**13. Causative factors in ALS**

that there are no target sites that could be detected in 3′ UTR for mRNAs expressed in midbrain for *CASP1*, *GRIA1*, *GPX1*, *DAXX*, *CAT*, *SOD1*, *NEFM*, *NEFL* and *NEFH* genes. Furthermore 5′ UTR lack any target sites of same miRNAs in *CASP1*, *GRIA1*, *GPX1*, *DAXX*, *CAT*, *SOD1*, *NEFM*, *NEFL*, *NEFH*, *RAC1* and *TNF* genes. The miRNAs expressed in midbrain showed the least number of target sites in *RAC1* and *CASP1* genes, whereas 29 target sites were predicted in *NOS1*. Similar results were there for the miRNAs expressed in the cerebellum for the same genes. For instance, *RAC1* has on one target site whereas *NOS1* has 11 target sites. For miRNAs expressed in the hippocampus, *CASP1* and *GRIA1* genes have a few numbers of predicted sites [4] and *MAP3K5* was predicted to possess maximum target sites [30]. Similarly, *CASP1* and *SOD1* have two target sites each, while 23 target sites had been predicted for *TNFRSF1A* gene for miRNAs, expressed in the frontal cortex. On the other hand, target sites could not be predicted for six genes, namely, *GPX1*, *CYCS*, *CHP*, *CASP3*, *SOD1* and *RAB5A*, for miRNAs expressed in the cerebellum. No target sites could be predicted in 3′ UTR of *ALS2*, *BID*, *CASP1*, *GRIA1*, *DAXX*, *CCS*, *CAT*, *CASP9*, *TOMM40*, *TNF*, *SOD1*, *RAC1*, *NEFM* and *NEFL* genes. No target site could be predicted in *BCL2*, *CASP1*, *GRIA1*, *DAXX*, *CYCS*, *CASP3*, *TNF*, *RAC1*, *NEFM* and *NEFH* genes for miRNAs expressed in the cerebellum. These results demonstrate that the majority of the ALS genes lack any target site for miRNAs expressed in the cerebellum. miRNA targets were found in most of the genes, but surprisingly few genes, *CASP1*, *GRIA1*, *DAXX*, *CAT*, *SOD1* and *NEFM*, lack any miRNA target site in their 3′ UTR. This shows that complexity of regulations and numerous members of this play give the brain another level of

**Figure 3.** Percentage of miRNAs, expressed in the brain. From this diagram we can have an idea that the regulation of

genes by miRNAs is widespread in various parts of the human brain (adapted from Ref. [9]).

entanglement [9] (**Figure 4**).

144 Update on Amyotrophic Lateral Sclerosis

Most of the cases are sporadic ALS [sALS]. Only 10–15% are usually inherited as an autosomal dominant trait and defined as familial [fALS]. They show comparable etiopathology and they affect same neuronal population [31]. Several ALS genes (*SOD1*, *Alsin*, *SETX*, *SPG11*, *FUS/ TLS*, *VAPB*, *ANG*, *TARDBP*, *FIG4*, *OPTN*, *ATXN2*, *VCP*, *UBQLN2*, *SIGMAR1*, *CHMP2B*, *PFN1*, *SQSTM1*, *C9ORF72*) and additional chromosomal loci have been identified [32, 33]. Almost two-thirds of the familial cases are due to mutations of *C9ORF72*, *SOD1*, *TARDBP* and *FUS* genes. Twenty percent of fALS is because of the mutation of *SOD1* gene, coding for the superoxide dismutase one mitochondrial and cytoplasmic copper/zinc enzyme, responsible for metabolizing naturally occurring, but harmful, superoxide radicals to molecular oxygen and hydrogen peroxide. A massive hexanucleotide-repeat expansion (GGGGCC)n in the first intron of *C9ORF72* gene on the chromosome 9p21is the most frequent cause of ALS identified to date [30, 33]. After identifying several causal genes, it has been hard to find the clear evidence of the pathogenic role of some mutations. The most extensively studied gene, SOD1, still lacks pathogenic feature of the reported missense, nonsense and deletion mutations. Studies suggest that there are multiple genetic factors which contribute to develop ALS ultimately or show phenotypical features. Though the contributions of environmental factors have not been proved, ALS is a combination of genetic and environmental factors. It has been clearly noted that with increasing age, one develops higher risk of ALS. Some reports tell that heavy metals, pesticides and exposure to electromagnetic field might cause ALS [34]. The use of neurotrophic factors like IGF-I, GDNF, VEGF, ADNF-9, colivelin and angiogenin showed potential in therapy of ALS. Talampanel (AMPA antagonist), ceftriaxone (antibiotic), pramipexole/ dexpramipexole (dopamine agonists) and arimoclomol (activator of molecular chaperons' protein repair pathway) are amongst the existing drugs to treat ALS. Lately stem cell and immune therapies have been suggested [35]. A comprehensive listing of trials in the USA can be found at ClinicalTrials.gov.

A wealth of evidence supports that ALS is a multifactorial and multisystemic disease, charac‐ terized by overlapping different mechanisms, transduction pathways and multicellular crosstalk. Transgenic animals expressing human mutant SOD1 (mSOD1) have helped to reveal that perishing of motor neurons is not the only reason behind ALS but there is active contri‐ bution of non-neuronal cells like microglia, astrocytes and muscle and T cells, which differently participate to the different phases of the disease [36, 37]. Expression of mSOD1 within the most vulnerable motor neurons primarily causes for disease onset; synthesis of the mutant protein by interneurons also positively participates to disease initiation [38, 39]. Speeding of disease progression is caused by moSOD-1-mediated injury and neighbouring astrocytes and micro‐ glia. Schwann [40] and muscle cells play as direct partners of the injured motor axons, either working as recipients or initiators of the initial damage. These characteristics thus properly earned to ALS the definition of non-cell-autonomous disease [41]. Chimeric mice formed by expressing both mutated and non-mutated SOD1 proved that wild-type nonneuronal cells prolong the survival of motor neurons carrying mSOD1 protein. Neighbouring astrocytes, microglia, oligodendrocytes and Schwann cells can cause the degeneration of motor neurons [42, 43]. Compelling evidence indicates that primary mSOD1-expressing astrocytes from mouse, rat and humans cause motor neurons death by releasing toxic factors into the media while sparing interneurons [44–46]. Astrocytes deriving from neuronal progenitor cells, gathered from fALS and sALS cases, exhibit features of non-cell-autonomous toxicity [47]. There are very few studies of non-cell-autonomous toxicity. Astrocytes expressing mutant TDP43 [TDP43-M337V] participate in ALS pathology only through cell-autonomous process‐ es. It has been studied that conditioned medium from astrocytes that express SOD1-G93A, or SOD1-G86R, or TDP43-A315T leads to extensive motor neuron death via non-cell-autonomous mechanisms. Still, there is a long way left to travel and to unravel mystifying stories behind ALS [21] (**Figures 1** and **3**).

#### **14. The miRNAs involved in ALS regulation**

#### **14.1. Effects of miRNA biogenesis related but mutated factors in ALS**

Several factors are there, which can affect CNS homeostasis and thereby facilitate neurodege‐ nerative and neuroinflammatory diseases. Perturbation in intricate miRNA network can disrupt neuronal function in cell-autonomous or non-cell-autonomous manner [48–50]. It has been shown that there is cell-type-specific deletion of miRNA-processing enzyme, Dicer in cerebellar Purkinje, or striatal, retinal, spinal and cortical neurons, which leads to degeneration and ataxia [51–54]. Neurodegeneration also occurs after targeted deletion of Dicer in astro‐

cytes, oligodendrocytes and Schwann cells, perhaps because the glial cells have some effects on neuronal survival [55–58] (**Table 1**). Experimental results show that deletion of Dicer directly in spinal motor neurons mimics most of the clinical (e.g. progressive paralysis) and pathological (e.g. astrocytosis and signs of axonopathy) traits of ALS [54]. Several groups have proved that TDP43 and FUS/TLS are involved in multilayered steps of RNA processing and miRNA biogenesis pathway. It has been investigated recently they have role in miRNA-related ALS [59–62]. FUS, nuclear factor complexes with Drosha along with DGCR8. This association is indispensible in miRNA processing in nucleus. Cytoplasmic TDP-43 associates with Dicer, containing TRBP. This interaction facilitates the processing of the specific pre-miRNAs by Dicer, generally a subset of the pre-miRNAs whose yield in the nucleus is regulated by TDP-43, by direct binding to their terminal loops. It was proved that TDP-43 promotes neuronal outgrowth by facilitating miRNA production. Further studies show that TDP-43 has its role in both nucleus and cytoplasm. It associates with DGCR8 containing nuclear Drosha complex in RNA-dependent and RNA-independent fashion. In nucleus, TDP-43 binds to a selected primiRNAs, and in cytoplasm, it binds to terminal loops of pre-miRNAs. TDP-43 facilitates neuronal outgrowth through the regulation of miRNA processing [60].

factors like IGF-I, GDNF, VEGF, ADNF-9, colivelin and angiogenin showed potential in therapy of ALS. Talampanel (AMPA antagonist), ceftriaxone (antibiotic), pramipexole/ dexpramipexole (dopamine agonists) and arimoclomol (activator of molecular chaperons' protein repair pathway) are amongst the existing drugs to treat ALS. Lately stem cell and immune therapies have been suggested [35]. A comprehensive listing of trials in the USA can

A wealth of evidence supports that ALS is a multifactorial and multisystemic disease, charac‐ terized by overlapping different mechanisms, transduction pathways and multicellular crosstalk. Transgenic animals expressing human mutant SOD1 (mSOD1) have helped to reveal that perishing of motor neurons is not the only reason behind ALS but there is active contri‐ bution of non-neuronal cells like microglia, astrocytes and muscle and T cells, which differently participate to the different phases of the disease [36, 37]. Expression of mSOD1 within the most vulnerable motor neurons primarily causes for disease onset; synthesis of the mutant protein by interneurons also positively participates to disease initiation [38, 39]. Speeding of disease progression is caused by moSOD-1-mediated injury and neighbouring astrocytes and micro‐ glia. Schwann [40] and muscle cells play as direct partners of the injured motor axons, either working as recipients or initiators of the initial damage. These characteristics thus properly earned to ALS the definition of non-cell-autonomous disease [41]. Chimeric mice formed by expressing both mutated and non-mutated SOD1 proved that wild-type nonneuronal cells prolong the survival of motor neurons carrying mSOD1 protein. Neighbouring astrocytes, microglia, oligodendrocytes and Schwann cells can cause the degeneration of motor neurons [42, 43]. Compelling evidence indicates that primary mSOD1-expressing astrocytes from mouse, rat and humans cause motor neurons death by releasing toxic factors into the media while sparing interneurons [44–46]. Astrocytes deriving from neuronal progenitor cells, gathered from fALS and sALS cases, exhibit features of non-cell-autonomous toxicity [47]. There are very few studies of non-cell-autonomous toxicity. Astrocytes expressing mutant TDP43 [TDP43-M337V] participate in ALS pathology only through cell-autonomous process‐ es. It has been studied that conditioned medium from astrocytes that express SOD1-G93A, or SOD1-G86R, or TDP43-A315T leads to extensive motor neuron death via non-cell-autonomous mechanisms. Still, there is a long way left to travel and to unravel mystifying stories behind

be found at ClinicalTrials.gov.

146 Update on Amyotrophic Lateral Sclerosis

ALS [21] (**Figures 1** and **3**).

**14. The miRNAs involved in ALS regulation**

**14.1. Effects of miRNA biogenesis related but mutated factors in ALS**

Several factors are there, which can affect CNS homeostasis and thereby facilitate neurodege‐ nerative and neuroinflammatory diseases. Perturbation in intricate miRNA network can disrupt neuronal function in cell-autonomous or non-cell-autonomous manner [48–50]. It has been shown that there is cell-type-specific deletion of miRNA-processing enzyme, Dicer in cerebellar Purkinje, or striatal, retinal, spinal and cortical neurons, which leads to degeneration and ataxia [51–54]. Neurodegeneration also occurs after targeted deletion of Dicer in astro‐




*Ni*, not investigated in the study (adapted from Ref. [21] with the permission from Bentham Science Publishers).

**Table 1.** Summarization of dysregulated miRNA in ALS.

**Tissue/cells miRNAs Changes Biological**

Spinal cord miR-9 ↑ Neuronal

Spinal cord miR-124a ↓ Glutamate

miR-508-5p, miR-373\*, miR-551a, miR-506, miR-518a-5p, miR-518e\*,

CSF miR-143-5p, miR-

148 Update on Amyotrophic Lateral Sclerosis

574-5p

Spinal cord miR-558, miR-16-2\*, miR-146a\*,

miR-890

miR-524-5p, miR-548a-5p, miR-606, miR-612, miR-647 miR-155, miR-17, miR-19b, miR-20a, miR-24-2\*, miR-106a, miR-142-3p, miR-142-5p, miR-146a, miR-146b, miR-223

Spinal cord miR-155, miR-17, miR-19b, miR-20a,

142-3p, miR-142- 5p, miR-146a, miR-146b, miR-155

miR-451, miR-1275, miR-328-5P, miR-638, miR-149, miR-

miR-23a, miR-29b, miR-206

665

miR-455

Skeletal muscle

Spinal cord miR-24-2\*, miR-

miR-24-2\*, miR-106a, miR-142-3p, miR-142-5p, miR-146a, miR-146b

Blood leukocytes miR-338-3p ↑ *Ni* sALS human patients

↓ *Ni*

↑ Mitochondrial function

Spinal cord miR-624, miR-520,

CSF miR-132-5p, miR-132-3p, miR-143-3p ↓ *Ni* sALS patients

Neurons miR-9 ↓ *Ni TARDBP* A90V,

**function**

differentiation

↑ NFL stability sALS human patients

*Ni* SOD1-G93A mice

*Ni* Human patients

Human patients

transport

↓ NFL stability

↑ *Ni*

**Model**

*TARDBP* M337V patients

SOD1-G93A mice

SOD1-G93A mice

One group has reported the changes that occur in the miRNA population followed by TDP-43 knockdown in cultured cells. It was observed that *let-7b* and *miR-663* expression levels are down- and upregulated, respectively (**Table 1**). It was found that both miRNAs can bind to TDP-43 directly in different positions: within the miRNA sequence itself (i.e. let-7b) or in the hairpin precursor (miR-663). Using microarray data and q-PCR, candidate transcripts are identified, whose expression levels are discriminately affected by these TDP-43–miRNA interactions. TDP-43 gets increased in ALS [61] leading to neurotoxicity from both gain and loss of functions. Neurodegeneration and ALS phenotypes are caused by a partial loss of TDP-43 function. TDP-43 toxicity in neighbouring cells has effects on motor neurons. Both dysregulation and dysfunction of TDP-43 are relevant to ALS [53]. Researchers have reported that TDP43 depletion could affect the levels of specific miRNAs in human hepatocarcinoma cells, by potentially binding to their sequence and/or precursor elements [61]. Regulation of miRNA by TDP43 is conserved amongst mammals. TDP43 associating with both nuclear and cytoplasmic microprocessor complexes actively participates in the production of miRNAs indispensable for neuronal outgrowth. Finally, it was noted that the carboxyl-terminal of TDP43, where most ALS mutations reside, is crucial for the interaction with the miRNAprocessing complexes [60]. Recent discovery of mutations in the genes, encoding for the RNAbinding proteins TDP43 and FUS/TLS, has proved the key role of regulatory RNA in the pathogenesis of ALS. Both proteins are generally localized within the nucleus, but their mutated forms delocalize and form neuronal inclusions and dystrophic neurites, as well as glial cytoplasmic inclusions. The sensory organ precursor cells of peripheral nervous system of *Drosophila melanogaster* were used as a model to demonstrate that hTDP43 clinical ALS mutations influence early neurogenesis and neuronal specification, through the regulation of miR-9a biogenesis. Further studies in human cells show that TDP43 is an essential element in miRNA biogenesis during neuronal differentiation by controlling the stability of Drosha and thus affecting miRNA production. Similar inferences were drawn after FUS/TLS silencing in human neuronal cell lines. FUS/TLS downregulation affects the biogenesis of a large class of miRNA and several of them have important roles in neuronal differentiation and synaptogenesis. Specific miRNAs, in particular miR-9, miR-125b, miR-132 and miR-143, are regulated by both TDP43 and FUS/TLS ALS-related proteins. In recent reports, TDP43 was shown to bind to and regulate the incorporation into the RISC complex of specific mature miRNAs, in both Drosophila and human systems. TDP43 interacting with miR-1/miR-206 family decreases the activity of these miRNAs, by the disruption of their association with the RISC complex. As this miRNA family is involved in muscle development and homeostasis, either depletion or overexpression of TDP43, by altering miR-1/miR-206 balance, might be involved in muscle pathogenesis of ALS. Nine specific miRNAs were selected and their levels were analysed in ALS patients, on the basis of their known misregulation after TDP43 depletion in cell lines. Samples of cerebrospinal fluid (CSF), serum and immortalized lymphoblastoid cell lines from fALS and sALS patients were analysed, and it was confirmed that there is dysregulation of miR-132, miR-143 and miR-574, whose processing is indeed regulated by TDP43 and/or FUS/TLS proteins. The sequencing of the 3′UTR region of the FUS/ TLS gene in 420 ALS patients highlighted a mutation in two ALS patients with severe consequence, leading to FUS/TLS protein accumulation. This mutation maps to the seed sequence recognized by miR-141 and miR-200a in the 3′-UTR of FUS/TLS and that FUS/TLS is linked to these miRNAs by a feedforward regulatory loop, where FUS/TLS upregulates miR-141/200a, which in turn affects the FUS/TLS protein synthesis [21].

### **15. CNS-related miRNA dysregulations in ALS pathogenesis**

miR-9, an evolutionarily conserved and multifunctional neuronal miRNA, is involved in the selection of neuronal precursors from the neuroepithelium in flies and in the specification of midbrain-hindbrain boundaries in vertebrates. Both miR-124 and miR-9 participates together in neuronal differentiation. Perturbation in their function results in the susceptibility to neuronal diseases. miR-9/miR-9\* expression is significantly reduced in patients with Hun‐ tington's disease. miR-9 and miR-132 are downregulated in Alzheimer-affected brain. miR-9 has role in ALS also. There are several miRNAs, which are specific for neural cell fate and cell cycle regulation. After inducing neuronal differentiation, miR-9, miR-124a, miR-19a and miR-19b are differentially expressed in SOD1-G93A epSPCs, while miR-9 gets upregulated in SOD1-G93A differentiated cells, with respect to WT-SOD1 and control mice. Expression analysis of the predicted miRNA targets leads to identification of a functional network of Hes1. Pten, Socs1 and Stat3 genes are important for controlling epSPC fate and showing neurogenic potential in vitro. A time-course analysis and cellular distribution pattern of miR-9 were tested.

It was inferred that miR-9 expression is spatially and temporally controlled in SOD1-G93A spinal cord and there is upregulation at presymptomatic stage through early symptomatic stages, mainly in the ventral horns of grey matter, where neurodegeneration is known to occur. There is a role of miR-9 in pathogenesis in induced pluripotent stem cell differentiated into postmitotic neurons and derived from ALS patients with the TDP43 A90V and M337V mutations. In all these cases, the levels of miR-9 are decreased with respect to control neurons. In *Drosophila* TDP43 mutants, miR-9a expression is significantly inhibited; it supports the concept that TDP43 acts through miR-9a to control neuronal specification and to assure the robustness of genetic control programmes. The steady-state level of miR-9 is required for normal neuronal functions, as both up- and downregulation can lead to neurodegeneration. Mutant SOD1 deregulates neurofilament (NF) balance in motor neurons [63]. NF proteins are one of the major intermediate filaments of neuronal cytoskeleton. They provide the structural integrity of neurons and help in maintenance of cell shape and axonal calibre. In CSF of ALS patients, low-molecular-weight NF is in abundance, which leads to a significant correlation between CSF NFL levels and disease progression. NF might be used as a marker of disease progression in ALS [64]. In ALS spinal motor neurons undergo a selective reduction in the steady-state level of NFL mRNA, which results in alteration of the stoichiometry of NF expression [63]. miR-9 is an upstream regulator of NF mRNAs and there are no such evidences in determination of regulation of NFL by miR-9 in ALS [54]. The q-PCR of sALS ventral lumbar spinal cord tissue shows that a set of additional miRNAs, predicted to control NFL, was deregulated. miR-146a\*, miR-524-5p and miR-582-3p are capable of interacting with NFL mRNA 3′ UTR in a manner that is steady with the suppressed steady-state mRNA levels observed for NFL in spinal motor neurons in ALS. This demonstrates the fact that miRNAs are involved in the suppression of NFL mRNA and ALS spinal motor neuron neurofilamen‐ tous form aggregates [65]. A recent study was performed on a RNA library derived from control and sALS spinal cords, and it was shown that a panel of novel miRNAs whose sequence is comprised in the miRNA response elements (MREs) within the NFL mRNA 3′ UTR was recognized to be differentially expressed in ALS, compared to control [66]. Functional analysis shows that miR-b1336 and miR-b2403 are significantly downregulated and both stabilise NFL mRNA. In motor neurons there are several miRNAs, which have the capability of controlling NF expression at post-transcriptional level. Dysregulation of miRNA expression occurs not only in motor neurons but also in spinal cord microglia isolated from SOD1-G93A mice. Scientists did whole expression profiling of miRNA and mRNA at presymptomatic stage and at onset of symptoms, and end stage of disease and pathway analysis was used to hypothesize a correlation between top ten altered inflammatory miRNAs and dysregulated inflammatory genes in ALS [65]. To characterize the relationship between miRNA/target deregulations functionally in ALS inflammation, microarray of miRNA from neonatal SOD1-G93A mouse brain primary microglia was done and it was found that the expression of mutant SOD1is able to increase about 50% of the expressed miRNAs [67]. There is significant increase in selected immune-enriched miRNAs, miR-22, miR-155, miR-125b and miR-146b amongst those most highly modulated. By luciferase assays and lentiviral infections, a group of researchers confirmed the increase in miR-365 and miR-125b, the last known to be involved in stimulation of pathogenic inflammatory responses and neurodegeneration by inhibiting neurotrophism-

glial cytoplasmic inclusions. The sensory organ precursor cells of peripheral nervous system of *Drosophila melanogaster* were used as a model to demonstrate that hTDP43 clinical ALS mutations influence early neurogenesis and neuronal specification, through the regulation of miR-9a biogenesis. Further studies in human cells show that TDP43 is an essential element in miRNA biogenesis during neuronal differentiation by controlling the stability of Drosha and thus affecting miRNA production. Similar inferences were drawn after FUS/TLS silencing in human neuronal cell lines. FUS/TLS downregulation affects the biogenesis of a large class of miRNA and several of them have important roles in neuronal differentiation and synaptogenesis. Specific miRNAs, in particular miR-9, miR-125b, miR-132 and miR-143, are regulated by both TDP43 and FUS/TLS ALS-related proteins. In recent reports, TDP43 was shown to bind to and regulate the incorporation into the RISC complex of specific mature miRNAs, in both Drosophila and human systems. TDP43 interacting with miR-1/miR-206 family decreases the activity of these miRNAs, by the disruption of their association with the RISC complex. As this miRNA family is involved in muscle development and homeostasis, either depletion or overexpression of TDP43, by altering miR-1/miR-206 balance, might be involved in muscle pathogenesis of ALS. Nine specific miRNAs were selected and their levels were analysed in ALS patients, on the basis of their known misregulation after TDP43 depletion in cell lines. Samples of cerebrospinal fluid (CSF), serum and immortalized lymphoblastoid cell lines from fALS and sALS patients were analysed, and it was confirmed that there is dysregulation of miR-132, miR-143 and miR-574, whose processing is indeed regulated by TDP43 and/or FUS/TLS proteins. The sequencing of the 3′UTR region of the FUS/ TLS gene in 420 ALS patients highlighted a mutation in two ALS patients with severe consequence, leading to FUS/TLS protein accumulation. This mutation maps to the seed sequence recognized by miR-141 and miR-200a in the 3′-UTR of FUS/TLS and that FUS/TLS is linked to these miRNAs by a feedforward regulatory loop, where FUS/TLS upregulates

150 Update on Amyotrophic Lateral Sclerosis

miR-141/200a, which in turn affects the FUS/TLS protein synthesis [21].

**15. CNS-related miRNA dysregulations in ALS pathogenesis**

miR-9, an evolutionarily conserved and multifunctional neuronal miRNA, is involved in the selection of neuronal precursors from the neuroepithelium in flies and in the specification of midbrain-hindbrain boundaries in vertebrates. Both miR-124 and miR-9 participates together in neuronal differentiation. Perturbation in their function results in the susceptibility to neuronal diseases. miR-9/miR-9\* expression is significantly reduced in patients with Hun‐ tington's disease. miR-9 and miR-132 are downregulated in Alzheimer-affected brain. miR-9 has role in ALS also. There are several miRNAs, which are specific for neural cell fate and cell cycle regulation. After inducing neuronal differentiation, miR-9, miR-124a, miR-19a and miR-19b are differentially expressed in SOD1-G93A epSPCs, while miR-9 gets upregulated in SOD1-G93A differentiated cells, with respect to WT-SOD1 and control mice. Expression analysis of the predicted miRNA targets leads to identification of a functional network of Hes1. Pten, Socs1 and Stat3 genes are important for controlling epSPC fate and showing neurogenic potential in vitro. A time-course analysis and cellular distribution pattern of miR-9 were tested.

relevant genes. miR-365 and miR-125b suppress the IL-6/STAT3 pathway in ALS microglia, by targeting IL-6 and STAT3, respectively, and they have a role in overall increase of TNFα mRNA levels. TNFα treatment increases miR-125b levels in microglia; thus it results in abnormal TNFα release. IL-6, a marker of activated microglia, is downregulated after symptom onset in ALS animal models and TNFα is upregulated in G93A mice and in ALS patients. miR-365 and miR-125b dysregulations might have a hand in pathological cytokine profile of ALS. People suggest that the pathogenesis of ALS involves astroglial dysfunction, with a dramatic loss of the excitatory amino acid transporter-2/glial transporter1 (EAAT2/GLT1) in both ALS patients and animal models [68–71]. The EAAT2/GLT1, is an important atroglial transporter, dynamically regulated by neurons, and it is involved in maintenance of extracel‐ lular glutamate concentration below neurotoxic level [72, 73]. The exosomes containing miR-124a and released from neurons can be directly internalized into astrocytes, which results in increase in miR-124a and GLT1 protein levels. miR-124a is downregulated in spinal cord of mutant SOD1 mouse models at end stage of disease, and in vivo injection of miR-124a oligonucleotides into spinal cord of ALS mice results in 30% increase in EAAT2/GLT1 expression. There is very little information about miRNA expression in ALS-induced muscle impairment. There is miR-23 upregulation in skeletal muscle biopsies, collected from ALS patients, and it is correlated with reduction in peroxisome proliferator-activated receptor coactivator-1 (PGC-1alpha) mRNA and protein, observed in both mouse and human muscle diseased samples. This coactivator is involved in muscle mitochondrial biogenesis and function. There is reduction in skeletal muscle mitochondrial function in ALS. It has been suggested that there might be a relation in miR-23a inhibition of PGC-1α and its downstream signalling [74, 75].

## **16. Role of miR-206 in disease impediment**

It was reported that miR-206, a skeletal muscle-specific miRNA, strongly induced in ALS mouse model is in coincidence with the onset of neurological symptoms. It is a modifier of disease pathogenesis. In G93A-SOD1 mice, loss of miR-206 expression does not affect disease onset apparently, but accelerates disease progression, by skeletal muscle atrophy, kyphosis and paralysis, and reduces survival. In miR-206−/−/G93A-SOD1 mice, neuromuscular junctions (NMJs) are disorganized and reinnervation of denervated muscles by motor axons is post‐ poned in the absence of miR-206. Transcript derived from the *miR-206/133b* locus, which was originally identified as a synapse-associated non-coding RNA called 7H4, has a role in encoding components of the postsynaptic apparatus. The reported 7H4 sequence does not include miR-206; RT-PCR shows that miR-206 sequences are included in this synapse-enriched transcript. Histone deacetylase 4 (HDAC4) mRNA is one of the strongest computationally predicted targets of miR-206. HDAC4 protein expression is upregulated in skeletal muscle of miR-206−/− animals in comparison with wild-type controls after denervation event. *Hdac4* mRNA levels remain unchanged in miR-206−/− mice. It suggests that miR-206 acts in this case by translational inhibition rather than by mRNA destabilization [21].
