Amyotrophic lateral sclerosis (ALS) is a relentless fatal paralytic disorder confined to the voluntary motor system.
Its prevalence is about three to five in 100,000 individuals, making it the most frequent paralytic disease in adults.
Although ALS can strike anyone at any age, generally the onset of the disease is in the fourth or fifth decade of life.
Common clinical features of ALS include muscle weakness, fasciculation, brisk (or depressed) reflexes, and extensor plantar responses.
Even though motor deficit usually predominates in the limbs, bulbar enervation can also be severely affected, leading to atrophy of the tongue, dysphagia, and dysarthria.
Other cranial nerves (e.g., oculomotor nerves) are usually spared.
The progressive decline of muscular function results in paralysis, speech and swallowing disabilities, emotional disturbance, and, ultimately, respiratory failure causing death among the vast majority of ALS patients within 2-5 years after the onset of the disease.
Pathologically, ALS is characterized by a loss of upper motor neurons in the cerebral cortex and of the lower motor neurons in the spinal cord.
Often, there is also a profound degeneration of the corticospinal tracts, which is most evident at the level of the spinal cord.
The few remaining motor neurons are generally atrophic, and many demonstrate abnormal accumulation of neurofilament, in both their cell bodies and axons.
To date, only a few approved treatments (e.g., mechanical ventilation and riluzole) prolong survival in ALS patients to some extent.
However, the development of more effective neuroprotective therapies remains impeded by our limited knowledge of the actual mechanisms by which neurons die in ALS, and of how the disease progresses and propagates.
ALS, like other common neurodegenerative disorders, is sporadic in the vast majority of patients, and familial in only a few.
The clinical and pathological expressions of ALS are almost indistinguishable between the familial and sporadic forms, although often in the former the age at onset is younger, the course of the disease more rapid, and the survival after diagnosis shorter.
The cause of sporadic ALS remains unknown, while that of at least some familial forms has been identified.
Although the identified gene defects responsible for ALS account for a minute fraction of cases, most experts believe that unraveling the molecular basis by which those mutant gene products cause neurodegeneration may shed light on the etiopathogenesis of the common sporadic form of ALS.
Genetic forms of ALS
Although familial ALS is often referred to as a single entity, genetic evidence actually reveals at least four different types that have been assigned to distinct loci of the human genome.
This review will focus on a form that is responsible for the disease in approximately 20% of all familial cases and that is linked to mutations in the gene for the cytosolic free radical-scavenging enzyme superoxide dismutase-1 (SOD1).
To date, approximately 100 different point mutations in SOD1 throughout the entire gene have been identified in ALS families, and all but one is dominant.
Many of these mutations lead to the substitution of an amino acid within regions of the enzyme with very distinct structural and functional roles.
It is thus fascinating to note that so many discrete SOD1 alterations share a similar clinical phenotype, even though the disease duration and, to a lesser extent, age at onset varies among patients with different SOD1 mutations.
Also astonishing is the fact that SOD1 mutations, which are present at birth and, by virtue of SOD1's ubiquitous expression, in all tissues, produce a rapidly progressive adult-onset degenerative condition in which motor neurons are almost exclusively affected.
Most of these mutations have apparently reduced enzymatic activity, a finding that has prompted investigators to test whether a loss of SOD1 activity can kill neurons.
It was unequivocally shown that reducing SOD1 activity to about 50% kills pheochromocytoma-12 cells and motor neurons in spinal cord organotypic cultures.
However, mutant mice deficient in SOD1 do not develop any motor neuron disease, and the transgenic expression of different SOD1 mutants in both mice and rats causes an ALS-like syndrome in these animals, whether SOD1 free radical-scavenging catalytic activity is increased, normal, or almost absent.
These observations provide compelling evidence that the cytotoxicity of mutant SOD1 is mediated not by a loss-of-function but rather by a gain-of-function effect.
Transgenic mutant SOD1 rodents unquestionably represent an excellent experimental model of ALS, one that has already generated valuable insights into the pathogenesis of ALS and opened new therapeutic avenues for this dreadful disease.
Hypothesis for mutant SOD1 cytotoxicity
Despite the explosion of ALS research engendered by the discovery of the SOD1 mutations, the actual nature of the gained function by which mutant SOD1 kills motor neurons in ALS remains elusive.
Multiple mechanisms have been implicated in the demise of motor neurons in ALS, but only a few may be directly relevant to the form linked to mutant SOD1.
For instance, the known free radical-scavenging function of SOD1 led researchers to believe that mutant SOD1-induced neurodegeneration was due to an oxidative stress.
This idea was, at least initially, received with enthusiasm due to the fact that a variety of markers of oxidative damage are indeed increased in ALS spinal cords.
Currently, it is thought that, if SOD1 mutants were to generate oxidative stress, they could do so by two distinct and not mutually exclusive mechanisms.
In the first mechanism, the point mutations would relax SOD1 conformation, hence allowing abnormal kinds or amounts of substrates to reach and react with the transitional metal - copper - contained in the catalytic site of the enzyme.
Among the aberrant substrates to be proposed are peroxynitrite and hydrogen peroxide, both of which can directly or indirectly mediate serious tissue damage.
In the second mechanism, it is speculated that SOD1 mutations are associated with a labile binding of zinc to the protein, and that, by having lost zinc, mutant SOD1, in the presence of nitric oxide, will catalyze the production of peroxynitrite, which can inflict serious oxidative damage to virtually all cellular elements.
Alternatively, mutant SOD1 cytotoxicity may result from the propensity of this mutant protein to form intracellular proteinaceous aggregates, which are a prominent pathological feature of several of the transgenic lines, and of various cultured cell types expressing mutant SOD1, including motor neurons.
As in other neurodegenerative disorders with intracellular inclusions, whether or not these proteinaceous aggregates are actually noxious remains uncertain.
Nevertheless, it may be speculated that their presence in the cytosol of motor neurons may be deleterious, by, for example, impairing the microtubule-dependent axonal transport of vital nutriments, or by perturbing the normal turnover of intracellular proteins.
Early on in the effort to determine the nature of mutant SOD1's gained function, it was discovered that transfected neuronal cells expressing mutant SOD1 cDNA were dying by apoptosis, a form of programmed cell death (PCD).
Morphology of dying motor neurons
In light of the presumed proapoptotic properties of mutant SOD1 observed in vitro, it may be wondered whether, in transgenic mutant SOD1 mice, dying spinal cord motor neurons would also exhibit features of apoptosis, whose morphological hallmarks include cytoplasmic and nuclear condensation, compaction of nuclear chromatin into sharply circumscribed masses along the inside of the nuclear membrane, and structural preservation of organelles (at least until the cell is broken into membrane-bound fragments called apoptotic bodies that are phagocytized).
In transgenic mutant SOD1 mice most of the sick neurons are atrophic, and their cytoplasm is occupied with vacuoles corresponding to dilated rough endoplasmic reticulum, Golgi apparatus, and mitochondria.
Sick neurons have diffusely condensed cytoplasm and nuclei and irregular shapes.
Although the actual type of this cell death remains to be determined, these dying neurons exhibit a rather nonapoptotic morphology with some features reminiscent of autophagic or cytoplasmic neuronal death.
Forms of PCD with morphological features distinct from apoptosis also exist, making it difficult to exclude the possibility that a nonapoptotic form of PCD underlies mutant SOD1-related cellular degeneration.
Thus, these findings suggest that, while degenerating neurons in both human ALS and its experimental models do exhibit some features reminiscent of apoptosis, the vast majority of dying cells cannot confidently be labeled as typical apoptotic.
Activation of apoptotic molecular pathways
More convincing approach to evaluate the role of apoptosis in ALS may be to determine whether the neurodegenerative process in transgenic mutant SOD1 mice, irrespective of the morphology of the dying cells, involves known molecular mediators of PCD, and whether targeting such key factors can affect the course of the disease.
In light of the presumed proapoptotic properties of mutant SOD1, it is tempting to suggest that the mutant protein may be a death-signaling molecule in itself, either directly, by setting in motion the PCD cascade, or indirectly, by interacting with a variety of intracellular targets such as trophic factors, Bcl-2 family members, or even mitochondria.
Mitochondria are a particularly appealing target, because they not only contain mutant SOD1 but are structurally and functionally altered in transgenic mutant SOD1 mice, and because they play a pivotal role in PCD.
Once the mutant SOD1-mediated neurodegenerative process has been initiated, several secondary alterations develop in spinal cords of transgenic mutant SOD1 mice, including microglial cell activation and T cell infiltration, both of which may release a plethora of cytokines and other pro-PCD mediators.
Accordingly, while the nature of the initial death signal in transgenic mutant SOD1 mice remains elusive, in a more advanced stage of the disease the increased expression of several extracellular inflammation-related factors such as IL-1β, IL-6, and TNF-a may amplify the death signals that are already reaching motor neurons in this mouse model of ALS, by activating death receptors such as Fas.
IL-1β content is also elevated in human ALS spinal cords.
Key molecular components of PCD are recruited in ALS.
While precious data on PCD in ALS have been obtained thanks to the study of postmortem human samples, information regarding the temporal relationships of these changes and their significance in the pathological cascade emanates essentially from the use of transgenic mutant SOD1 mouse models.
In light of the above-described PCD-related changes, it would appear that this active form of cell death is not the sole pathological mediator of cell demise in ALS but rather one key component within a coalition of deleterious factors ultimately responsible for the degenerative process.
However, the actual relationships between mutant SOD1 and the various other presumed culprits represented by protein aggregates, oxidant production, and PCD activation are still unknown, and a better understanding of the pathogenic cascade in ALS will require their elucidation.
In scientists' opinion, one of the most important take-home messages is that apoptotic morphology should not be used as the sole criterion of whether molecular pathways of PCD have been recruited.
Indeed, scientists cannot stress enough that the PCD molecular pathways may be activated in a neurodegenerative process such as that seen in ALS, even when the prevalent morphology of the dying cells is nonapoptotic.
Apart from the question of whether the morphology of dying neurons in ALS is apoptotic, but still relevant to this discussion, is the contrast between the paucity of morphologically identified dying cells and the rather robust spinal cord molecular PCD alterations.
How can this striking discrepancy be reconciled?
First, it is possible that the morphological expression of PCD is much more ephemeral than its molecular translation.
Therefore, since in ALS the degenerative process is asynchronous, small lasting differences in the expression of these markers may have significant impact on the total number of cells that exhibit a given marker at a given time point.
Second, it is also possible that, since apoptotic morphological features are confined to the cell body while PCD molecular alterations may be found not only in cell but also in cell processes, axons, and nerve terminals.
Thus, the detection of PCD morphology may be much more challenging than the detection of PCD molecular events.
Third, the molecular tools used see not only the rare cells that are truly dying but also the numerous sick cells that may or may not ultimately die and that thus may or may not show the typical apoptotic morphology.
Another important point that derives from the work in transgenic mutant SOD1 mice is that not only neurons but also glial cells appear to be the site of PCD-cascade activation.
This observation does not undermine the potential pathogenic role of PCD in the ALS death process, but it raises the possibility that PCD may not only kill neurons in this disease.
However, since SOD1 is expressed in all cells, not only in motor neurons, it is possible that the activation of PCD in both neurons and glia reflects the ubiquitous nature of the mutant protein expression.
Whether PCD is also activated in neuron and glial cells in the forms of ALS that are not linked to mutant SOD1 is unknown at this point.
Clearly, the overall mechanism of neurodegeneration in ALS is still incompletely known.
Nevertheless, the available evidence indicates that PCD is in play in ALS and thus warrants further investigation of the role of the PCD cascade in ALS pathogenesis and treatment.
The most effective therapeutic strategies tested so far in transgenic mutant SOD1 mice target very distinct molecular pathways.
Scientists can therefore imagine that, ultimately, the best therapy for ALS will come from a combination of several interventions and not from a single treatment.
In keeping with this view, unraveling the sequence of key PCD factors recruited during ALS neurodegeneration should enable scientists to identify the most significant molecules to be targeted by this therapeutic cocktail to produce optimal neuroprotection.