Inhibition of Prolyl Hydroxylase Protects against 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Neurotoxicity: MODEL FOR THE POTENTIAL INVOLVEMENT OF THE HYPOXIA-INDUCIBLE FACTOR PATHWAY IN PARKINSON DISEASE*

Hypoxia-inducible factor (HIF) plays an important role in cell survival by regulating iron, antioxidant defense, and mitochondrial function. Pharmacological inhibitors of the iron-dependent enzyme class prolyl hydroxylases (PHD), which target α subunits of HIF proteins for degradation, have recently been demonstrated to alleviate neurodegeneration associated with stroke and hypoxic-ischemic injuries. Here we report that inhibition of PHD by 3,4-dihydroxybenzoate (DHB) protects against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced nigral dopaminergic cell loss and up-regulates HIF-1α within these neurons. Elevations in mRNA and protein levels of HIF-dependent genes heme oxygenase-1 (Ho-1) and manganese superoxide dismutase (Mnsod) following DHB pretreatment alone are also maintained in the presence of MPTP. MPTP-induced reductions in ferroportin and elevations in nigral and striatal iron levels were reverted to levels comparable with that of untreated controls with DHB pretreatment. Reductions in pyruvate dehydrogenase mRNA and activity resulting from MPTP were also found to be attenuated by DHB. In vitro, the HIF pathway was activated in N27 cells grown at 3% oxygen treated with either PHD inhibitors or an iron chelator. Concordant with our in vivo data, the MPP⁺-elicited increase in total iron as well as decreases in cell viability were attenuated in the presence of DHB. Taken together, these data suggest that protection against MPTP neurotoxicity may be mediated by alterations in iron homeostasis and defense against oxidative stress and mitochondrial dysfunction brought about by cellular HIF-1α induction. This study provides novel data extending the possible therapeutic utility of HIF induction to a Parkinson disease model of neurodegeneration, which may prove beneficial not only in this disorder itself but also in other diseases associated with metal-induced oxidative stress.Parkinson disease (PD)² is a neurodegenerative disorder primarily associated with loss of dopaminergic (DAergic) neurons of the pars compacta region of the substantia nigra (SNpc). Dopaminergic neurons are particularly prone to oxidative damage due to high levels of inherent reactive oxygen species that are produced during dopamine synthesis or its breakdown by monoamine oxidases or autoxidation to quinones (1–3). Importantly, iron bound to neuromelanin within DAergic neurons can subsequently react with metabolically liberated hydrogen peroxide through the Fenton reaction to produce extremely toxic hydroxyl radicals. If not properly buffered, hydroxyl radicals can stimulate protein oxidation and lipid peroxidation, which is thought to contribute to macromolecular injury and neuronal death. Iron is the most abundant metal in the brain and some degree of accessible reactive iron is necessary for brain viability as it serves as a cofactor in DNA, RNA, and protein synthesis and for heme and non-heme enzymes involved in both mitochondrial respiration and neurotransmitter synthesis (4). Although iron deficiencies early in life are known to result in impairments in brain development (5), high concentrations of iron may result in cellular toxicity (6) in part due to its ability to catalyze the production of toxic oxygen radicals.An important family of enzymes that require iron as an essential cofactor are the prolyl 4-hydroxylases (PHDs), which serve to hydroxylate proline residues situated within hypoxia-inducible factor proteins (HIFs) (7). Under hypoxic or iron-lacking conditions, PHDs are prevented from hydroxylating proline residues within the alpha (α) subunits of the HIF protein, preventing the ubiquitination and proteasomal degradation of the protein. Stabilization of HIFα results in its accumulation within the cytosol and translocation to the nucleus where it binds HIFβ and then to hypoxia response elements found on a variety of genes including heme oxygenase-1 (Ho-1) and manganese superoxide dismutase (Mnsod).Previous studies have demonstrated that deferoxamine, an iron chelator, can activate HIF-1α and prevent neuronal death in both in vitro and in vivo models of ischemia likely via inhibition of PHDs (8, 9). PHD inhibitors have been demonstrated to prevent oxidative cell death and ischemic injury via HIF pathway activation (10). More recently, it has been shown that inactivation of HIF-1α in specific cortical and striatal neurons exacerbated tissue damage in a mouse model of ischemia (11). With increasing evidence of the protective effects of induction of HIF-dependent gene products involved in iron regulation, cell survival, and energy metabolism, PHD inhibitors have been implicated as targets for neuroprotection in the central nervous system. We demonstrate here that PHD inhibition increases induction of HIF and HIF-related genes, functionally impacts on parameters of iron homeostasis and metabolic function, and, most importantly, significantly reduces the extent of DAergic nigrostriatal injury observed in the well established murine MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) PD model.     

Abnormalities in oxygen sensing define early and late onset preeclampsia as distinct pathologies

Background

The pathogenesis of preeclampsia, a serious pregnancy disorder, is still elusive and its treatment empirical. Hypoxia Inducible Factor-1 (HIF-1) is crucial for placental development and early detection of aberrant regulatory mechanisms of HIF-1 could impact on the diagnosis and management of preeclampsia. HIF-1α stability is controlled by O₂-sensing enzymes including prolyl hydroxylases (PHDs), Factor Inhibiting HIF (FIH), and E3 ligases Seven In Absentia Homologues (SIAHs). Here we investigated early- (E-PE) and late-onset (L-PE) human preeclamptic placentae and their ability to sense changes in oxygen tension occurring during normal placental development.

Methods and Findings

Expression of PHD2, FIH and SIAHs were significantly down-regulated in E-PE compared to control and L-PE placentae, while HIF-1α levels were increased. PHD3 expression was increased due to decreased FIH levels as demonstrated by siRNA FIH knockdown experiments in trophoblastic JEG-3 cells. E-PE tissues had markedly diminished HIF-1α hydroxylation at proline residues 402 and 564 as assessed with monoclonal antibodies raised against hydroxylated HIF-1α P402 or P564, suggesting regulation by PHD2 and not PHD3. Culturing villous explants under varying oxygen tensions revealed that E-PE, but not L-PE, placentae were unable to regulate HIF-1α levels because PHD2, FIH and SIAHs did not sense a hypoxic environment.

Conclusion

Disruption of oxygen sensing in E-PE vs. L-PE and control placentae is the first molecular evidence of the existence of two distinct preeclamptic diseases and the unique molecular O₂-sensing signature of E-PE placentae may be of diagnostic value when assessing high risk pregnancies and their severity.     

ING1b negatively regulates HIF1α protein levels in adipose-derived stromal cells by a SUMOylation-dependent mechanism

Hypoxic niches help maintain mesenchymal stromal cell properties, and their amplification under hypoxia sustains their immature state. However, how MSCs maintain their genomic integrity in this context remains elusive, since hypoxia may prevent proper DNA repair by downregulating expression of BRCA1 and RAD51. Here, we find that the ING1b tumor suppressor accumulates in adipose-derived stromal cells (ADSCs) upon genotoxic stress, owing to SUMOylation on K193 that is mediated by the E3 small ubiquitin-like modifier (SUMO) ligase protein inhibitor of activated STAT protein γ (PIAS4). We demonstrate that ING1b finely regulates the hypoxic response by triggering HIF1α proteasomal degradation. On the contrary, when mutated on its SUMOylation site, ING1b failed to efficiently decrease HIF1α levels. Consistently, we observed that the adipocyte differentiation, generally described to be downregulated by hypoxia, was highly dependent on ING1b expression, during the early days of this process. Accordingly, contrary to what was observed with HIF1α, the absence of ING1b impeded the adipogenic induction under hypoxic conditions. These data indicate that ING1b contributes to adipogenic induction in adipose-derived stromal cells, and thus hinders the phenotype maintenance of ADSCs.Human mesenchymal stem/stromal cells (MSCs) are able to self-renew and differentiate into various cell types. Recently, MSCs have been developed as tools for tissue engineering and cell-based therapies¹ in particular owing to their trophic and immunosuppressive activities.² Conventionally, the bone marrow MSCs (BM-MSCs) and the adipose-derived stem/stromal cells (ADSCs) have constituted the main sources of MSCs for clinical use. These cells are expanded in vitro prior to their application; however, this long-term culture may allow the emergence of senescence and phenotypic alterations, rendering MSCs unsuitable for clinical purposes.³To overcome these issues, MSC culture in conditions mimicking hypoxic niches has been tested.⁴ Low O₂ tensions promote MSC growth, survival and maintain their self-renewing multipotent state.⁵ However, how hypoxia (1% O₂) affects MSC behavior is unclear. Responses to hypoxia are mainly mediated by hypoxia inducible factors (HIFs). HIF1, 2 and 3α subunits, are constitutively degraded in normoxia and stabilized in hypoxia. Consequently, when stabilized they can dimerize with HIF1β, and then translocate into the nucleus to modulate the expression of selected genes. HIF1α is highly expressed in MSCs, controls their metabolic fate and maintains them in an undifferentiated state.⁶ HIF1α has also been shown to delay the occurrence of senescence in MSCs, by repressing E2A and p21 expression.⁷The inhibitors of growth (ING) family genes act as readers of the epigenetic histone code. Among them, ING1 has been described as a type II tumor suppressor, regulating cell growth, DNA repair, apoptosis, chromatin remodeling and senescence.⁸ To some extent, ING1 and HIF might have opposite effects, (e.g. on tumor progression). Indeed, HIF1α, unlike ING1 that inhibits angiogenesis, promotes angiogenesis.⁹ Furthermore, p53, a well-known ING1b interactor, and HIF1α have been shown in several studies to have antagonistic effects. Following DNA damage, p53 induces apoptosis and inhibits survival of cells by reducing activity and levels of HIF1α.^{10, 11}So far, ING4 has been shown as the only ING protein to regulate the hypoxic response. Indeed, by interacting with HIF prolyl hydroxylase 2 (HPH-2), ING4 has been described to repress some HIF1α activities under hypoxic conditions.¹² Here, we show that ING1b accumulates in ADSCs following DNA damage in hypoxia. According to the opposing roles of ING1b and HIF1α, we hypothesized that ING1b could interfere with HIF1α and participate in the conservation of the genomic integrity of MSCs. Mechanistically, we found that ING1b interacted with HIF1α and promoted its proteasomal degradation in hypoxia. SUMOylation of ING1b played a role since the unSUMOylated form of ING1b was unable to trigger HIF1α degradation. The E3 small ubiquitin-like modifier (SUMO) ligase protein inhibitor of activated STAT protein γ (PIAS4) participated in HIF1α degradation and ING1b accumulation following a genotoxic stress in 1% O₂. ING1b, subsequently, took part in decreasing PIAS4 levels after DNA damage. Finally, we report that ING1b by decreasing HIF1α level modulated ADSC differentiation potential. These data indicate that ING1b, according to its SUMOylation status, regulates the hypoxic response by contributing to the HIF1α degradation, and therefore may impede HIF1α-related effects on the maintenance of ADSCs stem cell character.     

Prolyl 4-Hydroxylation of ��-Fibrinogen: A NOVEL PROTEIN MODIFICATION REVEALED BY PLASMA PROTEOMICS*

Plasma proteome analysis requires sufficient power to compare numerous samples and detect changes in protein modification, because the protein content of human samples varies significantly among individuals, and many plasma proteins undergo changes in the bloodstream. A label-free proteomics platform developed in our laboratory, termed “Two-Dimensional Image Converted Analysis of Liquid chromatography and mass spectrometry (2DICAL),” is capable of these tasks. Here, we describe successful detection of novel prolyl hydroxylation of α-fibrinogen using 2DICAL, based on comparison of plasma samples of 38 pancreatic cancer patients and 39 healthy subjects. Using a newly generated monoclonal antibody 11A5, we confirmed the increase in prolyl-hydroxylated α-fibrinogen plasma levels and identified prolyl 4-hydroxylase A1 as a key enzyme for the modification. Competitive enzyme-linked immunosorbent assay of 685 blood samples revealed dynamic changes in prolyl-hydroxylated α-fibrinogen plasma level depending on clinical status. Prolyl-hydroxylated α-fibrinogen is presumably controlled by multiple biological mechanisms, which remain to be clarified in future studies.For comprehensive analysis of plasma proteins, it is necessary to compare a sufficient number of blood samples to avoid simple interindividual heterogeneity, because the protein content of human samples varies significantly among individuals. Also, the provision of sufficient power is needed to detect protein modification because many plasma proteins undergo changes in the bloodstream (1). Even though the proteomic technologies have advanced (2, 3), there remains room for improvement. Different isotope labeling and identification-based methods have been developed for quantitative proteomics technologies (4–6), but the number of samples that can be compared by the current isotope-labeling methods is limited, and identification-based proteomics is unable to capture information regarding unknown modifications.A label-free proteomics platform developed in our laboratory, termed “Two-Dimensional Image Converted Analysis of Liquid chromatography and mass spectrometry (2DICAL)² (7), simply compares the liquid chromatography and mass spectrometry (LC-MS) data and detects a protein modification by finding changes in the mass to charge ratio (m/z) and retention time (RT). Enhanced methods for accurate MS peak alignment across multiple LC runs have enabled the successful implementation of clinical studies requiring comparison of a large number of samples (8, 9). Using 2DICAL to analyze plasma samples of pancreatic cancer patients and healthy controls, novel prolyl hydroxylation of α-fibrinogen was successfully discovered.Fibrinogen and its modification has been investigated because of its clinical importance (10, 11). On the other hand, prolyl hydroxylation has attracted attention after the discovery of the hypoxia-inducible factor 1α (HIF1α) prolyl-hydroxylase and its role in switching of HIF1α functions (12). Prolyl hydroxylation in other proteins has been energetically sought, but only a few such proteins have been identified (13). Only one study has reported prolyl hydroxylation of fibrinogen at the β chain (14).Here, we report the detection of prolyl 4-hydroxylated α-fibrinogen by plasma proteome analysis, a protein modification that dynamically changes in plasma depending on the clinical status and is a candidate plasma biomarker.     

Roles of β-Tubulin Residues Ala428 and Thr429 in Microtubule Formation in Vivo

The C termini of β-tubulin isotypes are regions of high sequence variability that bind to microtubule-associated proteins and motors and undergo various post-translational modifications such as polyglutamylation and polyglycylation. Crystallographic analyses have been unsuccessful in resolving tubulin C termini. Here, we used a stepwise approach to study the role of this region in microtubule assembly. We generated a series of truncation mutants of human βI and βIII tubulin. Transient transfection of HeLa cells with the mutants shows that mutants with deletions of up to 22 residues from βIII and 16 from βI can assemble normally. Interestingly, removal of the next residue (Ala⁴²⁸) results in a complete loss of microtubule formation without affecting dimer formation. C-terminal tail switching of human βI and βIII tubulin suggests that C-terminal tails are functionally equivalent. In short, residues outside of 1–429 of human β-tubulins make no contribution to microtubule assembly. Ala⁴²⁸, in the C-terminal sequence motif N-QQYQDA⁴²⁸, lies at the end of helix H12 of β-tubulin. We hypothesize that this residue is important for maintaining helix H12 structure. Deletion of Ala⁴²⁸ may lead to unwinding of helix H12, resulting in tubulin dimers incapable of assembly. Thr⁴²⁹ plays a more complex role. In the βI isotype of tubulin, Thr⁴²⁹ is not at all necessary for assembly; however, in the βIII isotype, its presence strongly favors assembly. This result is consistent with a likely more complex function of βIII as well as with the observation that evolutionary conservation is total for Ala⁴²⁸ and frequent for Thr⁴²⁹.Microtubules are involved in a great variety of cellular functions. Their constituent protein tubulin is an αβ heterodimer, both α- and β-tubulin existing as multiple isotypes, encoded by different genes and differing in amino acid sequence (1). The differences among the isotypes are highly conserved in evolution. In mammals, the β isotypes are βIa, βIb, βII, βIII, βIVa, βIVb, βV, and βVI. There is evidence that the isotype differences have functional significance. For instance, the βIV isotype is found in all axonemes (2).Structurally, both α- and β-tubulin consist of a globular region of 427 amino acids followed by a C-terminal region of 17–24 amino acids (3–5). The C-terminal region is highly negatively charged, being especially rich in glutamate residues and lacking in basic residues, and is likely to project outward from the rest of the molecule, because of its high negative charge and the electrostatic repulsion among the glutamate residues (3). The three-dimensional structure of the globular domain has been determined by electron and x-ray crystallography (4, 5). However, the C-terminal region has never been localized in the three-dimensional reconstructions except by computer modeling. The probable reasons for this are 1) that, if the C-terminal region projects out from the rest of molecule, it is likely to be very flexible with respect to the rest of the molecule and 2) the C-terminal region undergoes post-translational modification. Both of these can lead to structural heterogeneity and cause the C terminus to be invisible to crystallographic techniques.In this work, we examine the role of the C termini of human β-tubulins to determine the minimal sequence requirement for microtubule incorporation through structure/function analyses. The human βI and βIII tubulin isotypes were utilized based on their high degree of sequence variability clustered at the C terminus (Fig. 1) and the fact that βI is broadly distributed among normal tissues, whereas βIII has a very narrow tissue distribution. These two isotypes share 92% sequence identity, with differences among these isotypes occurring in both the globular domain and the C-terminal region (1).Open in a separate windowFIGURE 1.Sequence alignment of human βIa and βIII tubulin isotypes. Human βIa and βIII tubulin isotypes were aligned with ClustalX 1.83 and processed with BioEdit. Hyphens denote identical residues between sequences.

TABLE 1

The C-terminal amino acid sequences of the human β-tubulin isotypes

The return of Dr Jekyll in cancer metastasis

Cancer Cell 22: 571–584Metastasis, the process whereby tumour cells disseminate and colonize distant organs, is the primary cause of cancer mortality. Diverse models have been proposed to explain how tumour cells acquire metastatic competency. Calon et al (2012) now provide insight into the molecular underpinnings of metastasis by describing a key stromal, non-cell autonomous role for Transforming Growth Factor-beta (TGFβ) in promoting the initiation of colonization in otherwise TGFβ-resistant colorectal cancer (CRC) cells.Growing tumour cells are surrounded by stroma, a heterogenous population of cells that includes fibroblasts, endothelial precursors and cells of the immune system (Sethi and Kang, 2011; Valastyan and Weinberg, 2011). This stroma engages in an active dialogue with the tumour cells to create a unique microenvironment that is conducive to the survival and progression of a growing tumour. In late stage tumours, productive metastases arise when the tumour cells leave the primary site to disseminate throughout the body and seed new secondary tumours in distant organs. How tumour cells leave behind their primary microenvironment to establish and successfully colonize secondary sites that might harbour tumour-hostile environments has been the subject of extensive research and speculation. A recent study by Calon et al (2012) provides new insights into this long-standing question with the discovery that Transforming Growth Factor-beta (TGFβ) produced by tumour cells critically promotes colorectal cancer (CRC) cell colonization through its actions on the stroma (Figure 1).Open in a separate windowFigure 1TGFβ acting on stromal cells in the primary tumour promotes metastasis. Colorectal cancer cells (CRCs) are frequently insensitive to TGFβ as a result of mutations in pathway components, including the TGFβ receptors (TBRs) and Smads. At the primary tumour site, CRCs that secrete high level of TGFβ induce expression of IL11 in the cancer-associated fibroblasts (CAFs) found in the adjacent stroma. The CRCs then respond to the IL11 via GP130 and this promotes tumour colonization of secondary sites.The secreted factor, TGFβ has been called the ‘Dr Jekyll and Mr Hyde'' of cancer (Bierie and Moses, 2006) due to paradoxical function as both a tumour suppressor and a tumour promoter. For instance, TGFβ inhibits the proliferation of epithelial cells, an activity that most tumour cells must learn to overcome during cancer progression. However, TGFβ also promotes the metastatic phenotype by enhancing tumour cell migration and promoting epithelial-to-mesenchymal transition (EMT).In human CRC, the majority of tumour cells display constitutive Wnt signalling, typically because of mutations in either the adenomas polyposis gene (APC) or β catenin. However, mutations in TGFβ signalling pathway components, including the cell-surface receptors or the intracellular Smad mediator proteins, also play a prominent role, consistent with a tumour suppressive function of TGFβ. Nevertheless, high levels of TGFβ are found in CRC patients and correlates with poor clinical outcome (Tsushima et al, 2001). This raises the question of how TGFβ might promote poor clinical outcome in cancers that have acquired insensitivity to TGFβ. To explore this, Calon et al (2012) examined TGFβ expression levels in a large cohort of CRC patients and noted a strong positive association between the level of TGFβ expression and the risk of cancer recurrence. Indeed, TGFβ expression level outperformed the American Joint Cancer Committee (AJCC) staging system in predictive power. Consistent with the frequent loss of TGFβ pathway mediators in CRC, staining of tumour sections for active TGFβ signalling showed much higher levels in stromal cells as compared to the epithelial CRC cells. Expression profiling for TGFβ-responsive gene signatures (TBRS) using isolated stromal cell populations corroborated this observation, with high levels of TGFβ signalling evident in all stromal cell types tested, including fibroblasts, endothelial and immune cells. However, analysis of in vivo gene expression patterns revealed that it was the TBRS associated with cancer-associated fibroblasts (CAFs) that was the main predictor of poor outcome after therapy.To provide direct evidence of a connection between stromal TGFβ signalling and disease progression, Calon et al (2012) first conducted an elegant series of in vivo experiments in mice, using several colorectal cell lines that have inactivated TGFβ signalling. Subcutaneous injection of variants of these lines engineered to overexpress TGFβ led to activation of TGFβ signalling in adjacent stroma. Calon et al (2012) next turned their attention to examining whether stromal TGFβ signalling might influence metastasis. Inoculation of the engineered CRC cells in the caecum or spleen enhanced the rate of metastasis to the lung and/or liver that was particularly pronounced within the first 24 h post inoculation and most notably was abolished by administration of LY2157299, a TGFβ receptor-selective inhibitor. Similarly, liver metastasis arising through intra-splenic injection of colon cancer stem cells isolated from a patient with TGFβ receptor mutations was abolished by systemic TGFβ receptor-inhibitor treatment. Thus, high levels of TGFβ act to enhance the colonization capability of CRC cells at the initial phase of metastasis.In breast cancer cells, which retain an intact TGFβ signalling pathway, a cell-autonomous role for secreted TGFβ in mediating organ-specific metastatic colonization has been delineated (Kang et al, 2003; Massague, 2008; Padua et al, 2008). However, since the CRC cells employed by Calon et al (2012) were insensitive to TGFβ, the authors focused on the question of how stromal TGFβ signalling might confer a metastatic phenotype to the tumour cells. They went on to show that the IL11, which is secreted by TGFβ-stimulated CAFs, acting through the GP130/STAT3 pathway in the CRC cells, was required for colonization, most likely by suppressing tumour cell apoptosis (Figure 1). This likely allows the tumour cells to survive in the relatively hostile metastatic environments they encounter on their way to distant sites. Indeed, CRC cells engineered to produce their own IL11 effectively colonized liver, lungs, distant lymph nodes and brain.The process of metastasis is extremely inefficient, so how a cell might undergo the genetic and/or epigenetic changes required to leave the primary site and colonize a different organ with a distinct microenvironment is a critical question. Calon et al (2012) now provide new insights into the process by showing that TGFβ signalling in CAFs directed by the tumour cells feeds back on the cancer cell in the primary site to fuel metastasis. These studies thus provide support for the notion that tumour cells acquire the necessary changes to adapt to a new environment while still residing in the primary tumour site. However, it is important to remember that the activity of TGFβ on immune cells is also a potent mechanism that regulates the tumour microenvironment to promote cancer progression (Yang et al, 2010). Although, the TBRS in CAFs was shown to be the most relevant for recurrence rates, the finding that all stromal cell types displayed TGFβ-induced changes raises additional questions for future investigations that explore the extent of cellular interplay in contributing to the tumour-promoting role of TGFβ. Understanding the ongoing dialogue between tumour cells and their microenvironment continues to yield a rich resource of new interventional targets that limit not only primary tumour growth, but also metastasis, the primary cause of cancer death.     

Autophagy--alias self-eating--appetite and ageing

Two articles—one published online in January and in the March issue EMBO reports—implicate autophagy in the control of appetite by regulating neuropeptide production in hypothalamic neurons. Autophagy decline with age in POMC neurons induces obesity and metabolic syndrome.Kaushik et al. EMBO reports, this issue doi:10.1038/embor.2011.260Macroautophagy, which I will call autophagy, is a critical process that degrades bulk cytoplasm, including organelles, protein oligomers and a range of selective substrates. It has been linked with diverse physiological and disease-associated functions, including the removal of certain bacteria, protein oligomers associated with neurodegenerative diseases and dysfunctional mitochondria [1]. However, the primordial role of autophagy—conserved from yeast to mammals—appears to be its ability to provide nutrients to starving cells by releasing building blocks, such as amino acids and free fatty acids, obtained from macromolecular degradation. In yeast, autophagy deficiency enhances death in starvation conditions [2], and in mice it causes death from starvation in the early neonatal period [3,4]. Two recent articles from the Singh group—one of them in this issue of EMBO reports—also implicate autophagy in central appetite regulation [5,6].Autophagy seems to decline with age in the liver [7], and it has thus been assumed that autophagy declines with age in all tissues, but this has not been tested rigorously in organs such as the brain. Conversely, specific autophagy upregulation in Caenorhabditis elegans and Drosophila extends lifespan, and drugs that induce autophagy—but also perturb unrelated processes, such as rapamycin—promote longevity in rodents [8].Autophagy literally means self-eating, and it is therefore interesting to see that this cellular ‘self-eating'' has systemic roles in mammalian appetite control. The control of appetite is influenced by central regulators, including various hormones and neurotransmitters, and peripheral regulators, including hormones, glucose and free fatty acids [9]. Autophagy probably has peripheral roles in appetite and energy balance, as it regulates lipolysis and free fatty acid release [10]. Furthermore, Singh and colleagues have recently implicated autophagy in central appetite regulation [5,6].The arcuate nucleus in the hypothalamus has received extensive attention as an integrator and regulator of energy homeostasis and appetite. Through its proximity to the median eminence, which is characterized by an incomplete blood–brain barrier, these neurons rapidly sense metabolic fluctuations in the blood. There are two different neuronal populations in the arcuate nucleus, which appear to have complementary effects on appetite (Fig 1). The proopiomelanocortin (POMC) neurons produce the neuropeptide precursor POMC, which is cleaved to form α-melanocyte stimulating hormone (α-MSH), among several other products. The α-MSH secreted from these neurons activates melanocortin 4 receptors on target neurons in the paraventricular nucleus of the hypothalamus, which ultimately reduce food intake. The second group of neurons contain neuropeptide Y (NPY) and Agouti-related peptide (AgRP). Secreted NPY binds to downstream neuronal receptors and stimulates appetite. AgRP blocks the ability of α-MSH to activate melanocortin 4 receptors [11]. Furthermore, AgRP neurons inhibit POMC neurons [9].Open in a separate windowFigure 1Schematic diagram illustrating the complementary roles of POMC and NPY/AgRP neurons in appetite control. AgRP, Agouti-related peptide; MC4R, melanocortin 4 receptor; α-MSH, α-melanocyte stimulating hormone; NPY, neuropeptide Y; POMC, proopiomelanocortin.The first study from Singh''s group started by showing that starvation induces autophagy in the hypothalamus [5]. This finding alone merits some comment. Autophagy is frequently assessed by using phosphatidylethanolamine-conjugated Atg8/LC3 (LC3-II), which is specifically associated with autophagosomes and autolysosomes. LC3-II levels on western blot and the number of LC3-positive vesicles strongly correlate with the number of autophagosomes [1]. To assess whether LC3-II formation is altered by a perturbation, its level can be assessed in the presence of lysosomal inhibitors, which inhibit LC3-II degradation by blocking autophagosome–lysosome fusion [12]. Therefore, differences in LC3-II levels in response to a particular perturbation in the presence of lysosomal inhibitors reflect changes in autophagosome synthesis. An earlier study using GFP-LC3 suggested that autophagy was not upregulated in the brains of starved mice, compared with other tissues where this did occur [13]. However, this study only measured steady state levels of autophagosomes and was performed before the need for lysosomal inhibitors was appreciated. Subsequent work has shown rapid flux of autophagosomes to lysosomes in primary neurons, which might confound analyses without lysosomal inhibitors [14]. Thus, the data of the Singh group—showing that autophagy is upregulated in the brain by a range of methods including lysosomal inhibitors [5]—address an important issue in the field and corroborate another recent study that examined this question by using sophisticated imaging methods [15].“…decreasing autophagy with ageing in POMC neurons could contribute to the metabolic problems associated with age”Singh and colleagues then analysed mice that have a specific knockout of the autophagy gene Atg7 in AgRP neurons [5]. Although fasting increases AgRP mRNA and protein levels in normal mice, these changes were not seen in the knockout mice. AgRP neurons provide inhibitory signals to POMC neurons, and Kaushik and colleagues found that the AgRP-specific Atg7 knockout mice had higher levels of POMC and α-MSH, compared with the normal mice. This indicated that starvation regulates appetite in a manner that is partly dependent on autophagy. The authors suggested that the peripheral free fatty acids released during starvation induce autophagy by activating AMP-activated protein kinase (AMPK), a known positive regulator of autophagy. This, in turn, enhances degradation of hypothalamic lipids and increases endogenous intracellular free fatty acid concentrations. The increased intracellular free fatty acids upregulate AgRP mRNA and protein expression. As AgRP normally inhibits POMC/α-MSH production in target neurons, a defect in AgRP responses in the autophagy-null AgRP neurons results in higher α-MSH levels, which could account for the decreased mouse bodyweight.In follow-up work, Singh''s group have now studied the effects of inhibiting autophagy in POMC neurons, again using Atg7 deletion [6]. These mice, in contrast to the AgRP autophagy knockouts, are obese. This might be accounted for, in part, by an increase in POMC preprotein levels and its cleavage product adrenocorticotropic hormone in the knockout POMC neurons, which is associated with a failure to generate α-MSH. Interestingly, these POMC autophagy knockout mice have impaired peripheral lipolysis in response to starvation, which the authors suggest might be due to reduced central sympathetic tone to the periphery from the POMC neurons. In addition, POMC-neuron-specific Atg7 knockout mice have impaired glucose tolerance.This new study raises several interesting issues. How does the autophagy defect in the POMC neurons alter the cleavage pattern of POMC? Is this modulated within the physiological range of autophagy activity fluctuations in response to diet and starvation? Importantly, in vivo, autophagy might fluctuate similarly (or possibly differently) in POMC and AgRP neurons in response to diet and/or starvation. Given the tight interrelation of these neurons, how does this affect their overall response to appetite regulation in wild-type animals?Finally, the study also shows that hypothalamic autophagosome formation is decreased in older mice. To my knowledge, this is the first such demonstration of this phenomenon in the brain. The older mice phenocopied aspects of the POMC-neuron autophagy null mice—increased hypothalamic POMC preprotein and ACTH and decreased α-MSH, along with similar adiposity and lipolytic defects, compared with young mice. These data are provocative from several perspectives. In the context of metabolism, it is tantalizing to consider that decreasing autophagy with ageing in POMC neurons could contribute to the metabolic problems associated with ageing. Again, this model considers the POMC neurons in isolation, and it would be important to understand how reduced autophagy in aged AgRP neurons counterbalances this situation. In a more general sense, the data strongly support the concept that neuronal autophagy might decline with age.Autophagy is a major clearance route for many mutant, aggregate-prone intracytoplasmic proteins that cause neurodegenerative disease, such as tau (Alzheimer disease), α-synuclein (Parkinson disease), and huntingtin (Huntington disease), and the risk of these diseases is age-dependent [1]. Thus, it is tempting to suggest that the dramatic age-related risks for these diseases could be largely due to decreased neuronal capacity of degrading these toxic proteins. Neurodegenerative pathology and age-related metabolic abonormalities might be related—some of the metabolic disturbances that occur in humans with age could be due to the accumulation of such toxic proteins. High levels of these proteins are seen in many people who do not have, or who have not yet developed, neurodegenerative diseases, as many of them start to accumulate decades before any sign of disease. These proteins might alter metabolism and appetite either directly by affecting target neurons, or by influencing hormonal and neurotransmitter inputs into such neurons.     

New Delhi metallo-��-lactamase-1: local acquisition in Ontario, Canada, and challenges in detection

New Delhi metallo-β-lactamase-1 (NDM-1) is a recently identified metallo-β-lactamase that confers resistance to carbapenems and all other β-lactam antibiotics, with the exception of aztreonam. NDM-1 is also associated with resistance to many other classes of antibiotics. The enzyme was first identified in organisms isolated from a patient in Sweden who had previously received medical treatment in India, but it is now recognized as endemic throughout India and Pakistan and has spread worldwide. The gene encoding NDM-1 has been found predominantly in Escherichia coli and Klebsiella pneumoniae. We describe the isolation NDM-1–producing organisms from two patients in Toronto, Ontario. To the best of our knowledge, this is the first report of an organism producing NDM-1 that was locally acquired in Canada. We also discuss the evidence that NDM-1 can affect bacterial species other than E. coli and K. pneumoniae, the limited options for treatment and the difficulty laboratories face in detecting organisms that produce NDM-1.New Delhi metallo-β-lactamase-1 (NDM-1) is a metallo-β-lactamase that confers resistance to carbapenems and all other β-lactam antibiotics, with the exception of aztreonam. It is predominantly found in the Enterobacteriaeceae. It was first identified in Escherichia coli and Klebsiella pneumoniae isolated from a patient in Sweden who had previously received medical treatment in India. It is now recognized as endemic throughout India and Pakistan and has spread worldwide due to travel, “medical tourism” and the ability of the genetic element encoding the enzyme to transfer between bacteria.^1–3 Three reports of organisms producing NDM-1 in Canada have been published to date. In each instance, the organisms were isolated from the urinary tracts of patients who had recently been admitted to hospitals in India. Two of the isolates were strains of K. pneumoniae and one was a strain of E. coli.^4–6 Additional reports of isolation of organisms producing NDM-1 from patients in Canada have been presented in the lay press.Organisms that produce NDM-1 have been associated with resistance to classes of antibiotics other than the β-lactams, thus severely limiting options for treatment.² Infection control guidance regarding the management of colonization by or infection with organisms that produce carbapenemases, such as NDM-1, have recently been published by Canadian and European authorities.^7–9 An essential component of these recommendations is the rapid and accurate identification of the organisms in a clinical microbiology laboratory. The Clinical Laboratory Standards Institute (CLSI) and the United States Centers for Disease Control and Prevention (CDC) recommend screening for the production of carbapenemase using the Modified Hodge Test.^10,11 If the result of that test is positive, then the presence and type of carbapenemase can be confirmed by polymerase chain reaction.⁴Herein, we summarize two additional instances in which organisms producing NDM-1 were isolated from patients in Canada and the first where the organism appears to have been acquired in Canada.