Pharmacological approaches to tackle NCLs

Neuronal ceroid lipofuscinoses, also collectively known as Batten disease, are a group of rare monogenic disorders caused by mutations in at least 13 different genes. They are characterized by the accumulation of lysosomal storage material and progressive neurological deterioration with dementia, epilepsy, retinopathy, motor disturbances, and early death [1]. Although the identification of disease-causing genes provides an important step for understanding the molecular mechanisms underlying neuronal ceroid lipofuscinoses, compared to other diseases, obstacles to the development of therapies for these rare diseases include less extensive physiopathology knowledge, limited number of patients to test treatments, and poor commercial interest from the industry. Current therapeutic strategies include enzyme replacement therapies, gene therapies targeting the brain and the eye, cell therapies, and pharmacological drugs that could modulate defective molecular pathways. In this review, we will focus in the emerging therapies based in the identification of small-molecules. Recent advances in high- throughput and high-content screening (HTS and HCS) using relevant cell-based assays and applying automation and imaging analysis algorithms, will allow the screening of a large number of compounds in lesser time. These approaches are particularly useful for drug repurposing for Batten disease, that takes the advantage to search for compounds that have already been tested in humans, thereby reducing significantly the resources needed for translation to clinics.     

Genetically modified immunomodulatory cell-based biomaterials in tissue regeneration and engineering

To date, the wide application of cell-based biomaterials in tissue engineering and regeneration is remarkably hampered by immune rejection. Reducing the immunogenicity of cell-based biomaterials has become the latest direction in biomaterial research. Recently, genetically modified cell-based biomaterials with immunomodulatory genes have become a feasible solution to the immunogenicity problem. In this review, recent advances and future challenges of genetically modified immunomodulatory cell-based biomaterials are elaborated, including fabrication approaches, mechanisms of common immunomodulatory genes, application and, more importantly, current preclinical and clinical advances. The fabrication approaches can be categorized into commonly used (e.g., virus transfection) and newly developed approaches. The immunomodulatory mechanisms of representative genes involve complicated cell signaling pathways and metabolic activities. Wide application in curing multiple end-term diseases and replacing lifelong immunosuppressive therapy in multiple cell and organ transplantation models is demonstrated. Most significantly, practices of genetically modified organ transplantation have been conducted on brain-dead human decedent and even on living patients after a series of experiments on nonhuman primates. Nevertheless, uncertain biosecurity, nonspecific effects and overlooked personalization of current genetically modified immunomodulatory cell-based biomaterials are shortcomings that remain to be overcome.     

神经变性构象病及其分子基础

构象病的概念被广泛用于命名与蛋白质的构象异常相关的疾病。随着生命科学的进步,人们对神经变性疾病发病的分子机制有了较好的认识,发现几乎所有的此类疾病,诸如阿尔采末病（AD）、帕金森病（PD）、亨廷顿病（HD）以及朊蛋白病（PrD）等都具有一个共同的特征,即病变细胞中蓄积有大量错误折叠并易于聚合的蛋白质,这符合构象病的特点,所以又派生了神经变性构象病的新概念。近年来,人们在神经变性构象病的蛋白质错误折叠和聚合以及其细胞毒性方面的认识越来越走向深入,这将对寻找有效的治疗方法起到极大的推动作用。     

细胞凋亡的结构生物学研究进展

在多细胞生物体内,细胞会发生编程性死亡(即细胞凋亡),使得细胞数量得到精确调控。细胞凋亡调控的异常与癌症、自身免疫病、神经退行性疾病等疾病密切相关。在过去的二十年里,人们详细地研究了参与细胞凋亡调控的分子机制。该文综述了近年来利用结构生物学手段,对参与细胞凋亡调控的分子,主要是Caspase和与Caspase活性调控直接相关的蛋白功能的研究进展。     

Docosahexaenoic acid prevents neuronal apoptosis induced by soluble amyloid-beta oligomers

A growing body of evidence supports the notion that soluble oligomers of amyloid-beta (Abeta) peptide interact with the neuronal plasma membrane, leading to cell injury and inducing death-signalling pathways that could account for the increased neurodegeneration occurring in Alzheimer's disease (AD). Docosahexaenoic acid (DHA, C22:6, n-3) is an essential polyunsaturated fatty acid in the CNS and has been shown in several epidemiological and in vivo studies to have protective effects against AD and cognitive alterations. However, the molecular mechanisms involved remain unknown. We hypothesized that DHA enrichment of plasma membranes could protect neurones from apoptosis induced by soluble Abeta oligomers. DHA pre-treatment was observed to significantly increase neuronal survival upon Abeta treatment by preventing cytoskeleton perturbations, caspase activation and apoptosis, as well as by promoting extracellular signal-related kinase (ERK)-related survival pathways. These data suggest that DHA enrichment probably induces changes in neuronal membrane properties with functional outcomes, thereby increasing protection from soluble Abeta oligomers. Such neuroprotective effects could be of major interest in the prevention of AD and other neurodegenerative diseases.     

Life is a journey: a genetic look at neocortical development

Although the basic principles of neocortical development have been known for quite some time, it is only recently that our understanding of the molecular mechanisms that are involved has improved. Such understanding has been facilitated by genetic approaches that have identified key proteins involved in neocortical development, which have been placed into signalling pathways by molecular and cell-biological studies. The challenge of current research is to understand the manner in which these various signalling pathways are interconnected to gain a more comprehensive picture of the molecular intricacies that govern neocortical development.     

Mechanisms of colorectal and lung cancer prevention by vegetables: a genomic approach

Colorectal cancer (CRC) and lung cancer (LC) occur at high incidence, and both can be effectively prevented by dietary vegetable consumption. This makes these two types of cancer highly suitable for elucidating the underlying molecular mechanisms of cancer chemoprevention. Numerous studies have shown that vegetables exert their beneficial effects through various different mechanisms, but effects on the genome level remain mostly unclear. This review evaluates current knowledge on the mechanisms of CRC and LC prevention by vegetables, thereby focusing on the modulation of gene and protein expressions. The majority of the effects found in the colon are changes in the expression of genes and proteins involved in apoptosis, cell cycle, cell proliferation and intracellular defense, in favor of reduced CRC risk. Furthermore, vegetables and vegetable components changed the expression of many more genes and proteins involved in other pathways for which biologic meaning is less clear. The number of studies investigating gene and protein expression changes in the lungs is limited to only a few in vitro and animal studies. Data from these studies show that mostly genes involved in biotransformation, apoptosis and cell cycle regulation are affected. In both colon and lungs, genomewide analyses of gene and protein expression changes by new genomics and proteomics technologies, as well as the investigation of whole vegetables, are few in number. Further studies applying these 'omics' approaches are needed to provide more insights on affected genetic/biologic pathways and, thus, in molecular mechanisms by which different chemopreventive compounds can protect against carcinogenesis. Particularly studies with combinations of phytochemicals and whole vegetables are needed to establish gene expression changes in the colon, but especially in the lungs.     

Unveiling the molecular crosstalk in a human induced pluripotent stem cell-derived cardiac model

In vitro cell-based models that better mimic the human heart tissue are of utmost importance for drug development and cardiotoxicity testing but also as tools to understand mechanisms related with heart disease at cellular and molecular level. Besides, the implementation of analytical tools that allow the depiction and comprehensive understanding of the molecular mechanisms of the crosstalk between the different cell types is also relevant. In this work, we implemented a human cardiac tissue-like in vitro model, derived from human-induced pluripotent stem cell (hiPSC), and evaluated the relevance of the cell–cell communication between the two of the most representative cell populations of the human heart: cardiomyocytes (hiPSC-CM) and endothelial cells (hiPSC-EC). We observed that heterotypic cell communication promotes: (a) structural maturation of hiPSC-CM and (b) deposition of several extracellular matrix components (such as collagens and fibronectin). Overall, the toolbox of analytical techniques used in our study not only enabled us to validate previous reports from the literature on the importance of the presence of hiPSC-EC on hiPSC-CM maturation, but also bring new insights on the molecular mechanisms involved in the communication between these two cell types when cocultured in vitro.     

Physio-pathological roles of transglutaminase-catalyzed reactions

Transglutaminases (TGs) are a large family of related and ubiquitous enzymes that catalyze post-translational modifications of proteins. The main activity of these enzymes is the cross-linking of a glutaminyl residue of a protein/peptide substrate to a lysyl residue of a protein/peptide co-substrate. In addition to lysyl residues, other second nucleophilic co-substrates may include monoamines or polyamines (to form mono- or bi-substituted /crosslinked adducts) or -OH groups (to form ester linkages). In the absence of co-substrates, the nucleophile may be water, resulting in the net deamidation of the glutaminyl residue. The TG enzymes are also capable of catalyzing other reactions important for cell viability. The distribution and the physiological roles of TG enzymes have been widely studied in numerous cell types and tissues and their roles in several diseases have begun to be identified. "Tissue" TG (TG2), a member of the TG family of enzymes, has definitely been shown to be involved in the molecular mechanisms responsible for a very widespread human pathology: i.e. celiac disease (CD). TG activity has also been hypothesized to be directly involved in the pathogenetic mechanisms responsible for several other human diseases, including neurodegenerative diseases, which are often associated with CD. Neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, supranuclear palsy, Huntington's disease and other recently identified polyglutamine diseases, are characterized, in part, by aberrant cerebral TG activity and by increased cross-linked proteins in affected brains. In this review, we discuss the physio-pathological role of TG-catalyzed reactions, with particular interest in the molecular mechanisms that could involve these enzymes in the physio-pathological processes responsible for human neurodegenerative diseases.     

High content screening in neurodegenerative diseases

The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be addressed as a matter of great urgency. Multiple complementary 'omics' approaches have provided clues as to the genetic risk factors and pathogenic mechanisms underlying numerous neurodegenerative diseases, but most findings still require functional validation. For example, a recent genome wide association study for Parkinson's Disease (PD), identified many new loci as risk factors for the disease, but the underlying causative variant(s) or pathogenic mechanism is not known. As each associated region can contain several genes, the functional evaluation of each of the genes on phenotypes associated with the disease, using traditional cell biology techniques would take too long. There is also a need to understand the molecular networks that link genetic mutations to the phenotypes they cause. It is expected that disease phenotypes are the result of multiple interactions that have been disrupted. Reconstruction of these networks using traditional molecular methods would be time consuming. Moreover, network predictions from independent studies of individual components, the reductionism approach, will probably underestimate the network complexity. This underestimation could, in part, explain the low success rate of drug approval due to undesirable or toxic side effects. Gaining a network perspective of disease related pathways using HT/HC cellular screening approaches, and identifying key nodes within these pathways, could lead to the identification of targets that are more suited for therapeutic intervention. High-throughput screening (HTS) is an ideal methodology to address these issues. but traditional methods were one dimensional whole-well cell assays, that used simplistic readouts for complex biological processes. They were unable to simultaneously quantify the many phenotypes observed in neurodegenerative diseases such as axonal transport deficits or alterations in morphology properties. This approach could not be used to investigate the dynamic nature of cellular processes or pathogenic events that occur in a subset of cells. To quantify such features one has to move to multi-dimensional phenotypes termed high-content screening (HCS). HCS is the cell-based quantification of several processes simultaneously, which provides a more detailed representation of the cellular response to various perturbations compared to HTS. HCS has many advantages over HTS, but conducting a high-throughput (HT)-high-content (HC) screen in neuronal models is problematic due to high cost, environmental variation and human error. In order to detect cellular responses on a 'phenomics' scale using HC imaging one has to reduce variation and error, while increasing sensitivity and reproducibility. Herein we describe a method to accurately and reliably conduct shRNA screens using automated cell culturing and HC imaging in neuronal cellular models. We describe how we have used this methodology to identify modulators for one particular protein, DJ1, which when mutated causes autosomal recessive parkinsonism. Combining the versatility of HC imaging with HT methods, it is possible to accurately quantify a plethora of phenotypes. This could subsequently be utilized to advance our understanding of the genome, the pathways involved in disease pathogenesis as well as identify potential therapeutic targets.     

Current advances of stem cell-based therapy for kidney diseases

Kidney diseases are a prevalent health problem around the world. Multidrug therapy used in the current routine treatment for kidney diseases can only delay disease progression. None of these drugs or treatments can reverse the progression to an end-stage of the disease. Therefore, it is crucial to explore novel therapeutics to improve patients’ quality of life and possibly cure, reverse, or alleviate the kidney disease. Stem cells have promising potentials as a form of regenerative medicine for kidney diseases due to their unlimited replication and their ability to differentiate into kidney cells in vitro. Mounting evidences from the administration of stem cells in an experimental kidney disease model suggested that stem cell-based therapy has therapeutic or renoprotective effects to attenuate kidney damage while improving the function and structure of both glomerular and tubular compartments. This review summarises the current stem cell-based therapeutic approaches to treat kidney diseases, including the various cell sources, animal models or in vitro studies. The challenges of progressing from proof-of-principle in the laboratory to widespread clinical application and the human clinical trial outcomes reported to date are also highlighted. The success of cell-based therapy could widen the scope of regenerative medicine in the future.     

Pathophysiology and spectrum of diseases caused by defects in lymphocyte cytotoxicity

In experimental settings, lymphocyte cytotoxicity has been recognized as a central mechanism for immune defense against infected and neoplastic cells. More recently, molecular determinants of lymphocyte cytotoxicity have been identified through studies of rare, inherited hyperinflammatory and lymphoproliferative syndromes that include hemophagocytic lymphohistiocytosis (HLH). These studies have unraveled a set of genes pivotal for the biogenesis and directed release of perforin-containing lysosomes that mediate target cell killing, in addition to other pathways including Fas that also contribute to induction of cell death. Furthermore, studies of such human primary immunodeficiencies have highlighted non-redundant roles of perforin for maintenance of immune homeostasis. Besides providing mechanistic insights to lymphocyte cytotoxicity, studies of individuals with rare hyperinflammatory diseases are highlighting the relevance of lymphocyte cytotoxicity to more common human diseases. It is increasingly recognized that mutations abrogating lymphocyte cytotoxicity not only cause HLH, but also are associated with susceptibility to cancer and autoimmune syndromes. In addition, patients may initially be present with neurological symptoms or severe infectious disease masquerading as variable immunodeficiency syndrome. Here, we highlight new knowledge regarding the molecular mechanisms regulating lymphocyte cytotoxicity and review how mutations in genes associated with HLH cause disease. We also discuss the wider implications of impairments in lymphocyte cytotoxicity for human disease predisposition.     

Tumor-induced immune dysfunction

Immune system-based approaches for the treatment of malignant disease over the past decades have often focused on cytolytic effector cells such as cytotoxic T lymphocytes (CTL), and natural killer (NK) cells. It has also been demonstrated that tumor-bearing mice can be cured using a wide variety of approaches, some of which involve cytokine-mediated enhancement of CTL and NK cell activity. However, the apparent success in mice stands in contrast to the current situation in the clinic, wherein only a minority of patients have thus far benefited from CTL- or NK cell-based antitumor approaches. The underlying causes of tumor-associated immune suppression of CTL and NK cell activity are discussed, and features of interest shared with HIV infection, leprosy, and rheumatoid arthritis are also be mentioned. Remarkable and very recent observations have shed more light upon the causes of dysfunctional alterations in CTL and NK cells often associated with these diseases, that in turn have suggested new immunotherapeutic approaches for cancer and infectious disease. Received: 20 March 1999 / Accepted: 3 May 1999     

MicroRNAs as important players in regulating cancer through PTEN/PI3K/AKT signalling pathways

Cancer being the leading cause of mortality has become a great threat worldwide. Current cancer therapeutics lack specificity and have side effects due to a lack of understanding of the molecular mechanisms and signalling pathways involved in carcinogenesis. In recent years, researchers have been focusing on several signalling pathways to pave the way for novel therapeutics. The PTEN/PI3K/AKT pathway is one of the important pathways involved in cell proliferation and apoptosis, leading to tumour growth. In addition, the PTEN/PI3K/AKT axis has several downstream pathways that could lead to tumour malignancy, metastasis and chemoresistance. On the other hand, microRNAs (miRNAs) are important regulators of various genes leading to disease pathogenesis. Hence studies of the role of miRNAs in regulating the PTEN/PI3K/AKT axis could lead to the development of novel therapeutics for cancer. Thus, in this review, we have focused on various miRNAs involved in the carcinogenesis of various cancer via the PTEN/PI3K/AKT axis.     

Role of quality control pathways in human diseases involving protein misfolding

Advances in connecting phenotype to genotype have led to new insights regarding the basis of human disease. Many inherited diseases are now known to arise due to specific mutations within a gene that then lead to a protein product unable to assume a stable conformation within the cell. Cellular machineries serving as "quality control monitors" recognize and target such abnormally folded proteins for rapid destruction. As a consequence, specific biochemical pathways requiring the protein of interest are adversely affected and lead to the disease phenotype. Yet in other cases, upon its misfolding the particular protein quickly aggregates, leading to the formation of inclusion bodies that eventually lead to cell demise. In what follows I discuss some classic examples of human diseases known to arise due to mutations that lead to altered protein folding, abnormal protein maturation and/or protein aggregation. In many cases simply altering the protein folding environment within the cell, via molecular or pharmacological approaches, can effectively rescue the maturation and stability of the mutant protein and thereby reduce the onset and/or progression of the disease phenotype. These new insights regarding the mechanisms underlying the disease phenotype, as well as new approaches to correct the protein folding defect, will undoubtedly prove to have a tremendous impact on clinical medicine.