Spatial resolution of phospholipid scramblase 1 (PLSCR1), caspase-3 activation and DNA-fragmentation in the human hippocampus after cerebral ischemia

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Abstract

Reports on non-neural cells have shown that enhanced activity of the Ca2+-dependent/ATP-independent phospholipid scramblase (PLSCR1) is, at least in part, responsible for surface exposure of phosphatidylserine and the collapse of plasma membrane asymmetry in injured or apoptotic cells. To shed some light on mechanisms with a potential to lead to apoptotic death of human neurones following ischemic/hypoxic injury, we examined the immunoreactivity of hippocampal neurones for PLSCR1, caspase-3, cytochrome c and DNA-fragmentation in 22 individuals with clinically symptomatic cerebral ischemia after cardiac arrest or severe hypotension.

We found: (1) significant differences in the percentage of PLSCR1-immunoreactive neurones between controls and short survivors; statistically strong differences between the frequency of immunoreactive neurones among the subfields studied with lowest levels in the CA3; preferential distribution of immunoreactive neurones in controls within the regio entorhinalis, subfield CA1, and hilum. Additionally, these areas exhibited staining of fibre bundles which probably correspond to perforant path, alvear path and collateral’s of Schaffer, (2) caspase-3 was upregulated in a region-specific manner with marked activation in the selectively vulnerable hippocampal areas, (3) cytochrome c was redistributed, (4) DNA-fragmentation represented by scattered TUNEL-positive cells increased predominantly during the first 3 days after ischemia, and particularly in the regions of greatest susceptibility to hypoxic injury.

This study presents the first evidence that PLSCR1, and probably remodelling of plasma membrane phospholipids (PL), plays a role in ischemic injury in the human hippocampus.

Introduction

Apoptosis, a fundamental process occuring in virtually all cell types, is characterised by distinct and separate biochemical and morphological changes (Kerr et al., 1972). The most prominent feature typically occurs at the level of the nucleus and includes chromatin condensation and DNA fragmentation. Cell surface alterations are critical for apoptotic cell removal and include exposure of phosphatidylserine (PS) on the outer leaflet of the plasma membrane (Sims, 1998, Sims and Wiedmer, 2001). The plasma membrane phospholipids (PL) are normally asymmetrically distributed, with phosphatidycholine (PC) and sphingomyelin located primarily in the outer leaflet and the aminophospholipids phosphatidylserine and phosphatidylethanolamine restricted to the cytoplasmic leaflet (Vanags et al., 1996, Fadeel et al., 1999; Fadok et al., 2001a, Fadok et al., 2001b). An increase in intracellular Ca2+ due to cell activation, cell injury, or apoptosis causes a rapid bi-directional movement of the plasma membrane PL between leaflets, resulting in exposure of the PS and the phosphatidyethanolamine at the cell surface. The exposure of plasma membrane aminophospholipids has been shown to promote assembly and activation of several key enzymes of the coagulation and complement systems, as well as to accelerate the clearance of injured or apoptotic cells by the reticuloendothelial system, suggesting that Ca2+ induced remodeling of plasma membrane PL is central to both vascular hemostatic and cellular clearance mechanisms (Comfurius et al., 1996, Pastorelli et al., 2001). Clearance of apoptotic cells before they lyse their toxic contents into the surrounding tissue represents an important mechanism for limiting tissue injury, therefore, it is critical to ensure the timely generation of this recognition signal.

Removal of dying cells before giving up inflammatory contents and damaging the surrounding tissue may represent an important mechanism for limiting injury within the CNS (Witting et al., 2000). The exposure of phosphatidylserine at the cell surface—if apoptosis-associated or not—represents in many cell types a specific recognition signal for clearance by macrophages and can be prevented, reversed or suppressed under the same conditions (macromolecular synthesis inhibition or overexpression of Bcl-2) like other morphological features of apoptosis (Bevers et al., 1998, Martin et al., 1995, Adayev et al., 1998, Maiese and Vincent, 2000, Ivins et al., 1998, Basse et al., 1996, Bevers et al., 1999, Fadok et al., 2000). Little is known about the relationship between PS exposure and apoptotic cell death in neurons. However, Hirt et al., 2000 showed that Ca2+ stressed neurons were phagocytosed in vitro by microglia starting at 30 min after stimulation, whereas protein kinase C inhibitors prevented both Ca2+-mediated PS exposure and phagocytosis.

Studies on non-neural cells have shown that enhanced activity of the Ca2+-dependent/ATP-independent phospholipid scramblase (PLSCR1) is, at least partially, responsible for surface exposure of phosphatidylserine and collapse of plasma membrane asymmetry in injured or apoptotic cells (Verhoven et al., 1999). PLSCR1 is a 35-kDa ubiquitously expressed plasma membrane protein that mediates the accelerated transbilayer migration of plasma membrane phospholipids in activated, injured, or apoptotic cells exposed to elevated intracellular Ca2+ (Sims and Wiedmer, 2001). The upregulation and activation of PLSCR1 in response to apoptotic signals seems to be regulated by interferon-alpha at the transcriptional level, and by direct phosphorylation by protein kinase C delta (Zhou et al., 2000, Frasch et al., 2000). Furthermore, a delayed and prolonged activation of PLSCR1 during apoptosis may be a result of the sustained activation of PKCdelta bei caspase-3 (Khwaja and Tatton, 1999, Frasch et al., 2000; Fadok et al., 2001a, Fadok et al., 2001b).

The objective of the present study was to compare different features of apoptosis, including activation of caspase-3, redistribution of cytochrome c, nuclear changes and to assess the commonality of these events in neurons undergoing death under ischemic conditions. In addition, we wished to address the role of protease activation and presence of phospholipid scramblase (PLSCR1) in the externalisation of PS. We examined the immunoreactivity of hippocampal neurons for PLSCR1, caspase-3, cytochrome c in 22 individuals with clinically symptomatic cerebral ischemia after cardiac arrest or severe hypotension, and we compared kinetics of nuclear DNA-fragmentation, and PLSCR1 activation.

The findings indicate that neuronal death in hippocampus after ischemia has some of the early biochemical features, and morphological changes of apoptosis and that DNA-cleavage is a relatively late phenomenon in the apoptotic cascade.

Section snippets

Samples

Brain samples were obtained from 23 autopsy cases with a past history of transient hypoxic attacks (unconscious for few minutes) (Table 1), age ranging from 38 to 91 years (mean age 70.6±12.7 years). All samples (taken at less that 10 h postmortem), were dehydrated and paraffin embedded. Sections from comparable portions of the paraffin embedded hippocampus were stained with hematoxylin and eosin and cresyl violet for general examination.

Immunohistochemistry

Immunohistochemistry was performed on serial 7 μm sections.

PLSCR1-immunostaining

The antibody against PLSCR1 stained perikarya of neurones in a punctate pattern, and long fibers or fiber bundles within the neuropil and the white matter. Fig. 1A–D shows the distribution pattern of immunoreactive neurones in the hippocampal formation. We found statistically strong differences between the frequency of immunoreactive neurones among the subfields studied with highest levels in CA1, and important distribution of immunoreactive neurones within the granule cell layer of the fascia

Discussion

The phagocytic clearance of apoptotic neurons by microglia requires specific surface signals to be present on those neurons, enabling binding to and engulfment by microglia without eliciting an inflammatory response. In spite of growing interest in the mechanisms leading to the engulfment of apoptotic cells, the molecular basis by which an apoptotic cell is recognised is not entirely understood.

In the peripheral immune system, a number of surface molecules have been shown to be involved in the

Acknowledgements

The authors are grateful to Mrs Angelika Langenhagen, Mrs Inge Szász-Jakobi for technical assistance. We also thank Dr. Jürgen Bohl from department of Neuropathology of Mainz for providing us with autopsy material. This study was supported by the Held & Hecker-Stiftung-2002 (Grant 8599621 to Dr. A. Rami).

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