RESUMO
Hit screening, which involves the identification of compounds or targets capable of modulating disease-relevant processes, is an important step in drug discovery. Some assays, such as image-based high-content screenings, produce complex multivariate readouts. To fully exploit the richness of such data, advanced analytical methods that go beyond the conventional univariate approaches should be employed. In this work, we tackle the problem of hit identification in multivariate assays. As with univariate assays, a hit from a multivariate assay can be defined as a candidate that yields an assay value sufficiently far away in distance from the mean or central value of inactives. Viewed another way, a hit is an outlier from the distribution of inactives. A method was developed for identifying multivariate hit in high-dimensional data sets based on principal components and robust Mahalanobis distance (the multivariate analogue to the Z- or T-statistic). The proposed method, termed mROUT (multivariate robust outlier detection), demonstrates superior performance over other techniques in the literature in terms of maintaining Type I error, false discovery rate and true discovery rate in simulation studies. The performance of mROUT is also illustrated on a CRISPR knockout data set from in-house phenotypic screening programme.
Assuntos
Ensaios de Triagem em Larga Escala , Análise Multivariada , Humanos , Ensaios de Triagem em Larga Escala/métodos , Descoberta de Drogas/métodos , Algoritmos , Análise de Componente Principal , Simulação por ComputadorRESUMO
Quality by Design (QbD) is an approach to assay development to determine the design space, which is the range of assay variable settings that should result in satisfactory assay quality. Typically, QbD is applied in manufacturing, but it works just as well in the preclinical space. Through three examples, we illustrate the QbD approach with experimental design and associated data analysis to determine the design space for preclinical assays.
RESUMO
Small airway epithelial cells (SAECs) play a central role in the pathogenesis of lung diseases and are now becoming a crucial cellular model for target identification and validation in drug discovery. However, primary cell lines such as SAECs are often difficult to transfect using traditional lipofection methods; therefore, gene editing using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is often carried out through ribonucleoprotein (RNP) electroporation. Here we have established a robust, scalable, and automated arrayed CRISPR nuclease (CRISPRn) screening workflow for SAECs which can be combined with a myriad of disease-specific endpoint assays.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Sistemas CRISPR-Cas/genética , Inativação Gênica , Pulmão , Células Epiteliais/metabolismoRESUMO
S100A4, belonging to a large multifunctional S100 protein family, is a Ca2+-binding protein with a significant role in stimulating the motility of cancer and immune cells, as well as in promoting pro-inflammatory properties in different cell types. In the CNS, there is limited information concerning S100A4 presence and function. In this study, we analyzed the expression of S100A4 and the effect of the S100A4 transcriptional inhibitor niclosamide in murine activated primary microglia. We found that S100A4 was strongly up-regulated in reactive microglia and that niclosamide prevented NADPH oxidase 2, mTOR (mammalian target of rapamycin), and NF-κB (nuclear factor-kappa B) increase, cytoskeletal rearrangements, migration, and phagocytosis. Furthermore, we found that S100A4 was significantly up-regulated in astrocytes and microglia in the spinal cord of a transgenic rat SOD1-G93A model of amyotrophic lateral sclerosis. Finally, we demonstrated the increased expression of S100A4 also in fibroblasts derived from amyotrophic lateral sclerosis (ALS) patients carrying SOD1 pathogenic variants. These results ascribe S100A4 as a marker of microglial reactivity, suggesting the contribution of S100A4-regulated pathways to neuroinflammation, and identify niclosamide as a possible drug in the control and attenuation of reactive phenotypes of microglia, thus opening the way to further investigation for a new application in neurodegenerative conditions.
Assuntos
Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/metabolismo , Microglia/citologia , Microglia/efeitos dos fármacos , Niclosamida/uso terapêutico , Proteína A4 de Ligação a Cálcio da Família S100/antagonistas & inibidores , Adulto , Esclerose Lateral Amiotrófica/imunologia , Animais , Western Blotting , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Masculino , Camundongos , Microglia/imunologia , Microglia/metabolismo , Microscopia de Fluorescência , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Fagocitose/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Proteína A4 de Ligação a Cálcio da Família S100/metabolismo , Superóxido Dismutase-1 , Serina-Treonina Quinases TOR/metabolismoRESUMO
S100B is a Ca2+ -binding protein mainly concentrated in astrocytes. Its levels in biological fluids (cerebrospinal fluid, peripheral and cord blood, urine, saliva, amniotic fluid) are recognized as a reliable biomarker of active neural distress. Although the wide spectrum of diseases in which the protein is involved (acute brain injury, neurodegenerative diseases, congenital/perinatal disorders, psychiatric disorders) reduces its specificity, its levels remain an important aid in monitoring the trend of the disorder. Mounting evidence now points to S100B as a Damage-Associated Molecular Pattern molecule which, when released at high concentration, through its Receptor for Advanced Glycation Endproducts, triggers tissue reaction to damage in a series of different neural disorders. This review addresses this novel scenario, presenting data indicating that S100B levels and/or distribution in the nervous tissue of patients and/or experimental models of different neural disorders, for which the protein is used as a biomarker, are directly related to the progress of the disease: acute brain injury (ischemic/hemorrhagic stroke, traumatic injury), neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis), congenital/perinatal disorders (Down syndrome, spinocerebellar ataxia-1), psychiatric disorders (schizophrenia, mood disorders), inflammatory bowel disease. In many cases, over-expression/administration of the protein induces worsening of the disease, whereas its deletion/inactivation produces amelioration. This review points out that the pivotal role of the protein resulting from these data, opens the perspective that S100B may be regarded as a therapeutic target for these different diseases, which appear to share some common features reasonably attributable to neuroinflammation, regardless their origin.
Assuntos
Biomarcadores , Doenças do Sistema Nervoso , Subunidade beta da Proteína Ligante de Cálcio S100 , Animais , HumanosRESUMO
TAR DNA-binding protein 43 (TDP-43) is an RNA-binding protein and a major component of protein aggregates found in amyotrophic lateral sclerosis and several other neurodegenerative diseases. TDP-43 exists as a full-length protein and as two shorter forms of 25 and 35 kDa. Full-length mutant TDP-43s found in amyotrophic lateral sclerosis patients re-localize from the nucleus to the cytoplasm and in part to mitochondria, where they exert a toxic role associated with neurodegeneration. However, induction of mitochondrial damage by TDP-43 fragments is yet to be clarified. In this work, we show that the mitochondrial 35 kDa truncated form of TDP-43 is restricted to the intermembrane space, while the full-length forms also localize in the mitochondrial matrix in cultured neuronal NSC-34 cells. Interestingly, the full-length forms clearly affect mitochondrial metabolism and morphology, possibly via their ability to inhibit the expression of Complex I subunits encoded by the mitochondrial-transcribed mRNAs, while the 35 kDa form does not. In the light of the known differential contribution of the full-length and short isoforms to generate toxic aggregates, we propose that the presence of full-length TDP-43s in the matrix is a primary cause of mitochondrial damage. This in turn may cause oxidative stress inducing toxic oligomers formation, in which short TDP-43 forms play a major role.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Mitocôndrias/metabolismo , Neurônios , Oligonucleotídeos/toxicidade , Isoformas de Proteínas/metabolismo , Linhagem Celular Transformada , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Chaperonina 60/genética , Chaperonina 60/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Citosol/ultraestrutura , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/ultraestrutura , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Imunoprecipitação , Microscopia Eletrônica , Mitocôndrias/efeitos dos fármacos , Mutação/efeitos dos fármacos , Mutação/genética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/ultraestrutura , Consumo de Oxigênio/efeitos dos fármacos , Isoformas de Proteínas/genética , Isoformas de Proteínas/ultraestrutura , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Mitocondrial/genética , RNA Mitocondrial/metabolismo , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , TransfecçãoRESUMO
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a non-cell autonomous motor neuron loss. While it is generally believed that the disease onset takes place inside motor neurons, different cell types mediating neuroinflammatory processes are considered deeply involved in the progression of the disease. On these grounds, many treatments have been tested on ALS animals with the aim of inhibiting or reducing the pro-inflammatory action of microglia and astrocytes and counteract the progression of the disease. Unfortunately, these anti-inflammatory therapies have been only modestly successful. The non-univocal role played by microglia during stress and injuries might explain this failure. Indeed, it is now well recognized that, during ALS, microglia displays different phenotypes, from surveillant in early stages, to activated states, M1 and M2, characterized by the expression of respectively harmful and protective genes in later phases of the disease. Consistently, the inhibition of microglial function seems to be a valid strategy only if the different stages of microglia polarization are taken into account, interfering with the reactivity of microglia specifically targeting only the harmful pathways and/or potentiating the trophic ones. In this review article, we will analyze the features and timing of microglia activation in the light of M1/M2 phenotypes in the main mice models of ALS. Moreover, we will also revise the results obtained by different anti-inflammatory therapies aimed to unbalance the M1/M2 ratio, shifting it towards a protective outcome.
RESUMO
Neuroinflammation is one of the major players in amyotrophic lateral sclerosis (ALS) pathogenesis, and astrocytes are significantly involved in this process. The astrocytic protein S100B can be released in pathological states activating the receptor for advanced glycation end products (RAGE). Different indications point to an aberrant expression of S100B and RAGE in ALS. In this work, we observed that S100B and RAGE are progressively and selectively upregulated in astrocytes of diseased rats with a tissue-specific timing pattern, correlated to the level of neurodegeneration. The expression of the full-length and soluble RAGE isoforms could also be linked to the degree of tissue damage. The mere presence of mutant SOD1 is able to increase the intracellular levels and release S100B from astrocytes, suggesting the possibility that an increased astrocytic S100B expression might be an early occurring event in the disease. Finally, our findings indicate that the protein may exert a proinflammatory role in ALS, since its inhibition in astrocytes derived from SOD1G93A mice limits the expression of reactivity-linked/proinflammatory genes. Thus, our results propose the S100B-RAGE axis as an effective contributor to the pathogenesis of the disease, suggesting its blockade as a rational target for a therapeutic intervention in ALS.
Assuntos
Receptor para Produtos Finais de Glicação Avançada/metabolismo , Subunidade beta da Proteína Ligante de Cálcio S100/metabolismo , Superóxido Dismutase-1/metabolismo , Animais , Animais Geneticamente Modificados , Astrócitos/metabolismo , Western Blotting , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Masculino , Microscopia de Fluorescência , Ratos , Receptor para Produtos Finais de Glicação Avançada/genética , Subunidade beta da Proteína Ligante de Cálcio S100/genética , Superóxido Dismutase-1/genéticaRESUMO
A common feature of non-coding repeat expansion disorders is the accumulation of RNA repeats as RNA foci in the nucleus and/or cytoplasm of affected cells. These RNA foci can be toxic because they sequester RNA-binding proteins, thus affecting various steps of post-transcriptional gene regulation. However, the precise step that is affected by C9orf72 GGGGCC (G4C2) repeat expansion, the major genetic cause of amyotrophic lateral sclerosis (ALS), is still poorly defined. In this work, we set out to characterise these mechanisms by identifying proteins that bind to C9orf72 RNA. Sequestration of some of these factors into RNA foci was observed when a (G4C2)31 repeat was expressed in NSC34 and HeLa cells. Most notably, (G4C2)31 repeats widely affected the distribution of Pur-alpha and its binding partner fragile X mental retardation protein 1 (FMRP, also known as FMR1), which accumulate in intra-cytosolic granules that are positive for stress granules markers. Accordingly, translational repression is induced. Interestingly, this effect is associated with a marked accumulation of poly(A) mRNAs in cell nuclei. Thus, defective trafficking of mRNA, as a consequence of impaired nuclear mRNA export, might affect translation efficiency and contribute to the pathogenesis of C9orf72 ALS.