**18. Hotspot identification**

MicroRNAs can bind to the same complementary target sites or it can bind to proximally located sites adjacent to other miRNA target sites. A hotspot is a stretch of nucleotide sequence, which is prone to target of several groups of miRNAs. miRNAs are regulated and expressed spatially and temporally inside cell (**Figure 5**). This type of regulation and expression gives miRNAs power to play a plethora of roles in various different biological processes. All the miRNAs occupying position in "hotspot" may not regulate a gene in the same pattern. Generally, predominant miRNA competitively outcasts other miRNAs and plays as a potential repressor. There is no clear evidence of the cause behind competitive outcasting and variable effectiveness. Various researchers are trying to use different standards and parameters like the same chromosomal location for more than two miRNAs, the same orientation, phylogenetic relationships and absence of interfering transcription unit.

miRNA targets are generally predicted using miRanda and then further analysed for miRNAprone regions in all the selected genes. Generally, a region is defined as hotspot, if it has a minimum of 10 nucleotides overlapping from the starting position and occurrence of three miRNA targets. It has been shown that there are 11 miRNA-prone regions in 35 genes associated to ALS. CYCS and MAP2K3 have one hotspot each in 3′ UTR, while single target site hotspot was in 5′ UTR in four genes (**Figure 5**), that is, BAX, DERL1, SLC1A2 and RAB5A. Studies have shown that MAP2K3 has two hotspots by miRNA cluster prediction. Target sites for three miRNAs, i.e. hsa-miR-107, hsa-miR-423-5p and hsa-miR-103, are congregated in another hotspot found in 3′ UTR. MAP2K3, having two such hotspots, is presumed to show more sensitivity towards gene regulation by microRNAs. The small number of hotspots makes the miRNAs more specific and stringent regulator. The variability in existence of such target site hotspots adds complexity in gene regulation [9] (**Figure 5**).

**Figure 5.** Schematic representation of susceptible 3′ hotspots of human MAP2K3 gene by various miRNAs like hsamiR-107 and hsa-miR-103. To detect the susceptible sites on human MAP2K3 gene, bioinformatic tools like miRanda, TargetScan and PicTar were used (adapted from Ref. [9]).

#### **19. Multifarious miRNA regulation**

MicroRNA-mediated gene regulation adds multilayered complexity to gene expression. Distribution of target sites over several genes of one miRNA makes the picture more intricate. In principle, one miRNA can bind to more than one gene (multiplicity), and one gene can be controlled by more than one miRNA (cooperativity) [79]. Relaxed base pairing gives rise to multiplicity property. Target sites are rarely distributed on a gene. Generally they are scattered and the range of occurrence varies in numbers in a gene [80]. Often, presence of more target sites per gene indicates efficient regulation. Studies have shown that miRNAs, related to ALS, have multiple targets. After calculating multiplicity and cooperativity, scientists have shown that hsa-miR-370 showed maximum multiplicity as it showed 65 interactions with 30 gene sequences while no target site could be predicted in five genes, namely, *CASP1*, *GPX1*, *DERL1*, *CCS* and *SOD1*. It is highly expected that the degree of repression by hsa-miR-370 would be considerably high. It has been shown in the papers that hsa-miR-874 have 52 interactions with 29 genes and have no target in *CASP1*, *CHP*, *SOD1*, *RAC1*, *RAB5A* and *MAP2K6* genes. None of the above-mentioned miRNAs display any interaction with SOD1. Documented evidences show that SOD1 is one of the core factors for ALS that encodes the free radical scavenging enzyme copper zinc superoxide dismutase in ALS pathway. Then these miRNAs might not be involved in the regulation of *SOD1* activity. Scientists have reported that *PRPH* showed high cooperativity followed by *TNFRSF1A* and *TOMM40*. It is really difficult to interpret the complex picture presented by these multifaceted interactions. *PRPH* is regulated by 10 miRNAs at 23 positions, so first 10 miRNAs demonstrate high cooperativity towards *PRPH*. Relatively very low cooperativity was shown in *BID*, *MAP2K6* and *DERL1* genes. It might be inferred that there exists a low sensitivity of these genes towards microRNA-mediated gene regulation. Simple process of selection of microRNA targets is endowed with inherent complexity and this led to the development of a complex network by two phenomena, that is, multiplicity and cooperativity. The multiplicity and cooperativity might be the deciding factors for the mode of miRNA action. How these factors work is a matter of mystery [9].

## **20. Complex interrelationship**

miRNA targets. It has been shown that there are 11 miRNA-prone regions in 35 genes associated to ALS. CYCS and MAP2K3 have one hotspot each in 3′ UTR, while single target site hotspot was in 5′ UTR in four genes (**Figure 5**), that is, BAX, DERL1, SLC1A2 and RAB5A. Studies have shown that MAP2K3 has two hotspots by miRNA cluster prediction. Target sites for three miRNAs, i.e. hsa-miR-107, hsa-miR-423-5p and hsa-miR-103, are congregated in another hotspot found in 3′ UTR. MAP2K3, having two such hotspots, is presumed to show more sensitivity towards gene regulation by microRNAs. The small number of hotspots makes the miRNAs more specific and stringent regulator. The variability in existence of such target

**Figure 5.** Schematic representation of susceptible 3′ hotspots of human MAP2K3 gene by various miRNAs like hsamiR-107 and hsa-miR-103. To detect the susceptible sites on human MAP2K3 gene, bioinformatic tools like miRanda,

MicroRNA-mediated gene regulation adds multilayered complexity to gene expression. Distribution of target sites over several genes of one miRNA makes the picture more intricate. In principle, one miRNA can bind to more than one gene (multiplicity), and one gene can be controlled by more than one miRNA (cooperativity) [79]. Relaxed base pairing gives rise to multiplicity property. Target sites are rarely distributed on a gene. Generally they are scattered and the range of occurrence varies in numbers in a gene [80]. Often, presence of more target sites per gene indicates efficient regulation. Studies have shown that miRNAs, related to ALS, have multiple targets. After calculating multiplicity and cooperativity, scientists have shown that hsa-miR-370 showed maximum multiplicity as it showed 65 interactions with 30 gene

site hotspots adds complexity in gene regulation [9] (**Figure 5**).

154 Update on Amyotrophic Lateral Sclerosis

TargetScan and PicTar were used (adapted from Ref. [9]).

**19. Multifarious miRNA regulation**

**Figure 6** conveys interaction map of miRNA and selected 35 genes. Interactions amongst genes and miRNAs are depicted with arrows, where 1 = hsa-miR-370, 2 = hsa-miR-874, 3 = hsa-

**Figure 6.** Interaction map of different miRNAs and selected 35 genes. Complex interrelationship amongst different genes and different miRNAs is depicted with the arrows. It is very evident from the diagram that one with miRNA can target 3′ UTR of several genes, thus adding layers to the gene regulation architecture (adapted from Ref. [9]).

miR-423-3p, 4 = hsa-miR-323-5p, 5 = hsa-miR-760, 6 = hsa-miR-149, 7 = hsa-miR-139-3p, 8 = hsamiR-744, 9 = hsa-miR-324-3p, 10 = hsa-miR-339-3p and 11 = hsa-miR-654-5p. From **Figure 6** we can understand the multilayered regulation [9].

## **21. Usage of miRNAs in therapeutics**

The human mind is not satisfied to know the causative factors of a disease. Our main target is to reduce ailment and to do that we need good diagnostic system along with good therapeutics. miRNAs are one of a kind. Being a regulator of gene expression, it has far-reaching effect on our physiology. They are dysregulated in several diseases. Researchers are now trying to generate therapeutic targets to treat ALS. Presently, riluzole is the only FDA-approved drug to treat ALS [81] and it modestly slows the disease progression. A phase I clinical trial of an antisense oligonucleotide [ISIS 333611] has been proven effective. This oligonucleotide targets SOD1 mRNA and represses the production of mutant SOD1 and this is effective when delivered to the CSF of patients with fALS [82]. The same kind of drug is there which targets the sense strand of the C9orf72 hexanucleotide repeat and reduces the toxicity by suppressing RNA foci formation both in vivo and in vitro [30, 33, 83]. Several delivery systems are being invented day in and day out to deliver miRNA formulations and drugs to the CNS through blood-brain barrier. The first miRNA-based therapeutic is the miR-122 antagonist SPC3649, which is currently being evaluated in phase II clinical trials. It targets the hepatitis C virus. So far, this agent has not exhibited any adverse effects.

There are two basic approaches of miRNA-based therapeutics. One is miRNA antagonists which impede endogenous miRNAs that have a harmful gain of function in diseased tissues and involve the use of an anti-miR – a chemically modified antisense RNA – to knockdown miRNA. In one of the first endeavours to use antagomirs in ALS, delivery of anti-miR-155 to *SOD1* Gly93Ala mice via ventricular osmotic pumps delayed mortality of the patients successfully. A downfall associated with this approach is the potential for nonspecific binding to other RNAs inside the cells. As one miRNA can regulate several mRNAs, it limits usage of miRNAs as therapeutics. In the second approach to miRNA therapeutics that involves miRNA mimics and miRNA replacement therapies, miRNAs are reintroduced into cells exhibiting downregulation, thus reactivating key pathways [84].

It is really difficult to dissect all the pathways, which are regulated by one miRNA. Before releasing drugs in clinics, there should be several clinical trials to eliminate any deleterious effect. Williams and group [85] showed that miRNA-206 delayed ALS progression and promoted regeneration of neuromuscular synapses effectively in mice. This group investigat‐ ed pathological modifications in motor axons and nerve terminals that precede motor neuron degeneration and clinical symptoms and the role of the skeletal muscle-specific miR-206 in motor neuron-skeletal muscle fibre signalling. This miRNA is significantly upregulated in SOD1-G93A mouse model of ALS. The genetic ablation of miR-206 in these ALS mice accel‐ erates the disease progression. miR26 might be needed for compensatory regeneration of neuromuscular synapses after acute nerve injury and thus it decelerates ALS progression. miR-206 represses the translation of HDAC4 mRNA and counteracts its negative influence. This miRNA is also involved in neuromuscular gene expression and synapse formation. miR-206 decelerates ALS progression by detecting motor neuron injury and boosting com‐ pensatory regeneration of neuromuscular synapses by specifically inhibiting HDAC4 protein synthesis. HDAC4 has a deleterious role in ALS patients and it has been confirmed. It was shown in experiments that low progression rate in patients with ALS is associated to greater compensatory reinnervation and low HDAC4 levels. miR-133b is present in the same transcript encoding miR206, which is upregulated after denervation in the NMJs. The genetic ablation of miR-133b was studied in SOD1-G93A mice and it was noticed that the lack of miR-133b did not modify NMJ development or reinnervation after nerve injury and, overall, disease progression was not affected. miR-206 plays an important role in NMJ reinnervation specifi‐ cally. miR-155 expression can be efficiently inhibited by anti-miR delivery to the CNS and periphery of SOD1-G93A mouse [86]. Inhibition of miR-155 can improve disease progression in ALS. Single intracerebro-ventricular injection of miR-29a-specific antagomir inhibiting CNS-related miR-29a expression in CNS can prolong the survival of ALS patients, thus suggesting miR-29a as a possible marker for disease progression. Anti-miR can be used as therapeutics in treating ALS but we should have an eye on the bystander effects [21].
