Margarita Glazova

Apoptotic signaling proteins: possible participation in the regulation of vasopressin and catecholamines biosynthesis in the hypothalamus

The role of apoptotic signaling proteins for long-lived neurons in the mature brain is poorly understood. Recently, we have shown that water deprivation leads to the activation of vasopressin (VP) secretion and expression of Bcl-2 and caspase-9 apototic proteins in the hypothalamus of the rat brain. In the present work, we continued to study a possible relationship between the functional activity of neurosecretory cells of the hypothalamus and apoptosis related proteins. We found that water deprivation leads to simultaneous activation of synthesis of VP and p53 and Bcl-2 apoptotic proteins in the mouse brain. To study a possible effect of apoptotic proteins on the functional state of hypothalamic neurons, the VP and tyrosine hydroxylase (TH) synthesis were analyzed in p53, p21Waf1/Cip1 and Bcl-2 deficient mice. Loss of p53 and Bcl-2 significantly reduced VP synthesis in paraventricular and supraoptic nuclei and TH expression in arcuat, periventricular and zona incerta nuclei of the hypothalamus. Surprisingly, in contrast with the loss of p53, the inactivation of p21Waf1/Cip1 up-regulates the expression of VP and TH. These data indicate that p53, p21Waf1/Cip1 and Bcl-2 proteins, besides affecting cell cycle, tumor suppression and apoptosis, may act as modulators of neurosecretory activity of hypothalamic neurons; however, this problem remains to be determined more detailed.

Role of neuronal nitric oxide in the regulation of vasopressin expression and release in response to inhibition of catecholamine synthesis and dehydration

We used neuronal nitric oxide synthase (nNOS) gene knockout mice to study the effects of catecholamines and neuronal nitric oxide on vasopressin expression in the hypothalamic neurosecretory centers. nNOS gene deletion did not change the level of vasopressin mRNA in the supraoptic or paraventricular nuclei. In contrast, vasopressin immunoreactivity was lower in nNOS deficient mice than in wild-type animals. Dehydration increased vasopressin mRNA levels and decreased vasopressin immunoreactivity in both wild-type and nNOS knockout mice, but these responses were more marked in the nNOS knockout mice. Treatment with alpha-mpt, a pharmacologic inhibitor of catecholamine synthesis, resulted in increased vasopressin mRNA levels in wild-type mice and in reduced vasopressin immunoreactivity in both wild-type and nNOS knockout mice. From these results, we conclude: (1) neuronal nitric oxide suppresses vasopressin expression under basal conditions and during activation of the vasopressinergic system by dehydration; (2) catecholamines limit vasopressin expression; (3) nNOS is required for the effects of catecholamines on vasopressin expression.

Neurogenic potential of spinal cord organotypic culture.

There are several neurogenic niches in the adult mammalian central nervous system. In the central nervous system, neural stem cells (NSC) localize not only to the periventricular area, but are also diffusely distributed in the parenchyma. Here, we assessed neurogenic potential of organotypic cultures prepared from adult mouse spinal cord. Slices were placed on Millipore inserts for organotypic culture and incubated in neurobasal media supplemented with B27 and N2 for up to 9 weeks. After 3-4 weeks, the cells aggregates formed in the slices. The aggregates cells were BrdU-uptake, nestin and alkaline phosphatase positive. At the later stage of incubation, we observed Oct3/4 in the inner mass of the neurospheres as well as expression of Dppa1, which is an Oct-4 downstream target gene and a marker for pluripotency. To check differentiation, the formed neurospheres were isolated and cultured for several days in differentiation media. The obtained data demonstrated the cells from isolated neurospheres differentiate into astrocytes and MAP2-positive neurons. Immunostaining for HB9 and Lim2 revealed subsequent differentiation of MAP2-positive cells into motor neurons and interneurons, respectively. We hypothesized neuronal loss and/or long-term culturing of spinal cord slices may trigger a reset of the internal cell program and promote proliferation and further differentiation of NSC.

Embryonic stem cells inhibit expression of erythropoietin in the injured spinal cord.

Recent observations have demonstrated neuroprotective role of erythropoietin (Epo) and Epo receptor in the central nervous system. Here we examined Epo function in the murine spinal cord after transplantation of pluripotent mouse embryonic stem (ES) cells pre-differentiated towards neuronal type following spinal cord injury. Expression of Epo was measured at both mRNA and protein levels in the ES cells as well as in the spinal cords after 1 and 7 days. Our data demonstrated that expression of Epo mRNA, as well as its protein content, in ES cells was significantly decreased after differentiation procedure. In the spinal cords, analysis showed that Epo mRNA level was significantly decreased after 1 day of ES cell injections in comparison to media-injected control. Epo protein level detected by Western blot was diminished as well. Examination of Epo production in the injured spinal cords after media or ES cells injections by indirect immunofluorescence showed increased Epo-immunopositive staining after media injections 1 day after injection. In contrast, ES cell transplantation did not induce Epo expression. Seven days after ES cell injections, Epo-immunopositive cells distribution in the ipsilateral side was not changed, while the intensity of immunostaining on the contralateral side was increased, approaching levels in control media-injected tissues. Our data let us to presume that previously described immediate positive effects of ES cells injected into the injured zone of spinal cord are not based on Epo, but on other factors or hormones, which should be elucidated further.

Apoptosis and proliferation in the inferior colliculus during postnatal development and epileptogenesis in audiogenic Krushinsky–Molodkina rats

It is known that audiogenic seizure (AGS) expression is based on the activation of the midbrain structures such as the inferior colliculus (IC). It was demonstrated that excessive sound exposure during the postnatal developments of the IC in rats led to AGS susceptibility in adulthood, which correlated with underdevelopment of the IC. In adult rodents, noise overstimulation induced apoptosis in the IC. The purpose of this study was to investigate postnatal development of the IC in rats genetically prone to AGS and to check if audiogenic kindling would activate apoptosis and/or proliferation in the IC. In our study, we used inbred audiogenic Krushinsky-Molodkina (KM) rats, which are characterized by age-dependent seizure expression. Analysis of postnatal development showed the increased number of proliferating cells in the IC central nucleus of KM rats on the 14th postnatal day (P14) in comparison with those of Wistar rats. Moreover, we also observed increased apoptosis level and decreased general cell population in the IC central nucleus. These data pointed towards a delayed development of the IC in KM rats. Analysis of the IC central nucleus of KM rat after audiogenic kindling for a week, with one AGS per day, demonstrated dramatically increased cell death, which was accompanied with a reduction of general cell population. Audiogenic kindling also decreased proliferation in the IC central nucleus. However, a week after the last AGS, the number of proliferating cells was increased, which supposes a certain compensatory mechanism to prevent cell loss.

Apoptotic signaling proteins as the factors control neural differentiation

B cancer stem cells (BCSCs) constitute a subpopulation of tumor cells that express stem cell associated markers and have a high capacity for tumor generation in vivo. Identification of BCSCs from tumor samples or breast cancer cell lines has been based mainly on CD44+/CD24−/low or ALDH+ phenotypes. BCSCs isolation has allowed the analysis of the molecular mechanisms involved in their origin, self-renewal, differentiation into tumor cells, resistance to radiation therapy and chemotherapy, and invasiveness and metastatic ability. Molecular genetic analysis using knockout animals and inducible transgenics has identified NF-[1]B, c-Jun, p21CIP1, and Forkhead-like-protein Dach1 involvement in BCSC expansion and fate. Clinical analyses of BCSCs in breast tumors have found a correlation between the proportion of BCSCs and poor prognosis. Therefore, new therapies that specifically target BCSCs are an urgent need. We summarize recent evidence that partially explain the biological characteristics of BCSCsA signaling proteins play the critical role during development of CNS. These proteins mainly control the size of developing neuronal populations. However, accumulating data demonstrated that apoptosis-related proteins participate in the regulation of ESC differentiation. For example, cell death-relevant proteins are functionally involved in differentiation of a wide range of cell types. We analyzed development of hypothalamus nuclei in Bcl-2 and p53 knockout mice. Obtained data demonstrated that Bcl-2 and p53 deficienciesaffected not only on the functional activity of neuroendocrine cells of hypothalamus, but also differentially changed the cell’s populations. These observation let us supposed that apoptosis-relevant proteins may participate in the regulation of differentiation of neuroendocrine cells of hypothalamus or to be involved in proliferation and/or differentiation of neural progenitor cells (NPC). We have studied differentiation/proliferation of neural progenitorsisolated from hippocampus of new-born mice. Inhibition of Bcl-2 with HA14-1 during differentiation initiated glia formation, while pifithrinalpha treatments (inhibitor of p53 activity) led to inhibition of neural differentiation but upregulated of NPC self-renewal. Thus, our data demonstrated that apoptosis signaling proteins may regulate the mechanisms of NPC differentiation.This work was supported by grants from Russian Foundation for Fundamental Research (RFBR 10-04-00127-a, and RFBR 11-04-00648-a)P brain injury remains a major cause of cerebral palsy and other adverse neurological outcomes. Although therapeutic hypothermia is now established to improve recovery from hypoxia–ischemia (HI) at term, many infants continue to survive with disability, and hypothermia has not yet been tested in preterm infants. Novel data indicates that the mechanisms of brain injury from hypoxia-ischemia and injury from exposure to intrauterine inflammation (as in cases of preterm birth) may follow converging mechanisms. There is increasing evidence from in vitro and in vivo trials that stem cells may have multiple beneficial effects on outcome after hypoxic–ischemic injury. Stem cell therapies have shown great promise in animal studies in decreasing neurological impairment. Although the mechanisms of action of stem cells, the optimal type, dose, and method of administration remain surprisingly unclear, cell based interventions after completion of the majority of secondary cell death appear to have potential to improve functional outcome for neonates after HI. Evaluation and understanding of mechanisms that lead to the improvement of perinatal brain injury and consideration cell therapy use for the inflammation-induced perinatal brain injury, as in cases of preterm birth, are our main goals to bring cell therapy from bench to bedsideI is critical to develop new therapeutic approaches for prostate cancer, since there is no effective treatment for patients in the advanced stages of this disease. And although many gene therapy approaches have been evaluated to date, clinical responses unfortunately remain poor. We have examined the potential of developing more effective cell-based gene therapies for preventing prostate cancer progression, using adipose-derived mesenchymal stem cells (ASC) as vehicles. ASC have gained interest as promising tools for delivering cancer therapy. Adipose tissue can be obtained readily in amounts sufficient for ASC isolation, which can be expanded rapidly, allowing its use at low passage numbers, and can be transduced by viral and nonviral means. And since current techniques allow isolation of ASC from an individual’s own adipose tissue, this could help prevent immune reaction, suggesting that ASC could be far more efficient as a gene delivery vehicle than viral vectors currently in clinical trials. The present study reports our observations using this novel gene therapy modality. In this study we have examined the potential of new antiangiogenesis and proapoptotic therapies delivered by ASC and evaluated their ability to reduce prostate tumor growth rate in vitro and in vivo. We expressed in ASC several antitumor cytokine therapies, and all were found to be highly effective in preventing tumor progression in vivo using prostate and breast cancer xenograft models. Overall, ASC-delivered PEDF prevented HUVEC tube formation in vitro and induced strong apoptosis of cocultured cancer cells, suggesting a potential bystander effect that might be useful for therapeutic applications. Similar antitumor findings were observed when Mda7 or IFNb were delivered by ASC, with induction of strong cell cycle arrest, and viability reduction by an increase in the apoptosis of cocultured tumor cells as assessed by Caspase 3/7 detection. The transgene expression was overall non-toxic and did not appear to interfere with the normal differentiation potential of ASC (osteogenesis or adipogenesis). We also observed that ASC retained their innate ability to migrate towards tumor cells in co-culture and this ability could be blocked by inhibition of CXCR4 signaling. ASC were found to be non-tumorigenic in vitro using a soft agar assay, as well as in vivo, utilizing two prostate cancer xenograft models. PEDF was the most promising therapeutic cytokine delivered, and completely prevented prostate tumor establishment in vivo of both the TC2Ras and PC3 highly aggressive prostate cancer models. In conclusion, ASC expressing antitumor cytokines could effectively reduce prostate tumor growth in vivo, suggesting ASC-cytokine therapies might have translational applications. Future studies will aim to target cytokines to tumor tissues by virtue of peptide targeting moieties to enhance cytokine therapy effectsT use of stem cells in cell replacement therapy for neurodegenerative diseases has received a great deal of scientific and public interest in recent years. Oxidative stress, toxic byproducts, which prevails in the microenvironment during the diseased condition, may limit the survival of the transplanted stem cells affecting tissue regeneration and even longevity. Recently, functional neural stem cells with transgenes proved to be a promising therapeutic strategy for various stress models both in vitro and in vivo. Our investigations aimed to clarify whether constitutive retroviral expression of arginine decarboxylase (ADC) genes induces differentiation of NSCs in vitro and transplantation of these functional stem cells could enhance their survival and increase ROS scavenging capacity, thereby suppress the pathogenesis in different CNS injury models. The spinal cord injured animals which received ADC-NSCs showed improvements in walking ability. Immunohistochemical staining results demonstrated that transplanted ADC-NSCs enhanced neurogenesis within the injury site, and these neurons extended their processes and formed synaptic structures. ADC-NSCs might attribute partly to a reduction in tissue loss from secondary injury processes as well as diminished glial scarring. In experimental stroke, transplantation of ADC-NSCs contributed for the increased functional recovery and decreased gliar scar formation. The ADC-NSCs transplanted at the penumbra area regulated the proliferation and differentiation at the ischemic injury site. These effects were confirmed by Ki67, BrdU, Tuj1, DCX, GFAP and VEGF immunohistochemical staining. Our study evaluated the ADC-NSCs are a potential source for transplantable material for therapy of spinal cord injury and experimental stroke models in vivoH play a central and crucial role for the production and elimination of serum cholesterol and lipid homeostasis and their proper function is of key importance for cardiovascular health. In particular, hepatocytes produce large amounts of endogenously-synthesized cholesterol, which is secreted into circulating blood in the form of apolipoprotein particles. Cholesterol-secreting hepatocytes are also clinically-relevant cells targeted by statin treatment in vivo. At present, the study of cholesterol homeostasis is largely restricted to the use of animal models and immortalized cell lines that fail to recapitulate many key aspects of normal human hepatocyte function. Hepatocyte-like cells (HLCs) derived from human embryonic and induced pluripotent stem cells (hESCs and hiPSCs) can potentially provide a cell culture model for the study of cholesterol homeostasis, dyslipidemias, and the action of pharmaceuticals important for cardiovascular health. This seminar will present research showing that differentiated cells resembling hepatocytes can be readily produced from hESCs and hiPSCs. The resulting HLCs exhibit many features of human hepatocytes in vivo, to include acivities related to serum lipid homeostasis. Overall, the research shows that HLCs derived from human pluripotent cells provide a robust cell culture system for the investigation of the hepatic contribution to cholesterol homeostasis at both cellular and molecular levels