Study In Effects Of Seipin Deficiency In A?-induced Neurotoxicity And The Underlying Mechanisms

BackgroundCongenital generalized lipodystrophy type 2(CGL2)is characterized by a near total loss of adipose tissue,severe metabolic disorders and cognitive deficit or mental retardation.CGL2 is caused by loss-of-function of seipin gene.Studies by in situ hybridization and immunohistochemistry reported that seipin is selectively expressed in cerebral cortex,hippocampus,hypothalamus,brainstem and cerebellum.The hippocampal pyramidal cells and astrocytes highly expressed seipin.Through building neuronal seipin knockout(seipin-KO)mouse model,we did not observe the changes in the brain structure and the loss of neuronal cells.Our recent study has reported that neuronal seipin depletion reduces the expression of peroxisome proliferator-activated receptor gamma(PPAR?).PPAR? is known to be an important ligand-activated transcription factor.the PPARy down-regulation is known to elevate neuro-inflammatory.Alzheimer's disease(AD)is the most common form of dementia,and is characterized by progressive loss of memory and cognition.The activation of PPARy can improve hippocampus-dependent cognitive deficits in AD mouse models.The PPARy activation has been recently shown to mitigate the neuronal inflammation in chronic and acute neurological insults.The PPAR? has received increasing attention in AD due to its anti-inflammatory function.Therefore,it is proposed that the neuronal seipin deficiency through reducing PPAR? can facilitate the A?-induced neuroinflammation and neurotoxicity.ObjectiveTo determine influence of the neuronal seipin deficiency on the A?-induced neuroinflammation and neurotoxicity.Materials and Methods(1)Animal models:neuron-specific seipin knockout(seipin-KO)mice were provided by Professor George Liu from Peking University.The genotypes were identified by PCR using genomic DNA from their tail.(2)Preparation of AD animal model:Institute of Cancer Research(ICR)male mice were intracerebroventriculy(i.c.v.)injected with the "aggregated" A?25-35(1.2 nmo 1/2 ?l)or A?1-42(0.1 nmol/2 ?l)to prepare the A?25-35-mice and A?1-42-mice.This experiment contains six group mice:WT mice,A?25-35-injected WT mice,A?1-42-injected WT mice,seipin-KO mice,A?25-35-injected seipin-KO mice and A?1-42-injected mice.The control mice were injected with the same volume of dissolvent.(3)Behavioral examination:Morris water maze task and Y-maze task were used to test the cognitive performance.(4)Histological examination:the pyramidal cells in hippocampal CA1 region was assessed by Toluidine blue staining.Hoechst staining was used to examine the number of apoptotic cells.(5)GFAP and IBal immuno-staining were used to examine the number of astrocytes and microglia.(6)Levels of IL-6 and TNF-a were assayed by enzyme linked immunosorbent assay(ELISA).(7)Levels of glycogen synthase kinase-3?(GSK-3?)and signal transducer and activator of transcription(STAT3)protein and phosphorylation,and PPARy protein in the hippocampus were examined by Western blot.Results(1)Compared with WT mice,seipin-KO mice spent longer escape-latency to reach the hidden platform in Morris water maze and shorter swimming time in the target quadrant and less the alternation rate in Y-maze,indicating that the seipin deficiency causes the cognitive deficit.(2)The injection(i.c.v)of A?25-35(1.2 nmol/2 ?l)in WT mice did not cause the changes in cognitive function,but in seipin-KO mice further increased escape-latency in Morris water maze and reduced swimming time in the target quadrant and the alternation rate in Y-maze,indicating that the seipin deficiency aggravates the A?-induced cognitive deficit.(3)Level of PPARy protein in seipin-KO mice was decreased in the hippocampus compared with controls.Treatment of seipin-KO mice with the PPARy agonist rosiglitazone could correct the increase in the escape-latency in Morris water maze and the decreases in the swimming time of the target quadrant and the alternation rate in Y-maze,which were blocked by the PPAR? antagonist GW9962.Furthermore,the administration of rosiglitazone in seipin-KO mice could prevent the A?-induced cognitive deficit,which was sensitive to GW9962,indicating that the seipin deficiency through reducing PPAR? aggravates the A?-induced cognitive deficit.(4)The number of pyramidal cells in hippocampal CA1 regions had no significant difference between WT mice and seipin-KO mice.The injection(i.c.v)of A?25-35 or A?1-42(0.1 nmol/2 ?l)caused approximately 30-35%loss of pyramidal cells and a significant increase in Hoechst-positive cells in seipin-KO mice,but not in WT mice,which could be prevented by the administration of rosiglitazone,indicating that the seipin deficiency through reducing PPARy potentiates the A?-induced neurotoxicity.(5)We observed that GFAP+ astrocytes expressed highly the seipin protein in the hippocampal CA1 region,but Ibal+ microglia did not.The number of GFAP+astrocytes and Ibal+ microglia had no significant difference between WT mice and seipin-KO mice,but the levels of IL-6 and TNF-a were higher in seipin-KO mice than those in WT mice,which could be corrected by the administration of rosiglitazone,indicat:ing that the seipin deficiency through reducing PPARy increases the levels of pro-inflammatory cytokines.(6)The injection(i.c.v)of A?25-35 or A?1-42 increased the number of GFAP+ astrocytes and Ibal+ microglia,and the levels of IL-6 and TNF-? in sepin-KO mice,but not in WT mice,which could be blocked by the administration of rosiglitazone,indicating that the seipin deficiency through reducing PPARy increases the sensitivity of A?-induced neuroinflammation.(7)In the seipin-KO mice,the level of glycogen synthase kinase-3?(GSK-3?)phosphorylation at Tyr216 was elevated,while at Ser9,it was reduced compared to the WT mice,which were corrected by the rosi treatment but were unaffected by the A?25-35.Similarly,the level of hippocampal STAT3 phosphorylation in the seipin-KO mice was elevated compared with that in WT mice,which was further increased by the administration of rosiglitazone,indicating that the seipin deficiency through reducing PPARy increases the GSK-3? activity.ConclusionSeipin deficiency in astrocytes increases GSK-3? activity and levels of IL-6 and TNF-a through reducing PPARy,which can facilitate A?-induced neuroinflammation to cause the death of neuronal cells and cognitive deficits.Clinical significanceThe present study provides a possibility for novel therapeutic application of PPAR,y agonist for cognitive deficits in CGL2.