TrkB Receptor Agonist R13 Ameliorated The Behavioral Ability Of ALS And AD Mice Model Via Improved Mitochondrial Function

Background:Neurodegenerative diseases are characterized by progressive loss of neurons,which including amyotrophic lateral sclerosis(Amyotrophic lateral sclerosis,ALS),Alzheimer's disease(Alzheimer's disease,AD)and Parkinson's disease(Parkinson's disease,PD),etc.ALS,which is one of motor neuron disease,is characterized by the degeneration of both upper and lower motoneurons,muscle weakness and paralysis,finally death.ALS is a rare disease with a global incidence of 3 per100,000.ALS patients usually die of respiratory failure or pneumonia within 2 to 5 years after diagnosis.Although some ALS patients carry a variety of gene mutations related to neuronal function,the pathogenesis of most ALS patients is still unclear.The treatment of ALS patients is symptomatic therapy,such as the use of muscle relaxants for spasticity and speech therapy for speech disorders.Known as Alzheimer's disease commonly,AD's main pathological features are senile plaques formed by?-amyloid protein deposition and neurofibrillary tangles formed by hyperphosphorylation of tau protein.The main clinical manifestations of AD are memory impairment,loss of learning ability,personality and behavior changes and so on.With the aging of the population,the incidence of AD is gradually increasing,and it has become the fifth leading cause of death in our country.The drugs used in clinical treatment of ALS and AD can only alleviate the symptoms of the disease,but can not achieve the cure effect.Mitochondria are organelles for energy supply,and their dysfunction is closely related to the onset of ALS and AD.Prevoius studies on animal models of ALS have shown that the oligomer of mutant SOD1 binds to mitochondria,resulting in a decrease in the activity of the complex of the mitochondrial electron transport chain.In ALS patients and animal models,defects in respiratory chain function are associated with mitochondrial protein oxidative damage and lipid peroxidation.At the same time,the imbalance of energy metabolism may also lead to motor neuron dysfunction in ALS.In AD patients,ATP levels and mitochondrial biogenesis are significantly decreased.The protein expression and activity of mitochondrial complex were decreased in patients with mild cognitive impairment.The loss of mitochondrial DNA is related to the decrease of mitochondrial complex activity.Brain-derived neurotrophic factor(brain-derivedneurotrophicfactor,BDNF)plays key roles in the survival,maintenance and regeneration of specific groups of neurons in the adult brain,and regulating neurogenesis,hippocampal synaptic plasticity and learning and memory.BDNF promotes mitochondrial biogenesis and oxidative metabolism by enhancing the expression of PGC-1?,thereby increasing mitochondrial mass and activity to maintain muscle function and delay muscle atrophy.BDNF increases glucose utilization by cultured cortical neurons,enhances mitochondrial energy production and promotes synaptic plasticity.BDNF exerts its biological function mainly by binding low-affinity p75^NTR receptors and high-affinity TrkB receptors.BDNF/TrkB receptor signaling occurs through three main tyrosine kinase-mediated pathways:MAPK-ERK pathway,PI3K-AKT pathway and PLC?1-PKC pathway.It has been reported that recombinant BDNF protein is used to treat ALS and AD.However,its application is greatly limited because of its short half-life and difficulty passing through the blood-brain barrier(BBB).In the present study,we explore the effect of a TrkB receptor agonist R13 on ALS animal model SOD1^G93A mice and AD animal model 5×FAD mice.and provide a new candidate drug for the treatment of ALS and AD.Objective:To explore the improvement effects of the TrkB receptor agonist R13 on ALS animal model SOD1^G93A mice and AD animal model 5×FAD mice and its molecular mechanismMethods:Intragastric administration of 40-day-old SOD1^G93A mice with R13 for 80 days,the exercise ability of mice was analysed by behavior test(pole climbing,rotarod test,grip and suspension).Mitochondrial related proteins were detected by western blots.The effect of R13treatment on the pathology of gastrocnemius muscle or spinal cord in the SOD1^G93A mice was detected by hematoxylin-eosin staining or immunohistochemistry,respectively.ATP and MDA kits were used to detect the levels of ATP and MDA in the spinal cord and medulla oblongata.The proteomics platform of our laboratory was used to test the effect of R13 on the protein expression profile in the spinal cord and medulla oblongata of mice.The ability of learning and memory of5-month-old 5×FAD mice was tested by morris water maze after continued oral administration of R13 for 3 months.Mitochondrial related proteins were detected by western blots.The pathology of A?in the 5×FAD mice was detected by immunohistochemistry and dot blot hybridization.Proteomics was used to detect the effects of R13 on mitochondrial related protein expression profile in the 5×FAD mice.Results:1 The improvement effects of R13 on SOD1^G93Amice:?R13 treatment enhanced the exercise balance ability of SOD1^G93A mouse detected by rod climbing and rotating rod test.?R13 activated TrkB receptor and its downstream signal proteins p-Akt and p-AMPKa in the medulla oblongata of SOD1^G93A mice.?R13 promoted the expression of mitochondrial biogenesis-related protein PGC-1?in medulla and spinal cord tissues of SOD1^G93A mice,and also increased the expression levels of mitochondrial dynamic proteins Drp1 and Fis1.R13 treatment promoted the expression level of OXPHOS-related proteins including Uqcrfs1,ATP5a,Cytc SDHB and Cox5a.The ATP production and lipid peroxidation levels in the ALS mice were reversed by R13 treatment.?R13treatment alleviated the pathological changes in ALS mice including gastrocnemius fibrosis,spinal cord motor neuron loss,spinal cord astrocytes and microglia proliferation.?Proteomics revealed the differentially expressed protein between the model group and the R13 treatment group in spinal cord tissue and medulla oblongata.These proteins were divided into three categories by cluster analysis,which mainly related to muscle contraction,electron transport chain,gliosis,etc.?In the mitochondrial differential protein expression profile of spinal cord and medulla oblongata,we found that R13 restored the abnormally increased or decreased mitochondrial protein expression in ALS mice.2 The improvement effects of R13 on 5×FAD mice:?The escape latency of 5×FAD mice were descreased by R13 treatment.?R13 activated TrkB receptor in the hippocampus,and promoted the TrkB receptor downstream signal p-Akt and p-ERK protein expression levels.?R13 increased the level of ATP in the hippocampus of 5×FAD mice,and promoted OXPHOS related proteins including complex I(NDUFA10),complex II(SDHB),complex III(UQCRFS1)and complex IV(COX5B)expression level.?The relative copy number of mitochondrial DNA(mt DNA)in hippocampus of 5×FAD mice was increased by R13 treatment.R13 promoted the expression of mitochondrial-related proteins Cyt c,PDH and VDAC1.Moreover,R13 increased mitochondrial biogenesis-related proteins(p-AMPK?,AMPK,p-CERB,CREB,PGC-1?,NRF1 and TFAM)expression level.?R13 treatment reduced the level of A?amyloid in the prefrontal cortex of5×FAD mice and the phosphorylation level of tau in the hippocampus(p S404 and p S396).?The139 differentially expressed proteins located in mitochondria in the hippocampus were detected by Proteomics.R13 reversed the abnormally expressed mitochondrial-related proteins in 5×FAD mice.MCODE analysis found that these differential proteins are mainly related to the OXPHOS module.Conclusion:(1)R13 improved mitochondrial function and pathological changes of SOD1^G93Amice by activating TrkB receptor,thus improving motor ability of SOD1^G93A mice.(2)R13 treatment alleviated learning and memory impairments in 5×FAD mice by improving mitochondrial function and reducing A?deposition and tau phosphorylation.