Regular paperLactate dyscrasia: a novel explanation for amyotrophic lateral sclerosis
Introduction
Amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease) is a debilitating disease that is characterized by progressive neurodegeneration of motoneurons in the brain and spinal cord. Initial manifestations are weakness of limbs, or weakness in the bulbar region leading to abnormalities of speech, swallowing difficulties, and facial weakness (Schmidt et al., 2009). Eventually the loss of motoneurons results in paralysis of voluntary muscles and to death by respiratory failure within 1–5 years of onset of the disease.
ALS is the most common form of motoneuron disease in humans: a little over 5600 people in the USA are diagnosed with ALS each year (incidence of 1–2 per 100,000 per year). Although most cases of ALS typically develop between the ages of 40 and 70, it is often overlooked as being an age-related disease with ethnic and gender predilections. ALS is slightly more prevalent in men (60%) (Wijesekera and Leigh, 2009).
The etiological mechanisms that underlie ALS are unclear, however 5%–10% of ALS cases are familial (FALS) and in those families, there is a 50% chance that each offspring will inherit the genetic mutation and develop the disease (Beghi et al., 2006, Mitchell and Borasio, 2007). However, the underlying gene defect in most patients with FALS is unknown and 90% of all cases have no family history of ALS and are considered sporadic ALS (SALS). The pathophysiology of ALS has been postulated to involve immune mechanisms (neuroinflammation and T-cell responses), glutamate-mediated excitotoxicity, oxidative stress, mitochondrial dysfunction, apoptosis, protein aggregation, and aberrant axonal transport (Holmoy, 2008, Mantovani et al., 2009; Pasinelli and Brown, 2006, Seksenyan et al., 2009; Shaw, 2005, Wang et al., 2004).
The neuropathology of ALS has been characterized from postmortem analyses (Leigh et al., 1995). The major pathological features of ALS include: (1) degeneration of the corticospinal tracts and extensive loss of lower motoneurons or anterior horn cells (Garofalo et al., 1995, Ghatak et al., 1986, Hughes, 1982); (2) degeneration and loss of Betz cells and other pyramidal cells in the primary motor cortex (Hammer et al., 1979, Maekawa et al., 2004, Udaka et al., 1986); and (3) reactive gliosis in the motor cortex and spinal cord (Ekblom et al., 1994, Kawamata et al., 1992; Murayama et al., 1991, Schiffer et al., 1996).
In addition to the loss of neurons, various types of inclusion bodies have been identified in degenerating neurons and surrounding reactive astrocytes and are well demonstrated hallmarks of ALS (Barbeito et al., 2004). Ubiquitinated inclusions found in lower motoneurons of the spinal cord and brainstem are the most common and specific type of inclusion in ALS (Matsumoto et al., 1993) and in corticospinal upper motoneurons (Sasaki and Maruyama, 1994). The exact composition of such inclusions, classified as “Lewy body-like inclusions”, “Skein-like inclusions” (He and Hays, 2004, Kawashima et al., 1998), and Bunina bodies (Wada et al., 1999) is not known. However, the proteins identified so far can include ubiquitin (Leigh et al., 1991, Murayama et al., 1989), Cu/Zn superoxide dismutase 1 (SOD1) (Shibata et al., 1994, Shibata et al., 1996), peripherin (He and Hays, 2004), Dorfin (a RING-finger type E3 ubiquitin ligase) (Niwa et al., 2002), and more rarely synuclein (Sone et al., 2005). Various studies conducted in ALS postmortem tissue in the early nineties found accumulations of intermediate filament proteins (hyperphosphorylated neurofilament subunits and peripherin) in hyaline conglomerate inclusions and axonal “spheroids” in spinal cord motoneurons (Corbo and Hays, 1992, Munoz et al., 1988, Sobue et al., 1990), and pyramidal cells of the motor cortex (Troost et al., 1992). Moreover, cystatin C-containing Bunina bodies are found in the cell bodies of motoneurons in ALS (Okamoto et al., 1993, Sasaki and Maruyama, 1994). Some breakdown products of abnormal proteins caused by oxidative stress called ubiquitinated inclusion bodies (UIBs), are also implied in the pathogenesis of ALS (Alves-Rodrigues et al., 1998). Fragmentation of the Golgi apparatus (Fujita et al., 2000, Fujita et al., 2002, Gonatas et al., 1998), mitochondrial vacuolization (Okamoto et al., 1990) and ultrastructural abnormalities of synaptic terminals (Sasaki and Iwata, 1996) are other neuropathological features of ALS.
Approximately 20% of ALS patients also have signs and symptoms of frontotemporal dementia such as cortical atrophy including the frontal and temporal lobes (Nakano, 2000), hippocampus and amygdala (Wilhelmsen et al., 2004), spongiform change in the neocortex, and UIBs in the substantia nigra (Al-Sarraj et al., 2002). Furthermore, the presence of crescent shaped inclusion-type UIBs in the neostriatum has been found to be a feature specific to ALS-frontotemporal dementia, and not occurring in a variety of other neurodegenerative disorders including Pick's disease, Parkinson's disease, and Alzheimer's disease (Kawashima et al., 1998). UIBs are found in ALS patients in the dentate gyrus, frontal and parietal neocortices, anterior cingulate gyrus, hippocampus, parahippocampal gyrus, amygdale and neostriatum. The density and distribution of these inclusions was higher in cognitively-impaired ALS patients (as defined by poor performance on neuropsychological testing) than in unimpaired individuals (Kawashima et al., 1998; Wilson et al., 2001). The cognitively impaired patients also had UIBs in the temporal, occipital, and entorhinal cortices, posterior cingulate gyrus, caudate, and putamen. Computerized morphometry revealed a 25% reduction in the pyramidal neuronal density in layer V of the premotor cortex, dorsolateral prefrontal cortex, and anterior cingulate cortex compared with age-matched controls (Maekawa et al., 2004). This is particularly relevant in the context of findings from positron emission tomography neuroimaging which identified decreased binding of the GABAergic ligand (11C)-flumazenil in the prefrontal cortex (Lloyd et al., 2000, Turner et al., 2005) and increased microglial activation (implicated in mechanisms of neuronal cell death) in the dorsolateral prefrontal cortex (Turner et al., 2004).
Dissecting the spatiotemporal changes in pathology is key to understanding the molecular mechanism(s) involved in ALS. From a spatial perspective, the notion that ALS affects only the motoneurons while sparing the central nervous system was refuted when neuropathological examination showed ubiquitin-immunoreactive but tau-negative inclusions in the frontotemporal cortex, hippocampus, and dentate gyrus (Jackson and Lowe, 1996). To determine where and when the pathological changes of motoneuron disease begins, Fischer and colleagues (Fischer et al., 2004) performed a comprehensive spatiotemporal analysis of disease progression in SOD1G93A mice. Quantitative pathological analysis was performed in the same mice at multiple ages at neuromuscular junctions (NMJs), ventral roots, and spinal cord. Mice became clinically weak at 80 days and died at 131 ± 5 days. At 47 days, 40% of end plates were denervated whereas there was no evidence of ventral root or cell body loss. At 80 days, 60% of ventral root axons were lost but there was no loss of motoneurons. Motoneuron loss was well underway by 100 days. Microglial and astrocytic activation around motoneurons was not identified until after the onset of distal axon degeneration. Thus, in this animal model of human ALS, motoneuron pathology begins at the distal axon and proceeds in a “dying back” pattern. This is supported by the denervation and reinnervation changes in muscle but normal-appearing distal motoneurons following autopsy of a reported ALS patient (Fischer et al., 2004).
The basic unit of movement is comprised of skeleton, muscles connected to skeleton, and nerves connected to the muscles. A motor unit consists of 1 motor neuron in the anterior horn of the spinal cord, its axon, and all the muscle fibers innervated by the branches of the axon (Fig. 1). The axon of the nerve terminates on the muscle fibers at the NMJ. The number of motor units that are active in a muscle at any 1 time determines the level of performance of the muscle. Thus each functional NMJ determines the motor ability.
It has been demonstrated that neuromuscular deficits in ALS do not result from motoneuron cell death but rather from loss of axonal integrity. As mentioned above, in the SOD1G93A transgenic mouse model, motor unit numbers in fast-twitch tibialis anterior, extensor digitorum longus, and medial gastrocnemius muscles decline from 40 days of age, 40–50 days before reported overt symptoms and motoneuron loss (Hegedus et al., 2007, Kennel et al., 1996). Motor unit numbers fall after overt symptoms in the slow-twitch soleus muscle (Hegedus et al., 2007). Similarly, the fact that end plates are denervated much earlier than the axons and the cell body loss during the pathogenesis of ALS as described in SOD1G93A mice (Fischer et al., 2004), gives ample indication that the degenerative process in ALS starts at the NMJ. In canine motoneuron diseases, functional motor unit failure precedes neuromuscular degeneration (Balice-Gordon et al., 2000).
Early muscle-specific decline has been correlated to selective preferential vulnerability of large, fast motor units, innervated by large motoneurons. Large motoneurons appear to be the most vulnerable in ALS with die-back occurring prior to overt symptoms (Hegedus et al., 2007). Subsequently, it was found that disease progression in fast-twitch muscles of SOD1G93A mice involves parallel processes: (1) gradual selective motor axon die-back of the fast fatigable motor units that contain large type IIB muscle fibers, and of fatigue-intermediate motor units that innervate type IID/X muscle fibers; and (2) activity-dependent conversion of motor units to those innervated by smaller motor axons innervating type IIA fatigue-resistant muscle fibers (Hegedus et al., 2009).
Studies that have used strategies to preserve the NMJ in ALS models invariably show a delay in disease progression (Dobrowolny et al., 2005, Ferri et al., 2003; Gifondorwa et al., 2007, Li et al., 2007; Rouaux et al., 2007, Storkebaum et al., 2005; Suzuki and Svendsen, 2008, Suzuki et al., 2007, Suzuki et al., 2008). Conversely, prevention of neurite outgrowth promotes denervation in an ALS model (Jokic et al., 2006).
There is conflicting evidence as to whether degenerative signals emanate from the motoneuron itself, or from the muscle. It has been shown that suppression of hSOD1G93A expression within muscle alone is insufficient to maintain grip strength or affect disease onset or survival, but that suppression of hSOD1G93A expression in both motor neurons and muscle is sufficient to maintain grip strength (Miller et al., 2006). Moreover, this group found that follistatin induced inhibition of myostatin, produced sustained increases in muscle mass, myofiber number, and fiber diameter, but these increases did not affect survival. Conversely, a mouse model of muscle restricted mitochondrial defect (similar to the hypermetabolism found in ALS) has been shown to generate motor neuron degeneration (Dupuis et al., 2009). Transgenic mice with muscular overexpression of uncoupling protein 1, a potent mitochondrial uncoupler, displayed age-dependent deterioration of the NMJ that correlated with progressive signs of denervation, grip strength, and a mild late-onset motor neuron pathology.
Together, these data indicate that understanding the molecular events that promote the degeneration and dismantling of NMJ is crucial to understanding the underlying cause of ALS and is a prerequisite for identifying appropriate treatment strategies. The important question therefore is what molecular events lead to the deterioration of the motoneuron terminals at the NMJ? Factors produced by either the muscles or motoneurons that impact the NMJ might be considered prime candidates in the molecular pathology of the disease. What are these factors, and how are they regulated and how do they affect the normal molecular signaling and trafficking at the NMJs?
Section snippets
The lactate dyscrasia hypothesis of ALS
What is interesting about many patients with ALS is that the function of the eye muscles is spared. Understanding what is different about the eye muscles compared with other muscles might therefore throw light on what causes skeletal muscles to dysfunction. One noticeable characteristic of the ocular nerves and muscles is that they use lactic acid as a metabolic substrate to sustain function and therefore do not become fatigued by high lactic acid, unlike skeletal muscles (Andrade and McMullen,
What derails lactate homeostasis leading to neuromuscular junction toxicity?
ALS is an age-related disease; the cause of ALS must therefore be related to the aging process. Because the downstream cause of ALS is the loss of NMJ and motoneurons, understanding what signals regulate the balance between the formation of motoneurons (innervation) and loss of motoneurons (denervation) is critical to identifying the upstream signals that promote ALS. It is known that denervated muscle is readily reinnervated, whereas innervated muscle cannot be hyperinnervated (Frank et al.,
A multidrug therapy for the treatment of ALS
Based on the above model along with the other evidence described above, therapeutic strategies for the treatment ALS should incorporate drugs that: (1) maintain lactate homeostasis in NMJs; (2) maintain mitochondrial function; (3) halt damage to peripheral nerves; and (4) promote regeneration of peripheral nerves. Thus, combinations of drugs that inhibit lactate accumulation at the NMJ, enhance respiratory chain function, and that are neurotrophic should be most effective at halting the
Conclusions
We present a novel molecular model for the molecular pathogenesis of ALS that involves an ATP-dependent MNLS to maintain lactate homeostasis at the NMJ by tightly regulating the flow of lactate from muscle to neurons and visa versa. Failure of this shuttle is proposed to lead to lactate assimilation in the NMJ leading to cellular stress, toxicity, and subsequent degeneration. Future studies should focus on the identification and characterization of the MNLS and the mutational and endocrine
Disclosure statement
There are no actual or potential conflicts of interest.
Acknowledgements
The development of this new model of ALS was inspired by discussions with Susan Grossberg and Steve Saling and the dire need to find therapies for this disease. This is VA manuscript #2010-12.
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