Transgenic mice with elevated basal level of calcium ions in neurons as a model of aged-induced neurodegeneration of sporadic Alzheimer’s disease

Alzheimer’s Disease (AD) - the most common form of dementia - is a progressive and irreversible neurodegenerative disorder that leads to cognitive, memory and behavioral impairments. Historically, the disease has been defined by the extracellular deposition of neuritic plaques of beta-amyloid peptide, intracellular hyperphosphorylated neurofibrillary tangles of tau protein that lead to selective neuronal loss. AD is an increasing public health concern among the ageing population.

Majority of animal models of AD are based on beta-amyloid/tau hypothesis. They overexpress either mutated Amyloid Precursor Protein or presenilins, which are responsible for early onset of familial AD (FAD), or mutated tau mimicking phosphorylated protein. FAD models represent less than 5% of human cases and seem to have little value to understand the mechanisms of sporadic AD (SAD). One of the arguments to support such view is the failure of clinical trials aimed to affect the disease by decreasing the level of beta-amyloid in the brain. Our project aims to create an alternative model.

We plan to generate and characterize transgenic mice, which will have dysregulated Ca2+ homeostasis by overexpression of STIM proteins involved in Store Operated Calcium Entry (SOCE). According to “Ca hypothesis of ageing”, formulated independently by Khachaturian and by Thibault, brain ageing is a result of a subtle, but long-lasting, dysregulation of Ca2+ homeostasis. The major risk factor in SAD is age, during which Ca2+ homeostasis becomes less effective leading to increased Ca2+ level and sustained increases upon neuronal stimulations. We expect that dysregulation of neuronal Ca2+ homeostasis in the proposed model will have the consequences for neurons similar to those occurring during ageing or produced by a large increases of Ca2+ during excitotoxicity.

The proposal is based on our original observations (Klejman 2009; Gruszczynska-Biegala 2011). We showed that the basal Ca2+ level in neurons can be modulated by overexpression of STIM proteins. Neurons expressing either YFP-STIM1 or YFP-STIM2 had lower resting Ca2+ levels than non-transfected cells, whereas cells co-expressing YFP-STIM2 and ORAI1 had higher level of resting Ca2+. Based on these and other data we expect to see  similar effects in brain neurons of transgenic mice. Thus, the elevation of basal Ca2+ level will create conditions, which will predispose neurons to the pathological changes observed in human SAD.

Two types of lines of double transgenic mice will be generated: one with constitutive and another one with inducible expression. Animals overexpressing each protein separately (ORAI1 or STIM1 or STIM2) under control of Thy1.2 promoter will be crossed to obtain double transgenic offspring. The lines overexpressing single protein will serve as controls. The generation of single transgenic lines has started and they might be available at the beginning of 2012.

The second type of transgenic lines will have inducible expression under CaM Kinase II promoter. Using a conditional binary gain–off function transgenic approach in combination with an inducible neuronal CRE transgenic line we will be able to activate our genes of interests (STIM1, STIM2 or ORAI1) at different time points. Offspring of them with double inducible expression will be studied.

We will analyse generated mice models at the level of cell, organ and organism. Ca2+-homeostasis and sensitivity to ageing and different stress factors will be compared in neurons. The activation of Ca2+-dependent pathways such as CREB signaling, changes in LTP and LTD, appearance of histopathological markers and neuronal loss will be described using cultured neurons as well as fixed brain sections and fresh slices. We will also compare ageing and behavior of young and old mice.

Specifically, we will analyze Ca2+ level and its influx into cytoplasm using FURA-2AM and single cell imaging in primary neuronal cultures. We will also estimate the effects of prolonged cell culture for the changes in gene expression, survival and sensitivity to toxic agents. The levels of mRNA of target genes, transcription of which depends on either CREB, DREAM or NFAT as well as on CHOP, will be evaluated using Real Time PCR and custom made arrays. We used these methods before to estimate the effects of beta-catenin on the expression of its potential gene targets (Wisniewska 2010; Misztal 2011).

Brains of mice line with elevated Ca2+ level in neurons will be compared with brains of other mice. We will stain sections of perfused mice brains obtained at different ages with specific antibodies against various  proteins (Bcl-2, CREB, c-fos, DREAM, P-tau and total tau, nitric oxide synthase), and other such as those involved in cell cycle regulation. The level of P-CREB and calcineurin translocation to nuclei, as signs of the activation of the respective signaling pathways, will be determined. We will be looking for possible pathological changes such as neurodegeneration by staining with FluoroJade. Cellular localization of superoxide production will be examined in fixed brain slices by monitoring dihydroethidium fluorescence using confocal microscopy.

Multiphoton microscopic analysis will be performed in slices of fresh brain to compare Ca2+ homeostasis in neurons of experimental and control mice lines. In acute brain slices standard tetanization protocols will be used to elicit LTP (high frequency stimulation) or low frequency stimulation to evoke LTD. We will also analyse synaptic plasticity in neuronal cultures in which LTP and LTD will be induced chemically. Young adult and old mice, which exhibit elevated basal Ca2+, will be analyzed for accelerated mortality, signs of neurological pathologies, by specific behavioral tests, and changes in their brains using magnetic resonance imaging. They should help us to estimate the extent of ageing and possible memory and learning deficits.  

The project will deliver lines of transgenic mice, papers in journals of high impact, and application for the protection of intellectual property rights for mice line(s) with the phenotype resulting from dysregulated Ca2+-homeostasis. This mice model will be useful to determine changes occurring in the ageing brain, which predisposes it to pathologies of SAD. The project is worthy to pursue also because it will help to understand the role of STIM proteins and ORAI1 in SOCE and its significance for neurons.