Lessons from a Recovering Academic.

The conversion of basic biology into new therapeutics requires scientific activities in both academia and industry. Successful drug discovery projects span disciplines, sectors, and institutions and tightly couple laboratory and clinical experiments. Here, Ehlers describes conceptions and misconceptions about how science is conducted in industry versus academia.

Local zones of endoplasmic reticulum complexity confine cargo in neuronal dendrites.

Following synthesis, integral membrane proteins dwell in the endoplasmic reticulum (ER) for variable periods that are typically rate limiting for plasma membrane delivery. In neurons, the ER extends for hundreds of microns as an anastomosing network throughout highly branched dendrites. However, little is known about the mobility, spatial scales, or dynamic regulation of cargo in the dendritic ER. Here, we show that membrane proteins, including AMPA-type glutamate receptors, rapidly diffuse within the continuous network of dendritic ER but are confined by increased ER complexity at dendritic branch points and near dendritic spines. The spatial range of receptor mobility is rapidly restricted by type I mGluR signaling through a mechanism involving protein kinase C (PKC) and the ER protein CLIMP63. Moreover, local zones of ER complexity compartmentalize ER export and correspond to sites of new dendritic branches. Thus, local control of ER complexity spatially scales secretory trafficking within elaborate dendritic arbors.

Syntaxin-4 defines a domain for activity-dependent exocytosis in dendritic spines.

Changes in postsynaptic membrane composition underlie many forms of learning-related synaptic plasticity in the brain. At excitatory glutamatergic synapses, fusion of intracellular vesicles at or near the postsynaptic plasma membrane is critical for dendritic spine morphology, retrograde synaptic signaling, and long-term synaptic plasticity. Whereas the molecular machinery for exocytosis in presynaptic terminals has been defined in detail, little is known about the location, kinetics, regulation, or molecules involved in postsynaptic exocytosis. Here, we show that an exocytic domain adjacent to the postsynaptic density (PSD) enables fusion of large, AMPA receptor-containing recycling compartments during elevated synaptic activity. Exocytosis occurs at microdomains enriched in the plasma membrane t-SNARE syntaxin 4 (Stx4), and disruption of Stx4 impairs both spine exocytosis and long-term potentiation (LTP) at hippocampal synapses. Thus, Stx4 defines an exocytic zone that directs membrane fusion for postsynaptic plasticity, revealing a novel specialization for local membrane traffic in dendritic spines.

TGF-beta signaling specifies axons during brain development.

In the mammalian brain, the specification of a single axon and multiple dendrites occurs early in the differentiation of most neuron types. Numerous intracellular signaling events for axon specification have been described in detail. However, the identity of the extracellular factor(s) that initiate neuronal polarity in vivo is unknown. Here, we report that transforming growth factor beta (TGF-beta) initiates signaling pathways both in vivo and in vitro to fate naive neurites into axons. Neocortical neurons lacking the type II TGF-beta receptor (TbetaR2) fail to initiate axons during development. Exogenous TGF-beta is sufficient to direct the rapid growth and differentiation of an axon, and genetic enhancement of receptor activity promotes the formation of multiple axons. Finally, we show that the bulk of these TGF-beta-dependent events are mediated by site-specific phosphorylation of Par6. These results define an extrinsic cue for neuronal polarity in vivo that patterns neural circuits in the developing brain.

Diametric neural ensemble dynamics in parkinsonian and dyskinetic states.

Loss of dopamine in Parkinson's disease is hypothesized to impede movement by inducing hypo- and hyperactivity in striatal spiny projection neurons (SPNs) of the direct (dSPNs) and indirect (iSPNs) pathways in the basal ganglia, respectively. The opposite imbalance might underlie hyperkinetic abnormalities, such as dyskinesia caused by treatment of Parkinson's disease with the dopamine precursor L-DOPA. Here we monitored thousands of SPNs in behaving mice, before and after dopamine depletion and during L-DOPA-induced dyskinesia. Normally, intermingled clusters of dSPNs and iSPNs coactivated before movement. Dopamine depletion unbalanced SPN activity rates and disrupted the movement-encoding iSPN clusters. Matching their clinical efficacy, L-DOPA or agonism of the D2 dopamine receptor reversed these abnormalities more effectively than agonism of the D1 dopamine receptor. The opposite pathophysiology arose in L-DOPA-induced dyskinesia, during which iSPNs showed hypoactivity and dSPNs showed unclustered hyperactivity. Therefore, both the spatiotemporal profiles and rates of SPN activity appear crucial to striatal function, and next-generation treatments for basal ganglia disorders should target both facets of striatal activity.

Ubiquitination in postsynaptic function and plasticity.

Neurons are highly specialized cells whose connectivity at synapses subserves rapid information transfer in the brain. Proper information processing, learning, and memory storage in the brain requires continuous remodeling of synaptic networks. Such remodeling includes synapse formation, elimination, synaptic protein turnover, and changes in synaptic transmission. An emergent mechanism for regulating synapse function is posttranslational modification through the ubiquitin pathway at the postsynaptic membrane. Here, we discuss recent findings implicating ubiquitination and protein degradation in postsynaptic function and plasticity. We describe postsynaptic ubiquitination pathways and their role in brain development, neuronal physiology, and brain disorders.

Myosin Vb mobilizes recycling endosomes and AMPA receptors for postsynaptic plasticity.

Learning-related plasticity at excitatory synapses in the mammalian brain requires the trafficking of AMPA receptors and the growth of dendritic spines. However, the mechanisms that couple plasticity stimuli to the trafficking of postsynaptic cargo are poorly understood. Here we demonstrate that myosin Vb (MyoVb), a Ca2+-sensitive motor, conducts spine trafficking during long-term potentiation (LTP) of synaptic strength. Upon activation of NMDA receptors and corresponding Ca2+ influx, MyoVb associates with recycling endosomes (REs), triggering rapid spine recruitment of endosomes and local exocytosis in spines. Disruption of MyoVb or its interaction with the RE adaptor Rab11-FIP2 abolishes LTP-induced exocytosis from REs and prevents both AMPA receptor insertion and spine growth. Furthermore, induction of tight binding of MyoVb to actin using an acute chemical genetic strategy eradicates LTP in hippocampal slices. Thus, Ca2+-activated MyoVb captures and mobilizes REs for AMPA receptor insertion and spine growth, providing a mechanistic link between the induction and expression of postsynaptic plasticity.

Arc ubiquitination in synaptic plasticity.

The activity-regulated cytoskeleton-associated protein (Arc) is a neuron-expressed activity regulated immediate early gene (IEG) product that is essential for memory consolidation and serves as a direct readout for neural activation during learning. Arc contributes to diverse forms of synaptic plasticity mediated by the trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Notably, Arc protein expression abruptly increases and then rapidly decreases following augmented network activity. A large body of work has focused on Arc transcription and translation. Far fewer studies have explored the relevance of Arc protein stability and turnover. Here, we review recent findings on the mechanisms controlling Arc degradation and discuss its contributions to AMPA receptor trafficking and synaptic plasticity.

Identification of Phosphorylation Consensus Sequences and Endogenous Neuronal Substrates of the Psychiatric Risk Kinase TNIK.

Traf2- and Nck-interacting kinase (TNIK) is a serine/threonine kinase highly expressed in the brain and enriched in the postsynaptic density of glutamatergic synapses in the mammalian brain. Accumulating genetic evidence and functional data have implicated TNIK as a risk factor for psychiatric disorders. However, the endogenous substrates of TNIK in neurons are unknown. Here, we describe a novel selective small molecule inhibitor of the TNIK kinase family. Using this inhibitor, we report the identification of endogenous neuronal TNIK substrates by immunoprecipitation with a phosphomotif antibody followed by mass spectrometry. Phosphorylation consensus sequences were defined by phosphopeptide sequence analysis. Among the identified substrates were members of the delta-catenin family including p120-catenin, δ-catenin, and armadillo repeat gene deleted in velo-cardio-facial syndrome (ARVCF), each of which is linked to psychiatric or neurologic disorders. Using p120-catenin as a representative substrate, we show TNIK-induced p120-catenin phosphorylation in cells requires intact kinase activity and phosphorylation of TNIK at T181 and T187 in the activation loop. Addition of the small molecule TNIK inhibitor or knocking down TNIK by two shRNAs reduced endogenous p120-catenin phosphorylation in cells. Together, using a TNIK inhibitor and phosphomotif antibody, we identify endogenous substrates of TNIK in neurons, define consensus sequences for TNIK, and suggest signaling pathways by which TNIK influences synaptic development and function linked to psychiatric and neurologic disorders.

Molecular genetics and imaging technologies for circuit-based neuroanatomy.

Brain function emerges from the morphologies, spatial organization and patterns of connectivity established between diverse sets of neurons. Historically, the notion that neuronal structure predicts function stemmed from classic histological staining and neuronal tracing methods. Recent advances in molecular genetics and imaging technologies have begun to reveal previously unattainable details about patterns of functional circuit connectivity and the subcellular organization of synapses in the living brain. This sophisticated molecular and genetic 'toolbox', coupled with new methods in optical and electron microscopy, provides an expanding array of techniques for probing neural anatomy and function.

The Angelman syndrome protein Ube3a/E6AP is required for Golgi acidification and surface protein sialylation.

Angelman syndrome (AS) is a severe disorder of postnatal brain development caused by neuron-specific loss of the HECT (homologous to E6AP carboxy terminus) domain E3 ubiquitin ligase Ube3a/E6AP. The cellular role of Ube3a remains enigmatic despite recent descriptions of synaptic and behavioral deficits in AS mouse models. Although neuron-specific imprinting is thought to limit the disease to the brain, Ube3a is expressed ubiquitously, suggesting a broader role in cellular function. In the current study, we demonstrate a profound structural disruption and cisternal swelling of the Golgi apparatus (GA) in the cortex of AS (UBE3A(m-/p+)) mice. In Ube3a knockdown cell lines and UBE3A(m-/p+) cortical neurons, the GA is severely under-acidified, leading to osmotic swelling. Both in vitro and in vivo, the loss of Ube3a and corresponding elevated pH of the GA is associated with a marked reduction in protein sialylation, a process highly dependent on intralumenal Golgi pH. Altered ion homeostasis of the GA may provide a common cellular pathophysiology underlying the diverse plasticity and neurodevelopmental deficits associated with AS.

RE104: Synthesis and Activity of a Novel Serotonergic Psychedelic Prodrug of 4-Hydroxy-N,N-diisopropyltryptamine.

Results from randomized clinical trials of psilocybin in depressive disorders highlight the therapeutic potential of serotonergic psychedelic compounds in mental health disorders. The synthetic 5-hydroxytryptamine 2A receptor agonist 4-hydroxy-N,N-diisopropyltryptamine (4-OH-DiPT) is structurally similar to psilocin but is reported to have a shorter duration (2-3 h) of psychedelic effects, suggesting the potential for psilocybin-like therapeutic activity with reduced clinical resource burden. Here, we describe the preclinical and translational characterization of RE104, a 4-OH-DiPT prodrug comprising a glutarate moiety designed to cleave rapidly in situ and thus provide reasonable bioavailability of the active drug. Plasma concentration of 4-HO-DiPT over time in PK experiments in rats was correlated with head-twitch intensity. The half-life of 4-OH-DiPT was 40 min after subcutaneous administration of RE104 in rats. In a forced swim test, a single dose of RE104 (1 mg/kg) significantly reduced mean immobility time at 1 week compared with vehicle (P < 0.001), confirming translational antidepressant potential. Taken together, these data with RE104 show that the glutarate ester can act as an efficient prodrug strategy for 4-HO-DiPT, a unique short-duration psychedelic with potential in depressive disorders.

Synaptic dysfunction and abnormal behaviors in mice lacking major isoforms of Shank3.

SHANK3 is a synaptic scaffolding protein enriched in the postsynaptic density (PSD) of excitatory synapses. Small microdeletions and point mutations in SHANK3 have been identified in a small subgroup of individuals with autism spectrum disorder (ASD) and intellectual disability. SHANK3 also plays a key role in the chromosome 22q13.3 microdeletion syndrome (Phelan-McDermid syndrome), which includes ASD and cognitive dysfunction as major clinical features. To evaluate the role of Shank3 in vivo, we disrupted major isoforms of the gene in mice by deleting exons 4-9. Isoform-specific Shank3(e4-9) homozygous mutant mice display abnormal social behaviors, communication patterns, repetitive behaviors and learning and memory. Shank3(e4-9) male mice display more severe impairments than females in motor coordination. Shank3(e4-9) mice have reduced levels of Homer1b/c, GKAP and GluA1 at the PSD, and show attenuated activity-dependent redistribution of GluA1-containing AMPA receptors. Subtle morphological alterations in dendritic spines are also observed. Although synaptic transmission is normal in CA1 hippocampus, long-term potentiation is deficient in Shank3(e4-9) mice. We conclude that loss of major Shank3 species produces biochemical, cellular and morphological changes, leading to behavioral abnormalities in mice that bear similarities to human ASD patients with SHANK3 mutations.

Rapid blue-light-mediated induction of protein interactions in living cells.

Dimerizers allowing inducible control of protein-protein interactions are powerful tools for manipulating biological processes. Here we describe genetically encoded light-inducible protein-interaction modules based on Arabidopsis thaliana cryptochrome 2 and CIB1 that require no exogenous ligands and dimerize on blue-light exposure with subsecond time resolution and subcellular spatial resolution. We demonstrate the utility of this system by inducing protein translocation, transcription and Cre recombinase-mediated DNA recombination using light.

Dendritic trafficking for neuronal growth and plasticity.

Among the largest cells in the body, neurons possess an immense surface area and intricate geometry that poses many unique cell biological challenges. This morphological complexity is critical for neural circuit formation and enables neurons to compartmentalize cell-cell communication and local intracellular signalling to a degree that surpasses other cell types. The adaptive plastic properties of neurons, synapses and circuits have been classically studied by measurement of electrophysiological properties, ionic conductances and excitability. Over the last 15 years, the field of synaptic and neural electrophysiology has collided with neuronal cell biology to produce a more integrated understanding of how these remarkable highly differentiated cells utilize common eukaryotic cellular machinery to decode, integrate and propagate signals in the nervous system. The present article gives a very brief and personal overview of the organelles and trafficking machinery of neuronal dendrites and their role in dendritic and synaptic plasticity.

Secrets of the secretory pathway in dendrite growth.

Dendrites and axons exhibit different morphologies and patterns of growth. This difference in neuronal structure is controlled by evolutionarily conserved directed trafficking through the secretory pathway.

Diffusional trapping of GluR1 AMPA receptors by input-specific synaptic activity.

Synaptic activity regulates the postsynaptic accumulation of AMPA receptors over timescales ranging from minutes to days. Indeed, the regulated trafficking and mobility of GluR1 AMPA receptors underlies many forms of synaptic potentiation at glutamatergic synapses throughout the brain. However, the basis for synapse-specific accumulation of GluR1 is unknown. Here we report that synaptic activity locally immobilizes GluR1 AMPA receptors at individual synapses. Using single-molecule tracking together with the silencing of individual presynaptic boutons, we demonstrate that local synaptic activity reduces diffusional exchange of GluR1 between synaptic and extraynaptic domains, resulting in postsynaptic accumulation of GluR1. At neighboring inactive synapses, GluR1 is highly mobile with individual receptors frequently escaping the synapse. Within the synapse, spontaneous activity confines the diffusional movement of GluR1 to restricted subregions of the postsynaptic membrane. Thus, local activity restricts GluR1 mobility on a submicron scale, defining an input-specific mechanism for regulating AMPA receptor composition and abundance.

Postsynaptic positioning of endocytic zones and AMPA receptor cycling by physical coupling of dynamin-3 to Homer.

Endocytosis of AMPA receptors and other postsynaptic cargo occurs at endocytic zones (EZs), stably positioned sites of clathrin adjacent to the postsynaptic density (PSD). The tight localization of postsynaptic endocytosis is thought to control spine composition and regulate synaptic transmission. However, the mechanisms that situate the EZ near the PSD and the role of spine endocytosis in synaptic transmission are unknown. Here, we report that a physical link between dynamin-3 and the postsynaptic adaptor Homer positions the EZ near the PSD. Disruption of dynamin-3 or its interaction with Homer uncouples the PSD from the EZ, resulting in synapses lacking postsynaptic clathrin. Loss of the EZ leads to a loss of synaptic AMPA receptors and reduced excitatory synaptic transmission that corresponds with impaired synaptic recycling. Thus, a physical link between the PSD and the EZ ensures localized endocytosis and recycling by recapturing and maintaining a proximate pool of cycling AMPA receptors.

In vivo light-induced activation of neural circuitry in transgenic mice expressing channelrhodopsin-2.

Channelrhodopsin-2 (ChR2) is a light-gated, cation-selective ion channel isolated from the green algae Chlamydomonas reinhardtii. Here, we report the generation of transgenic mice that express a ChR2-YFP fusion protein in the CNS for in vivo activation and mapping of neural circuits. Using focal illumination of the cerebral cortex and olfactory bulb, we demonstrate a highly reproducible, light-dependent activation of neurons and precise control of firing frequency in vivo. To test the feasibility of mapping neural circuits, we exploited the circuitry formed between the olfactory bulb and the piriform cortex in anesthetized mice. In the olfactory bulb, individual mitral cells fired action potentials in response to light, and their firing rate was not influenced by costimulated glomeruli. However, in piriform cortex, the activity of target neurons increased as larger areas of the bulb were illuminated to recruit additional glomeruli. These results support a model of olfactory processing that is dependent upon mitral cell convergence and integration onto cortical cells. More broadly, these findings demonstrate a system for precise manipulation of neural activity in the intact mammalian brain with light and illustrate the use of ChR2 mice in exploring functional connectivity of complex neural circuits in vivo.

Genetic control of neuronal activity in mice conditionally expressing TRPV1.

Here we describe a knock-in mouse model for Cre-loxP-based conditional expression of TRPV1 in central nervous system neurons. Expression of Cre recombinase using biolistics, lentivirus or genetic intercrosses triggered heterologous expression of TRPV1 in a cell-specific manner. Application of the TRPV1 ligand capsaicin induced strong inward currents, triggered action potentials and activated stereotyped behaviors, allowing cell type-specific chemical genetic control of neuronal activity in vitro and in vivo.