Brg1 is a member of the SWI/SNF chromatin-remodeling complex, and in some organisms Brg1 has been shown to interact with ␤-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 oligonucleotidemediated 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-morpholinoinjected 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. Developmental Dynamics 235:2722-2735, 2006.

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 (APP␤sw) leads to selective appearance of insoluble amyloid ␤42 (A␤42), suggesting an essential role for Hsp110 in APP processing and A␤ 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.

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 ROS-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 of 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.

We have carried out a site-directed mutagenic analysis that is consistent with ATP binding to a palmate motif rather than to a Walker A͞B motif in domains B and C. To accommodate our present findings, as well as the other recent findings of structural and functional equivalence, we are proposing a novel mechanism for CPS. In this mechanism utilization of ATP bound to domain C is coupled to carbamoylphosphate synthesis at domain B via a nucleotide switch, with the energy of ATP hydrolysis at domain C allowing domain B to cycle between two alternative conformations.Carbamoyl-phosphate (CP) is a high-energy biological compound that plays a key role in the introduction of both ammonia and single carbon units into the metabolic pool. CP may serve either as the precursor for synthesis of pyrimidine nucleotides or, in an alternative pathway, as the precursor for arginine. In addition to serving as a required component of almost all proteins, arginine is used in the liver for ammonia detoxification via the urea cycle and, under appropriate physiological conditions, it is also used in a variety of tissues for nitric oxide formation. Depending on the physiological role, CP synthetases (CPSs) vary in mode of regulation and subunit composition (1). However, all of the CPSs thus far sequenced display marked primary sequence identity (2, 3), and all catalyze the formation of CP, P i , and two ADP from HCO 3 Ϫ , NH 3 , and two ATP. Sequence analysis demonstrated regions of internal duplication within CPS and suggested, on the basis of similarities to the ATP-binding sequence motifs of Walker A͞B proteins (i.e., proteins involving a ␤␣␤ nucleotide binding fold; ref. 4), that each region of internal duplication contains one ATP site (5, 6). Subsequent studies (7-10) demonstrated that the synthetase duplications correspond to independently folded domains (B and C) and that binding of one ATP molecule is localized to each of the two synthetase domains (6,(11)(12)(13). A great deal of experimental evidence (14-23) has established that the role of one ATP is to form the enzyme-bound intermediate carboxy-phosphate (CxP) and that domain B binds this ATP (ATP B ) whereas domain C binds the other ATP molecule (ATP C ).When we carried out the presently described studies, further definition of the utilization of ATP B and ATP C by CPS could not be based on a solved three-dimensional structure because there was none available for any CPS at that time. However, we found that domains B and C of CPS could each be fit to the structural coordinates for the biotin carboxylase (BN) component of Escherichia coli acetyl Co-A carboxylase (24). This BN homology modeling seemed appropriate for the following reasons. First, there is significant sequence identity shared by portions of BN (as well as other biotin-dependent enzymes) and each of the CPS domains B and C (2). Also, both CPS and BN couple cleavage of ATP to ADP with the activation of HCO 3 Ϫ to yield CxP, and both catalyze reaction of the activated carboxyl group with an amino group (NH 3 for...

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 β-galactosidase (SA-β-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-β-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.

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.

i. Summary/Abstract 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 1–6. To understand the physiological roles of Hsf1, Hsf2 and Hsf4 in vivo, we generated knockout mouse lines for these factors 7–9. Number of other laboratories have also generated Hsf1 10, 11, Hsf2 12, 13 and Hsf4 14 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.

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.