Prospective Approach to Deciphering the Impact of Intercellular Mitochondrial Transfer from Human Neural Stem Cells and Brain Tumor-Initiating Cells to Neighboring Astrocytes.

The communication between neural stem cells (NSCs) and surrounding astrocytes is essential for the homeostasis of the NSC niche. Intercellular mitochondrial transfer, a unique communication system that utilizes the formation of tunneling nanotubes for targeted mitochondrial transfer between donor and recipient cells, has recently been identified in a wide range of cell types. Intercellular mitochondrial transfer has also been observed between different types of cancer stem cells (CSCs) and their neighboring cells, including brain CSCs and astrocytes. CSC mitochondrial transfer significantly enhances overall tumor progression by reprogramming neighboring cells. Despite the urgent need to investigate this newly identified phenomenon, mitochondrial transfer in the central nervous system remains largely uncharacterized. In this study, we found evidence of intercellular mitochondrial transfer from human NSCs and from brain CSCs, also known as brain tumor-initiating cells (BTICs), to astrocytes in co-culture experiments. Both NSC and BTIC mitochondria triggered similar transcriptome changes upon transplantation into the recipient astrocytes. In contrast to NSCs, the transplanted mitochondria from BTICs had a significant proliferative effect on the recipient astrocytes. This study forms the basis for mechanistically deciphering the impact of intercellular mitochondrial transfer on recipient astrocytes, which will potentially provide us with new insights into the mechanisms of mitochondrial retrograde signaling.

The Impact of Social Isolation and Environmental Deprivation on Blood Pressure and Depression-Like Behavior in Young Male and Female Mice.

Background: Social isolation (SI) and loneliness are major adult and adolescent health concerns, particularly in the coronavirus disease 2019 (COVID-19) era. Recent prospective cohort studies indicate that older women who experienced both SI and loneliness had a significantly higher risk of cardiovascular disease (CVD). Hypertension, a well-established risk factor for CVD, is more prevalent in elderly women than men. Furthermore, a lack of social relationships is strongly associated with an increased risk of hypertension in middle-aged and elderly women compared to men. Although this has not been extensively studied, adolescents and young adults who experience loneliness or SI may also be at risk for CVD and depression. The purpose of this study was to examine the effect of SI on blood pressure and depression-like behavior in young male and female mice. Methods: Weaned C57BL/6 mice were randomly assigned (n = 6/group/sex) to either group housing (GH) or SI. Animals in the SI group were housed in individual cages for 8 weeks with no view of other animals. The cages were kept in ventilated racks to prevent pheromone exposure and socially isolated animals had no cage enrichment. Results: SI increased systolic, diastolic, and mean arterial blood pressure in females and elevated heart rate in both sexes. Body weight gain was dramatically increased in socially isolated females but tended to decrease in socially isolated males. In the forced swim test, which detects depression-like behavior, there was no difference between groups in total immobility time. The latency to immobility, however, was significantly decreased in socially isolated females. Serum concentrations of corticosterone and metanephrine did not differ between socially isolated and group-housed females, but corticosterone levels were significantly reduced in socially isolated males. Conclusions: Our results indicate that 8 weeks of SI leads to significant changes in blood pressure and heart rate and mild changes in depression-like behavior in young mice, with females affected more than males.

Inorganic polyphosphate as an energy source in tumorigenesis.

Cancer cells have high demands for energy to maintain their exceedingly proliferative growth. However, the mechanism of energy expenditure in cancer is not well understood. We hypothesize that cancer cells might utilize energy-rich inorganic polyphosphate (polyP), as energetic reserve. PolyP is comprised of orthophosphates linked by phosphoanhydride bonds, as in ATP. Here, we show that polyP is highly abundant in several types of cancer cells, including brain tumor-initiating cells (BTICs), i.e., stem-like cells derived from a mouse brain tumor model that we have previously described. The polymer is avidly consumed during starvation of the BTICs. Depletion of ATP by inhibiting glycolysis and mitochondrial ATP-synthase (OXPHOS) further decreases the levels of polyP and alters morphology of the cells. Moreover, enzymatic hydrolysis of the polymer impairs the viability of cancer cells and significantly deprives ATP stores. These results suggest that polyP might be utilized as a source of phosphate energy in cancer. While the role of polyP as an energy source is established for bacteria, this finding is the first demonstration that polyP may play a similar role in the metabolism of cancer cells.

Characterization of brain tumor initiating cells isolated from an animal model of CNS primitive neuroectodermal tumors.

CNS Primitive Neuroectodermal tumors (CNS-PNETs) are members of the embryonal family of malignant childhood brain tumors, which remain refractory to current therapeutic treatments. Current paradigm of brain tumorigenesis implicates brain tumor-initiating cells (BTIC) in the onset of tumorigenesis and tumor maintenance. However, despite their significance, there is currently no comprehensive characterization of CNS-PNETs BTICs. Recently, we described an animal model of CNS-PNET generated by orthotopic transplantation of human Radial Glial (RG) cells - the progenitor cells for adult neural stem cells (NSC) - into NOD-SCID mice brain and proposed that BTICs may play a role in the maintenance of these tumors. Here we report the characterization of BTIC lines derived from this CNS-PNET animal model. BTIC's orthotopic transplantation generated highly aggressive tumors also characterized as CNS-PNETs. The BTICs have the hallmarks of NSCs as they demonstrate self-renewing capacity and have the ability to differentiate into astrocytes and early migrating neurons. Moreover, the cells demonstrate aberrant accumulation of wild type tumor-suppressor protein p53, indicating its functional inactivation, highly up-regulated levels of onco-protein cMYC and the BTIC marker OCT3/4, along with metabolic switch to glycolysis - suggesting that these changes occurred in the early stages of tumorigenesis. Furthermore, based on RNA- and DNA-seq data, the BTICs did not acquire any transcriptome-changing genomic alterations indicating that the onset of tumorigenesis may be epigenetically driven. The study of these BTIC self-renewing cells in our model may enable uncovering the molecular alterations that are responsible for the onset and maintenance of the malignant PNET phenotype.

Stabilization of HIF-1α and HIF-2α, up-regulation of MYCC and accumulation of stabilized p53 constitute hallmarks of CNS-PNET animal model.

Recently, we described a new animal model of CNS primitive neuroectodermal tumors (CNS-PNET), which was generated by orthotopic transplantation of human Radial Glial (RG) cells into NOD-SCID mice's brain sub-ventricular zone. In the current study we conducted comprehensive RNA-Seq analyses to gain insights on the mechanisms underlying tumorigenesis in this mouse model of CNS-PNET. Here we show that the RNA-Seq profiles derived from these tumors cluster with those reported for patients' PNETs. Moreover, we found that (i) stabilization of HIF-1α and HIF-2α, which are involved in mediation of the hypoxic responses in the majority of cell types, (ii) up-regulation of MYCC, a key onco-protein whose dysregulation occurs in ~70% of human tumors, and (iii) accumulation of stabilized p53, which is commonly altered in human cancers, constitute hallmarks of our tumor model, and might represent the basis for CNS-PNET tumorigenesis in this model. We discuss the possibility that these three events might be interconnected. These results indicate that our model may prove invaluable to uncover the molecular events leading to MYCC and TP53 alterations, which would be of broader interest considering their relevance to many human malignancies. Lastly, this mouse model might prove useful for drug screening targeting MYCC and related members of its protein interaction network.

SPARC overexpression combined with radiation retards angiogenesis by suppressing VEGF-A via miR­410 in human neuroblastoma cells.

Neuroblastoma (NB) is the most common extra-cranial solid tumor in children and despite aggressive therapy survival rates remain low. One of the contributing factors for low survival rates is aggressive tumor angiogenesis, which is known to increase due to radiation, one of the standard therapies for neuroblastoma. Therefore, targeting tumor angiogenesis can be a viable add-on therapy for the treatment of neuroblastomas. In the present study, we demonstrate that overexpression of secreted protein acidic and rich in cysteine (SPARC) suppresses radiation induced angiogenesis in SK-N­BE(2) and NB1691 neuroblastoma cells. We observed that overexpression of SPARC in SK-N-BE(2) and NB1691 cells reduced radiation induced angiogenesis in an in vivo mouse dorsal skin model and an ex vivo chicken CAM (chorioallantoic-membrane) model and also reduced tumor size in subcutaneous mouse tumor models of NB. We also observed that SPARC overexpression reduces VEGF-A expression, in SK-N-BE(2) and NB1691 NB cells via miR-410, a VEGF-A targeting microRNA. SPARC overexpression alone or in combination with miR-410 and radiation was shown to be effective at reducing angiogenesis. Moreover, addition of miR-410 inhibitors reversed SPARC mediated inhibition of VEGF-A in NB1691 cells but not in SK-N-BE(2) NB cells. In conclusion, the present study demonstrates that the overexpression of SPARC in combination with radiation reduced tumor angiogenesis by downregulating VEGF-A via miR-410.

Enhanced catalytic site thermal stability of cold-adapted esterase EstK by a W208Y mutation.

Hydrophobic interactions are known to play an important role for cold-adaptation of proteins; however, the role of amino acid residue, Trp, has not been systematically investigated. The extracellular esterase, EstK, which was isolated from the cold-adapted bacterium Pseudomonas mandelii, has 5 Trp residues. In this study, the effects of Trp mutation on thermal stability, catalytic activity, and conformational change of EstK were investigated. Among the 5 Trp residues, W(208) was the most crucial in maintaining structural conformation and thermal stability of the enzyme. Surprisingly, mutation of W(208) to Tyr (W(208)Y) showed an increased catalytic site thermal stability at ambient temperatures with a 13-fold increase in the activity at 40°C compared to wild-type EstK. The structure model of W(208)Y suggested that Y(208) could form a hydrogen bond with D(308), which is located next to catalytic residue H(307), stabilizing the catalytic domain. Interestingly, Tyr was conserved in the corresponding position of hyper-thermophilic esterases EstE1 and AFEST, which are active at high temperatures. Our study provides a novel insight into the engineering of the catalytic site of cold-adapted enzymes with increased thermal stability and catalytic activity at ambient temperatures.