Chronic hypobaric hypoxia induced apoptosis in CA1 region of hippocampus: A possible role of NMDAR mediated p75^NTR upregulation

Abstract

Hypobaric hypoxia has been implicated with neural degeneration and memory loss. Though there has been considerable knowledge on the role of the p75^NTR in triggering apoptosis, the occurrence of a similar mechanism in hypoxic stress still remains to be explored. We, in the present study, have tried to explore the role of p75^NTR in mediating apoptosis in hypobaric hypoxia. Male Sprague Dawley rats were exposed to an altitude of 7,620 m for different durations. To study the contribution of apoptosis to hypobaric hypoxia induced cell death in the hippocampus, rat brains were examined for the occurrence of apoptosis by determining the number of cells showing DNA breaks using terminal deoxynucleotidyl transferase-mediated UTP nick end labeling (TUNEL) assay and chromatin condensation using Hoechst staining along with estimation of caspase activity and expression of active Caspase 3. Expression of p75^NTR was studied to determine its possible role in triggering apoptosis in hypobaric hypoxia. Exposure to hypobaric hypoxia was found to progressively increase the number of TUNEL positive and Hoechst positive cells along with increase in caspase activity, thus suggesting apoptotic mode of cell death. p75^NTR was found to be upregulated on prolonged exposure to hypobaric hypoxia corresponding to the increase in the number of apoptotic cells. Further, reduced expression of p75^NTR expression by antisense nucleotide administration significantly decreased apoptosis in the CA1 region of hippocampus. Blocking of NMDA receptors by MK801 interestingly decreased p75^NTR expression and the number of TUNEL positive cells as compared to hypoxic animals. These findings suggest the regulation of p75^NTR by NMDA receptors and its role in inducing apoptosis in hypoxia.

Introduction

Hypobaric hypoxia has been reported to cause memory impairment and cognitive dysfunctions (Kramer et al., 1993, Shukitt-Hale et al., 1998). Altered neurotransmitter synthesis, uptake and release (Benveniste et al., 1984, Rossi et al., 2000), free radical generation (Askew, 2002) and changes in gene expression and protein functions (Pellegrini-Giampietro et al., 1992, Gorter et al., 1997, Chandel et al., 1998) are typically associated with hypoxia and ischemia (Hartman et al., 2005). We have previously shown the occurrence of oxidative stress in hypobaric hypoxia (Maiti et al., 2006) that might play a key role in causing memory impairment. However limited neuroprotection has been reported on antioxidant supplementation in hypoxic and ischemic stress indicating involvement of other complex molecular mechanisms that might lead to neural degeneration in hypobaric hypoxia (Adcock et al., 2002). There also has been ample evidence on the occurrence of glutamate excitotoxicity in hypoxic stress and ischemia (Goldberg et al., 1987, Won et al., 2002). The CA1 region of the hippocampus in particular has been earlier reported to be vulnerable to hypoxic insult (Maiti et al., 2007). We have earlier reported on the altered expression of NMDA and AMPA receptor subunits in hypobaric hypoxia and the occurrence of excitotoxicity in the hippocampus (Hota et al., in press). However the events downstream of glutamate receptor activation in hypobaric hypoxia still remains an enigma. The role of neurotrophins in mediating neuronal damage in hypoxic stress has been a matter of debate in recent days. Though there has been reports on the neuroprotective role of neurotrophins and their receptors in hypoxic and ischemic stress (Narumiya et al., 1998), the findings on potentiation of neuronal injury by the neurotrophins (Behrens et al., 1999) has also drawn considerable attention.

Neurotrophins are a paradigmal family of neurotrophic factors that are crucial in the development and maintenance of the nervous system (Barde, 1989, Kaplan and Stephens, 1994, Lewin and Barde, 1996). The intracellular signaling cascades of neurotrophins are mediated by two discrete receptor subtypes. The agonist-selective high affinity trk family of neurotrophin receptors that selectively bind to different members of the neurotrophin family, with NGF binding to trkA, BDNF and neurotrophin-4/5 to trkB and neurotrophin 3 binding to trkC, mediate neuronal growth and survivability (Kaplan and Miller, 1997). The agonist-nonselective low affinity p75^NTRs that form the second subtype on the other hand appear to be quite paradoxical and the signaling cascades pertaining to its activation still remain to be characterized.

The p75^NTR is a 75-kDa glycoprotein with an extracellular domain containing four cysteine-rich repeats required for neurotrophin binding, a single transmembrane domain, and an intracellular domain that contains a Type II death domain and lacks catalytic activity (Dechant and Barde, 2002, Hempstead, 2002). Over expression of p75^NTR induces apoptosis in neuronal cells (Frade et al., 1996, Roux et al., 1999, Friedman, 2000). In vivo over expression of the intracellular domain of p75^NTR in transgenic mouse neurons leads to widespread apoptosis of peripheral and central neurons (Majdan et al., 1997). Similarly, disruption of ligand binding to p75^NTR reduces apoptosis in the retina (Frade et al., 1996). Expression of p75^NTR has also been reported to elicit death of hippocampal neurons (Carter and Lewin, 1997, Friedman, 2000). There also has been evidence on upregulation of these receptors after damage in several neuronal populations.

Caspase 3 activation has been shown to be central to the apoptotic pathway triggered by p75^NTR over expression (Troy et al., 2002). This is further supported by the complete lack of Caspase 3 activation in the hippocampus of p75-null animals after pilocarpine treatment induced seizures (Troy et al., 2002). Though the molecular mechanisms involved in transcriptional regulation still remain to be elucidated, cell swelling and nitric oxide (NO) signaling cascades have been proposed to elicit p75^NTR expression in conditions of neural damage (Peterson and Bogenmann, 2003). Similar upregulation of neuronal nitric oxide synthase (nNOS) in excitotoxic conditions therefore indicates towards a calcium mediated regulation of p75^NTR expression.

In view of the above findings the present study aimed at exploring the occurrence of apoptotic neuronal death in the CA1 region of the hippocampus of rats on exposure to different durations of hypobaric hypoxia at a simulated altitude of 7620 m by Terminal deoxynucleotidyl transferase-mediated UTP nick end labeling (TUNEL), Hoechst staining, and estimation of Caspase 3 activity. The expression of p75^NTR on different durations of hypoxic exposure was also studied. Regulation of p75^NTR expression by NMDAR activation was explored by administering the selective NMDAR antagonist MK801 and studying its effect on p75^NTR expression during hypobaric hypoxia.