Investigation Of SLC33A1Gene Function And Its Role In Hereditary Spastic Paraplegia Using Zebrafish Model

SLC33A1gene, also known as AT-1(the acetyl-CoA transporter1), was mapped to human chromosome3q25. SLC33A1encodes the acetyl-CoA transporter, which transports acetyl-CoA into the lumen of the Golgi apparatus, where acetyl-CoA serves as the substrate of acetyltransferases to modify the sialyl residues of gangliosides. Recently, SLC33A1was reported to regulate the acetylation status of ER-transiting proteins, includingβ-site APP cleaving enzyme1(BACEl), amyloid precursor protein (APP) and low density lipoprotein receptor (LDLR). And the post-translational events that occur in the early secretory pathway are crucial for the correct assembly of membrane and secreted proteins.SLC33A1was implicated in the pathogenesis of four neurodegenerative diseases. It was upregulated in the residual motor neurons of patients affected by sporadic amyotrophic lateral sclerosis (ALS). Alzheimer’s disease (AD) patients displayed an approximately three-fold increase in the mRNA levels of SLC33A1compared to normal controls. Moreover, the patients affected by autosomal dominant spastic paraplegia-42(SPG42) carried the p.S113R mutation. Recent work has also shown that mutations in SLC33A1cause a lethal autosomal-recessive disorder with congenital cataracts, hearing loss, and low serum copper and ceruloplasmin. The patients displayed cerebellar hypoplasia and hypomyelination, suggesting that SLC33A1plays an important role in both physiological and pathological conditions.Hereditary spastic paraplegias (HSPs, also known as SPG) are a clinically and genetically heterogeneous group of neurological disorders characterized by progressive spasticity and weakness of lower extremities. As an upper motor neuron disease, the main pathological feature is retrograde degeneration of the nerve fibres in the corticospinal tracts and posterior columns. ALS is a form of adult onset motor neuron disease caused by the degeneration of upper and lower neurons. AD is characterized by irreversible neuronal cell death. The implication of SLC33A1in these diseases suggests that SLC33A1is vital to neuronal cells and axon maintenance. However, how dysfunction of SLC33A1leads to these neurodegenerative disorders is not clearly elucidated.Biomedical research depends on the use of animal models to understand the pathogenesis of human disease at cellular and molecular level, and to provide systems for developing and testing new therapies. The zebrafish (Danio rerio) has become an attractive model for developmental, genetic, and disease model studies because it is a vertebrate with conserved organization of common organs and tissues including the brain and the spinal cord. Due to its well-characterized nervous system and relatively simple organization, it is well suited for analysis of neurodegenerative diseases. Morpholino antisense oligos knockdown technology is widely used in zebrafish system. It interferes with progression of the ribosomal initiation complex, thus prevents translation of the targeted transcript. Many zebrafish models of motor neuron diseases have already been generated through Morpholino antisense oligo knockdown technology, including ALS1, ALS2, spinal muscular atrophy (SMA), SPG4, SPG8, SPG11and SPG3A. This allows an enhanced understanding of the basic cell biological processes that underlie the disease phenotype, suggesting that zebrafish is amenable for studies on motor neuron diseases.In this study, we used zebrafish as an in vivo model to investigate the function of SLC33A1in the development of zebrafish embryos, especially in the motor neuron development.Part I. Characterization of zebrafish slc33al gene and its expression patternIn order to investigate the function of Slc33al in vivo using zebrafish model, we first examined the protein structure and expression pattern of zebrafish slc33al.1) As showed by sequence alignment, zebrafish and human SLC33A1proteins are69%identical; zebrafish and mouse SLC33A1proteins are69%identical too. This showed that SLC33A1is conserved among vertebrate organisms.2) Since SLC333A1is a membrane protein, the SOSUI software was used to predict the secondary structure. We found that the secondary structure of zebrafish Slc33a1was similar to human homolog, and it contains12transmembrane domains, suggesting the conservation of SLC33A1function between human and zebrafish. Therefore, zebrafish is suitable for function analysis of SLC33A1.3) The slc33al mRNA levels of zebrafish at different developmental stages were determined by quantitative real-time PCR. Slc33al mRNA appeared immediately after fertilization in the zygote and throughout the whole developmental stages, while peaked in the adults. Furthermore, according to the whole mount in situ hybridization of slc33al mRNA provided by zfin.org, slc33al is expressed ubiquitously.Taken together, these results indicate that zebrafish was suitable for modeling human diseases caused by SLC33A1mutations.Part II. Phenotype analysis of the slc33al knockdown zebrafishMorpholino antisense oligonucleotides knockdown gene expression by sterically blocking the translation initiation complex. In this part, we used the morpholino based knockdown technology to generate the zebrafish model to investigate the function of SLC33A1:1) An antisense Morpholino was designed to specifically inhibit the translation of zebrafish slc33a1mRNA, and a5-base mismatch oligonucleotide was used as a control. Slc33a1MO or control MO was injected into embryos at1-4cell stage. The specificity and validity of slc33a1MO were determined by Western blot and TNT Quick Coupled Transcription/Translation System. We found that the protein level of Slc33a1was significantly decreased by slc33al MO both in vivo and in vitro.2) When Slc33al levels were reduced throughout the entire embryo, a curly tail phenotype and lethality was observed. And this is similar to the morphological changes of the zebrafish models of SPG4, SPG8, SPG11and ALS2. Although HSP is a childhood onset disorder, and ALS is an adult onset disease, these data indicated SLC33A1was vital to the embryo development.3) In parallel to the reduced motility of motor neuron disease patients, knockdown slc33al disrupted the motility of zebrafish. At48hpf, while the control MO injected zebrafish embryos normally responded to a touch with contractions that initiates a bout of swimming, the diminished touch-evoked behaviors were observed in slc33al knockdown embryos, which responded to touch without contractions or with contractions but no swimming. The motility defects observed in slc33a1MO embryos were similar to the zebrafish models of SPG4, SPG11, SPG3A, ALS1and ALS2.4) Slc33al knockdown disrupted outgrowth and pathfinding of both primary and secondary motor axon, while other sensory and interneuron axons were unaffected. Moreover, the aberrant distribution of motor neurons at spinal cord and hindbrain was observed. These results revealed that Slc33al functions in motor axon maintenance and the migration of motor neurons. These are common phenotypes to all the known zebrafish models of ALS and HSP.Part Ⅲ. p.S113R mutation damages SLC33A1functionIn2008, the p.S113R mutation in SLC33A1was reported to cause autosomal dominant spastic paraplegia (SPG42). Cross-species alignment showed the serine at position113is highly conserved among vertebrates. And bioinformatics analysis predicated the mutation would dislodge the transmembrane domain, leading to the dysfunction of SLC33A1.In order to uncover the property and impact of the mutation, we used the zebrafish model to conduct the third part of experiments as follows:1) We first constructed the wild-type human SLC33A1expression vector, and then the p.S113R mutation was introduced by site-directed mutagenesis. The constructs were linearized and capped RNAs were transcribed in vitro from the linear construct using mMESSAGE mMachine SP6kit for zebrafish rescue experiments;2) The rescue experiment by coinjection of slc33a1MO with wild-type or mutant human SLC33A1mRNA was performed. We found that the wild-type human SLC33A1mRNA was able to rescue the curly tail and motor axon defects caused by slc33a1knockdown in zebrafish, while the mutant was not, suggesting that S113R mutation damages SLC33A1function.3) When S113R mutant mRNA was injected into the embryos alone, no developmental defect was observed, indicating that S113R mutation is a loss-of-function mutation.Part IV. SLC33A1regulates motor axon outgrowth via BMP pathwayRecent studies suggested that abnormal BMP signaling probably be a unifying mechanism to certain kinds of hereditary spastic paraplegia. At least four proteins encoded by HSP genes were inhibitors of BMP signaling, including NDPA1, ATLASTIN1, SPASTIN and SPARTIN. Studies of the fly homologues of genes that cause neurodegenerative diseases have revealed the implications for reduced BMP/TGF-β signaling in ALS, SMA, Huntington’s disease (HD) and increased signaling in multiple sclerosis (MS). Moreover, BMP signaling regulates microtubules and axon transport in Drosophila melanogaster, and inhibits axonal regeneration in the adult spinal cord in rats. All these data confirmed the implication of BMP signaling in the maintenance of axonal homoeostasis and in the pathogenic processes of neurodegenerative diseases. Since motor axon outgrowth defects were observed in the SLC33A1zebrafish model, we searched for the possible impact of SLC33A1in BMP signaling:1) Western blot analyses embryos of different developmental stages (12hpf,24hpf,48hpf and60hpf) showed that slc33al knockdown resulted in significant increase of P-Smadl/5/8which is the major indicator of BMP signaling. Furthermore, coinjection of human SLC33A1mRNA could downregulate the increased BMP signaling caused by slc33a1knockdown, suggesting SLC33A1is an inhibitor of BMP pathway.2) Slc33a1MO injected embryos were dechorionated and incubated in embryo medium with3μM,9μM or18μM Dorsomorphin (DM), which can inhibit the kinase activity of BMP receptor, thus downregulate the BMP pathway. We found that9μM DM treatment alleviates the knockdown phenotypes, including morphology, axon and motility defects; further validating that the defects observed in slc33al knockdown embryos are a result of defective regulation of BMP signaling.3) Increased P-Smadl/5/8was also observed in cultured fibroblasts from the patients with S113R mutation in SLC33A1gene, further confirming that SLC33A1functions as an inhibitor of BMP pathway.In summary, we used the zebrafish model to investigate SLC33A1function in vivo. Slc33al knockdown zebrafish displayed a series of phenotypes including disrupted tail development, motor axon defects and reduced motility. And p.S113R mutation was confirmed to be a loss-of-function mutation. Furthermore, the BMP signaling pathway was upregulated in these embryos, and pharmacological inhibition of BMP signaling with dorsomorphin rescued the knockdown phenotypes, illustrating the implication of SLC33A1in motor axon maintenance and BMP signaling. The insights gained here may also be of relevance to more common neurological disorders in which axonopathy is a contributing feature and provide a new potential therapeutic target.