Implication of the Kallikrein-Kinin System in Neurological Disorders: Quest for Potential Biomarkers and Mechanisms

Neurological disorders represent major health concerns in terms of comorbidity and mortality worldwide. Despite a tremendous increase in our understanding of the pathophysiological processes involved in disease progression and prevention, the accumulated knowledge so far resulted in relatively moderate translational benefits in terms of therapeutic interventions and enhanced clinical outcomes. Aiming at specific neural molecular pathways, different strategies have been geared to target the development and progression of such disorders. The kallikrein-kinin system (KKS) is among the most delineated candidate systems due to its ubiquitous roles mediating several of the pathophysiological features of these neurological disorders as well as being implicated in regulating various brain functions. Several experimental KKS models revealed that the inhibition or stimulation of the two receptors of the KKS system (B1R and B2R) can exhibit neuroprotective and/or adverse pathological outcomes. This updated review provides background details of the KKS components and their functions in different neurological disorders including temporal lobe epilepsy, traumatic brain injury, stroke, spinal cord injury, Alzheimer’s disease, multiple sclerosis and glioma. Finally, this work will highlight the putative roles of the KKS components as potential neurotherapeutic targets and provide future perspectives on the possibility of translating these findings into potential clinical biomarkers in neurological disease

Correction: Characterization of the Kallikrein-Kinin System Post Chemical Neuronal Injury: An In Vitro Biochemical and Neuroproteomics Assessment.

Traumatic Brain Injury (TBI) is the result of a mechanical impact on the brain provoking mild, moderate or severe symptoms. It is acknowledged that TBI leads to apoptotic and necrotic cell death; however, the exact mechanism by which brain trauma leads to neural injury is not fully elucidated. Some studies have highlighted the pivotal role of the Kallikrein-Kinin System (KKS) in brain trauma but the results are still controversial and inconclusive. In this study, we investigated both the expression and the role of Bradykinin 1 and 2 receptors (B1R and B2R), in mediating neuronal injury under chemical neurotoxicity paradigm in PC12 cell lines. The neuronal cell line PC12 was treated with the apoptotic drug Staurospor-ine (STS) to induce cell death. Intracellular calcium release was evaluated by Fluo 4-AM staining and showed that inhibition of the B2R prevented calcium release following STS treatment. Differential analyses utilizing immunofluorescence, Western blot and Real-time Polymerase Chain Reaction revealed an upregulation of both bradykinin receptors occur-ring at 3h and 12h post-STS treatment, but with a higher induction of B2R compared to B1R. This implies that STS-mediated apoptosis in PC12 cells is mainly conducted throug

Characterization of the Kallikrein-Kinin System Post Chemical Neuronal Injury: An <i>In Vitro</i> Biochemical and Neuroproteomics Assessment

<div><p>Traumatic Brain Injury (TBI) is the result of a mechanical impact on the brain provoking mild, moderate or severe symptoms. It is acknowledged that TBI leads to apoptotic and necrotic cell death; however, the exact mechanism by which brain trauma leads to neural injury is not fully elucidated. Some studies have highlighted the pivotal role of the Kallikrein-Kinin System (KKS) in brain trauma but the results are still controversial and inconclusive. In this study, we investigated both the expression and the role of Bradykinin 1 and 2 receptors (B1R and B2R), in mediating neuronal injury under chemical neurotoxicity paradigm in PC12 cell lines. The neuronal cell line PC12 was treated with the apoptotic drug Staurosporine (STS) to induce cell death. Intracellular calcium release was evaluated by Fluo 4-AM staining and showed that inhibition of the B2R prevented calcium release following STS treatment. Differential analyses utilizing immunofluorescence, Western blot and Real-time Polymerase Chain Reaction revealed an upregulation of both bradykinin receptors occurring at 3h and 12h post-STS treatment, but with a higher induction of B2R compared to B1R. This implies that STS-mediated apoptosis in PC12 cells is mainly conducted through B2R and partly via B1R. Finally, a neuroproteomics approach was conducted to find relevant proteins associated to STS and KKS in PC12 cells. Neuroproteomics results confirmed the presence of an inflammatory response leading to cell death during apoptosis-mediated STS treatment; however, a “survival” capacity was shown following inhibition of B2R coupled with STS treatment. Our data suggest that B2R is a key player in the inflammatory pathway following STS-mediated apoptosis in PC12 cells and its inhibition may represent a potential therapeutic tool in TBI.</p></div

Fluo-4 AM staining of PC12 cells 3h post-STS treatment depicting intracellular calcium release.

<p>Immunofluorescent images of <b>(a)</b> PC12 cells control, <b>(b)</b> STS treated, <b>(c)</b> B2A pre-treated, <b>(d)</b> B2A pre-treated and STS treated, <b>(e)</b> B2I pre-treated, and <b>(f)</b> B2I pre-treated and STS treated. Scale bar = 10 000nm. STS: Staurosporine; B2A: Bradykinin 2 Receptor activated; B2I: Bradykinin 2 Receptor inhibited.</p

Immunofluorescent analysis of Bradykinin 1 and 2 receptors expression in PC12 cells.

<p>Immunofluorescent images of <b>(a)</b> B1R and <b>(b)</b> B2R in PC12 cells in response to 3h, 12h and 24h of STS treatment. Scale bar = 10μm. B1R: Bradykinin 1 receptor; B2R: Bradykinin 2 receptor; STS: Staurosporine. Blue: DAPI (4´,6-diamidino-2-phenylindole), Red: B1R pAb (upper panel) or B2R pAb (lower panel) with Alexa-Fluor 488-conjugated goat anti-mouse IgG.</p

B2R protein expression upon 3h STS treatment of PC12 cells.

<p><b>(a)</b> Western Blot of protein extracts from PC12 cells. <b>(b)</b> Histogram representing densitometry analysis of B2R expression using Image J software (n = 3). Error bars represent standard errors of the mean. Symbols indicate statistical difference: B2R vs. B2R-Ctrl, (***) p<0.001 (one-way ANOVA with Bonferroni correction for multiple comparisons).</p

Pathways study of STS-induced altered proteins.

<p>Global Interaction Proteome of <b>(a)</b> STS treated cells and <b>(b)</b> B2I+STS treated cells. The green rectangles, violet rectangles and blue hexagons are reflective of biological processes, disease processes and functional classes, respectively. The different colors of proteins reflect their alteration: upregulated (red), downregulated (orange), downregulated and unique to the treated group (yellow). The green highlights represent the detrimental effects observed following STS or B2I+STS treatment and the pink highlight represents the protective effect observed following B2I+STS treatment. STS: Staurosporine, B2I: Bradykinin 2 receptor inhibited. Creatine kinase B-type [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128601#pone.0128601.ref015" target="_blank">15</a>], designated by blue highlight is shown to be upregulated in the STS+ B2I treatment where cell survival pathway was detected.</p

Expression of the B1R and B2R genes in PC12 cells (a) 3h and (b) 12h post-STS treatment, using RT-PCR assay.

<p>(n = 3, triplicate). Each gene was normalized to the housekeeping gene GAPDH, and the control was used as a reference for comparative analysis. Error bars represent standard errors of the mean. Symbols indicate statistical difference: B1R vs B1R-Ctrl, (###) p<0.001, (##) p<0.01, (#) p<0.05; B2R vs B2R-Ctrl, (***) p<0.001 (one-way ANOVA with Bonferroni correction for multiple comparisons). B1R vs. B2R, (++) p<0.1, (+) p<0.05 (Paired t test).</p