Biology subjects

Neuzil, J.

Publications and source records attributed to Neuzil, J..

10 recordsLinked to original sources

bioRxiv2026

TRANSSULFURATION LINKS ASPARTATE-ASPARAGINE METABOLISM AND REDOX HOMEOSTASIS TO DRIVE TUMOR GROWTH

Cytosolic redox balance is tightly coupled to aspartate synthesis through the malate-aspartate shuttle, and limiting the malate-aspartate shuttle has been proposed to constrain tumor growth by restricting aspartate availability. Here we show that tumors derived from cancer cells lacking GOT1 and GOT2, the cytosolic and mitochondrial aspartate aminotransferases essential for as-partate production and malate-aspartate shuttle function, grow despite impaired canonical as-partate synthesis. This is because cytosolic redox state, not aspartate supply, is the primary metabolic bottleneck in GOT1/GOT2 knockout cells. Using single-cell transcriptomics, metabo-lite tracing, and a loss-of-function CRISPR screen, we find that these tumors engage an adaptive bypass in which availability of asparagine, a product of aspartate, enables serine- and methio-nine-dependent transsulfuration to generate -ketobutyrate, whose reduction regenerates cy-tosolic NAD and restores redox homeostasis. Pharmacological inhibition or genetic ablation of transsulfuration abrogates this asparagine-driven rescue. These findings define asparagine as a regulator of cytosolic NAD/NADH balance and reveal a link between amino acid metabolism and redox control that suggests transsulfuration as a targetable vulnerability in tumor redox maintenance. Significance statementAspartate synthesis and cytosolic redox balance are both coupled through the malate-aspartate shuttle. We show that the cytosolic NAD/NADH ratio, not aspartate supply, is a critical output of the malate-aspartate shuttle for tumor growth. Availability of asparagine, a product of aspar-tate, enables serine- and methionine-dependent transsulfuration to restore cytosolic NAD/NADH balance, proliferation and tumor growth independently of canonical aspartate pro-duction by the malate-aspartate shuttle. This defines asparagine as a regulator of cytosolic re-dox and identifies transsulfuration as a targetable vulnerability in tumor redox maintenance.

cancer biology↗

bioRxiv2026

α-Synuclein Triggers Intercellular Nanotubes Formation to Prevent Apoptosis in Astroglia by Promoting Stemness

Astrocytes play a significant role in neuroprotection by internalizing neurodegenerative aggregates and facilitating their degradation. Recent studies indicate that -Synuclein (-SYN) protofibrils promote the transfer of pathogenic aggregates and dysfunctional mitochondria between astroglia via tunneling nanotubes (TNTs), which enhances cell survival and resistance to apoptosis. However, the underlying mechanism of TNT-driven apoptosis resistance remains unclear. We find that -SYN protofibrils induce aberrant mitochondria with decreased membrane potential ({Psi}m) and promote dynamic actin remodeling by relocating phosphorylated focal adhesion kinase (pFAK) to the nucleus, which triggers TNT formation in human astrocytoma cell lines and primary murine astrocytes. The important novel finding of this study is that pFAK in the nucleus co-localizes with Nanog, a crucial transcription factor for preserving stemness, and the interaction between pFAK and Nanog is critical for promoting p53 degradation via Mdm2-mediated ubiquitination and upregulating autophagy, thereby supporting the survival of astroglia exposed to toxic -SYN protofibrils. ROCK inhibitor y-27632 also drives TNT-formation via pFAK translocation to the nucleus, colocalizes with Nanog, and enhances stemness-related gene expression. Inhibiting TNT with the actin depolymerizing agent cytochalasin-D prevents pFAK co-localization with Nanog in the nucleus and fails to protect cells from -SYN-induced apoptosis. Nanog knockdown does not degrade p53 and hinders cell rescue from apoptosis. Furthermore, these transient TNTs transfer mitochondria to adjacent cells, potentially helping maintain metabolic stability. This study reveals that the TNT formation pathway promotes pFAK-Nanog interaction in the nucleus, leading to p53 degradation, which protects astroglia against -SYN proteotoxicity and prevents apoptosis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/727344v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@18d8675org.highwire.dtl.DTLVardef@76a383org.highwire.dtl.DTLVardef@e92202org.highwire.dtl.DTLVardef@1b7ee4b_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

Restoration of mitochondrial function to metabolically stressed astrocytes by fusion with mesenchymal stromal cells.

Mitochondrial networks in eukaryotic cells are dynamic, undergoing fusion, fission, biogenesis and transfer between cells to manage cellular requirements for energy production and biosynthetic components. Mitochondrial transfer has been observed in response to cellular injury and can happen by a variety of mechanisms. To examine whether mitochondria could be restored to injured astrocytes, we established a model of discrete astrocyte-specific, mitochondrial-specific injury in a mixed culture model. Immortalized {rho}0 astrocytes were generated, then put under lethal metabolic stress by withdrawal of pyruvate and uridine. When cells stressed in this way were grown in the presence of mesenchymal stromal cells, enhanced survival of the {rho}0 cells was seen, accompanied by the rapid acquisition of an entire mitochondrial networks. Gap-fill ligation and rolling circle amplification for cell-specific mitochondrial genome SNP confirmed that the mitochondrial networks were derived from the mesenchymal stromal donor cells, and both mitochondrial DNA and mitochondrial function were restored to the p0 cells long-term. The presence of two nuclei in the {rho}0 cells in the early stages of mitochondrial transfer and recovery was observed. This demonstrated that in this instance, the mechanism of mitochondrial rescue was fusion of the {rho}0 astrocyte and the mesenchymal stromal cell. In support of this, other neural cells were similarly shown to fuse with stressed {rho}0 astrocytes, suggesting that whole organelles may be donated to restore critical mitochondrial function.

cell biology↗

bioRxiv2024

Bioenergetics of human spermatozoa in patients with testicular germ cell tumour

In testicular germ cell tumour (TGCT) patients, sperm cryopreservation prior to anti-cancer treatment represents the main fertility preservation approach. However, it is associated with low sperm recovery rate after thawing. Since sperm is a high-energy demanding cell, which is supplied by glycolysis and oxidative phosphorylation (OXPHOS), mitochondrial dysfunctionality can directly result in sperm anomalies. In this study, we investigated the bioenergetic pattern of cryopreserved sperm of TGCT patients in comparison with normozoospermic samples using two state-of-the-art methods; the Extracellular Flux Analyzer (XF Analyzer) and Two-Photon Fluorescence Lifetime imaging (2P-FLIM), in order to assess the contributions of OXPHOS and glycolysis to energy provision. A novel protocol for combined measurement of OXPHOS (Oxygen Consumption Rate - OCR) and glycolysis (Extracellular Acidification Rate - ECAR) using the XF Analyzer was developed together with a unique customized AI-based approach for semiautomated processing of 2P-FLIM images. Our study delivers optimized Low-HEPES modified Human Tubal Fluid media (mHTF) for sperm handling during pre-analytical and analytical phases to maintain sperm physiological parameters and optimal OCR, equivalent of OXPHOS. The negative effect of cryopreservation was signified by deterioration of both bioenergetic pathways represented by modified OCR and ECAR curves and the derived parameters. This was true for normozoospermic as well as TGCT samples, which showed even a stronger damage within the respiratory chain compared to the level of glycolytic activity impairment. The impact of cryopreservation and pathology are supported by 2P-FLIM analysis showing a significant decrease in bound NADH in contrast to unbound NAD(P)H which reflects decreased metabolic activity in samples from TGCT patients. Our study provides novel insight into the impact of TGCT on sperm bioenergetics and delivers a verified protocol to be used for assessment of human sperm metabolic activity, which can be a valuable tool for further research and clinical andrology. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=182 SRC="FIGDIR/small/595824v2_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@130b9eeorg.highwire.dtl.DTLVardef@1fec841org.highwire.dtl.DTLVardef@d401forg.highwire.dtl.DTLVardef@1eccff8_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

Characterization of regeneration initiating cells during Xenopus laevis tail regeneration

Embryos are regeneration and wound healing masters. They not only rapidly close their wounds, remodel injured tissue without a scar, but also regenerate body parts. Many animal models with variable regenerative capabilities have already been studied. Additionally, with the introduction of high throughput techniques, novel regeneration mechanisms including genes and signaling pathways, and specialized cell types required for regeneration control in spatial and temporal aspects have been identified. Until now our knowledge has been limited to primarily the late phases of regeneration (> 1 day post injury). In this paper, we reveal the critical steps for regeneration initiation. We have discovered Regeneration Initiating Cells (RICs) using single cell and spatial transcriptomic analyses during tail regeneration in Xenopus laevis. RICs are formed transiently from the basal epidermal cells and are critical for the modification of the surrounding extracellular matrix to allow for migration of other cell types such as regeneration organizing cells that further promote regeneration. Absence or deregulation of RICs leads to excessive extracellular matrix deposition and regeneration defects.

developmental biology↗

bioRxiv2022

Repurposing of mitochondria-targeted tamoxifen: Novel anti-cancer drug exhibits potent activity against major protozoan and fungal pathogens.

Many of the currently available anti-parasitic and anti-fungal frontline drugs have severe limitations, including adverse side effects, complex administration, and increasing occurrence of resistance. The discovery and development of new therapeutic agents is a costly and lengthy process. Therefore, repurposing drugs with already established clinical application offers an attractive, fast-track approach for novel treatment options. In this study, we show that the anti-cancer drug MitoTam, a mitochondria-targeted analog of tamoxifen, efficiently eliminates a wide range of evolutionarily distinct pathogens in vitro, including pathogenic fungi, Plasmodium falciparum, and several species of trypanosomatid parasites, causative agents of debilitating neglected tropical diseases. MitoTam treatment was also effective in vivo and significantly reduced parasitemia of two medically important parasites, Leishmania mexicana and Trypanosoma brucei, in their respective animal infection models. Functional analysis in the bloodstream form of T. brucei showed that MitoTam rapidly altered mitochondrial functions, particularly affecting cellular respiration, lowering ATP levels, and dissipating mitochondrial membrane potential. Our data suggest that the mode of action of MitoTam involves disruption of the inner mitochondrial membrane, leading to rapid organelle depolarization and cell death. Altogether, MitoTam is an excellent candidate drug against several important pathogens, for which there are no efficient therapies and for which drug development is not a priority. Author SummaryMitoTam, a mitochondrially targeted analog of tamoxifen, is a promising anti-cancer candidate drug acting by accumulating in and destabilizing cell mitochondria. In this study, we analyze its effect on a wide range of evolutionarily distinct and medically important pathogens. These include a) pathogenic fungi, Candida albicans, and Cryptococcus neoformans, ubiquitous opportunistic pathogens that cause life-threatening diseases in immunocompromised or immunologically deficient individuals; b) Plasmodium falciparum, the causative agent of human malaria; and c) several species of trypanosomatid parasites such as Trypanosoma cruzi, responsible for deadly Chagas disease in South America, Trypanosoma brucei, the causative agent of sleeping sickness in Africa, and Leishmania, the etiological agent of leishmaniasis, a spectrum of diseases ranging from usually self-healing but potentially disfiguring cutaneous and mucocutaneous leishmaniasis, to visceral leishmaniasis, which is invariably fatal if left untreated. We show that MitoTam efficiently kills these parasites in laboratory conditions and in the case of trypanosomes and leishmaniases suppress or at least slow down the infection in mouse model.

microbiology↗

bioRxiv2022

Genetic Impairment of Succinate Metabolism Disrupts Bioenergetic Sensing in Adrenal Neuroendocrine Tumor

Metabolic dysfunction mutations can impair energy sensing and cause cancer. Loss of function of mitochondrial TCA cycle enzyme, succinate dehydrogenase B (SDHB) results in various forms of cancer typified by pheochromocytoma (PC). Here we delineate a signaling cascade where the loss of SDHB induces the Warburg effect in PC tumors, triggers dysregulation of Ca2+ homeostasis, and aberrantly activates calpain and the protein kinase Cdk5, through conversion of its cofactor from p35 to p25. Consequently, aberrant Cdk5 initiates a cascade of phospho- signaling where GSK3 inhibition inactivates energy sensing by AMP-kinase through dephosphorylation of the AMP-kinase {gamma} subunit, PRKAG2. Overexpression of p25-GFP in mouse adrenal chromaffin cells also elicits this phosphorylation signaling and causes PC tumor formation. A novel Cdk5 inhibitor, MRT3-007, reversed this phospho-cascade, invoking an anti- Warburg effect, cell cycle arrest, and senescence-like phenotype. This therapeutic approach halted tumor progression in vivo. Thus, we reveal an important novel mechanistic feature of metabolic sensing and demonstrate that its dysregulation underlies tumor progression in PC and likely other cancers. HighlightsO_LILoss of SDHB function in pheochromocytoma causes Ca2+ dysregulation, calpain activation, and aberrant activation of the protein kinase Cdk5. C_LIO_LIHyperactive Cdk5 deregulates a GSK3/PRKAG2/AMPK signaling cascade. C_LIO_LIp25 overexpression and consequent aberrant Cdk5 activity in chromaffin cells causes pheochromocytoma. C_LIO_LIInhibition of Cdk5 activates the PRKAG2/AMPK/p53 signaling to rescue cell senescence and block PC tumor progression. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=175 SRC="FIGDIR/small/475410v2_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@16f0a48org.highwire.dtl.DTLVardef@10c1c7aorg.highwire.dtl.DTLVardef@21b100org.highwire.dtl.DTLVardef@1eb8674_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2021

Quantitative analysis of neuronal mitochondrial movement reveals patterns resulting from neurotoxicity of rotenone and 6-hydroxydopamine

Alterations in mitochondrial dynamics, including their trafficking, can present early manifestation of neuronal degeneration. However, current methodologies used to study mitochondrial trafficking events rely on parameters that are mostly altered in later stages of neurodegeneration. Our objective was to establish a reliable computational methodology to detect early alterations in neuronal mitochondrial trafficking. We propose a novel quantitative analysis of mitochondria trajectories based on innovative movement descriptors, including straightness, efficiency, anisotropy, and kurtosis. Using biological data from differentiated SH-SY5Y cells treated with mitochondrial toxicants 6-hydroxydopamine and rotenone, we evaluated time and dose-dependent alterations in trajectory descriptors. Mitochondrial movement was analyzed by total internal reflection fluorescence microscopy followed by computer modelling to describe the process. The stacks of individual images were analyzed by an open source MATLAB algorithm (www.github.com/kandelj/MitoSPT) and to characterize mitochondria trajectories, we used the Python package trajpy (https://github.com/ocbe-uio/trajpy/). Our results confirm that this computational approach is effective and accurate in order to study mitochondrial motility and trajectories in the context of healthy and diseased neurons in different stages.

bioinformatics↗

bioRxiv2020

Mitochondria-adaptor TRAK1 promotes kinesin-1 driven transport in crowded environments

Intracellular trafficking of organelles, driven by kinesin-1 stepping along microtubules, underpins essential processes including neuronal activity. In absence of other proteins on the microtubule surface, kinesin-1 performs micron-long runs. Under protein crowding conditions, however, kinesin-1 motility is drastically impeded. It is thus unclear how kinesin-1 acts as an efficient transporter in crowded intracellular environments. Here, we demonstrate that TRAK1 (Milton), an adaptor protein essential for mitochondrial trafficking, activates kinesin-1 and increases its robustness of stepping in protein crowding conditions. Interaction with TRAK1 i) facilitated kinesin-1 navigation around obstacles, ii) increased the probability of kinesin-1 passing through cohesive envelopes of tau and iii) increased the run length of kinesin-1 in cell lysate. We explain the enhanced motility by the observed direct interaction of TRAK1 with microtubules, providing an additional anchor for the kinesin-1-TRAK1 complex. We propose adaptor-mediated tethering as a mechanism regulating kinesin-1 motility in various cellular environments.

cell biology↗

bioRxiv2019

Marizomib suppresses triple-negative breast cancer via proteasome and oxidative phosphorylation inhibition

Lacking effective targeted therapies, triple-negative breast cancer (TNBCs) is highly aggressive with development of metastasis especially brain, and remains clinically challenging breast cancer subtype to treat. Despite the survival dependency on the proteasome pathway genes, FDA-approved proteasome inhibitors induced minimal clinical response in breast cancer patients due to weak proteasome inhibition. Here, we show that a potent proteasome inhibitor Marizomib (Mzb) inhibits multiple proteasome catalytic activities and induces a better anti-tumor response in TNBC cell lines and patient-derived xenografts alone and in combination with the standard-of-care chemotherapy. Mechanistically, Mzb inhibits oxidative phosphorylation (OXPHOS) via PGC-1 suppression in conjunction with proteasome inhibition in TNBC cells. Mzb reduces lung and brain metastases by reducing the number of circulating tumor cells and the expression of multiple genes involved in the epithelial-to-mesenchymal transition. Furthermore, Mzb-induced OXPHOS inhibition upregulates glycolysis to meet the energetic demands of TNBC cells and, hence, combined inhibition of glycolysis with Mzb exposure leads to a synergistic anti-cancer activity. Collectively, our data provide a strong rationale for a clinical evaluation of Mzb in primary and metastatic TNBC patients.\n\nOne Sentence SummaryMarizomib inhibits primary tumor growth, and also reduces lung and brain metastases in pre-clinical models of triple-negative breast cancer.

cancer biology↗