Motor neuron disease

The curious ways of ALS

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That mutations in the SOD1 enzyme underlie inherited forms of a motor neuron disease known as ALS is clear. But the question of what the consequences of such mutations are seems to have more than one answer.

Amyotrophic lateral sclerosis (ALS) is a neurological disorder characterized by the selective premature degeneration and death of motor neurons, which control voluntary actions such as breathing and walking. The disease starts in adult life, and the ensuing progressive paralysis is typically fatal within a few years, usually owing to failure of the respiratory system. Reporting in Genes & Development, Nishitoh et al.1 provide new insight into the molecular events that provoke ALS, linking its pathogenesis to a well-established stress pathway in an intracellular organelle called the endoplasmic reticulum.

Although most cases of ALS are sporadic, in roughly 10% of instances the disease is dominantly inherited — that is, carrying even one copy of the disease-associated mutation is sufficient to cause this fatal paralysis. A landmark discovery2 in 1993 ushered in the molecular era of ALS research, with the finding that mutations in the gene encoding the enzyme superoxide dismutase 1 (SOD1) are responsible for 20% of inherited ALS cases. (Normal SOD1 is a crucial intracellular antioxidant, facilitating the clearance of the potentially toxic superoxide radical.)

Mice expressing various ALS-related mutations in SOD1 recapitulate the fatal paralysis seen in human patients. Studies in these animals have established that ALS is caused by the toxic activity of the SOD1 mutation rather than reduced activity of this enzyme3. Selective silencing of the mutant gene in various cell types has shown that the ubiquitously expressed mutant SOD1 damages motor neurons and non-neuronal neighbouring cells such as astrocytes and microglia. Whereas damage to motor neurons is associated with the onset of ALS, damage to astrocytes and microglia — the innate immune cells of the spinal cord — sharply accelerates the progress of the disease4,5. But it is a sobering reality that, 15 years after the discovery of the SOD1 mutation in familial ALS, no consensus has emerged as to the molecules and pathways that are directly affected by mutant SOD1.

Nishitoh and colleagues1 now show that the mutant enzyme inhibits ERAD, the cell's machinery for eliminating proteins that fail to fold properly in the endoplasmic reticulum (ER), following up on earlier claims that ER stress is a pathogenic component in ALS6. The first crucial step in the 'ERADication' of misfolded proteins is their transport from the lumen of the ER to the cytoplasm, where their covalent attachment to the protein ubiquitin (a process known as ubiquitination) marks them for degradation by the proteasome complex. The authors find that three different SOD1 mutants, but not the normal enzyme, can interact with an ERAD component called Derlin-1, which is instrumental in both the transport of misfolded proteins from the ER lumen to the cytoplasm and their degradation. By binding to Derlin-1, mutant SOD1 inhibits the ERAD pathway, causing both defective functioning of the ER (ER stress) and activation of a protein kinase called ASK1, which is involved in driving programmed cell death (Fig. 1a, overleaf). These observations provide a putative direct molecular link between mutations in SOD1 and a main pathological hallmark of ALS — the presence of ubiquitinated aggregates of misfolded proteins in the cytoplasm.

Figure 1: Proposed mechanisms of toxicity in SOD1-mediated ALS.

a, Nishitoh et al.1 show that mutant SOD1 interacts with Derlin-1, a protein that is essential for transporting misfolded proteins from the lumen of the endoplasmic reticulum (ER) to the cytoplasm for degradation. Consequently, misfolded proteins accumulate in the ER lumen, and the resulting ER stress activates ASK1 — a protein involved in programmed cell death — by an unidentified mechanism. b, Near the cell membrane, mutant SOD1 also interacts strongly with Rac1 (ref. 6), keeping the Nox enzyme active. The resulting increased production of the superoxide radical, and its release into the extracellular space, is toxic to neighbouring cells. The precise cells in which mutant SOD1 interacts with Derlin-1 or Rac1 are unknown.

The precise cells in which Derlin-1 and mutant SOD1 interact are not known, although the most likely candidates are astrocytes rather than motor neurons, because deletion of the ASK1 gene prolongs survival of a SOD1 mutant mouse by slowing disease progress, without affecting its onset. But a perplexing finding is that the interaction between Derlin-1 and mutant SOD1 is not detectable until symptoms of the disease appear, suggesting that it is secondary to some unidentified initiating event.

Nishitoh and colleagues' observations follow several other recent findings. Earlier this year, it was reported7 that both normal and mutant SOD1 associate with Rac1, an activator of the enzyme NADPH oxidase (Nox) found at the cell membrane. Normally, Nox mediates the production of superoxide — in immune cells such as microglia — to kill bacteria and other pathogens. It emerges that the association of normal SOD1 with Rac1 is part of a tightly regulated mechanism that, under chemically reducing conditions, activates Nox. But mutant SOD1 interacts with Rac1 with a higher affinity than normal, 'locking' Nox in its active, superoxide-producing form, even under oxidizing conditions. This results in a tenfold increase in superoxide production and its release into the extracellular space (Fig. 1b).

Paradoxically, therefore, instead of carrying out its normal job of removing intracellular superoxide, mutant SOD1 might increase extracellular levels of this radical and so damage motor neurons and other cells. A Nox inhibitor improved survival in a SOD1 mutant mouse almost exclusively by delaying the onset of ALS7. As the presence of mutant SOD1 in motor neurons, but not microglia, is one of the factors that determine the timing of disease onset, this finding suggests that the interaction between mutant SOD1 and Rac1 triggers disease by activating a Nox variant (Nox1) found in motor neurons, rather than another variant of this enzyme (Nox2) that occurs in microglia.

So, are ER stress1,6 and the increased production of superoxide7 — both potential triggers of death for motor neurons and neighbouring cells — the whole story of the molecular events underlying ALS? Almost certainly not. Observations in SOD1 mutant mice and in tissue samples from patients with sporadic ALS suggest that one contributory factor to the damage is excitotoxicity (excessive firing of motor neurons that occurs when the stimulatory neurotransmitter glutamate is not rapidly removed from the synaptic junctions between two neurons). Excessive stimulation of the glutamate receptors on neuronal membranes, and the accompanying increase in calcium-ion influx, can trigger a cascade of toxic events in the neuron, including damage to both mitochondria (the cell's powerhouses) and the ER (the main reservoir of intracellular calcium). Loss of the glutamate transporter EAAT2, a synaptic 'vacuum cleaner' for glutamate, from neighbouring astrocytes contributes to this phenomenon8, but a direct role — if any — for mutant SOD1 in this mechanism remains unclear.

Mutant SOD1 has also been proposed9 to interact directly with chromogranins (components of vesicles secreted by neurons) and to co-secrete with them. This in turn may trigger damage to motor neurons and neighbouring cells.

And there is more. Mitochondrial dysfunction has long been implicated in the damage to motor neurons seen in ALS. Misfolded mutant SOD1 is deposited on the cytoplasmic face of the outer mitochondrial membrane in cells of the spinal cord10. Moreover, mitochondrial dysfunction in motor neurons11 carrying SOD1 mutations is associated with the release of cytochrome c, a potent trigger of cell death. Finally, mutant SOD1 provokes oxidative stress in astrocyte mitochondria12, which could accelerate death in neighbouring neurons.

With all these potential contributors, the proposed pathways of pathogenesis in inherited ALS might strike some as “curiouser and curiouser”, much as Alice proclaimed of the strange happenings in Lewis Carroll's Alice's Adventures in Wonderland. Among the biggest challenges will be to distinguish mechanisms that cause the disease from those that are only epiphenomena. In our view, the most likely possibility is that every set of observations discussed here is partly right, with age-dependent, selective toxicity requiring a convergence of damage to motor neurons and non-neurons alike. So stay tuned: the main pieces of this puzzle are yet to be revealed.


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