The Function And Mechanisms ZDHHC16 And ZDHHC17-the Members Of DHHC Protein Family During Neural System Development

Protein activity relies on several modifications after translation in cytoplasm that include ubiquitination, phosphorylation, glycosylation and lipid modification.The lipid modification increases protein hydrophobicity, facilitates protein interactions with lipid bilayers, and can markedly alter protein transportation, sorting and function. One out of five lipid systems is recently discovered for forty years and important for protein activity.Palmitoylation, defined as the addition of saturated 16-carbon palmitic acid to specific cysteine residues. As reported, from yeast to mammal, hundreds of protein proved to get palmitoylated after translation. The thioester linkages of S-palmitoylation are the most common type in vivo comparing to N-palmitoylation. Importantly, palmitoylation is a unique lipid modification in that it is reversible. It could dynamically alter protein-palmitoylation levels, and this provides an important mechanism for the regulation of protein transportation, infusion with plasm membrane system and signalling. The recent discovery of palmitoyltransferases provide a breakthrough progress in palmitoylation and substrate proteins research although the palmitoylation has been found in more than 30 years ago. The identification of eight genes including Erf2 and Akrl as acyltransferases in yeast brought into focus on a family of Proteins with a DHHC consensus sequence and a cysteine-rich domain. In 2004, Bred DS identified 23 genes in mammalian genomes (human and mouse), and the development of new proteomic and imaging methods have accelerated Palmitoylation analysis.In recent years, a large number of studies have indicated that the palmitoylation is mainly occurred in the neuronal proteins. The identification of palmitoylated substrate-DHHC enzyme pairs has facilitated detailed studies to clarify the roles of palmitoylation. It is becoming clear that palmitoylation is important for neuronal development and neurological disorders. Many proteins that are involved in the formation of neuronal processes and spines are palmitoylated. Palmitoylation of NCAM 140 targets it to the lipid raft of the growth cone membrane and is required for NCAM-mediated neurite outgrowth. Of the 23 DHHC proteins, DHHC7 and DHHC3, show PAT activity for NCAM140.Palmitoylation has a particularly important role in NSCs self-renew and determination. A series of key proteins that participate in NSCs development are palmitoylated such as wnt3a and shh. The latest research shows that DHHC5 palmitoylates flotillin-2 to induce the NSCs differentiation. Moreover, presynaptic and postsynaptic membranes are both characteristically enriched in palmitoylated proteins such as SNAP25 and PSD95. It is reported that several members of DHHC family including DHHC2, DHHC3, DHHC 15, DHHC 17 palmitoylate those proteins to regulate the sorting, transporting and enrichment on pre-post-synapse. The transduction of synapse relays on DHHC family due to the palmitoylation of ligand-gated and voltage-dependent ion channels. Most importantly, Genetic evidence in humans and mice suggests that genes encoding DHHCs are associated with several neurological disorders. DHHC8 linked to a high risk of developing schizophrenia. An analysis of a X-linked mental retardation family shows that DHHC 15 and DHHC9 are the strong candidates for the disease.DHHC17 was originally isolated as a huntingtin-interacting protein and associated with Huntington’s disease. The latest studies in vitro have also demonstrated that DHHC 12 is associated with the metabolism of APP, suggesting the involvement of DHHC 12 in Alzheimer’s.All these research progress indicate that DHHC family play an important role in neural development and function. Thus, research of DHHC family is valuable and significant in clinical treatment. However, there are still many unsolved questions about the specific functions and mechanisms of DHHC family on neural development needed to be elucidated although the transgenic mice of DHHCS,8,17 were established. At the same time, the development model of mice also makes us to find another suitable model to observe the role of DHHC family during embryonic development, especially neural development. The zebrafish model, with the advantage of its fast development and transparent body, has been widely used in neuronal development studies. Here, we used the zebrafish as a model to understand the functional roles of DHHC family in neural development in vivo by reverse-genetic technology.Chapter I Function and mechanism of ZDHHC16 during NSCs proliferation BackgroudZDHHC16, one of the characterized DHHC family members, is highly conserved from invertebrates to vertebrates. Zebrafish zdhhcl6 encoding a 387-amino acid protein comprises 13 exons. Comparing with other members of the family, the analysis of ZDHHC16 is few and still stays on in vitro. Recent report shows that ZDHHC16 interacted with JAB1 and participated in the activation of c-Abl to activate the cell apoptosis program in vitro. The latest research progress in vitro indicated the importance of ZDHHC16 in NSCs survival when exposed to high glucose. However, the precise role of ZDHHC16 in NSCs and neural development remain largely elusive.ResultsFirstly, we investigated the spatiotemporal expression pattern of ZDHHC16 in zebrafish by using WISHand RT-PCR. Our results indicated that the ZDHHC16 transcripts were highly expressed in the developing anterior neural plate since tailbud stage. Thus, the expression pattern of ZDHHC16 emphasizes its importance in central nervous system (CNS) development and particular, in forebrain formation.To investigate the function of ZDHHC16 during zebrafish neural development, we disrupted its translation by injecting antisense morpholino oligonucleotides into 1-cell stage embryos. At 10hpf, ZDHHC16 morphants showed defects at the anterior neural plate, whereas controls developed normally. By 24 hpf, the morphogenesis defects were more pronounced; in fact, the embryos showed small head and absence of telencEphalon. Moreover, we tested several of neural markers to identify the function of ZDHHC16 in neural development. In accordance with the phenotypes, the expression pattern of telencephalon marker, foxgl, emx2, dlx2, were significantly decreased at 24 hpf in ZDHHC16-morphants. Moreover, the expression of acetylated a-tubulin which marked neurons of brain was severely decreased at telencEphalon in ZDHHC16-morphants. ZDHHC16 regulate early telencephalic pattern as early as neural-ectoderm formation. The markers of the neural plate, such as sox3 and rx3, showed reduced expression levels at primitive telencephalon in ZDHHC16 morphants during early neural-ectoderm formation(75% Epiboly stage).Therefore, these results indicate that ZDHHC16 might participate in NSCs activity.We performed experiments to explore the potential role of ZDHHC16 in NSCs development processes both in vivo and in vitro. Using RT-PCR and in situ hybridization, we found that the expression of sox2 was reduced in ZDHHC16 morphants. This result suggests the number of NSCs was decreased in ZDHHC16 morphants. The results of BrdU, TUNEL and expression of pcna and mcm5 showed reduced proliferation of NSCs in ZDHHC16 morphants with no distinct change in the number of apoptosis cell. Moreover, the conclusion from results of experiments in vitro was similar with those from in vivo.To investigate the molecular mechanism of ZDHHC16 in NSCs proliferation, we tested a series of transcription factor involved with NSCs proliferation. The expression levels of erm and pea3 were reduced after ZDHHC16 knockdown. Moreover, the levels of ERK1/2 phosphorylation were strongly reduced following telencephalon development in ZDHHC16 morphants compared to controls. However, the expression of ligands and receptors were not impaired. These results suggest one possibility that activity of a kinase in ERK pathway is regulated by ZDHHC16, thus, the ERK phosphorylation is impacted. Next, we attempted to understand the mechanism of ZDHHC16 in regulating ERK pathway. Thus, we perform the rescue experiment by co-injecting ZDHHC16△DHHC expression vector with zdhhc16MO. The ZDHHC16 ADHHC could not rescue the decreased levels of MO-induced ERK 1/2 comparing to full length ZDHHC16. Taken together, we elucidated that ZDHHC16 promotes NSCs proliferation in the zebrafish telencEphalon and regulates FGF/ERK transduction that dEpends on PATs activity.ConclusionNeural stem cells (NSCs) comprise a relatively undifferentiated population of cells which could sustain certain numbers and differentiate into neurons and glial cells under the control of a complex array of intrinsic and extrinsic factors. The maintenance of NSCs is a tightly regulated process and is crucial for proper brain development and homeostasis. Our research aimed to explore the role and molecular mechanism of ZDHHC16 in NSCs proliferation.Our results suggest that ZDHHC16 activity is essential for early NSCs proliferation where it acts to activate the FGF/ERK network, allowing for the initiation of proliferarion-regulated gene expression programs, depends on PATs activity. These studies highlight the growing importance of DHHC family and their PAT activity during neurodevelopment. More importantly, future studies will provide new clues towards mechanism detection and clinical treatment of neural tube malformations related to telencephalon.Chapter II Function and mechanism of ZDHHC17 during axon outgrowthBackgroudZDHHC17, a member of DHHC protein family, consists of 633 amino acid residues, including 6-8 ankyrin repeats in the N-terminal region followed by five predicted transmembrane helices. The protein contains a signature DHHC palmitoyl transferase motif located close to the fourth predicted transmembrane helix. ZDHHC17 is detected far more frequently at both mRNA and protein levels in neural system. This suggests that ZDHHC17 might be particularly important in neural development and neural system function. ZDHHC17 is implicated in occurance of Huntington’s disease since mice with reduced ZDHHC17 levels displayed the similar phenotype. In addition, other roles of ZDHHC17 are just beginning to be appreciated, including the mediation of Mg2+ transport, regulation of Ca2+channel functions and activation of c-Jun N-terminus kinase (JNK) pathways. However, the roles and molecular mechanisms of ZDHHC17 in neural development are not yet fully understood. Here we used zdhhc17 morpholino (MO) in zebrafish and zdhhcl 7-specific siRNA in NSCs and NGF-induced PC 12 cells to demonstrate the function and mechanisms of ZDHHC17 during neural development.ResultsTo investigate the function of ZDHHC17 during neural development, we first injected MO oligonucleotides to target against the translation initiation site and mouse ZDHHC17 mRNA in one-cell stage embryos. After injection, the MO-injected embryos appeared to develop normally compared with control MO-injected siblings. The motility of morphants was markedly lower than that of control embryos from 3 days post fertilization (dpf) onwards. In response to touch, morphant larvae moved significantly slower and swam shorter distances than control embryos. The results of immunolabeled the spinal neuron with Znpl and Islet 1 at 28hpf and 22hpf respectively showed that the spinal neuron axonal tracts of morphants were truncated, however, the numbers of spinal differentiated neurons between control and morphant embryos was not significant difference. This indicated that ZDHHC17 may affect spinal neuron outgrowth. To further analyzing the role of ZDHHC17 during neuron differentiation, we used isolated mouse NSCs and NGF-induced PC 12 as model in vitro, transfected with zdhhc17 siRNA and observed neuronal generation and outgrowth to identifying the role of ZDHHC17 during neuron differentiation. The results were consistent with those from experiments in vivo.Next, we further explored the mechanisms of ZDHHC17 in regulating the neuronal outgrowth. Using Western blot, we detected the critical factor of mitogen-activated protein kinase (MAPK) classes which has been shown to play important role in neuronal differentiation. Our results demonstrated that ZDHHC17 promoted ERK1/2 phosphorylation and activity in NGF induced PC12 cells with no influence on the phosphorylation of p38 and JNK.In order to investigate the mechanism of ZDHHC17 in ERK1/2 phosphorylation, we searched the database for protein structure. Since ZDHHC17 does not have a canonical kinase domain, we further attempted to clarify whether TrkA and cAMp-dependent protein kinase A (PKA), possible upstream kinases in the ERK1/2 signaling pathway, are involved. Coimmunoprecipitation experiments revealed that ZDHHC17 associated with TrkA but not PKA. Meanwhile, ZDHHC17 was linked to the trafficking capabilities of the TrkA. In zdhhc17 siRNA-treated induced PC 12 cells, the TrkA-positive endosomes exhibited perinuclear localization rather than peripheral localization as observed in control cells. These results suggest that the abnormalities in endosomal dynamics might contribute to the dysregulation of recEptor signaling in the endocytic pathway. Since TrkA could be phosphorylated, we speculated that ZDHHC17 might regulate the transport of TrkA-positive endosomes. As reported, the TrkA-positive endosome traffics along microtubules, and also affects microtubule dynamics. Thus, using immunoprecipitation, we observed abnormal downregulated of TrkA-tubulin complex formation in vitro and in vivo. Moreover, the association between TrkA and tubulin was exceedingly enhanced in the presence of ZDHHC17. Therefore, these results indicate that ZDHHC17 recruits TrkA and tubulin to form a trafficking module for TrkA recycling and signal transmission during neurite formation.At last, we explored the mechanisms of ZDHHC17 in TrkA-tubulin formation. We constructed DHHC17 △ANK to transfection NGF-induced PC12cells.The result shows that the effect of ZDHHC17was dependent on the ankyrin domain, because ZDHHC17△ did not clearly upregulate ERK1/2 phosphorylation as observed with wild-type ZDHHC17.ConclusionCorrect axonal growth during nervous system development is critical for synaptic transduction and nervous system function. Successful axon outgrowth and formation primarily depend on a permissive environment and the expression of growth-associated proteins (GAPs) in neurons. Our results demonstrate that ZDHHC17 is crucial for axon growth via a mechanism independent on its PAT activity by regulating the TrkA-tubulin complex formation specifically. Our results extend the function and molecular mechanism of ZDHHC17 during neural development, provide new clues to fully understand the biological mechanism of DHHC family members in vivo.