Targeting chondroitinase ABC to axons enhances the ability of chondroitinase to promote neurite outgrowth and sprouting.

BACKGROUND: There is currently no effective treatment for promoting regeneration of injured nerves in patients who have sustained injury to the central nervous system such as spinal cord injury. Chondroitinase ABC is an enzyme, which promotes neurite outgrowth and regeneration. It has shown considerable promise as a therapy for these conditions. The aim of the study is to determine if targeting chondroitinase ABC expression to the neuronal axon can further enhance its ability to promote axon outgrowth. Long-distance axon regeneration has not yet been achieved, and would be a significant step in attaining functional recovery following spinal cord injury. METHODOLOGY/PRINCIPAL FINDINGS: To investigate this, neuronal cultures were transfected with constructs encoding axon-targeted chondroitinase, non-targeted chondroitinase or GFP, and the effects on neuron outgrowth and sprouting determined on substrates either permissive or inhibitory to neuron regeneration. The mechanisms underlying the observed effects were also explored. Targeting chondroitinase to the neuronal axon markedly enhances its ability to promote neurite outgrowth. The increase in neurite length is associated with an upregulation of ß-integrin staining at the axonal cell surface. Staining for phosphofocal adhesion kinase, is also increased, indicating that the ß-integrins are in an activated state. Expression of chondroitinase within the neurons also resulted in a decrease in expression of PTEN and RhoA, molecules which present a block to neurite outgrowth, thus identifying two of the pathways by which ChABC promotes neurite outgrowth. CONCLUSIONS / SIGNIFICANCE: The novel finding that targeting ChABC to the axon significantly enhances its ability to promote neurite extension, suggests that this may be an effective way of promoting long-distance axon regeneration following spinal cord injury. It could also potentially improve its efficacy in the treatment of other pathologies, where it has been shown to promote recovery, such as myocardial infarction, stroke and Parkinson's disease.

Polyphenol Effects on Cholesterol Metabolism via Bile Acid Biosynthesis, CYP7A1: A Review.

Atherosclerosis, the main contributor to coronary heart disease, is characterised by an accumulation of lipids such as cholesterol in the arterial wall. Reverse cholesterol transport (RCT) reduces cholesterol via its conversion into bile acids (BAs). During RCT in non-hepatic peripheral tissues, cholesterol is transferred to high-density lipoprotein (HDL) particles and returned to the liver for conversion into BAs predominantly via the rate-limiting enzyme, cholesterol 7 α-hydroxylase (CYP7A1). Numerous reports have described that polyphenol induced increases in BA excretion and corresponding reductions in total and LDL cholesterol in animal and in-vitro studies, but the process whereby this occurs has not been extensively reviewed. There are three main mechanisms by which BA excretion can be augmented: (1) increased expression of CYP7A1; (2) reduced expression of intestinal BA transporters; and (3) changes in the gut microbiota. Here we summarise the BA metabolic pathways focusing on CYP7A1, how its gene is regulated via transcription factors, diurnal rhythms, and microRNAs. Importantly, we will address the following questions: (1) Can polyphenols enhance BA secretion by modulating the CYP7A1 biosynthetic pathway? (2) Can polyphenols alter the BA pool via changes in the gut microbiota? (3) Which polyphenols are the most promising candidates for future research? We conclude that while in rodents some polyphenols induce CYP7A1 expression predominantly by the LXRα pathway, in human cells, this may occur through FXR, NF-KB, and ERK signalling. Additionally, gut microbiota is important for the de-conjugation and excretion of BAs. Puerarin, resveratrol, and quercetin are promising candidates for further research in this area.

Inhibitors of DAG metabolism suppress CCR2 signalling in human monocytes.

BACKGROUND AND PURPOSE: CCL2 is an inflammatory chemokine that stimulates the recruitment of monocytes into tissue via activation of the GPCR CCR2. EXPERIMENTAL APPROACH: Freshly isolated human monocytes and THP-1 cells were used. Fura-2 loaded cells were used to measure intracellular Ca2+ responses. Transwell migration to measure chemotaxis. siRNA-mediated gene knock-down was used to support pharmacological approaches. KEY RESULTS: CCL2 evoked intracellular Ca2+ signals and stimulated migration in THP-1 monocytic cells and human CD14+ monocytes in a CCR2-dependent fashion. Attenuation of DAG catabolism in monocytes by inhibiting DAG kinase (R59949) or DAG lipase (RHC80267) activity suppressed CCL2-evoked Ca2+ signalling and transwell migration in monocytes. These effects were not due to a reduction in the number of cell surface CCR2. The effect of inhibiting DAG kinase or DAG lipase could be mimicked by addition of the DAG analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) but was not rescued by application of exogenous phosphatidylinositol 4,5-bisphosphate. Suppressive effects of R59949, RHC80267, and OAG were partially or fully reversed by Gö6983 (pan PKC isoenzyme inhibitor) but not by Gö6976 (PKCα and PKCß inhibitor). RNAi-mediated knock-down of DAG kinase α isoenzyme modulated CCL2-evoked Ca2+ responses in THP-1 cells. CONCLUSIONS AND IMPLICATIONS: Taken together, these data suggest that DAG production resulting from CCR2 activation is metabolised by both DAG kinase and DAG lipase pathways in monocytes and that pharmacological inhibition of DAG catabolism or application suppresses signalling on the CCL2-CCR2 axis via a mechanism dependent upon a PKC isoenzyme that is sensitive to Gö6983 but not Gö6976.

Validation of control genes and a standardised protocol for quantifying gene expression in the livers of C57BL/6 and ApoE-/- mice.

The liver plays a critical role in food and drug metabolism and detoxification and accordingly influences systemic body homeostasis in health and disease. While the C57BL/6 and ApoE-/- mouse models are widely used to study gene expression changes in liver disease and metabolism, currently there are no validated stably expressed endogenous genes in these models, neither is it known how gene expression varies within and across liver lobes. Here we show regional variations in the expression of Ywhaz, Gak, Gapdh, Hmbs and Act-ß endogenous genes across a liver lobe; Using homogeneous samples from the four liver lobes of 6 C57BL/6 mice we tested the stability of 12 endogenous genes and show that Act-ß and Eif2-α are the most stably expressed endogenous genes in all four lobes and demonstrate lobular differences in the expression of Abca1 cholesterol efflux gene. These results suggest that sampling from a specified homogeneous powdered liver lobe is paramount in enhancing data reliability and reproducibility. The stability of the 12 endogenous genes was further tested using homogeneous samples of left liver lobes from 20 ApoE-/- mice on standard or high polyphenol diets. Act-ß and Ywhaz are suitable endogenous genes for gene expression normalisation in this mouse model.

Trafficking and processing of bacterial proteins by mammalian cells: Insights from chondroitinase ABC.

BACKGROUND: There is very little reported in the literature about the relationship between modifications of bacterial proteins and their secretion by mammalian cells that synthesize them. We previously reported that the secretion of the bacterial enzyme Chondroitinase ABC by mammalian cells requires the strategic removal of at least three N-glycosylation sites. The aim of this study was to determine if it is possible to enhance the efficacy of the enzyme as a treatment for spinal cord injury by increasing the quantity of enzyme secreted or by altering its cellular location. METHODOLOGY/PRINCIPAL FINDINGS: To determine if the efficiency of enzyme secretion could be further increased, cells were transfected with constructs encoding the gene for chondroitinase ABC modified for expression by mammalian cells; these contained additional modifications of strategic N-glycosylation sites or alternative signal sequences to direct secretion of the enzyme from the cells. We show that while removal of certain specific N-glycosylation sites enhances enzyme secretion, N-glycosylation of at least two other sites, N-856 and N-773, is essential for both production and secretion of active enzyme. Furthermore, we find that the signal sequence directing secretion also influences the quantity of enzyme secreted, and that this varies widely amongst the cell types tested. Last, we find that replacing the 3'UTR on the cDNA encoding Chondroitinase ABC with that of ß-actin is sufficient to target the enzyme to the neuronal growth cone when transfected into neurons. This also enhances neurite outgrowth on an inhibitory substrate. CONCLUSION/SIGNIFICANCE: Some intracellular trafficking pathways are adversely affected by cryptic signals present in the bacterial gene sequence, whilst unexpectedly others are required for efficient secretion of the enzyme. Furthermore, targeting chondroitinase to the neuronal growth cone promotes its ability to increase neurite outgrowth on an inhibitory substrate. These findings are timely in view of the renewed prospects for gene therapy, and of direct relevance to strategies aimed at expressing foreign proteins in mammalian cells, in particular bacterial proteins.

Family Group Decision Making (FGDM) with Lakota families in two tribal communities: tools to facilitate FGDM implementation and evaluation.

This article describes an adapted Family Group Decision Making (FGDM) practice model for Native American communities, the FGDM family and community engagement process, and FGDM evaluation tools as one example for other native communities. Challenges and successes associated with the implementation and evaluation of these meetings are also described in the context of key historical and cultural factors, such as intergenerational grief and trauma, as well as past misuse of data in native communities.