Molecular Mechanisms Of REEP1-mediated Neurodegeneration

BackgroundAmyotrophic lateral sclerosis(ALS)is one of the most common motor neuron diseases characterized by progressive degeneration of motor neurons(MNs)in the brainstem and spinal cord.The molecular mechanisms of MN loss in ALS remain unknown.So far,there is no effective treatment for ALS.Many ALS-related proteins,including SOD1,TDP-43,FUS,and C9ORF72,are closely associated with mitochondrial dysfunction.Evidence collected from in vitro and in vivo disease models and ALS patients suggests that mitochondrial dysfunction play a central role in the disease progression of ALS.Mitochondrial dysfunction and reduced ATP levels are prevalent in ALS and animal models of ALS.The activity of mitochondrial oxidative phosphorylation complexes(OXPHOS)Ⅰ,Ⅱ,Ⅲ,and Ⅳ was significantly reduced in the spinal cord of ALS patients and in SOD1G93A(G93A)mice.However,the mechanisms underlying mitochondrial dysfunction in ALS remain elusive.Receptor expression-enhancing protein l(REEP1)is closely related to another motor neuron disease,hereditary spastic paraplegia(HSP).It is a member of the REEP family,localized on the tubular endoplasmic reticulum membrane and regulates endoplasmic reticulum morphology by binding to tubulin.And it is also located on mitochondria.It is unknown whether there is a link between members of the REEP family and other motor neuron diseases.Other groups have found that HSP caused by REEP1 mutation can cause mitochondrial dysfunction,which is closely related to ALS.This study aims to clarify the association between REEP1 and ALS,as well as the function of REEP1 localization on mitochondria.Objectives1.To explore the expression levels of REEP family proteins in ALS patients and ALS-related animal models.2.To study the effect of increasing REEP1 protein expression on motor impairment and survival of G93 A mice.3.To study the effect of increasing REEP1 protein expression on motor neuron death and neuroinflammation of G93 A mice.4.To investigate REEP1 deletion on mitochondria distribution and function.5.To explore the mechanism of REEP1 regulating mitochondrial function.6.To examine whether REEP1 protects motor neuron survival by enhancing mitochondrial function.Methods1.Study the expression level changes of REEPs in ALS patients and ALS mice Western blot and immunohistochemical staining methods were used to detect the expression levels of REEP family proteins in the spinal cord of age-matched control and ALS patients,as well as NTG and G93 A transgenic mice of different ages.2.Study the effect of REEP1 overexpression on neurodegeneration in G93A mice G93A mice were injected with AAV1-GFP or AAV 1-hREEP1-Flag at the L1 segment of spinal cord by stereotaxic injection.Animal behavioral testing was performed to investage the motor function of these mice.Meanwhile,the lower limb muscle function of mice was studied by electrophysiology and muscle anatomy.The number of neuromuscular junctions,the level of neuroinflammation and the death of motor neurons in the spinal cord of mice were detected by immunofluorescence staining.3.To explore whether loss of REEP1 affects mitochondrial distribuation and function Using the CRISPR/Cas9 method,REEP1 was knocked out in Lenti-X 293T and SH-SY5Y cells.In order to analyze mitochondrial function.We tested the mitochondrial membrane potential and ATP levels in these cells.Also analyzed mitochondrial distribution by immunofluorescence staining with Tom20 antibody.To study the assembly of CIV,we performed Blue-native PAGE electrophoresis and immunoblot analysis.Finally,the activity of mitochondrial CIV in WT and REEP1KO cells was determined.4.Study the molecular mechanism by which REEP1 regulates mitochondrial function We first identify REEP 1-interacting proteins through quantitative mass spectrometry analysis and co-immunoprecipitation.Identified the domains of REEP 1 interacting with mitochondrial CIV subunit NDUFA4.Using lenti-viral over-expression system,we re-overexpressed REEP1 wild type and mutations in Lenti-X 293T REEP1KO and SH-SY5Y REEP1KO cells.Immunofluorescent staining,mitochondrial membrane potential test,ATP measurement,bluenative PAGE electrophoresis and CIV activity assays were used to study the effects of different REEP1 on mitochondrial function.5.The relationship between REEP1 and NDUFA4 protein levels Using western blot analysis,we detected the levels of NDUFA4 in ALS patients and ALS mice.In wild type and REEP1 knockout cells,mRNA and protein levels of NDUFA4 were analyzed by the real-time PCR and immunoblot.6.Effects of REEP1 deletion on neuronal morphology and viabilitySH-SY5Y was differentiated to neurons.Cell count and MTT assay were used to analyze the survival of neurons.Immunofluorescent staining was used to study the length as well as the number of neuronal dendrites.To study the effect of REEP1 on neuronal survival under stress conditions,we treated neurons with tunicamycin and hydrogen peroxide to induce endoplasmic reticulum stress and oxidative stress.Results1.Reduced expression of REEP1 in ALS patients and G93A miceThe expression level of REEP1 was significantly decreased in spinal cord samples from ALS patients.However,the expression of other REEP family members,such as REEP2,REEP3,REEP5,was unchanged.The expression of REEP 1 in the spinal cord of G93 A transgenic mice for ALS was also significantly decreased,which was in an age-dependent manner.2.Forced expression of REEP1 abolishes skeletal muscle atrophy in G93A miceOverexpression of REEP 1 in the spinal cord neurons of G93A mice extended its lifespan.Meanwhile,the motor performance of G93A mice was also greatly improved by upregulation of REEP 1 expression.In addition,upregulation of REEP 1 partially restored neuromuscular junction denervation and motor neuron death.The proliferation of microglial cells and astrogliosis in the spinal cord of G93A/REEP1 mice was significantly reduced compared with that of G93 A/GFP mice.3.REEP1 depletion impairs mitochondrial function40%of REEP1KO cells demonstrated pre-nuclear aggregated mitochondria,while mitochondria were distributed evenly in wild type cells.Mitochondrial membrane potential and intracellular ATP levels were significantly reduced in REEP1KO cells compared with WT cells.Blue native electrophoresis and activity analysis revealed abnormal assembly and reduced activity of CIV in REEP1KO cells.4.REEP1 binds CIV subunit NDUFA4 and regulate mitochondrial functionWe found 101-110 amino acids of REEP1 are required for its binding to NDUFA4.Deletion of 101-110 amino acids in REEP1 could not rescue mitochondrial dysfunction caused by REEP1 deletion.5.REEP1 stabilizes NDUFA4Immunoblot analysis showed that NDUFA4 expression was greatly reduced in the spinal cord of ALS patients compared with age-matched healthy individuals.Similarly,the protein level of NDUFA4 in the spinal cord of G93A mice and REEP1KO cells were also significantly reduced.6.Loss of REEP1 in neurons induces mitochondrial CIV dysfunctionNDUFA4 expression and CIV deficiency were consistently observed in REEP1KO neurons.Although loss of REEP1 did not affect neuronal survival,REEP1KO neurons exhibited shorter dendrites compared to WT neurons.Under stress condition,REEP1 deletion resulted in increased sensitivity of neurons to oxidative stress and ER stress.ConclusionHere we report that REEP1 acts as an important regulator of CIV assembly,which is pivotal to preserve motor neurons in G93A mice.The REEP1 reduction observed in ALS and ALS mouse models is an important contributor to the impairment of mitochondrial morphology and function in ALS.The REEP1-NDUFA4 pathway may be a potential novel therapeutic target for ALS and other motor neuron diseases,which needs further validation in other disease models.