Tau and HMW tau phosphorylation and compartmentalization in apoptotic neuronal PC12 cells

Abstract

In the Alzheimer disease brain, the microtubule-associated protein tau is hyperphosphorylated. There is also evidence that apoptotic-like processes may contribute to the neuronal loss in AD. In an apoptotic model that involves replating neuronal PC12 cells without serum and nerve growth factor (NGF), tau was hyperphosphorylated. During replating, however, neurites are removed. Here, differentiated cells were maintained in serum-free media before growth factor removal, thus maintaining neuritic processes during the apoptotic process and allowing for evaluation of neuritic changes. Tau phosphorylation, evaluated by immunoblotting and immunocytochemistry, was compared with various measures of cell death. Compared with control, NGF-deprived cells exhibited gradual and consistent increases of lactate dehydrogenase release over a 5-day period and a peak of caspase-3 activity at Day 2 after NGF removal. Nuclear staining demonstrated chromatin condensation in NGF-deprived cells. Apoptotic cells had thickened, tortuous, and shortened neuritic processes compared with control cells. Immunoblotting showed an increase in both tau and high molecular weight (HMW) tau phosphorylation during the apoptotic process. Immunoreactivity of both tau isoforms shifted from the detergent insoluble cytoskeleton to the detergent soluble compartment in the apoptotic cells. The microtubule binding of both tau isoforms from apoptotic cells also was impaired. Immunoblotting of purified plasma membrane showed preferential association of HMW tau with the plasma membrane during apoptosis. Also, plasma membrane-associated HMW tau was more phosphorylated during apoptosis. Immunocytochemistry demonstrated increased tau phosphorylation in most apoptotic cells, especially in the neurites. Tau was, however, dephosphorylated cells in the last stages of apoptosis.

Role of Cytoskeleton Proteins in the Morphological Changes During Apoptotic Cell Death of Cerebellar Granule Neurons

“…In all cases there were a correlation between the morphological alterations and the cytoskeleton changes induced by these apoptotic conditions. A large body of evidence suggests that cytoskeleton participates in morphological changes during apoptotic progression induced in different cell death models [5,6,8,10]; however, limited information exists about the specific changes of cytoskeleton protein in apoptotic death, particularly in cell death of CGC induced by K5 and STS treatment. In the present study, actin and a-tubulin showed changes in all tested conditions.…”

“…Apoptosis can be distinguished from other types of cell death by some morphological and biochemical characteristics, including cell volume reduction, nuclear condensation, DNA fragmentation and the activation of a group of proteases called caspases, among others [3]. Several studies have been focused for understanding the mechanisms involved in the morphological changes during the apoptotic cell death [4][5][6]. Most of the morphological changes observed seem to be regulated by caspases, which act on many substrates [7], including the cytoskeleton molecules [8].…”

“…Some cytoskeleton proteins have been reported to be substrate for proteases during apoptosis induced by several conditions [5,6]. Particularly, in cerebellar and hippocampal granule cells, apoptotic cell death is associated with cytoskeleton disruption [22][23][24][25][26][27][28][29].…”

Does the term ‘trophic’ actually mean anti-amyloidogenic? The case of NGF

“…55 An early tau hyperphosphorylation, with a reduction of its microtubule binding affinity has been found in NGF-deprived differentiated PC12 cells. 56 In addition, although other research groups have reported that Ab antibodies can reduce tau hyperphosphorylation in vitro and in vivo, 57,58 the functional relationship between endogenous overproduced Ab, tau hyperphosphorylation/cleavage and apoptotic signaling in the same neuronal model has not been investigated before. Similar NGF-dependent modifications of site-specific tau phosphorylation have been found in the AD11 animal model, 54 in correlation with the temporal appearance of Ab peptide species.…”

Nerve growth factor as a paradigm of neurotrophins related to Alzheimer's disease

“…This NT controls the endogenous tau synthesis either directly, by up-regulation of gene transcription (Drubin et al, 1985;Sadot et al, 1996), or indirectly, by its ubiquitination and proteosomal degradation (Babu et al, 2005). The phosphorylation state of tau-which critically controls its physiopathology leading to selfaggregation and/or reduced assembly and stability of microtubules used as tracks for axonal trafficking (Stoothoff et al, 2005)-is also regulated by NGF deprivation in vitro (Nuydens et al, 1997;Shelton and Johnson, 2001) as well as in vivo Capsoni et al, 2002a,b). Moreover, since tau controls the bidirectionality of axonal motor-driven transport in a concentration-dependent manner and differentially modulates the kinesin and dynein activity along microtubule tracks (Dixit et al, 2008), defective intracellular trafficking of cargoes, including NTFs, could be due to an increased expression level of this protein (Ebneth et al, 1998;Stamer et al, 2002;Mandelkow et al, 2003) or to its altered intracellular localization (Thies et al, 2007) or hyperphosphorylation (Tatebayashi et al, 2004;Alonso et al, 1997).…”