Neuroimmunology of Amyotrophic Lateral Sclerosis

The Corticospinal Tract
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Figure 1: The corticospinal tract sustains damage
from upper motor neurons degeneration in ALS. These
neurons project from the cortex to the spinal cord.
Adapted form Wikimedia Commons.

Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative motor neuron disease that involves the deterioration and eventual death of both cortical and spinal motor neurons. Its symptoms vary from muscle weakness, fasciculation, and spasticity to severe muscle atrophy and impeded eating, moving, and breathing. After initial onset, the prognosis of ALS is usually comprised of a rapid decline of motor control, coupled with cognitive impairments, and culminates in death within three to five years[1]. Due to the aggressive nature of this disease, many studies have been conducted to further understand its etiology, although no exact mechanism for the initial cause of the disease has been delineated. Genetic analyses and transgenic model manipulations have highlighted important genes, namely SOD1, that are implicated with the disease. It is apparent that a SOD1 mutation in ALS confers gain of function that is toxic to affected motor neurons, but an exact mechanism that links the mutation to cell death is still unclear[2]. Recent, promising studies have began to investigate the immunological component of ALS: how altered microglial function and expression contributes to an inflammatory response and the degeneration of motor neurons. Evidence suggests that microglia are “activated” in individuals with the SOD1 mutation and confer toxicity to motor neurons and surrounding glial cell populations[3]. Current investigations into the immunology of this disease aim to ascertain the conditions and mechanisms that enable the conversion of microglia into their active disease state, and what consequence this transition has on the cellular and molecular functions of cells in the central nervous system.

1. Disease Progression and Symptoms

ALS is one of five motor neuron diseases, which arise from the selective degeneration of either Upper Motor Neurons (UMN) or Lower Motor Neurons (LMN)[4]. UMNs are large pyramidal cells whose main efferents originate in the cortex, namely layer V of the primary motor area. The axons of these cells project either to the spinal cord where they synapse on LMNs in the ventral horn in various regions of the cord or to the brainstem where they synapse on cranial nerve nuclei that are associated with cranial nerves that control movement[5]. The entirety of voluntary movement is made possible through UMN and LMN connectivity, with LMNs innervating skeletal muscle and facilitating their contraction through transduction of motor signals that originate in the cortex[6]. Degradation of UMNs and LMNs manifests in a progressively deteriorating control of voluntary movement. Denervation of muscles leads to muscular atrophy and eventual difficulties with moving, eating, speaking, and breathing.

1.1 Onset and Diagnosis

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Figure 2: Denervation or lack of use causes
muscles to atrophy and deteriorate.
Adapted form Wikimedia Commons.

Initial evidence of motor neuron degradation presents as subtle complications or difficulties with motor activity. The earliest observable symptoms include muscle spasticity, fasciculation, and rigidity that, in most cases begins in the limbs. These symptoms begin to affect voluntary motion in fine muscle control and dexterity involved in various everyday tasks. These small impediments eventually progress into muscle denervation and atrophy as the disease progresses, and more motor neurons are lost[7].
The disease eventually progresses to a point of muscle atrophy in which the individual begins to experience problems with motility, dyspnea (trouble breathing), dysphagia (trouble eating), dysarthria (trouble speaking), and hyperreflexia (an exaggeration of reflexive responses). Since ALS is comprised of damage to both upper and lower motor neurons, diagnostic criteria for the disease includes indicators that degeneration has impacted both the UMNs and LMNs. Diagnosis of progressed ALS usually relies on the observation of both muscle atrophy, signifying LMN damage, and hyperreflexia, which is indicative of UMN damage. Simple reflex tests, such as the Babinski sign, can be uses to asses UMN health. Further evidence for ALS can be derived from an electromyography (EMG) test or nerve conduction test, which measure electrical activity in muscles and nerves respectively. This allows for the exclusion of simple peripheral nerve or muscle damage[8].

1.2 Disease Progression

Patients of ALS experience rapid decline in their ability to use their bodily muscles, resulting in progressively increasing severity of their symptoms, which include dyspnea, dysphagia, dysarthria, as well as overall motility. Regardless of the form or location of onset symptoms, spreading motor neuron degradation leads to diffuse progressive muscle atrophy, of which the most pressing complications with direct health consequences are dyspnea and dysphagia. Once the disease has progressed far enough, the individual loses the inability to control the muscles that enable swallowing, and cannot properly ingest food resulting in a high risk of choking. This leads to health factors such as weight and malnutrition becoming an issue. Furthermore, internal muscles of the torso that facilitate breathing also progressively deteriorate to the point that overall lung function is compromised. Combined, these factors make a feeding tube and respirator a necessity during later phases of the disease. In addition to an overall inability to walk, stand, or even move limbs voluntarily, the combination of these severe symptoms renders an individual with ALS in need of constant care in the progressed stages of the disease[9].
Despite severe deterioration in some parts of the brain, many ALS patients maintain their cognitive functioning throughout the progression of the disease. Combined with the fact that all sensory perception and autonomic function remains intact, perhaps one of the most horrific aspects of this disease, is that most ALS patients are completely cognizant of their functional decline. This results in high rates of comorbidity with instances of depression and anxiety within ALS patients. Recent work has shown that a larger than previously anticipated (approximately 50%) of ALS patients also show executive dysfunction, as well as working memory and consolidation deficits. A small proportion of about 5% of ALS patients develop severe cognitive impairments through frontotemporal dementia in which executive control, general social behaviour, attentional regulation, and effectual experience are severely impaired or distorted. The expression of frontotemporal dementia with ALS is indicative of very severe neuronal degradation and shortens life expectancy of an individual by at least a year[10].
Since ALS is a disease of rapid neurological decline, most individuals with the disease die within 3-5 years of being diagnosed. Only 4% of those diagnosed live with the disease for more than 10 years. The most prevalent cause of death for ALS patients is respiratory failure from complete atrophy of the diaphragm muscles[11].

2. Immunological Etiology

The direct causal factor for ALS is still unknown. 95% of instances of ALS arise sporadically, while the remaining 5% of instances can be attributed to familial heredity[12]. Though the mechanism for sporadic ALS remains a mystery, its similarity to familial ALS suggests that there are common factors involved in its etiology, namely mutations of a specific set of genes which confer a toxicity to motor neurons. Recent investigations into ALS have observed other various cell types and processes to be correlated with the progression of the disease, leading to its conceptualization as that of a multifactorial etiology. The apparent correlation of cytokines, chemokines, and other various pro-inflammatory factors, suggests that there is an immunological component to the progression of the disease[13].

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Figure 3: The Cu/Zn superoxide dismutase enzyme (SOD1 enzyme).
Mutations to this enzyme result in neurotoxicity to motor neurons.
Adapted from Wikimedia Commons.

2.1 Genetics

2.1.1 SOD1

ALS was shown to be strongly correlated to mutations in the SOD1 gene on chromosome 21in the early 1990’s[14]. This gene is involved in the production of the Cu/Zn superoxide dismutase enzyme, an antioxidant protein whose function is to remove harmful free radicals that are a by-product of cellular metabolism. Recent studies have demonstrated that the neurodegenerative effects of the SOD1 mutation can be impeded by silencing the mutant SOD1 gene using interfering RNA (RNAi)[15]. This suggests two significant facts pertaining to the etiology of ALS: that mutated SOD1 activity is directly correlated with the expression of ALS, and that the conferred toxicity of the mutation is the consequence of a gain of function rather than a loss of function.

2.2 Implication of Microglia

Microglia are a type of macrophage and are the local immune cells in the central nervous system that are responsible for protection of both the brain and spinal cord. Microglia comprise 10-15% of the glial population of the central nervous system, and respond to inflammation and pro-inflammatory agents. Recent investigations into the immunological component of ALS suggest microgliosis as a mediative mechanism that connects the SOD1 mutation to the degeneration of motor neurons. A observable feature of ALS is a high incidence of microglia in areas of motor neuron death. The current literature holds that microglia are “activated” in the instance of a SOD1 mutation, which invokes a gain of function, bringing them into a state in which they exert toxic effects upon motor neurons, subsequently leading to their death.

2.2.1 Microgliosis

Microgliosis, or the accumulation of microglia at the site of neuronal damage or inflammation, has been shown to occur in all sorts of neurodegenerative diseases[16]. Both microgliosis and microglial activation in brain tissue and the spinal cord have been shown to occur to a far greater degree in cases of ALS when compared to a non diseased phenotype, where instances of microglia expressing the disease state increase with disease progression[17]. Genetic analyses have also revealed that, specifically, microglia with a SOD1 mutation are harmful to neuronal health, as they infiltrate motor neuronal axons during disease progression and confer toxicity, providing evidence of a correlation between the mutation and neuronal death[18]. Furthermore, activated microglia in ALS display a distinctly different phenotype and transcriptional profile when compared to the non-mutated form. Accumulated SOD1 microglia express upregulated neurotoxic factors which contribute to the damage they exert on motor neurons. Upregulated microglial neurotoxic factors in ALS include Mpm12, which is associated with increasing toxicity in the end stage of the disease, Optineurin (Optn), which activates apoptotic an inflammatory pathways, and NADPH Oxidase (Nox2), which is responsible for creating superoxide free radicals from NADPH electrons, whose buildup results in severe toxicity. ALS microgliosis is also correlated with an increase in cytokine-related gene activity. Tumour Necrosis Factor alpha (TNFα) and various forms of Interleukin are among those whose activity is greatly increased, further facilitating the dysregulated immune response[19]. These cytokines’ primary function is the activation of immune cells and the stimulation of apoptosis and inflammatory responses. These factors suggest that SOD1 is able to exert its toxic effects through the accumulation of activated microglia around motor neurons, and by up regulating factors that promote neuronal deterioration. Furthermore, these toxic effects elicit further cytokine release and aggravation of the inflammatory response through activation of more microglia. Indeed the amount of activated microglia in the CNS is indicative of the progression and severity of the disease[20].

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Figure 4: Higher levels of mutated SOD1 are shown to be correlated with progressed ALS,
with the amount of mutated SOD1 (shown in red) being positively correlated with disease severity
and the amount of activated microglia.
Adapted from Chiu et al 2013.

2.2.2 Microglial Activation

Under normal conditions, microglia are quick to become activated and alter gene expression in response to environmental imbalances, and are able to confer either degenerative or trophic factors to nearby cells in a tightly regulated manner, depending on the stimuli responsible for their activation. Dysregulation of this system results in uncontrolled activation of microglia, and progressive decline into neurodegenerative disease states. Little attention however, has been paid to the mechanisms through which microglia are initially activated, especially in instances of ALS. Recent evidence suggests that activation is mediated by complex cytokine interactions and the inflammatory effects of purinergic P2 receptors[21]. TNFα and IL-6

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Figure 5: When compared to the non-diseased state,
levels of TNFα are significantly higher in instances of ALS,
suggesting an immune response.
Adapted from Parisi et al. 2013

One characteristic component of ALS is increased levels of pro-inflammatory cytokines such as TNFα. TNFα serves to exacerbate the inflammatory immune response by activating additional microglia. Recent evidence however, suggests that the mechanism of microglial activation is dependent on a balance of TNFα and IL-6 levels. In non ALS microglia, IL-6 activates the STAT3 pathway which confers a neurotrophic effect to help reduce the inflammatory response, repress further TNFα release and promote cell health[22]. However, in ALS microglia, in the over abundance of TNFα, this pathways is inhibited by TNFα which thus facilitates its own transcription[23]. This leads to a dysregulated cycle of TNFα and exacerbation of the immune response through further activation of microglia. This complex interplay of cytokine activity mirrors the potential for an inflammatory response to be both damaging as well as beneficial, with the immunological component of ALS arising from a deregulated system that comes from a toxicity that is conferred by the SOD1 mutation and culminates in an abnormal expression of cytokines in the central nervous system. Thus, forming an ever-worsening cycle of activation and degeneration. P2X7r

Activated ALS microglia also show an upregulation of P2X7r, an ionotropic purinergic P2 receptor that binds ATP and produces many pro-inflammatory downstream effects[24]. Specifically, activation of P2X7r in ALS microglia results in upregulation of the transcriptional facilitators of TNFα, along with other pro-inflammatory factors. This feeds forward into the previously mentioned dysregulation of cytokine balance by further facilitating toxicity conferred by microglia as well as increasing TNFα production, and a greater rate of microglial activation as a result. Concurrent with this evidence, P2X7r activity is positively correlated with disease progression[25].

2.3 Role of miRNA

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Figure 6:
The mechanism of interaction between miR-365 and miR-125b in the IL-6/STAT3 pathway.
Repression of the IL6/STAT3 pathway increases the activation of TNFa gene transcription.
TNFa is able to upregulate miR-125b, thereby enhancing its own transcription.
Adapted from Parisi et al. 2013.

Current emerging research suggests that the timing and control of cytokine equilibrium and activation of microglia in ALS is fine tuned by miRNA activity which can act as a repressor or activator of gene transcription[26]. In particular significance to the TNFα, IL-6, STAT3 system, it has been shown that the transcription of each is mediated by the interactions of miR-365 and miR-125b. Over expression of miR-365 and miR-125b is caused by activation of the microglia via TNFα. miR-125b inhibits STAT3 mRNA, resulting in its decreased production, while miR-365 inhibits IL-6 mRNA, resulting in its reduced expression in ALS microglia. Together, the effects of these miRNA serve to inhibit transcriptional repression exerted on TNFα, resulting in its increased production and initiation of the microglia into the dysequlibrated state of abnormal cytokine production that is indicative of its activation and neurotoxicity in ALS[27]. Furthermore, activation of P2X7r is able to upregulate TNFα production because activation of the receptor serves to further upregulate miR-125b, which severs to further suppresses the IL-6/STAT3 pathway and directly allow TNFα to facilitate its own production, thus exacerbating the immune response and further activating more microglia[28].

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