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).
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.
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- Materials and methods
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.