MCell4 with BioNetGen: A Monte Carlo simulator of rule-based reaction-diffusion systems with Python interface.

Biochemical signaling pathways in living cells are often highly organized into spatially segregated volumes, membranes, scaffolds, subcellular compartments, and organelles comprising small numbers of interacting molecules. At this level of granularity stochastic behavior dominates, well-mixed continuum approximations based on concentrations break down and a particle-based approach is more accurate and more efficient. We describe and validate a new version of the open-source MCell simulation program (MCell4), which supports generalized 3D Monte Carlo modeling of diffusion and chemical reaction of discrete molecules and macromolecular complexes in solution, on surfaces representing membranes, and combinations thereof. The main improvements in MCell4 compared to the previous versions, MCell3 and MCell3-R, include a Python interface and native BioNetGen reaction language (BNGL) support. MCell4's Python interface opens up completely new possibilities for interfacing with external simulators to allow creation of sophisticated event-driven multiscale/multiphysics simulations. The native BNGL support, implemented through a new open-source library libBNG (also introduced in this paper), provides the capability to run a given BNGL model spatially resolved in MCell4 and, with appropriate simplifying assumptions, also in the BioNetGen simulation environment, greatly accelerating and simplifying model validation and comparison.

A spatial model of autophosphorylation of CaMKII in a glutamatergic spine suggests a network-driven kinetic mechanism for bistable changes in synaptic strength.

Activation of N-methyl-D-aspartate-type glutamate receptors (NMDARs) at synapses in the CNS triggers changes in synaptic strength that underlie memory formation in response to strong synaptic stimuli. The primary target of Ca2+ flowing through NMDARs is Ca2+/calmodulin-dependent protein kinase II (CaMKII) which forms dodecameric holoenzymes that are highly concentrated at the postsynaptic site. Activation of CaMKII is necessary to trigger long-term potentiation of synaptic strength (LTP), and is prolonged by autophosphorylation of subunits within the holoenzyme. Here we use MCell4, an agent-based, stochastic, modeling platform to model CaMKII holoenzymes placed within a realistic spine geometry. We show how two mechanisms of regulation of CaMKII, 'Ca2+-calmodulin-trapping (CaM-trapping)' and dephosphorylation by protein phosphatase-1 (PP1) shape the autophosphorylation response during a repeated high-frequency stimulus. Our simulation results suggest that autophosphorylation of CaMKII does not constitute a bistable switch. Instead, prolonged but temporary, autophosphorylation of CaMKII may contribute to a biochemical-network-based 'kinetic proof-reading" mechanism that controls induction of synaptic plasticity.

Amplification of neurotoxic HTTex1 assemblies in human neurons.

Huntington's disease (HD) is a genetically inherited neurodegenerative disorder caused by expansion of a polyglutamine (polyQ) repeat in the exon-1 of huntingtin protein (HTT). The expanded polyQ enhances the amyloidogenic propensity of HTT exon 1 (HTTex1), which forms a heterogeneous mixture of assemblies with a broad neurotoxicity spectrum. While predominantly intracellular, monomeric and aggregated mutant HTT species are also present in the cerebrospinal fluids of HD patients, however, their biological properties are not well understood. To explore the role of extracellular mutant HTT in aggregation and toxicity, we investigated the uptake and amplification of recombinant HTTex1 assemblies in cell culture models. We find that small HTTex1 fibrils preferentially enter human neurons and trigger the amplification of neurotoxic assemblies; astrocytes or epithelial cells are not permissive. The amplification of HTTex1 in neurons depletes endogenous HTT protein with non-pathogenic polyQ repeat, activates apoptotic caspase-3 pathway and induces nuclear fragmentation. Using a panel of novel monoclonal antibodies and genetic mutation, we identified epitopes within the N-terminal 17 amino acids and proline-rich domain of HTTex1 to be critical in neural uptake and amplification. Synaptosome preparations from the brain homogenates of HD mice also contain mutant HTT species, which enter neurons and behave similar to small recombinant HTTex1 fibrils. These studies suggest that amyloidogenic extracellular mutant HTTex1 assemblies may preferentially enter neurons, propagate and promote neurodegeneration.

A sex difference in the response of the rodent postsynaptic density to synGAP haploinsufficiency.

SynGAP is a postsynaptic density (PSD) protein that binds to PDZ domains of the scaffold protein PSD-95. We previously reported that heterozygous deletion of Syngap1 in mice is correlated with increased steady-state levels of other key PSD proteins that bind PSD-95, although the level of PSD-95 remains constant (Walkup et al., 2016). For example, the ratio to PSD-95 of Transmembrane AMPA-Receptor-associated Proteins (TARPs), which mediate binding of AMPA-type glutamate receptors to PSD-95, was increased in young Syngap1+/-mice. Here we show that only females and not males show a highly significant correlation between an increase in TARP and a decrease in synGAP in the PSDs of Syngap1+/-rodents. The data reveal a sex difference in the adaptation of the PSD scaffold to synGAP haploinsufficiency.

A multi-state model of the CaMKII dodecamer suggests a role for calmodulin in maintenance of autophosphorylation.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) accounts for up to 2 percent of all brain protein and is essential to memory function. CaMKII activity is known to regulate dynamic shifts in the size and signaling strength of neuronal connections, a process known as synaptic plasticity. Increasingly, computational models are used to explore synaptic plasticity and the mechanisms regulating CaMKII activity. Conventional modeling approaches may exclude biophysical detail due to the impractical number of state combinations that arise when explicitly monitoring the conformational changes, ligand binding, and phosphorylation events that occur on each of the CaMKII holoenzyme's subunits. To manage the combinatorial explosion without necessitating bias or loss in biological accuracy, we use a specialized syntax in the software MCell to create a rule-based model of a twelve-subunit CaMKII holoenzyme. Here we validate the rule-based model against previous experimental measures of CaMKII activity and investigate molecular mechanisms of CaMKII regulation. Specifically, we explore how Ca2+/CaM-binding may both stabilize CaMKII subunit activation and regulate maintenance of CaMKII autophosphorylation. Noting that Ca2+/CaM and protein phosphatases bind CaMKII at nearby or overlapping sites, we compare model scenarios in which Ca2+/CaM and protein phosphatase do or do not structurally exclude each other's binding to CaMKII. Our results suggest a functional mechanism for the so-called "CaM trapping" phenomenon, wherein Ca2+/CaM may structurally exclude phosphatase binding and thereby prolong CaMKII autophosphorylation. We conclude that structural protection of autophosphorylated CaMKII by Ca2+/CaM may be an important mechanism for regulation of synaptic plasticity.

In memoriam: John Lisman - commentaries on CaMKII as a memory molecule.

Shortly before he died in October 2017, John Lisman submitted an invited review to Molecular Brain on 'Criteria for identifying the molecular basis of the engram (CaMKII, PKMζ)'. John had no opportunity to read the referees' comments, and as a mark of the regard in which he was held by the neuroscience community the Editors decided to publish his review as submitted. This obituary takes the form of a series of commentaries on Lisman's review. At the same time we are publishing as a separate article a longer response by Todd Sacktor and André Fenton entitled 'What does LTP tell us about the roles of CaMKII and PKMζ in memory?' which presents the case for a rival memory molecule, PKMζ.

Phosphorylation of synaptic GTPase-activating protein (synGAP) by polo-like kinase (Plk2) alters the ratio of its GAP activity toward HRas, Rap1 and Rap2 GTPases.

SynGAP is a Ras and Rap GTPase-activating protein (GAP) found in high concentration in the postsynaptic density (PSD) fraction from mammalian forebrain where it binds to PDZ domains of PSD-95. Phosphorylation of pure recombinant synGAP by Ca2+/calmodulin-dependent protein kinase II (CaMKII) shifts the balance of synGAP's GAP activity toward inactivation of Rap1; whereas phosphorylation by cyclin-dependent kinase 5 (CDK5) has the opposite effect, shifting the balance toward inactivation of HRas. These shifts in balance contribute to regulation of the numbers of surface AMPA receptors, which rise during synaptic potentiation (CaMKII) and fall during synaptic scaling (CDK5). Polo-like kinase 2 (Plk2/SNK), like CDK5, contributes to synaptic scaling. These two kinases act in concert to reduce the number of surface AMPA receptors following elevated neuronal activity by tagging spine-associated RapGAP protein (SPAR) for degradation, thus raising the level of activated Rap. Here we show that Plk2 also phosphorylates and regulates synGAP. Phosphorylation of synGAP by Plk2 stimulates its GAP activity toward HRas by 65%, and toward Rap1 by 16%. Simultaneous phosphorylation of synGAP by Plk2 and CDK5 at distinct sites produces an additive increase in GAP activity toward HRas (∼230%) and a smaller, non-additive increase in activity toward Rap1 (∼15%). Dual phosphorylation also produces an increase in GAP activity toward Rap2 (∼40-50%), an effect not produced by either kinase alone. As we previously observed for CDK5, addition of Ca2+/CaM causes a substrate-directed doubling of the rate and stoichiometry of phosphorylation of synGAP by Plk2, targeting residues also phosphorylated by CaMKII. In summary, phosphorylation by Plk2, like CDK5, shifts the ratio of GAP activity of synGAP to produce a greater decrease in active Ras than in active Rap, which would produce a shift toward a decrease in the number of surface AMPA receptors in neuronal dendrites.

The Protein Biochemistry of the Postsynaptic Density in Glutamatergic Synapses Mediates Learning in Neural Networks.

The strength of each excitatory synapse in the central nervous system is regulated by its prior activity in a process called synaptic plasticity. The initiation of synaptic plasticity occurs when calcium ions enter the postsynaptic compartment and encounter a subcellular structure called the postsynaptic density (PSD). The PSD is attached to the postsynaptic membrane just underneath the concentrated plaque of neurotransmitter receptors. It is comprised of a core set of 30-60 proteins, approximately 20 of which are scaffold proteins. The rest include protein kinases and phosphatases, some of which respond to calcium ion; small GTPases and their regulators; chaperones; ubiquitins; and proteases. The assembly of the PSD involves competitive binding among a variety of specific protein binding sites to form a dynamic network. A biochemical challenge for the future is to understand how the dynamic regulation of the structure, composition, and activity of the PSD mediates synaptic plasticity and how mutations in PSD proteins lead to mental and neurodegenerative diseases.

Densin-180 Controls the Trafficking and Signaling of L-Type Voltage-Gated Cav1.2 Ca2+ Channels at Excitatory Synapses.

Voltage-gated Cav1.2 and Cav1.3 (L-type) Ca2+ channels regulate neuronal excitability, synaptic plasticity, and learning and memory. Densin-180 (densin) is an excitatory synaptic protein that promotes Ca2+-dependent facilitation of voltage-gated Cav1.3 Ca2+ channels in transfected cells. Mice lacking densin (densin KO) exhibit defects in synaptic plasticity, spatial memory, and increased anxiety-related behaviors-phenotypes that more closely match those in mice lacking Cav1.2 than Cav1.3. Therefore, we investigated the functional impact of densin on Cav1.2. We report that densin is an essential regulator of Cav1.2 in neurons, but has distinct modulatory effects compared with its regulation of Cav1.3. Densin binds to the N-terminal domain of Cav1.2, but not that of Cav1.3, and increases Cav1.2 currents in transfected cells and in neurons. In transfected cells, densin accelerates the forward trafficking of Cav1.2 channels without affecting their endocytosis. Consistent with a role for densin in increasing the number of postsynaptic Cav1.2 channels, overexpression of densin increases the clustering of Cav1.2 in dendrites of hippocampal neurons in culture. Compared with wild-type mice, the cell surface levels of Cav1.2 in the brain, as well as Cav1.2 current density and signaling to the nucleus, are reduced in neurons from densin KO mice. We conclude that densin is an essential regulator of neuronal Cav1 channels and ensures efficient Cav1.2 Ca2+ signaling at excitatory synapses.SIGNIFICANCE STATEMENT The number and localization of voltage-gated Cav Ca2+ channels are crucial determinants of neuronal excitability and synaptic transmission. We report that the protein densin-180 is highly enriched at excitatory synapses in the brain and enhances the cell surface trafficking and postsynaptic localization of Cav1.2 L-type Ca2+ channels in neurons. This interaction promotes coupling of Cav1.2 channels to activity-dependent gene transcription. Our results reveal a mechanism that may contribute to the roles of Cav1.2 in regulating cognition and mood.

Biochemistry and neuroscience: the twain need to meet.

Neuroscience has come to mean the study of electrophysiology of neurons and synapses, micro and macro-scale neuroanatomy, and the functional organization of brain areas. The molecular axis of the field, as reflected in textbooks, often includes only descriptions of the structure and function of individual channels and receptor proteins, and the extracellular signals that guide development and repair. Studies of cytosolic 'molecular machines', large assemblies of proteins that orchestrate regulation of neuronal functions, have been neglected. However, a complete understanding of brain function that will enable new strategies for treatment of the most intractable neural disorders will require that in vitro biochemical studies of molecular machines be reintegrated into the field of neuroscience.

A model for regulation by SynGAP-α1 of binding of synaptic proteins to PDZ-domain 'Slots' in the postsynaptic density.

SynGAP is a Ras/Rap GTPase-activating protein (GAP) that is a major constituent of postsynaptic densities (PSDs) from mammalian forebrain. Its α1 isoform binds to all three PDZ (PSD-95, Discs-large, ZO-1) domains of PSD-95, the principal PSD scaffold, and can occupy as many as 15% of these PDZ domains. We present evidence that synGAP-α1 regulates the composition of the PSD by restricting binding to the PDZ domains of PSD-95. We show that phosphorylation by Ca2+/calmodulin-dependent protein kinase II (CaMKII) and Polo-like kinase-2 (PLK2) decreases its affinity for the PDZ domains by several fold, which would free PDZ domains for occupancy by other proteins. Finally, we show that three critical postsynaptic signaling proteins that bind to the PDZ domains of PSD-95 are present in higher concentration in PSDs isolated from mice with a heterozygous deletion of synGAP.

Computational reconstitution of spine calcium transients from individual proteins.

We have built a stochastic model in the program MCell that simulates Ca(2+) transients in spines from the principal molecular components believed to control Ca(2+) entry and exit. Proteins, with their kinetic models, are located within two segments of dendrites containing 88 intact spines, centered in a fully reconstructed 6 × 6 × 5 µm(3) cube of hippocampal neuropil. Protein components include AMPA- and NMDA-type glutamate receptors, L- and R-type voltage-dependent Ca(2+) channels, Na(+)/Ca(2+) exchangers, plasma membrane Ca(2+) ATPases, smooth endoplasmic reticulum Ca(2+) ATPases, immobile Ca(2+) buffers, and calbindin. Kinetic models for each protein were taken from published studies of the isolated proteins in vitro. For simulation of electrical stimuli, the time course of voltage changes in the dendritic spine was generated with the desired stimulus in the program NEURON. Voltage-dependent parameters were then continuously re-adjusted during simulations in MCell to reproduce the effects of the stimulus. Nine parameters of the model were optimized within realistic experimental limits by a process that compared results of simulations to published data. We find that simulations in the optimized model reproduce the timing and amplitude of Ca(2+) transients measured experimentally in intact neurons. Thus, we demonstrate that the characteristics of individual isolated proteins determined in vitro can accurately reproduce the dynamics of experimentally measured Ca(2+) transients in spines. The model will provide a test bed for exploring the roles of additional proteins that regulate Ca(2+) influx into spines and for studying the behavior of protein targets in the spine that are regulated by Ca(2+) influx.

Protein purification using PDZ affinity chromatography.

PDZ domains function in nature as protein-binding domains within scaffold and membrane-associated proteins. They comprise approximately 90 residues and undergo specific, high-affinity interactions with complementary C-terminal peptide sequences, other PDZ domains, and/or phospholipids. We have previously shown that the specific, strong interactions of PDZ domains with their ligands make them well suited for use in affinity chromatography. This unit provides protocols for the PDZ affinity chromatography procedure that are applicable for the purification of proteins that contain PDZ domains or PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We detail the preparation of affinity resins composed of PDZ domains or PDZ domain peptide ligands coupled to solid supports. These resins can be used to purify proteins containing endogenous or genetically introduced PDZ domains or ligands, eluting the proteins with free PDZ domain peptide ligands.

Phosphorylation of synaptic GTPase-activating protein (synGAP) by Ca2+/calmodulin-dependent protein kinase II (CaMKII) and cyclin-dependent kinase 5 (CDK5) alters the ratio of its GAP activity toward Ras and Rap GTPases.

synGAP is a neuron-specific Ras and Rap GTPase-activating protein (GAP) found in high concentrations in the postsynaptic density (PSD) fraction from the mammalian forebrain. We have previously shown that, in situ in the PSD fraction or in recombinant form in Sf9 cell membranes, synGAP is phosphorylated by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), another prominent component of the PSD. Here, we show that recombinant synGAP (r-synGAP), lacking 102 residues at the N terminus, can be purified in soluble form and is phosphorylated by cyclin-dependent kinase 5 (CDK5) as well as by CaMKII. Phosphorylation of r-synGAP by CaMKII increases its HRas GAP activity by 25% and its Rap1 GAP activity by 76%. Conversely, phosphorylation by CDK5 increases r-synGAP's HRas GAP activity by 98% and its Rap1 GAP activity by 20%. Thus, phosphorylation by both kinases increases synGAP activity; CaMKII shifts the relative GAP activity toward inactivation of Rap1, and CDK5 shifts the relative activity toward inactivation of HRas. GAP activity toward Rap2 is not altered by phosphorylation by either kinase. CDK5 phosphorylates synGAP primarily at two sites, Ser-773 and Ser-802. Phosphorylation at Ser-773 inhibits r-synGAP activity, and phosphorylation at Ser-802 increases it. However, the net effect of concurrent phosphorylation of both sites, Ser-773 and Ser-802, is an increase in GAP activity. synGAP is phosphorylated at Ser-773 and Ser-802 in the PSD fraction, and its phosphorylation by CDK5 and CaMKII is differentially regulated by activation of NMDA-type glutamate receptors in cultured neurons.

Multi-state modeling of biomolecules.

Multi-state modeling of biomolecules refers to a series of techniques used to represent and compute the behavior of biological molecules or complexes that can adopt a large number of possible functional states. Biological signaling systems often rely on complexes of biological macromolecules that can undergo several functionally significant modifications that are mutually compatible. Thus, they can exist in a very large number of functionally different states. Modeling such multi-state systems poses two problems: the problem of how to describe and specify a multi-state system (the "specification problem") and the problem of how to use a computer to simulate the progress of the system over time (the "computation problem"). To address the specification problem, modelers have in recent years moved away from explicit specification of all possible states and towards rule-based formalisms that allow for implicit model specification, including the κ-calculus, BioNetGen, the Allosteric Network Compiler, and others. To tackle the computation problem, they have turned to particle-based methods that have in many cases proved more computationally efficient than population-based methods based on ordinary differential equations, partial differential equations, or the Gillespie stochastic simulation algorithm. Given current computing technology, particle-based methods are sometimes the only possible option. Particle-based simulators fall into two further categories: nonspatial simulators, such as StochSim, DYNSTOC, RuleMonkey, and the Network-Free Stochastic Simulator (NFSim), and spatial simulators, including Meredys, SRSim, and MCell. Modelers can thus choose from a variety of tools, the best choice depending on the particular problem. Development of faster and more powerful methods is ongoing, promising the ability to simulate ever more complex signaling processes in the future.

PDZ affinity chromatography: a general method for affinity purification of proteins based on PDZ domains and their ligands.

PDZ (PSD-95, DiscsLarge, ZO1) domains function in nature as protein binding domains within scaffold and membrane-associated proteins. They comprise ∼90 residues and make specific, high affinity interactions with complementary C-terminal peptide sequences, with other PDZ domains, and with phospholipids. We hypothesized that the specific, strong interactions of PDZ domains with their ligands would make them well suited for use in affinity chromatography. Here we describe a novel affinity chromatography method applicable for the purification of proteins that contain PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We created a series of affinity resins comprised of PDZ domains from the scaffold protein PSD-95, or from neuronal nitric oxide synthase (nNOS), coupled to solid supports. We used them to purify heterologously expressed neuronal proteins or protein domains containing endogenous PDZ domain ligands, eluting the proteins with free PDZ domain peptide ligands. We show that Proteins of Interest (POIs) lacking endogenous PDZ domain ligands can be engineered as fusion products containing C-terminal PDZ domain ligand peptides or internal, N- or C-terminal PDZ domains and then can be purified by the same method. Using this method, we recovered recombinant GFP fused to a PDZ domain ligand in active form as verified by fluorescence yield. Similarly, chloramphenicol acetyltransferase (CAT) and ß-Galactosidase (LacZ) fused to a C-terminal PDZ domain ligand or an N-terminal PDZ domain were purified in active form as assessed by enzymatic assay. In general, PDZ domains and ligands derived from PSD-95 were superior to those from nNOS for this method. PDZ Domain Affinity Chromatography promises to be a versatile and effective method for purification of a wide variety of natural and recombinant proteins.

Subcellular organization of camkii in rat hippocampal pyramidal neurons.

Calcium/calmodulin-dependent protein kinase II (CaMKII) plays a key role in N-methyl-D-aspartate (NMDA) receptor-dependent long-term synaptic plasticity; its location is critical for signal transduction, and may provide clues that further elucidate its function. We therefore examined the subcellular localization of CaMKII in CA1 stratum radiatum of adult rat hippocampus, by using immuno-electron microscopy after chemical fixation. When tissue was fixed quickly, the concentration of CaMKIIα (assessed by pre-embedding immunogold) was significantly higher in dendritic shafts than in spine heads. However, when tissue was fixed 5 minutes after perfusion with normal saline, the density of labeling decreased in dendritic shaft while increasing in spine heads, implying rapid translocation into the spine during brief perimortem stress. Likewise, in quickly fixed tissue, CaMKII within spine heads was found at comparable concentrations in the "proximal" half (adjacent to the spine neck) and the "distal" half (containing the postsynaptic density [PSD]), whereas after delayed fixation, label density increased in the distal side of the spine head, suggesting that CaMKII within the spine head moves toward the PSD during this interval. To estimate its distribution at the synapse in vivo, we performed postembedding immunogold staining for CaMKII in quick-fixed tissue, and found that the enzyme did not concentrate primarily within the central matrix of the PSD. Instead, labeling density peaked ∼40 nm inside the postsynaptic membrane, at the cytoplasmic fringe of the PSD. Labeling within 25 nm of the postsynaptic membrane concentrated at the lateral edge of the synapse. This lateral "PSD core" pool of CaMKII may play a special role in synaptic plasticity.

Synaptic Signaling in Learning and Memory.

Learning and memory require the formation of new neural networks in the brain. A key mechanism underlying this process is synaptic plasticity at excitatory synapses, which connect neurons into networks. Excitatory synaptic transmission happens when glutamate, the excitatory neurotransmitter, activates receptors on the postsynaptic neuron. Synaptic plasticity is a higher-level process in which the strength of excitatory synapses is altered in response to the pattern of activity at the synapse. It is initiated in the postsynaptic compartment, where the precise pattern of influx of calcium through activated glutamate receptors leads either to the addition of new receptors and enlargement of the synapse (long-term potentiation) or the removal of receptors and shrinkage of the synapse (long-term depression). Calcium/calmodulin-regulated enzymes and small GTPases collaborate to control this highly tuned mechanism.