Gary J. Iacobucci

Organically Modified Silica Nanoparticles Are Biocompatible and Can Be Targeted to Neurons In Vivo

The application of nanotechnology in biological research is beginning to have a major impact leading to the development of new types of tools for human health. One focus of nanobiotechnology is the development of nanoparticle-based formulations for use in drug or gene delivery systems. However most of the nano probes currently in use have varying levels of toxicity in cells or whole organisms and therefore are not suitable for in vivo application or long-term use. Here we test the potential of a novel silica based nanoparticle (organically modified silica, ORMOSIL) in living neurons within a whole organism. We show that feeding ORMOSIL nanoparticles to Drosophila has no effect on viability. ORMOSIL nanoparticles penetrate into living brains, neuronal cell bodies and axonal projections. In the neuronal cell body, nanoparticles are present in the cytoplasm, but not in the nucleus. Strikingly, incorporation of ORMOSIL nanoparticles into the brain did not induce aberrant neuronal death or interfered with normal neuronal processes. Our results in Drosophila indicate that these novel silica based nanoparticles are biocompatible and not toxic to whole organisms, and has potential for the development of long-term applications.

prickle modulates microtubule polarity and axonal transport to ameliorate seizures in flies.

Significance Mutations in prickle genes from flies to humans cause epilepsy, a disorder that affects approximately 1% of the population. Although Prickle has been studied for many years, its molecular function is unknown. We now show that Prickle modulates transport of vesicles in fruit fly neurons. Additionally, we show that prickle mutants have electrophysiological defects consistent with epilepsy, and merely altering the balance of Prickle isoforms causes seizures. Finally, we demonstrate that reducing the level of vesicle transport motor proteins can suppress prickle-mediated seizures, revealing a previously unidentified pathway in the pathophysiology of epilepsy. Recent analyses in flies, mice, zebrafish, and humans showed that mutations in prickle orthologs result in epileptic phenotypes, although the mechanism responsible for generating the seizures was unknown. Here, we show that Prickle organizes microtubule polarity and affects their growth dynamics in axons of Drosophila neurons, which in turn influences both anterograde and retrograde vesicle transport. We also show that enhancement of the anterograde transport mechanism is the cause of the seizure phenotype in flies, which can be suppressed by reducing the level of either of two Kinesin motor proteins responsible for anterograde vesicle transport. Additionally, we show that seizure-prone prickle mutant flies have electrophysiological defects similar to other fly mutants used to study seizures, and that merely altering the balance of the two adult prickle isoforms in neurons can predispose flies to seizures. These data reveal a previously unidentified pathway in the pathophysiology of seizure disorders and provide evidence for a more generalized cellular mechanism whereby Prickle mediates polarity by influencing microtubule-mediated transport.

NMDA receptors: linking physiological output to biophysical operation

NMDA receptors are preeminent neurotransmitter-gated channels in the CNS, which respond to glutamate in a manner that integrates multiple external and internal cues. They belong to the ionotropic glutamate receptor family and fulfil unique and crucial roles in neuronal development and function. These roles depend on characteristic response kinetics, which reflect the operation of the receptors. Here, we review biologically salient features of the NMDA receptor signal and its mechanistic origins. Knowledge of distinctive NMDA receptor biophysical properties, their structural determinants and physiological roles is necessary to understand the physiological and neurotoxic actions of glutamate and to design effective therapeutics.

Presenilin influences glycogen synthase kinase-3 β (GSK-3β) for kinesin-1 and dynein function during axonal transport

Within axons, molecular motors transport essential components required for neuronal growth and viability. Although many levels of control and regulation must exist for proper anterograde and retrograde transport of vital proteins, little is known about these mechanisms. We previously showed that presenilin (PS), a gene involved in Alzheimers disease (AD), influences kinesin-1 and dynein function in vivo. Here, we show that these PS-mediated effects on motor protein function are via a pathway that involves glycogen synthase kinase-3β (GSK-3β). PS genetically interacts with GSK-3β in an activity-dependent manner. Excess of active GSK-3β perturbed axonal transport by causing axonal blockages, which were enhanced by reduction of kinesin-1 or dynein. These GSK-3β-mediated axonal defects do not appear to be caused by disruptions or alterations in microtubules (MTs). Excess of non-functional GSK-3β did not affect axonal transport. Strikingly, GSK-3β-activity-dependent axonal transport defects were enhanced by reduction of PS. Collectively, our findings suggest that PS and GSK-3β are required for normal motor protein function. Our observations propose a model, in which PS likely plays a role in regulating GSK-3β activity during transport. These findings have important implications for our understanding of the complex regulatory machinery that must exist in vivo and how this system is coordinated during the motility of vesicles within axons.

Probing the Structural Dynamics of the NMDA Receptor Activation by Coarse-Grained Modeling

N-Methyl-D-aspartate (NMDA) receptors are glutamate-gated excitatory channels that play essential roles in brain functions. High-resolution structures have been solved for an allosterically inhibited and agonist-bound form of a functional NMDA receptor; however, other key functional states (particularly the active open-channel state) were only resolved at moderate resolutions by cryo-electron microscopy (cryo-EM). To decrypt the mechanism of the NMDA receptor activation, structural modeling is essential to provide presently missing information about structural dynamics. We performed systematic coarse-grained modeling using an elastic network model and related modeling/analysis tools (e.g., normal mode analysis, flexibility and hotspot analysis, cryo-EM flexible fitting, and transition pathway modeling) based on an active-state cryo-EM map. We observed extensive conformational changes that allosterically couple the extracellular regulatory and agonist-binding domains to the pore-forming trans-membrane domain (TMD), and validated these, to our knowledge, new observations against known mutational and functional studies. Our results predict two key modes of collective motions featuring shearing/twisting of the extracellular domains relative to the TMD, reveal subunit-specific flexibility profiles, and identify functional hotspot residues at key domain-domain interfaces. Finally, by examining the conformational transition pathway between the allosterically inhibited form and the active form, we predict a discrete sequence of domain motions, which propagate from the extracellular domains to the TMD. In summary, our results offer rich structural and dynamic information, which is consistent with the literature on structure-function relationships in NMDA receptors, and will guide in-depth studies on the activation dynamics of this important neurotransmitter receptor.

Spatial and Temporal Characteristics of Normal and Perturbed Vesicle Transport

Efficient intracellular transport is essential for healthy cellular function and structural integrity, and problems in this pathway can lead to neuronal cell death and disease. To spatially and temporally evaluate how transport defects are initiated, we adapted a primary neuronal culture system from Drosophila larval brains to visualize the movement dynamics of several cargos/organelles along a 90 micron axonal neurite over time. All six vesicles/organelles imaged showed robust bi-directional motility at both day 1 and day 2. Reduction of motor proteins decreased the movement of vesicles/organelles with increased numbers of neurite blocks. Neuronal growth was also perturbed with reduction of motor proteins. Strikingly, we found that all blockages were not fixed, permanent blocks that impeded transport of vesicles as previously thought, but that some blocks were dynamic clusters of vesicles that resolved over time. Taken together, our findings suggest that non-resolving blocks may likely initiate deleterious pathways leading to death and degeneration, while resolving blocks may be benign. Therefore evaluating the spatial and temporal characteristics of vesicle transport has important implications for our understanding of how transport defects can affect other pathways to initiate death and degeneration.

Kinetic Models for Activation and Modulation of NMDA Receptor Subtypes

NMDA receptors are a diverse family of excitatory channels with critical roles in central synaptic transmission, development, and plasticity. Controlled expression of seven subunits and their combinatorial assembly into tetrameric receptors produces a range of molecularly distinct receptor subtypes. Despite relatively similar atomic structures, each subtype has input-output functions with unique biophysical and pharmacologic profiles. Here, we briefly summarize recent advances in understanding how gating and allosteric modulation are similar or distinct across NMDA receptor isoforms and identify open questions that will focus research in this area going forward.

Ethanol stimulates the in vivo axonal movement of neuropeptide dense-core vesicles in Drosophila motor neurons

Proper neuronal function requires essential biological cargoes to be packaged within membranous vesicles and transported, intracellularly, through the extensive outgrowth of axonal and dendritic fibers. The precise spatiotemporal movement of these cargoes is vital for neuronal survival and, thus, is highly regulated. In this study we test how the axonal movement of a neuropeptide‐containing dense‐core vesicle (DCV) responds to alcohol stressors. We found that ethanol induces a strong anterograde bias in vesicle movement. Low doses of ethanol stimulate the anterograde movement of neuropeptide‐DCV while high doses inhibit bi‐directional movement. This process required the presence of functional kinesin‐1 motors as reduction in kinesin prevented the ethanol‐induced stimulation of the anterograde movement of neuropeptide‐DCV. Furthermore, expression of inactive glycogen synthase kinase 3 (GSK‐3β) also prevented ethanol‐induced stimulation of neuropeptide‐DCV movement, similar to pharmacological inhibition of GSK‐3β with lithium. Conversely, inhibition of PI3K/AKT signaling with wortmannin led to a partial prevention of ethanol‐stimulated transport of neuropeptide‐DCV. Taken together, we conclude that GSK‐3β signaling mediates the stimulatory effects of ethanol. Therefore, our study provides new insight into the physiological response of the axonal movement of neuropeptide‐DCV to exogenous stressors.

Local Ca2+ Nanodomains Initiate Ca2+/Calmodulin-Dependent Inactivation of NMDA Receptors

NMDA receptors (NRs) are glutamate- and glycine- gated non-selective excitatory channels that are expressed throughout the central nervous system. They have large (50 - 70 pS) unitary conductance and a large fraction of the current (10 - 20 %) is carried by Ca2+. Specifically the NR-mediated Ca2+ influx drives critical physiological processes including synaptic plasticity and apoptosis and also reduces NR gating, a process termed Ca2+-dependent inactivation (CDI). CDI requires Ca2+ binding to calmodulin (CaM) and is typically measured as increased macroscopic current desensitization. To investigate mechanisms underlying CDI we combined mathematical models of Ca2+ diffusion with one-channel and macroscopic current recordings from HEK293 transfected with GluN1-2a and GluN2A NR subunits. First, to investigate whether CDI is produced by local rises in intracellular Ca2+, we calibrated the extent of CDI observed extracellular Ca2+ flux through the pore under conditions of high intracellular buffering (BAPTA) to the extent of CDI observed with controlled dialysis of intracellular [Ca2+]. Using a model for intracellular Ca2+ diffusion based on Neher-Stern theory and the experimentally measured CDI produced by NR-mediated Ca2+ influx, we estimated the Ca2+-sensor responsible for CDI (i.e. CaM) resides within 5 nm distance of the Ca2+ source. This is consistent with previous biochemical evidence supporting the hypothesis that CaM preassociates with the C-terminal domain of GluN1 on the C0 cassette. In addition, single-channel current recordings combined with kinetic modeling suggest that Ca2+/CaM decreases NR activity by increasing the occupancy of desensitized states. Together, these results suggests CDI is initiated by an influx of Ca2+ which activates a local population of CaM to potentiate desensitization of NRs.