Alix is required for activity-dependent bulk endocytosis at brain synapses.

In chemical synapses undergoing high frequency stimulation, vesicle components can be retrieved from the plasma membrane via a clathrin-independent process called activity-dependent bulk endocytosis (ADBE). Alix (ALG-2-interacting protein X/PDCD6IP) is an adaptor protein binding to ESCRT and endophilin-A proteins which is required for clathrin-independent endocytosis in fibroblasts. Alix is expressed in neurons and concentrates at synapses during epileptic seizures. Here, we used cultured neurons to show that Alix is recruited to presynapses where it interacts with and concentrates endophilin-A during conditions triggering ADBE. Using Alix knockout (ko) neurons, we showed that this recruitment, which requires interaction with the calcium-binding protein ALG-2, is necessary for ADBE. We also found that presynaptic compartments of Alix ko hippocampi display subtle morphological defects compatible with flawed synaptic activity and plasticity detected electrophysiologically. Furthermore, mice lacking Alix in the forebrain undergo less seizures during kainate-induced status epilepticus and reduced propagation of the epileptiform activity. These results thus show that impairment of ADBE due to the lack of neuronal Alix leads to abnormal synaptic recovery during physiological or pathological repeated stimulations.

Systemic Delivery of a Brain-Penetrant TrkB Antagonist Reduces Cocaine Self-Administration and Normalizes TrkB Signaling in the Nucleus Accumbens and Prefrontal Cortex.

UNLABELLED: Cocaine exposure alters brain-derived neurotrophic factor (BDNF) expression in the brain. BDNF signaling through TrkB receptors differentially modulates cocaine self-administration, depending on the brain regions involved. In the present study, we determined how brain-wide inhibition of TrkB signaling affects cocaine intake, the motivation for the drug, and reinstatement of drug taking after extinction. To overcome the inability of TrkB ligands to cross the blood-brain barrier, the TrkB antagonist cyclotraxin-B was fused to the nontoxic transduction domain of the tat protein from human immunodeficiency virus type 1 (tat-cyclotraxin-B). Intravenous injection of tat-cyclotraxin-B dose-dependently reduced cocaine intake, motivation for cocaine (as measured under a progressive ratio schedule of reinforcement), and reinstatement of cocaine taking in rats allowed either short or long access to cocaine self-administration. In contrast, the treatment did not affect operant responding for a highly palatable sweet solution, demonstrating that the effects of tat-cyclotraxin-B are specific for cocaine reinforcement. Cocaine self-administration increased TrkB signaling and activated the downstream Akt pathway in the nucleus accumbens, and had opposite effects in the prefrontal cortex. Pretreatment with tat-cyclotraxin-B normalized protein levels in these two dopamine-innervated brain regions. Cocaine self-administration also increased TrkB signaling in the ventral tegmental area, where the dopaminergic projections originate, but pretreatment with tat-cyclotraxin-B did not alter this effect. Altogether, our data show that systemic administration of a brain-penetrant TrkB antagonist leads to brain region-specific effects and may be a potential pharmacological strategy for the treatment of cocaine addiction. SIGNIFICANCE STATEMENT: Brain-derived neurotrophic factor (BDNF) signaling through TrkB receptors plays a well established role in cocaine reinforcement. However, local manipulation of BDNF signaling yields divergent effects, depending on the brain region, thereby questioning the viability of systemic TrkB targeting for the treatment of cocaine use disorders. Our study provides first-time evidence that systemic administration of a brain-penetrant TrkB antagonist (tat-cyclotraxin-B) reduces several behavioral measures of cocaine dependence, without altering motor performance or reinforcement by a sweet palatable solution. In addition, although cocaine self-administration produced opposite effects on TrkB signaling in the nucleus accumbens and prefrontal cortex, tat-cyclotraxin-B administration normalized these cocaine-induced changes in both brain regions.

Balancing the basal ganglia circuitry: a possible new role for dopamine D2 receptors in health and disease.

Current therapies for treating movement disorders such as Parkinson's disease are effective but limited by undesirable and intractable side effects. Developing more effective therapies will require better understanding of what causes basal ganglia dysregulation and why medication-induced side effects develop. Although basal ganglia have been extensively studied in the last decades, its circuit anatomy is very complex, and significant controversy exists as to how the interplay of different basal ganglia nuclei process motor information and output. We have recently identified the importance of an underappreciated collateral projection that bridges the striatal output direct pathway with the indirect pathway. These bridging collaterals are extremely plastic in the adult brain and are involved in the regulation of motor balance. Our findings add a new angle to the classical model of basal ganglia circuitry that could be exploited for the development of new therapies against movement disorders. In this Scientific Perspective, we describe the function of bridging collaterals and other recent discoveries that challenge the simplicity of the classical basal ganglia circuit model. We then discuss the potential implication of bridging collaterals in the pathophysiology of Parkinson's disease and schizophrenia. Because dopamine D2 receptors and striatal neuron excitability have been found to regulate the density of bridging collaterals, we propose that targeting these projections downstream of D2 receptors could be a possible strategy for the treatment of basal ganglia disorders. © 2015 International Parkinson and Movement Disorder Society.

Striatal D2 receptors regulate dendritic morphology of medium spiny neurons via Kir2 channels.

Structural plasticity in the adult brain is essential for adaptive behaviors and is thought to contribute to a variety of neurological and psychiatric disorders. Medium spiny neurons of the striatum show a high degree of structural plasticity that is modulated by dopamine through unknown signaling mechanisms. Here, we demonstrate that overexpression of dopamine D2 receptors in medium spiny neurons increases their membrane excitability and decreases the complexity and length of their dendritic arbors. These changes can be reversed in the adult animal after restoring D2 receptors to wild-type levels, demonstrating a remarkable degree of structural plasticity in the adult striatum. Increased excitability and decreased dendritic arborization are associated with downregulation of inward rectifier potassium channels (Kir2.1/2.3). Downregulation of Kir2 function is critical for the neurophysiological and morphological changes in vivo because virally mediated expression of a dominant-negative Kir2 channel is sufficient to recapitulate the changes in D2 transgenic mice. These findings may have important implications for the understanding of basal ganglia disorders, and more specifically schizophrenia, in which excessive activation of striatal D2 receptors has long been hypothesized to be of pathophysiologic significance.

Brain-derived neurotrophic factor stimulates growth of pituitary melanotrope cells in an autocrine way.

Brain-derived neurotrophic factor (BDNF) is expressed in the mammalian pituitary gland, in both the anterior and intermediate lobes, where its functional significance is unknown. Melanotrope cells in the intermediate pituitary lobe of the amphibian Xenopus laevis also produce BDNF, which co-exists in secretory granules with α-melanophore-stimulating hormone (α-MSH), a peptide that causes pigment dispersion in dermal melanophores during adaptation of the toad to a dark background. Xenopus melanotropes are highly plastic, undergoing very strong growth to support the high biosynthesis and release of α-MSH in black-adapted animals. In this study we have tested our hypothesis that this enhanced growth of the melanotrope is maintained by autocrine release of BDNF. Furthermore, since the extracellular-regulated kinase (ERK) pathway is a major component of BDNF signaling in neuronal plasticity, we investigated its involvement in melanotrope cell growth. For these purposes melanotropes were treated for 3 days in vitro, with either an anti-BDNF serum or a recombinant tropomyosin-receptor kinase B (TrkB) receptor fragment to eliminate released BDNF, or with the ERK inhibitor U0126. We also applied a novel inhibitor of the TrkB receptor, cyclotraxin-B, to test this receptor's involvement in melanotrope cell growth regulation. All treatments markedly reduced melanotrope cell growth. Therefore, we conclude that autocrine release of BDNF and subsequent TrkB-dependent ERK-mediated signaling is important for melanotrope cell growth during its physiologically induced activation.

An integrated microfluidic/microelectrode array for the study of activity-dependent intracellular dynamics in neuronal networks.

In the central nervous system, neurons are organized in specific neural networks with distinct electrical patterns, input integration capacities, and intracellular dynamics. In order to better understand how neurons process information, it is crucial to keep the complex organization of brain circuits. However, performing subcellular investigations with high spatial and temporal resolution in vivo is technically challenging, especially in fine structures, such as axonal projections. Here, we present an on-a-chip system that combines a microfluidic platform with a dedicated matrix of electrodes to study activity-dependent dynamics in the physiological context of brain circuits. Because this system is compatible with high-resolution video-microscopy, it is possible to simultaneously record intracellular dynamics and electrical activity in presynaptic axonal projections and in their postsynaptic neuronal targets. Similarly, specific patterns of electrical activity can be applied to both compartments in order to investigate how intrinsic and network activities influence intracellular dynamics. The fluidic isolation of each compartment further allows the selective application of drugs at identified sites to study activity-dependent synaptic transmission. This integrated microfluidic/microelectrode array (microMEA) platform is a valuable tool for studying various intracellular and synaptic dynamics in response to neuronal activity in a physiologically relevant context that resembles in vivo brain circuits.

Reconstituting Corticostriatal Network on-a-Chip Reveals the Contribution of the Presynaptic Compartment to Huntington's Disease.

Huntington's disease (HD), a devastating neurodegenerative disorder, strongly affects the corticostriatal network, but the contribution of pre- and postsynaptic neurons in the first phases of disease is unclear due to difficulties performing early subcellular investigations in vivo. Here, we have developed an on-a-chip approach to reconstitute an HD corticostriatal network in vitro, using microfluidic devices compatible with subcellular resolution. We observed major defects in the different compartments of the corticostriatal circuit, from presynaptic dynamics to synaptic structure and transmission and to postsynaptic traffic and signaling, that correlate with altered global synchrony of the network. Importantly, the genetic status of the presynaptic compartment was necessary and sufficient to alter or restore the circuit. This highlights an important weight for the presynaptic compartment in HD that has to be considered for future therapies. This disease-on-a-chip microfluidic platform is thus a physiologically relevant in vitro system for investigating pathogenic mechanisms and for identifying drugs.

Neuronal network maturation differently affects secretory vesicles and mitochondria transport in axons.

Studying intracellular dynamics in neurons is crucial to better understand how brain circuits communicate and adapt to environmental changes. In neurons, axonal secretory vesicles underlie various functions from growth during development to plasticity in the mature brain. Similarly, transport of mitochondria, the power plant of the cell, regulates both axonal development and synaptic homeostasis. However, because of their submicrometric size and rapid velocities, studying the kinetics of these organelles in projecting axons in vivo is technically challenging. In parallel, primary neuronal cultures are adapted to study axonal transport but they lack the physiological organization of neuronal networks, which in turn may bias observations. We previously developed a microfluidic platform to reconstruct a physiologically-relevant and functional corticostriatal network in vitro that is compatible with high-resolution videorecording of axonal trafficking. Here, using this system we report progressive changes in axonal transport kinetics of both dense core vesicles and mitochondria that correlate with network development and maturation. Interestingly, axonal flow of both types of organelles change in opposite directions, with rates increasing for vesicles and decreasing for mitochondria. Overall, our observations highlight the need for a better spatiotemporal control for the study of intracellular dynamics in order to avoid misinterpretations and improve reproducibility.

Dopamine D2 receptors regulate the anatomical and functional balance of basal ganglia circuitry.

Structural plasticity in the adult brain is essential for adaptive behavior. We have found a remarkable anatomical plasticity in the basal ganglia of adult mice that is regulated by dopamine D2 receptors (D2Rs). By modulating neuronal excitability, striatal D2Rs bidirectionally control the density of direct pathway collaterals in the globus pallidus that bridge the direct pathway with the functionally opposing indirect pathway. An increase in bridging collaterals is associated with enhanced inhibition of pallidal neurons in vivo and disrupted locomotor activation after optogenetic stimulation of the direct pathway. Chronic blockade with haloperidol, an antipsychotic medication used to treat schizophrenia, decreases the extent of bridging collaterals and rescues the locomotor imbalance. These findings identify a role for bridging collaterals in regulating the concerted balance of striatal output and may have important implications for understanding schizophrenia, a disease involving excessive activation of striatal D2Rs that is treated with D2R blockers.

TrkB inhibition as a therapeutic target for CNS-related disorders.

The interaction of brain-derived neurotrophic factor (BDNF) with its tropomyosin-related kinase receptor B (TrkB) is involved in fundamental cellular processes including neuronal proliferation, differentiation and survival as well as neurotransmitter release and synaptic plasticity. TrkB signaling has been widely associated with beneficial, trophic effects and many commonly used psychotropic drugs aim to increase BDNF levels in the brain. However, it is likely that a prolonged increased TrkB activation is observed in many pathological conditions, which may underlie the development and course of clinical symptoms. Interestingly, genetic and pharmacological studies aiming at decreasing TrkB activation in rodent models mimicking human pathology have demonstrated a promising therapeutic landscape for TrkB inhibitors in the treatment of various diseases, e.g. central nervous system (CNS) disorders and several types of cancer. Up to date, only a few selective and potent TrkB inhibitors have been developed. As such, the use of crystallography and in silico approaches to model BDNF-TrkB interaction and to generate relevant pharmacophores represent powerful tools to develop novel compounds targeting the TrkB receptor.

Cyclotraxin-B, a new TrkB antagonist, and glial blockade by propentofylline, equally prevent and reverse cold allodynia induced by BDNF or partial infraorbital nerve constriction in mice.

UNLABELLED: Several lines of evidence indicate that brain-derived neurotrophic factor (BDNF) plays a key role as a central pronociceptive modulator of pain, acting through postsynaptic TrkB receptors that trigger intracellular signaling cascades leading to central sensitization. The overall aim of this study was to investigate to what extent BDNF could participate in the generation and maintenance of trigeminal neuropathic pain. The results showed that acute intracisternal administration of nanogram doses of BDNF in naïve mice elicited long-lasting, dose-related, cold allodynic responses to topical application of acetone onto vibrissal pad skin. The systemic administration of cyclotraxin-B (CTX-B), a new TrkB receptor antagonist, or propentofylline, an inhibitor of glial activation, was able to either prevent or reverse the effects of intracisternal BDNF on cold nociception. In addition, the blockade of TrkB receptor by CTX-B inhibited the mechanisms that either initiate or maintain cold allodynia in the ipsilateral vibrissal pad skin after unilateral constriction of the infraorbital nerve. These observations raise the possibility that BDNF is capable on its own of conveying many features of the signaling mechanisms that underlie central sensitization caused by nerve constriction. PERSPECTIVE: Although further studies are necessary to examine in detail the mechanisms underlying the strong anti-allodynic action of CTX-B, this compound may represent an interesting lead for the development of novel therapeutic strategies aimed at preventing and/or suppressing central sensitization associated with neuropathic pain.

Identification of a low-molecular weight TrkB antagonist with anxiolytic and antidepressant activity in mice.

The neurotrophin brain-derived neurotrophic factor (BDNF) and its receptor tropomyosin-related kinase B (TrkB) have emerged as key mediators in the pathophysiology of several mood disorders, including anxiety and depression. However, therapeutic compounds that interact with TrkB receptors have been difficult to develop. Using a combination of structure-based in silico screening and high-capacity functional assays in recombinant and neuronal cells, we identified a low-molecular weight TrkB ligand (ANA-12) that prevented activation of the receptor by BDNF with a high potency. ANA-12 showed direct and selective binding to TrkB and inhibited processes downstream of TrkB without altering TrkA and TrkC functions. KIRA-ELISA analysis demonstrated that systemic administration of ANA-12 to adult mice decreased TrkB activity in the brain without affecting neuronal survival. Mice administered ANA-12 demonstrated reduced anxiety- and depression-related behaviors on a variety of tests predictive of anxiolytic and antidepressant properties in humans. This study demonstrates that structure-based virtual screening strategy can be an efficient method for discovering potent TrkB-selective ligands that are active in vivo. We further propose that ANA-12 may be a valuable tool for studying BDNF/TrkB signaling and may constitute a lead compound for developing the next generation of therapeutic agents for the treatment of mood disorders.

Cyclotraxin-B, the first highly potent and selective TrkB inhibitor, has anxiolytic properties in mice.

In the last decades, few mechanistically novel therapeutic agents have been developed to treat mental and neurodegenerative disorders. Numerous studies suggest that targeting BDNF and its TrkB receptor could be a promising therapeutic strategy for the treatment of brain disorders. However, the development of potent small ligands for the TrkB receptor has proven to be difficult. By using a peptidomimetic approach, we developed a highly potent and selective TrkB inhibitor, cyclotraxin-B, capable of altering TrkB-dependent molecular and physiological processes such as synaptic plasticity, neuronal differentiation and BDNF-induced neurotoxicity. Cyclotraxin-B allosterically alters the conformation of TrkB, which leads to the inhibition of both BDNF-dependent and -independent (basal) activities. Finally, systemic administration of cyclotraxin-B to mice results in TrkB inhibition in the brain with specific anxiolytic-like behavioral effects and no antidepressant-like activity. This study demonstrates that cyclotraxin-B might not only be a powerful tool to investigate the role of BDNF and TrkB in physiology and pathology, but also represents a lead compound for the development of new therapeutic strategies to treat brain disorders.