Abstract
BackgroundCerebral inflammation is a hallmark of neuronal degeneration. Dipeptidyl peptidase IV, aminopeptidase N as well as the dipeptidyl peptidases II, 8 and 9 and cytosolic alanyl-aminopeptidase are involved in the regulation of autoimmunity and inflammation. We studied the expression, localisation and activity patterns of these proteases after endothelin-induced occlusion of the middle cerebral artery in rats, a model of transient and unilateral cerebral ischemia.MethodsMale Sprague-Dawley rats were used. RT-PCR, immunohistochemistry and protease activity assays were performed at different time points, lasting from 2 h to 7 days after cerebral ischemia. The effect of protease inhibitors on ischemia-dependent infarct volumes was quantified 7 days post middle cerebral artery occlusion. Statistical analysis was conducted using the t-test.ResultsQualitative RT-PCR revealed these proteases in ipsilateral and contralateral cortices. Dipeptidyl peptidase II and aminopeptidase N were up-regulated ipsilaterally from 6 h to 7 days post ischemia, whereas dipeptidyl peptidase 9 and cytosolic alanyl-aminopeptidase were transiently down-regulated at day 3. Dipeptidyl peptidase 8 and aminopeptidase N immunoreactivities were detected in cortical neurons of the contralateral hemisphere. At the same time point, dipeptidyl peptidase IV, 8 and aminopeptidase N were identified in activated microglia and macrophages in the ipsilateral cortex. Seven days post artery occlusion, dipeptidyl peptidase IV immunoreactivity was found in the perikarya of surviving cortical neurons of the ipsilateral hemisphere, whereas their nuclei were dipeptidyl peptidase 8- and amino peptidase N-positive. At the same time point, dipeptidyl peptidase IV, 8 and aminopeptidase N were targeted in astroglial cells. Total dipeptidyl peptidase IV, 8 and 9 activities remained constant in both hemispheres until day 3 post experimental ischemia, but were increased (+165%) in the ipsilateral cortex at day 7. In parallel, aminopeptidase N and cytosolic alanyl-aminopeptidase activities remained unchanged.ConclusionsDistinct expression, localization and activity patterns of proline- and alanine-specific proteases indicate their involvement in ischemia-triggered inflammation and neurodegeneration. Consistently, IPC1755, a non-selective protease inhibitor, revealed a significant reduction of cortical lesions after transient cerebral ischemia and may suggest dipeptidyl peptidase IV, aminopeptidase N and proteases with similar substrate specificity as potentially therapy-relevant targets.
Highlights
Cerebral inflammation is a hallmark of neuronal degeneration
To further examine the function of Dipeptidyl peptidase IV (DPIV), Aminopeptidase N (APN) and/or DPIV-/APN-like peptidases in ischemia-dependent neurodegeneration, we have studied these target peptidases in an in vivo model of transient, unilateral cerebral ischemia due to endothelin-induced occlusion of the middle cerebral artery
Results mRNA expression of dipeptidyl peptidase II (DPII), DPIV, dipeptidyl peptidase 8 (DP8), dipeptidyl peptidase 9 (DP9), APN and cytosolic alanyl-aminopeptidase (cAAP) at different time points after Endothelin-induced occlusion of the middle cerebral artery (eMCAO) Sprague-Dawley rats were exposed to a transient focal cerebral ischemia due to eMCAO. 2 h, 6 h, 24 h, 3 d, and 7 d post eMCAO, cortices of the affected ipsilateral hemisphere and the non-ischemic contralateral side were separately collected and analyzed by qualitative RT-PCR
Summary
Cerebral inflammation is a hallmark of neuronal degeneration. Anti-inflammatory treatments of the ischemic brain may constitute a therapeutic option to target delayed pathophysiological processes and to manage neuronal degeneration and cerebral damage. Peptidases like dipeptidyl peptidase IV (DPIV, CD26, E.C. 3.4.14.5) and aminopeptidase N (APN, CD13, E.C. 3.4.11.2) are known to regulate a variety of biological processes related to inflammation such as T cell activation, immune responses and inflammation-related diseases [8,9,10,11]. DPIV, a 110 kD type II transmembrane glycoprotein, identical with the T cell antigen CD26, belongs to the group of post-proline dipeptidyl aminopeptidases, consisting of five DPIV gene family proteases, i.e. DPIV, fibroblast activation protein (FAP), DP8, DP9, and DPII (E.C.3.4.14.2) [10,11,12,13]. It has been shown that an additional binding site in the central pore of DPIV is responsible for the cellular effects of ligands of this enzyme with respect to growth regulation and cytokine production [17]
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