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. Author manuscript; available in PMC: 2022 Apr 24.
Published in final edited form as: Brain Res. 2016 Apr 30;1643:10–17. doi: 10.1016/j.brainres.2016.04.071

Membrane lipid peroxidation in neurodegeneration: Role of thrombin and proteinase-activated receptor-1

Bruce A Citron a,b,1, Syed Ameenuddin a,2, K Uchida f, William Z Suo a,b, Karen SantaCruz a,d,3, Barry W Festoff a,b,c,e,*
PMCID: PMC9035224  NIHMSID: NIHMS1797303  PMID: 27138068

Abstract

Thrombin and membrane lipid peroxidation (MLP) have been implicated in various central nervous system (CNS) disorders from CNS trauma to stroke, Alzheimer's (AD) and Parkinson's (PD) diseases. Because thrombin also induces MLP in platelets and its involvement in neurodegenerative diseases we hypothesized that its deleterious effects might, in part, involve formation of MLP in neuronal cells. We previously showed that thrombin induced caspase-3 mediated apoptosis in motor neurons, via a proteinase-activated receptor (PAR1). We have now investigated thrombin's influence on the oxidative state of neurons leading to induction of MLP-protein adducts. Translational relevance of thrombin-induced MLP is supported by increased levels of 4-hydroxynonenal-protein adducts (HNEPA) in AD and PD brains. We now report for the first time that thrombin dose-dependently induces formation of HNEPA in NSC34 mouse motor neuron cells using anti-HNE and anti-acrolein monoclonal antibodies. The most prominent immunoreactive band, in SDS-PAGE, was at ~54 kDa. Membrane fractions displayed higher amounts of the protein-adduct than cytosolic fractions. Thrombin induced MLP was mediated, at least in part, through PAR1 since a PAR1 active peptide, PAR1AP, also elevated HNEPA levels. Of interest, glutamate and Fe2SO4 also increased the ~54 kDa HNEPA band in these cells but to a lesser extent. Taken together our results implicate the involvement of thrombin and MLP in neuronal cell loss observed in various CNS degenerative and traumatic pathologies.

Keywords: Membrane lipid peroxidation, Reactive oxygen, Apoptosis, Neurodegenerative diseases, Spinal cord injury

1. Introduction

Thrombin is a serine protease widely known for its role in hemostasis. However, over the last several years thrombin has increasingly been implicated in a wide variety of central nervous system (CNS) functions and disorders. These include neuronal cell shape, development and death, neurodegenerative diseases and CNS trauma (Festoff et al., 1996; Festoff et al., 2001; Suo et al., 2004; Turgeon and Houenou, 1997; Wang and Reiser, 2003; Xi et al., 2003). In addition to both brain (TBI) and spinal cord (SCI) injury thrombin signaling has been implicated in Alzheimer's (AD) (Grammas et al., 2006; Ho et al., 1994) and Parkinson's (PD) diseases. Thrombin and PAR peptides cause neurodegeneration in the basal ganglia (Suo et al., 2003a), thrombin causes neurofibrillary tangle (NFT) formation in mice (Suo et al., 2003b) and both thrombin and its precursor prothrombin are produced and increased in neuritic plaques in AD brains (Arai et al., 2006). Hallmarks of these conditions include increased levels of reactive oxygen species (ROS) and oxidative stress (Choi, 1995; Markesbery and Carney, 1999) as well as cell death by apoptosis and or necrosis (Krantic et al., 2005; LeBlanc, 2005; Przedborski, 2005). Neurons are particularly vulnerable to the increased oxidative stress.

The presence of membrane lipid peroxidation (MLP) products associated with the oxidative stress in neurodegenerative diseases has prompted numerous mechanistic studies on lipid peroxidative products like malonaldehyde (MDA), acrolein and 4-hydroxynonenal (HNE) leading to protein adducts in neuronal and glial cell cultures. Recent studies have reported the presence of MLP protein adducts in AD and PD brains (Ando et al., 1998; Rofina et al., 2004; Sayre et al., 1997). Formation of MLP may be involved in the earliest stages of AD pathogenesis that may provide a window for therapeutic intervention (Butterfield et al., 2006; Volkel et al., 2006). Several groups including our own have implicated thrombin, acting through one or more of its proteinase-activated receptors (PARs), as an extracellular signal that activates intracellular pathways which, if prolonged, culminate in apoptosis (Donovan et al., 1997; LeBlanc, 2005; Smirnova et al., 1998a; Smirnova et al., 1998b; Thirumangalakudi et al., 2009; Turgeon and Houenou, 1997).

We have also shown that certain modulators of these pathways may protect against thrombin neurotoxicity even when relatively high concentrations of the protease persist (Smirnova et al., 2001) These same PARs, upon activation by thrombin, are responsible for platelet aggregation, which is accompanied by an increase in HNE production (Hurst et al., 1987; Malle et al., 1995). In the present study, we utilized monoclonal antibodies (MAbs) directed against two different aldehyde protein adducts, HNE and acrolein, and studied AD and PD brain cytosolic and membrane fractions, comparing them with age matched control brain samples. Further translational studies were carried out in a murine motor neuron cell line to define potential mechanisms related to thrombin and MLP. Parts of this work were presented earlier in poster form1.

Evidence that MLP occurs with frequency in acute and chronic disorders of the central nervous system (CNS) has prompted numerous studies in model systems. A number of reports indicate that both MDA and HNE induce MLP in neurons and glial cells in culture (Abarikwu et al., 2012; Blanc et al., 1998a; Blanc et al., 1998b; Blanc et al., 1997; Bruce-Keller et al., 1998; Kabuta et al., 2015a; Siddiqui et al., 2012). An additional relationship between HNE, MLP and β-amyloid (Aβ peptide) in AD (Mark et al., 1997a; Mark et al., 1997b; Montine et al., 1998) has also been identified. Of interest, the coagulation protease, thrombin (Smith-Swintosky et al., 1995), like HNE (Mattson and Pedersen, 1998), lowers the threshold of neuronal vulnerability to added Aβ. In turn, thrombin has been shown to stimulate the formation of both MDA and HNE in isolated rat platelets in a time and dose-dependent fashion that occurred in parallel with thromboxane B2 production and platelet aggregation (Gorog and Kovacs, 1995; Ilic et al., 2011; Nosal et al., 1993). By contrast, direct addition of HNE prevented aggregation of platelets induced by ADP or arachidonic acid but not by thrombin (Hurst et al., 1987).

To determine if a relationship existed between thrombin-mediated neurotoxicity and MLP injury we also compared, in parallel experiments, the effects of α-thrombin and a PAR1 active peptide, PAR1AP (TRAF-6), on mouse hybrid motor neurons previously found to have HNE protein adducts induced by Fe2SO4 (Pedersen et al., 1999). Our results indicate clear evidence of MLP in membrane fractions of murine neurons treated with nanomolar α-thrombin or micromolar PAR1AP. MLP was also detected in membranes of neurons exposed to conventional oxidative stress (Fe2SO4) but not with excitotoxic agents (glutamate). Of particular significance, we found that the principal antigenic band(s) induced by thrombin were in the ~50 kDa range, greater in membrane than in cytosolic proteins. One of these bands co-migrated with the murine neuronal PAR1, the principal G-protein-coupled thrombin receptor in these cells.

2. Materials and methods

2.1. Materials

We purchased fetal bovine sera (FBS), Dulbecco’s essential medium (DMEM), N1 neuronal defined medium (consisting of 0.5 g/L insulin, 0.5 g/L human transferrin, 0.5 mg/L sodium selenite, 1.6 g/L putrescine, and 0.73 mg/L progesterone) and Lubrol-PX from Sigma (St. Louis, MO). Tissue culture plates (6 well) were bought from Costar (Corning, NY). Pre-cast sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) gels were purchsed from Novex (San Diego, CA). ECF Western blotting kit was from Amersham (Pittsburgh, PA, U.S.A), where as the Antimouse IgG fluorescein-linked antibody and anti-fluorescein alkaline phosphatase conjugate were from Molecular Probes (Eugene, OR, U.S.A) Life Science (Arlington Heights, IL., U.S.A). Bicinchoninic acid (BCA) protein assay reagent were obtained from Pierce (Rockford, IL, U.S.A.). Protease inhibitor cocktail tablets were purchased from Boehringer Mannheim (Germany). Purified human α-thrombin, and thrombin receptor active peptide (TRAP-6; SFLLRN) were generous gifts of Drs. John Fenton, II, pH.D. (Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, NY) and Thomas Anderson (Albany Medical College, Albany, NY), respectively. Antibody to murine PAR1 was a generous gift of Drs. Patricia Andrade-Gordon and Michael D’Andrea (R. W. Johnson Pharmaceutical Research Institute, Spring House, PA).

2.2. Tissue culture

As in our previously-published studies (Citron et al., 1997; Smirnova et al., 1998a; Smirnova et al., 1998b), we used motor neuron-like NSC19 (or NSC34) cells (initially a gift from Dr. N. Cashman, Montreal Neurologic Hospital, Montreal, Canada), a mouse-mouse neural hybrid cell line produced through fusion of the aminopterin-sensitive neuroblastoma N18TG2 with motor neuron-enriched embryonic day 12-14 spinal cord cells, (Cashman et al., 1992). Relevant to our experiments, a prior report utilized these same cells to estimate the effects of HNE on glutamate transport (Pedersen et al., 1999). Following our previous experience with these cells, we grew them in 10% FBS/DMEM at 5% CO2 and 37 °C. For 24 hours prior to addition of agents, we maintained the cells in the absence of serum using the N1 medium supplement for neural cell cultures (Bottenstein and Sato, 1979).

2.3. Human brain specimens

AD, PD and control human brain specimens were obtained from the University of Kansas Medical Center Alzheimer’s Disease Center (ADC). Affected and aged matched controls ranged from 69-93 years of ages. Brain samples displayed prevalent Lewy bodies. AD samples were from patients diagnosed with AD prior to death and confirmed by plentiful neuritic plaques and neurofibrillary tangles in the cortex and hippocampus. The postmortem interval was less than 8 hours and samples were rapidly frozen in liquid nitrogen. All neuropathologic diagnoses were confirmed according to published criteria (Braak and Braak, 1994; Hyman and Trojanowski, 1997). All work was conducted in accordance with the VA Medical Center and University research oversight committees and programs.

2.4. Membrane and cytosol preparation

Following treatment of cultured motor neurons, washed monolayers were scraped in polycarbonate centrifuge tubes and then sonicated (Fisher Scientific 60 Sonic dismembrator) on ice with 3 bursts at setting 5 for 5 sec each time. Similarly, human brain specimens were homogenized in 10 volumes of ice cold homogenization buffer (10 mM Tris, 10 mM EDTA, 2 mM EGTA, 250 mM sucrose, 2 mM DTT pH 7.8) with three bursts of 15 sec each on ice using a Polytron PT 1200 (Brinkmann Instruments). Homogenates were centrifuged at 600 rpm for 5 min (Beckman GS-15R) at 4 °C to remove cellular debris. Supernatants were transferred to tubes in a near vertical rotor, loaded into a benchtop ultracentrifuge (Optima TLX Beckman Instruments), and centrifuged at 374,000 g for 30 min. Supernatants (cytosol) were removed, aliquoted and snap-frozen in liquid N2 and stored at −70 °C. Pellets (membranes) were re-suspended in 1% Lubrol-PX, aliquoted and frozen as above. Protein concentration was determined using the BCA method (Smith et al., 1985).

2.5. SDS-PAGE and Western blotting

SDS-PAGE was performed on pre-cast 12% gels (Novex, San Diego, CA), and ran in a Novex power supply at 125 V for 90 min. Cytosol and membrane fraction proteins, whether cultured motor neurons or brain samples from mid-frontal cortex of AD and control and substantia nigra from PD brains, were loaded at 5 μg per well. Following electrophoresis proteins were transferred onto PVDF membranes using a Novex transfer apparatus and procedures recommended by the manufacturer. Membranes were blocked with 10% non-fat dry milk and then incubated with anti-HNE or anti-acrolein MAbs at 1:1000 and 1:4000 dilutions, respectively (Uchida et al., 1998a; Uchida et al., 1998b; Uchida et al., 1993). Following primary Mab incubation membranes were incubated in fluorescein-conjugated rabbit anti-mouse IgG secondary antibody at a dilution of 1:600 and the signal was amplified with anti-fluorescein alkaline phosphatase conjugate, according to the manufacturer's suggestions. The membranes were visualized with the ECF substrate, and then scanned using 450 nm excitation on the STORM 860 Phosphoflouroimager (Molecular Dynamics), and analyzed by ImageQuant software.

2.6. Statistical analysis

Data shown are means ± SEM of at least 3 replicates. Significant differences compared with control values (*** p < 0.001, ** p < 0.01, * p < 0.05) were arrived at by ANOVA with Scheffe's post hoc test and unpaired Student's t-test using StatView 4.5 software (Abacus Concepts, Berkeley, CA).

3. Results

3.1. Detection of HNEPAs in AD and PD human brains

We probed AD and PD brain protein samples with the anti-HNE monoclonal antibody and the results are presented in Figs. 1 and 2. Dramatically, the relative immunoreactivity with anti-HNE was significantly higher in both AD and PD extracts as compared to aged-matched control brains (p < 0.01). Of interest, when we probed human brain fractions with HNE antibody a larger number of intermediate bands were observed between 50 and 75 kDa (Fig. 1A). The most prominent bands observed were in the same range for anti-acrolein (Fig. 1B). In contrast, MLP-adduct bands immunoreactive for either HNE or acrolein were diminished or not detected in cytosolic or membrane fractions of normal brains. From densitometric analysis (Figs. 1C and 2C), the relative immunoreactivity for HNE was also significantly greater in PD than AD as compared to controls (p < 0.01).

Fig. 1. Human brain cytoplasmic fractions probed with anti-HNE (A), anti-acrolein (B) and densitometric analysis (C) show increased oxidative stress damage.

Fig. 1.

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Neurodegenerative diseased human brain cytosol extracts were separated by SDS-PAGE and probed with anti-HNE (A) or anti-acrolein MAb (B). We show Western blot analysis of human brain cytosol showing increase with HNE and acrolein protein adducts in AD and PD disease brains. Densitometric analysis of HNE and acrolein protein conjugates were six and three fold higher, respectively, than controls (C). Values are mean ± SEM **(p < 0.01), *(p < 0.05) vs control by ANOVA with Scheffe's post hoc tests. A larger number of intermediate bands were detected between 50-75 kDa with anti-HNE (A). Most prominent bands in a similar range was also seen with anti-acrolein (B). MLP-adduct bands immunoreactive for either HNE or acrolein were diminished or not detected in cytosolic or membrane fractions of normal brains.

Fig. 2. Evidence of increased ROS damage found in human brain membrane fractions probed with anti-HNE and anti-acrolein Mabs.

Fig. 2.

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Western blot analysis of human brain membrane fractions as in Fig. 1 and Materials and Methods indicated an increase in (A) HNE and (B) acrolein protein adducts in Alzheimer's and Parkinson's disease brains. (C) Densitometric analysis of HNE and acrolein protein conjugates were two and three fold higher, respectively, than controls. Values are mean ± SEM **(p < 0.01), *(p < 0.05) vs control by ANOVA with Scheffe's post hoc tests.

3.2. Formation of HNEPAs with thrombin

We next sought to explore the mechanistic basis of MLP in neurodegeneration. To do this we evaluated the effects of thrombin, a PAR peptide (TRAP-6 or PAR1AP), glutamate and FeSO4 separately on the formation of HNEPA are shown in Fig. 3. HNE-protein adducts in both cytosol and membrane fractions of MNC were detected, using anti HNE monoclonal antibody. In the initial experiments cells were treated with thrombin (500 nM), PAR1AP (750 μM), glutamate (100 μM) or FeSO4.

Fig. 3. Thrombin and PAR1AP induce both HNE and acrolein protein adducts in motor neurons.

Fig. 3.

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Enhanced immunoreactivity indicated the treatment effect of α-thrombin (500 nM), PAR1AP (750 μM), glutamate (100 mM), and FeSO4 (500 μM) compared to controls on production of HNE protein conjugates in cytosolic and membrane fractions of NSC34 cultured motoneurons. Densitometric analysis displayed at least a two fold relative increase in MLP compared to untreated motoneurons. Thrombin and PAR1AP affected membrane and cytosol compartments equally compared with glutamate and FeSO4.

Western blots of the protein extracts clearly revealed increased levels of HNE-protein adducts of different size bands following thrombin treatment. The most prominent band was found to be ~54 kDa protein based on its SDS-PAGE electrophoretic mobility. Since this band was so prominent, we focused on it and performed densitometric quantitation. The relative density of the band was as an indicator of the formation of HNEPA and also an indicator of oxidative stress.

Quantitative analysis of the blots showed a significant increase in the HNE-protein adduct following thrombin treatment when compared to untreated cells, both in cytosol (p < 0.001), and membrane fractions (p < 0.001), The increase in the HNEPA ~54 kDa band was several fold higher (Fig. 3).

Relevant to this here we also report for the first time that PAR1AP (TRAP-6), a six amino acid active peptide for PAR1 (750 μM), significantly increased the levels of the ~54 kDa protein band in cytosol (p < 0.001) and membrane fractions (p < 0.001). The observed elevation with the PAR1AP is comparable to the thrombin mediated increase in HNEPA (Fig. 3), although at > 1000 fold concentration, as is known for other effects of thrombin and PAR1.

3.3. Neurotoxins glutamate and Fe2SO4 also cause formation of neuronal HNEPA

We tested whether known neurotoxic molecules glutamate, an excitotoxic neurotransmitter and FeSO4, known inducer of oxidative stress also increased HNEPA in model motor neurons. Although both glutamate and FeSO4 significantly increased the formation of HNEPA in both cell fractions (p < 0.05) when compared to the controls, the increase with either glutamate or FeSO4 was lower than the increase found with thrombin in the membrane fractions (Fig. 3).

3.4. Dose dependent effect of thrombin

Dose related effects of thrombin on the formation of HNEPA are shown in Fig. 4. Accumulation of HNEPA was found to increase up to 1.0 μM of thrombin. The observed increase with 1.0 μM of thrombin was over 4.5 fold higher as compared to the effect found with 0.1 of μM of thrombin.

Fig. 4. Dose-dependent induction of MLP in motor neurons by thrombin.

Fig. 4.

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NSC34 motoneurons treated with varying amounts of □-thrombin displayed a linear increase in membrane HNE (r2=0.94). The PAR-1 active peptide (PAR1AP), at a 1000 fold higher concentration, also caused an increase in membrane HNE. Results are mean ± SEM. *** (p < 0.001), ** (p < 0.01), * (p < 0.05) Student's t test thrombin vs. control or PAR1AP vs. control.

3.5. NSC34 membrane proteins stained with anti-PAR-1 antibody

We have previously used a well-characterized antibody (PAR-1C) to identify PAR1 protein in these NSC cells (Smirnova et al., 1998a). We used this same antibody to show an increase in PAR1 expression in motor neurons of the autosomal recessive wobbler mutant mouse model of motor neuron disease (Festoff et al., 2000). Using this antibody in Western blots we found an increase in thrombin-treated samples compared with control NSC34 cell membranes (Fig. 5). When compared with either anti-HNE or anti-acrolein gels, this band co-migrated with aldehyde adducts induced by thrombin or PAR1AP.

Fig. 5. PAR-1 levels after insult of NSC34 model motor neurons.

Fig. 5.

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Western immunoblots indicated that thrombin treatment (along with FeSO4 and glutamate insults) resulted in an eight fold increase in PAR-1 in the cytoplasmic fraction compared to a four fold increase in the membrane fraction. The activating peptide PAR1AP induced the largest increase (four fold) in the membrane fraction.

4. Discussion

In the present study, we have investigated whether thrombin concentrations that induce apoptosis in motor neurons could produce HNE-protein adducts (HNEP) in these cells. We used the well-characterized NSC34 mouse motor neuron cell line (Cashman et al., 1992; Citron et al., 1997), which we have also shown responds to low doses of thrombin in a dramatic neurotoxic manner (Smirnova et al., 1998b). Since motor neuronal loss is a major component following SCI, and that both thrombin and its principal receptor, PAR1, are significantly increased early after SCI (Citron et al., 2000), such studies potentially offer mechanistic understanding for SCI-related apoptosis. Here, for the first time we demonstrate that thrombin can induce formation of HNE-protein adducts in cultured motor neurons.

A variety of proinflammatory, damage-associated molecules (DAMPs) □□□□□, such as high mobility group box protein 1 (HMGB1), S100B, as well as Aβ (Clark and Vissel, 2015). Furthermore, increasing evidence indicates that thrombin contributes to oxidative damage in CNS disease (Krenzlin et al., 2016). These results are consonant with previous studies in platelets demonstrating that thrombin can induce formation of both MDA and HNE in platelet membranes (Buczynski et al., 1993; Gorog and Kovacs, 1995; Nosal et al., 1993). As such, the current results provide this information in neural cells, which are much more sensitive to oxidative stress. In this regard, formation of ROS, excitotoxicity and lipid peroxidation are operative in the pathogenesis of various neurodegenerative (Barnham et al., 2004; Zarkovic, 2003), and neurotraumatic diseases, both TBI and SCI (Gard et al., 2001; Xu et al., 2005), and in CNS disorders in general (Juurlink and Paterson, 1998; Lewen et al., 2000; Murphy et al., 2003; Okonkwo and Stone, 2003). As major cytotoxic products, found in various tissues both in normal and pathological conditions, HNE and acrolein can form adducts with cellular and extracellular proteins that could be detected by the use of monoclonal antibodies (Uchida et al., 1998a; Uchida et al., 1998b; Uchida et al., 1993). Accumulation of MLP-protein adducts were reported in the CNS of amyotrophic lateral sclerosis (ALS) (Kabuta et al., 2015b; Pedersen et al., 1998) and prion diseases (Freixes et al., 2006) as well as in AD and PD (Sayre et al., 1997; Yoritaka et al., 1996) suggesting a role for MLP related protein adducts in neurodegeneration. Further, SCI was also shown to be associated with accumulation of both HNE and acrolein protein adducts (Luo et al., 2005; Springer et al., 1997; Xu et al., 2005).

Thrombin is generated from the proteolytic cleavage of prothrombin and has emerged as a pleiotropic protease with prominent extravascular functions (Festoff et al., 1996; Gabazza et al., 2004; Narayanan, 1999; Rohatgi et al., 2004), especially in the CNS, where activation of one of several G-protein-coupled receptors now known as as PARs appears to be the mechanism (Ben Shimon et al., 2015; Chapman, 2013; Luo et al., 2007; Rohatgi et al., 2004; Suo et al., 2004; Wang and Reiser, 2003). Most cellular functions of thrombin are now thought to be mediated via activation of one or more of these PARs, principally PAR1 and PAR4 (Camerer et al., 2006; Kataoka et al., 2003; Ludeman et al., 2005). Thrombin mediates apoptotic neuronal death in susceptible subpopulations at nanomolar concentrations while it provides neuroprotection at picomolar concentrations in cultured astrocytes (Beecher et al., 1994) and hippocampal neurons (Suo et al., 2003b). Motor neurons are amongst the most vulnerable neurons and rapidly undergo neurite retraction and mobilization of calcium followed by caspase activation and apoptosis when exposed to nanomolar thrombin (Smirnova et al., 1998a; Smirnova et al., 1998b). Motor neurons are also dependent on PAR1 in vivo and express high levels of this PAR both normally (Niclou et al., 1994; Weinstein et al., 1995) and abnormally after injury (Niclou et al., 1998) or in a neurodegenerative model, the wobbler mouse (Festoff et al., 2000). Both CNS trauma and neurodegeneration are closely associated with ROS and MLP (Farooqui and Horrocks, 1998; Liu et al., 2003; Markesbery and Carney, 1999; Mattson, 1998; Sayre et al., 1997; Zarkovic, 2003).

Thrombin is also a potent proinflammatory mediator (Dugina et al., 2002; Steinhoff et al., 2005; Suo et al., 2004), including roles in microglia (Hanisch et al., 2004; Ji et al., 2004; Nicole et al., 2005; Ryu et al., 2000; Suo et al., 2002; Suo et al., 2003a; Weinstein et al., 2005; Yang et al., 2004). Other functions in the CNS have to do with mechanical injury as well as ischemia and stroke (Bednar, 2000; Figueroa et al., 1998; Gingrich and Traynelis, 2000; Karabiyikoglu et al., 2004; Kario et al., 1999; Kataoka et al., 2000). Such observations are coupled with the capability of the CNS in synthesizing prothrombin in developing brain (Dihanich et al., 1991) and spinal cord (Citron et al., 2000) and after global ischemia (Riek-Burchardt et al., 2002) and SCI (Citron et al., 2000). Furthermore, the presence of PARs as well as the most potent tissue inhibitor of thrombin, the serpin, protease nexin I (PN-I), emphasize the important physiological as well as pathophysiological roles for thrombin signaling in CNS (Krenzlin et al., 2016).

In addition to HNE, acrolein is rapidly incorporated into proteins preferentially reacting with lysine residues to form acrolein adducts (Uchida, 2015). Such are particularly prominent in microtubule-associated tau protein in AD (Uchida et al., 1998b). Relative to our current results, studies in AD transgenic mice elucidated a close topographic relationship between tau-immunoreactive dystrophic neurites surrounding Aβ deposits and ROS markers, including HNE (Mattson et al., 1997; Pappolla et al., 1998; Smith et al., 1998) and redox proteomics in AD brains (Perluigi et al., 2009).

Our results show that thrombin induces unambiguous HNE MAb-positive ~54 kDa bands in a dose-dependent response in cultured NSC34 motor neurons (Fig. 4). Likewise the PAR1AP synthetic peptide (SFLLRN) showed the same HNE MAb-positive ~54 kDa bands as well. Furthermore, the co-migration of this MLP adduct band with PAR1 suggests that this G-protein-coupled receptor may be particularly sensitive to thrombin-mediated MLP. Although the mechanism underlying formation of HNEPA or acrolein adducts in MLP is unclear, their generation and accumulation in the CNS may contribute to the increasing neuronal damage beginning at the cell membrane. Furthermore, our results suggest that at least one potential membrane target is PAR1, activated by thrombin that gains access through a compromised blood-brain barrier in neurotrauma or is locally synthesized in reactive glial populations in aging or neurodegeneration.

Acknowledgements

We thank Drs. N. Cashman, J.W. Fenton, II, Thomas Anderson and Michael D’Andrea for providing NSC34 cell lines, human α-thrombin, PAR1AP and PAR1C antibody, respectively. The authors are grateful to Michael Lucido and Sonia Malburg for expert technical assistance. This work was supported, in part, by the Department of Veterans Affairs (Veterans Health Administration, Office of Research and Development, Biomedical Laboratory Research and Development), the Lied Fund of the University of Kansas Medical Center Research Institute, Inc., the Alzheimer's Association, and the ALS Association.

Footnotes

Conflict of interest

Dr. Festoff is founder and CEO of PHLOGISTIX LLC. The other authors declare that they have no conflict of interests.

Disclaimer

The contents do not represent the views of the Department of Veterans Affairs or the United States Government.

1

Ameenuddin et al., Membrane lipid peroxidation (MLP) in Alzheimer's (AD) and Parkinson's (PD) brains and role of protease activated receptor-1 (PAR-1). American Society for Neurochemistry, Chicago, IL, March 25, 2000

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