Investigation Of NTE Gene Function And Its Role In Neurodegenerative Diseases Using Zebrafish Model

Neural target esterase (NTE) also known as PNPLA6, is a member of patatin-like phospholipase (PNPLA) family. NTE is anchored in ER via its transmembrane domain, and it has two distinct functional domains, a N-terminal regulatory domain and a C-terminal catalytic domain. NTE has been demonstrated to be a phospholipase B promoting phosphatidylcholine deacylated to glycerophosphoc-holine in mammalian cells. OP-induced neuropathy syndrome (OPIDN) is a neurological disorder characterized by paralysis of the lower limbs and degeneration of long axons in the spinal cord and peripheral nerves appear about1-3weeks or later after exposure to certain OP. In research for OPIDN, NTE was identified as the substrate of OPs.NTE was reported to be responsible for an autosomal recessive hereditary spastic paraplegia(HSP). HSPs are a group of clinical and genetic heterogeneity neurological disorders characterised by the presence of lower limb spasticity and weakness. The main pathological feature of these disorders is retrograde degeneration of the longest motor axons in the corticospinal tracts and posterior columns. HSPs can be divided into pure or complicated HSPs depending on the presence of other neurological features in addition to spastic paraparesis, for example muscle atrophy, muscle tension obstacles, ataxia, epilepsy, mental retardation, dementia optic atrophy or hearing impairment. Since symptom onset age of HSP is mainly in childhood or adolescence, and there is no effective treatment in clinic, which represents a great deal of pain and a heavy burden for both patients and their families.Swiss cheese (sws) is the orthologous gene of NTE in Drosophila. Sws mutant induced degenerative changes in Drosophila nervous system, including glial hyperwrapping,vacuolization and apoptosis. Since disruption of Nte in mice results in embryonic lethality, it isn’t suitable for the investigation of the role of NTE in neural development and HSP. Brain-specific deletion of Nte in mice results in disruption of ER, abnormal reticular aggregation, and vacuolization of nerve cell bodies.Although studies on animal models including mice and drosophila have extended our understanding of NTE, its role in neural development and HSP is not clearly understood. We established a vertebrates model of Nte insufficiency using morpholino oligonucleotide knockdown in zebrafish. Nte insufficiency results in emboys curly tail, and other developmental abnormalities. Foremore, Nte insufficiency also leaded to motor neuron defects including axon truncation and branching. Activity of esterase is necessary for the biology function of NTE. We also found that the Nte insufficiency cause bone morphogenetic proteins (BMPs) signals over-activated, which may be responsible for the defects of developmental abnormalities and motor neuron defects.PART ONEKnockdown of nte expression leads to morphological abnormalities in zebrafish embryosAs a vertebrate model animal, zebrafish showed a lot of advantages. Zebrafish Nte protein exhibits73%identity compared to its analogous protein in human. In this study, we investigated the function of NTE in zebrafish development and its roles in neurodegenerative diseases using zebrafish as the model animal. 1) The zebrafish Nte coding sequence contains4026bps which encode1342amino acids. We constructed the zebrafish NTE eukaryotic expression vector pcDNA3.1A-nte.2) Using the RT-PCR and real-time quantitative PCR analysis, we detected that zebrafish Nte was expressed in early stages of embryo development and sustained throughout the whole process of embryo development3) To determin the function of Nte in zebrafish development, we used the morpholino antisense oligonucleotides to knockdown the expression of nte in zebrafish embryos and observed the phenotypes of Nte insufficiency in the frist5days of embryo development.4) The most prominent phenotype observed in nte MO-injected embryos is curly tail.53.7%of367nte MO-injected embryos appeared curly-tail phenotype, while only16.2%of333control MO-injected embryos appeared this tail abnormality.5) To determine the effect of nte knockdown on motility, embryos were allowed to develop to5days post-fertilization and were assigned into there different groups according to the degree of motility, as normal, impaired and immotile. The results showed that54.1%of133nte MO-injected embryos had motility defects, while only23.7%of133control MO-injected embryos had motility defects. The motility of nte MO-injected embryos was significantly reduced.6) In early stages of embryos development (4-somite stage and6-somite stage), the distance between head and tail of nte MO-injected embryos was greater than that in the control MO-injected embryos, and the anterior-posterior (AP) of nte MO-injected embryos was significantly shorter than the control MO-injected embryo.7) The nte MO-injected embryos also showed some other defects including reduced eyes and otic vesicle size, midbrain-hindbrain boundary (MHB) defects, swelling of the pericardium, diminished heart rate and blood flow.In conclusion, we constructed the zebrafish nte eukaryotic expression vector pcDNA3.1A-nte, and established the zebrafish model of nte knockdown in this section. Nte knockdown zebrafish embryos appeared in multiple developmental abnormalities. The most notable abnormalities we found were tail curl and reduced motility. NTE plays an important role in zebrafish development, and the zebrafish model of nte knockdown we established is a suitable for the investigation of Nte function and its role in neurodegenerative diseases.PART TWOThe biological function of NTE is dependent on its esterase activityNTE comprises a N-terminal regulatory domain and a C-terminal catalytic domain. The active site of NTE contains Ser966and two Asp (Asp960and Asp1086) which are essential for enzymatic activity. Mutation of Ser966abolished the esterase activity of NTE and mutation of Asp60or Asp reduced esterase activity of NTE10,000-fold. In order to verify the specificity of nte knockdown, rescue experiment with human NTE mRNA was performed.1) We constructed the human NTE expression vector pCS2-NTE and produced mRNA by in vitro transcription. When human WT NTE mRNA was co-injected with nte MO, only14.2%of373those embryos showed curly tails at36hpf, and70.1%of124those embryos exhibited normal motility. Obviously, the human NTE mRNA can rescue the zebrafish nte knockdown phenotype,2) However, none of the three mutant mRNA was able to rescue the abnormal phenotypes of the nte knockdown embryos.In conclusion, we determined that functions of Nte in the zebrafish development depends on its esterase activity, and Nte without esterase activity couldn’t play its normal biological functions. PART THREENte is required for primary motor neurons and axon developmentSince NTE is the molecular target of OPs in motor neuron disease OPIDN and the mutations of enzyme active site of NTE lead to upper motor neuron disease HSP, NTE may plays an important role in motor neuron growth and maintenance. Zebrafish neurons in the spinal cord are mainly divided into motor neurons, sensory neurons and interneurons by function.1) We used the anti-Znpl, a specific marker for primary motor neuron axons, for immunostaining in26hpf and36hpf embryos. Axons in nte MO-injected embryos showed branching and truncation (failing to join the ventral muscles). At36hpf, the incidence of axon branching and truncation (10axons per side) in nte MO-injected embryos was higher than that in controls.69.2%side of embryos exhibited at least one truncated axon in nte MO injected embryos, only31.3%in control embryos.And65.2%embryos exhibited at least one branching axons in nte MO-injected embryos, while only29.2%in control embryos. We also did analysis of axon growth of secondary motor neuron at72hpf using the Znl2antibody staining and found branching and truncation defects also appeared in secondary motor neuron axons.2) In order to confirm whether the axonal growth defects in nte knockdown embryos is caused by cruly tial, the spinal cord cells of nte MO-injected embryos and control MO-injected embryos in18-somite stage were cultured in vitro, and the growth of motoneuron were analyzed. The motor neurons from nte MO-injected embryos appeared significantly more bifurcation and axonal length of them were obviously shorter than those from the control group. A group of cells with RLDx label from the early nte MO-injected embryos and control group embryos at4hpf were transplantated into wild-type zebrafish embryos at10hpf respectively. The motor neurons from nte MO-injected embryos appeared significantly more bifurcation and their axonal length were obviously shorter than them from control group.3) Using acetylation α-tubulin antibodies, Rohon-Beard (RB) and lateral line sensory neurons axons in36hpf embryos were immunostained.3A10antibody were used to labeling Mauthner neuron and axon in36hpf embryos. Both of those two types of neuron axons had no obvious abnormality in nte MO-injected embryos.4) Using HuC/D, mature neurons were immunostained, and no obvious abnormality was found. Immunostaining with39.4D5antibody against zebrafish motoneurons in30hpf embryos showed that the number of motoneurons in nte MO-injected embryos was significantly less than those in controls.5) Using acridine orange staining to detect apoptotic cells, it is found that there were more apoptotic cells in nte MO-injected embryos compared to controls, especially heads and tails. The results were similar to the followed TUNEL detection.6) By ultrastructure observation of nte MO knockdown embryos, we found intracellular vacuoles, abnormal membrane structure accumulation, abnormal endoplasmic mitochondria and increased autophagy. And we identified increased autophagy in nte knockdown embryos by the Western Blot analysis with anti-LC3-II.In conclusion, Nte plays an important role in zebrafish motoneuron growth and maintenance, while other types of neurons were not significantly affected. And these phenotypes may be caused by abnormal membrane structures resulting in induced autophagy and apoptosis.PART FOURNte knockdown up-regulated the BMP signaling pathwayAxon growth and guidance are regulated by a variety of cell signaling pathway. Inhibition of BMP signals was thought to be required for axon growth. To determine whether BMP signals were affected, we detected the levels of P-Smad1/5/8, the marker molecules of BMP signals. The results showed that the level of P-Smadl/5/8in nte MO injected embryos was striking increased, implying up-regulation of BMP signals.. Moreover, treated with6μM BMP signals inhibitor Dorsomorphin at10hpf, nte insufficiency phenotype in nte MO-injected embryos could be rescued, including curly-tails and movement motility defects. In conclusion, the phenotypes of nte insufficiency are likely to be caused by up-regulation of BMP signals.Taking together, we generated a vertebrate zebrafish model of nte knockdown by morpholino oligonucleotide technique. Zebrafish nte knockdown resulted in developmental abnormalities in early stages of embryos, including curly tail, reduced motility and defects of motor neuron growth. The phenotypes in nte knockdown embryos could be rescued by introduction of wide type, but not mutant, human NTE mRNA. Our results also revealed nte knockdown can up-regulating BMP signals. Our investigation for the NTE is important for understanding NTE function and its role in neurodegenerative diseases.