Title

 

Authors

A. Grolla, D. Lim, G. Balzaretti, G. Fakhfouri, E. Marcello, M. Di Luca, P.L. Canonico, A.A. Genazzani

Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche (DiSCAFF), Università del Piemonte Orientale, via Bovio 6, 28100 Novara

 

Abstract

Alzheimer’s disease is the most common age-related neurological disorder, characterized by progressive loss of memory, cognitive impairment and dementia leading eventually to death. Histological hallmark of the disease is the extracellular deposition of plaques of Amyloid b(Aβ plaques) in different regions of the brain, especially in hippocampus. The toxic form of Ab(Ab_1-42) is produced by cleavage of the amyloid precursor protein (APP) by concerted action of two secretases, β-secretase (BACE) and γ-secretase. A third protease which processes APP, ADAM10  α-secretase (a disintegrin and metalloproteinase 10), is involved in the phisiological non-amyloidogenic APP processing. The beneficial activity of ADAM10 is mediated by interaction with the synapse-associated protein-97 (SAP97), which mediates ADAM10 trafficking to the plasma membrane and promotes its activity (Marcello et al. 2007).
Deregulation of Ca²⁺ homeostasis, especially in endoplasmic reticulum and at the synaptic level, plays an important role in pathogenesis of AD (Berridge et al. 2010; Supnet and Bezprozvanny 2010). Moreover, the abnormalities of the glutamatergic synaptic signaling occur early in the course of the disease, which is reflected in a loss of the synaptic plasticity exacerbated by the loss of function by the astrocytes at this level (Hamilton and Attwell, 2010).
Recently an innovative, non-transgenic model of Alzheimer’s disease has been developed, in which the interaction between ADAM10 and SAP97 is disrupted using a cell-penetrating Tat-peptide fused to ADAM10 proline-rich domains (Tat-Pro ADAM10^709-729). Tat-peptide injected intraperitoneally in mice or put on primary neurons is able to shift the metabolism of the amyloid precursor protein towards amyloidogenesis and allows the reproduction of initial phases of sporadic Alzheimer's disease (Epis et al., 2010).
In this context, we were interested in investigating alterations of the calcium signaling in primary co-cultures of rat hippocampal neurons and glial cells treated with Tat-Pro ADAM10^709-729 peptide. We have analyzed some key components of calcium signaling at both transcription and protein levels such as the Group I metabotropic glutamate receptors (mGluR1 and mGluR5), the ionotropic glutamate receptors (NMDA) and the neuronal type IP₃ receptor (IP₃R1) and we found that these receptors were significantly increased after the treatment with Tat-Pro ADAM10^709-729. The up-regulation of these components was the cause of the deregulation of calcium signaling observed in fluorescence microscopy experiments. In particular, we focused our attention in investigating the involvement of astroglial cells in the first phases of this pathology, showing that probably these changes appeared first in glial cells, which then affect the neurons. In this regard, we confirmed the possibility for glial cells to produce Ab_1-42 peptide and we verified their impact on the survival of the neurons themselves. Moreover, direct application of Ab_1-42 mimicked the effect of Tat-Pro on Ca²⁺ homeostasis.FK506, a specific inhibitor of phosphatase calcineurin, was able to reduce the effects of Tat-Pro peptide, suggesting a possible involvement of this Ca²⁺-dependent enzyme in deregulation of Ca²⁺-related genes.
Taking together, our results demonstrate that Tat-Pro ADAM10^709-729 model can be used for investigation of the role of alterations of calcium homeostasis in glial cells in the early stage of sporadic AD.