Neurodegenerative disorders, such as Alzheimer’s disease (AD), Parkinson’s disease (PD) and frontotemporal dementias (FTD), are considered distinct entities, however, there is increasing evidence of an overlap from the clinical, pathological and genetic points of view. All neurodegenerative diseases are characterized by neuronal loss and death in specific areas of the brain, for example, hippocampus and cortex for AD, midbrain for PD, frontal and temporal lobes for FTD. Loss of neurons is a relatively late event in the progression of neurodegenerative diseases that is typically preceded by other events such as metabolic changes, synaptic dysfunction and loss, neurite retraction, and the appearance of other abnormalities, such as axonal transport defects. The brain’s ability to compensate for these dysfunctions occurs over a long period of time and results in late clinical manifestation of symptoms, when successful pharmacological intervention is no longer feasible. Currently, diagnosis of AD, PD and different forms of dementia is based primarily on analysis of the patient’s cognitive function. It is therefore important to find non-invasive diagnostic methods useful to detect neurodegenerative diseases during early, preferably asymptomatic stages, when a pharmacological intervention is still possible. Altered expression of microRNAs (miRNAs) in many disease states, including neurodegeneration, and increasing relevance of miRNAs in biofluids in different pathologies has prompted the study of their possible application as neurodegenerative diseases biomarkers in order to identify new therapeutic targets. Here, we review what is known about the role of miRNAs in the pathogenesis of neurodegeneration and the possibilities and challenges of using these small RNA molecules as a signature for neurodegenerative conditions.

Spermatogenesis is maintained by a pool of spermatogonial stem cells (SSCs). Analyses of the molecular profile of SSCs have revealed the existence of subsets, indicating that the stem cell population is more heterogeneous than previously believed. However, SSC subsets are poorly characterized. In rodents, the first steps in spermatogenesis have been extensively investigated, both under physiological conditions and during the regenerative phase that follows germ cell damage. In the widely accepted model, the SSCs are type Asingle (As) spermatogonia. Here, we tested the hypothesis that As spermatogonia are phenotypically heterogeneous by analyzing glial cell line-derived neurotrophic factor (GDNF) family receptor α1 (GFRA1) expression in whole-mounted seminiferous tubules, via cytofluorimetric analysis and in vivo colonogenic assays. GFRA1 is a coreceptor for GDNF, a Sertoli cell-derived factor essential for SSC self-renewal and proliferation. Morphometric analysis demonstrated that 10% of As spermatogonia did not express GFRA1 but were colonogenic, as shown by germ cell transplantation assay. In contrast, cells selected for GFRA1 expression were not colonogenic in vivo. In human testes, GFRA1 was also heterogeneously expressed in Adark and in Apale spermatogonia, the earliest spermatogonia. In vivo 5-bromo-2′-deoxyuridine administration showed that both GFRA1+ and GFRA1− As spermatogonia were engaged in the cell cycle, a finding supported by the lack of long-term label-retaining As spermatogonia. GFRA1 expression was asymmetric in 5% of paired cells, suggesting that As subsets may be generated by asymmetric cell division. Our data support the hypothesis of the existence of SSC subsets and reveal a previously unrecognized heterogeneity in the expression profile of As spermatogonia in vivo. Disclosure of potential conflicts of interest is found at the end of this article.

In mice and other mammals, spermatogenesis is maintained by spermatogonial stem cells (SSCs), a cell population belonging to undifferentiated type A spermatogonia. In the accepted model of SSC self-renewal, Asingle (As) spermatogonia are the stem cells, whereas paired (Apaired (Apr)) and chained (Aaligned (Aal)) undifferentiated spermatogonia are committed to differentiation. This model has been recently challenged by evidence that As and chained (Apr and Aal), undifferentiated spermatogonia are heterogeneous in terms of gene expression and function. The expression profile of several markers, such as GFRA1 (the GDNF co-receptor), is heterogeneous among As, Apr and Aal spermatogonia. In this study, we have analysed and quantified the distribution of GFRA1-expressing cells within the different stages of the seminiferous epithelial cycle. We show that in all stages, GFRA1+ chained spermatogonia (Apr to Aal) are more numerous than GFRA1+ As spermatogonia. Numbers of chained GFRA1+ spermatogonia are sharply reduced in stages VII–VIII when Aal differentiate into A1 spermatogonia. GFRA1 expression is regulated by GDNF and in cultures of isolated seminiferous tubules, we found that GDNF expression and secretion by Sertoli cells is stage-dependent, being maximal in stages II–VI and decreasing thereafter. Using qRT-PCR analysis, we found that GDNF regulates the expression of genes such as Tex14, Sohlh1 and Kit (c-Kit) known to be involved in spermatogonial differentiation. Expression of Kit was upregulated by GDNF in a stage-specific manner. Our data indicate that GDNF, besides its crucial role in the self-renewal of stem cells also functions in the differentiation of chained undifferentiated spermatogonia.

MicroRNAs (miRNAs) are emerging as key regulators of complex biological processes in several cardiovascular diseases, including atrial fibrillation (AF). Reverse transcription-quantitative polymerase chain reaction is a powerful technique to quantitatively assess miRNA expression profile, but reliable results depend on proper data normalization by suitable reference genes. Despite the increasing number of studies assessing miRNAs in cardiac disease, no consensus on the best reference genes has been reached. This work aims to assess reference genes stability in human cardiac tissue with a focus on AF investigation. We evaluated the stability of five reference genes (U6, SNORD48, SNORD44, miR-16, and 5S) in atrial tissue samples from eighteen cardiac-surgery patients in sinus rhythm and AF. Stability was quantified by combining BestKeeper, delta-C_q, GeNorm, and NormFinder statistical tools. All methods assessed SNORD48 as the best and U6 as the worst reference gene. Applications of different normalization strategies significantly impacted miRNA expression profiles in the study population. Our results point out the necessity of a consensus on data normalization in AF studies to avoid the emergence of divergent biological conclusions.

Cyclin-dependent kinase 5 regulatory subunit 1 (CDK5R1) encodes p35, the main activatory subunit of cyclin-dependent kinase 5 (CDK5). The p35/CDK5 active complex plays a fundamental role in brain development and functioning, but its deregulated activity has also been implicated in various neurodegenerative disorders, including Alzheimer’s disease (AD). CDK5R1 displays a large and highly evolutionarily conserved 3′-untranslated region (3′-UTR), a fact that has suggested a role for this region in the post-transcriptional control of CDK5R1 expression. Our group has recently demonstrated that two miRNAs, miR-103 and miR-107, regulate CDK5R1 expression and affect the levels of p35. MiR-103 and miR-107 belong to the miR-15/107 family, a group of evolutionarily conserved miRNAs highly expressed in human cerebral cortex. In this work, we tested the hypothesis that other members of this group of miRNAs, in addition to miR-103 and miR-107, were able to modulate CDK5R1 expression. We provide evidence that several miRNAs belonging to the miR-15/107 family regulate p35 levels. BACE1 expression levels were also found to be modulated by different members of this family. Furthermore, overexpression of these miRNAs led to reduced APP phosphorylation levels at the CDK5-specific Thr668 residue. We also show that miR-15/107 miRNAs display reduced expression levels in hippocampus and temporal cortex, but not in cerebellum, of AD brains. Moreover, increased CDK5R1 mRNA levels were observed in AD hippocampus tissues. Our results suggest that the downregulation of the miR-15/107 family might have a role in the pathogenesis of AD by increasing the levels of CDK5R1/p35 and consequently enhancing CDK5 activity.

MicroRNAs as lung cancer biomarkers