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The serine protease inhibitor TLCK attenuates intrinsic death pathways in neurons upstream of mitochondrial demise

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Abstract

It is well-established that activation of proteases, such as caspases, calpains and cathepsins are essential components in signaling pathways of programmed cell death (PCD). Although these proteases have also been linked to mechanisms of neuronal cell death, they are dispensable in paradigms of intrinsic death pathways, e.g. induced by oxidative stress. However, emerging evidence implicated a particular role for serine proteases in mechanisms of PCD in neurons. Here, we investigated the role of trypsin-like serine proteases in a model of glutamate toxicity in HT-22 cells. In these cells glutamate induces oxytosis, a form of caspase-independent cell death that involves activation of the pro-apoptotic protein BH3 interacting-domain death agonist (Bid), leading to mitochondrial demise and ensuing cell death. In this model system, the trypsin-like serine protease inhibitor Nα-tosyl-l-lysine chloromethyl ketone hydrochloride (TLCK) inhibited mitochondrial damage and cell death. Mitochondrial morphology alterations, the impairment of the mitochondrial membrane potential and ATP depletion were prevented and, moreover, lipid peroxidation induced by glutamate was completely abolished. Strikingly, truncated Bid-induced cell death was not affected by TLCK, suggesting a detrimental activity of serine proteases upstream of Bid activation and mitochondrial demise. In summary, this study demonstrates the protective effect of serine protease inhibition by TLCK against oxytosis-induced mitochondrial damage and cell death. These findings indicate that TLCK-sensitive serine proteases play a crucial role in cell death mechanisms upstream of mitochondrial demise and thus, may serve as therapeutic targets in diseases, where oxidative stress and intrinsic pathways of PCD mediate neuronal cell death.

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Abbreviations

AD:

Alzheimer’s disease

AIF:

Apoptosis-inducing-factor

Bax:

Bcl-2-associated X protein

Bcl-2:

B-cell lymphoma 2

Bid:

BH3 interacting-domain death agonist

BODIPY:

4,4-Difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-sindacene-3-undecanoic acid

CCCP:

Carbonylcyanide-3-chlorophenylhydrazone

HtrA2:

High temperature requirement factor A2

MMP:

Mitochondrial membrane potential

MTT:

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NF-κB:

Nuclear factor kappa B

OCR:

Oxygen consumption rate

PCD:

Programmed cell death

PD:

Parkinson’s disease

ROS:

Reactive oxygen species

TLCK:

Nα-tosyl-l-lysine chloromethyl ketone hydrochloride

TPCK:

N-p-tosyl-l-phenylalanine chloromethyl ketone

TMRE:

Tetramethylrhodamine ethyl ester

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Acknowledgments

We thank the excellent technical support by Mrs. Katharina Elsässer and Eileen Daube and the support by our student Lucia von Wachter for the mitochondrial counting. We thank Wei Wan and Shuna Wang for their technical support. Furthermore, we thank Mrs. Emma Esser for careful editing of the manuscript and Roche Diagnostics GmbH for providing support with the xCELLigence system.

Conflict of interests

The authors declare that they have no conflict of interests.

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  1. C. Reuther

    Present address: Institut für Schlaganfall- und Demenzforschung (ISD), Klinikum der Universität München, Max-Lebsche-Platz 30, 81377, Munich, Germany

Authors and Affiliations

  1. Institut für Pharmakologie und Klinische Pharmazie, Fachbereich Pharmazie, Philipps-Universität Marburg, Karl-von-Frisch-Straße 1, 35032, Marburg, Germany

    C. Reuther, G. K. Ganjam, A. M. Dolga & C. Culmsee

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  1. C. Reuther
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Correspondence to C. Culmsee.

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Reuther, C., Ganjam, G.K., Dolga, A.M. et al. The serine protease inhibitor TLCK attenuates intrinsic death pathways in neurons upstream of mitochondrial demise. Apoptosis 19, 1545–1558 (2014). https://doi.org/10.1007/s10495-014-1027-7

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