Microtubule Integrity Is Associated with the Functional Activity of Mitochondria in HEK293

Mitochondria move along the microtubule network and produce bioenergy in the cell. However, there is no report of a relationship between bioenergetic activity of mitochondria and microtubule stability in mammalian cells. This study aimed to investigate this relationship. We treated HEK293 cells with microtubule stabilizers (Taxol and Epothilone D) or a microtubule disturber (vinorelbine), and performed live-cell imaging to determine whether mitochondrial morphology and bioenergetic activity depend on the microtubule status. Treatment with microtubule stabilizers enhanced the staining intensity of microtubules, significantly increased ATP production and the spare respiratory capacity, dramatically increased mitochondrial fusion, and promoted dynamic movement of mitochondria. By contrast, bioenergetic activity of mitochondria was significantly decreased in cells treated with the microtubule disturber. Our data suggest that microtubule stability promotes mitochondrial functional activity. In conclusion, a microtubule stabilizer can possibly recover mitochondrial functional activity in cells with unstable microtubules.

Ixabepilone: Overview of Effectiveness, Safety, and Tolerability in Metastatic Breast Cancer

Treatment algorithms for metastatic breast cancer describe sequential treatment with chemotherapy and, if appropriate, targeted therapy for as long as the patient receives benefit. The epothilone ixabepilone is a microtubule stabilizer approved as a monotherapy and in combination with capecitabine for the treatment of metastatic breast cancer in patients with demonstrated resistance to anthracyclines and taxanes. While chemotherapy and endocrine therapy form the backbone of treatment for metastatic breast cancer, the epothilone drug class has distinguished itself for efficacy and safety among patients with disease progression during treatment with chemotherapy. In phase III trials, ixabepilone has extended progression-free survival and increased overall response rates, with a manageable toxicity profile. Recent analyses of subpopulations within large pooled datasets have characterized the clinical benefit for progression-free survival and overall survival for ixabepilone in special populations, such as patients with triple-negative breast cancer or those who relapsed within 12 months of prior treatment. Additional investigation settings for ixabepilone therapy discussed here include adjuvant therapy, weekly dosing schedules, and ixabepilone in new combinations of treatment. As with other microtubule stabilizers, ixabepilone treatment can lead to peripheral neuropathy, but evidence-based management strategies may reverse these symptoms. Dose reductions did not appear to have an impact on the efficacy of ixabepilone plus capecitabine. Incorporation of ixabepilone into individualized treatment plans can extend progression-free survival in a patient population that continues to represent an unmet need.

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

Role of microtubules in Piezo2 mechanotransduction of mouse Merkel cells

Piezo2 channels are expressed in Merkel cells to mediate mechanotransduction leading to the sense of touch. Here we determined the role of microtubules in regulating Piezo2-mediated mechanotransduction in Merkel cells. Piezo2-mediated currents in Merkel cells are potentiated by microtubule stabilizer paclitaxel but reduced by microtubule destabilizer vincristine. Mechanically evoked afferent impulses are also enhanced by microtubule stabilizers and suppressed by microtubule destabilizers. Microtubules may play an essential role in Piezo2 mechanotransduction in Merkel cells.

Investigation of Low Dose Cabazitaxel Potential as Microtubule Stabilizer in Experimental Model of Alzheimer's Disease: Restoring Neuronal Cytoskeleton

Background: Neuronal Microtubule (MT) tau protein, providing cytoskeleton to neuronal cells, plays a vital role, including maintenance of cell shape, intracellular transport, and cell division. Tau hyperphosphorylation mediated MT destabilization results in axonopathy, additionally neurotransmitter deficit and ultimately causing Alzheimer's disease. Pre-clinically, streptozotocin (3mg/kg, 10μl/ unilateral, ICV) stereotaxically mimics the behavioral and neurochemical alterations similar to Alzheimer's tau pathology resulting in MT assembly defects further lead to neuropathological cascades. Objective: Clinically approved medications such as Donepezil (DNP), rivastigmine, and Memantine (MEM) are responsible for symptomatic care only, but there is no specific pharmacological intervention that directly interacts with the neuronal microtubule destabilization. Methods: The current study focused on the involvement of anti-cancer agent microtubule stabilizer cabazitaxel at a low dose (0.5 and 2 mg/kg) alone and in combination with standard drugs DNP (5 mg/kg), MEM (10 mg/kg) and microtubule stabilizer Epothilone D (EpoD) (3 mg/kg) in the prevention of intracerebroventricular streptozotocin (ICV-STZ) intoxicated microtubule-associated tau protein hyperphosphorylation. Results: Chronic treatment of CBZ at a low dose alone and in combination with standard drugs showing no side effect and significantly improve the cognitive impairment, neurochemical alterations along with reducing the level of hyperphosphorylated tau by preventing the breakdown of the neuronal cytoskeleton, respectively. Conclusion: The above findings suggested that CBZ at low dose show neuroprotective effects against ICV-STZ induced microtubule-associated tau protein hyperphosphorylation in rats and may be an effective agent for the preventive treatment of AD.

Preservation of the microtubule network to beat atrial fibrillation: role of SR-mitochondrial contacts

Background Atrial fibrillation (AF), the most common tachyarrhythmia, is a progressive disease characterized by electrophathology, which is defined as the structural remodeling of atrial cardiomyocytes that underlies electrophysiological remodeling and contractile dysfunction and consequently AF progression. We recently discovered that disruption of microtubule network, by HDAC6 activation, is a key factor underlying structural remodeling and AF promotion in experimental models for AF and patients with AF. However, the molecular mechanism how microtubule disruption induces contractile dysfunction in AF is unclear. Sarcoplasmic reticulum (SR) and mitochondria are the central organelles for normal cardiomyocyte contraction by controlling the Ca2+ and energy (ATP) homeostasis. The crosstalk between SR and mitochondria via contacts, termed SR-mitochondria contacts (SMCs), is essential for normal mitochondrial and cardiac function. Interestingly, functional SMCs are highly dependent on intact microtubule network. This study aims to whether preservation of microtubule protects against AF via SMC pathway. Methods and results Tachypacing of HL-1 cardiomyocytes significantly impaired calcium transient (CaT) amplitude compared to normal paced cardiomyocytes. Pretreatment with microtubule stalilizer taxol and acetyl-CoA inducer β-hydroxybutyrate (βOHB) significantly protected against tachypacing-induced CaT loss. Moreover, by using immunoflurecent staining, mitochondrial associated membrane isolation and seahorse, we showed that tubacin, Taxol and βOHB also significantly inhibited the SMC reduction, attenuated tachypacing-induced mitochondrial dysfunction. Consistently, these microtubule stabilizer drugs also prevented tachypacing-induced contractile dysfunction in the Drosophila model for AF. Conclusions Preservation of microtubule network prevented the reduction of SMCs and the consequent mitochondrial and contractile dysfunction in HL-1 cardiomyocytes and Drosophila models for AF. Therefore, the microtubule-SMC pathway is a novel central therapeutic target for AF. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Dutch Heart Foundation

The RepID–CRL4 ubiquitin ligase complex regulates metaphase to anaphase transition via BUB3 degradation

The spindle assembly checkpoint (SAC) prevents premature chromosome segregation by inactivating the anaphase promoting complex/cyclosome (APC/C) until all chromosomes are properly attached to mitotic spindles. Here we identify a role for Cullin–RING ubiquitin ligase complex 4 (CRL4), known for modulating DNA replication, as a crucial mitotic regulator that triggers the termination of the SAC and enables chromosome segregation. CRL4 is recruited to chromatin by the replication origin binding protein RepID/DCAF14/PHIP. During mitosis, CRL4 dissociates from RepID and replaces it with RB Binding Protein 7 (RBBP7), which ubiquitinates the SAC mediator BUB3 to enable mitotic exit. During interphase, BUB3 is protected from CRL4-mediated degradation by associating with promyelocytic leukemia (PML) nuclear bodies, ensuring its availability upon mitotic onset. Deficiencies in RepID, CRL4 or RBBP7 delay mitotic exit, increase genomic instability and enhance sensitivity to paclitaxel, a microtubule stabilizer and anti-tumor drug.