NRF2 Contributes To The Protection Of Parkinson-like Symptoms In Mice After TBI

IntroductionParkinson’s disease(PD)is a common neurodegenerative disease worldwide that is typical charactered by motor dysfunction,which mainly occurs in the elderly.As the progressive degeneration of dopaminergic neurons(DA)in the substantia nigra pars compacta(SNpc),patients with PD have severe symptoms.In the early stages of disease development,patients may have decreased sense of smell,decreased sleep,anxiety,depression and other nonmotor symptoms.When substantial dopaminergic neuron loss occurs in the substantia nigra,the patient appears static tremor,slow movement,myotonia and other motor symptoms.As the disease progresses further,patients begin to develop gait disturbances,postural instability,autonomic dysfunction,dysphagia,dementia,and eventually death.However,there is no clear explanation for the cause of PD.Traumatic brain injury(TBI)is defined as temporary or permanent structural and functional impairment of the brain,caused by an external force.In the epidemiology,TBI has become a social concern mainly owing to high incidence and prevalence.Currently,human studies have shown that TBI is one of the risk factors for PD.Nrf2(nuclear factor erythroid 2 related factor 2),a key transcription,plays a regulator role in cellular antioxidant response.Under the physiological condition,Nrf2 resides in cytosol and binds to the cytoplasmic protein companion molecule Keap1(Kelch-like ECH associated protein1)to form a stable complex,which is degraded by ubiquitination and proteolysis.Under the condition of oxidative stress or other toxins,Nrf2 is decoupled with Keap1 by phosphorylation and translocated to the nucleus,where it binds to small Maf proteins to form heterodimer.Antioxidant response element(ARE)sequences are recognized by heterodimers to induce target gene expression of glutathione peroxidase(GSH-Px),heme oxygenase1(HO-1)and NAD(P)H:quinine oxidoreductase1(NQO1),which can activate pathways including REDOX regulation,inflammatory response,apoptosis,and mitochondrial function.Studies have shown that Nrf2 plays an important role in brain injury and neurodegenerative diseases.To establish chronic TBI models by using wild-type mice in different postinjured intervals,and to explore the pathophysiological changes of Parkinsonlike behavior.To establish a chronic TBI model using wild-type mice and Nrf2knockout mice,and to explore the effect of Nrf2-knockout on Parkinson-like behavior in mice.To establish a mouse TBI model of Nrf2 overexpression by intraperitoneal administration of dimethyl fumarate(DMF).The TBI model of Nrf2 overexpression mice and wild-type mice to explore the protective effect of Nrf2 overexpression on Parkinson-like behavior in chronic TBI mice.Materials and MethodsTwelve-week-old healthy male C57BL/6J(WT)and Nrf2-knockout(Nrf2-KO)mice(18-22g)were used in our study.Controlled cortical impact(CCI)models were applied to induce a moderate traumatic brain injuryPart Ⅰ:WT mice were randomly divided into Sham group and TBI group.After behavioral tests at 0 month post injury(mpi),1 mpi,3mpi and 6mpi,the mice were sacrificed by excessive inhalation of isoflurane and brain tissue was taken from the mice in all groups.H&E and Nissl staining were applied to detect the changes of substantia nigra cells after injury.Terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)was used to detect the apoptosis of dopaminergic neurons.The protein expression of tyrosine hydroxylase(TH)and 4-HNE at different time after TBI was detected by Western blot and immunofluorescence staining.The expression of a-synapse nucleoprotein(asyn)and TDP-43 in dopaminergic neurons was detected by immunofluorescence staining.The mRNA expression of Nrf2 downstream genes(Hmox-1,Nqo1,Gclc and Gclm)was detected by qPCR.Oxidative stress was detected by ELISA(SOD and MDA levels were determined).The protein and mRNA expressions of inflammatory cytokines(IL-1β,IL-6,TNF-α)were detected by ELISA and qPCR.The expression of glial cells was detected by Western blot and immunofluorescence staining.Part Ⅱ:All mice were randomly divided into following groups:WT-Sham,WT-TBI,Nrf2-KO-Sham,Nrf2-KO-TBI,WT-TBI+Veh and WT-TBI+DMF.WTSham group and WT-TBI group mice were sacrificed by excessive inhalation of isoflurane at 1 days post injury(dpi),7dpi,14dpi and 1 months post injury(mpi),and brain tissue was taken.After behavioral tests at 6mpi,the mice were sacrificed by excessive inhalation of isoflurane and brain tissue was taken from the mice in all groups.The protein expression of NRF2 at different time after TBI was detected by Western blot and immunofluorescence staining.Exercise ability of mice was measured by muscle strength test,open field test and rotating test.The expression of a-syn in dopaminergic neurons was detected by immunofluorescence staining.The mRNA expression of Nrf2 downstream gene(Hmox-1,Nqo1)was detected by qPCR.Oxidative stress was detected by ELISA(SOD and MDA levels were determined).The protein and mRNA expressions of inflammatory cytokines(IL-1 β,IL-6,TNF-α)were detected by ELISA and qPCR.The expression of glial cells was detected by Western blot and immunofluorescence staining.ResultsPart Ⅰ:The Parkinson-like symptoms were observed in the mice on the chronic TBI models,including depression,impaired motor ability,impaired balance and abnormal gait.Dopaminergic neurons in the SN were apoptotic,and the number of neurons was progressively reduced after TBI.The deposition of a-syn on neurons was increased progressively.TDP-43 was mislocated in the cytoplasm at 6mpi.Oxidative stress and neuroinflammatory reactions were observed in the SN.The activation and morphological complexity of astrocytes peaked at 3mpi,the activation of microglia after TBI peaked at 6mpi,and microglia differentiated in the pro-inflammatory(M1)direction.Part Ⅱ:In the WT groups,we observed that NRF2 expression was increased at 1dpi,7dpi,14dpi and 1mpi.In dopaminergic neurons,NRF2 was detected in the nucleus at 1dpi,7dpi and 14dpi after injury.The proportion of NRF2 in the nucleus gradually decreased,and the expression of NRF2 was mainly detected in the cytoplasm at 1 mpi.Compared with the WT group,Nrf2 knockout resulted in severe PD-like behavior.Muscle strength and balance ability were decreased in the Nrf2-KOTBI group.Our study showed that the degree of apoptosis of dopaminergic neurons was aggravated,the number of neurons was significantly decreased,and a-syn deposition was increased in dopaminergic neurons after Nrf2 knockout.Moreover,oxidative stress and neuroinflammation were shown as dramatic change.The activation and morphological complexity of astrocytes were significantly increased.The activation of microglia cells was significantly increased,presenting amoeba-like changes.In the WT-TBI+DMF group,the mice showed improvements in motor ability,muscle strength and balance.The number of neurons increased,a-syn deposition on neurons decreased.Oxidative stress and neuroinflammation were alleviated,and activation of astrocytes and microglia cells was reduced.Conclusion1、PD-like behavior is associated with massive dopaminergic neuronal death and abnormal protein deposition in the chronic TBI mice,which due to persistent oxidative stress and neuroinflammation.2、Activation of Nrf2 pathway occurred only in acute and subacute TBI mice.3、NRF2 has a neuroprotective effect on PD-like behavior in chronic TBI mice.The application of DMF can relieve neuroinflammation and improve the damage of dopaminergic neurons effectively.