The Effect Of Sigma-1 Receptor Deletion On Dopamine Neurons And Its Mechanism

Sigma-1 receptor（σ₁R）,a G protein receptor（223 amino acids）,is expressed in neurons and glial cells in central nervous system.The activation of σ₁R in plasma membrane enhances the Ca2+ influx across NMDA receptor（NMDAr）and release of pre-synaptic glutamic acid and dopamine.In addition,the σ₁R is a ligand-operated endoplasmic reticulum（ER）-localized chaperone protein,notably enriched in mitochondrion-associated ER membranes.The activation of σ₁R directly affects inositol-1,4,5 trisphosphate receptor（IP3R）-gated ER Ca2+ release and mobilization.Parkinson’s disease（PD）,characterized by resting tremor and hypokinesia,is a progressive neurodegenerative disorder.The main pathological hallmarks of PD are loss of dopaminergic neurons in the substantia nigra pars compacta（SNpc）and the presence of Lewy bodies.Using positron emission tomography,the σ₁R binding sites are found to be reduced in the brains of early-phase PD patients.In addition,σ₁R knockout（σ₁R-/-）mice showed an age-related impairment of motor coordination.Theσ₁R deficiency has been reported to cause the degeneration of motor neuron in spinal cord.However,the influence of σ₁R deficiency in dopaminergic neurons has not yet been reported.Aggregation and fibrillation of α-synuclein（αSyn）are known to be a critical step in the pathogenesis of Lewy bodies.The blockade of σ₁R induces ER stress with decline of proteasomal activity through disturbing Ca2+ homeostasis.ER stress can enhance aggregation of αSyn.Proteasomal inhibition can increase the phosphorylation（S129）of αSyn.On the other hand,the inhibition of σ₁R causes a increase in the location of ganglioside GM1 at lipid rafts.The direct interaction between helical-αSyn and the sialic acid and carbohydrate moieties of GM1 leads to the accumulation of αSyn.Because the oligomerization and phosphorylation of αSyn are neurotoxic,it is proposed that the reduction or dysfunction of aiR in the brains of PD patients through enhancing the oligomerization and accumulation of αSyn leads to the loss of dopaminergic neurons.The σ₁R is expressed in astrocytes.The activation of glial and microglial are enhanced in the brains of PD patients with the elevation of pro-inflammatory factors,such as tumor necrosis factor-α（TNF-α）,interleukin-6（IL-6）.Neurotoxin MPTP or 6-OHDA can cause death of dopaminergic neurons with activation of astrocytes and microglias leading to increase in the pro-inflammatory cytokines.Methamphetamine-mediated activation of astrocytes involves the activation of σ₁R through ERK-CREB signaling pathway.Furthermore,methamphetamine facilitates the release of dopamine through increasing levels of the dopamine transporter（DAT）,the vesicular monoamine transporter 2（VMAT2）or NMDAr mediated Ca2+ influx.The aiR deficiency depresses the phosphorylation of NR2B to reduce the Ca2+ influx across NMDAr.However,the effects of the σ₁R deficiency on MPTP-induced death of dopaminergic neurons have not yet been reported.Objective1.To investigate the influence of aiR deficiency on dopaminergic neurons and underlying molecular mechanisms.2.To explore the effects of aiR deficiency on MPTP-induced loss of dopaminergic neurons.The First PartInfluence of σ₁R deficiency on dopaminergic neuronsMaterials and Methods1.The genotype of σ₁R-/-mice was identified by PCR method.Three,six and nine-month-old（3,6,9-M-old）male σ₁R-/-mice were used at the beginning of all experiments.2.Motor coordination and balance function were examined using constant and accelerating rotarod tests.3.The immunostaiming of σ₁R,tyrosine hydroxylase（TH）or Hochest was performed.The number of TH-positive（TH+）cell bodies and Hochest positive（Hochest+）cells in SNpc were count.4.Levels of σ₁R,soluble and insoluble aSyn phosphorylation,eIF2a phosphorylation and CHOP in SN were examined by Western blotting analysis.5.Activity of proteasome was measured by fluorometric assay.Results1.In comparison with age-matched WT mice,the latency stayed on the rotorod with both constant and accelerating speeds in either 6-M-old or 9-M-old σ₁R-/-mice was markedly reduced,but in 3-M-old σ₁R-/-mice had no difference,indicating that the aiR deficiency causes an age-related deficits in the motor coordination.2.The σ₁R protein was observed in the dopaminergic neurons of the SNpc in WT mice.In comparison with WT mice,6-M-old and 9-M-old σ₁R-/-mice exhibited a decrease in the number of TH+ cells with increase of Hoechst+ cells,whereas 3-M-old σ₁R-/-mice did not,indicating that the σ₁R deficiency causes an age-related death of dopaminergic neurons in the SNpc.3.The levels of insoluble aSyn oligomers were elevated in 6-M-old and 9-M-oldσ₁R-/-mice compared to WT mice,but the soluble aSyn monomer had not significant differences between WT mice and σ₁R-/-mice.The aSyn fibril content was increased in dopaminergic cells of 9-M-old σ₁R-/-mice,indicating that theσ₁R deficiency increases age-dependently the aggregation and fibrillation of aSyn in dopaminergic cells.4.Levels of monomers or oligomers aSyn phosphorylation were elevated in 6-M-old and 9-M-old σ₁R-/-mice,which accompanied by the decline of proteasome activities,indicating that the σ₁R deficiency probably through reducing proteasome activity enhances the aSyn phosphorylation.5.In comparison with WT mice,the phosphorylation eukaryotic initiation factor 2A（phosphor-eIF2a）and the level of CHOP were observably increased in 6-M-old and 9-M-old σ₁R-/-mice,but not in 3-M-old σ₁R-/-mice,indicating that the aiR deficiency induces the ER stress.6.The treatment with the GM1 binder rifampicin（Rif）for 30 days in 5-M-old and 8-M-old σ₁R-/-mice could attenuate the levels of αSyn oligomers without the changes in the aSyn phosphorylation and proteasome activity.The administration of the ER stress inhibitor salubrinal for 30 days in 5-M-old mice corrected the increase in αSyn oligomers and phosphorylation,and the decrease in the proteasome activity.Although salubrinal inhibited the increased aSyn phosphorylation in 8-M-old σ₁R--" mice,had no effects on the aSyn aggregation.The results indicate that（1）the aiR deficiency → ER stress → reduced proteasome activity → increased aSyn phosphorylation → promoted aSyn aggregation;（2）the σ₁R deficiency → increased GM1-binding to aSyn →increased aSyn aggregation.7.The administration of salubrinal for 30 days in 5-M-old or 8-M-old σ₁R-/-mice could prevent the death of TH+ cells,which was companied by the improvement of motor coordination.In 8-M-old σ₁R-/-mice,the treatment with Rif could partially reduce the loss of TH+ cells and the damage of motor coordination.SummaryThe σ₁R deficiency causes age-related aggregation and phosphorylation of aSyn leading to loss of dopaminergic neurons and hypokinesia（summary diagram）.The Second PartEffects of σ₁R deficiency on MPTP-neurotoxicityMaterials and Methods1.The mice received a subcutaneous（s.c.）injection of MPTP（20 mg/kg）for 5 weeks.The primary cultured astrocytes and microglia were incubated in MPP+（500 μM）.2.The beam walking test and rotarod test were used to evaluate motor coordination and balance function.3.Immunostaining of TH,glial fibrillary acidic protein（GFAP）,ionized calcium-binding adapter molecule 1（Ibal）and Hochest was performed.The number of TH+ cells,Hochest+ cells,GFAP-positive（GFAP＋）cells and Ibal-positive（Ibal＋）cells in SNpc were count.4.Enzyme linked immunosorbent assay（ELISA）was used to examine levels of IL-6,IL-1βandTNF-α.5.Levels of DAT,VMAT2,GFAP,Ibal and phosphorylation of NR2B,STAT3 and ERK1/2 were examined by Western blotting analysis.Results1.On days 5-11 after the last MPTP-injection,WT mice treated with MPTP（MPTP-mice）showed the prolongation of walking time to traverse the beam and the shorten latency on the rotated rod,but σ₁R-/-mice or σ₁R+/-mice treated with MPTP（MPTP-σ₁R-/-mice or MPTP-σ₁R+/-mice）did not.The σ₁R antagonist NE100 could attenuate the impairment of motor coordination in MPTP-WT mice.Following the treatment with the aiR agonist PRE084,MPTP-σ₁R+/-mice appeared the motor coordination.The results indicate that the aiR deficiency reduces MPTP-induced deficits in motor coordination.2.Similarly,the decrease of TH+ cells and increase of Hoechst+ cells were observed in MPTP-mice,but not in MPTP-σ₁R-/-mice.The treatment of MPTP-mice with NE100 attenuated the loss of TH+ cells,while the administration of PRE084 caused 30%loss of TH+ cells in MPTP-aiR+/-mice.The results indicate that theσ₁R deficiency reduces MPTP-induced death of dopaminergic neurons.3.In the SNpc of WT mice,the σ₁R protein was observed in GFAP+ cells,but not in Ibal+ cells.The number of GFAP+ and Ibal+ cells in σ₁R-/-mice did not differ from WT mice.In comparison with WT mice,the number of Iba1+ cells in both MPTP-mice and MPTP-σ₁R-/-mice was increased approximately 30-40%.The activated GFAP+ cell showed thick processes.The number of activated GFAP+cells was increased near 2-fold in MPTP-mice,while only 1.3-fold in MPTP-σ₁R-/-mice.The treatment of MPTP-mice with NE100 attenuated the number of activated GFAP+ cells,indicating that the σ₁R deficiency prevents MPTP-induced astrocyte activation.4.In primary mouse astrocytes and microglia,the application of MPP+ increased the levels of GFAP and Ibal expression with elevation of the IL-6,IL-1β and TNF-a release.The treatment with NE100 could prevent the MPP+-increased GFAP expression and IL-6,IL-lβ and TNF-a release in astrocytes but not in microglia.5.The application of MPP+increased the ERK1/2 phosphorylation in astrocytes,but in microglia did not.6.In primary mouse astrocytes,the application of MPP+ increased the ERK1/2 phosphorylation,which was sensitive to NE100.The MEK inhibitor U0126 prevented the production and release of IL-6,IL-1β and TNF-a in astrocytes.7.In primary mouse microglias,MPP+ increased the phosphorylation of ERK1/2,which was insensitive to NE100.U0126 had no effect on the MPP+-increased GFAP expression and release of IL-6,IL-1β and TNF-a.8.The level of NR2B phosphorylation in SN of σ₁R-/-mice was lower than that in WT mice.The amplitude of increased NR2B phosphorylation in MPTP-mice was higher than MPTP-σ₁R-/-mice.The NR2B inhibitor Ro25-6981 prevented the death of TH+ cells in MPTP-mice.The NMDAr agonist NMDA caused the loss of TH+ cells in MPTP-σ₁R-/-mice.9.In comparison with WT mice,the level of DAT protein was lower in aσ₁R-/-mice,which was corrected by the administration of NMDA.The proportion of decreased DAT protein in MPTP-mice was higher than MPTP-σ₁R-/-mice.10.The levels of VMAT2 protein in σ₁R-/-mice had no significant difference from WT mice.The decline of VMAT2 level in MPTP-mice could be rescued by NE100,indicating that the death of dopaminergic neurons leads to the reduction ofVMAT2.SummaryThe σ₁R deficiency reduces MPTP-induced death of dopaminergic neurons and motor deficits through inhibiting astrocytes-related inflammatory and DAT expression（summary diagram）.Conclusion and clinical significance:The results in the present study indicate that the reduction or dysfunction of σ₁R in early stage PD patients reduces death of dopaminergic neurons through inhibiting astrocytes-related inflammatory and DAT expression,but in middle-late stage PD patients increases aggregation and phosphorylation of a-synuclein to cause loss of dopaminergic neurons.