Mechanism Of PINK1 Loss-Induced Alteration In Glia-Mediated Neuro-Inflammation

Parkinson disease?PD?is a common neurodegenerative disorder caused by a slow but progressive degeneration of dopaminergic neurons in the substantia nigra.Mutations in PTEN-induced kinase 1?PINK1?cause recessive familial PD.PINK1regulates mitochondrial function through its effect on mitochondrial respiration a nd Parkin-dependent mitophagy.Currently,there is no detailed characterization of the effects of PINK1 loss on inflammatory gene expression in glia.To better understand how PINK1 deficiency affects experimentally-induced glia-mediated inflammatory responses and physiological neuroprotective functions of glia,we aim to study the following questions by using primary glia,specifically mixed glia,astrocytes and microglia,isolated from WT and PINK1-deficient mouse brain.Therefore,the overall goal of this project is to study the importance of PINK1 in glial cell physiology with special emphasis on glia-mediated neuroinflammation,and to determine the requirement of PINK1 for the protection against neuronal death caused by experimentally?LPS/IFN-??induced inflammation.The specific experiments were designed to explore whether PINK1 could serve as a future?drug?target for modulation of inflammation in neurodegenerative disorders.Firstly,this study isolate and culture mixed glia from mouse brains and the mixed glia cells were treated with LPS/IFN-?to study the function of PINK1 in inflammation in a population of cells that most closely resembles the cellular context of the brain.Among the pro-inflammatory changes that occurred due to lack of PINK1,increased expression of inducible nitric oxide synthase?iNOS?and cyclooxygenase-2?COX-2?was associated with elevated production of nitric oxide?NO?in response to LPS/IFN-?.Moreover,loss of PINK1 resultes in increased LPS/IFN-?induced activity of the NF-k?signaling pathway,which stemmed mostly from PINK1^-/-astrocytes that accounted for the majority of the cells in mixed glial cultures.However,reduced expression of TNF-?and IL1-?were observed in LPS/IFN-?treated PINK1-deficient mixed glia.Importantly,the combined neuroinflammatory abnormalities render PINK1-deficient mixed glia more toxic towards co-cultured primary wildtype neurons upon treatment with LPS/IFN-?.The enhanced neuronal toxicity depends mainly on increased NO production in LPS/IFN-?treated PINK1-deficient mixed glia.Secondly,this study characterizes the cellular mechanisms responsible for the altered neuroinflammatory profile observed in mixed glia.To tackle this question,it is necessary to analyze astrocytes and microglia separately.These experiments reveal that PINK1 regulated inflammation in a glia type-specific manner.Specifically,loss of PINK1 enhances pro-inflammatory signals in LPS/IFN-?treated astrocytes,thereby increasing NO production and expression of iNOS and COX2 as well as elevated levels of TNF-?and IL1-?.By contrast,lack of PINK1 reduces microglia-mediated inflammation by decreasing the production of TNF-?and IL1-?in LPS/IFN-?treated pure microglia,showing that the same two cytokines are regulated in opposite directions in astrocytes and microglia as a result of PINK1 loss.Importantly,this study also shows that the levels astrocyte-derived TGF-?and microglia-derived IL-10,two critical cytokines that reciprocally regulate the neuroinflammatory response of the other glia type,are changed in directions that can explain the specific inflammatory abnormalities observed in each individual type of glia cell.Finally,to study how PINK1 loss affects?-synuclein induced neuroinflammation,two different forms of?-synuclein?purified?-synuclein monomer and preformed?-synuclein fibrils,or?S-PFFs?were used in this project for induction of inflammation response in glia.Neither?-synuclein nor IFN-?alone induces measurable concentrations of NO.However,when?S-PFFs were combined with IFN-???S-PFFs/IFN-??,they induce significantly more NO production in PINK1^-/-than in WT mixed glia.This is consistent with increased expression of iNOS in PINK1-deficient mixed glia under the same conditions.In contrast,expression of COX-2,IL-6,TNF-?and IL1-?are similar in?S-PFFs/IFN-?treated WT and PINK1^-/-mixed glia.Although PINK1-deficient mixed glia produces more NO than WT mixed glia in response to treatment with?S-PFF/IFN-?,this difference is not apparent when pure microglia were analyzed.Therefore,the increased NO production in PINK1^-/-mixed glia is indirect through astrocytes present in mixed glia,possibly through similar mechanisms as those observed after treatment of mixed glia with LPS/IFN-??see above?.Instead,loss of PINK1 attenuates the inflammatory phenotype of microglia in response to?S-PFF/IFN-?,as shown by reduced expression of the pro-inflammatory cytokines IL-6,TNF-?and IL1-?.In summary,this study suggests that loss of PINK1 resulted in abnormal innate immune responses in glia?mixed glia,astrocytes and microglia?,mainly by increasing the pro-inflammatory phenotype of astrocytes in mixed glia,while attenuating inflammation by microglia.Neuron death is mediated by enhanced astrocyte-derived NO production in mixed glia lacking PINK1.These results from this project provide new insights into the mechanisms underlying the pathogenesis of PD.Specifically,in addition to previously reported mitochondrial dysfunctio n in PINK1 deficiency,glia-mediated and NO-dependent neuroinflammation have been indentified as an additional mechanism that may contribute to dopamine neuron loss in PINK1-related familial Parkinsonism.