition of the p110δ isoform of PI3-kinase by IC87114 promotes a transient increase in PtdIns(4,5)P(2), leading to a potentiation of exocytosis in chromaffin cells. We then exploit this pathway to examine the effect of a transient PtdIns(4,5)P(2) increase on neurosecretory vesicles behaviour, outside the context of a secretagogue stimulation. Our results demonstrate that a rise in PtdIns(4,5)P(2) is sufficient to promote the mobilization and recruitment of secretory vesicles to the plasma membrane via Cdc42-mediated actin reorganization. PtdIns(4,5)P(2), therefore, orchestrates the actin-based conveyance of secretory vesicles to the plasma membrane.
Small molecules demonstrate the role of dynamin as a bi-directional regulator of the exocytosis fusion pore and vesicle release
Publisher: Springer Science and Business Media LLC
Date: 05-05-2015
DOI: 10.1038/MP.2015.56
Abstract: Hormones and neurotransmitters are stored in specialised vesicles and released from excitable cells through exocytosis. During vesicle fusion with the plasma membrane, a transient fusion pore is created that enables transmitter release. The protein dynamin is known to regulate fusion pore expansion (FPE). The mechanism is unknown, but requires its oligomerisation-stimulated GTPase activity. We used a palette of small molecule dynamin modulators to reveal bi-directional regulation of FPE by dynamin and vesicle release in chromaffin cells. The dynamin inhibitors Dynole 34-2 and Dyngo 4a and the dynamin activator Ryngo 1-23 reduced or increased catecholamine released from single vesicles, respectively. Total internal reflection fluorescence (TIRF) microscopy demonstrated that dynamin stimulation with Ryngo 1-23 reduced the number of neuropeptide Y (NPY) kiss-and-run events, but not full fusion events, and slowed full fusion release kinetics. Amperometric stand-alone foot signals, representing transient kiss-and-run events, were less frequent but were of longer duration, similarly to full erometric spikes and pre-spike foot signals. These effects are not due to alterations in vesicle size. Ryngo 1-23 action was blocked by inhibitors of actin polymerisation or myosin II. Therefore, we demonstrate using a novel pharmacological approach that dynamin not only controls FPE during exocytosis, but is a bi-directional modulator of the fusion pore that increases or decreases the amount released from a vesicle during exocytosis if it is activated or inhibited, respectively. As such, dynamin has the ability to exquisitely fine-tune transmitter release.
Need for speed: Super-resolving the dynamic nanoclustering of syntaxin-1 at exocytic fusion sites
Publisher: Elsevier BV
Date: 06-2020
DOI: 10.1016/J.NEUROPHARM.2019.02.036
Abstract: Communication between cells relies on regulated exocytosis, a multi-step process that involves the docking, priming and fusion of vesicles with the plasma membrane, culminating in the release of neurotransmitters and hormones. Key proteins and lipids involved in exocytosis are subjected to Brownian movement and constantly switch between distinct motion states which are governed by short-lived molecular interactions. Critical biochemical reactions between exocytic proteins that occur in the confinement of nanodomains underpin the precise sequence of priming steps which leads to the fusion of vesicles. The advent of super-resolution microscopy techniques has provided the means to visualize in idual molecules on the plasma membrane with high spatiotemporal resolution in live cells. These techniques are revealing a highly dynamic nature of the nanoscale organization of the exocytic machinery. In this review, we focus on soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) syntaxin-1, which mediates vesicular fusion. Syntaxin-1 is highly mobile at the plasma membrane, and its inherent speed allows fast assembly and disassembly of syntaxin-1 nanoclusters which are associated with exocytosis. We reflect on recent studies which have revealed the mechanisms regulating syntaxin-1 nanoclustering on the plasma membrane and draw inferences on the effect of synaptic activity, phosphoinositides, N-ethylmaleimide-sensitive factor (NSF), α-soluble NSF attachment protein (α-SNAP) and SNARE complex assembly on the dynamic nanoscale organization of syntaxin-1. This article is part of the special issue entitled 'Mobility and trafficking of neuronal membrane proteins'.
Inhibition of the PtdIns(5) kinase PIKfyve disrupts intracellular replication of Salmonella
Publisher: Wiley
Date: 18-03-2010
Super-resolved trajectory-derived nanoclustering analysis using spatiotemporal indexing
Publisher: Springer Science and Business Media LLC
Date: 08-06-2023
DOI: 10.1038/S41467-023-38866-Y
Abstract: Single-molecule localization microscopy techniques are emerging as vital tools to unravel the nanoscale world of living cells by understanding the spatiotemporal organization of protein clusters at the nanometer scale. Current analyses define spatial nanoclusters based on detections but neglect important temporal information such as cluster lifetime and recurrence in “hotspots” on the plasma membrane. Spatial indexing is widely used in video games to detect interactions between moving geometric objects. Here, we use the R-tree spatial indexing algorithm to determine the overlap of the bounding boxes of in idual molecular trajectories to establish membership in nanoclusters. Extending the spatial indexing into the time dimension allows the resolution of spatial nanoclusters into multiple spatiotemporal clusters. Using spatiotemporal indexing, we found that syntaxin1a and Munc18-1 molecules transiently cluster in hotspots, offering insights into the dynamics of neuroexocytosis. Nanoscale spatiotemporal indexing clustering (NASTIC) has been implemented as a free and open-source Python graphic user interface.
Tyrosine kinases compete for growth hormone receptor binding and regulate receptor mobility and degradation
Publisher: Elsevier BV
Date: 05-2023
Presynaptic targeting of botulinum neurotoxin type A requires a tripartite PSG‐Syt1 ‐SV2 plasma membrane nanocluster for synaptic vesicle entry
Publisher: EMBO
Date: 25-05-2023
Abstract: The unique nerve terminal targeting of botulinum neurotoxin type A (BoNT/A) is due to its capacity to bind two receptors on the neuronal plasma membrane: polysialoganglioside (PSG) and synaptic vesicle glycoprotein 2 (SV2). Whether and how PSGs and SV2 may coordinate other proteins for BoNT/A recruitment and internalization remains unknown. Here, we demonstrate that the targeted endocytosis of BoNT/A into synaptic vesicles (SVs) requires a tripartite surface nanocluster. Live‐cell super‐resolution imaging and electron microscopy of catalytically inactivated BoNT/A wildtype and receptor‐binding‐deficient mutants in cultured hippoc al neurons demonstrated that BoNT/A must bind coincidentally to a PSG and SV2 to target synaptic vesicles. We reveal that BoNT/A simultaneously interacts with a preassembled PSG‐synaptotagmin‐1 (Syt1) complex and SV2 on the neuronal plasma membrane, facilitating Syt1‐SV2 nanoclustering that controls endocytic sorting of the toxin into synaptic vesicles. Syt1 CRISPRi knockdown suppressed BoNT/A‐ and BoNT/E‐induced neurointoxication as quantified by SNAP‐25 cleavage, suggesting that this tripartite nanocluster may be a unifying entry point for selected botulinum neurotoxins that hijack this for synaptic vesicle targeting.
Author Reply to Peer Reviews of Multiple animal viruses, including SARS-CoV-2 and its variants, can use alternative cell entry mechanisms for infection
Publisher: EMBO
Date: 03-10-2023
Fyn nanoclustering requires switching to an open conformation and is enhanced by FTLD-Tau biomolecular condensates
Publisher: Springer Science and Business Media LLC
Date: 18-10-2022
DOI: 10.1038/S41380-022-01825-Y
Abstract: Fyn is a Src kinase that controls critical signalling cascades and has been implicated in learning and memory. Postsynaptic enrichment of Fyn underpins synaptotoxicity in dementias such as Alzheimer’s disease and frontotemporal lobar degeneration with Tau pathology (FTLD-Tau). The FLTD P301L mutant Tau is associated with a higher propensity to undergo liquid–liquid phase separation (LLPS) and form biomolecular condensates. Expression of P301L mutant Tau promotes aberrant trapping of Fyn in nanoclusters within hippoc al dendrites by an unknown mechanism. Here, we used single-particle tracking photoactivated localisation microscopy to demonstrate that the opening of Fyn into its primed conformation promotes its nanoclustering in dendrites leading to increased Fyn/ERK/S6 downstream signalling. Preventing the auto-inhibitory closed conformation of Fyn through phospho-inhibition or through perturbation of its SH3 domain increased Fyn’s nanoscale trapping, whereas inhibition of the catalytic domain had no impact. By combining pharmacological and genetic approaches, we demonstrate that P301L Tau enhanced both Fyn nanoclustering and Fyn/ERK/S6 signalling via its ability to form biomolecular condensates. Together, our findings demonstrate that Fyn alternates between a closed and an open conformation, the latter being enzymatically active and clustered. Furthermore, pathogenic immobilisation of Fyn relies on the ability of P301L Tau to form biomolecular condensates, thus highlighting the critical importance of LLPS in controlling nanoclustering and downstream intracellular signalling events.
VAMP2 Binding to Munc18-1 Domain 3a Controls the Nanoscale Reorganization of the Plasma Membrane and Vesicle Interface During Vesicular Priming
Publisher: Elsevier BV
Date: 2019
DOI: 10.2139/SSRN.3362260
Modular transient nanoclustering of activated β2-adrenergic receptors revealed by single-molecule tracking of conformation-specific nanobodies
Publisher: Proceedings of the National Academy of Sciences
Date: 19-11-2020
Abstract: Proteins moving freely on the plasma membrane can become transiently trapped in functionally essential clusters. This capability is likely to be influenced by subtle conformational states of the protein promoting or preventing such confinement. The downside of conventional imaging of overexpressed tagged proteins is that it precludes selective tracking of inherently minor albeit functionally essential conformer populations. Intracellular expression of single-chain nanobodies allowed us to track endogenous proteins in highly specific conformational states in live cells and small organisms. We unveiled the full scope of nanoclustering behavior of β 2 -adrenergic receptors in various conformations, along with their transient nature. This technique is broadly applicable to other proteins and will help unravel essential dynamics and organization of nanoclusters.
Subdiffractional tracking of internalized molecules reveals heterogeneous motion states of synaptic vesicles
Publisher: Rockefeller University Press
Date: 24-10-2016
Abstract: Our understanding of endocytic pathway dynamics is severely restricted by the diffraction limit of light microscopy. To address this, we implemented a novel technique based on the subdiffractional tracking of internalized molecules (sdTIM). This allowed us to image anti–green fluorescent protein Atto647N-tagged nanobodies trapped in synaptic vesicles (SVs) from live hippoc al nerve terminals expressing vesicle-associated membrane protein 2 (VAMP2)–pHluorin with 36-nm localization precision. Our results showed that, once internalized, VAMP2–pHluorin/Atto647N–tagged nanobodies exhibited a markedly lower mobility than on the plasma membrane, an effect that was reversed upon restimulation in presynapses but not in neighboring axons. Using Bayesian model selection applied to hidden Markov modeling, we found that SVs oscillated between diffusive states or a combination of diffusive and transport states with opposite directionality. Importantly, SVs exhibiting diffusive motion were relatively less likely to switch to the transport motion. These results highlight the potential of the sdTIM technique to provide new insights into the dynamics of endocytic pathways in a wide variety of cellular settings.
Alternative cell entry mechanisms for SARS-CoV-2 and multiple animal viruses
Publisher: Cold Spring Harbor Laboratory
Date: 03-07-2023
DOI: 10.1101/2023.07.02.547368
Abstract: The cell entry mechanism of SARS-CoV-2, the causative agent of the COVID-19 pandemic, is not fully understood. Most animal viruses hijack cellular endocytic pathways as an entry route into the cell. Here, we show that in cells that do not express serine proteases such as TMPRSS2, genetic depletion of all dynamin isoforms blocked the uptake and strongly reduced infection with SARS-CoV-2 and its variant Delta. However, increasing the viral loads partially and dose-dependently restored infection via a thus far uncharacterized entry mechanism. Ultrastructural analysis by electron microscopy showed that this dynamin-independent endocytic processes appeared as 150-200 nm non-coated invaginations and was efficiently used by numerous mammalian viruses, including alphaviruses, influenza, vesicular stomatitis, bunya, adeno, vaccinia, and rhinovirus. Both the dynamin-dependent and dynamin-independent infection of SARS-CoV-2 required a functional actin cytoskeleton. In contrast, the alphavirus Semliki Forest virus, which is smaller in diameter, required actin only for the dynamin-independent entry. The presence of TMPRSS2 protease rescued SARS-CoV-2 infection in the absence of dynamins. Collectively, these results indicate that some viruses such as canine parvovirus and SARS-CoV-2 mainly rely on dynamin for endocytosis-dependent infection, while other viruses can efficiently bypass this requirement harnessing an alternative infection entry route dependent on actin.
Hericerin derivatives from Hericium erinaceus exert BDNF-like neurotrophic activity in central hippocampal neurons and enhance memory
Publisher: Cold Spring Harbor Laboratory
Date: 28-08-2020
DOI: 10.1101/2020.08.28.271676
Abstract: The traditional medicinal mushroom Hericium erinaceus has long been known for enhancing the peripheral nerve regeneration through targeting nerve growth factor (NGF) neurotrophic activity. It was also reported to protect against ageing-dependent cognitive decline in wildtype and in Alzheimer’s disease mouse models suggesting a yet to be defined action on neurons of the central nervous system. Here, we purified and identified biologically active compounds from H. erinaceus , based on their ability to promote neurite outgrowth in hippoc al neurons. N -de phenylethyl isohericerin (NDPIH), an isoindoline compound from this mushroom together with its hydrophobic derivative hericene A, were highly potent in inducing extensive axon outgrowth and neurite branching in the absence of serum demonstrating high neurotropic activity. NDPIH also induced enlarged growth cones suggestive of a brain-derived neurotrophic factor (BDNF)-like activity. Pharmacological inhibition of tropomyosin receptor kinase B (TrkB) by ANA12 prevented NDPIH-induced neurotrophic activity providing evidence that NDPIH acts via TrkB receptors to mediate its neurotrophic effect in central neurons. Finally, in vivo treatment with H. erinaceus crude extract and hericene A significantly increased BDNF and downstream pathway and enhanced learning and memory in the novel object recognition memory test. Our results suggest that hericene A can promote BDNF-like activity in neurons in vitro and in vivo thereby enhancing recognition memory.
Non–beta blocker enantiomers of propranolol and atenolol inhibit vasculogenesis in infantile hemangioma
Publisher: American Society for Clinical Investigation
Date: 02-2022
DOI: 10.1172/JCI151109
Actin- and Dynamin-Dependent Maturation of Bulk Endocytosis Restores Neurotransmission following Synaptic Depletion
Publisher: Public Library of Science (PLoS)
Date: 22-05-2012
Erratum: In vivo single-molecule imaging of syntaxin1A reveals polyphosphoinositide- and activity-dependent trapping in presynaptic nanoclusters
Publisher: Springer Science and Business Media LLC
Date: 09-02-2017
DOI: 10.1038/NCOMMS14492
Abstract: Nature Communications 7: Article number: 13660 (2016) Published: 16 December 2016 Updated: 9 February 2017 This Article was originally published with an incorrect publication date. The paper was due to be published on 3 Jan 2017, but due to an error was published earlier on the 16 Dec 2016. The publication date in both the PDF and HTML versions of the paper has been updated to reflect this.
Complement receptor C3aR1 controls neutrophil mobilization following spinal cord injury through physiological antagonism of CXCR2
Publisher: American Society for Clinical Investigation
Date: 02-05-2019
Correction: Publisher Correction: In vivo single-molecule imaging of syntaxin1A reveals polyphosphoinositide- and activity-dependent trapping in presynaptic nanoclusters
Publisher: Springer Science and Business Media LLC
Date: 10-04-2018
DOI: 10.1038/NCOMMS16191
Abstract: Nature Communications 7: Article number: 13660 (2016) Published: 16 December 2016 Updated: 10 April 2018 The original HTML version of this Article had an incorrect volume number of 8 it should have been 7. This has now been corrected in the HTML the PDF version of the Article was correct from the time of publication.
DDHD2 interacts with STXBP1 to mediate long-term memory via the generation of myristic acid
Publisher: Cold Spring Harbor Laboratory
Date: 11-05-2023
DOI: 10.1101/2023.05.11.540316
Abstract: The phospholipid and free fatty acid (FFA) composition of neuronal membranes plays a crucial role in learning and memory, but the mechanisms through which neuronal activity affects the brain’s lipid landscape remain largely unexplored. Saturated FFAs, particularly myristic acid (C14:0), strongly increase during neuronal stimulation and memory acquisition, suggesting the involvement of phospholipase A1 (PLA1) activity in synaptic plasticity. Here, we show that genetic ablation of the DDHD2 isoform of PLA1 in mice reduced memory performance in reward-based learning and spatial memory models prior to the development of neuromuscular deficits, and markedly reduced saturated FFAs across the brain. DDHD2 was shown to bind to the key synaptic protein STXBP1. Using STXBP1/2 knockout neurosecretory cells and a haploinsufficient STXBP1 +/- mouse model of STXBP1 encephalopathy that is also associated with intellectual disability and motor dysfunction, we show that STXBP1 controls the targeting of DDHD2 to the plasma membrane and the generation of saturated FFAs in the brain. Our findings suggest key roles for DDHD2 and STXBP1 in the lipid metabolism underlying synaptic plasticity, learning and memory.
Spatial redistribution of neurosecretory vesicles upon stimulation accelerates their directed transport to the plasma membrane
Publisher: Public Library of Science (PLoS)
Date: 16-03-2022
DOI: 10.1371/JOURNAL.PONE.0264521
Abstract: Through the integration of results from an imaging analysis of intracellular trafficking of labelled neurosecretory vesicles in chromaffin cells, we develop a Markov state model to describe their transport and binding kinetics. Our simulation results indicate that a spatial redistribution of neurosecretory vesicles occurs upon secretagogue stimulation leading vesicles to the plasma membrane where they undergo fusion thereby releasing adrenaline and noradrenaline. Furthermore, we find that this redistribution alone can explain the observed up-regulation of vesicle transport upon stimulation and its directional bias towards the plasma membrane. Parameter fitting indicates that in the deeper compartment within the cell, vesicle transport is asymmetric and characterised by a bias towards the plasma membrane.
Ancestral SARS-CoV-2, but not Omicron, replicates less efficiently in primary pediatric nasal epithelial cells
Publisher: Public Library of Science (PLoS)
Date: 08-2022
DOI: 10.1371/JOURNAL.PBIO.3001728
Abstract: Children typically experience more mild symptoms of Coronavirus Disease 2019 (COVID-19) when compared to adults. There is a strong body of evidence that children are also less susceptible to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection with the ancestral viral isolate. However, the emergence of SARS-CoV-2 variants of concern (VOCs) has been associated with an increased number of pediatric infections. Whether this is the result of widespread adult vaccination or fundamental changes in the biology of SARS-CoV-2 remain to be determined. Here, we use primary nasal epithelial cells (NECs) from children and adults, differentiated at an air–liquid interface to show that the ancestral SARS-CoV-2 replicates to significantly lower titers in the NECs of children compared to those of adults. This was associated with a heightened antiviral response to SARS-CoV-2 in the NECs of children. Importantly, the Delta variant also replicated to significantly lower titers in the NECs of children. This trend was markedly less pronounced in the case of Omicron. It is also striking to note that, at least in terms of viral RNA, Omicron replicated better in pediatric NECs compared to both Delta and the ancestral virus. Taken together, these data show that the nasal epithelium of children supports lower infection and replication of ancestral SARS-CoV-2, although this may be changing as the virus evolves.
Dynamin Inhibition Blocks Botulinum Neurotoxin Type A Endocytosis in Neurons and Delays Botulism
Publisher: Elsevier BV
Date: 10-2011
G-protein-coupled inwardly rectifying potassium (GIRK) channel activation by the p75 neurotrophin receptor is required for amyloid beta toxicity
Publisher: Frontiers Media SA
Date: 08-08-2017
p75 neurotrophin receptor mediates neuronal cell death by activating GIRK channels through phosphatidylinositol 4,5-bisphosphate
Publisher: Society for Neuroscience
Date: 02-01-2008
DOI: 10.1523/JNEUROSCI.2699-07.2008
Abstract: The pan neurotrophin receptor p75 NTR signals programmed cell death both during nervous system development and after neural trauma and disease in the adult. However, the molecular pathways by which death is mediated remain poorly understood. Here, we show that this cell death is initiated by activation of G-protein-coupled inwardly rectifying potassium (GIRK/Kir3) channels and a consequent potassium efflux. Death signals stimulated by neurotrophin-mediated cleavage of p75 NTR activate GIRK channels through the generation and binding of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 /PIP2] to GIRK channels. Both GIRK channel activity and p75 NTR -mediated neuronal death are inhibited by sequestration of PtdIns(4,5)P 2 and application of GIRK channel inhibitors, whereas pertussis toxin treatment has no effect. Thus, p75 NTR activates GIRK channels without the need for G i/o -proteins. Our results demonstrate a novel mode of activation of GIRK channels, representing an early step in the p75 NTR -mediated cell death pathway and suggesting a function for these channels during nervous system development.
Arachidonic acid potentiates exocytosis and allows neuronal SNARE complex to interact with Munc18a
Publisher: Wiley
Date: 11-2006
DOI: 10.1111/J.1471-4159.2006.04286.X
Abstract: Neuronal communication relies on the fusion of neurotransmitter-containing vesicles with the neuronal plasma membrane. Recent genetic studies have highlighted the critical role played by polyunsaturated fatty acids in neurotransmission, however, there is little information available about which fatty acids act on exocytosis and, more importantly, by what mechanism. We have used permeabilized chromaffin cells to screen various fatty acids of the n-3 and n-6 series for their acute effects on exocytosis. We have demonstrated that an n-6 series polyunsaturated fatty acid, arachidonic acid, potentiates secretion from intact neurosecretory cells regardless of the secretagogue used. We have shown that arachidonic acid dose dependently increases soluble NSF attachment protein receptor complex formation in chromaffin cells and bovine cortical brain extracts and that a non-hydrolysable analogue of arachidonic acid causes a similar increase in SNARE complex formation. This prompted us to examine the effect of arachidonic acid on SNARE protein interactions with Munc18a, a protein known to prevent Syntaxin1a engagement into the SNARE complex in vitro. In the presence of arachidonic acid, we show that Munc18a can interact with the neuronal SNARE complex in a dose-dependent manner. We further demonstrate that arachidonic acid directly interacts with Syntaxin1a.
Dynamin1 long- and short-tail isoforms exploit distinct recruitment and spatial patterns to form endocytic nanoclusters
Publisher: Research Square Platform LLC
Date: 14-03-2023
DOI: 10.21203/RS.3.RS-2641489/V1
Abstract: Endocytosis requires a coordinated framework of molecular interactions that ultimately lead to the fission of nascent endosomes. How cytosolic proteins, such as dynamin, timely concentrate at discrete sites that are sparsely distributed across the plasma membrane remains poorly understood. Two dynamin 1 (Dyn1) major splice variants differ by the length of their C-terminal proline-rich region (short-tail and long-tail). Using sptPALM in PC12 cells, neurons and MEF cells, we demonstrate that short-tail Dyn1 isoforms Dyn1ab and Dyn1bb display an activity-dependent recruitment to the membrane, promptly followed by concentration into nanoclusters. These nanoclusters were sensitive to both Calcineurin and Dyn1 GTPase inhibitors, and were larger, denser, and more numerous than that of long-tail isoform Dyn1aa. Spatiotemporal modelling confirmed that Dyn1 isoforms perform distinct search patterns and undergo dimensional reduction to generate endocytic nanoclusters, with short-tail isoforms more robustly exploiting lateral trapping in the generation of nanoclusters compared to long-tail isoform Dyn1aa.
Frontotemporal dementia mutant tau (P301L) locks Fyn in an open, active conformation conducive to nanoclustering
Publisher: Cold Spring Harbor Laboratory
Date: 06-09-2020
DOI: 10.1101/2020.09.04.282855
Abstract: Fyn is a Src kinase that controls critical signalling cascades and its postsynaptic enrichment underpins synaptotoxicity in Alzheimer’s disease (AD) and frontotemporal dementia (FTLD-tau). Previously, we found that pathogenic FTLD tau mutant (P301L) expression promotes aberrant trapping of Fyn in nanoclusters within hippoc al dendrites via an unknown mechanism (Padmanabhan et al., 2019). Here, we imaged Fyn-mEos2 using single particle tracking photoactivated localization microscopy (sptPALM) to demonstrate that nanoclustering of Fyn in hippoc al dendrites is promoted by Fyn’s open, primed conformation. Disrupting the auto-inhibitory, closed conformation of Fyn through phospho-inhibition, and perturbation of Fyn’s SH3 domain increases, Fyn’s nanoscale trapping. However, inhibition of Fyn’s catalytic domain has no impact on its mobility. Tau-P301L promotes Fyn lateral trapping via Fyn opening and ensuing increased catalytic activation. Pathogenic tau may therefore drive synaptotoxicity by locking Fyn in an open, catalytically active conformation, leading to postsynaptic entrapment and aberrant signalling cascades.
Flux of signalling endosomes undergoing axonal retrograde transport is encoded by presynaptic activity and TrkB
Publisher: Springer Science and Business Media LLC
Date: 30-09-2016
DOI: 10.1038/NCOMMS12976
Abstract: Axonal retrograde transport of signalling endosomes from the nerve terminal to the soma underpins survival. As each signalling endosome carries a quantal amount of activated receptors, we hypothesized that it is the frequency of endosomes reaching the soma that determines the scale of the trophic signal. Here we show that upregulating synaptic activity markedly increased the flux of plasma membrane-derived retrograde endosomes (labelled using cholera toxin subunit-B: CTB) in hippoc al neurons cultured in microfluidic devices, and live Drosophila larval motor neurons. Electron and super-resolution microscopy analyses revealed that the fast-moving sub-diffraction-limited CTB carriers contained the TrkB neurotrophin receptor, transiently activated by synaptic activity in a BDNF-independent manner. Pharmacological and genetic inhibition of TrkB activation selectively prevented the coupling between synaptic activity and the retrograde flux of signalling endosomes. TrkB activity therefore controls the encoding of synaptic activity experienced by nerve terminals, digitalized as the flux of retrogradely transported signalling endosomes.
Location, Location, Location: Protein kinase nanoclustering for optimized signalling output
Publisher: Cold Spring Harbor Laboratory
Date: 29-09-2023
Activity-driven relaxation of the cortical actomyosin II network synchronizes Munc18-1-dependent neurosecretory vesicle docking
Publisher: Springer Science and Business Media LLC
Date: 24-02-2015
DOI: 10.1038/NCOMMS7297
Abstract: In neurosecretory cells, secretory vesicles (SVs) undergo Ca(2+)-dependent fusion with the plasma membrane to release neurotransmitters. How SVs cross the dense mesh of the cortical actin network to reach the plasma membrane remains unclear. Here we reveal that, in bovine chromaffin cells, SVs embedded in the cortical actin network undergo a highly synchronized transition towards the plasma membrane and Munc18-1-dependent docking in response to secretagogues. This movement coincides with a translocation of the cortical actin network in the same direction. Both effects are abolished by the knockdown or the pharmacological inhibition of myosin II, suggesting changes in actomyosin-generated forces across the cell cortex. Indeed, we report a reduction in cortical actin network tension elicited on secretagogue stimulation that is sensitive to myosin II inhibition. We reveal that the cortical actin network acts as a 'casting net' that undergoes activity-dependent relaxation, thereby driving tethered SVs towards the plasma membrane where they undergo Munc18-1-dependent docking.
Trapping of Syntaxin1a in Presynaptic Nanoclusters by a Clinically Relevant General Anesthetic
Publisher: Elsevier BV
Date: 2018
DOI: 10.1016/J.CELREP.2017.12.054
Abstract: Propofol is the most commonly used general anesthetic in humans. Our understanding of its mechanism of action has focused on its capacity to potentiate inhibitory systems in the brain. However, it is unknown whether other neural mechanisms are involved in general anesthesia. Here, we demonstrate that the synaptic release machinery is also a target. Using single-particle tracking photoactivation localization microscopy, we show that clinically relevant concentrations of propofol and etomidate restrict syntaxin1A mobility on the plasma membrane, whereas non-anesthetic analogs produce the opposite effect and increase syntaxin1A mobility. Removing the interaction with the t-SNARE partner SNAP-25 abolishes propofol-induced syntaxin1A confinement, indicating that syntaxin1A and SNAP-25 together form an emergent drug target. Impaired syntaxin1A mobility and exocytosis under propofol are both rescued by co-expressing a truncated syntaxin1A construct that interacts with SNAP-25. Our results suggest that propofol interferes with a step in SNARE complex formation, resulting in non-functional syntaxin1A nanoclusters.
MOLECULAR VIDEOGAMING: SUPER-RESOLVED TRAJECTORY-BASED NANOCLUSTERING ANALYSIS USING SPATIO-TEMPORAL INDEXING
Publisher: Cold Spring Harbor Laboratory
Date: 09-09-2021
DOI: 10.1101/2021.09.08.459552
Abstract: Single-molecule localization microscopy (SMLM) techniques are emerging as vital tools to unravel the nanoscale world of living cells. However, current analysis methods primarily focus on defining spatial nanoclusters based on detection density, but neglect important temporal information such as cluster lifetime and recurrence in “hotspots” on the plasma membrane. Spatial indexing is widely used in videogames to effectively detect interactions between moving geometric objects. Here, we use the R-tree spatial indexing algorithm to perform SMLM data analysis and determine whether the bounding boxes of in idual molecular trajectories overlap, as a measure of their potential membership in nanoclusters. Extending the spatial indexing into the time dimension allows unique resolution of spatial nanoclusters into multiple spatiotemporal clusters. We have validated this approach using synthetic and SMLM-derived data. Quantitative characterization of recurring nanoclusters allowed us to demonstrate that both syntaxin1a and Munc18-1 molecules transiently cluster in hotspots on the neurosecretory plasma membrane, offering unprecedented insights into the dynamics of these protein which are essential to neuronal communication. This new analytical tool, named Nanoscale Spatiotemporal Indexing Clustering (NASTIC), has been implemented as a free and open-source Python graphic user interface.
Engineering stable peptide toxins by means of backbone cyclization: Stabilization of the α-conotoxin MII
Publisher: Proceedings of the National Academy of Sciences
Date: 14-09-2005
Abstract: Conotoxins (CTXs), with their exquisite specificity and potency, have recently created much excitement as drug leads. However, like most peptides, their beneficial activities may potentially be undermined by susceptibility to proteolysis in vivo . By cyclizing the α-CTX MII by using a range of linkers, we have engineered peptides that preserve their full activity but have greatly improved resistance to proteolytic degradation. The cyclic MII analogue containing a seven-residue linker joining the N and C termini was as active and selective as the native peptide for native and recombinant neuronal nicotinic acetylcholine receptor subtypes present in bovine chromaffin cells and expressed in Xenopus oocytes, respectively. Furthermore, its resistance to proteolysis against a specific protease and in human plasma was significantly improved. More generally, to our knowledge, this report is the first on the cyclization of disulfide-rich toxins. Cyclization strategies represent an approach for stabilizing bioactive peptides while keeping their full potencies and should boost applications of peptide-based drugs in human medicine.
A dominant-negative SOX18 mutant disrupts multiple regulatory layers essential to transcription factor activity
Publisher: Cold Spring Harbor Laboratory
Date: 12-11-2020
DOI: 10.1101/2020.11.11.378968
Abstract: Few genetically dominant mutations involved in human disease have been fully explained at the molecular level. In cases where the mutant gene encodes a transcription factor, the dominant-negative mode of action of the mutant protein is particularly poorly understood. Here, we studied the genome-wide mechanism underlying a dominant-negative form of the SOX18 transcription factor (SOX18 RaOp ) responsible for both the classical mouse mutant Ragged opossum and the human genetic disorder Hypotrichosis-Lymphedema-Telangiectasia-Renal Syndrome. Combing three single-molecule imaging assays in living cells, we found that SOX18 RaOp disrupts the system through an accumulation of molecular interferences which impair several functional properties of the wild-type SOX18 protein, including its chromatin-binding dynamics. The dominant-negative effect is further lified by recruiting the interactome of its wild-type counterpart, which perturbs regulatory nodes such as SOX7 and MEF2C. Our findings explain in unprecedented detail the multi-layered process that underpins the molecular etiology of dominant-negative transcription factor function.
Inhibition of PIKfyve by YM-201636 Dysregulates Autophagy and Leads to Apoptosis-Independent Neuronal Cell Death
Publisher: Public Library of Science (PLoS)
Date: 27-03-2013
Saturated free fatty acids and association with memory formation
Publisher: Springer Science and Business Media LLC
Date: 08-06-2021
DOI: 10.1038/S41467-021-23840-3
Abstract: Polyunsaturated free fatty acids (FFAs) such as arachidonic acid, released by phospholipase activity on membrane phospholipids, have long been considered beneficial for learning and memory and are known modulators of neurotransmission and synaptic plasticity. However, the precise nature of other FFA and phospholipid changes in specific areas of the brain during learning is unknown. Here, using a targeted lipidomics approach to characterise FFAs and phospholipids across the rat brain, we demonstrated that the highest concentrations of these analytes were found in areas of the brain classically involved in fear learning and memory, such as the amygdala. Auditory fear conditioning led to an increase in saturated (particularly myristic and palmitic acids) and to a lesser extent unsaturated FFAs (predominantly arachidonic acid) in the amygdala and prefrontal cortex. Both fear conditioning and changes in FFA required activation of NMDA receptors. These results suggest a role for saturated FFAs in memory acquisition.
Author Correction: Super-resolved trajectory-derived nanoclustering analysis using spatiotemporal indexing
Publisher: Springer Science and Business Media LLC
Date: 25-07-2023
A growth hormone receptor SNP promotes lung cancer by impairment of SOCS2-mediated degradation
Publisher: Springer Science and Business Media LLC
Date: 02-10-2017
DOI: 10.1038/ONC.2017.352
Decatenation checkpoint-defective melanomas are dependent on PI3K for survival
Publisher: Wiley
Date: 23-06-2014
DOI: 10.1111/PCMR.12268
Abstract: Melanoma cell lines are commonly defective for the G2-phase cell cycle checkpoint that responds to incomplete catenation of the replicated chromosomes. Here, we demonstrate that melanomas defective for this checkpoint response are less sensitive to genotoxic stress, suggesting that the defective cell lines compensated for the checkpoint loss by increasing their ability to cope with DNA damage. We performed an siRNA kinome screen to identify kinases responsible and identified PI3K pathway components. Checkpoint-defective cell lines were three-fold more sensitive to small molecule inhibitors of PI3K. The PI3K inhibitor PF-05212384 promoted apoptosis in the checkpoint-defective lines, and the increased sensitivity to PI3K inhibition correlated with increased levels of activated Akt. This work demonstrates that increased PI3K pathway activation is a necessary adaption for the continued viability of melanomas with a defective decatenation checkpoint.
Increased Polyubiquitination and Proteasomal Degradation of a Munc18-1 Disease-Linked Mutant Causes Temperature-Sensitive Defect in Exocytosis
Publisher: Elsevier BV
Date: 10-2014
DOI: 10.1016/J.CELREP.2014.08.059
Abstract: Munc18-1 is a critical component of the core machinery controlling neuroexocytosis. Recently, mutations in Munc18-1 leading to the development of early infantile epileptic encephalopathy have been discovered. However, which degradative pathway controls Munc18-1 levels and how it impacts on neuroexocytosis in this pathology is unknown. Using neurosecretory cells deficient in Munc18, we show that a disease-linked mutation, C180Y, renders the protein unstable at 37°C. Although the mutated protein retains its function as t-SNARE chaperone, neuroexocytosis is impaired, a defect that can be rescued at a lower permissive temperature. We reveal that Munc18-1 undergoes K48-linked polyubiquitination, which is highly increased by the mutation, leading to proteasomal, but not lysosomal, degradation. Our data demonstrate that functional Munc18-1 levels are controlled through polyubiquitination and proteasomal degradation. The C180Y disease-causing mutation greatly potentiates this degradative pathway, rendering Munc18-1 unable to facilitate neuroexocytosis, a phenotype that is reversed at a permissive temperature.
VAMP2/Munc18-1 domain 3a interaction controls the nanoscale reorganization underpinning vesicular priming
Publisher: Cold Spring Harbor Laboratory
Date: 11-09-2023
Munc18-1 is a molecular chaperone for α-synuclein, controlling its self-replicating aggregation
Publisher: Rockefeller University Press
Date: 05-09-2016
Abstract: Munc18-1 is a key component of the exocytic machinery that controls neurotransmitter release. Munc18-1 heterozygous mutations cause developmental defects and epileptic phenotypes, including infantile epileptic encephalopathy (EIEE), suggestive of a gain of pathological function. Here, we used single-molecule analysis, gene-edited cells, and neurons to demonstrate that Munc18-1 EIEE-causing mutants form large polymers that coaggregate wild-type Munc18-1 in vitro and in cells. Surprisingly, Munc18-1 EIEE mutants also form Lewy body–like structures that contain α-synuclein (α-Syn). We reveal that Munc18-1 binds α-Syn, and its EIEE mutants coaggregate α-Syn. Likewise, removal of endogenous Munc18-1 increases the aggregative propensity of α-SynWT and that of the Parkinson’s disease–causing α-SynA30P mutant, an effect rescued by Munc18-1WT expression, indicative of chaperone activity. Coexpression of the α-SynA30P mutant with Munc18-1 reduced the number of α-SynA30P aggregates. Munc18-1 mutations and haploinsufficiency may therefore trigger a pathogenic gain of function through both the corruption of native Munc18-1 and a perturbed chaperone activity for α-Syn leading to aggregation-induced neurodegeneration.
Phosphoinositide 3-kinase δ regulates membrane fission of Golgi carriers for selective cytokine secretion
Publisher: Rockefeller University Press
Date: 13-09-2010
Abstract: Phosphoinositide 3-kinase (PI3K) p110 isoforms are membrane lipid kinases classically involved in signal transduction. Lipopolysaccharide (LPS)-activated macrophages constitutively and abundantly secrete proinflammatory cytokines including tumor necrosis factor-α (TNF). Loss of function of the p110δ isoform of PI3K using inhibitors, RNA-mediated knockdown, or genetic inactivation in mice abolishes TNF trafficking and secretion, trapping TNF in tubular carriers at the trans-Golgi network (TGN). Kinase-active p110δ localizes to the Golgi complex in LPS-activated macrophages, and TNF is loaded into p230-labeled tubules, which cannot undergo fission when p110δ is inactivated. Similar blocks in fission of these tubules and in TNF secretion result from inhibition of the guanosine triphosphatase dynamin 2. These findings demonstrate a new function for p110δ as part of the membrane fission machinery required at the TGN for the selective trafficking and secretion of cytokines in macrophages.
Targeting membrane trafficking in infection prophylaxis: dynamin inhibitors
Publisher: Elsevier BV
Date: 02-2013
DOI: 10.1016/J.TCB.2012.10.007
Abstract: Many pathogens hijack existing endocytic trafficking pathways to exert toxic effects in cells. Dynamin controls various steps of the intoxication process used by numerous pathogenic bacteria, viruses, and toxins. Targeting dynamin with pharmaceutical compounds may therefore have prophylactic potential. Here we review the growing number of pathogens requiring dynamin-dependent trafficking to intoxicate cells, outline the mode of internalization that leads to their pathogenicity, and highlight the protective effect of pharmacological and genetic approaches targeting dynamin function. We also assess the methodologies used to investigate the role of dynamin in the intoxication process and discuss the validity and potential pitfalls of using dynamin inhibitors (DIs) as therapeutics.
An Acto-Myosin II Constricting Ring Initiates the Fission of Activity-Dependent Bulk Endosomes in Neurosecretory Cells
Publisher: Society for Neuroscience
Date: 28-01-2015
DOI: 10.1523/JNEUROSCI.3228-14.2015
Abstract: Activity-dependent bulk endocytosis allows neurons to internalize large portions of the plasma membrane in response to stimulation. However, whether this critical type of compensatory endocytosis is unique to neurons or also occurs in other excitable cells is currently unknown. Here we used fluorescent 70 kDa dextran to demonstrate that secretagogue-induced bulk endocytosis also occurs in bovine chromaffin cells. The relatively large size of the bulk endosomes found in this model allowed us to investigate how the neck of the budding endosomes constricts to allow efficient recruitment of the fission machinery. Using time-lapse imaging of Lifeact–GFP-transfected chromaffin cells in combination with fluorescent 70 kDa dextran, we detected acto-myosin II rings surrounding dextran-positive budding endosomes. Importantly, these rings were transient and contracted before disappearing, suggesting that they might be involved in restricting the size of the budding endosome neck. Based on the complete recovery of dextran fluorescence after photobleaching, we demonstrated that the actin ring-associated budding endosomes were still connected with the extracellular fluid. In contrast, no such recovery was observed following the constriction and disappearance of the actin rings, suggesting that these structures were pinched-off endosomes. Finally, we showed that the rings were initiated by a circular array of phosphatidylinositol(4,5)bisphosphate microdomains, and that their constriction was sensitive to both myosin II and dynamin inhibition. The acto-myosin II rings therefore play a key role in constricting the neck of budding bulk endosomes before dynamin-dependent fission from the plasma membrane of neurosecretory cells.
Actomyosin-II Proactively Shields Axons of the Central Nervous System from Mild Mechanical Stress
Publisher: Cold Spring Harbor Laboratory
Date: 13-08-2023
DOI: 10.1101/2023.08.09.552549
Abstract: Pan et al found that actomyosin-II-driven radial contractility underpins the resilience of central axons to mild mechanical stress by suppressing the propagation and firing of injurious Ca 2+ waves. Boosting actomyosin-II activity alleviates axon degeneration in mice with traumatic brain injury. Traumatic brain injury (TBI) remains a significant and unmet health challenge. However, our understanding of how neurons, particularly their fragile axons, withstand the abrupt mechanical impacts within the central nervous system remains largely unknown. Using a microfluidic device applying discrete levels of transverse forces to axons, we identified the stress levels that most axons could resist and explored their instant responses at nanoscale resolution. Mild stress induces rapid and reversible axon beading, driven by actomyosin-II-dependent radial contraction, which restricts the spreading and bursting of stress-induced Ca 2+ waves. More severe stress causes irreversible focal swelling and Ca 2+ overload, ultimately leading to focal axonal swelling and degeneration. Up-regulating actomyosin-II activity prevented the progression of initial injury in vivo , protecting commissural axons from degeneration in a mice TBI model. Our study established a scalable axon injury model and uncovered the critical roles of actomyosin-II in shielding neurons against detrimental mechanical stress.
Surface SV2A-Syt1 nanoclusters act as a sequestration hub that limits dynamin-1 recruitment and targeting to recycling synaptic vesicles
Publisher: Cold Spring Harbor Laboratory
Date: 13-12-2022
DOI: 10.1101/2022.12.12.520075
Abstract: Following exocytosis, the recapture of plasma membrane-stranded vesicular proteins into recycling synaptic vesicles (SVs) is essential for sustaining neurotransmission. Surface clustering of vesicular proteins has been postulated as a ‘pre-assembly’ mechanism for endocytosis – ensuring high-fidelity retrieval. Here, we used single-molecule imaging to examine the nanoclustering of synaptotagmin-1 (Syt1) and synaptic vesicle protein 2A (SV2A) in hippoc al neurons. Syt1 forms surface nanoclusters through interaction of its C2B domain with SV2A, that are sensitive to mutations in this domain (Syt1 K326A/K328A ) and knocking down SV2A. SV2A co-cluster with Syt1 and blocking SV2A’s cognate interaction with Syt1 (SV2A T84A ) also decreased SV2A clustering. Surprisingly, impairing SV2A-Syt1 nanoclustering enhanced plasma membrane recruitment of key endocytic protein dynamin-1, leading to accelerated Syt1 endocytosis, altered intracellular sorting and decreased trafficking of Syt1 to Rab5-positive endocytic compartments. SV2A-Syt1 surface nanoclusters therefore negatively regulate the rate of their own re-entry into recycling SVs by controlling the recruitment of the endocytic machinery.
Brevenal Inhibits Pacific Ciguatoxin-1B-Induced Neurosecretion from Bovine Chromaffin Cells
Publisher: Public Library of Science (PLoS)
Date: 20-10-2008
Myosin VI small insert isoform maintains exocytosis by tethering secretory granules to the cortical actin
Publisher: Rockefeller University Press
Date: 04-02-2013
Abstract: Before undergoing neuroexocytosis, secretory granules (SGs) are mobilized and tethered to the cortical actin network by an unknown mechanism. Using an SG pull-down assay and mass spectrometry, we found that myosin VI was recruited to SGs in a Ca2+-dependent manner. Interfering with myosin VI function in PC12 cells reduced the density of SGs near the plasma membrane without affecting their biogenesis. Myosin VI knockdown selectively impaired a late phase of exocytosis, consistent with a replenishment defect. This exocytic defect was selectively rescued by expression of the myosin VI small insert (SI) isoform, which efficiently tethered SGs to the cortical actin network. These myosin VI SI–specific effects were prevented by deletion of a c-Src kinase phosphorylation DYD motif, identified in silico. Myosin VI SI thus recruits SGs to the cortical actin network, potentially via c-Src phosphorylation, thereby maintaining an active pool of SGs near the plasma membrane.
Nanoscale organization of the pre-synapse: Tracking the neurotransmitter release machinery
Publisher: Elsevier BV
Date: 08-2022
DOI: 10.1016/J.CONB.2022.102576
Abstract: Chemical communication is underpinned by the fusion of neurotransmitter-containing synaptic vesicles with the plasma membrane at active zones. With the advent of super-resolution microscopy, the door is now opened to unravel the dynamic remodeling of synapses underpinning learning and memory. Imaging proteins with conventional light microscopy cannot provide submicron information vital to determining the nanoscale organization of the synapse. We will first review the current super-resolution microscopy techniques available to investigate the localization and movement of synaptic proteins and how they have been applied to visualize the synapse. We discuss the new techniques and analytical approaches have provided comprehensive insights into synaptic organization in various model systems. Finally, this review provides a brief update on how these super-resolution techniques and analyses have opened the way to a much greater understanding of the synapse, the fusion and compensatory endocytosis machinery.
Towards a revised framework of modified time series InSAR for mapping land deformation
Publisher: Springer Science and Business Media LLC
Date: 09-2020
SARS-CoV-2 infection and viral fusogens cause neuronal and glial fusion that compromises neuronal activity
Publisher: American Association for the Advancement of Science (AAAS)
Date: 09-06-2023
Abstract: Numerous viruses use specialized surface molecules called fusogens to enter host cells. Many of these viruses, including the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), can infect the brain and are associated with severe neurological symptoms through poorly understood mechanisms. We show that SARS-CoV-2 infection induces fusion between neurons and between neurons and glia in mouse and human brain organoids. We reveal that this is caused by the viral fusogen, as it is fully mimicked by the expression of the SARS-CoV-2 spike (S) protein or the unrelated fusogen p15 from the baboon orthoreovirus. We demonstrate that neuronal fusion is a progressive event, leads to the formation of multicellular syncytia, and causes the spread of large molecules and organelles. Last, using Ca 2+ imaging, we show that fusion severely compromises neuronal activity. These results provide mechanistic insights into how SARS-CoV-2 and other viruses affect the nervous system, alter its function, and cause neuropathology.
The janus faces of botulinum neurotoxin: Sensational medicine and deadly biological weapon
Publisher: Wiley
Date: 2007
DOI: 10.1002/JNR.21171
Abstract: The botulinum neurotoxins are the most dangerous toxins known (BoNTs serotypes A-G) and induce profound flaccid neuromuscular paralysis by blocking nerve-muscle communication. Poisoned motoneurons react by emitting a sprouting network known to establish novel functional synapses with the abutting muscle fiber. Understanding how our motoneurons are capable of bypassing such transmission blockade, thereby overcoming paralysis, by an astonishing display of plasticity is one of the research goals that have numerous therapeutic ramifications. This Mini-Review aims at giving a brief update on the recent discoveries regarding the molecular mechanism of botulinum toxins intoxication. Curing botulism still is a challenge once the toxin has found his way inside motoneurons. In view of the potential use of botulinum toxins as biological weapon, more research is needed to find efficient ways of curing this disease.
p110δ PI3-Kinase Inhibition Perturbs APP and TNFα Trafficking, Reduces Plaque Burden, Dampens Neuroinflammation, and Prevents Cognitive Decline in an Alzheimer's Disease Mouse Model
Publisher: Society for Neuroscience
Date: 30-07-2019
DOI: 10.1523/JNEUROSCI.0674-19.2019
Abstract: Alzheimer's disease (AD) is associated with the cleavage of the amyloid precursor protein (APP) to produce the toxic amyloid-β (Aβ) peptide. Accumulation of Aβ, together with the concomitant inflammatory response, ultimately leads to neuronal death and cognitive decline. Despite AD progression being underpinned by both neuronal and immunological components, therapeutic strategies based on dual targeting of these systems remains unexplored. Here, we report that inactivation of the p110δ isoform of phosphoinositide 3-kinase (PI3K) reduces anterograde axonal trafficking of APP in hippoc al neurons and d ens secretion of the inflammatory cytokine tumor necrosis factor-alpha by microglial cells in the familial AD APP swe /PS1 ΔE9 (APP/PS1) mouse model. Moreover, APP/PS1 mice with kinase-inactive PI3Kδ (δ D910A ) had reduced Aβ peptides levels and plaques in the brain and an abrogated inflammatory response compared with APP/PS1 littermates. Mechanistic investigations reveal that PI3Kδ inhibition decreases the axonal transport of APP by eliciting the formation of highly elongated tubular-shaped APP-containing carriers, reducing the levels of secreted Aβ peptide. Importantly, APP/PS1/δ D910A mice exhibited no spatial learning or memory deficits. Our data highlight inhibition of PI3Kδ as a new approach to protect against AD pathology due to its dual action of d ening microglial-dependent neuroinflammation and reducing plaque burden by inhibition of neuronal APP trafficking and processing. SIGNIFICANCE STATEMENT During Alzheimer's disease (AD), the accumulation of the toxic amyloid-β (Aβ) peptide in plaques is associated with a chronic excessive inflammatory response. Uncovering new drug targets that simultaneously reduce both Aβ plaque load and neuroinflammation holds therapeutic promise. Using a combination of genetic and pharmacological approaches, we found that the p110δ isoform of phosphoinositide 3-kinase (PI3K) is involved in anterograde trafficking of the amyloid precursor protein in neurons and in the secretion of tumor necrosis factor-alpha from microglial cells. Genetic inactivation of PI3Kδ reduces Aβ plaque deposition and abrogates the inflammatory response, resulting in a complete rescue of the life span and spatial memory performance. We conclude that inhibiting PI3Kδ represents a novel therapeutic approach to ameliorate AD pathology by d ening plaque accumulation and microglial-dependent neuroinflammation.
Sustained synaptic-vesicle recycling by bulk endocytosis contributes to the maintenance of high-rate neurotransmitter release stimulated by glycerotoxin
Publisher: The Company of Biologists
Date: 04-2010
DOI: 10.1242/JCS.049296
Abstract: Glycerotoxin (GLTx), a large neurotoxin isolated from the venom of the sea worm Glycera convoluta, promotes a long-lasting increase in spontaneous neurotransmitter release at the peripheral and central synapses by selective activation of Cav2.2 channels. We found that GLTx stimulates the very high frequency, long-lasting (more than 10 hours) spontaneous release of acetylcholine by promoting nerve terminal Ca2+ oscillations sensitive to the inhibitor ω-conotoxin GVIA at the hibian neuromuscular junction. Although an estimate of the number of synaptic vesicles undergoing exocytosis largely exceeds the number of vesicles present in the motor nerve terminal, ultrastructural examination of GLTx-treated synapses revealed no significant change in the number of synaptic vesicles. However, we did detect the appearance of large pre-synaptic cisternae suggestive of bulk endocytosis. Using a combination of styryl dyes, photoconversion and horseradish peroxidase (HRP)-labeling electron microscopy, we demonstrate that GLTx upregulates presynaptic-vesicle recycling, which is likely to emanate from the limiting membrane of these large cisternae. Similar synaptic-vesicle recycling through bulk endocytosis also occurs from nerve terminals stimulated by high potassium. Our results suggest that this process might therefore contribute significantly to synaptic recycling under sustained levels of synaptic stimulation.
A HIV-1 Tat mutant protein disrupts HIV-1 Rev function by targeting the DEAD-box RNA helicase DDX1
Publisher: Springer Science and Business Media LLC
Date: 12-2014
Secretagogue Stimulation of Neurosecretory Cells Elicits Filopodial Extensions Uncovering New Functional Release Sites
Publisher: Society for Neuroscience
Date: 04-12-2013
DOI: 10.1523/JNEUROSCI.2634-13.2013
Abstract: Regulated exocytosis in neurosecretory cells relies on the timely fusion of secretory granules (SGs) with the plasma membrane. Secretagogue stimulation leads to an enlargement of the cell footprint (surface area in contact with the coverslip), an effect previously attributed to exocytic fusion of SGs with the plasma membrane. Using total internal reflection fluorescence microscopy, we reveal the formation of filopodia-like structures in bovine chromaffin and PC12 cells driving the footprint expansion, suggesting the involvement of cortical actin network remodeling in this process. Using exocytosis-incompetent PC12 cells, we demonstrate that footprint enlargement is largely independent of SG fusion, suggesting that vesicular exocytic fusion plays a relatively minor role in filopodial expansion. The footprint periphery, including filopodia, undergoes extensive F-actin remodeling, an effect abolished by the actomyosin inhibitors cytochalasin D and blebbistatin. Imaging of both Lifeact-GFP and the SG marker protein neuropeptide Y-mCherry reveals that SGs actively translocate along newly forming actin tracks before undergoing fusion. Together, these data demonstrate that neurosecretory cells regulate the number of SGs undergoing exocytosis during sustained stimulation by controlling vesicular mobilization and translocation to the plasma membrane through actin remodeling. Such remodeling facilitates the de novo formation of fusion sites.
Amyloid-β and tau complexity — towards improved biomarkers and targeted therapies
Publisher: Springer Science and Business Media LLC
Date: 15-12-2017
DOI: 10.1038/NRNEUROL.2017.162
Abstract: Most neurodegenerative diseases are proteinopathies, which are characterized by the aggregation of misfolded proteins. Although many proteins have an intrinsic propensity to aggregate, particularly when cellular clearance systems start to fail in the context of ageing, only a few form fibrillar aggregates. In Alzheimer disease, the peptide amyloid-β (Aβ) and the protein tau aggregate to form plaques and tangles, respectively, which comprise the histopathological hallmarks of this disease. This Review discusses the complexity of Aβ biogenesis, trafficking, post-translational modifications and aggregation states. Tau and its various isoforms, which are subject to a vast array of post-translational modifications, are also explored. The methodological advances that revealed this complexity are described. Finally, the toxic effects of distinct species of tau and Aβ are discussed, as well as the concept of protein 'strains', and how this knowledge can facilitate the development of early disease biomarkers for stratifying patients and validating new therapies. By targeting distinct species of Aβ and tau for therapeutic intervention, the way might be paved for personalized medicine and more-targeted treatment strategies.
In vivo single-molecule imaging of syntaxin1A reveals polyphosphoinositide- and activity-dependent trapping in presynaptic nanoclusters
Publisher: Springer Science and Business Media LLC
Date: 16-12-2016
DOI: 10.1038/NCOMMS13660
Abstract: Syntaxin1A is organized in nanoclusters that are critical for the docking and priming of secretory vesicles from neurosecretory cells. Whether and how these nanoclusters are affected by neurotransmitter release in nerve terminals from a living organism is unknown. Here we imaged photoconvertible syntaxin1A-mEos2 in the motor nerve terminal of Drosophila larvae by single-particle tracking photoactivation localization microscopy. Opto- and thermo-genetic neuronal stimulation increased syntaxin1A-mEos2 mobility, and reduced the size and molecular density of nanoclusters, suggesting an activity-dependent release of syntaxin1A from the confinement of nanoclusters. Syntaxin1A mobility was increased by mutating its polyphosphoinositide-binding site or preventing SNARE complex assembly via co-expression of tetanus toxin light chain. In contrast, syntaxin1A mobility was reduced by preventing SNARE complex disassembly. Our data demonstrate that polyphosphoinositide favours syntaxin1A trapping, and show that SNARE complex disassembly leads to syntaxin1A dissociation from nanoclusters. Lateral diffusion and trapping of syntaxin1A in nanoclusters therefore dynamically regulate neurotransmitter release.
Caveolin-1 and cavin1 act synergistically to generate a unique lipid environment in caveolae
Publisher: Rockefeller University Press
Date: 26-01-2021
Abstract: Caveolae are specialized domains of the vertebrate cell surface with a well-defined morphology and crucial roles in cell migration and mechanoprotection. Unique compositions of proteins and lipids determine membrane architectures. The precise caveolar lipid profile and the roles of the major caveolar structural proteins, caveolins and cavins, in selectively sorting lipids have not been defined. Here, we used quantitative nanoscale lipid mapping together with molecular dynamic simulations to define the caveolar lipid profile. We show that caveolin-1 (CAV1) and cavin1 in idually sort distinct plasma membrane lipids. Intact caveolar structures composed of both CAV1 and cavin1 further generate a unique lipid nano-environment. The caveolar lipid sorting capability includes selectivities for lipid headgroups and acyl chains. Because lipid headgroup metabolism and acyl chain remodeling are tightly regulated, this selective lipid sorting may allow caveolae to act as transit hubs to direct communications among lipid metabolism, vesicular trafficking, and signaling.
Radial contractility of actomyosin rings facilitates axonal trafficking and structural stability
Publisher: Rockefeller University Press
Date: 17-03-2020
Abstract: Most mammalian neurons have a narrow axon, which constrains the passage of large cargoes such as autophagosomes that can be larger than the axon diameter. Radial axonal expansion must therefore occur to ensure efficient axonal trafficking. In this study, we reveal that the speed of various large cargoes undergoing axonal transport is significantly slower than that of small ones and that the transit of erse-sized cargoes causes an acute, albeit transient, axonal radial expansion, which is immediately restored by constitutive axonal contractility. Using live super-resolution microscopy, we demonstrate that actomyosin-II controls axonal radial contractility and local expansion, and that NM-II filaments associate with periodic F-actin rings via their head domains. Pharmacological inhibition of NM-II activity significantly increases axon diameter by detaching the NM-II from F-actin and impacts the trafficking speed, directionality, and overall efficiency of long-range retrograde trafficking. Consequently, prolonged NM-II inactivation leads to disruption of periodic actin rings and formation of focal axonal swellings, a hallmark of axonal degeneration.
Mapping Organelle Motion Reveals a Vesicular Conveyor Belt Spatially Replenishing Secretory Vesicles in Stimulated Chromaffin Cells
Publisher: Public Library of Science (PLoS)
Date: 29-01-2014
Single-Molecule Imaging of Recycling Synaptic Vesicles in Live Neurons
Publisher: Springer US
Date: 2020
Increased neurovirulence of omicron BA.5 over BA.1 in human brain organoids and K18-hACE2 mice
Publisher: Research Square Platform LLC
Date: 31-03-2023
DOI: 10.21203/RS.3.RS-2702556/V1
Abstract: The reduced pathogenicity of the omicron BA.1 sub-lineage compared to earlier variants is well described, although whether such attenuation is retained for later variants like BA.5 remains controversial. We show that a BA.5 isolate was significantly more pathogenic in K18-hACE2 mice than a BA.1 isolate, with BA.5 infections showing increased neuroinvasiveness, resulting in brain infection and mortality, similar to that seen for original ancestral isolates. BA.5 also infected human cortical brain organoids to a greater extent than the BA.1 and original ancestral isolates. In the brains of mice neurons were the main target of infection, and in human organoids neuronal progenitor cells and immature neurons were infected. Evidence for brain infection and brain damage in certain COVID-19 patients is becoming compelling, with the results herein illustrating the increasing intrinsic neuropathogenic potential of evolving omicron variants.
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