Study In Influence Of Seipin Deficiency On Dopaminergic Neurons And The Underlying Mechanisms

Congenital generalized lipodystrophy type 2(CGL2)is an autosomal recessive disorder and characterized by a near total loss of adipose tissue and severe metabolic disorders.In addition,cognitive deficit and motor coordination impairment are observed in patients with CGL2.Thus,the underlying mechanisms remain to be elucidated..CGL2 is caused by loss-of-function of seipin gene.The seipin protein is the two transmembrane intrinsic protein of the endoplasmic reticulum,which is highly expressed in the central nervous system.Recently,the decrease of seipin expression is reported in substantia nigra of Parkinson’s disease(PD)patients.The dopamine neurons in the substantia nigra are important for regulating the motor coordination.The main pathological hallmarks of PD is the presence of Lewy bodies in the substantia nigra pars compacta(SNpc),resulting in the loss of dopaminergic neurons,The main pathological mechanism of Lewy bodies is known to be the aggregation and fibrillation of a-synuclein(aSyn).The results of preliminary experiment showed that dopaminergic neurons in substantia nigra expressed the seipin protein.Our studies have reported that the lack of seipin in hippocampal neurons suppresses the expression and activity of peroxisome proliferator-activated receptor gamma(PPARγ).The decrease of PPARy causes a increase in the activity of glycogen synthase kinase 3 beta(GSK3β).The activation of GSK3β promotes inflammatory responses in the brain and increases the phosphorylation of aSyn.Thus,it is proposed that seipin deficiency through reducing the expression of PPARγ affects oligomerization and phosphorylation of aSyn leading to the loss of dopaminergic neurons and motor coordination impairment.Research objectiveTo determine influence of seipin deficiency on dopaminergic neurons and the underlying mechanismsMethods1.Preparation of animal models:Systemic seipin knockout(seipin-sKO)mice,neuron-specific seipin knockout(seipin-nKO)mice and adipose-specific seipin knockout(seipin-aKO)mice were provided by Professor George Liu(Peking University).The genotypes were identified by PCR using genomic DNA from their tail.Three,eight and twelve-month-old(3,8,12-M-old)male seipin-nKO and wild-type mice(nWT),eight-month-old(8-M-old)male seipin-sKO and wild-type mice(sWT),seipin-aKO and wild-type mice(aWT)were used for all experiments.2.Motor coordination and balance function were examined using the beam walking test,and constant and accelerating rotarod tests.3.We performed the immunostaining of tyrosine hydroxylase(TH),and TH/seipin dual immunostaining.TH-positive(TH+)cells in the SNpc were count.4.Levels of seipin,aSyn,aSyn phosphorylation,PPARy,GRP78,CHOP,GSK3β,caspase-3 in the SN were examined by western blotting analysis.5.Enzyme linked immunosorbent assay(ELISA)was used to examine levels of IL-6 and TNF-a in the SN.6.Real time PCR was used to examine levels of PPARy mRNA in the SN.Results1.In comparison with age-matched nWT mice,8-M-old and 12-M-old seipin-nKO mice spent a longer time traversing the beam and remained on the rotarod for less time before falling off,but in 3-M-old seipin-nKO mice had no difference.Compared with age-matched sWT mice,8-M-old seipin-sKO mice spent a longer time traversing the beam and remained on the rotarod for less time,whereas 8-M-old seipin-aKO mice and age-matched aWT mice had no difference in the time traversing the beam and the latency stayed on the rotarod.The results show that the neuronal seipin deficiency causes an age-related progressive decline in motor coordination.2.Dopaminergic neurons expressed the seipin protein in SNpc of of nWT mice.In comparison with age-matched nWT mice,8-M-old and 12-M-old seipin-nKO mice exhibited a decrease in the number of TH+ cells,but 3-M-old seipin-nKO mice did not.The results show that the seipin deficiency causes an age-related progressive loss of dopaminergic neurons.3.The level of aSyn oligomers(70-100 kD)and aSyn oligomers phosphorylation were higher in 8-M-old seipin-nKO mice than that in age-matched nWT mice,but the levels of the aSyn monomer(19 kD)and phosphorylated aSyn monomer had no significant differences between nWT and seipin-nKO mice,the aSyn fibril formation was increased in dopaminergic neurons of SNpc.However,3-M-old seipin-nKO mice had no significant changes compared with nWT mice.The results indicated that the seipin deficiency enhances an age-related accumulation and phosphorylation of aSyn,and aSyn fibril formation.4.Compared with age-matched nWT mice,the level of PPARγ mRNA was reduced,the level of Tyr216 phospho-GSK3β was elevated and the level of Ser9 phospho-GSK3β was reduced in the SN of 3-M-old and 8-M-old seipin-nKO mice.8-M-old seipin-nKO mice exhibited elevation of IL-6 compared to nWT mice,but 3-M-old seipin-nKO mice did not.5.The treatment of 8-M-old seipin-nKO mice with the PPARy agonist rosiglitazone(Rosi)for 7 days could decrease the activity of GSK3β,The administration of Rosi or the GSK3P inhibitor AR-A014418(7 days)could inhibit the increase of IL-6.The results indicated that the seipin deficiency through reducing PPARγenhances age-dependent GSK3β activation leading to neuroinflammation.6.The treatment with Rosi for 28 days could correct the increase in the levels of aSyn oligomers and phosphorylation of aSyn in seipin-nKO mice.The administration of AR-A014418 in 8-M-old seipin-nKO mice could attenuate the phosphorylation of aSyn,but it had no effect on the oligomerization of aSyn.7.The level of cleaved caspase-3(c-caspase-3)in the SN of 8-M-old seipin-nKO mice was higher than that in nWT mice,whereas there was no significant difference between 3-M-old nWT mice and seipin-nKO mice.The treatment of seipin-nKO mice with Rosi or AR-A014418 prevented the increase in the level of c-caspase-3,the loss of dopaminergic neurons and could alleviate the deficits in motor coordination.ConclusionNeuronal seipin deficiency through reducing the expression of PPARγ and increasing the activity of GSK3β,enhanced aggregation and phosphorylation of aSyn or neuroinflammation causes the loss of dopaminergic neurons leading to deficits in motor coordination.