P–205 Epothilone D as an actin cytoskeleton stabilizer improved mitochondria bioenergenesis and blastocyst formation of mouse preimplantation embryo

Study question What is primary factor of bioenergetics product activity between microtubule instability and the functional activity of mitochondria in embryo? Summary answer The actin cytoskeleton instability is presumably the primary cause for the bioenergenesis of mitochondrial function to the preimplantation embryo development. What is known already Mitochondria are cellular organelles dynamically moving and morphological changes. It provides for homeostatic energy to the cell. The dynamic property of the mitochondria is associated with the microtubule network in the cell. However, the stability of the microtubule was clearly identified for preimplantation embryo development. Study design, size, duration This study is designed to assess the ATP productivity of the mitochondria, and specifically to observe what its primary factor is in terms of providing microtubule stability in mammalian cells. Additionally, we investigated the relationship between blastocyst formation and actin cytoskeleton stabilization by EpD with 2-cell mice. Participants/materials, setting, methods We prepared the microtubule stability regulation model with the HEK293 cell line by using the microtubule stabilizer as an Epothilone D (EpD). Then we analyzed the metabolic activity of the cells through oxidative phosphorylation (OXP) ratios analysis. Also, we performed confocal live imaging to observe mitochondria morphology depending on the cells’ microtubule. Next, we treated EpD to 2-cell culture media for the analysis of blastocyst development ratios. Main results and the role of chance EpD significantly increased fusion form. Also, EpD enhance bioenergy ratios like OXP in the mitochondria and functional activity related marker, like mTOR compared with the control. These results suggest that microtubule stabilization enhances mitochondrial metabolism by increasing oxygen consumption. Also, EpD in 2-cell culture media led to a significant increase in the speed of development and 50% higher hatched out blastocyst formation ratios compared to the control group. Limitations, reasons for caution This study had limited animal experiments. For the next study, we are planning with an aim to improve the quality and development ratios of human embryos by EpD. Wider implications of the findings: Microtubule stabilizer has a possibility to recover the mitochondria’s functional activity in the preimplantation embryo development. Mitochondrial functional activity along the actin cytoskeleton may play a pivotal role in determining the embryo quality and development ratios for archive pregnancy. Trial registration number non-clinical trials

Upregulation of Apol8 by Epothilone D facilitates the neuronal relay of transplanted NSCs in spinal cord injury

Background Microtubule-stabilizing agents have been demonstrated to modulate axonal sprouting during neuronal disease. One such agent, Epothilone D, has been used to treat spinal cord injury (SCI) by promoting axonal sprouting at the lesion site after SCI. However, the role of Epothilone D in the differentiation of neural stem cells (NSCs) in SCI repair is unknown. In the present study, we mainly explored the effects and mechanisms of Epothilone D on the neuronal differentiation of NSCs and revealed a potential new SCI treatment. Methods In vitro differentiation assays, western blotting, and quantitative real-time polymerase chain reaction were used to detect the effects of Epothilone D on NSC differentiation. Retrograde tracing using a pseudotyped rabies virus was then used to detect neuronal circuit construction. RNA sequencing (RNA-Seq) was valuable for exploring the target gene involved in the neuronal differentiation stimulated by Epothilone D. In addition, lentivirus-induced overexpression and RNA interference technology were applied to demonstrate the function of the target gene. Last, an Apol8-NSC-linear ordered collagen scaffold (LOCS) graft was prepared to treat a mouse model of SCI, and functional and electrophysiological evaluations were performed. Results We first revealed that Epothilone D promoted the neuronal differentiation of cultured NSCs and facilitated neuronal relay formation in the injured site after SCI. Furthermore, the RNA-Seq results demonstrated that Apol8 was upregulated during Epothilone D-induced neuronal relay formation. Lentivirus-mediated Apol8 overexpression in NSCs (Apol8-NSCs) promoted NSC differentiation toward neurons, and an Apol8 interference assay showed that Apol8 had a role in promoting neuronal differentiation under the induction of Epothilone D. Last, Apol8-NSC transplantation with LOCS promoted the neuronal differentiation of transplanted NSCs in the lesion site as well as synapse formation, thus improving the motor function of mice with complete spinal cord transection. Conclusions Epothilone D can promote the neuronal differentiation of NSCs by upregulating Apol8, which may provide a promising therapeutic target for SCI repair.

Engineering the acyltransferase domain of epothilone polyketide synthase to alter the substrate specificity

Background Polyketide synthases (PKSs) include ketone synthase (KS), acyltransferase (AT) and acyl carrier protein (ACP) domains to catalyse the elongation of polyketide chains. Some PKSs also contain ketoreductase (KR), dehydratase (DH) and enoylreductase (ER) domains as modification domains. Insertion, deletion or substitution of the catalytic domains may lead to the production of novel polyketide derivatives or to the accumulation of desired products. Epothilones are 16-membered macrolides that have been used as anticancer drugs. The substrate promiscuity of the module 4 AT domain of the epothilone PKS (EPOAT4) results in production of epothilone mixtures; substitution of this domain may change the ratios of epothilones. In addition, there are two dormant domains in module 9 of the epothilone PKS. Removing these redundant domains to generate a simpler and more efficient assembly line is a desirable goal. Results The substitution of module 4 drastically diminished the activity of epothilone PKS. However, with careful design of the KS-AT linker and the post-AT linker, replacing EPOAT4 with EPOAT2, EPOAT6, EPOAT7 or EPOAT8 (specifically incorporating methylmalonyl-CoA (MMCoA)) significantly increased the ratio of epothilone D (4) to epothilone C (3) (the highest ratio of 4:3 = 4.6:1), whereas the ratio of 4:3 in the parental strain Schlegelella brevitalea 104-1 was 1.4:1. We also obtained three strains by swapping EPOAT4 with EPOAT3, EPOAT5, or EPOAT9, which specifically incorporate malonyl-CoA (MCoA). These strains produced only epothilone C, and the yield was increased by a factor of 1.8 compared to that of parental strain 104-1. Furthermore, mutations of five residues in the AT domain identified Ser310 as the critical factor for MMCoA recognition in EPOAT4. Then, the mutation of His308 to valine or tyrosine combined with the mutation of Phe310 to serine further altered the product ratios. At the same time, we successfully deleted the inactive module 9 DH and ER domains and fused the ΨKR domain with the KR domain through an ~ 25-residue linker to generate a productive and simplified epothilone PKS. Conclusions These results suggested that the substitution and deletion of catalytic domains effectively produces desirable compounds and that selection of the linkers between domains is crucial for maintaining intact PKS catalytic activity.

Enhancing microtubule stabilization rescues cognitive deficits and ameliorates pathological phenotype in an amyloidogenic Alzheimer’s disease model

In Alzheimer’s disease (AD), and other tauopathies, microtubule destabilization compromises axonal and synaptic integrity contributing to neurodegeneration. These diseases are characterized by the intracellular accumulation of hyperphosphorylated tau leading to neurofibrillary pathology. AD brains also accumulate amyloid-beta (Aβ) deposits. However, the effect of microtubule stabilizing agents on Aβ pathology has not been assessed so far. Here we have evaluated the impact of the brain-penetrant microtubule-stabilizing agent Epothilone D (EpoD) in an amyloidogenic model of AD. Three-month-old APP/PS1 mice, before the pathology onset, were weekly injected with EpoD for 3 months. Treated mice showed significant decrease in the phospho-tau levels and, more interesting, in the intracellular and extracellular hippocampal Aβ accumulation, including the soluble oligomeric forms. Moreover, a significant cognitive improvement and amelioration of the synaptic and neuritic pathology was found. Remarkably, EpoD exerted a neuroprotective effect on SOM-interneurons, a highly AD-vulnerable GABAergic subpopulation. Therefore, our results suggested that EpoD improved microtubule dynamics and axonal transport in an AD-like context, reducing tau and Aβ levels and promoting neuronal and cognitive protection. These results underline the existence of a crosstalk between cytoskeleton pathology and the two major AD protein lesions. Therefore, microtubule stabilizers could be considered therapeutic agents to slow the progression of both tau and Aβ pathology.

Studies toward the Total Synthesis of Epothilone D: Synthesis of the Thiazole-Containing Northern Segment

Epothilone D, a microtubule-stabilizing macrolide, is an attractive synthetic target molecule as a potential anti-cancer drug candidate. As part of our ongoing synthetic studies of this natural product, this paper describes the synthesis of the thiazole-containing northern segment of epothilone D via an E-selective bromomethylenation and a Ni/Cr-mediated cross-coupling reaction.