The Metabolic Changes between Monolayer (2D) and Three-Dimensional (3D) Culture Conditions in Human Mesenchymal Stem/Stromal Cells Derived from Adipose Tissue.

INTRODUCTION: One of the key factors that may influence the therapeutic potential of mesenchymal stem/stromal cells (MSCs) is their metabolism. The switch between mitochondrial respiration and glycolysis can be affected by many factors, including the oxygen concentration and the spatial form of culture. This study compared the metabolic features of adipose-derived mesenchymal stem/stromal cells (ASCs) and dedifferentiated fat cells (DFATs) cultivated as monolayer or spheroid culture under 5% O2 concentration (physiological normoxia) and their impact on MSCs therapeutic abilities. RESULTS: We observed that the cells cultured as spheroids had a slightly lower viability and a reduced proliferation rate but a higher expression of the stemness-related transcriptional factors compared to the cells cultured in monolayer. The three-dimensional culture form increased mtDNA content, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), especially in DFATs-3D population. The DFATs spheroids also demonstrated increased levels of Complex V proteins and higher rates of ATP production. Moreover, increased reactive oxygen species and lower intracellular lactic acid levels were also found in 3D culture. CONCLUSION: Our results may suggest that metabolic reconfiguration accompanies the transition from 2D to 3D culture and the processes of both mitochondrial respiration and glycolysis become more active. Intensified metabolism might be associated with the increased demand for energy, which is needed to maintain the expression of pluripotency genes and stemness state.

Mitochondrial dynamics, elimination and biogenesis during post-ischemic recovery in ischemia-resistant and ischemia-vulnerable gerbil hippocampal regions.

Transient ischemic attacks (TIA) result from a temporary blockage in blood circulation in the brain. As TIAs cause disabilities and often precede full-scale strokes, the effects of TIA are investigated to develop neuroprotective therapies. We analyzed changes in mitochondrial network dynamics, mitophagy and biogenesis in sections of gerbil hippocampus characterized by a different neuronal survival rate after 5-minute ischemia-reperfusion (I/R) insult. Our research revealed a significantly greater mtDNA/nDNA ratio in CA2-3, DG hippocampal regions (5.8 ± 1.4 vs 3.6 ± 0.8 in CA1) that corresponded to a neuronal resistance to I/R. During reperfusion, an increase of pro-fission (phospho-Ser616-Drp1/Drp1) and pro-fusion proteins (1.6 ± 0.5 and 1.4 ± 0.3 for Mfn2 and Opa1, respectively) was observed in CA2-3, DG. Selective autophagy markers, PINK1 and SQSTM1/p62, were elevated 24-96 h after I/R and accompanied by significant elevation of transcription factors proteins PGC-1α and Nrf1 (1.2 ± 0.4, 1.78 ± 0.6, respectively) and increased respiratory chain proteins (e.g., 1.5 ± 0.3 for complex IV at I/R 96 h). Contrastingly, decreased enzymatic activity of citrate synthase, reduced Hsp60 protein level and electron transport chain subunits (0.88 ± 0.03, 0.74 ± 0.1 and 0.71 ± 0.1 for complex IV at I/R 96 h, respectively) were observed in I/R-vulnerable CA1. The phospho-Ser616-Drp1/Drp1 was increased while Mfn2 and total Opa1 reduced to 0.88 ± 0.1 and 0.77 ± 0.17, respectively. General autophagy, measured as LC3-II/I ratio, was activated 3 h after reperfusion reaching 2.37 ± 0.9 of control. This study demonstrated that enhanced mitochondrial fusion, followed by late and selective mitophagy and mitochondrial biogenesis might together contribute to reduced susceptibility to TIA.

Mitofusin 2 Integrates Mitochondrial Network Remodelling, Mitophagy and Renewal of Respiratory Chain Proteins in Neurons after Oxygen and Glucose Deprivation.

In attempts to develop effective therapeutic strategies to limit post-ischemic injury, mitochondria emerge as a key element determining neuronal fate. Mitochondrial damage can be alleviated by various mechanisms including mitochondrial network remodelling, mitochondrial elimination and mitochondrial protein biogenesis. However, the mechanisms regulating relationships between these phenomena are poorly understood. We hypothesized that mitofusin 2 (Mfn2), a mitochondrial GTPase involved in mitochondrial fusion, mitochondria trafficking and mitochondria and endoplasmic reticulum (ER) tethering, may act as one of linking and regulatory factors in neurons following ischemic insult. To verify this assumption, we performed temporal oxygen and glucose deprivation (OGD/R) on rat cortical primary culture to determine whether Mfn2 protein reduction affected the onset of mitophagy, subsequent mitochondrial biogenesis and thus neuronal survival. We found that Mfn2 knockdown increased neuronal susceptibility to OGD/R, prevented mitochondrial network remodelling and resulted in prolonged mitophagosomes formation in response to the insult. Next, Mfn2 knockdown was observed to be accompanied by reduced Parkin protein levels and increased Parkin accumulation on mitochondria. As for wild-type neurons, OGD/R insult was followed by an elevated mtDNA content and an increase in respiratory chain proteins. Neither of these phenomena were observed for Mfn2 knockdown neurons. Collectively, our findings showed that Mfn2 in neurons affected their response to mild and transient OGD stress, balancing the extent of defective mitochondria elimination and positively influencing mitochondrial respiratory protein levels. Our study suggests that Mfn2 is one of essential elements for neuronal response to ischemic insult, necessary for neuronal survival.

Mild palmitate treatment increases mitochondrial mass but does not affect EA.hy926 endothelial cells viability.

A dyslipidaemia-related increase of the concentration of long-chain fatty acids in the plasma is an important pathological factor substantially increasing risk of serious consequences in vascular endothelium. Inflammatory response, atherosclerosis and insulin resistance seem the most severe. Palmitate at excessive concentrations has been shown to have a harmful effect on endothelial cells impairing NO generation, stimulating reactive oxygen species (ROS) formation and affecting their viability. On the other hand we found that palmitate applied for 48 h at 100 µM concentration which is sufficient to induce inflammatory response, increase ROS generation and reduce insulin sensitivity of EA.hy926 cells, unexpectedly also stimulates NO synthesis and increases mitochondrial mass, suggesting a pro-survival rather than anti-survival effect. This finding unveils a potential protective mechanism allowing cells to maintain their energy homeostasis under conditions of a moderate deregulation of lipid metabolism.

The Effect of a Novel c.820C>T (Arg274Trp) Mutation in the Mitofusin 2 Gene on Fibroblast Metabolism and Clinical Manifestation in a Patient.

Charcot-Marie-Tooth disease type 2A (CMT2A) is an autosomal dominant axonal peripheral neuropathy caused by mutations in the mitofusin 2 gene (MFN2). Mitofusin 2 is a GTPase protein present in the outer mitochondrial membrane and responsible for regulation of mitochondrial network architecture via the fusion of mitochondria. As that fusion process is known to be strongly dependent on the GTPase activity of mitofusin 2, it is postulated that the MFN2 mutation within the GTPase domain may lead to impaired GTPase activity, and in turn to mitochondrial dysfunction. The work described here has therefore sought to verify the effects of MFN2 mutation within its GTPase domain on mitochondrial and endoplasmic reticulum morphology, as well as the mtDNA content in a cultured primary fibroblast obtained from a CMT2A patient harboring a de novo Arg274Trp mutation. In fact, all the parameters studied were affected significantly by the presence of the mutant MFN2 protein. However, using the stable model for mitofusin 2 obtained by us, we were next able to determine that the Arg274Trp mutation does not impact directly upon GTP binding. Such results were also confirmed for GTP-hydrolysis activity of MFN2 protein in patient fibroblast. We therefore suggest that the biological malfunctions observable with the disease are not consequences of impaired GTPase activity, but rather reflect an impaired contribution of the GTPase domain to other MFN2 activities involving that region, for example protein-protein interactions.

[Mitofusin 2 and mitochondrial dynamics in norm and pathology]. / Mitofuzyna 2 i dynamika mitochondriów w normie i patologii.

Results of an intensive research performed during last 25 years have revealed that an understanding of biochemical and molecular principles of oxidative phosphorylation has not finished the streak of ground-breaking discoveries of newly identified mitochondrial functions in numerous cellular processes. Among other things it has been shown that mitochondria undergo reversible fission and fusion processes, and may form a complex network which functionally and structurally interacts with the endoplasmic reticulum membranes and probably also other organelles. An organization of mitochondrial network is closely controlled and is of high importance for numerous intracellular processes to occur properly. In this review, mitofusin 2 - one of a few proteins involved in a maintenance of an appropriate mitochondrial architecture, and in the consequence in the regulation of mitochondrial metabolism and calcium signalling, the controlling of the mitochondrial DNA level, and the regulation of cell proliferation and differentiation is the focus. Mutations within mitofusin 2-encoding gene are a cause of Charcot-Marie-Tooh 2A - type neuropathies while an affected expression of this protein seems to be related to neoplasia, type 2 diabetes, or vascular hyperplasia. Numerous experimental data confirm pleiotropic effects of mitofisin 2 in animal cells.

Effects of the Hybridization of Opioid and Neurotensin Pharmacophores on Cell Survival in Rat Organotypic Hippocampal Slice Cultures.

Several neurotransmitter and neuromodulatory systems can control physiological glutamatergic activity. For example, opioid receptor ligands were shown to partially inhibit N-methyl-D-aspartic acid (NMDA) receptor-dependent glutamatergic excitotoxicity. Also, the endogenous tridecapeptide neurotensin (NT) was found to modulate excessive glutamate release and glutamate receptor activity in neurons. Alternatively to the one target-one drug approach, it has been well documented that hybrid compounds encompassing two pharmacophores in one molecular scaffold can represent more potent drugs. Moreover, such structures with dual activity can potentially enable a reduction of undesirable side effects and/or improved bioavailability. Herein, we describe the neuroprotective potential of an opioid-NT hybrid peptide (PK20), which was recently designed and synthesized within our group. The protective properties of PK20, assessed in an in vitro model of excitotoxic injury in organotypic hippocampal slice cultures subjected to NMDA, were compared to the effects caused by NT. Our results indicate that PK20 is a potent anti-neurodegenerative agent. Moreover, co-administered with NMDA, PK20 (25-100 ng/ml) dose-dependently reduced hippocampal cell death, determined by a decrease in the propidium iodide signal. We also report for the first time the significant NT-induced neuroprotective effect, as its application (50-100 ng/ml) to hippocampal slice cultures protected CA1 damage against neurotoxicity caused by NMDA.

Mitofusin 2 Deficiency Affects Energy Metabolism and Mitochondrial Biogenesis in MEF Cells.

Mitofusin 2 (Mfn2), mitochondrial outer membrane protein which is involved in rearrangement of these organelles, was first described in pathology of hypertension and diabetes, and more recently much attention is paid to its functions in Charcot-Marie-Tooth type 2A neuropathy (CMT2A). Here, cellular energy metabolism was investigated in mouse embryonic fibroblasts (MEF) differing in the presence of the Mfn2 gene; control (MEFwt) and with Mfn2 gene depleted MEFMfn2-/-. These two cell lines were compared in terms of various parameters characterizing mitochondrial bioenergetics. Here, we have shown that relative rate of proliferation of MEFMfn2-/- cells versus control fibroblasts depend on serum supplementation of the growth media. Moreover, MEFMfn2-/- cells exhibited significantly increased respiration rate in comparison to MEFwt, regardless of serum supplementation of the medium. This effect was correlated with increased level of mitochondrial markers (TOM20 and NAO) as well as mitochondrial transcription factor A (TFAM) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) protein levels and unchanged total ATP content. Interestingly, mitochondrial DNA content in MEFMfn2-/- cells was not reduced. Fundamentally, these results are in contrast to a commonly accepted belief that mitofusin 2 deficiency inevitably results in debilitation of mitochondrial energy metabolism. However, we suggest a balance between negative metabolic consequences of mitofusin 2 deficiency and adaptive processes exemplified by increased level of PGC-1α and TFAM transcription factor which prevent an excessive depletion of mtDNA and severe impairment of cell metabolism.

The organotypic longitudinal spinal cord slice culture for stem cell study.

The objective of this paper is to describe in detail the method of organotypic longitudinal spinal cord slice culture and the scientific basis for its potential utility. The technique is based on the interface method, which was described previously and thereafter was modified in our laboratory. The most important advantage of the presented model is the preservation of the intrinsic spinal cord fiber tract and the ventrodorsal polarity of the spinal cord. All the processes occurring during axonal growth, regeneration, synapse formation, and myelination could be visualized while being cultured in vitro for up to 4-5 weeks after the slices had been isolated. Both pups and adult animals can undergo the same, equally efficient procedures when going by the protocol in question. The urgent need for an appropriate in vitro model for spinal cord regeneration results from a greater number of clinical trials concerning regenerative medicine in the spinal cord injury and from still insufficient knowledge of the molecular mechanisms involved in the neuroreparative processes. The detailed method of organotypic longitudinal spinal cord slice culture is accompanied by examples of its application to studying biological processes to which both the CNS inhabiting and grafted cells are subjected.

Mitofusin 2 expression dominates over mitofusin 1 exclusively in mouse dorsal root ganglia - a possible explanation for peripheral nervous system involvement in Charcot-Marie-Tooth 2A.

Mitofusin 2 (Mfn2), a protein of the mitochondrial outer membrane, is essential for mitochondrial fusion and contributes to the maintenance and operation of the mitochondrial network. Mutations in the mitofusin 2 gene cause axonal Charcot-Marie-Tooth type 2A (CMT2A), an inherited disease affecting peripheral nerve axons. The precise mechanism by which mutations in MFN2 selectively cause the degeneration of long peripheral axons is not known. There is a hypothesis suggesting the involvement of reduced expression of a homologous protein, mitofusin 1 (Mfn1), in the peripheral nervous system, and less effective compensation of defective mitofusin 2 by mitofusin 1. We therefore aimed to perform an analysis of the mitofusin 1 and mitofusin 2 mRNA and protein expression profiles in different mouse tissues, with special attention paid to dorsal root ganglia (DRGs), as parts of the peripheral nervous system. Quantitative measurement relating to mRNA revealed that expression of the Mfn2 gene dominates over Mfn1 mainly in mouse DRG, as opposed to other nervous system samples and other tissues studied. This result was further supported by Western blot evaluation. Both these sets of data confirm the hypothesis that the cellular consequences of mutations in the mitofusin 2 gene can mostly be manifested in the peripheral nervous system.

Protein kinase C beta in postischemic brain mitochondria.

PKC is implicated in the regulation of mitochondrial metabolism. We examined the association of PKCß with mitochondria and followed postischemic changes in its amount in mitochondria isolated from ischemia-vulnerable (CA1) and ischemia-resistant (CA2-4,DG) hippocampus in gerbil model of transient brain ischemia. Our observations suggest that transient ischemic episode induces a significant, rapid and long lasting increase of PKCß in mitochondria in CA2-4,DG, which may bespeak neuroprotection. In organotypic hippocampal culture (OHC) model of neurodegeneration, PKCß inhibition imposed over NMDA toxicity extended the death area beyond the CA1. These results suggest that PKCß might have a protective effect against excitotoxic damage in rat OHC. The pull-down method and LC-MS/MS analysis revealed mitochondrial proteins that can bind directly with PKCßΙ. The proteins were parts of i) mitochondrial redox carriers forming the electron transport chain including ATP synthase and ii) MPTP: ANT and creatine kinase. PKCß acting through mitochondrial proteins could play a role in protecting the cells from death by e.g. influencing ROS and ATP production after ischemia in CA2-4,DG region of the hippocampus.

[Mitofusin 2 as a crucial peripheral nervous system protein and a common regulator of cell metabolism]. / Mitofuzyna 2--wazne bialko obwodowego ukladu nerwowego i powszechnie wystepujacy regulator metabolizmu.

Some of the metabolic disorders are manifested by a predominantly expressed symptoms from the single organ, however, they display discrete symptoms from other tissues. Charcot Marie Tooth disease (CMT) divided into demyelinating (CMT1) and axonal (CMT2) subtypes is characterized by a slowly progressive wasting of distal muscles. CMT2A form diagnosis requires identification of mutation in a gene coding for mitofusin 2 (MFN2). Mitofusin 2 is a protein of an outer mitochondrial membrane encoded in the nuclear genome and characterized by numerous biochemical functions. Mfn2 is involved mainly in the fusion of mitochondria and the cooperation between endoplasmic reticulum and mitochondria. It seems probably that Mfn2 possesses also some regulatory functions and takes part in a regulation of respiratory chain activity, transcription of several proteins and in intracellular signals transduction. Mfn2-linked pathology is also observed in diabetes and heart diseases. Here, we aim to show that mitofusin 2 is a protein crucial not only for peripheral nerve disorders but is a one of the common regulator of cell metabolism.

Neuroprotective potential of biphalin, multireceptor opioid peptide, against excitotoxic injury in hippocampal organotypic culture.

UNLABELLED: Biphalin is a dimeric opioid peptide that exhibits affinity for three types of opioid receptors (MOP, DOP and KOP). Biphalin is undergoing intensive preclinical study. It was recognized that activation of δ-opioid receptor elicits neuroprotection against brain hypoxia and ischemia. We compare the effect of biphalin and morphine and the inhibition of opioid receptors by naltrexone on survival of neurons in rat organotypic hippocampal cultures challenged with NMDA. FINDINGS: (1) 0.025-0.1 µM biphalin reduces NMDA-induced neuronal damage; (2) biphalin neuroprotection is abolished by naltrexone; (3) reduced number of dead cells is shown even if biphalin is applied with delay after NMDA challenge.