Amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS), initially known as Charcot’s sclerosis , was named after the French neurobiologist and physician Jean-Martin Charcot who first described this type of muscular atrophy in the early nineteenth century . In the United States, ALS became widely known as Lou Gehrig ‘ s disease after the famous baseball player who succumbed to the disease in the late 1930s. Currently, ALS is the most common motor neuron disease, with a worldwide incidence of 8 cases per 100,000 population per year . Familial forms constitute approximately 5% to 10% of all cases. Onset increases with age, with a peak in the seventh decade and a slight preponderance among men compared with women. Rapid progression of motor neuron loss leads to death an average of 3 to 5 years after symptom onset . The cause of ALS remains unknown and there is still no curative therapy.

The term amyotrophy refers to peripheral denervation producing lower motor neuron signs of weakness, muscular atrophy, and fasciculations. Upper motor neuron signs of spasticity and hyperreflexia are appreciated as the disease advances, because of sclerosis of the lateral corticospinal tracts. Symptom onset is typically characterized by weakness or clumsiness in an upper extremity with or without fasciculations. In a subset of cases, presenting weakness in the bulbar musculature leads to progressive dysphagia and dysarthria. Bulbar onset infers a rapidly poor prognosis with life expectancy 1 to 2 years after the onset of symptoms.

As the disease progresses, widespread limb and trunk weakness leads to a rapid decline in physical function. Regardless of the initial site of disease manifestation, weakness in the bulbar and respiratory muscles places patients at risk for aspiration pneumonia, which is often the eventual cause of death.

Although the events leading to neuronal toxicity are not clearly understood, ALS is now widely accepted to most likely be a multifactorial disease. Many factors, not necessarily related, have been shown to participate in motor neuron cell death in ALS, including glutamate toxicity, oxidative stress, neurofilament accumulation and neuroinflammation . These pathways are not mutually exclusive and may act individually or in unison to cause motor neuron disease .

Diverse neurologic disorders such as Alzheimer’s disease, Parkinson’s disease, HIV-associated dementia, and other neurodegenerative diseases share common pathologic themes. Understanding these commonalities may help in developing new neuroprotective strategies . One common feature is neuroinflammation.

Key players in neuroinflammation

Key players in neuroinflammation

Neuroinflammation in amyotrophic lateral sclerosis

Although microglia and neuroinflammation are implicated in nearly all disorders of the CNS, the role of microglial cells in ALS recently came into focus. In histopathologic studies of human ALS tissue, strong activation and proliferation of microglia have been described in the primary motor cortex, brainstem motor nuclei, and ventral horns of the spinal cord: all areas with motor neuron loss . Activated microglia were seen in areas with not only severe motor neuron death but also mild neuronal death . Furthermore, a recent positron emission tomographic (PET) scan study showed that microglial activation strongly correlated with clinical signs of upper motor neuron loss in sporadic ALS .

Evidence implicating activation of microglia in motor neuron disease was also seen in studies on mutant SOD1 (mtSOD1) transgenic mice, the predominant animal model of ALS. These studies showed that microglial activation occurred in the earliest stages of motor neuron degeneration, even before symptoms of weakness . Several groups reported expression of proinflammatory mediators, such as interleukin (IL)-1β, IL-6, cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), or tumor necrosis factor (TNF)-α, even preceding the development of clinical signs . The expression profile of these inflammatory molecules was microglia/macrophage-like.

Elevated levels of MCP-1 and IL-6, for example, were also reported in patients who had ALS . Motor neurons are exquisitely sensitive to neuroinflammation. Several studies have shown that mediators associated with neuroinflammation, such as TNF-α, IL-1β, or FAS ligand (FasL), can readily trigger motor neuron apoptosis .

In ALS, in contrast to other neurodegenerative diseases such as multiple sclerosis or Alzheimer’s disease, influx of peripheral immune cells such as lymphocytes or neutrophils is rare and mainly associated with end-stage disease . However, recent progress in understanding T-cell subsets has led to experts to question whether a neuroprotective T-cell response is initially mounted in ALS but fails because of unknown reasons . Therefore, in ALS, neuroinflammation is solely sustained by the interactions of microglia, neurons, and macroglia.

Growth factors are a group of proteins promoting the growth and survival of cells. Important neurotrophic growth factors include neural growth factor, brain-derived neurotrophic factor, insulin-like growth factor I, and vascular endothelial growth factor (VEGF) .

Dysregulation of several growth factors have been implicated in ALS . For example, deletions in the VEGF promoter caused ALS-like symptoms in mice , and patients who have ALS show reduced VEGF levels in cerebrospinal fluid. In the CNS, astrocytes and microglia—the main mediators of neuroinflammation—are also the main sources of neurotrophic factors . Neuroinflammation leads not only to an increase in inflammatory products but also is usually accompanied by the down-regulation of neurotrophic factors . This withdrawal of trophic support could add insult to injury and finally drive damaged motor neurons into apoptosis .

The increased expression of inflammatory factors or down-regulation of growth factors could also explain how damage to lower motor neurons (spinal cord) could propagate to upper motor neurons (cortex). Proapoptotic inflammatory signals (eg, TNF-α, FasL) in the spinal cord could be detected by the axonal projections of upper motor neurons and trigger apoptosis in these cells. Likewise, the down-regulation of VEGF, for example, in the target area of an upper motor neuron could lead to apoptosis of this cell from failure of trophic support ( Fig. 1 ) .

Neuroinflammatory view of glia–motor neuron interactions in the normal and ALS/mtSOD1 CNS. In the unperturbed CNS, microglia, astrocytes, and motor neurons interact through the release of soluble messengers, such as growth factors, cytokines, and cell–cell contacts. The cells are in a quiescent state. The expression of mtSOD1 or, in the case of sporadic ALS, an unknown trigger leads to activation of glial cells. Activated microglia produce proinflammatory cytokines and glutamate and down-regulate growth factor expression, the SOD1 mutation critically alters this expression profile. Astrocytes, by virtue of mtSOD1 expression and in response to the inflamed tissue, also down-regulate growth factor expression and release inflammatory mediators. Furthermore, they lose their capacity to buffer glutamate. The prolonged exposure of motor neurons to the inflammatory and excitotoxic microenvironment leads to degeneration through apoptosis. Green arrows , trophic support; red arrows , inflammatory mediators. All named factors are prominent examples only.

The (mis)conception of cell autonomous effects

Motor neuron cell loss was long seen from a neurocentric viewpoint as a “disease from within the motor neuron.” Recently, it became clear that cell–cell interactions must be considered part of the cascade leading to cell death. Therefore, motor neuron degeneration occurring from intrinsic dysfunction and was called cell autonomous , even though motor neuron–autonomous seems to be the more correct term. When surrounding cells contribute to the event leading to motor neuron loss, the effect would be called non–cell autonomous . Clearly, neuroinflammation would be a non–cell autonomous effect from the motor neuron viewpoint.

Compelling evidence from the mtSOD1 transgenic animal models of ALS showed the involvement of non–cell autonomous effects in the pathogenesis of motor neuron disease. In wild-type/mtSOD1 chimeric mice, wild-type motor neurons surrounded by mtSOD1 glia were damaged, whereas mtSOD1 neurons surrounded by wild-type glia were healthier . This finding strongly indicates a non–cell autonomous disease mechanism and the critical involvement of cell types other than neurons.

Very recently, using an elegant approach, mtSOD1 was selectively removed from microglial cells and macrophages through a CD11b-driven Cre/LoxP system. These animals showed a dramatically slowed disease progression. Removing mtSOD1 from motor neurons, however, delayed the onset of disease but had little influence on disease progression . Although some open questions remain about these data pertaining to limited recombination efficiency, the results clearly indicate a role for mtSOD1 within microglial cells in the disease process.

Similar data were obtained through crossing the mtSOD1 into the PU.1 knockout strain, which are unable to develop myeloid and lymphoid cells without bone marrow transplantation. Disease in mtSOD1/PU.1 ^−/− animals receiving wild-type bone marrow progressed considerably slower compared with mtSOD1 bone marrow controls . This article and an earlier report showed that the expression of mtSOD1 in microglial cells leads to an increase in their cytotoxic potential, because they release increased amounts of superoxide, nitrate, and TNF-α in vitro .

Clearly, microglial intracellular mtSOD1 leads to unidentified changes in microglial phenotype with relevance to disease progression. In contrast, the activation of microglial cells by extracellular mtSOD1 was recently shown . The authors showed that chaperone-associated mtSOD1 secreted from neuronal cells triggered microglial activation and in turn led to motor neuron death in vitro. Taken together, both pathways, intracellular and extracellular mtSOD1, could trigger a vicious neuroinflammatory circle with microglia at center stage.

Astrocytes have recently reappeared on the center stage of ALS . They have been implicated early on as key players in the glutamate toxicity hypothesis because their transport capacity seems hampered in ALS . However, recent data showed that mtSOD1 within astrocytes changes their properties and that they secrete an unknown factor leading to motor neuron degeneration .

Using their mtSOD1 CreLloxP system, the Cleveland group used a GFAP-Cre mouse to excise mtSOD1 from astrocytes. In contrast to the CD11b/microglial Cre model, the recombination efficiency was very high. Although genetic ablation of mtSOD1 in astrocytes did not affect the onset of disease, it significantly slowed its progression. The removal of mtSOD1 from astrocytes reduced the activation of microglial cells, as assessed using MAC-2 and iNOS staining. These indirect effects on microglia also show the close relationship of the two arms of the CNS’ immune system.

Non–cell autonomous has been used to define the involvement of other cells contributing to motor neuron death. Both microglia and astrocytes seem to play crucial roles in the motor neuron’s demise in ALS, although this may imply that mtSOD1 has a cell autonomous effect on glial cells. How microglia or astrocytes actually transmit these effects to motor neurons and induce their loss remains to be shown. The fact cannot be excluded that the mtSOD1 protein accumulation in microglia or astrocytes could also induce intrinsic loss of properties, such as a diminished neuroprotective function, which may be one mechanism contributing to the loss of motor neurons. Nevertheless, the inflammatory cascade doubtlessly plays a important role in ALS, lending credibility to the idea that therapeutic modulation of inflammation may help treat this currently incurable disease ( Table 1 ).

Current therapy in amyotrophic lateral sclerosis: neuroprotective, anti-inflammatory, or both?

Although a sizable number of medications have been tested in ALS, there is currently only one FDA-approved drug, riluzole, available for therapy . Riluzole, believed to exert its effect through presynaptic reduction of glutamate release, prolongs survival by a mere 3 months and does not clearly improve quality of life or functional parameters in patients who have ALS .

Although most studies indicate that riluzole works on neurons, some argue that microglia could be a target of the drug. Microglia can release high levels of glutamate, creating the possibility that riluzole could also slow down disease progression through an anti-inflammatory effect on microglia .

The advent of ALS animal models has prompted a surge in preclinical studies. The predictability of disease onset and progression makes these animals useful for pharmacologic trials . Although some concern has been expressed about the inherent limitations of mtSOD1 models (eg, they resemble only a small population of patients who have ALS, several pharmacologic results from these models do not hold true in humans ), these animals are the only models currently available and have already greatly advanced understanding of ALS .

The current list of compounds in preclinical and clinical studies is long and includes targets for all suspected causes (eg, glutamate toxicity, oxidative stress) . Most relevant for this article, however, are the group of drugs that are believed to exert their effects through an anti-inflammatory mode of action.