Dual roles of the chemorepellent axon guidance molecule RGMa in establishing pioneering axon tracts and neural fate decisions in embryonic vertebrate forebrain

Repulsive guidance molecule A (RGMa) is a glycosylphosphatidylinositol‐anchored plasma membrane protein that was originally identified based on its chemorepulsive activity during axon navigation in the developing nervous system. Knock down of RGMa has previously shown to perturb axon navigation in the developing Xenopus forebrain (Wilson and Key, 2006). In order to further understand the in vivo role of RGMa in axon guidance, we have adopted an in vivo gain‐of‐function approach. RGMa was mosaically overexpressed in the developing Xenopus embryo by the injection of mRNA into single blastomeres. Ectopic expression of RGMa affected the morphology and the topography of developing axon tracts in vivo. Pioneer axons misrouted or aberrantly projected in response to ectopic RGMa in the developing Xenopus forebrain, confirming the in vivo chemorepulsive activity of this ligand. In addition, we show here for the first time that overexpression of RGMa acts cell‐autonomously to generate ectopic neurons in the developing embryonic brain. Taken together, the current study reveals a pleiotropic role of RGMa in early vertebrate embryonic brain in the spatial organization of axon tracts, pioneer axon guidance, and neural cell differentiation. © 2011 Wiley Periodicals, Inc. Develop Neurobiol, 2012     

Saikosaponin-d,a novel SERCA inhibitor,induces autophagic cell death in apoptosis-defective cells

Autophagy is an important cellular process that controls cells in a normal homeostatic state by recycling nutrients to maintain cellular energy levels for cell survival via the turnover of proteins and damaged organelles. However, persistent activation of autophagy can lead to excessive depletion of cellular organelles and essential proteins, leading to caspase-independent autophagic cell death. As such, inducing cell death through this autophagic mechanism could be an alternative approach to the treatment of cancers. Recently, we have identified a novel autophagic inducer, saikosaponin-d (Ssd), from a medicinal plant that induces autophagy in various types of cancer cells through the formation of autophagosomes as measured by GFP-LC3 puncta formation. By computational virtual docking analysis, biochemical assays and advanced live-cell imaging techniques, Ssd was shown to increase cytosolic calcium level via direct inhibition of sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase pump, leading to autophagy induction through the activation of the Ca²⁺/calmodulin-dependent kinase kinase–AMP-activated protein kinase–mammalian target of rapamycin pathway. In addition, Ssd treatment causes the disruption of calcium homeostasis, which induces endoplasmic reticulum stress as well as the unfolded protein responses pathway. Ssd also proved to be a potent cytotoxic agent in apoptosis-defective or apoptosis-resistant mouse embryonic fibroblast cells, which either lack caspases 3, 7 or 8 or had the Bax-Bak double knockout. These results provide a detailed understanding of the mechanism of action of Ssd, as a novel autophagic inducer, which has the potential of being developed into an anti-cancer agent for targeting apoptosis-resistant cancer cells.     

Phosphoproteomic analysis reveals site-specific changes in GFAP and NDRG2 phosphorylation in frontotemporal lobar degeneration

Frontotemporal lobar degeneration (FTLD) is a progressive neurodegenerative disease characterized by behavioral abnormalities, personality changes, language dysfunction, and can co-occur with the development of motor neuron disease. One major pathological form of FTLD is characterized by intracellular deposition of ubiquitinated and phosphorylated TAR DNA binding protein-43 (TDP-43), suggesting that dysregulation in phosphorylation events may contribute to disease progression. However, to date systematic analysis of the phosphoproteome in FTLD brains has not been reported. In this study, we employed immobilized metal affinity chromatography (IMAC) followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify phosphopeptides from FTLD and age-matched control post-mortem human brain tissue. Using this approach, we identified 786 phosphopeptides in frontal cortex (control and FTLD), in which the population of phosphopeptides represented approximately 50% of the total peptides analyzed. Label-free quantification using spectral counts revealed six proteins with significant changes in the FTLD phosphoproteome. N-myc-Downstream regulated gene 2 (NDRG2) and glial fibrillary acidic protein (GFAP) had an increased number of phosphospectra in FTLD, whereas microtubule associated protein 1A (MAP1A), reticulon 4 (RTN4; also referred to as neurite outgrowth inhibitor (Nogo)), protein kinase C gamma (PRKCG), and heat shock protein 90 kDa alpha, class A member 1(HSP90AA1) had significantly fewer phosphospectra compared to control brain. To validate these differences, we examined NDRG2 phosphorylation in FTLD brain by immunoblot analyses, and using a phosphoserine-13 (pSer13) GFAP monoclonal antibody we show an increase in pSer13 GFAP levels by immunoblot concomitant with increased overall GFAP levels in FTLD cases. These data highlight the utility of combining proteomic and phosphoproteomic strategies to characterize post-mortem human brain tissue.     

Neuronal LR11 Expression Does Not Differentiate between Clinically-Defined Alzheimer's Disease and Control Brains

Alzheimer's disease (AD) is the leading cause of dementia in the elderly. Because the pathological changes underlying this disease can begin decades prior to the onset of cognitive impairment, identifying the earliest events in the AD pathological cascade has critical implications for both the diagnosis and treatment of this disease. We previously reported that compared to autopsy confirmed healthy control brain, expression of LR11 (or SorLA) is markedly reduced in AD brain as well as in a subset of people with mild cognitive impairment (MCI), a prodromal clinical stage of AD. Recent studies of the LR11 gene SORL1 have suggested that the association between SORL1 single nucleotide polymorphisms (SNPs) and AD risk may not be universal. Therefore, we sought to confirm our earlier findings in a population chosen solely based on clinical criteria, as in most genetic studies. Quantitative immunohistochemistry was used to measure LR11 expression in 43 cases from the Religious Orders Study that were chosen based on a final pre-mortem clinical diagnosis of MCI, mild/moderate AD or no cognitive impairment (NCI). LR11 expression was highly variable in all three diagnostic groups, with no significant group differences. Low LR11 cases were identified using the lowest tertile of LR11 expression observed across all cases as a threshold. Contrary to previous reports, low LR11 expression was found in only 29% of AD cases. A similar proportion of both the MCI and NCI cases also displayed low LR11 expression. AD-associated lesions were present in the majority of cases regardless of diagnostic group, although we found no association between LR11 levels and pathological variables. These findings suggest that the relationship between LR11 expression and the development of AD may be more complicated than originally believed.     

Invasiveness of transformed human breast epithelial cell lines is related to cathepsin B and inhibited by cysteine proteinase inhibitors

The activities'of the lysosomal cysteine proteinases cathepsin B and L are regulated by their endogenous inhibitors, stefins A and B, and cystatin C, and their imbalance may be associated with increased invasiveness and development of the malignant cell phenotype. The aim of this study was to investigate mRNA, protein and activity levels of the above proteins in relation to in vitro invasiveness and to the reported in vivo tumorigenicity of four human breast tumor cell lines: the spontaneously immortalized cell line MCF10A, its c-Ha-ras transfectant MCF10AT, and two tumorigenic derivative cell lines, MCF10AT-Ca1a and MCF10AT-Ca1d. Invasiveness did not correlate with tumorigenicity, since the MCF10AT cell was the most invasive and the remaining three were at about half of its level. Cathepsin B expression paralleled the in vitro invasiveness through matrigel at all levels of expression, but cathepsin L did not. Stefin levels were elevated several-fold in the tumorigenic cell lines, but not in MCF10AT. The hypothesis that cathepsin B plays an active role in the invasion of breast cancer cell lines was confirmed by the fact that synthetic cysteine proteinase inhibitors, particularly those selective for cathepsin B, significantly reduced the invasion of the MCF10AT cells.     

TGF-β induces the expression of SAP30L, a novel nuclear protein

Background  

We have previously set up an in vitro mesenchymal-epithelial cell co-culture model which mimics the intestinal crypt villus axis biology in terms of epithelial cell differentiation. In this model the fibroblast-induced epithelial cell differentiation from secretory crypt cells to absorptive enterocytes is mediated via transforming growth factor-β (TGF-β), the major inhibitory regulator of epithelial cell proliferation known to induce differentiation in intestinal epithelial cells. The aim of this study was to identify novel genes whose products would play a role in this TGF-β-induced differentiation.