A conformational switch driven by phosphorylation regulates the activity of the evolutionarily conserved SNARE Ykt6.

Ykt6 is a soluble N-ethylmaleimide sensitive factor activating protein receptor (SNARE) critically involved in diverse vesicular fusion pathways. While most SNAREs rely on transmembrane domains for their activity, Ykt6 dynamically cycles between the cytosol and membrane-bound compartments where it is active. The mechanism that regulates these transitions and allows Ykt6 to achieve specificity toward vesicular pathways is unknown. Using a Parkinson's disease (PD) model, we found that Ykt6 is phosphorylated at an evolutionarily conserved site which is regulated by Ca2+ signaling. Through a multidisciplinary approach, we show that phosphorylation triggers a conformational change that allows Ykt6 to switch from a closed cytosolic to an open membrane-bound form. In the phosphorylated open form, the spectrum of protein interactions changes, leading to defects in both the secretory and autophagy pathways, enhancing toxicity in PD models. Our studies reveal a mechanism by which Ykt6 conformation and activity are regulated with potential implications for PD.

Orai1 Channels Are Essential for Amplification of Glutamate-Evoked Ca2+ Signals in Dendritic Spines to Regulate Working and Associative Memory.

Store-operated Orai1 calcium channels function as highly Ca2+-selective ion channels and are broadly expressed in many tissues including the central nervous system, but their contributions to cognitive processing are largely unknown. Here, we report that many measures of synaptic, cellular, and behavioral models of learning are markedly attenuated in mice lacking Orai1 in forebrain excitatory neurons. Results with focal glutamate uncaging in hippocampal neurons support an essential role of Orai1 channels in amplifying NMDA-receptor-induced dendritic Ca2+ transients that drive activity-dependent spine morphogenesis and long-term potentiation at Schaffer collateral-CA1 synapses. Consistent with these signaling roles, mice lacking Orai1 in pyramidal neurons (but not interneurons) exhibit striking deficits in working and associative memory tasks. These findings identify Orai1 channels as essential regulators of dendritic spine Ca2+ signaling, synaptic plasticity, and cognition.

Discovery and characterization of novel small-molecule CXCR4 receptor agonists and antagonists.

The chemokine CXCL12 (SDF-1) and its cognate receptor CXCR4 are involved in a large number of physiological processes including HIV-1 infectivity, inflammation, tumorigenesis, stem cell migration, and autoimmune diseases. While previous efforts have identified a number of CXCR4 antagonists, there have been no small molecule agonists reported. Herein, we describe the identification of a novel series of CXCR4 modulators, including the first small molecules to display agonist behavior against this receptor, using a combination of structure- and ligand-based virtual screening. These agonists produce robust calcium mobilization in human melanoma cell lines which can be blocked by the CXCR4-selective antagonist AMD3100. We also demonstrate the ability of these new agonists to induce receptor internalization, ERK activation, and chemotaxis, all hallmarks of CXCR4 activation. Our results describe a new series of biologically relevant small molecules that will enable further study of the CXCR4 receptor and may contribute to the development of new therapeutics.

Regulation of neurogenesis by calcium signaling.

Calcium (Ca(2+)) signaling has essential roles in the development of the nervous system from neural induction to the proliferation, migration, and differentiation of neural cells. Ca(2+) signaling pathways are shaped by interactions among metabotropic signaling cascades, intracellular Ca(2+) stores, ion channels, and a multitude of downstream effector proteins that activate specific genetic programs. The temporal and spatial dynamics of Ca(2+) signals are widely presumed to control the highly diverse yet specific genetic programs that establish the complex structures of the adult nervous system. Progress in the last two decades has led to significant advances in our understanding of the functional architecture of Ca(2+) signaling networks involved in neurogenesis. In this review, we assess the literature on the molecular and functional organization of Ca(2+) signaling networks in the developing nervous system and its impact on neural induction, gene expression, proliferation, migration, and differentiation. Particular emphasis is placed on the growing evidence for the involvement of store-operated Ca(2+) release-activated Ca(2+) (CRAC) channels in these processes.

Store-operated CRAC channels regulate gene expression and proliferation in neural progenitor cells.

Calcium signals regulate many critical processes during vertebrate brain development including neurogenesis, neurotransmitter specification, and axonal outgrowth. However, the identity of the ion channels mediating Ca(2+) signaling in the developing nervous system is not well defined. Here, we report that embryonic and adult mouse neural stem/progenitor cells (NSCs/NPCs) exhibit store-operated Ca(2+) entry (SOCE) mediated by Ca(2+) release-activated Ca(2+) (CRAC) channels. SOCE in NPCs was blocked by the CRAC channel inhibitors La(3+), BTP2, and 2-APB and Western blots revealed the presence of the canonical CRAC channel proteins STIM1 and Orai1. Knock down of STIM1 or Orai1 significantly diminished SOCE in NPCs, and SOCE was lost in NPCs from transgenic mice lacking Orai1 or STIM1 and in knock-in mice expressing the loss-of-function Orai1 mutant, R93W. Therefore, STIM1 and Orai1 make essential contributions to SOCE in NPCs. SOCE in NPCs was activated by epidermal growth factor and acetylcholine, the latter occurring through muscarinic receptors. Activation of SOCE stimulated gene transcription through calcineurin/NFAT (nuclear factor of activated T cells) signaling through a mechanism consistent with local Ca(2+) signaling by Ca(2+) microdomains near CRAC channels. Importantly, suppression or deletion of STIM1 and Orai1 expression significantly attenuated proliferation of embryonic and adult NPCs cultured as neurospheres and, in vivo, in the subventricular zone of adult mice. These findings show that CRAC channels serve as a major route of Ca(2+) entry in NPCs and regulate key effector functions including gene expression and proliferation, indicating that CRAC channels are important regulators of mammalian neurogenesis.