A rise in NAD precursor nicotinamide mononucleotide (NMN) after injury promotes axon degeneration

NAD metabolism regulates diverse biological processes, including ageing, circadian rhythm and axon survival. Axons depend on the activity of the central enzyme in NAD biosynthesis, nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2), for their maintenance and degenerate rapidly when this activity is lost. However, whether axon survival is regulated by the supply of NAD or by another action of this enzyme remains unclear. Here we show that the nucleotide precursor of NAD, nicotinamide mononucleotide (NMN), accumulates after nerve injury and promotes axon degeneration. Inhibitors of NMN-synthesising enzyme NAMPT confer robust morphological and functional protection of injured axons and synapses despite lowering NAD. Exogenous NMN abolishes this protection, suggesting that NMN accumulation within axons after NMNAT2 degradation could promote degeneration. Ectopic expression of NMN deamidase, a bacterial NMN-scavenging enzyme, prolongs survival of injured axons, providing genetic evidence to support such a mechanism. NMN rises prior to degeneration and both the NAMPT inhibitor FK866 and the axon protective protein Wld^S prevent this rise. These data indicate that the mechanism by which NMNAT and the related Wld^S protein promote axon survival is by limiting NMN accumulation. They indicate a novel physiological function for NMN in mammals and reveal an unexpected link between new strategies for cancer chemotherapy and the treatment of axonopathies.Axon degeneration in disease shares features with the progressive breakdown of the distal segment of severed axons as described by Augustus Waller in 1850 and named Wallerian degeneration.¹ The serendipitous discovery of Wallerian degeneration slow (Wld^S) mice, where transected axons survive 10 times longer than in wild types (WTs),² suggested that axon degeneration is a regulated process, akin to apoptosis of the cell bodies but distinct in molecular terms.^3,4 This process appears conserved in rats, flies, zebrafish and humans.^{5, 6, 7, 8} Wld^S blocks axon degeneration in some disease models, indicating a mechanistic similarity.³ Therefore understanding the pathway it influences is an excellent route towards novel therapeutic strategies.Wld^S is a modified nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1) enzyme, whose N-terminal extension partially relocates NMNAT1 from nuclei to axons, conferring gain of function.^9,10 In mammals, three NMNAT isoforms, nuclear NMNAT1, cytoplasmic NMNAT2 and mitochondrial NMNAT3, catalyse nicotinamide adenine dinucleotide (NAD) synthesis from nicotinamide mononucleotide (NMN) and adenosine triphosphate (ATP; Figure 1a).^11,12 Several reports indicate Wld^S protects injured axons by maintaining axonal NMNAT activity.^{13, 14, 15} In WT injured axons, without Wld^S, NMNAT activity falls when the labile, endogenous axonal isoform, NMNAT2, is no longer transported from cell bodies.¹⁶ NMNAT2 is required for axon maintenance¹⁶ and for axon growth in vivo and in vitro,^17,18 and modulation of its stability by palmitoylation¹⁹ or ubiquitin-dependent processes both in mice or when ectopically expressed in Drosophila^{19, 20, 21} has a corresponding effect on axon survival.Open in a separate windowFigure 1FK866 acts within axons to delay degeneration after injury. (a) The salvage pathway of NAD biosynthesis from nicotinamide (Nam) and nicotinic acid (Na). Only NAD biosynthesis from Nam is sensitive to FK866, which potently inhibits NAMPT while having no effect on nicotinic acid phosphoribosyltransferase (NaPRT).²⁹ The reaction catalysed by bacterial NMN deamidase is also shown. (b) SCG explants were treated with 100 nM FK866 for the indicated times, and then the whole explants (top panel) or the cell bodies (bottom left panel) and neurite fractions (bottom right panel) were separately collected. NAD was determined with an HPLC-based method (see Materials and Methods; n=3, mean and S.D. shown). (c) SCG neurites untreated (top panels) or treated with 100 nM FK866 the day before transection (bottom panels) and imaged after transection at the indicated time points. (d) SCG explants were treated with 100 nM FK866 1 day before or at the indicated times after cutting their neurites. Degeneration index was calculated from three fields in 2–4 independent experiments. The effect of treatment is highly significant when the drug is preincubated or added at 0–4 h after cut (mean ±S.E.M., n=6–12, one-way ANOVA followed by Bonferroni''s post-hoc test, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, compared with untreated)Wld^S partially colocalizes with mitochondria^14,22 and was shown to increase mitochondria motility and Ca²⁺-buffering capacity.²³ Inhibiting mitochondrial permeability transition pore protects degenerating axons.²⁴ However, Wld^S is protective in axons devoid of mitochondria,⁸ and targeting a cytosolic variant of NMNAT2 to mitochondria abolished its protective effect,¹⁹ suggesting a late mitochondrial involvement in Wallerian degeneration.Despite the importance of NMNAT activity in axon survival and degeneration, the molecular players remain elusive. Although NMNAT activity is required for protection,¹³ the hypothesis that increased NAD levels are responsible^25,26 does not fit some data.^27,28While further investigating the role of NAD, we found that blocking nicotinamide phosphoribosyltransferase (NAMPT, the enzyme preceding NMNAT, Figure 1a), was surprisingly axon-protective despite lowering NAD. NAMPT catalyses the synthesis of NMNAT-substrate NMN, the rate-limiting step in the NAD salvage pathway from nicotinamide (Nam) (Figure 1a). Here, we show that NMN accumulates after axon injury, and we provide genetic and pharmacological evidence supporting a role for this NMN increase in axon degeneration when NMNAT2 is depleted. We reveal an unexpected new direction for research into the degenerative mechanism, a novel class of protective proteins and new players in an axon-degeneration pathway sensitive to drugs under development for cancer.     

Functional Characterization of the Plastidial 3-Phosphoglycerate Dehydrogenase Family in Arabidopsis

This work contributes to unraveling the role of the phosphorylated pathway of serine (Ser) biosynthesis in Arabidopsis (Arabidopsis thaliana) by functionally characterizing genes coding for the first enzyme of this pathway, 3-phosphoglycerate dehydrogenase (PGDH). We identified two Arabidopsis plastid-localized PGDH genes (3-PGDH and EMBRYO SAC DEVELOPMENT ARREST9 [EDA9]) with a high percentage of amino acid identity with a previously identified PGDH. All three genes displayed a different expression pattern indicating that they are not functionally redundant. pgdh and 3-pgdh mutants presented no drastic visual phenotypes, but eda9 displayed delayed embryo development, leading to aborted embryos that could be classified as early curled cotyledons. The embryo-lethal phenotype of eda9 was complemented with an EDA9 complementary DNA under the control of a 35S promoter (Pro-35S:EDA9). However, this construct, which is poorly expressed in the anther tapetum, did not complement mutant fertility. Microspore development in eda9.1eda9.1 Pro-35S:EDA9 was arrested at the polarized stage. Pollen from these lines lacked tryphine in the interstices of the exine layer, displayed shrunken and collapsed forms, and were unable to germinate when cultured in vitro. A metabolomic analysis of PGDH mutant and overexpressing plants revealed that all three PGDH family genes can regulate Ser homeostasis, with PGDH being quantitatively the most important in the process of Ser biosynthesis at the whole-plant level. By contrast, the essential role of EDA9 could be related to its expression in very specific cell types. We demonstrate the crucial role of EDA9 in embryo and pollen development, suggesting that the phosphorylated pathway of Ser biosynthesis is an important link connecting primary metabolism with development.Plant primary metabolism is a complex process where many interacting pathways must be finely coordinated and integrated in order to achieve proper plant development and acclimation to the environment. An example of such complexity is the biosynthesis of the amino acid l-Ser, which takes place in at least two different organelles and by different pathways. This amino acid is essential for the synthesis of proteins and other biomolecules needed for cell proliferation, including nucleotides and Ser-derived lipids, such as phosphatidylserine and sphingolipids. Additionally, d-Ser has been attributed a signaling function in male gametophyte-pistil communication (Michard et al., 2011).Despite the important role played by Ser in plants, the biological significance of the coexistence of several Ser biosynthetic pathways and how they interact to maintain amino acid homeostasis in cells is not yet understood. Three different Ser biosynthesis pathways have been described in plants (Kleczkowski and Givan, 1988; Ros et al., 2013; Fig. 1). One is the glycolate pathway, which takes place in mitochondria and is associated with photorespiration (Tolbert, 1980, 1997; Douce et al., 2001; Bauwe et al., 2010; Maurino and Peterhansel, 2010). In this pathway, two molecules of Gly are converted to one molecule of Ser in a reaction catalyzed by the Gly decarboxylase complex and Ser hydroxymethyltransferase (Fig. 1). Ser synthesis through the glycolate pathway is obtained in green tissues during daylight hours (Tolbert, 1980, 1985; Douce et al., 2001), suggesting that alternative Ser biosynthesis pathways may be required in the dark and/or in nonphotosynthetic organs. In this respect, Ser can be synthesized through two nonphotorespiratory pathways (Kleczkowski and Givan, 1988), the plastidial phosphorylated pathway (Ho et al., 1998, 1999a, 1999b; Ho and Saito, 2001) and the so-called glycerate pathway, which synthesizes Ser by the dephosphorylation of 3-phosphoglycerate (3-PGA; Kleczkowski and Givan, 1988; Fig. 1). This latter pathway includes the reverse sequence of the section of the oxidative photosynthetic carbon cycle linking 3-PGA to Ser (3-PGA-glycerate-hydroxypyruvate-Ser), these reactions being catalyzed by putative enzymes such as 3-PGA phosphatase, glycerate dehydrogenase, Ala-hydroxypyruvate aminotransferase, and Gly hydroxypyruvate aminotransferase. Although the existence of enzymatic activities of this pathway has been demonstrated (Kleczkowski and Givan, 1988), its functional significance is unknown and genes coding for the specific enzymes of the pathway have not been characterized to date.Open in a separate windowFigure 1.Schematic representation of Ser biosynthesis in plants. The enzymes participating in each Ser biosynthetic pathway are listed separately. Photorespiratory pathway (glycolate pathway): GDC, Gly decarboxylase; SHMT, Ser hydroxymethyltransferase. Glycerate pathway: PGAP, 3-phosphoglycerate phosphatase; GDH, glycerate dehydrogenase; AH-AT, Ala-hydroxypyruvate aminotransferase. Phosphorylated pathway: PSAT, 3-phosphoserine aminotransferase; PSP, 3-phosphoserine phosphatase. Abbreviations used for metabolites are as follows: 3-PHP, 3-phosphohydroxypyruvate; 3-PS, 3-phosphoserine; THF, tetrahydrofolate; 5,10-CH₂-THF, 5,10-methylene-tetrahydrofolate. This figure is adapted from Cascales-Miñana et al. (2013).The plastidial phosphorylated pathway of serine biosynthesis (PPSB; Fig. 1), which is conserved in mammals and plants, synthesizes Ser via 3-phosphoserine utilizing 3-PGA as a precursor (Kleczkowski and Givan, 1988). Evidence for the existence of this pathway in plants stems from the isolation and characterization of its enzyme activities (Handford and Davies, 1958; Slaughter and Davies, 1968; Larsson and Albertsson, 1979; Walton and Woolhouse, 1986). The PPSB involves three enzymes catalyzing sequential reactions: 3-phosphoglycerate dehydrogenase (PGDH), 3-phosphoserine aminotransferase, and 3-phosphoserine phosphatase (PSP; Fig. 1). Genes coding for some isoforms of these enzymes have been cloned and biochemically characterized in Arabidopsis (Arabidopsis thaliana; Ho et al., 1998, 1999a, 1999b; Ho and Saito, 2001).In humans, the PPSB plays a crucial role in cell proliferation control and oncogenesis (Bachelor et al., 2011; Locasale et al., 2011; Pollari et al., 2011; Possemato et al., 2011). The functional significance of the PPSB in plants has recently been unraveled by providing evidence for the crucial role of PSP1, the last enzyme of the pathway in embryo, pollen, and root development (Cascales-Miñana et al., 2013). However, the PPSB still requires further characterization. In order to gain a complete understanding of the PPSB function in plants, precise molecular, metabolic, and genetic knowledge of all the enzymes and genes of the pathway is needed. In this work, we follow a gain- and loss-of-function approach in Arabidopsis to characterize a family of genes coding for putative isoforms of PGDH, the first enzyme of the PPSB. Here, we identify the essential gene of this family and provide evidence for its crucial function in embryo and pollen development.     

Endemic Viruses of Squirrel Monkeys (Saimiri spp.)

Nonhuman primates are the experimental animals of choice for the study of many human diseases. As such, it is important to understand that endemic viruses of primates can potentially affect the design, methods, and results of biomedical studies designed to model human disease. Here we review the viruses known to be endemic in squirrel monkeys (Saimiri spp.). The pathogenic potential of these viruses in squirrel monkeys that undergo experimental manipulation remains largely unexplored but may have implications regarding the use of squirrel monkeys in biomedical research.Abbreviations: HTLV1, human T-cell leukemia virus type 1; HVS, herpesvirus saimiri; IPF, idiopathic pulmonary fibrosis; SaHV, Saimiriine herpesvirus; SFV, simian foamy virus; SM-CMV, squirrel monkey cytomegalovirus; SMPyV, squirrel monkey polyomavirus; SMRV, squirrel monkey retrovirusThe similarity between the nonhuman primate and human immune systems is a key advantage in the use of nonhuman primates compared with other mammalian models of human disease.^{13,71,88,94,103,113,125} In addition, the diversity of environmental and infectious disease agents encountered by primates is similar to that of humans, providing nonhuman primates a comparable level of biologic complexity.¹ Old World primates, such as macaques and baboons, and New World primates, including squirrel monkeys and marmosets, are commonly used in biomedical research. Squirrel monkeys (Saimiri spp.) are neotropical primates native to the forests of Central and South America. Of the 7 species of squirrel monkey, 3 (S. oerstedii, S. vanzolinii, and S. ustus) are classified as endangered, vulnerable to extinction in the wild, or near threatened, whereas the remaining 4 (S. boliviensis, S. cassiquiarensis, S. macrodon, and S. sciureus) are not endangered, although the S. cassiquiarensis albigena subspecies is near threatened^52,81 (Figure 1). In South America, where squirrel monkeys are indigenous, breeding colonies of S. sciureus have been maintained at the Pasteur Institute in French Guiana and at the Oswaldo Cruz Foundation in Brazil.^7,12 In the United States, the Squirrel Monkey Breeding and Research Resource, an NIH-sponsored national research resource, maintains breeding colonies for S. boliviensis boliviensis, S. sciureus sciureus, and S. boliviensis peruviensis.Open in a separate windowFigure 1.Taxonomy of Saimiri species with associated IUCN designations.^52,81Squirrel monkeys adapt easily to laboratory housing and can be housed in smaller spaces than can Old World primates.¹ Unlike when working with Old World primates, particularly macaques, no additional personnel protective equipment is necessary when working with squirrel monkeys beyond that recommended for working with other New World primates.⁹² Their small size, combined with the reduced need for personnel protective equipment during handling, make squirrel monkeys attractive species for model development and for studies of viral pathogenesis, which cost approximately 30% to 40% less than comparable studies in macaques.¹ The likelihood of zoonotic transmission of infectious pathogens is considerably less than that associated with macaques and the risk of Macacine herpesvirus 1 (B virus) is nonexistent, given that neotropical primates do not harbor this lethal virus.¹ These factors are increasingly important in the current climate of limited grant funding for biomedical research and emphasis on safety for laboratory personnel. The limited availability of immunologic reagents with specificity for neotropical primates has hindered broader use of squirrel monkeys in biomedical research, compared with that of the more commonly used Old World primates. In addition, the small size of neotropical primates limits the volume of blood that can be collected at any one time. To abrogate these limitations, the NIH Nonhuman Primate Reagent Resource (www.nhpreagents.org) provides an increasing repertoire of agents that have been characterized for immunologic studies of neotropical primates.⁸⁹Squirrel monkeys are used in numerous aspects of biomedical research, including studies of viral persistence, neuroendocrinology, infectious diseases, cancer treatments, vaccine development, gene expression, and reproductive physiology.¹¹⁷ The similarity between the squirrel monkey immune system and that of humans means that, as with macaques, there is a high likelihood that research outcomes will recapitulate what occurs in human diseases.^13,71,87,94 This is particularly true for the study of several notable infectious diseases, including malaria, Creutzfeldt–Jakob disease, and human T-cell leukemia virus type 1 (HTLV1) infection.^19,56,128 For these diseases, squirrel monkeys are the model system of choice for studying pathogenesis, experimental treatments, and strategies for prevention.Squirrel monkeys are recognized as some of the most susceptible nonhuman primate species for the experimental transmission of Creutzfeldt–Jakob disease and other transmissible spongiform encephalopathies that cause chronic wasting disease.^11,72,98,130 The experimental infection of squirrel monkeys with HTLV1 has led to their use in vaccine development and chemotherapy research directed against HTLV1.^44,57,58,82 In addition, squirrel monkeys are an important model for studying the immunology of malaria and for testing vaccines against several Plasmodium species.^19,20,68,114 Furthermore, squirrel monkeys have been used in pharmacologic research to raise HDL levels to prevent atherosclerosis and reduce the risk of coronary heart disease.⁶ As the use of squirrel monkeys increases, especially for infectious disease research, accurate information about the endemic viral infections of squirrel monkeys is needed because of the potential for zoonotic transfer of these viruses to humans (and vice versa) and to understand the potential influence these agents may have on research involving other infectious pathogens diseases and immunosuppressive drugs.     

Keratins and protein synthesis: the plot thickens

In addition to protecting epithelial cells from mechanical stress, keratins regulate cytoarchitecture, cell growth, proliferation, apoptosis, and organelle transport. In this issue, Vijayaraj et al. (2009. J. Cell Biol. doi:10.1083/jcb.200906094) expand our understanding of how keratin proteins participate in the regulation of protein synthesis through their analysis of mice lacking the entire type II keratin gene cluster.Keratins are members of the intermediate filament family. They form intricate cytoskeletal networks via the assembly and organization of 10–12-nm filaments, whose formation is initiated through coiled-coil interactions between a type I keratin (e.g., K18 and -19) and a type II keratin (e.g., K8; Kim and Coulombe, 2007). Most keratin proteins have been shown to contribute to the protection of cells and tissues from mechanical and nonmechanical stresses (Toivola et al., 2005; Kim and Coulombe, 2007). Whereas the large number of keratin genes (n = 54; Schweizer et al., 2006) and the heteropolymerization-based assembly of their protein products should in part serve the purpose of modulating the viscoelastic properties of keratin networks to assist various cellular needs, common sense dictates that there should be additional roles for these proteins. Not surprisingly, efforts over the last decade have implicated keratin proteins in several nontraditional functions, including cytoarchitecture, proliferation and growth, apoptosis, and organelle transport, to name a few (Toivola et al., 2005; Kim and Coulombe, 2007). Yet, the high homology between several keratin proteins along with their overlapping distribution in epithelia has limited researchers'' progress toward uncovering the full range of keratin function in vivo (Baribault et al., 1994; Tamai et al., 2000; McGowan et al., 2002; Kerns et al., 2007). In this issue, Vijayaraj et al. report on the ultimate bypass of redundancy by eliminating all keratin filaments via the generation of a mouse strain lacking all type II keratins (KtyII^−/− mice). The study of these mice, which are viable until embryonic day 9.5, led to the discovery of a novel mechanism through which keratin proteins regulate protein synthesis and cell growth (Kim et al., 2006, 2007; Galarneau et al., 2007). The authors'' findings also showcase the recent conceptual and technical advances of chromosome engineering in the mouse genome.For over a decade, the Cre-loxP site-specific recombination system has been a popular method to generate targeted conditional knockout embryonic stem (ES) cells and mice. Although recombination efficiency is inversely proportional to the distance between loxP sites, larger chromosomal rearrangements have been successfully engineered into mouse ES cells using Cre-loxP (Ramírez-Solis et al., 1995). Generating such targeting vectors is cumbersome using traditional cloning methods. This said, DNA recombineering eliminates many of the constraints of finding unique restriction enzyme sites in genomic DNA sequences (Liu et al., 2003). Also, an Sv129 bacterial artificial chromosome (BAC) library generated from AB2.2 ES cells makes it easier to obtain large genomic sequences or even target ES cells directly with loxP-containing BACs (Liu et al., 2003; Adams et al., 2005). Finally, the Mutagenic Insertion and Chromosome Engineering Resource (MICER), a library of ready-made targeting vectors spread throughout the mouse genome, is now available (Adams et al., 2004). Vijayaraj et al. (2009) used MICER vectors to remove the entire 0.68-Mb keratin type II cluster on mouse chromosome 15 (Fig. 1 A). Owing to the interdependency of type I and II keratins for 10-nm filament assembly (Fig. 1 B), the resulting KtyII^−/− mice represent the first successful elimination of all keratin filaments from an organism as complex as a mouse.Open in a separate windowFigure 1.Genome organization, assembly, and epithelial function of keratins. (A) Arrangement of keratin clusters in the mouse genome. Human keratin genes that have not been identified or annotated in the mouse genome are shown on the bottom side and marked with a question mark. The arrows mark the boundaries of the region deleted by Vijayaraj et al. (2009) on mouse chromosome 15. (B) Summary of the multistep pathway through which type I and II keratin protein monomers polymerize to form 10-nm filaments. The antiparallel docking of the lollipop-shaped coiled-coiled dimers along their lateral surfaces generates structurally apolar tetramers and accounts for the lack of polarity of assembled keratin intermediate filaments. For all steps in the pathway, the forward (assembly promoting) reaction is heavily favored in vitro (Kim and Coulombe, 2007). (C) Keratins influence the localization and function of many cellular components. As highlighted here, keratins interact with and modulate the mTOR pathway in several ways, both in skin keratinocytes and gut epithelial cells, and regulate the localization of microtubules, γ-tubulin, and GLUT transporters in polarized epithelia. Components are not drawn to scale in this schematic.KtyII^−/− embryos display severe growth retardation and die midgestation (Baribault et al., 1993; Hesse et al., 2000; Tamai et al., 2000). Smaller cell size has been observed previously in K17^−/− skin keratinocytes and K8^−/− liver hepatocytes, correlating with altered Akt/mammalian target of rapamycin (mTOR) signaling (Fig. 1 C) and a reduction in bulk protein synthesis (Kim et al., 2006; Galarneau et al., 2007). Although K17 appears to modulate the mTOR pathway through its physical interaction with 14-3-3–σ in keratinocytes (Fig. 1 C; Kim et al., 2006), the mechanism for how K8 influences protein synthesis in hepatocytes is less clear but appears to integrate responses to both insulin and integrin stimulation (Galarneau et al., 2007). Loss of K8 is also associated with an increase in Akt activity (Galarneau et al., 2007), which is contrary to the findings in the K17^−/− setting (Kim et al., 2006), calling into question whether the two settings use the same mechanism to modulate mTOR signaling. Vijayaraj et al. (2009) uncover yet another path through which keratins are able to influence protein synthesis. The authors find that loss of all keratin filaments causes mislocalization of GLUT transporters and disruption of glucose homeostasis through AMP kinase (AMPK) activation. In addition, the authors report that in the absence of the keratin network, AMPK phosphorylates Raptor, which then interacts with mTOR to repress protein synthesis and hamper cell growth (Fig. 1 C). These findings further the evidence for an important role of keratin proteins (or filaments) in the regulation of translation and epithelial cell growth. However, they also raise the question of whether keratins affect mTOR signaling via an as of yet unknown, common denominator or whether several mechanisms come together, perhaps in a cell type– and context-dependent fashion, to achieve the same downstream effect.Unlike actin and microtubules, keratin filaments are not believed to possess intrinsic polarity (Fig. 1 B). However, K8/K18 and/or K8/K19 filaments play a significant role in maintaining apicobasal compartmentalization in simple epithelial linings in both the small intestine (Ameen et al., 2001; Oriolo et al., 2007) and colon of adult mice (Toivola et al., 2004) and have also been implicated in organelle transport (Toivola et al., 2005; Kim and Coulombe, 2007). The mechanism or mechanisms accounting for this surprising influence of keratins on the establishment and maintenance of spatial order in epithelial cells are unknown. Ameen et al. (2001) and Oriolo et al. (2007) recently made a dent in this mystery by showing that K8/K18 filaments are necessary for the proper localization of γ-tubulin to the apical compartment in polarized epithelial cells, thereby participating in the organization of noncentrosomic microtubules (note: the interested reader should examine a recent study by Bocquet et al. [2009], which shows a role for neuronal intermediate filaments in tubulin polymerization in axons). Similar to previous observations made in K8^−/− mice (Ameen et al., 2001; Toivola et al., 2004), Vijayaraj et al. (2009) show that apical proteins, particularly GLUT1 and -3, are mislocalized in KtyII^−/− embryonic epithelia. However, in this instance, microtubule organization appears to be intact. Although the authors'' experimental findings again nicely demonstrate a role for keratin proteins in the establishment of polarity in simple epithelial settings, the underlying mechanism or mechanisms still need to be ascertained.The mouse model generated by Vijayaraj et al. (2009) has important implications for the field of keratin biology and intermediate filaments in general. It will allow researchers to address central questions about the contributions of keratins during development and tissue homeostasis unencumbered by the redundancy of properties and functions among members of this large family. The availability of tissue- or cell type–specific promoters makes it possible to express the Cre recombinase in specific epithelial settings, thereby promoting the elimination of keratins in a more restricted fashion. It will be interesting to see how the total loss of keratin filaments affects different tissues and subpopulations of cells, highlighting essential functions and perhaps uncovering previously unappreciated roles for keratins in complex cellular processes.     

Isolation of Helicobacter spp. from Mice with Rectal Prolapses

Enterohepatic Helicobacter species (EHS) often are associated with typhlocolitis and rectal prolapse in mice. We sought to describe rectal prolapses histologically, relate lesions to mouse genotype and EHS infection status, and characterize EHS pathogens on our campus. Our mouse population was housed among 6 facilities on our main campus and a seventh, nearby facility. We investigated cases of rectal prolapse over 1 y and included 76 mice, which were broadly categorized according to genotype. Microscopically, lesions ranged from mild to severe typhlocolitis, often with hyperplastic and dysplastic foci. Neoplastic foci tended to occur at the ileocecal–colic junction. Lesions were most severe in strains that had lower-bowel inflammatory disease, notably IL10, Rag1, and Rag2 knockout strains; prolapses occurred in these strains when housed both in areas with endemic EHS and in our Helicobacter-free barrier facility. Most mice with rectal prolapses were immunocompromised genetically modified mice; however, the most frequently sampled strain, the lamellipodin knockout, was noteworthy for its high incidence of rectal prolapse, localized distal colonic and rectal lesions, and lack of known immunodeficiency. This strain is being explored as a model of rectal carcinoma. Most of the colons examined tested PCR-positive for EHS, often with coinfections. Although H. bilis is prevalent on our campus, we did not find this organism in any mice exhibiting clinical signs of rectal prolapse. Identification of H. apodemus in 22% of cases has fueled increased surveillance on our campus to characterize this organism and differentiate it from the closely related H. rodentium.Abbreviations: EHS, enterohepatic Helicobacter species; IBD, inflammatory bowel disease; RFLP, restriction-fragment–length polymorphism; RP, rectal prolapseRectal prolapse (RP) occurs commonly in laboratory mice and is often associated with lower-bowel inflammation. Mice have a relatively short and poorly supported distal colon, which lacks a serosal covering.³⁰ This anatomic weakness, coupled with a microbial insult, toxic injury, or space-occupying neoplastic masses within the gastrointestinal tract, are the predisposing factors for tenesmus and RP (Figure 1). In the context of microbial insults, the pathogenesis involves diffuse or multifocal inflammation in the more proximal segments of colon or distal colon, which can result in thickened edematous tissue and tenesmus, triggering a prolapse.^6,30,40 Bacteria most often associated with this condition are the enterohepatic Helicobacter species (EHS) and Citrobacter rodentium; although in theory any pathogenic bacteria causing colitis may predispose mice to RP.^1,11,13,38Open in a separate windowFigure 1.Mouse rectal prolapse. An example of the clinical presentation of rectal prolapse in laboratory mice. Note the attachment of bedding and nesting material in the film of mucous that frequently is seen covering the exposed rectal tissue. Generally the tissue becomes severely erythematous, as can be appreciated in this photograph.Although the clinical presentation of RP may occur in immunocompetent mice, it is most often associated with mice that have a spontaneous or transgenic mutation causing immunodeficiency.^11,13,38 Indeed, these naturally occurring murine pathogens are used to model inflammatory bowel disease in strains that are highly susceptible to typhlocolitis with EHS infection; examples include Il10^−/− and Rag-deficient mice.^{3,5,8,9,13,16,19,20,22,40} In addition, H. hepaticus and other EHS including H. typhlonius, H. rodentium, and H. bilis, which are known to persistently colonize the intestinal crypt of the lower bowel, have been shown to induce colitis-associated cancer in susceptible immunodeficient strains of mice.^{4,7,9,23,24,27,29,31}In 1999, our institution introduced a rodent importation policy to reduce the introduction of murine pathogens. As part of this program, all approved commercial vendors were screened to ensure animals were SPF for EHS. Any random-source mice (typically imported from other academic institutions for collaborative projects) were required to be rederived by embryo transfer. In comparing PCR data between 1999 (prior to implementing the ET policy) and 2009, we found that after more than a decade of strict rederivation and husbandry practices that reduce fecal–oral transmission, EHS prevalence was markedly reduced.²¹ Despite this success, these practices did not completely eradicate rodent EHS. Of particular note, 2 facilities on campus house well-established long-term breeding colonies, many of which are unique transgenic lines with various immunodeficiencies, that are used primarily for immunology and cancer research. Rederivation of each of these strains was considered to be cost-prohibitive; thus EHS has remained endemic in these breeding colonies for more than a decade, as evident by our recent surveillance for EHS prevalence.²¹ The species known to be prevalent on our campus prior to the current study included H. hepaticus, H. rodentium, H. typhlonius, and H. bilis; in a few isolated areas, H. mastomyrinus was identified also.²¹Although EHS infections often are subclinical, we sought to correlate the presence of EHS-endemic areas with clinical lower-bowel inflammation (evident by rectal prolapse). In this survey of laboratory mice at our institution, we identified patterns in mouse strain susceptibility to RP, RP association with EHS, and histopathologic findings and correlated specific EHS species with clinical disease. Because we sought to study spontaneous infections, we excluded any mice on study with experimentally induced inflammatory bowel disease (IBD), including Helicobacter-induced IBD and chemically induced colitis models.From July 2011 to July 2012, a total of 63 mice with RP from these 6 facilities at our institution were necropsied as part of this investigation. In addition, 13 mice with RP were identified at a nearby research institute housing mice known to have endemic EHS.     

Dendritic cells are stressed out in tumor

A recently paper published in Cell reports that dendritic cells (DCs) are dysfunctional in the tumor environment. Tumor impairs DC function through induction of endoplasmic reticulum stress response and subsequent disruption of lipid metabolic homeostasis.Tumors develop diverse strategies to escape tumor-specific immunity. Tumor-infiltrating dendritic cells (tDCs) are dysfunctional and/or mediate immune suppression¹. Cubillos-Ruiz et al.² showed in their recent Cell paper that tDCs exhibit an activation of the unfolded protein response (UPR), as indicated by the presence of high levels of spliced XPB1, and this may be attributed to reactive oxygen species (ROS) in tumor, which induces lipid peroxidation, leading to the endoplasmic reticulum (ER) stress in tDCs. Furthermore, they demonstrated that UPR activation in tDCs results in poor DC function, which is accompanied by impaired lipid metabolism and subsequent reduction of T cell anti-tumor immunity. Thus, these observations present a novel mechanism for tDC malfunction.ER stress is evoked by the presence of unfolded or chemically modified proteins. In short, the presence of damaged proteins is sensed by the proteins in the ER membrane. Of these, IRE1α can remove a short nucleotide sequence from mRNA encoding XBP1 protein. This splicing event facilitates XBP1 translation. XBP1 protein binds to consensus sequences in target genes and activates their expression³. Many XBP1 target genes are fatty acid synthesis enzymes. Enhanced production of fatty acids leads to the formation of lipid droplets inside the cytoplasm and extension of the ER compartment due to efficient intracellular membrane formation³. Therefore, this mechanism is a form of adaptation of the cell to the harsh environment, which sustains the production of functional proteins. In such a context, the article by Cuillos-Ruiz et al.² shows intriguing data on the XBP1 pathway in silencing DC function in the tumor environment.First, Cubillos-Ruiz et al.² observed that tDCs express high levels of spliced XBP1, its direct target genes and other markers for the ER stress response. Targeted deletion of XBP1 in CD11c⁺ DCs reveals an association of the XBP1-dependent ER stress response with immunosuppressive properties of tDCs. DC-specific deletion of XBP1 not only inhibits tumor growth and prolongs animal survival, but also reduces tumor peritoneal metastasis, ascites accumulation, and splenomegaly in an ovarian cancer-bearing mouse model.Next, the authors elucidated why the XBP1 pathway in tDCs is highly activated. Unexpectedly, neither typical tumor-associated cytokines nor hypoxia can efficiently stimulate XBP1 activation. Interestingly, tDCs contain high levels of lipid peroxidation byproducts bound to the proteins, which is associated with the production of ROS. Microarray analysis revealed that DC-specific XBP1 deletion downregulates both UPR pathway-dependent genes and lipid metabolism, which results in lower total lipid production, loss of lipid droplets in the cytoplasm and decreased production of triacylglycerides. This phenotype can be recapitulated by chemical inhibition of ROS formation or IRE1α and XBP1 signaling. Furthermore, XBP1-deficient DCs are potent stimulators of OT-1 T cells. Adoptive transfer of T cells isolated from metastatic tumor-bearing mice with DC-specific XBP1 deletion also shows their superior ability to control tumor growth.On the basis of these observations, the authors tested the effects of therapeutic intervention with the usage of nanocomplexes containing XBP1 siRNA. The size of lipid particles or nanocomplexes determines the anatomical location of specific drug delivery⁴. Additionally, they previously optimized the nanocomplexes for selective engulfing by DCs⁵. They found that administration of the nanoparticles containing XBP1 siRNA causes potent T cell activation, which is accompanied by reduced cancer metastatic foci and improved animal survival.The paper by Cubillos-Ruiz et al.² provides an important insight into DC biology in general. Since the identification of Toll-like receptor (TLR) signaling, the main direction in DC biology has been associated with PAMP and DAMP recognition in various degrees (Figure 1). However, the link between cellular metabolism in specific microenvironment and DC biology is poorly explored (Figure 1). The ER stress response has previously been observed in DCs^6,7. It is thought that in response to ER stress, XBP1 is crucial for DC generation, survival, and function^6,7. The conceptual link between XBP1 signaling and DC biology is as follows⁸: (i) stimulation of DCs leads to ER stress; (ii) ER stress activates UPR/XBP1 pathway; (iii) XBP1 activates lipid synthesis genes; (iv) lipids are used for the extension of the ER and Golgi compartment, which are of importance for cytokine production and secretion^9,10. Now, the authors challenged this concept in the context of cancer. The authors demonstrated that XBP1 signaling is strongly associated with poor T cell activation and limitation of XBP1 signaling leads to improved T cell function in the tumor environment (Figure 1). Thus, the paper sheds a new light on DC biology.Open in a separate windowFigure 1Double-faced role of XBP1 signaling pathway in DCs. Left: under immunostimulatory conditions, DCs receive signals from Toll-like receptors. NF-κB signaling induces a XBP1-dependent ER stress response, which enhances lipid metabolism. Formation of new membranes expands ER and Golgi compartments, which enhances cytokine production and secretion⁷. Right: In the tumor microenvironment, DCs are exposed to ROS, which also results in ER stress. However, in this case DC lipid metabolism is impaired and DCs acquire an immunosuppressive phenotype².Of course, as with any interesting work, the article raises more questions than answers. For example, which immune-related properties of DCs are “selectively” affected by the XBP1 pathway? Is there an actual cause-and-effect relationship between aberrant lipid accumulation and the immunosuppressive phenotype of tDCs? The authors observed no obvious change in PD-L1 (B7-H1) expression in DCs, but noticed reduced surface levels of peptide-loaded MHC-I complexes in wild-type tDCs as compared to XBP-deficient tDCs. However, it remains unknown whether and how DC cross-presentation is involved in tDC function regulated by ER stress, XBP1 activation, and lipid metabolism. Human ovarian cancer-associated DCs express high levels of B7-H1 and limited IL-12¹¹. It would be interesting to thoroughly examine the cytokine profile, and the B7 and TNF family members, along with lipid pathway manipulation in XBP1^−/− DCs. It is well known that ER morphology and function is substantial for the synthesis of membrane proteins and cytokines, which would be affected by the XBP1 pathway. Paradoxically, a restricted ER stress response can help immune reaction, which requires the expansion of the ER compartment. Another question is how and which lipid synthesis and metabolism pathway is targeted by XBP1 in DCs (or/and tumor cells in the same environment). Obviously, future studies are warranted to address these important questions.In conclusion, the paper by Cubillos-Ruiz et al.² opens an interesting chapter for scientifically and therapeutically exploring DC biology in a specific metabolic environment. Given that silencing XBP1 signaling in DCs enhances tumor immunity, and XBP1 is an intrinsic pro-tumor factor, it is reasonable to assume that targeting this pathway may be beneficial in patients with cancer and could kill two birds with one stone.     

Identification of a Novel Abscisic Acid-Regulated Farnesol Dehydrogenase from Arabidopsis

In Arabidopsis (Arabidopsis thaliana), farnesylcysteine is oxidized to farnesal and cysteine by a membrane-associated thioether oxidase called farnesylcysteine lyase. Farnesol and farnesyl phosphate kinases have also been reported in plant membranes. Together, these observations suggest the existence of enzymes that catalyze the interconversion of farnesal and farnesol. In this report, Arabidopsis membranes are shown to possess farnesol dehydrogenase activity. In addition, a gene on chromosome 4 of the Arabidopsis genome (At4g33360), called FLDH, is shown to encode an NAD⁺-dependent dehydrogenase that oxidizes farnesol more efficiently than other prenyl alcohol substrates. FLDH expression is repressed by abscisic acid (ABA) but is increased in mutants with T-DNA insertions in the FLDH 5′ flanking region. These T-DNA insertion mutants, called fldh-1 and fldh-2, are associated with an ABA-insensitive phenotype, suggesting that FLDH is a negative regulator of ABA signaling.Isoprenylated proteins are modified at the C terminus via cysteinyl thioether linkage to either a 15-carbon farnesyl or a 20-carbon geranylgeranyl group (Clarke, 1992; Zhang and Casey, 1996; Rodríguez-Concepción et al., 1999; Crowell, 2000; Crowell and Huizinga, 2009). These modifications mediate protein-membrane and protein-protein interactions and are necessary for the proper localization and function of hundreds of proteins in eukaryotic cells. In Arabidopsis (Arabidopsis thaliana), the PLURIPETALA (PLP; At3g59380) and ENHANCED RESPONSE TO ABA1 (At5g40280) genes encode the α- and β-subunits of protein farnesyltransferase (PFT), respectively (Cutler et al., 1996; Pei et al., 1998; Running et al., 2004). These subunits form a heterodimeric zinc metalloenzyme that catalyzes the efficient transfer of a farnesyl group from farnesyl diphosphate to protein substrates with a C-terminal CaaX motif, where “C” is Cys, “a” is an aliphatic amino acid, and “X” is usually Met, Gln, Cys, Ala, or Ser (Fig. 1). The PLP and GERANYLGERANYL-TRANSFERASE BETA (At2g39550) genes encode the α- and β-subunits of protein geranylgeranyltransferase type 1 (PGGT1), respectively (Running et al., 2004; Johnson et al., 2005). These subunits form a distinct heterodimeric zinc metalloenzyme that catalyzes the efficient transfer of a geranylgeranyl group from geranylgeranyl diphosphate to protein substrates with a C-terminal CaaL motif, where “C” is Cys, “a” is an aliphatic amino acid, and “L” is Leu. A third protein prenyltransferase, called protein geranylgeranyltransferase type II or RAB geranylgeranyltransferase, catalyzes the dual geranylgeranylation of RAB proteins with a C-terminal XCCXX, XXCXC, XXCCX, XXXCC, XCXXX, or CCXXX motif, where “C” is Cys and “X” is any amino acid. However, RAB proteins must be associated with the RAB ESCORT PROTEIN to be substrates of RAB geranylgeranyltransferase. Plant protein prenylation has received considerable attention in recent years because of the meristem defects of Arabidopsis PFT mutants and the abscisic acid (ABA) hypersensitivity of Arabidopsis PFT and PGGT1 mutants (Cutler et al., 1996; Pei et al., 1998; Running et al., 1998, 2004; Johnson et al., 2005).Open in a separate windowFigure 1.Proposed metabolism of farnesal and farnesol as it relates to protein prenylation. The portion of the cycle shown in red is the subject of this article.Proteins that are prenylated by either PFT or PGGT1 undergo further processing in the endoplasmic reticulum (Crowell, 2000; Crowell and Huizinga, 2009). First, the aaX portion of the CaaX motif is removed by proteolysis (Fig. 1). This reaction is catalyzed by one of two CaaX endoproteases, which are encoded by the AtSTE24 (At4g01320) and AtFACE-2 (At2g36305) genes (Bracha et al., 2002; Cadiñanos et al., 2003). Second, the prenylated Cys residue at the new C terminus is methylated by one of two isoprenylcysteine methyltransferases (Fig. 1), which are encoded by the AtSTE14A (At5g23320) and AtSTE14B (ICMT; At5g08335) genes (Crowell et al., 1998; Crowell and Kennedy, 2001; Narasimha Chary et al., 2002; Bracha-Drori et al., 2008). A specific isoprenylcysteine methylesterase encoded by the Arabidopsis ICME (At5g15860) gene has also been described, demonstrating the reversibility of isoprenylcysteine methylation (Deem et al., 2006; Huizinga et al., 2008).Like all proteins, prenylated proteins have a finite half-life. However, unlike other proteins, prenylated proteins release farnesylcysteine (FC) or geranylgeranylcysteine (GGC) upon degradation. Mammals possess a prenylcysteine lyase enzyme that catalyzes the oxidative cleavage of FC and GGC (Zhang et al., 1997; Tschantz et al., 1999; Tschantz et al., 2001; Beigneux et al., 2002; Digits et al., 2002). This FAD-dependent thioether oxidase consumes molecular oxygen and generates hydrogen peroxide, Cys, and a prenyl aldehyde product (i.e. farnesal or geranylgeranial). In Arabidopsis, a similar lyase exists. However, the Arabidopsis enzyme, which is encoded by the FCLY (At5g63910) gene, is specific for FC (Fig. 1; Crowell et al., 2007; Huizinga et al., 2010). GGC is metabolized by a different mechanism.Plant membranes have been shown to contain farnesol kinase, geranylgeraniol kinase, farnesyl phosphate kinase, and geranylgeranyl phosphate kinase activities (Fig. 1; Thai et al., 1999). These membrane-associated kinases differ with respect to nucleotide specificity, suggesting that they are distinct enzymes (i.e. farnesol kinase and geranylgeraniol kinase can use CTP, UTP, or GTP as a phosphoryl donor, whereas farnesyl phosphate kinase and geranylgeranyl phosphate kinase exhibit specificity for CTP as a phosphoryl donor). However, it remains unclear if farnesol kinase is distinct from geranylgeraniol kinase or if farnesyl phosphate kinase is distinct from geranylgeranyl phosphate kinase. Nonetheless, it is clear that these kinases convert farnesol and geranylgeraniol to their monophosphate and diphosphate forms for use in isoprenoid biosynthesis, including sterol biosynthesis and protein prenylation.Because plants have the metabolic capability to generate farnesal from FC and farnesyl diphosphate from farnesol, we considered the possibility that plant membranes also contain an oxidoreductase capable of catalyzing the reduction of farnesal to farnesol and/or the oxidation of farnesol to farnesal (Fig. 1; Thai et al., 1999; Crowell et al., 2007). To date, the only reports of such an oxidoreductase are from the corpora allata glands of insects, where it participates in juvenile hormone synthesis, and black rot fungus-infected sweet potato (Ipomoea batatas; Baker et al., 1983; Inoue et al., 1984; Sperry and Sen, 2001; Mayoral et al., 2009). Insect farnesol dehydrogenase is an NADP⁺-dependent oxidoreductase that is encoded by a subfamily of short-chain dehydrogenase/reductase (SDR) genes (Mayoral et al., 2009). Farnesol dehydrogenase from sweet potato is a 90-kD, NADP⁺-dependent homodimer with broad specificity for prenyl alcohol substrates and is induced by wounding and fungus infection of potato roots (Inoue et al., 1984).Here, we extended previous work in which [1-³H]FC was shown to be oxidized to [1-³H]farnesal, and [1-³H]farnesal reduced to [1-³H]farnesol, in the presence of Arabidopsis membranes (Crowell et al., 2007). The reduction of [1-³H]farnesal to [1-³H]farnesol was abolished by pretreatment of Arabidopsis membranes with NADase, suggesting that sufficient NAD(P)H is present in Arabidopsis membranes to support the enzymatic reduction of farnesal to farnesol. In this report, we demonstrate the presence of farnesol dehydrogenase activity in Arabidopsis membranes using [1-³H]farnesol as a substrate. Moreover, we identify a gene on chromosome 4 of the Arabidopsis genome (At4g33360), called FLDH, that encodes an NAD⁺-dependent dehydrogenase with partial specificity for farnesol as a substrate. FLDH expression is repressed by exogenous ABA, and fldh mutants exhibit altered ABA signaling. Taken together, these observations suggest that ABA regulates farnesol metabolism in Arabidopsis, which in turn regulates ABA signaling.     

Ovine Model for Critical-Size Tibial Segmental Defects

A segmental tibial defect model in a large animal can provide a basis for testing materials and techniques for use in nonunions and severe trauma. This study reports the rationale behind establishing such a model and its design and conclusions. After ethics approval of the study, aged ewes (older than 5 y; n = 12) were enrolled. A 5-cm mid diaphyseal osteoperiosteal defect was made in the left tibia and was stabilized by using an 8-mm stainless-steel cross-locked intramedullary nail. Sheep were euthanized at 12 wk after surgery and evaluated by using radiography, microCT, and soft-tissue histology techniques. Radiology confirmed a lack of hard tissue callus bridging across the defect. Volumetric analysis based on microCT showed bone growth across the 16.5-cm³ defect of 1.82 ± 0.94 cm³. Histologic sections of the bridging tissues revealed callus originating from both the periosteal and endosteal surfaces, with fibrous tissue completing the bridging in all instances. Immunohistochemistry was used to evaluate the quality of the healing response. Clinical, radiographic, and histologic union was not achieved by 12 wk. This model may be effective for the investigation of surgical techniques and healing adjuncts for nonunion cases, where severe traumatic injury has led to significant bone loss.Abbreviations: BMP2, bone morphogenic protein 2; CATK, cathepsin K; VEGF, vascular endothelial growth factorThe human tibia is the most frequently broken long bone, often with significant bone loss.⁴ Segmental tibial defects can occur as a result of large tumor removal, trauma such as motor vehicle accidents, and more recently, blast injuries as seen with the escalating number of global conflicts. Treatment of these large bone and surrounding soft tissue defects is an ongoing, costly, and challenging clinical problem; no surgical technique has currently achieved preeminence.⁴ The general consensus on factors that affect healing include concomitant disease, age, and degree of trauma.⁵ When the first 2 factors, which are patient-related, are removed from the equation, healing is influenced by the size, anatomic location, and soft-tissue coverage of the defect. The ability to study these situations in a well-controlled, robust, and reproducible preclinical model would be advantageous to help establish effective surgical techniques and evaluate implants and materials.A literature review revealed that many ovine models for bone defects have been used, but all have limitations^{6,12,14,15,20,21,24,25,27,31,37,39,40} (Figure 1). Variations in protocols, such as age of the animals, size of the defect, and the bone and stabilization techniques used, limit meaningful comparison between studies.^33,34 Although some studies have investigated material performance in the healing of defects, they did not rigorously quantify control defects,^17,20 and others used no controls at all.³⁹ There is often no explanation regarding the use of a particular defect size, leading to the question of whether the defect size was critical.²⁴ The choice of bone used has been also varied; the femur,¹⁵ tibia,³⁷ and metatarsus⁴⁰ have all been studied. A noncritical-size defect implies that healing would eventually occur without the presence of any graft materials. One study,¹² for example, used a 3-cm defect at an average of 1.8 times the diameter of the tibias in question and found that empty controls achieved as much as 26% of the stiffness of an intact tibia after 12 wk. Stabilization methods include plating,^21,40 external fixtures,²⁰ intramedullary nails,^6,16 and a combination of intramedullary nails and plating.³⁷Open in a separate windowFigure 1.A limited summary of the many studies where a segmental tibial has been used with their references.The criteria used in the present study for a critical-size segmental tibial defect model were based on the following factors. The ovine tibia closely resembles that of the human tibia in terms of size, shape, and physical properties and is commonly used when studying human orthopedic diseases.^26,34 Intramedullary nailing has become the most commonly used method of tibial fracture fixation in human orthopedic surgery.^8,22 An 8-mm intramedullary nail is commonly used in the treatment of human fractures, further confirming the size similarity between the ovine and human tibiae.¹⁹The aim of this study was to establish and characterize a preclinical ovine 5-cm osteoperiosteal critical-size tibial segmental defect model in mature sheep. The endpoints included those commonly used clinically, such as radiography and microCT. Histology to investigate the degree of healing and immunohistochemistry to characterize the healing process were included to complete the evaluation process.