Wilmon F. Grant

Activity-Dependent Dendritic Arborization Mediated by CaM-Kinase I Activation and Enhanced CREB-Dependent Transcription of Wnt-2

Members of the Wnt signaling family are important mediators of numerous developmental events, including activity-dependent dendrite development, but the pathways regulating expression and secretion of Wnt in response to neuronal activity are poorly defined. Here, we identify an NMDA receptor-mediated, Ca2+-dependent signaling pathway that couples neuronal activity to dendritic arborization through enhanced Wnt synthesis and secretion. Activity-dependent dendritic outgrowth and branching in cultured hippocampal neurons and slices is mediated through activation by CaM-dependent protein kinase kinase (CaMKK) of the membrane-associated gamma isoform of CaMKI. Downstream effectors of CaMKI include the MAP-kinase pathway of Ras/MEK/ERK and the transcription factor CREB. A serial analysis of chromatin occupancy screen identified Wnt-2 as an activity-dependent CREB-responsive gene. Neuronal activity enhances CREB-dependent transcription of Wnt-2, and expression of Wnt-2 stimulates dendritic arborization. This novel signaling pathway contributes to dynamic remodeling of the dendritic architecture in response to neuronal activity during development.

Regulation of axonal extension and growth cone motility by calmodulin-dependent protein kinase I.

Calcium and calmodulin (CaM) are important signaling molecules that regulate axonal or dendritic extension and branching. The Ca2+-dependent stimulation of neurite elongation has generally been assumed to be mediated by CaM-kinase II (CaMKII), although other members of the CaMK family are highly expressed in developing neurons. We have examined this assumption using a combination of dominant–negative CaMKs (dnCaMKs) and other specific CaMK inhibitors. Here we report that inhibition of cytosolic CaMKI, but not CaMKII or nuclear CaMKIV, dramatically decreases axonal outgrowth and branching in cultured neonatal hippocampal and postnatal cerebellar granule neurons. CaMKI is found throughout the cell cytosol, including the growth cone. Growth cones of neurons expressing dnCaMI or dnCaMKK, the upstream activator of CaMKI, exhibit collapsed morphology with a prominent reduction in lamellipodia. Live-cell imaging confirms that these morphological changes are associated with a dramatic decrease in growth cone motility. Treatment of neurons with 1,8-naphthoylene benzimidazole-3-carboxylic acid (STO-609), an inhibitor of CaMKK, causes a similar change in morphology and reduction in growth cone motility, and this inhibition can be rescued by transfection with an STO-609-insensitive mutant of CaMKK or by transfection with constitutively active CaMKI. These results identify CaMKI as a positive transducer of growth cone motility and axon outgrowth and provide a new physiological role for the CaMKK–CaMKI pathway.

Maternal High Fat Diet Is Associated with Decreased Plasma n–3 Fatty Acids and Fetal Hepatic Apoptosis in Nonhuman Primates

To begin to understand the contributions of maternal obesity and over-nutrition to human development and the early origins of obesity, we utilized a non-human primate model to investigate the effects of maternal high-fat feeding and obesity on breast milk, maternal and fetal plasma fatty acid composition and fetal hepatic development. While the high-fat diet (HFD) contained equivalent levels of n-3 fatty acids (FAs) and higher levels of n-6 FAs than the control diet (CTR), we found significant decreases in docosahexaenoic acid (DHA) and total n-3 FAs in HFD maternal and fetal plasma. Furthermore, the HFD fetal plasma n-6∶n-3 ratio was elevated and was significantly correlated to the maternal plasma n-6∶n-3 ratio and maternal hyperinsulinemia. Hepatic apoptosis was also increased in the HFD fetal liver. Switching HFD females to a CTR diet during a subsequent pregnancy normalized fetal DHA, n-3 FAs and fetal hepatic apoptosis to CTR levels. Breast milk from HFD dams contained lower levels of eicosopentanoic acid (EPA) and DHA and lower levels of total protein than CTR breast milk. This study links chronic maternal consumption of a HFD with fetal hepatic apoptosis and suggests that a potentially pathological maternal fatty acid milieu is replicated in the developing fetal circulation in the nonhuman primate.

Regulation of Agouti-Related Protein Messenger Ribonucleic Acid Transcription and Peptide Secretion by Acute and Chronic Inflammation

Agouti-related protein (AgRP) is an orexigenic neuropeptide produced by neurons in the hypothalamic arcuate nucleus (ARC) that is a key component of central neural circuits that control food intake and energy expenditure. Disorders in energy homeostasis, characterized by hypophagia and increased metabolic rate, frequently develop in animals with either acute or chronic diseases. Recently, studies have demonstrated that proopiomelanocortin-expressing neurons in the ARC are activated by the proinflammatory cytokine IL-1beta. In the current study, we sought to determine whether inflammatory processes regulate the expression of AgRP mRNA and to characterize the response of AgRP neurons to IL-1beta. Here, we show by real-time RT-PCR and in situ hybridization analysis that AgRP mRNA expression in rodents is increased in models of acute and chronic inflammation. AgRP neurons were found to express the type I IL-1 receptor, and the percentage of expression was significantly increased after peripheral administration of lipopolysaccharide. Furthermore, we demonstrate that IL-1beta inhibits the release of AgRP from hypothalamic explants. Collectively, these data indicate that proinflammatory signals decrease the secretion of AgRP while increasing the transcription of the AgRP gene. These observations suggest that AgRP neurons may participate with ARC proopiomelanocortin neurons in mediating the anorexic and metabolic responses to acute and chronic disease processes.

A Postnatal Pax7+ Progenitor Gives Rise to Pituitary Adenomas

Pituitary adenomas are classified into functioning and nonfunctioning (silent) tumors on the basis of hormone secretion. However, the mechanism of tumorigenesis and the cell of origin for pituitary adenoma subtypes remain to be elucidated. Employing a tamoxifen-inducible mouse model, we demonstrate that a novel postnatal Pax7(+) progenitor cell population in the pituitary gland gives rise to silent corticotroph macro-adenomas when the retinoblastoma tumor suppressor is conditionally deleted. While Pax transcriptional factors are critical for embryonic patterning as well as postnatal stem cell renewal for many organs, we have discovered that Pax7 marks a restricted cell population in the postnatal pituitary intermediate lobe. This Pax7(+) early progenitor cell population is overlapping but ontologically downstream of the Nestin(+) pituitary stem cell population, yet upstream of another newly discovered Myf6(+) late progenitor cell population. Interestingly, the Pax7(+) progenitor cell population is evolutionarily conserved in primates and humans, and Pax7 expression is maintained not only in murine tumors but also in human functioning and silent corticotropinomas. Taken together, our results strongly suggest that human silent corticotroph adenomas may in fact arise from a Pax7 lineage of the intermediate lobe, a region of the human pituitary bearing closer scientific interest as a reservoir of pituitary progenitor cells.

Genetic and Pharmacologic Blockade of Central Melanocortin Signaling Attenuates Cardiac Cachexia in Rodent Models of Heart Failure

The central melanocortin system plays a key role in the regulation of food intake and energy homeostasis. We investigated whether genetic or pharmacologic blockade of central melanocortin signaling attenuates cardiac cachexia in mice and rats with heart failure. Permanent ligation of the left coronary artery (myocardial infarction (MI)) or sham operation was performed in wild-type (WT) or melanocortin-4 receptor (MC4R) knockout mice. Eight weeks after surgery, WT-Sham mice had significant increases in lean body mass (LBM; P<0·05) and fat mass (P<0·05), whereas WT-MI did not gain significant amounts of LBM or fat mass. Resting basal metabolic rate (BMR) was significantly lower in WT-Sham mice compared to WT-MI mice (P<0·001). In contrast, both MC4-Sham and MC4-MI mice gained significant amounts of LBM (P<0·05) and fat mass (P<0·05) over the study period. There was no significant difference in the BMR between MC4-Sham and MC4-MI mice. In the second experiment, rats received aortic bands or sham operations, and after recovery received i.c.v. injections of either artificial cerebrospinal fluid (aCSF) or the melanocortin antagonist agouti-related protein (AGRP) for 2 weeks. Banded rats receiving AGRP gained significant amount of LBM (P<0·05) and fat mass (P<0·05) over the treatment period, whereas banded rats receiving aCSF did not gain significant amounts of LBM or fat mass. These results demonstrated that genetic and pharmacologic blockade of melanocortin signaling attenuated the metabolic manifestations of cardiac cachexia in murine and rat models of heart failure.

Administration of IL-1β to the 4th ventricle causes anorexia that is blocked by agouti-related peptide and that coincides with activation of tyrosine-hydroxylase neurons in the nucleus of the solitary tract

Inflammation-associated cachexia is associated with multiple chronic diseases and involves activation of appetite regulating centers in the arcuate nucleus of the hypothalamus (ARH). The nucleus of the solitary tract (NTS) in the brainstem has also been implicated as an important nucleus involved in appetite regulation. We set out to determine whether the NTS may be involved in inflammation-associated anorexia by injecting IL-1 beta into the 4th ventricle and assessing food intake and NTS neuronal activation. Injection of IL-1 beta produced a decrease in food intake at 3 and 12h after injection which was ameliorated at the 12h time point by a sub-threshold dose of agouti-related peptide (AgRP). Investigation into neuron types in the NTS revealed that IL-1 beta injection was associated with an increase in c-Fos activity in NTS neurons expressing tyrosine hydroxylase (TH). Additionally, injection of IL-1 beta into the 4th ventricle did not produce c-Fos activation of neurons expressing pro-opiomelanocortin (POMC) in the ARH, cells known to be involved in producing anorexia in response to systemic inflammation. Double-label in situ hybridization revealed that TH neurons did not express IL-1 receptor I (IL1-RI) transcript, demonstrating that c-Fos activation of TH neurons in this setting was not via direct stimulation of IL-1 beta on TH neurons themselves. We conclude that IL-1 beta injection into the 4th ventricle produces anorexia and is accompanied by an increase in activation in TH neurons in the NTS. This provides evidence that the brainstem may be an important mediator of anorexia in the setting of inflammation.

Pancreatic inflammation and increased islet macrophages in insulin-resistant juvenile primates.

Chronic high caloric intake has contributed to the increased prevalence of pediatric obesity and related morbidities. Most overweight or obese children, however, do not present with frank metabolic disease but rather insulin resistance or subclinical precursors. The innate immune system plays a role in the pathophysiology of type 2 diabetes but how it contributes to early metabolic dysfunction in children on chronic high-fat diet (HFD) is unclear. We hypothesize that such inflammation is present in the pancreas of children and is associated with early insulin resistance. We used nonhuman primate (NHP) juveniles exposed to chronic HFD as a model of early pediatric metabolic disease to demonstrate increased pancreatic inflammatory markers before the onset of significant obesity or glucose dysregulation. Pancreata from 13-month-old Japanese macaques exposed to a HFD from in utero to necropsy were analyzed for expression of cytokines and islet-associated macrophages. Parameters from an intravenous glucose tolerance test were correlated with cytokine expression. Before significant glucose dysregulation, the HFD cohort had a twofold increase in interleukin 6 (IL6), associated with decreased first-phase insulin response and a sexually dimorphic (male) increase in IL1β correlating with increased fasting glucose levels. The number of islet-associated macrophages was also increased. Pancreata from juvenile NHP exposed to HFD have increased inflammatory markers and evidence of innate immune infiltration before the onset of significant obesity or glucose dysregulation. Given the parallel development of metabolic disease between humans and NHPs, these findings have strong relevance to the early metabolic disease driven by a chronic HFD in children.

Perinatal Exposure to a High-Fat Diet Is Associated with Reduced Hepatic Sympathetic Innervation in One-Year Old Male Japanese Macaques

Our group recently demonstrated that maternal high-fat diet (HFD) consumption is associated with non-alcoholic fatty liver disease, increased apoptosis, and changes in gluconeogenic gene expression and chromatin structure in fetal nonhuman primate (NHP) liver. However, little is known about the long-term effects that a HFD has on hepatic nervous system development in offspring, a system that plays an important role in regulating hepatic metabolism. Utilizing immunohistochemistry and Real-Time PCR, we quantified sympathetic nerve fiber density, apoptosis, inflammation, and other autonomic components in the livers of fetal and one-year old Japanese macaques chronically exposed to a HFD. We found that HFD exposure in-utero and throughout the postnatal period (HFD/HFD), when compared to animals receiving a CTR diet for the same developmental period (CTR/CTR), is associated with a 1.7 fold decrease in periportal sympathetic innervation, a 5 fold decrease in parenchymal sympathetic innervation, and a 2.5 fold increase in hepatic apoptosis in the livers of one-year old male animals. Additionally, we observed an increase in hepatic inflammation and a decrease in a key component of the cholinergic anti-inflammatory pathway in one-year old HFD/HFD offspring. Taken together, these findings reinforce the impact that continuous exposure to a HFD has in the development of long-term hepatic pathologies in offspring and highlights a potential neuroanatomical basis for hepatic metabolic dysfunction.

Endosomal MR1 Trafficking Plays a Key Role in Presentation of Mycobacterium tuberculosis Ligands to MAIT Cells

Mucosal-Associated Invariant T (MAIT) cells, present in high frequency in airway and other mucosal tissues, have Th1 effector capacity positioning them to play a critical role in the early immune response to intracellular pathogens, including Mycobacterium tuberculosis (Mtb). MR1 is a highly conserved Class I-like molecule that presents vitamin B metabolites to MAIT cells. The mechanisms for loading these ubiquitous small molecules are likely to be tightly regulated to prevent inappropriate MAIT cell activation. To define the intracellular localization of MR1, we analyzed the distribution of an MR1-GFP fusion protein in antigen presenting cells. We found that MR1 localized to endosomes and was translocated to the cell surface upon addition of 6-formyl pterin (6-FP). To understand the mechanisms by which MR1 antigens are presented, we used a lentiviral shRNA screen to identify trafficking molecules that are required for the presentation of Mtb antigen to HLA-diverse T cells. We identified Stx18, VAMP4, and Rab6 as trafficking molecules regulating MR1-dependent MAIT cell recognition of Mtb-infected cells. Stx18 but not VAMP4 or Rab6 knockdown also resulted in decreased 6-FP-dependent surface translocation of MR1 suggesting distinct pathways for loading of exogenous ligands and intracellular mycobacterially-derived ligands. We postulate that endosome-mediated trafficking of MR1 allows for selective sampling of the intracellular environment.