Reversible intracellular translocation of KRas but not HRas in hippocampal neurons regulated by Ca2+/calmodulin

The Ras/MAPK pathway regulates synaptic plasticity and cell survival in neurons of the central nervous system. Here, we show that KRas, but not HRas, acutely translocates from the plasma membrane (PM) to the Golgi complex and early/recycling endosomes in response to neuronal activity. Translocation is reversible and mediated by the polybasic-prenyl membrane targeting motif of KRas. We provide evidence that KRas translocation occurs through sequestration of the polybasic-prenyl motif by Ca2+/calmodulin (Ca2+/CaM) and subsequent release of KRas from the PM, in a process reminiscent of GDP dissociation inhibitor–mediated membrane recycling of Rab and Rho GTPases. KRas translocation was accompanied by partial intracellular redistribution of its activity. We conclude that the polybasic-prenyl motif acts as a Ca2+/CaM-regulated molecular switch that controls PM concentration of KRas and redistributes its activity to internal sites. Our data thus define a novel signaling mechanism that differentially regulates KRas and HRas localization and activity in neurons

Editorial: Imaging synapse structure and function

These are the glory days for imaging synapse structure and function. Thanks to recent advances in both optical and electron microscopy, it is now possible to image individual synapses with unprecedented spatial and temporal resolution. The parallel development of a wide range of genetically-encoded synaptic reporters enables all-optical recording of synaptic activity in genetically-defined neuronal populations. These engineering breakthroughs allow neuroscientists to interrogate the brain in ways that were inconceivable just a few years ago. The ability to image synaptic structure and activity in large functional circuits is beginning to yield key insights into how the brain stores, processes, and computes information. This research topic consists of eleven articles (methods, primary research papers, and reviews) that provide an overview of the latest developments in synapse imaging. Rather than attempting an exhaustive list of synaptic reporters and microscopy techniques, our collection emphasizes approaches that merge technical advances from diverse areas to extract a rich palette of novel information from individual synapses. Watanabe presents a method that combines optogenetics and rapid freezing (Flash-and-Freeze) to visualize the synaptic vesicle (SV) cycle at the ultrastructural level with millisecond resolution Watanabe. This revolutionary approach revealed ultra-fast endocytosis of SVs at central synapses and neuromuscular junctions (Watanabe et al., 2013a,b) and promises to uncover many new kinetic aspects of synapse dynamics. Begemann and Galic review recent efforts to image neuronal preparations with both light and electron microscopy, with a series of hybrid techniques referred to as Correlative Light Electron Microscopy (CLEM). Jackson and Burrone describe the first genetically-encoded fluorescent reporter (sypHy-RGECO) that enables concurrent monitoring of calcium dynamics and SV fusion. sypHy-RGECO will undoubtedly be a powerful means of examining calcium triggering of SV exocytosis at the level of individual presynaptic boutons. Using similar probes, Tang et al. show that the mental disease gene DISC1 (Disrupted-In-Schizophrenia-1) accelerates SV exocytosis by facilitating calcium influx through N-type voltage-gated Ca2+ channels. Calcium transients at synapses are also shaped by both mobilization and sequestration of calcium by intracellular stores. Kwon et al. report on the latest advances in organelle-specific calcium sensors and review the contribution of the endoplasmic reticulum and mitochondria to calcium dynamics and synaptic transmission/plasticity. Until recently, one major impediment to imaging of synaptic activity has been our inability to directly measure membrane potential with adequate signal/noise ratio. This is rapidly changing with the recent improvement of a wide range of genetically-encoded voltage indicators (GEVIs) that now are capable of monitoring both single action potentials and even subthreshold synaptic potentials, both in vitro and in vivo Nakajima et al. Three papers describe recent advances on the localization, dynamics and function of postsynaptic receptors and scaffolds. Using a combination of single-molecule tracking (uPAINT) and photoactivated localization microscopy (PALM), Li and Blanpied assess the diffusion properties of membrane proteins within the postsynaptic density (PSD). The same authors recently used localization microscopy to demonstrate the existence of transsynaptic nanocolumns that align the neurotransmitter release machinery to postsynaptic receptors (Tang et al., 2016a). Dosemeci et al. review a series of EM studies that reveal the presence of a dense lamina—the “pallium”—just beneath the core layer of the PSD, and discuss how translocation of signaling proteins and scaffolds in and out of the pallium may shape activity-induced changes in dendritic spines. In keeping with the theme of postsynaptic signaling, Dore et al. discuss evidence for metabotropic functions of NMDARs, based on time-resolved FRET and other imaging approaches. Finally, at the level of synaptic circuits, two reviews describe the use of genetically-encoded synaptic labels to trace neural circuits in a variety of different model systems, ranging from C. elegans to mammals (Hong and Park; Lee et al.). Overall, we hope that the fine collection of papers contained within this research topic highlights a useful synapse imaging toolkit for the neuroscience community. The next big challenge in brain imaging will be to scale up these synaptic measurements to large ensembles of neurons to comprehend how circuits compute. This will require synaptic reporters that operate in a synaptically-relevant time scale (milliseconds), along with improved genetic targeting strategies, further advances in automated high-speed microscopy, and refined bioinformatics tools for analysis of the resulting large datasets

The small GTPase HRas shapes local PI3K signals through positive feedback and regulates persistent membrane extension in migrating fibroblasts

Self-amplification of phosphoinositide 3-kinase (PI3K) signaling is believed to regulate asymmetric membrane extension and cell migration, but the molecular organization of the underlying feedback circuit is elusive. Here we use an inducible approach to synthetically activate PI3K and interrogate the feedback circuitry governing self-enhancement of 3'-phosphoinositide (3-PI) signals in NIH3T3 fibroblasts. Synthetic activation of PI3K initially leads to uniform production of 3-PIs at the plasma membrane, followed by the appearance of asymmetric and highly amplified 3-PI signals. A detailed spatiotemporal analysis shows that local self-amplifying 3-PI signals drive rapid membrane extension with remarkable directional persistence and initiate a robust migratory response. This positive feedback loop is critically dependent on the small GTPase HRas. Silencing of HRas abrogates local amplification of 3-PI signals upon synthetic PI3K activation and results in short-lived protrusion events that do not support cell migration. Finally, our data indicate that this feedback circuit is likely to operate during platelet-derived growth factor-induced random cell migration. We conclude that positive feedback between PI3K and HRas is essential for fibroblasts to spontaneously self-organize and generate a productive migratory response in the absence of spatial cues

Neuronal SOCE: myth or reality?

Store-operated Ca2+ entry (SOCE) is the primary Ca2+ influx pathway in non-excitable cells. Long thought to be absent in nerve cells, neuronal SOCE is gaining popularity. We argue here that the evidence for SOCE in neurons remains contentious, mostly because SOCE imaging assays are inadequate in these cells

Autism-associated CHD8 keeps proliferation of human neural progenitors in check by lengthening the G1 phase of the cell cycle

De novo mutations (DNMs) in chromodomain helicase DNA binding protein 8 (CHD8) are associated with a specific subtype of autism characterized by enlarged heads and distinct cranial features. The vast majority of these DNMs are heterozygous loss-of-function mutations with high penetrance for autism. CHD8 is a chromatin remodeler that preferentially regulates expression of genes implicated in early development of the cerebral cortex. How CHD8 haploinsufficiency alters the normal developmental trajectory of the brain is poorly understood and debated. Using long-term single-cell imaging, we show that disruption of a single copy of CHD8 in human neural precursor cells (NPCs) markedly shortens the G1 phase of the cell cycle. Consistent with faster progression of CHD8+/− NPCs through G1 and the G1/S checkpoint, we observed increased expression of E cyclins and elevated phosphorylation of Erk in these mutant cells – two central signaling pathways involved in S phase entry. Thus, CHD8 keeps proliferation of NPCs in check by lengthening G1, and mono-allelic disruption of this gene alters cell-cycle timing in a way that favors self-renewing over neurogenic cell divisions. Our findings further predict enlargement of the neural progenitor pool in CHD8+/− developing brains, providing a mechanistic basis for macrocephaly in this autism subtype

Late Endosomal Cholesterol Accumulation Leads to Impaired Intra-Endosomal Trafficking

Background Pathological accumulation of cholesterol in late endosomes is observed in lysosomal storage diseases such as Niemann-Pick type C. We here analyzed the effects of cholesterol accumulation in NPC cells, or as phenocopied by the drug U18666A, on late endosomes membrane organization and dynamics. Methodology/Principal Findings Cholesterol accumulation did not lead to an increase in the raft to non-raft membrane ratio as anticipated. Strikingly, we observed a 2–3 fold increase in the size of the compartment. Most importantly, properties and dynamics of late endosomal intralumenal vesicles were altered as revealed by reduced late endosomal vacuolation induced by the mutant pore-forming toxin ASSP, reduced intoxication by the anthrax lethal toxin and inhibition of infection by the Vesicular Stomatitis Virus. Conclusions/Significance These results suggest that back fusion of intralumenal vesicles with the limiting membrane of late endosomes is dramatically perturbed upon cholesterol accumulation

STIM2 regulates AMPA receptor trafficking and plasticity at hippocampal synapses

STIM2 is an integral membrane protein of the endoplasmic reticulum (ER) that regulates the activity of plasma membrane (PM) channels at ER-PM contact sites. Recent studies show that STIM2 promotes spine maturation and surface expression of the AMPA receptor (AMPAR) subunit GluA1, hinting at a probable role in synaptic plasticity. Here, we used a Stim2 cKO mouse to explore the function of STIM2 in Long-Term Potentiation (LTP) and Depression (LTD), two widely-studied models of synaptic plasticity implicated in information storage. We found that STIM2 is required for the stable expression of both LTP and LTD at CA3-CA1 hippocampal synapses. Altered plasticity in Stim2 cKO mice is associated with subtle alterations in the shape and density of dendritic spines in CA1 neurons. Further, surface delivery of GluA1 in response to LTP-inducing chemical manipulations was markedly reduced in excitatory neurons derived from Stim2 cKO mice. GluA1 endocytosis following chemically-induced LTD was also impaired in Stim2 cKO neurons. We conclude that STIM2 facilitates synaptic delivery and removal of AMPARs and regulates activity-dependent changes in synaptic strength through a unique mode of communication between the ER and the synapse

Robust neuronal symmetry breaking by Ras-triggered local positive feedback

Neuronal polarity is initiated by a symmetry-breaking event whereby one out of multiple minor neurites undergoes rapid outgrowth and becomes the axon [1]. Axon formation is regulated by phosphatidylinositol 3-kinase (PI3K)-related signaling elements [2-10] that drive local actin [11] and microtubule reorganization [3, 12], but the upstream signaling circuit that causes symmetry breaking and guarantees the formation of a single axon is not known. Here, we use live FRET imaging in hippocampal neurons and show that the activity of the small GTPase HRas, an upstream regulator of PI3K, markedly increases in the nascent axonal growth cone upon symmetry breaking. This local increase in HRas activity results from a positive feedback loop between HRas and PI3K, locally reinforced by vesicular transport of HRas to the axonal growth cone. Recruitment of HRas to the axonal growth cone is paralleled by a decrease in HRas concentration in the remaining neurites, suggesting that competition for a limited pool of HRas guarantees that only one axon forms. Mathematical modeling demonstrates that local positive feedback between HRas and PI3K, coupled to recruitment of a limited pool of HRas, generates robust symmetry breaking and formation of a single axon in the absence of extrinsic spatial cues

Stimulation of synaptic vesicle exocytosis by the mental disease gene DISC1 is mediated by N-Type voltage-gated calcium channels

Lesions and mutations of the DISC1 (Disrupted-in-schizophrenia-1) gene have been linked to major depression, schizophrenia, bipolar disorder and autism, but the influence of DISC1 on synaptic transmission remains poorly understood. Using two independent genetic approaches-RNAi and a DISC1 KO mouse-we examined the impact of DISC1 on the synaptic vesicle (SV) cycle by population imaging of the synaptic tracer vGpH in hippocampal neurons. DISC1 loss-of-function resulted in a marked decrease in SV exocytic rates during neuronal stimulation and was associated with reduced Ca(2+) transients at nerve terminals. Impaired SV release was efficiently rescued by elevation of extracellular Ca(2+), hinting at a link between DISC1 and voltage-gated Ca(2+) channels. Accordingly, blockade of N-type Cav2.2 channels mimics and occludes the effect of DISC1 inactivation on SV exocytosis, and overexpression of DISC1 in a heterologous system increases Cav2.2 currents. Collectively, these results show that DISC1-dependent enhancement of SV exocytosis is mediated by Cav2.2 and point to aberrant glutamate release as a probable endophenotype of major psychiatric disorders