Michael A. Colicos

Actin-Dependent Regulation of Neurotransmitter Release at Central Synapses

Depolymerization of actin by latrunculin A transiently promotes neurotransmitter release. The mean rate of mEPSCs increases by a Ca2+-independent process, without a concomitant change in the mean amplitude. The readily releasable vesicle pool size and the rate of refilling of the readily releasable pool remain unaltered by latrunculin treatment. Evoked neurotransmitter release also increases in a manner consistent with an increase in vesicle release probability. The observed enhancement of neurotransmitter release is specific to actin depolymerization mediated by latrunculin A and is not caused by cytochalasin D. Our findings indicate that actin participates in a regulatory mechanism that restrains fusion of synaptic vesicles at the active zone.

Remodeling of Synaptic Actin Induced by Photoconductive Stimulation

Use-dependent synapse remodeling is thought to provide a cellular mechanism for encoding durable memories, yet whether activity triggers an actual structural change has remained controversial. We use photoconductive stimulation to demonstrate activity-dependent morphological synaptic plasticity by video imaging of GFP-actin at individual synapses. A single tetanus transiently moves presynaptic actin toward and postsynaptic actin away from the synaptic junction. Repetitive spaced tetani induce glutamate receptor-dependent stable restructuring of synapses. Presynaptic actin redistributes and forms new puncta that label for an active synapse marker FM5-95 within 2 hr. Postsynaptic actin sprouts projections toward the new presynaptic actin puncta, resembling the axon-dendrite interaction during synaptogenesis. Our results indicate that activity-dependent presynaptic structural plasticity facilitates the formation of new active presynaptic terminals.

A Preformed Complex of Postsynaptic Proteins Is Involved in Excitatory Synapse Development

Nonsynaptic clusters of postsynaptic proteins have been documented; however, their role remains elusive. We monitored the trafficking of several candidate proteins implicated in synaptogenesis, when nonsynaptic clusters of scaffold proteins are most abundant. We find a protein complex consisting of two populations that differ in their content, mobility, and involvement in synapse formation. One subpopulation is mobile and relies on actin transport for delivery to nascent and existing synapses. These mobile clusters contain the scaffolding proteins PSD-95, GKAP, and Shank. A proportion of mobile clusters that exhibits slow movement and travels short distances contains neuroligin-1. The second group consists of stationary nonsynaptic scaffold complexes that mainly contain neuroligin-1, can recruit synaptophysin-containing axonal transport vesicles, and are readily transformed to functional presynaptic contacts that recycle the vital dye FM 4-64. These results postulate a mechanism whereby preformed scaffold protein complexes serve as predetermined postsynaptic hotspots for establishment of new functional excitatory synapses.

Activity-Dependent Regulation of Synaptic AMPA Receptor Composition and Abundance by β3 Integrins

At synapses, cell adhesion molecules (CAMs) provide the molecular framework for coordinating signaling events across the synaptic cleft. Among synaptic CAMs, the integrins, receptors for extracellular matrix proteins and counterreceptors on adjacent cells, are implicated in synapse maturation and plasticity and memory formation. However, little is known about the molecular mechanisms of integrin action at central synapses. Here, we report that postsynaptic beta3 integrins control synaptic strength by regulating AMPA receptors (AMPARs) in a subunit-specific manner. Pharmacological perturbation targeting beta3 integrins promotes endocytosis of GluR2-containing AMPARs via Rap1 signaling, and expression of beta3 integrins produces robust changes in the abundance and composition of synaptic AMPARs without affecting dendritic spine structure. Importantly, homeostatic synaptic scaling induced by activity deprivation elevates surface expression of beta3 integrins, and in turn, beta3 integrins are required for synaptic scaling. Our findings demonstrate a key role for integrins in the feedback regulation of excitatory synaptic strength.

Targeted polyphosphatase expression alters mitochondrial metabolism and inhibits calcium-dependent cell death

Polyphosphate (polyP) consists of tens to hundreds of phosphates, linked by ATP-like high-energy bonds. Although polyP is present in mammalian mitochondria, its physiological roles there are obscure. Here, we examine the involvement of polyP in mitochondrial energy metabolism and ion transport. We constructed a vector to express a mitochondrially targeted polyphosphatase, along with a GFP fluorescent tag. Specific reduction of mitochondrial polyP, by polyphosphatase expression, significantly modulates mitochondrial bioenergetics, as indicated by the reduction of inner membrane potential and increased NADH levels. Furthermore, reduction of polyP levels increases mitochondrial capacity to accumulate calcium and reduces the likelihood of the calcium-induced mitochondrial permeability transition, a central event in many types of necrotic cell death. This confers protection against cell death, including that induced by β-amyloid peptide, a pathogenic agent in Alzheimers disease. These results demonstrate a crucial role played by polyP in mitochondrial function of mammalian cells.

Imaging of cAMP Levels and Protein Kinase A Activity Reveals That Retinal Waves Drive Oscillations in Second-Messenger Cascades

Recent evidence demonstrates that low-frequency oscillations of intracellular calcium on timescales of seconds to minutes drive distinct aspects of neuronal development, but the mechanisms by which these calcium transients are coupled to signaling cascades are not well understood. Here we test the hypothesis that spontaneous electrical activity activates protein kinase A (PKA). We use live-cell indicators to observe spontaneous and evoked changes in cAMP levels and PKA activity in developing retinal neurons. Expression of cAMP and PKA indicators in neonatal rat retinal explants reveals spontaneous oscillations in PKA activity that are temporally correlated with spontaneous depolarizations associated with retinal waves. In response to short applications of forskolin, dopamine, or high-potassium concentration, we image an increase in cAMP levels and PKA activity, indicating that this second-messenger pathway can be activated quickly by neural activity. Depolarization-evoked increases in PKA activity were blocked by the removal of extracellular calcium, indicating that they are mediated by a calcium-dependent mechanism. These findings demonstrate for the first time that spontaneous activity in developing circuits is correlated with activation of the cAMP/PKA pathway and that PKA activity is turned on and off on the timescale of tens of seconds. These results show a link between neural activity and an intracellular biochemical cascade associated with plasticity, axon guidance, and neural differentiation.

Metabotropic NMDA receptor signaling couples Src family kinases to pannexin-1 during excitotoxicity

Overactivation of neuronal N-methyl-D-aspartate receptors (NMDARs) causes excitotoxicity and is necessary for neuronal death. In the classical view, these ligand-gated Ca2+-permeable ionotropic receptors require co-agonists and membrane depolarization for activation. We report that NMDARs signal during ligand binding without activation of their ion conduction pore. Pharmacological pore block with MK-801, physiological pore block with Mg2+ or a Ca2+-impermeable NMDAR variant prevented NMDAR currents, but did not block excitotoxic dendritic blebbing and secondary currents induced by exogenous NMDA. NMDARs, Src kinase and Panx1 form a signaling complex, and activation of Panx1 required phosphorylation at Y308. Disruption of this NMDAR-Src-Panx1 signaling complex in vitro or in vivo by administration of an interfering peptide either before or 2 h after ischemia or stroke was neuroprotective. Our observations provide insights into a new signaling modality of NMDARs that has broad-reaching implications for brain physiology and pathology.

Altered synchrony and connectivity in neuronal networks expressing an autism-related mutation of neuroligin 3

The neuroligin (NL) gene family codes for brain specific cell adhesion molecules that play an important role in synaptic connectivity. Recent studies have identified NL mutations linked to patients with autism spectrum disorders (ASD). Cognitive deficits seen in autistic patients are hypothesized to arise from altered synchronicity both within and between brain regions. Here we show how the expression of autism-associated neuroligin mutation R471C-NL3 affects synchrony in dissociated cultures of rat hippocampal neurons. Spontaneous network activity patterns of cultures expressing wild type and mutant NL3 were measured by optical techniques. Firing events were quantified and compared by cross-correlation analysis. Our results suggest that NL3 overexpression enhances synchrony of spontaneous activity patterns, however, this ability is reduced with the R471C-NL3 mutation. We investigated the structural basis of this phenomenon using fractal dimension analysis to characterize the arrangement of axon trajectories. R471C-NL3 cultures were associated with lower fractal dimensions and higher lacunarity values, indicating a decrease in the complexity of axonal architecture. Transfection of R471C-NL3 into a subpopulation of cells in a network resulted in neuronal degeneration. This degeneration likely affected the inhibitory population of neurons, as there were half as many (P<0.01, n=12) glutamate decarboxylase (GAD) 65 expressing cells in R471C-NL3 cultures compared to wild type NL3 and control cultures. Electrophysiological recordings showed a reduction of inhibitory activity in networks carrying the mutation in comparison to networks overexpressing wild-type NL3. Together, these data support the hypothesis that the autism-associated NL3 mutation affects information processing in neuronal networks by altering network architecture and synchrony.

Astrocytic Ca2+ Waves Guide CNS Growth Cones to Remote Regions of Neuronal Activity

Activity plays a critical role in network formation during developmental, experience-dependent, and injury related remodeling. Here we report a mechanism by which axon trajectory can be altered in response to remote neuronal activity. Using photoconductive stimulation to trigger high frequency action potentials in rat hippocampal neurons in vitro, we find that activity functions as an attractive cue for growth cones in the local environment. The underlying guidance mechanism involves astrocyte Ca(2+) waves, as the connexin-43 antagonist carbenoxolone abolishes the attraction when activity is initiated at a distance greater than 120 microm. The asymmetric growth cone filopodia extension that precedes turning can be blocked with CNQX (10 microM), but not with the ATP and adenosine receptor antagonists suramin (100 microM) and alloxazine (4 microM), suggesting non-NMDA glutamate receptors on the growth cone mediate the interaction with astrocytes. These results define a potential long-range signalling pathway for activity-dependent axon guidance in which growth cones turn towards directional, temporally coordinated astrocyte Ca(2+) waves that are triggered by neuronal activity. To assess the viability of the guidance effect in an injury paradigm, we performed the assay in the presence of conditioned media from lipopolysaccharide (LPS) activated purified microglial cultures, as well as directly activating the glia present in our co-cultures. Growth cone attraction was not inhibited under these conditions, suggesting this mechanism could be used to guide regeneration following axonal injury.

Paralemmin-1, a Modulator of Filopodia Induction Is Required for Spine Maturation

Dendritic filopodia are thought to participate in neuronal contact formation and development of dendritic spines; however, molecules that regulate filopodia extension and their maturation to spines remain largely unknown. Here we identify paralemmin-1 as a regulator of filopodia induction and spine maturation. Paralemmin-1 localizes to dendritic membranes, and its ability to induce filopodia and recruit synaptic elements to contact sites requires protein acylation. Effects of paralemmin-1 on synapse maturation are modulated by alternative splicing that regulates spine formation and recruitment of AMPA-type glutamate receptors. Paralemmin-1 enrichment at the plasma membrane is subject to rapid changes in neuronal excitability, and this process controls neuronal activity-driven effects on protrusion expansion. Knockdown of paralemmin-1 in developing neurons reduces the number of filopodia and spines formed and diminishes the effects of Shank1b on the transformation of existing filopodia into spines. Our study identifies a key role for paralemmin-1 in spine maturation through modulation of filopodia induction.