Francisco J. Monje

Constant Darkness Induces IL-6-Dependent Depression-Like Behavior through the NF-κB Signaling Pathway

Substantial experimental evidence indicates a major role for the circadian system in mood disorders. Additionally, proinflammatory cytokines have been proposed to be involved in the pathogenesis of depression. However, the molecular elements determining the functional interplay between these two systems in depression have not been described as yet. Here we investigate whether long-term light deprivation in the constant darkness (DD) paradigm affects depression-like behavior in mice and concomitantly modulates the levels of proinflammatory cytokines. We find that after 4 weeks of DD, mice display depression-like behavior, which is paralleled by reduced hippocampal cell proliferation. This chronobiologically induced depressive state is associated with elevated levels of plasma IL-6 (interleukin-6) and IL-6 and Il1-R1 (interleukin 1 receptor, type I) protein levels in the hippocampus and also alters hippocampal protein levels of the clock genes per2 and npas2. Using pharmacological blockers of the NF-κB pathway, we provide evidence that the effects of DD on depression-like behavior, on hippocampal cell proliferation, on altered expressional levels of brain and plasma IL-6, and on the modulation of clock gene expression are mediated through NF-κB signaling. Moreover, NF-κB activity is enhanced in hippocampal tissue of DD mice. Mice with a deletion of IL-6, one of the target genes of NF-κB, are resistant to DD-induced depression-like behavior, which suggests a pivotal role for this cytokine in the constant darkness mouse model of depression. We here first describe some of the molecular elements bridging chronobiological and inflammatory processes in the constant darkness mouse model of depression.

Rodent models in depression research: Classical strategies and new directions

Abstract Depression, among other mood disorders, represents one of the most common health problems worldwide, with steadily increasing incidence and major socio-economic consequences. However, since the knowledge about the underlying pathophysiological principles is still very scanty, depression and other mood disorders are currently diagnosed solely on clinical grounds. Currently used treatment modalities would therefore benefit enormously from the development of alternative therapeutic interventions. The implementation of proper animal models is a prerequisite for increasing the understanding of the neurobiological basis of mood disorders and is paving the way for the discovery of novel therapeutic targets. In the past thirty years, since the seminal description of the Forced Swim Test as a system to probe antidepressant activity in rodents, the use of animals to model depression and antidepressant activity has come a long way. In this review we describe some of the most commonly used strategies, ranging from screening procedures, such as the Forced Swim Test and the Tail Suspension Test and animal models, such as those based upon chronic stress procedures, to genetic approaches. Finally we also discuss some of the inherent limitations and caveats that need to be considered when using animals as models for mental disorders in basic research.

Activation Mechanism for CRAC Current and Store-operated Ca2+ Entry CALCIUM INFLUX FACTOR AND Ca2+-INDEPENDENT PHOSPHOLIPASE A2β-MEDIATED PATHWAY

Here we tested the role of calcium influx factor (CIF) and calcium-independent phospholipase A2 (iPLA2) in activation of Ca2+ release-activated Ca2+ (CRAC) channels and store-operated Ca2+ entry in rat basophilic leukemia (RBL-2H3) cells. We demonstrate that 1) endogenous CIF production may be triggered by Ca2+ release (net loss) as well as by simple buffering of free Ca2+ within the stores, 2) a specific 82-kDa variant of iPLA2β and its corresponding activity are present in membrane fraction of RBL cells, 3) exogenous CIF (extracted from other species) mimics the effects of endogenous CIF and activates iPLA2β when applied to cell homogenates but not intact cells, 4) activation of ICRAC can be triggered in resting RBL cells by dialysis with exogenous CIF, 5) molecular or functional inhibition of iPLA2β prevents activation of ICRAC, which could be rescued by cell dialysis with a human recombinant iPLA2β, 6) dependence of ICRAC on intracellular pH strictly follows pH dependence of iPLA2β activity, and 7) (S)-BEL, a chiral enantiomer of suicidal substrate specific for iPLA2β, could be effectively used for pharmacological inhibition of ICRAC and store-operated Ca2+ entry. These findings validate and significantly advance our understanding of the CIF-iPLA2-dependent mechanism of activation of ICRAC and store-operated Ca2+ entry.

Learning not to Fear: Neural Correlates of Learned Safety

The ability to recognize and properly respond to instances of protection from impending danger is critical for preventing chronic stress and anxiety—central symptoms of anxiety and affective disorders afflicting large populations of people. Learned safety encompasses learning processes, which lead to the identification of episodes of security and regulation of fear responses. On the basis of insights into the neural circuitry and molecular mechanisms involved in learned safety in mice and humans, we describe learned safety as a tool for understanding neural mechanisms involved in the pathomechanisms of specific affective disorders. This review summarizes our current knowledge on the neurobiological underpinnings of learned safety and discusses potential applications in basic and translational neurosciences.

STAT3 controls IL6-dependent regulation of serotonin transporter function and depression-like behavior

Experimental evidence suggests a role for the immune system in the pathophysiology of depression. A specific involvement of the proinflammatory cytokine interleukin 6 (IL6) in both, patients suffering from the disease and pertinent animal models, has been proposed. However, it is not clear how IL6 impinges on neurotransmission and thus contributes to depression. Here we tested the hypothesis that IL6-induced modulation of serotonergic neurotransmission through the STAT3 signaling pathway contributes to the role of IL6 in depression. Addition of IL6 to JAR cells, endogenously expressing SERT, reduced SERT activity and downregulated SERT mRNA and protein levels. Similarly, SERT expression was reduced upon IL6 treatment in the mouse hippocampus. Conversely, hippocampal tissue of IL6-KO mice contained elevated levels of SERT and IL6-KO mice displayed a reduction in depression-like behavior and blunted response to acute antidepressant treatment. STAT3 IL6-dependently associated with the SERT promoter and inhibition of STAT3 blocked the effect of IL6 in-vitro and modulated depression-like behavior in-vivo. These observations demonstrate that IL6 directly controls SERT levels and consequently serotonin reuptake and identify STAT3-dependent regulation of SERT as conceivable neurobiological substrate for the involvement of IL6 in depression.

Store-Operated Orai1 and IP3 Receptor-Operated TRPC1 Channel: Separation of the Siamese Twins

Store-operated channels (SOC) are known to be physiologically activated following agonist-induced IP3 production and depletion of Ca2+ stores. Here we present molecular, biophysical and mechanistic evidence that two ubiquitously expressed plasma membrane channels may be responsible for creating a complex and sometimes controversial SOC image: one being a real SOC encoded by Orai1 and activated exclusively upon depletion of Ca2+ stores (via iPLA2β -dependent pathway), while the second one is an IP3 receptor-operated channel (IP3ROC) encoded by TRPC1 and activated via its conformational coupling with IP3 receptor. In RBL-2H3 cells endogenously expressing Orai1 and TRPC1, we unmasked and characterized whole-cell current through IP3ROC channels that was hiding behind some familiar fingerprints of ICRAC, a current through the classical Ca2+-selective SOC (CRAC) channels. We discriminated these currents by their molecular identity, selectivity and different requirements for store depletion, IP3, iPLA2β and conformational coupling to IP3 receptor. New knowledge on the properties and co-existence of Orai1-encoded SOC and TRPC1-encoded IP3ROC, and the use of experimental approaches introduced in this manuscript should help avoid further confusion about these channels, and open new exciting possibilities for their independent studies.

Focal Adhesion Kinase Regulates Neuronal Growth, Synaptic Plasticity and Hippocampus-Dependent Spatial Learning and Memory

The focal adhesion kinase (FAK) is a non-receptor tyrosine kinase abundantly expressed in the mammalian brain and highly enriched in neuronal growth cones. Inhibitory and facilitatory activities of FAK on neuronal growth have been reported and its role in neuritic outgrowth remains controversial. Unlike other tyrosine kinases, such as the neurotrophin receptors regulating neuronal growth and plasticity, the relevance of FAK for learning and memory in vivo has not been clearly defined yet. A comprehensive study aimed at determining the role of FAK in neuronal growth, neurotransmitter release and synaptic plasticity in hippocampal neurons and in hippocampus-dependent learning and memory was therefore undertaken using the mouse model. Gain- and loss-of-function experiments indicated that FAK is a critical regulator of hippocampal cell morphology. FAK mediated neurotrophin-induced neuritic outgrowth and FAK inhibition affected both miniature excitatory postsynaptic potentials and activity-dependent hippocampal long-term potentiation prompting us to explore the possible role of FAK in spatial learning and memory in vivo. Our data indicate that FAK has a growth-promoting effect, is importantly involved in the regulation of the synaptic function and mediates in vivo hippocampus-dependent spatial learning and memory.

Association of polyalanine and polyglutamine coiled coils mediates expansion disease-related protein aggregation and dysfunction

The expansion of homopolymeric glutamine (polyQ) or alanine (polyA) repeats in certain proteins owing to genetic mutations induces protein aggregation and toxicity, causing at least 18 human diseases. PolyQ and polyA repeats can also associate in the same proteins, but the general extent of their association in proteomes is unknown. Furthermore, the structural mechanisms by which their expansion causes disease are not well understood, and these repeats are generally thought to misfold upon expansion into aggregation-prone β-sheet structures like amyloids. However, recent evidence indicates a critical role for coiled-coil (CC) structures in triggering aggregation and toxicity of polyQ-expanded proteins, raising the possibility that polyA repeats may as well form these structures, by themselves or in association with polyQ. We found through bioinformatics screenings that polyA, polyQ and polyQA repeats have a phylogenetically graded association in human and non-human proteomes and associate/overlap with CC domains. Circular dichroism and cross-linking experiments revealed that polyA repeats can form—alone or with polyQ and polyQA—CC structures that increase in stability with polyA length, forming higher-order multimers and polymers in vitro. Using structure-guided mutagenesis, we studied the relevance of polyA CCs to the in vivo aggregation and toxicity of RUNX2—a polyQ/polyA protein associated with cleidocranial dysplasia upon polyA expansion—and found that the stability of its polyQ/polyA CC controls its aggregation, localization and toxicity. These findings indicate that, like polyQ, polyA repeats form CC structures that can trigger protein aggregation and toxicity upon expansion in human genetic diseases.

Drebrin depletion alters neurotransmitter receptor levels in protein complexes, dendritic spine morphogenesis and memory-related synaptic plasticity in the mouse hippocampus.

Drebrin an actin‐bundling key regulator of dendritic spine genesis and morphology, has been recently proposed as a regulator of hippocampal glutamatergic activity which is critical for memory formation and maintenance. Here, we examined the effects of genetic deletion of drebrin on dendritic spine and on the level of complexes containing major brain receptors. To this end, homozygous and heterozygous drebrin knockout mice generated in our laboratory and related wild‐type control animals were studied. Level of protein complexes containing dopamine receptor D1/dopamine receptor D2, 5‐hydroxytryptamine receptor 1A (5‐HT1AR), and 5‐hydroxytryptamine receptor 7 (5‐HT7R) were significantly reduced in hippocampus of drebrin knockout mice whereas no significant changes were detected for GluR1, 2, and 3 and NR1 as examined by native gel‐based immunoblotting. Drebrin depletion also altered dendritic spine formation, morphology, and reduced levels of dopamine receptor D1 in dendritic spines as evaluated using immunohistochemistry/confocal microscopy. Electrophysiological studies further showed significant reduction in memory‐related hippocampal synaptic plasticity upon drebrin depletion. These findings provide unprecedented experimental support for a role of drebrin in the regulation of memory‐related synaptic plasticity and neurotransmitter receptor signaling, offer relevant information regarding the interpretation of previous studies and help in the design of future studies on dendritic spines. We examined effect of genetic deletion of drebrin, which an actin‐bundling key regulator of dendritic spine genesis and morphology, on dendritic spine density, maturity, level of complexes containing major brain receptors and also, in synaptic plasticity. These findings support for a role of drebrin in the regulation of memory‐related synaptic plasticity and neurotransmitter receptors signaling in dendritic spines.

The learned safety paradigm as a mouse model for neuropsychiatric research.

Fear conditioning is one of the most widely used animal models for studying the neurobiological basis of fear and anxiety states. Conditioned inhibition of fear (or learned safety), however, is a relatively unexplored behavioral paradigm addressing the aspect of regulation of fear, which is central to survival and mental health. Although fear conditioning is achieved by pairing a previously neutral, conditioned stimulus (CS) with an aversive, unconditioned stimulus (US), learned safety training consists of a series of explicitly unpaired CS–US presentations. Animals are trained for 3 d, one session per day, and learn to associate the CS with protection from the impending danger of the aversive events. The entire procedure can be completed within 7 d. The protocol has been successfully used to study the molecular underpinnings of a behavioral intervention for depression. This paradigm complements currently used animal tests in neuropsychiatric research addressing the dysregulation of emotional behaviors in genetic, pharmacological or environmental mouse models of human affective disorders.