The Modulatory Effects Of Uncoupling Protein2on Depression

Major depressive disorder (MDD) is characterized by depressed mood, lowself-esteem, anhedonia, and disrupted sleeping, eating, and cognition, and has nowbeen recognized to have an overall impact on global illness that is projected to besecond only to ischemic heart disease in social and economical burden by2020.About21%of the world’s population is affected by depression. The variouspathological factors involved in depression are: monoamine hypothesis,neurotransmitter receptor hypothesis, neurotrophic factor hypothesis, hypothalamic-pituitary-adrenal (HPA) dysregulation, oxidative stress, cytokine hypothesis. Recentdrug therapies used to treat depression include: selective serotonin re-uptakeinhibitors, norepinephrine and dopamine re-uptake inhibitors and tricyclicantidepressants. However, approximately one third of MDD patients do not respondto traditional pharmacological medications. Thus, there is a major unmet need for thedevelopment of novel therapeutic target and more efficacious therapeutic agents.Uncoupling proteins2(UCP2) is a member of mitochondrial anion-carrier proteinsthat are located on the inner mitochondrial membrane. It functions as a facilitator ofproton leak. UCP2-mediated ROS attenuation is thought to occur via dissipation of mitochondrial protonmotive force, again consistent with its role as an uncouplingprotein. UCP2is thought to have a cytoprotective role by stimulating mitochondrialbiogeneis and preventing induction of cell death by decreasing membrane potentialand calcium influx into mitochondria. Mammalian UCP2is found in the kidney,pancreas, spleen, immune cells and the central nervous system. UCP2has beenhypothesized to participate in several metabolic processes involved in, for example,the regulation of food intake, insulin secretion and immune responses. NeuronalUCP2can directly influence neurotransmission, synaptic plasticity andneurodegenerative processes.Our previous study has demonstrated that lack of UCP2increased the sensitivity toCMS-induced depression. However, the penetrating mechanism which accounted forthe relationship between UCP2and depression is unclear. Recently, the inflammationhypothesis of depression posits that depression is related to the activation of immunesystem. IL-1β, produced by NLRP3inflammasome activation, is a key factor in thepro-inflammatory response to psychological stress. Mitochondrial ROS is essentialfor NLRP3inflammasome activation. Whether UCP2is involved in the activation ofNLRP3inflammasome via reducing the production of ROS and oxidative stress isstill unknown. Growing evidence has implicated astrocytes are the major sites forROS production and oxidative stress as well as innate immune responses, astrocytedysfunction is critical in the pathophysiology of mood disorders. Thus, wehypothesized that UCP2regulates NLRP3inflammasome activation in mouseastrocytes and participate in the pathogenesis of depression.Wild-type and UCP2knockout mice were used to establish chronic mild stress(CMS)-induced anhedonia model of depression. We first explore the roles and theinvolved mechanisms of UCP2knockout in depression-like behaviors andneurogenesis. Then, the primary cultured astrocytes were used to study the role of UCP2on NLRP3inflammasome activation in mouse astrocyte as well as theinvolved mechanisms.AIM: UCP2knockout mice were used to investigate the role and the mechanism ofuncoupling protein2in depression and elucidate the critical role of UCP2in NLRP3inflammasom activation in astrocytes.METHODS:1. Chronic mild stress (CMS) was used to establish anhedonia modelof depression.2. The depression-like symptoms were evaluated by sucrose preferencetest, tail suspension test (TST) and forced swimming test (FST).3.Immunohistochemistry was taken for5-bromo-2-deoxyuridine (BrdU), doublecortin(DCX) and glial fibrillary acidic protein (GFAP). The total numbers of BrdU-positivecells in the subgranular zone (SGZ), and DCX-positive cells and GFAP-positive cellsin the dentate gyrus (DG) were obtained stereologically using the optical fractionatormethod.4. The level of ROS (reactive oxygen species) in hippocampus wasdetermined by immunofluorescence method.5. The levels of NF-κB (nuclear factorkappa B) p65, the phosphoralation of JNK (c-Jun NH2-terminal kinase), NLRP3(nucleotide-binding oligomerization domain (NOD)-like receptor3), caspase-1, IL-1β(interleukin-1beta), Grp78(78-kilodalton glucose regulated protein), CHOP(C/EBP-homologous protein), caspase-12, BDNF (brain-derived neurotrophic factor),FGF-2(fibroblast growth factor-2) and the phosphoralation of CREB (cAMPresponse element binding protein) were determined by Western blotting. The plasmaIL-1β level was analyzed by ELISA kit.6. Primary astrocyte cultures were preparedfrom cortex and hippocampal tissues of postnatal (P1-P2) UCP2+/+and UCP2-/-C57BL/6J mice, and were exposed to ATP (5mM) or MSU (250μg/mL). Westernblotting and ELISA were used to analysis the expression of NLRP3, caspase-1and IL-1β in astrocytes.7. Flow cytometric analysis was used to determine themitochondrial dysfunction in astrocytes. Immunofluorescence was used to analyse theROS production in astrocytes. Co-immunoprecipitation and Western blotting wereused to determine the direct association of NLRP3with TXNIP in astrocytes.Transfection of plasmid pcDNA3.1-hUCP2was used to augment the expression ofUCP2protein in mouse astrocytes.RESULTS:1. UCP2knockout mice showed aggravated depressive responsesUCP2knockout exacerbated CMS-induced reductions of body weight and sucrosepreference (P <0.05), and the degradation of the physical state of the coat (P <0.05).UCP2knockout increased the immobility times in TST and FST compared withwild-type mice under basal conditions (P <0.05). UCP2knockout aggravatedCMS-induced expansion of the immobility times in TST and FST (P <0.05),increasing by16%in TST and by92%in FST compared with UCP2+/+mice.2. UCP2knockout mice showed aggravated neurogenesis suppression andastrocytes injuryUCP2knockout significantly decreased the numbers of proliferated cells and newlyborn neurons in SGZ in CMS model (P <0.05), reducing by17%in BrdU+cells andby33%in DCX+cells compared with UCP2+/+mice. UCP2knockout aggravatedCMS-induced astrocytes loss in hippocampus (P <0.05), reducing by21%in GFAP+cells compared with UCP2+/+mice.3. UCP2knockout exacerbated CMS-induced activation of NLRP3inflammasome in hippocampus by increasing ROS production and JNKactivationUCP2knockout elevated CMS-induced ROS production (P <0.05), and significantly increased CMS-induced p65and pJNK expression in hippocampus (P <0.05). UCP2knockout aggravated CMS-induced augmention of NLRP3, caspase-1,IL-1β expression in hippocampus (P <0.05), as well as increased IL-1β secretion inplasma (P <0.01).4. UCP2knockout aggravated CMS-induced endoplasmic reticulum stressand suppression of neurotrophic factors expression in hippocampusUCP2knockout increased CHOP expression in hippocampus (P <0.05), but notGrp78and caspase-12expression (P>0.05) compared with wild-type mice underbasal condition. UCP2knockout aggravated CMS-induced upregulation of Grp78,CHOP, caspase-12expression in hippocampus (P <0.05). UCP2knockoutexacerbated CMS-induced reduction of BDNF, FGF-2expression, via inhibition ofCREB phosphorylation in hippocampus (P <0.05).5. UCP2deficiency exacerbated NLRP3inflammasome activation in primarycultured mouse astrocytesUCP2deficiency remarkably promoted p65, NLRP3, caspase-1and Il-1βexpression in primary cultured mouse astrocytes in response to NLRP3inflammasome activators, such as ATP or MSU (P <0.05). Caspase-1inhibitorz-YVAD-fmk significantly suppressed the secretion of IL-1β induced by ATP or MSUin both genotypes (P <0.01), and abolished the elevation of IL-1β secretion inUCP2-/-astrocytes. Pharmacological inhibition of UCP2aggravated MSU-inducedupregulation of NLRP3, caspase-1and Il-1β expression in UCP2+/+astrocytes (P <0.05), but not in UCP2-/-astrocytes (P>0.05).6. UCP2knockout aggravated NLRP3inflammasome activation via increasingROS generation and p-JNK in mouse astrocytesUCP2knockout increased the number of dysfunctional mitochondrial and ROSproduction under basal condition (P <0.05) in mouse astrocytes. UCP2deficiencyincreased MSU-induced mitochondrial dysfunction, and elevated MSU-induced ROSproduction in astrocytes (P <0.05). UCP2knockout enhanced the direct associationof NLRP3with TXNIP in astrocytes (P <0.05). ROS inhibitor APDC reduced the direct association of NLRP3with TXNIP (P <0.05), suppressed MSU-inducedincreased expression of NLRP3, caspase-1and IL-1β (P <0.05), abolished excesscaspase-1acitvation and IL-1β production in UCP2-/-mouse astrocytes. UCP2knockout aggravated the phosphorylation of JNK in primary cultured mouseastrocytes (P <0.05). JNK inhibitor SP600125abolished upregulation of NLRP3,caspase-1, Il-1β expression in mouse astrocytes (P <0.05).7. Overexpression of UCP2reduced ROS generation and significantlyattenuated NLRP3inflammasome activation in mouse astrocytesOverexpression of UCP2attenuated mitochondrial dysfunction in mouse astrocytesand significantly reduced MSU-induced ROS elevation (P <0.05), reducing by29%in UCP2+/+astrocytes and by44%in UCP2-/-astrocytes. Overexpression of UCP2expression in astrocytes attenuated MSU-induced excess NLRP3, caspase-1andIL-1β expression (P <0.05).CONCLUSIONS:1. UCP2knockout aggravated depressive symptoms via activation of NLRP3inflammasome in mouse model of depression.2. UCP2knockout exacerbated inflammatory responses via aggravating activationof NLRP3inflammasome in astrocytes.The major contributions of the present study lie in:1. The results provide direct evidence that UCP2participate in the initation andprogress of depression,.2. Our study demonstrates that UCP2is a key regulator of NLRP3inflammasomeactivation in astrocytes, which contributes to the pathogenesis of depression.3. UCP2is a crucial modulator of NLRP3inflammasome activation, whichprovides a new approach for targeting NLRP3in meta-inflammatory diseases.