J. Neurochem. (2010) 114, 291–301.
In the present study, we evaluated the therapeutic efficacy of acetyl-l-carnitine (ALC) administration on mitochondrial dysfunction following tenth thoracic level contusion spinal cord injury (SCI) in rats. Initial results from experiments in vitro with naïve mitochondria showed that, in the absence of pyruvate, ALC can be used as an alternative substrate for mitochondrial respiration. Additionally, when added in vitro to mitochondria isolated from 24 h injured cords, ALC restored respiration rates to normal levels. For administration studies in vivo, injured rats were given i.p. injections of saline (vehicle) or ALC (300 mg/kg) at 15, 30 or 60 min post-injury, followed by one booster after 6 h. Mitochondria were isolated 24 h post-injury and assessed for respiration rates, activities of NADH dehydrogenase, cytochrome c oxidase and pyruvate dehydrogenase. SCI significantly (p < 0.05) decreased respiration rates and activities of all enzyme complexes, but ALC treatment significantly (p < 0.05) maintained mitochondrial respiration and enzyme activities compared with vehicle treatment. Critically, ALC administration in vivo at 15 min and 6 h post-injury versus vehicle, followed once daily for 7 days, significantly (p < 0.05) spared gray matter. In summary, ALC treatment maintains mitochondrial bioenergetics following contusion SCI and, thus, holds great potential as a neuroprotective therapy for acute SCI.
pyruvate dehydrogenase complex
respiratory control ratio
reactive oxygen species
spinal cord injury
Approximately 262 000 Americans are living with the typically devastating neurological deficits that are secondary to spinal cord injury (SCI) (see https://www.nscisc.uab.edu). Most injuries do not involve actual physical transection of the spinal cord, but rather blunt trauma because of contusion, compression or stretch injuries (see reviews; Hall and Springer 2004; Rabchevsky and Smith 2001). Much of the spinal tissue degeneration that occurs following these types of SCI is as a result of secondary injury processes that are triggered by the primary mechanical trauma (see reviews; Hall and Springer 2004; Rabchevsky and Smith 2001). Mitochondria serve as the powerhouse of the cell by maintaining ratios of ATP:ADP that thermodynamically favor the hydrolysis of ATP to ADP + Pi; yet a byproduct of this process is the generation of reactive oxygen species (ROS). In fact, mitochondria have been shown to play a key role in the ensuing neuronal death cascade (Stout et al. 1998), and mitochondrial dysfunction and oxidative stress have been directly linked to increased excitotoxicity following SCI (Luo et al. 2004; Sullivan et al. 2005; McEwen et al. 2007). Accordingly, it is our working hypothesis that maintaining mitochondrial homeostasis and bioenergetics is crucial to promoting cell survival following SCI. This is based on our documentation of the progressive nature of mitochondrial dysfunction over 24 h following contusion SCI (Sullivan et al. 2007), and that pharmacological interventions which mitigate impaired mitochondrial bioenergetics lead to significantly reduced ROS production and promote neuroprotection (Patel et al. 2009).
Acetyl-l-carnitine (ALC) is a constituent of the inner mitochondrial membrane that contains acetyl and carnitine moieties, and is an ester of the trimethylated amino acid, L-carnitine that is synthesized in the human brain, liver, and kidneys by the enzyme ALC transferase (Anonymous 1999). Studies have shown that ALC readily crosses the blood-brain barrier and undergoes limited metabolism and is subsequently excreted in the urine via renal tubular reabsorption (Parnetti et al. 1992). Because of multiple effects of ALC, it is used clinically for age-related neurodegenerative conditions such as Alzheimer’s dementia, memory-related problems, depression, age-related disorders, diabetic neuropathy/cataracts, and in cerebral ischemia and reperfusion (Bonavita 1986; Tempesta et al. 1987; Rai et al. 1990; Spagnoli et al. 1991; Sano et al. 1992; Lowitt et al. 1995; Onofrj et al. 1995; Swamy-Mruthinti and Carter 1999). ALC has many neuromodulatory and neurotrophic actions, which include facilitating the uptake of acetyl CoA into the mitochondria during fatty acid oxidation, enhancing acetylcholine production, and stimulating protein and phospholipid synthesis required for membrane formation and integrity (see review; Pettegrew et al. 2000).
Studies in rats have shown that chronic ALC treatment increases life-span, improves cognitive behavior in aged animals and improves long-term memory performance; one of the suggested mechanism(s) of action of ALC is by improving mitochondrial bioenergetics which allows neurons to produce ATP necessary to maintain normal membrane potential (Ghirardi et al. 1989; Barnes et al. 1990; Markowska et al. 1990; Carta and Calvani 1991; McDaniel et al. 2003). In addition, ALC also reported to play a role in partial prevention of overoxidation and/or accumulation of the overoxidized form of specific liver mitochondrial enzymes from aged rats; however the mechanism is still uncertain (Musicco et al. 2009). Treatment with ALC has also shown beneficial therapeutic effects for a variety of chronic neurological diseases (Puca et al. 1990; Sima et al. 1996; Pettegrew et al. 2000; Chiechio et al. 2002; Tomassini et al. 2004; Calabrese et al. 2005; Traina et al. 2006). Moreover, because endogenous ALC contributes to the bioenergetic processes, it plays a pivotal role in diseases correlated with metabolic compromise, such as mitochondrial-related disorders (Pettegrew et al. 2000; Dhitavat et al. 2002; Virmani and Binienda 2004; Di Cesare Mannelli et al. 2007, 2009). In addition to acting as an acetyl-CoA precursor, carbon from the acetyl group of ALC is also used to produce the antioxidant GSH; thereby reducing oxidative damage and protecting cells against lipid peroxidation (Aureli et al. 1999). Reports have also shown that ALC regulates sphingomyelin levels and provides the essential substrate pools for mitochondrial energy production, thus stabilizing cell membrane fluidity and preventing excessive neuronal cell death in aging humans (Anonymous 1999).
In the present study, we specifically targeted mitochondrial dysfunction following contusion SCI by administering ALC in vivo to evaluate its neuroprotective efficacy. Our hypothesis was that providing such a compound which serves as a biofuel for mitochondria as well as promotes antioxidant systems will preserve their bioenergetics and foster neuroprotection following SCI. To determine the effects of ALC administration at various time points post-injury, we assessed total mitochondrial bioenergetics (mix of synaptic and non-synaptic populations) in terms of respiratory control ratio (RCR), respiration rates, and activities of key mitochondrial enzyme complexes from acutely injured spinal cords, with and without ALC treatment at 24 h post-injury. Moreover, we tested whether prolonged, daily ALC treatment increased spinal cord tissue sparing at 1 week post-injury.
Materials and methods
Spinal cord injury and treatments
Female Sprague-Dawley rats (n = 132) (Harlan Labs, Indianapolis, IN, USA) weighing 200–250 g were housed in the animal facility, Biomedical and Biological Sciences Research Building, University of Kentucky and allowed ad libitum access to water and food. All animal housing conditions, surgical procedures, and post-operative care techniques were conducted according to the University of Kentucky Institutional Animal Care and Use Committee and the National Institutes of Health animal care guidelines. Prior to surgeries, rats were anesthetized with ketamine (80 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) and hair on dorsal surface was shaven and skin disinfected. A dorsal laminectomy was performed at the tenth thoracic level to expose the spinal cord. Contusions (200 kdyn) were performed with the Infinite Horizon impactor (PSI, LLC, Lexington, KY, USA), as previously described (Sullivan et al. 2007; Patel et al. 2009). Injured rats demonstrated total hind limb paralysis 24 h following contusion injuries. Sham rats received a dorsal laminectomy only and had normal hind limb locomotion post-surgery. Injured rats were treated with either vehicle (saline) or 300 mg/kg ALC (Sigma, St Louis, MO, USA) (i.p.), at 15 min, 30 min or 1 h after contusion SCI. A booster injection of ALC was given at 6 h post-SCI.
In preliminary experiments for in vivo studies, we conducted a ALC dose-response study using 100, 200 or 300 mg/kg body weight bolus injections based on previously published reports (Petruzzella et al. 1992; Paradies et al. 1999). We found marginally improved mitochondrial bioenergetics with 100 mg/kg and significantly improved bioenergetics with both 200 and 300 mg/kg dosages. As the higher dosages had almost identical beneficial effects on mitochondrial bioenergetics 24 h following SCI, we continued employing the highest dosage to get maximum bioenergetic effects without producing overt negative side effects. Moreover, this dosage (300 mg/kg, i.p.) has been reported to improve the bioenergetics of cerebral and cardiac mitochondria isolated from aged compared with young rats (Petruzzella et al. 1992; Paradies et al. 1999). Importantly, these rats were killed 3 h after ALC treatment, demonstrating a rapid beneficial effect of this dosage.
For the tissue sparing experiments, injured rats (n = 6 per group) received i.p. injection of vehicle or 300 mg/kg ALC 15 min post-SCI and a booster after 6 h. In addition, rats received i.p. injection of either vehicle or 300 mg/kg ALC once daily up to 7 days post-SCI. At 24 h after the last injection of vehicle or ALC, the rats were euthanatized and perfused to isolate spinal cord tissue for histology.
At 24 h following sham operation or injury with drug treatment, rats were killed with CO2 and decapitated for isolation and characterization of mitochondria, as described previously (Jin et al. 2004; Sullivan et al. 2007). The spinal cords were rapidly removed and placed on an ice cold dissecting plate containing isolation buffer with 1 mM EGTA (215 mM mannitol, 75 mM sucrose, 0.1% bovine serum albumin, 20 mM HEPES, 1 mM EGTA and pH is adjusted to 7.2 with KOH). Based on pilot studies, mitochondria from two spinal cords were pooled in 350 μL of isolation buffer containing EGTA to augment protein concentration for reliable mitochondrial respiration during experiments. Each spinal cord was dissected into 2 cm segments centered on the injury site (within the sight of laminectomy) and homogenized in 2 mL of ice cold isolation buffer with EGTA. The homogenate was then centrifuged twice at 1300 g for 3 min at 4°C and the resulting supernatant was removed and centrifuged at 13 000 g for 10 min at 4°C. The resuspended mitochondrial/synaptosomal pellets were burst in a nitrogen cell disruption chamber (1200 psi, 10 min) that was cooled to 4°C. The resulting crude fractions were then placed atop a discontinuous Ficoll gradient (7.5%/10%) and centrifuged at 100 000 g for 30 min at 4°C. The sedimented mitochondrial pellet was resuspended in isolation buffer without EGTA and centrifuged for 10 min at 10 000 g. The final mitochondrial pellet was resuspended in EGTA-free isolation buffer at a concentration of ∼ 10 mg/mL and stored on ice until further use. The protein concentration was determined using the BCA protein assay kit (Fisher Scientific, Fair Lawn, NJ, USA) by measuring absorbance at 560 nm with a Biotek Synergy HT plate reader (Winooski, VT, USA).
Assessment of ALC effects in vitro
The mitochondria were isolated 24 h after either sham operation or contusion injury. To determine whether ALC can be used as alternative substrate for mitochondrial respiration (ATP production), 5 mM ALC (final concentration) was added, in the absence of pyruvate, to the respiration chamber containing naïve spinal cord mitochondria and respiration buffer containing 2.5 mM malate. Subsequently, other substrates and specific complex inhibitors were added, as described below. On the contrary, to assess the effects of ALC on either sham or injured spinal cord mitochondria in vitro, 5 mM ALC (final concentration) was added to the respiration chamber immediately after pyruvate + malate; thereafter, mitochondrial respiration was carried out as described above. The mitochondrial respiration rates were calculated as nmols oxygen/min/mg protein. For these in vitro studies, we first characterized naïve spinal cord mitochondria and found that dosages below 5 mM ALC were ineffective for mitochondrial respiration whereas dosages above 5 mM ALC did not have added beneficial effects. Therefore, we conducted all subsequent in vitro studies with mitochondria isolated from sham and injured spinal cords using a final dosage of 5 mM ALC.
Measurement of mitochondrial function
Mitochondrial respiration was assessed using a miniature Clark-type electrode (Hansatech Instruments, Norfolk, UK) in a sealed, thermostatically controlled chamber at 37°C as described previously (Sullivan et al. 2003; Patel et al. 2009). Mitochondria were added to the chamber to yield a final protein concentration of ∼ 200–300 μg/mL respiration buffer (125 mM KCl, 2 mM MgCl2, 2.5 mM KH2PO4, 20 mM HEPES and 0.1% bovine serum albumin, pH 7.2). Respiration was initiated by the addition of oxidative substrates pyruvate (5 mM) and malate (2.5 mM) which is designated as State II respiration. This was followed by the addition of 120 nmol ADP (State III respiration) and the addition of 1 μM oligomycin to induce State IV respiration. The mitochondrial uncoupler carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (1 μM) was added to the chamber to asses NADH dehydrogenase (complex I) driven maximum electron transport, which is designated as State V-Complex I respiration. The complex I inhibitor rotenone (0.8 μM) was then added to the chamber followed by the addition of 10 mM succinate to allow succinate dehydrogenase (complex II) driven maximum electron transport, which is designated as State V-Complex II respiration. The mitochondrial respiration rates were calculated as nmols oxygen/min/mg protein. As detailed previously (Sullivan et al. 2003; Patel et al. 2009), the RCR was calculated by dividing the slope of the response of isolated mitochondria to State III respiration (presence of ADP) by the slope of the response to State IV respiration (presence of 1 μM oligomycin and absence of ADP).
Activities of mitochondrial complexes
The NADH dehydrogenase (complex I) and cytochrome c oxidase (complex IV) assays were performed as described with modifications (Smith 1955; Patel et al. 2009). Measurement of total pyruvate dehydrogenase complex (PDHC) activity was carried out as described with modification (Starkov et al. 2004; Opii et al. 2007). In summary, mitochondria were freeze thawed and briefly sonicated for three cycles in 10 mM phosphate buffer (pH 7.4). Six microgram of isolated mitochondrial protein was added into the buffer containing a final concentration of 50 mM KCl, 10 mM HEPES, pH 7.4, 0.3 mM thiamine pyrophosphate, 10 μM CaCl2, 0.2 mM MgCl2, 5 mM pyruvate, 1 μM rotenone and 0.2 mM NAD+. Reaction was started by addition of 0.14 mM CoA and the assay was performed at Ex λ 340 nm/Em λ 460 nm. Increases in NADH fluorescence were observed at 1-min intervals at 30°C using BioTek Synergy HT plate reader (Winooski). The total PDHC activity units were expressed as nmol/min/mg protein.
Spinal cord tissue processing for histology
Rats were overdosed with sodium pentobarbital and transcardially perfused with 0.1 M phosphate-buffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde in PBS. To maintain consistent sampling, each spinal cord was transected at the rostral T6 spinal root and a 30 mm segment of spinal cord (∼ L1) was immediately dissected and post-fixed for 2 h at 4°C before cryoprotecting in 20% sucrose/PBS at 4°C and embedding as described (Rabchevsky and Smith 2001). Frozen spinal cords were then serially cryosectioned at 20 μm and processed for stereological assessment of tissue sparing after staining with eriochrome cyanine (Sigma) for myelin followed by cresyl violet (Sigma) for cell bodies before rinsing (Rabchevsky et al. 2001). Stained sections were maintained in Citrisolv (Fisher Scientific, Fair Lawn, NJ, USA) to clear excess cresyl violet and subsequently coverslipped with Permount (Fisher Scientific) mounting medium.
Quantification of lesion volumes and tissue sparing
Quantitative image analysis was performed on each series of eriochrome cyanine/cresyl violet-stained cross-sections using Scion Imaging software (Scion Corporation, Frederick, MD, USA) on a Nikon Eclipse 400 microscope mounted with a DAGE-MTI CCD-100 camera (Dage-MTI, Inc, Michigan City, IN, USA). Histological analyses were assessed blindly with respect to treatment. Spared tissue was based on positive staining for myelin or if the gray matter cytoarchitecture approximated that seen in uninjured tissue. Employing the Cavalieri method (Michel and Cruz-Orive 1988), the cross-sectional area containing necrotic or damaged tissue was carefully circumscribed and the entire volume of injured tissue calculated from a series of 12 evenly spaced (1 mm) sections centered on the injury site. Similarly, cross-sectional area measurements of spared gray matter and white matter were each quantified separately to calculate their respective volumes in the injured spinal cords (Rabchevsky et al. 2001).
Representative sections were photographed with an Olympus ‘Magnafire’ digital camera mounted on an Olympus BX51 microscope (Olympus Corp., Melville, NY, USA). All sections were photographed with the same objective magnification (× 4; × 10 eye piece) and with the same exposure settings. Photomicrographs were optimized for final production by adjusting only the brightness and contrast using Adobe Photoshop 7.0 (Adobe Systems Inc., San Jose, CA, USA). All graphs were created with DeltaGraph 5.4 (Red Rock Software, Inc., Salt Lake City, UT, USA).
For both in vitro and in vivo experiments, differences among sham, injured vehicle-treated, and injured ALC-treated groups at the various time-points of administration post-injury were investigated using anova and the Newman–Keuls post hoc when warranted. Significance was set at p < 0.05 for all analyses.
In vitro studies
To investigate the potential role of ALC as an alternative substrate for mitochondrial energy production, we replaced pyruvate with ALC and measured mitochondrial respiration (oxygen consumption). As shown in Fig. 1, in the presence of ALC (absence of pyruvate) mitochondrial respiration was comparable to that seen in the presence of pyruvate, illustrating that ALC can be utilized for mitochondrial ATP synthesis. Next, we designed experiments in vitro to assess the effects of ALC addition on respiration rates of mitochondria isolated from contused spinal cords versus shams. For these experiments, after addition of pyruvate + malate to the respiration buffer, we added 5 mM ALC (final concentration) to the respiration chamber containing either mitochondria from sham or injured spinal cords. Respiration rates were calculated in terms of nmols oxygen/min/mg protein and are shown in Fig. 2. Typical respiration traces for mitochondria isolated from sham and injured spinal cords (with and without ALC addition) are shown in Fig. 2a. Significantly compromised mitochondrial respiration was observed 24 h after contusion SCI compared with shams (Fig. 2b), as we previously reported (Sullivan et al. 2007). However, in the presence of ALC, State III and State V-Complex I respiration rates in mitochondria isolated from injured spinal cords increased significantly compared with respiration without ALC addition (25.21% and 25.47%, respectively). Addition of ALC to sham mitochondria resulted in no significant changes in State III or State V-Complex I respiration rates. Interestingly, compared with sham mitochondria without ALC, State III respiration rates were not significantly different from injured mitochondria with ALC addition (Fig. 2b). This indicates that ALC addition to injured mitochondria in vitro increased respiration rates to near sham levels. However, State V-Complex I respiration rates in injured mitochondria, with or without ALC addition in vitro, were significantly compromised compared with shams without ALC (Fig. 2b).
In vivo studies
In an alternative approach, we assessed the protective efficacy of ALC administration in vivo on mitochondrial bioenergetics following acute SCI. ALC or saline was injected (i.p.) at 15 min, 30 min or 1 h post-injury versus sham operation. Mitochondria were isolated 24 h after injury or sham operation and subjected to assessments of mitochondrial bioenergetics in terms of respiration rates and activities of key mitochondria enzyme complexes. Mitochondrial bioenergetics was assessed by measuring oxygen consumption of isolated mitochondria under various experimental conditions at 24 h post-injury. Typical respiration traces for mitochondria isolated from sham rats versus treatments at 15 min post-injury with either saline (vehicle) or ALC are shown in Fig. 3. Compromised mitochondrial respiratory activity was observed in vehicle-treated injured rats compared with shams (Fig. 3). Treatment with ALC after 15 min (Fig. 3), 30 min and 1 h (traces not shown) significantly maintained mitochondrial respiration rates at near normal levels. The results showed that for all mitochondrial parameters examined at all three time points of administration, vehicle-treated injured groups were not significantly different. Data from vehicle-treated injured groups did not show significant variation over time post-injection; therefore we used two animals per injection time point to establish a single vehicle-treated injured group (Figs 4 and 5). The RCR indicates how well the electron transport system is coupled to ATP synthesis, an important measure of mitochondrial function/integrity. Compared with shams, there were significant reductions in the RCR for all vehicle-treated injured groups [F(4,22) = 21.558, p < 0.0001] at 15 min, 30 min and 1 h post-injury (Fig. 4a). In contrast, ALC administration at 15 min, 30 min and 1 h post-injury significantly improved mitochondrial function compared with vehicle-treated injured group (Fig. 4a), although the RCR values remained significantly lower compared with shams. No significant differences in state IV respiration were measured, indicating that the changes in RCR values observed were due entirely to alterations in state III respiration rates and not changes in proton conductance across the inner membrane (State IV respiration).
Quantification of mitochondrial respiration rates showed a significant decrease in state III [F(4, 22) = 23.704, p <0.0001] and State V-Complex I [F(4,22) = 20.035, p < 0.0001)] respiration rates in all injured groups compared with shams (Fig. 4b). Post hoc analysis showed a significant decrease in both State III and State V-Complex I respiration in vehicle-treated injured spinal cord mitochondria compared with shams. Conversely, treatment with ALC at 15 min, 30 min and 1 h post-injury significantly maintained State III and State V-Complex I respiration rates compared with vehicle-treated injured rats (Fig. 4b). However, the values remained significantly lower than shams. State V-Complex II respiration rates were not statistically different across the groups (Fig. 4b), as reported previously (Sullivan et al. 2007).
In vehicle-treated rats, direct measurement of mitochondrial electron transport system complex enzymatic activities at 24 h after injury revealed significant reductions in complex I [F(4,11) = 6.737, p < 0.005], complex IV [F(4,11) = 9.341, p < 0.001] and PDHC activities [F(4,11) = 31.404, p < 0.0001] compared with sham rats (Fig. 5). However, ALC administered 15 and 30 min after injury significantly maintained complex I, IV and total PDHC activities compared with vehicle-treated injured rats (Fig. 5).
Effect of ALC treatment on spinal cord tissue sparing
Qualitative evaluation of the histopathology seen throughout spinal cord cross-sections from ALC – versus vehicle-treated injured rats (Fig. 6) demonstrated more sparing of tissue with ALC treatment, notably the gray matter both rostral and caudal of the injury site at 1 week post-SCI. Compared with the vehicle-treated injured group, daily ALC administration significantly increased tissue sparing 7 days post-injury (Fig. 7). Results showed significantly greater lesion volumes in the vehicle-treated group compared with the ALC-treated group (Fig. 7a). Moreover, ALC treatment resulted in significantly increased gray matter sparing, along with marginally increased white matter sparing (Fig. 7b). Additionally, the ALC-treated group showed significant increases in tissue sparing at the injury epicenter compared with the vehicle-treated group (Fig. 7c and d).
While the present study confirms that mitochondrial dysfunction plays a key role in the development of secondary pathophysiology following contusion SCI (Sullivan et al. 2007; Patel et al. 2009), this is the first demonstration of the therapeutic efficacy of ALC treatment to stabilize mitochondrial bioenergetics and, in turn, significantly spare injured spinal cord tissue. Despite various experimental approaches to promote neuroprotection following SCI (see review; Hall and Springer 2004; Onose et al. 2009), the only compound reported to show modest beneficial effects following acute SCI in human clinical trials is methylprednisolone (Bracken et al. 1997), which is reported to act by inhibiting post-traumatic lipid peroxidation (see review; Hall and Springer 2004). The significance of the current study is that our novel therapeutic strategy targeted the main source of free radical production, the mitochondria. Based on our recent report that mild uncoupling of mitochondria after contusion SCI significantly reduced oxidative damage (Patel et al. 2009), the current findings indicate that supplementing an alternative biofuel, ALC, resulted in improved bioenergetics and increased tissue sparing, likely because of reduced secondary oxidative damage.
Collectively, our data signify that mitochondrial dysfunction is a pivotal target for pharmacological interventions to promote neuroprotection following acute contusion SCI. Our novel findings in a SCI model are supported by previous reports that ALC treatment prevents or reduces oxidative damage, and improves mitochondrial function in different models of aging, age-related diseases and chemically-induced oxidative stress. For example, similar to the current findings, long-term ALC feeding to aged rats partially or completely ameliorates activities of complexes I, IV and V in brain mitochondria and oxidative damage to levels seen in young rats (Long et al. 2009). Also, pre-treatment with ALC for 2 weeks has demonstrated neuroprotection against 3,4-methylenedioximethamphetamine-induced neurotoxicity in rat brain mitochondria by improving the expression of the electron transport system components, decreasing mtDNA deletion, and reducing carbonyl formation (Alves et al. 2009). ALC additionally prevents beta-amyloid (Abeta)-induced neuronal cell death by reducing ROS production and ATP depletion in differentiated SH-SY-5Y human neuroblastoma cells (Dhitavat et al. 2002). Finally, in a chronic rotenone-induced cellular model of Parkinson’s disease, 4 weeks pre-treatment with ALC effectively protected SK-N-MC human neuroblastoma cells against mitochondrial dysfunction, oxidative damage (Zhang et al. 2010). Notably, the underlying mechanism is thought to be up-regulation of the peroxisome proliferators-activated receptor gamma co-activator 1α, which is a key regulator of mitochondrial biogenesis and respiration.
Earlier studies have reported that ALC can act as a substrate for efficient mitochondrial respiration in other tissues/organ systems (Clark and Nicklas 1970; Nicklas et al. 1971; Storey 1980). The results from this study show that ALC can, in fact, be used as an alternative substrate for normal respiration of naïve spinal cord mitochondria to produce energy. We subsequently found that addition of ALC in vitro significantly improved oxygen consumption of mitochondria isolated from acutely injured spinal cords. These data indicate that mitochondria in the injured spinal cord can successfully utilize an alternative substrate/biofuel such as ALC to maintain ATP levels following SCI. Remarkably, the administration of ALC in vivo up to 1 h post-SCI also significantly maintained mitochondrial bioenergetics at 24 h post-injury. Moreover, prolonged ALC treatment in vivo (7 days) significantly increased spinal cord tissue sparing following contusion SCI, supported by previous report in rats that extended ALC treatment improves mitochondrial function and reduces oxidative damage in aging (Long et al. 2009).
Consistent with our previous studies characterizing total mitochondria (combined synaptic and non-synaptic populations) (Sullivan et al. 2007), as well as synaptic and non-synaptic mitochondria from injured spinal cords (Patel et al. 2009), we found total mitochondria from vehicle-treated injured spinal cords demonstrated compromised mitochondrial bioenergetics at 24 h post-injury. Importantly, ALC administration up to 1 h post-injury significantly maintained mitochondrial bioenergetics in terms of respiration rates and mitochondrial enzyme activities; notably, NADH dehydrogenase (complex I), cytochrome c oxidase (complex IV) and PDHC, all of which are highly susceptible to oxidative damage (Fiskum et al. 2004; Martin et al. 2005; Opii et al. 2007; Vazquez-Memije et al. 2008; Alves et al. 2009; Roy Chowdhury et al. 2009; Wei et al. 2009). Consistent with our previous reports, State III and State V-Complex I respiration rates and NADH dehydrogenase activity were compromised while State V–Complex II respiration rates and activity of succinate dehydrogenase were unchanged following SCI (Sullivan et al. 2007; Patel et al. 2009). This indicates, therefore, that the injury-induced loss of mitochondrial bioenergetics is most likely because of compromised NADH-linked respiration and/or complex I driven electron transport. However, this finding coupled with our data demonstrating that ALC can restore state III respiration in mitochondria from the injured spinal cord seems to indicate a pivotal role for PDHC dysfunction in SCI pathophysiology. The same enzymatic machinery is used by pyruvate and ALC to produce reducing equivalents for complex I driven electron transport, with the notable exception of PDHC. Our finding that in vivo administration of ALC ameliorates SCI-induced reductions in mitochondrial enzyme activities is supported by a previous report that ALC increases endogenous antioxidant GSH levels, thereby protecting key mitochondrial enzymes (complex I, IV and PDHC) from oxidative damage (Aureli et al. 1999).
Therefore, to further establish the role of PDHC in the pathophysiology of SCI, we directly assessed its activity, along with those of Complex I and IV, all of which have been shown to be susceptible to oxidative damage. To our knowledge, this is the first study to demonstrate decreased PDHC activity in mitochondria isolated from contused spinal cords. PDHC catalyzes the conversion of pyruvate to acetyle-CoA, NADH and CO2 (Reed 1981, 2001). Acetyl CoA enters the citric acid cycle and forms NADH which are oxidized in the mitochondrial respiratory electron transfer chain and donate electrons for ATP synthesis; thus making this enzyme the key link between glycolysis and the tricarboxylic acid cycle (Zaidan et al. 1998; Martin et al. 2005). Thus, compromised PDHC activity could lead to a decline in mitochondrial respiration, notably because of reduced NADH levels. In contrast, production of acetyl-CoA is markedly increased by ALC which, in turn, increases the pool of reducing equivalent NADH (see review; Pettegrew et al. 2000). This indicates that ALC supplementation can specifically override injury-induced dysfunction of the critical enzyme, PDHC. In fact, this is what we demonstrated in our in vitro studies showing that ALC addition restores state III respiration in mitochondria isolated 24 h following SCI. Importantly, the inactivation and dysfunction of PDHC leads to metabolic failure and increased oxidative stress in several neurodegenerative disorders and CNS injury models (Sorbi et al. 1983; Sheu et al. 1985; De Meirleir et al. 1993; Zaidan et al. 1998; Bogaert et al. 2000; Pocernich and Butterfield 2003; Rosenthal and Henderson 2003; Richards et al. 2006; Opii et al. 2007; Robertson et al. 2007).
It has been reported in different injury models and abnormal conditions that oxidative stress at the cellular level results in structural modifications which, in turn, render inactivation/dysfunction of several important mitochondrial proteins (Bogaert et al. 2000; Fiskum et al. 2004; Martin et al. 2005; Opii et al. 2007; Long et al. 2009). Few studies have targeted mitochondrial dysfunction following contusion SCI using different pharmacological agents, such as mitochondrial uncouplers and/or anti-oxidants (McEwen et al. 2007; Patel et al. 2009). The current study is the first to demonstrate that the use of an alternative biofuel for mitochondria, ALC, maintains their bioenergetic homeostasis and, consequently, affords neuroprotection. Notably, intermittent, daily ALC treatments significantly increased gray matter tissue sparing and marginally spared white matter. We hypothesize that such neuroprotection directly stemmed from the maintenance of mitochondrial bioenergetics with ALC treatment, and thereby reducing oxidative damage.
To further elucidate the possible mechanism(s) of the beneficial effects of ALC, studies on oxidative markers (3-nitrotyrosine, 4-hydroxynonenal, and protein carbonyl] must be further pursued at different time points post-injury, as we have detailed previously (Patel et al. 2009). Moreover, long-term behavioral studies and tissue sparing analyses following more prolonged, intermittent ALC treatment following SCI will provide a solid foundation to validate this neuroprotective strategy as a beneficial therapeutic for acute and, potentially, chronic SCI.
We are thankful for the expert technical assistance of Christopher R. O’Dell. Grant support: KSCHIRT #8-13 (AGR), NIH/NINDS P3 NS051220.