Center of Excellence on Neurodegenerative Diseases
[Scientific staff | Publications within DiMI | References]
The Center of Excellence of the University of Milan was founded by the Ministry of public Health in 2001. The aim of the center is to foster multidisciplinary research on the molecular basis of neurodegenerative diseases to study innovative diagnostic tools and therapies. The Center of Excellence has established a technological platform on animal and cell engineering that will be involved in the present project. In the past years the technological platform has been concentrating on the generation of cellular and animal models for “in vivo” detection of gene expression and on the development of novel concepts for animal transgenesis. Initially, we focussed on means to generate transgenes allowing ubiquitous expression of selected reporter genes. As a proof of principle we generated a reporter mouse to monitor the activity of estrogen receptors (ER) by whole mouse optical imaging . The results obtained indicate that the generated plasmid system is suitable for obtaining the ubiquitous expression of a selected gene marker, and using the technology generated we are constructing a series of mice suitable for the in vivo detection of the activity of intracellular receptors. We are presently generating a series of second-generation vectors (and transgenic animals) using reporters suitable for PET imaging analysis (dopamine D2 receptor and thymidine kinase genes). In this second-generation vector project, by using IRES sequences, the same promoter will transcribe bicistronic mRNA coding for two different reporters. The expression of two reporter genes will be monitored at the same time using two different methodologies (e.g., by photon imaging on expressed luciferase enzyme and PET scanning on the dopamine D2 receptor). Most importantly, all these vectors are made with a cassette strategy in order to extend their utility to any transcription factor or any imaging methodology, simply by exchanging the promoter or reporter of use.
In the last few years we have studied the role of glia in glutamate signaling and in the mechanisms of microglia activation and regulation. In particular, we have discovered a TNFalpha- and prostaglandin-dependent glutamate release process from glial cells which is critical in normal brain communication but, when de-regulated, may also cause neurodegeneration. Indeed, we have directly demonstrated its relevance to the development of AIDS neuropathology.
Based on this background, our group is currently investigating, by using in vitro and in vivo complementary approaches, whether glia actively participate to the cascade of events leading to neuronal death in amyotrophic lateral sclerosis (ALS) and prion diseases. In particular, mice expressing the ALS-linked mutant SOD1 G93A enzyme [Tg(SOD1-G93A)] are available in the lab and are presently being used either as a source of brain cell cultures for in vitro mechanistic studies or for crossings to various cytokines or cytokine receptor knockout animals (e.g. TNF-/-) to study the impact of a null proinflammatory cytokine background on disease progression. In parallel, we plan to study the dynamics of astroglial alterations during disease progression by mating Tg(SOD1-G93A) animals to mice expressing the enhanced green fluorescent protein (eGFP) under the control of the human glial fibrillary acidic protein (GFAP).
With regard to the mechanisms of microglia activation, we have recently proved that estradiol may prevent microglia activation and we are in the process of studying the molecular mechanisms underlying this effect and its relevance in neurodegenerative disorders like Alzheimer’s. disease (AD). To this aim we are utilizing APP23 transgenic mice has been previously described . These mice express the human APP751 cDNA with the Swedish double mutation under control of the neuron-specific mouse Thy-1 promoter fragment. Overexpression of the mutated form of APP leads to the formation of the typical neuropahtologic signs of AD, with -amiloid deposits rich in hyperphosphorylated Tau proteinFor the future research we plan to generate two reporter animals to study brain inflammatory processes. The first will be a reporter mouse expressing ubiquitously a reporter to monitor for the activity of peroxisome proliferator receptors (PPAR). This will represent a valuable model for the study of drugs potentially useful in the treatment of adenoleukodystophy and a double reporter mouse for activation of a transcription factor associated to inflammatory processes. Such promoter will direct the transcription of a single transcript coding for two reporter genes with IRES (ribosome entry) sequences interposed between them. As reporter genes, we will utilize a gene encoding a fluorescent protein (GFP or its mutants) and a gene encoding a protein able to bind labelled radioligands (either viral timidine kinase (TK) or mutated dopamine D2 receptor lacking signal-transduction capabilities). The two reporter genes are suited to detect inflammatory processes by in vivo imaging with different technologies, two-photon and PET/SPECT. Animal PET with radioligands for either the TK or the D2 gene has been already successfully performed.