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A multitude of studies have elucidated the crucial roles that miRNA play in the development of the brain and CNS with miRNAs emerging as critical regulators of the process (reviewed in 203). In addition to being highly abundant in the brain204, one of the earliest studies in this field highlighted the importance of miRNA in neuronal development through use of a mutated

version of the Dicer enzyme in zebrafish205. This mutation alone led to significant morphological defects in brain development due to irregular brain patterning and morphogenesis that was rescued through the introduction of a single miRNA mimic miR- 340205. Early lethality during embryogenesis and severe malformations have also been reported in Dicer knockout mice176. The development of transgenic mice that deleted Dicer in early adulthood shed some further insight into the important roles that miRNA play, with these mice having a diseased phenotype displaying neuronal death, memory loss, tremor, and spontaneous seizure activity176,206-211.

In addition to CNS development, the maintenance of health and homeostasis is also strongly mediated by miRNAs. MiR-26a has been implicated in many studies to be involved in a variety of neuronal processes such as the regulation of neuron morphology, axon regeneration and synapse development and plasticity212-215.

1.5.1.1. MicroRNA in the retina

In the retina, tight gene regulation is required for maintenance of visual health in what is an incredibly demanding physiological environment 64,65,67,94. Amongst the non-coding RNA involved in retinal gene regulation, miRNA remain the most well-studied (reviewed in 216). With over 300 identified miRNAs that are differentially expressed in developing and mature mouse retina217,218, the importance of miRNA in retinal development and physiology is becoming increasingly evident primarily through studies of the impact of their impairment in specific retinal cell types and development stages.

Due to the differential or selective regulation of miRNA among tissue, highly expressed retinal miRNA seem to have specific spatial expression patterns in the retina219-222. In 2010,

Karali et al., performed high-resolution expression atlas that allowed for the characterisation of the spatiotemporal distribution of 221 miRNAs in the mouse eye, further demonstrating notably distinct enrichment patterns in the retina219. More than 100 of these miRNAs also displayed restricted expression domains at varying developmental stages219.

Similar to the brain, there have been several studies that demonstrate the repercussions in the retina following the ablation of the Dicer enzyme, which as mentioned previously, is key to the biogenesis of miRNA223-226. Global perturbation of miRNA biogenesis has been shown to disrupt the normal development of the retina, lens, cornea and optic chiasm223,224,226. Dicer deletion has also been shown to impair the developmental transition of retinal progenitors, lead to the disorganisation of the photoreceptor OS, and result in abnormal retinal morphology promoting retinal degeneration227. Specifically, miR-125, miR-9 and let-7 have been revealed to control the transition from early to late stage retinal progenitors225. Further, it was demonstrated that Dicer1 ablation in retinal Müller glia led to the interference of the normal migration of Müller cells, causing them to be displaced to other retinal layers, which disrupted the normal retinal architecture and reduced visual acuity228. Similar effects were seen in specific Dicer ablation in RPE that resulted in improper RPE differentiation and function229.

The retina-enriched miRNA cluster, miR-183/96/182, is a well-studied subset of miRNA for photoreceptor function. They have been shown to be highly abundant in photoreceptors and strongly regulated by light218,220,230. It has been shown that inactivation in this cluster in mice resulted in photoreceptor dysfunction and defective synaptic transmissions, which led to progressive retinal degeneration231. When miRNAs were specifically depleted in cone

photoreceptors, the supplementation of miR-182 and miR-183 preserved cone outer segments and restored light responses in vitro232.

The miR-204/miR-211 family is one of the most abundant in the retina with both being expressed very strongly in all neuronal layers of the retina233, and having highly similar sequences and targeting capabilities. Loss of miR-204 induced apoptosis and altered the expression of photoreceptor markers, whereas miR-211 disturbances led to progressive dystrophy of the cone photoreceptors complemented by loss of the photoreceptors and subsequent alterations in visual function233.

These previously mentioned miRNA families and clusters are perhaps the most well characterised retinal miRNAs thus far, but the importance of others in maintaining tissue health and homeostasis cannot be discounted. Table 1.1 summarises those miRNAs that are shown to be highly expressed in the retina and where they are found.

Table 1.1. Highly expressed microRNA in the retina

MicroRNA Retinal location Species References

miR-9 INL – Müller cells Mouse 220

miR-23a RPE Mouse 234

miR-29b GCL, INL Mouse 235

miR-29c INL, ONL Mouse 220

miR-124 GCL, INL, ONL Mouse 220,236,237

miR-204/miR-211 RPE, INL Human, mouse 233,242-244

Let-7d INL Mouse 197

1.5.1.2. miR-124 – A neuronal-enriched miRNA

MiR-124, has been observed to be the most abundant miRNA in the brain accounting for 25- 48% of all brain miRNAs221. By suppressing hundreds of non-neural genes miR-124 contributes to both the acquisition and maintenance of neuronal identity245,246. In an early study, the administration of miR-124 in HeLa cells resulted in a shift of the gene expression of the cells to resemble that of the brain246_{. It was demonstrated that miR-124 promotes} neuronal differentiation through the suppression of anti-neural factor SCP1 and PTPB2, which culminates in the transition to neuron-specific transcriptome splicing247. miR-124 also is suggested to promote neurite outgrowth with the blocking of its function delaying the process248. Further, miR-124 is involved in the regulation of processes in maintaining glutamate homeostasis249_{, and suppressing microglial activation and astrocyte reactivity}250,251_, therefore having a role in the regulation of neuroinflammation (to be discussed further in Chapter 5).

MiR-124 levels are also enriched in the retina and shown to be largely neuronal-specific with high levels found in the photoreceptors236,237,252. Its importance in retinal development was shown in Rncr3-/- _{mice where the main primary transcript of miR-124 is disrupted}237_{. This} revealed that through targeting of the gene Lhx2, miR-124 is pivotal for the maturation and survival of cone photoreceptors237. However, despite its prominence in the retina, highly conserved nature (from C. elegans to humans221), and function in retinal development, its presence and activity in retinal degenerations, if any, has yet to be elucidated and only

putative links have been made specifically to its role in microglia and neural inflammation250,253,254.