The Role Of PERK Signaling Mediated A1 Reactive Astrocyte In Manganese-induced Neurotoxicity

BackgroundManganese is an essential trace metal,but excessive exposure to it can lead to manganese toxicity,which is called manganism.Excess manganese is mainly deposited in the basal ganglia,causing movement disorders.The typical clinical manifestation of manganism is parkinsonism.However,the specific mechanism of manganese neurotoxicity remains to be discovered.Recent studies found that glial cells may play a critical role in inducing manganese neurotoxicity.An increasing quantity of evidence has shown that astrocytes involve in the pathogenesis of central nervous system diseases.Astrocytes are activated in response to abnormal events in the CNS.According to the diverse proteomic,transcriptomic,and functional characteristics of activated astrocytes,they can be classified into A1 type and A2 type,of which A1 type was neurotoxic.A1 reactive astrocytes have been found in various neurodegenerative diseases such as Parkinson’s disease,suggesting that it may drive neurodegeneration.Manganese in the central nervous system is mainly distributed in astrocytes.However,excessive manganese can cause astrocyte activation.Nevertheless,the type of activation and its role in manganese neurotoxicity is unclear.Therefore,the present study aimed to elucidate the phenotype of astrocyte activation following manganese exposure and its specific role in manganese neurotoxicity.The PERK（Protein kinase RNA-like endoplasmic reticulum kinase）pathway,as a branch of the unfolded protein response,is involved in various processes including apoptosis,innate immunity,metabolism,and cell differentiation;whether it participates in cell activation is uncertain.PERK is enriched in the mitochondria-associated endoplasmic reticulum（ER）membranes and is involved in ER-mitochondrial interactions.It is not clear whether PERK is associated with mitochondrial disorders following manganese exposure.Aims1.To examine the phenotype of reactive astrocytes under manganese treatment.2.To reveal the effects of manganese-induced A1 reactive astrocytes on neurons.3.To demonstrate the role of the PERK signaling in manganese-induced A1 reactive astrocytes.4.To explore the effects of PERK on mitochondrial structure and function under manganese exposure.Methods1.The manganese exposure mouse models were established by subcutaneous injection of Mn Cl₂·4H₂O.The blood and brain tissue manganese levels were determined by atomic absorption spectroscopy.The effects of manganese exposure on the motor ability were examined by open field test,climbing pole test,and rotarod test.And the effects of manganese exposure on dopaminergic neurons in the basal ganglia were examined by TH immunofluorescence staining.2.Primary astrocytes were cultured and purified by magnetic bead sorting.Flow cytometry was used to detect the purity of primary astrocytes.An MTT assay was used to detect the effect of manganese exposure on the survival rate of primary astrocytes.3.In vivo and in vitro,changes in morphology,protein expression,and transcriptional characteristics of astrocytes were examined by RT-q PCR and immunofluorescence staining.Single-cell extraction and sorting of astrocytes were performed on adult mouse brain tissues.RT-q PCR was performed to detect the transcriptional characteristics of astrocytes in the substantia nigra and striatum.4.In vitro,the effect of manganese-induced A1 reactive astrocytes on neurons was examined by culturing primary neurons with the astrocyte conditioned medium.In vivo,the role of A1-reactive astrocytes in manganese neurotoxicity was investigated by the block of A1astrocyte conversion.5.In vivo and in vitro,the morphological changes of the endoplasmic reticulum were observed by transmission electron microscopy and ER-Tracker staining.And the change in unfolded protein response signaling pathways was measured by RT-q PCR and Western blot.6.In vivo and in vitro,the activation of PERK signaling was inhibited by AAV viral interference or small molecule inhibitors.And the role of the PERK pathway in manganese-induced astrocyte activation was clarified by RT-q PCR and immunofluorescence staining to detect changes in astrocyte transcriptional characteristics and protein profiles.7.In vivo and in vitro,the mitochondrial morphological changes of astrocytes after manganese exposure were observed by transmission electron microscopy and Mito-Tracker staining.In vitro,the changes in oxygen consumption rate were detected by the O2K analyzer,and the changes in reactive oxygen species were detected by DCFH-DA staining.The relationship between PERK and mitochondrial morphology and function was investigated by inhibiting PERK with small molecule inhibitors.Results1.Manganese exposure affected the motor ability of mice.Manganese exposure significantly increased blood and brain tissue manganese levels,decreased motor ability,and reduced the number of dopaminergic neurons in the basal ganglia（P<0.05）.2.Manganese exposure induced A1-type activation of astrocytes in the substantia nigra and striatum.In vivo,results of RT-q PCR showed significant elevation of PAN and A1-specific markers in the substantia nigra and striatum after manganese exposure（P<0.01）,and no significant elevation of A2 markers.Purification of astrocytes from the substantia nigra and striatum,RT-q PCR analysis showed that PAN and A1-specific markers were significantly elevated in astrocytes after manganese exposure（P<0.01）,while A2-specific markers were not significantly elevated.Immunofluorescence staining showed an increase in the volume and branching of astrocytes in the substantia nigra and striatum and a significant increase in the number of C3-positive astrocytes.In vitro,RT-q PCR showed a significant increase in PAN and A1-specific markers after manganese exposure（P<0.05）,and no significant increase in A2markers.3.Manganese-induced A1 reactive astrocytes could damage neurons.In vitro,PI/DAPI staining showed that manganese-induced A1 reactive astrocytes had no significant effect on the survival rate of primary neurons（P>0.05）.SNAP25/PSD95 immunofluorescence staining showed that manganese-induced A1 reactive astrocytes resulted in a significant reduction in the number of neuronal synapses（P<0.01）.In vivo,the block of A1 astrocyte conversion improved the motor ability and attenuated the reduction in the number of dopaminergic neurons in the substantia nigra and striatum after manganese exposure.4.Manganese exposure activated the PERK signaling in astrocytes.Transmission electron microscopy and ER-tracker staining revealed that the endoplasmic reticulum structure of astrocytes was disturbed after manganese exposure.RT-q PCR showed that ATF4,a transcription factor of the PEKR pathway,was most significantly elevated by manganese exposure（P<0.001）.Western blot analysis showed that P-PERK and P-e IF2αwere significantly elevated after manganese exposure（P<0.01）,and global protein synthesis rates were significantly decreased.5.Inhibition of the PERK signaling partially blocked manganese-induced A1 reactive astrocytes conversion.In vitro,using small molecule compounds to inhibit the PERK pathway,RT-q PCR analysis showed a significant decrease in some PAN-related markers and most A1-specific markers（P<0.01）,with no significant change in A2-specific markers.In vivo,after inhibition of the PERK pathway,RT-q PCR of the substantia nigra and striatum showed a significant decrease in some PAN-related markers and most A1-specific markers（P<0.01）,and no significant changes in A2-specific markers.The results of immunofluorescence staining showed a significant decrease in the number of C3-positive astrocytes.6.PERK affected mitochondrial morphology and function in astrocytes after manganese exposure.Analysis by transmission electron microscopy,Mito-Tracker staining,O2K energy analysis,and ROS assay revealed that manganese exposure caused mitochondrial fragmentation,inhibited mitochondrial respiratory function,and increased reactive oxygen species in astrocytes.Inhibition of PERK phosphorylation attenuated mitochondrial fragmentation,improved mitochondrial respiratory function,and reduced reactive oxygen species in astrocytes after manganese exposure.ConclusionsIn summary,this present study found that Manganese exposure induces A1-type activation of astrocytes in the substantia nigra and striatum;A1 reactive astrocyte plays a key role in manganese neurotoxicity;The activation of PERK signaling is an important mechanism of manganese-induced A1 reactive astrocytes;PERK is involved in the regulation of mitochondrial morphology and function after manganese exposure.Through the exploration of the pathogenesis,this study provides a novel intervention target for the prevention and treatment of manganese neurotoxicity.