Mini reviewNeuroprotection by astrocytes in brain ischemia: Importance of microRNAs
Introduction
Cerebral ischemia is a key pathological event in several disease states; stroke, cardiac arrest and resuscitation, and head trauma being the most common. Stroke is one of the leading causes of death worldwide and the leading cause of long-term neurological disability. Despite many clinical stroke trials being conducted, the only efficacious treatment to date is thrombolysis [5]. Similarly, clinically effective treatment for the cerebral injury resulting from cardiac arrest and resuscitation is limited to hypothermia [4], [56]. Lack of consideration of the role of astrocytes, the importance of cell–cell interactions, the complex interplay among signaling pathways, and poorly defined treatment windows for specific targets are thought to be factors in the clinical failure of many potential neuroprotective strategies.
Astrocytes play many key roles both in normal and pathological central nervous system functioning. Astrocytes are central to K+ homeostasis, neurotransmitter uptake, synapse formation and regulation of the blood brain barrier. Astrocytes are the most abundant brain cell type, and in addition to multiple important homeostatic roles, they are essential to central nervous system development, helping to organize the structural architecture of the brain and modulate neuronal plasticity (for recent reviews see [3], [12], [54]).
In focal cerebral ischemia, modeled commonly in rodents as transient middle cerebral artery occlusion (MCAO), the neurons die within hours in the center of the ischemic territory (ischemic core) forming the initial area of infarction (see image in Fig. 1A) while the neurons in the adjacent peri-ischemic (penumbral) area may either survive via induction of pro-survival signaling pathways, or die at a later period of reperfusion (delayed neuronal death) via initiation of pro-apoptotic pathways [16]. Brief global cerebral ischemia, as seen with cardiac arrest and resuscitation, and modeled in rodents as forebrain ischemia or four vessel occlusion, causes delayed loss of neurons, with cornu ammonis 1 (CA1) pyramidal neurons being most vulnerable (image in Fig. 1B), whereas the nearby dentate gyrus (DG) neurons are relatively resistant.
It has been shown that loss of key astrocytic proteins and likely astrocyte cell death precede delayed neuronal death in permanent focal [31] but not in global [45] cerebral ischemia. Astrocytic glial fibrillary acidic protein (GFAP) messenger RNA (mRNA) declined more quickly than neuronal glucose transporter 3 (GLUT3) mRNA in the ischemic core [31]. Our group showed that loss of glutamate transport activity and immunoreactivity for the astrocyte-specific glutamate transporter-1 (GLT-1) in astrocytes occurred at early reperfusion times, hours to days before the death of CA1 neurons [45].
Recently several strategies for protecting neurons by manipulating astrocytes have been shown to be effective, and the roles of astrocytes as both neuroprotective and neuron endangering in stroke were reviewed recently [66]. Astrocytes have been shown to protect neurons from oxidative stress by increasing neuronal glutathione levels, and by the interleukin 6 (IL6) pathway [19], [37], [50], and preconditioned astrocytes were shown to release protective factors, including erythropoietin, that protected neurons [60]. In vivo stimulation of the astrocyte specific purinergic P2Y1 receptor protected neurons from photothrombotic ischemia [67]. Glial supporting cells were recently shown to protect neighboring hair cell neurons by release of heat shock protein 70 (Hsp70) [33], and induction of brain-derived neurotrophic factor (BDNF) in astrocytes by galectin-1 reduced neuronal apoptosis in the ischemic boundary zone and improved functional recovery [49]. Ceftriaxone treatment, which induces GLT-1 expression, reduces CA1 delayed neuronal injury in hippocampal slice culture and in transient forebrain ischemia [45]. Finally, protection by pyruvate against glutamate neurotoxicity is mediated by astrocytes through a glutathione-dependent mechanism [34]. Thus multiple strategies targeting astrocytes have been found effective in protecting neurons, encompassing several distinct mechanisms.
Enhancing astrocyte resistance to ischemic stress by overexpressing a heat shock protein or an antioxidant enzyme resulted in improved survival of CA1 neurons following forebrain ischemia [61]. These protective proteins, HSP72, or mitochondrial superoxide dismutase, were genetically targeted for expression in astrocytes using the astrocyte-specific human GFAP promoter. In both cases we found protection was accompanied by preservation of the astrocytic glutamate transporter GLT-1, and reduced oxidative stress in the CA1 region [61]. Similarly, selective overexpression of GLT-1 in astrocytes provided neuroprotection from moderate hypoxia-ischemia [59].
MicroRNAs (miRNAs) are a novel and abundant class of 19- to 22-nucleotide (nt) noncoding RNAs that control gene expression at the post-transcriptional level. miRNAs bind target messenger RNAs (mRNA) and induce mRNA degradation or repression of translation. Because the targeting or seed sequence is relatively short, individual miRNAs have many targets, and single mRNAs can be targeted by multiple miRNAs. Increasing evidence supports a role for miRNAs in the response to cerebral ischemia, for review see [40], [44]. Changes in miRNAs with ischemic brain injury were first identified using miRNA profiling techniques in focal cerebral ischemia [14], [25], [32], in forebrain ischemia [64], and in stroke patients [55]. Recently studies have evaluated the significance of individual miRNAs and their regional expression in ischemic brain damage. miRNAs participate in synapse regulation and neuronal activity. The role of miRNAs in excitotoxicity and normal physiology was recently reviewed [15]. The faster post-transcriptional effect of miRNAs, and their ability to simultaneously regulate many target genes, suggests they may have greater therapeutic potential for stroke than therapies targeting a single gene by direct transcriptional control. Numerous miRNAs are expressed in a cell-specific or cell enriched manner, some specifically in astrocytes [46], [53].
The importance of astrocytes for neuroprotection after cerebral ischemia has been reviewed by our group and others recently [3], [54], [66]. This mini review focuses on the novel regulation of astrocytes by astrocyte-enriched miRNAs in this setting.
Section snippets
Astrocyte-enriched miRNAs and their targets
We demonstrated recently that two brain-enriched miRNAs, miR-181a [42] and miR-29a [46], are involved in the regulation of outcome following cerebral ischemia (Fig. 1) (see Sections 3.1 Astrocyte-enriched miRNA-181a, 3.2 miR-29a and miR-29b). Interestingly, the literature and our experiments suggest that both the miR-181 and miR-29 families are astrocyte-enriched [22], [46].
To define the role of a miRNA, determining its molecular targets and cellular expression are the first critical steps.
Targeting protection of astrocytes to protect neurons: the potential of miRNAs
Because of the importance of astrocytes in the brain and their early changes after cerebral ischemia, they are now recognized as important targets for manipulation of delayed neuronal injury after both focal and global cerebral ischemia. Indeed our studies and those of other laboratories have demonstrated that this can be a successful strategy. Here we examine possible uses of miRNAs in this context.
Future directions
Strategies to improve the neuronal supportive functions of astrocytes have been used successfully in animal and in vitro studies. We speculate that miRNAs may have greater therapeutic potential as candidates for the treatment of stroke than therapies targeting induction of a single gene because of their faster post-transcriptional effect and their ability to simultaneously regulate many target genes. Several astrocyte-enriched miRNAs such as miR-181a and miR-29a have been demonstrated to
Acknowledgments
Grant sponsor: NIH; Grant numbers: GM049831, NS053898, NS080177. The authors thank William Magruder for help preparing the manuscript. The authors have no conflicting financial interests.
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