An Zhou

Polycomb Group Proteins as Epigenetic Mediators of Neuroprotection in Ischemic Tolerance

Polycomb group proteins protect neurons from injury through a mechanism involving decreased potassium channel function. Promoting Tolerance A temporary or permanent interruption of blood flow (ischemia) to the brain can lead to brain injury, with potentially devastating consequences, such as stroke. Intriguingly, previous exposure to a brief period of ischemia that is not itself sufficient to cause brain injury can protect against a later, more prolonged period of ischemia. This phenomenon, called ischemic tolerance, is associated with a general suppression of gene expression in the tolerant brain. Paradoxically, the tolerant state depends on protein synthesis. Stapels et al. performed proteomic analyses of mouse brains subjected to various ischemic conditions and found, in ischemic-tolerant brains, an increase in the abundance of polycomb group proteins, which act as transcriptional repressors. Further analyses of both mouse brain and cultured cells indicated that the polycomb proteins SCMH1 and BMI1 inhibited potassium channel abundance and activity and that this represented a mechanism underlying ischemic tolerance. A further understanding of the mechanisms underlying tolerance could open the door to new therapies for ischemic stroke. Exposing the brain to sublethal ischemia affects the response to a subsequent, otherwise injurious ischemia, resulting in transcriptional suppression and neuroprotection, a response called ischemic tolerance. Here, we show that the proteomic signature of the ischemic-tolerant brain is characterized by increased abundance of transcriptional repressors, particularly polycomb group (PcG) proteins. Knocking down PcG proteins precluded the induction of ischemic tolerance, whereas in an in vitro model, overexpressing the PcG proteins SCMH1 or BMI1 induced tolerance to ischemia without preconditioning. We found that PcG proteins are associated with the promoter regions of genes encoding two potassium channel proteins that show decreased abundance in ischemic-tolerant brains. Furthermore, PcG proteins decreased potassium currents in cultured neuronal cells, and knocking down potassium channels elicited tolerance without preconditioning. These findings reveal a previously unknown mechanism of neuroprotection that involves gene repressors of the PcG family.

Altered Expression of the Neuropeptide-Processing Enzyme Carboxypeptidase E in the Rat Brain after Global Ischemia

Carboxypeptidase E, an exoprotease involved in the processing of bioactive peptides released by a regulated secretory pathway, was identified in a subtractive complementary DNA library derived from an ischemic rat brain by differential screening. In situ hybridization and immunocytochemical analysis showed the presence of carboxypeptidase E messenger RNA and protein in the cerebral cortex, thalamus, striatum, and hippocampus of a healthy rat brain. After 15 minutes of transient global ischemia followed by 8 hours of reperfusion, increased levels of carboxypeptidase E messenger RNA and protein were observed in the hippocampal CA1 and CA3 regions and in the cortex, as detected by Northern and Western blot analyses and in situ hybridization. After extended reperfusion (24 to 72 hours), both carboxypeptidase E messenger RNA and protein levels were decreased. The ischemia-induced changes in carboxypeptidase E expression suggest that this enzyme may play a role in modulating the brains response to ischemia.

Altered biosynthesis of neuropeptide processing enzyme carboxypeptidase E after brain ischemia: molecular mechanism and implication.

In this study, using both in vivo and in vitro ischemia models, the authors investigated the impact of brain ischemia on the biosynthesis of a key neuropeptide-processing enzyme, carboxypeptidase E (CPE). The response to brain ischemia of animals that lacked an active CPE was also examined. Combined in situ hybridization and immunocytochemical analyses for CPE showed reciprocal changes of CPE mRNA and protein, respectively, in the same cortical cells in rat brains after focal cerebral ischemia. Western blot analysis revealed an accumulation of the precursor protein of CPE in the ischemic cortex in vivo and in ischemic cortical neurons in vitro. Detailed metabolic labeling experiments on ischemic cortical neurons showed that ischemic stress caused a blockade in the proteolytic processing of CPE. When mice lacking an active CPE protease were subjected to a sublethal episode of focal cerebral ischemia, abundant TUNEL-positive cells were seen in the ischemic cortex whereas only a few were seen in the cortex of wild-type animals. These findings suggest that ischemia has an adverse impact on the neuropeptide-processing system in the brain and that the lack of an active neuropeptide-processing enzyme exacerbates ischemic brain injury.

Ero1‐L, an ischemia‐inducible gene from rat brain with homology to global ischemia‐induced gene 11 (Giig11), is localized to neuronal dendrites by a dispersed identifier (ID) element‐dependent mechanism

Many changes in neuronal gene expression occur in response to ischemia, and these may play a role in determining the fate of ischemic neurons. To identify genes induced in the rat brain following cerebral ischemia, a strategy was used that combines subtractive hybridization and differential screening. Among the genes identified was one referred to as global ischemia‐inducible gene 11(Giig11). Sequence analysis indicated that Giig11 exhibited 97% and 91% identity to the known Ero1‐L (S. cereviseae ero1‐like oxidoreductase) of mouse and human origin, which is involved in oxidative endoplasmic reticulum protein folding. Rat Ero1‐L/Giig11 also contains a l07‐bp sequence that is nearly identical (> 95%) to the known dispersed repetitive identifier (ID), but which is lacking in mouse and human Ero1‐L. Northern blotting showed that expression of the ID element and Ero1‐L/Giig11 mRNA increased after global cerebral ischemia. In situ hybridization demonstrated increased expression of Ero1‐L/Giig11 in the brain following ischemic injury, with the highest levels in the vulnerable hippocampal CA1 pyramidal neurons. Transfection of cultured primary hippocampal neurons with a plasmid containing green fluorescent protein (gfp) and Ero1‐L/Giig11 cDNA (with and without the ID element) produced a gfp‐Ero1‐L/Giig11 fusion protein, and more fusion protein was localized into dendrites in the presence of the ID element, suggesting that the ID element promotes Ero1‐L/Giig11 protein localization to dendrites. Therefore, Ero‐1L/Giig11 may have a role in ischemia‐induced neuronal repair or survival mechanisms directed at counteracting abnormalities in protein folding, maturation and distribution.

Nascent Proteomes in Peripheral Blood Mononuclear Cells as a Novel Source for Biomarker Discovery in Human Stroke

Background and Purpose— The proteome of newly synthesized proteins (nascent proteome) in peripheral blood mononuclear cells (PBMCs) can be a novel source of stroke biomarkers. Changes in the PBMC nascent proteome after stroke reflect the dynamic response-in-action not detectable in the total proteome (all existing proteins) in blood. Here, we test the application of nascent proteomics as a novel approach for stroke biomarker discovery. Methods— The PBMC nascent proteome in human blood was determined by metabolic labeling of fresh PBMC cultures with azidohomoalanine (an azide-containing methionine surrogate), followed by mass spectrometry detection and quantification of azidohomoalanine-labeled proteins. The PBMC nascent and total proteomes were compared between patients with stroke and matched controls. Results— Both PBMC nascent and total proteomes showed differences between stroke patients and controls. Results of hierarchical clustering analysis of proteomic data revealed greater changes in the nascent than in the total PBMC proteomes, supporting the usefulness of the PBMC nascent proteome as a novel source of stroke biomarkers. Conclusions— Nascent proteomes in PBMC can be a novel source for biomarker discovery in human stroke.

Nascent proteomes of ischemic-injured and ischemic-tolerant neuronal cells

In a recent study on ischemic rodent brains, we quantitatively characterised and compared brain proteomes under ischemic-preconditioned or injured or tolerant conditions. We discovered an enriched presence of repressive transcriptional regulator proteins with essential roles as epigenetic regulators in ischemic-tolerant brains (Stapels et al., 2010). We further showed their robust, dynamic and differential changes under different ischemic conditions in brains and in cultured neuronal cells. In the present work, using neuronal cell cultures, we aimed to characterise the nascent proteome, the proteome that presents early when the cells receive an ischemic insult. These would be the proteomic changes of newly synthesised proteins. Identification of effectors of this phase of response to ischemia bears the best promise of identifying therapeutic targets for treating acute stroke when patients present to hospital. We compared these nascent proteomes across different ischemic conditions using bioinformatic tools.

Dynamic changes in proprotein convertase 2 activity in cortical neurons after ischemia/reperfusion and oxygen-glucose deprivation.

In this study, a rat model of transient focal cerebral ischemia was established by performing 100 minutes of middle cerebral artery occlusion, and an in vitro model of experimental oxygen-glucose deprivation using cultured rat cortical neurons was established. Proprotein convertase 2 activity gradually decreased in the ischemic cortex with increasing duration of reperfusion. In cultured rat cortical neurons, the number of terminal deoxynucleotidyl transferase-mediated 2’-deoxyuridine 5’-triphosphate-biotin nick end labeling-positive neurons significantly increased and proprotein convertase 2 activity also decreased gradually with increasing duration of oxygen-glucose deprivation. These experimental findings indicate that proprotein convertase 2 activity decreases in ischemic rat cortex after reperfusion, as well as in cultured rat cortical neurons after oxygen-glucose deprivation. These changes in enzyme activity may play an important pathological role in brain injury.