Intermittent Theta Burst Stimulation Attenuates Mitochondrial Dysfunction In The Brain Of APP/PS1 Mice And Its Underlying Mechanisms

BackgroundAlzheimer’s disease（AD）,characterized by progressive impairment of multi-cognitive domains,is deemed as a globally public health problem to be addressed principally due to its high incidence and lack of effective therapy.Currently,clinical trials about therapeutic strategies targeting the major pathogenic substances such asβ-amyloid（Aβ）have always been frustrated.Thus,it is imperative to explore alternative therapies to halt the early neurodegeneration of AD.A growing body of evidence suggests that mitochondrial dysfunction is likely a vital target for the therapy of AD.Importantly,certain mitochondria-related cellular physiological processes can be modulated by magnetic fields.Intermittent theta burst stimulation（iTBS）,a non-invasive brain stimulation technique,regulates the activity of neural network through pulsed magnetic fields.Clinical studies have shown that iTBS facilitates multi-cognitive domains in the AD patients,suggesting that it may be a potential non-drug therapy for AD.Iron-sulfur cluster assembly 1（ISCA1）belongs to the evolutionarily conserved A-type proteins family and plays an important role in the maintenance of optimal mitochondrial function.Previous studies suggest that ISCA1 can respond to magnetic stimulation,suggesting that it may be an important target for magnetic modulation of mitochondria.Thus,the present study aims to explore whether iTBS modulates mitochondrial function to attenuate cognitive deficits and related pathologies in AD brain,as well as the role of ISCA1 in iTBS-induced mitochondrial modulation in AD.Materials and methods1.Effects of iTBS on cognitive function and pathologies in AD mice（1）Effects of iTBS on cognition and behaviors:6-month-old APP/PS1 mice（Tg）and C57BL/6J mice（Wt）were used and divided into three groups:Wt with sham stimulation,Tg with sham stimulation and Tg with iTBS treatment.The mice were administrated with one session iTBS per day for 21 consecutive days.At the age of 7 months,Morris water maze test,open field test and Y maze test were used to measure the cognitive behaviors of mice.（2）Effects of iTBS on the AD-related pathologies:After behavioral tests,the brains of mice were collected.Histological staining,western blotting and ELISA were used to detect the pathological changes in the brain of AD mice,including Aβdeposition,Tau phosphorylation,neurodegeneration,synaptic plasticity and neuroinflammation.2.Effects of iTBS on mitochondrial function and neuronal activity in AD mice（1）Effects of iTBS on mitochondrial functions in AD:¹⁸F-FDG PET was used to detect glucose metabolism in the brain of mice,and immunohistochemistry,western blotting and biochemical analysis were used to measure oxidative stress damage and mitochondrial dynamics.（2）Effects of iTBS on the neuronal activity in AD:The electrophysiology test was used to detect neuronal excitability.At the age of 7 months,the mice were decapitated directly,and300μm brain slices were prepared.The whole-cell mode was used to record the firing pattern of cortical neurons.Neu N and c-FOS immunofluorescent co-staining was used to detect neuronal activity in the brain of mice.3.The role of ISCA1 in the modulation of mitochondrial function by iTBS in AD（1）Effects of iTBS on ISCA1 and mitochondrial iron-sulfur cluster assembly:The SH-SY5Y cells carrying human APP gene（SH-SY5Y-APP695）were subjected to sham stimulation or various pulses of stimulation（150,300 and 600）for 5 days,and western blotting was used to detect the expression of ISCA1.In animal studies,6-month-old APP/PS1mice were divided into sham stimulation group and iTBS group.The mice were administrated with one session iTBS per day for 21 consecutive days.Western blotting was used to detect the levels of ISCA1,late iron sulfur cluster assembly-related proteins and mitochondrial iron sulfur proteins.（2）The impact of ISCA1 depletion on iTBS-induced mitochondrial modulation:The ISCA1-deficient SH-SY5Y-APP695 cell model was constructed through si RNA-induced ISCA1 knockdown in SH-SY5Y-APP695 cell.The cells were divided into four groups:si NC sham,si NC iTBS,si ISCA1 sham and si ISCA1 iTBS.The cells were administrated with one session iTBS per day for 5 consecutive days in vitro.The levels of late iron-sulfur cluster assembly-related proteins and mitochondrial iron-sulfur proteins were measured using western blotting.The mitochondrial respiration and membrane potentials were detected using Seahorse analysis and JC-1 analysis（flow cytometry）,respectively.The mitochondrial dynamics was measured using mitochondrial staining.（3）The impact of ISCA1 depletion on iTBS-induced anti-Aβeffect:The SH-SY5Y-APP695 cells and ISCA1-deficient SH-SY5Y-APP695 cells were divided into four groups:si NC sham,si NC iTBS,si ISCA1 sham and si ISCA1 iTBS.The cells were treated with one session iTBS or sham stimulation per day for 5 consecutive days in vitro,and Aβ40and Aβ42 levels were measured using ELISA.Results1.Effects of iTBS on cognitive function and pathologies in AD mice（1）iTBS improved the cognitive impairment in AD mice:Compared with the Tg sham group,the cognitive and behavioral deficits of AD mice were significantly reduced by iTBS treatment as reflected by better behavioral performance in Morris water maze test,open field test and Y maze test.（2）iTBS alleviated AD-related pathologies:Compared with Tg sham group,the Aβpathologies in the brain of AD mice were significantly reduced by iTBS treatment,accompanied by improvement in other pathological changes,including Tau phosphorylation,neurodegeneration,synaptic plasticity and neuroinflammation.2.Effects of iTBS on mitochondrial function and neuronal activity in AD mice（1）iTBS attenuated mitochondrial dysfunction in AD:Compared with Wt mice,the7-month-old AD mice displayed a significant decrease in glucose uptake in multiple brain regions.Importantly,the glucose uptake was significantly restored by iTBS treatment in the brain of AD mice.In addition,iTBS alleviated oxidative stress damage in the brain of AD mice as reflected by the reduction of protein peroxide（3-NT）and lipid peroxide（MDA）and the enhancement of antioxidant system（SOD,GSH and T-AOC）.Finally,iTBS restored the dynamic balance of mitochondria in AD mice,which was manifested by increased expression of mitochondrial fusion proteins and decreased mitochondrial fission proteins.（2）iTBS enhanced the neuronal activity in AD brain:Compared with Wt mice,the excitability of cortical neurons in 7-month-old AD mice were significantly decreased,and iTBS treatment significantly inhibited the hypoexcitability of cortical neurons.In addition,iTBS also inhibited the downregulation of c-FOS expression in hippocampal and cortical neurons of AD mice,thereby increasing neuronal activity.3.The role of ISCA1 in the modulation of mitochondrial function by iTBS in AD（1）iTBS regulated ISCA1 and mitochondrial iron-sulfur cluster assembly:Compared with Wt mice,the expression of ISCA1 in the brain of AD mice was significantly decreased.In SH-SY5Y-APP695 cells,the expression of ISCA1 increased gradually with the increase of iTBS pulses.Moreover,the expression of ISCA1 was upregulated by iTBS treatment in the brain of AD mice.Additionally,iTBS upregulated the levels of late iron-sulfur cluster assembly-related proteins and mitochondrial iron-sulfur proteins in the brain of AD mice.（2）ISCA1 depletion abolished iTBS-induced mitochondrial modulation:Consistent with animal studies,iTBS elevated the levels of ISCA1,late iron-sulfur cluster assembly-related proteins and mitochondrial iron-sulfur proteins in SH-SY5Y-APP695 cells.Interestingly,ISCA1 knockdown eliminated the regulatory effects of iTBS on the iron-sulfur cluster assembly pathway.Moreover,iTBS increased the mitochondrial respiration capacity,mitochondrial membrane potentials and mitochondrial fusion in SH-SY5Y-APP695 cells.These above effects of iTBS disappeared when the expression of ISCA1 was interfered by si RNA.（3）ISCA1 depletion abolished the inhibitory effect of iTBS on Aβ:iTBS treatment decreased the levels of Aβ40 and Aβ42 in the lysates of SH-SY5Y-APP695 cells.Moreover,ISCA1 knockdown eliminated the inhibitory effect of iTBS on Aβ.ConclusioniTBS may alleviate cognitive deficits and AD-type pathologies through improving mitochondrial dysfunction in the brain of AD.ISCA1 is involved in the process of iTBS-induced mitochondrial modulation,and is a potentially therapeutic target for transcranial magnetic stimulation in AD.