Cytosolic calcium: Judge, jury and executioner of neurodegeneration in Alzheimer's disease and beyond.

This review discusses the driving principles that may underlie neurodegeneration in dementia, represented most dominantly by Alzheimer's disease (AD). While a myriad of different disease risk factors contribute to AD, these ultimately converge to a common disease outcome. Based on decades of research, a picture emerges where upstream risk factors combine in a feedforward pathophysiological cycle, culminating in a rise of cytosolic calcium concentration ([Ca2+ ]c ) that triggers neurodegeneration. In this framework, positive AD risk factors entail conditions, characteristics, or lifestyles that initiate or accelerate self-reinforcing cycles of pathophysiology, whereas negative risk factors or therapeutic interventions, particularly those mitigating elevated [Ca2+ ]c , oppose these effects and therefore have neuroprotective potential.

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.

miR-27b shapes the presynaptic transcriptome and influences neurotransmission by silencing the polycomb group protein Bmi1.

BACKGROUND: MicroRNAs (miRNAs) are short non-coding RNAs that are emerging as important post-transcriptional regulators of neuronal and synaptic development. The precise impact of miRNAs on presynaptic function and neurotransmission remains, however, poorly understood. RESULTS: Here, we identify miR-27b-an abundant neuronal miRNA implicated in neurological disorders-as a global regulator of the presynaptic transcriptome. miR-27b influences the expression of three quarters of genes associated with presynaptic function in cortical neurons. Contrary to expectation, a large majority of these genes are up-regulated by miR-27b. This stimulatory effect is mediated by miR-27b-directed silencing of several transcriptional repressors that cooperate to suppress the presynaptic transcriptome. The strongest repressive activity appears to be mediated by Bmi1, a component of the polycomb repressive complex implicated in self-renewal of neural stem cells. miR-27b knockdown leads to reduced synaptogenesis and to a marked decrease in neural network activity, which is fully restored by RNAi-mediated silencing of Bmi1. CONCLUSIONS: We conclude that silencing of Bmi1 by miR-27b relieves repression of the presynaptic transcriptome and supports neurotransmission in cortical networks. These results expand the repressive activity of Bmi1 to genes involved in synaptic function and identify a unique post-transcriptional circuitry that stimulates expression of synaptic genes and promotes synapse differentiation.

Liprin-α1, ERC1 and LL5 define polarized and dynamic structures that are implicated in cell migration.

Cell migration during development and metastatic invasion requires the coordination of actin and adhesion dynamics to promote protrusive activity at the front of the cell. The knowledge of the molecular mechanisms required to achieve such coordination is fragmentary. Here, we identify a new functional complex that drives cell motility. ERC1a (an isoform of ERC1) and the LL5 proteins LL5α and LL5ß (encoded by PHLDB1 and PHLDB2, respectively) are required, together with liprin-α1, for effective migration and tumor cell invasion, and do so by stabilizing the protrusive activity at the cell front. Depletion of either protein negatively affects invasion, migration on extracellular matrix, lamellipodial persistence and the internalization of active integrin ß1 receptors needed for adhesion turnover at the front of the cell. Liprin-α1, ERC1a and LL5 also define new highly polarized and dynamic cytoplasmic structures uniquely localized near the protruding cell edge. Our results indicate that the functional complex and the associated structures described here represent an important mechanism to drive tumor cell migration.

Pharmacological modulation of septins restores calcium homeostasis and is neuroprotective in models of Alzheimer's disease.

Abnormal calcium signaling is a central pathological component of Alzheimer's disease (AD). Here, we describe the identification of a class of compounds called ReS19-T, which are able to restore calcium homeostasis in cell-based models of tau pathology. Aberrant tau accumulation leads to uncontrolled activation of store-operated calcium channels (SOCCs) by remodeling septin filaments at the cell cortex. Binding of ReS19-T to septins restores filament assembly in the disease state and restrains calcium entry through SOCCs. In amyloid-ß and tau-driven mouse models of disease, ReS19-T agents restored synaptic plasticity, normalized brain network activity, and attenuated the development of both amyloid-ß and tau pathology. Our findings identify the septin cytoskeleton as a potential therapeutic target for the development of disease-modifying AD treatments.

Asian promise: the state and future of collaborations in neuroscience.

As investment in science and technology continues to grow in Asia, countries such as China, Japan and Singapore are witnessing great improvements in their neuroscience research environments; this is reflected in the opening of new research institutions and in the influx of neuroscientists trained abroad. Collaborative projects between researchers in these countries and laboratories in the United States and Europe are not only helping to shape these institutions, they are also leading to a surge in high-quality publications in both basic and translational neuroscience, resulting in increasing international recognition. Nature Reviews Neuroscience asks four neuroscientists about their collaborative experiences and the impact that such collaborations are having on neuroscience research.

The Mood Stabilizer Lithium Slows Down Synaptic Vesicle Cycling at Glutamatergic Synapses.

Lithium is a mood stabilizer broadly used to prevent and treat symptoms of mania and depression in people with bipolar disorder (BD). Little is known, however, about its mode of action. Here, we analyzed the impact of lithium on synaptic vesicle (SV) cycling at presynaptic terminals releasing glutamate, a neurotransmitter previously implicated in BD and other neuropsychiatric conditions. We used the pHluorin-based synaptic tracer vGpH and a fully automated image processing pipeline to quantify the effect of lithium on both SV exocytosis and endocytosis in hippocampal neurons. We found that lithium selectively reduces SV exocytic rates during electrical stimulation, and markedly slows down SV recycling post-stimulation. Analysis of single-bouton responses revealed the existence of functionally distinct excitatory synapses with varying sensitivity to lithium-some terminals show responses similar to untreated cells, while others are markedly impaired in their ability to recycle SVs. While the cause of this heterogeneity is unclear, these data indicate that lithium interacts with the SV machinery and influences glutamate release in a large fraction of excitatory synapses. Together, our findings show that lithium down modulates SV cycling, an effect consistent with clinical reports indicating hyperactivation of glutamate neurotransmission in BD.

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.

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.

Quantitative Determination of Primary Cilia Protein Distribution Using Immunofluorescence Staining and MATLAB Analysis.

Primary cilia are microtubule-based sensory organelles surrounded by membrane. They can detect mechanical and chemical stimuli. The last few years have uncovered cilia as unique signaling hubs that host a number of receptors and effector molecules. Thus, defining how specific proteins localize and are distributed along the cilium is critical to understanding its function. Quantitative immunofluorescence can be used to accurately assess the localization of receptors and signaling molecules within the primary cilia. However, image analysis can be time consuming, and there are limited programs that can accurately determine staining intensity along the cilia. To overcome these issues, we developed a series of MATLAB scripts to accurately measure staining intensity along the length of the cilia, in both a semi-automated and automated fashion. Here, we describe the scripts and include a protocol for image analysis for each. With these scripts, the protocols can be used to analyze the distribution of any ciliary protein using immunofluorescence images.

Parkin protects against LRRK2 G2019S mutant-induced dopaminergic neurodegeneration in Drosophila.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are currently recognized as the most common genetic cause of parkinsonism. Among the large number of LRRK2 mutations identified to date, the G2019S variant is the most common. In Asia, however, another LRRK2 variant, G2385R, appears to occur more frequently. To better understand the contribution of different LRRK2 variants toward disease pathogenesis, we generated transgenic Drosophila over-expressing various human LRRK2 alleles, including wild type, G2019S, Y1699C, and G2385R LRRK2. We found that transgenic flies harboring G2019S, Y1699C, or G2385R LRRK2 variant, but not the wild-type protein, exhibit late-onset loss of dopaminergic (DA) neurons in selected clusters that is accompanied by locomotion deficits. Furthermore, LRRK2 mutant flies also display reduced lifespan and increased sensitivity to rotenone, a mitochondrial complex I inhibitor. Importantly, coexpression of human parkin in LRRK2 G2019S-expressing flies provides significant protection against DA neurodegeneration that occurs with age or in response to rotenone. Together, our results suggest a potential link between LRRK2, parkin, and mitochondria in the pathogenesis of LRRK2-related parkinsonism.

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.

Spines and neurite branches function as geometric attractors that enhance protein kinase C action.

Ca2+ and diacylglycerol-regulated protein kinase Cs (PKCs; conventional PKC isoforms, such as PKCgamma) are multifunctional signaling molecules that undergo reversible plasma membrane translocation as part of their mechanism of activation. In this article, we investigate PKCgamma translocation in hippocampal neurons and show that electrical or glutamate stimulation leads to a striking enrichment of PKCgamma in synaptic spines and dendritic branches. Translocation into spines and branches was delayed when compared with the soma plasma membrane, and PKCgamma remained in these structures for a prolonged period after the response in the soma ceased. We have developed a quantitative model for the translocation process by measuring the rate at which PKCgamma crossed the neck of spines, as well as cytosolic and membrane diffusion coefficients of PKCgamma. Our study suggests that neurons make use of a high surface-to-volume ratio of spines and branches to create a geometric attraction process for PKC that imposes a delayed enhancement of PKC action at synapses and in peripheral processes.

Feedback-mediated neuronal competition for survival cues regulates innervation of a target tissue.

Proper wiring of the nervous system requires tight control of the number of nerve terminals that innervate a target tissue. Recent work by Deppmann et al., now suggests that this is achieved by feedback-mediated neuronal competition for target-derived survival cues. The authors' model is inspired by the theory for pattern formation based on self-activation and lateral inhibition, proposed by Meinhardt and Gierer more than 30 years ago.

Specific localization and timing in neuronal signal transduction mediated by protein-lipid interactions.

A large number of signaling proteins translocate from the cytosol to the plasma membrane in response to receptor and electrical stimuli. The site of translocation to the plasma membrane and the "on" and "off" rates of the translocation process are critical for defining the specificity of the signaling response. In addition to targeting mechanisms based on protein-protein interactions, signaling proteins have evolved a large repertoire of covalent lipid modifications and lipid binding protein modules that regulate reversible membrane association. The time constants of these membrane interactions range from milliseconds to several hours. Here we discuss how diversity in lipid-based membrane anchoring and targeting motifs contributes to plasticity in neuronal signaling by providing local and regional control mechanisms as well as a means to transduce and integrate signals over a broad range of different time scales.

Generation and characterisation of two D2A1 mammary cancer sublines to model spontaneous and experimental metastasis in a syngeneic BALB/c host.

Studying the complex mechanisms underlying breast cancer metastasis and therapy response necessitates relevant in vivo models, particularly syngeneic models with an intact immune system. Two syngeneic spontaneously metastatic sublines, D2A1-m1 and D2A1-m2, were generated from the poorly metastasising BALB/c-derived D2A1 cell line by serial in vivo passaging. In vivo and in vitro analyses revealed distinct and shared characteristics of the metastatic D2A1-m1 and D2A1-m2 sublines. In particular, D2A1-m1 cells are more aggressive in experimental metastasis assays, while D2A1-m2 cells are more efficient at disseminating from the primary tumour in spontaneous metastasis assays. Surprisingly, classical metastasis-associated in vitro phenotypes, such as enhanced proliferation, migration and invasion, are reduced in the sublines compared to the parental cell line. Further, evasion of immune control cannot fully explain their enhanced metastatic properties. By contrast, both sublines show increased resistance to apoptosis when cultured in non-adherent conditions and, for the D2A1-m2 subline, increased 3D tumour spheroid growth. Moreover, the enhanced spontaneous metastatic phenotype of the D2A1-m2 subline is associated with an increased ability to recruit an activated tumour stroma. The metastatic D2A1-m1 and D2A1-m2 cell lines provide additional syngeneic models for investigating the different steps of the metastatic cascade and thereby represent valuable tools for breast cancer researchers. Finally, this study highlights that morphology and cell behaviour in 2D cell-based assays cannot be used as a reliable predictor of metastatic behaviour in vivo.

Dynamics of GPI-anchored proteins on the surface of living cells.

Rather than being distributed homogeneously on the cell surface, proteins are probably aggregated in clusters or in specific domains. Some of these domains (lipid rafts) have lipid compositions, which differ from their surrounding membrane. They have been implicated in cell signaling, cell adhesion, and cholesterol homeostasis. Estimates of their size vary from 40 to 350 nm in diameter depending on the study and cell type used. Rafts are enriched in glycosphingolipids and cholesterol and appear to be in a more ordered lipid phase. Although there is some knowledge of their function in cell signaling, less is known about their assembly and dynamics in cells at various temperatures. We use image correlation spectroscopy and dynamic image correlation spectroscopy to study the clustering and diffusion of glycosylphosphatidylinositol (GPI)-anchored proteins within the plasma membrane of living cells at various temperatures. We find that GPI-anchored proteins occur both as monomers and in clusters at the cell surface. The propensities to cluster as well as the diffusion coefficient of these clusters are strongly temperature dependent. At 37 degrees C the GPI-anchored proteins are highly dynamic with a lower state of clustering than at lower temperatures.

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.

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.

Impaired spatial memory and enhanced long-term potentiation in mice with forebrain-specific ablation of the Stim genes.

Recent findings point to a central role of the endoplasmic reticulum-resident STIM (Stromal Interaction Molecule) proteins in shaping the structure and function of excitatory synapses in the mammalian brain. The impact of the Stim genes on cognitive functions remains, however, poorly understood. To explore the function of the Stim genes in learning and memory, we generated three mouse strains with conditional deletion (cKO) of Stim1 and/or Stim2 in the forebrain. Stim1, Stim2, and double Stim1/Stim2 cKO mice show no obvious brain structural defects or locomotor impairment. Analysis of spatial reference memory in the Morris water maze revealed a mild learning delay in Stim1 cKO mice, while learning and memory in Stim2 cKO mice was indistinguishable from their control littermates. Deletion of both Stim genes in the forebrain resulted, however, in a pronounced impairment in spatial learning and memory reflecting a synergistic effect of the Stim genes on the underlying neural circuits. Notably, long-term potentiation (LTP) at CA3-CA1 hippocampal synapses was markedly enhanced in Stim1/Stim2 cKO mice and was associated with increased phosphorylation of the AMPA receptor subunit GluA1, the transcriptional regulator CREB and the L-type Voltage-dependent Ca(2+) channel Cav1.2 on protein kinase A (PKA) sites. We conclude that STIM1 and STIM2 are key regulators of PKA signaling and synaptic plasticity in neural circuits encoding spatial memory. Our findings also reveal an inverse correlation between LTP and spatial learning/memory and suggest that abnormal enhancement of cAMP/PKA signaling and synaptic efficacy disrupts the formation of new memories.