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Drug-induced gene expression profiles can identify potential mechanisms of toxicity. We focus on obtaining signatures for cardiotoxicity of FDA-approved tyrosine kinase inhibitors (TKIs) in human induced-pluripotent-stem-cell-derived cardiomyocytes, using bulk transcriptomic profiles. We use singular value decomposition to identify drug-selective patterns across cell lines obtained from multiple healthy human subjects. Cellular pathways affected by cardiotoxic TKIs include energy metabolism, contractile, and extracellular matrix dynamics. Projecting these pathways to published single cell expression profiles indicates that TKI responses can be evoked in both cardiomyocytes and fibroblasts. Integration of transcriptomic outlier analysis with whole genomic sequencing of our six cell lines enables us to correctly reidentify a genomic variant causally linked to anthracycline-induced cardiotoxicity and predict genomic variants potentially associated with TKI-induced cardiotoxicity. We conclude that mRNA expression profiles when integrated with publicly available genomic, pathway, and single cell transcriptomic datasets, provide multiscale signatures for cardiotoxicity that could be used for drug development and patient stratification., (© 2024. The Author(s).)

Glioblastoma Multiforme (GBM) remains a particularly difficult cancer to treat, and survival outcomes remain poor. In addition to the lack of dedicated drug discovery programs for GBM, extensive intratumor heterogeneity and epigenetic plasticity related to cell-state transitions are major roadblocks to successful drug therapy in GBM. To study these phenomenon, publicly available snRNAseq and bulk RNAseq data from patient samples were used to categorize cells from patients into four cell states (i.e. phenotypes), namely: (i) neural progenitor-like (NPC-like), (ii) oligodendrocyte progenitor-like (OPC-like), (iii) astrocyte- like (AC-like), and (iv) mesenchymal-like (MES-like). Patients were subsequently grouped into subpopulations based on which cell-state was the most dominant in their respective tumor. By incorporating phosphoproteomic measurements from the same patients, a protein-protein interaction network (PPIN) was constructed for each cell state. These four-cell state PPINs were pooled to form a single Boolean network that was used for in silico protein knockout simulations to investigate mechanisms that either promote or prevent cell state transitions. Simulation results were input into a boosted tree machine learning model which predicted the cell states or phenotypes of GBM patients from an independent public data source, the Glioma Longitudinal Analysis (GLASS) Consortium. Combining the simulation results and the machine learning predictions, we generated hypotheses for clinically relevant causal mechanisms of cell state transitions. For example, the transcription factor TFAP2A can be seen to promote a transition from the NPC-like to the MES-like state. Such protein nodes and the associated signaling pathways provide potential drug targets that can be further tested in vitro and support cell state-directed (CSD) therapy., Competing Interests: Competing interests All authors declare no financial or non-financial competing interests.

Cancer therapy continues to be plagued by modest therapeutic advances. This is particularly evident in glioblastoma multiforme (GBM) wherein treatment failures are attributed to intratumoral heterogeneity (ITH), a dynamic process of cell state transitions or plasticity. To address ITH, we introduce the concept of cell state-directed (CSD) therapy through a quantitative systems pharmacology model of temozolomide (TMZ), a cornerstone of GBM drug therapy. The model consisting of multiple modules incorporated an epigenetic-based gene transcription-translation module that enabled CSD therapy. Numerous model simulations were conducted to demonstrate the potential impact of CSD therapy on TMZ activity. The simulations included those based on global sensitivity analyses to identify fragile nodes - MDM2 and XIAP - in the network, and also how an epigenetic modifier (birabresib) could overcome a mechanism of TMZ resistance. The positive results of CSD therapy on TMZ activity supports continued efforts to develop CSD therapy as a new anticancer approach., (© 2023 The Authors. CPT: Pharmacometrics & Systems Pharmacology published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Three proteins act in concert to cause neuronal pathology.

There is a demand for scientists trained in quantitative systems pharmacology (QSP) that has yet to be met by changes in graduate education. The multidisciplinary nature of QSP is not unlike its predecessor, pharmacokinetics (PKs) and pharmacodynamics (PDs) that have now become firmly established in many educational programs. A hindrance to the evolution of educational programs for QSP is explored and suggestions to move QSP into its proper position as a unique discipline are presented., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Transactive response DNA binding protein 43 (TDP-43) is an RNA processing protein central to the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Nuclear TDP-43 mislocalizes in patients to the cytoplasm, where it forms ubiquitin-positive inclusions in affected neurons and glia. Physiologically, cytoplasmic TDP-43 is associated with stress granules (SGs). Here, we explored TDP-43 cytoplasmic accumulation and stress granule formation following osmotic and oxidative stress. We show that sorbitol drives TDP-43 redistribution to the cytoplasm, while arsenite induces the recruitment of cytoplasmic TDP-43 to TIA-1 positive SGs. We demonstrate that inducing acute oxidative stress after TDP-43 cytoplasmic relocalization by osmotic shock induces poly (ADP-ribose) polymerase (PARP) cleavage, which triggers cellular toxicity. Recruitment of cytoplasmic TDP-43 to polyribosomes occurs in an SH-SY5Y cellular stress model and is observed in FTD brain lysate. Moreover, the processing body (P-body) marker DCP1a is detected in TDP-43 granules during recovery from stress. Overall, this study supports a central role for cytoplasmic TDP-43 in controlling protein translation in stressed cells., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Objective: Despite considerable research, the goal of finding nonsurgical remedies against thoracic aortic aneurysm and acute aortic dissection remains elusive. We sought to identify a novel aortic PK (protein kinase) that can be pharmacologically targeted to mitigate aneurysmal disease in a well-established mouse model of early-onset progressively severe Marfan syndrome (MFS)., Approach and Results: Computational analyses of transcriptomic data derived from the ascending aorta of MFS mice predicted a probable association between thoracic aortic aneurysm and acute aortic dissection development and the multifunctional, stress-activated HIPK2 (homeodomain-interacting protein kinase 2). Consistent with this prediction, Hipk2 gene inactivation significantly extended the survival of MFS mice by slowing aneurysm growth and delaying transmural rupture. HIPK2 also ranked among the top predicted PKs in computational analyses of DEGs (differentially expressed genes) in the dilated aorta of 3 MFS patients, which strengthened the clinical relevance of the experimental finding. Additional in silico analyses of the human and mouse data sets identified the TGF (transforming growth factor)-β/Smad3 signaling pathway as a potential target of HIPK2 in the MFS aorta. Chronic treatment of MFS mice with an allosteric inhibitor of HIPK2-mediated stimulation of Smad3 signaling validated this prediction by mitigating thoracic aortic aneurysm and acute aortic dissection pathology and partially improving aortic material stiffness., Conclusions: HIPK2 is a previously unrecognized determinant of aneurysmal disease and an attractive new target for antithoracic aortic aneurysm and acute aortic dissection multidrug therapy.

Drug resistance is a significant obstacle to successful and durable anti-cancer therapy. Targeted therapy is often effective during early phases of treatment; however, eventually cancer cells adapt and transition to drug-resistant cells states rendering the treatment ineffective. It is proposed that cell state can be a determinant of drug efficacy and manipulated to affect the development of anticancer drug resistance. In this work, we developed two stochastic cell state models and an integrated stochastic-deterministic model referenced to brain tumors. The stochastic cell state models included transcriptionally-permissive and -restrictive states based on the underlying hypothesis that epigenetic instability mitigates lock-in of drug-resistant states. When moderate epigenetic instability was implemented the drug-resistant cell populations were reduced, on average, by 60%, whereas a high level of epigenetic disruption reduced them by about 90%. The stochastic-deterministic model utilized the stochastic cell state model to drive the dynamics of the DNA repair enzyme, methylguanine-methyltransferase (MGMT), that repairs temozolomide (TMZ)-induced O6-methylguanine (O6mG) adducts. In the presence of epigenetic instability, the production of MGMT decreased that coincided with an increase of O6mG adducts following a multiple-dose regimen of TMZ. Generation of epigenetic instability via epigenetic modifier therapy could be a viable strategy to mitigate anticancer drug resistance., Competing Interests: The authors have declared that no competing interests exist.

A novel coronavirus, severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) or coronavirus disease 2019 (COVID-19), has caused a pandemic that continues to cause catastrophic health and economic carnage and has escalated the identification and development of antiviral agents. Remdesivir (RDV), a prodrug and requires intracellular conversions to the active triphosphate nucleoside (TN) has surfaced as an active anti-SARS-CoV-2 drug. To properly design therapeutic treatment regimens, it is imperative to determine if adequate intracellular TN concentrations are achieved in target tissues, such as the lungs. Because measurement of such concentrations is unrealistic in patients, a physiologically-based pharmacokinetic (PBPK) model was developed to characterize RDV and TN disposition. Specifically, a hybrid PBPK model was developed based on previously reported data in humans. The model represented each tissue as a two-compartment model-both extracellular and intracellular compartment wherein each intracellular compartment contained a comprehensive metabolic model to the ultimate active metabolite TN. Global sensitivity analyses and Monte-Carlo simulations were conducted to assess which parameters and how highly sensitive ones impacted peripheral blood mononuclear cells and intracellular lung TN profiles. Finally, clinical multiple-dose regimens indicated that minimum lung intracellular TN concentrations ranged from ~ 9 uM to 4 uM, which suggest current regimens are effective based on in vitro half-maximal effective concentration values. The model can be used to explore tissue drug disposition under various conditions and regimens, and expanded to pharmacodynamic models., (© 2021 The Authors. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of the American Society for Clinical Pharmacology and Therapeutics.)

Kinase inhibitors (KIs) represent an important class of anti-cancer drugs. Although cardiotoxicity is a serious adverse event associated with several KIs, the reasons remain poorly understood, and its prediction remains challenging. We obtain transcriptional profiles of human heart-derived primary cardiomyocyte like cell lines treated with a panel of 26 FDA-approved KIs and classify their effects on subcellular pathways and processes. Individual cardiotoxicity patient reports for these KIs, obtained from the FDA Adverse Event Reporting System, are used to compute relative risk scores. These are then combined with the cell line-derived transcriptomic datasets through elastic net regression analysis to identify a gene signature that can predict risk of cardiotoxicity. We also identify relationships between cardiotoxicity risk and structural/binding profiles of individual KIs. We conclude that acute transcriptomic changes in cell-based assays combined with drug substructures are predictive of KI-induced cardiotoxicity risk, and that they can be informative for future drug discovery.

Reliably predicting in vivo efficacy from in vitro data would facilitate drug development by reducing animal usage and guiding drug dosing in human clinical trials. However, such prediction remains challenging. Here, we built a quantitative pharmacokinetic/pharmacodynamic (PK/PD) mathematical model capable of predicting in vivo efficacy in animal xenograft models of tumor growth while trained almost exclusively on in vitro cell culture data sets. We studied a chemical inhibitor of LSD1 (ORY-1001), a lysine-specific histone demethylase enzyme with epigenetic function, and drug-induced regulation of target engagement, biomarker levels, and tumor cell growth across multiple doses administered in a pulsed and continuous fashion. A PK model of unbound plasma drug concentration was linked to the in vitro PD model, which enabled the prediction of in vivo tumor growth dynamics across a range of drug doses and regimens. Remarkably, only a change in a single parameter-the one controlling intrinsic cell/tumor growth in the absence of drug-was needed to scale the PD model from the in vitro to in vivo setting. These findings create a framework for using in vitro data to predict in vivo drug efficacy with clear benefits to reducing animal usage while enabling the collection of dense time course and dose response data in a highly controlled in vitro environment., (© 2019 F. Hoffmann-La Roche Ltd. Clinical and Translational Science published by Wiley Periodicals, Inc. on behalf of the American Society for Clinical Pharmacology and Therapeutics.)

Marfan syndrome (MFS) is associated with mutations in fibrillin-1 that predispose afflicted individuals to progressive thoracic aortic aneurysm (TAA) leading to dissection and rupture of the vessel wall. Here we combined computational and experimental approaches to identify and test FDA-approved drugs that may slow or even halt aneurysm progression. Computational analyses of transcriptomic data derived from the aortas of MFS patients and MFS mice (Fbn1mgR/mgR mice) predicted that subcellular pathways associated with reduced muscle contractility are key TAA determinants that could be targeted with the GABAB receptor agonist baclofen. Systemic administration of baclofen to Fbn1mgR/mgR mice validated our computational prediction by mitigating arterial disease progression at the cellular and physiological levels. Interestingly, baclofen improved muscle contraction-related subcellular pathways by upregulating a different set of genes than those downregulated in the aorta of vehicle-treated Fbn1mgR/mgR mice. Distinct transcriptomic profiles were also associated with drug-treated MFS and wild-type mice. Thus, systems pharmacology approaches that compare patient- and mouse-derived transcriptomic data for subcellular pathway-based drug repurposing represent an effective strategy to identify potential new treatments of human diseases.

Accumulation and aggregation of TDP-43 is a major pathological hallmark of amyotrophic lateral sclerosis and frontotemporal dementia. TDP-43 inclusions also characterize patients with GGGGCC (G4C2) hexanucleotide repeat expansion in C9orf72 that causes the most common genetic form of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD). Functional studies in cell and animal models have identified pathogenic mechanisms including repeat-induced RNA toxicity and accumulation of G4C2-derived dipeptide-repeat proteins. The role of TDP-43 dysfunction in C9ALS/FTD, however, remains elusive. We found G4C2-derived dipeptide-repeat protein but not G4C2-RNA accumulation caused TDP-43 proteinopathy that triggered onset and progression of disease in Drosophila models of C9ALS/FTD. Timing and extent of TDP-43 dysfunction was dependent on levels and identity of dipeptide-repeat proteins produced, with poly-GR causing early and poly-GA/poly-GP causing late onset of disease. Accumulating cytosolic, but not insoluble aggregated TDP-43 caused karyopherin-α2/4 (KPNA2/4) pathology, increased levels of dipeptide-repeat proteins and enhanced G4C2-related toxicity. Comparable KPNA4 pathology was observed in both sporadic frontotemporal dementia and C9ALS/FTD patient brains characterized by its nuclear depletion and cytosolic accumulation, irrespective of TDP-43 or dipeptide-repeat protein aggregates. These findings identify a vicious feedback cycle for dipeptide-repeat protein-mediated TDP-43 and subsequent KPNA pathology, which becomes self-sufficient of the initiating trigger and causes C9-related neurodegeneration.

The microtubule-associated protein tau regulates myriad neuronal functions, such as microtubule dynamics, axonal transport and neurite outgrowth. Tauopathies are neurodegenerative disorders characterized by the abnormal metabolism of tau, which accumulates as insoluble neuronal deposits. The adult human brain contains equal amounts of tau isoforms with three (3R) or four (4R) repeats of microtubule-binding domains, derived from the alternative splicing of exon 10 (E10) in the tau transcript. Several tauopathies are associated with imbalances of tau isoforms, due to splicing deficits. Here, we used a trans-splicing strategy to shift the inclusion of E10 in a mouse model of tauopathy that produces abnormal excess of 3R tau. Modulating the 3R/4R ratio in the prefrontal cortex led to a significant reduction of pathological tau accumulation concomitant with improvement of neuronal firing and reduction of cognitive impairments. Our results suggest promising potential for the use of RNA reprogramming in human neurodegenerative diseases., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

An intronic GGGGCC (G4C2) hexanucleotide repeat expansion inC9orf72 is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD). Repeat-associated non-AUG (RAN) translation of G4C2 RNA can result in five different dipeptide repeat proteins (DPR: poly GA, poly GP, poly GR, poly PA, and poly PR), which aggregate into neuronal cytoplasmic and nuclear inclusions in affected patients, however their contribution to disease pathogenesis remains controversial. We show that among the DPR proteins, expression of poly GA in a cell culture model activates programmed cell death and TDP-43 cleavage in a dose-dependent manner. Dual expression of poly GA together with other DPRs revealed that poly GP and poly PA are sequestered by poly GA, whereas poly GR and poly PR are rarely co-localised with poly GA. Dual expression of poly GA and poly PA ameliorated poly GA toxicity by inhibiting poly GA aggregation both in vitro and in vivo in the chick embryonic spinal cord. Expression of alternative codon-derived DPRs in chick embryonic spinal cord confirmed in vitro data, revealing that each of the dipeptides caused toxicity, with poly GA being the most toxic. Further, in vivo expression of G4C2 repeats of varying length caused apoptotic cell death, but failed to generate DPRs. Together, these data demonstrate that C9-related toxicity can be mediated by either RNA or DPRs. Moreover, our findings provide evidence that poly GA is a key mediator of cytotoxicity and that cross-talk between DPR proteins likely modifies their pathogenic status in C9ALS/FTD., (© The Author 2017. Published by Oxford University Press.)

A large GGGGCC hexanucleotide repeat expansion in the first intron or promoter region of the C9orf72 gene is the most common genetic cause of familial and sporadic Amyotrophic lateral sclerosis (ALS), a devastating degenerative disease of motor neurons, and of Frontotemporal Dementia (FTD), the second most common form of presenile dementia after Alzheimer's disease. C9orf72 -associated ALS/FTD is a multifaceted disease both in terms of its clinical presentation and the misregulated cellular pathways contributing to disease progression. Among the numerous pathways misregulated in C9orf72 -associated ALS/FTD, altered RNA processing has consistently appeared at the forefront of C9orf72 research. This includes bidirectional transcription of the repeat sequence, accumulation of repeat RNA into nuclear foci sequestering specific RNA-binding proteins (RBPs) and translation of RNA repeats into dipeptide repeat proteins (DPRs) by repeat-associated non-AUG (RAN)-initiated translation. Over the past few years the true extent of RNA misprocessing in C9orf72 -associated ALS/FTD has begun to emerge and disruptions have been identified in almost all aspects of the life of an RNA molecule, including release from RNA polymerase II, translation in the cytoplasm and degradation. Furthermore, several alterations have been identified in the processing of the C9orf72 RNA itself, in terms of its transcription, splicing and localization. This review article aims to consolidate our current knowledge on the consequence of the C9orf72 repeat expansion on RNA processing and draws attention to the mechanisms by which several aspects of C9orf72 molecular pathology converge to perturb every stage of RNA metabolism.

CNS Anticancer Drug Discovery and Development, 16-17 November 2016, Scottsdale, AZ, USA The 2016 second CNS Anticancer Drug Discovery and Development Conference addressed diverse viewpoints about why new drug discovery/development focused on CNS cancers has been sorely lacking. Despite more than 70,000 individuals in the USA being diagnosed with a primary brain malignancy and 151,669-286,486 suffering from metastatic CNS cancer, in 1999, temozolomide was the last drug approved by the US FDA as an anticancer agent for high-grade gliomas. Among the topics discussed were economic factors and pharmaceutical risk assessments, regulatory constraints and perceptions and the need for improved imaging surrogates of drug activity. Included were modeling tumor growth and drug effects in a medical environment in which direct tumor sampling for biological effects can be problematic, potential new drugs under investigation and targets for drug discovery and development. The long trajectory and diverse impediments to novel drug discovery, and expectation that more than one drug will be needed to adequately inhibit critical intracellular tumor pathways were viewed as major disincentives for most pharmaceutical/biotechnology companies. While there were a few unanimities, one consensus is the need for continued and focused discussion among academic and industry scientists and clinicians to address tumor targets, new drug chemistry, and more time- and cost-efficient clinical trials based on surrogate end points.

Photo cross-linking of proteins with short RNA oligomers is a classical method to study RNA-protein interactions that are implicated in many aspects of RNA metabolism and function. Most commonly, this involves the use of [γ- 32 P]-labeled RNA probes. Although very sensitive, these procedures are complicated by the safety issues associated with the use of radioisotopes. Here, we describe a modified UV cross-linking method using oligonucleotide probes end labelled with the infrared dye IRDye®800. After UV cross-linking, proteins are separated by SDS-PAGE and cross-linked products are visualized with the Odyssey ® Infrared Imaging system. This end labelling approach provides a streamlined alternative to random labelling which reduces the efficiency of in-vitro transcription. End labelling is also independent of the length of the probe, thus facilitating quantitative comparisons. To validate the method, we have confirmed the binding of HuD to the 3'-UTR of the mRNA for the microtubule-associated protein tau, implicated in the pathogenesis of Alzheimer's disease. UV cross-linking of HuD with a labeled 21-mer probe was successfully performed using a recombinant purified glutathione- S -transferase-HuD fusion protein as well as with lysates from CHO cells transfected with HuD cDNA. UV cross-linking combined with infrared imaging offers a convenient and robust strategy to analyse RNA-protein interactions and their emerging importance in disease., (© The Author 2017. Published by Oxford University Press.)

Tau, as a microtubule (MT)-associated protein, participates in key neuronal functions such as the regulation of MT dynamics, axonal transport, and neurite outgrowth. Alternative splicing of exon 10 in the tau primary transcript gives rise to protein isoforms with three (3R) or four (4R) MT binding repeats. Although tau isoforms are balanced in the normal adult human brain, imbalances in 3R:4R ratio have been tightly associated with the pathogenesis of several neurodegenerative disorders, yet the underlying molecular mechanisms remain elusive. Several studies exploiting tau overexpression and/or mutations suggested that perturbations in tau metabolism impair axonal transport. Nevertheless, no physiological model has yet demonstrated the consequences of altering the endogenous relative content of tau isoforms over axonal transport regulation. Here, we addressed this issue using a trans-splicing strategy that allows modulating tau exon 10 inclusion/exclusion in differentiated human-derived neurons. Upon changes in 3R:4R tau relative content, neurons showed no morphological changes, but live imaging studies revealed that the dynamics of the amyloid precursor protein (APP) were significantly impaired. Single trajectory analyses of the moving vesicles showed that predominance of 3R tau favored the anterograde movement of APP vesicles, increasing anterograde run lengths and reducing retrograde runs and segmental velocities. Conversely, the imbalance toward the 4R isoform promoted a retrograde bias by a significant reduction of anterograde velocities. These findings suggest that changes in 3R:4R tau ratio has an impact on the regulation of axonal transport and specifically in APP dynamics, which might link tau isoform imbalances with APP abnormal metabolism in neurodegenerative processes., Significance Statement: The tau protein has a relevant role in the transport of cargos throughout neurons. Dysfunction in tau metabolism underlies several neurological disorders leading to dementia. In the adult human brain, two tau isoforms are found in equal amounts, whereas changes in such equilibrium have been associated with neurodegenerative diseases. We investigated the role of tau in human neurons in culture and found that perturbations in the endogenous balance of tau isoforms were sufficient to impair the transport of the Alzheimer's disease-related amyloid precursor protein (APP), although neuronal morphology was normal. Our results provide evidence of a direct relationship between tau isoform imbalance and defects in axonal transport, which induce an abnormal APP metabolism with important implications in neurodegeneration., (Copyright © 2017 the authors 0270-6474/17/370059-12$15.00/0.)

The prevalence of mutant isocitrate dehydrogenase 1 (IDH1) brain tumors has generated significant efforts to understand the role of the mutated enzyme product d-2-hydroxyglutarate (D2HG), an oncometabolite, in tumorigenesis, as well as means to eliminate it. Glymphatic clearance was proposed as a pathway that could be manipulated to accelerate D2HG clearance and dictated the study design that consisted of two cohorts of mice bearing U87/mutant IDH1 intracerebral tumors that underwent two microdialysis - providing D2HG interstitial fluid concentrations - sampling periods of awake and asleep (activate glymphatic clearance) in a crossover manner. Glymphatic clearance was found not to have a significant effect on D2HG brain tumor interstitial fluid concentrations that were 126.9 ± 74.8 μM awake and 117.6 ± 98.6 μM asleep. These concentrations, although low relative to total brain tumor concentrations of 6.8 ± 3.6 mM, were considered sufficient to be transported by interstitial fluid and taken up into normal cells to cause deleterious effects. A model of D2HG CNS distribution supported this contention and was further supported by in vitro studies that showed D2HG could interfere with immune cell function. The study provides insight into the compartmental distribution of D2HG in the brain, wherein the interstitial fluid serves as a dynamic pathway for D2HG to enter normal cells and contribute to tumorigenesis.

Tau proteins, which stabilize the structure and regulate the dynamics of microtubules, also play important roles in axonal transport and signal transduction. Tau proteins are missorted, aggregated, and found as tau inclusions under many pathological conditions associated with neurodegenerative disorders, which are collectively known as tauopathies. In the adult human brain, tau protein can be expressed in six isoforms due to alternative splicing. The aberrant splicing of tau pre-mRNA has been consistently identified in a variety of tauopathies but is not restricted to these types of disorders as it is also present in patients with non-tau proteinopathies and RNAopathies. Tau mis-splicing results in isoform-specific impairments in normal physiological function and enhanced recruitment of excessive tau isoforms into the pathological process. A variety of factors are involved in the complex set of mechanisms underlying tau mis-splicing, but variation in the cis-element, methylation of the MAPT gene, genetic polymorphisms, the quantity and activity of spliceosomal proteins, and the patency of other RNA-binding proteins, are related to aberrant splicing. Currently, there is a lack of appropriate therapeutic strategies aimed at correcting the tau mis-splicing process in patients with neurodegenerative disorders. Thus, a more comprehensive understanding of the relationship between tau mis-splicing and neurodegenerative disorders will aid in the development of efficient therapeutic strategies for patients with a tauopathy or other, related neurodegenerative disorders. [BMB Reports 2016; 49(8): 405-413].

Background/aim: The alkylating agent, temozolomide (TMZ), is considered the standard-of-care for high-grade astrocytomas -known as glioblastoma multiforme (GBM)- an aggressive type of tumor with poor prognosis. The therapeutic benefit of TMZ is attributed to formation of DNA adducts involving the methylation of purine bases in DNA. We investigated the effects of TMZ on arginine and lysine amino acids, histone H3 peptides and histone H3 proteins., Materials and Methods: Chemical modification of amino acids, histone H3 peptide and protein by TMZ was performed in phosphate buffer at physiological pH. The reaction products were examined by mass spectrometry and western blot analysis., Results: Our results showed that TMZ following conversion to a methylating cation, can methylate histone H3 peptide and histone H3 protein, suggesting that TMZ exerts its anticancer activity not only through its interaction with DNA, but also through alterations of protein post-translational modifications., Conclusion: The possibility that TMZ can methylate histones involved with epigenetic regulation of protein indicates a potentially unique mechanism of action. The study will contribute to the understanding the anticancer activity of TMZ in order to develop novel targeted molecular strategies to advance the cancer treatment., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Leber congenital amaurosis (LCA) is an inherited retinal dystrophy that causes childhood blindness. Photoreceptors are especially sensitive to an intronic mutation in the cilia-related gene CEP290, which causes missplicing and premature termination, but the basis of this sensitivity is unclear. Here, we generated differentiated photoreceptors in three-dimensional optic cups and retinal pigment epithelium (RPE) from iPSCs with this common CEP290 mutation to investigate disease mechanisms and evaluate candidate therapies. iPSCs differentiated normally into RPE and optic cups, despite abnormal CEP290 splicing and cilia defects. The highest levels of aberrant splicing and cilia defects were observed in optic cups, explaining the retinal-specific manifestation of this CEP290 mutation. Treating optic cups with an antisense morpholino effectively blocked aberrant splicing and restored expression of full-length CEP290, restoring normal cilia-based protein trafficking. These results provide a mechanistic understanding of the retina-specific phenotypes in CEP290 LCA patients and potential strategies for therapeutic intervention., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: The most common forms of amyotrophic lateral sclerosis and frontotemporal dementia are caused by a large GGGGCC repeat expansion in the first intron of the C9orf72 gene. The repeat-containing intron should be degraded after being spliced out, however GGGGCC repeat-containing RNA species either accumulate in nuclear foci or are exported to the cytoplasm where they are translated into potentially toxic dipeptide repeat proteins by repeat-associated non-AUG-initiated (RAN) translation., Results: In order to determine the mechanisms of repeat-containing intron misprocessing, we have analyzed C9orf72 transcripts in lymphoblasts from C9orf72 expansion carriers (n = 15) and control individuals (n = 15). We have identified polyadenylated C9orf72 RNA species retaining the repeat-containing intron and in which downstream exons are spliced correctly resulting in a C9orf72 mRNA with an enlarged 5'-UTR containing the GGGGCC repeats. Intron-retaining transcripts are produced from both wild-type and mutant alleles. Intron-retaining C9orf72 transcripts were also detected in brain with a 2.7 fold increase measured in the frontal cortex from heterozygous expansion carriers (n = 11) compared to controls (n = 10). The level of intron-retaining transcripts was increased 5.9 fold in a case homozygous for the expansion. We also show that a large proportion of intron 1-retaining C9orf72 transcripts accumulate in the nucleus., Conclusions: Retention of the repeat-containing intron in mature C9orf72 mRNA can potentially explain nuclear foci formation as well as nuclear export of GGGGCC repeat RNA and suggests that the misprocessing of C9orf72 transcripts initiates the pathogenic process caused by C9orf72 hexanucleotide repeat expansions as well as provides the basis for novel therapeutic strategies.

A proportion of Alzheimer's disease cases displays inclusions of the RNA-binding protein, TDP-43. Considering the pathogenic role of tau mis-splicing, we compared tau isoform expression between Alzheimer's disease cases with or without TDP-43 inclusions. The average ratio of tau isoforms containing or lacking exon 10 (4R/3R ratio) or the total level of tau mRNA was not significantly different between cases with or without TDP-43 pathology in any of the brain regions examined. Although TDP-43 functions may be affected, TDP-43 does not critically regulate expression or splicing of tau in Alzheimer's disease suggesting that TDP-43 contributes to Alzheimer's disease through mechanisms independent of tau., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Pharmacokinetics (PKs) and pharmacodynamics (PDs) have always been integral to the design of rational drug dosing regimens. Early on PK-driven approaches came under the auspices of therapeutic drug monitoring that progressed into population-based PK and PK/PD modeling analyses. As the availability of tissue samples for measurement of drug concentrations is limited in patients, the bulk of such model-based methods relied on plasma drug concentrations to both build models and monitor therapy. The continued advances in systems biology and the spawning of systems pharmacology propelled the creation of enhanced PD (ePD) models. One of the main characteristic of ePD models is that they are derived from mechanistically grounded biochemical reaction networks. These models are commonly represented as systems of coupled ordinary differential equations with the ability to tailor each reaction and protein concentration to an individual's genomic/proteomic profile. As patient genomic analyses become more common, many genetic and protein abnormalities can be represented in the ePD models, and thus offer a path toward personalized anticancer therapies. By linking PK models to ePD models, a full spectrum of pharmacological simulation tools is available to design sophisticated multidrug regimens. However, ePD models are not a panacea and face challenges in model identifiability, scaling and parameter estimation. Nonetheless, as new technologies evolve and are coupled with fresh ideas on model implementation, it is likely that ePD and PK/ePD models will be considered a viable enterprise to customize anticancer drug therapy., (© 2015 Wiley Periodicals, Inc.)

Introduction: Cytoplasmic TDP-43 inclusions are the pathological hallmark of amyotrophic lateral sclerosis (ALS) and tau-negative frontotemporal lobar dementia (FTLD). The G4C2 repeat mutation in C9ORF72 is the most common cause of ALS and FTLD in which, in addition to TDP-43 inclusions, five different di-peptide repeat (DPR) proteins have been identified. Di-peptide repeat proteins are translated in a non-canonical fashion from sense and antisense transcripts of the G4C2 repeat (GP, GA, GR, PA, PR). DPR inclusions are abundant in the cerebellum, as well as in the frontal and temporal lobes of ALS and FTLD patients and some are neurotoxic in a range of cellular and animal models, implying that DPR aggregation directly contributes to disease pathogenesis. Here we sought to quantify inclusions for each DPR and TDP-43 in ALS cases with and without the C9ORF72 mutation. We characterised the abundance of DPRs and their cellular location and compared this to cytoplasmic TDP-43 inclusions in order to explore the role of each inclusion in lower motor neuron degeneration., Results: Spinal cord sections from ten cases positive for the C9ORF72 repeat expansion (ALS-C9+ve) and five cases that were not were probed by double immunofluorescence staining for individual DPRs and TDP-43. Inclusions immunoreactive for each of the DPRs were present in the spinal cord but they were rare or very rare in abundance (in descending order of frequency: GA, GP, GR, PA and PR). TDP-43 cytoplasmic inclusions were 45- to 750-fold more frequent than any DPR, and fewer than 4 % of DPR inclusions colocalized with TDP-43 inclusions. In motor neurons, a single cytoplasmic DPR inclusion was detected (0.1 %) in contrast to the 34 % of motor neurons that contained cytoplasmic TDP-43 inclusions. Furthermore, the number of TDP-43 inclusions in ALS cases with and without the C9ORF72 mutation was nearly identical., Conclusions: For all other neurodegenerative diseases, the neurotoxic protein aggregates are detected in the affected population of neurons. TDP-43 cytoplasmic aggregation is the dominant feature of ALS spinal cord pathology irrespective of C9ORF72 mutation status. The near absence of DPR inclusions in spinal cord motor neurons challenges their contribution to lower motor neuron degeneration in ALS-C9+ve cases.

The overlapping clinical and neuropathologic features between late-onset apparently sporadic Alzheimer's disease (LOAD), familial Alzheimer's disease (FAD), and other neurodegenerative dementias (frontotemporal dementia, corticobasal degeneration, progressive supranuclear palsy, and Creutzfeldt-Jakob disease) raise the question of whether shared genetic risk factors may explain the similar phenotype among these disparate disorders. To investigate this intriguing hypothesis, we analyzed rare coding variability in 6 Mendelian dementia genes (APP, PSEN1, PSEN2, GRN, MAPT, and PRNP), in 141 LOAD patients and 179 elderly controls, neuropathologically proven, from the UK. In our cohort, 14 LOAD cases (10%) and 11 controls (6%) carry at least 1 rare variant in the genes studied. We report a novel variant in PSEN1 (p.I168T) and a rare variant in PSEN2 (p.A237V), absent in controls and both likely pathogenic. Our findings support previous studies, suggesting that (1) rare coding variability in PSEN1 and PSEN2 may influence the susceptibility for LOAD and (2) GRN, MAPT, and PRNP are not major contributors to LOAD. Thus, genetic screening is pivotal for the clinical differential diagnosis of these neurodegenerative dementias., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Reaction kinetics were studied to quantify the effects of polar aprotic organic solvents on the acid-catalyzed conversion of xylose into furfural. A solvent of particular importance is γ-valerolactone (GVL), which leads to significant increases in reaction rates compared to water in addition to increased product selectivity. GVL has similar effects on the kinetics for the dehydration of 1,2-propanediol to propanal and for the hydrolysis of cellobiose to glucose. Based on results obtained for homogeneous Brønsted acid catalysts that span a range of pKa values, we suggest that an aprotic organic solvent affects the reaction kinetics by changing the stabilization of the acidic proton relative to the protonated transition state. This same behavior is displayed by strong solid Brønsted acid catalysts, such as H-mordenite and H-beta., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Mutations of isocitrate dehydrogenase 1 (IDH1) are frequently found in certain cancers such as glioma. Different from the wild-type (WT) IDH1, the mutant enzymes catalyze the reduction of α-ketoglutaric acid to d-2-hydroxyglutaric acid (D2HG), leading to cancer initiation. Several 1-hydroxypyridin-2-one compounds were identified to be inhibitors of IDH1(R132H). A total of 61 derivatives were synthesized, and their structure-activity relationships were investigated. Potent IDH1(R132H) inhibitors were identified with Ki values as low as 140 nM, while they possess weak or no activity against WT IDH1. Activities of selected compounds against IDH1(R132C) were found to be correlated with their inhibitory activities against IDH1(R132H), as well as cellular production of D2HG, with R(2) of 0.83 and 0.73, respectively. Several inhibitors were found to be permeable through the blood-brain barrier in a cell-based model assay and exhibit potent and selective activity (EC50 = 0.26-1.8 μM) against glioma cells with the IDH1 R132H mutation.

The microtubule-associated protein tau is predominantly localized in the axonal compartment over the entire length of the axon in neurons. The mechanisms responsible for the localization of tau in axons at long distance from the cell body are not properly understood. Using fluorescence in situ hybridization, we show that tau mRNA is present in the central and distal parts of the axons of cultured rat cortical neurons. Axonal tau mRNA is associated with granules which are distributed throughout the entire length of the axon, including the growth cone. We also show that tau mRNA-containing axonal particles are associated with elongation factor 1A, a component of the protein translation machinery. The presence of tau mRNA in axons might be at least part of the process by which tau is localized to distal axons., (Copyright © 2013 Elsevier B.V. All rights reserved.)

ON123300 is a low molecular weight multikinase inhibitor identified through a series of screens that supported further analyses for brain tumor chemotherapy. Biochemical assays indicated that ON123300 was a strong inhibitor of Ark5 and CDK4, as well as growth factor receptor tyrosine kinases such as β-type platelet-derived growth factor receptor (PDGFRβ). ON123300 inhibited U87 glioma cell proliferation with an IC(50) 3.4 ± 0.1 μmol/L and reduced phosphorylation of Akt, yet it also unexpectedly induced Erk activation, both in a dose- and time-dependent manner that subsequently was attributed to relieving Akt-mediated C-Raf S259 inactivation and activating a p70S6K-initiated PI3K-negative feedback loop. Cotreatment with the EGFR inhibitor gefitinib produced synergistic cytotoxic effects. Pursuant to the in vitro studies, in vivo pharmacokinetic and pharmacodynamic studies of ON123300 were completed in mice bearing intracerebral U87 tumors following intravenous doses of 5 and 25 mg/kg alone, and also at the higher dose concurrently with gefitinib. ON123300 showed high brain and brain tumor accumulation based on brain partition coefficient values of at least 2.5. Consistent with the in vitro studies, single agent ON123300 caused a dose-dependent suppression of phosphorylation of Akt as well as activation of Erk in brain tumors, whereas addition of gefitinib to the ON123300 regimen significantly enhanced p-Akt inhibition and prevented Erk activation. In summary, ON123300 demonstrated favorable pharmacokinetic characteristics, and future development for brain tumor therapy would require use of combinations, such as gefitinib, that mitigate its Erk activation and enhance its activity.

Methotrexate (MTX) is the cornerstone of chemotherapy for primary central nervous system lymphoma, yet how the blood-brain barrier (BBB) efflux transporters ABCG2 and ABCC4 influence the required high-dose therapy is unknown. To evaluate their role, we used four mouse strains, C57BL/6 (wild-type; WT), Abcg2(-/-), Abcc4(-/-), and Abcg2(-/-);Abcc4(-/-) (double knockout; DKO) to conduct brain microdialysis studies after single intravenous MTX doses of 50 mg/kg. When the area under the concentration-time curve for plasma (AUC(plasma)) was used to assess systemic exposure to MTX, the rank order was Abcc4(-/-) < WT < Abcg2(-/-) < Abcg2(-/-)Abcc4(-/-). Only the DKO exposure was significantly higher than that of the WT group (P < 0.01), a reflection of the role of Abcg2 in biliary excretion and Abcc4 in renal excretion. MTX brain interstitial fluid concentrations obtained by microdialysis were used to calculate the area under the concentration-time curve for the brain (AUC(brain)), which found the rank order of exposure to be WT < Abcc4(-/-) < Abcg2(-/-) < Abcg2(-/-)Abcc4(-/-) with the largest difference being 4-fold: 286.13 ± 130 μg*min/ml (DKO) versus 66.85 ± 26 (WT). Because the transporters affected the systemic disposition of MTX, particularly in the DKO group, the ratio of the AUC(brain)/AUC(plasma) or the brain/plasma partition coefficient Kp was calculated, revealing that the DKO strain had a significantly higher value (0.23 ± 0.09) than the WT strain (0.11 ± 0.05). Both Abcg2 and Abcc4 limited BBB penetration of MTX; however, only when both drug efflux pumps were negated did the brain accumulation of MTX significantly increase. These findings indicate a contributory role of both ABCG2 and ABCC4 to limiting MTX distribution in patients.

TDP-43 is found in cytoplasmic inclusions in 95% of amyotrophic lateral sclerosis (ALS) and 60% of frontotemporal lobar degeneration (FTLD). Approximately 4% of familial ALS is caused by mutations in TDP-43. The majority of these mutations are found in the glycine-rich domain, including the variant M337V, which is one of the most common mutations in TDP-43. In order to investigate the use of allele-specific RNA interference (RNAi) as a potential therapeutic tool, we designed and screened a set of siRNAs that specifically target TDP-43(M337V) mutation. Two siRNA specifically silenced the M337V mutation in HEK293T cells transfected with GFP-TDP-43(wt) or GFP-TDP-43(M337V) or TDP-43 C-terminal fragments counterparts. C-terminal TDP-43 transfected cells show an increase of cytosolic inclusions, which are decreased after allele-specific siRNA in M337V cells. We then investigated the effects of one of these allele-specific siRNAs in induced pluripotent stem cells (iPSCs) derived from an ALS patient carrying the M337V mutation. These lines showed a two-fold increase in cytosolic TDP-43 compared to the control. Following transfection with the allele-specific siRNA, cytosolic TDP-43 was reduced by 30% compared to cells transfected with a scrambled siRNA. We conclude that RNA interference can be used to selectively target the TDP-43(M337V) allele in mammalian and patient cells, thus demonstrating the potential for using RNA interference as a therapeutic tool for ALS.

GGGGCC (G4C2) hexanucleotide repeat expansion in chromosome 9 open reading frame 72 (C9ORF72) has been identified as the most common genetic abnormality in both frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). To investigate the role of C9ORF72-related G4C2 repeat expansion in ALS and FTLD, several animal and cell culture models have been generated that reveal initial insights into the disease pathogenesis of C9 ALS/FTLD. These models include neurons differentiated from patient-derived pluripotent stem cells as well as genetically engineered cells and organisms that knock down C9ORF72 orthologues or express G4C2 repeats. Targeted reduction or knockdown of C9ORF72 homologues in zebrafish and mice so far produced conflicting results which neither rule out, nor confirm reduced expression of C9ORF72 as a pathogenic mechanism in C9 ALS/FTLD. In contrast, studies using patient-derived cells, as well as Drosophila and zebrafish models overexpressing disease-related hexanucleotide expansions, can cause repeat length-dependent formation of RNA foci, which directly and progressively correlate with cellular toxicity. RNA foci formation is accompanied by sequestration of specific RNA-binding proteins (RBPs), including Pur-alpha, hnRNPH and ADARB2, suggesting that G4C2-mediated sequestration and functional depletion of RBPs are cytotoxic and thus directly contribute to disease. Moreover, these studies provide experimental evidence that repeat-associated non-ATG translation of repeat-containing sense and antisense RNA leads to dipeptide-repeat proteins (DPRs) that can accumulate and aggregate, indicating that accumulation of DPRs may represent another pathogenic pathway underlying C9 ALS/FTLD. These studies in cell and animal models therefore identify RNA toxicity, RBP sequestration and accumulation of DPRs as emerging pathogenic pathways underlying C9 ALS/FTLD.

The GGGGCC (G4C2) intronic repeat expansion within C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Intranuclear neuronal RNA foci have been observed in ALS and FTD tissues, suggesting that G4C2 RNA may be toxic. Here, we demonstrate that the expression of 38× and 72× G4C2 repeats form intranuclear RNA foci that initiate apoptotic cell death in neuronal cell lines and zebrafish embryos. The foci colocalize with a subset of RNA binding proteins, including SF2, SC35, and hnRNP-H in transfected cells. Only hnRNP-H binds directly to G4C2 repeats following RNA immunoprecipitation, and only hnRNP-H colocalizes with 70% of G4C2 RNA foci detected in C9ORF72 mutant ALS and FTD brain tissues. We show that expanded G4C2 repeats are potently neurotoxic and bind hnRNP-H and other RNA binding proteins. We propose that RNA toxicity and protein sequestration may disrupt RNA processing and contribute to neurodegeneration., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Like many solid tumors, glioblastomas are characterized by intratumoral biologic heterogeneity that may contribute to a variable distribution of drugs and their associated pharmacodynamic responses, such that the standard pharmacokinetic approaches based on analysis of whole-tumor homogenates may be inaccurate. To address this aspect of tumor pharmacology, we analyzed intratumoral pharmacokinetic/pharmacodynamic characteristics of the EGFR inhibitor gefitinib in mice with intracerebral tumors and developed corresponding mathematical models. Following a single oral dose of gefitinib (50 or 150 mg/kg), tumors were processed at selected times according to a novel brain tumor sectioning protocol that generated serial samples to measure gefitinib concentrations, phosphorylated extracellular signal-regulated kinase (pERK), and immunohistochemistry in 4 different regions of tumors. Notably, we observed up to 3-fold variations in intratumoral concentrations of gefitinib, but only up to half this variability in pERK levels. As we observed a similar degree of variation in the immunohistochemical index termed the microvessel pericyte index (MPI), a measure of permeability in the blood-brain barrier, we used MPI in a hybrid physiologically-based pharmacokinetic (PBPK) model to account for regional changes in drug distribution that were observed. Subsequently, the PBPK models were linked to a pharmacodynamic model that could account for the variability observed in pERK levels. Together, our tumor sectioning protocol enabled integration of the intratumoral pharmacokinetic/pharmacodynamic variability of gefitinib and immunohistochemical indices followed by the construction of a predictive PBPK/pharmacodynamic model. These types of models offer a mechanistic basis to understand tumor heterogeneity as it impacts the activity of anticancer drugs.

A series of novel (E)-N-aryl-2-arylethenesulfonamides (6) were synthesized and evaluated for their anticancer activity. Some of the compounds in this series showed potent cytotoxicity against a wide spectrum of cancer cell-lines (IC50 values ranging from 5 to 10 nM) including all drug resistant cell-lines. Nude mice xenograft assays with compound (E)-N-(3-amino-4-methoxyphenyl)-2-(2',4',6'-trimethoxyphenyl)ethenesulfonamide (6t) showed dramatic reduction in tumor size, indicating their in vivo potential as anticancer agents. A preliminary drug development study with compound 6t is predicted to have increased blood-brain barrier permeability relative to many clinically used antimitotic agents. Mechanistic studies indicate that 6t and some other analogues disrupted microtubule formation, formation of mitotic spindles, and arrest of cells in mitotic phase. Compound 6t inhibited purified tubulin polymerization in vitro and in vivo and circumvented drug resistance mediated by P-glycoprotein. Compound 6t specifically competed with colchicine binding to tubulin and with similar avidity as podophylltoxin, indicating its binding site on tubulin.