Thioridazine reverts the phenotype in cellular and Drosophila models of amyotrophic lateral sclerosis by enhancing TDP-43 aggregate clearance.

Brain inclusions mainly composed of misfolded and aggregated TAR DNA binding protein 43 (TDP-43), are characteristic hallmarks of amyotrophic lateral sclerosis (ALS). Irrespective of the role played by the inclusions, their reduction represents an important therapeutic pathway that is worth exploring. Their removal can either lead to the recovery of TDP-43 function by removing the self-templating conformers that sequester the protein in the inclusions, and/or eliminate any potential intrinsic toxicity of the aggregates. The search for curative therapies has been hampered by the lack of ALS models for use in high-throughput screening. We adapted, optimised, and extensively characterised our previous ALS cellular model for such use. The model demonstrated efficient aggregation of endogenous TDP-43, and concomitant loss of its splicing regulation function. We provided a proof-of-principle for its eventual use in high-throughput screening using compounds of the tricyclic family and showed that recovery of TDP-43 function can be achieved by the enhanced removal of TDP-43 aggregates by these compounds. We observed that the degradation of the aggregates occurs independent of the autophagy pathway beyond autophagosome-lysosome fusion, but requires a functional proteasome pathway. The in vivo translational effect of the cellular model was tested with two of these compounds in a Drosophila model expressing a construct analogous to the cellular model, where thioridazine significantly improved the locomotive defect. Our findings have important implications as thioridazine cleared TDP-43 aggregates and recovered TDP-43 functionality. This study also highlights the importance of a two-stage, in vitro and in vivo model system to cross-check the search for small molecules that can clear TDP-43 aggregates in TDP-43 proteinopathies.

Alternative splicing and liver disease.

Alternative splicing produces complex and dynamic changes in the protein isoforms that are necessary for the proper biological functioning of the metabolic pathways involved in liver development and hepatocyte homeostasis. Changes in the physiological state of alternatively spliced forms are increasingly linked to liver pathologies. This may occur when the expression or function of the set of proteins controlling the alternative splicing processes are altered by external effectors such as oxidative stress and other environmental variations. Studies addressing these modifications reveal a complex interplay between the expression levels of different proteins that regulate the alternative splicing process as well as the changes in alternative splicing. This interplay results in a cascade of different protein isoforms that correlate with the progression of non-alcoholic fatty liver disease, hepatocellular carcinoma, and alcoholic liver disease. However, research on the detailed molecular mechanism underlying the production of these isoforms is needed. It is imperative to identify the physiological processes affected by the differentially spliced isoforms and confirm their role on the onset and maintenance of the pathology. This is required to design potential therapeutic approaches targeting the key splicing changes to revert the pathological condition as well as identify prognostic markers. In this review, we describe the complexity of the splicing process through an example to encourage researchers to go down this path. Subsequently, rather than a catalog of splicing events we have hand-picked and discuss a few selected studies of specific liver pathologies and suggested ways to focus research on these areas.

TDP-43 affects splicing profiles and isoform production of genes involved in the apoptotic and mitotic cellular pathways.

In recent times, high-throughput screening analyses have broadly defined the RNA cellular targets of TDP-43, a nuclear factor involved in neurodegeneration. A common outcome of all these studies is that changing the expression levels of this protein can alter the expression of several hundred RNAs within cells. What still remains to be clarified is which changes represent direct cellular targets of TDP-43 or just secondary variations due to the general role played by this protein in RNA metabolism. Using an HTS-based splicing junction analysis we identified at least six bona fide splicing events that are consistent with being controlled by TDP-43. Validation of the data, both in neuronal and non-neuronal cell lines demonstrated that TDP-43 substantially alters the levels of isoform expression in four genes potentially important for neuropathology: MADD/IG20, STAG2, FNIP1 and BRD8. For MADD/IG20 and STAG2, these changes could also be confirmed at the protein level. These alterations were also observed in a cellular model that successfully mimics TDP-43 loss of function effects following its aggregation. Most importantly, our study demonstrates that cell cycle alterations induced by TDP-43 knockdown can be recovered by restoring the STAG2, an important component of the cohesin complex, normal splicing profile.

Antimycobacterial activity of new N(1)-[1-[1-aryl-3-[4-(1H-imidazol-1-yl)phenyl]-3-oxo]propyl]-pyridine-2-carboxamidrazone derivatives.

N(1)-[1-[1-aryl-3-[4-(1H-imidazol-1-yl)phenyl]-3-oxo]propyl]-pyridine-2-carboxamidrazone derivatives were design, synthesized and tested for their in vitro antimycobacterial activity. The new compounds showed a moderate antimycobacterial activity against the tested strain of Mycobacterium tuberculosis H37Ra and a significant antimycobacterial activity against several mycobacteria other than tuberculosis strains.

Exonic splicing signals impose constraints upon the evolution of enzymatic activity.

Exon splicing enhancers (ESEs) overlap with amino acid coding sequences implying a dual evolutionary selective pressure. In this study, we map ESEs in the placental alkaline phosphatase gene (ALPP), absent in the corresponding exon of the ancestral tissue-non-specific alkaline phosphatase gene (ALPL). The ESEs are associated with amino acid differences between the transcripts in an area otherwise conserved. We switched out the ALPP ESEs sequences with the sequence from the related ALPL, introducing the associated amino acid changes. The resulting enzymes, produced by cDNA expression, showed different kinetic characteristics than ALPL and ALPP. In the organism, this enzyme will never be subjected to selection because gene splicing analysis shows exon skipping due to loss of the ESE. Our data prove that ESEs restrict the evolution of enzymatic activity. Thus, suboptimal proteins may exist in scenarios when coding nucleotide changes and consequent amino acid variation cannot be reconciled with the splicing function.

Predominance of spliceosomal complex formation over polyadenylation site selection in TDP-43 autoregulation.

TDP-43 is a nuclear protein involved in many aspects of RNA metabolism. To ensure cellular viability, its expression levels within cells must be tightly regulated. We have previously demonstrated that TDP-43 autoregulation occurs through the activation of a normally silent intron in its 3'-UTR sequence that results in the use of alternative polyadenylation sites. In this work, we analyse which is the dominant event in autoregulation: the recognition of the splice sites of 3'-UTR intron 7 or the intrinsic quality of the alternative polyadenylation sites. A panel of minigene constructs was tested for autoregulation functionality, protein production and subcellular messenger RNA localization. Our data clearly indicate that constitutive spliceosome complex formation across intron 7 does not lead to high protein production but, on the contrary, to lower TDP-43 messenger RNA and protein levels. This is due to altered nucleocytoplasmic distribution of the RNA that is mostly retained in the nucleus and degraded. This study provides a novel in-depth characterization of how RNA binding proteins can autoregulate their own levels within cells, an essential regulatory process in maintaining cellular viability.

TDP-43 regulates its mRNA levels through a negative feedback loop.

TAR DNA-binding protein (TDP-43) is an evolutionarily conserved heterogeneous nuclear ribonucleoprotein (hnRNP) involved in RNA processing, whose abnormal cellular distribution and post-translational modification are key markers of certain neurodegenerative diseases, such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration. We generated human cell lines expressing tagged forms of wild-type and mutant TDP-43 and observed that TDP-43 controls its own expression through a negative feedback loop. The RNA-binding properties of TDP-43 are essential for the autoregulatory activity through binding to 3' UTR sequences in its own mRNA. Our analysis indicated that the C-terminal region of TDP-43, which mediates TDP-43-hnRNP interactions, is also required for self-regulation. TDP-43 binding to its 3' UTR does not significantly change the pre-mRNA splicing pattern but promotes RNA instability. Moreover, blocking exosome-mediated degradation partially recovers TDP-43 levels. Our findings demonstrate that cellular TDP-43 levels are under tight control and it is likely that disease-associated TDP-43 aggregates disrupt TDP-43 self-regulation, thus contributing to pathogenesis.

Antisense-based RNA therapy of factor V deficiency: in vitro and ex vivo rescue of a F5 deep-intronic splicing mutation.

Antisense molecules are emerging as a powerful tool to correct splicing defects. Recently, we identified a homozygous deep-intronic mutation (F5 c.1296+268A>G) activating a cryptic donor splice site in a patient with severe coagulation factor V (FV) deficiency and life-threatening bleeding episodes. Here, we assessed the ability of 2 mutation-specific antisense molecules (a morpholino oligonucleotide [MO] and an engineered U7 small nuclear RNA [snRNA]) to correct this splicing defect. COS-1 and HepG2 cells transfected with a F5 minigene construct containing the patient's mutation expressed aberrant messenger RNA (mRNA) in excess of normal mRNA. Treatment with mutation-specific antisense MO (1-5 µM) or a construct expressing antisense U7snRNA (0.25-2 µg) dose-dependently increased the relative amount of correctly spliced mRNA by 1 to 2 orders of magnitude, whereas control MO and U7snRNA were ineffective. Patient-derived megakaryocytes obtained by differentiation of circulating progenitor cells did not express FV, but became positive for FV at immunofluorescence staining after administration of antisense MO or U7snRNA. However, treatment adversely affected cell viability, mainly because of the transfection reagents used to deliver the antisense molecules. Our data provide in vitro and ex vivo proof of principle for the efficacy of RNA therapy in severe FV deficiency, but additional cytotoxicity studies are warranted.

Mutually exclusive splicing regulates the Nav 1.6 sodium channel function through a combinatorial mechanism that involves three distinct splicing regulatory elements and their ligands.

Mutually exclusive splicing is a form of alternative pre-mRNA processing that consists in the use of only one of a set of two or more exons. We have investigated the mechanisms involved in this process for exon 18 of the Na(v) 1.6 sodium channel transcript and its significance regarding gene-expression regulation. The 18N exon (neonatal form) has a stop codon in phase and although the mRNA can be detected by amplification methods, the truncated protein has not been observed. The switch from 18N to 18A (adult form) occurs only in a restricted set of neural tissues producing the functional channel while other tissues display the mRNA with the 18N exon also in adulthood. We demonstrate that the mRNA species carrying the stop codon is subjected to Nonsense-Mediated Decay, providing a control mechanism of channel expression. We also map a string of cis-elements within the mutually exclusive exons and in the flanking introns responsible for their strict tissue and temporal specificity. These elements bind a series of positive (RbFox-1, SRSF1, SRSF2) and negative (hnRNPA1, PTB, hnRNPA2/B1, hnRNPD-like JKTBP) splicing regulatory proteins. These splicing factors, with the exception of RbFox-1, are ubiquitous but their levels vary during development and differentiation, ensuing unique sets of tissue and temporal levels of splicing factors. The combinatorial nature of these elements is highlighted by the dominance of the elements that bind the ubiquitous factors over the tissue specific RbFox-1.

Tailored antisense oligonucleotides designed to correct aberrant splicing reveal actionable groups of mutations for rare genetic disorders.

Effective translation of rare disease diagnosis knowledge into therapeutic applications is achievable within a reasonable timeframe; where mutations are amenable to current antisense oligonucleotide technology. In our study, we identified five distinct types of abnormal splice-causing mutations in patients with rare genetic disorders and developed a tailored antisense oligonucleotide for each mutation type using phosphorodiamidate morpholino oligomers with or without octa-guanidine dendrimers and 2'-O-methoxyethyl phosphorothioate. We observed variations in treatment effects and efficiencies, influenced by both the chosen chemistry and the specific nature of the aberrant splicing patterns targeted for correction. Our study demonstrated the successful correction of all five different types of aberrant splicing. Our findings reveal that effective correction of aberrant splicing can depend on altering the chemical composition of oligonucleotides and suggest a fast, efficient, and feasible approach for developing personalized therapeutic interventions for genetic disorders within short time frames.

The role of TDP-43 in the pathogenesis of ALS and FTLD.

TDP-43 (TAR DNA-binding protein 43) is an hnRNP (heterogeneous nuclear ribonucleoprotein) protein whose role in cellular processes has come to the forefront of neurodegeneration research after the observation that it is the main component of brain inclusions in ALS (amyotrophic lateral sclerosis) and FTLD (frontotemporal lobar degeneration) patients. Functionally, this aberrant aggregation and mislocalization implies that, in the affected neurons, transcripts regulated by TDP-43 may be altered. Since then, a considerable amount of data has been gathered on TDP-43 interactions and on the genes that are influenced by its absence or overexpression. At present, however, most of these data come from high-throughput searches, making it problematic to separate the direct effects of TDP-43 from secondary misregulations occurring at different levels of the gene expression process. Furthermore, our knowledge of the biochemistry of TDP-43, its RNA-binding characteristics, its nuclear and cytoplasmic targets, and the details of its interactions with other proteins is still incomplete. The understanding of these features could hold the key for uncovering TDP-43's role in ALS and FTLD pathogenesis. We describe in the present paper our work on TDP-43 RNA binding, self-regulation and aggregation processes, and attempt to relate them to the neurodegenerative pathologies.

Age-Related Alternative Splicing: Driver or Passenger in the Aging Process?

Alternative splicing changes are closely linked to aging, though it remains unclear if they are drivers or effects. As organisms age, splicing patterns change, varying gene isoform levels and functions. These changes may contribute to aging alterations rather than just reflect declining RNA quality control. Three main splicing types-intron retention, cassette exons, and cryptic exons-play key roles in age-related complexity. These events modify protein domains and increase nonsense-mediated decay, shifting protein isoform levels and functions. This may potentially drive aging or serve as a biomarker. Fluctuations in splicing factor expression also occur with aging. Somatic mutations in splicing genes can also promote aging and age-related disease. The interplay between splicing and aging has major implications for aging biology, though differentiating correlation and causation remains challenging. Declaring a splicing factor or event as a driver requires comprehensive evaluation of the associated molecular and physiological changes. A greater understanding of how RNA splicing machinery and downstream targets are impacted by aging is essential to conclusively establish the role of splicing in driving aging, representing a promising area with key implications for understanding aging, developing novel therapeutical options, and ultimately leading to an increase in the healthy human lifespan.

Emerging Trends in the Field of Inflammation and Proteinopathy in ALS/FTD Spectrum Disorder.

Proteinopathy and neuroinflammation are two main hallmarks of neurodegenerative diseases. They also represent rare common events in an exceptionally broad landscape of genetic, environmental, neuropathologic, and clinical heterogeneity present in patients. Here, we aim to recount the emerging trends in amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD) spectrum disorder. Our review will predominantly focus on neuroinflammation and systemic immune imbalance in ALS and FTD, which have recently been highlighted as novel therapeutic targets. A common mechanism of most ALS and ~50% of FTD patients is dysregulation of TAR DNA-binding protein 43 (TDP-43), an RNA/DNA-binding protein, which becomes depleted from the nucleus and forms cytoplasmic aggregates in neurons and glia. This, in turn, via both gain and loss of function events, alters a variety of TDP-43-mediated cellular events. Experimental attempts to target TDP-43 aggregates or manipulate crosstalk in the context of inflammation will be discussed. Targeting inflammation, and the immune system in general, is of particular interest because of the high plasticity of immune cells compared to neurons.

Complexities of 5'splice site definition: implications in clinical analyses.

In higher eukaryotes, the 5' splice site (5'ss) is initially recognized through an RNA-RNA interaction by U1 small nuclear ribonucleoprotein (U1 snRNP). This event represents one of the key steps in initial spliceosomal assembly and many disease-associated mutations in humans often disrupt this process. Beside base pair complementarity, 5'ss recognition can also be modified by additional factors such as RNA secondary structures or the specific binding of other nuclear proteins. In this work, we have focused on investigating a few examples of changes detected within the 5'ss in patients, that would not be immediately considered "disease causing mutations". We show that the splicing outcome of very similar mutations can be very different due to variations in trans-acting factor(s) interactions and specific context influences. Using several NF1 donor sites and SELEX approaches as experimental models, we have examined the binding properties of particular sequence motifs such as GGGU found in donor sites, and how the sequence context can change their interaction with hnRNPs such as H/F and A1/A2. Our results clearly show that even minor differences in local nucleotide context can differentially affect the binding ability of these factors to the GGGU core. Finally, using a previously identified mutation in KCNH2 that resulted in intron retention we show how very similar 5'ss mutations found in patients can have a very different splicing outcome due to the neighbouring sequence context, thus highlighting the general need to approach splicing problems with suitable experimental approaches.

Physiological tissue-specific and age-related reduction of mouse TDP-43 levels is regulated by epigenetic modifications.

The cellular level of TDP-43 (also known as TARDBP) is tightly regulated; increases or decreases in TDP-43 have deleterious effects in cells. The predominant mechanism responsible for the regulation of the level of TDP-43 is an autoregulatory negative feedback loop. In this study, we identified an in vivo cause-effect relationship between Tardbp gene promoter methylation and specific histone modification and the TDP-43 level in tissues of mice at two different ages. Furthermore, epigenetic control was observed in mouse and human cultured cell lines. In amyotrophic lateral sclerosis, the formation of TDP-43-containing brain inclusions removes functional protein from the system. This phenomenon is continuous but compensated by newly synthesized protein. The balance between sequestration and new synthesis might become critical with ageing, if accompanied by an epigenetic modification-regulated decrease in newly synthesized TDP-43. Sequestration by aggregates would then decrease the amount of functional TDP-43 to a level lower than those needed by the cell and thereby trigger the onset of symptoms.

Force-feeding malignant mesothelioma stem-cell like with exosome-delivered miR-126 induces tumour cell killing.

Malignant pleural mesothelioma (MPM) is an aggressive tumour resistant to treatments. It has been postulated that cancer stem cells (CSCs) persist in tumours causing relapse after multimodality treatment. In the present study, a novel miRNA-based therapy approach is proposed. MPM-derived spheroids have been treated with exosome-delivered miR-126 (exo-miR) and evaluated for their anticancer effect. The exo-miR treatment increased MPM stem-cell like stemness and inhibited cell proliferation. However, at a prolonged time, the up taken miR-126 was released by the cells themselves through exosomes; the inhibition of exosome release by an exosome release inhibitor GW4869 induced miR-126 intracellular accumulation leading to massive cell death and in vivo tumour growth arrest. Autophagy is involved in these processes; miR-126 accumulation induced a protective autophagy and the inhibition of this process by GW4869 generates a metabolic crisis that promotes necroptosis, which was associated with PARP-1 over-expression and cyt-c and AIF release. Here, for the first time, we proposed a therapy against CSCs, a heterogeneous cell population involved in cancer development and relapse.

Influence of Friedreich ataxia GAA noncoding repeat expansions on pre-mRNA processing.

The intronic GAA repeat expansion in the frataxin (FXN) gene causes the hereditary neurodegenerative disorder Friedreich ataxia. Although it is generally believed that GAA repeats block transcription elongation, direct proof in eukaryotic systems is lacking. We tested in hybrid minigenes the effect of GAA and TTC repeats on nascent transcription and pre-mRNA processing. Unexpectedly, disease-causing GAA(100) repeats did not affect transcriptional elongation in a nuclear HeLa Run On assay, nor did they affect pre-mRNA transcript abundance. However, they did result in a complex defect in pre-mRNA processing. The insertion of GAA but not TTC repeats downstream of reporter exons resulted in their partial or complete exclusion from the mature mRNAs and in the generation of a variety of aberrant splicing products. This effect of GAA repeats was observed to be position and context dependent; their insertion at different distances from the reporter exons had a variable effect on splice-site selection. In addition, GAA repeats bind to a multitude of different splicing factors and induced the accumulation of an upstream pre-mRNA splicing intermediate, which is not turned over into mature mRNA. When embedded in the homologous frataxin minigene system, the GAA repeats did not affect the pre-mRNA transcript abundance but did significantly reduce the splicing efficiency of the first intron. These data indicate an association between GAA noncoding repeats and aberrant pre-mRNA processing because binding of transcribed GAA repeats to a multitude of trans-acting splicing factors can interfere with normal turnover of intronic RNA and thus lead to its degradation and a lower abundance of mature mRNA.

Characterization of the human TARDBP gene promoter.

The expression of TDP-43, the main component of neuronal intracellular inclusions across a broad spectrum of ALS and FTD disorders, is developmentally regulated and studies in vivo have shown that TDP-43 overexpression can be toxic, even before observation of pathological aggregates. Starting from these observations, the regulation of its expression at transcriptional level might represent a further key element for the pathogenesis of neurodegenerative diseases. Therefore, we have characterized the human TARDBP promoter, in order to study the transcriptional mechanisms of expression. Mapping of cis-acting elements by luciferase assays in different cell outlined that the activity of the promoter seems to be higher in SH-SY5Y, Neuro2A, and HeLa than in HEK293. In addition, we tested effects of two SNPs found in the promoter region of ALS patients and observed no significant effect on transcription levels in all tested cell lines. Lastly, while TDP-43 overexpression did not affect significantly the activity of its promoter (suggesting that TDP-43 does not influence its own transcription), the presence of the 5'UTR sequence and of intron-1 splicing seem to impact positively on TDP-43 expression without affecting transcript stability. In conclusion, we have identified the region spanning nucleotides 451-230 upstream from the transcription start site as the minimal region with a significant transcription activity. These results lay an important foundation for exploring the regulation of the TARDBP gene transcription by exogenous and endogenous stimuli and the implication of transcriptional mechanisms in the pathogenesis of TDP-43 proteinopathies.

High Throughput miRNA Screening Identifies miR-574-3p Hyperproductive Effect in CHO Cells.

CHO is the cell line of choice for the manufacturing of many complex biotherapeutics. The constant upgrading of cell productivity is needed to meet the growing demand for these life-saving drugs. Manipulation of small non-coding RNAs-miRNAs-is a good alternative to a single gene knockdown approach due to their post-transcriptional regulation of entire cellular pathways without posing translational burden to the production cell. In this study, we performed a high-throughput screening of 2042-human miRNAs and identified several candidates able to increase cell-specific and overall production of Erythropoietin and Etanercept in CHO cells. Some of these human miRNAs have not been found in Chinese hamster cells and yet were still effective in them. We identified miR-574-3p as being able, when overexpressed in CHO cells, to improve overall productivity of Erythropoietin and Etanercept titers from 1.3 to up to 2-fold. In addition, we validated several targets of miR-574-3p and identified p300 as a main target of miR-574-3p in CHO cells. Furthermore, we demonstrated that stable CHO cell overexpressing miRNAs from endogenous CHO pri-miRNA sequences outperform the cells with human pri-miRNA sequences. Our findings highlight the importance of flanking genomic sequences, and their secondary structure features, on pri-miRNA processing offering a novel, cost-effective and fast strategy as a valuable tool for efficient miRNAs engineering in CHO cells.