María Celina Rodicio

Analytical aspects of microRNA in diagnostics: a review.

MicroRNAs (miRNA) are short (∼22 nucleotides) non-coding RNA molecules that regulate gene expression at the post-transcriptional level. Their expression is specific to cells and tissues and is temporally regulated. miRNAs are known to be involved in developmental and physiological processes, and their dysregulation leads to development of diseases. Since their profiles reflect pathological processes, miRNAs have recently been proposed as being useful in diagnostics as biomarkers of the onset, prognosis and risk of diseases, as well as in the classification of different types of cancer. The establishment of miRNA profiles that are representative of diseases and the detection of different types and levels of miRNA in samples are therefore critical milestones in diagnostics. miRNAs can be detected in blood and body fluids as well as in tissues, thus making non-invasive collection of samples possible. For a method to be useful in diagnostics, it should be simple, inexpensive and highly sensitive. Here, we will review current methods of detecting miRNAs and indicate the advantages and disadvantages of each techniques. We will then summarize some of the clinical evidence for the potential application of miRNAs as biomarkers in diagnostics. We conclude providing some general perspectives on the use of miRNAs in clinical situations, including therapeutic applications.

Rapid in situ codetection of noncoding RNAs and proteins in cells and formalin-fixed paraffin-embedded tissue sections without protease treatment

Noncoding RNAs (ncRNAs) comprise a diverse group of RNAs that function in essential cellular processes such as pre-mRNA splicing and mRNA translation and also regulate various aspects of gene expression in physiology and development. Methods of subcellular and tissue localization of ncRNAs are essential to understand their biological roles and their contribution to disease. We describe a rapid fluorescent (FISH) or chromogenic (CISH) in situ hybridization protocol for localization of ncRNAs (including microRNAs (miRNAs), small nucleolar RNAs (snoRNAs), small nuclear RNAs (snRNAs), piwi-associated RNAs (piRNAs) and ribosomal RNAs (rRNAs)) in formalin-fixed, paraffin-embedded (FFPE) tissues and cultured cells, using locked nucleic acid (LNA)-modified oligonucleotides. In this protocol, sections are heated in citrate buffer, which eliminates the need for protease treatment, thus preserving optimal morphology and protein epitopes, and allowing the simultaneous detection of proteins with immunofluorescence staining (IF). LNA–FISH requires 5 h, or between 10 and 36 h when combined with IF; LNA–CISH requires 2 d.

Ontogeny of γ‐aminobutyric acid‐immunoreactive neuronal populations in the forebrain and midbrain of the sea lamprey

Although brain organization in lampreys is of great interest for understanding evolution in vertebrates, knowledge of early development is very scarce. Here, the development of the forebrain and midbrain γ‐aminobutyric acid (GABA)‐ergic systems was studied in embryos, prolarvae, and small larvae of the sea lamprey using an anti‐GABA antibody. Ancillary immunochemical markers, such as proliferating cell nuclear antigen (PCNA), calretinin, and serotonin, as well as general staining methods and semithin sections were used to characterize the territories containing GABA‐immunoreactive (GABAir) neurons. Differentiation of GABAir neurons in the diencephalon begins in late embryos, whereas differentiation in the telencephalon and midbrain was delayed to posthatching stages. In lamprey prolarvae, the GABAir populations appear either as compact GABAir cell groups or as neurons interspersed among GABA‐negative cells. In the telencephalon of prolarvae, a band of cerebrospinal fluid‐contacting (CSF‐c) GABAir neurons (septum) was separated from the major GABAir telencephalic band, the striatum (ganglionic eminence) primordium. The striatal primordium appears to give rise to most GABAir neurons observed in the olfactory bulb and striatum of early larval stages. GABAir populations in the dorsal telencephalon appear later, in 15–30‐mm‐long larvae. In the diencephalon, GABAir neurons appear in embryos, and the larval pattern of GABAir populations is recognizable in prolarvae. A small GABAir cluster consisting mainly of CSF‐c neurons was observed in the caudal preoptic area, and a wide band of scattered CSF‐c GABAir neurons extended from the preoptic region to the caudal infundibular recess. A mammillary GABAir population was also distinguished. Two compact GABAir clusters, one consisting of CSF‐c neurons, were observed in the rostral (ventral) thalamus. In the caudal (dorsal) thalamus, a long band extended throughout the ventral tier. The nucleus of the medial longitudinal fascicle contained an early‐appearing GABAir population. The paracommissural pretectum of prolarvae and larvae contained a large group of non‐CSF‐c GABAir neurons, although it was less compact than those of the thalamus, and a further group was found in the dorsal pretectum. In the midbrain of larvae, several groups of GABAir neurons were observed in the dorsal and ventral tegmentum and in the torus semicircularis. The development of GABAergic populations in the lamprey forebrain was similar to that observed in teleosts and in mouse, suggesting that GABA is a very useful marker for understanding evolution of forebrain regions. The possible relation between early GABAergic cell groups and the regions of the prosomeric map of the lamprey forebrain (Pombal and Puelles [ 1999 ] J. Comp. Neurol. 414:391–422) is discussed in view of these results and information obtained with ancillary markers. J. Comp. Neurol. 446:360–376, 2002.

Detection methods for microRNAs in clinic practice

MicroRNAs (miRNA) are short non-coding RNA molecules that regulate gene expression. miRNAs profiles are specific for cell lineages and tissues, and their changes reflect pathological processes. This fact introduces the possibility of their use in diagnostics. The application of miRNAs in diagnostics is critically dependent on the establishment of miRNA profiles that can discriminate patients from normal healthy individuals with good sensitivity and specificity and on the development of methods for their accurate and high-throughput quantification. In this review, we present an overview of some of the different techniques and methods currently used to detect miRNAs. We focus on methods that can be employed in routine clinic diagnostics indicating their advantages as well as their shortcomings, with special attention being paid to the most innovative ones. Since disease-specific miRNAs can be found in blood serum, we also present emerging methods for the detection of circulating miRNAs as a way of fast, reliable and non-invasive diagnostic.

Early development of the retina and pineal complex in the sea lamprey: Comparative immunocytochemical study

Lampreys have a complex life cycle, with largely differentiated larval and adult periods. Despite the considerable interest of lampreys for understanding vertebrate evolution, knowledge of the early development of their eye and pineal complex is very scarce. Here, the early immunocytochemical organization of the pineal complex and retina of the sea lamprey was studied by use of antibodies against proliferating cell nuclear antigen (PCNA), opsin, serotonin, and γ‐aminobutyric acid (GABA). Cell differentiation in the retina, pineal organ, and habenula begins in prolarvae, as shown by the appearance of PCNA‐negative cells, whereas differentiation of the parapineal vesicle was delayed until the larval period. In medium‐sized to large larvae, PCNA‐immunoreactive (‐ir) cells were numerous in regions of the lateral retina near the differentiated part of the larval retina (central retina). A late‐proliferating region was observed in the right habenula. Opsin immunoreactivity appears in the pineal vesicle of early prolarvae and 3 or 4 days later in the retina. In the parapineal organ, opsin immunoreactivity was observed only in large larvae. In the pineal organ, serotonin immunoreactivity was first observed in late prolarvae in photoreceptive (photoneuroendocrine) cells, whereas only a few of these cells appeared in the parapineal organ of large larvae. No serotonin immunoreactivity was observed in the larval retina. GABA immunoreactivity appeared earlier in the retina than in the pineal complex. No GABA‐ir perikaryon was observed in the retina of larval lampreys, although a few GABA‐ir centrifugal fibers innervate the inner retina in late prolarvae. First GABA‐ir ganglion cells occur in the pineal organ of 15–17 mm larvae, and their number increases during the larval period. The only GABA‐ir structures observed in the parapineal ganglion of larvae were afferent fibers, which appeared rather late in development. The time sequence of development in these photoreceptive structures is rather different from that observed in teleosts and other vertebrates. This suggests that the unusual development of the three photoreceptive organs in lampreys reflects specialization for their different functions during the larval and adult periods. J. Comp. Neurol. 442:250–265, 2002.

Presence of glutamate, glycine, and γ‐aminobutyric acid in the retina of the larval sea lamprey: Comparative immunohistochemical study of classical neurotransmitters in larval and postmetamorphic retinas

The neurochemistry of the retina of the larval and postmetamorphic sea lamprey was studied via immunocytochemistry using antibodies directed against the major candidate neurotransmitters [glutamate, glycine, γ‐aminobutyric acid (GABA), aspartate, dopamine, serotonin] and the neurotransmitter‐synthesizing enzyme tyrosine hydroxylase. Immunoreactivity to rod opsin and calretinin was also used to distinguish some retinal cells. Two retinal regions are present in larvae: the central retina, with opsin‐immunoreactive photoreceptors, and the lateral retina, which lacks photoreceptors and is mainly neuroblastic. We observed calretinin‐immunostained ganglion cells in both retinal regions; immunolabeled bipolar cells were detected in the central retina only. Glutamate immunoreactivity was present in photoreceptors, ganglion cells, and bipolar cells. Faint to moderate glycine immunostaining was observed in photoreceptors and some cells of the ganglion cell/inner plexiform layer. No GABA‐immunolabeled perikarya were observed. GABA‐immunoreactive centrifugal fibers were present in the central and lateral retina. These centrifugal fibers contacted glutamate‐immunostained ganglion cells. No aspartate, serotonin, dopamine, or TH immunoreactivity was observed in larvae, whereas these molecules, as well as GABA, glycine, and glutamate, were detected in neurons of the retina of recently transformed lamprey. Immunoreactivity to GABA was observed in outer horizontal cells, some bipolar cells, and numerous amacrine cells, whereas immunoreactivity to glycine was found in amacrine cells and interplexiform cells. Dopamine and serotonin immunoreactivity was found in scattered amacrine cells. Amacrine and horizontal cells did not express classical neurotransmitters (with the possible exception of glycine) during larval life, so transmitter‐expressing cells of the larval retina appear to participate only in the vertical processing pathway. J. Comp. Neurol. 499:810–827, 2006.

Ontogeny of γ-aminobutyric acid–immunoreactive neurons in the rhombencephalon and spinal cord of the sea lamprey

The development of neurons expressing γ‐aminobutyric acid (GABA) in the rhombencephalon and spinal cord of the sea lamprey (Petromyzon marinus) was studied for the first time with an anti‐GABA antibody. The earliest GABA‐immunoreactive (GABAir) neurons appear in late embryos in the basal plate of the isthmus, caudal rhombencephalon, and rostral spinal cord. In prolarvae, the GABAir neurons of the rhombencephalon appear to be distributed in spatially restricted cellular domains that, at the end of the prolarval period, form four longitudinal GABAir bands (alar dorsal, alar ventral, dorsal basal, and ventral basal). In the spinal cord, we observed only three GABAir longitudinal bands (dorsal, intermediate, and ventral). The larval pattern of GABAir neuronal populations was established by the 30‐mm stage, and the same populations were observed in premetamorphic and adult lampreys. The ontogeny of GABAergic populations in the lamprey rhombencephalon and spinal cord is, in general, similar to that previously described in mouse and Xenopus. J. Comp. Neurol. 464:17–35, 2003.

Ganglion cells and retinopetal fibers of the larval lamprey retina: An HRP ultrastructural study

The ultrastructure of ganglion cells and centrifugal fibers of the larval lamprey retinas were studied using horseradish peroxidase (HRP) as a marker. Larval ganglion cells were found both in the inner nuclear layer and the inner plexiform layer of the differentiated retina, and also were present in the undifferentiated retina. Direct photoreceptor-ganglion cell contacts and the presence of centrifugal fibers are described for the first time in the lamprey. The centrifugal fibers contact directly with ganglion cells in this species.

Biochemical and genetic evidence for a role of IGHMBP2 in the translational machinery

The human motor neuron degenerative disease spinal muscular atrophy with respiratory distress type 1 (SMARD1) is caused by loss of function mutations of immunoglobulin mu-binding protein 2 (IGHMBP2), a protein of unknown function that contains DNA/RNA helicase and nucleic acid-binding domains. Reduced IGHMBP2 protein levels in neuromuscular degeneration (nmd) mice, the mouse model of SMARD1, lead to motor neuron degeneration. We report the biochemical characterization of IGHMBP2 and the isolation of a modifier locus that rescues the phenotype and motor neuron degeneration of nmd mice. We find that a 166 kb BAC transgene derived from CAST/EiJ mice and containing tRNA genes and activator of basal transcription 1 (Abt1), a protein-coding gene that is required for ribosome biogenesis, contains the genetic modifier responsible for motor neuron rescue. Our biochemical investigations show that IGHMBP2 associates physically with tRNAs and in particular with tRNA(Tyr), which are present in the modifier and with the ABT1 protein. We find that transcription factor IIIC-220 kDa (TFIIIC220), an essential factor required for tRNA transcription, and the helicases Reptin and Pontin, which function in transcription and in ribosome biogenesis, are also part of IGHMBP2-containing complexes. Our findings strongly suggest that IGHMBP2 is a component of the translational machinery and that these components can be manipulated genetically to suppress motor neuron degeneration.

Calbindin and calretinin immunoreactivity in the retina of adult and larval sea lamprey.

The presence of calretinin and calbindin immunoreactivity is studied in the retina of larval and adult lamprey and their respective distributions are compared. Calretinin distribution is also studied in the retina of transforming stages. Western blot analysis in brain extracts showed a 29-kDa band with both polyclonal anti-calbindin and anti-calretinin antibodies. Calbindin and calretinin immunoreactivity has shown a partially different distribution. In the adult retina large and small bipolar cells, with respectively stratified or diffuse axons, the inner row of horizontal cells and ganglion cells and/or some amacrine cells were labeled with anti-calretinin antibody. The anti-calbindin antibody labels the same cell types except most of ganglion cells, but the label was less conspicuous. Therefore, the possible existence of these two calcium-binding proteins in the central nervous system of the sea lamprey could be discussed. In the differentiated central retina of larval lampreys, numerous calretinin immunoreactive bipolar and ganglion cells were observed, while, in the lateral retina, only ganglion cells were labeled, accordingly with the lack of differentiation of other neural cell types. CR-ir bipolar cells appeared in the retina by the stage 5 of transformation, i.e. about the time when differentiation of photoreceptors occurs. The comparison of the distribution of calretinin and calbindin between adult and larval central retina of lampreys shows striking differences that could be related to the different functionality of eyes in these two stages of the life cycle of lampreys. In addition, this is the first report on the presence of calcium-binding proteins in the larval and transforming lamprey retina, on the presence of calretinin- and calbindin-immunoreactive horizontal cells in adult lamprey retinas and on the differential stratification of bipolar cell terminals.