Tricyclic and tetracyclic antidepressants upregulate VMAT2 activity and rescue disease-causing VMAT2 variants.

Vesicular monoamine transporter 2 (VMAT2) is an essential transporter that regulates brain monoamine transmission and is important for mood, cognition, motor activity, and stress regulation. However, VMAT2 remains underexplored as a pharmacological target. In this study, we report that tricyclic and tetracyclic antidepressants acutely inhibit, but persistently upregulate VMAT2 activity by promoting VMAT2 protein maturation. Importantly, the VMAT2 upregulation effect was greater in BE(2)-M17 cells that endogenously express VMAT2 as compared to a heterologous expression system (HEK293). The net sustained effect of tricyclics and tetracyclics is an upregulation of VMAT2 activity, despite their acute inhibitory effect. Furthermore, imipramine and mianserin, two representative compounds, also demonstrated rescue of nine VMAT2 variants that cause Brain Vesicular Monoamine Transport Disease (BVMTD). VMAT2 upregulation could be beneficial for disorders associated with reduced monoamine transmission, including mood disorders and BVMTD, a rare but often fatal condition caused by a lack of functional VMAT2. Our findings provide the first evidence that small molecules can upregulate VMAT2 and have potential therapeutic benefit for various neuropsychiatric conditions.

Lung inflammation is associated with lipid deposition.

Lung inflammation, pneumonia, is an acute respiratory disease of varying etiology that has recently drawn much attention during the COVID-19 pandemic as lungs are among the main targets for SARS-CoV-2. Multiple other etiological agents are associated with pneumonias. Here, we describe a newly-recognized pathology, namely abnormal lipid depositions in the lungs of patients who died from COVID-19 as well as from non-COVID-19 pneumonias. Our analysis of both semi-thin and Sudan III-stained lung specimens revealed extracellular and intracellular lipid depositions irrespective of the pneumonia etiology. Most notably, lipid depositions were located within vessels adjacent to inflamed regions, where they apparently interfere with the blood flow. Structurally, the lipid droplets in the inflamed lung tissue were homogeneous and lacked outer membranes as assessed by electron microscopy. Morphometric analysis of lipid droplet deposition area allowed us to distinguish the non-pneumonia control lung specimens from the macroscopically intact area of the pneumonia lung and from the inflamed area of the pneumonia lung. Our measurements revealed a gradient of lipid deposition towards the inflamed region. The pattern of lipid distribution proved universal for all pneumonias. Finally, lipid metabolism in the lung tissue was assessed by the fatty acid analysis and by expression of genes involved in lipid turnover. Chromato-mass spectrometry revealed that unsaturated fatty acid content was elevated at inflammation sites compared to that in control non-inflamed lung tissue from the same individual. The expression of genes involved in lipid metabolism was altered in pneumonia, as shown by qPCR and in silico RNA-seq analysis. Thus, pneumonias of various etiologies are associated with specific lipid abnormalities; therefore, lipid metabolism can be considered to be a target for new therapeutic strategies.

The habenular volume and PDE7A allelic polymorphism in major depressive disorder: preliminary findings.

OBJECTIVES: The habenula is a brain structure implicated in depression, yet with unknown molecular mechanisms. Several phosphodiesterases (PDEs) have been associated with a risk of depression. Although the role of PDE7A in the brain is unknown, it has enriched expression in the medial habenula, suggesting that it may play a role in depression. METHODS: We analysed: (1) habenula volume assessed by 3-T magnetic resonance imaging (MRI) in 84 patients with major depressive disorder (MDD) and 41 healthy controls; (2) frequencies of 10 single nucleotide polymorphisms (SNPs) in PDE7A gene in 235 patients and 41 controls; and (3) both indices in 80 patients and 27 controls. The analyses considered gender, age, body mass index and season of the MRI examination. RESULTS: The analysis did not reveal habenula volumetric changes in MDD patients regardless of PDE7A SNPs. However, in the combined group, the carriers of one or more mutations among 10 SNPs in the PDE7A gene had a lower volume of the left habenula (driven mainly by rs972362 and rs138599850 mutations) and consequently had the reduced habenular laterality index in comparison with individuals without PDE7A mutations. CONCLUSIONS: Our findings suggest the implication of the PDE7A gene into mechanisms determining the habenula structure.

Neuronal lack of PDE7a disrupted working memory, spatial learning, and memory but facilitated cued fear memory in mice.

BACKGROUND: PDEs regulate cAMP levels which is critical for PKA activity-dependent activation of CREB-mediated transcription in learning and memory. Inhibitors of PDEs like PDE4 and Pde7 improve learning and memory in rodents. However, the role of PDE7 in cognition or learning and memory has not been reported yet. METHODS: Therefore, we aimed to explore the cognitive effects of a PDE7 subtype, PDE7a, using combined pharmacological and genetic approaches. RESULTS: PDE7a-nko mice showed deficient working memory, impaired novel object recognition, deficient spatial learning & memory, and contextual fear memory, contrary to enhanced cued fear memory, highlighting the potential opposite role of PDE7a in the hippocampal neurons. Further, pharmacological inhibition of PDE7 by AGF2.20 selectively strengthens cued fear memory in C57BL/6 J mice, decreasing its extinction but did not affect cognitive processes assessed in other behavioral tests. The further biochemical analysis detected deficient cAMP in neural cell culture with genetic excision of the PDE7a gene, as well as in the hippocampus of PDE7a-nko mice in vivo. Importantly, we found overexpression of PKA-R and the reduced level of pPKA-C in the hippocampus of PDE7a-nko mice, suggesting a novel mechanism of the cAMP regulation by PDE7a. Consequently, the decreased phosphorylation of CREB, CAMKII, eif2a, ERK, and AMPK, and reduced total level of NR2A have been found in the brain of PDE7a-nko animals. Notably, genetic excision of PDE7a in neurons was not able to change the expression of NR2B, BDNF, synapsin1, synaptophysin, or snap25. CONCLUSION: Altogether, our current findings demonstrated, for the first time, the role of PDE7a in cognitive processes. Future studies will untangle PDE7a-dependent neurobiological and molecular-cellular mechanisms related to cAMP-associated disorders.

Proteomic and electron microscopy study of myogenic differentiation of alveolar mucosa multipotent mesenchymal stromal cells in three-dimensional culture.

Myocyte differentiation is featured by adaptation processes, including mitochondria repopulation and cytoskeleton re-organization. The difference between monolayer and spheroid cultured cells at the proteomic level is uncertain. We cultivated alveolar mucosa multipotent mesenchymal stromal cells in spheroids in a myogenic way for the proper conditioning of ECM architecture and cell morphology, which induced spontaneous myogenic differentiation of cells within spheroids. Electron microscopy analysis was used for the morphometry of mitochondria biogenesis, and proteomic was used complementary to unveil events underlying differences between two-dimensional/three-dimensional myoblasts differentiation. The prevalence of elongated mitochondria with an average area of 0.097 µm2 was attributed to monolayer cells 7 days after the passage. The population of small mitochondria with a round shape and area of 0.049 µm2 (p < 0.05) was observed in spheroid cells cultured under three-dimensional conditions. Cells in spheroids were quantitatively enriched in proteins of mitochondria biogenesis (DNM1L, IDH2, SSBP1), respiratory chain (ACO2, ATP5I, COX5A), extracellular proteins (COL12A1, COL6A1, COL6A2), and cytoskeleton (MYL6, MYL12B, MYH10). Most of the Rab-related transducers were inhibited in spheroid culture. The proteomic assay demonstrated delicate mechanisms of mitochondria autophagy and repopulation, cytoskeleton assembling, and biogenesis. Differences in the ultrastructure of mitochondria indicate active biogenesis under three-dimensional conditions.

Vocal and physical phenotypes of calsyntenin2 knockout mouse pups model early-life symptoms of the autism spectrum disorder.

This study discovered a novel acoustic phenotype in Calsyntenin2 deficient knockout (Clstn2-KO) pups in the neurodevelopment period of 5-9 postnatal days (PND 5-9). The narrowband ultrasonic calls (nUSVs) were less complex (mostly one-note, shorter in duration and higher in peak frequency) in Clsnt2-KO than in wild-type (WT) C57BL/6 J pups. The wideband ultrasonic calls (wUSVs) were produced substantially more often by Clstn2-KO than WT pups. The clicks were longer in duration and higher in peak frequency and power quartiles in Clstn2-KO pups. The elevated discomfort due to additional two-minute maternal separation coupled with experimenter's touch, resulted in significantly higher call rates of both nUSVs and clicks in pups of both genotypes and sexes compared to the previous two-minute maternal separation, whereas the call rate of wUSVs was not affected. In Clstn2-KO pups, the prevalence of emission of wUSVs retained at both sex and both degrees of discomfort, thus providing a reliable quantitative acoustic indicator for this genetic line. Besides the acoustic differences, we also detected the increased head-to-body ratio in Clstn2-KO pups. Altogether, this study demonstrated that lack of such synaptic adhesion protein as calsyntenin2 affects neurodevelopment of vocalization in a mouse as a model organism.

The increased density of the habenular neurons, high impulsivity, aggression and resistant fear memory in Disc1-Q31L genetic mouse model of depression.

Mood disorders affect nearly 300 million humans worldwide, and it is a leading cause of death from suicide. In the last decade, the habenula has gained increased attention due to its major role to modulate emotional behavior and related psychopathologies, including depression and bipolar disorder, through the modulation of monoamines' neurotransmission. However, it is still unclear which genetic factors may directly affect the function of the habenula and hence, could contribute to the psychopathological mechanisms of mood disorders. Disrupted-In-Schizophrenia-1 (DISC1) gene is among robust gene-candidates predisposing to major depression, bipolar disorder and schizophrenia in humans. DISC1-Q31L, a well-established genetic mouse model of depression, offers a unique opportunity for translational studies. The current study aimed to probe morphological features of the habenula in the DISC1-Q31L mouse line and detect novel behavioral endophenotypes, including the increased emotionality in mutant females, high aggression in mutant males and deficient extinction of fear memory in DISC1 mutant mice of both sexes. The histological analysis found the increased neural density in the lateral and medial habenula in DISC1-Q31L mice regardless of sex, hence, excluding direct association between the habenular neurons and emotionality in mutant females. Altogether, our findings demonstrated, for the first time, the direct impact of the DISC1 gene on the habenular neurons and affective behavior in the DISC1-Q31L genetic mouse line. These new findings suggest that the combination of the DISC1 genetic analysis together with habenular neuroimaging may improve diagnostics of mood disorder in clinical studies.

Lack of synaptic protein, calsyntenin-2, impairs morphology of synaptic complexes in mice.

Calsyntenin-2 (Clstn2) is the synaptic protein, which belongs to the superfamily of cadherins, playing an important role in learning and memory. We recently reported that Clstn2 knockout mice (Clstn2-KO) have a deficit of GABAergic interneurons, associated with hyperactivity, deficient spatial memory, and social behavior. Therefore, we sought to characterize morphometric features of the ultrastructure of synaptic complexes of hippocampal and cortical neurons in Clstn2-KO mice, using high magnification electron microscopy. Morphometric analysis revealed a reduction of symmetric (inhibitory) synaptic density, length of synaptic contacts, and postsynaptic density in neurons of Clstn2-KO mice. Moreover, cortical neurons of Clstn2-KO mice were characterized by the predominance of the simplified type of synapses with the emergence of negative curvature of the synaptic zone in Clstn2-KO mice. Notably, presynaptic zones of cortical neurons of Clstn2-KO mice were characterized by the increased number of synaptic vesicles in opposite to the decreased number of synaptic vesicles in the presynaptic zones of hippocampal neurons. Overall, we found that lack of calsyntenin-2 leads to the striking architectonic alterations of synaptic complexes in the mouse brain, disrupting synaptic density, shape, and connectivity.

Conditioned stimulus presentations alter anxiety level in fear-conditioned mice.

It is generally believed that fear is rapidly triggered by a distinct cue while anxiety onset is less precise and not associated with a distinct cue. Although it has been claimed that both processes can be measured with certain independence of each other, it is unclear how exactly they differ. In this study, we measured anxiety in mice that received discriminative fear conditioning using behavioral, heart rate and calcium (Ca2+) responses in the ventral hippocampal CA1 (vCA1) neurons. We found that the occurrence of fear significantly interfered with anxiety measurements under various conditions. Diazepam reduced basal anxiety level but had no effect during the presentation of conditioned stimulus (CS). Injection of an inhibitory peptide of PKMzeta (ZIP) into the basolateral amygdala almost entirely abolished CS-triggered fear expression and reduced anxiety to basal level. Heart rate measures suggested a small reduction in anxiety during CS-. Calcium responses in the lateral hypothalamus-projecting vCA1 neurons showed a steady decay during CS suggesting a reduced anxiety. Thus, under our experimental conditions, CS presentations likely reduce anxiety level in the fear-conditioned mice.

Uncoupling DISC1 × D2R Protein-Protein Interactions Facilitates Latent Inhibition in Disc1-L100P Animal Model of Schizophrenia and Enhances Synaptic Plasticity via D2 Receptors.

Both Disrupted-In-Schizophrenia-1 (DISC1) and dopamine receptors D2R have significant contributions to the pathogenesis of schizophrenia. Our previous study demonstrated that DISC1 binds to D2R and such protein-protein interaction is enhanced in patients with schizophrenia and Disc1-L100P mouse model of schizophrenia (Su et al., 2014). By uncoupling DISC1 × D2R interaction (trans-activator of transcription (TAT)-D2pep), the synthesized TAT-peptide elicited antipsychotic-like effects in pharmacological and genetic animal models, without motor side effects as tardive dyskinesia commonly seen with typical antipsychotic drugs (APDs), indicating that the potential of TAT-D2pep of becoming a new APD. Therefore, in the current study, we further explored the APD-associated capacities of TAT-D2pep. We found that TAT-D2pep corrected the disrupted latent inhibition (LI), as a hallmark of schizophrenia associated endophenotype, in Disc1-L100P mutant mice-a genetic model of schizophrenia, supporting further APD' capacity of TAT-D2pep. Moreover, we found that TAT-D2pep elicited nootropic effects in C57BL/6NCrl inbred mice, suggesting that TAT-D2pep acts as a cognitive enhancer, a desirable feature of APDs of the new generation. Namely, TAT-D2pep improved working memory in T-maze, and cognitive flexibility assessed by the LI paradigm, in C57BL/6N mice. Next, we assessed the impact of TAT-D2pep on hippocampal long-term plasticity (LTP) under basal conditions and upon stimulation of D2 receptors using quinpirole. We found comparable effects of TAT-D2pep and its control TAT-D2pep-scrambled peptide (TAT-D2pep-sc) under basal conditions. However, under stimulation of D2R by quinpirole, LTP was enhanced in hippocampal slices incubated with TAT-D2pep, supporting the notion that TAT-D2pep acts in a dopamine-dependent manner and acts as synaptic enhancer. Overall, our experiments demonstrated implication of DISC1 × D2R protein-protein interactions into mechanisms of cognitive and synaptic plasticity, which help to further understand molecular-cellular mechanisms of APD of the next generation.

The role of the habenula in the transition from reward to misery in substance use and mood disorders.

The habenula (Hb) is an evolutionary well-conserved structure located in the epithalamus. The Hb receives inputs from the septum, basal ganglia, hypothalamus, anterior cingulate and medial prefrontal cortex, and projects to several midbrain centers, most importantly the inhibitory rostromedial tegmental nucleus (RMTg) and the excitatory interpeduncular nucleus (IPN), which regulate the activity of midbrain monoaminergic nuclei. The Hb is postulated to play a key role in reward and aversion processing across species, including humans, and to be implicated in the different stages of transition from recreational drug intake to addiction and co-morbid mood disorders. The Hb is divided into two anatomically and functionally distinct nuclei, the lateral (LHb) and the medial (MHb), which are primarily involved in reward-seeking (LHb) and misery-fleeing (MHb) behavior by controlling the RMTg and IPN, respectively. This review provides a neuroanatomical description of the Hb, discusses preclinical and human findings regarding its role in the development of addiction and co-morbid mood disorders, and addresses future directions in this area.

Specific Inhibition of Phosphodiesterase-4B Results in Anxiolysis and Facilitates Memory Acquisition.

Cognitive dysfunction is a core feature of dementia and a prominent feature in psychiatric disease. As non-redundant regulators of intracellular cAMP gradients, phosphodiesterases (PDE) mediate fundamental aspects of brain function relevant to learning, memory, and higher cognitive functions. Phosphodiesterase-4B (PDE4B) is an important phosphodiesterase in the hippocampal formation, is a major Disrupted in Schizophrenia 1 (DISC1) binding partner and is itself a risk gene for psychiatric illness. To define the effects of specific inhibition of the PDE4B subtype, we generated mice with a catalytic domain mutant form of PDE4B (Y358C) that has decreased ability to hydrolyze cAMP. Structural modeling predictions of decreased function and impaired binding with DISC1 were confirmed in cell assays. Phenotypic characterization of the PDE4B(Y358C) mice revealed facilitated phosphorylation of CREB, decreased binding to DISC1, and upregulation of DISC1 and ß-Arrestin in hippocampus and amygdala. In behavioral assays, PDE4B(Y358C) mice displayed decreased anxiety and increased exploration, as well as cognitive enhancement across several tests of learning and memory, consistent with synaptic changes including enhanced long-term potentiation and impaired depotentiation ex vivo. PDE4B(Y358C) mice also demonstrated enhanced neurogenesis. Contextual fear memory, though intact at 24 h, was decreased at 7 days in PDE4B(Y358C) mice, an effect replicated pharmacologically with a non-selective PDE4 inhibitor, implicating cAMP signaling by PDE4B in a very late phase of consolidation. No effect of the PDE4B(Y358C) mutation was observed in the prepulse inhibition and forced swim tests. Our data establish specific inhibition of PDE4B as a promising therapeutic approach for disorders of cognition and anxiety, and a putative target for pathological fear memory.

Cognitive Deficits in Calsyntenin-2-deficient Mice Associated with Reduced GABAergic Transmission.

Calsyntenin-2 has an evolutionarily conserved role in cognition. In a human genome-wide screen, the CLSTN2 locus was associated with verbal episodic memory, and expression of human calsyntenin-2 rescues the associative learning defect in orthologous Caenorhabditis elegans mutants. Other calsyntenins promote synapse development, calsyntenin-1 selectively of excitatory synapses and calsyntenin-3 of excitatory and inhibitory synapses. We found that targeted deletion of calsyntenin-2 in mice results in a selective reduction in functional inhibitory synapses. Reduced inhibitory transmission was associated with a selective reduction of parvalbumin interneurons in hippocampus and cortex. Clstn2(-/-) mice showed normal behavior in elevated plus maze, forced swim test, and novel object recognition assays. However, Clstn2(-/-) mice were hyperactive in the open field and showed deficits in spatial learning and memory in the Morris water maze and Barnes maze. These results confirm a function for calsyntenin-2 in cognitive performance and indicate an underlying mechanism that involves parvalbumin interneurons and aberrant inhibitory transmission.

Ultrasonic Vocalizations in Mice During Exploratory Behavior are Context-Dependent.

While rat ultrasonic vocalizations (USVs) are known to vary with anticipation of an aversive vs. positive stimulus, little is known about USVs in adult mice in relation to behaviors. We recorded the calls of adult C57BL/6J male mice under different environmental conditions by exposing mice to both novel and familiar environments that varied in stress intensity through the addition of bright light or shallow water. In general, mouse USVs were significantly more frequent and of longer duration in novel environments. Particularly, mice in dimly-lit novel environments performed more USVs while exhibiting unsupported rearing and walking behavior, and these calls were mostly at high frequency. In contrast, mice exhibited more low frequency USVs when engaging in supported rearing behavior in novel environments. These findings are consistent with data from rats suggesting that low-frequency calls are made under aversive conditions and high-frequency calls occur in non-stressful conditions. Our findings increase understanding of acoustic signals associated with exploratory behaviors relevant to cognitive and motivational aspects of behavior.

Self-directed exploration provides a Ncs1-dependent learning bonus.

Understanding the mechanisms of memory formation is fundamental to establishing optimal educational practices and restoring cognitive function in brain disease. Here, we show for the first time in a non-primate species, that spatial learning receives a special bonus from self-directed exploration. In contrast, when exploration is escape-oriented, or when the full repertoire of exploratory behaviors is reduced, no learning bonus occurs. These findings permitted the first molecular and cellular examinations into the coupling of exploration to learning. We found elevated expression of neuronal calcium sensor 1 (Ncs1) and dopamine type-2 receptors upon self-directed exploration, in concert with increased neuronal activity in the hippocampal dentate gyrus and area CA3, as well as the nucleus accumbens. We probed further into the learning bonus by developing a point mutant mouse (Ncs1(P144S/P144S)) harboring a destabilized NCS-1 protein, and found this line lacked the equivalent self-directed exploration learning bonus. Acute knock-down of Ncs1 in the hippocampus also decoupled exploration from efficient learning. These results are potentially relevant for augmenting learning and memory in health and disease, and provide the basis for further molecular and circuit analyses in this direction.

A dopamine D2 receptor-DISC1 protein complex may contribute to antipsychotic-like effects.

Current antipsychotic drugs primarily target dopamine D2 receptors (D2Rs), in conjunction with other receptors such as those for serotonin. However, these drugs have serious side effects such as extrapyramidal symptoms (EPS) and diabetes. Identifying a specific D2R signaling pathway that could be targeted for antipsychotic effects, without inducing EPS, would be a significant improvement in the treatment of schizophrenia. We report here that the D2R forms a protein complex with Disrupted in Schizophrenia 1 (DISC1) that facilitates D2R-mediated glycogen synthase kinase (GSK)-3 signaling and inhibits agonist-induced D2R internalization. D2R-DISC1 complex levels are increased in conjunction with decreased GSK-3α/ß (Ser21/9) phosphorylation in both postmortem brain tissue from schizophrenia patients and in Disc1-L100P mutant mice, an animal model with behavioral abnormalities related to schizophrenia. Administration of an interfering peptide that disrupts the D2R-DISC1 complex successfully reverses behaviors relevant to schizophrenia but does not induce catalepsy, a strong predictor of EPS in humans.

Disrupted-In-Schizophrenia-1 (DISC1) interactome and mental disorders: impact of mouse models.

Disrupted-In-Schizophrenia-1 (DISC1) has captured much attention because it predisposes individuals to a wide range of mental illnesses. Notably, a number of genes encoding proteins interacting with DISC1 are also considered to be relevant risk factors of mental disorders. We reasoned that the understanding of DISC1-associated mental disorders in the context of network principles will help to address fundamental properties of DISC1 as a disease gene. Systematic integration of behavioural phenotypes of genetic mouse lines carrying perturbation in DISC1 interacting proteins would contribute to a better resolution of neurobiological mechanisms of mental disorders associated with the impaired DISC1 interactome and lead to a development of network medicine. This review also makes specific recommendations of how to assess DISC1 associated mental disorders in mouse models and discuss future directions.

α7nAchR/NMDAR coupling affects NMDAR function and object recognition.

The α7 nicotinic acetylcholine receptor (nAchR) and NMDA glutamate receptor (NMDAR) are both ligand-gated ion channels permeable to Ca2+ and Na+. Previous studies have demonstrated functional modulation of NMDARs by nAchRs, although the molecular mechanism remains largely unknown. We have previously reported that α7nAchR forms a protein complex with the NMDAR through a protein-protein interaction. We also developed an interfering peptide that is able to disrupt the α7nAchR-NMDAR complex and blocks cue-induced reinstatement of nicotine-seeking in rat models of relapse. In the present study, we investigated whether the α7nAchR-NMDAR interaction is responsible for the functional modulation of NMDAR by α7nAchR using both electrophysiological and behavioral tests. We have found that activation of α7nAchR upregulates NMDAR-mediated whole cell currents and LTP of mEPSC in cultured hippocampal neurons, which can be abolished by the interfering peptide that disrupts the α7nAchR-NMDAR interaction. Moreover, administration of the interfering peptide in mice impairs novel object recognition but not Morris water maze performance. Our results suggest that α7nAchR/NMDAR coupling may selectively affect some aspects of learning and memory.