Astrocyte Kir6.1/K-ATP Channel Regulates Mitochondrial Functions To Participate In Parkinson's Disease

Parkinson's disease?PD?is the second most common neurodegenerative disease in the world after Alzheimer's disease.PD has a chronic development characterized by progressive movement disorders,psychiatric symptoms,and cognitive impairment.Cardinal movement dysfunctions of Parkinson's disease include tremor,stiffness,slowness of movement,and postural instability.The typical pathological characteristic of PD is progressively loss of dopaminergic neurons accompanying with striatal DA?Dopamine?depletion.Presently,clinical treatment strategy for PD is L-DOPA?levodopa?replacement therapy,although most can relieve the symptoms of PD patients,but it cannot prevent progressive death of dopaminergic neurons,and long-term use can produce the serious complications such as motor fluctuations and dyskinesia.A growing number of researchs indicate that the environmental factors,genetic factors and aging are together involving in initiation and progression of PD.It is generally accepted that PD pathology is complicated because there are multiple mechanisms including excitatory toxicity,mitochondrial dysfunction,inflammation changes,oxidative stress and the processing of misfolded proteins can be contributing factors in the initiation and progress in PD.However,according to the current hypothesis,variety of drugs such as NMDA receptor antagonist,antioxidant and apoptosis inhibitors showed no effective neural protective effect.Although the pathological mechanism of PD is unclear,a consistent understanding is that neuroinflammation,mainly the activation of microglia and astrocytes,plays a key role in this disease.Astrocytes are the largest number of cells in the central nervous system.Astrocytes have a range of functions,many of which are crucial for maintaining neuronal normal functions.They provide structural,neutrient and metabolic support,and regulate synaptic transmission,water transport,and blood flow within the brain.The neural networks,formed by neurons-astrocytes-microglias,have dynamic equilibrium.Astrocytes change their phenotypes,from resting state to reactive state,according to the diversity of signals around them.In this course,the reactive-astrocytes response machinery includes secretion of pro-inflammatory cytokines,release of neurotrophins,and clearance of dead cells.These set of intracellular events involved in cell repair,remodeling of extracellular matrix,and other molecular events.In terms of above functions,astrocytes exhibit high energy requirements,therefore,the astrocytes mitochondrial homeostasis may play a critical role in determine the process of PD.Therefore,to modulate the function of astrocytes in PD by targeting the mitochondrial function constitutes an innovative approach to interfere in the PD process.ATP-sensitive potassium channel?ATP-sensitive channel,K-ATP channel?is a kind of special potassium ion channel with coupling cell metabolism and electrical activity and non-voltage dependence.K-ATP channels highly express in the substantia nigra and striatum associated with Parkinson's disease,and participate in regulating release of a variety of PD related neurotransmitters such as DA and glutamate.Some groups including our lab demonstrated that K-ATP channel opener has neuroprotective effects in various toxins induced animal PD models,involving mechanisms are restoration of mitochondrial function,anti-excitatory toxicity,relief apoptosis and inhibition of neuroinflammation.The DA neurons expressing Kir6.2/SUR1 were most sensitive to rotenone toxicity.Kir6.2 knockout can alleviate the damage effect of MPTP combined with the probenecid induced DA neurons loss in PD model mice.These studies indicate that Kir6.1/K-ATP channel may protect DA neurons in initiation and progress of PD.A recent report of our laboratory demonstrated that the microglial Kir6.1/K-ATP channel was involved in regulating microglia phenotype and participating protection effect in PD.The above results suggest that the Kir6.1/ K-ATP channel may be a promising target for ideal neuroprotective agent.The expression of Kir6.1/KATP channels has also been suggested in the plasma and mitochondrial inner membrane of astrocytes widely distributed throughout the central nervous system.However,it has not been clearly elucidated how this type of channel to participate in PD development and what is the precise mechanism.Therefore,in the first part of this study,we used Kir6.1^loxp/loxp and Kir6.1^loxp/loxpGFAP^cre/+ mice to induce LPS,MPTP acute PD model and MPTP/p chronic PD model to investigate the regulation of Kir6.1/K-ATP channel in mouse model of PD.Our results revealed that Kir6.1/K-ATP channel in astrocytes plays an important role in PD.The depletion of astrocyte Kir6.1 gene exacerbated PD-like neural injury including decreased dopamine content in the striatum of MPTP/p chronic PD model,promoted ER stress and inflammation,activation of apoptotic pathways,inhibition autophagy.Previous evidence has been shown that MPP+ stimulation can result in damage of astrocytes and mitochondrial dysfunction in mice substantia nigra.In addition,opening astrocytes K-ATP channel could relieve MPTP/p-induced mitochondrial related apoptosis of astrocytes and maintained ATP level.Combining the first part of this study,the astrocyte kir6.1 gene knockout caused a wide range of cellular stress responses.In the second part of this work,we firstly cultured primary middle brain astrocytes using Kir6.1^loxp/loxp and Kir6.1^loxp/loxpGFAP^cre/+ mice.Secondly,we focused on the role of astrocyte Kir6.1/K-ATP channel in MPP+-induced astrocyte dysfunctions.We observed aggregated apoptosis and inflammation responds in astrocyte with Kir6.1 depletion after MPP+ treatment indicating that the function of astrocytes was in further impaired.Further study demonstrated that the astrocyte Kir6.1 knockout,especially those in mitochondria,enhanced mitochondrial function damage and then deteriorated primary middle brain DA neuron injury by inhibiting mitophagy.Part I Effects of astrocyte Kir6.1 gene depletion in neural injury in mouse model of PDAIM: To investigate the effect of astrocyte Kir6.1/K-ATP channel on toxicants-induced PD mouse model.METHODS: 3-4 months old male Kir6.1^loxp/loxp and Kir6.1^loxp/loxpGFAP^cre/+ miec were treated with LPS or MPTP to establish acute model of PD.??1?LPS acute model of PD:5.0mg LPS was injected into substantia nigra of midbrain according Paximas and Watson atlas.?2?MPTP acute model of PD: 20 mg/kg MPTP in saline was injected subcutaneously,4 times a day,and 1 h interval between two injection.?Immunohistochemistry was taken for TH,GFAP and IBA-1 expression.The quantitative analysis of DA neurons,astrocytes and microglia in SNc of mice was carried out by using stereological method.3-4 months old male Kir6.1^loxp/loxp and Kir6.1^loxp/loxpGFAP^cre/+ mice were treated with MPTP combined with the probenecid to establish chronic model of PD: 20 mg/kg MPTP in saline was injected subcutaneously,and 250 mg/kg probenecid in DMSO was injected intraperitoneally every day for 5.5 weeks.Immunohistochemistry and stereological method were used to evaluate neuronal damage in MPTP/p model of astrocytic Kir6.1 knockout mice.The behavior tests such as the rotarod test,locomotor activity test and pole test were taken to monitor the parkinsonian movement function.HPLC was used to detect the metabolism of MPTP in two transgenicmice mice: 20 mg/kg MPTP in saline was injected subcutaneously,and 250 mg/kg probenecid in DMSO was injected intraperitoneally.MPP+ level in serum,striatum,liver or kidney was measured by HPLC 90 min after MPTP single injection into both transgenicmice of mice.Application of ELISA was performed to detect the enzyme activity of monoamine oxidase B?MAO B?in serum and brain tissue.HPLC method was used to detect the changes in the levels of monoamine transmitters and amino acid neurotransmitters in two genotypes mice.ELISA was used for estimation the secretion of serum pro-inflammatory cytokines?IL-1,IL-6 and TNF-??.For estimation the stress response to chronic MPTP intoxication protocol in two phenotypes mice,Western blot was applied to detect following proteins: Inflammatory response proteins including p65,NLRP1,AIM2,NLRP3,NLRC4,pro-caspase-1,Caspase-1,pro IL-1?,IL-1?,complement C3,GSDMD,GSDMD-N.ER Stress related proteins:GRP78,CHOP,Caspase12,Autophagy associated proteins : p62,LC3II/LC3 I.Apoptosis-related proteins:Bax,AIF,Bcl-2.In addition,Western blot were used to detect the expression of BDNF,GDNF and FGF-2.RESULTS:1)There was no significant difference of TH neurons in SNc in saline group between Kir6.1^loxp/loxpand Kir6.1^loxp/loxpGFAP^cre/+ mice.Compared with Kir6.1^loxp/loxp mice,Kir6.1^loxp/loxpGFAP^cre/+ mice showed significant reduction of TH positive neurons in both LPS and MPTP acute PD models.2)Astrocyte Kir6.1 knockout aggravated SNc glial activation and proliferation in both LPS and MPTP acute PD models,including astrocytes and microglia.3)There was no significant difference of MPTP metabolism in plasma,striatum,liver and kidney of two transgenicmice mice.4)There was no significant difference of enzyme activity of MAO-B between two transgenicmice mice in saline group and chronic MPTP/p group.5)There was no significant difference of DA and its metabolites in saline group.After MPTP/p model was successfully prepared,levels of striatum DA,DOPAC,HVA and 5-HT were significantly decreased in two transgenicmice mice.Compared with Kir6.1^loxp/loxp mice,Kir6.1^loxp/loxpGFAP^cre/+ mice showed more fiercely reduction concentration of striatum DA.In addition,there was no significant difference in the level of amino acid neurotransmitters in the striatum of two transgenicmice mice,whether in the basic state or in the MPTP/p model.6)There was no significant difference on movement function between two transgenicmice mice in saline group.Kir6.1^loxp/loxpGFAP^cre/+ mice showed increased dysfunction of movement.7)Astrocyte Kir6.1 knockout aggravated SNc loss of DA neurons and glial activation and proliferation in MPTP/p PD model.8)Astrocyte Kir6.1 knockout promoted secretion of inflammatory factors including IL-1? and TNF-?.9)After MPTP/p treatment,astrocyte Kir6.1 knockout mice showed significant increase of inflammatory response.10)The astrocyte Kir6.1 knockout aggravated activation of ER stress.11)Autophagy was inhibited in Kir6.1^loxp/loxpGFAP^cre/+ mice after MPTP/p treatment.12)Apoptosis related-proteins including Bax and AIF were significantly up-regulated,while bcl-2 was significantly down-regulated in Kir6.1^loxp/loxpGFAP^cre/+ mice after MPTP/p treatment.13)The depletion of the astrocyte Kir6.1 gene did not affects the m RNA and protein expression of BDNF,GDNF,FGF-2 in midbrain and striatum of two transgenicmice mice.CONCLUSIONS: 1)Astrocyte Kir6.1 knockout exacerbates PD-intoxication protocol induced the loss of DA neurons and aggregates the proliferation and activation of astrocyte and microglia in middle brain.2)Astrocyte Kir6.1 depletion has significant effect on set of cellular stress events and shows neural injury in PD mouse model.3)Kir6.1/K-ATP channel in astrocyte plays a critical role in PD.Part II The role and mechanism of astrocyte Kir6.1/K-ATP channel in regulation of mitochondrial functionsAIM: To investigate the role and mechanism of astrocyte Kir6.1 gene deletion in modulation of mitochondrial homeostasis.METHODS: Primary mesencephalic astrocytes were cultured from new-born Kir6.1^loxp/loxp and Kir6.1^loxp/loxpGFAP^cre/+ mice within 3 days of birth.Two genotype astrocytes were treated with 100?M for 48 h.The supernatants and cells were collected for subsequent experiments.After MPP+ stimulation,we used H2DCF-DA fluorescent probe to analysis the influence of Kir6.1 deficiency on ROS generation in two phenotype primary astrocytes.Immunofluorescence,flow cytometry and Western blotting methods were used to detect the effect of astrocyte Kir6.1 deletion on MPP+ induced apoptosis in two phenotype primary astrocytes.Western blotting was used to detect the expression of BDNF,GDNF and FGF2 in two genotype astrocytes.Western blotting was used to detect the secretion of IL-1?,TNF-?,IL-6.The inflammation signal related proteins?P65,NLRP3,complement C3,caspase-1?in two genotype astrocytes were detected by Western blotting.The mitochondrial mass of two genotype astrocytes was quantified by flow cytometry using the Mito Tracker Green,mitochondrial fluorescence probe.After stimulation with 100?M MPP+,two genotype astrocytes were co-stained with Mito Tracker Green and Mito Deep-red or labeled with JC-1,after then the membrane potential was detected by Immunofluorescence and flow cytometry.Mito SOX was applied to label mitochondrial ROS detected by immunofluorescence and flow cytometry.Two transgenicmice primary astrocytes were co-transfected with GFP-LC3 Plasmid and mito DSRed2 Plasmid,the co-localization of mitochondria and autophagosomes were observed by fluorescence microscope.Mitochondrial proteins of two transgenicmice astrocytes were isolated by mitochondrial separation kit combing with Western blotting to analysis mitophagy related proteins?PINK1/PARKIN?P62?LC3II/I?.In order to identify the distribution of Kir6.1 in primary astrocytes,we used immunofluorescence co-staining of TOM20 and Kir6.1 at 48 h after MPP+ treatment.After treatment two transgenicmice primary astrocytes with different agents,including the mitochondrial K-ATP specific blocker 5-HD,the opener diazoxide,or the autophagy inducer Rapamycin,we detected the change of ROS and membrane potential by Immunofluorescence and flow cytometry.The supernatants of different groups from two transgenicmice astrocytes were then assayed for inflammatory factors using ELISA.After MPP+ administration for 48 h,we collected supernatants of different groups from two transgenicmice astrocytes as conditional medium.Subsequently mixed with neurobasal in portion of 1:2,the conditional medium was added into primary middle brain neurons.TH immunohistochemistry was used to observation the role of kir6.1/K-ATP channel on DA neuronal survival and axon length.RESULTS: 1)Following the treatment with MPP+,a significant increase in the ROS level was recorded in Kir6.1 depletion group.2)Compared with control group,astrocyte Kir6.1 knockout group showed significant activation of MPP+-induced apoptosis.3)Stimulation of two phenotypes cells with MPP+ increase the Astrocyte Kir6.1 knockout group aggravated expression of inflammation related proteins?P65,C3,Caspase-1,IL-1?,TNF-??.4)Following addition of MPP+,compared with control group,the mitochondrial membrane potential of the astrocyte Kir6.1 knockout group significantly decreased.5)Astrocyte Kir6.1 deficiency aggravated MPP+-induced generation of ROS.6)Astrocyte Kir6.1 depletion aggravated MPP+-induced increase of mitochondrial mass.7)Double immunofluresence of Kir6.1 and TOMM20 results proved that Kir6.1 subunit was distributed in the mitochondria.8)After astrocytes from Kir6.1^loxp/loxp mice were pre-incubated with 5-HD and then treated with MPP+,a significant decrease of the mitochondrial membrane potential?MMP?was recorded.But pre-incubating 5-HD and Diazoxide and then treatment with MPP+ could not restore the MMP.Following pre-incubating with 5-HD and then MPP+,a significant increase of the mitochondrial original ROS was detected.But pre-incubating 5-HD and Diazoxide and then MPP+ could not restore the mitochondrial original ROS level.9)Co-transfected with plasmid GFP-LC3 and mito-DSRed2,then treated with MPP+,significant inhibition of the mitophagy was recorded in astrocyte Kir6.1 knockout group by immunofluorescence.9)The cytoplasmic and mitochondrial protein of two transgenicmice astrocytes were isolated and Western blot results showed that the astrocyte Kir6.1 gene deficiency suppressed PINK1/parkin mediated mitophagy.12)Culture astrocytes from Kir6.1^loxp/loxpGFAP^cre/+ mice,following pre-incubating with Rapamycin and then MPP+,a significant recovery in MMP,the mitochondrial original ROS and inflammatory cytokines level were detected.13)When middle brain DA neurons were stimulated with conditional medium from both transgenicmice astrocytes,Kir6.1 deficiency astrocytes group significantly inhibited the number and axon length of TH positive neurons.Pre-incubating with Rapamycin reversed the damage to neurons.CONCLUSIONS: 1)Astrocyte Kir6.1 gene deletion aggravates MPP+ induced the damage of astrocytes and mitochondrial dysfunction.2)Astrocyte Kir6.1 deficiency suppress MPP+ induced mitophage which promote inflammation and exaggerate DA neuron damage.3)Astrocyte Kir6.1/K-ATP channels,especially on mitochondrial,exert important role on regulating mitophagy.The major contributions of the present study lie in:1.Kir6.1/K-ATP channel in astrocyte played an important role in PD.Astrocytes Kir6.1 knockout exacerbated loss of TH positive DA neurons,activation glial cells in PD toxicants induced mouse model.Further studies showed that astrocytes Kir6.1 deficiency significantly decreased DA level and induced multiple intracellular stress events including activation of ER stress,inflammation,apoptotic pathways and inhibition of autophagy.Together these results provide important insights into targeting astrocytes Kir6.1/K-ATP channel in drug discovery for PD therapy.2.Astrocyte Kir6.1/K-ATP channel mediated mitochondrial homeostasis via mitophagy involving in PD process.This study reveals that astrocytes Kir6.1 gene deficiency increase MPP+ induced damage of astrocytes and loss of the mitochondrial function,then exacerbation of the systemic inflammatory response,which can aggravate DA neurons injury.Furthermore,Astrocyte Kir6.1/K-ATP channels,especially on mitochondrial,exert important role on regulating mitophagy involving in regulating the mitochondrial homeostasis of astrocytes.These data can develop neuroprotective agents targeting the astrocytes mitochondrial function for PD.