Michela A. Denti

Involvement of the intracellular ion channel CLIC1 in microglia-mediated beta-amyloid-induced neurotoxicity

It is widely believed that the inflammatory events mediated by microglial activation contribute to several neurodegenerative processes. Alzheimers disease, for example, is characterized by an accumulation of β-amyloid protein (Aβ) in neuritic plaques that are infiltrated by reactive microglia and astrocytes. Although Aβ and its fragment 25-35 exert a direct toxic effect on neurons, they also activate microglia. Microglial activation is accompanied by morphological changes, cell proliferation, and release of various cytokines and growth factors. A number of scientific reports suggest that the increased proliferation of microglial cells is dependent on ionic membrane currents and in particular on chloride conductances. An unusual chloride ion channel known to be associated with macrophage activation is the chloride intracellular channel-1 (CLIC1). Here we show that Aβ stimulation of neonatal rat microglia specifically leads to the increase in CLIC1 protein and to the functional expression of CLIC1 chloride conductance, both barely detectable on the plasma membrane of quiescent cells. CLIC1 protein expression in microglia increases after 24 hr of incubation with Aβ, simultaneously with the production of reactive nitrogen intermediates and of tumor necrosis factor-α (TNF-α). We demonstrate that reducing CLIC1 chloride conductance by a specific blocker [IAA-94 (R(+)-[(6,7-dichloro-2-cyclopentyl-2,3-dihydro-2-methyl-1-oxo-1H-inden-5yl)-oxy] acetic acid)] prevents neuronal apoptosis in neurons cocultured with Aβ-treated microglia. Furthermore, we show that small interfering RNAs used to knock down CLIC1 expression prevent TNF-α release induced by Aβ stimulation. These results provide a direct link between Aβ-induced microglial activation and CLIC1 functional expression.

Circulating miRNAs as Biomarkers for Neurodegenerative Disorders

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.

Long-Term Benefit of Adeno-Associated Virus/Antisense-Mediated Exon Skipping in Dystrophic Mice

Many mutations and deletions in the dystrophin gene, responsible for Duchenne muscular dystrophy (DMD), can be corrected at the posttranscriptional level by skipping specific exons. Here we show that long-term benefit can be obtained in the dystrophic mouse model through the use of adeno-associated viral vectors expressing antisense sequences: persistent exon skipping, dystrophin rescue, and functional benefit were observed 74 weeks after a single systemic administration. The therapeutic benefit was sufficient to preserve the muscle integrity of mice up to old age. These results indicate a possible long-term gene therapy treatment of the DMD pathology.

miR-205 Expression Levels in Nonsmall Cell Lung Cancer Do Not Always Distinguish Adenocarcinomas From Squamous Cell Carcinomas.

Accurate classification of nonsmall cell lung cancers is of paramount clinical relevance, as novel chemotherapeutic agents show different efficacy in adenocarcinomas (ADCs) compared with squamous cell carcinomas (SQCCs). Cyto and histomorphology may sometimes be insufficient for this distinction and immunohistochemistry may improve diagnostic accuracy. The measurement of miR-205 may be another tool for the distinction between ADC and SQCC. The aim of our study was to compare morphologic and immunohistochemical classification with the relative quantification of miR-205 and miR-21 insurgically resected and well-characterized lung tumors (25 ADCs, 24 SQCCs, 1 adenosquamous). The miR-21 relative levels were similar in SQCC and ADC, whereas the miR-205 relative levels were lower in ADC (P<0.0001). The miR-205 sample score value, determined according to Lebanony et al, was higher in ADC (range, 2.8 to 9.08) compared with SQCC (range, −4.17 to 2.445) (P<0.0001). Accordingly, 22 tumors were classified as ADC and 28 tumors as SQCC, although 8 cases (2 SQCCs and 6 ADCs) were in the range of “near cutoff values.” Four cases classified as SQCC (according to the sample score method) corresponded to cases classified as ADC on the basis of morphoimmunohistochemical evaluation. In conclusion, the relative quantification of miR-205 and miR-21 seems to be a promising diagnostic tool. However, the molecular approach is still not completely satisfactory as it may misclassify a non-negligible percentage of cases. Therefore, it cannot be used as a substitute of accurate morphologic and immunophenotypical characterization of tumors, but could be used as an adjunctive diagnostic criterion in selected cases.

MicroRNAs as lung cancer biomarkers

Lung cancer is the leading cause of cancer mortality worldwide. Its high mortality is due to the poor prognosis of the disease caused by a late disease presentation, tumor heterogeneities within histological subtypes, and the relatively limited understanding of tumor biology. Importantly, lung cancer histological subgroups respond differently to some chemotherapeutic substances and side effects of some therapies appear to vary between subgroups. Biomarkers able to stratify for the subtype of lung cancer, prognosticate the course of disease, or predict the response to treatment are in high demand. In the last decade, microRNAs (miRNAs), measured in resected tumor samples or in fine needle aspirate samples have emerged as biomarkers for tumor diagnosis, prognosis and prediction of response to treatment, due to the ease of their detection and in their extreme specificity. Moreover, miRNAs present in sputum, in plasma, in serum or in whole blood have increasingly been explored in the last five years as less invasive biomarkers for the early detection of cancers. In this review we cover the increasing amounts of data that have accumulated in the last ten years on the use of miRNAs as lung cancer biomarkers.

Exon-Skipping Antisense Oligonucleotides to Correct Missplicing in Neurogenetic Diseases

Alternative splicing is an important regulator of the transcriptome. However, mutations may cause alteration of splicing patterns, which in turn leads to disease. During the past 10 years, exon skipping has been looked upon as a powerful tool for correction of missplicing in disease and progress has been made towards clinical trials. In this review, we discuss the use of antisense oligonucleotides to correct splicing defects through exon skipping, with a special focus on diseases affecting the nervous system, and the latest stage achieved in its progress.

Heterogeneity of large cell carcinoma of the lung: an immunophenotypic and miRNA-based analysis.

Large cell carcinomas (LCCs) of the lung are heterogeneous and may be of different cell lineages. We analyzed 56 surgically resected lung tumors classified as LCC on the basis of pure morphologic grounds, using a panel of immunophenotypic markers (adenocarcinoma [ADC]-specific, thyroid transcription factor-1, cytokeratin 7, and napsin A; squamous cell carcinoma [SQCC]-specific, p63, cytokeratin 5, desmocollin 3, and Δnp63) and the quantitative analysis of microRNA-205 (microRNA sample score [mRSS]). Based on immunoprofiles 19 (34%) of the cases were reclassified as ADC and 14 (25%) as SQCC; 23 (41%) of the cases were unclassifiable. Of these 23 cases, 18 were classified as ADC and 5 as SQCC according to the mRSS. Our data show that an extended panel of immunohistochemical markers can reclassify around 60% of LCCs as ADC or SQCC. However, a relevant percentage of LCCs may escape convincing immunohistochemical classification, and mRSS could be used for further typing, but its clinical relevance needs further confirmation.

Identification of new p53 target microRNAs by bioinformatics and functional analysis

BackgroundThe tumor suppressor p53 is a sequence-specific transcription factor that regulates an extensive network of coding genes, long non-coding RNAs and microRNAs, that establish intricate gene regulatory circuits influencing many cellular responses beyond the prototypical control of cell cycle, apoptosis and DNA repair.MethodsUsing bioinformatic approaches, we identified an additional group of candidate microRNAs (miRs) under direct p53 transcriptional control. To validate p53 family-mediated responsiveness of the newly predicted target miRs we first evaluated the potential for wild type p53, p63β and p73β to transactivate from p53 response elements (REs) mapped in the miR promoters, using an established yeast-based assay.ResultsThe REs found in miR-10b, -23b, -106a, -151a, -191, -198, -202, -221, -320, -1204, -1206 promoters were responsive to p53 and 8 of them were also responsive to p63β or p73β. The potential for germline p53 mutations to drive transactivation at selected miR-associated REs was also examined. Chromatin Immuno-Precipitation (ChIP) assays conducted in doxorubicin-treated MCF7 cells and HCT116 p53+/+ revealed moderate induction of p53 occupancy at the miR-202, -1204, -1206, -10b RE-containing sites, while weak occupancy was observed for the miR-23b-associated RE only in MCF7 cells. RT-qPCR analyses cells showed modest doxorubicin- and/or Nutlin-dependent induction of the levels of mature miR-10b, -23b, -151a in HCT116 p53+/+ and MCF7 cells. The long noncoding RNA PVT1 comprising miR-1204 and −1206 was weakly induced only in HCT116 p53+/+ cells, but the mature miRs were not detected. miR-202 expression was not influenced by p53-activating stimuli in our cell systems.ConclusionsOur study reveals additional miRs, particularly miR-10b and miR-151a, that could be directly regulated by the p53-family of transcription factors and contribute to the tuning of p53-induced responses.

Delivery is key: lessons learnt from developing splice‐switching antisense therapies

The use of splice‐switching antisense therapy is highly promising, with a wealth of pre‐clinical data and numerous clinical trials ongoing. Nevertheless, its potential to treat a variety of disorders has yet to be realized. The main obstacle impeding the clinical translation of this approach is the relatively poor delivery of antisense oligonucleotides to target tissues after systemic delivery. We are a group of researchers closely involved in the development of these therapies and would like to communicate our discussions concerning the validity of standard methodologies currently used in their pre‐clinical development, the gaps in current knowledge and the pertinent challenges facing the field. We therefore make recommendations in order to focus future research efforts and facilitate a wider application of therapeutic antisense oligonucleotides.

A Cross-Platform Comparison of Affymetrix and Agilent Microarrays Reveals Discordant miRNA Expression in Lung Tumors of c-Raf Transgenic Mice

Non-coding RNAs play major roles in the translational control of gene expression. In order to identify disease-associated miRNAs in precursor lesions of lung cancer, RNA extracts from lungs of either c-Raf transgenic or wild-type (WT) mice were hybridized to the Agilent and Affymetrix miRNA microarray platforms, respectively. This resulted in the detection of a range of miRNAs varying between 111 and 267, depending on the presence or absence of the transgene, on the gender, and on the platform used. Importantly, when the two platforms were compared, only 11–16% of the 586 overlapping genes were commonly detected. With the Agilent microarray, seven miRNAs were identified as significantly regulated, of which three were selectively up-regulated in male transgenic mice. Much to our surprise, when the same samples were analyzed with the Affymetrix platform, only two miRNAs were identified as significantly regulated. Quantitative PCR performed with lung RNA extracts from WT and transgenic mice confirmed only partially the differential expression of significant regulated miRNAs and established that the Agilent platform failed to detect miR-433. Finally, bioinformatic analyses predicted a total of 152 mouse genes as targets of the regulated miRNAs of which 4 and 11 genes were significantly regulated at the mRNA level, respectively in laser micro-dissected lung dysplasia and lung adenocarcinomas of c-Raf transgenic mice. Furthermore, for many of the predicted mouse target genes expression of the coded protein was also repressed in human lung cancer when the publically available database of the Human Protein Atlas was analyzed, thus supporting the clinical significance of our findings. In conclusion, a significant difference in a cross-platform comparison was observed that will have important implications for research into miRNAs.