The Role And Mechanisms Of Laminin β1 In Regulating The Comorbidity Of Neuropathic Pain And Psychiatric Disorders

BackgroundNeuropathic pain is universal,and frequently comorbid with psychiatric disorders,such as sleep disorders,weakness,and even anxiety,depression and other mental illnesses.Particularly,aberrant psychiatric conditions may lead to an exaggerated duration and intensity of pain and drive a vicious cycle between pain and emotional aversion.Despite the presence of various analgesics and antidepressants,incomplete understanding of the mechanisms underlying psychiatric comorbidity induced by neuropathic pain hampered its effective treatment.Thus,exploiting a new and effective treatment for comorbidity of psychiatric disorders in neuropathic pain remains a major challenge.Cellular plasticity in the anterior cingulate cortex(ACC)is assumed as a critical interface for pain perception and emotion.But so far,mounting efforts thus far have been focused on intracellular mechanisms of plasticity rather than the extracellular alterations that might trigger and facilitate intracellular changes in these cellular functions.The extracellular matrix(ECM)in the central nervous system(CNS)is associated with the regulation of synaptic plasticity and various pathophysiological processes.Laminin is a key element of ECM consisting of one α-,β-and γ-chain and implicated in several pathophysiological processes.However,little is known about whether and how laminins participate in the comorbidity of psychiatric disorders in neuropathic pain,and if so,which extracellular-intracellular interacting processes underlie these actions.Aims1.To investigate the role and effect of Lamb1 expression in ACC on the comorbidity of neuropathic pain and psychiatric disorders in mice;2.To further explore the regulatory effects of Lamb1 on cingulate synaptic plasticity and related molecular mechanisms;3.To explore the effect of drug intervention on the expression of Lamb1 on the comorbidity of chronic pain and psychiatric disorders.Methods1.Expression profile of Lamb1 in ACC and its plastic changes in neuropathic pain associated with anxiety-and depression-like behavior.The mice were treated with unilateral spared nerve injury(SNI)surgery.The pain threshold at different time points were measured by von Frey filaments and thermal radiation.Meanwhile,the open field test(OFT),elevated-plus maze(EPM),tail suspension test(TST)and sucrose preference test(SPT)were applied to test the effect of chronic pain on anxiety-and depression-like behavior.RNA sequencing,quantitative real-time PCR(q PCR)and Western blot were used to detect the transcriptome level in the ACC of mice with chronic pain associated with anxiety-and depression-like behavior,and to further clarify the expression level of Lamb1.The expression profile of Lamb1 in the ACC was observed by immunofluorescence staining.2.Effects of Lamb1 knockdown in ACC on pain sensitivity and associated anxietyand depression-like behavior.Lamb1 interference virus was constructed,and the interference efficiency was verified by q PCR,Western blot and immunofluorescence staining.After Lamb1 interference virus was injected into the contralateral ACC brain region of SNI-induced chronic pain and anxiety-and depression-like mice,mechanical and thermal sensitivity as well as anxiety-and depression-like behavior(EPM,TST and SPT)were observed.3.The effect of Lamb1 on the structural and functional plasticity of synapses in ACC.After 56 days of SNI surgery,the dendritic processes and spines of ACC pyramidal neurons were examined using Golgi staining methods in mice expressing sh Lamb1 and scrambled sh RNA.Sholl analysis was performed by Imaris software to observe the changes of dendritic complexity.Density and classification of dendritic spines were further analyzed.The pyramidal neuronal excitability,synaptic transmission and its underlying mechanisms were analyzed by whole-cell patch-clamp recording after injection of Lamb1 interference virus in the ACC of wild-type mice.4.The potential mechanisms underlying the regulation of the comorbidity of neuropathic pain and psychiatric disorders by Lamb1 in ACC.The changes of F-actin and postsynaptic membrane protein PSD95 in ACC and cultured cortical neurons were observed by phalloidin staining.The interaction between Lamb1 and Integrin β1(Itg β1)and the effect of Lamb1 on Src/Rho A/LIMK/cofilin signaling pathway were detected by Immunoprecipitation,Pulldown and Western blot.The effects of Lamb1 on AMPA receptors,NMDA receptors and PSD95 in plasma membrane were further detected by nuclear membrane separation and Western blot.Fiber photometry was used to record the activity of GCa MP6s-expressing pyramidal neurons in the ACC during tail suspension by unilateral intracingulate injection of AAV vector containing Ca MKII-GCa MP6s.5.The effect and mechanism of Lamb1-integrin β1 pathway activation on the comorbidity of neuropathic pain and aversive emotion.Bilateral ACC injection of integrinβ1 activator Pyrinintegrin were performed in SNI-subjected mice,the pain threshold was detected by mechanical and thermal stimulation,and the anxiety-and depression-like behavior of mice was detected by EPM,TST and SPT.Western blot and cellular phalloidin staining were used to investigate the potential regulatory mechanism of Pyrintegrin.6.The effect of other depression models and gender differences on the expression of Lamb1 in the ACC.The chronic restraint stress model(CRS)and chronic exposure to corticosterone(CORT)were used to establish the depression model.Lamb1 expression level was detected by western blot.Unilateral SNI operation was performed in female mice,and Lamb1 levels were detected by western blot.Results1.Mechanical and thermal sensitivity were performed at 3,7,14,28 and 56 days after surgery,respectively.We observed that the mice displayed stable mechanical and thermal hyperalgesia on day 7 after SNI,and long-lasting exaggerated pain response was developed throughout the whole testing period.Meanwhile,the mice showed anxiety-like behavior at day 28 after SNI.Moreover,the mice exhibited anxiety-and depression-like behavior at day56 after surgery,suggesting that SNI mice showed comorbidities of psychiatric disorders under the condition of continuous hyperalgesia.ACC was further analyzed using RNA sequencing(RNA-seq)technique at day 56 after SNI.A total of 565 differentially expressed genes were detected,which were closely related to ECM receptor interaction and focal adhesion related pathways.Of particular,Lamb1,a type of ECM,displayed a dramatic transcriptional downregulation following SNI,and was verified at both m RNA and protein levels by q PCR and western blotting analysis.Furthermore,Lamb1 was mainly expressed in pyramidal neurons in ACC by immunofluorescence staining analysis.2.After knockdown of Lamb1 in ACC,the thresholds of ipsilateral and contralateral hindpaw in response to von Frey hairs were significantly reduced in both Sham-and SNItreated mice,suggesting that down-regulation of Lamb1 in ACC led to exaggerated mechanical allodynia in comparison with scrambled sh RNA.Furthermore,Lamb1 knockdown in ACC resulted in anxiety-and depression-like behavior in sham group.3.Golgi staining showed that the absence of Lamb1 led to spine remodeling in the apical dendrites of ACC pyramidal neurons,manifested by the increased density of dendritic spines and morphological changes,such as the increased density of stubby-and mushroomshaped apical spines.Further electrophysiological analysis demonstrated that Lamb1 deficiency drove ACC pyramidal neurons into an hyperexcitable state,characterized by the increased firing frequency and decreased rheobase.Meanwhile,AMPARs-mediated synaptic transmission was significantly enhanced after Lamb1 knockdown.Further analysis indicated that the above synaptic regulation of Lamb1 was related to presynaptic and postsynaptic mechanisms.4.F-actin labeling with phalloidin in both in vivo ACC tissue or in vitro cultured cortical neurons revealed a significant enhancement in neurons expressing sh Lamb1 in comparison with those expressing scrambled sh RNA,indicating that absence of Lamb1 results in abnormal actin organization at dendritic spines.Lamb1 strongly interacted with integrin β1 and disruption of Lamb1 largely eliminated this interaction by Immunoprecipitation assay,confirming the potential regulatory relationship between Lamb1 and integrin β1.By further testing Lamb1-integrin β1 downstream signal cascade activation,we found that a significant reduction of phosphorylation of Src after knockdown of Lamb1 in the ACC,thereby reduced the level of tyrosine-phosphorylated p190 Rho GAP,increased level of active Rho A,and further enhanced ROCK2 and LIMK2 level,and finally inactivated cofilin by increasing the phosphorylation.Thus,Lamb1 knockdown decreased levels of active cofilin,eventually resulting in rearrangement of actin.On the other hand,Glu R1 in the membrane fraction of ACC tissues were dramatically enhanced in Lamb1knockdown mice,and PSD95 was also significantly upregulated.In the tail suspension experiment of Lamb1 knockout mice,the activity of pyramidal neurons expressing GCAMP6s in ACC was increased by optical fiber calcium imaging.5.Delivery of integrin β1 activator,Pyrinintegrin in ACC significantly relieved the pain hypersensitivity and anxiety-and depression-like behavior induced by SNI,as compared to vehicle delivery.The protein expression of ACC was further detected,and found that Pyrintegrin reduced the phosphorylation level of cofilin by blocking the decrease of Src phosphorylation,and further inhibited F-actin polymerization induced by Lamb1 knockdown.6.No significant changes of Lamb1 was detected in the ACC of mice subjected to either chronic CORT or CRS depression models.Additionally,the change of ACC Lamb1 in SNI did not show sex difference because female mice displayed reduced Lamb1 level in the ACC as well following SNI.Conclusions1.After establishment of anxiety and depression-like behavior associated with chronic pain,it was found that Lamb1 in ACC is significantly downregulated and mainly distributed in pyramidal neurons.This suggests that Lamb1 in ACC plays a potential role in the comorbidities of chronic pain and psychiatric disorders.2.Exogenously knocking down Lamb1 in ACC by virus infection exacerbates hyperpathia and induces anxiety-and depression-like behavior,suggesting that Lamb1 in ACC is closely related to pain hypersensitivity and associated anxiety and depression-like behavior in mice.3.Loss of Lamb1 produces prominent spine remodeling of apical dendrites in ACC pyramidal neurons,manifesting as increased spine densities as well as spine morphological changes,and drives ACC pyramidal neurons into a hyperexcitable state.The AMPARsmediated synaptic transmission is markedly potentiated by silence of Lamb1.Further mechanistic analysis revealed that both pre-and post-synaptic mechanisms are involved in the above synaptic modulation by Lamb1.These results suggest that loss of function in Lamb1 orchestrates structural and functional plasticity of pyramidal neurons in the ACC.4.Lamb1 is involved in integrin β1-dependent-modulation of actin cytoskeleton via Src/Rho A/LIMK/cofilin signaling pathway,which further leads to synaptic remodeling and synaptic transport of AMPARs,thereby enhancing synaptic function.5.The integrin β1 agonist Pyrintegrin may alleviate the F-actin polymerization induced by Lamb1 knockdown via Src/cofilin signaling pathway,and further significantly ameliorate the production of pain hypersensitivity and associated anxiety and depressionlike behaviors.Therefore,this study further confirmed the molecular mechanisms of Lamb1-integrin β1 regulating synaptic plasticity.Furthermore,it suggests that Lamb1 as ECM might represent a promising novel target in treating the comorbidity of neuropathic pain and related psychiatric disorders.6.Lamb1 in ACC may not be associated with non-pain-related depression,and there is no sex difference in the regulation of pain associated with psychiatric disorders.