| 【Background】 Hereditary spastic paraplegia(HSP)is a group of central nervous system degenerative monogenic diseases with high clinical and genetic heterogeneity.Progressive spastic paraplegia and myasthenia in the lower extremities are main clinical features of HSP patients.HSP can be clinically classified into two types based upon whether it is associated with additional neurological or extra-neurological signs: pure HSP and complicated HSP.Single gene mutation is the main cause of HSP.By far,88 pathogenic genes and more than 100 pathogenic mutations of HSP have been identified.In our previous study,a new homozygous missense mutation of FARS2,c.424G>T(p.D142Y),was identified in a Chinese consanguineous family as a pathogenic mutation of pure HSP.And for the first time,FARS2 was confirmed to be a pathogenic gene of HSP.The mutation was then included in the Online Mendelian Inheritance in Man(OMIM)database and named as SPG77 FARS2 encodes mitochondrial phenylalanyl tRNA synthetase(mt-Phe RS),which is a member of the mitochondrial aminoacyl-tRNA synthetase(mt-aa RS)family.The classic function of mt-aa RS is to charge 20 amino acids to their homologous mitochondrial tRNA molecules,and promote mitochondrial protein translation.Mutations of other family members including DARS2(encoding mitochondrial aspartyl-tRNA synthetase 2),EARS2(encoding mitochondrial glutamyl-tRNA synthetase 2),MARS2(encoding mitochondrial methionyl-tRNA synthetase 2)and RARS2(encoding mitochondrial arginyl-tRNA synthetase 2)genes have been reported to cause human mitochondrial diseases.Such patients often have significant central nervous system lesions,while other organs or systems like heart,kidney and bone can also be involved.While current studies show that FARS2 variations only relate to neuropathy in patients.Infantile-onset epileptic encephalopathy,later-onset spastic paraplegia and juvenile onset refractory epilepsy are three main phenotypes exhibited by patients harboring FARS2 mutations.Somatic biochemical indicators in FARS2 mutant patients generally indicated abnormal levels of oxidative phosphorylation.What’s more,a large number of in vitro experiments of FARS2 pathogenic mutations and enzymic tests of patient-derived cells showed reduced or completely disappeared mt-Phe RS aminoacylation activity,suggesting that loss of enzymatic function caused by FARS2 mutation might be the main cause of human diseases.However,the underlying pathogenesis of neuropathy-associated Fars2 deficiency remains unclear and needs further study.【Purpose】 We aim to mimic the pathophysiology of FARS2 patients by establishing both in vivo andin vitro models with Fars2 deficiency;by using the established models,we aim to studywhether Fars2 deficiency leads to neurodegenerative phenotypes by inducing mitochondrialdysfunction,and how Fars2 deficiency leads to mitochondrial dysfunction;finally,we aimto explore the effect of Fars2 deficiency on downstream pathways during neuronaldevelopment.【Methods】 1.Global Fars2 knockout and Fars2 c.424G>T(p.D142Y)mutant mouse models were established by using CRISPR/Cas9.Homozygous embryos at early developmental stages were obtained by time-mating,and morphological characteristics of homozygous embryos were observed by using a stereomicroscope or by HE staining;q-PCR was used to identify the expression pattern of markers in three germ layers.2.Conditional Fars2 knockout mouse model was established by using CRISPR/Cas9.By mating with Nestin-Cre mouse,neuron-specific Fars2 knockout(c KO)mouse was obtained.The structural changes of embryo brain were detected by Nissl staining and immunofluorescent staining.TUNEL staining and Western blotting were used for investigating apoptosis signaling in the tissue.3.Morpholinos were injected into fertilized zebrafish ova of hb9-EGFP transgenic line to obtain fars2 knockdown zebrafish.Morphological phenotypes were observed under the microscope;trajectory tracking and stereotypic escape response were recorded to evaluate behavioral changes.4.In vitro experiments were carried out by transferring Fars2-targeted shRNA lentivirus into primary cultured mouse neurons.Immunofluorescent staining was used to observe the outgrowth of neurites;TUNEL staining and Western blotting were used to detect apoptosis cells.5.Northern blotting was used to evaluate the aminoacylation state of tRNAPhe in c KO mouse cortical tissue,Western blotting was carried out to detect the expression levels of mitochondrial complexes and mitochondrial genome encoded protein subunits;qPCR was carried out to measure mitochondrial mRNA levels.6.Mitochondrial membrane potential,ATP,ROS and NAD+ level of c KO mouse cortex,Fars2 knockdown neurons and PC12 cell lines were measured to reveal mitochondrial function;ultrastructure changes of mitochondria were observed under a transmission electron microscope;the level of mitochondrial aerobic respiration was measured in PC12 cells by seahorse assay,and cell apoptosis was detected by Annexin V/ PI.7.Transcriptomic sequencing was performed at various developmental stages in Fars2 knockdown neurons to enrich differentially expressed genes and analyze the downstream pathways and molecules.【Results】 1.The development of global Fars2 knockout mouse and p.D142 Y mutant mouse embryos stopped at very early stage and before neurogenesis.2.Neural-specific Fars2 knockout mouse model died at late stage of embryonic development.Thinner cortex and enlarged ventricle induced by neuron apoptosis were observed in c KO mouse brain which was consist with the MRI results revealing cortical atrophy and reduced cerebral white matter volume in human patients.3.fars2 knockdown zebrafish model showed developmental malformation,shortened motor neuron axon and disordered neurite arborization;reduced locomotor capacity and less responsive to stimuli were also observed in fars2 knockdown zebrafish which restored the spastic paraplegia phenotype in human patients.4.Fars2 knockdown neurons showed delayed and disrupted development of neurite outgrowth and neuron apoptosis that in accord with the phenotype in animal models.5.The interrupted Fars2 function disturbs the aminoacylation process of mt-tRNAPhe and mitochondrial protein synthesis in Fars2 deficient organisms,and thus resulted in the reduced levels of mitochondrial respiratory chain complex I,III and IV.6.Reduced ATP and NAD+ level among with increased ROS production,reduced mitochondrial membrane potential and oxygen consumption,with increased number of apoptotic cells were observed in Fars2 deficient organisms;observation of mitochondrial ultrastructure showed interrupted mitochondrial cristae and mitochondrial swelling.7.Transcriptomic analysis suggested that Fars2-deficient induced neuronal dysplasia may closely related to the activation of cell cycle pathways,axon sheathing process and NF-κB signaling pathway during neuron development.Moreover,Dctn3,a dynein activating protein subunit,were found to be a potential downstream molecule of Fars2 defects in neurons.【Conclusion】 In this study,we successfully covered the neuropathy phenotype and pathological changes found in human patients by establishing multiple animal models with Fars2 deficiency,and together with the in vitro cell models,the pathogenic mechanism of FARS2 gene mutation leading to hereditary spastic paraplegia was revealed: Fars2 deficiency disruptes the phenylalanine aminoacylation process of tRNA in mitochondria,thereby leads to impaired mitochondrial respiratory chain complex levels and disrupted mitochondrial function by inhibiting mitochondrial protein translation,and abnormal mitochondrial function inhibits the normal development and growth of neuronal processes,induces neuroinflammation and neuronal apoptosis.Transcriptomic analysis at multiple time points suggests that cell cycle,axonal myelination related pathways,NF-κB signaling pathway and the potential downstream molecule,Dctn3,play important roles in neuronal degeneration induced by Fars2 defect,which might provide evidence for studying potential therapeutic target for FARS2 mutant patients. |
