MICRO RNAS – DEPENDENT REGULATORY PATHWAYS INVOLVED IN NEURONAL DEVELOPMENT AND NERVOUS SYSTEM DISEASE

ABSTRACT: 

Brain development and neural differentiation are processes regulated by complex molecular mechanisms. During the last years, it was discovered that small noncoding RNA molecules called microRNAs are implicated in this processes. MicroRNAs are RNA molecules of about 22 nucleotides long, with partial or full complementarity with target mRNA sequences. They block Mrna translation after associating with its target sequence, through the mediation of a ribonucleoprotein complex called RISC (RNA induced silencing complex). This mechanism ensures a quick and precise transition between the gene expression pattern of one development al stage to the next. Hereditary or acquired nervous system diseases, either degenerative or not, they were associated with changes in microRNAs expression. These changes have effects on disease onset or evolution. Future studies will try to reveal the precise mechanism for microRNA action and the ways of how these molecules can be used for treatment.

INTRODUCTION

Differentiation, development and proper functioning of the nervous system depend on a strict regulation of gene expression, especially of those that have specific expression according to tissue, stage of development or to the function performed. Most of the genes involved in controlling gene expression encode proteins with affinity for nucleic acids, in particular for regulating the level of DNA sequences. In recent years a large number of small molecules of RNA (ribonucleic acid) as reglators1,2 have been discovered. They have been involved in controlling key cellular processes such as: differentiation, development, proliferation, apoptosis, stress response, and in triggering or influencing the development of diseases such as cancer or neurodegenerative diseases.

These RNA molecules with a regulating role have been called microRNAs (miRNA) due to their very small size of only 22 to 25 nucleotides in length compared to a molecule of messenger RNA (mRNA) which can be made up of several thousand nucleotides. The miRNA pathway resembles another regulatory pathway involving small non-coding RNA molecules: the interference path through RNA (RNAi). The difference is that the latter involves the formation of bicatenary RNA molecules (siRNA – small interfering RNA) which control the specific mRNA degradation. This mechanism is currently used in most functional molecular studies.

Over 1,400 different types of miRNA have been described in humans3 and included in the Mirbeau database hosted by the Faculty of Natural Sciences, University of Manchester3. Some miRNA present in humans are phylogenetically preserved starting from mice and even from other inferior organisms such as Caernorhabditis elegans and Drosophila. Some miRNA are specific to humans 2, 4. Current data suggest that over 30% of genes are regulated by mechanisms dependent on miRNA.

MIRNA SYNTHESIS AND ACTION MECHANISMS

miRNA encoding genes are located either in the introns of certain protein coding genes or in intergenic regions, singly or in groups (clusters) of miRNA5. Some miRNA are expressed constituently in all cells, others only in certain tissues or stages of deve-lopment5.

Mature miRNA synthesis is achieved in a sequence of steps that begin in the nucleus and finish in the cytoplasm5. The process of generating mature miRNA is illustrated in Figure 1.

Figure 1. The synthesis of miRNA.

Transcription is made by RNA polymerase II, which is involved in the transcription of RNAm; the primary transcript (pri-miRNA) is immediately processed through the detachment of miRNA precursor (pre-miRNA) by the proteins complex called microprocessor , which includes Drosha and DGCR8 (DiGeorge Syndrome Critical Region gene 8), the precursor has a characteristic structure „in hairpin” that allows recognition and processing to the next steps; The export of the precursor in the cytoplasm – is actively mediated by a protein called exportina V; Isolation of mature miARN is processed in the cytoplasm at the level of the protein complex called RISC (RNA Interference silencing complex) where is detached and built the single-stranded molecule miARN mature,the rest of molecule precursor being directed to degradation. The RISC complex directs the miARN molecule to target (changed by Schratt, G. MicroRNAs at the synapse. Nature Reviews Neuroscience 10, 8-2009).

MicroRNA genes that are part of a cluster of genes are transcribed together, in the same primary molecule, while their separation takes place later; often, members of a cluster have a coordinated action5.

Four proteins have an essential role in the synthesis and action of miRNA, a fact that the functional studies have taken advantage of5:

-Drosha – an enzyme belonging to the ribonuclease III family which carries out the separation of the precursor pre-miRNA from the molecule of the primary transcript;

-DGCR8 – DiGeorge Syndrome Critical Region gene 8 found in a chromosomal region affected in the DiGeorge syndrome; it interacts with Drosha and facilitates linking to pre-miRNA molecule;

-Dicer – an enzyme from the same family as the enzyme Drosha, with a role in the cytoplasmic processing of the pre- miRNA precursor to the mature miRNA;

-Ago2 (Argonaut) – in humans it has the official name EIF2C2 (eukaryotic translation initiation factor 2C, 2); it interacts with Dicer and contributes to the recognition and separation of mature miRNA;

MicroRNAs’ role is to fine-tune, specifically, the expression of certain important genes for the development of cellular processes. This is achieved by blocking the translation of mRNA containing the recognition sequence (target) in its terminal portion. MiRNA is attached to the recognition sequence located in its terminal region, preventing the advancement of the translational complex. Depending on the degree of complementarity of miRNA with the target sequence, the effect can only be the blocking of translation or the total degradation of mRNA.

This mechanism of regulating gene expression has the advantage of being more rapid than the one based on the stimulatory action of certain transcription factors and the de novo synthesis of a messenger RNA and of the corresponding protein. This rapid intervention is essential especially for the transition between different stages of development and differentiation or for the cellular response to stress exerted by environmental factors.

NOMENCLATURE 

Genes encoding microRNAs are named by associating the prefix combination mir (mi from the microRNA and r from RNA) with a figure indicating the order number received at its inclusion in the database: for example, mir-1.

Table 1. Differentiated expression of miRNA during neuronal development

Mature microRNA molecule is named in a similar way but with the capitalized R: e.g. miR-1. If two or more mature miRNA molecules have similar sequences, they will receive the same number plus a letter: for example miR-1a 3,6. An exception to this rule are genes let-7 and lin-4 that have kept the name under which they were described initially 1,2,6.

ROLE OF MIRNA IN NEURONAL DIFFERENTIATION

Neuronal cells result from a complex process of transformation of multipotent progenitor cells, under the action of complex regulating systems involving the expression of genes specific to each stage.

Based on the critical role of miRNA in the development of the body, numerous studies have been initiated to elucidate their role in neuronal differentiation and in the development of the nervous system. All these studies have shown that the most important stages of these processes are dependent on the miRNA pathway7. Due to the very small molecule and to the imperfect complementarity with the target sequence, in silico identification of mRNA subjected to regulation by miRNA is very difficult, despite the complex protocols developed to date5.

Researchers have described miRNA molecules specifically expressed in the brain, with a role in neurogenesis, cell employing in one of the directions of cell differentiation, ensuring tissue specificity and survival, morphogenesis and synaptic transmission 8-14.

The process of converting stem cells into neurons has been evaluated experimentally as having five successive stages, with the expression of a specific miRNA set in each stage:

– Stem cell stage;

– Embryonic body stage;

– Neural precursor stage;

– Neural differentiation stage.

Among miRNA species with an essential role in determining cell destination during neural development one must mention: miR-131, which is mainly expressed in the embryonic stage rather than in the adult one, miR-9 and miR-19b which are more intensely expressed during neurogenesis and miR-124 and 128 which are expressed later towards the adult stage 11. Table I presents a summary of key miRNA molecules that have been associated with regulating various stages of development.

Neural progenitor cells, whose pathway has been blocked by inhibiting the expression of miRNA Dicer enzyme, express proteins more intensively, which maintain their undifferentiated state. Among them stand Hmga2 and Prominina-1 involved in maintaining stem cells. Hmga2 is expressed in neural stem cells, early embryonic period, and serves to inhibit the expression of cell cycle inhibitors, which help maintain the proliferative capacity of neural stem cells. As the process of differentiation is advancing, the expression of Hmga2 decreases due to the dampening effect of the microRNA belonging to let-7 family.

Let-7 is one of the first microRNA molecules discovered and one of the most widely expressed miRNAs in all cells and tissues, in almost all organisms. Originally described as an activator of the transition between stages of development in C. elegans2 and then connected to the pupa stage and that of adult in D. melanogaster1. Later it was involved in the modulation of cell proliferation, tumour transformation or evolution of some cancers. In humans, let-7 family has 11 members3.

When using inducers of differentiation of neuronal cells whose path was blocked by inhibiting miRNA Dicer, apoptosis is affected before initiating the process of differentiation. In return, the function of self-renewing neural progenitor cells is less affected by the absence of miRs, even if the pace of cell division is slowed down 15.

With regard to glial differentiation, it can be initiated even in the event of miRNA pathway blockade but it cannot be finalised 15.

It is interesting that these effects are reversible if the level of cellular microRNA is restored either by supplying those molecules necessary for the initiation and completion of differentiation or by restoring the expression of Dicer 15. Even small amounts of miRNA are sufficient for the process of differentiation. In addition, although normally the lifetime of a RNA molecule is short, miRNA molecules persist in a cell for several days after their production, a fact which makes their production inhibiting effect visible only after a few days 15.

Studies on zebrafish have shown that brain formation is poor if the miRNA pathway is blocked during the embryonic stage. miR-430a, miR-430b and miR430c have been involved, which have a role in the degradation of mRNA of maternal origin and in the transition from zygote to embryo 16.

Some miRNA molecules are expressed in specific brain regions (e.g., miR-222 in telencephalon) or in types of cells (miR-218a in motor neurons) 9.

Abolition of miRNA causes abnormalities of the mesencephalon and cerebellum and the failure of the differentiation of dopaminergic neurons; abnormalities of the dorsal root ganglia, enteric nervous system and sympathetic ganglia 17.

An interesting finding was the association of miRNA molecules known for their role in apoptosis with different stages of neuronal differentiation. The expression of miR-16, let-7a and miR-34a is increased during neuronal development. Anti-proliferative role of these molecules is consistent with the transition from the state of mitotic active precursor to the one of terminally differentiated cell, such as the mature neuron. The expression of anti-apoptotic miRNAs correlates with that of proteins with similar function: protein p53, caspase and Bcl-2 protein are also increased.

Other miRNAs such as miR-19a and miR-20a, with the incentive role of cell proliferation, are inhibited during differentiation, except for miR-19a which is expressed more intensely in the early stages, when cells are still active mitotically 18.

MIRNA AND NEUROLOGICAL DISORDERS

The importance of processes under the control of miRNA pathway suggests the possibility of their involvement in the neurologic pathology 19. Although functional studies are still insufficient, existing data support the direct or indirect involvement of small non-coding RNA molecules in conditions such as: Parkinson’s disease, Alzheimer disease, spinal amyotrophy, mental retardation.

FRAGILE  X SYNDROME 

The protein FMR1 (Fragile X Mental Retardation Protein), whose mutations cause fragile X syndrome, was one of the first proteins involved in neurological disorders associated with the functioning of miRNA pathway20 after its interaction with Dicer, miRNA, Argonaut protein (Ago2) and translationally active ribosomes has been revealed 11, 20, suggesting involvement of the miRNA pathway in the regulating mechanism dependent on FMR1.

FMRP protein has a RNA-binding domain involved in the regulation of translation of attached mRNA molecules. Protein expression is ubiquitous, more intense in the brain, where it is associated with about 400 mRNAs 21. It intervenes in the performance of complex functions: memory, learning, cognitive functions 21.

Change through phosphorylation of FMR1 results in its attachment to numerous pre-miRNA molecules while blocking the association with Dicer, which prevents mRNA translation and also the processing of miRNA precursors 22. The final effect is the alteration of neural functions dependent on molecules processed with the help of FMR1.

Table 2. Differentiated expression of miRNA during neuronal development

SPINAL  ATROPHY

Spinal muscular atrophy is an autosomal recessive genetic disease caused by deficiency of SMN (survival motor neuron) protein. The genetic defect consists in the mutation of both alleles of a ubiquitous expressed gene, called neuronal survival gene 1 (SMN1). The SMN protein, as inferred from its name, is essential for cell viability23,24. The two SMN genes, 1 and 2, have sequences that differ by several nucleotides. SMN2 gene expression product is shorter due to the absence of exon 7. In approximately 95% of cases the disease is caused by mutations in the gene SMN1 and SMN2 gene determines the severity of the disease 23. Complete elimination of both SMN genes is lethal early, during the embryo stage 24.

The SMN complex is composed of SMN protein that interacts with other proteins of the same type and with proteins Gemin 2-8 and unrip. MiRNAs are constant components of the ribonucleoproteic complex and interact directly with the Gemine-SMN protein group. A total of 40 miRNAs was isolated from ribonucleoproteins containing the proteins Gemin 3, Gemin 4, eIF2C2 25.

The main function of SMN is to contribute, together with other components of the SMN complex to the RNA metabolism by assembling small nuclear ribonucleoproteins (snRNP) involved in splice. In addition, SMN proteins have a role in transcription and control of axonal transport 26, 27. MicroRNA activity is necessary for the survival of post mitotic spinal motor neurons in vivo 25.

Lack of SMN in spinal amyotrophy determines the alteration of splice process of protein synthesis necessary to cell functions 27.

The reason why a protein with widespread distribution in the body and with a function in all cell types specifically affects the peripheral motor neuron is still not known with precision. Recent studies have shown that the pathogenic elements could be the altered ribonucleoproteins assembly 28 and the impaired neuromuscular synapses 29.

The fact is certain that disease severity correlates with the alteration degree of the formation of SMN and snRNP complex in the spinal cord, as observed in mice used as a model of SMA. Most affected are the components Gemin 2, 6 and 8 of the SMN complex, especially Gemin 8 27.

PARKINSON’S DISEASE

MiRNAs are essential for the formation 17 and maintaining of dopaminergic neurons. Altered miRNA pathway generates a phenotype similar to Parkinson’s disease in affected mice 30.

Two molecules of miRNA, miR-7 and miR-133 regulate the post-transcriptional levels of alpha-synuclein, a protein that accumulates in Parkinson’s disease. The two miRNA are expressed predominantly in the brain in inverse proportion to synuclein. It is considered that the action of the two miRNA could be the basis of new therapeutic methods in Parkinson’s disease 31.

ONCOLOGY

Since the early years of their discovery, miRNAs have been associated with neoplasia pathology 32. Brain cancer studies revealed abnormal expression of certain miRNAs:

-miR-21 overexpression in the glioblastomas, which acts oncogenetically, as well as in other forms of cancer;

-miR-124 and miR-137 are under-expressed in anaplastic astrocytomas; miR-137 target is CDK6, a stimulator of cellular cycle which thus remains active;

-Let-7 is involved in most cancers studied to date.

The existence of certain miRNA molecules with inhibitory effect on uncontrolled cell proliferation allowed the initiation of trials in order to find certain therapeutic protocols.

CONCLUSIONS

Most existing data to date support the association between certain species of miRNA and disease onset or development, including the diseases of the nervous system. The importance of miRNA for the development of the nervous system is confirmed by the severe effects of their depletion but the precise mechanisms of their intervention still require numerous clarifications. The pace of discoveries in this area supports the inclusion of such microRNA markers in the future standard disease diagnosis and evolution monitoring protocols. Currently, researchers are trying to identify certain plasmatic markers for non-invasive diagnosis of disease. Improvement of miRNA-based therapeutic means could correct some defects with greater specificity than conventional treatments that are used currently.

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