Biomarkers and molecular mechanisms of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease in adults involving non-demyelinating motor disorders. About 90% of ALS cases are sporadic, while 10–12% of cases are due to some genetic reasons. Mutations in superoxide dismutase 1 (SOD1), TAR, c9orf72 (chromosome 9 open reading frame 72) and VAPB genes are commonly found in ALS patients. Therefore, the mechanism of ALS development involves oxidative stress, endoplasmic reticulum stress, glutamate excitotoxicity and aggregation of proteins, neuro-inflammation and defective RNA function. Cholesterol and LDL/HDL levels are also associated with ALS development. As a result, sterols could be a suitable biomarker for this ailment. The main mechanisms of ALS development are reticulum stress, neuroinflammation and RNA metabolism. The multi-nature development of ALS makes it more challenging to pinpoint a treatment.


Introduction
ALS, like Parkinson's disease (PD) and Alzheimer's Disease (AD), is known as a nondemyelinating neurodegenerative disease, first described by Dr. Jean-Martin Charcot in 1869 [1] This disease is associated with selective and progressive loss of corticosteroid motor neurons and spinal and bulbar motor neurons. As a result, the symptoms of ALS are muscle cramps, weakness, hyporeflexia and ultimately frontotemporal dementia (DFT), and it eventually leads to death.
A study showed that ALS affects 223,000 people worldwide, and this number may increase by 69% in next 20 years [2]. Therefore, having an understanding and knowledge of early biomarkers and patient follow-ups may improve the prognosis of ALS.

Etiology
The etiology of ALS remains an enigma, but several genetic, environmental and pathologic clues hold some promise. One finding is that 5% to 10% of patients seem to have inherited ALS in an autosomal dominant pattern. Some of them-2% of the total ALS patients-carry a mutation of a gene on chromosome 21 (Cu, Zn superoxide dismutase [SOD1]) that normally assists in detoxifying superoxide free radicals [3]. ALS is mostly sporadic, however, familial ALS is linked to monogenic causes, such as mutations in C9orf72, SOD1, or other genes [4,5]. Besides, in a study it was shown that tobacco use can also increase the ALS risk by almost four-fold. Other environmental factors such as heavy metals, ambient aromatic hydrocarbons, pesticides and cyanotoxins, as well as head injury, also appear to be a risk factor for ALS [6][7][8]. It therefore appears that genetic as well as environmental factors together or separately may cause the ALS disease [9-12].

Biomarkers
In fact, there are no such reliable biomarkers of ALS, to date [13]. However, mutations in phosphorylated neurofilament heavy chain (pNfH) were found to be linked to ALS development [14]. In fact, cerebrospinal fluid (CSF) and blood from victims with ALS and other neurodegenerative diseases showed elevated levels of NFs [14][15][16][17][18][19][20]. Neurofilament levels actually rise in the blood and CSF ahead of the appearance of disease symptoms in people carrying a mutation in the SOD1 gene [21]. Levels of both neurofilament light chain (NfL) and phosphorylated neurofilament heavy chain (pNfH) are elevated with poor prognosis in ALS patients [17,[22][23][24]. However, both nonclinical studies with transgenic rodents and clinical studies with familial ALS patients indicate that neuroinflammation and immune dysregulation are related to the pathogenesis and heterogeneity of the ALS disease [6,25].
Further, activated astrocytes, microglia and monocytes were detected in the motor cortex of ALS patients [26]. Similarly, levels of ferritin, creatine kinase, interleukins, and TNF-, in plasma of ALS patients were elevated compared to controls, pointing towards the T-cell-affected neuromuscular pathology in ALS [27]. In addition, C-reactive protein (CRP), an inflammation marker is also elevated in the serum of Pre-ALS and correlates with rapid progression of the disease [28]. Table 1 displays the different biomarkers that are related to different phenotypic abnormalities found in ALS.

Genetic factors in ALS
More than 20 genes have been described for familial ALS (fALS) cases. However, those gene products are very different in their functions and make it difficult to find a clue for the onset of ALS disease. In most cases, the cause of sALS is not known, but it generally starts at an older age [9-11]. Several fALS genes such as SOD, TDP-43, FUS and C9ORF72 have also been reported in sALS cases [74].

Other Rare Occurring Mutant Genes in fALS:
 A missense mutation in the D-amino acid oxidase (DAO) gene has been reported in several families with ALS disease [75]. DAO mutations decrease the cell viability, increase the ubiquitinated aggregates and enhance the apoptosis of primary motor neurons in culture [75,76].
 In one case, a genetic subtype ALS7 is found to be linked to chromosome 20ptel-p13 and shows the signs of onset of fALS [10]. Tables 2 and 3 display the responsible genes involved for fALS and sALS disease, respectively.

Molecular mechanisms of amyotrophic lateral sclerosis
The common ALS genes, listed in Fig. 1, define three primary actions in ALS pathophysiology:  Protein conformational instability and its degradation: Loss of antioxidant defense (SOD 1 function) causes the accumulation of free radicals and generates oxidative stress [78,88]. Aggregation of proteins, SOD1 [present only in the fALS] [77,129], TDP-43 [130], FUS [131,132], Optineurin (Optn), Ataxin-2 and Ubiquilin-2 [129] are involved in causing ALS.  Impaired trafficking of RNA: Mutation of multiple ALS genes showed disturbances in RNA-binding proteins, RNA synthesis, its function and metabolism. Mutations in the TDP-43, FUS and C9orf72 genes develop stress granules in the cytoplasm, toxicity to neurons and disturbance of the splicing activity [133].  Altered axonal and cytoskeletal biology: Cytoskeletal dynamics are altered in ALS.
Mutations in profilin-1 are likely to impair energy-dependent extension of filamentous actin and elongation of growth cones, a process that is enhanced by a reduction in signaling from ephrin A4. Tubulin mutations compromise the structure of microtubules. Mutations in dynactin are predicted to impair retrograde transport along the microtubule backbone. All those above disturbances culminate to multiple secondary, downstream pathologic processes, including activation of endoplasmic reticulum (ER) stress and autophagy, proteasomal as well as mitochondrial dysfunction, disturbed axonal transport, altered dendritic morphology and excitotoxicity.
 Reticulum stress: It is induced by the accumulation of abnormal proteins due to mutations of SOD1 in ALS [134,135].  Structure and Functioning of Mitochondria: Alterations in the vacuolization and mitochondrial swelling decreases in the activity of the respiratory chain and causes ALS [136].  Glutamate Excitotoxicity: Glutamate is a powerful neurotransmitter, synthesized at the presynaptic terminal and is diffused to activate post-synaptic neuron AMPA (α-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid) and NMDA (N-methyl-D-aspartate). In ALS patients, glutamate levels were abnormally high in their plasma compared to healthy subjects. This phenomenon may cause neuronal toxicity and cell death in ALS [137][138][139].  Neuroinflammation: As the disease progresses, microglial cells acquire an M1 phenotype and secrete ROS, pro-inflammatory cytokines and neurotoxic molecules, and ultimately promote motor neuron death [140,141]. Therefore the proposed pathogenic mechanisms may include either protein aggregation, oxidative stress, mitochondrial dysfunction, glutamate receptor-mediated excitotoxicity or neuroinflammation [2,4,5,9,10]. In Fig. 1, we have shown by a schematic diagram how and where the genes are involved in ALS pathology. Conformational instability and aggregation of proteins, impaired trafficking of RNA and altered axonal and cytoskeletal dynamics are the primary ones of all the responsible genes mutations These result on multiple secondary, downstream pathologic processes such as activation of endoplasmic reticulum (ER) stress and autophagy, proteasomal excitotoxicity, altered mitochondrial function, disturbed axonal transport, altered dendritic morphology, and neuroinflammation.

Management oof ALS cases
The diverse pathophysiology in ALS limits the treatment strategies for the management of the disease and therefore demands the cohort treatment through neurologists, pneumologists, physiotherapists, nutritionists, etc. FDA (U.S. Food and Drug Administration) has approved, so far, only two drugs to be applied for the treatment of ALS patients. One is Riluzole, one of whose action is to inhibit the release of glutamic acid from neurons, in vivo, and thus blocks the postsynaptic effects [142]. It may be partly due to inactivation of voltage-dependent sodium channels on glutamatergic nerve terminals, as well as activation of a G-protein-dependent signal transduction process or noncompetitive blockade of N-methyl-D-aspartate (NMDA) receptors [142]. In vivo, riluzole actually showed neuroprotective, anticonvulsant, and sedative properties. It improves survival by a couple of months, only [143][144][145][146][147][148], whereas another drug, Edaravone, which is a free radical scavenger, reduces oxidative stress and inhibits neuronal death in animal models [149]. In clinical trial, this drug, Edaravone, showed promising results in decreasing the death rate ofan ALS ALS patient by 35-40% and leading to approval in the United States in 2017 [150].
Very recently (Sept. 2022) the U.S. Food and Drug Administration approved Relyvrio (sodium phenylbutyrate/taurursodiol) to treat patients with fatal ALS disease despite of uncertainty about its effectiveness (https://www.cnn.com/2022/09/29/health/als-drug-relyvrio). Relyvrio targets both endoplasmic reticulum (ER) and mitochondria of motor neurons in ALS patients. Vitamin E (tocopherols and tocotrienols) as an antioxidant can slow down the onset, and also the progression of ALS disease [151].

Discussion
ALS is a neurodegenerative disease that starts due to defective function or non-function of motor neurons in the spinal cord and in the brain. Symptomatically the disease is characterized by progressive muscular atrophy, slow speech, paralysis, swallowing disturbances and respiration problems [152]. In most cases, death occurs typically 3-5 years after the diagnosis of the disease as the failure of the respiratory system becomes prominent, although in some cases survival could be longer [153]. From a genetic point of view, the majority of ALS cases are sporadic (sALS), and approximately 10% of cases can be considered familial (fALS). ALS is a complex disorder, and the biological mechanisms are still not completely understood as it involves different pathways including abnormal RNA metabolism, altered mitochondrial function and regulation of oxidative balance, modulation of neuronal excitability, axonal transport, control of the inflammatory response and protein folding and degradation, in the disease pathogenesis [154,155].
In ALS, as in other neurodegenerative diseases, there is an urgent need for sensitive, reliable diagnostic and disease-progression biomarkers for early detection and treatment of the disease. Peripheral blood inflammatory cytokines as they are increased in other neuro-degenerative disease, cannot be considered as a specific diagnostic marker for ALS.
Many anti-inflammatory molecules have been used against ALS over the past 3 years with some success, but a cure is still far away. The limitations of sample collections for diagnostic marker studies are as follows:  (1) Collection of disease samples and controls should be with the same demographic characteristics  (2) The collection of samples at different days rather than at a single time point on any single day should be better as biomarkers of disease progression.  (3) The sensitivity of the used technique, other than ELISA, should be considered to detect the minimal concentrations of the molecule suspected for the disease. Plasma cytokines are elevated in ALS patients and are still considered as a disease marker for progression and for disease severity [156], however, more knowledge are needed to investigate a possible role of some other inflammatory cytokines those could be used for diagnosis of the disease as well its prognosis. However, blood biomarkers might not reflect the motor neuron defects as those present in the CSF [157]. In fact, the blood-brain barrier could inhibit the crossing of disease biomarkers towards the systemic compartment. Since frequent collection of CSF is hazardous we have to rely on blood samples as an ideal source of biomarkers.

Conclusion
The genetic spectrum of fALS and sALS is heterogeneous. Several genes in ALS are known to cause many other neurodegenerative diseases, such as alsin with primary lateral sclerosis (PLS), and infantile onset ascending hereditary spastic paralysis (IAHSP), senataxin with SCAR1 or AOA2, spatacsin with HSP, VAPB with SMA,