Pregnane X receptor (PXR) activation: A mechanism for neuroprotection in a mouse model of Niemann-Pick C disease

Niemann–Pick type C1 (NPC1) disease is a fatal neurodegenerative disease characterized by neuronal lipid storage and progressive Purkinje cell loss in the cerebellum. We investigated whether therapeutic approaches to bypass the cholesterol trafficking defect in NPC1 disease might delay disease progression in the npc1−/− mouse model. We show that the neurosteroid allopregnanolone (ALLO) and T0901317, a synthetic oxysterol ligand, act in concert to delay onset of neurological symptoms and prolong the lifespan of npc1−/− mice. ALLO and T0901317 therapy preserved Purkinje cells, suppressed cerebellar expression of microglial-associated genes and inflammatory mediators, and reduced infiltration of activated microglia in the cerebellar tissue. To establish whether the mechanism of neuroprotection in npc1−/− mice involves GABAA receptor activation, we compared treatment of natural ALLO and ent-ALLO, a stereoisomer that has identical physical properties of natural ALLO but is not a GABAA receptor agonist. ent-ALLO provided identical functional and survival benefits as natural ALLO in npc1−/− mice, strongly supporting a GABAA receptor-independent mechanism for ALLO action. On the other hand, the efficacy of ALLO, ent-ALLO, and T0901317 therapy correlated with the ability of these compounds to activate pregnane X receptor-dependent pathways in vivo. These findings suggest that treatment with pregnane X receptor ligands may be useful clinically in delaying the progressive neurodegeneration in human NPC disease

Transcriptome response to heavy metal stress in Drosophila reveals a new zinc transporter that confers resistance to zinc

All organisms are confronted with external variations in trace element abundance. To elucidate the mechanisms that maintain metal homeostasis and protect against heavy metal stress, we have determined the transcriptome responses in Drosophila to sublethal doses of cadmium, zinc, copper, as well as to copper depletion. Furthermore, we analyzed the transcriptome of a metal-responsive transcription factor (MTF-1) null mutant. The gene family encoding metallothioneins, and the ABC transporter CG10505 that encodes a homolog of ‘yeast cadmium factor’ were induced by all three metals. Zinc and cadmium responses have similar features: genes upregulated by both metals include those for glutathione S-transferases GstD2 and GstD5, and for zinc transporter-like proteins designated ZnT35C and ZnT63C. Several of the metal-induced genes that emerged in our study are regulated by the transcription factor MTF-1. mRNA studies in MTF-1 overexpressing or null mutant flies and in silico search for metal response elements (binding sites for MTF-1) confirmed novel MTF-1 regulated genes such as ferritins, the ABC transporter CG10505 and the zinc transporter ZnT35C. The latter was analyzed in most detail; biochemical and genetic approaches, including targeted mutation, indicate that ZnT35C is involved in cellular and organismal zinc efflux and plays a major role in zinc detoxification

EVER Proteins, Key Elements of the Natural Anti-Human Papillomavirus Barrier, Are Regulated upon T-Cell Activation

Human papillomaviruses (HPV) cause a variety of mucosal and skin lesions ranging from benign proliferations to invasive carcinomas. The clinical manifestations of infection are determined by host-related factors that define the natural anti-HPV barrier. Key elements of this barrier are the EVER1 and EVER2 proteins, as deficiency in either one of the EVER proteins leads to Epidermodysplasia Verruciformis (EV), a genodermatosis associated with HPV-induced skin carcinoma. Although EVERs have been shown to regulate zinc homeostasis in keratinocytes, their expression and function in other cell types that may participate to the anti-HPV barrier remain to be investigated. In this work, we demonstrate that EVER genes are expressed in different tissues, and most notably in lymphocytes. Interestingly, in contrast to the skin, where EVER2 transcripts are hardly detectable, EVER genes are both abundantly expressed in murine and human T cells. Activation of CD4+ and CD8+ T cells via the TCR triggers a rapid and profound decrease in EVER expression, accompanied by an accumulation of free Zn2+ ions. Thus, EVER proteins may be involved in the regulation of cellular zinc homeostasis in lymphocytes. Consistent with this hypothesis, we show that the concentration of Zn2+ ions is elevated in lymphoblastoid cells or primary T cells from EVER2-deficient patients. Interestingly, we also show that Zn2+ excess blocks T-cell activation and proliferation. Therefore, EVER proteins appear as key components of the activation-dependent regulation of Zn2+ concentration in T cells. However, the impact of EVER-deficiency in T cells on EV pathogenesis remains to be elucidated

Corneal Alterations during Combined Therapy with Cyclodextrin/Allopregnanolone and Miglustat in a Knock-Out Mouse Model of NPC1 Disease

BACKGROUND: Niemann Pick disease type C1 is a neurodegenerative disease caused by mutations in the NPC1 gene, which result in accumulation of unesterified cholesterol and glycosphingolipids in the endosomal-lysosomal system as well as limiting membranes. We have previously shown the corneal involvement in NPC1 pathology in form of intracellular inclusions in epithelial cells and keratocytes. The purpose of the present study was to clarify if these inclusions regress during combined substrate reduction- and by-product therapy (SRT and BPT). METHODOLOGY/PRINCIPAL FINDINGS: Starting at postnatal day 7 (P7) and thereafter, NPC1 knock-out mice (NPC1(-/-)) and wild type controls (NPC1(+/+)) were injected with cyclodextrin/allopregnanolone weekly. Additionally, a daily miglustat injection started at P10 until P23. Starting at P23 the mice were fed powdered chow with daily addition of miglustat. The sham group was injected with 0.9% NaCl at P7, thereafter daily starting at P10 until P23, and fed powdered chow starting at P23. For corneal examination, in vivo confocal laser-scanning microscopy (CLSM) was performed one day before experiment was terminated. Excised corneas were harvested for lipid analysis (HPLC/MS) and electron microscopy. In vivo CLSM demonstrated a regression of hyperreflective inclusions in all treated NPC1(-/-)mice. The findings varied between individual mice, demonstrating a regression, ranging from complete absence to pronounced depositions. The reflectivity of inclusions, however, was significantly lower when compared to untreated and sham-injected NPC1(-/-) mice. These confocal findings were confirmed by lipid analysis and electron microscopy. Another important CLSM finding revealed a distinct increase of mature dendritic cell number in corneas of all treated mice (NPC1(-/-) and NPC1(+/+)), including sham-treated ones. CONCLUSIONS/SIGNIFICANCE: The combined substrate reduction- and by-product therapy revealed beneficial effects on the cornea. In vivo CLSM is a non-invasive tool to monitor disease progression and treatment effects in NPC1 disorder

MTF-1-Mediated Repression of the Zinc Transporter Zip10 Is Alleviated by Zinc Restriction

The regulation of cellular zinc uptake is a key process in the overall mechanism governing mammalian zinc homeostasis and how zinc participates in cellular functions. We analyzed the zinc transporters of the Zip family in both the brain and liver of zinc-deficient animals and found a large, significant increase in Zip10 expression. Additionally, Zip10 expression decreased in response to zinc repletion. Moreover, isolated mouse hepatocytes, AML12 hepatocytes, and Neuro 2A cells also respond differentially to zinc availability in vitro. Measurement of Zip10 hnRNA and actinomycin D inhibition studies indicate that Zip10 was transcriptionally regulated by zinc deficiency. Through luciferase promoter constructs and ChIP analysis, binding of MTF-1 to a metal response element located 17 bp downstream of the transcription start site was shown to be necessary for zinc-induced repression of Zip10. Furthermore, zinc-activated MTF-1 causes down-regulation of Zip10 transcription by physically blocking Pol II movement through the gene. Lastly, ZIP10 is localized to the plasma membrane of hepatocytes and neuro 2A cells. Collectively, these results reveal a novel repressive role for MTF-1 in the regulation of the Zip10 zinc transporter expression by pausing Pol II transcription. ZIP10 may have roles in control of zinc homeostasis in specific sites particularly those of the brain and liver. Within that context ZIP10 may act as an important survival mechanism during periods of zinc inadequacy

Human and mouse neuroinflammation markers in Niemann‐Pick disease, type C1

Niemann‐Pick disease, type C1 (NPC1) is an autosomal recessive lipid storage disorder in which a pathological cascade, including neuroinflammation occurs. While data demonstrating neuroinflammation is prevalent in mouse models, data from NPC1 patients is lacking. The current study focuses on identifying potential markers of neuroinflammation in NPC1 from both the Npc1 mouse model and NPC1 patients. We identified in the mouse model significant changes in expression of genes associated with inflammation and compared these results to the pattern of expression in human cortex and cerebellar tissue. From gene expression array analysis, complement 3 (C3) was increased in mouse and human post‐mortem NPC1 brain tissues. We also characterized protein levels of inflammatory markers in cerebrospinal fluid (CSF) from NPC1 patients and controls. We found increased levels of interleukin 3, chemokine (C‐X‐C motif) ligand 5, interleukin 16 and chemokine ligand 3 (CCL3), and decreased levels of interleukin 4, 10, 13 and 12p40 in CSF from NPC1 patients. CSF markers were evaluated with respect to phenotypic severity. Miglustat treatment in NPC1 patients slightly decreased IL‐3, IL‐10 and IL‐13 CSF levels; however, further studies are needed to establish a strong effect of miglustat on inflammation markers. The identification of inflammatory markers with altered levels in the cerebrospinal fluid of NPC1 patients may provide a means to follow secondary events in NPC1 disease during therapeutic trials.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/147148/1/jimd0083.pd

Allopregnanolone Promotes Regeneration and Reduces β-Amyloid Burden in a Preclinical Model of Alzheimer's Disease

Previously, we demonstrated that allopregnanolone (APα) promoted proliferation of rodent and human neural progenitor cells in vitro. Further, we demonstrated that APα promoted neurogenesis in the hippocampal subgranular zone (SGZ) and reversed learning and memory deficits in the male triple transgenic mouse model of Alzheimer's (3xTgAD). In the current study, we determined the efficacy of APα to promote the survival of newly generated neural cells while simultaneously reducing Alzheimer's disease (AD) pathology in the 3xTgAD male mouse model. Comparative analyses between three different APα treatment regimens indicated that APα administered 1/week for 6 months was maximally efficacious for simultaneous promotion of neurogenesis and survival of newly generated cells and reduction of AD pathology. We further investigated the efficacy of APα to impact Aβ burden. Treatment was initiated either prior to or post intraneuronal Aβ accumulation. Results indicated that APα administered 1/week for 6 months significantly increased survival of newly generated neurons and simultaneously reduced Aβ pathology with greatest efficacy in the pre-pathology treatment group. APα significantly reduced Aβ generation in hippocampus, cortex, and amygdala, which was paralleled by decreased expression of Aβ-binding-alcohol-dehydrogenase. In addition, APα significantly reduced microglia activation as indicated by reduced expression of OX42 while increasing CNPase, an oligodendrocyte myelin marker. Mechanistic analyses indicated that pre-pathology treatment with APα increased expression of liver-X-receptor, pregnane-X-receptor, and 3-hydroxy-3-methyl-glutaryl-CoA-reductase (HMG-CoA-R), three proteins that regulate cholesterol homeostasis and clearance from brain. Together these findings provide preclinical evidence for the optimal treatment regimen of APα to achieve efficacy as a disease modifying therapeutic to promote regeneration while simultaneously decreasing the pathology associated with Alzheimer's disease

EMPOWERING A TUCSON SMALL BUSINESS WITH MIS

The aim for this thesis was to apply my learned MIS skills for a small business in Tucson. This was accomplished by applying the principles and concepts I learned in my studies. I researched Tucson small businesses and connected with Antigone Books because of my passion for reading. I felt their website was a prime candidate for improvement. I dedicated hours to learning how to navigate their Drupal system with no prior experience and collected data on the most important focus areas through online research on current usability trends. I applied principles and practices from my courses to address their concerns. While many modern technology solutions and features were proposed for this company, the owners do not have the capacity or need to implement all ideas. Antigone Books made selective decisions to prioritize design to encourage in person book shopping. By adapting the website’s user-friendliness, I completed my MIS goals and fulfilled the three owners’ site goals. This research is significant because I am directing attention to a widespread problem: the lack of time and resources that many small businesses experience with respect to technology. I assisted an organization in identifying their priorities for their online presence and in-store experience. My thesis makes a significant contribution to the small business community by dedicating attention to the fast-paced technology industry