**5. ALS: neuroinflammation and neurodegeneration**

proinflammatory cytokines such as IL-6 and TNF-α secreted by microglia and astrocytes,

In clinical studies, comparative analyzes were performed in the brains of cognitively normal patients chronically using NSAIDs over age versus those not using NSAIDs that revealed no changes in the appearance of senile plaques, but there was a threefold decrease in the number of activated microglia in the brains of chronic users of NSAIDs [65]. AD patients who used NSAIDs compared with another group of patients who did not use NSAIDs showed a significantly slower progression of disease [66]. These findings are correlated with the above and suggest that the protection provided by the chronic use of NSAIDs in AD patients may be

Despite all these favorable results, we cannot overlook the fact that clinical trials of NSAIDs for patients with cognitive impairment and AD did not show clear results, and the observed effects vary depending on the cognitive instrument that is used. For example, the results indicate that the NSAID naproxen reduced cognitive decline in some patients but caused acceleration in cognitive decline in other patients. Conversely, celecoxib (another NSAID) appears to have similar, but attenuated effects in AD patients [67]. Therefore, it is still prema‐ ture to make clinical recommendations, despite the positive results. However, positive findings open new avenues of research with significant clinical potential in order to develop an effective treatment for AD and other diseases with neuroinflammatory components.

On the other hand, as a result of the lack of efficacy of current treatments for AD, and based on the positive results obtained in patients taking anti-inflammatory drugs, a new possibility has opened up the study of the association of inflammatory processes and pathophysiology

A new form of prevention against the neuroinflammatory process, and thus also an interesting way to prevent neurodegenerative brain damage, is based on changes in diet and the con‐

An interesting example of such food supplements is a new naturally occurring compound with high concentrations of antioxidants and anti-inflammatory properties called Andean Com‐ pound (initially called as Shilajit Andino). The Andean Compound is a very complex mixture of humic substances, generated by the decomposition of ancient plant material; it is originated as an endemic natural product of the Andes Mountains. Its main active principle is fulvic acid [68]. According to studies by Cornejo et al., fulvic acid is able to block tau self-aggregation affecting the length and morphology of PHFs generated *in vitro*, projecting as a good support for the treatment of AD. Also, after exposure of preformed tau fibrils to fulvic acid, a decrease in the length of PHFs can be detected [69]. So, this compound emerge as a novel nutraceutical

The formation of tangles has been identified as a key and convergent event among many of the factors involved in the neurodegenerative process. Our multidisciplinary research group is currently working on a new nutraceutical containing Andean Compound plus B vitamins (B6, B9, and B12 vitamins) named Brain-Up 10®. Patients who have participated in a pilot clinical trial showed a trend toward lower cognitive impairment, a reduction in neuropsy‐

sumption of nutritional supplements, functional foods, and nutraceuticals.

with potential uses against neurodegenerative brain disorders [69].

derived at least partially from the attenuation of microglial activation [58].

avoiding proinflammatory activity of these cells [27, 64].

of AD.

26 Update on Dementia

ALS is another neurodegenerative disease whose hallmark is a combination of degeneration of upper motor neurons in the brain stem and motor cortex and lower motor neuron death from spinal cord. This causes progressive muscle atrophy and paralysis, leading to death of the patient 3–5 years after the diagnosis. Although there are some variations, it is considered a late-onset disease, because visible symptoms appear around 55–60 years, including weakness in hands or legs, speech difficulties, and dysphagia [70]. The global incidence of ALS is 2–3 per 100,000 people, affecting more men than women [71]. The primary mechanism of disease still remains unknown, although there is evidence of calcium deregulation, mitochondrial damage, RNA alterations, protein misfolding and aggregation, ROS imbalance, and inflam‐ mation, among others [72, 73].

There are two subtypes of ALS: sporadic (sALS) that represents between 90 and 95% of total cases and familial ALS (fALS) that represents the remaining percentage (10–5%). It has been reported that fALS can be triggered by mutations in more than 24 different genes, associated with very diverse cellular functions. Superoxide dismutase 1 (SOD1) has been the most characterized gene, accounting for ∼20% of total of fALS cases with more than 150 different mutations associated with the disease [74, 75]. Transactive response DNA binding protein 43 (TDP-43) is another remarkable gene in the disease, affecting both fALS and sALS [76, 77]. This protein is also linked with the development of frontotemporal dementia (FTD), both diseases sharing the deposition of TDP-43. This protein was identified as a major component of the ubiquitinated neuronal cytoplasmic inclusions deposited in cortical neurons in FTD and in upper and lower motor neurons in ALS, coinciding with an overlap in clinical development of FTD with ALS. This kind of overlapping syndrome may be expected since both diseases affect neurons in frontal cortex [78, 79]. In recent years, there are many reports of hexanucleo‐ tide repeat expansions in the chromosome 9 open reading frame 72 gene (C9ORF72) that has also been associated with FTD and ALS, being present in around 30% of familial cases [80].

Recent evidence suggests that motor neuron degeneration in ALS is not an autonomous process; instead it includes astrocyte and microglia participation as discussed below. The observation that nonneuronal cells contribute to neuron death in transgenic model of mice carrying SOD1G37R mutation, was broadly supported by different groups that saw the same effect on *in vitro* studies observing that astrocytes from human ALS patients and transgenic SOD1MUT mice induce motor neuron death [81–84]. In addition to astrocytes, an active contribution of microglia expressing SOD1MUT was evidenced in motor neuron degeneration [85] and recently was demonstrated that microglia rather than astrocytes induce neuronal death through NF-κB, major regulator of inflammation in SOD1G93A mouse model [86].

#### **5.1. Inflammation and neuroimmunomodulation: microglial signs**

A lot of evidence, from animal models as well as patients from familial and sporadic cases, has been observed related to microglia involvement in ALS pathogenesis. In the last time, the microglia role in ALS went from being considered as a consequence of the pathogenic process to being considered as a key factor in the progress of disease, existing two different stages associated to opposite functions of microglia: first in a protective mode in early stages of disease and a later stage with neurotoxic participation [87]. In lumbar spinal cord from 11 weeks old (disease onset) SOD1G93A mice, microglial cells show an M2 phenotype and improve motor neuron survival, while microglia from end stage SOD1G93A mice display an M1 phenotype producing motor neuron death [88, 89]. Anti-inflammatory profile in ALS is documented by release of cytokines such as interleukin 4 (IL-4) and neurotrophic factors such as insulin-like growth factor 1 (IGF-1) and significantly increased expression in microglia from spinal cord of presymptomatic SOD1G93A mice [90]. Recently, through a technique that allows the *in vivo* following of activated microglia in SOD1G93A and SOD1G37R ALS mice, the overexpression of IL-10, an important regulator that would control the anti-inflammatory profile in the pre‐ symptomatic stage of disease, was demonstrated [91]. On the other hand, proinflammatory phenotype in ALS is evidenced by the increased expression of interleukin-1 beta (IL-1β) and tumor necrosis factor α (TNF-α) in spinal cord of SOD1G93A of advanced stages of disease [92– 94]. Another consequence of neuroinflammatory process is ROS release and, in microglia from spinal cord of SOD1G93A mice, is that genes of enzymes that regulate the nitric oxide production, Arg1 and iNOS, are upregulated [95] contributing with more evidence to support the neuro‐ inflammatory theory for ALS pathogenesis. Moreover, astrocytes from ALS murine models including SOD1G37R and SOD1G93A have shown an increase in the expression of proinflamma‐ tory genes too, as diverse interleukins (IL-1β; IL-18), prostaglandin E2, interferon gamma (interferon-γ), and TNF-α, among others, which could also potentiate the activation of microglia, participating in a vicious circle [96–98].

Meanwhile, in ALS patients, microgliosis also has been seen in the ventral horn of spinal cord, together with T cells near to corticospinal tract, in CSF, and in other regions of central nervous system at autopsy [99]. In addition to that, through a new technology used in other neurode‐ generative diseases, such as AD or Huntington disease, which utilizes a specific ligand for positron emission tomography (PET) that detects only activated microglia, *in vivo* microgliosis was observed in diverse areas of the brain such as motor and dorsolateral prefrontal cortex and thalamus, in a heterogeneous population of ALS patients, existing a correlation between the intensity of microgliosis and disease progression [100–102].

In AD, it has been demonstrated that after neuronal death, aggregated tau can induce micro‐ glial activation and generate a neuroinflammatory cascade resulting in the expression of damage signals [28], surging the possibility that in ALS, SOD1 and TDP43 aggregates (hall‐ mark of disease) could have a similar effect on inflammatory process. However, recent evidence shows that, in fact, inflammatory process through LPS and TNF-α stimulation induces the formation of TDP43 aggregates and its mislocalization in a motor neuron cellular model and primary culture of microglia and astrocytes from hTDP43A315T transgenic model, as in spinal cord from the same mice [103], presenting new data to this possible vicious cycle between neuroinflammation and aggregates in the disease.

#### **5.2. Other microglial evidence**

[85] and recently was demonstrated that microglia rather than astrocytes induce neuronal death through NF-κB, major regulator of inflammation in SOD1G93A mouse model [86].

A lot of evidence, from animal models as well as patients from familial and sporadic cases, has been observed related to microglia involvement in ALS pathogenesis. In the last time, the microglia role in ALS went from being considered as a consequence of the pathogenic process to being considered as a key factor in the progress of disease, existing two different stages associated to opposite functions of microglia: first in a protective mode in early stages of disease and a later stage with neurotoxic participation [87]. In lumbar spinal cord from 11 weeks old (disease onset) SOD1G93A mice, microglial cells show an M2 phenotype and improve motor neuron survival, while microglia from end stage SOD1G93A mice display an M1 phenotype producing motor neuron death [88, 89]. Anti-inflammatory profile in ALS is documented by release of cytokines such as interleukin 4 (IL-4) and neurotrophic factors such as insulin-like growth factor 1 (IGF-1) and significantly increased expression in microglia from spinal cord of presymptomatic SOD1G93A mice [90]. Recently, through a technique that allows the *in vivo* following of activated microglia in SOD1G93A and SOD1G37R ALS mice, the overexpression of IL-10, an important regulator that would control the anti-inflammatory profile in the pre‐ symptomatic stage of disease, was demonstrated [91]. On the other hand, proinflammatory phenotype in ALS is evidenced by the increased expression of interleukin-1 beta (IL-1β) and tumor necrosis factor α (TNF-α) in spinal cord of SOD1G93A of advanced stages of disease [92– 94]. Another consequence of neuroinflammatory process is ROS release and, in microglia from spinal cord of SOD1G93A mice, is that genes of enzymes that regulate the nitric oxide production, Arg1 and iNOS, are upregulated [95] contributing with more evidence to support the neuro‐ inflammatory theory for ALS pathogenesis. Moreover, astrocytes from ALS murine models including SOD1G37R and SOD1G93A have shown an increase in the expression of proinflamma‐ tory genes too, as diverse interleukins (IL-1β; IL-18), prostaglandin E2, interferon gamma (interferon-γ), and TNF-α, among others, which could also potentiate the activation of

Meanwhile, in ALS patients, microgliosis also has been seen in the ventral horn of spinal cord, together with T cells near to corticospinal tract, in CSF, and in other regions of central nervous system at autopsy [99]. In addition to that, through a new technology used in other neurode‐ generative diseases, such as AD or Huntington disease, which utilizes a specific ligand for positron emission tomography (PET) that detects only activated microglia, *in vivo* microgliosis was observed in diverse areas of the brain such as motor and dorsolateral prefrontal cortex and thalamus, in a heterogeneous population of ALS patients, existing a correlation between

In AD, it has been demonstrated that after neuronal death, aggregated tau can induce micro‐ glial activation and generate a neuroinflammatory cascade resulting in the expression of damage signals [28], surging the possibility that in ALS, SOD1 and TDP43 aggregates (hall‐ mark of disease) could have a similar effect on inflammatory process. However, recent evidence shows that, in fact, inflammatory process through LPS and TNF-α stimulation

**5.1. Inflammation and neuroimmunomodulation: microglial signs**

28 Update on Dementia

microglia, participating in a vicious circle [96–98].

the intensity of microgliosis and disease progression [100–102].

Other possible link between microglia, neuroinflammation and ALS corresponds to hemi‐ channels. The communication between glial cells mainly occurs through gap junctions (GJ) [104]. These are intercellular channels that connect the cytoplasmic compartment of neighbor‐ ing cells, allowing the pass of ions and small molecules up to 1000 Da [105, 106]. Every GJ is composed of two hemichannels, and each hemichannel is formed by six subunit proteins called connexins [107, 108]. In general, hemichannels are closed in physiologic states; however under pathologic conditions, they present a higher activity and opening, which could be triggered by metabolic inhibition, inflammatory mediators, or connexin mutations [109, 110]. In addition to that, it has been proposed that in pathologic environments, activated microglia is capable of releasing proinflammatory molecules that increment the opening of hemichannels, reducing the communication between astrocytes; depriving neurons of protective role of glia and reduce the neuronal viability [109]. Otherwise, different inflammatory treatments such as TNF-α and interferon-γ enhance connexin-43 (Cx43) expression in activated microglia, establishing a possible mechanism of activation after inflammatory stimulus in ALS [104].

In AD, it has been observed that exposition of amyloid-beta peptide increases the activity of hemichannels in astrocytes, microglia, and neurons and that hemichannel blockers prevent death of hippocampal neurons [111, 112]. It was also demonstrated that a hemichannel blocker, capable of crossing the blood-brain barrier, INI-0602, alleviates AD symptoms in a transgenic model of disease [112].

In ALS, although there are few antecedents about hemichannels and the disease, the same publication shows that SOD1G93A transgenic mice treated with the INI-0602 blocker increment‐ ed the life span in comparison to the nontreated group, preventing axonal lost and diminishing the atrophy and improving muscular size [112].
