| BackgroundStrumpell first described hereditary forms of spastic paraplegia in 1883,with Lorrain later providing more extensive details.Hereditary spastic paraplegia(HSP)is not a single disease entity;it is a group of clinically and genetically diverse disorders that share a primary feature.Statistical studies have shown that the incidence of HSP is about 2-10/100000,and the clinical symptoms are diverse.Different patients with different manifestations,usually accompanied by cerebral palsy,optic nerve injury,mental retardation and other neurological symptoms Up to now,76 SPG causative genes have been located,and 54 genes are fully identified.SPG4 is the most common dominantly inherited HSP gene,representing approximately 40%of such cases.De novo mutations in ATAD3A were recently found to cause a neurological syndrome with developmental delay,hypotonia,spasticity,optic atrophy,axonal neuropathy,and hypertrophic cardiomyopathy.Using whole-exome sequencing,we identified a dominantly inherited heterozygous variant c.1064G>A(p.G355D)in ATAD3A in a mother presenting with HSP and axonal neuropathy and her son with dyskinetic cerebral palsy,both with disease onset in childhood.HSP is a clinically and genetically heterogeneous disorder of the upper motor neurons.Symptoms began in early childhood may resemble spastic cerebral palsy.The function of ATAD3A is yet undefined.The dominant-negative patient mutation affects the Walker A motif,which is responsible for ATP binding in the AAA module of ATAD3A,and we show that the recombinant mutant ATAD3A protein has a markedly reduced ATPase activity.We further show that overexpression of the mutant ATAD3 A fragments the mitochondrial network and induces lysosome mass.Similarly,we observed altered dynamics of the mitochondrial network and increased lysosomes in patient fibroblasts and neurons derived through differentiation of patient-specific induced pluripotent stem cells.These alterations were verified in patient fibroblasts to associate with upregulated basal autophagy through mTOR inactivation,which resembling starvation.Mutations in ATAD3A can thus be dominantly inherited and underlie variable neurological phenotypes,including HSP,with intrafamiliar variability.This finding extends the group of mitochondrial inner membrane AAA proteins associated with spasticity.Objectives1.Firstly,to collect the symptoms of our targeted patients with HSP,and to find the potential disease causing mutation by whole-exome sequencing.2.To observe the abnormal phenotype induced by ATADA3A mutant in the over-expressing cell model.3.To explore unbalanced mitochondrial network phenotype through primary fibroblasts cells from the patient’s skin tissue.4.To observe the mitochondrial network dynamics in neurons derived through differentiation of patient-specific induced pluripotent stem cells.Methods1.To identify the genetic cause of the dominantly inherited condition in this family,we performed whole-exome sequencing on the DNA samples of the affected mother and son.2.For whole-exome sequencing,the exome targets of the patients’ DNA were captured with the Agilent Clinical Research Exome followed by Illumina HiSeq 2500 sequencing.The variants were annotated with the ANNOVAR program and added annotations for predicted deleteriousness.3.An ATP molecule was docked into the ATP-binding site of the model using the Discovery studio 4.5 software(BIOVIA).4.The ATPase assay was performed on wild type ATAD3A and the mutants by calculating the amount of phosphate released.5.Overexpression of ATAD3A in primary fibroblasts,cells for immunocytochemistry and western blot were co-transfected with mitochondria-targeted red fluorescent protein(mito-Red).6.Western blot and immunocytochemistry were approached in patients’ fibroblast cell model,and cells were incubated with MitoTracker-Red and Lyso-Tracker-Green for live cell imaging.7.Primary fibroblasts were reprogrammed using the combination of six factors(OCT3/4,SOX2,KLF4,L-MYC,LIN28 and T53 shRNA)in three episomal plasmids on matrigel coated plates to induce iPS cells.8.The cells were differentiated into neurons as adherent cultures on Poly-D-lysine and Laminin coated plates using an established differentiation protocol.9.Electronic microscopy,live cell imaging and immunocytochemistry were approached in neurons derived through differentiation of patient-specific induced pluripotent stem cells.Results1.A deformity in the lower extremities observed in patient and Magnetic Resonance Imaging shows thinning and atrophies of the spinal cord.The glycine in the Walker A motif affected by the patient mutation G355D is invariant in ATAD3 proteins between species,in human mitochondrial proteins with AAA modules and in the human ATAD family.2.We performed whole-exome sequencing on the DNA samples of the affected mother and son.Filtering for variants in known HSP genes did not reveal potential disease-causing variants.But we found an ATAD3A c.1064G>A(p.G355D)heterozygous mutation.3_A structural in silico analysis suggest that the mutation introduces a negative charge at a distance of less than 4 A from the y-phosphate of an incoming ATP in the substrate-binding site,which is predicted to result in decreased affinity for ATP.4.ATPase activities of wild type and mutant proteins containing the amino acid changes G355D or K358A(the invariant lysine of the Walker A motif)showed that the ATPase activities of the Walker A mutant proteins were markedly reduced in comparison to the wild type recombinant protein.Moreover,the reduced ATP binding of the mutant ATAD3A had a strong dominant-negative effect on the ATPase function.5.Both mutants induced fragmentation significantly more frequently than the overexpression of the wild type ATAD3A.As tested by western blot,all transfected ATAD3A constructs were expressed at similar levels.We also detected increased amounts of lysosomes by LysoTracker staining in the cells containing fragmented mitochondria.6.While ATAD3 A protein levels were normal in patient cells,we noticed an increase in the ratio of respiratory complex Ⅳ and Ⅱ subunits,and a reduction in the level of the mitochondrial fission protein Drpl in patient cells compared to controls.Also,patient mitochondria were hyperpolarized,which has been shown to be a feature of hyperfused mitochondria.7.In addition,large amounts of lysosomes were seen in patient cells by staining with LysoTracker Green,and by electron microscopy.Collectively,these data indicate that the mutant ATAD3A induced mitochondrial elongation in patient fibroblasts,which was associated with mTOR inhibition leading to the induction of basal macroautophagy.8.Immunohistochemistry of OCT4,SSEA4 and TRA-1-60 shows that all iPSC lines express endogenous pluripotent stem cell surface markers.Quantitative PCR results show that all clones express the endogenous stem cell markers.RT-PCR was used to assess the removal of transgene vectors and shows that OriP and EBNA1 were absent from all lines.9.The differentiation process of the patient cells was similar compared with the controls,and morphologically patient neurons appeared indistinguishable from controls.Confocal images suggested that patient neurons had unbalanced dynamics of mitochondria.Lysotracker staining and electron microscopy showed an increase in lysosomes in patient neurons compared with controls.No changes in ATAD3A protein level or mtDNA copy number nor mtDNA deletions were observed in samples taken from the mixed neuronal cμLtures.Conclusions1.The recombinant mutant ATAD3 A protein has a markedly reduced ATPase activity.2.Overexpression of the mutant ATAD3A fragments the mitochondrial network and induces autophagy and lysosomes.3.Similarly,unbalanced mitochondrial network dynamics are seen in patient fibroblasts and neurons derived through differentiation of patient-specific induced pluripotent stem cells.4.Patient cells were were in a starvation-like state with upregulated autophagy.Dominant-negative mutations in ATAD3A can thus underlie variable neurological phenotypes,including HSP,with intrafamiliar variability. |
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