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.

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.

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.

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.

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.

A patient-based model of RNA mis-splicing uncovers treatment targets in Parkinson's disease.

Parkinson's disease (PD) is a heterogeneous neurodegenerative disorder with monogenic forms representing prototypes of the underlying molecular pathology and reproducing to variable degrees the sporadic forms of the disease. Using a patient-based in vitro model of PARK7-linked PD, we identified a U1-dependent splicing defect causing a drastic reduction in DJ-1 protein and, consequently, mitochondrial dysfunction. Targeting defective exon skipping with genetically engineered U1-snRNA recovered DJ-1 protein expression in neuronal precursor cells and differentiated neurons. After prioritization of candidate drugs, we identified and validated a combinatorial treatment with the small-molecule compounds rectifier of aberrant splicing (RECTAS) and phenylbutyric acid, which restored DJ-1 protein and mitochondrial dysfunction in patient-derived fibroblasts as well as dopaminergic neuronal cell loss in mutant midbrain organoids. Our analysis of a large number of exomes revealed that U1 splice-site mutations were enriched in sporadic PD patients. Therefore, our study suggests an alternative strategy to restore cellular abnormalities in in vitro models of PD and provides a proof of concept for neuroprotection based on precision medicine strategies in PD.

The splicing code.

This issue dedicated to the code of life tackles very challenging and open questions in Biology. The genetic code, brilliantly uncovered over 50 years ago is an example of a univocal biological code. In fact, except for very few and marginal variations, it is the same from bacteria to man, the RNA stretch: 5' GUGUUC 3' reads as the dipeptide: Val-Phe in bacteria, in yeast, in Arabidopsis, in zebra fish, in mouse and in human. A degree of ambiguity is possible if mutations are introduced in the tRNAs in a way that the anticodon reads one amino acid but the aminoacyl-transferase attaches a different one onto the tRNA. These were the very useful suppressor genes that aided greatly the study of bacterial genetics. Other biological codes however, are more akin to social codes and are less amenable to an unambiguous deciphering. Legal and ethical codes, weather we like it or not, are flexible and depend on the structure and history of the society that has produced them, as well as a specific point in time. The codes that govern RNA splicing have similar characteristics. In fact, the splicing code depends on a myriad of different factors that in part are influenced by the background in which they are read such as different cells, tissues or developmental stages. Given the complexity of the splicing process, the construction of an algorithm that can define exons or their fate with certainty has not yet been achieved. However a substantial amount of information towards the deciphering of the splicing code has been gathered and in this manuscript we summarize the point reached.

New routes in frontotemporal dementia drug discovery.

INTRODUCTION: Research into the pathogenic mechanisms behind frontotemporal dementia (FTD) has yielded several new targets for therapeutic intervention; such targets include specific new pathways uncovered by mutations as well as targets involving the modulation, formation and degradation of protein aggregates. Areas covered: Herein, the authors outline the principal molecular causes underlying FTD to date and the research that has been performed in these areas with respect to an eventual corrective strategy. Expert opinion: While it is worthwhile targeting pathways affected by specific mutations with a causative loss of function linked to FTD, research still has to contend with issues including the remaining presence of protein aggregates or that treatments are rarely universally applicable. Aiming to recover function in a downstream target caused by the protein aggregates will likely be insufficient due to the large cascade of events affected. It is our belief that the clearance of these aggregates and the inhibition of protein misfolding are more appropriate and direct routes to an eventual therapy.

Neurodegeneration and RNA-binding proteins.

In the eukaryotic nucleus, RNA-binding proteins (RBPs) play a very important role in the life cycle of both coding and noncoding RNAs. As soon as they are transcribed, in fact, all RNA molecules within a cell are bound by distinct sets of RBPs that have the task of regulating its correct processing, transport, stability, and function/translation up to its final degradation. These tasks are particularly important in cells that have a complex RNA metabolism, such as neurons. Not surprisingly, therefore, recent findings have shown that the misregulation of genes involved in RNA metabolism or the autophagy/proteasome pathway plays an important role in the onset and progression of several neurodegenerative diseases. In this article, we aim to review the recent advances that link neurodegenerative processes and RBP proteins. WIREs RNA 2017, 8:e1394. doi: 10.1002/wrna.1394 For further resources related to this article, please visit the WIREs website.

A Simplified and Efficient Process for Insulin Production in Pichia pastoris.

A significant barrier to insulin is affordability. In this manuscript we describe improvements to key steps in the insulin production process in Pichia pastoris that reduce cost and time. The strategy for recovery and processing of human insulin precursor has been streamlined to two steps from bioreactor to the transpeptidation reaction. In the first step the insulin precursor secreted during the methanol induction phase is recovered directly from the culture broth using Tangential Flow Filtration with a Prostak™ module eliminating the laborious and time-consuming multi-step clarification, including centrifugation. In the second step the protein is applied at very high loadings on a cation exchange resin and eluted in a mixture of water and ethanol to obtain a concentrated insulin precursor, suitable for use directly in the transpeptidation reaction. Overall the yield from insulin precursor to human insulin was 51% and consisted of three purification chromatography steps. In addition we describe a method for recovery of the excess of H-Thr(tBu)-OtBu from the transpeptidation reaction mixture, one of the more costly reagents in the process, along with its successful reuse.

An Amyloid-Like Pathological Conformation of TDP-43 Is Stabilized by Hypercooperative Hydrogen Bonds.

TDP-43 is an essential RNA-binding protein forming aggregates in almost all cases of sporadic amyotrophic lateral sclerosis (ALS) and many cases of frontotemporal lobar dementia (FTLD) and other neurodegenerative diseases. TDP-43 consists of a folded N-terminal domain with a singular structure, two RRM RNA-binding domains, and a long disordered C-terminal region which plays roles in functional RNA regulatory assemblies as well as pernicious aggregation. Evidence from pathological mutations and seeding experiments strongly suggest that TDP-43 aggregates are pathologically relevant through toxic gain-of-harmful-function and/or harmful loss-of-native-function mechanisms. Recent, but not early, microscopy studies and the ability of TDP-43 aggregates to resist harsh treatment and to seed new pathological aggregates in vitro and in cells strongly suggest that TDP-43 aggregates have a self-templating, amyloid-like structure. Based on the importance of the Gln/Asn-rich 341-367 residue segment for efficient aggregation of endogenous TDP-43 when presented as a 12X-repeat and extensive spectroscopic and computational experiments, we recently proposed that this segment adopts a beta-hairpin structure that assembles in a parallel with a beta-turn configuration to form an amyloid-like structure. Here, we propose that this conformer is stabilized by an especially strong class of hypercooperative hydrogen bonding unique to Gln and Asn sidechains. The clinical existence of this conformer is supported by very recent LC-MS/MS characterization of TDP-43 from ex vivo aggregates, which show that residues 341-367 were protected in vivo from Ser phosphorylation, Gln/Asn deamidation and Met oxidation. Its distinct pattern of SDS-PAGE bands allows us to link this conformer to the exceptionally stable seed of the Type A TDP-43 proteinopathy.

TDP-43 aggregation mirrors TDP-43 knockdown, affecting the expression levels of a common set of proteins.

TDP-43 protein plays an important role in regulating transcriptional repression, RNA metabolism, and splicing. Typically it shuttles between the nucleus and the cytoplasm to perform its functions, while abnormal cytoplasmic aggregation of TDP-43 has been associated with neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). For the purpose of this study we selected a set of proteins that were misregulated following silencing of TDP-43 and analysed their expression in a TDP-43-aggregation model cell line HEK293 Flp-in Flag-TDP-43-12x-Q/N F4L. Following TDP-43 sequestration in insoluble aggregates, we observed higher nuclear levels of EIF4A3, and POLDIP3ß, whereas nuclear levels of DNMT3A, HNRNPA3, PABPC1 and POLDIP3α dropped, and cytoplasmic levels of RANBP1 dropped. In addition, immunofluorescence signal intensity quantifications showed increased nuclear expression of HNRNPL and YARS, and downregulation of cytoplasmic DPCD. Furthermore, cytoplasmic levels of predominantly nuclear protein ALYREF increased. In conclusion, by identifying a common set of proteins that are differentially expressed in a similar manner in these two different conditions, we show that TDP-43 aggregation has a comparable effect to TDP-43 knockdown.

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.

Functional Analysis of Mutations in Exon 9 of NF1 Reveals the Presence of Several Elements Regulating Splicing.

Neurofibromatosis type 1 (NF1) is one of the most common human hereditary disorders, predisposing individuals to the development of benign and malignant tumors in the nervous system, as well as other clinical manifestations. NF1 is caused by heterozygous mutations in the NF1 gene and around 25% of the pathogenic changes affect pre-mRNA splicing. Since the molecular mechanisms affected by these mutations are poorly understood, we have analyzed the splicing mutations identified in exon 9 of NF1, which is particularly prone to such changes, to better define the possible splicing regulatory elements. Using a minigene approach, we studied the effect of five splicing mutations in this exon described in patients. These highlighted three regulatory motifs within the exon. An in vivo splicing analysis of an extensive collection of changes generated in the minigene demonstrated that the CG motif at c.910-911 is critical for the recognition of exon 9. We also found that the GC motif at c.945-946 is involved in exon recognition through SRSF2 and that this motif is part of a Composite Exon Splicing Regulatory Element made up of physically overlapping enhancer and silencer elements. Finally, through an in vivo splicing analysis and in vitro binding assays, we demonstrated that the c.1007G>A mutation creates an Exonic Splicing Silencer element that binds the hnRNPA1 protein. The complexity of the splicing regulatory elements present in exon 9 is most likely responsible for the fact that mutations in this region represent 25% of all exonic changes that affect splicing in the NF1 gene.

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.

Characterization of variegate porphyria mutations using a minigene approach.

Porphyrias are a group of metabolic diseases that affect the skin and/or nervous system. In 2008, three unrelated patients were diagnosed with variegate porphyria at the CIPYP (Centro de Investigaciones sobre Porfirinas y Porfirias). Sequencing of the protoporphyrinogen oxidase gene, the gene altered in this type of porphyria, revealed three previously undescribed mutations: c.338+3insT, c.807G>A, and c.808-1G>C. As these mutations do not affect the protein sequence, we hypothesized that they might be splicing mutations. RT-PCRs performed on the patient's mRNAs showed normal mRNA or no amplification at all. This result indicated that the aberrant spliced transcript is possibly being degraded. In order to establish whether they were responsible or not for the patient's disease by causing aberrant splicing, we utilized a minigene approach. We found that the three mutations lead to exon skipping; therefore, the abnormal mRNAs are most likely degraded by a mechanism such as nonsense-mediated decay. In conclusion, these mutations are responsible for the disease because they alter the normal splicing pathway, thus providing a functional explanation for the appearance of disease and highlighting the use of minigene assays to complement transcript analysis.