Novel therapeutic targets for amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is an untreatable and fatal neurodegenerative disease that is identified by the loss of motor neurons in the spinal cord, brain stem, and motor cortex which theatrically reduces life expectancy. Although the primary cause of ALS remains unclear, its heterogeneity put forward for consideration of association with various factors, including endogenous and/or environmental ones, which may be involved in progressive motor neuron stress that causes activation of different cell death pathways. It is hypothesized that this disease is triggered by factors related to genetic, environmental, and age-dependent risk. In spite of large neurobiological, molecular and genetic research, at the beginning of the 21st century, ALS still remains one of the most devastating neurodegenerative diseases because of the lack of effective therapeutic targets. It is a challenge for the clinical and scientific community. A better understanding of the etiology of ALS is necessary to develop specific targets of this progressive neurodegenerative disease. This review states about the current knowledge of targets in ALS research. This review provides an overview of the contribution of different targets like mitochondrial dysfunction, glutamate transport and excitotoxicity, protein accumulation, Oxidative stress, neuromuscular junction, microglia, and other molecular targets in the pathogenesis of ALS. Copyright:

Introduction

Amyotrophic lateral sclerosis (ALS) also known as “Lou Gehrig's disease,” is a chronic neurodegenerative disease.[1] This disease can present in the form of loss of lower or upper motor neurons or both. Symptoms such as dysarthria and difficulty in swallowing can also occur, resulting in regurgitation and aspiration pneumonia. Nevertheless, this condition does not affect the motor neurons that regulate the sphincter and extraocular muscles, as well as muscles, the autonomic and sensory neurons of the viscera. Respiratory failure occurs in most of the patients within 2–5 years of diagnosis as a result of the involvement of respiratory muscles. Some patients may have cognitive impairment further adding to the damage. Only 10% of the patients survive with 10 years of life expectancy.[2]

Recent population-based studies have recorded an ALS prevalence of 4.1–8.4 per 100,000 people.[3] Around 90%–95% cases are sporadic (sALS) and 5%–10% are inherited (fALS).[1] In India, the prevalence is 4–5/100,000.[45] Any age group can be affected in ALS but most commonly presents in 50–75 years of age group.[6] The etiology of ALS is still a mystery and the exact mechanisms of its pathogenesis are yet to be identified. Like other neurodegenerative diseases, it is highly multifactorial.[7] Signatures of this condition are some pathological mechanisms that include neuroinflammation, various proteinopathies, glutamate-induced excitotoxicity, microglial activation, apoptosis, oxidative stress, and ion channel and mitochondrial dysfunction [Figure 1].[89] Till now ALS is uncurable with no effective treatment. Relieving symptoms and improving the quality of life remains the focus of the current treatment regimens.[10] Riluzole, the first approved therapy available for the patients with ALS, supposed to act by various mechanisms like by blocking presynaptic release of glutamate, reducing hyperexcitability by inactivating voltage-dependent Na+ channels, slowing down inactivation of K+ channels, inhibition of protein kinase C, and interfering with excitatory transmitter induced intracellular events demonstrates a modest efficacy in slowing the disease progression especially when given early in the disease course.[11] Edaravone is another approved drug, appeared to be acting by scavenging H2O2 and by upregulating peroxiredoxin-2, downregulating isomerase A3, and inhibiting apoptosis, scavenges H2O2 and protects the cells against oxidative stress was also found to be modestly effective but only in a small group of people with early-stage ALS.[12] In this review article, we have tried to identify various novel targets that can be useful in the treatment of ALS [Figure 1].

Figure 1

Diagrammatic representation of novel targets of ALS. Novel drug targets for amyotrophic lateral sclerosis: (1) Mitochondiral activity modulator prevents mitochondrial DNA damage; (2) Glutamate inhibitors decreases excitotoxicity and improve motor function; (3) Protein aggregation inhibitor prevent protein misfolding and accumulation; (4) MicroRNA modulator regulates apoptosis, Necroptosis, and inflammation; (5) Axonal transport modulator regulates posttranslational modifications of RNA binding proteins and other proteins; (6) Macrophage regulator prevents neuroinflammation and subsequent motor neurons degeneration; (7) C90rf72 inhibitor prevent modulation of downstream pathways; (8) Reactive oxygen species inhibitor prevent mitochondrial dysfunction and cell damage; (9) Apoptotic inhibitor prevent apoptosis and cell damage; (10) SOD1 inhibitor prevents protein aggregation; (11) Creatine kinase 1 inhibitor prevents phosphorylation of TDP-43 binding protein; (12) Neuromuscular junction modulator prevent loss of neuromuscular junction integrity

Novel Therapeutic Targets in Amyotrophic Lateral Sclerosis

Targeting mitochondrial dysfunction

Mitochondrial dysfunction is the key marker for various neurodegenerative diseases such as ALS. The previous study shows that mitochondria play a pivotal role in the pathogenesis of ALS.[13] Dysfunctional mitochondria lead to altered energy production in the neurons and ultimately causes neuronal cell death. Mitochondrial perturbations make neurons more prone to oxidative stress and thus causes neuronal cell damage. One of the important biomarkers of mitochondrial dysfunction is succinate dehydrogenase and cytochrome c oxidase. In this regard, a study carried out on patient samples of ALS has found the deficiency of cytochrome c oxidase enzyme.[14] This study proved multiple DNA damage in mitochondria of neuronal cells in patients with ALS.

Dextromethorphan currently in use for Parkinson's disease is an opioid that belongs to the benzothiazanate family. The study shows that it works by inhibiting leaky conductance of mitochondria by means of decreasing consumption of oxygen and increasing the production of ATP which ultimately stabilizes the damaged cells and reduces the further neuronal cell death. Dextromethorphan is used in combination with quinidine. This combination blocks the P450 cytochrome enzyme activity by causing its demethylation. The clinical trial of this combination therapy has shown effective treatment modality for pseudobulbar and bulbar-type symptoms of ALS.[15] Successful clinical trials of dextromethorphan with high safety and efficacy have been reported for the treatment of ALS (as per clinicaltrials.gov data).

Olesoxime belongs to a cholesteroloxime compound family. It acts via modulating the mitochondrial membrane pore (mPTP). Olesoxime binds to two outer membrane proteins voltage-dependent anion-selective channel and translocator protein (TSPO), which ultimately alters the mitochondrial permeability transition pore.[1] Various preclinical studies have shown its effect on the survival of the neuronal cells and preventing further damage to the neurons under stress conditions.[16] In vivo studies in SOD1(G93A) mice has indicated improvement in microglia activation and a decrease in motor neuron cell death.[17] GNX4728, a mitochondrial pore modulator shows a two-time increase in survival in ALS mice. This small modulator increases the calcium retention capacity of mitochondria by inhibiting the mitochondrial pore.[18] The Phase-I and Phase-II clinical trials of olesoxime were successful with tolerable toxicity; unfortunately, Phase-III trial was not successful.

Cutamesine, a neuronal sigma-1-receptor (S1R) agonist, acts via binding to ion channels and proteins presents on endoplasmic reticulum and plasma membrane, respectively. S1R agonist stabilizes the mitochondrial associated membrane domain by regulating calcium flux. Thus, maintains the cellular bioenergetics in a various neurodegenerative disease like ALS.[19] In addition, cutamesine decreases the reactive oxygen species (ROS) production, oxidative stress, inflammation and ultimately prevents the mitochondrial dysfunction in motor neuron cells. In vivo studies in ALS transgenic mice (SOD1G93A) indicates its effectiveness against ALS-related neuronal cell death (anti-apoptotic) and mitochondria stabilization via affecting the PI3K-AKT signaling pathway.[19] A clinical trial of cutamesine may help in targeting the dysfunctional mitochondria.

Targeting glutamate transport and excitotoxicity

Glutamate is an excitatory neurotransmitter present in the central nervous system (CNS) that is released via synaptic vesicles and the released glutamate binds to various inotropic (ligand-gated ion channels) or metabotropic receptors (GPCR). Glutamate clearance in the synapse is one of the crucial steps in normal brain physiology. Astrocytes in the brain have glutamate transporter i.e., excitatory amino acid transporter 2 (EAAT2; Na+ dependent high-affinity transporter).[20] Various studies have shown low levels of these transporters in the spinal cord and cortex of ALS patients due to aberrant processing of EAAT2 mRNA transcript. This altered expression of EAAT2 causes an increase in glutamate leading to motor neuron death and degeneration.[21]

Glutamate levels are comparatively high in the CNS as compared to other parts of the body. If the clearance of glutamate in the synaptic vesicles exceeds above the normal range, it causes the over activation of glutamate transporters which ultimately leads to excitotoxicity (glutamate-induced excitotoxicity). The persistently high glutamate concentration alters the cell expressing these high-affinity glutamate transporters and make these cells more susceptible to cell death.[22] Another mechanism of excitotoxicity is through N-methyl D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. These receptors regulate the Ca2+ flux to maintain intracellular calcium concentration. When the calcium concentration is high, it propagates the death-inducing signals which ultimately cause cell death. The imbalance in calcium influx also causes an alteration in the activity of certain enzymes such as endonucleases, proteases, phosphatases, and phospholipases. These activated enzymes further cause cell damage.[23] Preclinical studies found the activated calcium-permeable AMPA receptor causes motor neuron damage and death in the case of in vitro culture system[24] and activation of AMPA without the increase glutamate concentration in case of in vivo rat model.[25]

Topiramate is a drug which is mainly used to prevent migraine and to treat epilepsy. The study has found that it works by blocking the AMPA receptors which may lead to a decrease in excitotoxicity and improvement in motor functions. Preclinical studies found positive results but the clinical trial was not successful.[26] A randomized control trial at a dose of 800 mg/day exhibited high toxicity and low efficiency, therefore low doses should be explored for the effective treatment of ALS. Therefore, there is a need for new drugs for targeting and regulating AMPA activation.

Certain beta-lactam antibiotics have also shown positive results for the improvement of ALS symptoms. Ceftriaxone is a beta-lactam family antibiotic, it has been found that it causes the upregulation of glutamate transporter (GLT1) by binding to its promoter sequence.[27] Other than this it also regulates the survival motor neuron protein, Nrf2 and decrease in glutamate concentration and decreased glutamate-induced toxicity which ultimately decreases the inflammation and motor neuron damage.[28] Successful clinical trials have also been done for beta-lactam antibiotics.[29] Phase-III trial of ceftriaxone indicated an overall increase in survival of patients with ALS.

Talampanel is a noncompetitive AMPA antagonist. Its structure is quite similar to 2,3-benzodiazepines, this drug inhibits the AMPA receptors and causes a decrease in neuro-excitotoxicity in motor neuronal cells. Preclinical studies in SOD1 mice have shown a decrease in the levels of calcium in the motor neurons and also a decrease in excitotoxicity.[30] It indicated positive Phase-I and Phase-II clinical trials and a safe pharmacokinetic profile.[31]

Some natural extracts also show good preclinical profiles for targeting excitotoxicity such as aquilariae lignum as it works by decreasing the concentration of glutamate and oxidative stress which in turn decreases the cell damage. It also causes the activation of BDNF (brain-derived neurotrophic factor) and down-regulation of apoptotic pathways.[32]

Targeting protein aggregation

ALS is characterized by misfolded proteins. These misfolded proteins form aggregates which in turn alters the motor neuron function. Some molecular components or proteins such as optineurin, TDP43, UBQLN2 (ubiquilin), and fused in sarcoma (FUS) are related to cellular protein aggregation in ALS.[33] These molecules are a type of RNA binding proteins (RBPs) which alters the protein expression and causes ubiquitinated protein aggregates. These aggregates formed due to altered autophagy or proteasomal degradation (UPS). These aggregates alter the motor neuron function and also cause neuronal cell death and altered RNA granule (stress granules) formation which ultimately leads to neurodegenerative disorders such as ALS. Heat shock proteins regulate the proper folding of the proteins. Any alteration in HSP expression leads to protein misfolding and accumulation of proteins. The exact relationship of protein aggregation and its effect on motor neurons in ALS is still under research.[34]

Arimoclomol is a derivative of hydroxylamine, and it induces the heat shock protein response and thereby decreasing the protein misfolding and accumulation. Preclinical studies in the SOD1 mouse model have demonstrated its positive effect on motor functions in mouse and low protein aggregates formation. This drug has shown positive results on both early as well as late stages of the disease.[35] Arimoclomol indicated a positive effect on motor functions and overall survival of the participants involved in the study with low toxicity. Phase-3 trial is under process for the further indication of arimoclomol as a potential therapeutic drug for ALS.

Targeting dysregulated RNA processing

One more mechanism that acts as a potential target for ALS is altered RNA metabolism and processing.[36] Dysregulated RNA metabolism is linked with protein aggregation as mentioned above. Various aggregation-prone RBPs like Ataxin2, TDP43, hnRNPs, FET (FUS, EWSR1, TAF15) get mislocalized and ultimately form aggregation complex. These aggregates alter RNA Splicing, capping, polyadenylation and transport of target RNA which disrupts the downstream pathways of cellular morphology and function. These alterations have been studied in ALS which suggests their role as potential targets in ALS.[37]

Dysregulated RNA metabolism and processing also affects the noncoding RNAs such as microRNAs (miRNAs). These miRNAs regulate the gene expression of various processes such as apoptosis, necroptosis, and inflammation. Certain miRNAs have been found in ALS that control the apoptosis, necroptosis, and autophagy of motor neurons. Preclinical studies in SOD1 transgenic mice found the high expression of miR125b which regulates the mediators of apoptosis such as BCL2.[38] Thus, miRNAs have evolved as novel therapeutic targets for the treatment of ALS.

Targeting axonal transport

Axonal transport is important for the movement of cargos and posttranslational modifications of RBPs and other proteins.[39] As neurons long with extreme axons or dendrites, transport is crucial to export proteins to each part of the neurons. The first evidence of altered axonal transport was observed in postmortem cases of ALS. The study found the accumulation of neurofilaments (phosphorylated), lysosomes, and mitochondrial in the proximal part of the axons of motor neurons which further shows the role of transport vesicles in regulating the axonal transport.[4041] Increased mitochondrial damage along with altered calcium homeostasis leads to disrupted binding of motor proteins (kinesin-1) to microtubules (tubulin) which decreases the microtubule stability and ultimately alters the axonal transport. The other mechanism is an alteration in neuron-specific pathways of retrograde endosome trafficking signaling (dynein). Histone deacetylase (HDAC) is also involved in dysregulation of axonal transport as HDAC decreases the acetylation of microtubules which further decreases the Kinesin-1 mediated axonal transport. Preclinical studies in SODI transgenic mice have shown that the deletion of HDAC6 decreases the progression of ALS by regulating axonal transport.[42]

Various drugs such as noscapine, GSK-3 β inhibitor, and lithium along with valproate have shown positive results in the SOD1 transgenic mouse model.[41] New drugs or gene editing can explore the axonal transport pathway as a potential drug target for the treatment of ALS.

Targeting microglia or macrophage

Immunohistochemical findings in ALS patients suggested that the infiltration of the gray and white matter of spinal cords by macrophages. These macrophages expressed cyclo-oxygenase-2 and inducible nitric oxide synthase in a huge amount. The dying neuronal cells were surrounded and seemed to be phagocytized by the infiltrating macrophages. Cerebrospinal fluid (CSF) also showed increased levels of monocyte chemoattractant protein-1 in both sporadic and familial cases of ALS. Raised levels of macrophage colony-stimulating factors also seen in tissues and CSF of ALS patients. High tumor necrosis factor alpha levels in ALS patients also correlate with macrophage activation. All these findings support the role of the macrophage in causing neuroinflammation and subsequent motor neuron degeneration in ALS. Blood spinal cord barrier also got compromised very early in the affected areas which might increase the macrophage infiltration into the CNS and hence can possibly play an important role in the early course of the disease. It is postulated that by targeting macrophage activation, inflammation in CNS can be decreased and disease progression can be modulated. A novel molecule NP001 found to be a regulator of these macrophages. Phase 1 and 2 studies showed promising results in terms of safety, tolerability and halting the disease progression in the patients showing marked macrophage induced neuroinflammation.[43] Autophagy also played an important role in regulating the activation/inactivation of the immune system. Targeting autophagy as well as macrophage activation can be considered as a feasible option in the treatment of ALS.

Targeting C9ORF72

The gain of function mutations in the gene C9ORF72 can lead to hexanucleotide repeats in ALS patients and can affect various downstream pathways which include DNA damage, dysfunctional nucleolus, nucleo-cytoplasmic transport deficits, endoplasmic reticulum stress, dysfunction of autophagy mechanism, inhibition of translation, proteasome inhibition, and alteration in dynamics of stress granules. Various pathways in this can be the potential targets. Like CRISPR mediated genome editing to remove C9ORF72 hexanucleotide repeats; by inhibiting the transcription products by targeting transcription elongation factors SUPT4H and PAF1C; by targeting repeats containing RNA by antisense oligonucleotides or RNA interference; by reducing the export of toxic repeat-containing RNA to the cytoplasm by selective inhibitors like KTP350.[44]

Targeting oxidative stress

Oxidative phosphorylation inside the cell is responsible for the production of ROS like O2−, H2O2, OH−. The reaction of O2− with NO produces peroxynitrite. These ROS can cause damage to the cells. Apoptosis gets activated inside such cells and prevent large scale damage. Apoptosis is tightly regulated by mitochondria. It is found that the mutant mitochondrial SOD1 product binds with anti-apoptotic protein Bcl-2 and decreases its function.[45] This mitochondrial dysfunction can be targeted to slow down the disease progression in ALS patients. Other strategies can include decreasing ROS production and inhibition of the apoptotic pathway.

Targeting SOD1 mutation

Superoxide dismutase plays an important role in detoxifying superoxide radicals generated during the metabolic processes. It gets activated after binding with cofactors like Zn+2 and Cu+2 ions. Variation in this binding can accelerate protein aggregation and reduce the stability of SOD enzyme. This aggregation and stabilization are being targeted while designing new drugs for ALS. Through a computer-based approach, mutant SOD and dopamine complex were used to search new lead molecules that can inhibit aggregation.[46] Docking studies showed 2 molecules viz. 2,354-tetrahydrostilbene and hesperidin showing higher affinity towards SOD for binding thus can be used as a potential agent to design and develop new drugs in the treatment of ALS. The inhibiting oligomerization of SOD can also be used as an effective target in screening and developing novel therapeutics against SOD associated ALS for which currently there is no cure.

Targeting apoptosis (caspase 3)

Glutamate action in the motor neurons is terminated by its uptake from EAAT-2. This transporter is cleaved by caspase-3 causing inhibition of this transporter and high glutamate levels. Glutamate induced excitotoxicity is one of the proposed mechanisms in the pathogenesis of ALS. Through molecular docking studies, efforts have been made to search for natural inhibitors of plant origin which are nonpeptidyl against this transporter. 2 lead compounds curcumin and rosmarinic acid found to mimic the effects of known peptidyl inhibitors. These can be used to further generate new lead compounds to inhibit EAAT-2.[46] Furthermore, studies have shown the important role of CASP-3, CASP-8, andCASP-9 in neurodegenerative disorders which can also be used as a target in the treatment of ALS.

Targeting phosphorylation

Creatine kinase 1 (CK-1) found to be upregulated in the neurons of ALS patients. This enzyme causes the phosphorylation of TDP-43 binding protein, which is found to be accumulating in the affected motor neurons. Docking studies with CK-1 δ and N-benzothiozolyl-2-phenyl acetamide were carried out and 2 compounds were found to have the lowest binding energies which when evaluated further found to have good BBB penetration.[46] Thus, these can become a potential candidate drug and similarly other novel molecules can be explored.

Targeting neuromuscular junctions

One of the pathological hallmarks of ALS is disrupted neuromuscular junctions (NMJs). Alterations in Na+ channels and Na+-K+ in the muscle cause disrupted muscle activation which leads to damage in mitochondria. Dysfunctional mitochondria in muscle lead to the production of ROS along with altered calcium homeostasis which causes apoptosis or necroptosis of motor neurons and hence, muscle atrophy occurs.[47] Therefore, loss of NMJ integrity is a pathological marker of ALS and this can act as a potential target for the treatment of ALS.

The Nav1.6 channel can also be targeted in the novel drug designing process against ALS. In silico studies showed the interaction of Riluzole with this channel and its residues. Thus, this knowledge can be exploited to look for newer molecules.

Food and Drug Administration-Approved Drugs for Amyotrophic Lateral Sclerosis and Their Limitations

Riluzole acts in several biological systems and reduced the neurotransmission like glutamatergic. Riluzole works to stabilize the sodium channels i.e., voltage-dependent as the form of inactivated abidance. Riluzole activates the GPCR associated pathways and inhibit the secretion of glutamate, it also inhibits the NMDA receptor-mediated postsynaptic events. These actions of riluzole help to stimulate the inhibition of the excitatory process, so it shows a good neuroprotective activity.[48]

Edaravone has the same properties as that of vitamin C and vitamin E. It scavenges the free radical form that is water-soluble and fat-soluble like peroxy radicals (lipid peroxyl and peroxynitrite). The free radical scavenging property of Edaravone comes from its electron-donating property. Edaravone prevents oxidative stress in different brain cells like microglia, glia, neurons, etc., and reduced the inflammatory response. Merely, there is no definite mechanism of action known for Edaravone in ALS treatment. Edaravone has a strong potential in neuron protection as compared to direct action on motor neurons.[49]

Riluzole and Edaravone both are FDA approved drugs for ALS treatment but both are showed several side effects. Firstly, Riluzole is showed a toxicity for anti-glutamate related processes. Riluzole alters the respiratory functions and action potential by inhibiting the sodium channels.[1] Secondly, Edaravone showed several side effects such as anaphylactic symptoms, allergic reactions for sulfite, and hypersensitivity. It also causes internal injuries, headache, eczema, gait disturbance and dermatitis in more than 10% of patients of ALS.[49] Various promising drugs for the treatment of ALS are under clinical trials as summarized in the Table 1.

Table 1

Various newer and older molecules are tried which are under various phases of development

Phase I	Phase II	Phase III
Newer molecules	Drugs	BIIB067
AP101	Biotin	Deferiprone
DNL747	rhEPO	CannTrust CBD Oil
BIIB078	Inosine	Methylcobalmin
BIIB100	Deferiprone	Taurourso
GD0134	L-serine	deoxycholic Acid
IPL344	Rapamycin	Levosimendan
Cu (II) ATSM	Pimozide	Arimoclomol
MN166	Lithium and
FDA approved drugs	valproate
Betamethasone	Lacosamide
sodium phosphate/	IL-2
betamethasone acetate	Fasudil
Dextromethorphan HBr	Memantine
and quinidine sulfate	Dextromethorphan
Lacosamide	HBr and Quinidine
rhEPO	sulfate
Darunavir	Perampanel
Ritonavir	Ranolazine
Dolutegravir
Tenofovir alafenamide

IL=Interleukin, FDA=Food and Drug Administration, rhEPO=recombinant human erythropoietin, CBD=Cannabidiol, HBr=hydrobromide

Conclusion

In spite of numerous studies in ALS, the exact mechanism of the disease progression is largely unknown

There is a challenge in drug translational research in ALS since most of the drugs failed under clinical trials. Despite a decade of research, only two drugs, i.e., Riluzole and Edaravone, have approved by the FDA. There are numerous reasons for the slow progress of drug development in ALS. First, there is a dearth of suitable animal models for ALS which can recapitulate the true disease condition. Second, ALS is a disease with multifactorial etiology, therefore all factors should be taken into consideration to avoid any fallacious results

Although the precise pathogenic mechanism is still unknown, the use of different drugs has given some insight into the various pathways involving in this disease process. There is an immediate need to look for newer molecules as well as already known drugs with newer indications to be used as a therapy for ALS. Targeting one pathway alone or in combination can be a way out for this incurable disease.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.