Sarah Stanley

Acute infection and macrophage subversion by Mycobacterium tuberculosis require a specialized secretion system

Although many bacterial pathogens use specialized secretion systems for virulence, no such systems have been described for Mycobacterium tuberculosis, a major pathogen of humans that proliferates in host macrophages. In a screen to identify genes required for virulence of M. tuberculosis, we have discovered three components and two substrates of the first Sec-independent secretion pathway described in M. tuberculosis, which we designate the Snm pathway. Here we demonstrate that the proteins Snm1, -2, and -4 are required for the secretion of ESAT-6 and CFP-10, small proteins previously identified as major T cell antigens. Snm2, a member of the AAA ATPase family, interacts with substrates and with Snm1, another AAA ATPase. We show that M. tuberculosis mutants lacking either the Snm system or these substrates exhibit defects in bacterial growth during the acute phase of a mouse infection and are attenuated for virulence. Strikingly, snm mutants fail to replicate in cultured macrophages and to inhibit macrophage inflammatory responses, two well established activities of wild-type M. tuberculosis bacilli. Thus, the Snm secretion pathway works to subvert normal macrophage responses and is a major determinant of M. tuberculosis virulence.

Radio-Wave Heating of Iron Oxide Nanoparticles Can Regulate Plasma Glucose in Mice

Gene Expression by Remote Control Techniques that allow remote, noninvasive activation of specific genes in specific tissues could one day be applied to regulate expression of therapeutic proteins in a clinical setting. In a proof-of-concept study, Stanley et al. (p. 604) showed that heating of iron oxide nanoparticles by radiowaves can remotely activate insulin gene expression in cultured cells and in a mouse model. Heating of membrane-targeted nanoparticles induced opening of a temperature-sensitive membrane channel in the cells and triggered calcium entry. The intracellular calcium signal in turn stimulated expression of an engineered insulin gene, leading to the synthesis and release of insulin. In experiments with mice bearing tumors that expressed the engineered insulin gene, exposure to radiowaves promoted secretion of insulin from the tumors and lowered blood glucose levels in the animals. Gene expression in mice can be activated remotely and noninvasively by radio-wave heating of nanoparticles. Medical applications of nanotechnology typically focus on drug delivery and biosensors. Here, we combine nanotechnology and bioengineering to demonstrate that nanoparticles can be used to remotely regulate protein production in vivo. We decorated a modified temperature-sensitive channel, TRPV1, with antibody-coated iron oxide nanoparticles that are heated in a low-frequency magnetic field. When local temperature rises, TRPV1 gates calcium to stimulate synthesis and release of bioengineered insulin driven by a Ca2+-sensitive promoter. Studying tumor xenografts expressing the bioengineered insulin gene, we show that exposure to radio waves stimulates insulin release from the tumors and lowers blood glucose in mice. We further show that cells can be engineered to synthesize genetically encoded ferritin nanoparticles and inducibly release insulin. These approaches provide a platform for using nanotechnology to activate cells.

Identification of neuronal subpopulations that project from hypothalamus to both liver and adipose tissue polysynaptically

The autonomic nervous system regulates fuel availability and energy storage in the liver, adipose tissue, and other organs; however, the molecular components of this neural circuit are poorly understood. We sought to identify neural populations that project from the CNS indirectly through multisynaptic pathways to liver and epididymal white fat in mice using pseudorabies virus strains expressing different reporters together with BAC transgenesis and immunohistochemistry. Neurons common to both circuits were identified in subpopulations of the paraventricular nucleus of the hypothalamus (PVH) by double labeling with markers expressed in viruses injected in both sites. The lateral hypothalamus and arcuate nucleus of the hypothalamus and brainstem regions (nucleus of the solitary tract and A5 region) also project to both tissues but are labeled at later times. Connections from these same sites to the PVH were evident after direct injection of virus into the PVH, suggesting that these regions lie upstream of the PVH in a common pathway to liver and adipose tissue (two metabolically active organs). These common populations of brainstem and hypothalamic neurons express neuropeptide Y and proopiomelanocortin in the arcuate nucleus, melanin-concentrating hormone, and orexin in the lateral hypothalamus and in the corticotrophin-releasing hormone and oxytocin in the PVH. The delineation of this circuitry will facilitate a functional analysis of the possible role of these potential command-like neurons to modulate autonomic outflow and coordinate metabolic responses in liver and adipose tissue.

A role for arcuate cocaine and amphetamine-regulated transcript in hyperphagia, thermogenesis, and cold adaptation.

We have recently shown that injection of the hypothalamic peptide cocaine and amphetamine regulated transcript (CART) into discrete hypothalamic nuclei stimulates food intake. This stimulation was particularly marked in the arcuate nucleus. Here we show that twice daily intra‐arcuate injection of 0.2 nmole CART peptide for 7 days was associated with a 60% higher daytime food intake, an 85% higher thermogenic response to the β3 agonist BRL 35135, and a 60% increase in brown adipose tissue UCP‐1 mRNA. In a separate study, using stereotactically targeted gene transfer, a CART transgene was delivered by using polyethylenimine to the arcuate nucleus of adult rats. Food intake was increased significantly during ad libitum feeding and following periods of food withdrawal and food restriction in CART over‐expressing animals. CART over‐expressing animals lost 12% more weight than controls following a 24‐h fast. Brown adipose tissue uncoupling protein‐1 (UCP‐1) mRNA levels (collected Day 25) were 80% higher in CART over‐expressing animals. Finally, by using quantitative in situ hybridization, we found that chronic cold exposure (20 days at 4oC) increased arcuate nucleus CART mRNA by 124%. Together with the orexigenic and thermogenic effects of CART, this finding suggests a role for arcuate nucleus CART in cold adaptation.

Effects of acute and chronic relaxin-3 on food intake and energy expenditure in rats

The effects of acute and repeated intraparaventricular (iPVN) administration of human relaxin-3 (H3) were examined on food intake, energy expenditure, and the hypothalamo-pituitary thyroid axis in male Wistar rats. An acute high dose iPVN injection of H3 significantly increased food intake 1 h post-administration [0.4+/-0.1 g (vehicle) vs 1.6+/-0.5 g (180 pmol H3), 2.4+/-0.5 g (540 pmol H3) and 2.2+/-0.5 g (1,620 pmol H3), p<0.05 for all doses vs vehicle]. Repeated iPVN H3 injection (180 pmol/twice a day for 7 days) significantly increased cumulative food intake in ad libitum fed animals compared with vehicle [211.8+/-7.1 g (vehicle) vs 261.6+/-6.7 g (ad libitum fed H3), p<0.05]. Plasma leptin was increased in the H3 ad libitum fed group. Plasma thyroid stimulating hormone was significantly decreased after acute and repeated administration of H3. These data suggest H3 may play a role in long-term control of food intake.

Remote regulation of glucose homeostasis in mice using genetically encoded nanoparticles

Means for temporally regulating gene expression and cellular activity are invaluable for elucidating underlying physiological processes and would have therapeutic implications. Here we report the development of a genetically encoded system for remote regulation of gene expression by low-frequency radio waves (RFs) or a magnetic field. Iron oxide nanoparticles are synthesized intracellularly as a GFP-tagged ferritin heavy and light chain fusion. The ferritin nanoparticles associate with a camelid anti-GFP–transient receptor potential vanilloid 1 fusion protein, αGFP-TRPV1, and can transduce noninvasive RF or magnetic fields into channel activation, also showing that TRPV1 can transduce a mechanical stimulus. This, in turn, initiates calcium-dependent transgene expression. In mice with stem cell or viral expression of these genetically encoded components, remote stimulation of insulin transgene expression with RF or a magnet lowers blood glucose. This robust, repeatable method for remote regulation in vivo may ultimately have applications in basic science, technology and therapeutics.

Bidirectional electromagnetic control of the hypothalamus regulates feeding and metabolism.

Targeted, temporally regulated neural modulation is invaluable in determining the physiological roles of specific neural populations or circuits. Here we describe a system for non-invasive, temporal activation or inhibition of neuronal activity in vivo and its use to study central nervous system control of glucose homeostasis and feeding in mice. We are able to induce neuronal activation remotely using radio waves or magnetic fields via Cre-dependent expression of a GFP-tagged ferritin fusion protein tethered to the cation-conducting transient receptor potential vanilloid 1 (TRPV1) by a camelid anti-GFP antibody (anti-GFP–TRPV1). Neuronal inhibition via the same stimuli is achieved by mutating the TRPV1 pore, rendering the channel chloride-permeable. These constructs were targeted to glucose-sensing neurons in the ventromedial hypothalamus in glucokinase–Cre mice, which express Cre in glucose-sensing neurons. Acute activation of glucose-sensing neurons in this region increases plasma glucose and glucagon, lowers insulin levels and stimulates feeding, while inhibition reduces blood glucose, raises insulin levels and suppresses feeding. These results suggest that pancreatic hormones function as an effector mechanism of central nervous system circuits controlling blood glucose and behaviour. The method we employ obviates the need for permanent implants and could potentially be applied to study other neural processes or used to regulate other, even dispersed, cell types.

Relaxin-3 stimulates the hypothalamic-pituitary-gonadal axis

The hypothalamus plays a key role in the regulation of both energy homeostasis and reproduction. Evidence suggests that relaxin-3, a recently discovered member of the insulin superfamily, is an orexigenic hypothalamic neuropeptide. Relaxin-3 is thought to act in the brain via the RXFP3 receptor, although the RXFP1 receptor may also play a role. Relaxin-3, RXFP3, and RXFP1 are present in the hypothalamic paraventricular nucleus, an area with a well-characterized role in the regulation of energy balance that also modulates reproductive function by providing inputs to hypothalamic gonadotropin-releasing hormone (GnRH) neurons. Other members of the relaxin family are known to play a role in the regulation of reproduction. However, the effects of relaxin-3 on reproductive function are unknown. We studied the role of relaxin-3 in the regulation of the hypothalamo-pituitary-gonadal (HPG) axis. Intracerebroventricular (5 nmol) and intraparaventricular (540–1,620 pmol) administration of human relaxin-3 (H3) in adult male Wistar rats significantly increased plasma luteinizing hormone (LH) 30 min postinjection. This effect was blocked by pretreatment with a peripheral GnRH antagonist. Central administration of human relaxin-2 showed no significant effect on plasma LH. H3 dose-dependently stimulated the release of GnRH from hypothalamic explants and GT1-7 cells, which express RXFP1 and RXFP3, but did not influence LH or follicle-stimulating hormone release from pituitary fragments in vitro. We have demonstrated a novel role for relaxin-3 in the stimulation of the HPG axis, putatively via hypothalamic GnRH neurons. Relaxin-3 may act as a central signal linking nutritional status and reproductive function.

Hypothalamic Interactions Between Neuropeptide Y, Agouti‐Related Protein, Cocaine‐ and Amphetamine‐Regulated Transcript and Alpha‐Melanocyte‐Stimulating Hormone In Vitro in Male Rats

A number of neuropeptides implicated in the hypothalamic regulation of appetite are synthesized in the arcuate nucleus (Arc). Neuropeptide Y (NPY) and agouti‐related protein (Agrp) are orexigenic. The pro‐opiomelanocortin (POMC) product alpha‐melanocyte‐stimulating hormone (α‐MSH) is anorectic. Intracerebroventricular administration of cocaine‐ and amphetamine‐regulated transcript (CART) decreases food intake. However, recent results show that CART is orexigenic when injected into discrete hypothalamic nuclei. There is almost complete coexpression of NPY and Agrp mRNA in Arc neurones, and the majority of CART‐containing neurones in the Arc also contain POMC mRNA. We investigated possible interactions between these neuropeptides in vitro using a rat hypothalamic explant system. Administration of 1, 10 and 100 nm of NPY to hypothalamic explants significantly increased release of Agrp(83‐132)‐immunoreactivity (IR). NPY (10 and 100 nm) significantly increased the release of CART(55‐102)‐IR and α‐MSH‐IR from hypothalamic explants. Agrp(83‐132) (10 nm) administered to hypothalamic explants significantly increased the release of NPY‐IR. Agrp(83‐132) (10 and 100 nm) significantly decreased the release of CART(55‐102)‐IR from hypothalamic explants. Administration of 1, 10 and 100 nm CART(55‐102) to hypothalamic explants resulted in a significant increase in NPY‐IR release. Administration of 10 nm CART(55‐102) to hypothalamic explants significantly increased the release of Agrp(83‐132)‐IR. NDP‐MSH (10 nm) administered to hypothalamic explants significantly increased the release of NPY‐IR. NDP‐MSH (10 and 100 nm) significantly increased the release of Agrp(83‐132)‐IR from hypothalamic explants. These data suggest that orexigenic neuropeptides in the arcuate nucleus stimulate the release of each other, perhaps reinforcing orexigenic behaviour via a positive‐feedback loop. Our results are also in keeping with the possibility that the melanocortin‐3 receptor in the arcuate nucleus may influence the release of arcuate neuropeptides.

Profiling of Glucose-Sensing Neurons Reveals that GHRH Neurons Are Activated by Hypoglycemia

Comprehensive transcriptional profiling of glucose-sensing neurons is challenging because of low expression levels of glucokinase (Gck) and other key proteins that transduce a glucose signal. To overcome this, we generated and validated transgenic mice with a neuronal/endocrine-specific Gck promoter driving cre expression and mated them to mice with cre-dependent expression of an EGFP-tagged ribosomal protein construct (EEF1A1-LSL.EGFPL10) that can be used to map and profile cells. We found significant Gck expression in hypothalamic and limbic regions in cells that are activated following administration of glucose or 2-deoxyglucose. Transcriptional profiling from Gck-cre/EEF1A1-LSL.EGFPL10 mice enriched known and previously unknown glucose-sensing populations including neurons expressing growth hormone releasing hormone (GHRH). Electrophysiological recordings show that hypoglycemia activates GHRH neurons, suggesting a mechanistic link between hypoglycemia and growth hormone release. These studies provide a means for mapping glucose-sensitive neurons and for generating transcriptional profiles from other cell types expressing cre in a cell-specific manner.