Faculty Bibliography

Transmeningeal pharmacotherapy for cerebral cortical disorders requires drug delivery through the subdural/subarachnoid space, ideally with a feedback controlled mechanism. We have developed a device suitable for this function. The first novel component of the apparatus is a silicone rubber strip equipped with (a) fluid-exchange ports for both drug delivery and local cerebrospinal fluid (CSF) removal, and (b) EEG recording electrode contacts. This strip can be positioned between the dura and pia maters. The second novel component is an implantable dual minipump that directs fluid movement to and from the silicone strip and is accessible for refilling and emptying the drug and CSF reservoirs, respectively. This minipump is regulated by a battery-powered microcontroller integrating a bi-directional radiofrequency (RF) communication module. The entire apparatus was implanted in 5 macaque monkeys, with the subdural strip positioned over the frontal cortex and the minipump assembly secured to the cranium under a protective cap. The system was successfully tested for up to 8months for (1) transmeningeal drug delivery using acetylcholine (ACh) and muscimol as test compounds, (2) RF-transmission of neocortical EEG data to assess the efficacy of drug delivery, and (3) local CSF removal for subsequent diagnostic analyses. The device can be used for (a) monitoring neocortical electrophysiology and neurochemistry in freely behaving nonhuman primates for more than 6months, (b) determining the neurobiological impact of subdural/subarachnoid drug delivery interfaces, (c) obtaining novel neuropharmacological data on the effects of central nervous system (CNS) drugs, and (d) performing translational studies to develop subdural pharmacotherapy devices

Regulation of the hypothalamic-pituitary-adrenal (HPA) axis is critical for adaptation to environmental changes. The principle regulator of the HPA axis is corticotrophin-releasing hormone (CRH), which is made in the parventricular nucleus and is an important target of negative feedback by glucocorticoids. However, the molecular mechanisms that regulate CRH are not fully understood. Disruption of normal HPA axis activity is a major risk factor of neuropsychiatric disorders in which decreased expression of the glucocorticoid receptor (GR) has been documented. To investigate the role of the GR in CRH neurons, we have targeted the deletion of the GR, specifically in the parventricular nucleus. Impairment of GR function in the parventricular nucleus resulted in an enhancement of CRH expression and an up-regulation of hypothalamic levels of BDNF and disinhibition of the HPA axis. BDNF is a stress and activity-dependent factor involved in many activities modulated by the HPA axis. Significantly, ectopic expression of BDNF in vivo increased CRH, whereas reduced expression of BDNF, or its receptor TrkB, decreased CRH expression and normal HPA functions. We find the differential regulation of CRH relies upon the cAMP response-element binding protein coactivator CRTC2, which serves as a switch for BDNF and glucocorticoids to direct the expression of CRH.

Preserved hair has been increasingly used as an ancient DNA source in high throughput sequencing endeavors, and it may actually offer several advantages compared to more traditional ancient DNA substrates like bone. However, cold environments have yielded the most informative ancient hair specimens, while its preservation, and thus utility, in temperate regions is not well documented. Coprolites could represent a previously underutilized preservation substrate for hairs, which, if present therein, represent macroscopic packages of specific cells that are relatively simple to separate, clean and process. In this pilot study, we report amplicons 147-152 base pairs in length (w/primers) from hair shafts preserved in a south Chilean coprolite attributed to Darwin's extinct ground sloth, Mylodon darwinii. Our results suggest that hairs preserved in coprolites from temperate cave environments can serve as an effective source of ancient DNA. This bodes well for potential molecular-based population and phylogeographic studies on sloths, several species of which have been understudied despite leaving numerous coprolites in caves across of the Americas

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing, inflammation, and host defense. PGRN also functions as a neurotrophic factor, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel, extracellular matrix protein, and growth factor. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor.

An in-depth understanding of the molecular and cellular complexity of angiogenesis continues to advance as new stimulators and inhibitors of blood vessel formation are uncovered. Gaining a more complete understanding of the response of blood vessels to both stimulatory and inhibitory molecules will likely contribute to more effective strategies to control pathological angiogenesis. Here, we provide evidence that endothelial cell interactions with structurally altered collagen type IV may suppress the expression of insulin-like growth factor binding protein-4 (IGFBP-4), a well documented inhibitor of the IGF-1/IGF-1R signaling axis. We report for the first time that IGFBP-4 differentially inhibits angiogenesis induced by distinct growth factor signaling pathways as IGFBP-4 inhibited FGF-2- and IGF-1-stimulated angiogenesis but failed to inhibit VEGF-induced angiogenesis. The resistance of VEGF-stimulated angiogenesis to IGFBP-4 inhibition appears to depend on sustained activation of p38 MAPK as blocking its activity restored the anti-angiogenic effects of IGFBP-4 on VEGF-induced blood vessel growth in vivo. These novel findings provide new insight into how blood vessels respond to endogenous inhibitors during angiogenesis stimulated by distinct growth factor signaling pathways.

The molecular basis for primary hereditary hypertriglyceridemia has been identified in fewer than 5% of cases. Investigation of monogenic dyslipidemias has the potential to expose key metabolic pathways. We describe a hitherto unreported disease in ten individuals manifesting as moderate to severe transient childhood hypertriglyceridemia and fatty liver followed by hepatic fibrosis and the identification of the mutated gene responsible for this condition. We performed SNP array-based homozygosity mapping and found a single large continuous segment of homozygosity on chromosomal region 12q13.12. The candidate region contained 35 genes that are listed in Online Mendelian Inheritance in Man (OMIM) and 27 other genes. We performed candidate gene sequencing and screened both clinically affected individuals (children and adults with hypertriglyceridemia) and also a healthy cohort for mutations in GPD1, which encodes glycerol-3-phosphate dehydrogenase 1. Mutation analysis revealed a homozygous splicing mutation, c.361-1G>C, which resulted in an aberrantly spliced mRNA in the ten affected individuals. This mutation is predicted to result in a truncated protein lacking essential conserved residues, including a functional site responsible for initial substrate recognition. Functional consequences of the mutation were evaluated by measuring intracellular concentrations of cholesterol and triglyceride as well as triglyceride secretion in HepG2 (hepatocellular carcinoma) human cells lines overexpressing normal and mutant GPD1 cDNA. Overexpression of mutant GPD1 in HepG2 cells, in comparison to overexpression of wild-type GPD1, resulted in increased secretion of triglycerides (p = 0.01). This finding supports the pathogenicity of the identified mutation.