Permeation of individual cryoprotectants and their different combinations into mouse liver tissue.

Development of successful tissue cryopreservation methods requires specific knowledge regarding tissue permeation of individual cryoprotective agents (CPAs) and their combinations. The present study assessed the permeation of dimethyl sulfoxide, ethylene glycol, and propylene glycol into liver tissue, and addressed whether the diffusion coefficient of individual CPAs changes when combining CPAs. To do this, mouse liver slices were exposed at room temperature to 3.5 mol/L concentrations of CPAs individually or in combination for 15, 30, 45, and 60 min. Subsequently, tissue CPA concentrations were determined using a gas chromatography/mass spectrometry (GC/MS) method. Our results show that (1) the GC/MS method allows measurement of multiple CPA concentrations in a single small tissue sample, (2) dimethyl sulfoxide has a higher diffusion coefficient than ethylene glycol and propylene glycol, and (3) the CPA diffusivity appears to decrease in mixtures with multiple CPAs. These findings may help the development of effective tissue cryopreservation methods.

Dusp26 phosphatase regulates mitochondrial respiration and oxidative stress and protects neuronal cell death.

The dual specificity protein phosphatases (Dusps) control dephosphorylation of mitogen-activated protein kinases (MAPKs) as well as other substrates. Here, we report that Dusp26, which is highly expressed in neuroblastoma cells and primary neurons is targeted to the mitochondrial outer membrane via its NH2-terminal mitochondrial targeting sequence. Loss of Dusp26 has a significant impact on mitochondrial function that is associated with increased levels of reactive oxygen species (ROS), reduction in ATP generation, reduction in mitochondria motility and release of mitochondrial HtrA2 protease into the cytoplasm. The mitochondrial dysregulation in dusp26-deficient neuroblastoma cells leads to the inhibition of cell proliferation and cell death. In vivo, Dusp26 is highly expressed in neurons in different brain regions, including cortex and midbrain (MB). Ablation of Dusp26 in mouse model leads to dopaminergic (DA) neuronal cell loss in the substantia nigra par compacta (SNpc), inflammatory response in MB and striatum, and phenotypes that are normally associated with Neurodegenerative diseases. Consistent with the data from our mouse model, Dusp26 expressing cells are significantly reduced in the SNpc of Parkinson's Disease patients. The underlying mechanism of DA neuronal death is that loss of Dusp26 in neurons increases mitochondrial ROS and concurrent activation of MAPK/p38 signaling pathway and inflammatory response. Our results suggest that regulation of mitochondrial-associated protein phosphorylation is essential for the maintenance of mitochondrial homeostasis and dysregulation of this process may contribute to the initiation and development of neurodegenerative diseases.

Photobiomodulation has rejuvenating effects on aged bone marrow mesenchymal stem cells.

The plasticity and proliferative capacity of stem cells decrease with aging, compromising their tissue regenerative potential and therapeutic applications. This decline is directly linked to mitochondrial dysfunction. Here, we present an effective strategy to reverse aging of mouse bone marrow mesenchymal stem cells (BM-MSCs) by restoring their mitochondrial functionality using photobiomodulation (PBM) therapy. Following the characterization of young and aged MSCs, our results show that a near-infrared PBM treatment delivering 3 J/cm2 is the most effective modality for improving mitochondrial functionality and aging markers. Furthermore, our results unveil that young and aged MSCs respond differently to the same modality of PBM: whereas the beneficial effect of a single PBM treatment dissipates within 7 h in aged stem cells, it is lasting in young ones. Nevertheless, by applying three consecutive treatments at 24-h intervals, we were able to obtain a lasting rejuvenating effect on aged MSCs. Our findings are of particular significance for improving autologous stem cell transplantation in older individuals who need such therapies most.

Probing lasting cryoinjuries to oocyte-embryo transcriptome.

Clinical applications of oocytes cryopreservation include preservation of future fertility of young cancer patients, substitution of embryo freezing to avoid associated legal and ethical issues, and delaying childbearing years. While the outcome of oocyte cryopreservation has recently been improved, currently used vitrification method still suffer from increased biosafety risk and handling issues while slow freezing techniques yield overall low success. Understanding better the mechanism of cryopreservation-induced injuries may lead to development of more reliable and safe methods for oocyte cryopreservation. Using the mouse model, a microarray study was conducted on oocyte cryopreservation to identify cryoinjuries to transcriptionally active genome. To this end, metaphase II (MII) oocytes were subjected to standard slow freezing, and then analyzed at the four-cell stage after embryonic genome activation. Non-frozen four-cell embryos served as controls. Differentially expressed genes were identified and validated using RT-PCR. Embryos produced from the cryopreserved oocytes displayed 200 upregulated and 105 downregulated genes, associated with the regulation of mitochondrial function, protein ubiquitination and maintenance, cellular response to stress and oxidative states, fatty acid and lipid regulation/metabolism, and cell cycle maintenance. These findings reveal previously unrecognized effects of standard slow oocyte freezing on embryonic gene expression, which can be used to guide improvement of oocyte cryopreservation methods.

HSF1-Mediated Control of Cellular Energy Metabolism and mTORC1 Activation Drive Acute T-Cell Lymphoblastic Leukemia Progression.

Deregulated oncogenic signaling linked to PI3K/AKT and mTORC1 pathway activation is a hallmark of human T-cell acute leukemia (T-ALL) pathogenesis and contributes to leukemic cell resistance and adverse prognosis. Notably, although the multiagent chemotherapy of leukemia leads to a high rate of complete remission, options for salvage therapy for relapsed/refractory disease are limited due to the serious side effects of augmenting cytotoxic chemotherapy. We report that ablation of HSF1, a key transcriptional regulator of the chaperone response and cellular bioenergetics, from mouse T-ALL tumors driven by PTEN loss or human T-ALL cell lines, has significant therapeutic effects in reducing tumor burden and sensitizing malignant cell death. From a mechanistic perspective, the enhanced sensitivity of T-ALLs to HSF1 depletion resides in the reduced MAPK-ERK signaling and metabolic and ATP-producing capacity of malignant cells lacking HSF1 activity. Impaired mitochondrial ATP production and decreased intracellular amino acid content in HSF1-deficient T-ALL cells trigger an energy-saving adaptive response featured by attenuation of the mTORC1 activity, which is coregulated by ATP, and its downstream target proteins (p70S6K and 4E-BP). This leads to protein translation attenuation that diminishes oncogenic signals and malignant cell growth. Collectively, these metabolic alterations in the absence of HSF1 activity reveal cancer cell liabilities and have a profound negative impact on T-ALL progression. IMPLICATIONS: Targeting HSF1 and HSF1-dependent cancer-specific anabolic and protein homeostasis programs has a significant therapeutic potential for T-ALL and may prevent progression of relapsed/refractory disease.

Targeted Deletion of Hsf1, 2, and 4 Genes in Mice.

Heat shock transcription factors (Hsfs) regulate transcription of heat shock proteins as well as other genes whose promoters contain heat shock elements (HSEs). There are at least five Hsfs in mammalian cells, Hsf1, Hsf2, Hsf3, Hsf4, and Hsfy (Wu, Annu Rev Cell Dev Biol 11:441-469, 1995; Morimoto, Genes Dev 12:3788-3796, 1998; Tessari et al., Mol Hum Repord 4:253-258, 2004; Fujimoto et al., Mol Biol Cell 21:106-116, 2010; Nakai et al., Mol Cell Biol 17:469-481, 1997; Sarge et al., Genes Dev 5:1902-1911, 1991). To understand the physiological roles of Hsf1, Hsf2, and Hsf4 in vivo, we generated knockout mouse lines for these factors (Zhang et al., J Cell Biochem 86:376-393, 2002; Wang et al., Genesis 36:48-61, 2003; Min et al., Genesis 40:205-217, 2004). Numbers of other laboratories have also generated Hsf1 (Xiao et al., EMBO J 18:5943-5952, 1999; Sugahara et al., Hear Res 182:88-96, 2003), Hsf2 (McMillan et al., Mol Cell Biol 22:8005-8014, 2002; Kallio et al., EMBO J 21:2591-2601, 2002), and Hsf4 (Fujimoto et al., EMBO J 23:4297-4306, 2004) knockout mouse models. In this chapter, we describe the design of the targeting vectors, the plasmids used, and the successful generation of mice lacking the individual genes. We also briefly describe what we have learned about the physiological functions of these genes in vivo.

Therapeutic inducers of the HSP70/HSP110 protect mice against traumatic brain injury.

Traumatic brain injury (TBI) induces severe harm and disability in many accident victims and combat-related activities. The heat-shock proteins Hsp70/Hsp110 protect cells against death and ischemic damage. In this study, we used mice deficient in Hsp110 or Hsp70 to examine their potential requirement following TBI. Data indicate that loss of Hsp110 or Hsp70 increases brain injury and death of neurons. One of the mechanisms underlying the increased cell death observed in the absence of Hsp110 and Hsp70 following TBI is the increased expression of reactive oxygen species-induced p53 target genes Pig1, Pig8, and Pig12. To examine whether drugs that increase the levels of Hsp70/Hsp110 can protect cells against TBI, we subjected mice to TBI and administered Celastrol or BGP-15. In contrast to Hsp110- or Hsp70i-deficient mice that were not protected following TBI and Celastrol treatment, there was a significant improvement of wild-type mice following administration of these drugs during the first week following TBI. In addition, assessment of neurological injury shows significant improvement in contextual and cued fear conditioning tests and beam balance in wild-type mice that were treated with Celastrol or BGP-15 following TBI compared to TBI-treated mice. These studies indicate a significant role of Hsp70/Hsp110 in neuronal survival following TBI and the beneficial effects of Hsp70/Hsp110 inducers toward reducing the pathological consequences of TBI. Our data indicate that loss of Hsp110 or Hsp70 in mice increases brain injury following TBI. (a) One of the mechanisms underlying the increased cell death observed in the absence of these Hsps following TBI is the increased expression of ROS-induced p53 target genes known as Pigs. In addition, (b) using drugs (Celastrol or BGP-15) to increase Hsp70/Hsp110 levels protect cells against TBI, suggesting the beneficial effects of Hsp70/Hsp110 inducers to reduce the pathological consequences of TBI.

An essential role for heat shock transcription factor binding protein 1 (HSBP1) during early embryonic development.

Heat shock factor binding protein 1 (HSBP1) is a 76 amino acid polypeptide that contains two arrays of hydrophobic heptad repeats and was originally identified through its interaction with the oligomerization domain of heat shock factor 1 (Hsf1), suppressing Hsf1's transcriptional activity following stress. To examine the function of HSBP1 in vivo, we generated mice with targeted disruption of the hsbp1 gene and examined zebrafish embryos treated with HSBP1-specific morpholino oligonucleotides. Our results show that hsbp1 is critical for preimplantation embryonic development. Embryonic stem (ES) cells deficient in hsbp1 survive and proliferate normally into the neural lineage in vitro; however, lack of hsbp1 in embryoid bodies (EBs) leads to disorganization of the germ layers and a reduction in the endoderm-specific markers (such as α-fetoprotein). We further show that hsbp1-deficient mouse EBs and knockdown of HSBP1 in zebrafish leads to an increase in the expression of the neural crest inducers Snail2, Tfap2α and Foxd3, suggesting a potential role for HSBP1 in the Wnt pathway. The hsbp1-deficient ES cells, EBs and zebrafish embryos with reduced HSBP1 levels exhibit elevated levels of Hsf1 activity and expression of heat shock proteins (Hsps). We conclude that HSBP1 plays an essential role during early mouse and zebrafish embryonic development.

Inactivation of heat shock factor Hsf4 induces cellular senescence and suppresses tumorigenesis in vivo.

Studies suggest that Hsf4 expression correlates with its role in cell growth and differentiation. However, the role of Hsf4 in tumorigenesis in vivo remains unexplored. In this article, we provide evidence that absence of the Hsf4 gene suppresses evolution of spontaneous tumors arising in p53- or Arf-deficient mice. Furthermore, deletion of hsf4 alters the tumor spectrum by significantly inhibiting development of lymphomas that are normally observed in the majority of mice lacking p53 or Arf tumor suppressor genes. Using mouse embryo fibroblasts deficient in the hsf4 gene, we have found that these cells exhibit reduced proliferation that is associated with induction of senescence and senescence-associated ß-galactosidase (SA-ß-gal). Cellular senescence in hsf4-deficient cells is associated with the increased expression of the cyclin-dependent kinase inhibitors, p21 and p27 proteins. Consistent with the cellular senescence observed in vitro, specific normal tissues of hsf4(-/-) mice and tumors that arose in mice deficient in both hsf4 and p53 genes exhibit increased SA-ß-gal activity and elevated levels of p27 compared with wild-type mice. These results suggest that hsf4 deletion-induced senescence is also present in vivo. Our results therefore indicate that Hsf4 is involved in modulation of cellular senescence, which can be exploited during cancer therapy.

Targeted deletion of Hsf1, 2, and 4 genes in mice.

Heat-shock transcription factors (Hsfs) regulate transcription of heat-shock proteins as well as other genes whose promoters contain heat-shock elements. There are at least five Hsfs in mammalian cells, Hsf1, Hsf2, Hsf3, Hsf4, and Hsfy. To understand the physiological roles of Hsf1, Hsf2, and Hsf4 in vivo, we generated knockout mouse lines for these factors. In this chapter, we describe the design of the targeting vectors, the plasmids used, and the successful generation of mice lacking the individual genes. We also briefly describe what we have learned about the physiological functions of these genes in vivo.

Loss of Hsp110 leads to age-dependent tau hyperphosphorylation and early accumulation of insoluble amyloid beta.

Accumulation of tau into neurofibrillary tangles is a pathological consequence of Alzheimer's disease and other tauopathies. Failures of the quality control mechanisms by the heat shock proteins (Hsps) positively correlate with the appearance of such neurodegenerative diseases. However, in vivo genetic evidence for the roles of Hsps in neurodegeneration remains elusive. Hsp110 is a nucleotide exchange factor for Hsp70, and direct substrate binding to Hsp110 may facilitate substrate folding. Hsp70 complexes have been implicated in tau phosphorylation state and amyloid precursor protein (APP) processing. To provide evidence for a role for Hsp110 in central nervous system homeostasis, we have generated hsp110(-)(/)(-) mice. Our results show that hsp110(-)(/)(-) mice exhibit accumulation of hyperphosphorylated-tau (p-tau) and neurodegeneration. We also demonstrate that Hsp110 is in complexes with tau, other molecular chaperones, and protein phosphatase 2A (PP2A). Surprisingly, high levels of PP2A remain bound to tau but with significantly reduced activity in brain extracts from aged hsp110(-)(/)(-) mice compared to brain extracts from wild-type mice. Mice deficient in the Hsp110 partner (Hsp70) also exhibit a phenotype comparable to that of hsp110(-)(/)(-) mice, confirming a critical role for Hsp110-Hsp70 in maintaining tau in its unphosphorylated form during aging. In addition, crossing hsp110(-)(/)(-) mice with mice overexpressing mutant APP (APPßsw) leads to selective appearance of insoluble amyloid ß42 (Aß42), suggesting an essential role for Hsp110 in APP processing and Aß generation. Thus, our findings provide in vivo evidence that Hsp110 plays a critical function in tau phosphorylation state through maintenance of efficient PP2A activity, confirming its role in pathogenesis of Alzheimer's disease and other tauopathies.

Critical role of Brg1 member of the SWI/SNF chromatin remodeling complex during neurogenesis and neural crest induction in zebrafish.

Brg1 is a member of the SWI/SNF chromatin-remodeling complex, and in some organisms Brg1 has been shown to interact with beta-catenin and positively control the TCF/LEF transcription factor that is located downstream of the Wnt signal transduction pathway. During development, TCF/LEF activity is critical during neurogenesis and head induction. In zebrafish, Brg1-deficient embryos exhibit retinal cell differentiation and eye defects; however, the role of Brg1 in neurogenesis and neural crest cell induction remains elusive. We used zebrafish deficient in Brg1 (yng) or Brg1 specific-morpholino oligonucleotide-mediated knockdown to analyze the embryonic requirements of Brg1. Our results indicate that reduction in Brg1 expression leads to the expansion of the forebrain-specific transcription factor, six3, and marked reduction in expression of the mid/hind-brain boundary and hind-brain genes, engrailed2 and krox20, respectively. At 12 hpf, the expression of neural crest specifiers are severely affected in Brg1-morpholino-injected embryos. These results suggest that Brg1 is involved in neural crest induction, which is critical for the development of neurons, glia, pigment cells, and craniofacial structures. Brg1 is a maternal factor, and brg1-deficient embryos bearing the yng mutation derived from heterozygote intercrosses exhibit lesser effects on neural crest-specific gene expression, but show defects in neurogenesis and neural crest cell differentiation. This is exhibited by the aberrant brain patterning, a reduction in the sensory neurons, and craniofacial defects. These results further elucidate the critical role for Brg1 in neurogenesis, neural crest induction, and differentiation.

Mutational analysis of ATP-grasp residues in the two ATP sites of Saccharomyces cerevisiae carbamoyl phosphate synthetase.

The ATP-grasp fold is found in enzymes that catalyze the formation of an amide bond and occurs twice in carbamoyl phosphate synthetase. We have used site-directed mutagenesis to further define the relationship of these ATP folds to the ATP-grasp family and to probe for distinctions between the two ATP sites. Mutations at D265 and D810 severely diminished activity, consistent with consensus ATP-grasp roles of facilitating the transfer of the gamma-phosphate group of ATP. H262N was inactive whereas H807N, the corresponding mutation in the second ATP domain, exhibited robust activity, suggesting that these residues were not involved in the ATP-grasp function common to both domains. Mutations at I316 were somewhat catalytically impaired and were structurally unstable, consistent with a consensus role of interaction with the adenine and/or ribose moiety of ATP. L229G was too unstable to be purified and characterized. S228A showed essentially wild-type behavior.

Unmasking a functional allosteric domain in an allosterically nonresponsive carbamoyl-phosphate synthetase.

Although carbamoyl-phosphate synthetases (CPSs) share sequence identity, multidomain structure, and reaction mechanism, they have varying physiological roles and allosteric effectors. Escherichia coli CPS (eCPS) provides CP for both arginine and pyrimidine nucleotide biosynthesis and is allosterically regulated by metabolites from both pathways, with inhibition by UMP and activation by IMP and ornithine. The arginine-specific CPS from Saccharomyces cerevisiae (sCPS), however, apparently responds to no allosteric effectors. We have designed and analyzed a chimeric CPS (chCPS, in which the C-terminal 136 residues of eCPS were replaced by the corresponding residues of sCPS) to define the structural basis for the allosteric nonresponsiveness of sCPS and thereby provide insight into the mechanism for allosteric selectivity and responsiveness in the other CPSs. Surprisingly, ornithine and UMP each had a significant effect on chCPS activity, and did so at concentrations that were similar to those effective for eCPS. We further found that sCPS bound both UMP and IMP and that chCPS bound IMP, although none of these interactions led to changes in enzymatic activity. These findings strongly suggest that the nonresponsive sCPS is not able to communicate occupancy of the allosteric site to the active site but does contain a latent allosteric interaction domain.