Downregulation of CD47 and CD200 in patients with focal cortical dysplasia type IIb and tuberous sclerosis complex.

BACKGROUND: Focal cortical dysplasia type IIb (FCD IIb) and tuberous sclerosis complex (TSC) are well-recognized causes of chronic intractable epilepsy in children. Accumulating evidence suggests that activation of the microglia/macrophage and concomitant inflammatory response in FCD IIb and TSC may contribute to the initiation and recurrence of seizures. The membrane glycoproteins CD47 and CD200, which are highly expressed in neurons and other cells, mediate inhibitory signals through their receptors, signal regulatory protein α (SIRP-α) and CD200R, respectively, in microglia/macrophages. We investigate the levels and expression pattern of CD47/SIRP-α and CD200/CD200R in surgically resected brain tissues from patients with FCD IIb and TSC, and the potential effect of soluble human CD47 Fc and CD200 Fc on the inhibition of several proinflammatory cytokines associated with FCD IIb and TSC in living epileptogenic brain slices in vitro. The level of interleukin-4 (IL-4), a modulator of CD200, was also investigated. METHODS: Twelve FCD IIb (range 1.8-9.5 years), 13 TSC (range 1.5-10 years) patients, and 6 control cases (range 1.5-11 years) were enrolled. The levels of CD47/SIRP-α and CD200/CD200R were assessed by quantitative real-time polymerase chain reaction and western blot. The expression pattern of CD47/SIRP-α and CD200/CD200R was investigated by immunohistochemical analysis, and the cytokine concentrations were measured by enzyme-linked immune-sorbent assays. RESULTS: Both the messenger RNA and protein levels of CD47, SIRP-α, and CD200, as well as the mRNA level of IL-4, were downregulated in epileptogenic lesions of FCD IIb and TSC compared with the control specimens, whereas CD200R levels were not significantly changed. CD47, SIRP-α, and CD200 were decreasingly expressed in dysmorphic neuron, balloon cells, and giant cells. CD47 Fc and CD200 Fc could inhibit IL-6 release but did not suppress IL-1ß or IL-17 production. CONCLUSIONS: Our results suggest that microglial activation may be partially caused by CD47/SIRP-α- and CD200/CD200R-mediated reductions in the immune inhibitory pathways within FCD IIb and TSC cortical lesions where chronic neuroinflammation has been established. Upregulation or activation of CD47/SIRP-α and CD200/CD200R may have therapeutic potential for controlling neuroinflammation in human FCD IIb and TSC.

PGAM5-CypD pathway is involved in bromocriptine-induced RIP3/MLKL-dependent necroptosis of prolactinoma cells.

Bromocriptine, the most commonly used dopamine (DA) receptor agonists for prolactinoma, can effectively reduce tumor size of prolactinoma, but the mechanism was not fully understood. Apoptosis had been well-recognized to contribute to the tumor mass regression caused by bromocriptine. However, whether other types of non-apoptotic cell death involved in the bromocriptine-induced prolactinoma shrinkage had not been fully clarified. The newly discovered molecular mechanism of necroptosis provides the possibility to examine this programmed necrosis in the pharmacological function of bromocriptine. The aim of present study was to evaluate and investigate the underlying mechanism of necroptosis in involution of prolactinoma induced by bromocriptine. By immunohistochemistry, we found that the numbers of receptor-interacting serine-threonine kinase 3(RIP3) and phosphorylated mixed lineage kinase domain-like protein (pMLKL)-positive cells and their expression intensities were increased in patients with prolactinoma after bromocriptine therapy. For further exploring the mechanism of bromocriptine, prolactinoma cell line (MMQ cells) was adopted to study the mechanism of necroptosis in vitro. Cell viability and ATP level of MMQ cells were decreased, while reactive oxygen species (ROS) level was increased after bromocriptine treatment. The above effects could be partially reversed by Necrostatin-1, an inhibitor of necroptosis. Ultrastructural study further confirmed the necroptosis of MMQ cells, which was characterized by ruptured membrane, dissolved cytoplasm and especially the dramatically swollen mitochondria. Furthermore, we demonstrated that bromocriptine induced RIP3/MLKL-dependent necroptosis of prolactinoma cells and phosphoglycerate mutase family 5(PGAM5)/ Cyclophilin D (CypD) pathway was involved. The results suggested that necroptosis might be a promising target for clinical therapy for prolactinoma.

Upregulations of CRH and CRHR1 in the Epileptogenic Tissues of Patients with Intractable Infantile Spasms.

AIM: Infantile spasms (IS) are an age-specific epileptic syndrome with specific clinical symptom and electroencephalogram (EEG) features, lacking treatment options, and a poor prognosis. Excessive endogenous corticotropin-releasing hormone (CRH) in infant brain might result in IS. However, the data from human IS are limited. In our study, we investigated the expressions of CRH and its receptor type 1 (CRHR1) in surgical tissues from patients with IS and autopsy controls. METHODS: Specimens surgically removed from 17 patients with IS, and six autopsy controls were included in the study. Real-time PCR, Western blotting, and immunostaining were used to detect the expressions of mRNA, protein expression, and distribution. The correlation between variates was analyzed by Spearman rank correlation. RESULTS: The expressions of CRH and CRHR1 were significantly upregulated in the epileptogenic tissues of IS patients compared with the control group. CRH was distributed mainly in neurons, while CRHR1 was distributed in neurons, astrocytes, and microglia. The expression levels of CRH and CRHR1 were positively correlated with the frequency of epileptic spasms. Moreover, the expression of protein kinase C (PKC), which was an important downstream factor of CRHR1, was significantly upregulated in the epileptogenic tissues of patients with IS and was positively correlated with the CRHR1 expression levels and the frequency of epileptic spasms. CONCLUSION: These results suggest that the CRH signal transduction pathway might participate in the epileptogenesis of IS, supporting the hypothesis that CRH is related to the pathogenesis of IS.

Expression of TRPC6 and BDNF in Cortical Lesions From Patients With Focal Cortical Dysplasia.

Focal cortical dysplasia (FCD) likely results from abnormal migration of neural progenitor cells originating from the subventricular zone. To elucidate the roles in molecules that are involved in neural migration pathway abnormalities in FCDs, we investigated the expression patterns of transient receptor potential canonical channel 6 (TRPC6) and brain-derived neurotrophic factor (BDNF) in cortical lesions from FCD patients and in samples of normal control cortex. TRPC6 and BDNF mRNA and protein levels were increased in FCD lesions. By immunohistochemistry, they were strongly expressed in microcolumns, heterotopic neurons, dysmorphic neurons, and balloon cells (BCs). Colocalization assays revealed that most of the misshapen TRPC6-positive or heterotopic cells had a neuronal lineage with the exception of TRPC6-positive FCDiib patient BCs, which had both neuronal and glial features. Most TRPC6-positive cells were glutamatergic neurons. There was also greater expression of calmodulin-dependent kinase IV (CaMKIV), the downstream factor of TRPC6, in FCD lesions, suggesting that TRPC6 expression promoted dendritic growth and the development of dendritic spines and excitatory synapses via the CaMKIV-CREB pathway in FCD. Thus, overexpression of BDNF and TRPC6 and activation of the TRPC6 signal transduction pathway in cortical lesions of FCD patients may contribute to FC pathogenesis and epileptogenesis.

Increased Expression of Transient Receptor Potential Vanilloid 4 in Cortical Lesions of Patients with Focal Cortical Dysplasia.

AIM: Focal cortical dysplasia (FCD) represents a well-known cause of medically intractable epilepsy. Studies found that transient receptor potential vanilloid receptor 4 (TRPV4) may participate in the occurrence of seizures. This study investigated the expression patterns of TRPV4 in FCD and the cascade that regulate functional state of TRPV4 in cortical neurons. METHODS: Thirty-nine surgical specimens from FCD patients and 10 age-matched control samples from autopsies were included in this study. Protein expression and distribution were detected by Western blot, immunohistochemistry, and immunofluorescence staining. Calcium imaging was used to detect the TRPV4-mediated Ca(2+) influx in cortical neurons. RESULTS: (1) The protein levels of TRPV4 and of an upstream factor, protein kinase C (PKC), were markedly elevated in FCD. (2) TRPV4 staining was stronger in the dysplastic cortices of FCD and mainly observed in neuronal microcolumns and malformed cells. (3) The activation of TRPV4 was central for [Ca(2+)]i elevation in cortical neurons, and this activity of TRPV4 in cortical neurons was regulated by the PKC, but not the PKA, pathway. CONCLUSION: The overexpression and altered cellular distribution of TRPV4 in FCD suggest that TRPV4 may potentially contribute to the epileptogenesis of FCD.

Increased Expression and Cellular Localization of P2X7R in Cortical Lesions of Patients With Focal Cortical Dysplasia.

Focal cortical dysplasias (FCDs) are major brain malformations that commonly lead to medically intractable epilepsy. The purinergic ionotropic P2X7 receptor (P2X7R) is an atypical P2X subtype that gates calcium and sodium ions. Previous animal studies have suggested that P2X7R is a contributing factor in epileptogenesis. This study aimed to define the distribution and expression of P2X7R in 35 FCD patient-surgical-resection specimens relative to autopsy control samples (n = 8). Immunohistochemical colocalization assays revealed that P2X7R was primarily expressed in neurons, astrocytes, and microglia. In FCD samples, P2X7R protein levels were increased in abnormal cell types such as dysmorphic neurons and balloon cells, which are characteristic of FCD. By real-time PCR and Western blotting, P2X7R mRNA and protein expression levels were elevated in FCD patient samples vs control samples; P2X7R expression was also higher in FCDII vs FCDIa patient samples. Because interleukin-1ß is a downstream factor of the P2X7R signaling pathway, we determined that there was also moderate-to-strong interleukin-1ß expression in the dysmorphic neurons, balloon cells, and microglia in FCD patient lesions. These results indicate that increasing P2X7R levels may contribute to the pathogenesis of human FCD and that P2X7R represents a potential anti-epileptogenic target.

Expression of TRPV1 in cortical lesions from patients with tuberous sclerosis complex and focal cortical dysplasia type IIb.

Tuberous sclerosis complex (TSC) and focal cortical dysplasia type IIb (FCDIIb) are recognized as causes of intractable epilepsy. Transient receptor potential vanilloid receptor 1 (TRPV1), a member of the transient receptor potential family, is the capsaicin receptor and is known to be involved in peripheral nociception. Recent evidence suggested that TRPV1 may be a contributing factor in epileptogenicity. Here, we evaluated the expression of TRPV1 in the cortical lesions of TSC and FCDIIb relative to normal control cortex. TRPV1 was studied in epilepsy surgery cases with TSC (cortical tubers; n=12) and FCDIIb (n=12) using immunocytochemistry, confocal analysis, and Western blotting (WB). Immunohistochemical location of the TRPV1 was predominately detected in the abnormal cell types, such as dysmorphic neurons, balloon cells (BCs) and giant cells. Co-localization assays further revealed that cells expressing TRPV1 mainly had a neuronal lineage, apart from some BCs in FCDIIb, which obviously were of astrocytic lineage. The increased TRPV1 expression within the dysplastic cortex of TSC and FCDIIb was confirmed by WB. Interestingly, both immunohistochemical and WB data indicated that TRPV1 might have both cytoplasm and nuclear distribution, suggesting a potential nuclear role of TRPV1. The over-expression of TRPV1 in cortical lesions of TSC and FCDIIb suggested the possible involvement of TRPV1 in the intrinsic and increased epileptogenicity of malformations of cortical development associated epilepsy diseases and may represent a potential antiepileptogenic target. However, the current data are merely descriptive, and further electrophysiological investigation is needed in the future.