A Preliminary Research On The Effect Of Hereditary Spastic Paraplegia Causatave Gene SLC3A1 Missense Mutation On Mitochondria

Hereditary spastic paraplegia is a group of rare neurodegenerative disorders which is clinically and genetically heterogeneous.The main clinical symptoms of HSP were progressive muscle weakness and increased muscle tone of the bilateral lower limbs.The pathological changes of HSP include axonal degeneration and demyelination,which sometimes accompanied by neuron loss.Since the pathogenesis of HSP has not been clarified and there are no effective therapies for HSP,it is important to identify the causative genes of HSP and to study their function.Up to now,more than 70 HSP pathogenic sites and 59 corresponding HSP causative genes have been identified.Among them,SPG42 causative gene SLC33A1 was identified by our laboratory using linkage analysis method by studying a HSP family from Qingdao.We found that all the patients from this pedigree carried SLC33A1 c.339T>G missense mutation,resulting in SLC33A1 protein at the 113th position of the serine is replaced by arginine,this missense mutation led to HSP.In order to study the pathogenesis of SLC33A1S113R missense mutation,our Lab successfully generated the Slc33a1S113R knock-in mouse model in 2013.Previous results show that the heterozygous mutant mice(Slc33a1wt/mut)are able to survive,while homozygous mutant mice(Slc33a1wt/wt)died in early embryonic developmental stage,which indicates that SLC33A1 is essential for early embryonic development in mice.In addition,transmission electron microscopy of the 12-month-old Slc33a1wt/mut mouse spinal cord gray matter showed that there were obvious ultrastructural abnormalities and degeneration in nerve cells of Slc33a1wt/mut mice.Especially the mitochondria,there are obvious swollen and degenerated mitochondria with cristae deficiency and other morphological changes,such as abnormal endoplasmic reticulum、increased free ribosomes and swollen Golgi apparatus.All of which indicates that Slc33al plays an important role in mitochondrial morphology and functionalmaintenance.Therefore,we studied the role of Slc33a1S113R mutation in mitochondria by using skin fibroblasts derived from SPG42 patients and normal human,SLC33A1 high expression HeLa cells,and mouse cerebral cortical neurons derived from wild type and Slc33a1S113R knock-in heterozygous mice.Mitochondria as the "power plant" in the cell,is the most important energy supplier.Since Mitochondria can continuously and steadily provide energy for the neurons,normal mitochondrial structure and function is vital for neuronal survival.Under normal circumstances,the mitochondria were short rod-like,granular or linear,but the results of transmission electron microscopy(TEM)in the spinal cord gray matter of Slc33a1wt/mut mice showed that the mitochondria occur obvious swelling,local cavitation and other forms of abnormal.In addition,at the cellular level in vitro,we were cultured for 6 days of mouse cerebral cortical neurons with axon-specific marker Tau protein and dendritic specific marker Map2 protein immunofluorescence staining.the results showed that part of axons appears swelling in Slc33a1wt/mut mutant mice.At the same time,we transfected mitochondrial expression vector pDsRed2-Mito into the primary cultured cortical neurons,and found that there were obvious swelling and accumulated mitochondria in the axons of neurons derived of Slc33a1wt/mut mice.Subsequently,we examined studied the effect of Slc33a1 mutation in the axonal transport of mitochondria,the mitochondrial axonal transport capacity of Slc33a1wt/mut mutant heterozygous mice was found to be significantly decreased,suggesting that the effect of Slc33a1S113R missense mutation on mitochondria can occur at a single cell level.Moreover,it is affecting the transport of mitochondria in neuronal axons,resulting in mitochondrial accumulation,which in turn affects mitochondrial energy supply to neuronal cells.In vivo,we analyzed the retrograde axonal transport capacity of Slc33a1 mutant mice by injecting CTB into the gastrocnemius muscle,and the CTB retrograde tracer experiments showed that the retrograde axonal transport of Slc33a1wt/mut mice was abnormal.However,by immunofluorescence study,detection of α-Tubulin,thecytoskeleton was normal.It indicated that Slc33a1S113R mutations do not affect axonal transport by affecting the cytoskeleton.In addition,the mitochondrial membrane potential levels of different genotype cells were examined by JC-1 method.It was found that compared with the control group,the mitochondrial membrane potential of Slc33a1wt/mut mouse cortical neurons and SPG42 skin fibroblasts was significantly lower,indicating that the Slc33a1 mutation could lead to mitochondrial function dysfunction.In addition,we also detected the ATP concentration of different genotype cells.It was found that compared with the control group,the ATP concentration of skin fibroblasts in SPG42 patients was significantly lower,indicating that Slc33a1S113R mutation resulted in Slc33a1S113R mice abnormal ATP synthesis in mitochondrial.For further discussion about that how SLC33A1 affects the morphology and function of mitochondria,primarily we study the subcellular localization of SLC33A1.It has been reported that as an acetyl-CoA transporter which located in the endoplasmic reticulum,SLC33A1 can affected its function through multiple protein acetylation regulation such as APP cleavaged protein BACE1,Low Density Lipoprotein Receptor(LDLR)and autophagy related protein ATG9A,thereby participated in many crucial cell life activities.We first determined that SLC33A1 not only co-localized with the endoplasmic reticulum,but also partially co-localized with mitochondria,we speculated that SLC33A1 was also participated in the acetylation of mitochondrial-associated proteins,thus affecting the maintenance of its function.In conclusion,we identified the abnormal mitochondrial morphology of spinal cord in the Slc33als113R knocked-in heterozygous mouse.Moreover,the Slc33a1S113R mutations caused mitochondrial axonal transport abnormalities,resulting in mitochondria accumulation and axon swelling.In addition,mitochondrial membrane potential and ATP synthesis capacity were influenced.By immunofluorescence localization experiment,we identified SLC33A1 and mitochondrial displayed a strong co-localization in the cell.These results provided new clues for further understanding about the function of SLC33A1 and exploration of the mechanism of Slc33a1S113R missense mutation which generated the pathogenesis of HSP.