S113R Mutation In Slc33a1 Leads To Hereditary Spastic Paraplegia And Augmented BMP Signaling In A Mouse Model

The acetyl-CoA transporter,SLC33A1 is a multiple transmembrane protein that consists of 549 amino acids.Cytosolic acetyl-CoA is transported into the lumen of the ER/Golgi apparatus by the ER membrane transporter SLC33A1.Acetyl-CoA is then utilized for acetylation of ER-resident and-transiting proteins.In 2008,we reported that a heterozygous missense mutation in SLC33A1,p.Serll3Arg(S113R)(c.339T>G)(MIM#603690.0001),causes autosomal-dominant spastic paraplegia type 42(SPG42,MIM#612539)in a large Chinese family in Qingdao.In 2015,we surveyed all the pathogenic variants in the SPG42 family using whole exome sequencing and further determined SLC33A1 mutation as the most plausible causal variant in this family.Animal models can be used to study the pathogenesis and progressiveness of some certain human diseases in vivo,and to provide mechanistic insight necessary for designing novel therapeutic strategies.The mouse genome is highly homologous to that of human,mouse models are widely used to study the function of human genes and the correlation between mutant genes and certain diseases.To better understand the function of SLC33A1,we generated Slc33alS113R knock-in mice in 2013 and found that homozygous SIc33alS113R mutant(Slc33almut/mut)mice were embryonic lethal.Heterozygous Slc33al mutant(Slc33alwt/mut)mice bred normally and showed no overt phenotypic abnormalities.We here present the results obtained from Slc33alwt/mut mice,mainly focusing on analysing their nervous system pathology and the pathogenesis of SPG42.In addition,using a sciatic nerve crush injury model in vivo and dorsal root ganglion(DRG)culture in vitro we studied the capacity of injury-induced neurites regeneration in Slc33alwt/mut mice.Part I.Phenotype analysis of Slc33alwt/mut miceHereditary spastic paraplegia(HSP)is a clinically and genetically heterogeneous group of neurodegenerative or developmental disorders in which progressive lower extremity weakness and spasticity are the predominant clinical symptoms.HSP is usually caused by developmental failure or distal degeneration of motor axons in the corticospinal tract.To determine whether Slc33alwt/mut mouse model can recapitulate human SPG42 disease,we focused on studying the motor function and the nervous system of the aged mice.When examined at 8-24 months using a Hind-leg clasping reflex test,Slc33alwt/mut mice showed no overt phenotypic abnormalities.However,both rotarod and exercise tolerance tests showed that the 12 months old Slc33alwt/mut mice exhibited a decline in locomotion and significant hindlimb weakness compared with age-matched wild-type(WT)littermates.We then determined,by immunofluorescence staining,Toluidine Blue staining,Luxol Fast Blue staining and transmission electron microscopy,whether there was axonal degeneration in spinal cords of Slc33alwt/mut mice.We observed a significant reduction in the density of axons in the corticospinal tracts of the lumber spinal cord of 12-month-old Slc33alwt/mut mice,and many axons were demyelinated and irregularly shaped,when compared with WT littermates.Transmission electron microscopy revealed degeneration within the grey matter of lumber spinal cord of 12-month-old Slc33alwt/mut mice,such as swollen and degenerated mitochondria with cristae deficiency,increased free ribosomes,abnormal endoplasmic reticulum,swollen Golgi apparatus cisternae,and nuclear pocket formation in both glial cells and neurons of the spinal cord.In contrast,examination of sciatic nerves revealed no significant difference between Slc33alwt/mut mice and WT littermates.In addition,examination of the gastrocnemius muscles also revealed no significant difference between WT and Slc33alwt/mut mice,indicating that there is no obvious deficit in the peripheral nervous system of Slc33alwt/mut mice.In addition,congenital cataracts,hearing loss,and low serum copper and ceruloplasmin phenotypes reported to be present in other SLC33A1 mutations were not detected in the Slc33alwt/mnt mice.Altogether,we demonstrated that the Slc33alwt/mut mice with an S113R mutation developed behavioral abnormalities and central neurodegeneration consistent with pure HSP phenotypes,which are similar to those in individuals with HSP carrying this mutation.Thus,our knock-in mouse model can be used to study the pathogenesis and therapeutic strategies of HSP.Part Ⅱ.Augmented BMP signaling in Slc33a1wt/mut miceHSP-associated mutations are found in at least five proteins-atlastin-1(SPG3A),spastin(SPG4),NIPA1(SPG6),spartin(SPG20)and PNPLA6(SPG39)-that function as inhibitors of BMP signaling.Thus,BMP signaling is a compelling candidate that is implicated in HSP.We previously reported a significant elevation of BMP signaling in Slc33al knockdown zebrafish and in fibroblasts derived from an SPG42 individual,indicating that SLC33A1S113R mutation likely causes SPG42 via affecting BMP signaling.We thus assessed whether the Slc33a1S113R mutation would impact BMP signaling in mice by Western blotting.Results showed that there was a significant increase in the level of BMPR1A protein in the spinal cords of 12 months old SIc33a1wt/mut mice.Since there is an injury-induced regulation of BMP-signaling pathway components,the increase of BMPRIA observed in 12 months old mice could be a consequence of the neurodegenerative process.Therefore,we also evaluated the BMPR1A levels in newborn and 3-month-old Slc33alwt/mut mice.The results also showed an upregulation of BMPRIA level in these undiseased mice.We were unable to detect the baseline levels of pSmadl/5/8 in spinal cords by Western blotting analysis.However,levels of pSmadl/5/8 and BMPRIA were significantly increased in mouse embryonic fibroblasts(MEF)and primary cultured neurons derived from Slc33alwt/mut mice.In addition,we also detected an increase in BMPR2 levels in the brains and spinal cords of Slc33alwt/mut mice,but not in MEF,which indicated that BMPRIA upregulation is cell autonomous but BMPR2 increase is a subsequent effect.Part Ⅲ.Slc33alS113R accelerated injury-induced sciatic nerve regeneration via BMP signalingSeveral lines of evidence suggest that BMP signaling may be involved in neuronal regeneration.Thus,we hypothesized that SLC33A1 mutation may affect PNS regeneration via upregulating BMP signaling.We therefore used a sciatic nerve crush model in vivo to address the effect of Slc33alS113R on axonal regeneration in the PNS.The sciatic functional index(SFI)and histological analysis were used to detect functional and histological nerve recovery.SFI value showed that the functional nerve recovery following crush injury was obviously accelerated in Slc3Jalwt/mut mice compared with WT littermates.Histological analysis showed many small-diameter axons with a thin myelin sheath in specimens of the crushed sciatic nerve from both Slc33alwt/mut mice and their WT littermates.However,the number of myelinated axons in Sc33alwt/mut mice was significantly higher than that in WT littermates.These results indicate that nerve regeneration following crush injury was accelerated in Slc33alwtlmut mice.We next tested whether the accelerated axonal regeneration following sciatic nerve injury in Slc33alwt/mut mice was a result of increased BMP signaling.Indeed,we found that the BMPR1A level in the sciatic nerves of Slc33alwt/mut mice was higher than that in WT littermates.Moreover,crush injury induced a further increase in BMPR1A level from Slc33a1wt/mut mice.Consistent with the negative regulation of BMP signaling by SLC33A1,there was a significant decrease in the level of SLC33A1 protein in the sciatic nerves at day 2 after injury compared with non-injured controls.To determine whether inhibition of BMP signaling could attenuate the accelerated axonal regeneration following injury,we injected BMP signaling antagonist,Noggin,into the sciatic nerve of Slc33a1wt/mut mice immediately prior to sciatic nerve crush.Nerve regrowth in the sciatic nerve following crush injury was assayed via both immunofluorescent evaluation of the growth-associated protein,GAP-43,expressing fibers and by a functional measure,and the pinch test.Results indicated that functional nerve recovery following crush injury was accelerated in Slc33alwt/mut mice.Noggin treatment can efficiently attenuate the accelerated injury-induced axonal regeneration in Slc33alwt/mut mice,which suggested that the accelerated injury-induced axonal regeneration in Slc33alwt/mut mice is at least partially mediated through upregulation of BMP signaling.Part IV.Slc33a1S113R accelerated axonal growth in cultured DRG neurons via BMP signalingDRG neurons are a favored model system to study sensory axonal regeneration.The dissociated neuron cultures allow assessment of neurite formation in individual cells,while the cultured whole explanted DRG maintain important interactions between resident neurons and supporting cells.It had been reported that BMP signaling could markedly enhance DRG axonal growth capacity.Thus,we turned to DRG cultures to gain insight into the influence of SLC33A1 mutation in peripheral sensory axonal regeneration.Results showed that DRG neurons derived from Slc33alwt/mut mice were able to extend much longer axons than adult naive neurons in both cultured neonatal whole DRG explants and dissociated DRG neurons,which indicates that the process is neuron-autonomous.Immunofluorescence analysis showed increased levels of BMPRlA and pSmadl/5/8 in the DRG neurons from Slc33alwt/mut mice compared with WT littermates.Exposure to Noggin attenuated the accelerated neurite outgrowth in both DRG explants and DRG neurons from Slc33alwt/mut mice.These data demonstrated that Slc33alS113R mutation enhances BMP-dependent neurite formation in DRG neurons in vitro and that Noggin treatment can attenuate the enhanced neurite outgrowth of DRG derived from Slc33alwt/mut mice.Taken together,in this study,we demonstrated that the phenotype of Slc33alwt/mut mice with S113R variant can recapitulate the clinical features of HSP,such as progressive impairments in motor function and axonal degeneration.Slc3alwt/mut mice can be used to study the pathogenesis and therapeutic strategies of HSP.Upregulation of BMP signaling by SLC33A1 mutation is involved in the pathogenesis of SPG42.Compared with WT littermates,Slc33alS113R variant lead to accelerated neurite extension in vitro and axonal regeneration after sciatic nerve injury in vivo through up-regulating BMP signaling.