Research report
Delayed, but prolonged increases in astrocytic clusterin (ApoJ) mRNA expression following acute cortical spreading depression in the rat: evidence for a role of clusterin in ischemic tolerance

https://doi.org/10.1016/S0169-328X(03)00124-4Get rights and content

Abstract

Clusterin is a sulfated glycoprotein produced by neurons and by resting and activated astrocytes that has several putative functions, including protective responses to brain injury. Cortical spreading depression (CSD) is a powerful yet largely benign stimulus that acutely is capable of providing long-lasting ischemic tolerance. The current study investigated possible alterations in expression of clusterin mRNA in the cerebral cortex of the rat at various times after unilateral CSD. Using semiquantitative in situ hybridization histochemistry, significant increases (30–100%; P≤0.05) in clusterin mRNA were detected in layers I–III and IV–VI of the ipsilateral cortex at 1, 2, 7 and 14 (layers I–III only) days after CSD. Transcript levels in the ipsilateral cortex were again equivalent to contralateral (control) levels at 28 days after CSD. These molecular anatomical studies also revealed that both neurons and nonneuronal cells (presumed reactive astrocytes) increased their expression of clusterin mRNA following CSD. Notably the time-course of increases in clusterin mRNA after CSD (1–14 days) overlaps that during which CSD reportedly provides neuroprotection against subsequent cerebral ischemia. These findings along with other evidence suggest that increased clusterin production and secretion, particularly by astrocytes, could be neuroprotective—perhaps via one or more of its putative actions that include inhibition of complement activation and cytolysis, effects on chemotaxis and apoptosis, and actions as an anti-stress protein chaperone.

Introduction

Cortical spreading depression (CSD) is classically characterized as a slowly-propagating wave (1–6 mm/min) of neuronal and astrocytic depolarization that results in transient suppression of electroencephalographic activity. In the lissencephalic cortex, unilateral CSD spreads across the entire ipsilateral hemisphere and is associated with glutamate-induced N-methyl-d-aspartate receptor activation and reversible changes in local blood flow, metabolism and cellular ion balance, including the concentration of K+ and Ca2+ [34], [55]. Despite the increased metabolic load placed on tissue, CSD alone is a relatively benign event that does not cause any apparent neuronal damage or loss, even when repeatedly elicited over several hours [40]. In contrast, when CSD is induced acutely in metabolically or hemodynamically compromised brain tissue, the additional metabolic stress induced by CSD potentiates the amount of cell damage, both in vivo and in vitro [2], [4], [41]. However, several groups have also reported that an episode of CSD elicited 1–15 days prior to an ischemic insult significantly attenuates subsequent resultant ischemic damage [28], [31], [35], [69]. The precise mechanisms underlying this and other forms of ischemic preconditioning are not yet known, although protection is not due to changes in local blood flow during the subsequent ischemic insult [35], [69]. Instead, it appears more likely to be related to a complex cascade of genetic and biochemical events induced by CSD or similar stimuli, in both neurons and glial cells (including resting/activated astrocytes and microglia) [11], [20], [33], [36], [51], [52], [53], [54].

Clusterin, also known as sulphated glycoprotein-2 and apolipoprotein J (ApoJ), is a major secretory glycoprotein produced by a variety of species (including birds and mammals) in a number of tissues, including kidney, liver, testis and brain [26], [43], [66]. Clusterin has numerous putative roles that include inhibition of complement activation and resultant cytolysis, effects on chemotaxis and cell survival/apoptosis, and actions as an anti-stress protein chaperone, although such physiological functions are still being validated (e.g. [23], [46]).

In normal rat brain, clusterin expression is widespread, but there is a differential regional distribution and abundance of clusterin mRNA [10], [43]. Ependymal cells lining the cerebral ventricles and the choroid plexus contain the highest relative densities of clusterin mRNA along with cells in the glial limitans. Pyramidal neurons in the hippocampal formation and neurons in the paraventricular and ventromedial hypothalamic nuclei, the habenular complex and several brainstem nuclei also contain high transcript levels. In contrast, areas such as the basal ganglia (caudate putamen/globus pallidus) and cerebral cortex contain very low to moderate levels, respectively (see [10], [43] for review). Early immunohistochemical studies revealed that clusterin protein was only detected in limited subgroups of neurons in particular regions of the brain which included the midbrain red nucleus, the trigeminal motor and other pontine nuclei, some cerebellar nuclei and Purkinje cells in the cerebellar cortex [43].

However, alterations in brain clusterin expression occur after numerous experimental perturbations and in association with different experimental models of disease. For example, following cerebral ischemia (and/or hypoxia), clusterin mRNA and protein are upregulated in glia in rat, mouse and human brain [37], [58], [64], [70], [72], while a recent molecular genetic study suggested that clusterin might play a protective role during cerebral ischemia ( [63]; but see [19]). Thus, while it is generally agreed that clusterin contributes to plasticity following injurious or ‘stressful’ perturbations of nervous tissue, precise functional details are unclear.

Therefore, as part of a continuing series of investigations into the molecular plasticity of cerebrocortical neurons and glia produced by CSD [52], [53], [54], the current study assessed possible spatiotemporal changes in clusterin mRNA expression following unilateral CSD in the rat [65]. As an indication of CSD-induced astrocytic and neuronal activation, the levels of glial fibrillary acidic protein (GFAP) [11], [20] and dynein light-chain (DLC) [18], [24] mRNAs were also studied. Our findings provide further evidence for a possible neuroprotective action of clusterin, particularly against cerebral ischemia.

Section snippets

Animals and induction of CSD

All animal-related procedures were conducted with the approval of the Austin and Repatriation Medical Centre and Howard Florey Institute Animal Welfare Committees in accordance with guidelines issued by the National Health and Medical Research Council.

Male Sprague–Dawley rats (250–300 g, from the Biological Research Laboratories, Austin and Repatriation Medical Centre) were anesthetized with a pentobarbitone–brietal mixture (30:32 mg/kg, i.p.) and secured in a stereotaxic frame. CSD was induced

CSD activates both astrocytes and neurons

In sham control rats, GFAP mRNA expression by astrocytes in regions of the forebrain examined was of low abundance in all grey matter areas, relative to higher levels observed in white matter tracts such as the optic nerve and corpus callosum (Fig. 1A), consistent with previous reports [11], [68]. GFAP mRNA was also expressed by astrocytes in the glial limitans on the brain surface and cells lining the cerebral ventricles. Previous studies have demonstrated that unilateral CSD produces

Differential neuronal and astrocytic expression of clusterin mRNA following CSD

Neurons, astroglia and ependymal cells constitutively express clusterin mRNA in the rat and mouse brain ( [10], [43], [58]; current study). However, previous studies of injury models suggest that the severity and type of insult determine the cell types that preferentially increase their expression of clusterin [48], [61]. Following several forms of brain injury, reactive astrocytes increase their expression of clusterin. This has been demonstrated in the peri-infarct rim after permanent middle

Summary and conclusions

The present study primarily examined alterations in cortical levels of clusterin mRNA at various times following CSD in rats. In accordance with previous studies, acute, experimentally-induced CSD caused the marked activation of cortical astrocytes and neurons with a resultant cascade of molecular/neurochemical alterations, illustrated here by an increase in GFAP and DLC mRNA levels, respectively that occurred throughout all regions and layers of the cerebral cortex. Under these conditions, CSD

Acknowledgements

This research was supported by National Health and Medical Research Council of Australia Grant no. 990761 and by grants from the Austin Hospital Medical Research Foundation. During the course of these studies AKW was the recipient of a postgraduate Melbourne Research Scholarship from the University of Melbourne.

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