Defective Neuropeptide Processing and Ischemic Brain Injury: A Study on Proprotein Convertase 2 and its Substrate Neuropeptide in Ischemic Brains

Abstract

Using a focal cerebral ischemia model in rats, brain ischemia-induced changes in expression levels of mRNA and protein, and activities of proprotein convertase 2 (PC2) in the cortex were examined. In situ hybridization analyses revealed a transient upregulation of the mRNA level for PC2 at an early reperfusion hour, at which the level of PC2 protein was also high as determined by immunocytochemistry and western blotting. When enzymatic activities of PC2 were analyzed using a synthetic substrate, a significant decrease was observed at early reperfusion hours at which levels of PC2 protein were still high. Also decreased at these reperfusion hours were tissue levels of dynorphin-A(1–8) (DYN-A(1–8)), a PC2 substrate, as determined by radioimmunoassay. Further examination of PC2 protein biosynthesis by metabolic labeling in cultured neuronal cells showed that in ischemic cells, the proteolytic processing of PC2 was greatly attenuated. Finally, in mice, an intracerebroventricular administration of synthetic DYN-A(1–8) significantly reduced the extent of ischemic brain injury. In mice those lack an active PC2, exacerbated brain injury was observed after an otherwise non-lethal focal ischemia. We conclude that brain ischemia attenuates PC2 and PC2-mediated neuropeptide processing. This attenuation may play a role in the pathology of ischemic brain injury.

Neuroprotective Role of a Brain-Enriched Tyrosine Phosphatase, STEP, in Focal Cerebral Ischemia

“…A 6-0 monofilament (Doccol) was advanced from the CCA through the internal carotid artery to a length of 10 -11 mm from the bifurcation to occlude the middle cerebral artery (Wetzel et al, 2008). Depending on the experiment, the occluding filament was kept in place for 10 -30 min, and animals were immediately killed, or the filament was gently retracted after 30 min to allow reperfusion for various durations (3 or 24 h).…”

“…Primary neuronal cultures were obtained from 16-to 17-d-old rat embryos as described previously (Paul et al, 2003). Briefly, the striatum and the adjoining cortex were dissected, the tissue dissociated mechanically and resuspended in DMEM/F-12 (1:1) containing 5% FCS.…”

“…Male Sprague Dawley (SD) and Wistar rats and pregnant female SD rats were from Harlan Laboratories. STEP knock-out mice (KO or STEP Ϫ/Ϫ ) were developed on a C57BL6 background (Venkitaramani et al, 2009) and were bred at the University of New Mexico Animal Care Facility. Antibodies used were as follows: polyclonal anti-p38, rabbit monoclonal anti-phospho-p38 (T P EY P ) from Cell Signaling Technology, monoclonal anti-myc from Santa Cruz Biotechnology, and monoclonal anti-STEP (recognizes all STEP isoforms) from Novus Biologicals.…”

“…Dephosphorylation of this residue occurs after stimulation of NR1/NR2B subunit-containing NMDARs (NR2B-NMDARs) and subsequent activation of a Ca 2ϩ -dependent phosphatase, calcineurin (Paul et al, 2003;Paul and Connor, 2010). Dephosphorylation of this residue renders STEP active in terms of its ability to bind to its substrates and inhibit their activity (Paul et al, 2003;Poddar et al, 2010). This dephosphorylated form of STEP is referred to as the "active" form hereafter.…”

“…STEP fails to bind to its substrates after phosphorylation of this residue by dopamine/D1 receptormediated activation of the cAMP/protein kinase A (PKA) pathway (Paul et al, 2000). Dephosphorylation of this residue occurs after stimulation of NR1/NR2B subunit-containing NMDARs (NR2B-NMDARs) and subsequent activation of a Ca 2ϩ -dependent phosphatase, calcineurin (Paul et al, 2003;Paul and Connor, 2010). Dephosphorylation of this residue renders STEP active in terms of its ability to bind to its substrates and inhibit their activity (Paul et al, 2003;Poddar et al, 2010).…”