Mutazioni nel gene tau associate ad instabilità cromosomica: un nuovo ruolo della proteina tau. Studio nel topo transgenico

Tau is a major microtubule-associated protein of neurons that promotes assembly and stabilization of cytoskeleton microtubules. Under physiological conditions, tau is mainly located to the axons of neurons and is critical in the process of neural outgrowth and axonal integrity. The protein is also present in non-neural cells, such as fibroblasts and lymphocytes. Under pathological conditions, tau becomes hyperphosphorylated, which means a higher degree of phosphorylation at physiological sites, as well as de novo phosphorylation at additional, pathological sites. Phosphorylation decreases the binding of tau to microtubules and its translocation to the somatodendritic compartment where it forms filaments, which assemble into macroscopically visible structures called neurofibrillary tangles (NFTs). Tauophaties comprise a diverse group of adult-onset diseases that are characterized by disruption of cytoskeleton and deposition of hyperphosphorilated tau filaments in neurons and sometimes in glia, resulting in cerebral atrophy and dementia. Tauophaties of genetic origin are caused by dominant mutations in the tau gene; the most studied is the P301L mutation, associated with frontotemporal dementia (FTD) phenotype. Transgenic mice expressing human tau with P301L mutation develop NFTs composed of filamentous tau, similar to straight filaments in the human neurodegenerative disease, that are concentrated in the spinal cord, brain stem, cerebellar deep nuclei, diencephalon and basal telencephalon. In addition there is neural loss (almost 50% in the spinal cord), reactive gliosis, axonal degeneration and glial inclusions. The aim of this study was to investigate the role of tau protein in chromosome stability, by means of its interaction with both microtubules and chromatin, through cytogenetic analysis on cells derived from central nervous system (subventricular zone) and from other tissues (splenic lymphocytes and fibroblasts) of transgenic mice expressing human tau with P301L mutation. We performed standard cytogenetic analysis on splenic lymphocytes and, most notably, on neural stem cells (NSC) from transgenic mice expressing the human tau with the P301L mutation (strain JNPL3). Transgenic mice expressing wild-type human tau (strain JN25) and non transgenic mice of the same strain were analyzed as controls. Analysis of splenic lymphocytes showed a significant difference in the level of aneuploidy between JNPL3 and JN25 mice as well as in non-transgenic controls (Student’s t-test, p<0.01). A significant difference was also present between homozygous and hemizygous transgenic animals, indicating a gene dosage effect. Cytogenetic analysis of JNPL3 skin fibroblast showed an high level of hyperdiploid cells, that seems to increase in passages of culture, but preliminary results need to be compared with fibroblasts culture of control mice. An advantage of the studies on animal models is the possibility to isolate neural stem cells to evaluate the role of the protein in the Central Nervous System (CNS). These cells can be obtained from subventricular zone (SVZ) of mice and can be used both for cytogenetic analysis and to establish a cellular line. The use of these cells enables us to study the effect of the mutation directly on cells obtained from CNS and processed immediately or after different periods of culture in vitro to evaluate the functions of the protein. First, we analyzed cells derived from 5-10 days JNPL3 mice after a short period of culture (5 hours). The results showed that, under physiological conditions, more than half of the neural cells was hypodiploid (53%). This data can be correlated with those reported in literature on aneuploid neuroprogenitors isolated from normal mouse SVZ (Rehen et al., 2001, Kaushal et al., 2003), showing a percentage of aneuploidy (33%) which is significantly different compared with our data. Analysis of cultured neural stem cells is in progress; preliminary results showed a higher level of chromosome aberrations in JNPL3 mice, particularly regarding structural aberrations. The present findings support the role of tau in chromosome stability, opening new insights into tau biology and new perspectives for therapeutic approaches.