An acto-myosin II constricting ring initiates the fission of activity-dependent bulk endosomes in neurosecretory cells

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

Secretagogue stimulation of neurosecretory cells elicits filopodial extensions uncovering new functional release sites

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

Activity-driven relaxation of the cortical actomyosin II network synchronizes Munc18-1-dependent neurosecretory vesicle docking

In neurosecretory cells, secretory vesicles (SVs) undergo Ca2(+)-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

Munc18-1 is a molecular chaperone for α-synuclein, controlling its self-replicating aggregation

Munc 18-1 is a key component of the exocytic machinery that controls neurotransmitter release. Munc 18-1 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 Munc 18-1 EIEE-causing mutants form large polymers that coaggregate wild-type Munc 18-1 in vitro and in cells. Surprisingly, Munc 18-1 EIEE mutants also form Lewy body like structures that contain a-synuclein (alpha-Syn). We reveal that Munc 18-1 binds alpha-Syn, and its EIEE mutants coaggregate alpha-Syn. Likewise, removal of endogenous Munc 18-1 increases the aggregative propensity of alpha-Syn(wT) and that of the Parkinson's disease-causing a-Syn(A30P) mutant, an effect rescued by Munc18-1(WT) expression, indicative of chaperone activity. Coexpression of the alpha-Syn(A30P) mutant with Munc 18-1 reduced the number of alpha-Syn(A30P) aggregates. Munc 18-1 mutations and haploinsufficiency may therefore trigger a pathogenic gain of function through both the corruption of native Munc 18-1 and a perturbed chaperone activity for a-Syn leading to aggregation-induced neurodegeneration

Myosin VI small insert isoform maintains exocytosis by tethering secretory granules to the cortical actin.

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 Ca(2+)-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

Myosin VI small insert isoform maintains exocytosis by tethering secretory granules to the cortical actin.

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 Ca(2+)-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

ENA/VASP proteins regulate exocytosis by mediating myosin VI-dependent recruitment of secretory granules to the cortical actin network

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

p110δ PI 3-kinase inhibition perturbs APP and TNFα trafficking, reduces plaque burden, dampens neuroinflammation and prevents cognitive decline in an Alzheimer's disease mouse model

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 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 hippocampal neurons and dampens secretion of the inflammatory cytokine tumor necrosis factor alpha (TNFα) by microglial cells in the familial AD APP/PS1 (APP/PS1) mouse model. Moreover, APP/PS1 mice with kinase-inactive PI3Kδ (δ) had reduced Aβ peptides levels and plaques in the brain and an abrogated inflammatory response compared to 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 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 dampening microglial-dependent neuroinflammation and reducing plaque burden by inhibition of neuronal APP trafficking and processing.During Alzheimer's disease, the accumulation of the toxic 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 promises. Using a combination of genetic and pharmacological approaches, we found that the p110δ isoform of PI 3-kinase (PI3Kδ) is involved in anterograde trafficking of the amyloid precursor protein (APP) in neurons, and in the secretion of TNFα 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 dampening plaque accumulation and microglial-dependent neuroinflammation