Our previous study screened the 1040‐compound library for inhibition of release of cytochrome c and found that N‐acetyl‐tryptophan ranked 13th in the purified mitochondria system and inhibited mutant‐huntingtin‐induced ST14A striatal cell death (Wang et al . 2008 ). However, little is known about the molecular mechanisms underlying NAT's neuroprotection. Here we assess the neuroprotective ability of N‐acetyl‐tryptophan in NSC‐34 motoneurons and primary motor neurons, which is consistent with the results of chemical structure and activity relationship analysis. Our study evaluates the mechanisms of action of L‐NAT on motor neuron cell death and suggests that L‐NAT may be an effective therapeutic agent for ALS.

Multiple mechanisms have been implicated in ALS pathogenesis, including mitochondrial dysfunction, apoptosis, and inflammation dysfunction of protein degradation, oxidative stress, and glutamate excitotoxicity (Pandya et al . 2012 ; Zhu et al . 2015 ). Among these, mitochondrial dysfunction is an early event in motor neuron degeneration, and the mitochondrial apoptotic death pathway is crucial in both motor neuron death and in the pathogenesis of ALS (Liu et al . 2002 ; Wang et al . 2007 ). Release of cytochrome c from mitochondria triggers a set of biochemical changes that lead to the activation of neuronal cell death in mSOD1 G93A mice and in NSC‐34 motoneurons (Zhu et al . 2002 ; Friedlander 2003 ; Chi et al . 2007 ). In addition to proteolysis of IL‐1β during the immune response, caspase‐1 initiates the cell death pathway in neurodegenerative diseases (Friedlander 2003 ; Zhang et al . 2003 ; Wang 2009 ). During disease progression in mSOD1 G93A mice, caspase‐1 acts as a chronic initiator and caspase‐3 acts as the final effector of motor neuron death (Li et al . 2000 ; Pasinelli et al . 2000 ). Expression of a dominant negative mutant of caspase‐1 attenuates mitochondrial release of cytochrome c and the ensuing activation of caspase‐9 and ‐3 in the spinal cord of transgenic mutant SOD1 mice (Guegan et al . 2002 ).

N‐acetyl‐tryptophan (NAT), a NK‐1R antagonist, disrupts the binding of SP to the NK‐1R. NAT has been included in the library of the Neurodegeneration Drug Screening Consortium of 1040 compounds assembled by the National Institute of Neurological Disorders and Stroke, indicating its potential as a therapeutic agent. There are three isomers of NAT: N‐acetyl‐l‐tryptophan (L‐NAT), N‐acetyl‐DL‐tryptophan (DL‐NAT), and N‐acetyl‐d‐tryptophan (D‐NAT). L‐NAT significantly improved motor and cognitive outcomes in models of Parkinson's diseases (Thornton and Vink 2012 ; Thornton et al . 2014 ), as well as reduced brain edema and axonal injury in experimental traumatic brain injury (Vink et al . 2004 ; Donkin et al . 2009 , 2010 ) and stroke (Turner et al . 2005 ). However, there are no reports on the neuroprotective effects of L‐NAT on ALS.

Substance P (SP) belongs to a group of neurokinins, collectively called tachykinins, and is the natural high‐affinity ligand of the neurokinin‐1 receptor (NK‐1R). As a bioactive neuropeptide composed of 11 amino acids (RPKPEEFFGLM‐NH2), SP is an important excitatory neurotransmitter and/or neuromodulator in the peripheral and central nervous systems. SP mediates various biological functions such as neuronal excitation, smooth muscle contraction, pain transmission (Macdonald and Nowak 1981 ; Mantyh 2002 ; Zhang et al . 2004 ), and regulation of cell apoptosis (Bang et al . 2004 ; Peters et al . 2005 ) and survival (Harrison and Geppetti 2001 ; Caioli et al . 2011 ). SP has been suggested to play a role in neurodegenerative diseases including Alzheimer's disease (Herpfer and Lieb 2003 ), Parkinson's disease (Zhang et al . 2008 ; Thornton et al . 2010 ), ALS (Caioli et al . 2011 ), Huntington's disease (Kowall et al . 1993 ), and spinal cord injury (Leonard et al . 2013 ). Inhibition of SP activity, either by preventing SP release or by antagonism of NK‐1R using NK‐1R antagonist, consistently results in marked improvements in functional outcome (Vink and van den Heuvel 2010 ). In other words, the inhibition of binding of SP to the NK‐1R, using NK‐1R antagonist may be a useful treatment for Parkinson's disease (Thornton et al . 2010 ; Zacest et al . 2010 ), traumatic brain injury (Vink et al . 2004 ), cerebral ischemia (Yu et al . 1997 ), affective disorders (Czeh et al . 2006 ), spinal cord injury (Leonard and Vink 2013 ), or other central nervous system (CNS) disorders (Saria 1999 ). Interestingly, SP may play an important role in the pathophysiology of ALS, as elevated levels of SP have been reported in the cerebrospinal fluid of ALS patients (Matsuishi et al . 1999 ). However, there are no reports on the inhibition or neuroprotective effects of SP release using NK‐1R antagonist L‐NAT on ALS.

Densitometric quantification was performed with the Quantity One Program (Bio‐Rad Laboratories, Hercules, CA, USA). Statistical significance was evaluated by t ‐test: p < 0.05 were considered significant and are indicated (*), ** and *** indicate p < 0.01 and p < 0.001, respectively. Drug analysis, including IC 50 and maximum protection, was performed using the Prism 5.0 program (GraphPad Software Inc., San Diego, CA, USA).

The previously developed model of NK‐1R, kindly provided by Dr Gerhard Klebe (Evers and Klebe 2004 ), was used to evaluate its binding with L‐NAT and D‐NAT. InsightII and Discovery_3 (Accerlrys Inc., San Diego, CA, USA) were used for modeling. The software was run on a Silicon Graphics computer Oxygen 2. Energy calculations were performed in a vacuum using Consistent Valence Forcefield as the force field. Before the binding experiments, L‐NAT and D‐NAT were constructed using the molecular building tools in InsightII, and their energies were minimized under the same conditions. The similarity of NAT with the molecules studied (Evers and Klebe 2004 ) was the ring moiety and the scaffold containing hydrogen bond acceptor, and the same domain of NK‐1R composed of amino acid residues Gln144, Glu172, His171, Ile183, His244, and Try251 was used as the binding domain for L‐NAT and D‐NAT. To simplify the calculations, these residues were left free while other amino acid residues of NK‐1R were fixed during the energy minimization process. L‐NAT was manually placed in the binding domain with the ring moiety and the hydrogen bond acceptor scaffold oriented at the similar position, as performed previously by Evers et al . (Evers and Klebe 2004 ). The energy of the L‐NAT/NK‐1R complex was optimized, and the resulting energy value was compared with the binding energy of the D‐NAT/NK‐1R complex. To achieve this goal, optimized D‐NAT was also manually placed into the binding domain of NK‐1R, with its ring moiety superimposed on that of L‐NAT in its optimized complex model. Similarly, energy minimization of the D‐NAT/NK‐1R complex was carried out and the total energy was measured.

NSC‐34 motoneurons were harvested and lysed on ice under hypotonic conditions in a buffer containing 25 mM HEPES (pH 7.5), 5 mM MgCl 2, 5 mM EDTA, and 5 mM dithiothreitol for 10 min. Cell lysates were then incubated and centrifuged at 14 000 rpm for 3–5 min at 4°C. The supernatants were incubated with three fluorogenic substrates (Suc‐LLVY‐MCA, Boc‐LRR‐AMC, and Z‐LLE‐AMC from Biomol) for the determination of chymotrypsin‐like, trypsin‐like, and caspase‐like proteasome activity, respectively. Briefly, cell lysates (20 μg protein) were incubated with each substrate at 37°C for 3 h in assay buffer (50 mM Tris/HCl, pH 7.5, 25 mM KCl, 10 mM NaCl, 1 mM MgCl 2 ) in a 96‐well clear‐bottom/black plate. Sodium dodecyl sulfate (0.03%) was added to the assay buffer to activate chymotrypsin‐like activity. Fluorescence intensity was measured by excitation at 360 nm and emission at 460 nm in a fluorescence plate reader (Victor 3 1420 Multilabel Counter, PerkinElmer, Waltham, MA, USA).

Cell extracts and enzyme assays in NSC‐34 cells were performed with the ApoAlert caspase fluorescence assay kit as previously described (Wang et al . 2008 ). Caspase‐3‐like substrate Ac‐DEVD‐AFC was purchased from BD Pharmingen, and caspase‐9‐like substrate Ac‐LEHD‐AFC and Caspase‐1‐like substrate Ac‐YVAD‐AFC were purchased from Calbiochem, Billerica, MA, USA. Released AFC was quantified in a Bio‐Rad Versa Fluorometer (excitation at 400 nm and emission at 505 nm).

NSC‐34 cells were treated with H2O2, and small drops (5 μL) of culture supernatants were spotted onto polyvinylidene difluoride membranes. Blots were blocked in 5% milk in PBS and incubated with an SP antibody in 5% milk, 0.1% Tween 20, 150 nM NaCl, and 50 mM Tris‐HCl, pH 7.5, for indicated time points at 4°C. Antibody to SP was purchased from Abcam (Cambridge, MA, USA). After serial washes, the blots were incubated with an horseradish peroxidase‐conjugated secondary antibody for 2 h at 20‐26°C. The amount of SP released was assessed, using enhanced chemiluminescence reagent .

NSC‐34 cells were exposed to H2O2 with or without test drugs in a time‐course manner. Particulates were removed and the supernatants were collected and subjected to the Parameter Substance P Immunoassay. Concentrations of substance P were determined by Enzyme‐Linked Immunosorbent Assay (ELISA), using Substance P Parameter Assay Kit (R&D Systems, Minneapolis, MN, USA).

NSC‐34 cells were treated on glass coverslips precoated with poly‐d‐lysine/Lamanin (BD Bioscience, San Jose, CA, USA). Cells were fixed in 4% paraformaldehyde for 15 min, 0.1 M glycine for 15 min, and 1% Triton X‐100 for 30 min. Blocking was done in 5% bovine serum albumin in PBS for 30 min. Cells were then incubated with active‐caspase‐3 antibodies (Cell Signaling Technology, Danvers, MA, USA) and FITC‐conjugated secondary antibodies. 4',6‐diamidino‐2‐phenylindole (DAPI) staining was performed and images taken with a Nikon (Tokyo, Japan) Eclipse TE 200 fluorescence microscope and processed, using IP Lab software (Spectra Services, Webster, NY, USA).

Cytosolic and mitochondrial fractionation was performed as described (Wang et al . 2006 , 2007 ; Chi et al . 2007 ). NSC‐34 cells were scraped from dishes using a rubber policeman. The samples were homogenized in a Dounce homogenizer by 10–15 strokes on ice in a homogenization buffer [10 mM HEPES, pH 7.4, 250 mM sucrose, 10 mM KCl, 1.5 mM MgCl 2 , 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol plus protease inhibitor cocktail (Roche)], followed by 700 g centrifugation for 5 min at 4°C; the supernatant was centrifuged at 15 000 g for 25 min at 4°C and used as the cytosolic component. Pellets were lysed with ristocetin‐induced platelet agglutination buffer for 10 min on ice, and supernatants were added to the sample buffer to obtain the membrane fraction containing mitochondria. Antibody to cytochrome c was purchased from PharMingen (San Diego, CA, USA), Smac/Diablo antibody was purchased from Novus Biologicals (Littleton, CO, USA), and antibody to AIF was purchased from Sigma.

NSC‐34 cells were exposed to H2O2 with or without test drugs. Cells were collected in lysis buffer [20 mM Tris, pH 8.0/137 mM NaCl/10% glycerol/1% Nonidet P‐40/2 mM EDTA with 5 mM Na2VO4, protease inhibitor mixture (Roche Molecular Biochemicals)/0.2 mM phenylmethylsulfonyl fluoride] on ice, centrifuged at 19 720 g for 10 min at 4°C, and directly analyzed by western blot. Antibodies to caspase‐3 and ‐9 were purchased from Cell Signaling Technology (Beverly, MA, USA), antibody to caspase‐1 was a gift from Dr Junying Yuan (Harvard Medical School), antibody to β‐actin was from Sigma, and secondary antibodies and enhanced chemiluminescence reagents were from Amersham Pharmacia Biotech, Piscataway, NJ, USA.

Released Lactate dehydrogease (LDH) activity was measured as described previously (Wang et al . 2009 ) and according to the protocol provided by the manufacturer (Roche Molecular Biochemicals, Indianapolis, IN, USA). Briefly, NSC‐34 motoneurons were grown in 96‐well plates, and reaction mixture (100 mL) was added to conditioned media (100 mL) and then removed from 96‐well plates after centrifugation at 250 g for 10 min. Absorbance of samples at 490 nm was measured in an ELISA reader after 60 min of incubation at 20‐26°C. The same volume of medium was used as the background control.

Primary motor neurons were cultured as described previously (Lv et al . 2012 ; Skorupa et al . 2012 ). Briefly, spinal neurons were isolated from spinal cord segments of embryonic day 14 (E14) mice (Charles River Laboratories Inc., Wilmington, MA, USA). The meninges were removed, and the trypsinized cells were suspended in neurobasal medium, supplemented with B27 and glutamine. The neurons were then cultured on plates coated with 100 μg/mL poly‐D‐lysine (Sigma, St Louis, MO, USA) and 8 μg/mL laminin (Sigma) and kept in a cell culture incubator at 37°C with a humidified atmosphere of 5% CO 2 and 95% air. The medium was changed every 3 days. Mixed primary motor neuron cultures were used for experiments on days 6–7 of culture.

NSC‐34 motoneurons were purchased from Cedarlane Laboratories Ltd., Burlington, Ontario, Canada. NSC‐34 motoneurons are a mouse neural hybrid cell line produced by fusion of motor neuron‐enriched, embryonic mouse spinal‐cord neurons and mouse neuroblastoma cells. These cells resemble motor neurons in many ways, e.g., generation of action potentials, expression of neurofilament proteins, and acetylcholine synthesis, storage, and release (Cashman et al . 1992 ). These immortalized motor neuron‐like cells have been widely used as a cellular model of ALS (Weishaupt et al . 2006 ). NSC‐34 motoneurons were cultured in a humidified atmosphere of 95% air/5% CO2 in a 37°C incubator in Dulbecco's modified Eagle's medium supplemented with 10% heat‐inactivated fetal bovine serum and penicillin/streptomycin (Cashman et al . 1992 ; Bishop et al . 1999 ). Cell culture medium was usually replaced every 3–4 days.

Results

N‐acetyl‐ l ‐tryptophan is neuroprotective in NSC‐34 motoneurons and primary motor neurons NAT has been reported to offer neuroprotection in experimental models of neurodegenerative diseases, including Huntington's disease (Wang et al. 2008) and Parkinson's disease (Thornton and Vink 2012) by us and other researchers. To determine on the cellular level whether NAT offers neuroprotection in models of ALS, we evaluated the neuroprotective abilities of three isomers of NAT: L‐NAT, DL‐NAT, and D‐NAT in NSC‐34 motoneurons and primary motor neurons, the proposed cellular models of ALS. We used different concentrations of H2O2 to induce cell death in NSC‐34 motoneurons. A direct measurement of cell death by trypan blue dye exclusion assay shows dose‐dependent NSC‐34 cell death (Fig. 1a). In addition, the MTS assay, a measure of mitochondrial function and thus cell survival, provides confirmation complementary to the trypan blue results in NSC‐34 cells (Fig. 1b). Furthermore, the LDH assay, based on the measurement of lactate dehydrogenase activity, provides a measure of cell death in primary motor neurons. Figure 1 Open in figure viewer PowerPoint H2O2 induces cell death in NSC‐34 motoneurons and primary motor neurons in a dose‐dependent manner, while L‐NAT inhibits cell death. (a, b) NSC‐34 motoneurons were treated with a series of concentrations of H2O2 for 18 h. Cell death was evaluated by either the trypan blue dye exclusion assay (a) or the MTS, inner salt assay (b). C‐H, NSC‐34 cells (c‐e) and primary motor neurons (f, g) were treated in 96‐well plates by exposing them to 50 μM H2O2 for 18 h, either with or without a series of concentrations of L‐NAT (c, g), DL‐NAT (d), or D‐NAT (e, g) varying by three orders of magnitude: 0, 0.001, 0.01, 0.1, 1, 10 nM, 0.1, 1, 10, 30, 200, 300 μΜ. The test drug was preincubated in the medium for 2 h before 50 μM H2O2 treatment. Cell death was evaluated by the MTS (c‐e) and Lactate dehydrogease (LDH) (f, g) assays. The extent of cell death is always normalized relative to what is measured in the absence of both death stimulus and test drugs (white bar). The black bar corresponds to the extent of cell death in response to the respective stress without the test drug. Grey bars correspond to the extent of cell death with stress and the indicated concentration of the test drug. The cell death induced by H2O2 is defined as 100%. The relative percentage of cell death resulting from the incubation of different concentrations of test drug under H2O2 is recorded as relative cell death, which is displayed graphically. The resulting curves (plotted semi‐logarithmically) define the IC 50 and maximum protection of the test drug. Results are the average of three to five experiments, *p < 0.05, **p < 0.01 and ***p < 0.001. The IC 50 , maximum protection, and toxicity of each compound are tabulated (h). The chemical structures of L‐NAT (c), DL‐NAT (d), and D‐NAT (e) are shown. Incubation with either L‐NAT or DL‐NAT at indicated doses resulted in statistically significant inhibition of H2O2‐mediated NSC‐34 motoneuron cell death (Fig. 1c and d). To determine the relative potencies of the two drugs, we measured the extent of cell death as a function of drug concentration. The resulting curves (plotted semi‐logarithmically) define the IC 50 and maximum protection afforded by L‐NAT (0.3 μM and 47%, respectively, Fig. 1c and h) and DL‐NAT (1.0 μM and 22%, respectively, Fig. 1d and h). However, D‐NAT exhibits no significant inhibition of cell death (toxicity ≥ 30 μM, Fig. 1e and h). Our study demonstrates that L‐NAT and DL‐NAT, but not D‐NAT, are neuroprotective in NSC‐34 motoneurons. We next set out to confirm the above findings in primary motor neuron cultures. Indeed, L‐NAT at the indicated doses also resulted in statistically significant prevention of H2O2‐mediated primary motor neuron cell death (IC 50 : 16 nM and maximum protection 70%, respectively, Fig. 1f). But D‐NAT did not significantly inhibit cell death (Fig. 1g). Taken together, our data demonstrate that the L‐NAT, but not its isomer D‐NAT, offers neuroprotection in cellular models of ALS. We further tested the toxicity of L‐NAT in NSC‐34 cells and primary motor neurons. Our study demonstrates that the toxicity of L‐NAT is extremely low in these cultured cells. L‐NAT begins to induce NSC‐34 motoneuronal death in a very high concentration of 10 mM (Fig. 2a) and primary motor neuronal death in a higher concentration of 30 mM (Fig. 2b). Figure 2 Open in figure viewer PowerPoint The toxicities of L‐NAT in NSC‐34 cells and primary motor neurons. NSC‐34 motoneurons (a) and primary motor neurons (b) were treated for 18 h with a series of concentrations of L‐NAT: 0, 0.1, 1, 10, 100, 1000, 10000, 20000, 30000, 50000, 75000. Cell death was evaluated by the MTS assay (a) or the Lactate dehydrogease (LDH) assay (b), respectively. *p < 0.05 and *** p < 0.001.

N‐acetyl‐ l ‐tryptophan offers better neuroprotection than N‐acetyl‐ d ‐tryptophan: structure‐bioactivity relationship To provide additional insight into our findings on the neuroprotective function of L‐NAT, but not D‐NAT, in cellular models of ALS, we extended the study of the structure‐bioactivity relationship using molecular modeling analysis. The finding that L‐NAT, a naturally occurring form, provides biologically neuroprotective function, is strongly supported by structural analyses of the corresponding complexes of L‐NAT and D‐NAT with NK‐1R. We analyzed the docking energy changes for the complexes of L‐NAT and D‐NAT with the receptor NK‐1R, respectively, as shown in Fig. 3. The upper panel of Fig. 3 shows the positions of L‐NAT (left) and D‐NAT (right) when binding with the NK‐1R. The detailed binding environment is enlarged and is shown in the lower panel of Fig. 3 for L‐NAT (left) and D‐NAT (right). The residue ILE 183 was close to the hydrogen bond acceptor scaffold, similar to some of the molecules studied previously (Evers and Klebe 2004). The ring moiety has a π–π stacking effect with HIS176 and TYR251, as well as with HIS244. These interactions are likely the basis of the binding. The major difference between the L‐NAT and D‐NAT complexes is the orientation of the carboxyl groups relating to GLN144. The smaller distance to the carboxyl group of L‐NAT may contribute to stronger binding to the NK‐1R. By comparing the orientation of the carboxyl groups on L‐NAT and D‐NAT, it can be seen from the lower panels in Fig. 3 that interaction differences lie between the carboxyl group of the ligands and the pocket formed by the residues HIS176, GLN144, and ILE183 of the receptor. While the carboxyl group of L‐NAT points into the displaying plane, that of D‐NAT points out of the plane. This varying orientation is the reason for the energy differences when they bind the receptor NK‐1R. Figure 3 Open in figure viewer PowerPoint Molecular modeling and energy minimization analysis of L‐NAT and D‐NAT docking to NK‐1 receptor. The binding position of L‐NAT (upper left) or D‐NAT (upper right) (colored stick model) with NK‐1R (ribbon presentation) are shown. The grey stick models represent the relevant amino acid residues of the binding pocket. The lower panel illustrates the orientation of L‐NAT (left) and D‐NAT (right) (colored stick model) in the binding pocket (grey stick model) of NK‐1R. The binding strength of L‐NAT and D‐NAT to NK‐1R can be evaluated by comparing the total energies of L‐NAT/NK‐1R and D‐NAT/NK‐1R complexes. The final optimized energy of the L‐NAT/NK‐1R complex was found to be 7.95 kcal/mol lower than that of the D‐NAT/NK‐1R complex, suggesting that L‐NAT binds more efficiently with NK‐1R, taking into account the total energy difference for L‐NAT and D‐NAT described above. The modeling results indicate more stable binding between L‐NAT and NK‐1R, suggesting that L‐NAT could be a more potent antagonist of NK‐1R than D‐LAT in inhibiting the binding of SP to the NK‐1R; therefore it would be capable of stronger neuroprotection in ALS, which is consistent with our finding in NSC‐34 cells and primary motor neurons that L‐NAT is a better neuroprotective agent than D‐NAT (Fig. 1). Analysis of chemical structure and activity relationships therefore confirms that L‐NAT, which functions as the best neuroprotective agent in models of ALS, generated the most stable complex with the NK‐1R.

L‐NAT inhibits the secretion of Substance P and IL‐1β and the activation of caspase‐1 To provide further proof of principle for the effectiveness of NAT, we chose naturally occurring and stable L‐NAT for additional in‐depth evaluation. Elevated SP production can directly result in cultured cell death (Castro‐Obregon et al. 2004; Thornton et al. 2010). To test whether SP plays a role in ALS, we evaluated whether exogenous SP induces NSC‐34 motoneuronal death and oxidative stress induces the endogenous secretion of SP in NSC‐34 motoneurons. First, we used different concentrations of SP to induce cell death in NSC‐34 motoneurons. An MTS assay shows dose‐dependent NSC‐34 cell death (Fig. 4a). Second, the presence of SP was detected in NSC‐34 cells exposed to H2O2 by ELISA analysis. In time course experiments, we demonstrated gradually increased release of SP at the protein level at 0 min, 15 min, 2 h, and 6 h (Fig. 4b). Third, to further verify SP expression at the protein level, dot blot analysis conducted with SP antibodies confirmed the presence and increased release of SP in a time‐dependent manner (Fig. 4b). Together the above results provide strong evidence that SP induces NSC‐34 motoneuronal cell death and that SP is released in NSC‐34 motoneuron insulted from oxidative stress. Figure 4 Open in figure viewer PowerPoint L‐NAT prevents the secretion of Substance P, the release of IL‐1β, and the activation of caspase‐1. NSC‐34 cells were treated by 50 μM H2O2 for 15 min, 30 min, and 1, 2, and 6 h in the presence or absence of L‐NAT. The supernatants were collected for MTS assay (a), dot blot analysis (b), or ELISA assay (c). The contents of SP (pg/mL) were normalized as the fold changes compared to the control sample (a and c). ELISA data on SP release were plotted, and the results are the mean ± SD of three‐four independent experiments (c). Cell death was induced by subjecting NSC‐34 motoneurons to 50 μM H2O2 for 18 h with or without 10 μM L‐NAT. Conditioned medium was collected and assayed for mature IL‐1β release (d). *p < 0.05, **p < 0.01 and ***p < 0.001. Alternatively, whole cells were lysed, and the complement of caspase‐1 activity was evaluated by fluorogenic assay (e). The results are the mean ± SD of three independent experiments. Experimental controls include cells treated with neither a cell death stimulus nor test drugs (white bars). Others received a death stimulus but were not treated with L‐NAT (black bars), or were exposed to L‐NAT and subjected to a death stimulus (gray bars). Alternatively, whole‐cell lysates were extracted for western blot with caspase‐1 antibody. Each sample contained 50 μg of protein. β‐actin was used as a loading control (f). Inhibiting the binding of SP to the NK‐1R, using NK‐1R antagonists including L‐NAT has been suggested as a treatment for CNS disorders (Saria 1999). Furthermore, L‐NAT treatment protected organotypic co‐culture and dopaminergic neurons from cell death, reduced neuroinflammation, and significantly improved motor function (Thornton et al. 2010), raising the question of whether SP release can be blocked by L‐NAT in this cellular model of ALS. Indeed, our data shows that the SP antagonist L‐NAT can prevent the secretion of SP in NSC‐34 cells (Fig. 4c). In contrast, one neuroprotective drug (melatonin) not belonging to the family of SP antagonists did not successfully block the release of SP, showing the specificity of L‐NAT's action (Fig. 4c). SP is known to regulate the inflammatory response and inflammation that play an important role in ALS. There is evidence for elevated levels of proinflammatory cytokine IL‐1β in transgenic mouse and rat models of ALS (Monk and Shaw 2006), and in NSC‐34 motoneurons treated with apoptotic insult (Li et al. 2007). IL‐1β induces the release of SP from primary neuronal cells (Guegan et al. 2001). In this study, we found that not only the release of SP (Fig. 4a), but also the release of IL‐1β and activation of caspase‐1 in NSC‐34 motoneurons, were challenged by oxidative stress (Fig. 4d–f). Caspase‐1 is the key mediator of IL‐1β processing to release mature IL‐1β, while the NK‐1R antagonist L‐NAT has been reported to markedly attenuate the withdrawal latency of inflamed rat paws (Gao et al. 2010). IL‐1β also interacts with NK‐1R agonists in mediating neutrophil accumulation during inflammatory disease (Pinter et al. 2002). We evaluated whether L‐NAT effectively inhibits the processing of IL‐1β and the activation of caspase‐1 in NSC‐34 cells. Indeed, along with the inhibition of SP secretion, administration of L‐NAT effectively prevents the secretion of IL‐1β (Fig. 4d), the activation of caspase‐1 (Fig. 4e), the cleavage of caspase‐1 (Fig. 4f), and cell death (Fig. 4c and h) in NSC‐34 cells exposed to H2O2. We speculate that H2O2 treatment induces not only the release of SP that mediates NSC‐34 cell death, but also the release of IL‐1β that may further cause the secretion of SP, which may mediate a feedback signal to stimulate extra secretion of IL‐1β and the activation of caspase‐1 to induce the program of whole cell death. Thus, these results demonstrate that L‐NAT prevents SP secretion, IL‐1β processing, and caspase‐1 activation. L‐NAT may be effective agent in the treatment of ALS.

Rescue of mitochondrial collapse and apoptotic pathways by L‐NAT Having established that L‐NAT inhibits SP and IL‐1β release and caspase‐1 activation in NSC‐34 motoneurons, the question that arises as to how L‐NAT exerts its neuroprotective effects. Higher concentrations of SP can interrupt mitochondrial oxygen consumption (Del Signore et al. 2009), suggesting that mitochondrial dysfunction may be at least partly due to elevated levels of SP under pathological conditions. It is well known that damaged mitochondria trigger mitochondria‐dependent apoptosis and inappropriate activation of apoptosis, including the activation of caspase‐1 in motor neurons, drives the progression of ALS, and SP has been shown to aggravate cell death (Fig. 4a) and apoptotic signals (Bang et al. 2004; Peters et al. 2005). Given that L‐NAT was determined through a mitochondrial assay to inhibit the activation of caspase‐1 in cultured NSC‐34 cells, we evaluated whether L‐NAT exerts its neuroprotective properties through inhibiting mitochondial apoptotic pathways. We investigated the ability of L‐NAT to prevent the release of cytochrome c and two other apoptogenic mitochondrial factors (Smac and AIF). As demonstrated through western blotting, cytochrome c/Smac/AIF was released from the mitochondria to the cytosolic fraction of NSC‐34 cells exposed to H2O2 (Fig. 5a). Furthermore, L‐NAT indeed effectively inhibited the release of cytochrome c/Smac/AIF from mitochondria into the cytoplasm (Fig. 5a). Figure 5 Open in figure viewer PowerPoint L‐NAT forestalls the release of mitochondrial apoptogenic factors, slows dissipation of the mitochondrial potential gradient, and prevents the activation of caspase–3/‐9. Cell death was induced in NSC‐34 motoneurons by subjecting them to 50 μM H2O2 with or without 10 μM L‐NAT for 2 h (a) or 6 h (b), or 18 h (c–e). Subsequently, cells were either extracted and cytosolic and mitochondrial components (a) or whole lysate (c) were obtained or stained with Rh 123 (b), or immunostained with active‐caspase‐3 antibody (d). NSC‐34 cells were immunostained with active‐caspase‐3 antibody and FITC‐conjugated secondary antibody and then stained with DAPI (d). The samples, each of which contained 50 μg of protein, were analyzed by western blot using antibodies to cytochrome c, AIF, Smac, caspase‐9, or ‐3. β‐actin was used as loading control for whole cells or cytosolic components, while COX IV was used as loading control for mitochondrial components (a). The arrows indicate the dissipation of ΔΨm (b). Densitometry was performed to quantify the intensity of the bands from three independent experiments (c, right panels). Cells were lysed and the complement of caspase‐9 and ‐3 activities was evaluated by a fluorogenic assay (e). *p < 0.05, **p < 0.01. The collapse of the mitochondrial transmembrane potential (ΔΨ m ) is a central event in neuronal cell death in models of neurodegeneration (Jordan et al. 2003; Wang et al. 2003, 2007). Consequently, we stained living NSC‐34 cells with rhodamine 123 (Rh 123) to determine the electrostatic charge of the mitochondria exposed to H2O2 in the presence or absence of L‐NAT (Fig. 5b). Staining showed that the dissipation of ΔΨ m strongly correlates with increased cell death of NSC‐34 cells exposed to H2O2; while L‐NAT inhibits the collapse of ΔΨ m (Fig. 5b). L‐NAT treatment preserved the punctate distribution of Rh 123 fluorescence in the presence of cellular stress (arrows in Fig. 5b), a property that correlates with the ability of compounds to reduce the extent of cell death. We propose that L‐NAT's mechanism of action is its ability to target mitochondria, resulting in inhibition of cytochrome c/AIF/Smac release and preservation of ΔΨ m . Cytochrome c is a molecule that facilitates self‐assembly of a complex of caspase‐9 dimers upon a scaffold of Apaf‐1 molecules, resulting in caspase‐9 activation. Caspase‐9 in turn activates caspase‐3, which plays a critical role as a cell death effector (Reed 2000). To further investigate the mechanism of action of L‐NAT, in parallel studies in NSC‐34 motoneurons, we tested whether L‐NAT also inhibits the proteolysis of procaspases‐9 and ‐3 to active enzymes. Indeed, caspase‐9 and ‐3 were activated in NSC‐34 motoneurons following H2O2 treatment as determined by western blots (Fig. 5c), fluorogenic assay (Fig. 5e), and immunocytochemistry for active caspase‐3 (Fig. 5d). Furthermore, L‐NAT inhibits procaspase‐9 and ‐3 activation in all of these assays (Fig. 5c–e). Our data demonstrated that L‐NAT inhibits both caspase‐dependent (cytochrome c, Smac, caspase‐9, and caspase‐3) and caspase‐independent (AIF) mitochondrial cell death pathways in cellular models of ALS.