Overexpression of amyloid precursor protein increases copper content in HEK293 cells

Amyloid precursor protein (APP) is a transmembrane glycoprotein widely expressed in mammalian tissues and plays a central role in Alzheimer's disease. However, its physiological function remains elusive. Cu2+ binding and reduction activities have been described in the extracellular APP135-156 region, which might be relevant for cellular copper uptake and homeostasis. Here, we assessed Cu2+ reduction and 64Cu uptake in two human HEK293 cell lines overexpressing APP. Our results indicate that Cu2+ reduction increased and cells accumulated larger levels of copper, maintaining cell viability at supra-physiological levels of Cu2+ ions. Moreover, wild-type cells exposed to both Cu2+ ions and APP135-155 synthetic peptides increased copper reduction and uptake. Complementation of function studies in human APP751 transformed Fre1 defective Saccharomyces cerevisiae cells rescued low Cu2+ reductase activity and increased 64Cu uptake. We conclude that Cu2+ reduction activity of APP facilitates co

Genome-wide identification of new Wnt/beta-catenin target genes in the human genome using CART method

Background: The importance of in silico predictions for understanding cellular processes is now widely accepted, and a variety of algorithms useful for studying different biological features have been designed. In particular, the prediction of cis regulatory modules in non-coding human genome regions represents a major challenge for understanding gene regulation in several diseases. Recently, studies of the Wnt signaling pathway revealed a connection with neurodegenerative diseases such as Alzheimer's. In this article, we construct a classification tool that uses the transcription factor binding site motifs composition of some gene promoters to identify new Wnt/beta-catenin pathway target genes potentially involved in brain diseases

Recent rodent models for Alzheimer's disease : clinical implications and basic research

Alzheimer's disease (AD) is the most common origin of dementia in the elderly. Although the cause of AD remains unknown, several factors have been identified that appear to play a critical role in the development of this debilitating disorder. In particular, amyloid precursor protein (APP), tau hyperphosphorylation, and the secretase enzymes, have become the focal point of recent research. Over the last two decades, several transgenic and non-transgenic animal models have been developed to elucidate the mechanistic aspects of AD and to validate potential therapeutic targets. Transgenic rodent models over-expressing human β-amyloid precursor protein (β-APP) and mutant forms of tau have become precious tools to study and understand the pathogenesis of AD at the molecular, cellular and behavioural levels, and to test new therapeutic agents. Nevertheless, none of the transgenic models of AD recapitulate fully all of the pathological features of the disease. Octodon degu, a South American rodent has been recently found to spontaneously develop neuropathological signs of AD in old age. This review aims to address the limitations and clinical relevance of transgenic rodent models in AD, and to highlight the potential for O. degu as a natural model for the study of AD neuropathology.23 page(s

Effect of Fzd5 overexpression on neuronal morphology is partially prevented by a JNK inhibitor.

<p><b>A</b>. Representative images showing hippocampal neurons, transfected with the empty vector PCS2+ plus GFP as a control or Fzd5-HA plus GFP, at 2 DIV, and treated with or without the JNK inhibitor, TAT-TI-JIP. Immunodetection of the axonal marker Tau1 (upper panels) and merge (lower panels) are shown. GFP was used to detect transfected neurons and white arrows mark the quantified axons. <b>B</b>, <b>C</b>. Quantification of total axonal length (B) and axonal branching (C) of control and Fzd5-overexpressing polarized neurons, with or without treatment. Neurons overexpressing Fzd5 with polarized axonal markers showed a significant increase in axonal branching and a significant decrease in axonal length compared to control and treated control neurons. These effects were prevented by TAT-TI-JIP treatments. <b>D</b>, <b>E</b>. Quantification of total dendritic length (D) and primary, secondary and tertiary dendritic branching (E) of control and Fzd5-overexpressing polarized neurons, with or without TAT-TI-JIP treatment. Neurons overexpressing Fzd5 showed a significant increase in total dendrite length and in the number of tertiary neurites compared to that of the control and treated control neurons. TAT-TI-JIP prevented these effects and in fact decreased the number of secondary and tertiary neurites. Scale bar: 20 µm. * p< 0.05; ** p< 0.01; *** p< 0.001; τ, significantly different from the control; τ p< 0.05; ττ p< 0.01; τττ p< 0.001. Error bars indicate standard error of the mean of three independent experiments for the treated neurons and five independent experiments for the neurons without treatment.</p

Fzd5 polarizes towards the axon.

<p><b>A</b>. Immunodetection of Fzd5 in neurons at developmental stages 3 and 4. MAP1B-P was used to identify the axonal processes and Phalloidin staining to observe F-actin distribution and identify growth cones. Scale bar: 20 µm. <b>B</b>. Quantification of Fzd5-positive axonal and non-axonal processes in neurons at stages 3 and 4. All axons were positive for Fzd5 (Fzd5+) in both stages, but the percentage of non-axonal Fzd5+ processes declined throughout development. Scale bar: 10µm. Bars represent mean ±SEM of three independent experiments. ** p< 0.01; *** p< 0.001.</p

Loss-of-function of Fzd5 alters the polarity of hippocampal neurons.

<p><b>A</b>. Immunodetection of the axonal marker MAP1B-P and the somato-dendritic marker MAP2 in neurons 48 h after transfection with the scramble or shFzd5 vectors. GFP was used to detect transfected neurons. Representative images, shown in the upper panels, of a control neuron expressing the axonal marker distributed through the axon (arrows) and in the lower panels, a neuron transfected with the shFzd5 expressing no axonal marker (arrows). Scale bar: 20 µm. <b>B</b>. Quantification of transfected neurons with polarized axonal marker. Polarization of axonal markers was significantly lower in neurons transfected with the shFzd5 vectors compared to that of control neurons. Bars represent mean ±SEM of three independent experiments. * p< 0.05.</p

Fzd5 overexpression in neurons that have already developed the axon alters the morphology of hippocampal neurons.

<p><b>A</b> Immunodetection of transfected (GFP+) hippocampal neurons with the axonal marker Tau1 (upper panels) and the somato-dendritic marker MAP2 (middle panels) in neurons transfected with the empty vector or overexpressing Fzd5 at 3 DIV. In the lower panels a merge of both markers with GFP is shown. <b>B</b>-<b>D</b>. Quantification of axonal length (D), total axonal branching (E), and total dendritic length (F). Neurons overexpressing Fzd5 with polarized axonal markers distribution showed a significant decrease in axonal length and a significant increase in axonal branching and dendritic length, compared to control neurons. <b>E</b>. Quantification of primary, secondary and tertiary dendritic branching. Neurons overexpressing Fzd5, with non-polarized axonal markers, showed a significant increase in secondary and tertiary dendritic branching compared to control neurons. Polarized neurons overexpressing Fzd5 showed significantly more tertiary branching than control neurons. <b>F</b>. Quantification of branching points of the neurites. Neurons overexpressing Fzd5 showed a significant increase in branching points compared to control neurons. Scale bar: 20 µm. Bars represent mean ±SEM of three independent experiments. * p< 0.05; ** p< 0.01; *** p< 0.001.</p

Overexpression of Fzd5 activates JNK in HEK293 cells and the effects of Fzd5 overexpression on neuronal polarity are partially prevented by the JNK inhibiton.

<p><b>A</b>-<b>C</b>. Detection of main effectors of canonical Wnt pathway in homogenates form HEK293 cells transfected with Fzd5-HA or the empty vector pCS2+. Activated GSK-3β (pGSK-3βTyr216), inhibited GSK-3β (pGSK-3βSer9), total GSK-3β (A), c-jun (B) and p-CRMP (C) levels were very similar in control and overexpressing Fzd5 cells. <b>D</b>-<b>E</b>. Detection of activated JNK levels in homogenates form HEK293 cells transfected with Fzd5-HA or the empty vector pCS2+. Cells overexpressing Fzd5 presented increased levels of activated JNK (pJNK) isoforms (D) and p-c-Jun (E) compared to control cells. <b>F</b>. Detection of HA and GFP in HEK293 homogenates as control of transfections. <b>G</b>. Quantification of neurons with a Tau1 polarized distribution after treatment with JNK inhibitor. The percentage of cells with polarized axonal markers was significantly lower in neurons overexpressing Fzd5 compared to controls, and was in part prevented by treatment with TAT-TI-JIP. <b>H</b>. Quantification of neurons with polarized distribution of Tau1 after treatment with Rho inhibitor. The percentage of neurons with polarized axonal markers was significantly lower in cells overexpressing Fzd5 compared to controls;Y27362 treatment had no effect. Bars represent mean ±SEM of four independent experiments. In the graphs, JNK2/3: 55 kDa and JNK1: 46 kDa.</p

Fzd5 overexpression alters the polarity of hippocampal neurons.

<p><b>A</b>. Immunodetection of the axonal markers MAP1B-P and Tau1 in neurons 24 h after transfection with the empty vector PCS2+/GFP as a control or Fzd5-HA/GFP at 2 DIV. GFP was used to identify transfected neurons. Representative images, shown in panels a and b, of control neurons expressing the axonal markers distributed in one neurite and in panels c and d, neurons that overexpress Fzd5 with the axonal marker distributed in the whole cell. <b>B</b>. Quantification of neurons transfected on 1-3 DIV with polarized distribution of an axonal marker. The percentage of neurons with polarized axonal markers was significantly lower in neurons overexpressing Fzd5 than in control neurons, in a DIV-dependent manner. Scale bar: 20 µm. Bars represent mean ±SEM of three independent experiments. *** p< 0.001.</p