A spontaneous mutation in contactin 1 in the mouse

Mutations in the gene encoding the immunoglobulin-superfamily member cell adhesion molecule contactin1 (CNTN1) cause lethal congenital myopathy in human patients and neurodevelopmental phenotypes in knockout mice. Whether the mutant mice provide an accurate model of the human disease is unclear; resolving this will require additional functional tests of the neuromuscular system and examination of Cntn1 mutations on different genetic backgrounds that may influence the phenotype. Toward these ends, we have analyzed a new, spontaneous mutation in the mouse Cntn1 gene that arose in a BALB/c genetic background. The overt phenotype is very similar to the knockout of Cntn1, with affected animals having reduced body weight, a failure to thrive, locomotor abnormalities, and a lifespan of 2-3 weeks. Mice homozygous for the new allele have CNTN1 protein undetectable by western blotting, suggesting that it is a null or very severe hypomorph. In an analysis of neuromuscular function, neuromuscular junctions had normal morphology, consistent with previous studies in knockout mice, and the muscles were able to generate appropriate force when normalized for their reduced size in late stage animals. Therefore, the Cntn1 mutant mice do not show evidence for a myopathy, but instead the phenotype is likely to be caused by dysfunction in the nervous system. Given the similarity of CNTN1 to other Ig-superfamily proteins such as DSCAMs, we also characterized the expression and localization of Cntn1 in the retinas of mutant mice for developmental defects. Despite widespread expression, no anomalies in retinal anatomy were detected histologically or using a battery of cell-type specific antibodies. We therefore conclude that the phenotype of the Cntn1 mice arises from dysfunction in the brain, spinal cord or peripheral nervous system, and is similar in either a BALB/c or B6;129;Black Swiss background, raising a possible discordance between the mouse and human phenotypes resulting from Cntn1 mutations.     

Hyperglycemia-induced O-GlcNAcylation and truncation of 4E-BP1 protein in liver of a mouse model of type 1 diabetes

4E-BP1 is a protein that, in its hypophosphorylated state, binds the mRNA cap-binding protein eIF4E and represses cap-dependent mRNA translation. By doing so, it plays a major role in the regulation of gene expression by controlling the overall rate of mRNA translation as well as the selection of mRNAs for translation. Phosphorylation of 4E-BP1 causes it to release eIF4E to function in mRNA translation. 4E-BP1 is also subject to covalent addition of N-acetylglucosamine to Ser or Thr residues (O-GlcNAcylation) as well as to truncation. In the truncated form, it is both resistant to phosphorylation and able to bind eIF4E with high affinity. In the present study, Ins2(Akita/+) diabetic mice were used to test the hypothesis that hyperglycemia and elevated flux of glucose through the hexosamine biosynthetic pathway lead to increased O-GlcNAcylation and truncation of 4E-BP1 and consequently decreased eIF4E function in the liver. The amounts of both full-length and truncated 4E-BP1 bound to eIF4E were significantly elevated in the liver of diabetic as compared with non-diabetic mice. In addition, O-GlcNAcylation of both the full-length and truncated proteins was elevated by 2.5- and 5-fold, respectively. Phlorizin treatment of diabetic mice lowered blood glucose concentrations and reduced the expression and O-GlcNAcylation of 4E-BP1. Additionally, when livers were perfused in the absence of insulin, 4E-BP1 phosphorylation in the livers of diabetic mice was normalized to the control value, yet O-GlcNAcylation and the association of 4E-BP1 with eIF4E remained elevated in the liver of diabetic mice. These findings provide insight into the pathogenesis of metabolic abnormalities associated with diabetes.     

Seasonal photosynthetic gas exchange and water-use efficiency in a constitutive CAM plant, the giant saguaro cactus (Carnegiea gigantea)

Crassulacean acid metabolism (CAM) and the capacity to store large quantities of water are thought to confer high water use efficiency (WUE) and survival of succulent plants in warm desert environments. Yet the highly variable precipitation, temperature and humidity conditions in these environments likely have unique impacts on underlying processes regulating photosynthetic gas exchange and WUE, limiting our ability to predict growth and survival responses of desert CAM plants to climate change. We monitored net CO₂ assimilation (A _net), stomatal conductance (g _s), and transpiration (E) rates periodically over 2 years in a natural population of the giant columnar cactus Carnegiea gigantea (saguaro) near Tucson, Arizona USA to investigate environmental and physiological controls over carbon gain and water loss in this ecologically important plant. We hypothesized that seasonal changes in daily integrated water use efficiency (WUE_day) in this constitutive CAM species would be driven largely by stomatal regulation of nighttime transpiration and CO₂ uptake responding to shifts in nighttime air temperature and humidity. The lowest WUE_day occurred during time periods with extreme high and low air vapor pressure deficit (D _a). The diurnal with the highest D _a had low WUE_day due to minimal net carbon gain across the 24 h period. Low WUE_day was also observed under conditions of low D _a; however, it was due to significant transpiration losses. Gas exchange measurements on potted saguaro plants exposed to experimental changes in D _a confirmed the relationship between D _a and g _s. Our results suggest that climatic changes involving shifts in air temperature and humidity will have large impacts on the water and carbon economy of the giant saguaro and potentially other succulent CAM plants of warm desert environments.     

Mammalian Sir2 homolog SIRT3 regulates global mitochondrial lysine acetylation

Homologs of the Saccharomyces cerevisiae Sir2 protein, sirtuins, promote longevity in many organisms. Studies of the sirtuin SIRT3 have so far been limited to cell culture systems. Here, we investigate the localization and function of SIRT3 in vivo. We show that endogenous mouse SIRT3 is a soluble mitochondrial protein. To address the function and relevance of SIRT3 in the regulation of energy metabolism, we generated and phenotypically characterized SIRT3 knockout mice. SIRT3-deficient animals exhibit striking mitochondrial protein hyperacetylation, suggesting that SIRT3 is a major mitochondrial deacetylase. In contrast, no mitochondrial hyperacetylation was detectable in mice lacking the two other mitochondrial sirtuins, SIRT4 and SIRT5. Surprisingly, despite this biochemical phenotype, SIRT3-deficient mice are metabolically unremarkable under basal conditions and show normal adaptive thermogenesis, a process previously suggested to involve SIRT3. Overall, our results extend the recent finding of lysine acetylation of mitochondrial proteins and demonstrate that SIRT3 has evolved to control reversible lysine acetylation in this organelle.     

Ena/VASP Is Required for neuritogenesis in the developing cortex

Mammalian cortical development involves neuronal migration and neuritogenesis; this latter process forms the structural precursors to axons and dendrites. Elucidating the pathways that regulate the cytoskeleton to drive these processes is fundamental to our understanding of cortical development. Here we show that loss of all three murine Ena/VASP proteins, a family of actin regulatory proteins, causes neuronal ectopias, alters intralayer positioning in the cortical plate, and, surprisingly, blocks axon fiber tract formation during corticogenesis. Cortical fiber tract defects in the absence of Ena/VASP arise from a failure in neurite initiation, a prerequisite for axon formation. Neurite initiation defects in Ena/VASP-deficient neurons are preceded by a failure to form bundled actin filaments and filopodia. These findings provide insight into the regulation of neurite formation and the role of the actin cytoskeleton during cortical development.     

The habitat functional response links seasonal third‐order selection to second‐order landscape characteristics

Determining how animals respond to differences in resource availabilities across spatiotemporal extents is critical to our understanding of organism distributions. Variations in resource distribution leading to changes in spatial arrangements across landscapes are indicative of a habitat functional response. Our goal was to assess how resource availabilities influenced both second‐order (i.e., home ranging behavior) and third‐order (i.e., habitat or resource selection) selection by feral pigs (Sus scrofa) in an agricultural landscape. We defined agriculturally based seasons to estimate home range characteristics using autocorrelated kernel density estimation within each season. We then modeled home range size as a function of resource availability (i.e., resource selection analyses) to determine whether individual behaviors were predicted by shifts in home ranging behavior. Both home range analyses and resource selection analyses indicated seasonal differences in selection for agricultural resources as availabilities changed, suggesting second‐ and third‐order selection is mechanistically linked through a habitat functional response.     

Mutations in a p-type ATPase gene cause axonal degeneration

Neuronal loss and axonal degeneration are important pathological features of many neurodegenerative diseases. The molecular mechanisms underlying the majority of axonal degeneration conditions remain unknown. To better understand axonal degeneration, we studied a mouse mutant wabbler-lethal (wl). Wabbler-lethal (wl) mutant mice develop progressive ataxia with pronounced neurodegeneration in the central and peripheral nervous system. Previous studies have led to a debate as to whether myelinopathy or axonopathy is the primary cause of neurodegeneration observed in wl mice. Here we provide clear evidence that wabbler-lethal mutants develop an axonopathy, and that this axonopathy is modulated by Wld(s) and Bax mutations. In addition, we have identified the gene harboring the disease-causing mutations as Atp8a2. We studied three wl alleles and found that all result from mutations in the Atp8a2 gene. Our analysis shows that ATP8A2 possesses phosphatidylserine translocase activity and is involved in localization of phosphatidylserine to the inner leaflet of the plasma membrane. Atp8a2 is widely expressed in the brain, spinal cord, and retina. We assessed two of the mutant alleles of Atp8a2 and found they are both nonfunctional for the phosphatidylserine translocase activity. Thus, our data demonstrate for the first time that mutation of a mammalian phosphatidylserine translocase causes axon degeneration and neurodegenerative disease.     

Decreased hypertrophic differentiation accompanies enhanced matrix formation in co-cultures of outer meniscus cells with bone marrow mesenchymal stromal cells

Introduction

The main objective of this study was to determine whether meniscus cells from the outer (MCO) and inner (MCI) regions of the meniscus interact similarly to or differently with mesenchymal stromal stem cells (MSCs). Previous study had shown that co-culture of meniscus cells with bone marrow-derived MSCs result in enhanced matrix formation relative to mono-cultures of meniscus cells and MSCs. However, the study did not examine if cells from the different regions of the meniscus interacted similarly to or differently with MSCs.

Methods

Human menisci were harvested from four patients undergoing total knee replacements. Tissue from the outer and inner regions represented pieces taken from one third and two thirds of the radial distance of the meniscus, respectively. Meniscus cells were released from the menisci after collagenase treatment. Bone marrow MSCs were obtained from the iliac crest of two patients after plastic adherence and in vitro culture until passage 2. Primary meniscus cells from the outer (MCO) or inner (MCI) regions of the meniscus were co-cultured with MSCs in three-dimensional (3D) pellet cultures at 1:3 ratio, respectively, for 3 weeks in the presence of serum-free chondrogenic medium containing TGF-β1. Mono-cultures of MCO, MCI and MSCs served as experimental control groups. The tissue formed after 3 weeks was assessed biochemically, histochemically and by quantitative RT-PCR.

Results

Co-culture of inner (MCI) or outer (MCO) meniscus cells with MSCs resulted in neo-tissue with increased (up to 2.2-fold) proteoglycan (GAG) matrix content relative to tissues formed from mono-cultures of MSCs, MCI and MCO. Co-cultures of MCI or MCO with MSCs produced the same amount of matrix in the tissue formed. However, the expression level of aggrecan was highest in mono-cultures of MSCs but similar in the other four groups. The DNA content of the tissues from co-cultured cells was not statistically different from tissues formed from mono-cultures of MSCs, MCI and MCO. The expression of collagen I (COL1A2) mRNA increased in co-cultured cells relative to mono-cultures of MCO and MCI but not compared to MSC mono-cultures. Collagen II (COL2A1) mRNA expression increased significantly in co-cultures of both MCO and MCI with MSCs compared to their own controls (mono-cultures of MCO and MCI respectively) but only the co-cultures of MCO:MSCs were significantly increased compared to MSC control mono-cultures. Increased collagen II protein expression was visible by collagen II immuno-histochemistry. The mRNA expression level of Sox9 was similar in all pellet cultures. The expression of collagen × (COL10A1) mRNA was 2-fold higher in co-cultures of MCI:MSCs relative to co-cultures of MCO:MSCs. Additionally, other hypertrophic genes, MMP-13 and Indian Hedgehog (IHh), were highly expressed by 4-fold and 18-fold, respectively, in co-cultures of MCI:MSCs relative to co-cultures of MCO:MSCs.

Conclusions

Co-culture of primary MCI or MCO with MSCs resulted in enhanced matrix formation. MCI and MCO increased matrix formation similarly after co-culture with MSCs. However, MCO was more potent than MCI in suppressing hypertrophic differentiation of MSCs. These findings suggest that meniscus cells from the outer-vascular regions of the meniscus can be supplemented with MSCs in order to engineer functional grafts to reconstruct inner-avascular meniscus.