Expression Of LILRB2(PirB) Signaling Pathway In Temporal Lobe Epilepsy

Temporal lobe epilepsy(TLE)is the most common form of focal epilepsy affecting a cerebral network comprising the hippocampus proper and extrahippocampal regions(i.e.,entorhinal and perirhinal cortices and the amygdala).Numerous TLE patient seizures cannot be controlled well by antiepileptic drugs(AEDs)and may respond to surgical resection of the epileptogenic foci.Therefore,understanding the biochemical mechanisms of intractable TLE is very urgent.The main pathological changes of TLE,including neuron degeneration,neurogenesis,gliosis,mossy fiber sprouting and synaptic reorganization,have been observed in the epileptic zone.Obviously,aberrant excitatory neural networks provide the anatomic basis for epilepsy.For instance,it has been confirmed that increased excitatory synaptic input to granule cells from CA3 and hilar areas composing the positive-feedback circuits might contribute to epileptogenesis in a rat model of TLE.However,the mechanism of abnormal neural network formation remains obscure.LILRB2(Leukocyte immunoglobulin like receptor B2)is a member of the leukocyte immunoglobulin-like receptor(LIR)family,which contains immunoreceptor tyrosine-based inhibitory motifs(ITIMs)that are predominantly expressed on cells of the myelomonocytic lineage.Moreover,several recent studies have indicated that LILRB2 also plays an important role in the central nervous system(CNS).PirB,as the human LILRB2 homolog,was originally identifled in the cells of the immunologic system and is also widely expressed in the central nervous system.LILRB2 or PirB participates in the process of synaptic plasticity and neurite growth in the central nervous system(CNS),suggesting a potential role of LILRB2 in epilepsy.However,the expression pattern of LILRB2(Pir B)and the downstream molecular signal in intractable TLE remains poorly understood.In this study we investigated the expression of LILRB2 and its important downstream factors POSH,SHROOM3,and ROCK1 / 2 in the temporal neocortex of patients with TLE and control,and further analyzed the correlation between the protein levels of the LILRB2 and the clinical features of TLE,to understand their role in TLE.In addition,we also detected the expression pattern of Pir B in the pilocarpine mouse model of TLE.Our major results:1.The expression of LILRB2 in the temporal neocortex of TLE patients.Western blotting and optical density analysis demonstrated enhanced immunoreactive protein levels of LILRB2 in the temporal neocortex of TLE patients compared with the control samples(P<0.01).Strong staining for LILRB2 was observed in the membrane and cytoplasm of neurons and glial cells in the TLE group.However,LILRB2 displayed weak to moderate staining in the corresponding regions of neurons and glial cells in the control group.The immunoreactivity(IR)scores of LILRB2 in TLE patients were dramatically higher than those in the control samples(P<0.05).2.LILRB2 exhibited a wide distribution in the temporal neocortex of patients with TLE.Double-labeled immunofluorescence demonstrated that LILRB2 was colocalized with NeuN,a marker of mature neurons and the astrocytes marker GFAP(glial fibrillary acidic protein),but not with Iba1 in microglia.3.Expression of POSH,SHROOM3,ROCK1 and ROCK2 in TLE patientsWestern blotting analyses revealed that the levels of POSH(P<0.05),SHROOM3(P<0.01),ROCK1(P<0.05),and ROCK2(P<0.05)were signiflcantly greater in temporal cortex of intractable TLE patients versus control samples.Immunohistochemistry revealed weak or intermediate immunolabeling of POSH,SHROOM3,ROCK1,and ROCK2 in normal neurons and glial cells of control samples.However,moderate and strong POSH,SHROOM3,ROCK1,and ROCK2 IR were observed in temporal cortices of intractable TLE patients.The IR scores of POSH,SHROOM3,ROCK1,and ROCK2 in TLE were higher compared with the CTX samples(P<0.05).Using double immunofluorescence labeling,we found that POSH,SHROOM3,ROCK1,and ROCK2 were colocalized with the neuron marker NeuN and the astrocyte marker GFAP.4.Correlation between the protein levels of the LILRB2 and the clinical features of TLE.The protein levels of LILRB2 in TLE were negatively correlated with the frequency of seizures before surgical resection.There were no significant correlations between the protein levels of LILRB2 and other clinical variables such as age at surgery and the course of epilepsy.5.Dynamic expression of Pir B in the temporal cortex and hippocampus in the TLE mouse model.Western blotting and semiquantitative densitometric analysis demonstrated that the Pir B expression in the hippocampus of the epileptic mice began to increase at 12 h post-SE(P<0.05),reached a peak at 7 days(P<0.01)and then maintained a signiflcantly high level until 60 days(P<0.01).In the temporal cortex,we observed a remarkable increase in PirB expression at 1 day(P<0.05),7 days(P<0.01)and 30 days(P<0.05)post-SE.6.The expression pattern of Pir B in experimental TLE mice.We used immunohistochemical staining to detect the distribution of Pir B.The positive staining was faint and scattered in control normal mice.In pilocarpine-induced epileptic mice,we observed strong positive staining on the hippocampal dentate gyrus,hilus,CA3,CA1 and temporal cortex neurons and glial cells.The IR scores of PirB in 7-day post-SE mice groups were increased compared with the controls(P<0.05).Next,in the hippocampus and temporal cortex of epileptic mice and controls,double-labeled immunofluorescence showed that Pir B was co-expressed with NeuN in neurons and with GFAP in astrocytes.Taken together,our results indicate that LILRB2(Pir B)signaling pathway may play an antiepileptic role.