Both secreted and the cellular levels of BDNF attenuated due to tau hyperphosphorylation in primary cultures of cortical neurons

https://doi.org/10.1016/j.jchemneu.2016.11.007Get rights and content

Highlights

  • OKA-induced tau hyperphosphorylation resulted in disruption of neurites.

  • This process lead to reduction in the levels of BDNF release in cortical neurons.

  • BDNF reduction might depend on the structural alterations in the neurites.

  • Altered neurotrophin levels in tauopathies might be associated with neurite structure.

Abstract

Intracellular aggregation of hyperphosphorylated tau in neurofibrillary tangles (NFTs) is a major neuropathological hallmark of taupathies such as Alzheimer’s disease. Okadaic acid (OKA) is a potent inhibitor of PP2A, leading to abnormal tau phosphorylation. Brain-derived neurotrophic factor (BDNF) is a neurotrophin that is selectively downregulated in AD. In this study, we investigated the effects of OKA induced tau hyperphosphorylation on secreted and cellular levels of BDNF in primary cortical neurons that were treated with 25 nM OKA. Tau phosphorylation at threonine 231 (Thr231) sites was assessed by Western blot using antibodies against phospho-Thr231. Non-phosphorylated tau protein was detected with the Tau-1 antibody. Levels of BDNF secreted to the culture medium were determined by ELISA at the 8th and 24th hours of treatment. Cellular localization and protein expression of BDNF and tau were assessed by immunofluorescent labeling and fluorescent intensity measurements at 24 h of treatment. Tau hyperphosphorylation was confirmed with increase in Thr231 and the decrease in Tau-1 signals after 8 h of OKA treatment, compared with the control groups, secreted BDNF levels in the OKA-treated group were significantly lower after 24 h of treatment but were not significantly different at 8 h of treatment. BDNF immunoreactivity was seen in cytoplasm and neurites of the neurons in control group. BDNF immunoreactivity significantly decreased in the OKA treated group and this attenuation was significant especially at neurites. Our results suggest that the decrease in BDNF secretion and the BDNF expression might depend on the disruption of microtubule structure caused by tau hyperphosphorylation.

Introduction

Alzheimer’s disease (AD) is the most common type of dementia and is a progressive neurodegenerative disease characterized by serious neurodegenerative changes such as cerebral atrophy, loss of synapses and neurons and selective reduction of neurotransmitters in cerebral cortex and certain subcortical regions. Two major pathological hallmarks of AD are extracellular senile plaques (SPs) composed of amyloid beta (Aβ) peptide and intracellular neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau (Kamat et al., 2013). Tau is a microtubule-associated protein (MAP) found abundantly in the central nervous system (CNS), especially in neurons (Weingarten et al., 1975). Under physiological conditions, it plays a key role in microtubule stability, axonal transport and neurite outgrowth; abnormal phosphorylation of tau leads to decrease in its interaction with microtubules and causes destabilization of the neuronal cytoskeleton, disruption of axonal transport and finally neuron death in AD brains (Kamat et al., 2013, Martin et al., 2011a, Martin et al., 2011b). Tau phosphorylation is controlled by two major enzymes: GSK-3β (glycogen synthase kinase-3β) and PP2A (protein phosphatase-2A). Changes in kinase and phosphatase activity causes the upregulation of GSK-3β and the downregulation of PP2A have been connected with tau hyperphosphorylation in tauopathies such as AD. Activity of PP2A is selectively reduced in AD brains by approximately 20% (Martin et al., 2011a, Martin et al., 2011b, Xing et al., 2006).

Okadaic acid (OKA) is one of the most effective inhibitor for PP2A and PP1 because of its involvement in protein phosphorylation and it is also a specific inhibitor for protein synthesis by hyperphosphorylation of elongation factor EF2 (Kamat et al., 2013). Studies using primary cortical neurons have shown that PP2A inhibition by OKA triggered tau hyperphosphorylation at several tau phosphorylation sites (Martin et al., 2011a, Martin et al., 2011b).

Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family and is involved in neuronal survival, neurite outgrowth and synaptic plasticity through its interaction with the receptors TrkB and p75NTR (Diniz and Teixeira, 2011, Aid et al., 2007). BDNF is active in hippocampus, cortex and basal forebrain, crucial brain areas for learning, memory and thinking (Yamada and Nabeshima, 2003). It has been shown that BDNF mRNA levels are significantly reduced in the hippocampus in AD patients. BDNF protein levels also reduced in the hippocampus and in cortical regions. As mentioned above, tau protein plays important roles in axonal transport and help maintain microtubule stabilization. Hyperphosphorylation of tau protein disrupts axonal transport and causes neuronal death. The BDNF-TrkB interaction activates the PI3K/Akt signaling pathway and causes tau dephosphorylation. Therefore, tau and BDNF are suggested to have a very important relationship (Elliott et al., 2005). However, there have been no reports discussing how BDNF expression and secretions affected by tau hyperphosphorylation.

The main goals of the present study were first to establish a tau hyperphosphorylation model in primary cortical cultures and then to investigate and understand the direct response of cortical neurons by means of BDNF expression, secretion and cellular localization.

Section snippets

Preparation of primary cortical neuron cultures

Primary cortical neurons were prepared from the cerebral cortex of embryonic day 16 (E16) Sprague-Dawley rat embryos, according to our previous studies (Gezen-Ak et al., 2011, Dursun et al., 2011, Dursun et al., 2013). Briefly, embryos were removed, and the cortex, excluding the olfactory cortex and the hippocampus, was dissected and freed of meninges. The cells were plated at a density of 6 × 105 cells per dish in Leibovitz 15 (L15) media (GibcoBRL 11415-064, Invitrogen Inc., New York, USA)

Cytotixicity

The cytotoxic effect of 25 nM okadaic acid treatment for 24 h was determined by measuring the level of LDH released into the culture medium. The level of LDH was significantly increased in the OKA-treated group compared with the control and vehicle groups (p < 0.0001) (Fig. 1). Although LDH release was increased mean cell number counts for control groups was 21.8 ± 7.57; for OKA treated groups was 16.0 ± 7.73; and for vehicle groups was 22.08 ± 7.25 at the 24th hours of treatments. The difference in the

Discussion

In this study, we addressed the effects of tau hyperphosphorylation via inhibition of PP2A by OKA on the release of BDNF by primary cortical neurons. Aβ accumulation is one of the two pathological hallmarks of AD, and many clinical studies aim to reverse the Aβ pathology. However, in recent years, there has been significant progress in understanding the molecular mechanisms of neurofibrillary degeneration, the other hallmark of the disease, and tau stands out as the target molecule for the

Conflict of interest

The authors declare that they have no conflict of interest.

Ethics statement

The study was approved by the Animal Welfare and Ethics Committee of Istanbul University, number 2012/84. The procedures involving experimentation on animal subjects were performed in accordance with both the guidelines of Istanbul University and with the National Research Council’s guidelines for the care and use of laboratory animals.

Acknowledgment

The present study was supported by the Research Fund of Istanbul University (Project No: 21345).

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