Currently, around 58 genes related to ALS have been
described. Of these 58 related genes, 32 are considered “main” or “causative”
genes; 7 were related as phenotype alterers, and 19 genes were related as
susceptibility genes [17,18]. The causative genes’ pathways are distinct; some
are related to mRNA processing, oxidative stress, endosome traffic, cell
signalling (VAPB, protein degradation pathways, AND chromatin remodelling [19].
SOD1 (Superoxide dismutase 1) Nowadays, more than 180 pathogenic mutations in
the superoxide dismutase gene (SOD1) have been described in patients with
Amyotrophic Lateral Sclerosis [20]. A great diversity of clinical evolution is
also observed among these genetic variants. For example, patients with the A4V
heterozygous mutation quickly develop an aggressive form of ALS, leading to
death within about a year [21]. Other variants, however, appear to have milder
phenotypic manifestations, such as the D90A homozygous mutation. This, which
causes an autosomal recessive form of ALS, evolves in a slower progression,
which can take up to a decade [22].
Some forms of ALS result from a mutation in the gene
encoding the antioxidant enzyme Cu+2/Zn+2 superoxide dismutase (SOD1). This
gene is located on chromosome 21 (21q22.1), spans 11 kb in length in
chromosomal DNA, and consists of 5 exons interrupted by four introns. The exons
encode a protein of 153 amino acids [23]. This protein is located in the
cytoplasm, nucleus, lysosomes, and mitochondrial intermembrane space. It has
the function of capturing copper and zinc ions and forming a homodimer, in
which it will carry out the dismutase function, removing dangerous superoxide
radicals and metabolizing them into molecules of oxygen and hydrogen peroxide,
which are converted into water and oxygen by the enzymes glutathione peroxidase
and catalase [24]. The SOD1 protein is highly conserved: horizontal gene
transfer is programmed to occur early in eukaryotic evolution. Indeed, the
human SOD1 protein is at least 50% homologous with non-human SOD1 proteins from
other mammalian species. The three-dimensional structure of the SOD1 protein is
very similar to that of immunoglobulin: the folding pattern (Greek key) in SOD1
resembles the hypervariable region (antigen binding) folds present in
immunoglobulin [23]. In different populations, the proportions are 12% to 23%
of patients diagnosed with Familial ALS and 2% to 7% of sporadic ALS carriers
carrying the mutation in the SOD1 gene. Due to the production of altered SOD1
proteins originating from these mutations, the protein encoded by the wild-type
allele has regular activity but is reduced in patients with Familial ALS.
Mutant SOD1 enzyme activity levels in Familial ALS patients are generally
reduced by 25.3% to 93% of the activity compared to normal individuals.
The consequence of
this is that the altered protein cannot wholly remove superoxide radicals, and
the oxidative stress generated by these superoxide radicals plays a role in the
toxic effect on neurons. Ticozzi presents studies that show that mutant SOD1 is
prone to incorrect folding and formation of cytoplasmic aggregates, and, in
turn, these aggregates can lead to cell death by kidnapping other cytoplasmic
proteins essential for neuronal survival by overloading and blocking the system
ubquitin/proteasome, by mitochondrial disruption, cytoskeletal disruption or
axonal transport [25-26]. Among the gain-of-function theories of how the mutant
SOD1 gene contributes to motor neuron death associated with Familial ALS, four
main hypotheses have been postulated: hydroxyl radical (OH-) toxicity,
nitration toxicity, copper toxicity, and aggregation toxicity [23]. However,
the exact mechanism by which SOD1 mutations lead to ALS pathologies is unknown,
although numerous hypotheses have been proposed to explain the mediation of
mutant SOD1 with toxicity, such as folding protein associated with aggregation,
oxidative stress, mitochondrial dysfunction, endoplasmic reticulum stress,
glutamate excitotoxicity, microglial inflammation and activation, and
abnormalities in axonal transport [27]. SOD1 dismutase activity depends on the
formation of homodimers associated with Cu+ and Zn2+ ions. Functional studies
with proteins carrying pathogenic alterations, such as C6S, N90A, and A89V,
demonstrated that their enzymatic activity is preserved in some mutant models.
This suggests that the loss of protein function does not play a significant
role in the neurodegenerative process of SOD1 in ALS [28]. These mutations are
believed to cause a toxic gain of function in the protein. Its progressive
accumulation in the cytosol would interfere with several cellular processes,
such as vesicle traffic and mitochondrial activity, leading to cell death
[29,30]. Initially, the hypothesis arose that the mutations could impair the
protein’s enzymatic activity, resulting in increased cellular levels of types
of reactive oxygen, oxidative stress, and neural death [25]. According to
Oliveira and Pereira26, 20% of mutations in Familial ALS in SOD1 cause folding
protein and formation of intracellular inclusions. Oxidative stress induces
SOD1 normally to monomerize as an intermediate in the aggregate formation
process, and it is known that SOD1 does not have this superoxide activity in
this way when mutated. Motor neurons are known to be particularly sensitive to
oxidative stress, which makes this process potentially more expressed in this
cell type. Specific changes in SOD1 messenger RNA half-life and the effect of
SOD1 aggregation and monomerization on superoxide activity raise the
possibility of decreased SOD1 activity in affected neurons. Six of the SOD1
mutations in patients with familial ALS (A4V; L38V; L106V; I113T; L144F, and
V148G) are likely to destabilize the folding loop subunit or contact dimers,
changing the structure of standard proteins [23]. The most frequent mutation in
the SOD1 gene is D90A. This is an enigmatic mutation because it keeps Cu+2/Zn+2
-SOD1 erythrocytic activity practically every day and has intact specific
activity and preserved stability under denaturing conditions. While some cases
are heterozygous for the D90A mutation, with dominantly inherited lineages and
an aggressive and variable phenotype, all patients are homozygous for the D90A
mutation, which in most cases is inherited as a recessive trait, have shown the
same phenotypic feature of slow ascending paresis. , starting asymmetrically at
the lower extremities. At four years, the first distal symptoms appear in the
upper extremities, along with the first bulbar symptoms. Months or even years
precede the onset of paresis in the lower extremities. Other atypical features
in homozygous D90A patients include bladder abnormality, the urgency to void
and difficulty initiating voiding, the periodic feeling of heat, and
significantly prolonged central motor latency recorded after transcranial
magnetic stimulation [26-31]. The superoxide activity of SOD1 can be measured
in two ways: by the intrinsic activity of SOD1, which reflects the enzymatic
efficiency of the protein, and by the measure of the recombinant activity of
the SOD1 protein normalized for its quantity. This activity is measured in at
least eight protein mutations, giving various results ranging from 0 to 150% in
the wild-type human allele for SOD1 activity; or by the complete activity
within the tissue sample, which can be affected by various factors in the
cellular environment, and this is obtained by the regular superoxide activity
for the tissue amount. This activity is an unbiased measure that takes into
account known and unknown influences on SOD1 enzymatic activity. Intrinsic
activity influences the complete training, but only 8 in one of the
determinants; the others are any factors that affect the quantity, biological
availability, and functionality of SOD1 [24]. SOD1 activity is generally
reduced by half in patients with Familial ALS, as measured in red blood cells,
lymphoblasts, and fibroblasts. Indirect evidence raises the possibility that a
severe reduction may occur in susceptible tissues and specific cell types due
to the reduced half-life of the mutant SOD1 messenger RNA in the Central
Nervous System and the possible effects of folding and aggregation of the SOD1
protein. SOD1 activity is regular or only slightly reduced in two mutations:
D90A in both homozygous and heterozygous patients; and L117V in heterozygous
patients, although the measurement of a homozygous patient showed a 67%
reduction in SOD1 activity when compared to control subjects [24]. Mutations in
SOD1 are characterized by an important variability of interfamilial and
interfamilial phenotypes concerning age and place of onset of clinical
manifestations and disease duration. An exception is the A4V mutation, which is
most often seen in lineages of ALS1 cases and consistently associated with high
penetrance, younger ages at onset, the prevalence of lower motor neuron signs,
and very rapid disease progression, usually in 12 months [25]. The penetrance
of SOD1 mutations is variable, being almost complete for A4V and less than 30%
at age 70 for I113T. However, most of the variants described so far are private
mutations. Thus, few of them can safely describe the genotype and phenotype
correlation. There is no specific therapy for patients with a mutation in the
SOD1 gene. Still, there are many ongoing studies to develop new techniques
(RNAi, antisense therapy) for the inactivation of this mutation, preventing the
cytotoxic synthesis caused by this gene. In the area of immunotherapy, an
antibody called SEDI (SOD1-Exposed-Dimer-Interface) was developed, with peptide
sequencing corresponding to the dimeric interface of SOD1, which recognizes the
enveloping or monomeric SOD1 proteins with this exposed interface [26]. In
patients with ALS, regardless of age at disease onset, increased DNA
methylation in whole blood was observed, constituting a marker of epigenetic
dysfunction in ALS [27]. This high global DNA methylation is also seen in
carriers of SOD1 mutations not fully penetrants (p.Asn65Ser, p.Gly72Ser,
p.Gly93Asp, and p.Gly130_Glu133del), contributing to the ALS phenotype [28].
VAPB (Vesicle-associated
membrane protein-associated protein B/C)
VAPB consists of a multifunctional protein originating
from a family (VAP) of vesicles associated with a protein membrane related to
endoplasmic reticulum proteins. VAP proteins (VAPA, VAPB, and VAPC) and their
homologs seem to be involved in different cellular processes, such as
intracellular traffic and signalling, microtubule organization, mitochondrial
localization, calcium homeostasis, lipid metabolism, and tumour proliferation
[30]. The VAPB is composed of 3 domains the Major Sperm Protein, the MSP is
composed of the first 150 amino acids, and the p56s mutation is found [31]. The
VAP proteins are expressed ubiquitously and located between the endoplasmic
reticulum region and the Golgi complex. They are involved with several
molecular pathways, and some are directly related to the activities of motor
neurons. The VAPB Protein has 16 highly conserved amino acids in its MSP domain
[17]. The VAPB gene is located on chromosome 20q13.3, covers 57.7 kb in length
in genomic DNA and is composed of six exons, and encodes the protein VAMP
(Vescicle-associated membrane protein-associated protein B). It is an integral
protein of the endoplasmic reticulum membrane, which has several functions,
such as in intracellular vesicle trafficking, lipid transport, and protein
unfolding response. These proteins are associated with intracellular membranes,
including both the endoplasmic reticulum and the Golgi apparatus [25-33]. The
VAPB gene encodes a protein located on the outer surface of the endoplasmic
reticulum (ER). This comprises two coiled-coil domains, an MSP domain (Major
Sperm Domain) and a transmembrane domain, which anchors it in the ER membrane
[33]. Interestingly, the VAPB protein has been described as located at sites of
contact between the ER. ER and other organelles, especially mitochondria,
endosomes, and lipid vesicles. It is through these intersections that different
cellular processes take place, such as autophagy, calcium flux regulation,
endosome maturation, unfolded protein response, and protein synthesis [34-36].
Mutations in the VAPB gene have been primarily associated with an autosomal
dominant familial subtype of Amyotrophic Lateral Sclerosis, ALS8. Patients with
this condition presented, in their initial description, two distinct
manifestations of ALS, classified as “typical” and “atypical” ALS,” which were
distinguished by the presence of tremors only in the atypical form. In
addition, manifestations of spinal muscular atrophy were observed among some
individuals in the genealogy with a mutation in the VAPB gene. This phenomenon
reinforces the tremendous phenotypic variability already described in the
phenotypes associated with ALS and suggests that VAPB plays a fundamental role
in eminently spinal circuits [31-37]. A mutation that causes the replacement of
proline 56 by serine in the MSP domain (P56S) disrupts the three-dimensional
structure and favours the aggregation of this protein. Data suggest that this
mutation is responsible for a variable form of motor neuron diseases found in
several families, mainly in Brazil. Due to the interaction of VAPB with other
proteins, the mutation can evolve into a less stable interaction of endoplasmic
reticulum proteins with at least two other proteins: tubulin and GAPDH [25-31].
The VAPB gene acts during transport and secretion in the Endoplasmic Reticulum
(ER) and the Golgi complex. The P56S mutation may disrupt this function leading
to the accumulation of transport intermediates in the form of cytosolic
membranous aggregates. However, expression of the mutant form of VAPB does not
alter the structure of the ER, and the possibility that alterations in the
membrane system of the Golgi complex and ER are occurring in cells that express
the altered form of VAPB is not ruled out. Thus, the VAPB mutant protein can
compromise intracellular membrane transport and secretion and lead to loss of
trophic signals or alteration of intracellular processes resulting in motor
neuron death. It is also possible that VAPB is present in distinct structures
of the ER membrane and the Golgi complex and that the mutation affects the
accumulation of proteins in these sites [31]. ALS8 was subsequently discovered
to be caused by this single mutation. Hypotheses suggest that the dominant
inheritance of ALS8 is due to the dominant negative effect of the mutant
protein. VAPB is ubiquitously expressed, yet the P56S mutation affects motor
neurons. This selective vulnerability also occurs with ALS1-SOD1, ALS2-Alsin,
and SETX mutations. Different cell types may not require the same amount of
VAPB for survival, or VAPB may have another, as yet unknown, specific function
in neurons. The P56S mutation can interfere with the stability of the VAPB
protein complex, and a failure or gain-of-function mechanism could result in
neurotoxicity and, consequently, motor neuron death [31]. Mutations in dominant
VAPB lead to VAMP aggregation within immobile clumps in the endoplasmic
reticulum (ER), which causes low protein levels, resulting in a diminished
endoplasmic reticulum with anchored proteins containing lipid bonds and motor
neuron degeneration. After insertion of the protein into the endoplasmic
reticulum membrane, the P56S mutation in the VAPB gene causes the rapid
assembly to generate paired cisternae in the ER, which give rise to a deeply
restructured and non-aggregated domain of cytosolic proteins, which would be
normal [33-38]. In addition to the loss of function mechanism, by sequestering
potentially functional proteins in inclusion bodies, evidence for a toxic gain of
function of mutant VAPB has also been reported. Mutant VAPB inclusions are
ubiquitin-positive in transfected cells and motor neurons from transgenic
animals. When overexpressed, wild and mutant types have been observed to
decrease proteasome activity. This fact suggests that VAPB inclusions can alter
proteasomes and act to alter protein degradation pathways, associated with an
important pathogenic mechanism by the toxicity of malformed protein aggregates
in both sporadic and familial ALS. Another mechanism involved in inhibiting
mitochondrial transport affects the kinesin anterograde motor regulation
[26-38]. So far, most individuals affected by ALS8 are Brazilian, Caucasian,
and carriers of the same mutation in exon 2 of the VAPB gene (c.166C>T; p.P56S
VAPB). A founder effect was then postulated for this mutation in Brazil, which
most likely arrived via Portuguese colonization 25 generations ago [31]. Later,
other individuals with European ancestry but not carriers of the same VAPB
haplotype were described in Germany, suggesting that the P56S mutation also
arose independently. Patients in China, the United Kingdom, and the United
States have also been reported with the P56S variant. More recently, different
mutations in this gene, p.T46I and P56H have also been associated with ALS8. A
third mutation (p.V234I) identified in a 43-year-old Dutch patient with joint
expansions at C9ORF72 was also recently identified [39-40].
Main limitations arising
from Amyotrophic Lateral Sclerosis and therapeutic treatment
It is a disease identified by presenting difficulties
in speech; in view of this, as soon as it is diagnosed in the first stages,
immediate intervention is necessary in order to soften the evolution of the
disease and adapt the treatments to meet the need according to the demand of
each patient [41]. Studies claim that the patient's mobility is impaired as the
disease progresses, compromising their functional performance and leading them
to depend on someone else's care. In this way, living together creates a
powerful bond, thus establishing respect for the patient, and therefore
physical fatigue does not prevent caring for the patient [42]. The main
complications resulting from this pathology are structural and motor, such as
weakness, contractures, and spasticity. It courses with impairment of speech,
swallowing, and breathing muscles. With its progression, the affected person
presents deformities and progressive paralysis, as well as the need for
ventilator support, which is the main cause of death [43]. With its rapid
progression resulting in a loss of the subject's autonomy in carrying out their
simple day-to-day activities, many times they ask for help to complete them, in
the future making them unable to carry them out independently, being forced to
depend on them the care of another person. And with that, there is a change in
the patient's and family's lifestyle and routine [42]. The loss of physical
integrity and the absence of a cure bring with it fears about death; this has
to do with being aware of how the disease progresses, which causes even more
fear and consequently interferes with the patient's quality of life [42]. The
emotional function is slightly related to the well-being of the individual, and
with the discovery of ALS, several feelings come to appear, among them, sadness
and despair are the most common; this has to do with the lack of freedom and
independence caused by the rapid disease progression [42]. There are several
possibilities of support for the patient with ALS; below are the leading
measures adopted in the face of the clinical problem presented.
Sialorrhea:
Symptom that causes social embarrassment, and may progress to aspiration
pneumonia, the most common cause of death in ALS, after respiratory failure per
se. Many patients wear bibs or insert tissues into their mouths to absorb
saliva. The American Academy of Neurology recommends non-pharmacological
measures, such as aspiration/suction, as well as pharmacological measures, such
as anticholinergics, glycopyrrolate, and amitriptyline. The application of
botulinum toxin has emerged as a new therapy against sialorrhea in these
patients, with satisfactory results. In refractory cases, radiotherapy can be
proposed [2-45].
Pseudo-bulbar
effects: Affects between 20-50% of carriers,
especially those with the bulbar form of the disease. These effects include
uncontrollable crying and laughing. Selective serotonin inhibitors, tricyclic
antidepressants, and serotonin-epinephrine reuptake inhibitors may be used. A
new combination of dextromethorphan and quinidine sulphate was shown to be
effective in a multicentre randomized phase 3 trial [2-47].
Sleep
disorders: Anxiety and depression are constant
conditions in ALS patients. Nocturnal hypoventilation also makes up for this
resting difficulty, reducing total sleep time. Postural change and the use of
pneumatic mattresses can help the patient. Mirtazapine is especially effective.
Other benzodiazepines can also be used, as well as hypnotics such as zolpidem
[2-48].
Respiratory
Failure: Patients with ALS can progress to frank
respiratory failure due to loss of strength and tonus of the diaphragmatic and
intercostal muscles, making it necessary for pulmonary assessment of patients
with the entity every three months, especially through spirometry. When the
volume forced expiratory force exceeds 50% of the expected, non-invasive
ventilation should be started [2-53].
Fatigue:
Found in 50-80% of carriers and has a multifactorial etiology, including sleep
disorders, nocturnal complaints such as nocturia and cramps, nutritional
status, vital capacity, depression, and use of medications, including riluzole
[2-56]. Modafinil showed significantly significant results when compared to
placebo in reducing fatigue in ALS patients [57].
Pain:
Symptom described in 60-70% of patients and usually involves extremities, neck,
trunk, and back. Anti-inflammatories, non-opiate agents, opiate agents, muscle
relaxants, quinine, gabapentin, steroids, botulinum toxin, and physical therapy
can be used [2-59].
Spasticity:
It can be a limitation of mobility and function for the patient. Studies
explicitly investigating spasticity in ALS patients are scarce. The most
commonly used drugs for this symptom include baclofen, tizanidine,
benzodiazepines, and dantrolene. Hydrotherapy, cryotherapy, heat, and shock
waves can also be used in muscle spasticity [60,61]. Laryngospasm is understood
as the sudden feeling that air cannot enter or leave the airways, usually
lasting a few seconds and accompanied by inspiratory stridor, audible breaths, and
forced rapid contractions of the laryngeal adductor muscles.
Non-pharmacological measures can help the patient: change to the orthostatic
position, fixation of the arms to stabilize the trunk, nasal breathing, and
repeated swallowing. Benzodiazepines can be used as adjuvants in this therapy
[2-64].
Constipation
and urinary urgency: Symptoms in up to 30%
of patients usually involve the genitourinary and digestive tract. Its
multifactorial etiology is related to reduced mobility, reduced fluid and solid
intake, use of medications, and weakness in the abdominal muscles. Treatment
begins with implementing a diet rich in fiber, liquids, and laxative juices.
Stimulants and laxatives such as Senna, Cascara Sagrada, and Bisacodyl should
be used with care, in low doses, avoiding their chronic use. Lactulose and
polyethylene glycol can be used as osmotic agents. Increased urinary frequency
is very common in ALS patients [65].
Non-pharmacological
measures include: avoiding caffeine and
alcohol and use of a Foley catheter. Anticholinergic drugs such as oxybutynin,
tolderodine, darifenacin, and solifenacin can be used [2-66]. There is no cure
for ALS; therefore, treatment is aimed at minimizing the symptoms, recommending
the action of a multidisciplinary team. Among the main medical approaches to
managing the disease, the administration of medications and/or surgical
approaches are included, whose objective is to minimize the symptoms and
limitations imposed by this health condition. Although speech therapy therapeutic
strategies are indicated for voice, speech, and swallowing management in
patients with ALS, drug and surgical treatments can also impact the mentioned
functions [41]. It should also be noted that the definition of palliative care,
according to the WHO, is an approach that improves the quality of life of
patients (adults and children) and their families who face problems associated
with life-threatening illnesses. As well as, according to a consensus-based
definition, palliative care is active, holistic care for individuals in all age
groups, offering serious health-related suffering resulting from serious
illnesses and, above all, those close to the end of life. This type of
treatment improves the quality of life of patients, family members, and
caregivers. With this, the adoption of palliative care contributes positively
to patients with ALS [67]. It should be mentioned that ALS is currently a
disease without curative treatment. However, a drug proven to be effective in
its treatment (Riluzole) increases the life expectancy of people with the
disease. In contrast, other drugs (i.e., tamoxifen and edaravone) are still
being studied; further analyses are needed to define their effectiveness. Of
these medications [68]. Pharmacological treatment is one of the treatment
possibilities for people with ALS. This approach aims to improve the survival
of these patients and help maintain functions related to communication and
eating, among others. The use of riluzole, for example, can increase the
survival of patients with ALS by up to six months. However, the literature does
not mention direct and positive effects on voice, speech, and swallowing.
Another drug, edaravone, effectively reduces functional limitations in people
at the onset of the disease. Nuexdeta had an effect on improving bulbar
function in patients with ALS, including the effect on self-perception related
to speech and swallowing functions [41]. For the treatment of Riluzole®, it is
the only drug approved by the Food and Drug Administration (FDA). It is a
benzothiazole capable of reducing the toxic action of glutamate on motor
neurons, increasing patient survival by up to 6 months; however, due to unknown
factors, it loses its functionality approximately 18 months after starting
treatment. Symptomatic treatment is indicated for a variety of symptoms and
consists of improving the patient's quality of life [69]. There is no evidence
that riluzole reverses already-established neuronal damage. Patients using it
should be monitored for possible kidney damage, with an elevation of
aminotransferases, nausea and vertigo, granulocytopenia, and asthenia [70].
Finally, palliative therapy is essential for managing patients with ALS, as it
helps prevent complications and promotes a better quality of life [68].
